Dendrimeric Sulfanyl Porphyrazines: Synthesis, Physico-Chemical Characterization, and Biological Activity for Potential Applications in Photodynamic Therapy

Article abstract of DOI:10.1002/cplu.201600051

Sulfanyl porphyrazines substituted at their periphery with different dendrimeric moieties up to their first generation were synthesized and characterized by photochemical and biological methods. The presence of a dendrimeric periphery enhanced the spectral properties of the porphyrazines studied. The singlet-oxygen-generation quantum yield of the obtained macrocycles ranged from 0.02 to 0.20 and was strongly dependent on the symmetry of the compounds and the terminal groups of the dendritic outer shell. The in vitro biological effects of three most promising tribenzoporphyrazines were examined; the results indicated their potential as photosensitizers for photodynamic therapy (PDT) against two oral squamous cell carcinoma cell lines derived from the tongue. The highest photocytotoxicity was found for sulfanyl tribenzoporphyrazine that possessed 4-[3,5-di(hydroxymethyl)phenoxy]butyl substituents with nanomolar IC₅₀ values at 10 and 42 nm against CAL 27 and HSC-3 cell lines, respectively. Topic of cancer: Sulfanyl porphyrazines and tribenzoporphyrazines substituted at their periphery with different dendrimeric moieties up to their first generation were synthesized and characterized by photochemical and biological methods. The in vitro biological effects of three most promising tribenzoporphyrazines were examined and found to exhibit nanomolar IC₅₀ values (see figure).

Full text of DOI:10.1002/cplu.201600051

DOI: 10.1002/cplu.201600051
Full Papers
Dendrimeric Sulfanyl Porphyrazines: Synthesis, Physico-
Chemical Characterization, and Biological Activity for
Potential Applications in Photodynamic Therapy
Dariusz T. Mlynarczyk,[a] Sebastian Lijewski,[a] Michal Falkowski,[a] Jaroslaw Piskorz,[b]
Wojciech Szczolko,[a] Lukasz Sobotta,[b] Magdalena Stolarska,[a, c] Lukasz Popenda,[d]
Stefan Jurga,[d, e] Krystyna Konopka,[f] Nejat Düzgünes¸,[f] Jadwiga Mielcarek,[b] and
Tomasz Goslinski*[a]
Sulfanyl porphyrazines substituted at their periphery with dif-
ferent dendrimeric moieties up to their first generation were
synthesized and characterized by photochemical and biological
methods. The presence of a dendrimeric periphery enhanced
the spectral properties of the porphyrazines studied. The sin-
glet-oxygen-generation quantum yield of the obtained macro-
cycles ranged from 0.02 to 0.20 and was strongly dependent
on the symmetry of the compounds and the terminal groups
of the dendritic outer shell. The in vitro biological effects of
three most promising tribenzoporphyrazines were examined;
the results indicated their potential as photosensitizers for
photodynamic therapy (PDT) against two oral squamous cell
carcinoma cell lines derived from the tongue. The highest pho-
tocytotoxicity was found for sulfanyl tribenzoporphyrazine that
possessed 4-[3,5-di(hydroxymethyl)phenoxy]butyl substituents
with nanomolar IC50 values at 10 and 42 nm against CAL 27
and HSC-3 cell lines, respectively.
Introduction
Porphyrazines (Pzs) are macrocyclic compounds that consist of
four pyrrole units linked together with azamethine groups in
place of methine bridges present in porphyrins.[1] The spectro-
scopic, photochemical, and electrochemical properties of Pzs
have been studied widely. These properties render them suita-
ble for potential applications in photodynamic therapy (PDT)
and photodynamic diagnosis (PDD) as photosensitizers, in
chemical catalysis, and in analytical chemistry as sensors.[2–5]
Porphyrazines that contain sulfanyl substituents reveal good
solubility in common organic solvents and significant photocy-
totoxicities.[6–9] Many years of research on sulfanyl porphyra-
zines led to the expansion of their periphery with various
groups; for example, ether/thioether,[10,11] calixarene,[12] (tetra-
thiafulvalene)sulfanylethyl,[13] 2’-(4-pyridoxy)ethyl,[14] 1-naph-
thylmethyl,[8] 2’-(ferrocenecarboxy)ethyl,[15] and 4-(4-nitroimida-
zol-1-yl)butyl.[16]
[a] D. T. Mlynarczyk, S. Lijewski, M. Falkowski, Dr. W. Szczolko, M. Stolarska,
Dr. T. Goslinski
Department of Chemical Technology of Drugs
Poznan University of Medical Sciences
Grunwaldzka 6, 60-780 Poznan (Poland)
E-mail: tomasz.goslinski@ump.edu.pl
[b] Dr. J. Piskorz, Dr. L. Sobotta, Dr. J. Mielcarek
Department of Inorganic and Analytical Chemistry
Poznan University of Medical Sciences
Grunwaldzka 6, 60-780 Poznan (Poland)
[c] M. Stolarska
The possibility to add and modify dendrimer substituents at
the periphery of macrocyclic rings has been applied so far for
porphyrins and phthalocyanines.[17,18] It has been shown that
dendrimeric moieties around the porphyrinoid core significant-
ly affect their photophysical characteristics.[19] Dendrimers by
themselves are highly branched polymers with unique physi-
co-chemical and biological properties.[20,21] They have been
considered drug-delivery systems because of their ability to
transport various molecules within the structure of their den-
drons. So far, dendrimers have been tested to be successful
carriers of anticancer drugs (e.g., paclitaxel, doxorubicin), gado-
linium compounds for diagnostic purposes, poorly water-solu-
ble antimicrobials, and many others.[22] Dendrimers have also
been investigated for PDT purposes.[21] The combination of
phthalocyanines with dendrimer substituents enabled the ex-
tension of the possible biological applications of porphyri-
Department of Pharmaceutical Technology
Poznan University of Medical Sciences
Grunwaldzka 6, 61-780 Poznan (Poland)
[d] Dr. L. Popenda, Prof. Dr. S. Jurga
NanoBioMedical Centre
Adam Mickiewicz University in Poznan
Umultowska 85, 61-614 Poznan (Poland)
[e] Prof. Dr. S. Jurga
Faculty of Physics
Adam Mickiewicz University in Poznan
Umultowska 85, 61-614 Poznan (Poland)
[f] Prof. Dr. K. Konopka, Prof. Dr. N. Düzgünes¸
Department of Biomedical Sciences
University of the Pacific
155 Fifth Street, San Francisco, CA 94103 (USA)
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cplu.201600051.
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noids, reduced aggregation, and therefore increased the effi-
ciency of singlet oxygen generation.[23] Dendrimers have also
revealed a potential to catalyze chemical reactions enantiose-
lectively. The bulky periphery of metalloporphyrinoids has
been found to mimic enzymes, and these molecules have
been applied in biomimetics.[24]
bonate led to the derivative 6, which was applied as the alky-
lating agent in the next step (Scheme 2). The alkylation reac-
tion of dimercaptomaleonitrile disodium salt 1 with 6 in dime-
thylformamide and in the presence of potassium carbonate led
to the novel maleonitrile derivative 7. This compound was
used subsequently in the Linstead macrocyclization reaction in
n-butanol in the presence of magnesium n-butanolate to
result in the symmetrical porphyrazine derivative 8.[27] In anoth-
er approach, 7 and 1,2-dicyanobenzene were used in the
mixed Linstead macrocyclization reaction with Hoffman–Barrett
modification in n-butanol and in the presence of magnesium
n-butanolate, thus leading to the novel tribenzoporphyrazine
derivative 9.[29]
Herein, we report the synthesis of a series of dendrimer-sub-
stituted sulfanyl porphyrazines. They were obtained as a part
of our research on sulfanyl porphyrazines that bear isophtha-
loxybutyl substituents on their periphery.[25,26] The novel com-
pounds were characterized in terms of their photophysical
properties. Selected sulfanyl tribenzoporphyrazines were sub-
jected to photocytotoxicity studies.
