Photochemical and photophysical properties of phthalocyanines modified with optically active alcohols

Article abstract of DOI:10.3390/molecules200813575

Three phthalocyanine derivatives were synthesized and characterized: one modified with a racemic mixture of 1-(4-bromophenyl)ethanol and two other macrocycles modified with each one of the enantioenriched isomers (R)-1-(4-bromophenyl)ethanol and (S)-1-(4-bromophenyl)ethanol. The compounds were characterized by1 H-NMR spectroscopy, mass spectrometry, UV-Vis absorption, and excitation and emission spectra. Additionally, partition coefficient values and the quantum yield of the generation of oxygen reactive species were determined. Interestingly, the phthalocyanine containing a (R)-1-(4-bromophenyl)ethoxy moiety showed higher quantum yield of reactive oxygen species generation than other compounds under the same conditions. In addition, the obtained fluorescence microscopy and cell viability results have shown that these phthalocyanines have different interactions with mammary MCF-7 cells. Therefore, our results indicate that the photochemical and biological properties of phthalocyanines with chiral ligands should be evaluated separately for each enantiomeric species.

Full text of DOI:10.3390/molecules200813575

Molecules 2015, 20, 13575-13590; doi:10.3390/molecules200813575
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Photochemical and Photophysical Properties of Phthalocyanines
Modified with Optically Active Alcohols
Aline A. Ramos, Francisco B. Nascimento, Thaiza F. M. de Souza, Alvaro T. Omori,
Tânia M. Manieri, Giselle Cerchiaro and Anderson O. Ribeiro *
Centro de Ciências Naturais e Humanas—CCNH, Universidade Federal do ABC—UFABC,
Avenida dos Estados, 5005, Bairro Bangu, Santo André—SP 09210-190, Brasil;
E-Mails: linearamos@gmail.com (A.A.R.); francisco.comunic@gmail.com (F.B.N.);
thaizamenegassi@gmail.com (T.F.M.S.); alvaro.omori@ufabc.edu.br (A.T.O.);
tania.manieri@gmail.com (T.M.M.); gicerchiaro@gmail.com (G.C.)
*
Author to whom correspondence should be addressed; E-Mail: anderson.ribeiro@ufabc.edu.br;
Tel.: +55-11-4996-0191.
Academic Editor: Giuseppe Mele
Received: 26 May 2015 / Accepted: 2 July 2015 / Published: 24 July 2015
Abstract: Three phthalocyanine derivatives were synthesized and characterized: one
modified with a racemic mixture of 1-(4-bromophenyl)ethanol and two other macrocycles
modified with each one of the enantioenriched isomers (R)-1-(4-bromophenyl)ethanol and
1
(
S)-1-(4-bromophenyl)ethanol. The compounds were characterized by H-NMR spectroscopy,
mass spectrometry, UV-Vis absorption, and excitation and emission spectra. Additionally,
partition coefficient values and the quantum yield of the generation of oxygen reactive species
were determined. Interestingly, the phthalocyanine containing a (R)-1-(4-bromophenyl)ethoxy
moiety showed higher quantum yield of reactive oxygen species generation than other
compounds under the same conditions. In addition, the obtained fluorescence microscopy
and cell viability results have shown that these phthalocyanines have different interactions
with mammary MCF-7 cells. Therefore, our results indicate that the photochemical and
biological properties of phthalocyanines with chiral ligands should be evaluated separately
for each enantiomeric species.
Keywords: chiral phthalocyanines; enzymatic resolution; photodynamic therapy;
photosensitizer; dyes

