Dendrimeric-like hexadecahydroxylated zinc phthalocyanine

Article abstract of DOI:10.1142/S1088424613500508

The design of a dendrimeric-like diglycerol-tetrasubstituted Zn(II) phthalocyanine resulted in a remarkably water-soluble compound due to the presence of 16 hydroxyls. Several parameters relevant to evaluate the photodynamic efficiency of a potentiel photosensitizer such as: aggregation behavior, fluorescence properties, singlet oxygen generation, binding to a carrier protein model (Bovine Serum Albumin) and partition coefficient have been measured. Biocompatibility was demonstrated by dark cytotoxicity in in vitro experiments. The absence of phototoxicity can be explained by an elevated hydrophilicity. All the collected data have confirmed that this new substitution pattern is promising to be used on phthalocyanines aiming at being photodynamic therapy agents.

Full text of DOI:10.1142/S1088424613500508

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2013; 17: 596–603
DOI: 10.1142/S1088424613500508
Published at http://www.worldscinet.com/jpp/
Dendrimeric-like hexadecahydroxylated zinc phthalocyanine.
Synthesis and evaluation of photodynamic efficiency
a
b,c,d,e
b,d,e,f
a
Serkan Alpugan , Guillaume Garcia
, Florent Poyer
, Mahmut Durmu s¸ ,
b,c,d,e◊
a◊
a◊
Philippe Maillard
, Vefa Ahsen and Fabienne Dumoulin*
a
Gebze Institute of Technology, Department of Chemistry, P.O. Box 141, Gebze, 41400 Kocaeli, Turkey
Institut Curie, Section de Recherche, Bât 110-112, Centre Universitaire, F-91405 Orsay, France
UMR 176 CNRS, Bât 110, Centre Universitaire, F-91405 Orsay, France
Université Paris-Sud, Centre Universitaire, F-91405 Orsay, France
CNRS GDR 3049 PHOTOMED, UMR 5623 Université Paul Sabatier, F-31062 Toulouse Cedex 9, France
U759 INSERM, Bât 112, Centre Universitaire, F-91405 Orsay, France
b
c
d
e
f
Dedicated to Professor Özer Bekaroğlu on the occasion of his 80th birthday
Received 8 March 2013
Accepted 31 March 2013
ABSTRACT: The design of a dendrimeric-like diglycerol-tetrasubstituted Zn(II) phthalocyanine
resulted in a remarkably water-soluble compound due to the presence of 16 hydroxyls. Several parameters
relevant to evaluate the photodynamic efficiency of a potentiel photosensitizer such as: aggregation
behavior, fluorescence properties, singlet oxygen generation, binding to a carrier protein model (Bovine
Serum Albumin) and partition coefficient have been measured. Biocompatibility was demonstrated by
dark cytotoxicity in in vitro experiments. The absence of phototoxicity can be explained by an elevated
hydrophilicity. All the collected data have confirmed that this new substitution pattern is promising to be
used on phthalocyanines aiming at being photodynamic therapy agents.
KEYWORDS: glycerol, phthalocyanine, photosensitizer, photodynamic therapy.
were elaborated to overcome the first drawbacks of the
INTRODUCTION
®
®
clinically used Photofrin and Visudyne , which are
badly characterized and exists under mixture forms.
Most of current research efforts are directed towards
the elaboration of third-generation photosensitizing
systems, such as theranostics combining imaging and
photodynamic agents. The development of optimized
second-generation photosensitizers is likely to be useful
for further conception of these advanced systems.
Due to their photophysical and photochemical pro-
perties, phthalocyanines are among the most attractive
tetrapyrrolic photosensitizing derivatives. Their maxi-
mum absorption is largely red-shifted compared to
porphyrins, and phthalocyanines are much more stable
than bacteriochlorins. Liposomal formulation of Zn
phthalocyanine (CGP55847) [2], Pc4 [3] and Photosens
Photodynamic therapy is
a
cancer treatment
alternative or complementary to surgery, radiotherapy
and chemotherapy. It is based on the interaction of
three factors: light at appropriate wavelength, molecular
oxygen and photosensitizer. The photosensitizer excited
by light returns to its ground state by transferring
the energy of its excited triplet to molecular oxygen,
subsequently converted into its toxic singlet species.