2,3-Bis[4-(3,5-dimethoxycarbonylphenoxy)butylsulfanyl]ma-
leonitrile (10) was synthesized according to previously pub-
lished procedures.[25,30] Briefly, the alkylation reaction of dimer-
captomaleonitrile disodium salt with dimethyl 5-(4-bromobu-
toxy)isophthalate led to 10. The tribenzoporphyrazine deriva-
tive 11 was obtained by means of the mixed Linstead macro-
cyclization reaction with the Hoffman–Barrett modification
using 10 and 1,2-dicyanobenzene in n-butanol in the presence
of magnesium n-butanolate (Scheme 3). The subsequent re-
duction reaction of 11 with lithium aluminium hydride was car-
ried out using the procedure reported earlier by Hçger[28] and
led to the formation of the porphyrazine derivative 12. The al-
kylation reaction of 12 was used in the divergent synthetic ap-
proach, which led to a tribenzoporphyrazine with G1 dendri-
meric moieties (13).
Results and Discussion
Synthesis and characterization
The alkylation reaction of dimercaptomaleonitrile disodium salt
1 with 3,5-bis(3,5-dimethoxybenzyloxy)benzyl bromide 2 in di-
methylformamide and with potassium carbonate as a base led
to the novel maleonitrile derivative 3. After its purification and
thorough characterization, 3 was utilized in the Linstead mac-
rocyclization reaction in n-butanol and in the presence of mag-
nesium n-butanolate as a base to form the novel porphyrazine
4 (Scheme 1).[27]
3,5-Bis[3,5-bis(methoxycarbonyl)phenoxymethyl]phenol (5)
was synthesized according to the Hçger procedure.[28] Briefly,
the hydroxyl group of a commercially available dimethyl 3-hy-
droxyisophtalate was protected in the reaction with silyl chlo-
ride. Next, ester groups were reduced to alcohol groups with
lithium aluminum hydride. The obtained diol was subjected to
the Mitsunobu reaction with dimethyl 5-hydroxyisophtalate to
form a branched ether, the terminal aromatic hydroxyl group
of which was deprotected using tetrabutylammonium fluoride
to give 5. The alkylation reaction of 5 with 1,4-dibromobutane
in dimethylformamide and in the presence of potassium car-
The novel sulfanyl porphyrazine derivatives were character-
ized using various methods, including UV/Vis, NMR, and mass
spectrometry. HPLC analysis performed in three different sol-
vent systems confirmed the purity of the macrocycles. NMR
1
spectroscopy—and the H–13C HMBC technique in particular—
was useful in elucidating the structure of 4. The presence of
correlation signals between C1, C4 pyrrole carbons at d=
158.5 ppm and C2, C3 pyrrole carbons at d=141.7 ppm with
protons at d=5.72 ppm allowed us to assign the SCH2 hydro-
gen atoms. Other correlations observed between the outer pe-
ripheral C3’’, C5’’ benzyl carbons at d=161.9 ppm to protons
of the methoxy substituent at d=3.68 ppm enabled us to dis-
tinguish between benzyl moieties of the outer and inner
sphere within the dendrimeric substituents. There were no
1
cross-peaks detected in the H–1H COSY spectrum of 4, unlike
1
in the H–1H COSY spectrum of 8, in which correlations in both
peripheral and linker groups were observed. That enabled the
assignment of the signals of aliphatic protons of 8 present in
1
the H NMR spectra. In addition, cross-peaks between C2’’ and
C4’’ of the outer sphere benzene rings were observed in 8.
1
Correlations observed in the H–13C HMBC between the butyl
chain protons of terminal esters and C3’’ aromatic carbons of
the outer dendrimeric sphere permitted differentiation be-
tween the outer and inner benzene substituents.
In the 1H–1H COSY spectrum of tribenzoporphyrazine 11,
correlations within aliphatic protons were found (two groups
of neighboring hydrogen atoms at d=3.85, 1.35, 1.16,
0.74 ppm and d=4.32, 3.75 1.97 ppm). The H–13C HMBC and
1H–13C HSQC spectra were required to assign the aromatic pro-
1
Scheme 1. Synthesis of compounds 3 and 4. Reagents and conditions:
(i) K2CO3, DMF, room temperature, 48 h; (ii) Mg(nOC4H9)2, nBuOH, reflux, 20 h.
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Scheme 2. Synthesis of compounds 6–9. Reagents and conditions: (i) Br(CH2)4Br, K2CO3, DMF, 508C, 22 h; (ii) K2CO3, DMF, 508C, 21 h; (iii) Mg(nOC4H9)2, nBuOH,
reflux, 22 h; (iv) 1,2-dicyanobenzene, Mg(nOC4H9)2, nBuOH, reflux, 20 h.
Scheme 3. Synthesis of compounds 11–13. Reagents and conditions: (i) 1,2-dicyanobenzene, Mg(nOC4H9)2, nBuOH, reflux, 20 h; (ii) LiAlH4, THF; (iii) dimethyl 5-
(4-bromobutoxy)benzene-1,3-dicarboxylate, Cs2CO3, DMF, 608C, 19 h. THF=tetrahydrofuran.
tons and carbon nuclei of isophthalate moieties present in the
periphery. These spectra showed that peaks at d=4.32 and
3.75 ppm correspond to OCH2 and SCH2 protons within the
butyl linker, respectively. Proton peaks of the tribenzo part ap-
peared in the range between d=8.10–9.29 ppm as a group of
peaks, which was also the case for other investigated tribenzo-
porphyrazines.[31]
Aliphatic protons and carbons in porphyrazine 12 were as-
1
1
signed using H–1H COSY and H–13C HSQC spectra. However,
with the use of 2D techniques it was not possible to identify
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protons and carbons of isophthalate and tribenzo fragments.
Similar problems were found in 13 because of the overlapping
peaks of aliphatic protons. For tribenzoporphyrazines 9 and
13, both substituted with G1-dendrimeric moieties, no correla-
tions in the 1H–1H COSY and 1H–13C HMBC spectra between
isophthalate and aliphatic moieties were observed. It ham-
pered the possibility of assigning the signals that belonged to
the inner and outer sphere of the dendrons.
Spectroscopic and singlet oxygen generation studies
The UV/Vis spectra of investigated porphyrazines show both
a short-wavelength Soret band between 290–420 nm and
a long-wavelength Q band between 550–750 nm. The first one
is a result of p–p* transitions from the molecular orbital (MO)
to the lowest unoccupied molecular orbital (LUMO). The
second band is also a consequence of p–p* transitions, but in
this case electrons are transferred from the highest occupied
molecular orbital (HOMO) to LUMO. The LUMO orbital is de-
generate or non-degenerate. Degeneration of the LUMO orbi-
tal depends on molecular symmetry.[32–34] In symmetrical por-
phyrazines this orbital is degenerate, and, as a consequence, in
the UV/Vis spectrum a sharp unsplit Q band appears, which
was the case for Pzs 4 and 8 (Figure 1). For tribenzoporphyra-
zines 9 and 13, which are molecules of low symmetry, the sit-
uation was quite different. Their non-degenerate LUMO orbi-
tals manifested themselves as a split band in the long-wave-
length UV/Vis region. As shown in the study by Leclaire
et al.,[19] the presence of bulky dendritic substituents in the pe-
riphery of a macrocycle is responsible for shielding the tetra-
pyrrolic core and thus for an increase in the Q-band intensity
with every dendrimer generation added. In our study, the rela-
tive absorbance of the Q band of tribenzoporphyrazines 9 and
13 with G0 and G1 dendrimeric substituents of different size
did not change significantly. However, important changes were
observed during the photochemical study discussed below.