Molecules 2015, 20
. Introduction
Phthalocyanines are a highly conjugated macrocycles with good thermal and chemical stability,
13576
1
properties that allow them to be applied in many different fields, such as use as photosensitizers in
photodynamic therapy [1].
However, because of the phthalocyanines’ planarity, a high self-aggregation in polar environments
is observed [2], which reduces their solubility and significantly affects their photochemical and
photophysical properties in these media. To circumvent this problem, the preparation and characterization
of substituted phthalocyanines is being broadly investigated [3,4]. The presence of a substituent on the
adjacent aromatic position could lead to a change in the phthalocyanine properties, such as its volume,
polarity and charge, and can therefore lead to changes in the aggregation process [5,6].
Several examples involving the synthesis and characterization of substituted phthalocyanines containing
chiral groups have been reported [7]. However, there is a lack of studies on chiral secondary alcohols
(
which can be elegantly obtained by biocatalysis) [8,9] as substituents.
The phthalocyanines modified with chiral secondary alcohols presented here are based on the
potential for obtaining different biological properties in structures modified with pure enantiomers.
In particular, there may be changes in their interaction with cells, toxicity, aggregation in biological
media and other properties, which can result in differences in their photodynamic activity, for example.
In photodynamic therapy, the photosensitizer is released to the target tissue and the region is exposed
to light of an appropriate wavelength [10]. Part of the energy is absorbed by the photosensitizer
and transferred to oxygen, generating reactive species that promotes cell death [11]. Consequently,
the solubility and the interaction with target cells are an important process to be considered for the
photosensitizer [12,13].
In this context, we describe the synthesis and characterization of phthalocyanines modified with
1
-(4-bromophenyl)ethanol, as both the racemic mixture and with each of its individual (R) or (S)
enantiomers and some biological studies to compare their efficiency as photosensitizers.
2
. Results and Discussion
2.1. Preparation and Structural Characterization
The first step of the synthesis (Scheme 1) is a well-known chiral resolution of 1-(4-bromophenyl)
ethanol [8]. Conditions used were similar to those of a published paper, which helped us to predict the
behavior of the isomers (R) and (S) in the chromatographic purification steps and to determine the
absolute configuration of the separated compounds (enantiomeric excess %ee
R
> 99 and %ee
S
= 98).
1
All synthesized structures (precursors and phthalocyanines 10–12) were confirmed by FTIR, H-NMR
1
and mass spectrometry. Figure 1 presents the structure of all synthesized macrocycles and the H-NMR
spectra obtained for phthalocyanine 10, as an example. The presence (and integration) of signals in
1
the region between 7 to 8 ppm in all H-NMR spectra confirmed the proportion of aromatic alcohol
derivative group attached to the phthalocyanine structure. The methylene and –CH– protons from alcohol
peripheral units are well defined in all spectra, with correct integrals, and were observed around 1.5
and 4.0 ppm, respectively. There are no significant differences in the positions and structure of peaks
between the spectra of optically active phthalocyanines and the racemic derivative one.

Molecules 2015, 20
13577
O
OH
O
CH3OH
K2CO3
(
R)
(
R)
O
OH
O
Br
Br
%ee > 99
3
5
O
MeOH
NaBH4
(
rac)
OH
+
CAL-B/ hexane
Br
Br
1
2
(
S)
Br
4
%ee = 98
O
R
O
R
N
CN
CN
CN
2
, 4 and 5
DMAE, 145°C, 8h
Zn(CH CO )
N
N
N
1
0 Pc - rac (63%)
DMF / K CO
N
Zn
N
11 Pc - S (77%)
12 Pc - R (60%)
2
3
3
2 2
O2N
*RO
CN
7
days
N
6
7
8
9
R = 2 (75%)
R = 4 (79%)
R = 5 (73%)
N
R
O
O
R
Scheme 1. Enzymatic resolution of 1-(4-bromophenyl)ethanol (1) and synthesis of
phthalocyanines containing chiral substituents.
1
0
11
12
7
.7
7.6
7.5
7.4
7.3
7.2
7.1
Chemical Shift (ppm)
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
Chem ic al Shift (ppm)
1
Figure 1. Chemical structure of all synthesized macrocycles and the H-NMR spectrum of
phthlalocyanine 10 in DMSO-d
6
.

Molecules 2015, 20
13578
The FTIR spectra of all compounds presented the absorption bands for C–H and C=C aromatic
stretching in the 3.000 and 1.600 cm region, the –CH
and –CH stretching between 2.900 to 2.850 cm−1
−
1
3
−1
and –C=N relative stretching in 1.340 and 1.220 cm , confirming the formation of the aromatic
phthalocyanine rings. Also, the ESI-TOF mass spectra of compounds presented a molecular ion peak at
1375.1 Da, what is in accordance with the proposed structures, as presented in Figure 2 for compound 10
as example.
1375.1
1
00
%
0
5
22.8
1797.2
Figure 2. ESI-TOF positive mode mass spectrum of phthlalocyanine 10.
In addition, the circular dicroism spectra of phthalocyanines 11and 12 (Figure 3) confirmed the
opposite optical activity properties of the aromatic macrocycle due to the presence of pure enantiomer
substituents [14].
5
A
0
-
5
B
3
00
400
500
600
700
W avelenght (nm)
Figure 3. Circular dichroism (CD) spectra of optically active phthalocyanines 11 (curve B)
and 12 (curve A).
2
.2. UV-Vis Behavior and Aggregation Processes
The absorption spectra of phthalocyanines 10, 11 and 12 presented absorption bands with maxima
around 350 nm and from 600 to 800 nm (Q bands), in DMSO (Table 1), which are typical for these
macrocycles in non-aggregated form [15,16]. It was observed that the maximum for the racemic
substituent are nearly the same as for the (R) and (S) configurations, which shows that, in non-aggregated
form (low concentrations in DMSO), the configuration of the alcohol moiety does not affect the
photophysical properties of the macrocycles.

