# Investigation of Zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) Porphyrazine for Application as Photosensitizer in Photodynamic Therapy of Cancer

Authors: Keiichi Sakamoto; Eiko Ohno-Okumura; Taku Kato; Masaki Watanabe; Michael J. Cook
Journal: Metal-Based Drugs
 (2008)

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## Abstract

The phthalocyanine analogue containing nonperipheral long alkyl-substituted benzenoid rings and pyridine rings, zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine, was synthesized.  Zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine reacted  with dimethyl sulfate and monochloroacetic acid to produce their quaternized products and  diethyl sulfate to produce the sulfo-substituted products. All quaternized and sulfo-substituted showed amphiphilic character. Identical peaks in cyclic voltammograms appeared for these products before and after quaternization. During  the evaluation of zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine for its photodynamic therapy of cancer (PDT) efficacy by cancer cell culture, the light exposed dimethyl sulfate quaternized zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines in IU-002 cells produce cell disruption that can be detected as a decrease in fluorescence.


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## Body

## 1. Introduction

Phthalocyanine derivatives have attracted attention as functional chromophores for
applications, especially organic charge carriers in photocopiers, as laser
light absorbers in data storage systems, as photoconductors in photovoltaic
cells, and in electrochromic displays [1–3]. Moreover, phthalocyanine derivatives can
be utilized as sensitizers in photodynamic therapy of cancer (PDT).Sensitizers for PDT require high photostability, high selectivity to
tumors, no dark cytotoxity, strong absorption in the region between 600 and 800 nm where penetration of tissue is good, a long triplet lifetime, and
satisfactory photosensitization of singlet oxygen. Phthalocyanine derivatives
are known to satisfy the aforementioned conditions [3–8].We previously synthesized the nonperipherally substituted phthalocyanine
derivatives, zinc alkylbenzopyridoporphyrazines, which possessed didecylbenzenoid and pyridinoid
moieties in the molecule and described regio isomer separation of one of the
alkylbenzopyridoporphyrazines [9]. We reported a fundamental study on PDT by
measuring for the triplet state lifetime of the alkylbenzopyridoporphyrazins
and regio isomers [10, 11]. As alkylbenzopyridoporphyrazine exhibited
solubility in organic solvents and was expected to have a higher tumor
affinity, quaternation of the pyridine nitrogen in the alkylbenzopyridoporphyrazine
was done to give solubility in an aqueous media, and to have bioavailability
and in vivo distribution [12]. Then, Nyokong et al. reported that
phthalocyanine analogues, tetra-2, 3-pyridoporphyrazine and its quaternized
compounds have excellent properties compared to zinc phthalocyanine-type
photosensitizer [13]. The amphiphilic phthalocyanine derivatives were concluded the best
compound for a new generation of photosensitizers for PDT [12]. In our previous
papers [9–12], the reported zinc bis(1,4-didecylbenzo)-bis(3,4-pyrido) porphyrazine
and its regio isomers were prepared by 1:1 mixture of 3,6-didecylphthalonitrile
and 3,4-pyridine dicarbonitrile.In the present study, another type, novel nonperipheral, substituted phthalocyanine
derivative, zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine was synthesized.In the case of related compounds, 2,3-py-rid-o-por-phy-ra-zi-nes are known to have not
only longer wavelength but stronger absorption intensity than corresponding
phthalocyanines and 3,4-pyridoporphyrazines [14]. In accordance with [14], it
is expected that zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine and its
quaternation compounds have stronger absorption intensities than that of zinc
bis(1,4-didecylbenzo)-bis(3,4-pyrido) porphyrazines reported before [9–12].
Therefore, the novel compound, zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine and its
quaternation compounds are expected to be excellent photosensitizer for PDT.