The resulting oxidative effect damages cell component,
leading to cell death. In the last four decades, much effort
led to significantly optimized photosensitizers. In order
to gather the ideal properties of photosensitizers [1] on
single molecule, second-generation photosensitizers
◊
SPP full member in good standing
[4] (a mixture of sulfonated aluminum phthalocyanines
*
Correspondence to: Fabienne Dumoulin, email: fdumoulin@
developed in Russia) are the best examples of phthalo-
cyanines clinically used or on advanced trial stages.
gyte.edu.tr, fax: +90 262-605-3101, tel: +90 262-605-3106
Copyright © 2013 World Scientific Publishing Company

DENDRIMERIC-LIKE HEXADECAHYDROXYLATED ZINC PHTHALOCYANINE
597
Nevertheless, except these latest examples,
phthalocyanines remain the poor relation
of tetrapyrrolic photosensitizers, probably
mainly due to their unjustified reputation
of poor water solubility. There are actually
a broad range of pathways to confer them
water-solubility [5]. Besides, phthalocyanines
exhibit most of the properties desired on
photosensitizers for PDT: a near-IR absorption
in the therapeutic window allowing deeper
tissue penetration, which is sought for most of
the treatments (except those of oesophagus and
ORL cases), no dark phototoxicity, suitable
body clearance, excellent stability especially
compared to bacteriochlorins absorbing
in the same range of wavelength, reproducible
syntheses in satisfying yields. Among water-
solubilizing substitution patterns, non-ionic
ones were developed after ionic ones but
are expected to allow better cell uptake and
different biodistributions. Non-ionic sub-
stitution patterns are based on polyoxo and
polyhydroxylated moieties. Among them,
symmetrical glycerol substitution gave
promising results, especially in non-peripheral
position [6]. Since then, many of our efforts
tendedtotheoptimizationofthephotodynamic
efficiency of glycerol-substituted phthalo-
cyanines, either by the introduction of
carbohydrates, grafting onto nanoparticles,
or tailored variations of the amphiphilicity of
the phthalocyanines. In the present work, we
focused on the development of a new glycerol-
based substituent: diglycerol moieties leading
to the dendrimeric-like water-soluble hexadec-
ahydroxylated Zn phthalocyanine 1 (Fig. 1),
the aim to increase the biocompatibility of this
potential new photosensitizer by conferring
suitable hydrophilicity and amphiphilicity.
The photodynamic potential of 1 was
investigated here, through the determination
of its photophysical and photochemical
parameters, as well as its in vitro dark
toxicity and phototoxicity.
OH
HO
HO
HO
O
O
O
OH
HO
O
N
N
HO
HO
N
Zn
N
O
N
N
OH
OH
O
O
O
O
N
N
OH
OH
O
O
HO
O
OH
OH
HO
1
Fig. 1. Structure of the dendrimeric-like diglycerol-substituted phthalo-
cyanine 1
O
OH
Cl
O
O
O
O
NaOH
+
OH
O
O
O
O
2
3
4
O
O
K2CO3
DMF
O N
2
CN
CN
O
O
O
O
4
+
O
CN
CN
5
6
Scheme 1. Preparation of phthalonitrile 6
symmetrically substituted secondary alcohols [7], and
already applied to solketal [8]. We slightly modified
the conditions, using solketal itself as the reaction bulk
solvent and sodium hydroxide as the base. Disolketal 4
was obtained in high yield (79%) and in large scale: up to
RESULTS AND DISCUSSION
1
20 g could be obtained in a single reaction. Nucleophilic
Syntheses and characterizations
displacement of the nitro group on 5 was achieved in
relatively moderate yield (44%), can be considered normal
as this is a secondary alcoholate, quite sterically hindered.
