Phthalocyanines functionalized with 2-methyl-5-nitro-1H-imidazolylethoxy and 1,4,7-trioxanonyl moieties and the effect of metronidazole substitution on photocytotoxicity

Article abstract of DOI:10.1016/j.jinorgbio.2013.06.012

Four novel magnesium(II) and zinc(II) phthalocyanines bearing 1,4,7-trioxanonyl, polyether and/or (2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy, heterocyclic substituents at their non-peripheral positions were synthesized and assessed in terms of physicochemical and biological properties. Magnesium phthalocyanine derivatives bearing polyether substituents (Pc-1), a mixed system of polyether and heterocyclic substituents (Pc-3), and four heterocyclic substituents (Pc-4), respectively, were synthesized following the Linstead macrocyclization reaction procedure. Zinc phthalocyanine (Pc-2) bearing polyether substituents at non-peripheral positions was synthesized following the procedure in n-pentanol with the zinc acetate, and DBU. Novel phthalocyanines were purified by flash column chromatography and characterized using NMR, MS, UV-Vis and HPLC. Moreover, two precursors in macrocyclization reaction phthalonitriles were characterized using X-ray. Photophysical properties of the novel macrocycles were evaluated, including UV-Vis spectra analysis and aggregation study. All macrocycles subjected to singlet oxygen generation and the oxidation rate constant measurements exhibited lower quantum yields of singlet oxygen generation in DMSO than in DMF. In addition, the Pc-2 molecule was found to be the most efficient singlet oxygen generator from the group of macrocycles studied. The photocytotoxicity evaluated on the human oral squamous cell carcinoma cell line, HSC-3, for Pc-3 was significantly higher than that for Pc-1, Pc-2, and Pc-4. Interestingly, Pc-3 was found to be the most active macrocycle in vitro although its ability to generate singlet oxygen was significantly lower than those of Pc-1 and Pc-2. However, attempts to encapsulate phthalocyanines Pc-1-Pc-3 in liposomal membranes were unsuccessful. The phthalocyanine-nitroimidazole conjugate, Pc-4 was encapsulated in phosphatidylglycerol:phosphatidylcholine unilamellar liposomes and subjected to photocytotoxicity study.

Full text of DOI:10.1016/j.jinorgbio.2013.06.012

Journal of Inorganic Biochemistry 127 (2013) 62–72
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Phthalocyanines functionalized with 2-methyl-5-nitro-1H-imidazolylethoxy
and 1,4,7-trioxanonyl moieties and the effect of metronidazole substitution
on photocytotoxicity
b
b
a
Marcin Wierzchowski a, , Lukasz Sobotta , Paulina Skupin-Mrugalska , Justyna Kruk ,
Weronika Jusiak a, Michael Yee c, Krystyna Konopka c, Nejat Düzgüneş c, Ewa Tykarska a,
Maria Gdaniec d, Jadwiga Mielcarek b, Tomasz Goslinski a
⁎
a
Department of Chemical Technology of Drugs, Faculty of Pharmacy, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland
Department of Inorganic and Analytical Chemistry, Faculty of Pharmacy, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland
Department of Biomedical Sciences, University of the Pacific, Arthur A. Dugoni School of Dentistry, San Francisco, CA 94115, USA
Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznan, Poland
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Four novel magnesium(II) and zinc(II) phthalocyanines bearing 1,4,7-trioxanonyl, polyether and/or (2-methyl-
5-nitro-1H-imidazol-1-yl)ethoxy, heterocyclic substituents at their non-peripheral positions were synthesized
and assessed in terms of physicochemical and biological properties. Magnesium phthalocyanine derivatives
bearing polyether substituents (Pc-1), a mixed system of polyether and heterocyclic substituents (Pc-3), and
four heterocyclic substituents (Pc-4), respectively, were synthesized following the Linstead macrocyclization re-
action procedure. Zinc phthalocyanine (Pc-2) bearing polyether substituents at non-peripheral positions was
synthesized following the procedure in n-pentanol with the zinc acetate, and DBU. Novel phthalocyanines
were purified by flash column chromatography and characterized using NMR, MS, UV–Vis and HPLC. Moreover,
two precursors in macrocyclization reaction phthalonitriles were characterized using X-ray. Photophysical prop-
erties of the novel macrocycles were evaluated, including UV–Vis spectra analysis and aggregation study. All
macrocycles subjected to singlet oxygen generation and the oxidation rate constant measurements exhibited
lower quantum yields of singlet oxygen generation in DMSO than in DMF. In addition, the Pc-2 molecule
was found to be the most efficient singlet oxygen generator from the group of macrocycles studied. The
photocytotoxicity evaluated on the human oral squamous cell carcinoma cell line, HSC-3, for Pc-3 was signifi-
cantly higher than that for Pc-1, Pc-2, and Pc-4. Interestingly, Pc-3 was found to be the most active macrocycle
in vitro although its ability to generate singlet oxygen was significantly lower than those of Pc-1 and Pc-2. How-
ever, attempts to encapsulate phthalocyanines Pc-1–Pc-3 in liposomal membranes were unsuccessful. The
phthalocyanine–nitroimidazole conjugate, Pc-4 was encapsulated in phosphatidylglycerol:phosphatidylcholine
unilamellar liposomes and subjected to photocytotoxicity study.
Received 15 March 2013
Received in revised form 21 June 2013
Accepted 21 June 2013
Available online 28 June 2013
Keywords:
Photocytotoxicity
Photosensitizer
Phthalocyanine
Singlet oxygen
Photodynamic therapy
© 2013 Elsevier Inc. All rights reserved.
1. Introduction
organelles and molecules, and tumor vasculature, and to complex in-
flammatory and immune responses [10,11].
Photodynamic therapy (PDT) is a relatively new therapeutic ap-
proach that has been applied successfully against tumors and derma-
tological lesions [1–4], like psoriasis [5], and age-related macular
degeneration [6]. Many of natural and synthetic compounds exhibit
photosensitizing potential, of which synthetic porphyrinoids, including
phthalocyanines are quite common [7–9]. The photodynamic reaction
involves the cooperative action of three components: light, oxygen
and the photosensitizer leading to the cytotoxic effects on subcellular
Novel third-generation photosensitizers, accompanied by delivery
systems have been investigated for PDT, and they aim to break some
limitations of the clinically used first- and second-generation photosen-
sitizers, like hematoporphyrin derivative (HpD) and temoporfin [12–15].
It seems that conjugation of porphyrinoids to nitroimidazoles and
related compounds, like metronidazole, misonidazole, etanidazole,
pimonidazole, nimorazole, and isometronidazole, which possess an-
ticancer activity in deep hypoxic conditions and are thus termed
“oxygen-mimetic” radiosensitizers [16–18] seeks to achieve this goal.
New pyropheophorbide-a derivatives possessing imidazole substituents
have revealed promising photodynamic activity towards A549 human
lung adenocarcinoma cells [19]. Additionally, cationic imidazolium
substituted metalloporphyrins have been studied using human HeLa
⁎
Corresponding author at: Department of Chemical Technology of Drugs, Poznan
University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland. Tel.: +48 61
854 66 37; fax: +48 61 854 66 39.
E-mail address: mwierzch@ump.edu.pl (M. Wierzchowski).