## 2. Results and Discussion

### 2.1. Synthesis and Quaternization of Phthalocyanine Derivative

The synthetic procedure used to prepare the
novel nonperipheral-substituted phthalocyanine derivative, zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine, was the same as that used
for the preparation of zinc bis(1,4-didecylbenzo)-bis(3,4-pyrido) porphyrazine
[9–12]. Zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine was synthesized in 80% yield
using equimolar amounts of 3,6-didecylphthalodinitrile and 2,3-pyridine
carbonitrile in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as basic catalyst (see Figure 1). The target
compound, zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine, and its
intermediates were studied using Fourier transformation infrared (IR), proton
nuclear magnetic resonance (1H-NMR), ultraviolet-visible
(UV-Vis) spectroscopy, and elemental analysis. The analytical data of the compound were
in good agreement with the proposed structure.Figure 1
Synthetic pathway of zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine.The synthesized zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine was anticipated to be a
mixture of products, with different numbers of pyridine rings in the molecule.
However, the target compound comprised only the proposed constituent as
confirmed by thin layer
chromatography (TLC). As the target compound had been purified by TLC using benzene as
eluent, only one blue-colored constituent was obtained. It is thought that the
desired compound was obtained in accordance with the mole ratio of the raw
materials used. The same phenomenon has been observed in the case of synthesis
of zinc bis(1,4-didecylbenzo)-bis(3,4-pyrido) porphyrazine [9–12].Zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido)
porphyrazine has two alkylbenzenoid and two pyridinoid rings in different
locations; thus, it has five regio isomers, three of which have rings adjacent
to the pyridinoido rings while the other two have opposed pyridinoid rings.
Although we previously reported the separation of regio isomers in
alkylbenzopyridoporphyrazine [9–12], no attempt
was made in this work to isolate the isomers of zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine.  Of course, the obtained blue-colored
constituent will be further
separated into five regio isomers
by using toluene-pyridine 7:3 eluent in accordance with [9–12].The zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine reacted with quaternizing
agents such as monochloroacetic acid (MCAA), diethyl sulfate (DES), and
dimethyl sulfate (DMS) inN,N-dimethylformamide (DMF) as a
solvent at 140°C for 2 hours.The respective products obtained were greenish-blue-colored
powders of which the yields
were 24, 21, and 25% for MCAA, DES, and DMS, respectively (see Figure3). Zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido)
porphyrazine was dissolved in toluene, chloroform, pyridine, and methanol but
not in water. After reacting with quaternizing agents, the products were also
soluble in water.In the cases of MCAA and DMS, analysis
revealed that the structures of the products were in good agreement with those
havingN-CH2COOH and N-CH3 groups, respectively.
Whereas when DES was used as a quaternizing agent, no  N-CH2CH3 singlet peak was present in the 1H-NMR spectrum, S=O stretching in the IR spectrum was observed.
Therefore, sulfonation but not quaternization was achieved  [12, 15].After reaction with the quaternizing agents, all compounds possessed amphiphilicproperties.


### 2.2. Spectroscopic and Electrochemical Properties

The UV-Vis spectrum of zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) por-phy-ra-zi-nes around 700 nm is
characteristic of phthalocyanine analogues, with the Q band attributable to the
difference between the highest occupied molecular orbital (HOMO) energy level
and the lowest unoccupied molecular orbital (LUMO) energy, that is, theπ-π* transition of the phthalocyanine ring.The quarternized derivatives of zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) por-phy-ra-zi-nes showed strongest absorption
at 676, 687, and 687 nm in water after reaction with DMS, DES, and MCAA,
respectively (Table1); these Q bands were bathochromic compared to the nonquaternized
parent compound. As the UV-Vis spectra of the quaternized compounds in water
showed very broad peaks, the amphiphilic compounds had excellent molecular
association tendency.Table 1
UV-Vis and fluorescence spectral data of quaternized zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine.
CompoundQ-bandFluorescenceλmax pyridine/nmλmax water/nmFmax pyridine/nmFmax water/nm1687—694—2665———21679, 6501687, 6476926881,2677, 262031693, 658, 628, 597708,1687, 6526986912673, 2645, 26054746,1673, 649, 606723,1676, 6466836882738, 1,2668, 2641, 26001Main peak.2In toluene.Zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine
and its quaternized compounds fluoresced on exposure to ultraviolet light. Although fluorescence
spectra generally were known to be mirror images of UV-Vis spectra at the longer
wavelengths, the Q bands nearly overlapped with the wavelengths at which fluorescence
occurs in the case of zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine
and its quaternized compounds, thus,  the
differences betweenλmax⁡ of UV-Vis and
the Fmax⁡ of fluorescence spectra, called the Storkes shift, were very small between 10 to 20 nm. These observations
are similar to that seen with the phthalocyanines zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido)
porphyrazine and its quaternized derivatives. These compounds are molecules
with high planarity which cannot change their configuration after quaternization.The important parameters of a cyclic
voltammetry (CV) are the reduction (Epc) and oxidation (Epa)
potential, the difference between reduction and oxidation potentials, (ΔEp), and formal reduction potential
(E°′) of the observed waves. [Note: Emid=E°′] (Table 2 and see Figure 3).Table 2
Potentials of quaternized zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine in DMF with
tetrabutylammonium perchlorate.Potentials of reversible wave are midpoint potential of anodic to cathodic peaks for each couple.
Potential (V versus Ag/AgCl)CompoundReduction (Epc)Oxidation (Epa)1–0.94*–0.62*–0.29*–0.03*0.32*0.48*0.97*ΔE**2–0.75*–0.52*–0.12*0.96*1.28*ΔE**3–0.83–0.51*–0.05*0.25*1.05*ΔE**0.144–1.15*–0.77–0.14*–0.05*0.50*ΔE**0.11*Irreversible peak.**The anodic peak to cathodic peak separation for reversible couple.The potential difference in CVs between the
reduction and oxidation correspond to the HOMO-LUMO energy gaps of the compound
[16]. Just as chemical reactions occur during the electron transfer between
HOMO and LUMO energy levels, photochemical reactions are also based on similar
phenomena of energy transfer. Before and after the quaternization, the HOMO-LUMO
energy gap of zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine was
unchanged. The shapes of CVs clearly showed that quaternized zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines had increased electron
responsibility.