Molecules 2015, 20
13579
Table 1. Absorption bands, quantum yield of fluorescence (Φ
F
) and of the generation of
oxygen reactive species (Φ ) for phthalocyanine synthesized (all in DMSO).
Δ
−
1
−1
Pc
λ
max (nm)
log Ɛ (cm ·M , 684 nm)
Log P
Φ
Δ
Φ
F
1
1
1
0
1
2
356, 615, 684
356, 615, 684
356, 615, 684
5.02
5.01
4.91
1.97 ± 0.01
2.37 ± 0.09
2.78 ± 0.02
0.67
0.65
0.76
0.10
0.10
0.12
However, in polar environments, the aggregation processes is intense, promoting a reduction in the
relative intensity of the Q bands and a red blue shift in the maximum (Figure 4). Our results have
shown that the substituents cannot completely prevent the aggregation process in intense polar media,
but this process is different for each phthalocyanine studied. Phthalocyanine 12, with a (R) resolution
chiral moiety, presented the absorption band around 680 nm with higher intensity related to the band
with maximum at 615 nm in up to 60% of water in DMSO. This higher intensity of the 680 nm band
indicates a major presence of monomers than oligomers at this polarity. The compounds 10 and 11,
however, presented a characteristic oligomeric species behavior in mixtures with 40% of water in
DMSO, with a maximum shifted to approximately 630 nm.
0
0
0
0
0
0
,25
,20
,15
,10
,05
,00
A
A
A
a
b
c
B
B
B
C
C
C
D
D
D
E
E
E
5
00
600
700
500
600
700
00
600
700
800
Wavelenght (nm)
Wavelenght (nm)
Wavelenght (nm)
Figure 4. UV-vis absorption spectra of compound 10 (a); 11 (b) and 12 (c), at same
concentration, in media with different proportions of DMSO and water (DMSO:water
1
00:0 (A), 80:20 (B), 60:40 (C), 40:60 (D) and 20:80 (E)).
This different behavior shows that the geometry of the substituent can influence the aggregation
process of the phthalocyanines, which is generally attributed to the self-facial arrangement of a range
of macrocycles [17]. In pure DMSO, the results also suggest differences in the aggregation process.
For different concentrations, the intensity of the maximum absorption band (684 nm) were plotted versus
the intensity of maximum fluorescence emission band (720 nm, λexc = 618 nm). For low absorption
−1
intensities (low phthalocyanine concentrations, from 0.1 to 2 µmol·L ), a linear relationship between
absorption and emission intensities were observed. However, for more concentrated solutions (absorption
maximum between 0.4 and 0.7), a nonlinear relationship were verified, suggesting the presence of
aggregation (Figure 5).
The phthalocyanine aggregation is due to J aggregates (head-to-tail aggregation) and H aggregates
(
face-to-face aggregation) [18,19], and the geometry of peripheral substituents can result in different
aggregation. Although not very discrepant, this difference can be sufficient to provide differences in
their photochemical and photophysical properties, and in their interaction with cells, such as MCF7.

Molecules 2015, 20
13580
900
800
700
600
500
400
300
200
100
0
0
.0
0.1
0.2
0.3
0.4
0.5
0.6
Absorption intensity at 684 nm
Figure 5. UV-vis absorption intensity at 684 nm and fluorescence emission intensity at
02 nm (λexc = 618 nm) under the same instrument conditions for compounds 10 (rac, ▲),
11, S, ●) and (12, R, ■), for different concentrations in DMSO, with higher stock solution
7
(
−1
5
μmol·L .
2.3. Generation of Oxygen Reactive Species and Fluorescence Quantum Yield
Another important aspect in the characterization of photosensitizers for photodynamic therapy is the
fluorescence quantum yield (Φ
F
) and its quantum yield (Φ ) of the generation of oxygen reactive
Δ
species under visible light irradiation [20,21]. To assess this property, we used an indirect method with
diphenylisobenzofuran (DPBF) as chemical quencher, comparing their rate of degradation promoted by
each of the synthesized phthalocyanine derivatives and by unsubstituted zinc(II)phthalocyanine (ZnPc)
under the same conditions [22–24]. The relationship between the rate of degradation of DPBF promoted
by each one of these phthalocyanine derivatives, under the same conditions, can be used as an indicator
of their photosensitizing properties. For this analysis, a mixture of each phthalocyanine (absorption
~
0.2 in 680 nm) and the DPBF (absorption ~0.9 in 418 nm) was irradiated with red LED lamp in
2
0 cycles of 6 s and the result obtained for compound 12 is showed in Figure 6 as example.
1
0
0
0
0
0
.0
.8
.6
.4
.2
.0
2
0 cycles of 6 seconds
3
00
400
500
600
700
800
Wavelength (nm)
Figure 6. UV-vis spectra of solution containing phthalocyanine (12) (absorption ~0.2 in
80 nm) and the DPBF (absorption ~0.9 in 418 nm), under constant irradiation by red LED
light, obtained after each 6 s period of irradiation.
6

