Photochemical studies and nanomolar photodynamic activities of phthalocyanines functionalized with 1,4,7-trioxanonyl moieties at their non-peripheral positions

Article abstract of DOI:10.1016/j.jinorgbio.2015.11.006

Manganese(III), cobalt(II), copper(II), magnesium(II), zinc(II) and metal-free phthalocyanines, possessing 1,4,7-trioxanonyl substituents, at their non-peripheral positions, were subjected to photochemical, photodynamic and biological activity studies. Demetallated phthalocyanine and its metallated d-block analogues, with copper(II), cobalt(II), manganese(III) chloride, were found to be less efficient singlet oxygen generators in comparison to the zinc(II) analogue and zinc(II) phthalocyanine reference. Irradiation of several phthalocyanines for short time periods resulted in a substantially increased cytostatic activity against both suspension (leukemic/lymphoma at 85 nM) and solid (cervix carcinoma at 72 nM and melanoma at 81 nM) tumour cell lines (up to 200-fold). Noteworthy is that enveloped viruses, such as for herpesvirus and influenza A virus, but not, non-enveloped virus strains, such as Coxsackie B4 virus and reovirus-1, exposed to irradiation in the presence of the phthalocyanines, markedly lost their infectivity potential.

Full text of DOI:10.1016/j.jinorgbio.2015.11.006

Journal of Inorganic Biochemistry 155 (2016) 76–81
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Photochemical studies and nanomolar photodynamic activities of
phthalocyanines functionalized with 1,4,7-trioxanonyl moieties at their
non-peripheral positions
Lukasz Sobotta a,1, Marcin Wierzchowski b,1, Michal Mierzwicki b, Zofia Gdaniec c, Jadwiga Mielcarek a,
Leentje Persoons d, Tomasz Goslinski b, Jan Balzarini d,
⁎
a
Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland
Department of Chemical Technology of Drugs, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12, 61-704 Poznan, Poland
Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 July 2015
Received in revised form 18 September 2015
Accepted 10 November 2015
Available online 14 November 2015
Manganese(III), cobalt(II), copper(II), magnesium(II), zinc(II) and metal-free phthalocyanines, possessing 1,4,7-
trioxanonyl substituents, at their non-peripheral positions, were subjected to photochemical, photodynamic and
biological activity studies. Demetallated phthalocyanine and its metallated d-block analogues, with copper(II),
cobalt(II), manganese(III) chloride, were found to be less efficient singlet oxygen generators in comparison to
the zinc(II) analogue and zinc(II) phthalocyanine reference. Irradiation of several phthalocyanines for short
time periods resulted in a substantially increased cytostatic activity against both suspension (leukemic/lympho-
ma at 85 nM) and solid (cervix carcinoma at 72 nM and melanoma at 81 nM) tumour cell lines (up to 200-fold).
Noteworthy is that enveloped viruses, such as for herpesvirus and influenza A virus, but not, non-enveloped virus
strains, such as Coxsackie B4 virus and reovirus-1, exposed to irradiation in the presence of the phthalocyanines,
markedly lost their infectivity potential.
Keywords:
Antiviral photodynamic therapy
Cytostatic photodynamic therapy
Photosensitizer
Phthalocyanine
Singlet oxygen
© 2015 Elsevier Inc. All rights reserved.
1. Introduction
cross-switching to a relatively long live triplet excited state. The transfer
of energy from the photosensitizer to molecular oxygen, which exists in
Phthalocyanines (Pcs) are macrocyclic molecules consisting of four
isoindole units linked by aza nitrogen atoms [1,2]. The aromatic
character of Pcs impacts their spectral and electrochemical properties. Ap-
plications of Pcs have been increasing. Although, historically, they have
been mainly used in the dye industry with copper phthalocyanine as
the well-known pigment [3]. Nowadays, their applications are extended
towards energy production (solar cells), electronics (laser printers, LCD
displays, switches, recordable CDs) and medicine (photosensitizers)
[4–8].
