Synthesis, properties and in vitro photodynamic activity of water-soluble azaphthalocyanines and azanaphthalocyanines

Article abstract of DOI:10.1111/j.1751-1097.2009.00647.x

In this work zinc azaphthalocyanines (AzaPcs) from the group of tetrapyrazinoporphyrazines and zinc azanaphthalocyanines from the group of tetra[6,7]quinoxalinoporphyrazines (TQP) with eight diethylaminoethylsulfanyl substituents were synthesized. Tertiary amines were later quaternized with ethyl iodide to obtain water-soluble photosensitizers (PSs). Quaternized compounds showed high singlet oxygen quantum yields as determined in DMF by monitoring decomposition of 1,3-diphenylisobenzofuran. In water medium, quaternized AzaPc derivatives appeared in monomeric form in a wide range of concentrations while quaternized TQP derivatives showed aggregation at higher concentrations (over 1 μm). Photodynamic activity was tested on Hep2 cells using light of λ > 640 nm. Both quaternized dyes showed high photodynamic activity (IC ₅₀ = 104 and 220 nm for AzaPc and TQP, respectively). Dark toxicity was not detected even at the highest concentration used in in vitro tests (200 μm) which indicates a promising therapeutic index of these new substances. Tested compounds localized inside the cells mainly within the lysosomes thus suggesting an endocytic mechanism of cellular uptake. No localization within mitochondria was detected. A great advantage of TQP derivatives over other PSs is their very strong absorption at 747 nm that allows activation at wavelengths penetrating deeper into human tissues.

Full text of DOI:10.1111/j.1751-1097.2009.00647.x

Photochemistry and Photobiology, 2010, 86: 168–175
Synthesis, Properties and In Vitro Photodynamic Activity of Water-soluble
Azaphthalocyanines and Azanaphthalocyanines
Petr Zimcik*1, Miroslav Miletin1, Hana Radilova2, Veronika Novakova1, Kamil Kopecky1,
Jaroslav Svec1 and Emil Rudolf3
1Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy in Hradec Kralove,
Charles University in Prague, Hradec Kralove, Czech Republic
2GENERI BIOTECH s.r.o., Hradec Kralove, Czech Republic
3Department of Medical Biology and Genetics, Faculty of Medicine in Hradec Kralove, Charles University in
Prague, Hradec Kralove, Czech Republic
Received 29 May 2009, accepted 11 September 2009, DOI: 10.1111 ⁄ j.1751-1097.2009.00647.x
The clinical history of PDT started in 1991 by approval of
porfimer sodium (Photofrinꢀ) in Canada for treatment of
ABSTRACT
In this work zinc azaphthalocyanines (AzaPcs) from the group of
tetrapyrazinoporphyrazines and zinc azanaphthalocyanines from
the group of tetra[6,7]quinoxalinoporphyrazines (TQP) with
eight diethylaminoethylsulfanyl substituents were synthesized.
Tertiary amines were later quaternized with ethyl iodide to
obtain water-soluble photosensitizers (PSs). Quaternized com-
pounds showed high singlet oxygen quantum yields as determined
in DMF by monitoring decomposition of 1,3-diphenylisobenzofu-
ran. In water medium, quaternized AzaPc derivatives appeared
in monomeric form in a wide range of concentrations while
quaternized TQP derivatives showed aggregation at higher
concentrations (over 1 lM). Photodynamic activity was tested on
Hep2 cells using light of k > 640 nm. Both quaternized dyes
showed high photodynamic activity (IC50 = 104 and 220 nM for
AzaPc and TQP, respectively). Dark toxicity was not detected
even at the highest concentration used in in vitro tests (200 lM)
which indicates a promising therapeutic index of these new
substances. Tested compounds localized inside the cells mainly
within the lysosomes thus suggesting an endocytic mechanism of
cellular uptake. No localization within mitochondria was
detected. A great advantage of TQP derivatives over other
PSs is their very strong absorption at 747 nm that allows
activation at wavelengths penetrating deeper into human tissues.
bladder cancer. However, its photophysical and pharmakoki-
netic properties are not optimal. This complicated mixture of
oligomers absorbs light at only 630 nm with low extinction
coefficient (e ꢀ 2000 M)1cm)1) and causes prolonged (up to
6 weeks) skin photosensitivity. These aforementioned draw-
backs have prompted the development of new PSs and some of
these second-generation PSs have been already accepted in
clinical practice (verteporfin, temoporfin, talaporfin, aminolev-
ulinic acid, methyl- and hexyl-aminolevulinate) (2,6,7). The
second-generation compounds are defined chemical individuals
with stronger absorption at longer wavelengths, short-lasting
skin photosensitivity and low dark toxicity. Of particular
importance appears to be their longer wavelength of absorption
as lower wavelengths are strongly absorbed by endogenous
chromophores (e.g. hem) and, consequently, the penetration of
red light (ꢀ630 nm) is rarely greater than 5 mm while it can be
as high as 1–2 cm with longer wavelengths (£800 nm) (7).
Phthalocyanines (Pcs) are among the most promising
groups of new PSs receiving increased attention in the last
few years. They absorb strongly (e ꢀ 150 000–200 000
M
)1cm)1) at longer wavelengths (close to 700 nm) and are
characterized by good singlet oxygen quantum yields (FD)
which is why they were subject to a number of biological tests.
Photodynamic activity of several new zinc and silicon Pcs was
investigated in vitro on different cell lines (8–11) as well as
against pathogenic microorganisms (12,13) and results showed
promise. In addition, biological tests of sulfonated ZnPc in vivo
on Wistar rats were performed recently (14). Pilot studies with
a long time known silicon Pc4 have recently been conducted on
animals (15) as well as deep investigations into its activity and
mechanism of action have been carried out (16–19). Lastly, a
mixture of sulfonated aluminum Pcs (Photosens) has already
been approved in Russia for cancer treatment since 2001.
Although the photophysical and photochemical properties of
Pcs are very promising, their common problems are low
solubility in water and strong tendency of this planar macro-
cyclic system to aggregation. These problems are usually
solved by using different additives such as organic solvents (20)
or Cremophor EL (10) that are not, however, devoid of their
own toxicity.
INTRODUCTION
Photodynamic therapy (PDT) is a well-established method for
the treatment of malignant and nonmalignant diseases which
combines three basic components, i.e. photosensitizer (PS),
light and oxygen with each of them being harmless when
applied individually. The mechanism of action of PDT is based
on the absorption of the light by PS and transfer of the energy
to surrounding molecules, mainly oxygen (1–5). Cytotoxic
species produced after illumination, most important of them
being singlet oxygen (1O2), inflict damage to cells ultimately
leading to their death.