Molecules 2015, 20
13581
The values obtained for Φ
Δ
in DMSO and in DMSO-water media differ according to the kind of
substituent in each structure, and are in accordance with the demonstrated different tendency of
macrocycles to form dimers and oligomers in polar environments. The results have shown that the
macrocycle with the (R) enantiomer substituent presents a higher quantum generation yield, which is in
accordance with its lower tendency to form aggregates under the same conditions compared to the
others. This behavior is also observed in more polar environments, like in mixtures of DMSO:water
(
20:80 to 80:10), where the (R) compound also presents a greater rate of decomposition of DPBF
compared to others at same conditions.
2.4. In Vitro Study
The interaction of phthalocyanines with cells in culture was visualized using fluorescence
microscopy. In the fluorescence microscopy experiments, the nuclei of MCF-7 tumor cells were
labeled with DAPI (nucleolus) to evaluate the localization of phthalocyanine in the red fluorescence line
(Figure 7). This qualitative analysis showed that all three phthalocyanines synthesized here presented an
interaction with target cells, with some visual differences between then.
a1
b1
c1
a2
b2
c2
a3
b3
c3
Figure 7. Fluorescence images of the interaction between phthalocyanines (45.0 µM final
concentration) 10 (images a1–a3), 11 (images b1–b3) and 12 (images c1–c3) and MCF7 cells
4
2
(
grown on chamber slides at a density of 4 × 10 cells/cm and treated with 300 nM DAPI
solution). The images were produced by 365 nm excitation, using 420 nm (DAPI) and 590 nm
(
phthalocyanines) emission filter. DAPI was used to following the studied phthalocyanine
red fluorescence) according to the nuclei position. AXIO—observer A1 (Zeiss).
(

Molecules 2015, 20
13582
In detailed fluorescence images (Figure 8a,b), it is possible to visualize the interaction of compound
1
2 with the MCF7 cell membrane, in a clear red emission circulating the nucleolus labeled with DAPI
(
blue). Also, even after 10 min of red light PDT treatment (Figure 8c), it is possible to confirm the presence
of the macrocyclic compound, with little membrane damage, in agreement with our photodynamic results
Figure 9). The two other phthalocyanines presented the same behavior, accumulating only in MCF7
cell membrane.
(
Figure 8. Fluorescence images of the interaction between phthalocyanine 12 (50.0 µM
final concentration) and MCF7 cells (a,b). The images were produced by 365 nm excitation,
using 420 nm (DAPI) and 590 nm (phthalocyanine) emission filter. DAPI was used to
following the studied phthalocyanine (red fluorescence) according to the nuclei position.
(
(
c) Presents the same phthalocyanine cell interaction after 10 min of red light irradiation
660 nm, 8 W/cm2).
2
20
10
Control
1 µM
2
00
80
60
40
20
00
5
µM
25 µM
0 µM
1
5
1
1
1
1
80
60
40
20
0
Time (min)
2
20
220
200
180
160
140
120
100
80
11
Control
1 µM
12
Control
1 µM
2
00
80
60
40
20
00
5
µM
5 µM
1
25 µM
0 µM
25 µM
50 µM
5
1
1
1
1
8
0
60
60
40
40
20
20
0
0
Time (min)
Time (min)
Figure 9. Phototoxic effect of compounds 10, 11 and 12 on the MCF-7 cell line in different
irradiation times (50 μM, 25 μM, 5 μM and 1 μM; 2 h incubation). The control group did
not receive the light treatment, only the phthalocyanine.

Molecules 2015, 20
13583
The dark toxicity was obtained for all phthalocyanines, evaluating the MCF-7 cell line viability
after different incubation periods with or without irradiation. No significant toxicity was observed for
all compounds (Figure 10).
220
200
180
160
140
120
100
10
Control
1 µM
5
µM
25 µM
0 µM
5
8
0
0
0
0
0
6
4
2
Time (h)
2
2
1
1
1
1
1
20
00
80
60
40
20
00
220
200
180
160
140
120
100
80
11
Control
1 µM
12
Control
1 µM
5
µM
25 µM
0 µM
5 µM
25 µM
50 µM
5
8
6
4
2
0
0
0
0
60
40
20
0
0
Time (h)
Time (h)
Figure 10. Toxic effect of compounds 10, 11 and 12 on the MCF-7 cell line after different
incubation times and concentrations (50 μM, 25 μM, 5 μM and 1 μM). The control group
did not receive the treatment.
The phototoxicity was evaluated for a range of phthalocyanine concentrations (1 μM, 5 μM,
−
2
2
5 μM and 50 μM) under irradiation with a 660 nm LED light (8 W·cm ) for different times (0.5; 1; 5
and 10 min).
The results showed that compounds 11 and 12 presented similar phototoxicity for all concentrations
and irradiation times studied. Compound 10, however, presented a higher phototoxicity, promoting a
reduction in about 50% of cell viability after 10 min of irradiation at 50 μM. The observed results can be
related with the hydrophilic-hydrophobic difference between compounds, indicated by their partition
coefficient values and aggregation behavior in aqueous media.
In our biological protocol, the compounds were dissolved in DMSO and added to RPMI-1640 cell
medium to final concentrations ranging from 1 μM to 50 μM. This procedure leads to approximately
2
% final DMSO in the aqueous medium, which promotes, as demonstrated in our solubility studies, a
high aggregation for all phthalocyanines analyzed, so when we analyze the phototoxicity results, part
of better PDT results observed for compound 10 can be explained considering that the racemic mixture
has half of the concentration of each enantiomer, what can lead to a reduced aggregation combination
in biological medium for this compound when compared to 11 or 12 at similar concentrations.