The novel porphyrazines were subjected to photochemical
studies. Evaluation of singlet oxygen generation for the exam-
ined compounds was performed in DMF (N,N-dimethylforma-
mide) according to the methodology described previously.[35–40]
Figure 1. UV/Vis spectra of investigated compounds in DMF: (a) symmetrical
porphyrazines 4 and 8; (b) tribenzoporphyrazines 11 and 12; and (c) triben-
zoporphyrazines 9 and 13.
1,3-Diphenylisobenzofuran (DPBF) was used as
a singlet
oxygen chemical quencher, whereas unsubstituted zinc phtha-
locyanine (ZnPc) with a known singlet oxygen quantum yield
(0.56 and 0.67 in DMF and DMSO (dimethyl sulfoxide), respec-
tively) was used as a reference. Mixtures of DPBF and porphyr-
azines 4, 8, 9, and 11–13 were irradiated with light of an ap-
propriate wavelength and changes in the UV/Vis spectra were
studied (Figure 2a). DPBF, which is a known singlet oxygen
trapping agent, was converted during the experiment to
a very unstable endoperoxide according to the first-order ki-
netic model (Figure 2b); this manifested in the UV/Vis spectra
as a decrease in the intensity of the band at 417 nm. The Pzs
studied have low or moderate singlet-oxygen-generation effi-
cacy, with the values reaching one half of that observed for un-
substituted zinc phthalocyanine (Table 1). For tribenzoporphyr-
azines, this might be the result of the decrease in symmetry.
Table 1. Quantum yields of singlet-oxygen generation by Pzs 4, 8, 9, and
11–13 in DMF.
Pzs
Singlet-oxygen generation
Irradiation wavelength [nm] Singlet-oxygen quantum yield [FD][a]
4
8
9
11
12
13
674
672
690
690
688
686
0.03
0.02
0.15
0.05
0.20
0.09
[a] Errorꢀ0.01.
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In vitro photocytotoxicity of selected tribenzoporphyrazines
The photodynamic activity of Pzs 9, 11, and 12 was investigat-
ed in vitro using two oral squamous cell carcinoma cell lines
derived from the tongue (CAL 27, HSC-3). The dark toxicity of
Pzs 9, 11, and 12 was evaluated at the limited concentration
range of 0.1 to 10 mm. There was no significant dark toxicity
observed for all compounds tested on both CAL 27 and HSC-3
cell lines (Figure 3). Therefore, the light-induced toxicity was
examined at the same concentrations of Pzs as that used for
dark toxicity measurements (0.1, 1.0, 10.0 mm) (Figure 3, Ta-
bles S11 and S12 in the Supporting Information).
All the compounds at 10.0 mm reduced the viability of both
cell lines. Porphyrazine 11 at this concentration decreased the
viability of CAL 27 cells by 58% and HSC-3 by 51%. Pz 9 re-
duced the viability of CAL 27 cells by 84% and HSC-3 cells by
95%, whereas Pz 12 showed photocytotoxicities higher than
90% against both cell lines.
In addition, Pz 9 and 12 had cytotoxic effects at 1.0 mm. Pz 9
decreased CAL 27 cell viability by 24% and HSC-3 by 69%. The
highest reduction of CAL 27 and HSC-3 cell viabilities (above
95%) at 1.0 mm was obtained with Pz 12. Moreover, Pz 12 at
the lowest concentration used (0.1 mm) reduced the viability of
CAL 27 cells by 99% and of HSC-3 cells by 95% (Figure 3,
Table S11 and S12 in Supporting Information).
Further experiments with Pzs 9, 11, and 12 were performed
at six concentrations for each compound to evaluate the corre-
lation between the viability of the cancer cells and the concen-
tration of photosensitizers (Figure 4, Tables S13 and S14 in the
Supporting Information). Pz 9 and 11 were tested in the con-
centration range 0.1 to 10.0 mm, and Pz 12 was tested in the
range of 0.005–1.0 mm. Calculation of the IC50 values indicated
that Pz 12 had the highest photocytotoxicity with values of
10 nm for CAL 27 cells and 42 nm for HSC-3 cells.
Figure 2. (a) Changes in the UV/Vis spectrum during irradiation of 12 and
the DPBF mixture in DMF. (b) First-order plots for the oxidation of DPBF by
photosensitized compounds 4, 8, 9, and 11–13 in DMF.
In our previous in vitro photodynamic studies with oral squ-
amous cell carcinoma (OSCC) cell lines, other porphyrinoid
photosensitizers were investigated. The photosensitizing activi-
ty of two porphyrazines that possessed fluoroalkylthio and di-
etherthio substituents was examined using HSC-3 and H413
cells.[41] The fluorinated macrocycle showed about 30–35%
photocytotoxicity on H413 cells at both 1.0 and 5.0 mm. How-
ever, there was no significant photosensitizing activity on HSC-
3 cells. In another study, zinc phthalocyanine and its analogue
with ester-alkyloxy substituents were tested against HSC-3
cells.[42] Zinc phthalocyanine decreased cell viability by 30% at
0.1 mm and by 100% at 5.0 mm. The photodynamic activity of
the substituted phthalocyanine was significantly lower, with
only 15% reduction of the viability of HSC-3 cells at 5.0 mm.
Further experiments performed with magnesium(II) and zinc(II)
phthalocyanines equipped with polyether substituents, and
the magnesium(II) phthalocyanine–metronidazole conjugate
revealed high in vitro photodynamic activity against HSC-3
cells.[43] All three phthalocyanine derivatives caused 100% pho-
tocytotoxicity at 5.0 mm. Moreover, zinc(II) phthalocyanine and
the magnesium(II) phthalocyanine–metronidazole conjugate
decreased the viability of HSC-3 cells at 0.1 mm by 17 and 34%,
respectively. The photosensitizing activity toward HSC-3 cells
Tribenzoporphyrazine derivative 12, which is an analogue
(hydroxyl groups at the periphery instead of carboxy) of 11,
shows the highest ability to generate singlet oxygen (FD =
0.20) in DMF. The enlargement of the tribenzoporphyrazine pe-
riphery from G0 to G1 resulted in higher FD values. The yield of
singlet-oxygen generation increased three times from 0.05 for
11 to 0.15 for 9, which is consistent with the literature data.[23]
In 9 and 13, both tribenzoporphyrazines with G1 peripheral
substituents, singlet-oxygen-generation values were 0.09 and
0.15, respectively. This can be the result of the distance be-
tween the inner and outer sphere within dendrimeric substitu-
ents. For octakis-substituted Pzs 4 and 8, the singlet-oxygen-
generation efficacy was relatively low (FD =0.03 for 4 and
FD =0.02 for 8). No increase in singlet-oxygen-generation effi-
cacy was observed by enlargement of our recently published
G0 symmetrical porphyrazine[25] to 8, as both revealed low sin-
glet-oxygen-generation efficacies with yields of 0.02 in DMF.