*Corresponding author email: petr.zimcik@faf.cuni.cz (Petr Zimcik)
ꢁ 2009 The Authors. JournalCompilation. The AmericanSocietyof Photobiology 0031-8655/10
168

Photochemistry and Photobiology, 2010, 86 169
kmax (e) 652 (169 000), 592 (25 000), 376 (112 000); Anal. Calc. for
C88H152I8N24S8Zn+3H2O: C 35.98, H 5.42, N 11.44 Found: C 36.17,
H 5.52, N 11.20.
Our own research has been focused on the aza analogs of
Pcs—azaphthalocyanines (AzaPcs) from the group of tetra-
pyrazinoporphyrazines. We have uncovered several relation-
ships between the structure of AzaPcs and their photophysical
properties (21,22). AzaPc macrocycle can be also enlarged for
next benzene rings yielding azanaphthalocyanines (AzaNcs)
from the group of tetraquinoxalinoporphyrazines (TQP).
These compounds absorb light at wavelength around 750 nm
and thus can be very suitable as modern PSs with a deeper
therapeutic hit. Although the photophysical and other prop-
erties of AzaPcs in organic solvents have been largely
investigated by several groups (23–26), no photodynamic
activity on cells has been studied yet. Furthermore, TQPs are
still very rarely mentioned in the literature and only few
researchers have described the synthesis and some basic
photophysical properties of these interesting compounds
(27–29). We present here synthesis, characterization, aggrega-
tion behavior, photophysical and photochemical properties
and in vitro photodynamic tests on Hep2 cells of water-soluble
AzaPcs and TQPs.
2,3,11,12,20,21,29,30-Octakis(2-(diethylamino)ethylsulfanyl)tetra-[6,7]-
quinoxalinoporphyrazine zinc(II) (3)—magnesium turnings (196 mg,
8.16 mmol) were refluxed in dry butanol (15 mL) with a small crystal
of iodine for 3 h. Compound 12 (508 mg, 1.15 mmol) was added and
reflux continued for next 24 h. Butanol was evaporated and the
product extracted from the resulting solid using THF. After
evaporation of THF, the solid was washed thoroughly with hot
MeOH yielding a blue solid (200 mg, 39%). This magnesium TQP
was dissolved in 2% aqueous HCl (50 mL) with the use of ultrasound
and stirred at rt for 45 min. The solution was neutralized with
concentrated Na2CO3 solution and made basic with few drops of
NaOH. The green precipitate was collected and washed with water
and hot MeOH yielding metal-free TQP (184 mg, 93%). The metal-
free TQP was dissolved in pyridine (30 mL), anhydrous zinc acetate
(1.83 g, 10 mmol) was added and the mixture was refluxed for
45 min. Pyridine was evaporated and the mixture was washed with
water. The green solid was then dissolved in 2% aqueous HCl,
filtered and made basic with NaOH. The green precipitate was
collected and washed thoroughly with water and hot MeOH. Yield
180 mg (95%). 1H NMR (C5D5N) d = 9.81 (s, 8H, ArH), 4.54–3.83
(broad, 16H, S-CH2), 3.44–3.26 (broad, 16H, N-CH2), 2.95 (q, 32H,
J = 7 Hz, N-CH2), 1.37 (t, 48H, J = 7 Hz, CH3); 13C NMR
(C5D5N) d = 156.6, 152.5, 140.3, 136.9, 121.3, 52.1, 47.4, 30.0,
12.6; IR (KBr) t = 2996, 2800, 1633, 1568, 1515, 1452, 1384, 1340,
1309, 1255, 1131, 1022; MALDI-TOF m ⁄ z = 1832 [M]+, UV–Vis
(pyridine) kmax (e) 752 (164 000), 693 (87 000), 382 (150 000); Anal.
Calc. for C88H120N24S8Zn+3H2O: C 55.92, H 6.72, N 17.79 Found:
C 55.82, H 6.49, N 17.83.
2,3,11,12,20,21,29,30-Octakis(2-(triethylammonio)ethylsulfanyl)
tetra-[6,7]-quinoxalinoporphyrazine zinc(II) octaiodide (4)—com-
pound 3 (87 mg, 47 lm) was dissolved in ethyl iodide (11 mL) and
the solution was stirred at rt for 2 days. The green precipitate, which
appeared after 2 days, was dissolved after addition of N-meth-
ylpyrrolidinon (15 mL) and the reaction was stirred for next 5 days
at rt. The green solution was poured into diethylether and precipitate
collected, washed thoroughly with diethylether and acetone. The
product was then dissolved in MeOH, filtered and evaporated. The
product was recrystallized from MeOH ⁄ diethylether. Yield was
120 mg (90%) of a dark green solid. 1H NMR (D2O+C5D5N)
d = 10.0 (s, 8H, ArH), 5.08–4.85 (broad, 16H, S-CH2), 4.16–3.57 (m,
64H, N-CH2), 1.77–1.46 (m, 72H, CH3); 13C NMR (D2O+C5D5N)
d = 153.8, 151.8, 140.0, 121.4, 55.5, 53.4, 30.2, 7.5 (one aromatic
signal overlapped with signal of the solvent); IR (KBr) t = 2974,
1630, 1452, 1384, 1339, 1255, 1131, 1022; UV–Vis (DMF) kmax (e)
748 (372 000), 712 (43 000), 669 (43 000), 389 (154 000); Anal. Calc.
for C104H160I8N24S8Zn+4H2O: C 39.58, H 5.37, N 10.65 Found: C
39.19, H 5.28, N 10.50.
2,3-bis(2-(diethylamino)ethylsulfanyl)quinoxaline-6,7-dicarbonitrile
(12)—diethylaminoethanthiol hydrochloride (849 mg, 5 mmol) was
dissolved in water (10 mL), 1 M aqueous solution of NaOH (10 mL,
10 mmol) was added and the solution stirred at rt for 15 min.
2,3-dichloroquinoxaline-6,7-dicarbonitrile (11) was added (500 mg,
2 mmol) dissolved in THF (100 mL). The solution turned yellow
and was stirred at rt. THF was evaporated after 1 h of stirring, few
drops of NaOH were added (to basic reaction) to the remaining
water suspension and the solid was filtered and washed with water.
The yellow-brown solid was dissolved in diethylether and washed
with water which was made basic with few drops of NaOH.
