Photophysical properties and photocytotoxicity of novel phthalocyanines - Potentially useful for their application in photodynamic therapy

Article abstract of DOI:10.1016/j.poly.2011.03.003

Two novel zinc(II) phthalocyanines bearing non-peripheral ester-alkyloxy substituents (Pc-4 and Pc-5) were synthesized, by a two-step procedure starting from 2,3-dicyanohydroquinone. Both sensitizers show promising photophysical properties, including solv

Full text of DOI:10.1016/j.poly.2011.03.003

Polyhedron 30 (2011) 1538–1546
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Polyhedron
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Photophysical properties and photocytotoxicity of novel phthalocyanines –
potentially useful for their application in photodynamic therapy
Tomasz Goslinski a, Tomasz Osmalek b, Krystyna Konopka c, Marcin Wierzchowski a, Piotr Fita d,e
,
Jadwiga Mielcarek b,
⇑
a Department of Chemical Technology of Drugs, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland
b Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland
c Department of Biomedical Sciences, University of the Pacific, Arthur A. Dugoni School of Dentistry, San Francisco, USA
d Department of Physical Chemistry, University of Geneva, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland
e Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Hoza 69, 00-681 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Two novel zinc(II) phthalocyanines bearing non-peripheral ester-alkyloxy substituents (Pc-4 and Pc-5)
were synthesized, by a two-step procedure starting from 2,3-dicyanohydroquinone. Both sensitizers
show promising photophysical properties, including solvatochromic study, qualitative evaluation of
emission, aggregation behavior and singlet oxygen generation. It was proven that the photodynamic
activity of the compounds studied depends on the molecular oxygen level. Comparison of the quantum
yields of singlet oxygen generation as well as the oxidation rate constants using 1,3-diphenylisobenzofu-
rane before and after deaeration proved the photodynamic effect of Pc-4 and Pc-5 to be governed by the
photosensitization mechanism II. Changes in the quantities of the compounds exposed to irradiation
were also estimated. Upon their exposure to light the changes in intensity of the Q band were monitored.
The photodecomposition of Pc-4 and Pc-5 in DMSO or DMF solutions was found to proceed according to
the first order kinetic reaction in two stages.
Received 18 January 2011
Accepted 3 March 2011
Available online 17 March 2011
Keywords:
Photocytotoxicity
Photosensitizer
Phthalocyanine
Singlet oxygen
Solvatochromic effect
PDT
The photosensitizing effect of Pc-4 in HSC-3 cells was significantly lower than that of ZnPc. Pc-4 was
ineffective at 0.1 lM, while a low, approx. 15% photocytotoxicity was observed at 5 lM, at a distance of 5
and 10 cm. The efficacy of Pc-4 in photokilling of cultured cells is affected by hydrophobicity and the
aggregation status of the photosensitizer.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
tals, Langmuir–Blodgett films, gas sensors, optoelectronic devices
or catalytic materials. Phthalocyanines are also considered to be
There is a large variety of macrocyclic compounds in our envi-
ronment. They play an essential role in regulation of many biolog-
ical processes such as cell growth or electron transfer during
photorespiration. Metallophthalocyanines can be considered as
synthetic porphyrin analogs. They possess a conjugated system of
one of the most promising compounds in the field of nanotechnol-
ogy [6–12]. It turned out that photoinduced electron transfer prop-
erties of covalently linked phthalocyanine–fullerene dyads are
much more advantageous than classical porphyrin systems [13].
The photosensitizing activity provides the opportunity to em-
ploy phthalocyanines as active agents for photodynamic therapy
in medicine [14–17]. Efficient singlet oxygen generation, high sta-
bility toward temperature and irradiation, desirable spectroscopic
features and low dark toxicity contribute to the fact that phthalo-
cyanines are currently one of the most potent and promising
photosensitizers.
A very low solubility of phthalocyanines in common organic
solvents has been the main hindrance for their desired activities
[18,19]. Unsubstituted or metal-free analogs are almost insoluble
and new synthetic methods should be designed for derivatives
containing appropriate functional groups such as amide, carboxylic
or sulfonic [20–22]. The introduction of long-chain substituents to
the periphery is expected to prevent the aggregation [23]. On the
p-electrons and exhibit the ability to chelate metal ions in the cen-
ter of the ring [1–3].
The first phthalocyanine was synthesized in 1907 and for next
20 years copper and iron complexes were used mainly as green
and blue dyestuffs in polygraphic and textile industry. A remark-
able increase of interest in potential applications of metallopht-
halocyanines began in 1930. In addition to classical substitution
reactions, phthalocyanine ring can be modified by a variety of dif-
ferent functional groups [4,5]. Unique physicochemical properties
of new derivatives found applications in production of liquid crys-
⇑
Corresponding author. Tel.: +48 61 854 66 04; fax: +48 61 854 66 09.
E-mail address: jmielcar@ump.edu.pl (J. Mielcarek).
0277-5387/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.poly.2011.03.003

T. Goslinski et al. / Polyhedron 30 (2011) 1538–1546
1539
Scheme 1. Preparation of phthalonitriles 2, 3, and phthalocyanines Pc-4–Pc-7.
other hand, the low solubility of most phthalocyanines in water
can be bypassed by the use of liposomes, emulsions or nanoparticle
carriers [24–26].
and carbons within the structure of the ester-alkyloxy substituent
and phthalocyanine core (Tables 1S and 2S, Supplementary
material).
Because zinc phthalocyanines satisfy almost all requirements
for a ‘‘good photosensitizer’’, they are currently the most widely
tested dyes for Photodynamic Therapy (PDT) – (Scheme 1, inset).
This work reports on the photophysicochemical and biological
properties of novel phthalocyanines possessing non-peripheral es-
ter-alkyloxy substituents.
2.2. Photochemical and solvatochromic studies
The electronic absorption spectra of Pc-4 and Pc-5 revealed
characteristic features of the phthalocyanine spectra. They show
a low intensity Soret band (B band) in the range of 300–400 nm
and a much more intense Q band in the range of 600–900 nm.
2. Results and discussion
The significant intensity of this band has been a result of the p–
p⁄ transitions in macrocyclic system. According to the literature,
the Q band present in the UV–Vis spectra of phthalocyanines in
comparison with other porphyrinoids has been found significantly
red shifted and more intense in relation to that of the B band [19].