The photochemical data obtained for Pzs 4 and 8 comply with
those previously published for sulfanyl Pzs.[25,41]
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Figure 3. Light toxicity under 690 nm light irradiation (and dark control) of Pzs 9, 11, and 12 against: (a) CAL 27 and (b) HSC-3 cell lines. Data represent the
meanÆstandard deviation obtained from two independent experiments performed in triplicate. Statistical significance is indicated with asterisks: *p<0.001,
**p<0.0001.
of demetalled porphyrazine and magnesium(II) tribenzopor-
phyrazine that possessed a diazepine substituent was demon-
strated in another study.[2] Magnesium(II) tribenzoporphyrazine
reduced cell viability by 95% at 1.0 mm and by about 50% at
0.1 mm. Demetalled porphyrazine showed 87% photocytotoxic-
ity at 10.0 mm, but at 1.0 mm the viability of HSC-3 cells was un-
affected. In addition, tribenzoporphyrazine had a photocytotox-
ic effect on H413 cells at 0.025 and 1.0 mm. These data demon-
strate that Pz 12 exhibits the strongest photocytotoxicity
against oral cancer cells of all the porphyrinoids synthesized so
far by our research group.
experiments using YD10B cells revealed the photosensitizing
activity of the hexenyl ester of 5-aminolevulinic acid, which de-
creased the cell viability by 85% at 5.0 mm.[46] Non-porphyri-
noid photosensitizers were also tested on OSCC cells. Lim et al.
examined fifteen different boron bipyrromethene derivatives
using HSC-2 cells that originated from the oral floor.[47] Three
of them exhibited high photocytotoxicity, with IC50 values in
the range of 0.1 to 0.6 mm. Lucky et al. prepared titanium oxide
coated nanoparticles and examined their in vitro photocyto-
toxicity on CAL-27 cells.[48] It was found that pegylated TiO2
nanoparticles decreased cell viability by about 80% at 1.0 mm
and about 40% at 10.0 mm. It is worth noting that Pzs 9, 11,
and 12 at 10.0 mm showed higher photosensitizing effects at
the same concentration on CAL-27 cells. The results reported
in this study together with literature data led to the conclusion
that Pz 12 possesses one of the highest activities in vitro on
OSCC cells described so far. It is also worth noting that Pz 9
(G1 peripheral dendron generation) revealed significantly lower
IC50 values than that for Pz 11 (G0 peripheral dendron genera-
tion) (Table 2). These results can be analyzed together with the
above described singlet-oxygen-generation efficacy with
values decreasing in the following order: Pz 12>Pz 9>Pz 11
(Table 1).
Other porphyrinoid photosensitizers used in the in vitro
photodynamic therapy of OSCC cells should also be noted.
Thomas et al. investigated the photosensitizing activity of
a novel water-soluble porphyrin that possessed 4-sulfonato-
phenyl rings using SCC-131 and SCC-172 cells.[44] The novel
porphyrin showed photocytotoxicity against both cell lines
with IC50 values of 13 and 11 mm for SCC-131 and SCC-172, re-
spectively. The two other OSCC cell lines—YD10B derived from
the tongue and YD38 from the lower gingiva—were used to
examine the photocytotoxicity of pheophorbide b.[45] The use
of 0.3 mm of this photosensitizer led to the reduction of YD10B
cell viability by 70% and of YD38 cell viability by 60%. Other
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Conclusion
In summary, the synthesis and characterization of sulfanyl por-
phyrazines and tribenzoporphyrazines substituted in their pe-
riphery with different dendrimeric substituents up to their first
generation were performed. Under the Linstead macrocycliza-
tion reaction conditions, a symmetrical sulfanyl porphyrazine
with 3,5-bis(3,5-dimethoxybenzyloxy)benzyl substituents as
well as symmetrical porphyrazine and tribenzoporphyrazine
with (4-{3,5-bis[3,5-bis(methoxycarbonyl)phenoxymethyl]phe-
noxy}butylsulfanyl) substituents were obtained. Moreover,
novel sulfanyl tribenzoporphyrazine with 4-[3,5-di(butoxycar-
bonyl)phenoxy]butyl substituents was reduced to tribenzopor-
phyrazine with 4-[3,5-di(hydroxymethyl)phenoxy]butyl sub-
stituents. Further alkylation reaction of this compound with
the use of the divergent synthetic approach led to pseudo-G1
sulfanyl dendrimeric tribenzoporphyrazine with 4-(3,5-bis{[4-
(3,5-dimethoxycarbonylphenoxy)butoxy]methyl}phenoxy)butyl
substituents.
The macrocycles were subjected to photochemical studies in
terms of singlet oxygen generation and revealed low or mod-
erate singlet-oxygen-generation efficacy. The sulfanyl tribenzo-
porphyrazine analogue with terminal hydroxyl groups at the
periphery showed the highest ability to generate singlet
oxygen (FD =0.20).
The photodynamic activity of tribenzoporphyrazines was in-
vestigated in vitro using two oral squamous cell carcinoma cell
lines derived from the tongue (CAL 27, HSC-3). Dark toxicity of
tribenzoporphyrazines was not observed in the limited concen-
tration range of 0.1 to 10.0 mm. Therefore, the light-induced
toxicity was examined at the same concentrations as the dark
toxicity (0.1, 1.0, 10.0 mm). It was found that all compounds at
10.0 mm reduced the viability of both cell lines by 50–95%. Tri-
benzoporphyrazine with 4-[3,5-di(hydroxymethyl)phenoxy]bu-
tyl substituents revealed the highest reduction of the viabilities
of CAL 27 and HSC-3 cells above 95% at 1.0 mm. Moreover, the
aforementioned tribenzoporphyrazine with 4-(3,5-dibutoxycar-
bonylphenoxy)butyl substituents decreased the viability of
both cell lines at the lowest concentration used (0.1 mm). Fur-
ther experiments were performed for each compound to eval-
uate the correlation between cancer-cell viability and the con-
centration of the photosensitizers. The lowest nanomolar IC50
values were obtained with tribenzoporphyrazine with 4-[3,5-
di(hydroxymethyl)phenoxy]butyl substituents (10 nm and
42 nm for CAL 27 and HSC-3 cells, respectively). It is worth
noting that sulfanyl tribenzoporphyrazine with 4-{3,5-bis[3,5-
bis(butoxycarbonyl)phenoxymethyl]phenoxy}butyl substituents
(G1 peripheral dendron generation) revealed significantly lower
IC50 values than sulfanyl tribenzoporphyrazine with 4-(3,5-dibu-
toxycarbonylphenoxy)butyl substituents (G0 peripheral den-
dron generation). These results can be compared to the sin-
glet-oxygen-generation efficiency. According to the singlet-
oxygen-generation efficiency, the sulfanyl tribenzoporhyrazines
could be ordered in terms of the applied substituent: 4-[3,5-
di(hydroxymethyl)phenoxy]butyl>4-{3,5-bis[3,5-bis(butoxycar-
bonyl)phenoxymethyl]phenoxy}butyl>4-[3,5-di(butoxycarbo-
nyl)phenoxy]butyl. The excellent photocytotoxicity of sulfanyl
Figure 4. Phototoxic effect of Pzs 9, 11, and 12 on CAL 27 and HSC-3 cells.
Data represent the meanÆstandard deviation for experiments performed in
triplicate.
Table 2. IC50 values [mm] for the cytotoxicity of the studied
photosensitizers.
Pzs
CAL 27
HSC-3
9
11
12
3.13
6.66
0.01
0.64
10.6
0.042
The higher photosensitizing activity of tribenzoporphyrazine
12 relative to Pz 9 and 11 would appear to be connected with
its significantly higher singlet-oxygen-generation yield and/or
the presence of hydroxyl groups in its periphery, which signifi-
cantly facilitates its solubility and distribution, rather than with
its singlet-oxygen-generation efficacy.