The cyclotetramerization of 6 was achieved equally
in dimethylaminoethanol or pentanol/DBU systems in
the presence of zinc acetate to obtain the corresponding
protected phthalocyanine 7 in high yield (46%). The
acidic hydrolysis of the isopropylidene protecting groups
Phthalocyanine 1 was obtained in four steps. Starting
from solketal 2 and epichlorhydrin 3, disolketal 4 was
obtained and grafted on 4-nitrophthalonitrile 5 to yield
phthalonitrile 6. Subsequent cyclotetramerization of 6
and final acidic hydrolysis yielded 1 (Scheme 1).
The opening of epichlorhydrin by two molecules of
alcohols is a well-known reaction providing access to
Copyright © 2013 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2013; 17: 597–603

598
S. ALPUGAN ET AL.
Table 1. Q-band electronic absorption data for 1
Solvent
lmax Q-band, nm
Log e
5.18
DMSO
DMF
water
PBS
684
682
4.67
637, 675
638, 679.5
678
4.47, 4.22
4.64, 4.39
4.60
MeOH
was performed using trifluoroacetic acid, and the final
unprotected phthalocyanine was obtained pure in 80% yield
after reprecipitation from ethanol in dichloromethane and
ethylacetate, followed by thorough washing with acetone.
Photophysics and photochemistry
Parameters which allow to estimate the potential of a
molecule as a photosensitizer for photodynamic therapy
havebeendetermined:preliminarygroundstateabsorption
measurements and aggregation state in water permitted
to select the best solvent for further investigation, such as
the fluorescence and singlet oxygen generation behavior.
In addition, study of the photodegradation of 1, as well
as its binding to a carrier protein model were performed,
to provide a full picture of the potential use of 1 as a
photodynamic therapy agent on a photophysical and
photochemical point of view.
Fig. 2. (Top) electronic absorption spectra of 1 in DMSO, DMF,
methanol, water and phosphate buffer, at concentration: 10 mM,
and (bottom) linearity of the absorption in DMSO at different
concentrations
Ground state electronic absorption and aggregation
behavior in water. The shape of the electronic absorption
spectrum of phthalocyanine gives precious indication
of its aggregation state. This information is crucial for
further measurements that must be conducted in non-
aggregated states to avoid misleading interpretations. The
electronic absorption spectra of 1 were recorded in polar
solvents: DMSO, DMF, methanol, water and phosphate
buffer solution. Extinction coefficient values and Q-band
maximum absorption wavelengths in these solvents are
summarized in Table 1. Corresponding spectra at 10 mM
concentrations are presented in Fig. 2 (top). These results
prompted us to select DMSO as the solvent in which
to perform further photophysical and photochemical
measurements, as it appeared to be a solvent in which 1 is
not aggregated at all (see Fig. 2 (bottom) for the linearity
of the absorption towards the concentration), thus
avoiding artefact due to aggregation likely to interfere
with the interpretation of the measurements.
Fig. 3. Electronic absorption spectra of 1 in water and upon
addition of increasing quantities of Triton X100
In water, phthalocyanine is quite aggregated, despite
observations with naked eye showing that 1 readily
dissolves in water. To confirm that the shape of the Q-band
in water is due to aggregation, Triton X100, a detergent
inhibiting the aggregation of molecules in aqueous media,
was added. The Q-band significantly became sharp,
indicating the monomerization of the phthalocyanine
hydrophilicity compared to Lipinski rule [9]. The high
solubility in aqueous media, despite the aggregation,
is promising for further biological experiments. The
behavior in PBS, more similar to biological media than
pure water and in which phthalocyanine 1 is even less
aggregated than in water, is a good indication that further
biological works are relevant.
(
Fig. 3). The water/octanol partition coefficient was
determined to be 0.04 (log P -1.4), reflecting its elevated
Copyright © 2013 World Scientific Publishing Company
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DENDRIMERIC-LIKE HEXADECAHYDROXYLATED ZINC PHTHALOCYANINE
599
Fluorescence spectroscopy, quantum yields and
lifetimes. A photosensitizer exhibiting some fluorescence
properties allows to follow its in vitro repartition,
and Zn(II) phthalocyanines are even now used as
fluorescence probes [10]. As the emission of fluorescence
is concurrent with the singlet oxygen generation, an
ideal photosensitizer for PDT must have a rather low
fluorescence, as most of Zn(II) phthalocyanines. The two
important parameters to measure are the fluorescence
quantum yield and lifetime. These values for 1, as
well as those of the reference compound used for this
study (unsubstituted ZnPc) are summarized in Table 2.