The positions and intensities of some bands, especially Q band,
have been also affected by the type of solvent used [31]. As follows
from Fig. 1, for the majority of solvents, the Q band was split into a
few components of similar or of different intensities. The separa-
tion of the resulting sub-bands strongly depended on the proper-
ties of the solvent, which eliminated vibrational structure as the
origin of this splitting.
2.1. Synthesis
According to a modified literature procedure [27,28] phthalo-
nitriles 2 and 3 were prepared by the alkylation of the inexpensive,
commercially available 2,3-dicyanohydroquinone 1 with ethyl
bromoacetate and ethyl 4-bromobutyrate in the presence of
K2CO3 in dimethylformamide (DMF) at 70 °C for 24 h, respectively
(Scheme 1). While the alkylation of 1 was performed with ethyl 3-
bromopropionate, an elimination reaction within the alkylating
agent occurred. The resulting hydroquinone dialkylated ether 2
decomposed under macrocyclic reactions conditions. The dinitrile
3 was found to be unreactive under the normal Linstead macrocyc-
lization conditions (a suspension of magnesium butoxide in reflux-
ing butanol) [29]. Therefore it was necessary to use harsh
conditions by heating at 130 °C for 24–72 h in 1-pentanol or 1-hex-
anol in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
as a base with or without metal salt [30]. Thus reaction performed
with zinc acetate led to the desired zinc phthalocyanines Pc-4, Pc-
5, whereas the reaction performed without metal salt source led to
the demetallated phthalocyanines Pc-6, Pc-7. It is noteworthy that
during macrocyclization reaction parallel transesterification reac-
tion within the peripheral ester-alkyloxy chains occurred.
All macrocyclic products Pc-4–Pc-7 were purified by column
chromatography and isolated as dark green films in yields of
7.6%, 0.4%, 1.6% and 4.7%, respectively. HPLC analysis revealed
the purity of samples Pc-4 and Pc-5 to be at the level 97.5–97.7%
and 97.6%, respectively. Phthalocyanines Pc-6 and Pc-7 were found
unstable under standard conditions (room temperature and access
to day light) as the photobleaching took place very fast, which was
monitored by HPLC. The 1H NMR spectra of phthalocyanines Pc-4–
Pc-7 in pyridine-d5 showed 1 singlet at d 7.86–7.88 ppm for the
phthalocyanine b-ring protons. The connectivities found in COSY
(Correlation Spectroscopy), HMQC (Heteronuclear Multiple Quan-
tum Correlation) and HMBC (Heteronuclear Multiple Bond Coher-
ence) spectra of Pc-4 and Pc-7 enabled to attribute all protons
In the UV–Vis spectra of Pc-4 and Pc-5 recorded in 2-propa-
nol, triethylamine, dioxane, N,N-dimethylformamide (DMF),
N,N-dimethylacetamide (DMA) and dimethylsulfoxide (DMSO),
the intense Q band was slightly split into the main one and the
blue shifted (kmax = 655–665 nm) side Q bands (Table 1). At the
same time, the molar absorption coefficient of the rising edge
was over three times smaller than that of the main component
of the Q band. Among all the solvents considered, only in meth-
anol the Q band was split into three components of different
intensities, possibly as a result of the axial coordination of the
Zn(II) ion by methanol. The main Q band component was present
at kmax = 735 nm and the two small bands, one at each side of the
Q band, at kmax = 660 and 820 nm, respectively. The band at
kmax = 660 nm has been often referred to as the X band appearing
as a result of deformation of the phthalocyanine ring in some sol-
vents [33].
It was found that the Q band red shifts were the greatest when
chloroform and acetonitrile were used as solvents. The complex Q
band spectra of Pc-4 and Pc-5 in chloroform possess a broad rising
shoulder on which two components could be discerned at 761 and
806 nm, accompanied by an additional poorly developed band in
the range of 410–530 nm. These features could be explained by
strong solvation effects of the solvents. The spectra of Pc-4 and
Pc-5 recorded in ethyl ether, ethyl acetate, hexane and cyclohex-
ane revealed broad complex Q bands in the range of 600–

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T. Goslinski et al. / Polyhedron 30 (2011) 1538–1546
Fig. 1. Normalized UV–Vis spectra of Pc-4 in various solvents.
Table 1
UV–Vis spectral data of Pc-4 and Pc-5 in various organic solvents (dipole moments and refractive indices [32]).
Solvent
Refractive index (20 °C)
Dipole moment (D)
Pc-4
Pc-5
B-band (nm)
Q-band (nm)
B-band (nm)
Q-band (nm)
Methanol
Acetonitrile
Diethyl ether
Ethyl acetate
Hexane
2-Propanol
Triethylamine
THF
1,4-Dioxane
Cyclohexane
DMF
DMA
Chloroform
DMSO
1.328
1.344
1.352
1.372
1.375
1.377
1.401
1.407
1.422
1.426
1.430
1.438
1.446
1.478
1.70
3.92
1.15
1.78
0.00
1.56
0.66
1.69
0.00
0.00
3.82
3.70
1.04
1.80
326
327
328
328
327
322
318
325
326
323
326
325
326
327
660
735
736
730
731
733
729
727
733
734
733
736
736
761
743
820
811
796
799
779
326
328
326
323
329
327
336
327
326
326
327
323
330
327
660
736
735
767
730
732
732
729
727
734
734
733
733
736
762
743
820
811
797
799
780
655
651
655
654
802
782
801
781
660
661
660
660
661
660
806
806
665
665
870 nm, which were split into two components of similar intensi-
ties. As it could be seen in Table 1, small structural differences in
the periphery did not influence significantly the spectra and solva-
tochromic effects. The Soret band and Q band ranges were similar,
with minor differences in intensities and shifts.
The correlation between the Q band shift towards longer wave-
lengths and the refractive index of the solvent was tested to eval-
uate the solvatochromic effects [34]. The wavelength
corresponding to the maximum of the Q band, kmax, was plotted
against the rational function (n2 ꢀ 1)/(2n2 + 1) of the solvent’s

T. Goslinski et al. / Polyhedron 30 (2011) 1538–1546
1541
Fig. 4. Absorption and emission spectra of Pc-4 in DMSO; excitation wavelength at
670 nm.