Pcs as photosensitizers have been applied on a regular basis in
clinical and preclinical treatment for photodynamic diagnosis (PDD),
photodynamic inactivation of bacteria and viruses (PACT), photody-
namic treatment of skin disorders, and photodynamic treatment of can-
cer (PDT) [9]. In PDT, after the absorption of light, Pcs undergo excitation
to their singlet excited state and then revert back to the ground state
with the emission of fluorescence (PDD) or undergo intersystem
its ground triplet state, is accompanied by the conversion to its excited
singlet state and production of singlet oxygen. Singlet oxygen is a com-
mon reactive oxygen agent able to kill cancer cells and microorganisms
[10]. PDT seems to possess potential in the treatment of skin melanoma
with the destruction of melanoma cells and only minimal side-effects
to skin fibroblasts. The therein applied PDT was found to generate
photocytotoxicity against cancer and enhanced the immunological
response [11,12].
A successful PDT treatment depends on a proper dosimetry, which is
strictly connected with the use of light — defined wavelengths and their
intensity, and a fixed dose of photosensitizer [13]. There is a constant in-
terest in novel Pc photosensitizers for photodynamic therapy. The main
drawback is that the currently available Pcs in medicine have poor sol-
ubility in water, which limits their administration in vitro or in vivo.
Therefore, there is a continuing interest in novel Pcs with good solubility
in water and high singlet oxygen generation. One option in obtaining
useful photosensitizers for PDT is to develop a proper drug delivery
system [14,15]. Another possibility is to use peripheral substituents
increasing the Pcs solubility (e.g. sulphonic, carboxylic, pyridyloxy
groups) [16–18]. Lately, we reported the synthesis, characterization, as
well as photodynamic biological properties of the series of Pc analogues,
⁎
Corresponding author.
E-mail address: jan.balzarini@rega.kuleuven.be (J. Balzarini).
These authors contributed equally to the work presented here and should therefore be
1
regarded as equivalent authors.
http://dx.doi.org/10.1016/j.jinorgbio.2015.11.006
0162-0134/© 2015 Elsevier Inc. All rights reserved.

L. Sobotta et al. / Journal of Inorganic Biochemistry 155 (2016) 76–81
77
equipped with polyetheroxy substituents at their non-peripheral
positions [19].
3.65, 1.08 ppm and a quartet at 3.41 ppm, respectively. Protons of the
adjacent methylene groups and the methyl groups of the polyethylene
chains were assigned using 1H–1H COSY spectrum (Fig. 1). Singlet at
0.07 ppm was attributed to core pyrrole-H protons.
In our research, we focused on the photochemical, photodynamic
and the biological properties of novel polyetheroxy-substituted Pc ana-
logues. The demetallated phthalocyanine and its complexes with vari-
ous metal ions in the centre, were subjected to biological tests on
cancer cell lines and selected viruses.
Carbon resonances within a 13C NMR spectrum of Pc 4 were unam-
biguously assigned, using a combination of two-dimensional techniques
such as HSQC (Heteronuclear Single Quantum Coherence) and HMBC
(Heteronuclear Multiple Bond Correlation). 1H and 13C chemical shifts
of Pc 4, together with key HMBC and 1H–1H COSY, correlations are
shown in Fig. 1.