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reaction mixture was filtered through Celite, which was then
washed with CH2Cl2. The filtrate was evaporated and the solid resi-
due was chromatographed (CH2Cl2/methanol 100:1 to 50:1) to give
3 as a yellow oil (471 mg, 89%); Rf =0.55 (CH2Cl2/methanol 100:1);
1H NMR (800 MHz, [D6]DMSO): d=6.65 (d, 4J=2 Hz, 4H; C2’, C6’
ArH), 6.60 (t, 4J=2 Hz, 2H; C4’, ArH), 6.57 (d, 4J=2 Hz, 8H; C2’’,
C6’’, ArOCH3), 6.43 (t, 4J=2 Hz, 4H; C4’’, ArOCH3), 4.98 (s, 8H;
OCH2), 4.39 (s, 4H; SCH2), 3.72 ppm (s, 24H; OCH3); 13C NMR
(201 MHz, [D6]DMSO): d=160.5 (C3’’, C5’’, ArOCH3), 159.5 (C3’, C5’,
ArC), 139.1 (C1’’, ArOCH3), 137.3 (C1’, ArC), 120.9 (CS), 112.5 (CN),
108.1 (C2’, C6’, ArC), 105.4 (C2’’, C6’’, ArOCH3) 101.4 (C4’, ArC), 99.5
(C4’’, ArOCH3), 69.2 (OCH2) 55.1 (CH3), 38.2 ppm (SCH2); UV/Vis
(CH2Cl2): lmax (loge)=280 (4.33), 349 nm (4.23); MS (MALDI): m/z:
987 [M+H]+, 1009 [M+Na]+, 1025 [M+K]+; HRMS (ESI): m/z calcd.
(C54H55N2O12S2): 987.3196 [M+H]+; found: 987.3195 [M+H]+; ele-
mental analysis calcd. for C54H55N2O12S2 ”0.5H2O: C 65.11 H, 5.57, N
2.81, S 6.44; found: C 64.74, H 6.04, N 2.67, S 5.99.
tribenzoporphyrazine with 4-[3,5-di(hydroxymethyl)phenoxy]-
butyl substituents seems to be related more to the presence
of hydroxyl groups in its periphery, which significantly facili-
tates its solubility and distribution, than with its singlet-
oxygen-generation efficacy.
Experimental Section
General procedures
All reactions were conducted in oven-dried glassware under an
argon atmosphere using a Radleys Heat-On heating system unless
otherwise stated. All solvents were rotary evaporated at or below
508C under reduced pressure. All solvents and reagents were ob-
tained from commercial suppliers and used without further purifi-
cation unless otherwise stated. Dry flash column chromatography
was carried out on Merck silica gel 60 (particle size 40–63 mm) and
reversed-phase chromatography on Fluka C18 silica gel 90 (particle
size 40–63). Thin-layer chromatography (TLC) was performed on
silica gel Merck Kieselgel 60 F254 plates and visualized by means of
UV spectroscopy (lmax =254 or 365 nm). UV/Vis spectra were re-
corded with Hitachi UV/VIS U-1900 or Shimadzu PC160 spectro-
photometers. The NMR spectra were acquired at 298 K with an
Agilent DD2 800 NMR spectrometer operating at resonance fre-
2,3,7,8,12,13,17,18-Octakis[3,5-bis(3,5-dimethoxybenzyloxy)ben-
zylthio]porphyrazinato magnesium(II) (4): Magnesium turnings
(5.0 mg, 0.2 mmol) and a small crystal of iodine were heated to
reflux in n-butanol (2.0 mL) for 4 h. After cooling to room tempera-
ture, the reaction mixture was transferred using a syringe to a flask
that contained 3 (197 mg, 0.20 mmol). Then the reaction mixture
was heated under reflux conditions for another 20 h. Next, the re-
action mixture was cooled to room temperature, filtered through
Celite, then washed with toluene and CH2Cl2. Filtrates were evapo-
rated to dryness, and a dark blue residue was chromatographed
using silica gel (CH2Cl2/methanol 100:1 to 20:1) then reversed-
phase column chromatography (methanol, then CH2Cl2) to give 4
as a blue film (71 mg, 36%). M.p.>508C (mesophase behavior);
Rf =0.46 (CH2Cl2/methanol 50:1); 1H NMR (800 MHz, [D5]pyridine):
1
quencies of 799.903 and 201.146 MHz for H and 13C, respectively.
Chemical shifts (d) are quoted in parts per million (ppm) and are
referred to a residual solvent peak. Coupling constants (J) are
quoted in Hertz (Hz). The abbreviations s, p, t, h, and m refer to
1
singlet, pentet, triplet, hidden, and multiplet, respectively. The H
and 13C NMR spectroscopic resonances were assigned using a com-
bination of homo- and heteronuclear two-dimensional techniques
1
1
1
such as H–1H COSY, H–13C HSQC, and H–13C HMBC. Mass spectra
(ES, MALDI TOF, HRMS) and combustion analyses were carried out
by the Advanced Chemical Equipment and Instrumentation Facility
at the Faculty of Chemistry and the Wielkopolska Center for Ad-
vanced Technologies at Adam Mickiewicz University in Poznan, and
at the European Center of Bioinformatics and Genomics in Poznan.
Melting point was obtained using a “Stuart” Bibby apparatus and
is uncorrected. Analytical HPLC was carried out using an Agilent
1200 instrument equipped with a UV-DAD detector. The chromato-
graphic separation was achieved on an octadecylsilane-coated
column (150 mm”4.6 mm, 5 mm; Eclipse XDB-C18, Agilent) or on
hexaphenyl-coated columns (150 mm”4.6 mm, 5 mm or 250 mm”
4.6 mm, 5 mm; Gemini C6-Phenyl, Phenomenex) under isocratic
and linear gradient conditions.
4
4
d=7.28 (d, J=2 Hz, 16H; C2’, C6’, ArH), 6.83 (t, J=2 Hz, 8H; C4’,
ArH), 6.71 (d, 4J=2 Hz, 32H; C2’’, C6’’, ArOCH3), 6.59 (t, 4J=2 Hz,
16H; C4’’, ArOCH3), 5.72 (s, 32H; OCH2), 4.90 ppm (s, 16H; SCH2),
3.68 s, 96H; OCH3); 13C NMR (201 MHz, [D5]pyridine): d=161.9 (C3’’,
C5’’, ArOCH3), 161.2 (C3’, C5’, ArC), 158.5 (C1, C4, pyrrole C), 142.1
(C1’, ArC), 141.7 (C2, C3, pyrrole C), 140.3 (C1’’, ArOCH3), 109.5 (C2’,
C6’, ArC), 106.3 (C2’’, C6’’, ArOCH3), 102.5 (C4’, ArC) 100.8 (C4’’,
ArOCH3), 70.7 (OCH2), 55.8 (OCH3), 41.3 ppm (SCH2); UV/Vis
(CH2Cl2): lmax (loge)=280 (4.89), 370 (4.78), 676 nm (4.80); MS
(MALDI): m/z: 3995 [M+Na]+; HRMS (ESI): m/z calcd.
(C216H217MgN8O48S8): 3970.2401 [M+H]+; found: 3970.2151 [M+H]+.
For HPLC purity, see the Supporting Information.
Synthetic procedure leading to symmetrical porphyrazine 8
and tribenzoporphyrazine 9
Synthetic procedures
1-(4-Bromobutoxy)-3,5-bis[3,5-bis(methoxycarbonyl)phenoxyme-
thyl]benzene (6): 3,5-Bis[3,5-bis(methoxycarbonyl)phenoxymethyl]-
phenol (0.464 g, 0.86 mmol), 1,4-dibromobutane (0.31 mL,
2.59 mmol), and potassium carbonate (3.6 g, 25.9 mmol) in anhy-
drous DMF (6 mL) were stirred at 508C for 22 h. Next, the reaction
mixture was filtered through Celite, then washed with CH2Cl2. The
filtrate was evaporated and the solid residue was chromatograph-
ed (CH2Cl2) to give 6 as a white powder (434 mg, 75%). M.p.