Afterward, diethylether solution was washed with acidic solution
(water+HCl) and discarded. The acidic water solution was made
basic with NaOH, washed with diethylether several times and the
organic layer was dried (Na2SO4). Diethylether was evaporated and
a yellow solid recrystallized from MeOH yielding 560 mg (62%) of
yellow needles. M.p. 127.8–128.7ꢂC (MeOH); 1H NMR (CDCl3)
d = 8.23 (s, 2H, ArH), 3.45 (t, 4H, J = 7 Hz, S-CH2), 2.83 (t, 4H,
J = 7 Hz, N-CH2), 2.65 (q, 8H, J = 7 Hz, N-CH2), 1.09 (t, 12H,
J = 7 Hz, CH3); 13C NMR (CDCl3) d = 161.0, 140.2, 134.3, 115.3,
112.2, 50.9, 47.1, 28.9, 12.0; IR (KBr) t = 3097, 3073, 3027, 2970,
2934, 2874, 2798, 2237 (CN), 1507, 1470, 1386, 1290,1265, 1195,
1124, 1069, 1030. Compound 12 was converted to its hydrochloride
after dissolution in acetone ⁄ diethylether 1:3 and bubbling with
MATERIALS AND METHODS
General. All organic solvents used for the synthesis were of analytical
grade. Anhydrous dimethylformamide (DMF) was purchased from
Acros, diethylaminoethanthiol hydrochloride and 1,3-diph-
enylisobenzofuran (DPBF) from Aldrich. Zinc(II) phthalocyanine
(ZnPc) was obtained from Eastman Organic Chemicals (New York).
All chemicals were used as received except for zinc(II) acetate
dihydrate (Lachema, Czech Republic) which was dried at 78ꢂC under
reduced pressure (13 mbar) for 5 h. TLC was performed on Merck
aluminum sheets with silica gel 60 F254. Merck Kieselgel 60 (0.040–
0.063 mm) was used for column chromatography. Melting points were
measured on Electrothermal IA9200 Series Digital Melting point
Apparatus (Electrothermal Engineering Ltd., Southend-on-Sea, Essex,
UK) and are uncorrected. Infrared spectra were measured in KBr
pellets on IR-Spectrometer Nicolet Impact 400. 1H and 13C NMR
spectra were recorded on Varian Mercury-Vx BB 300 (299.95
MHz—1H; 75.43 MHz—13C). Chemical shifts reported are given
relative to internal Si(CH3)4. Elemental analysis was performed on
Elemental Analyser EA1110 (Carlo Erba Instruments). UV–Vis
spectra were recorded on spectrophotometer UV-2401PC (Shimadzu
Europa GmbH, Duisburg, Germany). MALDI-TOF mass spectra
were recorded in positive reflectron mode on a mass spectrometer
Voyager-DE STR (Applied Biosystems, Framingham, MA). For each
sample, 0.5 lL of the mixture was spotted onto the target plate, air-
dried and covered with 0.5 lL of matrix solution consisting of 10 mg
of a-cyano-4-hydroxycinnamic acid in 100 lL of 50% ACN in 0.1%
trifluoroacetic acid. The instrument was calibrated externally with a
five-point calibration using Peptide Calibration Mix1 (LaserBio Labs,
Sophia-Antipolis, France). Compounds 1 (30), 10 (31) and 11 (31) were
synthesized according to published procedures. For modified proce-
dure of synthesis of 9, see Supporting Information.
Synthesis. 2,3,9,10,16,17,23,24-Octakis(2-(triethylammonio)ethyl-
sulfanyl) tetrapyrazinoporphyrazine zinc(II) octaiodide (2)—com-
pound 1 (82 mg, 50 lm) was dissolved in ethyl iodide (10 mL) and
the solution was stirred at room temperature (rt) for 2 days. The green
precipitate, which appeared after 2 days, was dissolved after addition
of N-methylpyrrolidinon (14 mL) and the reaction was stirred for the
next 5 days at rt. The green solution was poured into diethylether,
resulting precipitate collected and washed thoroughly with diethylether
and acetone. Thereafter the product was dissolved in MeOH, filtered
and the solvent was evaporated. The product was recrystallized from
MeOH ⁄ diethylether. Yield was 137 mg (95%) of green solid. 1H NMR
(D2O+C5D5N) d = 4.74–4.50 (broad, 16H, S-CH2), 4.20–3.96
(broad, 16H, N-CH2), 3.94–3.66 (broad, 48H, N-CH2), 1.82–1.54
(broad, 72H, CH3); 13C NMR (D2O+C5D5N) d = 55.6, 53.2, 30.0,
7.4 (aromatic signals not detected); IR (KBr) t = 2976, 1728, 1658,
1517, 1452, 1395, 1304, 1250, 1175, 1108, 1094, 971; UV–Vis (H2O)

170 Petr Zimcik et al.
gaseous HCl. A white-yellow precipitate was collected and recrys-
tallized from MeOH ⁄ diethylether. M.p. slowly decomposed from
200ꢂC.
RESULTS AND DISCUSSION
Design of the molecules
Hydrochlorides of 1 and 3—general procedure. Dye 1 or 3 (1 lmol)
was dissolved or suspended in THF (5 mL) and MeOH (1 mL) and
concentrated HCl (0.5 mL) was added. Any remaining solid dissolved
after the addition of HCl. The mixture was stirred at rt for 30 min,
toluene (5 mL) was added and solvents were evaporated to dryness.
The sample was dried under deep vacuum (4 mbar) at 50ꢂC for 45 min
and used directly for preparation of solutions used for biological tests.
Fluorescence and singlet oxygen. Singlet oxygen quantum yields
were determined according to a previously published procedure using
decomposition of a chemical trap of singlet oxygen DPBF (32).
Absorption of the dyes in Q-band area during measurements was
approximately 0.1. ZnPc was used as the reference (FD = 0.56 in
DMF [30,33]). In some cases HCl was added to a final concentration of
5 · 10)4 M. This addition did not influence the quantum yields of
standard ZnPc in DMF as was shown before (30). For more details see
Supporting Information. Fluorescence quantum yields were deter-
mined by comparative method using ZnPc as the reference (FF = 0.20
in pyridine [34]). To minimize reabsorption effect, absorption of the
dyes in the Q-band area was kept below 0.05, excitation wavelength
was 375 nm.
Cell lines. For photodynamic activity, Hep2 (Human Negroid
cervix carcinoma) cells were obtained from the European Collection
of Cell Cultures (No. 86030501, Salisbury, UK). Cells were maintained
in Dulbecco’s modified Eagle’s medium (Cambrex, Walkersville, MD)
with L-glutamine and glucose, supplemented with 10% fetal bovine
serum (PAA Laboratories GmbH, Pasching, Austria) and gentamicin
(50 lg mL)1; Sigma-Aldrich, St. Louis, MO), and were grown at 37ꢂC
in an atmosphere containing 5% CO2. The cell line was tested for
mycoplasma contamination.