Fig. 2. Plot of wavelength kmax of the Q band vs. the refractive index of the solvent
described by the rational function F = (n2 ꢀ 1)/(2n2 + 1); where n stands for the
refractive index.
As it can be seen in Fig. 4 and Fig. 1S (Supplementary material),
the emission spectra of Pc-4 and Pc-5 are consistent with both the
Stokes rule and the rule of mirror symmetry between the absorp-
tion and fluorescence bands. The emission properties of Pc-4 and
Pc-5 in DMSO were evaluated by comparison with those of ZnPc
possessing no peripheral substituents. For ZnPc the Stokes shift
refractive index n (Fig. 2) according to the procedure [34]. This
dependence is linear even though the influence of the solvent’s
dielectric constant on the solvatochromic shift is neglected here.
Simultaneously, it was checked that for Pc-4 and Pc-5 the Q band
shift was not correlated with the dipole moments of the solvents
tested. Therefore it can be concluded that for 2-propanol, triethyl-
amine, dioxane, DMF, DMA and DMSO the fast solvation due to
solvent’s polarizability (described by the refractive index) prevails
over the slow solvation resulting from reorientation of the sol-
vent’s molecules (which would disturb the linear dependence
shown in Fig. 2 and should be correlated with the dipole moment
of the solvent’s molecules).
The aggregation behavior of Pc-4 and Pc-5 were studied at dif-
ferent concentrations in DMSO and DMF (Fig. 3). The absorption of
the Q band increased proportionally to the concentration of the
compounds and no new bands that could be attributed to the
aggregated forms were found [35]. The Beer–Lambert law was
obeyed for each compound in all solvents.
was small (Dk = 7 nm) which confirms that the geometry of the
molecule in the singlet excited state S1 does not differ much from
that in the ground state. The Stokes shift in the emission spectra of
phthalocyanines possessing peripheral substituents was some-
what greater.
Fluorescence quantum yields (UF) were determined by the
comparative method according to the formula [36]
R
FpðkÞ 1 ꢀ 10ꢀAqc
Uf;qc
R
Uf;p
¼
ꢁ
ꢁ
1 ꢀ 10ꢀAP
FqcðkÞ
where Uf,p is fluorescence quantum yield of the sample; Uf,qc is fluo-
rescence quantum yield of the standard; Fp is fluorescence intensity
of the sample; Fqc is fluorescence intensity of the standard; Ap is
absorbance of the sample at the excitation wavelength; and Aqc is
the absorbance of the standard at the excitation wavelength.
The emission spectra of Pc-4 and Pc-5, similarly to the absorp-
tion ones, depend on the presence of heteroatoms in the macrocy-
clic periphery (Table 2). Therefore a decrease of fluorescence
quantum yield to the level of Uf = 0.06 in comparison to that ob-
served for ZnPc (Uf = 0.2) was noticed [37]. This process has been
related to the presence of free electron pairs at the oxygen atoms
substituted to the non-peripheral positions of Pc-4 and Pc-5,
which facilitates the nonradiative quenching being the result of
2.3. Emission properties
Qualitative evaluation of emission, including quantitative anal-
ysis of the fluorescence spectra and determination of the quantum
yields (Uf) was performed for Pc-4 and Pc-5.
their coupling with the
ring.
p-electron system of the phthalocyanine
2.4. Singlet oxygen generation; U
D
Singlet oxygen quantum yields in DMSO were almost equal for
Pc-4 and Pc-5 but lower than that of ZnPc [15]. This proves that
Table 2
Emission wavelengths, Stokes shifts values and fluorescence quantum yields at
different excitation wavelengths for Pc-4 and Pc-5 in DMSO.
Compound Emission Stokes shift
Uf (excitation at Uf (excitation
kmax (nm)
Soret band)
at Q band)
D
k (nm) (cmꢀ1
)
ZnPc
Pc-4
Pc-5
678
761
761
7
18
18
150
350
350
0.20 0.03 [37]
0.066 0.011
0.067 0.011
0.05 0.02
0.07 0.02
Fig. 3. Representative absorption spectra of Pc-4 in DMSO; concentrations from
1.41 to 8.44 ꢂ 10ꢀ6 mol Lꢀ1
.

1542
T. Goslinski et al. / Polyhedron 30 (2011) 1538–1546
Fig. 6. Changes in the UV–Vis spectrum of Pc-4 in DMSO during irradiation. The
inset plot shows semilogarithmic dependence of ln A733 vs. time.
Fig. 5. Changes in the UV–Vis spectrum of DPBF and Pc-4 in DMSO and first-order
plots for oxidation of DPBF photosensitized by Pc-4, Pc-5 and ZnPc in DMSO under
aerobic conditions.
nm
mode of decomposition of the phthalocyanine molecule, accompa-
nied by systematic decoloration of the solutions, photobleaching,
was reflected by the changes observed in the UV–Vis spectra. As
shown in Fig. 6, on exposure of Pc-4 and Pc-5 solutions to irradia-
tion the intensity of the Q band decreased and later this band
disappeared.
Changes in the quantities of the compounds exposed to irradia-
tion were also estimated. Upon their exposure to light the changes
in intensity of the Q band were monitored. The photodecomposi-
tion of Pc-4 and Pc-5 in DMSO or DMF solutions was found to pro-
ceed according to the first order kinetic reaction in two stages.
The rate constants of the reaction (k1obs and k2obs) were found
from the equation
Table 3
Kinetic parameters describing relationships ln (A0/A) = f(t) and quantum yields
generation of singlet oxygen by phthalocyanine Pc-4 and Pc-5 in the presence of
oxygen and after deaeration.
Solvent Compound Aerobic conditions
After deaeration
104 ꢂ (k
4.34 0.19
4.29 0.21
5.83 0.47
D
k) (sꢀ1
)
U
104 ꢂ (k
D )
k) (sꢀ1
D
DMSO
DMF
Pc-4
Pc-5
ZnPc
0.50
0.49
2.90 0.30
2.59 0.37
0.67 [38] 3.77 0.14
Pc-4
Pc-5
ZnPc
7.10 0.25
5.76 0.34
4.99 0.18
0.80
0.65
4.36 0.59
4.84 0.47
0.56 [38] 3.04 0.21
lnðAt ꢀ A Þ ¼ lnðA0 ꢀ A Þ ꢀ kobs ꢁ t
1
1
non-peripheral substitution by eight long chain ester groups
slightly reduces the ability to photosensitization. Distinct differ-
ences were found in DMF, where Pc-4 exhibited stronger photo-
sensitizing ability than Pc-5 and ZnPc –Fig. 5.