1298C; Rf =0.36 (CH2Cl2/methanol 50:1); 1H NMR (800 MHz,
[D6]DMSO): d=8.06 (t, 4J=1 Hz, 2H; C4’, ArH), 7.73 (d, 4J=1 Hz,
4H; C2’, C6’, ArH), 7.12 (s, 1H; C4, ArH), 7.02 (s, 2H; C2, C6, ArH),
3,5-Bis(3,5-dimethoxybenzyloxy)benzyl bromide (2) was purchased
from TCI Chemicals (Tokio, Japan). 3,5-Bis[3,5-bis(methoxycarbonyl)-
phenoxymethyl]phenol (5) was synthesized according to the pro-
cedure described by Hçger.[28] 2,3-Bis[4-(3,5-dimethoxycarbonylphe-
noxy)butylsulfanyl] maleonitrile (10) was synthesized according to
the previously published procedure.[25]
Synthetic procedure leading to symmetrical porphyrazine 4
2,3-Bis[3,5-bis(3,5-dimethoxybenzyloxy)benzylsulfanyl]maleoni-
trile (3): Dimercaptomaleonitrile disodium salt (100 mg,
0.54 mmol), 3,5-bis(3,5-dimethoxybenzyloxy)benzyl bromide (2)
(673 mg, 1.34 mmol), and K2CO3 (296 mg, 2.15 mmol) in anhydrous
DMF (10 mL) were stirred at room temperature for 48 h. Next, the
3
5.23 (s, 4H; ArCH2O), 4.04 (t, J=6 Hz, 2H; CH2CH2O), 3.88 (s, 12H;
OCH3), 3.60 (t, 3J=7 Hz, 2H; BrCH2), 1.97 (m, 2H; BrCH2CH2),
1.84 ppm (m, 2H; CH2CH2O); 13C NMR (201 MHz, [D6]DMSO): d=
165.1 (C=O), 158.7 (C1, ArC), 158.4 (C1’, ArC), 138.2 (C3, C5, ArC),
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131.5 (C3’, C5’, ArC), 121.9 (C4’, ArC), 119.6 (C2’, C6’, ArC), 118.3 (C4,
ArC), 113.0 (C2, C6, ArC), 69.4 (ArCH2O), 66.7 (CH2CH2O), 52.5
(OCH3), 34.8 (BrCH2), 29.0 (BrCH2CH2), 27.3 ppm (CH2CH2O); UV/Vis
(CH2Cl2): lmax (loge)=286 (3.67), 307 nm (3.83); MS (ES+): m/z: 671,
673 [M+H]+, 695, 697 [M+Na]+, 711, 713 [M+K]+; MS (ESÀ): m/z:
707, 709 [M+Cl]À; elemental analysis calcd. for C32H33BrO11·H2O: C
55.58, H 5.10; found: C 55.53, H 5.14.
bottomed flask. After cooling, the contents of the flask were trans-
ferred with a syringe to another round-bottomed flask that con-
tained maleonitrile derivative 7 (136 mg, 0.102 mmol) and 1,2-di-
cyanobenzene (131 mg, 1.024 mmol), then it was heated to reflux
for another 20 h. The resulting blue product was filtered through
Celite with toluene, and the solvents were evaporated under re-
duced pressure. Compound 9 was purified by means of column
chromatography using silica gel and n-hexane/ethyl acetate (7:2)
to give 82 mg of a blue film (39% yield). M.p. 87–898C; Rf =0.15
2,3-Bis(4-{3,5-bis[3,5-bis(methoxycarbonyl)phenoxymethyl]phe-
noxy}butylsulfanyl)maleonitrile (7): Dimercaptomaleonitrile diso-
dium salt (52 mg, 0.28 mmol), 6 (434 mg, 0.64 mmol), and potassi-
um carbonate (300 mg, 2.17 mmol) were stirred in anhydrous DMF
(5 mL) at 508C for 21 h. The solvent was evaporated, and the resid-
ual oil was chromatographed (CH2Cl2/methanol 50:1 to 35:1) to
give 7 as a yellow oil (317 mg, 85% yield). Rf =0.11 (CH2Cl2/metha-
1
(CH2Cl2/MeOH 35:1); H NMR (800 MHz, [D5]pyridine): d=9.62–9.17
(m, 8H; tribenzo-H), 8.61–8.47 (m, 6H; isophthalo-H), 8.25–8.13 (m,
4H; tribenzo-H), 8.12–7.79 (m, 12H; isophthalo-H), 5.31–5.03 (m,
8H; OCH2Ph), 4.78 (brs, 4H; SCH2), 4.36–4.23 (m, 20H; OCH2CH2),
1.65–1.52 (m, 20H; SCH2CH2CH2), 1.39–1.25 (m, 20H; SCH2CH2CH2,
CH2CH3), 0.86–0.75 ppm (m, 24H; CH3); 13C NMR (201 MHz,
[D5]pyridine): d=167.9, 161.8, 161.6, 161.5, 152.6, 142.9, 141.4,
140.7, 138.2, 136.5, 136.4, 135.3, 135.2, 135.1, 133.9, 131.7, 126.2,
125.6, 122.7, 122.6, 122.4, 72.9, 72.6, 72.4, 70.6, 67.8, 41.5, 33.8,
33.3, 32.4, 31.6, 26.5, 25.6, 21.8, 16.2; UV/Vis (CH2Cl2): lmax (loge)=
694 (3.73), 651 (3.63), 359 (3.64), 316 nm (3.66); MS (MALDI TOF):
m/z: 2071.871 [M+H]+; HRMS (ESI): m/z: calcd. (C116H126MgN8O22S2):
2070.82786 [M]+; found: 2070.84590 [M]+. For HPLC purity, see the
Supporting Information.
1
4
nol 50:1); H NMR (800 MHz, [D6]DMSO): d=7.99 (d, J=1 Hz, 4H;
C4’’, ArH), 7.67 (t, 4J=1 Hz, 8H; C2’’, C6’’, ArH), 7.09 (s, 2H; C4’,
ArH), 6.96 (s, 4H; C2’, C6’, ArH), 5.15 (s, 8H; ArCH2O), 3.99 (t, J=
3
6 Hz, 4H; CH2CH2O), 3.85 (s, 24H; OCH3), 3.23 (t, 3J=7 Hz, 4H;
SCH2), 1.81 ppm (brs, 8H; CH2CH2CH2O); 13C NMR (201 MHz,
[D6]DMSO): d=165.0 (C=O), 158.7 (C1’, ArC), 158.3 (C1’’, ArC), 138.1
(C3’, C5’, ArC), 131.3 (C3’’, C5’’, ArC), 121.9 (CS), 121.4 (C4’’, ArC),
119.5, (C2’’, C6’’, ArC), 118.2 (C4’, ArC), 112.9 (C2’, C6’, ArC), 112.4
(NC), 69.4 (C3’CH2O), 66.8 (SCH2CH2CH2CH2), 52.4 (OCH3), 34.2
(SCH2), 27.2 (SCH2CH2), 26.1 ppm (SCH2CH2CH2); UV/Vis (CH2Cl2):
lmax (loge)=285 (4.14), 315 (4.30), 343 ppm (4.22); MS (MALDI): m/
z: 1327 [M+H]+, 1349 [M+Na]+, 1365 [M+K]+; elemental analysis
calcd. for C68H66N2O22S2: C 61.53, H 5.01, N 2.11, S 4.83; found: C
61.09, H 5.02, N 2.07, S 4.74.