Molecules 2015, 20
13584
However, our results demonstrated that compound 10 presented a higher phototoxicity than
observed for both combined pure enantiomers 11 and 12 at the same concentrations, mostly at 25 μM
and 50 μM. This result indicates that the presence and distribution of chiral ligands in enantiomeric
pure phthalocyanines promote different patterns of cell interaction for each compound, influencing this
important step in photodynamic treatment.
Further analyses should be undertaken to understand the influence of chiral ligands on the interactions
between phthalocyanines and cells. In addition, in order to minimize the solubility and aggregation
problem, these compounds could be encapsulated in bio-carriers to improve their biological properties.
In this case, our observation opens a field for investigation to find new photosensitizers for
photodynamic therapy.
3. Experimental Section
3.1. General Information
All reagents and starting materials were purchased from Sigma-Aldrich (St. Louis, MO, USA), except
1
for other specification, and used as received. For H-NMR spectra (recorded in CDCl
3
at 500 MHz NMR
instrument from Bruker Daltonics Inc. (Billerica, MA, USA) tetramethylsilane was used as internal
reference. Flash chromatography was carried out on silica gel (230–400 mesh). Mass spectrometry analyses
were recorded on an Electrospray-TOF (ESI-TOF) mass spectrometer from Bruker Daltonics Inc.
(Billerica, MA, USA). Conversions and enantiomeric excesses of the enzyme-catalyzed reactions were
determined with similar conditions described in [6], using a GC-17A gas chromatograph (Shimadzu
Corp, Kyoto, Japan) equipped with a chiral capillary column Chirasil-Dex CB b-cyclodextrin. Circular
dicroism spectra of phthalocyanines were recorded on a J-815 instrument (Jasco Inc., Easton, MD,
USA). Fluorescence images were produced by 340 nm excitation, using a 580 nm emission filter in a
AXIO-observer A1 fluorescence microscope (Carl Zeiss Group, Oberkochen, Germany). DAPI was
used its interaction with cell nuclei and its blue fluorescence, which allows following the positions of the
studied phthalocyanines (red fluorescence) with respect to the cell nuclei.
3.2. Synthesis
3.2.1. Kinetic Resolution of 1-(4-Bromophenyl)ethanol
To access the enantioenriched alcohols, we employed the enzymatic resolution of secondary
alcohols mediated by lipases (Scheme 1). Thus, racemic 1-(4-bromophenyl)ethanol (prepared from
-bromoacetophenone) was reacted with Candida antarctica lipase (CAL-B) and vinyl acetate in hexane.
4
The acetate 3 and the (S)-alcohol 4 (95% ee) were isolated in 51% and 37% yield, respectively. Ester 3
was then hydrolyzed, leading to the (R)-alcohol 5 with 99% ee.
Briefly, 1-(4-bromophenyl)-ethanol (2, 5.7 mmol), vinyl acetate (11.4 mmol), lipase CAL-B enzyme
(8 mg) and hexane (50 mL) were stirred under reflux at 70 °C for 2 h. After cooling, the reaction mixture
was concentrated under reduced pressure and the solid purified by column chromatography on silica gel
using hexane-ethyl acetate (9:1) and (4:1), yielding 51% of acetate 3 (0.87 mmol, 353.3 mg, relative to
the calculated stoichiometric expected mass) and 37% (1.05 mmol, 212.0 mg) of (S)-alcohol 4. The