Phototoxicity assay. Photosensitizers 1–4 were dissolved in PBS to
a stock concentration 100 lM for phototoxicity or 1 mM for dark
toxicity tests and diluted further with PBS. The stock solutions were
filtered through 0.2 lm filters. Cells were seeded at 1 · 104 cells ⁄ well
in a 96-well plate. The day after, the PSs were added and incubated
with cells for 15 min, 90 min or 4 h. After incubation, cells were
rinsed with PBS, covered with fresh medium and irradiated
(58 mW cm)2, 15 min). To avoid any important dilution of medium
with PBS, the amount of added PBS with dyes remained at 1%
(phototoxicity) or 20% (dark toxicity) with respect to the volume of
medium in the well. Maximal concentrations of the dyes in tests were
therefore 1 and 200 lM for phototoxicity and dark toxicity, respec-
tively. Irradiation was performed using a 250 W QTH (Quartz
Tungsten Halogen) lamp (Oriel Instruments). Light was filtered
through a heat filter and cut on 640 nm filter. Twenty-four hours
after irradiation, the cell viability was determined using Cell
Counting Kit-8 (ALEXIS Corporation, Lausen, Switzerland). The
experiments were performed in triplicate for each concentration. Cells
grown in the culture medium diluted with an appropriate amount of
PBS without any dye were taken as control (100% viability). Dark
toxicity was tested with 24 h incubation of dyes with cells and
without any irradiation and protected from light.
Fluorescence microscopy and image analysis. The 100 lM stock
solutions of compounds 1–4 in PBS were diluted in cultivation medium
to the final concentration of 10 lM and Hep2 cells grown in cytospin
chambers were exposed to them for 12 h. Prior to visualization of
fluorescence, cells were exposed to MitotrackerGreen (Invitrogen-
Molecular Probes, Inc., Carlsbad, CA; 50 nM, 25 min, 37ꢂC) or
LysotrackerBlue (Invitrogen-Molecular Probes, Inc.; 25 nM, 25 min,
37ꢂC), rinsed with PBS and mounted. The localization and intensity of
fluorescence were examined under wide-field fluorescence microscope
We designed our molecules according to the previously
discovered relationships which are further discussed in this
paragraph. As mentioned above, aggregation of AzaPc and
AzaNc is well known and may constitute a problem for
photodynamic action. Aggregated PSs fail in efficient photo-
sensitization as the excited states are relaxed rather through
internal conversion than through intersystem crossing. Singlet
oxygen quantum yields (FD) are therefore strongly lowered
and the compound loses its photodynamic potential. Aggre-
gation in organic solvents is usually suppressed by introducing
bulky substituents preventing self-association (22). A slightly
different approach is considered in water medium where
charged substituents are used for prevention of the stacking.
Thus our intention was to prepare AzaPc and TQP with
quaternary ammonium groups inhibiting the aggregation. In
the previous photophysical study we showed that eight
charged substituents are necessary for complete monomeriza-
tion of AzaPc in water (30). The heteroatom connecting the
macrocycle system considerably influences FD and we showed
that alkylsulfanyl derivatives are among the best singlet
oxygen producers (21). Considering the central metal, zinc
appears to be the most suitable because it increases the triplet
state lifetimes resulting in very high FD. Other central metals
(Si, Al, Ge, Sn [35]) or metal-free derivatives (36) were
investigated for photodynamic effect too and such Pcs showed
good outcome in spite of their much lower FD values.
However, zinc is the metal of choice in order to maximize
the photophysical and photochemical properties. AzaNcs of
the TQP group form two isomers considering the position of
nitrogen atoms in the structure—tetra[2,3]quinoxalinopor-
phyrazines and tetra[6,7]quinoxalinoporphyrazines. The latter
isomers showed a much stronger bathochromic shift in the
main absorption band (27,31) and therefore they were chosen
for this application. We then decided to synthesize and test
molecules 2 and 4 (Scheme 1).
Synthesis
Synthesis of TQP derivatives requires suitably substituted
quinoxaline-6,7-dicarbonitriles. Recently, we described the
synthesis of an important precursor 11 from 4,5-diaminopht-
halonitrile 9 (31). 4,5-diaminophthalonitrile 9 was prepared by
the modified Youngblood’s procedure (Scheme 1) (37). Chlo-
rines in compound 11 are easily exchangeable by nucleophiles
under mild conditions and thus its reaction with 2-(diethyla-
mino)ethanethiolate at rt gave rise to 12 in a good yield.
Compound 12 was stored in the form of hydrochloride due to
stability reasons described previously for similar pyrazine
homolog (30). Cyclotetramerization of 12 using magnesium
butoxide led to Mg complex of corresponding TQP which was
then converted to the metal-free derivative. Insertion of zinc
into the center of the macrocycle yielded compound 3.
Alkylation of 3 in ethyl iodide led to desired compound 4.
During alkylation, partially alkylated 3 precipitated from
ethyl iodide that was used as alkylating agent and solvent at
the same time. To ensure further alkylation in solution,
N-methylpyrrolidinon was added to the reaction after 2 days
(Nikon Eclipse
E 400; Nikon Corporation, Kanagawa, Japan)
equipped with the digital color matrix camera COOL 1300 (VDS;
Vosskuhler, Osnabruck, Germany), using Texas Red (EX 540-580 and
BA 600-660), Cy5-HYQ (EX 590-650 and BA 663-738) and UV (EX
330-380 and BA 420) specific filters. Photographs were taken inde-
pendently using LUCIA DI Image Analysis System LIM (Laboratory
Imaging Ltd., Prague, Czech Republic). Image and colocalization
analyses were carried out with help of Lucia NIS-Elements AR 2.30 or
Image J. Fluorescence was finally combined with phase contrast. For
the purpose of analysis, at least 2000 cells were scored at 400·
magnification.

Photochemistry and Photobiology, 2010, 86 171
Scheme 1. Scheme of synthesis.
to support solubilization of the partially alkylated TQP. It is
noteworthy that the complete synthetic procedure from 5 to
compound 4 (Scheme 1) is very efficient, work up is simple and
none of the reaction steps require chromatographic purifica-
tion. AzaPc 1 has been prepared and described by us previously
(30) and its alkylation to 2 followed the same procedure as for
its TQP homolog. All analytical methods confirmed the
structure and purity of the compounds. Compounds 1 and 3
were also converted to the corresponding hydrochlorides to
allow their water solubility for biological tests.
in water corresponded well to the spectrum in DMF (Fig. 2a), it
did not change its shape at higher concentrations and obeyed
Lambert–Beer law in a wide range of concentrations (Fig. 1a).