The emission of 1 proved to be of a satisfactory level
Fig. 4. Decrease of DPBF absorption upon singlet oxygen
generation by 1. Concentration 10 mM. Inset: plots of DPBF
absorbance vs. time
(
F 0.17) for further biological investigations, such as
F
quantification of cell uptake and subcellular localization.
Singlet oxygen generation and photodegradation.
A satisfying generation of singlet oxygen is a key
property of a good photosensitizer, and reflected by
the singlet oxygen generation quantum yield FD.
The disappearance of a singlet oxygen quencher
upon irradiation allows to calculate this yield, using
irradiation in conditions close to those of biological
experiments. On the other hand, the generation of
singlet oxygen is likely to damage the photosensitizer
itself. This phenomenon called photodegradation must
be measured as well to estimate the photostability
of the photosensitizer. An aered solution of the
photosensitizer is irradiated with stronger light and
without singlet oxygen quencher, and subsequent
modification of the UV spectrum reflects the photode-
gradation of the photosentizer. Relevant data for
the singlet oxygen generation and photodegradation
are summarized in Table 3. 1,3-Diphenylisobenzofuran
Table 4. Binding and fluorescence quenching data for interaction
of BSA with 1 in PBS
BSA
SV
5
-1
13
-1 -1
-6
-1
Compound
1
K
/10 , M k /10 , M .s K /10 , M
n
q
b
1.12
1.12
2.76
1.07
Thephotodegradationof1isreflectedbytheabsorption
changes upon irradiation (Table 3). The photodegradation
-5
is time-linear and has a quantum yield F of 5.3 × 10 . 1
d
can be considered as moderately stable, which is a good
point for PDT photosensitizer as it helps to avoid any
accumulation in organisms.
Interactions with a carrier protein model. Bovine
Serum Albumin (BSA) is a carrier protein model
frequently used to estimate the potential efficacy of new
medicines, as their blood circulation is directly related to
their ability to bind and unbind to carrier proteins.
Fluorescence of the BSA at 348 nm due to its
tryptophane moieties is quenched when the protein is
bound to quencher, such as for example phthalocyanine.
Following the fluorescence of BSA upon the addition of
phthalocyanine allows the quantification of the binding, as
it decreases linearly upon the addition of phthalocyanine,
until the saturation of the BSA (Fig. 5) by 1. Calculations
of relevant data are summarized in Table 4.
(
DPBF) is used as the organosoluble singlet oxygen
quencher, and its quenching upon singlet oxygen
generation by 1, reflected by the diminution of its proper
maximum absorption at 417 nm is illustrated in Fig. 4.
The diminution of the absorption is time-linear. The
corresponding singlet oxygen generation yield is 77%,
higher than the reference unsubstituted phthalocyanine.
This high value perfectly fits the expectations of a good
photosensitizer.
The slope of the plots (Fig. 5, top inset) gave Stern–
Table 2. Fluorescence data for 1 in DMSO
Volmer quenching constants (K ) value of 1. Using the
SV
reported fluorescence lifetime of BSA (10 ns) [13], the
Compound Excitation Emission Stokes shift FF tF, ns
bimolecular quenching constant (k ) was determined (see
q
1
687
672
694
682
10
10
0.17 1.41
0.20 1.22
relevant equation in Supporting information). This value
1
3
-1 -1
ZnPc [11]
is of the order of 10 M .s , which exceed the proposed
1
0
-1 -1
value of 10 M .s for diffusion-controlled (dynamic)
quenching (according to the Einstein–Smoluchowski
approximation) at room temperature [14]. This confirms
that the mechanism of BSA quenching by 1 is not
diffusion-controlled and that the quenching is static,
not dynamic. The slope of the plot of Fig. 5 (bottom),
gave n values and their intercepts of these plots gave Kb
Table 3. Singlet oxygen generation and
photodegradation data for 1 in DMSO
-5
Compound
FD
F (10 )
d
1
0.77
0.67
5.30
2.61
ZnPc [11a, 12]
values. The values of K and n are typical of MPc-BSA
b
Copyright © 2013 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2013; 17: 599–603