0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2013.06.012

M. Wierzchowski et al. / Journal of Inorganic Biochemistry 127 (2013) 62–72
63
and murine CT26 cancer cell lines [20], whereas conjugates of
2-nitroimidazole and manganese porphyrin have demonstrated selec-
tivity and good radiotherapeutic effect towards murine SCCVII oral
squamous cell carcinoma cell lines implanted in C3H/He mice [21]. In
the course of this research, we recently reported the synthesis of a
novel porphyrazine functionalized with nitroimidazolylbutylsulfanyl
groups, and described its photochemical properties and cytotoxicity
on two prostate human cancer cell lines, LNCaP and PC3, and one
melanoma-derived cell line, MeWo [22].
In the present paper we report the synthesis, characterization,
photochemical properties and photocytotoxicity of four novel zinc(II)
and magnesium(II) phthalocyanines bearing 1,4,7-trioxanonyl and/or
2-methyl-5-nitro-1H-imidazolylethoxy substituents (metronidazole
fragments) at non-peripheral positions. Phthalocyanines exhibit limit-
ed solubility in aqueous media and common organic solvents, therefore
peripheral substitution with polyether moieties was applied. For the
last 30 years peripheral and axial substitution of phthalocyanines with
crown ethers and polyether moieties has resulted in valuable com-
pounds useful for studies on supramolecular interactions, in catalysis,
and as sensors, photosensitizers and photovoltaic devices [23–31].
In this study we attempt to determine whether the combination of
the three potentially promising building units, i.e. phthalocyanine
macrocycle, polyether chains and metronidazole units results in novel
conjugates exhibiting interesting and useful physicochemical and bio-
logical properties.
Eos Xcalibur diffractometer using graphite-monochromated MoKα
radiation, and processed with the Algient Technologies CrysAlis Pro
software [33]. The structures were solved by direct methods and re-
fined by the full-matrix least-squares method based on F2 (SHELXS,
SHELXL) [34]. All non-hydrogen atoms were refined anisotropically.
The C-bound hydrogen atoms were generated geometrically with
C\H = 0.93–0.98 Å and refined as riding on their corresponding car-
bon atoms, with Uiso (H) = 1.2 Ueq (C) or 1.5 Ueq (methyl groups).
Summary of structure determination is given in Table 1. Selected tor-
sion angles are given in Tables S1 and S2 (Supplementary data).
2.3. Synthesis
2.3.1. 3,6-Bis(1,4,7-trioxanonyl)-1,2-benzenedicarbonitrile (2)
2-(2-Ethoxyethoxy)ethyl bromide (1.96 mL, 12.90 mmol) and an-
hydrous K2CO3 (1.04 g, 10.40 mmol) were added to a well-stirred slur-
ry of 3,6-dihydroxy-1,2-benzenedicarbonitrile (830 mg, 5.20 mmol) in
DMF (15 mL) and heated at 70 °C for 24 h. After cooling to room tem-
perature, the reaction contents were poured into water and ice mixture
(100 mL) and left for 2 h. The resulting precipitate was isolated by fil-
tration, washed three times with distilled water (3 × 100 mL) and crys-
tallized from ethanol (50 mL) to give honey colored crystals (2) (1.63 g,
80%). M.p. = 70–71 °C. Rf (CH2Cl2 10:1) 0.77. UV–Vis (CH2Cl2) λmax
[nm] (log ε): 346 (3.85). 1H NMR (400 MHz, pyridine-d5) δ: 7.42
(s, 2H), 4.26 (t, 3J = 4.5 Hz, 4H), 3.83 (t, 3J = 4.5 Hz, 4H), 3.71 (t, 3J =
4.5 Hz, m, 4H), 3.56 (t, 3J = 4.5 Hz, 4H), 3.42 (q, 3 J = 7.0 Hz, 1H), 1.11
(t, 3 J = 7.0 Hz, 6H).1H NMR (400 MHz, DMSO-d6) δ: 7.63 (s, 2H), 4.30
(t, 3J = 4.5 Hz, 4H), 3.77 (t, 3J = 5.0 Hz, 4H), 3.58–3.61 (m, 4H), 3.52–
3.60 (m, 4H), 3.46–3.49 (m, 4H), 3.41 (q, 3J = 7.0 Hz, 4H), 1.08
(t, 3J = 7.5 Hz, 6H). 13C NMR (100 MHz, pyridine-d5) δ: 155.7,
120.2, 114.1, 105.0, 71.3, 70.4, 70.2, 69.5, 66.5, 15.5. 13C NMR (100 MHz,
DMSO-d6) δ: 155.0, 120.8, 113.5, 103.0, 70.1, 69.6, 69.2, 68.6, 66.5, 15.0
(graphical presentation of signal-structure allocation — Supplementary
Data). MS (ESI): m/z 393.434 [M + H]+. Anal. Cal. for C20H28N2O6: C,
61.21; H, 7.19; N, 7.14. Found: C, 61.12; H, 7.06; N, 7.11. Crystal data for
2, CCDC 927434.
2. Experimental
2.1. Materials and methods
Relevant modifications to previously published procedures are in-
cluded [22,32]. All reactions were conducted in oven-dried glassware
under nitrogen. Reported reaction temperatures refer to external bath
temperatures. All reactions were performed under an inert atmosphere
of nitrogen. All solvents were rotary evaporated (under reduced pressure)
at or below 60 °C. Dichloromethane, methanol and tetrahydrofuran were
purified by distillation. Other solvents and all reagents were obtained
from commercial suppliers and used without further purification. Melting
points were obtained on a “Stuart” Bibby apparatus and are uncorrected.
Dry flash column chromatography was carried out on Merck silica gel 60,
particle size 40–63 μm and Fluka silica gel 90 C18-reversed phase. Thin
layer chromatography (TLC) was performed on silica gel Merck Kieselgel
60 F254 plates and DC Kieselgel 60 RP-18 F254 and visualized with UV illu-
mination (λmax 254 or 365 nm). Mass spectrometry experiments (ES,
MALDI TOF) and elemental analyses were carried out by the Advanced
Chemical Equipment and Instrumentation Facility at the Faculty of Chem-
istry, Adam Mickiewicz University in Poznan.
UV–visible (UV–Vis) spectra were recorded on Hitachi UV/VIS
U-1900 and Unicam 500 spectrometers; λmax [nm] (log ε). 1D and
2D NMR experiments were carried out using a Bruker 400 spectrometer
by the Laboratory of Biomolecular NMR at the Institute of Bioorganic
Chemistry Polish Academy of Sciences in Poznan. Chemical shifts (δ)
are quoted in parts per million (ppm) and are referred to a residual
solvent peak. Coupling constants (J) are quoted in Hertz (Hz) to the
nearest 0.5 Hz. The abbreviations Ar, Imid, s, d, t, h, q, ps, pt, and m
refer to aromatic, imidazole, singlet, doublet, triplet, hidden, quartet,
pseudo-singlet, pseudo-triplet and multiplet respectively. The tech-
niques used to allocate signals were 1H, 13C, 1H–1H COSY, HSQC, and
HMBC. Analytical HPLC was performed on an Agilent 1200 Series
instrument.
2.3.2. 1-[2-(2,3-Dicyanophenoxy)ethyl]-2-methyl-5-nitro-1H-imidazole (4)
Anhydrous K2CO3 (7.69 g, 57.8 mmol), was added to a well-
stirred slurry of 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethanol (4.94 g,
28.9 mmol) and 3-nitrophtalonitrile (5.00 g, 28.9 mmol) in DMF
(60 mL) and heated at 70 °C for 24 h. After cooling to room temperature,
Table 1
Crystal data and refinement details of phthalonitriles 2 and 4.