Synthetic procedure leading to tribenzoporphyrazines 11–
13
22,23-Bis[4-(3,5-dibutoxycarbonylphenoxy)butylthio]triben-
zo[b,g,l]porphyrazinato magnesium(II) (11): Magnesium turnings
(100 mg, 4.098 mmol) were heated to reflux in n-butanol (40 mL)
with a small crystal of iodine for 6 h in a round-bottomed flask.
After cooling, the contents of the flask were transferred with a sy-
ringe to another round-bottomed flask that contained maleonitrile
2,3,7,8,12,13,17,18-Octakis(4-{3,5-bis[3,5-bis(butoxycarbonyl)-
phenoxymethyl]phenoxy}butylthio)porphyrazinato
magne-
sium(II) (8): Magnesium turnings (6 mg, 0.25 mmol) and a small
crystal of iodine were heated to reflux in n-butanol (2.5 mL) for 2 h.
After cooling to room temperature, the reaction mixture was trans-
ferred using a syringe to a flask that contained maleonitrile 7
(334 mg, 0.25 mmol) and was heated under reflux conditions for
22 h. Next, the reaction mixture was cooled to room temperature,
filtered through Celite, then washed with toluene. Filtrates were
evaporated in a rotary evaporator, which resulted in a dark blue
residue, which was chromatographed using silica gel (CH2Cl2/meth-
anol, 50:1 v/v) and reversed-phase column chromatography (meth-
anol, than CH2Cl2), then silica gel once again (n-hexane/ethyl ace-
tate, 7:3 v/v) to give 8 as a dark blue film (95 mg; 23% yield).
M.p.>2358C decomp; Rf =0.28 (n-hexane/ethyl acetate, 7:3);
1H NMR (800 MHz, [D5]pyridine): d=8.57 (s, 16H; C4’’, ArH), 8.07 (s,
32H; C2’’, C6’’, ArH), 7.39 (s, 8H; C4’, ArH), 7.20 (sh, 16H; C2’, C6’,
derivative
6
(500 mg, 0.745 mmol) and 1,2-dicyanobenzene
(955 mg, 7.450 mmol), the heated to reflux for another 20 h. The
resulting blue product was filtered through Celite with toluene,
and the solvents were evaporated under reduced pressure. Com-
pound 11 was purified by means of column chromatography using
silica gel and n-hexane/ethyl acetate (7:2) to give 307 mg of a blue
film (33% yield). M.p. 138–1408C; Rf =0.07 (CH2Cl2/MeOH 50:1);
1H NMR (800 MHz, [D6]DMSO): d=9.29, 9.28 (2d, J=5 Hz, 2H; tri-
3
benzo-H), 9.24 (d, J=7 Hz, 2H; tribenzo-H), 9.14 (d, J=7 Hz, 2H;
tribenzo-H), 8.20, 8.19 (2d, J=5 Hz, 2H; tribenzo-H), 8.12, 8.10 (2t,
J=6 Hz, 4H; tribenzo-H), 7.23 (s, 2H; isophthalate-H), 6.69 (s, 4H;
isophthalate-H), 4.33 (t, 3J=7 Hz, 4H; PhOCH2), 3.85 (t, 3J=7 Hz,
8H; C(O)OCH2), 3.75 (t, 3J=6 Hz, 4H; SCH2), 1.97 (m, 8H;
SCH2CH2CH2), 1.35 (m, 8H; CH2CH2CH3), 1.16 (m, 8H; CH2CH3),
0.74 ppm (t, 3J=7 Hz, 12H; CH3); 13C NMR (201 MHz, [D6]DMSO):
d=167.8, 166.9 (C=O), 160.9 (isophthalate), 159.9, 159.4, 155.9,
154.9, 142.1, 142.0, 141.3, 139.9, 134.8, 133.6 (isophthalate), 133.4,
133.1, 132.5, 126.0, 125.9, 123.8 (isophthalate), 121.0 (isophthalate),
69.9 (SCH2), 67.5 (C(O)OCH2), 37.1 (PhOCH2), 33.0 (CH2CH2CH3), 30.4,
29.5 (SCH2CH2CH2), 21.6 (CH2CH3), 16.5 ppm (CH3); UV/Vis (CHCl3):
lmax (loge)=721 (3.62), 626 (3.54), 357 nm (3.76); MS (MALDI-TOF):
m/z: 1247.403 [M+H]+; HRMS (ESI): m/z: calcd. (C68H71MgN8O10S8):
1247.45795 [M+H]+; found: 1247.45862 [M+H]+. For HPLC purity,
see the Supporting Information.
3
ArH), 5.22 (s, 32H; C3’CH2O), 4.59 (s, 16H; SCH2), 4.37 (t, J=7 Hz,
64H; COOCH2), 4.19 (s, 16H; ArOCH2CH2), 2.37 (brs, 32H;
SCH2CH2CH2), 1.66 (m, 64H; COOCH2CH2), 1.37 (m, 64H; CH3CH2),
0.88 ppm (m, 96H; CH3); 13C NMR (201 MHz, [D5]pyridine): d=166.0
(C=O), 160.5 (C1’, ArC), 159.7 (C1’’, ArC), 139.3 (C3’, C5’, ArC), 133.2
(C3’’, C5’’, ArC), 123.7 (C4’’, ArC), 120.7 (C2’’, C6’’, ArC), 119.6 (C4’,
ArC), 114.2 (C2’, C6’, ArC), 70.8 (ArCH2O), 68.5 (ArOCH2CH2), 65.9
(COOCH2), 35.9 (SCH2), 31.4 (COOCH2CH2), 29.6 (SCH2CH2CH2), 28.1
(SCH2CH2), 19.9 (CH3CH2), 14.3 ppm (CH3); UV/Vis (CH2Cl2): lmax
(loge)=673 (4.50), 374 (4.62), 314 (4.67), 286 nm (4.52); MS
(MALDI): m/z: 6677 [M+H]+; HRMS (MALDI): m/z: calcd.
(C368H457MgN8O88S8): 6675.915; found: 6675.762. For HPLC purity,
see the Supporting Information.
22,23-Bis{4-[3,5-di(hydroxymethyl)phenoxy]butylthio}triben-
zo[b,g,l]porphyrazinato magnesium(II) (12): LiAlH4 (44 mg,
1.152 mmol) was suspended in THF (10 mL) and stirred for 0.5 h at
08C. After that, a solution of 11 (300 mg, 0.240 mmol) in THF
(6 mL) was added dropwise over 40 min. Next, the ice bath was re-
moved, and the reaction mixture was left to warm to room tem-
22,23-Bis(4-{3,5-bis[3,5-bis(butoxycarbonyl)phenoxymethyl]phe-
noxy}butylthio)tribenzo[b,g,l]porphyrazinato magnesium(II) (9):
Magnesium turnings (13 mg, 0.512 mmol) were heated to reflux in
n-butanol (15 mL) with a small crystal of iodine for 6 h in a round-
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perature and was stirred for 2 h. The reaction was quenched with
saturated NH4Cl solution (10 mL) and left for another 15 min. The
reaction mixture was filtered through Celite, then washed with
CH2Cl2. The filtrates were evaporated to dryness. Column chroma-
tography (CH2Cl2, then CH2Cl2/MeOH 20:1) led to 12 in the form of
a deep blue film (150 mg, 65% yield). M.p. 151–1538C; Rf =0.12
solution, l-glutamine, FBS, Trypsin-EDTA, phosphate buffered saline
(PBS), Dulbecco’s phosphate buffered saline (DPBS)—were ob-
tained from the UCSF Cell Culture Facility, San Francisco, USA. Pho-
tosensitizers were dissolved in dimethyl sulfoxide (Sigma–Aldrich)
and subsequently diluted in DMEM (without FBS and phenol red)
to obtain the desired concentration of photosensitizer used in the
experiments. The DMSO concentration in the final solution did not
exceed 0.5%.