2. Results and discussion
2.1. Chemistry
2.2. Photostability
The novel metal-free phthalocyanine 4 was obtained with 33% yield
by adapting the demetallation reaction conditions previously described
[20,21]. The standard reaction time of 3 with TFA was extended from 0.5
to 3 h. Interestingly, a decreased reactivity of compound 3 was probably
due to the presence of polyether peripheral chains impeding the access
of protonic acid to the macrocyclic core. Subsequent remetallation was
based on the reaction of 4 with metal chlorides in DMF at a temperature
of 70 °C for 24 h and led to the products 5a–5c with differentiated yields
(Scheme 1) [22]. Noteworthy, in the remetallation reaction, various salt
species revealed differentiated reactivity and can be ordered in terms of
their decreasing efficiency in the following way: MnCl2·4H2O (yield
69%), CoCl2·4H2O (yield 53%) and CuCl2 (yield 15%). All new macrocy-
clic compounds were purified on silica gel by flash column chromatog-
raphy in normal and reversed-phase system and on cross-linked
dextran gel (Sephadex) by size-exclusion chromatography. HPLC analy-
ses confirmed the purity of the macrocyclic compounds 4, 5a–5c at a
level above 95%. New metal-free phthalocyanine 4 was characterized
using 1H and 13C NMR spectroscopy. In the 1H NMR spectrum of the
symmetrical phthalocyanine 4, only one singlet at 8.02 ppm, corre-
sponding to the aromatic protons, was observed. In the aliphatic region
five resonances of methylene groups and one of the methyl groups of
the polyetheroxy chains were identified: triplets at 5.35, 4.38, 3.93,
Photosensitizers can undergo two types of photochemical reactions —
phototransformation and photobleaching, eventually both of them. The
nature of the decomposition process depends on many factors including
the chemical structure of the investigated compound, the solvent used
and the concentration of the molecular oxygen. The progress of
phototransformation may be studied in the UV–vis through the appear-
ance of new absorption bands. Opposite to this, in the photobleaching
process, a disappearance of absorption bands in the visible region can
be observed resulting in the loss of the dye colour [23]. Photobleaching
of 6 is shown in Fig. 2. According to the literature, the main photoprod-
uct, which is characteristic for phthalocyanine decomposition in
the presence of molecular oxygen is phthalimide. The phthalimide
formation, which stems from phthalocyanine, is a typical Diels-Alder
cycloaddition of singlet oxygen, which is generated by phthalocyanine
in the photodynamic reaction [23,24].
Studied Pcs underwent photobleaching in DMF and DMSO. There was
no evidence of any new bands in their absorption spectra during irradia-
tion, whereas a disappearance of Pc Soret and Q-band was observed.
Some compounds (Table 1S, Supplementary data) decomposed in two
stages, according to the first order kinetic reaction. The first step taken
in photobleaching may be due to the formation of the cycloadduct of Pc
indole fragment with an excited form of molecular oxygen. The second
one is the result of the adduct decomposition to phthalimide. Interesting-
ly, some of the investigated compounds showed photodegradation in one
stage, possibly due to the very fast first step of decomposition.
The quantum yields of photobleaching were determined according
to a method presented by Seotsanyana-Mokhosi et al. [25]. Quantum
yields of photobleaching observed in the compounds dissolved in
DMSO were much lower than that of the DMF (Table 2S, Supplementary
data), and can be explained through the coordination-interaction be-
tween the macrocycle core metal ion and DMSO [26,27]. A confirmation
of the stabilizing effect of DMSO in the photobleaching process is the
result obtained for 4 in comparison to that measured for the other
metallated analogues 3, 5a–5c and 6. It was found that the quantum
yields of photobleaching in DMF and DMSO differ less in the case of 4
as compared to the other macrocycles studied. It is probably due to
the absence of metal ions in the core of Pc 4. In addition, a little lower
quantum yield in DMSO of 4 was probably the result of the DMSO vis-
cosity or the dielectric constant. Photobleaching was performed both
in aerobic conditions and with limited access to air (nitrogen bubbling
in solution for 20 min). Measurements performed for 3, 4, 5a and 5c
showed a decrease in the photobleaching quantum yields under
conditions limiting access to air, in comparison to the results achieved
under aerobic conditions (Table 2S, Supplementary data). This is a result
of the limited access to molecular oxygen, more specifically to a lower
singlet oxygen formation. A particular phenomenon was observed for
6, which is the most effective singlet oxygen generator. Moreover, sim-
ilar quantum yields of photobleaching both in aerobic conditions and at
limited access to air were found for 6. Interestingly enough, 5b in DMF
decomposed much faster under limited access to air than in aerobic con-
ditions. This phenomenon commits radical photoreactions with DMF.
Furthermore, in DMSO solution Pc 5b revealed similar quantum yields
under both conditions used, (i) a limited access to air and (ii) aerobic
Scheme 1. Reagents and conditions: (i) Br(CH2CH2O)2CH2CH3, K2CO3, DMF, 70 °C, 24 h
(80%); (ii) 3 — Mg(OnBu)2, nBuOH, reflux, 20 h (33%), 6 — nPeOH, Zn(OAc)2, reflux,
24 h (11%); (iii) TFA, CH2Cl2, r.t., 3 h (34%); (iv) given metal salt, DMF, 70 °C, 24 h.