2
4
Empirical formula
Formula weight
Temperature, K
C20H28N2O6
392.44
130
C14H11N5O3
297.28
293
Wavelength, Å
0.71073
0.71073
Crystal system
Space group
Monoclinic
P 21/n
Orthorhombic
Pbcn
Unit cell dimensions, Å and °
a = 7.6535 (7)
b = 37.939 (3)
c = 7.9448 (6)
β = 117.95 (1)
2037.8 (3)
4
a = 25.5069 (6)
b = 7.5446 (2)
c = 14.0286 (3)
Volume, Å3
Z
2699.7(1)
8
Calculated density, g/cm3
Absorption coefficient, mm−1
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I N 2σ(I)]
1.279
0.095
13351
1.463
0.108
13887
3703 [Rint = 0.0372]
3703/0/255
1.047
R1 = 0.0421
wR2 = 0.0803
R1 = 0.0611
wR2 = 0.0879
0.17, −0.19
2466 [Rint = 0.0254]
2466/0/200
1.069
R1 = 0.0393
wR2 = 0.1004
R1 = 0.0536
wR2 = 0.1044
0.17, −0.15
2.2. X-ray
R indices (all data)
Single crystals of phthalonitriles 2 and 4 were obtained at room
temperature by slow evaporation of ethanol and DMF solutions, re-
spectively. Diffraction data were collected with an Oxford Diffraction
Largest peak and hole, eA−3

64
M. Wierzchowski et al. / Journal of Inorganic Biochemistry 127 (2013) 62–72
the reaction contents were poured into water and ice (300 mL) and
left for 2 h. The resulting precipitate was isolated by filtration, washed
with distilled water (3 × 100 mL) and heated under reflux in CH3OH
(200 mL) for 40 min. The suspension was cooled and the light gray
solid was filtered and dried to give 4 (5.56 g, 65%). M.p. = 217 °C; Rf
(CH2Cl2:CH3OH 10:1) 0.58. UV–Vis (DMF) λmax [nm] (log ε): 318
(4.23). 1H NMR (400 MHz, pyridine-d5) δ: 8.25 (s, 1H), 7.52–7.61
(mh, 1H), 7.39, 7.37 (2 × d, 3J = 8.0 Hz, 3J = 9.5 Hz, 2H), 4.80
(t, 3J = 4.5 Hz, 2H), 4.59 (t, 3J = 4.5 Hz, 2H), 2.82 (s, 3H). 13C NMR
(100 MHz, pyridine-d5) δ: 160.8, 152.6, 139.1 (not resolved due to
the presence of NO2, only spot found in HMBC), 135.4h, 134.0,
126.1, 117.7, 116.8, 116.1, 114.1, 104.4, 69.1, 45.7, 14.8 (graphical
presentation of signal-structure allocation — Supplementary Data).
MS (ESI pos): m/z 298 [M + H]+, 320 [M + Na]+. MS (ESI neg):
m/z 296 [M − H]−. Anal. Cal. for C14H11N5O3 C, 56.56, H, 3.73, N,
23.56. Found: C, 56.46, H, 3.85, N, 23.59. Crystal data for 4, CCDC
927435.
pressure with toluene (3 × 15 mL). The main products Pc-1 and
Pc-3 were separated by column chromatography (CH2Cl2:CH3OH
10:1). Pc-1 was repurified according to the chromatographic proce-
dure described in Section 2.3.3 to give 315 mg (31%) of the dark
green solid. Pc-3 was repurified by flash column chromatography
(CH2Cl2:CH3OH 10:1, next in EtOAc was applied to get rid of impuri-
ties. The pure compound was flushed down with CH2Cl2:CH3OH
10:1). Evaporation of collected eluates resulted in a dark green
solid (57 mg, 15%). M.p. N 50 °C (mesophase behavior). Rf (10:1
THF:CH3OH) 0.85. UV–Vis (CH2Cl2) λmax [nm] (log ε): 836 (4.59),
542 (3.86), 320 (4.28). MS (MALDI TOF pos): m/z [M + H]+
1499.7, [M + Na]+ 1521.7. MS (MALDI TOF neg): m/z [M − H]−
1497.2 1H NMR (400 MHz, pyridine-d5) δ: 9.64 (d), 9.41 (d), 8.21
(s), 8.20 (s), 8.12 (t), 8.03 (t), 7.99 (s), 7.97 (s), 7.82 (d), 7.77 (d),
7.68 (d), 5.65–5.72 (m), 5.39–5.49 (m), 5.32–5.39 (m), 5.02–5.13
(m), 4.53–4.63 (m), 4.34 (t), 4.26 (t), 4.11–4.22 (m), 3.90 (t), 3.79–
3.85 (m), 3.64 (t), 3.49 (q), 3.44 (q), 2.86 (s), 2.84 (s), 1.11 (t), 1.01
(t); 13C NMR (100 MHz, pyridine-d5) δ 156.4, 153.9, 153.6, 153.4, 153.2,
153.0, 153.0, 152.9, 152.9, 152.8, 152.6, 152.5, 152.4, 152.2, 151.8, 143.0,
139.4, 133.9, 130.4, 130.3, 130.2, 129.0, 127.8, 126.0, 121.0, 120.8, 120.4,
118.0, 116.6, 116.4, 73.4, 72.7, 72.6, 71.6, 71.2, 71.0, 70.9, 70.8, 70.5,
70.4, 66.6, 66.5, 47.3, 15.5, 15.4, 14.8, 14.3. HPLC (see Supplementary
data).
2.3.3. [1,4,8,11,15,18,22,25-Octakis(1,4,7-trioxanonyl)phthalocyanine]
magnesium(II) (Pc-1)
A rapidly stirred mixture of magnesium turnings (37 mg, 1.5 mmol),
n-butanol (25 mL), and I2 (1 crystal) was heated under reflux for 8 h.
After the mixture was cooled to room temperature, phthalonitrile 2
(600 mg, 1.50 mmol) was added, and the reaction mixture was heated
under reflux for further 20 h. After being allowed to cool to room temper-
ature, the reaction mixture was filtered through Celite and evaporated to
dryness under reduced pressure with toluene (3 × 15 mL). Column chro-
matography (CH2Cl2:CH3OH 4:1) was performed. Evaporation of collect-
ed eluates gave the dark green solid Pc-1 (200 mg, 34%). M.p. N 50 °C
(mesophase behavior). Rf (THF:CH3OH 10:1) 0.78. UV–Vis (CH2Cl2):
2.3.6. {1,8,18,25-Tetrakis[2-(2-methyl-5-nitro-1H-imidazol-1-yl)etoxy]
phthalocyanine}magnesium(II) (Pc-4)
A rapidly stirred mixture of magnesium turnings (28 mg, 1.2 mmol),
n-butanol (30 mL), and I2 (1 crystal) was heated under reflux for 8 h.