### 2.3. Cancer Cell Study

The uptake of DMS quaternized zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines was done in IU-002 cells. IU-002 cells were
incubated at 37°C. After incubation for 3 hours, cellular quaternized zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines was observed with a fluorescence microscope.A fluorescent substance was
noted when the uptake of DMS quaternized
zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines in IU-002 cells was
carried out.Cell rupture can
be detected. Intact cells selectively concentrated fluorescence. After exposure to halogen light for 10 minutes
showed damage and loss of fluorescence although fluorescence in cells occurred in
perinuclear area (see Figure 4).Concequently, the light exposed DMS
quaternized zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines in cells
produces cell disruption that can be detected as a decrease as
fluorescence.


### 2.4. Conclusions

Zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines were synthesized from an
equimolar mixture of 3,6-didecylphthalonitrile and 2,3-pyridine carbonitrile in
the presence of basic catalyst.Zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines having two pyridine and two
alkyl-substituted benzene rings reacted with DMS, DES, and MCAA as quaternizing agents.When MCAA and DMS were employed as
quaternizing agents, zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines
were changed to their quaternized derivatives. However, when DES was employed,
we showed that sulfonation but not quaternization was achieved.Electrochmeical characterization of zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine and its quaternized
derivatives were estimated by CV technique. The shapes of CVs clearly showed that quaternized
zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines had increased electron
responsibility.The uptake of DMS-quaternized zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines was done in IU-002 cells.The light-exposed DMS quaternized
zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines in cells produces cell
disruption that can be detected as a decrease in fluorescence.


## 2.1. Synthesis and Quaternization of Phthalocyanine Derivative

The synthetic procedure used to prepare the
novel nonperipheral-substituted phthalocyanine derivative, zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine, was the same as that used
for the preparation of zinc bis(1,4-didecylbenzo)-bis(3,4-pyrido) porphyrazine
[9–12]. Zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine was synthesized in 80% yield
using equimolar amounts of 3,6-didecylphthalodinitrile and 2,3-pyridine
carbonitrile in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as basic catalyst (see Figure 1). The target
compound, zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine, and its
intermediates were studied using Fourier transformation infrared (IR), proton
nuclear magnetic resonance (1H-NMR), ultraviolet-visible
(UV-Vis) spectroscopy, and elemental analysis. The analytical data of the compound were
in good agreement with the proposed structure.Figure 1
Synthetic pathway of zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine.The synthesized zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine was anticipated to be a
mixture of products, with different numbers of pyridine rings in the molecule.
However, the target compound comprised only the proposed constituent as
confirmed by thin layer
chromatography (TLC). As the target compound had been purified by TLC using benzene as
eluent, only one blue-colored constituent was obtained. It is thought that the
desired compound was obtained in accordance with the mole ratio of the raw
materials used. The same phenomenon has been observed in the case of synthesis
of zinc bis(1,4-didecylbenzo)-bis(3,4-pyrido) porphyrazine [9–12].Zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido)
porphyrazine has two alkylbenzenoid and two pyridinoid rings in different
locations; thus, it has five regio isomers, three of which have rings adjacent
to the pyridinoido rings while the other two have opposed pyridinoid rings.
Although we previously reported the separation of regio isomers in
alkylbenzopyridoporphyrazine [9–12], no attempt
was made in this work to isolate the isomers of zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine.  Of course, the obtained blue-colored
constituent will be further
separated into five regio isomers
by using toluene-pyridine 7:3 eluent in accordance with [9–12].The zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine reacted with quaternizing
agents such as monochloroacetic acid (MCAA), diethyl sulfate (DES), and
dimethyl sulfate (DMS) inN,N-dimethylformamide (DMF) as a
solvent at 140°C for 2 hours.The respective products obtained were greenish-blue-colored
powders of which the yields
were 24, 21, and 25% for MCAA, DES, and DMS, respectively (see Figure3). Zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido)
porphyrazine was dissolved in toluene, chloroform, pyridine, and methanol but
not in water. After reacting with quaternizing agents, the products were also
soluble in water.In the cases of MCAA and DMS, analysis
revealed that the structures of the products were in good agreement with those
havingN-CH2COOH and N-CH3 groups, respectively.
Whereas when DES was used as a quaternizing agent, no  N-CH2CH3 singlet peak was present in the 1H-NMR spectrum, S=O stretching in the IR spectrum was observed.
Therefore, sulfonation but not quaternization was achieved  [12, 15].After reaction with the quaternizing agents, all compounds possessed amphiphilicproperties.