78
L. Sobotta et al. / Journal of Inorganic Biochemistry 155 (2016) 76–81
Fig. 1. 1H and 13C chemical shifts [ppm] of 4 in pyridine-d5. Key HMBC and 1H–1H COSY correlations are marked with dashed arrows and bold lines, respectively.
conditions. The lowest quantum yields within the photodecomposition
process for all compounds studied, under aerobic conditions, were
found for 5b in DMF and 5a in DMSO. The highest values of quantum
yield decomposition were found for 3 in DMF solution and 4 in DMSO
solution.
is connected with the spin–orbit coupling phenomenon [26–28]. Addi-
tionally, further studies performed under conditions with limited access
to air resulted in a decrease of singlet oxygen generation ability, indicat-
ing its dependency on the oxygen presence (Table 3S, Supplementary
data).
2.3. Singlet oxygen
2.4. Biological studies
One of the most important properties of phthalocyanines is their
ability to generate various reactive oxygen species, including singlet
oxygen. Singlet oxygen possesses a high oxidizing potential and there-
fore, has been utilized in different areas of life such as: cancer treatment
using photodynamic therapy, photooxidation of toxic molecules in the
waste water treatment, and in chemical synthesis [28,29].
Within all the investigated Pcs, compound 6 was found to be the
most efficient singlet oxygen generator [19] (Table 1). Demetallated
Pc and its metallated d-block analogues, with copper(II), cobalt(II),
manganese(III) chloride, were found to be less efficient. This fact can
be explained through the metal ion influence on the electronic energy
levels of the molecule. Open shell coordinated metal ions, such as
Mn3+, Cu2+, or Co2+, influence the short living excited state of
macrocycle, as compared to closed shell metal ions, such as Mg2+ or
Zn2+. This phenomenon directly influences the singlet oxygen genera-
tion efficacy. Within closed shell metal ions incorporated to the core of
macrocycles, zinc is known to cause the highest values of singlet oxygen
generation. It is the result of the well-known “heavy atom effect”, which
In a first series of experiments, the cytostatic activity of a variety of
phthalocyanine derivatives was studied in three different human
tumour cell models including CD+4 T-lymphoblast CEM cells growing
in suspension, and the solid tumour-derived cervix carcinoma HeLa
and melanoma SK-MEL-5 cells growing in monolayers. When adminis-
tered to tumour cell cultures for 3 to 4 days of incubation time at 37 °C, a
moderate cytostatic activity in the test compounds was observed. A 50%
inhibitory concentration (IC50) against tumour cell proliferation was
within the same order of magnitude for each compound. Compounds
3, 5b, 5c and 6 that contain, respectively, Mg2+, Co2+, Cu2+ or Zn2+
in their core were the most cytostatic compounds (IC50: 4.8–7.7 μM
for CEM, 6.8–19 μM for HeLa and 5.7–6.6 μM for SK-MEL-5). Metal-
free 4 and phthalocyanine 5a containing Mn3+ in their core showed
poor, if any cytostatic activity (IC50: 20–N50 μM for the three tumour
cell lines) (Table 2).
The tumour cell cultures were irradiated at 735 nm for 30 min at the
start of the tumour cell incubation (day 0): day 1 (after 24 h) and, day 2
Table 1
Quantum yields of singlet oxygen generation of phthalocyanines 3–6 and reference com-
pound (ZnPc) [30,31].
Pcs
Solvent
Irradiation
wavelength
λ [nm]
Quantum yields of singlet oxygen
generation ΔΦ ΔΦ
Aerobic
conditions
Limited access to
air
DMF
DMSO
DMF
DMSO
DMF
DMSO
DMF
DMSO
DMF
DMSO
DMF
DMSO
DMF
DMSO
732
739
757
764
0.32 0.01
0.30 0.01
b0.01
b0.01
Insoluble
b0.01
0.02 b0.01
0.02 b0.01
0.02 b0.01
b0.01
0.46 0.02
0.47 0.02
0.56
0.27 0.01
0.22 0.01
b0.01
3 [19]
4
b0.01
5a
826
718
723
736
740
731
740
670
672
b0.01
b0.01
b0.01
b0.01
b0.01
0.35 0.02
0.40 0.02
5b
5c
6 [19]
Fig. 2. Changes in the absorption spectra of phthalocyanine 6 in DMF solution after expo-
sure to radiation.