UV–Vis absorption and aggregation
Compounds 1 and 3 are soluble in most organic solvents
while alkylated homologs 2 and 4 are well soluble in water and
polar organic solvents (methanol, DMF, cmax > 5 mM for
both solvents). Electronic absorption spectra of AzaPcs (1 and
2) showed typical B-band around 375 nm and sharp Q-band
with vibrational bands at 655 nm. TQP homologs (3 and 4)
absorb at wavelengths longer by 90 nm (Table 1). This
important redshift caused by enlargement of the macrocyclic
system allows them to absorb the light at wavelengths with
potential of deeper penetration through human tissues.
AzaPcs 1 and 2 do not aggregate in DMF or pyridine and 2 is
nonaggregated in water too, a feature which is only rarely seen
for ZnPcs and ZnAzaPcs. The shape of the absorption spectrum
Table 1. Photophysical and photochemical data of investigated com-
pounds in DMF.
kmax
(nm)
kem
(nm)
FD†
(DMF)
FD†
(DMF ⁄ HCl)
Compound
FF*
1
2
3
4
655
656
745
748
661
661
750
751
0.004‡
0.058
0.007§
0.027
0.039‡
0.196
0.410§
0.754
0.467‡
0.726
0.478§
0.677
Figure 1. Absorption spectra of compounds 2 (a) and 4 (b) in water at
different concentrations ranging from 10 lM to 0.04 lM. Insets:
dependence of e at Q-band maximum of monomer (652 nm for
2, 755 nm for 4) on the concentration.
*ZnPc reference (FF in pyridine 0.20 [34]). †ZnPc as reference (FD in
DMF 0.56 [30,33]). ‡Data from Ref. (30). §Aggregation.

172 Petr Zimcik et al.
Fluorescence quantum yields (FF) (Table 1) were relatively
low for all compounds, in particular for nonalkylated com-
pounds 1 and 3, that can be explained by efficient photoin-
duced electron transfer (PET) and aggregation (see below).
Singlet oxygen quantum yields (FD) were determined by
observing the decomposition of its chemical trap DPBF.
Results are summarized in Table 1. Proper interpretation of
the results is complicated because they are influenced by two
factors—PET and aggregation. As shown by other authors,
PET from amines to excited Pc macrocycle is responsible for
quenching of Pc excited states (40) and as a result quantum
yields of both singlet oxygen and fluorescence are lowered (9).
Compound 1 has therefore low FD in DMF which is, however,
increased after the addition of HCl into solution. Hydrochloric
acid protonates amines which are not further available as
donors for PET. Alkylated AzaPc 2 has higher FD in DMF
than 1 as a consequence of PET inhibition by quarternization
but after addition of HCl its FD reaches an even higher value
of 0.726 typical of alkylsulfanyl AzaPcs (30). It seems therefore
that some of the amines may not be alkylated, although we
have no analytical support for this fact. As the effect of PET is
very strong, it may involve just one or even less nonalkylated
amino group per one molecule of AzaPc. Still, it shall not
constitute a problem for proper biological evaluation of this
compound as one possible free amino group will be fully
protonated in water medium at pH 7.4 and therefore no PET
will occur. It should be noted that both 1 and 2 were fully
monomeric during all measurements and that is why aggrega-
tion did not account for changes in FD and FF values.
TQP enlarged system does not allow efficient PET due to
longer distance between donor and acceptor and therefore FD
value of 3 in DMF is 0.410. However, its FD values are lowered
by observed aggregation of this compound in both DMF and
DMF with HCl. Inefficient PET and monomeric state of 4 lead
to very high FD values of about 0.7 in both DMF and
DMF ⁄ HCl. The values for 4 are similar in both solvent
systems and a possible small difference may be attributed to
experimental error. The above-mentioned facts emphasize the
important role of quaternization (aside from an increase in
water solubility) in preserving the good photochemical prop-
erties of AzaPc PSs substituted with amines.
Figure 2. (a) Absorption spectra of 2 in DMF (dashed) and water
(full), (b) absorption spectra of 4 in DMF (dashed) and water (full), (c)
absorption (full), excitation (dashed) and emission (dotted) spectra of 2
in water, (d) absorption (full), excitation (dashed) and emission
(dotted) spectra of 4 in water.
On the other hand, the enlarged macrocyclic system of TQP 3
tends to aggregate even in DMF or pyridine. Its spectrum in
pyridine changes with dilution and with higher temperature that
is typical of the presence of aggregates (see Supporting
Information). Quaternization of 3 reduced aggregation in
organic solvents and compound 4 was found to be fully
monomeric in DMF or MeOH. However, its water solution
showed the presence of aggregates at higher concentrations as it
changed its spectrum in Q-band area with dilution and did not
follow Lambert–Beer law (no constant extinction coefficient,
Fig. 1b). Significant monomerization started below 1 lM
(Fig. 1b, inset). Higher temperature or addition of organic
solvent (pyridine, DMF or MeOH) into water helped in
monomerization too (see Supporting Information).
Photodynamic activity
Fluorescence and singlet oxygen
Photodynamic activity and toxicity were tested on Human
Negroid cervix carcinoma cells Hep2. Compounds 2 and 4 as
well as hydrochlorides of 1 and 3 were tested after dissolution
in PBS. No organic solvent or any other additives like
surfactants were added. As shown in Table 2 no dark toxicity
Fluorescence emission spectra of all the studied compounds
were of the typical shape for AzaPc and similar compounds
with only a small Stokes shift (Table 1). In the case when
compounds were present as monomers, their excitation spectra
corresponded well to absorption spectra (DMF solutions of 1,
2 and 4 and water solution of 2 [see Fig. 2a,c]). In the case
when aggregates were detected in UV–Vis spectra (3 in DMF
or pyridine, 4 in water [see Fig. 2b,d]) the excitation spectra
differed from UV–Vis absorption spectra. The aggregates
formed in solution alter significantly the absorption spectrum.
The fluorescence excitation spectrum is not influenced because
the aggregates usually do not fluoresce besides few exceptions
of J-dimers (38,39). Titration of aggregated water solution of 4
with pyridine resulted in a change in the electronic absorption
spectra shape but it had no effect on fluorescence emission and
excitation spectra. Only an increase in fluorescence intensity
was observed.