## 2.2. Spectroscopic and Electrochemical Properties

The UV-Vis spectrum of zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) por-phy-ra-zi-nes around 700 nm is
characteristic of phthalocyanine analogues, with the Q band attributable to the
difference between the highest occupied molecular orbital (HOMO) energy level
and the lowest unoccupied molecular orbital (LUMO) energy, that is, theπ-π* transition of the phthalocyanine ring.The quarternized derivatives of zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) por-phy-ra-zi-nes showed strongest absorption
at 676, 687, and 687 nm in water after reaction with DMS, DES, and MCAA,
respectively (Table1); these Q bands were bathochromic compared to the nonquaternized
parent compound. As the UV-Vis spectra of the quaternized compounds in water
showed very broad peaks, the amphiphilic compounds had excellent molecular
association tendency.Table 1
UV-Vis and fluorescence spectral data of quaternized zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine.
CompoundQ-bandFluorescenceλmax pyridine/nmλmax water/nmFmax pyridine/nmFmax water/nm1687—694—2665———21679, 6501687, 6476926881,2677, 262031693, 658, 628, 597708,1687, 6526986912673, 2645, 26054746,1673, 649, 606723,1676, 6466836882738, 1,2668, 2641, 26001Main peak.2In toluene.Zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine
and its quaternized compounds fluoresced on exposure to ultraviolet light. Although fluorescence
spectra generally were known to be mirror images of UV-Vis spectra at the longer
wavelengths, the Q bands nearly overlapped with the wavelengths at which fluorescence
occurs in the case of zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine
and its quaternized compounds, thus,  the
differences betweenλmax⁡ of UV-Vis and
the Fmax⁡ of fluorescence spectra, called the Storkes shift, were very small between 10 to 20 nm. These observations
are similar to that seen with the phthalocyanines zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido)
porphyrazine and its quaternized derivatives. These compounds are molecules
with high planarity which cannot change their configuration after quaternization.The important parameters of a cyclic
voltammetry (CV) are the reduction (Epc) and oxidation (Epa)
potential, the difference between reduction and oxidation potentials, (ΔEp), and formal reduction potential
(E°′) of the observed waves. [Note: Emid=E°′] (Table 2 and see Figure 3).Table 2
Potentials of quaternized zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine in DMF with
tetrabutylammonium perchlorate.Potentials of reversible wave are midpoint potential of anodic to cathodic peaks for each couple.
Potential (V versus Ag/AgCl)CompoundReduction (Epc)Oxidation (Epa)1–0.94*–0.62*–0.29*–0.03*0.32*0.48*0.97*ΔE**2–0.75*–0.52*–0.12*0.96*1.28*ΔE**3–0.83–0.51*–0.05*0.25*1.05*ΔE**0.144–1.15*–0.77–0.14*–0.05*0.50*ΔE**0.11*Irreversible peak.**The anodic peak to cathodic peak separation for reversible couple.The potential difference in CVs between the
reduction and oxidation correspond to the HOMO-LUMO energy gaps of the compound
[16]. Just as chemical reactions occur during the electron transfer between
HOMO and LUMO energy levels, photochemical reactions are also based on similar
phenomena of energy transfer. Before and after the quaternization, the HOMO-LUMO
energy gap of zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine was
unchanged. The shapes of CVs clearly showed that quaternized zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines had increased electron
responsibility.