ZnPc [30,31]
0.67

L. Sobotta et al. / Journal of Inorganic Biochemistry 155 (2016) 76–81
79
Table 2
Effect of tumour cell culture irradiation at λ = 735 nm.
IC50a (μM)
Compound
CEM
HeLa
SK-MEL-5
Dark
Dark
Irradiation at 735 nm
Dark
Irradiation at 735 nm
Irradiation at 735 nm
3
4
5a
5b
5c
6
7.7 0.2
1.5 0.2
0.085 0.011
14
6
3
0.85 0.41
0.32 0.29
N50
4.9 0.4
0.38 0.29
0.072 0.032
–
≥50
–
–
6.6 2.3
5.7 2.3
–
20
20
7
4
N50
N50
13
19 11
6.8 6.0
0.34 0.28
–
–
0.33 0.15
0.081 0.052
19
8
7.1 1.5
4.8 0.1
5.4 1.1
3.2 0.9
0.13 0.03
0.50 0.46
a
50% Inhibitory concentration or compound concentration required to inhibit tumour cell proliferation by 50%.
(after 48 h). Tumour cell growth was determined on day 3 (CEM) or day
4 (HeLa, SK-MEL-5).
virus A (H1N1, H3N2) (N25- to 180-fold) and B (2.5- to 7-fold) (2
independent experiments in duplicate) (Table 3). Thus, short irradia-
tion of phtalocyanine derivatives can substantially decrease in the
infectivity of enveloped viruses, such as for herpesvirus and influenza
A virus, but not for non-enveloped viruses such as Coxsackie B4 virus
and reovirus-1.
In a second series of experiments, the tumour cell cultures to which
the compounds were administered, were irradiated for 30 min at
735 nm with a light intensity of 4.5 mW/cm2 (total light dose for each
irradiation 8.1 J/cm2) at the beginning of the cell-drug cultivation (day
0), at day 1 and at day 2 of the experiment. Under these experimental
conditions, several compounds became exquisitely toxic to the tumour
cells. It should be noticed that the irradiation sequence used (3
subsequent days for 30 min) did not affect the tumour cell growth in
the absence of the compounds. The degree of increased cytostatic
potential upon irradiation of the compound-exposed tumour cell
cultures highly differed depending the nature of the compounds. For
example, whereas the irradiation of compound 5a had no effect on its
(poor) cytostatic activity, and only a slight effect (2- to 3-fold increased
cytostatic activity) on compound 5b was observed, a dramatic increase
of cytostatic activity was found for compounds 3, 4, 5c and 6 (Table 2).
Depending the nature of the tumour cell lines and the compound, cyto-
static activity was increased by 5- to 200-fold for CEM, 20- to 100-fold
for HeLa, and 20- to 100-fold for SK-MEL-5 cells. The most striking
activities were found for compound 4 where irradiation at λ 735 nm
caused a 100- to 200-fold increased antiproliferative activity irrespec-
tive of the tumour cell line evaluated (Table 2).
3. Conclusions
The findings here indicate that phthalocyanine derivatives are light-
dependent inhibitors of tumour cell proliferation and decrease the
infectivity of a wide variety of enveloped viruses. This opens-up an in-
teresting area in the development of a novel type of chemotherapeutic
class of agents. They may provide a selective basis for the treatment of
local (i.e. dermatological) diseases through the activation of medica-
tions through a light-exposed biological (i.e. skin) surface, with minimal
effects on the whole host organism. This approach is fundamentally dif-
ferent from other photochemotherapeutic approaches that target DNA
through direct drug interaction, as is the case for 8-methoxypsoralen
and proflavine, after being exposed to long-wave ultraviolet irradiation
[32,33]. The phthalocyanine irradiation, with wavelengths in the far-red
light spectrum, is much less damaging for near-by tissues of the object
being irradiated, and affords its activity by the local generation of
various reactive oxygen species, including singlet oxygen.