After the mixture was cooled to room temperature, phthalonitrile 4
(350 mg, 1.2 mmol) was added, and the reaction mixture was heated
under reflux for a further 24 h. After being allowed to cool to room tem-
perature, the reaction mixture was diluted with CH3OH (30 mL) and fil-
tered through Celite. The Celite layer was washed with CH3OH (30 mL)
and CH2Cl2 (30 mL). Collected filtrates were evaporated to dryness
under reduced pressure with toluene (3 × 15 mL) and the solid residue
was purified by flash column chromatography (CH2Cl2:MeOH 7:1 to
4:1). Evaporation of the collected eluates gave a pale blue solid (45 mg,
13%). M.p. N 350 °C. Rf (20:1 CH2Cl2:CH3OH) 0.19. MS (MALDI-TOF
pos): m/z [M + H]+ 1213.0. MS (MALDI-TOF neg): m/z [M − H]−
1211.6. 1H NMR (400 MHz, pyridine-d5) δ: 9.41–9.50 (Ar, m, 4H), 8.34
(Imid, s, 2H), 8.07–8.23 (Ar, m, 4H), 8.16 (Ar, sh, 2H), 7.70–7.80 (Ar,
m, 4H), 5.64 (\CH2CH2\, ps, 4H), 5.43 (\CH2CH2\, ps, 8H), 5.24
(\CH2CH2\, ps, 4H), 2.92 (CH3\, ps, 6H), 2.66 (CH3\, ps, 6H). 13C
NMR (100 MHz, pyridine-d5) δ 156.2, 156.1, 154.8, 154.5, 154.3,
154.1, 154.0, 153.9, 153.8, 152.9, 152.8, 152.7, 152.5, 152.3, 142.5,
139.4, 139.4, 134.0, 133.9, 131.3, 131.2, 131.1, 131.0, 127.8, 127.7,
127.3, 127.3, 118.3, 118.1, 117.7, 117.5, 117.2, 115.3, 115.3, 71.0, 71.0,
69.3, 69.2, 46.9, 14.9, 14.5. HPLC purity 99.43–100% (Supplementary
data).
λmax [nm] (log ε): 799 (4.52), 758 (4.32), 326 (4.16). MS (MALDI-TOF):
m/z [M + H]+ 1594.8. 1H NMR (400 MHz, pyridine-d5) δ: 7.97 (s, 8H),
5.38 (t, 3J = 5.0 Hz, 16H), 4.33 (t, 3J = 5.0 Hz, 16H), 3.87 (dd, 3J = 5.5,
3J = 4.0 Hz, 16H), 3.63 (dd, J = 5.5, 3J = 4.0 Hz, 2H), 3.41 (q, 3J =
7.0 Hz, 16H), 1.09 (t, 3J = 7.0 Hz, 24H). 13C NMR (101 MHz,
pyridine-d5) δ: 153.2, 152.3, 128.7, 120.7, 72.5, 71.2, 70.7, 70.3,
66.5, 15.5. HPLC purity 98.09–100% (Supplementary data).
2.3.4. [1,4,8,11,15,18,22,25-Octakis(1,4,7-trioxanonyl)phthalocyanine]
zinc(II) (Pc-2)
Zinc acetate (420 mg, 2.30 mmol) was added to a solution of 2
(1.80 g, 4.50 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
(678 μL, 4.50 mmol) in n-pentanol (9 mL). The reaction mixture was
vigorously stirred and heated at 130 °C for 24 h. Next, the solvent was
evaporated under reduced pressure with toluene (2 × 50 mL) and the
dry residue was purified by column chromatography (CH2Cl2:CH3OH
30:1 to 4:1). Evaporation of collected eluates produced the dark green
Pc-2 (200 mg, 11%). M.p. N 50 °C (mesophase behavior). Rf (CH2Cl2:
CH3OH 30:1) 0.5. UV–Vis (C2H5OH): λmax [nm] (log ε): 725 (4.72),
653 (4.07), 332 (4.12). MS (MALDI-TOF): m/z [M + H+] 1635.1. 1H
NMR (400 MHz, pyridine-d5) δ: 8.01 (s, 8H), 5.43 (t, 3J = 5.0 Hz,
16H), 4.37 (t, 3J = 5.0 Hz, 16H), 3.91, 3.90 (d, d, 3J = 6.0 Hz, 3J =
5.0 Hz, 16H), 3.65, 3.64 (d, d, 3J = 5.0 Hz, 3J = 6.0 Hz, 16H), 3.43
(q, 3J = 7.0 Hz, 16H), 1.11 (t, 3J = 7.0 Hz, 24H). 13C NMR (100 MHz,
pyridine-d5) δ: 153.1, 152.3, 129.3, 120.8, 72.6, 71.2, 70.7, 70.3, 66.5,
15.5. HPLC purity 98.75–100% (Supplementary data).
2.4. Measurement of singlet oxygen generation
All experiments were carried out in DMF and DMSO at ambient
temperature in air conditions (without bubbling oxygen or air via
solvent) or with limited access to air (nitrogen bubbling for 20 min)
using 1,3-diphenylisobenzofuran (DPBF, Aldrich) as a chemical quencher.
The absorbance of the photosensitizer and the quencher was adjusted to
ca. 0.5 and 0.65, respectively, to avoid DPBF side chain reaction proceed-
ing at higher concentrations. Irradiation was performed in a 1 cm path
length quartz cell equipped with a magnetic stir bar. A high-pressure
xenon lamp (150 W, Optel) was used as the irradiation source. A
monochromator (M250/1200/U with 2 nm/mm (Dk = 4 nm) dis-
persion, Optel) was used to select an appropriate wavelength
(corresponding to a Q band at maximum absorbance) and to avoid di-
rect photodegradation of DPBF under UV light irradiation. The light in-
tensity was measured by an RD 0.2/2 with TD probe, Optel radiometer
2.3.5. {1,4,8,11,15,18-Hexakis(1,4,7-trioxanonyl)-22-[2-(2-methyl-5-nitro-
1H-imidazol-1-yl)ethoxy]phthalocyanine}magensium(II) (Pc-3)
A rapidly stirred mixture of magnesium turnings (68 mg, 2.8 mmol),
n-butanol (50 mL), and I2 (1 crystal) was heated under reflux for 8 h.
After cooling to a room temperature, phthalonitrile derivatives 2
(1000 mg 2.55 mmol) and 4 (76 mg, 0.25 mmol) were added.
Resulting reaction mixture was heated under reflux for the next
20 h. After cooling to a room temperature, the reaction mixture
was filtered through Celite and evaporated to dryness under reduced

M. Wierzchowski et al. / Journal of Inorganic Biochemistry 127 (2013) 62–72
65
and adjusted to 0.5 mW/cm2. A Shimadzu UV-160 spectrophotometer
with PC 160 Plus software was used for monitoring the samples after
the irradiation [32,35,36].
high power LED MultiChip Emitter (60 high efficiency AlGaAs diode
chips, Roithner LaserTechnik GmbH, Vienna, Austria) was used for illu-
mination with a light dose of 3 mW/cm2 at 735 nm for 20 min (7.90 V,
0.158 A). The total spectral irradiance at the level of the cells was mea-
sured using a radiometer PM 100A (Thorlabs, Newtown, NJ, USA). 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 complete medium, and cells were incu-
bated for 24 h at 37 °C. Cell viability was quantified by the Alamar Blue
assay.
2.5. Biological activity
Part of the methodology presented below was described earlier
[37].
2.5.1. In vitro photodynamic activity
2.5.1.1. Cell cultures. Two human oral squamous cell carcinoma (OSCC)
cell lines were utilized in this study. HSC-3 cells, derived from SCCs of
the tongue [38], were provided by Dr. R. Kramer (University of California
San Francisco (UCSF), San Francisco USA). H413 cells, derived from
SCC of the buccal mucosa [39] were obtained from Dr. R. Jordan
(UCSF). HSC-3 cells were cultured in Dulbecco's Modified Eagle's
MEM (DMEM) medium supplemented with 10% (v/v) heat-inactivated
fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin
(100 μg/mL), and L-glutamine (4 mM) (DMEM/10). H413 cells were
maintained in DMEM/10 supplemented with Ham's Nutrient Mixture
F12 (DMEM/10/F12). Cells were incubated in tissue culture flasks
(Falcon, Becton Dickinson Labware, UK) at 37 °C in a humidified atmo-
sphere containing 5% CO2, and were passaged 1:6 twice a week using
a Trypsin–EDTA solution. All media, with or without phenol red,
penicillin–streptomycin solution, L-glutamine, FBS, Trypsin–EDTA,
phosphate buffered saline (PBS), and Dulbecco's phosphate buff-
ered saline (DPBS), were obtained from the UCSF Cell Culture Facil-
ity, San Francisco, CA, USA. Photosensitizers were solubilized in
dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) to a
final concentration of 10 mM, and subsequently diluted in DMEM
or DMEM/F12 (without FBS and phenol red) to a concentration of
50 μM. The 50 μM solutions were diluted in the appropriate media
to obtain the desirable concentration of photosensitizer used in
the experiments.