## 2.3. Cancer Cell Study

The uptake of DMS quaternized zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines was done in IU-002 cells. IU-002 cells were
incubated at 37°C. After incubation for 3 hours, cellular quaternized zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines was observed with a fluorescence microscope.A fluorescent substance was
noted when the uptake of DMS quaternized
zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines in IU-002 cells was
carried out.Cell rupture can
be detected. Intact cells selectively concentrated fluorescence. After exposure to halogen light for 10 minutes
showed damage and loss of fluorescence although fluorescence in cells occurred in
perinuclear area (see Figure 4).Concequently, the light exposed DMS
quaternized zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines in cells
produces cell disruption that can be detected as a decrease as
fluorescence.


## 2.4. Conclusions

Zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines were synthesized from an
equimolar mixture of 3,6-didecylphthalonitrile and 2,3-pyridine carbonitrile in
the presence of basic catalyst.Zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines having two pyridine and two
alkyl-substituted benzene rings reacted with DMS, DES, and MCAA as quaternizing agents.When MCAA and DMS were employed as
quaternizing agents, zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines
were changed to their quaternized derivatives. However, when DES was employed,
we showed that sulfonation but not quaternization was achieved.Electrochmeical characterization of zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine and its quaternized
derivatives were estimated by CV technique. The shapes of CVs clearly showed that quaternized
zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines had increased electron
responsibility.The uptake of DMS-quaternized zinc
bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines was done in IU-002 cells.The light-exposed DMS quaternized
zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines in cells produces cell
disruption that can be detected as a decrease in fluorescence.


## 3. Experimental

### 3.1. Equipment

IR spectra were recorded on a Shimadzu FT-IR
8100A spectrometer using potassium bromide (KBr) pellets. UV-Vis spectra were
measured on a Shimadzu UV-2400PC spectrometer; each sample was prepared at5.0×10−5 mol dm−3 in pyridine, toluene, and water. Fluorescence spectra were
recorded in toluene, pyridine, and water using either a Hitachi F-4500
fluorescence spectrometer or a Jasco (Nihon Bunko) FP-6600 spectrofluorometer. 1H-NMR
spectra were measured at 400 MHz on a Bruker Avance 400S in benzene-d6 (C6H6-d6) or chloroform-d (CHCl3-d)
using tetramethylsilane (TMS) as an internal standard. Elemental analyses were
carried out using a Perkin-Elmer 2400CHN instrument. Samples for elemental
analysis were purified by repeated sublimation; the instrument was calibrated
with copper phthalocyanine. CVs were recorded on a BAS CV-50 W voltammetric
analyzer at room temperature in 1×10−3 mol dm−3 acetonitrile
solution containing a 0.01 mol dm−3 tetrabutylammonium perchlorate (TBAP).
CVs were recorded by scanning the potential at a rate of 50 mV s−1.
The working and counter electrodes were platinum wires and the reference
electrode was a silver/silver chloride- (Ag/AgCl) saturated sodium chloride
electrode. The area of the working electrode was 2.0×10−2 cm2.