Control values are set as 100% infectivity upon titration and CCID50
determination.
In a third set of experiments, compound 5c (100 μM) was exposed to
several virus strains, irradiated for 30 min at 735 nm and after irradia-
tion the compound/virus was added to confluent human embryonic
lung (HEL), African green monkey kidney (Vero) or Madin-Darby ca-
nine kidney (MDCK) cell cultures. Whereas, the irradiation treatment
had no effect on the virus-induced cytopathicity for Coxsackie virus B4
and reovirus-1; there was a dramatic drop of the virus infectivity in
the cell cultures infected with herpes simplex virus type 1 (30- to
350-fold), parainfluenza virus-3 (20- to 25-fold), Punta Toro virus
(N25- to 200-fold), Sindbis virus (N70 to N125-fold), and influenza
4. Experimental
4.1. Materials and methods
All reactions were conducted in oven-dried glassware under nitro-
gen. Reported reaction temperatures refer to the external bath temper-
atures. All reactions were performed under an inert atmosphere of
nitrogen. All solvents were evaporated under reduced pressure using a
rotavap, below 60 °C. All solvents and reagents were obtained from com-
mercial suppliers and used without further purification. Melting points
Table 3
Infectivity potential of viruses after irradiation for 30 min at λ = 735 nm in the presence of 5c.
Infectivity upon 30′ irradiation in the presence
of compound 5c (percent of control)a
Average percentage of virus
infectivity versus control
Virus strain
Experiment 1
Experiment 2
Herpes simplex virus type 1 (KOS)
Parainfluenza virus-3
Punta Toro virus
Sindbis virus
3
3.9
b0.46
b0.80
0.27
5.2
b4
1.6
4.6
b2.2
b1.1
b1.4
Influenza A virus
H1N1
H3N2
Influenza B virus
Coxsackie virus B4
Reovirus-1
b2.7
b2.7
45
110
100
0.55
4
15
85
76
b1.6
b3.3
30
98
88

80
L. Sobotta et al. / Journal of Inorganic Biochemistry 155 (2016) 76–81
were obtained on a “Stuart” Bibby apparatus and were not corrected.
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. Size-exclusion chromatography was performed on Sephadex
G-25. Thin layer chromatography (TLC) was performed on silica gel
Merck Kieselgel 60 F 254 plates and DC Kieselgel 60 RP-18 F254 and vi-
sualized with UV illumination (λmax 254 or 365 nm). Mass spectrometry
experiments (ES, MALDI TOF) and elemental analyses were carried out
at the Advanced Chemical Equipment and Instrumentation Facility at
the Faculty of Chemistry, Adam Mickiewicz University in Poznan. The
UV–visible (UV–vis) spectra were recorded on Hitachi UV/VIS U-1900
and Shimadzu UV-160A spectrometers; λmax [nm] (log ε). 1D and 2D
NMR experiments were carried out using a Bruker 400 spectrometer.
Chemical shifts (δ) are quoted in parts per million (ppm) and refer to
a residual solvent peak. Coupling constants (J) are quoted in Hertz
(Hz) to the nearest 0.5 Hz. The abbreviations s, t, h and q refer to singlet,
triplet, hidden and quartet, respectively. 1H and 13C signals were
assigned using 1H–1H COSY, HSQC and HMBC experiments. Analytical
HPLC was performed on an Agilent 1200 Series instrument.
4.2.3. [1,4,8,11,15,18,22,25-octakis(1,4,7-trioxanonyl)phthalocyanine]
cobalt(II) (5b)
A rapidly stirred mixture of phthalocyanine 4 (105 mg 0.07 mmol)
and CoCl2∙6H2O (79 mg 0.33 mmol) in 20 mL of DMF were heated at
70 °C for 24 h. After being allowed to cool to room temperature, the re-
action mixture was filtered and evaporated to dryness under reduced
pressure with toluene (60 mL). Column chromatography (CH2Cl2:
CH3OH 15:1, EtOAc, CH2Cl2:CH3OH 4:1) and size-exclusion chromatog-
raphy (Sephadex G-25, methanol) were performed to give dark brown
solid 5b (56 mg, 53% yield). M.p. N 40 °C (mesophase behaviour).