2.5.1.4. Cell viability. Cell morphology was evaluated by inverted phase
contrast microscopy at 25× magnification. The number of viable cells
used for the experiments was determined by the Trypan Blue exclusion
assay, using a Countess Automated Cell Counter (Gibco–Invitrogen Cor-
poration, Carlsbad, CA, USA). Cell viability was quantified by the modi-
fied Alamar Blue assay [40,41]. Briefly, 1.0 mL of 10% (v/v) Alamar Blue
dye in the appropriate complete medium was added to each well.
After incubation at 37 °C for 2–3 h, 200 μL of the supernatant was
assayed by measuring the absorbance at 570 nm and 600 nm. Cell via-
bility (as a percentage of control cells) was calculated according to the
formula [(A570–A600) of test cells] ×100/[(A570–A600) of control
cells].
2.5.2. Liposome preparation and evaluation of photocytotoxicity
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and
L-α-phosphatidyl-DL-glycerol (chicken egg, PG) were purchased from
Avanti Polar Lipids Inc. (Alabaster, AL, USA). Liposomes were prepared
by a thin-film hydration method [42]. Appropriate volumes of lipid
(POPC — 20 mg/mL, PG — 25 mg/mL) and Pc-4 (0.4 mg/mL) solutions
in chloroform (Fisher Scientific, USA) were mixed in a bioguard hood
and then dried using a rotary evaporator. The film formed on the bottom
of the glass tube was kept overnight in a vacuum oven at room temper-
ature to evaporate any residual chloroform. Subsequently, the dried
thin-film was hydrated with HEPES buffered saline (10 mM HEPES,
pH = 7.49, 140 mM NaCl) to force encapsulation and was completely
dispersed by vortexing for ca. 5–10 min. The resulting liposome suspen-
sion was passed 21 times through polycarbonate membranes with a
pore diameter of 100 nm, using a syringe extruder (Avanti Polar Lipids)
to achieve a uniform size distribution. The molar ratio of the final lipo-
some composition was Pc (0.1), POPC (8) and PG (2). The liposome
size was determined by dynamic light scattering measurements using
a Coulter N4 Plus Particle Size Analyzer (Beckman, USA). Samples were
stored at 2-8 °C under argon and were protected from light.
The size of the extruded liposomes of Pc-4 encapsulated in PG:POPC
liposomes was 148.3 nm (SD = 58.8, PI = 0.425). The final concentra-
tion of Pc-4 achieved in the liposome suspension was 100 μM. The bio-
logical activity of the free-form Pc-4 and that encapsulated in liposomes
was examined. Stock solutions of Pc-4 and those in liposomal suspension
were diluted with appropriate media (DME or DME F12, without FBS) to
achieve solutions of 0.1, 1.0 and 10.0 μM concentration. In the case of
Pc-4, a high power LED MultiChip Emitter (60 high efficiency AlGaAs
diode chips, Roithner LaserTechnik GmbH, Vienna, Austria) was used
for illumination with a light dose of 3 mW/cm2 at 735 nm for 20 min
(7.90 V, 0.158 A). The total spectral irradiance at the level of the cells
was measured using a radiometer PM 100A (Thorlabs, Newtown, NJ,
USA).
2.5.1.2. Dark toxicity. One day before the experiment, HSC-3 and H413
cells were seeded in 48-well plates (BD Falcon™, Franklin Lakes, NJ,
USA) at a density of 1.8 × 105 and 1.4 × 105 cells per well, respectively,
in 1 mL of complete medium (with FBS and phenol red), and used at ap-
proximately 80% confluence. Subsequently, cells were pre-washed
twice with PBS (0.5 mL), and 1 mL of medium (without FBS and phenol
red, containing photosensitizer at a given concentration) was added to
each well except the controls. The FBS-free media were used to avoid
binding of photosensitizers to serum proteins. After the 24 h incubation
at 37 °C, cells were washed twice with PBS and 1 mL of complete medi-
um was added to each well. The cells were incubated subsequently for
24 h at 37 °C. Cell viability was quantified by the Alamar Blue assay.
Cells incubated either with medium alone or medium/0.5% DMSO
served as controls.
2.5.1.3. Light-dependent toxicity. One day before the experiment,
HSC-3 and H413 cells were seeded in 48-well plates at a density
1.8 × 105 and 1.4 × 105 cells per well, respectively, in 1 mL of com-
plete medium, and used at approximately 80% confluence. Cells were
pre-washed twice with PBS, and 1 mL of medium without FBS and phe-
nol red, containing photosensitizer at a given concentration, was added
to each well, except the controls. The 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 (600–850 nm, 550 lx) from a Dura Max light bulb (75 W Med
120 V A19 Cl/LL 20 W; Philips Electronics North America Corporation,
Andover, MA), at a distance of 10 cm from the light bulb to the plate,
for 20 min (Pc-1–Pc-3). Infrared radiation was minimized using a
1 cm water filter between the cell plates and the light source. In
the case of Pc-4 (non-encapsulated and encapsulated in liposomes), a
2.5.3. Statistical analysis
Data represent the mean
SD from two independent experi-
ments performed in duplicate (Pc-1–Pc-3) and from one experi-
ment performed in triplicate (Pc-4 and Pc-4 in liposomes). Data
were compared for statistical significance by the unpaired Student's
t-test, using StatView software (Brain Power Inc., Calabasas, CA). A
probability value (P) of less than 0.05 was considered significantly
different.

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M. Wierzchowski et al. / Journal of Inorganic Biochemistry 127 (2013) 62–72
3. Results and discussion
of isomers of which only the isomer of C2v symmetry was isolated and
characterized. It turned out to be impossible to isolate the other isomers
(Cs, D2h, C4v) whose formation could be expected, since only a very
small amount of another macrocyclic product was found in the
crude reaction mixture analyzed by TLC, not allowing for separation.
Zinc phthalocyanine (Pc-2) was synthesized adapting the procedure
described by Vacus and coworkers [48] with 11% yield. The
macrocyclization reaction of phthalonitrile 2 was carried out in
n-pentanol with the zinc acetate as a source of template, and DBU
as a base for 24 h at 130 °C. Phthalocyanines Pc-1–Pc-4 were purified
on silica gel in normal and reversed-phase system by flash column chro-
matography. HPLC analyses revealed the purity of phthalocyanine sam-
ples Pc-1, Pc-2 and Pc-4 at the level above 98%. The HPLC analysis of
Pc-3 performed in various solvent systems revealed the presence of ag-
gregated species, which appeared also during UV–Vis and NMR experi-
ments, and are discussed below.
3.1. Synthesis
2,3-Dicyanohydroquinone (1) was subjected to alkylation reac-
tion with 2-(2-ethoxyethoxy)ethyl bromide, adapting an approach
from the literature, leading to a novel phthalonitrile derivative,
3,6-bis(1,4,7-trioxanonyl)benzene-1,2-dicarbonitrile (2) [43,44].