Molecules 2015, 20
13585
fractions were separated from the (R) product and submitted to a hydrolysis reaction whereby 1-(4-
bromophenyl)-ethyl acetate (3, 0.82 mmol), potassium carbonate (0.72 mmol) and methanol (50 mL)
were added to a flask and stirred at room temperature for 24 h. After the end of the reaction, analyzed by
TLC, the methanol was evaporated and the product was extracted with dichloromethane and washed with
a saturated solution of ammonium chloride. The organic phase was dried with sodium sulfate, filtered
and evaporated. The product was subjected to silica gel chromatography using hexane-ethyl acetate (4:1)
to give an 85% yield (0.70 mmol, 140.1 mg) of (R)-alcohol 5.
3
.2.2. Reaction of 4-Nitrophthalonitrile with 1-(4-Bromophenyl)ethanol (R, S and Racemic Forms)
Each optically pure alcohol and the racemate were reacted with commercially available
4
-nitrophthalonitrile and an excess of K
2
CO in DMF, followed by cyclotetramerization to produce
3
phthalocyanine derivatives 10, 11 and 12 (Scheme 1). Thus, 1-(4-bromophenyl)ethanol (2, 4 or 5,
.25 mmol), 4-nitrophthalonitrile (6, 1.0 mmol), potassium carbonate (6.4 mmol) and dimethylformamide
DMF, 3 mL) were added to a reaction flask and agitated at room temperature for 7 days. At the end of
1
(
the reaction, it was analyzed by TLC. The reaction mixture was poured into cold water and extracted three
times with dichloromethane. The organic phase was dried with sodium sulfate and the excess solvent was
evaporated. The product was subjected to silica gel chromatography using dichloromethane–hexane
(
2:1) as eluent.
−1
4
-[1-(4-Bromophenyl)ethoxy]phthalonitrile (7) was isolated in 75% yield. FTIR (KBr) umax/cm 3072
Ar–H, w), 2994, 2062, 2890 (–CH, –CH , and –CH , w), 2231 (–C≡N, w), 1600, 1498 (-C=C-, s),
) 500 MHz: δ 7.62 (d, J = 8.73 Hz, 1H, H-13), 7.51 (d,
= 8.73 Hz,
= 6.39 Hz, 3H, H-8). ESI-TOF
(
2
3
1
1
255, 1033 (Ar-O-CH
2
-). H-NMR (CDCl
3
J = 8.58 Hz, 2H, H-6 and H-2), 7.22–7.17 (m, 3H, H-3, H-5 and H-10), 7.10 (dd, J
1
J
2
= 2.64 Hz, 1H, H-14), 5.34 (q, J
1
= 6.39 Hz, 1H, H-7), 1.67 (d, J
1
+
m/z calcd. for C14
H
14
N
2
O
3
[M + Na] 281.0902, found 281.0896.
4
-[S-1-(4-Bromophenyl)ethoxy]phthalonitrile (8) was isolated in 79% yield. FTIR (KBr) umax/cm−1
3
072 (Ar–H, w), 2994, 2062, 2890 (–CH, –CH
2
, and –CH
) 500 MHz: δ 7.62 (d, J = 8.73 Hz, 1H, H-13), 7.51 (d,
= 8.73 Hz,
= 6.39 Hz, 3H, H-8). ESI-TOF
3
, w), 2231 (–C≡N, w), 1600, 1498 (-C=C-,
1
s), 1255, 1033 (Ar-O-CH
2
-). H-NMR (CDCl
3
J = 8.58 Hz, 2H, H-6 and H-2), 7.22–7.17 (m, 3H, H-3, H-5 and H-10), 7.10 (dd, J
= 2.64 Hz, 1H, H-14), 5.34 (q, J = 6.39 Hz, 1H, H-7), 1.67 (d, J
1
J
2
1
1
+
m/z calcd for C14
H
14
N
2
O
3
[M + Na] 281.0902, found 281.0896.
4
-[R-1-(4-Bromophenyl)ethoxy]phthalonitrile (9) was isolated in 73% yield. FTIR (KBr) umax/cm−1
3
072 (Ar–H, w), 2994, 2062, 2890 (–CH, –CH
2
, and –CH
) 500 MHz: δ 7.62 (d, J = 8.73 Hz, 1H, H-13), 7.51 (d,
= 8.73 Hz,
= 6.39 Hz, 3H, H-8). ESI-TOF
3
, w), 2231 (–C≡N, w), 1600, 1498 (-C=C-,
1
s), 1255, 1033 (Ar-O-CH
2
-). H-NMR (CDCl
3
J = 8.58 Hz, 2H, H-6 and H-2), 7.22–7.17 (m, 3H, H-3, H-5 and H-10), 7.10 (dd, J
= 2.64 Hz, 1H, H-14), 5.34 (q, J = 6.39 Hz, 1H, H-7), 1.67 (d, J
1
J
2
1
1
+
m/z calcd for C14
H
14
N
2
O [M + Na] 281.0902, found 281.0896.
3