Rf (CH2Cl2: MeOH 15:1) 0.18. UV–vis (CH2Cl2) λmax [nm] (log ε) 727
(4.86), 655 (4.30), 374 (4.33), 321 (4.57). MS (MALDI-TOF) [m/z]
[M + H]+ 1629.3. IR ν [cm−1] 1115, 1214, 1268, 1499, 1601, 2869.
HPLC (Supplementary data) purity 95.81–100.00%.
4.2.4. [1,4,8,11,15,18,22,25-octakis(1,4,7-trioxanonyl)phthalocyanine]
cooper(II) (5c)
A rapidly stirred mixture of phthalocyanine 4 (128 mg 0.08 mmol)
and CuCl2 (57 mg 0.06 mmol) in DMF (20 mL) were heated at 70 °C
for 24 h. After being allowed to cool to room temperature, the reaction
mixture was filtered and evaporated to dryness under reduced pressure
with toluene (60 mL). Column chromatography (CH2Cl2:CH3OH 20:1,
EtOAc, CH2Cl2:CH3OH 15:1) was performed to give dark green solid 5c
(19 mg, 15% yield). M.p. N 50 °C. Rf (CH2Cl2: CH3OH 15:1) 0.16. UV–vis
(CH2Cl2) λmax [nm] (log ε) 740 (5.17), 662 (4.57), 438 (4.07) 326
(4.75). MS (MALDI-TOF) [m/z] [M + H]+ 1633.3. IR ν [cm−1] 1114,
1267, 1501, 1601, 2867; HPLC (Supplementary data) purity 98.38–
100.00%.
4.2. Synthesis
Known compounds, 2,3-dicyanohydroquinone 1, 3,6-bis(1,4,7-
trioxanonyl)-1,2-benzenedicarbonitrile 2, [1,4,8,11,15,18,22,25-
octakis(1,4,7-trioxanonyl)phthalocyanine]magnesium(II) 3, and
[1,4,8,11,15,18,22,25-octakis(1,4,7-trioxanonyl)phthalocyanine]zinc(II)
6 were synthesized following previously published procedures [19].
4.2.1. 1,4,8,11,15,18,22,25-octakis(1,4,7-trioxanonyl)phthalocyanine (4)
Suspension of [1,4,8,11,15,18,22,25-octakis(1,4,7-trioxanonyl)
phthalocyanine]magnesium(II) (3) (100 mg, 0.06 mmol) was rapidly
mixed in trifluoroacetic acid (25 mL) for 3 h. Next, reaction contents
were poured into water and ice mixture (200 mL) and neutralized
with concentrated solution of sodium bicarbonate. Resulting solution
was extracted with CH2Cl2 (200 mL) and combined organic layers
were after drying with anhydrous MgSO4 were evaporated to dryness.
Column chromatography (CH2Cl2:CH3OH 15:1) was performed to give
dark green solid 4 (34 mg, 33% yield). M.p. N 50 °C (mesophase behav-
iour). Rf (THF:CH3OH 15:1) 0.65. UV–vis (CH2Cl2): λmax [nm] (log ε):
757 (5.10), 670 (4.54), 396 (4.32), 333 (4.69). MS (MALDI-TOF): m/z
[M + H]+ 1572.7. 1H NMR (400 MHz, pyridine-d5) δ: 8.02 (s, 8 H),
5.35 (t, 3J = 4.5 Hz, 16 H), 4.38 (t, 3J = 4.5 Hz, 16 H), 3.93 (t, 3J =
5.0 Hz, 16 H), 3.65 (t, 3J = 5.0 Hz, 16 H), 3.41 (q, 3J = 7.0 Hz, 16 H),
1.08 (t, 3J = 7.0 Hz, 24 H), 0.07 (s, 2 H). 13C NMR (101 MHz, pyridine-
d5) δ: 151.3, 148.5h (present only as a cross-peak in HMBC), 126.3,
120.1, 71.4, 70.1, 69.5, 69.2, 65.3, 14.3. HPLC (Supplementary data)
purity 95.10–96.49%.