Phthalonitrile (4) functionalized with heterocyclic, 2-(2-methyl-
5-nitro-1H-imidazol-1-yl)ethoxy moieties, was synthesized by a
nitro-group displacement reaction starting from 3-nitrobenzene-1,2-
dicarbonitrile (3) (Scheme 1) [45].
Both phthalonitrile derivatives 2 and 4 were found to be reactive
in macrocyclization reactions. Magnesium phthalocyanine derivatives
bearing (at non-peripheral positions) polyether substituents (Pc-1), a
mixed system of polyether and nitroimidazolylethoxy substituents
(Pc-3), and four nitroimidazolylethoxy (Pc-4), respectively, were
synthesized with moderate yields (15–34%) following the Linstead
macrocyclization reaction procedure with further modifications [46,47].
Pleasingly, compound 2 revealed promising reactivity in macrocyclization
reactions, similarly to the other polyetheroxy phthalonitriles [23,28]. This
phthalonitrile was subjected to direct macrocyclization conditions,
omitting its activation step to the more active isoindole derivative. In
addition, the macrocyclization reaction of phthalonitrile 4 towards
tetrametronidazole-substituted phthalocyanine Pc-4 led to a mixture
3.2. X-ray structure, NMR and UV–Vis data
The structures of phthalonitriles 2 and 4 were determined by
X-ray crystallography (Fig. 1). The molecule 2 assumed an asymmet-
ric form because of different conformations of its two oxyethylene
chains attached to the benzene ring. Both chains were folded (Table
S1, Supplementary data) and located at the same site of the aromatic
ring.
Scheme 1. Reagents and conditions: (i) BrCH2CH2OCH2CH2OCH2CH3, K2CO3, DMF, 70 °C, 24 h (80%); (ii) 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethanol, K2CO3, DMF, 70 °C, 24 h
(65%); (iii) Mg(OnBu)2, nBuOH, reflux, 20 h (34%) or Zn(OAc)2, DBU, nPeOH, 130 °C, 24 h (11%); (iv) Mg(OnBu)2, nBuOH, reflux, 20 h (Pc-1 — 31%, Pc-3 — 15%); (v) Mg(OnBu)2,
nBuOH, reflux, 24 h (13%).

M. Wierzchowski et al. / Journal of Inorganic Biochemistry 127 (2013) 62–72
67
Fig. 1. The molecular structure and atom labeling scheme for: (a) compound 2, (b) compound 4. Displacement ellipsoids are drawn at the 50% probability level and H atoms are
shown as spheres of arbitrary radii.
In compound 4, the benzene and imidazole rings that are bridged
by one oxyethylene group are oriented nearly perpendicular with the
dihedral angle between their planes of 86.05(6)°. Both compounds 2
and 4 associated to form separate polar layers (Fig. 1S, supporting
data).
the plotted spectra might suggest the appearance of aggregated
species. They are manifested in the UV–Vis spectra by the splitting
of the Q band or additional signals appearing next to the Q band
(Fig. 5).
Our observation is consistent with that of Kameyama et al. [49], who
have reported on J-aggregate formation of self-coordinated Pc dimers
possessing Zn and Mg ions in the core and substituted in peripheral po-
sitions with imidazole moieties. A similar phenomenon has also been
described by Niu et al. [50] for zinc Pcs substituted with aryloxy moie-
ties in non-peripheral positions indicating that etheric oxygen atoms
inserted between ethylene groups may by engaged in the formation
of J-aggregates, similarly to that observed with the imidazole nitrogens
described above. Moreover, it has been proved that J-aggregate forma-
tion proceeded particularly easily in non-coordinating solvents, such
as chloroform and toluene. The reason for this phenomenon has been
found to be dependent on intermolecular interactions between Zn⋯O.
Noteworthy, this interaction has not been observed in coordinating sol-
vents such as DMF and ethanol.
Therefore, the aggregation of Pc-3 was subsequently studied by
UV–Vis in chloroform and pyridine (Fig. 4S, Supplementary data).
The statistical analysis of the linear correlations between absorbance
values of the Q band and the red-shifted Qx-sub-band, and concentra-
tions of the Pc-3 revealed that the Beer–Lambert law was not obeyed
in the range of all concentrations studied for chloroform. However,
when the analysis was repeated in pyridine, no formation of addition-
al sub-bands resulting from the formation of the aggregated form was
observed, and the Beer–Lambert law turned out to be obeyed in the
range of concentrations studied (Table S3, Supplementary data). This re-
sult encouraged us to choose this solvent for all the NMR experiments.
Nevertheless, the Pc-3 molecule revealed massive aggregation during
the NMR study and two aggregated species were found in NMR spectra
after data acquisition (see detailed description in Supplementary data).
It could thus be explained that Pc-3 in pyridine-d5 at higher concen-
trations during the NMR experiment exists partly in aggregated
forms, even though it can be avoided at lower concentrations during
the UV–Vis measurement described above. It is worth mentioning that
the effects of aggregation phenomena of phthalocyanines substituted
by polyether chains or octaalkynylphthalocyanines have been studied
by means of 1H NMR spectroscopy [51,52].
Two phthalonitriles and four phthalocyanines were analyzed in
solution using various NMR techniques. Phthalonitrile 2 and its deriva-
tive phthalocyanines Pc-1, and Pc-2 revealed some similarities. Diagnos-
tic peaks at 7.42 and at 7.97 or 8.01 ppm, respectively, confirm protons
belonging to the aromatic part. In 1H NMR spectra polyether moieties
represent six signals of complex and differentiated multiplicity (Fig. 2
and Fig. 2S Supplementary data). In addition, glycolic fragment signals
of polyether moieties substituted at the macrocycle periphery are down-
field shifted in comparison to that observed at the phthalonitrile. The
connectivities within glycolic moieties were analyzed using 1H–1H
COSY spectra, whereas HSQC and HMBC enabled the allocation of all car-
bon peaks present in the 13C NMR experiments.
The 1H NMR spectrum of the metronidazole–phthalonitrile conju-
gate 4 revealed a typical singlet signal at 8.25 ppm assigned to the
hydrogen at C4 of the (2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy
substituent.
The same hydrogens present at C4 of the imidazole rings consti-
tuting the periphery of Pc-4 (labeled as “d”) appeared as two singlet
signals at 8.34 and 8.16 ppm resulting from the C2v symmetry of the
molecule. Isomers Cs and C4, D2 isomers would be represented by
four signals or one signal, respectively. The symmetry of Pc-4 was con-
firmed by the UV–Vis study (see Supplementary data). Similarly, a sin-
glet signal at 2.82 ppm representing the methyl at C2 in 4, appeared as
two singlet signals for Pc-4 (labeled as “g”) at 2.92 ppm and 2.66 ppm.
Phthalonitrile 4 benzene ring signals were represented by the hidden
triplet at 7.52–7.61 ppm and two overlapping doublets at 7.39 and
7.37 ppm. The corresponding signals of aromatic protons present in
the Pc-4 periphery were shifted upfield and represented by multiplets
at: 9.41–9.50 ppm, 8.07–8.23 ppm and 7.70–7.80 ppm. The aliphatic
linker was represented in the structure of 4 by two triplets at 4.80 ppm
(O-CH2CH2-N) and 4.59 ppm (O-CH2CH2-N), whereas the O-CH2CH2-N
linker groups of Pc-4 were represented by four overlapping singlets
forming a pseudo-triplet at 5.64, 5.43 and 5.24 ppm, whose multiplicity
is influenced by a steric hindrance. Carbon atoms of 4 and Pc-4 presented
in Fig. 3 were annotated according to 2D NMR (HSQC and HMBC)
experiments.