Table 2. Biological activities of AzaPc and TQP on Hep2 cells.
Dark toxicity†
(IC50 in lM)
Phototoxicity‡
(IC50 in lM)
Compound
1*
2
3*
4
37
>200
>200
>200
>1.0
0.104
0.273
0.220
AzaPc = azaphthalocyanine; TQP = tetra[6,7]quinoxalinoporphyr-
azine. *Applied in the form of hydrochlorides. †Incubation for 24 h.
‡Irradiation after 4 h of incubation.

Photochemistry and Photobiology, 2010, 86 173
was detected after 24 h treatment up to 200 lM which was a
maximal concentration used in tests. It shows the very low
toxicity of prepared dyes in the absence of light. The only
exception was hydrochloride of 1 which showed IC50 at 37 lM.
The IC50 values are defined as the dye concentration required
to kill 50% of the cells.
the cytoplasm where it colocalizes mainly with lysosomes
as indicated by image analysis. Conversely, no significant
colocalization with mitochondria was found. In addition, the
observed topography of intracellular distribution of 2 seems to
The photodynamic activity of tested compounds was deter-
mined after 4 h of incubation. The uptake time was based on
the experiment where compound 3 and quaternary 4 were
incubated with cells for different times and irradiated
(k > 640 nm, 58 mW cm)2, 15 min). The determined IC50
values were 279, 117 and 220 nM for 4 and >1000, 238 and
273 nM for 3 for 15 min, 90 min and 4 h, respectively. Although
very high photodynamic activity was observed after 90 min
treatment, thus indicating rapid uptake, the 4 h incubation was
chosen to allow full incorporation. Results of photodynamic
activity tests are summarized in Table 2 and Fig. 3.
In accordance with the above discussed results of photo-
chemical measurements and aggregation properties, the stron-
gest effect was observed for quaternized compound 2 followed
by 4, i.e. compounds which were originally designed as the
optimal compounds. Lower activity of 4 can be attributed to
its higher tendency to aggregate in water medium when
compared with 2. Amino groups of 1 and 3 in water medium
(pH 7.4) should be found in equilibrium between ionized and
nonionized forms. According to the Henderson–Hasselbalch
equation (41) and estimated pKa value 9.38
0.25 of amino
groups (calculated using Advanced Chemistry Development
[ACD ⁄ Labs] Software V9.04 for Solaris), at least one of eight
amino groups will be nonionized. It may lead to quenching of
excited states of 1 by PET and to very low photodynamic
activity. As no PET occurs in 3, its photodynamic activity is
comparable to quaternized homolog 4.
Subcellular localization
There have been several reports on the localization of cationic
Pc in subcellular compartments, especially in lysosomes (8) or
mitochondria (9,42). The subcellular localization was studied
after 12 h incubation in dark and analyzed together with
organelle-specific probes for lysosomes and mitochondria. As
shown in Fig. 4 for compound 2, the localization of the specific
fluorescence signals suggests the diffuse accumulation of 2 in
Figure 3. Viability of Hep2 treated with a range of concentrations of
the photosensitizers 1 (h), 2 (d), 3 (D) and 4 ( ) after irradiation
(k > 640 nm) for 15 min at a fluence rate of 58 mW cm)2. Mean of
triplicate experiments. Error bars represent standard deviation.
Figure 4. Fluorescence and phase contrast microscopy of compound 2
in Hep2 cells, 400·. Bar 5 lm.

174 Petr Zimcik et al.
rule out any significant accumulation or specific binding of this
compound to the cell membrane. The localization of this highly
charged compound in lysosomes can be anticipated and shows
an endocytic mechanism of cellular uptake. The tests were
performed also with other dyes (1, 3 and 4) and the results
indicated a similar behavior (see Supporting Information).
Unfortunately, their low fluorescence signal, a consequence of
very low FF, did not allow unequivocal conclusions to be made.
Figure S5. Changes in fluorescence emission of 4 in water at
1.0 lM with increasing amount of pyridine added to solution
(from 0% to 15% vol ⁄ vol). Excitation wavelength 375 nm.
Absorption at excitation wavelength still remained the same
(see Fig. S4A).
Figure S6. Changes in absorption spectra of DPBF after
irradiation of its DMF solution containing compound 2 for 0,
15, 30, 80, 140 and 190 s. Inset: dependence of ln(A0 ⁄ At) on
irradiation time t. A0 is absorbance at 417 nm prior to
irradiation, At is absorbance at 417 nm after irradiation for
time t.
CONCLUSION
In conclusion, we synthesized quaternized AzaPc and TQP
that are well soluble in water and are found in this solution
mainly in the monomeric state at high concentrations (2) or at
low concentrations (4). Both dyes are characterized by high
singlet oxygen quantum yields that make them promising PSs
for PDT. Quaternization of peripheral amines appeared to be
crucial for good photodynamic activity of AzaPc molecules as
deduced from lower singlet oxygen quantum yields and hardly
detected photodynamic activity of nonquaternized AzaPc 1.
The reason for the lower activity is most likely based on
deactivation of AzaPc excited states by intramolecular PET.
Photodynamic activity of similar TQP macrocycle 3 is almost
not influenced as a consequence of ineffective PET. Also, we
present here the results of the first in vitro photodynamic tests
of these types of PSs. Their photodynamic activity on Hep2
cells was found to be strong and they did not require any
special solubilizing agent to be added. Compounds 2 and 4
possess promising therapeutic index as they were found to be
nontoxic in the dark up to the maximum used concentration
200 lM (i.e. more than 1000 times less effective than toxic
concentration) and localize inside the cells mainly in lyso-
somes. Compound 4 has strong absorption at long wave-
lengths close to 750 nm that allows excitation with light having
deeper penetration through human tissues and that is why
larger tumors could be treated. Therefore we believe that this
particular compound has high potential to become a very
attractive alternative to established PS.
Figure S7. Fluorescence and phase contrast microscopy of
compounds 1 and 2 in Hep2 cells, 400·. Bar 5 lm.
Figure S8. Fluorescence and phase contrast microscopy of
compounds 3 and 4 in Hep2 cells, 400·. Bar 5 lm.
Modified synthesis of 9 (Figs. S1–S5) showing changes in
absorption and fluorescence spectra of 3 and 4, details on
DPBF decomposition (Fig. S6) and figures of subcellular
localization (Figs. S7 and S8) can be found.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting information
supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author for
the article.