4.3. Photostability
The photostability of the investigated compounds was tested both
under aerobic conditions and with limited access to air. A high pressure
xenon lamp and filter cutting-off wavelengths below 450 nm were
applied. Experiments have been performed according to the method
previously described [34].
4.4. Singlet oxygen generation
The quantum yield of singlet oxygen generation of Pcs was assessed
using 1,3-diphenylisobenzofuran (DPBF, Aldrich). DPBF as a chemical
quencher of singlet oxygen. Pc and DPBF solution was irradiated at a
wavelength corresponding to the Q band of the tested macrocycle.
The experimental procedure has been previously described in details
[18,19,24,25,31,34].
4.5. Anti-proliferative assays
4.2.2. [1,4,8,11,15,18,22,25-octakis(1,4,7-trioxanonyl)phthalocyanine]
manganese(III) chloride (5a)
The cytostatic activity of the tested compounds on human CD4+
T-lymphocyte (CEM), human cervix carcinoma (HeLa) and human
melanoma SK-MEL-5 cells was evaluated as follows: an appropriate
number of tumour cells were suspended in RPMI-1640 growth
medium and were seeded in 200 μl-wells of 96-well microtiter
plates, in the presence (or absence for purposes of control) of vari-
able concentrations of the test compounds. The cell cultures were ir-
radiated with red light at 735 nm for 30 min with a light intensity of
4.5 mW/cm2 (light dose 8.1 J/cm2) (or not irradiated (control)) be-
fore incubation at 37 °C in a humidified CO2-controlled atmosphere
(day 0). Recorded on day 1 and day 2, the irradiated tumour cell
cultures were exposed again to red light irradiation at 735 nm for
30 min, after which the cultures continued to be incubated at 37 °C.
On day 3 (CEM) or day 4 (HeLa, SK-MEL-5), the number of cells
were counted on a Coulter counter. The IC50 value was defined as
the compound concentration required to inhibit tumour cell prolif-
eration by 50%.
A rapidly stirred mixture of phthalocyanine 4 (95 mg 0.06 mmol) and
MnCl2∙4H2O (57 mg 0.06 mmol) in 20 mL of DMF were heated at 70 °C for
24 h. After being allowed to cool to room temperature, the reaction mix-
ture was filtered and evaporated to dryness under reduced pressure with
toluene (60 mL). Dry residue was dissolved in CH2Cl2 (30 mL) and rapidly
mixed with 30 mL of concentrated solution of sodium chloride for 30 min.
Next, organic layer was separated, washed with water (60 mL) and dried
with anhydrous MgSO4. Organic solvent was evaporated under reduced
pressure and dry residue was subjected to column chromatography
(CH2Cl2:CH3OH 15:1, EtOAc, CH2Cl2:CH3OH 15:1) to give dark brown
solid 5a (66 mg, 69% yield). M.p. N 80 °C (mesophase behaviour). Rf
(CH2Cl2:CH3OH 15:1) 0.08. UV–vis (CH2Cl2) λmax [nm] (log ε) 811
(4.96), 729 (4.34), 556 (4.18), 345 (4.53). MS (MALDI-TOF) [m/z] M+
1658.09, [M-Cl]+ 1624.32. IR ν [cm−1] 1070, 1213, 1309, 1503, 2866;
HPLC (Supplementary data) purity 98.56–99.93%.

L. Sobotta et al. / Journal of Inorganic Biochemistry 155 (2016) 76–81
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Acknowledgements
The authors thank Lizette van Berckelaer and Beata Kwiatkowska for
excellent technical assistance. This study was supported by the National
Science Centre under Grant No. N N401 067238. ZG thanks the
European Fund for Regional Development for Grant No UDA-
POIG.02.01.00-30-182/09. LS is a scholarship holder within the project
“Scholarship support for Ph.D. students specializing in majors strategic
for Wielkopolska's development”, Sub-measure 8.2.2 Human Capital
Operational Programme, co-financed by EU under the European Social
Fund. LS acknowledges the Polish National Science Centre for granting
him with a scholarship within the project “Etiuda 1” no. 2013/08/T/
NZ7/00242. The biological experiments were performed with support
of the KU Leuven (GOA 15/19 TBA).
Appendix A. Supplementary data
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