Part A of the molecule in the 1H NMR is represented by an aromatic
signal at 7.97 ppm, whereas benzo-fused protons of isoindole fragment
of part B are represented by a doublet at 9.64 ppm, a triplet at 8.12 ppm
and a doublet at 7.68 ppm (Fig. 4). The linker O-CH2CH2-N is represented
by a multiplet at 5.32–5.39 ppm (hidden) and a triplet at 4.34 ppm. Part
E is represented by the following signals: a multiplet at 5.39–5.49 ppm;
triplets at 4.34 ppm, 3.90 ppm, 3.64 ppm; a quartet at 3.44 ppm, and a
triplet at 1.11 ppm. The 1H–1H COSY, HMQC and HMBC spectra of Pc-3
were necessary to resolve the connectivities within polyether chains
and in the aromatic region of part B.
The phthalocyanine–metronidazole conjugate Pc-3 possesses
1,4,7-trioxanonyl- and (2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy moi-
eties, and both types of peripheral substituents were represented in
the 1H and 13C spectra (Fig. 4). The UV–Vis spectra of Pc-3 were recorded
in a wide range of solvents, including benzene, acetonitrile, acetone,
chloroform, chlorobenzene, dichloromethane, 1-chloronaphthalene,
N,N-dimethylformamide, DMSO, 1,4-dioxane, ethyl acetate, methanol,
n-hexane, triethylamine, water, toluene, and pyridine. The profiles of

68
M. Wierzchowski et al. / Journal of Inorganic Biochemistry 127 (2013) 62–72
Fig. 2. Signals annotations of 2 and Pc-1 in pyridine-d5 from 1H, 13C, 1H–1H COSY, HSQC and HMBC spectra.
values were slightly lower than those under conditions of limited access
3.3. Singlet oxygen generation
to air. It might be caused by a lower concentration of oxygen in the ana-
lyzed samples. It seems advantageous that the macrocycles studied pos-
sess values of singlet oxygen generation quantum yields comparable to
that of photosensitizers used commercially in PDT.
Photodegradation of 1,3-diphenylisobenzofuran (DPBF) at its maxi-
mum band of 417 nm in the presence of the Pc-1–Pc-4 and light en-
ables the calculation of the level of singlet oxygen generation and
establishment of the kinetics of this process. DPBF, which is a specific
singlet oxygen quencher, is known to form endoperoxides upon singlet
oxygen cycloaddition (Fig. 6). Zinc phthalocyanine (ZnPc, Aldrich) was
used as a reference [35,36,53,54]. Fig. 6 presents spectral variation of ab-
sorption curve for exemplary mixture of DPBF and Pc-1 as a function of
irradiation time.
Measurements made in parallel for Pc-1–Pc-4 and the reference
ZnPc under the same conditions enabled quantum yield calculations
by direct comparison of kinetic parameters (Table S4, Supplementary
data) [35,55–58]. The 1O2-mediated photodynamic mechanism of cy-
totoxicity is a typical mode of action for most PSs nevertheless other
competing mechanisms might be considered [59]. It can be seen from
Table 2 that all compounds exhibit lower quantum yields of singlet
oxygen generation in DMSO than in DMF. The Pc-2 molecule was
found to be the most efficient singlet oxygen generator from the group
of macrocycles studied. It is well known that a “heavy atom effect”
leads to an increased efficacy of triplet state generation and is connected
with the atomic number of the metal cation incorporated to the macro-
cyclic core [60]. Moreover, it has been proved by Ishii et al. that there is a
relationship between the symmetry of the porphyrinic π-conjugated
systems and singlet oxygen yields [61]. In addition, the experiments
performed with limited access to air revealed that the quantum yield
3.4. Photocytotoxicity
3.4.1. Photodynamic activity in vitro
The photodynamic activity of Pc-1–Pc-4 was examined in vitro
using two human OSCC cell lines, HSC-3 and H413 cells derived from
the tongue and the buccal mucosa, respectively.
To determine the dark toxicity, the cells were incubated with
Pc-1–Pc-4 at concentrations of 0.01, 0.1, 1, 5, 10 and 50 μM, in
serum-free medium for 24 h at 37 °C. Interestingly, Pc-3 was found
significantly less toxic than Pc-1 and Pc-2. Moreover, the toxicity
of Pcs in H413 cells was higher than that in HSC-3 cells (Table S5,
Supplementary data). The viability of HSC-3 was not reduced by Pc-1
at 0.1–5 μM and by Pc-2 at 0.1–1 μM, whereas Pc-3 was toxic only at
50 μM. Interestingly, the viability of H413 cells was unaffected only by
Pc-3, at 0.01 and 0.1 μM (Fig. 7).
The light-induced toxicities were examined only at the concentra-
tions that had not caused dark toxicity. HSC-3 cells were incubated
with Pc-1 at concentrations of 0.1, 1 and 5 μM, Pc-2 at 0.1 and 1 μM,
and Pc-3 at 0.01–10 μM for 24 h and subsequently exposed to light
(600–850 nm) for 20 min at a distance of 10 cm from the light source.
H413 cells were incubated with Pc-3 at 0.01 and 0.1 μM and exposed

M. Wierzchowski et al. / Journal of Inorganic Biochemistry 127 (2013) 62–72
69
Fig. 3. Signal annotations of 4 and Pc-4 in pyridine-d5 from 1H (bold), 13C, 1H–1H COSY, HSQC and HMBC spectra. Annotation of 13C carbons signals (ppm): a — 118.3, 118.1, 117.7, b — 131.3,
131.2, 131.1, 131.0, c — 117.5, 117.2, 115.4, 115.3, d — 134.0, or 133.9, e — 46.9, f — 71.0, 71.0, 69.3, 69.2, 14.9 or 14.5, g — 14.9 or 14.5, h — 152.5, 152.3, i — 139.4, 139.4, j — 156.2,
156.1, k — 127.8, 127.7, 127.3, 127.3, l — 153.9, 153.8, 152.9, 152.8, m — 142.5, other quaternary carbons 154.8, 154.5, 154.3, 154.1, 154.0, 152.7.
Fig. 4. Signal annotations of Pc-3 (pyridine-d5) from 1H, 13C, 1H–1H COSY, HSQC and HMBC spectra.

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M. Wierzchowski et al. / Journal of Inorganic Biochemistry 127 (2013) 62–72
Table 2
Singlet oxygen quantum yields of Pc-1–Pc-4.
Compound Irradiation wavelength [nm] (Singlet oxygen quantum yield ΦΔ)
DMF/DMSO
DMF/DMSO
Aerobic conditions
Pc-1
Pc-2
Pc-3
Pc-4
732/739
731/740
724/756
699/704
(0.3182
(0.4644
(0.1548
(0.1542
0.0070)/(0.2973
0.0161)/(0.3397
0.0038)/(0.0113
0.0043)/(0.1605
0.0127)
0.0081)
0.0008)
0.0026)
Limited access to air
Pc-1
Pc-2
Pc-3
Pc-4
732/739
731/740
724/756
699/704
(0.2743
(0.3536
(0.1376
(0.1475
0.0066)/(0.2161
0.0153)/(0.3185
0.0029)/(0.0064
0.0036)/(0.1532
0.0059)
0.0077)
0.0004)
0.0025)
Reference [53,62]
ZnPc 670/672
(0.67)/(0.56)
Fig. 5. Selected UV–Vis spectra of Pc-3 in various solvents.
of free-form Pc-4 and that encapsulated in liposomes was examined
at concentrations of 0.1, 1.0 and 10.0 μM. In case of Pc-4, a high power
LED MultiChip Emitter was used for illumination with a light dose of
3 mW/cm2 at 735 nm for 20 min. Free-form Pc-4 and encapsulated in
liposomes did not exhibit dark toxicity towards HSC-3 and H413 cells
(Fig. 7). However, a moderate toxic effect appeared after illumination.