REFERENCES
1. Triesscheijn, M., P. Baas, J. H. M. Schellens and F. A. Stewart
(2006) Photodynamic therapy in oncology. Oncologist 11, 1034–
1044.
2. Zimcik, P. and M. Miletin (2009) Photodynamic therapy. In Dyes
and Pigments—New Research (Edited by A. R. Lang), pp. 1–62.
Nova Science Publishers, New York.
3. Castano, A. P., T. N. Demidova and M. R. Hamblin (2004)
Mechanisms in photodynamic therapy: Part one—Photosensitiz-
ers, photochemistry and cellular localization. Photodiagnosis
Photodyn. Ther. 1, 279–293.
4. Dougherty, T. J., C. J. Gomer, B. W. Henderson, G. Jori,
D. Kessel, M. Korbelik, J. Moan and Q. Peng (1998) Photody-
namic therapy. J. Natl Cancer Inst. 90, 889–905.
5. Qiang, Y. G., X. P. Zhang, J. Li and Z. Huang (2006) Photody-
namic therapy for malignant and non-malignant diseases: Clinical
investigation and application. Chin. Med. J. 119, 845–857.
6. Moreira, L. M., F. V. dos Santos, J. P. Lyon, M. Maftoum-Costa,
C. Pacheco-Soares and N. S. da Silva (2008) Photodynamic
therapy: Porphyrins and phthalocyanines as photosensitizers.
Aust. J. Chem. 61, 741–754.
7. Wainwright, M. (2008) Photodynamic therapy: The development
of new photosensitisers. Anti-Cancer Agents Med. Chem. 8, 280–
291.
Acknowledgements—This work was supported by Research Project
MSM0021620822, Czech Science Foundation Grant No. 203 ⁄ 07 ⁄ P445
and Czech Academy of Sciences, Prague, Grant No. KAN200100801.
Cytotoxic and photodynamic tests were performed by GENERI
BIOTECH s.r.o. and their help is highly acknowledged.
8. Li, H. R., T. J. Jensen, F. R. Fronczek and M. G. H. Vicente
(2008) Syntheses and properties of a series of cationic water-
soluble phthalocyanines. J. Med. Chem. 51, 502–511.
9. Lo, P. C., J. D. Huang, D. Y. Y. Cheng, E. Y. M. Chan,
W. P. Fong, W. H. Ko and D. K. P. Ng (2004) New amphiphilic
silicon(IV) phthalocyanines as efficient photosensitizers for
photodynamic therapy: Synthesis, photophysical properties, and
in vitro photodynamic activities. Chem. Eur. J. 10, 4831–4838.
10. Amerio, P., T. Gobello, C. Mazzanti, E. Giaculli, S. Ruggeri,
D. Sordi, M. Amatetti, L. Chinni and A. Tulli (2007) Photody-
namic therapy plus topical retinoids in Darier’s disease. Photodi-
agnosis Photodyn. Ther. 4, 36–38.
11. Fabris, C., M. Soncin, G. Miotto, L. Fantetti, G. Chiti, D. Dei,
G. Roncucci and G. Jori (2006) Zn(II)-phthalocyanines as
phototherapeutic agents for cutaneous diseases. Photosensitiza-
tion of fibroblasts and keratinocytes. J. Photochem. Photobiol. B,
Biol. 83, 48–54.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Changes in absorption spectra of 3 in pyridine at
1.0 lM with increasing temperature.
Figure S2. Changesin absorption spectra of 3 in pyridine with
dilution from 10 lM to 0.3 lM at rt. Inset: Values of extinction
coefficient at 752 nm in dependence on concentration.
Figure S3. Changes in absorption spectra of 4 in water at
2.2 lM with increasing temperature.
Figure S4. Changes in absorption spectra of 4 in water at
1.0 lM with increasing amount of pyridine (A), DMF (B) or
MeOH (C) added into solution. Insets: Changes in absorbance
at 756 nm.

Photochemistry and Photobiology, 2010, 86 175
12. Scalise, I. and E. N. Durantini (2005) Synthesis, properties, and
photodynamic inactivation of Escherichia coli using a cationic and
a noncharged Zn(II) pyridyloxyphthalocyanine derivatives. Bio-
org. Med. Chem. 13, 3037–3045.
13. Mantareva, V., V. Kussovski, I. Angelov, E. Borisova, L. Avramov,
G. Schnurpfeil and D. Wohrle (2007) Photodynamic activity
of water-soluble phthalocyanine zinc(II) complexes against patho-
genic microorganisms. Bioorg. Med. Chem. 15, 4829–4835.
14. Zhang, Z. B., H. T. Jin, J. Bao, F. Fang, J. F. Wei and A. P. Wang
(2006) Intravenous repeated-dose toxicity study of ZnPcS2P2-
based-photodynamic therapy in Wistar rats. Photochem. Photo-
biol. Sci. 5, 1006–1017.
derivatives containing bulky phenoxy substituents to prevent self-
association. Tetrahedron Lett. 48, 7358–7361.
27. Kudrevich, S. V., M. G. Galpern, E. A. Lukyanets and
J. E. vanLier (1996) Substituted tetra 2,3-pyrazinoporphyrazines.
1. Angular annelation of tetra-2,3-quinoxalinoporphyrazine. Can.
J. Chem. 74, 508–515.
28. Mitzel, F., S. FitzGerald, A. Beeby and R. Faust (2003) Acetylenic
quinoxalinoporphyrazines as photosensitisers for photodynamic
therapy. Chem. Eur. J. 9, 1233–1241.
29. Haas, M., S. X. Liu, A. Neels and S. Decurtins (2006) A synthetic
approach to asymmetric phthalocyanines with peripheral metal-
binding sites. Eur. J. Org. Chem., 2006, 5467–5478.
15. Farooq, F. T., J. Berlin, E. Baron, A. L. Faulx, J. M. Marks and
A. Chak (2007) Pilot animal study of the phthalocyanine Pc4 as a
topically-applied photosensitizer for PDT of Barrett’s esophagus.
Gastrointest. Endosc. 65, AB155.
16. Ahmad, N., K. Kalka and H. Mukhtar (2001) In vitro and in vivo
inhibition of epidermal growth factor receptor-tyrosine kinase
pathway by photodynamic therapy. Oncogene 20, 2314–2317.
17. Ke, M. S., L. Y. Xue, D. K. Feyes, K. Azizuddin, E. D. Baron,
T.S.McCormick,H.Mukhtar,A.Panneerselvam,M.D.Schluchter,
K. D. Cooper, N. L. Oleinick and S. R. Stevens (2008) Apoptosis
mechanisms related to the increased sensitivity of Jurkat T-cells vs
A431 epidermoid cells to photodynamic therapy with the phtha-
locyanine pc 4. Photochem. Photobiol. 84, 407–414.