It was also proven that the photodynamic activity of the com-
pounds studied depends on the molecular oxygen level, as bub-
bling with nitrogen via reaction vessel for 20 min led to distinct
inhibition of the 1,3-diphenylisobenzofurane (DPBF) oxidation –
Table 3.
where A is absorbance in t0 ? t ; kobs is the reaction rate constant
1
[sꢀ1], and t is the time [s].
The rate constant of the second stage of the reaction (k2obs) was
found by subtraction. Comparison of k1obs and k2obs presented in
Table 4 indicates that the first stage of the reaction is about 10
times faster than the second one.
2.6. Biological activity
Comparison of the quantum yields of singlet oxygen generation
as well as the oxidation rate constants using DPBF before and after
deoxygenation has proved the photodynamic effect of Pc-4 and Pc-
5 to be governed by the photosensitization mechanism II. After
absorption of a photon by the photosensitizer and the subsequent
intersystem crossing to the triplet state, the energy was transferred
directly onto the oxygen molecule leading to formation of singlet
oxygen with strong oxidizing properties.
HSC cells were incubated with ZnPc or Pc-4 for 24 h in serum
free medium. Cell viability was quantified by the Alamar Blue assay
and expressed as the percentage of control cells (DME/0.5% DMSO).
Data represent the mean SD from two independent experiments
performed in triplicate.
HSC-cells were incubated with ZnPc or Pc-4 for 24 h and ex-
posed to light as described in Section 4. Cell viability was quanti-
fied by the Alamar Blue assay and expressed as the percentage of
control cells not exposed to light. Data represent the mean SD
from two independent experiments performed in triplicate.
2.5. Photostability assay
Energies and lifetimes of the excited states of the porphyrin
molecules depend on the molecular structure of the complex with
metal ions and the nature of the solvent. The greatest effect on sta-
bility of excited molecules exerts the stability of bonds in the mac-
roring and the type of metal ion at the center of the molecule [39].
For Pc-4 and Pc-5 the redox processes involving Zn(II) ions should
be excluded and the changes caused by this process should be re-
lated only to the macroring transformations. The excitation of the
ring accompanied by oxidation process led to its decomposition
into small fragments that do not absorb light in the UV–Vis. This
Table 4
Rate constants values for particular stages of Pc-4 and Pc-5 photodecomposition in
DMF.
Compound
102 ꢂ (kobs
I
D
kobs) (sꢀ1
)
103 ꢂ (kobs
II
D )
kobs) (sꢀ1
Pc-4
1.22 0.10
1.49 0.01
Pc-5
I
II
1.20 0.09
1.43 0.09

T. Goslinski et al. / Polyhedron 30 (2011) 1538–1546
1543
Table 5
Therefore a decrease of fluorescence quantum yield to the level
of Uf = 0.06 in comparison to that observed for ZnPc (Uf = 0.2)
was noticed. This process has been related to the presence of free
electron pairs at the oxygen atoms substituted to the non-periph-
eral positions of Pc-4 and Pc-5, which facilitates the nonradiative
Dark toxicity of ZnPc and Pc-4 in HSC-3 cells.
Compound
Control
ZnPc 0.1
ZnPc 5
Cell viability (%)
Compound
Control
Cell viability (%)
100.0 3.6
103.3 2.8
96.9 2.8
100.0 3.8
110.8 1.5
101.1 4.9
l
M
M
Pc-4 0.1
Pc-4 5.0
l
l
M
M
l
quenching being the result of their coupling with the
p electron
system of the phthalocyanine ring. It was proven that the photody-
namic activity of the compounds studied depends on the molecular
oxygen level, as bubbling with nitrogen via reaction vessel for
20 min led to distinct inhibition of the 1,3-diphenylisobenzofurane
(DPBF) oxidation. Comparison of the quantum yields of singlet oxy-
gen generation as well as the oxidation rate constants using DPBF
before and after deaeration proved the photodynamic effect of Pc-
4 and Pc-5 to be governed by the photosensitization mechanism II.
Changes in the quantities of the compounds exposed to irradiation
were also estimated. Upon their exposure to light the changes in
intensity of the Q band were monitored. The photodecomposition
of Pc-4 and Pc-5 in DMSO or DMF solutions was found to proceed
according to the first order kinetic reaction in two stages.
Table 6
Light-dependent toxicity of ZnPc and Pc-4 in HSC-3 cells.
Distance (cm)
Time (min)
Control
ZnPc 0.1 lM
ZnPc 5 lM
5
0
10
0
20
20
100.0 8.7
111.8 8.7
113.1 7.9
100.0 10.0
70.5 6.2y
0
100.0 5.6
99.1 12.0
101.5 7.1
100.0 2.3
103.5 11.9
0
Control
100.0 2.3
99.2 6.5
92.8 6.5
100.0 6.5
92.2 1.8
87.8 1.8a
100.0 1.2
102.3 0.3
91.2 6.8
100.0 8.0
90.8 7.7
86.5 6.1x
Pc-4 0.1
Pc-4 5
l
M
M
l
aSignificantly different compared with contral : a– (P < 0.025); x – (P < 0.05); y –
(P < 0.005).
The photosensitizing effect of Pc-4 in HSC-3 cells was signifi-
cantly lower than that of ZnPc. Pc-4 was ineffective at 0.1
while a low, approx. 15% photocytotoxicity was observed at
M, at a distance of 5 and 10 cm. The efficacy of Pc-4 in photok-
lM,
5
l
illing of cultured cells is affected by hydrophobicity and the aggre-
gation status of the photosensitizer. Nevertheless, the behavior
observed in homogeneous solution is not always the case in cellu-
lar systems, where the biological microenvironment of the phthal-
ocyanines can induce significant changes in the photophysics of
the sensitizers [15,40].
Further synthetic and physicalchemical studies concerning
photosensitizers possessing reduced amount of ester-alkoxy sub-
stituents in the periphery of other groups of porphyrinoids are in
progress in our laboratory.