### 3.2. Materials

TLC was performed using Merck 60 F254silica gel on aluminium sheets. Merck Silica
gel 60, particle size 0.063–0.200 nm 7734 grade was used in chromatographic
separations.Reagents were purchased from Sigma-Aldrich Chemicals (Miss, USA) and
were used as received without further purification.3,6-Didecylphthalonitrile was synthesized from thiophene via
2,5-Didecylthiophene and 2,5-Didecylthiophene-1,1-dioxide, in accordance with
our previous reports [9–12]. 1H NMR (δ 400 MHz, CHCl3-d/
ppm) 0.88 (t, 6H), 1.26 (m, 32H), 2.85 (t, 4H), 7.46 (s, 2H); IR(ν KBr/cm−1) 2960 (νC–H), 2240 (νC≡N), 1560 (νC=C), 1460 (νC–C), 1410 (νC–C), 1230 (δC–H), 730 (δC–H); Anal Calcd. for C28H4N2:
C. 82.69: H. 10.85: N.6.86. Found: C.82.26: H. 10.84: N. 6.84.2,3-Pyridine dicarbonitrile was synthesized from
pyridine-2,3-dicarboxylic acid. Pyridine-2,3-dicarboxylic acid (15 g, 0.09 mol)
in 200 cm3 of ethanol was refluxed in 7.5 cm3 of
concentrated sulfonic acid for 48 hours. After the solvent was removed and
neutralized with 3 M NaOH solution, the organic layer was extracted with three
times of 75 cm3 of diethyl ether. The extract was dried on calcium
chloride and filtered and the solvent evaporated. The residue was distilled to
afford pyridine 2,3-dicarboxy ester. The ester was dissolved in concentrated
aqueous ammonia and the solution was stirred for 48 hours to afford a white-colored
precipitate. The precipitate was filtered off and the corresponding diamide was
obtained as m.p. 179–181°C. Thionyl chloride (9.6 g, 0.08 mol) was added
dropwise to the diamide (6.6 g, 0.04 mmol) in 56 g of DMF at −10°C. This mixture was allowed to warm to room
temperature and was stirred for 24 hours. The solvent was removed by
evaporation and water was added to make the thionyl chloride ineffective and
then filtered. The product was purified by chromatography over silica gel with
benzene-petroleum ether 1:1 as eluent, giving a colorless solid (1.97 g, 17%),
m.p. 265°C.1H NMR (δ 400 MHz, CHCl3-d/ppm) 7.26 (s, 1H),
7.75 (s, 1H), 9.09 (s, 1H); IR(ν KBr/cm−1)
3090 (νC–H), 2240 (νC–N), 1600 (νC–C), 1550 (νC–C), 1470 (νC–C), 1220 (δC–H), 750 (δC–H); Anal Calcd.
for C7H3N3: C. 65.11: H. 2.34: N.32.55. Found:
C.65.12: H. 2.34: N. 32.56.


### 3.3. Zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) Porphyrazine

3,6-Didecylphthalonitrile (0.12 g, 0.29 mmol)
and 2,3-dicyanopyridine (0.04 g, 0.29 mmol) (see Figure1) were dissolved in
pentanol (7 cm3) and zinc chloride (0.05 g) was added; the ensuing
mixture was heated for 4 hours in the presence of DBU as catalyst. After
cooling, the reaction mixture was dissolved in toluene (50 cm3) and
filtered; the solvent was removed by evaporation. The product was purified by
TLC (eluent : toluene) yielding a blue solid (0.13 g; yield 80%). 1H NMR (δ 400 MHz, C6H6-d6/ppm) 0.9 (m, 12H, CH3), 1.61–2.61 (m, 64H, CH2),
4.18–4.36 (m, 8H, α-CH2),
7.45 (m, 4H, arom), 8.26 (m, 6H, Py);
IR(ν KBr/cm−1) 2960 (νC–H), 1600 (νC–C), 1500 (νC–C), 1420 (νC–C), 1200 (δC–H), 1100 (δC–H), 750 (δC–H); UV-Vis [λmax toluene/nm
(log εmax)] 665 (5.494);
Anal Calcd. for C70H94N10Zn: C. 73.68: H.
8.30: N.12.28. Found: C.73.67: H. 8.30: N. 12.28.Typical structures of
quaternized zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines.
(a)(b)(c)CVs of zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine.
(a)(b)(c)(d)Fluorescence image of IU-002 cells. (a) Control and (b) Incubated with DMS
quaternized zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido)porphyrazine and
irradiated with halogen light for 10 minutes.
(a)(b)


### 3.4. Quaternization of Zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) Porphyrazine

Zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido)
porphyrazine (0.17 g, 0.15 mmol) reacted with MCAA (0.57 g, 6 mmol), DES (0.1 g,
0.6 mmol) and DMS (0.2 g, 1.5 mmol), respectively, inN,N-dimetylformamide (DMF) 140°C for 2 hours (see Figures 2
and 3). The reaction mixture was dissolved in acetone (20 cm3), cooled
to room temperature, and the resulting solution was filtered. The solvent was
removed and the product was purified by TLC (eluent : THF-toluene, 8:2); the
product was recovered from the TLC plate via dissolution in pyridine followed
by filtration and solvent removal.
MCAA:Yield 25%, 1H NMR (δ 400 MHz, C6H6-d6/
ppm) 0.87(m, 12H, CH3), 1.13–1.70(m, 56H, γ-CH2), 1.82–2.61(m, 8H, β-CH2), 4.11–4.38(m, 4H, α-CH2), 6.20(s, 2H, CH2),
7.14–7.27(m, 4H, Arom), 8.73–.16(m, 6H, Py): IR(ν KBr/cm−1) 3480(νO–H), 3050, 2970(νC–H), 1740(νC=O), 1600, 1500, 1400(νC=C), 1210, 1100, 940, 790,
690(δC–H); DES yield 21%,
(δ 400 MHz, C6H6-d6/ppm) 0.86(m, 12H, CH3), 1.02–1.63(m, 56H, γ-CH2), 1.88–2.61(m, 8H, β-CH2), 4.26–4.50(m, 4H, α-CH2), 7.37(m, 4H, Arom),
8.22(m, 4H, Py):
IR(ν KBr/cm−1)
3480(νO–H), 3050, 2960(νC–H), 1600, 1460, 1400(νC=C), 1350, 1150(νS=O), 1250, 920, 770(δC–H), 580(δC−S); DMS yield 25%,   (δ 400 MHz, C6H6-d6/ppm) 0.90(m, 12H,
CH3), 0.95–1.45(m, 56H, γ-CH2), 1.60–2.41(m, 8H, β-CH2), 4.05(s, 6H, CH3), 4.25–4.42(m, 4H, α-CH2), 7.45(m, 4H, Arom),
8.02(m, 6H, Py): IR(ν KBr/cm−1)
3070, 2980(νC–H), 1500, 1400(νC=C), 1250, 1100, 950, 810,
660(δC–H).