Interestingly, Pc-4 was not active after illumination at any concentra-
tion used in this study towards the H413 cell line. On the contrary, a de-
crease in the viability of HSC-3 cells was observed with Pc-4, but only at
10 μM. The phototoxic effect was two times higher for Pc-4 in PG:POPC
liposomes (34.8%) than that observed with free Pc-4 (15.6%).
to light. Cell viability was quantified by the Alamar Blue assay. Re-
sults are expressed as the percentage of control cells incubated with
medium/0.5% DMSO alone.
Phthalocyanines Pc-1 at 5 μM, Pc-2 at 1 μM and Pc-3 at 1, 5 and
10 μM, caused 100% photocytotoxicity in HSC-3 cells. Pc-1 at 1 μM,
Pc-2 and Pc-3 at 0.1 μM decreased the viability of HSC-3 cells by
65.7%, 17.3% and 34.0%, respectively. In H413 cells, Pc-3 at 0.1 μM de-
creased the viability by 34.8%.
Many phthalocyanines that are used as photosensitizers in photo-
dynamic therapy (PDT) are of hydrophobic nature. On the one hand,
hydrophobicity ensures rapid cellular uptake and localization at crit-
ical subcellular membranous organelles; on the other, it leads to poor
solubility of the dye, making it difficult to formulate for parenteral
administration [63]. Delivery systems such liposomes may be used
as carriers in PDT as they prevent photosensitizer aggregation and
thus increase photosensitizer photoactivity. Moreover, liposomes of
proper size and composition may benefit from the EPR (Enhanced
Permeability and Retention) effect [63,64]. Herein, an attempt to ob-
tain small unilamellar liposomes with encapsulated phthalocyanines,
Pc-1–Pc-4 possessing polyether functionalities, was undertaken. Un-
fortunately, the formulations aiming to obtain liposomes containing
Pc-1–Pc-3 were unsuccessful, as these macrocycles precipitated during
hydration of dried thin lipid films. Pleasingly, Pc-4 was encapsulated in
PG:POPC liposomes with an average size of extruded liposomes of
148.3 nm and a final concentration of 100 μM. The biological activity
4. Conclusions
Four novel magnesium(II) and zinc(II) phthalocyanines bearing
1,4,7-trioxanonyl, polyether and/or (2-methyl-5-nitro-1H-imidazol-
1-yl)ethoxy, heterocyclic substituents at their non-peripheral positions
were synthesized and assessed in terms of physicochemical and biolog-
ical properties. Novel phthalocyanines were purified on silica gel in nor-
mal and reversed-phase systems by flash column chromatography and
characterized using NMR, MS, UV–Vis and HPLC. Moreover, two precur-
sors in the macrocyclization reaction phthalonitriles were characterized
using X-ray crystallography. Photophysical properties of the novel
macrocycles were evaluated, including UV–Vis spectra analysis and ag-
gregation study. Singlet oxygen generation and the oxidation rate con-
stant measurements made in parallel for all phthalocyanines and the
reference zinc(II) phthalocyanine under the same conditions enabled
Fig. 6. Scheme showing singlet oxygen generation and quenching of DPBF. The plot presents spectra changes for Pc-1 in DMF (aerobic conditions) during singlet oxygen measure-
ment (kinetic curve of DPBF oxidation — inset). 1PS — photosensitizer in the ground singlet state (S0), 1PS* — photosensitizer in first excited singlet states (S1) or higher (Sn) states,
3PS — photosensitizer in the triplet state (T1), 3O2 — oxygen molecule in the ground state (3Σ−g ), 1O2 — oxygen molecule in the first excited singlet state (1Δg); DPBF
(1,3-diphenylisobenzofuran), ODBB (ortho-dibenzoylbenzene).

M. Wierzchowski et al. / Journal of Inorganic Biochemistry 127 (2013) 62–72
71
Fig. 7. (a) The light-induced toxicity was examined only at the concentrations that did not cause dark toxicity. Light toxicity of Pc-1–Pc-3 was studied at different concentrations towards
HSC3 cells; (b) dark and light toxicity of free-form (0.1, 1.0, 10.0 μM) and liposome-encapsulated (0.1 lip, 1.0 lip, 10.0 lip μM) Pc-4 towards HSC-3 cells. HSC-3 cells were incubated with
Pc-1–Pc-3 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 duplicate (Pc-1–Pc-3) and from one experiment performed in triplicate (Pc-4 and Pc-4 in liposomes).
quantum yield calculations by direct comparison of kinetic parameters.
All macrocycles exhibited lower quantum yields of singlet oxygen gener-
ation in DMSO than in DMF. In addition, the Pc-2 molecule was found to
be the most efficient singlet oxygen generator from the group of
macrocycles studied. The photocytotoxicity measured on HSC-3
cells for the mixed phthalocyanine-metronidazole conjugate Pc-3
was significantly higher than those for phthalocyanines peripherally
modified with polyether groups Pc-1 and Pc-2, and that for the
tetra-metronidazole phthalocyanine derivative Pc-4. In addition,
Pc-3 showed photocytotoxicity at 1 μM with 100% photokilling of
cultured cells. Noteworthy, Pc-3 was found to be the most active
macrocycle in vitro despite the fact that its ability to generate singlet
oxygen was significantly lower than those of Pc-1 and Pc-2. At-
tempts to encapsulate phthalocyanines possessing polyetheric func-
tionalities Pc-1–Pc-3 in liposomes were unsuccessful, in contrast to
Pc-4, which was encapsulated in extruded PG:POPC liposomes and
its photocytotoxicity was evaluated. The use of conjugates of photo-
sensitizers with nitroimidazole and its derivatives gave promising
results worthy of further investigation.
Acknowledgments
The authors acknowledge financial support for the project from the
Polish Ministry of Science and Higher Education/the National Scientific
Centre (N N401 067238). The authors thank Prof. Zofia Gdaniec from
the Institute of Bioorganic Chemistry, Polish Academy of Sciences in
Poznan for recording NMR spectra (supported from the Ministry of Sci-
ence and Higher Education and the European Fund for Regional Develop-
ment under priority axis 2 – Research and Development Infrastructure,
measure 2.1 – No UDA-POIG.02.01.00-30-182/09). The authors thank
Beata Kwiatkowska and Agnieszka Gielara-Korzanska for excellent tech-
nical assistance.
Appendix A. Supplementary data
The following publication refers to the experimental data contained
in the Supplementary data [65]. The crystallographic data in CIF format
have been deposited at the Cambridge Crystallographic Data Centre,
CCDC 927434 for phthalonitrile 2 and CCDC 927435 for phthalonitrile 4
can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/
retrieving.html or e-mail: deposit@ccdc.cam.ac.uk. Supplementary data
related to this article can be found online at http://dx.doi.org/10.1016/j.
jinorgbio.2013.06.012.
Abbreviations
Ar
aromatic
DPBF
EPR
ESI
1,3-diphenylisobenzofuran
enhanced permeability and retention
electrospray ionization; mass spectrometry technique
fetal bovine serum
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