The dark-toxicities of ZnPc and Pc-4 in HSC-3 cells are pre-
sented in Table 5. At concentrations of 0.1 and 5.0 lM, ZnPc and
Pc-4 did not aggregate, and did not reduce the viability of HSC-3
cells. Significant, dose-dependent aggregation of Pc-4 was ob-
served at 10 and 50
not shown).
lM, without the loss of cell viability (data
Light-induced toxicities of ZnPc and Pc-4 in HSC-3 cells are
shown in Table 6. The cells were incubated with ZnPc and Pc-4
(0.1 and 5.0 lM) for 24 h, and subsequently exposed to light
(650–850 nm) for 20 min with spectral irradiance at 89 and
36 mW/cm2 at a distance of 5 and 10 cm from the light source,
4. Experimental
respectively. Pc-4 was ineffective at 0.1
15% photocytotoxicity was observed at 5
and 10 cm. These values were significantly lower than the control
cells. ZnPc at 0.1 M decreased cell viability by 30% at a distance of
5 cm (P < 0.005), while 5 M ZnPc caused 100% photocytotoxicity
l
M, while a low, approx.
lM, at a distance of 5
4.1. Synthesis
l
All reactions were conducted in oven dried glassware under ar-
gon. Reaction temperatures reported refer to external bath temper-
atures. All reagents were obtained from commercial suppliers and
used without further purification. All solvents were rotary evapo-
rated at or below 50 °C under reduced pressure. Melting points
were obtained on a ‘‘Stuart’’ Bibby Sterlin LtdÒ melting point appa-
ratus and are uncorrected. Chromatography was carried out on
Merck silica (eluants are given in parentheses). Dry flash column
chromatography was carried out on silica gel 60, particle size
l
at a distance of 5 and 10 cm.
In conclusion, the photosensitizing effect of Pc-4 was signifi-
cantly lower than that of ZnPc. The efficacy of photosensitizer in
photokilling of cultured cells is affected by its hydrophilic/hydro-
phobic properties. The cellular uptake of photosensitizer, which in-
volves binding to the cell membrane receptor and subsequent
internalization, is affected by hydrophobicity and the aggregation
status of the photosensitizer. A very low phototoxicity of Pc-4 orig-
inated most likely from the presence of eight ester-alkyloxy sub-
stituents (–O–(CH2)3–CO–OC5H11) in its molecule, which might
lead to an increased hydrophobicity and tendency to aggregate. At-
tempts to encapsulate Pc-4 in PG:PC and PG:CL multilamellar lip-
osomes were unsuccessful (data not shown).
40–63 lm. Thin layer chromatography (TLC) was performed on sil-
ica gel 60 F254 plastic sheets and visualized with UV (kmax 254 or
365 nm). Elemental analyses were determined by the Advanced
Chemical Equipment and Instrumentation Facility at the Faculty
of Chemistry, Adam Mickiewicz University in Poznan and the Lon-
don Metropolitan Microanalytical Service. UV–Vis spectra were re-
corded on a Hitachi UV/VIS U-1900 and Shimadzu UV-160A. FT-IR
measurements were collected on a Bruker Victor 22 and a Mattson
5000 spectrometers. 1H and 13C NMR spectra were recorded on a
Bruker DRX-400 and 500 spectrometers. Chemical shifts (d) are
quoted in parts per million (ppm) and are referred to a residual sol-
vent peak. Coupling constants (J) are quoted in Hertz (Hz) to the
nearest 0.5 Hz. The abbreviations s, d, t, p, h, and m refer to singlet,
doublet, triplet, pentet, hidden, and multiplet respectively. Addi-
tional techniques (1H–1H COSY (COrrelation SpectroscopY),
1H–1H TOCSY (TOtal Correlated SpectroscopY), HMQC (Heteronu-
clear Multiple Quantum Coherence), and HMBC (Heteronuclear
Multiple Bond Correlation)) were used to assist allocation. Analyt-
3. Conclusions
In conclusion, two novel zinc(II) phthalocyanines bearing non-
peripheral ester-alkyloxy substituents (Pc-4 and Pc-5) were
synthesized, by
a two-step procedure starting from 2,3-
dicyanohydroquinone.
Both sensitizers show promising photophysical properties,
including solvatochromic study, qualitative evaluation of emission,
aggregation behavior and singlet oxygen generation. The emission
spectra of Pc-4 and Pc-5, similarly to the absorption ones, depend
on the presence of heteroatoms in the macrocyclic periphery.

1544
T. Goslinski et al. / Polyhedron 30 (2011) 1538–1546
ical HPLC was performed on a Agilent 1200 Series. Mass spectrom-
etry (EI, ESI, MALDI TOF) were recorded by both the Imperial Col-
lege London Department of Chemistry Mass Spectrometry and
the Institute of Bioorganic Chemistry of the Polish Academy of Sci-
ences in Poznan Services.
4.1.4. 1,4,8,11,15,18,22,25-Octakis[3-(hexyloxycarbonyl)
propyloxy]phthalocyaninato zinc(II) (Pc-5)
DBU (0.58 g, 3.9 mmol) was added to the solution of 3 (1.5 g,
3.9 mmol) and Zn(OAc)2 (0.37 g, 2.0 mmol) in 1-hexanol (4 mL).
The reaction mixture was stirred at 140 °C for 72 h. Next 1-hex-
anol was evaporated with toluene and dry residue was purified
by silica gel chromatography (CH2Cl2:CH3OH, 15:1 to 10:1) to
give black green Pc-5 (10 mg, 0.4%). Rf (CH2Cl2:MeOH, 10:1)
4.1.1. 3,6-Bis(ethyloxycarbonylmethyloxy)-1,2-benzenedicarbonitrile
(2)
0.44. UV–Vis (MeOH): kmax (log e) = 238 (5.14), 367 (5.10), 662
Ethyl bromoacetate (8.35 g, 50 mmol) was added to well-stirred
(3.84), 736 (4.51). 1H NMR (400 MHz, pyridine-d5) 7.88 (s, 8H,
ArH), 5.18 (t, 3J = 7.0 Hz, 16H, ArOCH2), 4.12 (t, 3J = 7.0 Hz, 16H,
OCH2CH2), 3.09 (t, 3J = 7.0 Hz, 16H, CH2CO), 2.71 (p, 3J = 7.0 Hz,
16H, CH2CH2CO), 1.50 (p, 3J = 7.0 Hz, 16H, OCH2CH2), 1.23–1.06
(bm, 48H, CH2CH2CH2CH3), 0.73 (t, 3J = 7.0 Hz, 24H, CH3). 13C
NMR (100 MHz, pyridine-d5) 173.7, 153.2, 151.9, 128.6, 119.2,
71.6, 64.6, 31.6, 31.1, 28.9, 25.8, 25.6, 22.7, 14.1. MS (MALDI)
MH+ 2066; HPLC: stationary phase – Eclipse XDB-C18, mobile
phase – MeOH:THF:CH2Cl2 = 80:10:10 (v/v/v); UV detection at
735 nm, retention time 8.61 min, purity 95.02%.