600
S. ALPUGAN ET AL.
Fig. 6. In vitro dark toxicity (blue) and phototoxicity (red) of 1
against Y79 (top) and HT-29 (bottom) cell lines: survival rate
(
ratio to untreated cells
Fig. 5. Top: fluorescence emission spectral changes of BSA (C =
3
0 mM) on addition of varying concentrations of 1 in PBS. [1]:
A = 0, B = 1.66 mM, C = 3.33 mM, D = 5 mM, E = 6.66 mM, F =
.3 mM, G = saturated with 1. Bottom: determination of 1-BSA
binding constant (and number of binding sites on BSA). [BSA] =
too hydrophilic to croos the amphiphilic cell membrane
and be internalized. Another possibility is the release
of the photosensitizer during the incubation time. A
third assumption could be that even if 1 is internalized,
it remains aggregated inside the cell, the aggregation
preventing the electronic events required for the singlet
oxygen generation leading to the cell death, or that it
does not go to the appropriate organelles. Different
incubation times and internalization and/or subcellular
localization attemps are being performed using confocal
microscopy.
8
-5
-6
-6
-6
3.00 × 10 M and [1] = 0, 1.66 × 10 , 3.33 × 10 , 5.00 × 10 ,
-6 -6
6.66 × 10 , 8.33 × 10 M in PBS
interactions in aqueous solutions [15]. n values of
near unity suggest that complex 1 forms 1:1 adducts
with BSA. The decrease in the intrinsic fluorescence
intensity of tryptophan with 1 concentration indicates
that this complex readily bind to BSA, which implies
that the phthalocyanine reaches the subdomains where
tryptophan residues are located in BSA.
EXPERIMENTAL
In vitro dark toxicity and phototoxicity
Material and methods
A mandatory property of a good photosensitizer is
its dark toxicity, as it must exhibit toxicity only upon
irradiation. It was evaluated against two cell lines, Y79
and HT-29, after 24 h of incubation (Fig. 6). The survival
rate, compared to the untreated cells, remains complete
even at elevated concentrations, up to 80 mM, which is
a good indication of the suitability of phthalocyanine 1
as a photodynamic therapy agent. Further tests are being
carried out to determine its cell uptake before evaluating
its phototoxicity.
4-Nitrophthalonitrile (5) was prepared following
the literature [16]. All reaction solvents were dried and
purified as described by Perrin and Armarego [17].
Optical spectra in the UV-visible region were recorded
with a Shimadzu 2101 UV spectrophotometer using
a 1 cm path length cuvette at room temperature. The
mass spectra were recorded on a LCQ-ion trap (Thermo
Finnigan, San Jose, CA, USA), equipped with an ES
(Electrospray) source and MALDI (matrix assisted laser
desorption ionization) BRUKER Microflex LT using
Upon irradiation, no cell death could be observed,
reflecting an absence of phototoxicity (Fig. 6). This
is not correlated with the singlet oxygen generation
ability. Several hypotheses may be emitted: 1 may be
1
13
2,5-dihydroxybenzoic acid as matrix. H and C NMR
spectra were recorded in deuterated solvents solutions on
a Varian 500 MHz spectrometer.
Copyright © 2013 World Scientific Publishing Company
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DENDRIMERIC-LIKE HEXADECAHYDROXYLATED ZINC PHTHALOCYANINE
601
1
Syntheses
of a dark blue wax. C H N O Zn, MW 1851.38. H
92 120 8 28
NMR (DMSO-d ): d, ppm 9.23 (m, 4H, aromatics), 8.95
6
1
,3-bis((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-
(
m, 4H, aromatics), 7.83 (m, 4H, aromatics), 5.29 (m,
propan-2-ol (4). Sodium hydroxide (38 g, 0.95 mol)
was added portion wise to solketal 2 (235 mL, 250 g,
.9 mol). The slurry was stirred at 80 °C for 2 h and
4
2
H, CH-OPc), 4.31 (m, 8H, CH), 4.05, 3.69 (2 m, 48H,
4 CH ), 1.34, 1.24 (2 m, 48H, 16 CH ). MS (MALDI-
2
3
1
+
TOF): m/z 1849.75 found 1850.87 [M + H] . Anal. calcd.
for C H N O Zn, 2H O: C 58.54, H 6.62, N 5.94%.
allowed to cool down. Epichlorhydrine (44 g, 38 mL)
was then added drop by drop at room temperature. The
resulting reaction mixture was stirred at 110 °C overnight.