### 3.5. Cell Culture

IU-002 cells were maintained in MEM medium supplemented 5% fetal calf
serum.Cells seeded into 96-well tissue culture plates and incubated to allow
attachment to the plates. The
sensitizer was added to the medium at concentration ranging from 0 to 2 mgcm3. Cells were
incubated for 3 hours. The medium was removed, the cells were washed with
phosphate-buffered saline (PBS), and fresh medium was added.  Cells were exposed halogen light for 10
minutes. Appearance of cells was observed used a fluorescence microscope.


## 3.1. Equipment

IR spectra were recorded on a Shimadzu FT-IR
8100A spectrometer using potassium bromide (KBr) pellets. UV-Vis spectra were
measured on a Shimadzu UV-2400PC spectrometer; each sample was prepared at5.0×10−5 mol dm−3 in pyridine, toluene, and water. Fluorescence spectra were
recorded in toluene, pyridine, and water using either a Hitachi F-4500
fluorescence spectrometer or a Jasco (Nihon Bunko) FP-6600 spectrofluorometer. 1H-NMR
spectra were measured at 400 MHz on a Bruker Avance 400S in benzene-d6 (C6H6-d6) or chloroform-d (CHCl3-d)
using tetramethylsilane (TMS) as an internal standard. Elemental analyses were
carried out using a Perkin-Elmer 2400CHN instrument. Samples for elemental
analysis were purified by repeated sublimation; the instrument was calibrated
with copper phthalocyanine. CVs were recorded on a BAS CV-50 W voltammetric
analyzer at room temperature in 1×10−3 mol dm−3 acetonitrile
solution containing a 0.01 mol dm−3 tetrabutylammonium perchlorate (TBAP).
CVs were recorded by scanning the potential at a rate of 50 mV s−1.
The working and counter electrodes were platinum wires and the reference
electrode was a silver/silver chloride- (Ag/AgCl) saturated sodium chloride
electrode. The area of the working electrode was 2.0×10−2 cm2.


## 3.2. Materials

TLC was performed using Merck 60 F254silica gel on aluminium sheets. Merck Silica
gel 60, particle size 0.063–0.200 nm 7734 grade was used in chromatographic
separations.Reagents were purchased from Sigma-Aldrich Chemicals (Miss, USA) and
were used as received without further purification.3,6-Didecylphthalonitrile was synthesized from thiophene via
2,5-Didecylthiophene and 2,5-Didecylthiophene-1,1-dioxide, in accordance with
our previous reports [9–12]. 1H NMR (δ 400 MHz, CHCl3-d/
ppm) 0.88 (t, 6H), 1.26 (m, 32H), 2.85 (t, 4H), 7.46 (s, 2H); IR(ν KBr/cm−1) 2960 (νC–H), 2240 (νC≡N), 1560 (νC=C), 1460 (νC–C), 1410 (νC–C), 1230 (δC–H), 730 (δC–H); Anal Calcd. for C28H4N2:
C. 82.69: H. 10.85: N.6.86. Found: C.82.26: H. 10.84: N. 6.84.2,3-Pyridine dicarbonitrile was synthesized from
pyridine-2,3-dicarboxylic acid. Pyridine-2,3-dicarboxylic acid (15 g, 0.09 mol)
in 200 cm3 of ethanol was refluxed in 7.5 cm3 of
concentrated sulfonic acid for 48 hours. After the solvent was removed and
neutralized with 3 M NaOH solution, the organic layer was extracted with three
times of 75 cm3 of diethyl ether. The extract was dried on calcium
chloride and filtered and the solvent evaporated. The residue was distilled to
afford pyridine 2,3-dicarboxy ester. The ester was dissolved in concentrated
aqueous ammonia and the solution was stirred for 48 hours to afford a white-colored
precipitate. The precipitate was filtered off and the corresponding diamide was
obtained as m.p. 179–181°C. Thionyl chloride (9.6 g, 0.08 mol) was added
dropwise to the diamide (6.6 g, 0.04 mmol) in 56 g of DMF at −10°C. This mixture was allowed to warm to room
temperature and was stirred for 24 hours. The solvent was removed by
evaporation and water was added to make the thionyl chloride ineffective and
then filtered. The product was purified by chromatography over silica gel with
benzene-petroleum ether 1:1 as eluent, giving a colorless solid (1.97 g, 17%),
m.p. 265°C.1H NMR (δ 400 MHz, CHCl3-d/ppm) 7.26 (s, 1H),
7.75 (s, 1H), 9.09 (s, 1H); IR(ν KBr/cm−1)
3090 (νC–H), 2240 (νC–N), 1600 (νC–C), 1550 (νC–C), 1470 (νC–C), 1220 (δC–H), 750 (δC–H); Anal Calcd.
for C7H3N3: C. 65.11: H. 2.34: N.32.55. Found:
C.65.12: H. 2.34: N. 32.56.