slurry
of
3,6-dihydroxy-1,2-benzenedicarbonitrile
(3.20 g,
20 mmol) and anh. K2CO3 (5.53 g, 40 mmol) in DMF (50 mL), and
heated at 70 °C for 24 h. The reaction contents were poured into
water (400 mL), and the suspension was vigorously stirred. The
resulting white solid was filtered under reduced pressure, washed
with water, dried, heated under reflux in MeOH (70 mL) for 15 min
to give white solid (2) (4.04 g, 60.8%). M.p. = 137–138 °C. Rf
(CH2Cl2) 0.15. UV–Vis (MeOH): kmax (log e) = 223 (4.37), 333
(4.08). 1H NMR (400 MHz, DMSO-d6) d = 7.57 (s, 2H, ArH), 5.06 (s,
4H, ArOCH2), 4.17 (q, 3J = 7.0 Hz, 4H, OCH2), 1.21 (t, 3J = 7.0 Hz,
6H, CH3). 13C NMR (100 MHz, DMSO-d6) d = 167.8 (C@O), 154.4
(Ar, C–O), 120.5 (Ar, C–H), 113.3 (CN), 103.2 (Ar, C–CN), 65.9 (Ar-
OCH2), 61.0 (OCH2), 13.9 (CH3). Anal. calcd for C16H16N2O6ꢁ0.5H2O:
C, 56.30; H, 5.02; N, 8.21. Found: C, 56.14; H, 4.81; N, 8.20%.
4.1.5. 1,4,8,11,15,18,22,25-Octakis[(3-(pentyloxycarbonyl)
propyloxy]phthalocyanine (Pc-6)
DBU (0.25 g, 1.9 mmol) was added to the solution of 3 (0.75 g,
1.9 mmol) in 1-pentanol (6 mL). The reaction mixture was stirred
at 135 °C for 24 h. Next 1-pentanol was evaporated with toluene
and dry residue was purified by silica gel chromatography
(CH2Cl2:CH3OH, 50:1 to 10:1, next n-hexane:EtOAc, 7:1 to 1:1) to
give green solid Pc-6 (0.014 g, 1.6%). Rf (n-hexane:EtOAc, 7:4)
4.1.2. 3,6-Bis[3-(ethyloxycarbonyl)propyloxy]-1,2-benzenedicarbo-
nitrile (3)
Ethyl 4-bromobutyrate (9.75 g, 50 mmol) was added to a well-
stirred slurry of 3,6-dihydroxy-1,2-benzenedicarbonitrile (3.20 g,
20 mmol) and anh. K2CO3 (5.53 g, 40 mmol) in DMF (50 mL), and
heated at 70 °C for 24 h. The reaction contents were poured into
water (400 mL), and the solution was vigorously stirred. The
resulting white solid was filtered under reduced pressure, washed
with water, dried, heated under reflux in MeOH (70 mL) for 15 min
to give white solid (3) (7.16 g, 92.2%). M.p. = 140–141 °C. Rf
0.32. UV–Vis (CH3OH): kmax (log e) = 233 (4.62), 323 (4.66), 740
(4.85). 1H NMR (400 MHz, pyridine-d5) 7.86 (s, 8H, ArH), 5.02 (th,
16H, ArOCH2), 4.07 (t, 3J = 7.0 Hz, 16H, OCH2CH2), 3.14 (t,
3J = 7.0 Hz, 16H, CH2CO), 2.69 (p, 3J = 7.0 Hz, 16H, CH2CH2CO),
1.43 (p, 3J = 7.0 Hz, 16H, OCH2CH2) 1.23–1.03 (bm, 32H,
CH2CH2CH3), 0.69 (t, 3J = 7.0 Hz, 24H, CH3), 0.18 (s, 2H, NH). MS
(MALDI) MH+ 1892.
(CH2Cl2) 0.22. UV–Vis (MeOH): kmax (log e) = 227 (4.31), 354.5
(3.59). 1H NMR (400 MHz, DMSO-d6) d = 7.62 (s, 2H, ArH), 4.19 (t,
3J = 6.5 Hz, 4H, ArOCH2), 4.08 (q, 3J = 7.0 Hz, 4H, OCH2), 2.49 (t,
3J = 7.0 Hz, 4H, CH2CO), 2.00 (p, 3J = 7.0 Hz, 4H, CH2CH2CO), 1.19
(t, 3J = 7.0 Hz, 6H, CH3). 13C NMR (100 MHz, DMSO-d6) d = 172.8
(C@O), 155.2 (Ar, C–O), 121.0 (Ar, C–H), 113.9 (CN), 103.4 (Ar, C–
CN), 69.3 (ArOCH2), 60.4 (OCH2CH3), 30.2 (CH2CH2CO), 24.3
(CH2CH2CO), 14.5 (CH3). HRMS (EI) calcd for C20H24N2O6 [M]+
388.1634, found 388.1637. MS (ESI) MH+ 389, MK+ 427. Anal. Calc.
for C20H24N2O6: C, 61.84; H, 6.23; N, 7.21. Found: C, 61.70; H, 6.55;
N, 7.23%.