1
(CH2Cl2/MeOH 10:1); H NMR (800 MHz, [D5]pyridine): d=9.57–8.84
(m, 4H; tribenzo-H, isophthalo-H), 8.64–7.57 (m, 12H; tribenzo-H,
isophthalo-H), 7.51–7.21 (m, 2H; tribenzo-H, isophthal-H), 4.34 (m,
4H; OCH2CH2), 4.07–3.81 (m, 4H; SCH2), 1.62 (m, 4H; S-CH2-CH2-
CH2), 1.33 (m, 4H; S-CH2-CH2), 0.83 ppm (m, 8H; CH2OH); 13C NMR
(201 MHz, [D5]pyridine): d=161.9, 156.0, 155.9, 154.8, 146.4, 132.0,
130.3, 129.9, 128.9, 126.0, 125.7, 120.0, 119.0, 116.1, 100.3, 63.1,
61.5, 27.1, 15.5, 10.0 ppm; UV/Vis (CH2Cl2): lmax (loge)=711 (3.81),
652 (3.26), 597 (3.29), 382 nm (3.55); MS (MALDI TOF): m/z 967.181
[M]+. For HPLC purity, see the Supporting Information.
Dark toxicity
One day before the experiment, the cells were seeded in 48-well
plates at a density 1.8”105 in 1 mL of medium in each well (with
FBS and phenol red) and used at approximately 80% confluence.
Subsequently, the cells were washed with PBS (1 mL), and the
medium (1 mL; without FBS and phenol red) that contained the
photosensitizer at a given concentration was added to each well
except for the controls. The FBS-free media were used to avoid
binding of photosensitizers to serum proteins. After a 24 h incuba-
tion at 378C, the cells were washed with PBS, then complete
medium (1 mL) was added to each well, and the cells were incu-
bated for 24 h at 378C. The cell viability was quantified by the
Alamar Blue assay. The cells incubated either with medium alone
or in medium/0.5% DMSO served as controls.
22,23-Bis[4-(3,5-bis{[4-(3,5-dimethoxycarbonylphenoxy)butoxy]-
methyl}phenoxy)butylthio]tribenzo[b,g,l]porphyrazinato magne-
sium(II) (13): Porphyrazine derivative 12 (74 mg, 0.076 mmol), di-
methyl 5-(4-bromobutoxy)benzene-1,3-dicarboxylate (132 mg,
0.382 mmol), and cesium carbonate (248 mg, 0.761 mmol) were
stirred in DMF (15 mL) at 608C for 19 h. After cooling, the reaction
mixture was filtered through Celite, then washed with toluene. The
filtrates were evaporated under reduced pressure to yield the dry
residue. Column chromatography was performed (CH2Cl2/MeOH
20:1) to give 10 as a dark blue film (31 mg, 20% yield). M.p. 133–
1358C; Rf =0.12 (CH2Cl2/MeOH 20:1); 1H NMR (800 MHz,
[D5]pyridine): d=9.57 (m, 6H; tribenzo-H), 8.55–8.39 (m, 6H; tri-
benzo-H), 8.13 (m, 6H; isophthal-H), 7.95–7.77 (m, 12H; isophthal-
H), 4.49–4.37 (m, 8H; PhÀCH2ÀO), 4.04–3.91 (m, 12H; PhÀOÀCH2),
3.83 (m, 24H; CH3), 2.30 (d, J=13 Hz, 4H; SÀCH2), 1.94–1.84, 1.30–
1.20 ppm (m, 24H; CH2ÀCH2ÀCH2ÀCH2); 13C NMR (201 MHz,
[D5]pyridine): d=166.4, 166.0, 159.9, 150.6, 136.2, 134.5, 132.7,
124.2, 123.3, 120.4, 68.6, 67.6, 65.8, 62.2, 52.8, 30.4, 26.5, 26.1 ppm;
UV/Vis (CHCl3): lmax (loge)=694 (3.37), 651 (3.63), 359 (3.64),
316 nm (3.66); MS (MALDI TOF): m/z: 2024.679 [M+H]+. For HPLC
purity, see the Supporting Information.
Light-dependent toxicity
One day before the experiment, the CAL 27 and HSC-3 cells were
seeded in 48-well plates at a density 1.8”105 cells per well, respec-
tively, in complete medium (1 mL), and used at approximately 80%
confluence. The cells were washed with PBS (1 mL). Next, the
medium (1 mL; without FBS and phenol red) that contained the
photosensitizer was added to each well except for the controls.
After washing, controls were treated with the same medium with-
out photosensitizers. The cells were incubated for 24 h at 378C,
washed with PBS, and irradiated for 20 min with light at a wave-
length of 690 nm from a High Power LED Multi Chip Emitter
(Roithner Lasertechnik, 9.8 V). The light intensity at the surface of
the plate was set to 3.0 mWcmÀ2 as measured by a Thorlabs
TM100A Optical Power Meter, and the total light dose was
3.6 JcmÀ2. One plate from each experiment was not exposed to
light and served as a control. Directly after light exposure, the
medium without FBS and phenol red was replaced with 1 mL of
complete medium, and the cells were incubated for 24 h at 378C.
Cell viability was quantified by the Alamar Blue assay.
Singlet-oxygen measurements and spectroscopic studies
The evaluation of singlet oxygen generation for the examined
compounds was performed in DMF according to the methodology
previously described.[35–37] 1,3-Diphenylisobenzofuran (DPBF) was
used as a singlet-oxygen chemical quencher (Sigma–Aldrich). Un-
substituted zincphthalocyanine (ZnPc) with known singlet-oxygen
quantum yields (0.56 and 0.67 in DMF and DMSO, respectively)
was used as a reference.
Biological studies
Cell culture
Cell viability
Oral squamous cell carcinoma cell lines derived from the tongue
(CAL 27, HSC-3) were purchased from ATCC (Teddington, UK) for
CAL 27 or provided by Dr. R. Kramer (University of California, San
Francisco, UCSF, USA) for HSC-3.[49] The cells were cultured in Dul-
becco’s modified Eagle medium (DMEM), supplemented with 10%
(v/v) heat-inactivated fetal bovine serum (FBS), penicillin
(100 UmLÀ1), streptomycin (100 mgmLÀ1), and l-glutamine (4 mm)
(DMEM/10). The cells were incubated in tissue culture flasks at
378C in a humidified atmosphere that contained 5% CO2, then
were passaged 1:6 (HSC-3) or 1:4 (CAL 27) twice a week using
a Trypsin-EDTA solution. All media—that is, penicillin-streptomycin
Cell morphology was evaluated using a Nikon TMS inverted-phase
contrast microscope at 100” magnification. The number of viable
cells used for the experiments was determined by the Trypan Blue
exclusion assay (Gibco-Invitrogen Corporation). The cell viability
was quantified by a modified Alamar Blue assay.[50,51] Briefly, 10%
(v/v) Alamar Blue dye (1 mL) in the appropriate complete medium
was added to each well. After incubation at 378C for 2–3 hours;
the supernatant (200 mL) was assayed by measuring the absorb-
ance at 570 and 600 nm. The cell viability (as a percentage of con-
trol cells) was calculated according to the formula [Eq. (1)]:
ChemPlusChem 2016, 81, 460 – 470
www.chempluschem.org
469
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This study was supported by the National Science Centre under
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Manuscript received: February 1, 2016
Accepted Article published: March 1, 2016
Final Article published: March 23, 2016
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