After cooling to room temperature, the reaction mixture
was diluted with water and extracted three times by
dichloromethane. Combined organic phase were dried on
sodium sulphate, filtered, concentrated, and the excess of
solketalwasdistilledundervacuum.Theexpectedproduct
was purified by silica gel column chromatography, eluent
hexane/ethyl acetate (1/2, v/v), yielding 120 g (79%) of a
colorless transparent oil. C H O , MW 320.38. H NMR
CDCl ): d, ppm 4.26–4.33 (m, 2H), 4.06–4.09 (m, 2H),
.99–4.02 (m, 1H, CHOH), 3.73–3.77 (m, 2H), 3.53–3.64
m, 8H, 2 CH OCH ), 1.45 (bs, 6H, 2 CH ), 1.39 (bs, 6H,
CH3). C NMR: d, ppm 109.09, 74.38, 74.36, 72.55,
2.52, 72.51, 72.15, 69.03, 66.24, 66.22, 26.42, 25.07.
9
2
120
8
28
2
Found C, 58.72; H, 6.50; N, 5.98%.
(3),9(10),16(17),23(24)-tetrakis-[1,3-bis(2,3-
2
dihydroxypropoxy)propan-2-yloxy]phthalocyaninato
Zn(II) (1). Phthalocyanine 7 (100 mg, 54 mmol) was
stirred at room temperature in aqueous 90% trifluoro-
acetic acid (5 mL) for 5 h, then the reaction mixture was
concentrated under vacuum. The resulting solid was
thoroughly washed with dichloromethane, ethyl acetate,
acetone,andfinallydissolvedinmethanolandreprecipitated
in ethyl acetate, yielding 66 mg (80%) of a dark blue solid.
1
1
5
28
7
(
3
(
2
7
3
1
C H N O Zn, MW 1530.87. H NMR (DMSO-d6):
68
88
8
28
d, ppm 9.31 (m, 4H, aromatics), 9.25 (m, 4H, aromatics),
.83 (m, 4H, aromatics), 5.27, 5.21 (m, 4H, CH–OPc), 4.69
2
2
3
7
1
3
(
m, 8H, CH), 3.93 (m, 48H, 24 CH ). MS (MALDI-TOF):
2
+
m/z 1528.50 found 1529.873 [M + H] , 1552.51 [M + H +
Na] . Anal. calcd. for C H N O Zn: C 53.35, H 5.79, N
MS (ESI): isotopic cluster peaking at m/z 343.17 found
43.25 [M + Na] . Anal. calcd. for C H O : C, 56.23; H,
+
+
68 88
8
28
3
8
1
5
28
7
7.32%. Found C, 53.72; H, 5.63; N, 7.11%.
.81%. Found C, 56.28; H, 8.74%.
-{1,3-bis[(2,2-dimethyl-1,3-dioxolan-4-yl)-
methoxy]propan-2-yloxy}phthalonitrile (6). Disolketal
4
Water/octanol partition coefficient calculation
4
(4.2 g, 13 mmol), 4-nitrophthalonitrile 5 (16 mmol,
An excess of 1 was stirred in a solution of octanol and
water in order to obtain saturated phases. The solution
was filtered. An aliquot of each phase was 20-fold diluted
by DMSO. The electronic absorption of each solution (in
which the phthalocyanine is monomeric) was measured.
The coefficient partition value was obtained by dividing
the DMSO-diluted octanol phase absorbance by the
DMSO-diluted water phase absorbance.