4.1.6. 1,4,8,11,15,18,22,25-Octakis[3-(hexyloxycarbonyl)
propyloxy]phthalocyanine (Pc-7)
DBU (304 mg, 2 mmol) was added to the solution of 3 (776 mg,
2 mmol) in 1-hexanol (2 mL). The reaction mixture was stirred at
140 °C for 24 h. Next 1-hexanol was evaporated with toluene and
dry residue was purified by silica gel chromatography
(CH2Cl2:CH3OH, 25:1 to 10:1) to give Pc-7 (47 mg, 4.7%). Rf
(CH2Cl2:CH3OH, 10:1) 0.86. UV–Vis (CH2Cl2:CH3OH, 25:1): kmax
(log e )
) = 328 (4.74), 401 (4.27), 762 (5.07), 852 (3.93); IR (cmꢀ1
4.1.3. 1,4,8,11,15,18,22,25-Octakis[(3-(pentyloxycarbonyl)propyloxy]-
phthalocyaninato zinc(II) (Pc-4)
3301, 2955, 2930, 2858, 1732, 1599, 1503, 1383, 1269, 1175,
1059. 1H NMR (500 MHz, pyridine-d5) 7.87 (s, 8H, ArH), 5.07 (t,
3J = 6.5 Hz, 16H), 4.09 (t, 3J = 7.0 Hz, 16H), 3.15 (t, 3J = 7.5 Hz,
16H), 2.70 (p, 3J = 7.0 Hz, 16H), 1.44 (p, 3J = 7.5 Hz, 16H), 1.13 (p,
3J = 7.5 Hz, 16H), 1.06 (m, 32H), 0.71 (t, 3J = 7.0 Hz, 24H), 0.19 (s,
2H). 13C NMR (125 MHz, pyridine-d5) 173.7, 152.0, 150.1h, 127.0,
119.4, 71.4, 64.4, 31.5, 31.1, 28.9, 25.8, 22.7, 14.3 (Table 2S, Supple-
mentary material). MS (MALDI) MH+ 2005.
DBU (598 mg, 4 mmol) was added to the solution of 3 (1.55 g,
4 mmol) and Zn(OAc)2 (367 mg, 2 mmol) in 1-pentanol (7 mL).
The reaction mixture was stirred at 140 °C for 24 h. Next 1-penta-
nol was evaporated with toluene and dry residue was purified by
silica gel chromatography (CH2Cl2:CH3OH, 50:1 to 35:1) to give
Pc-4 (149 mg, 7.6%). Rf (CH2Cl2:CH3OH, 10:1) 0.65. UV–Vis
(CH2Cl2:CH3OH): kmax (log e) = 328 (4.13), 489 (3.72), 768 (4.35),
810 (4.54). IR (cmꢀ1) 2927, 2856, 1732, 1498, 1383, 1265, 1192,
1054. 1H NMR (400 MHz, pyridine-d5) 7.87 (s, 8H), 5.16 (t,
3J = 6.5 Hz, 16H), 4.09 (t, 3J = 7.0 Hz, 16H), 3.08 (t, 3J = 7.0 Hz,
16H), 2.69 (p, 3J = 7.0 Hz, 16H), 1.48 (p, 3J = 7.0 Hz, 16H), 1.16 (m,
32H), 0.74 (t, 3J = 7.0 Hz, 24H). 13C NMR (100 MHz, pyridine-d5)
173.5, 153.0, 151.7, 128.3, 119.0, 71.4, 64.4, 30.9, 28.4, 28.0, 25.3,
22.3, 13.8 (Table 1S, Supplementary material). MS (MALDI) MH+
1953; HPLC: stationary phase – Eclipse XDB-C18, mobile phase –
MeOH:THF:CH2Cl2 = 80:10:10 (v/v/v), UV detection at 735 nm,
retention time 4.28 min, purity 96.81%.
4.2. Photochemical and solvatochromic studies
All measurements were carried out on Shimadzu UV-160 A
spectrophotometer with PC 160 Plus software. Solvents were ob-
tained from commercial suppliers and used without further purifi-
cation. UV–Vis spectra were recorded in the range of 300–900 nm.
Molar extinction coefficients values were similar for both Pcs in
DMSO and DMF (Table 7).

T. Goslinski et al. / Polyhedron 30 (2011) 1538–1546
1545
Table 7
Prior to exposition solutions of Pc-4, Pc-5 were transferred to a
quartz cylindrical cell (V = 2.7 mL, l = 1 cm). The initial value of the
absorbance at Q bands was between 0.7 and 0.8. The photostability
of the compounds was evaluated in DMSO, DMF and THF. During
exposure to light for certain periods of time, the UV–Vis spectra
were recorded in the range of 200–800 nm.
Maximum absorptions and molar absorption coefficients of Pc-4 and Pc-5 in DMSO
and DMF.
Solvent
DMSO
Compound
kmax (log e)
Pc-4
Pc-5
327 (4.60) 665 (4.51) 743 (5.20)
327 (4.67) 665 (4.56) 743 (5.25)
Photodecomposition of phthalocyanines as a result of separate
irradiation of Q bands was initially monitored qualitatively as ex-
plicit changes in the shape of the absorption spectra.
DMF
Pc-4
Pc-5
326 (4.60) 660 (4.49) 736 (5.20)
327 (4.61) 661 (4.52) 733 (5.20)
The process was also evaluated quantitatively. After certain
time intervals of exposition, absorption values at characteristic
wavelengths for each phthalocyanine were measured.
4.3. Emission properties
Fluorescence quantum yield measurements of the compounds
under study were carried out in solutions in dimethylsulfoxide
(DMSO for UV spectroscopy from Fluka, used as supplied). Absor-
bance of the solutions was kept below 0.1 at the maximum of
the Q-band absorption in a 1-cm-thick cell in order to avoid the
reabsorption of emitted photons. Zinc phthalocyanine (ZnPc, Sig-
ma–Aldrich) was used as the standard (Uf = 0.20 0.03) [37]. Sta-
tionary UV–Vis spectra were recorded using Cary Eclipse
fluorescence and Cary 50 Scan absorption spectrophotometers
from Varian.
4.6. Biological activity
4.6.1. Chemicals
Zinc phthalocyanine (ZnPc, Sigma–Aldrich) and Pc-4 were dis-
solved in DMSO (Sigma–Aldrich) to
9.8 mM. Dulbecco’s modified Eagle’s MEM medium with or with-
out phenol red, penicillin–streptomycin solution, -glutamine and
trypsin–EDTA were obtained from the University of California
San Francisco (UCSF) Cell Culture Facility (San Francisco, CA). Ala-
mar Blue dye (alamarBlue™) was obtained from Biosource Interna-
a final concentration of
L
tional, Inc. (Camarillo, CA). Lipids,
L
-
a
-phosphatidylcholine (Egg,
4.4. Singlet oxygen generation
Chicken) (PC),
L
-a-phosphatidylglycerol (Egg, Chicken) (sodium
salt) (PG), and cardiolipin (1,3-bis(sn-30-phosphatidyl)-sn-glyc-
erol) (CL) (Heart, Bovine-Disodium Salt) were obtained from Avanti
Polar Lipids (Birmingham, AL).