30. Zimcik, P., M. Miletin, Z. Musil, K. Kopecky, L. Kubza and
D. Brault (2006) Cationic azaphthalocyanines bearing aliphatic
tertiary amino substituents—Synthesis, singlet oxygen production
and spectroscopic studies. J. Photochem. Photobiol. A, Chem 183,
59–69.
31. Musil, Z., P. Zimcik, M. Miletin, K. Kopecky and J. Lenco (2007)
Synthesis, separation and UV ⁄ Vis spectroscopy of pyrazino-
quinoxalino-porphyrazine macrocycles. Eur. J. Org. Chem., 2007,
4535–4542.
32. Musil, Z., P. Zimcik, M. Miletin, K. Kopecky, M. Link, P. Petrik
and J. Schwarz (2006) Synthesis and singlet oxygen production of
azaphthalocyanines bearing functional derivatives of carboxylic
acid. J. Porphyr. Phthalocyanines 10, 122–131.
18. Sakharov, D. V., E. D. R. Elstak, B. Chernyak and K. W. A. Wirtz
(2005) Prolonged lipid oxidation after photodynamic treatment.
Study with oxidation-sensitive probe C11-BODIPY581 ⁄ 591. FEBS
Lett. 579, 1255–1260.
19. Bai, L., J. Guo, F. A. Bontempo and J. L. Eiseman (2009) The
relationship of phthalocyanine 4 (Pc 4) concentrations measured
noninvasively to outcome of Pc 4 photodynamic therapy in mice.
Photochem. Photobiol. 85, 1011–1019.
20. Hofman, J. W., F. van Zeeland, S. Turker, H. Talsma,
S. A. Lambrechts, D. V. Sakharov, W. E. Hennink and C. F. van
Nostrum (2007) Peripheral and axial substitution of phthalocya-
nines with solketal groups: Synthesis and in vitro evaluation for
photodynamic therapy. J. Med. Chem. 50, 1485–1494.
21. Zimcik, P., M. Miletin, M. Kostka, J. Schwarz, Z. Musil and
K. Kopecky (2004) Synthesis and comparison of photodynamic
activity of alkylheteroatom substituted azaphthalocyanines.
J. Photochem. Photobiol. A, Chem 163, 21–28.
33. Michelsen, U., H. Kliesch, G. Schnurpfeil, A. K. Sobbi and
D. Wohrle (1996) Unsymmetrically substituted benzonaphthopor-
phyrazines: A new class of cationic photosensitizers for the photo-
dynamic therapy of cancer. Photochem. Photobiol. 64, 694–701.
34. Ogunsipe, A., D. Maree and T. Nyokong (2003) Solvent effects on
the photochemical and fluorescence properties of zinc phthalocy-
anine derivatives. J. Mol. Struct. 650, 131–140.
35. Seotsanyana-Mokhosi, I., T. Kresfelder, H. Abrahamse and
T. Nyokong (2006) The effect of Ge, Si and Sn phthalocyanine
photosensitizers on cell proliferation and viability of human
oesophageal carcinoma cells. J. Photochem. Photobiol. B-Biol. 83,
55–62.
36. Feofanov, A., A. Grichine, T. Karmakova, N. Kazachkina,
E. Pecherskih, R. Yakubovskaya, E. Luyanets, V. Derkacheva,
M. Egret-Charlier and P. Vigny (2002) Chelation with metal is not
essential for antitumor photodynamic activity of sulfonated
phthalocyanines. Photochem. Photobiol. 75, 527–533.
22. Kostka, M., P. Zimcik, M. Miletin, P. Klemera, K. Kopecky and
Z. Musil (2006) Comparison of aggregation properties and pho-
todynamic activity of phthalocyanines and azaphthalocyanines.
J. Photochem. Photobiol. A, Chem 178, 16–25.
23. Mørkved, E. H., N. K. Afseth and P. Zimcik (2007) Azapht-
halocyanines with extended conjugation through heteroaryl and
aryl substituents. Photochemical and photophysical properties.
J. Porphyr. Phthalocyanines 11, 130–138.
24. Donzello, M. P., E. Viola, C. Bergami, D. Dini, C. Ercolani, M.
Giustini, K. M. Kadish, M. Meneghetti, F. Monacelli, A. Rosa
and G. Ricciardi (2008) Tetra-2,3-pyrazinoporphyrazines with
externally appended pyridine rings. 6. Chemical and redox prop-
erties and highly effective photosensitizing activity for singlet
oxygen production of penta- and monopalladated complexes in
dimethylformamide solution. Inorg. Chem. 47, 8757–8766.
25. Kobak, R. Z. U. and A. Gul (2009) Synthesis and solution studies
on azaphthalocyanines with quaternary aminoethyl substituents.
Color. Technol. 125, 22–28.
37. Youngblood, W. J. (2006) Synthesis of a new trans-A(2)B(2)
phthalocyanine motif as a building block for rodlike phthalocya-
nine polymers. J. Org. Chem. 71, 3345–3356.
38. Kameyama, K., M. Morisue, A. Satake and Y. Kobuke (2005)
Highly fluorescent self-coordinated phthalocyanine dimers.
Angew. Chem. Int. Ed. Engl. 44, 4763–4766.
39. Novakova, V., P. Zimcik, K. Kopecky, M. Miletin, J. Kunes and
ˇ
K. Lang (2008) Self-assembled azaphthalocyanine dimers with
higher fluorescence and singlet oxygen quantum yields than the
corresponding monomers. Eur. J. Org. Chem. 2008, 3260–3263.
40. Daraio, M. E., A. Volker, P. F. Aramendıa and E. San Roman
(1996) Reaction of zinc phthalocyanine excited states with amines
in cationic micelles. Langmuir 12, 2932–2938.
41. Block, J. H. and J. M. Beale Jr (2004). Wilson and Gisvold’s
Textbook of Organic Medicinal and Pharmaceutical Chemistry,
11th edn. Lippincott Williams & Wilkins, Baltimore.
42. Dummin, H., T. Cernay and H. W. Zimmermann (1997) Selective
photosensitization of mitochondria in HeLa cells by cationic Zn
(II) phthalocyanines with lipophilic side-chains. J. Photochem.
Photobiol. B, Biol. 37, 219–229.
26. Makhseed, S., F. Ibrahim, C. G. Bezzu and N. B. McKeown
(2007) The synthesis of metal-free octaazaphthalocyanine