2.72 g, 1.2 eq) and potassium carbonate (100 mmol, 15 g)
were stirred overnight at 80 °C in dry DMF. After cooling
to room temperature, the reaction mixture was filtered,
diluted with water and extracted by ethyl acetate. The
organicphasewasthenwashedwithwater,driedonsodium
sulphate, filtered and concentrated. The expected product
was purified by silica gel column chromatography, eluent
hexane/ethyl acetate (1/2, v/v), yielding 2.56 g (44%) of
1
a pale yellow wax. C H N O , MW 446.49. H NMR
23
30
2
7
Photophysics and photochemistry
(
CDCl ): d, ppm 7.67 (d, 1H, aromatic CH), 7.40 (dd,
3
1
1
3
H, aromatic CH), 7.30 (dd, 1H, aromatic CH), 4.49 (m,
Detailsregardingthephotophysicalandphotochemical
experiments are provided in the supplementary material
(see Supporting information).
H, CH–O–Ar), 4.21, 4.00 (2m, 4H, 2 CH -Osolketal),
.47–3.78 (2 m, 10H, 2 CH –CH–CH ), 1.39 (bs, 6H, 2
2
2
2
13
CH ), 1.34 (bs, 6H, 2 CH ). C NMR (CDCl ): d, ppm
3
3
3
1
1
7
62.08, 135.32, 121.11, 120.69, 117.29, 115.60, 109.57,
07.55, 78.21, 74.57, 74.23, 72.66, 72.61, 71.06, 71.02,
0.84, 70.81, 66.32, 26.71, 25.25. MS (ESI): isotopic
Cell culture conditions and in vitro photocytotoxicity
The suspension cell line Y79 (human retinoblastoma
cells) and adherent cell line HT-29 (human colorectal
adenocarcinoma cells) were obtained from the American
Type Culture Collection (Rockville, MD, USA, HTB-18
and HTB-38, respectively). Y79 and HT-29 cells were
culturedinDulbecco’smodifiedEagle’smedium(DMEM,
Eurobio, les Ulis, France) Glutamax™ supplemented with
20% and 10% fetal calf serum (FCS, Eurobio) respectively
+
cluster peaking at m/z 469.19 found 469.32 [M + Na] .
Anal. calcd. for C H N O : C, 61.87; H, 6.77; N, 6.27%.
Found C, 61.31; H, 6.72; N, 6.81%.
(3),9(10),16(17),23(24)-tetrakis-{1,3-bis[(2,2-
dimethyl-1,3-dioxolan-4-yl)methoxy]propan-2-
23
30
2
7
2
yloxy} phthalocyaninato Zn(II) (7). Phthalonitrile 6
(
250 mg, 0.56 mmol) and zinc acetate (0.28 mmol)
were stirred in refluxing N,N′-dimethylaminoethanol
5 mL) overnight. The reaction mixture was cooled down
and antibiotics in humidified atmosphere under 5% CO
2
5
(
in air at 37 °C. For Y79 cells and HT-29 cells, 5.10 and
4
and poured into water. The blue solid was filtered and
purified on a silica gel column chromatography, eluent
hexane/ethyl acetate (1/10, v/v), yielding 120 mg (46%)
5.10 cells/well in 1 mL respectively, were seeded into
24-microwell plates with the appropriate culture medium.
After 2–3 h of incubation at 37 °C for Y79 and 24 h for
Copyright © 2013 World Scientific Publishing Company
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602
S. ALPUGAN ET AL.
HT-29, tested compound, in DMSO solution, were added
in the dark at a final concentration ranging from 1.5 to
supplementary information. Supplementary material is
available free of charge via the Internet at http://www.
worldscinet.com/jpp/jpp.shtml.
75 mM in duplicate (1.5, 3, 7.5, 15, 30, 75 mM). Two plates
of each cell line were realized, one for dark cytotoxicity
and the other for photocytotoxicity. Illuminated control
cells received 7.5 mL of DMSO free of dye. After 24 h
of incubation with PS at 37 °C in the dark, the cells were
washed with phosphate buffered saline (PBS, Eurobio)
and fresh medium free of drug was added. Illumination
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CONCLUSION
Phthalocyanine 1 reported here possesses numerous
features desired for photodynamic therapy agents. The
molecular design lead to an elevated water-solubility.
Relevant fluorescence investigations showed sufficient
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in the conditions used. Anyway, the present works
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