Molecules 2015, 20
.2.3. Syntheses of Optically Active Phthalocyanine 10, 11 and 12
The cyclotetramerization was realized in DMAE (dimethylethanolamine) at 145 °C in a closed
13586
2
system. The phthalonitriles 7, 8 or 9, zinc acetate and dimethylethylamine (DMAE) were added to the
reaction flask, which was subjected to reflux in an inert atmosphere for 8 h. After this, the excess
solvent was evaporated and the product was subjected to silica gel chromatography using 2% methanol
in dichloromethane as eluent The product purity was confirmed by the presence of a single peak in the
corresponding HPLC chromatogram using methanol acetonitrile (1:1) as eluent.
{
tetrakis-[1-(4-Bromophenyl)ethoxy]phthalocyaninato}zinc(II) (10): 63% yield; FTIR (KBr) umax/cm−1
1
3
7
070, 2984, 2067, 2897, 2215, 1620, 1488, 1250, 1093, 742; H-NMR (DMSO-d
6
) 500 MHz: δ
.13–7.71 (4 m, 28H, Ar), 5.36–5.43 (q, 4H, -CH) and 1.67 (s, 12H, -CH
3
). ESI-TOF m/z calculated
C
64
H44Br
4
N O
8 4
Zn (M) 1374.1; found [M + H] 1375.1;
{
tetrakis-[S-1-(4-Bromophenyl)ethoxy]phthalocyaninato}zinc(II) (11): 77% yield; FTIR (KBr) umax/cm−1
1
3
068, 2980, 2065, 2894, 2210, 1622, 1489, 1250, 1090, 740; H-NMR (DMSO-d
6
) 500 MHz: δ
7
.13–7.71 (3 m, 28H, Ar), 5.36–5.43 (q, 4H, -CH) and 1.67 (s, 12H, -CH
3
). ESI-TOF m/z calculated
C
64
H44Br
4
N O
8 4
Zn (M) 1374.1; found [M + H] 1373.9;
{
tetrakis-[R-1-(4-Bromophenyl)ethoxy]phthalocyaninato}zinc(II) (12): 60% yield; FTIR (KBr) umax/cm−1
1
3
7
072, 2981, 2060, 2891, 2210, 1624, 1484, 1254, 1090, 739; H-NMR (DMSO-d
6
) 500 MHz: δ
.13–7.71 (4 m, 28H, Ar), 5.36–5.43 (q, 4H, -CH) and 1.67 (s, 12H, -CH
3
). ESI-TOF m/z calculated
C
64
H44Br
4
N O
8 4
Zn (M) 1374.1; found [M + H] 1375.3.
3
.3. Photophysical and Photochemical Studies
.3.1. Singlet Oxygen Quantum Yields
3
Photogeneration quantum yields of singlet oxygen (Φ ) were obtained by an indirect method, using
Δ
diphenylisobenzofuran (DBPF) [14,15] as chemical quencher. Typically, a mixture of the phthalocyanine
absorption ~0.2 at 680 nm in DMSO) and the DPBF (absorption ~0.9 at 418 nm in DMSO) was irradiated
(
with red LED lamp in 20 cycles of 6 s. The Φ ) values were determined using zinc phthalocyanine (ZnPc)
Δ
as standard (Equation (1)):
ꢀ
ꢁꢂꢃ
ꢄ. ꢅ
ꢆꢇꢈ
ꢀ
ꢁꢂꢃ
Φ = Φ∆
∆
(1)
ꢄꢀꢁꢂꢃ. ꢅꢆꢇꢈ
where (Φ
Δ
ZnPc) is the singlet oxygen quantum yield for the ZnPc (Φ
Δ
= 0.67 in DMSO [10]); w and
wZnPc is the DPBF photobleaching rates in the presence of the phthalocyanine and ZnPc, respectively;
abs and IabsZnPc are the light absorption values for phthalocyanines (λ = 660 nm) and ZnPc (λ =
I
6
60 nm), respectively.
3
.3.2. Fluorescence Quantum Yields
F
Fluorescence quantum yields (Φ ) of all compounds were determined by comparative method using
the following the equation (Equation (2)):