Solutions of diphenylisobenzofurane (DPBF) as singlet oxygen
chemical quencher and photosensitizers in DMSO and DMF were
irradiated in a 1 cm path length quartz cell with monochromatic
light (sensitizer absorbance at Q band ꢃ 0.5) by a 150 W high-pres-
sure Xe lamp (type XBO-150, Optel) through a monochromator
(Optel). The light intensity was adjusted to 1.0 mW/cm2 (Radiome-
ter RD 0.2/2 with TD probe, Optel). Photooxidation reaction of DPBF
was used to determine singlet molecular oxygen (1Dg) generation
by the photosensitizers. The kinetics of DPBF photooxidation were
studied by observing the decrease of the absorbance at
kmax = 417 nm. UV–Vis spectra were recorded on Shimadzu UV-
160 A spectrophotometer with PC 160 Plus software. Zinc phthalo-
cyanine (ZnPc, Sigma–Aldrich) was used as the standard. Measure-
ments of the sample and reference under the same conditions
enabled quantum yield calculation by direct comparison of the
slopes in the linear region of the semilogarithmic plots of ln A0/A
vs. irradiation time obtained for the samples and corresponding
slopes obtained for the reference. All the experiments were per-
formed at ambient temperature. To avoid chain reactions induced
by DPBF in the presence of singlet oxygen, the concentration of
DPBF was set at ꢃ3 ꢂ 10ꢀ5 mol Lꢀ1 and was kept the same for both
the standard and the samples. Molar absorption coefficients
(dm3 molꢀ1 cmꢀ1) of DPBF at k = 417 nm were determined as
4.6.2. Cell culture
HSC-3 human squamous carcinoma cells, derived from the ton-
gue [41], were provided by Dr. R. Kramer (UCSF). The cells were
cultured in Dulbecco’s modified Eagle’s MEM supplemented with
10% (v/v) heat-inactivated fetal bovine serum (FBS), penicillin
(100 U/mL), streptomycin (100 lg/mL) and L-glutamine (4 mM)
(DMEM/10). The cells were incubated at 37 °C in a humidified
atmosphere containing 5% CO2 and were passaged 1:6 twice a
week using trypsin–EDTA solution.
4.6.3. Dark toxicity
HSC-3 cells were seeded in 48-well plates at a density 1.8 ꢂ 105
cells per well in 1 ml of DME/10 1 day before the experiment, and
used at approx. 80% confluence. Subsequently, cells were washed
twice with phosphate buffer saline (PBS) and 1 mL of medium
without FBS and phenol red, containing phosensitizer at a given
concentration, was added to each well except controls. The FBS-
free medium was used to avoid binding of the photosensitizers
to serum proteins. After the 24 h incubation at 37 °C, cells were
washed twice with PBS and cell viability was quantified by the Ala-
mar Blue assay. Cells incubated either with DME medium alone or
DME/0.5% DMSO served as controls.
e
= 19 880 in DMSO and e = 21 821 in DMF. Solutions of the sensi-
tizer containing DPBF were prepared in the dark and irradiated in
the Q band region. Additional experiments were also performed for
each mixture of DPBF and photosensitizer after 20 min bubbling
with nitrogen.
4.6.4. Light-dependent toxicity
HSC-3 cells were seeded in 48-well plates at a density of
1.8 ꢂ 105 cells per well in 1 mL of DME/10 1 day before the exper-
iment and used at approx. 80–90% confluence. Cells were pre-
washed twice with PBS, and 1 mL of medium without FBS and phe-
4.5. Photostability assay
Samples were illuminated under aerobic conditions according
to the recommendations in the first version of the ICH Harmonised
Tripartite Guideline – Q1B. The source of light was 150 W high-
pressure Xe lamp (type XBO-150, Optel). The photodecomposition
process for each compound was performed for Q band using a yel-
low glass cut-off filter HCC16 with maximal transmission between
600 and 800 nm. The intensity of radiation was measured by a lux-
ometer (TES 1335, TES Electrical Electronic Corp.) and adjusted at
130 klux.
nol red, containing 0.1 or 5 lM of a given phosensitizer, was added
to each well except controls. Cells were incubated for 24 h at 37 °C,
washed twice with PBS and then 0.5 mL of medium without FBS
and phenol red was added. Subsequently, the cells were exposed
to light (650–850 nm) from the light bulb (Dura Max 75 W Med
120 V A19 Cl/LL 20 W; Philips Electronics North America Corporation,
Andover, MA), at a distance of 5 or 10 cm from the light bulb to the
plate, for 20 min. The total spectral irradiance at the level of cells,
and in the presence of a water filter, was 89 and 36 mW/cm2 (650–

1546
T. Goslinski et al. / Polyhedron 30 (2011) 1538–1546
850 nm), measured using a radiometer RD 0.2/2 with TD probe
(Optel). These measurements indicated that the irradiance was
constant over the small area occupied by the 48-well plates. Infra-
red radiation was minimized using a 2 cm water filter between the
cell plates and the light source. One plate from each experiment
was not exposed to light and served as a control. Directly after light
exposure, medium without FBS and phenol red was replaced with
1 mL of fresh DME/10, and cells were incubated for 24 h at 37 °C.
Cell viability was quantified by the Alamar Blue assay.
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Acknowledgments
This study was partially supported by grant from the Polish
Ministry of Science and Higher Education (N405 031 32/2052).
T.G. thanks Professor A.G.M. Barrett from the Imperial College in
London and Professor S. Sobiak from the Poznan University of Med-
ical Sciences for supporting his studies. P.F. thanks Professor E.
Vauthey from the University of Geneva for supporting his studies
and acknowledges the financial support from the Foundation for
Polish Science. The authors thank Beata Kwiatkowska and Michael
Yee for excellent technical assistance.
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.poly.2011.03.003.