Molecules 2015, 20
13587
ꢊ. ꢋꢀꢁꢂꢃ. ꢌ
ꢊꢀꢁꢂꢃ. ꢋ. ꢌ
Φ = Φꢉꢀ
ꢉ
ꢁꢂꢃ
(2)
ꢀ
ꢁꢂꢃ
were F and FZnPc are the areas under the fluorescence emission curves of the phthalocyanines and the
standard, respectively; A and AZnPc are the relative absorbance of the sample and standard at the excitation
2
2
wavelength, respectively; η and η ZnPc are the refractive indices of the solvents for the sample and
standard, respectively. The unsubstituted zinc phthalocyanine (ZnPc) was employed as standard. The
solutions were prepared in DMSO with absorbance ranged between 0.05 and 0.1 at the excitation
wavelength (λexc = 618 nm).
3
.3.3. Determination of Partition Coefficients (PO/W
)
Five milliliters of 10 µM solution from each phthalocyanine derivative was prepared in n-octanol. The
UV-Vis spectrum was sampled. Then, 5 mL of water was added to the solution and the container was
stirred for 30 min. Centrifugation (5 min at 5000 rpm) enabled a phase separation and the organic phase
was sampled again. The partition coefficient was obtained from the difference in the phthalocyanine
absorption intensity (in 650 nm region) in both stages. At least three independent measures were
performed and the corresponding PO/W value was taken as the overall average.
3
.4. Cell Interaction Study
3
.4.1. Cell Culture
2
The human breast carcinoma line (MCF-7) cells were cultured in 75 cm bottles with RPMI-1640
medium supplemented with 10% fetal bovine serum, 2 mL insulin purified from bovine pancreas, sodium
pyruvate (0.5 mM) and non essentials amino acids: L-alanine (0.89 g/L), L-asparagine H O (1.50 g/L),
L-aspartic acid (1.33 g/L), L-glutamic acid (1.47 g/L), glycine (0.75 g/L), L-proline (1.15 g/L) and L-serine
2
(
1.05 g/L). The samples were incubated at 37 °C in an atmosphere of 5% CO
2
and 80% on reaching
4
2
confluence were trypsinized and reinoculated onto plates at a density of 4 × 10 cells/cm (fluorescence
4
microscopy) and 2.48 × 10 (cell viability test) were plated for experiments.
3
.4.2. Fluorescence Microscopy
Human breast epithelial cells MCF-7 (adenocarcinoma, estrogen receptor +; ATCC) were incubated
in RPMI-1640 medium supplemented with 10% fetal bovine serum (Invitrogen Corp, Carlsbad, CA, USA),
0.0 μg/mL insulin purified from bovine pancreas, 100 U/mL penicillin and 10.0 µg/mL streptomycin.
1
4
2
MCF-7 cells were grown on chamber slides at a density of 4 × 10 cells/cm and then treated with
00 nM DAPI solution (4ʹ,6-diamidino-2-phenylindole, Invitrogen Corp, Carlsbad, CA, USA) at 37 °C
for 30 min. The cells were then washed with phosphate buffered saline (PBS: 137 mM NaCl and
.7 mM KCl in 10 mM phosphate buffer at pH 7.4) and incubated with 45 µM phthalocyanines
DMSO) in dark. After 1-h treatment, the cells were pre-fixed with 30% and 50% ethanol diluted in
3
2
(
cells medium (v/v) followed by a 100% ethanol post-fixation. The cells were then washed with
PBS. The fluorescence images were produced by 365 nm excitation, using 420 nm (DAPI) and 590 nm
(
phthalocyanines) emision filter in an AXIO—observer A1 (Zeiss) using a 40× objective and also

Molecules 2015, 20
13588
5
90–650 nm excitation and 662–733 nm emission filter in an AF6000 Leica fluorescence microscope
using a 63× oil immersion objective.
3
.4.3. Cell Viability—MTT
Cell viability assays are important to show the toxicity of a compound. We conducted a MTT
colorimetric test to assess cell viability. For this, cells were plated in 96-well plates with 2.48 × 104
density and incubated at 37 °C in an atmosphere of 5% CO . The phthalocyanines were added at 1, 5,
0, 25 and 50 μM, and the cell viability was analyzed in 2, 6, 24 and 48 h. For photodynamic tests, the
2
1
cells were incubated with phthalocyanines at 50 μM, for 2 h, and the plates were divided into control
2
(
no irradiation) and treated (irradiated). The red LED light irradiation (660 nm, 8 W/cm ) was applied
in period of 0.5, 1.5 and 10 min. The cells viability was analyzed after 2, 6 and 24 h. After the time set
for the treatment, was added 30 μL of MTT solution, and the cells were again incubated for one hour.
Thereafter the whole solution was removed from the wells and 150 μL of DMSO was added to solubilize
formazan crystals formed from the reduction of MTT salt. Viability was measured by ELISA reader
which measures the absorbance of each well of the plate. With the values obtained we performed the
following calculation: % viable cells = (absorbance control/treated absorbance) × 100.
4
. Conclusions
A phthalocyanine derivative with an (R)-1-(4-bromophenyl)ethanol substituent presented a lower
tendency to aggregation than similar phthalocyanine derivatives under the same conditions, resulting in
a higher yield in the generation of reactive oxygen species. The results obtained by fluorescence
microscopy and cell viabiliity have shown that these phthalocyanines interact in different ways with
mammary MCF-7 cells. Therefore, our results indicate that the photochemical and biological properties
of phthalocyanines with chiral ligands should be evaluated separately for each isomeric species.
Acknowledgments
The authors thank FAPESP (2011/22561-9), UFABC, CNPq and CAPES for their financial support
of this research.
Author Contributions
A.A.R., F.B.N., T.F.M.S. and T.M.M. performed research and analyzed the data; A.T.O., G.C. and
A.O.R. performed research, analyzed the data and wrote the paper. All authors read and approved the
final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.

Molecules 2015, 20
13589
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©
2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/4.0/).
(