## 3.3. Zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) Porphyrazine

3,6-Didecylphthalonitrile (0.12 g, 0.29 mmol)
and 2,3-dicyanopyridine (0.04 g, 0.29 mmol) (see Figure1) were dissolved in
pentanol (7 cm3) and zinc chloride (0.05 g) was added; the ensuing
mixture was heated for 4 hours in the presence of DBU as catalyst. After
cooling, the reaction mixture was dissolved in toluene (50 cm3) and
filtered; the solvent was removed by evaporation. The product was purified by
TLC (eluent : toluene) yielding a blue solid (0.13 g; yield 80%). 1H NMR (δ 400 MHz, C6H6-d6/ppm) 0.9 (m, 12H, CH3), 1.61–2.61 (m, 64H, CH2),
4.18–4.36 (m, 8H, α-CH2),
7.45 (m, 4H, arom), 8.26 (m, 6H, Py);
IR(ν KBr/cm−1) 2960 (νC–H), 1600 (νC–C), 1500 (νC–C), 1420 (νC–C), 1200 (δC–H), 1100 (δC–H), 750 (δC–H); UV-Vis [λmax toluene/nm
(log εmax)] 665 (5.494);
Anal Calcd. for C70H94N10Zn: C. 73.68: H.
8.30: N.12.28. Found: C.73.67: H. 8.30: N. 12.28.Typical structures of
quaternized zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines.
(a)(b)(c)CVs of zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine.
(a)(b)(c)(d)Fluorescence image of IU-002 cells. (a) Control and (b) Incubated with DMS
quaternized zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido)porphyrazine and
irradiated with halogen light for 10 minutes.
(a)(b)


## 3.4. Quaternization of Zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) Porphyrazine

Zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido)
porphyrazine (0.17 g, 0.15 mmol) reacted with MCAA (0.57 g, 6 mmol), DES (0.1 g,
0.6 mmol) and DMS (0.2 g, 1.5 mmol), respectively, inN,N-dimetylformamide (DMF) 140°C for 2 hours (see Figures 2
and 3). The reaction mixture was dissolved in acetone (20 cm3), cooled
to room temperature, and the resulting solution was filtered. The solvent was
removed and the product was purified by TLC (eluent : THF-toluene, 8:2); the
product was recovered from the TLC plate via dissolution in pyridine followed
by filtration and solvent removal.
MCAA:Yield 25%, 1H NMR (δ 400 MHz, C6H6-d6/
ppm) 0.87(m, 12H, CH3), 1.13–1.70(m, 56H, γ-CH2), 1.82–2.61(m, 8H, β-CH2), 4.11–4.38(m, 4H, α-CH2), 6.20(s, 2H, CH2),
7.14–7.27(m, 4H, Arom), 8.73–.16(m, 6H, Py): IR(ν KBr/cm−1) 3480(νO–H), 3050, 2970(νC–H), 1740(νC=O), 1600, 1500, 1400(νC=C), 1210, 1100, 940, 790,
690(δC–H); DES yield 21%,
(δ 400 MHz, C6H6-d6/ppm) 0.86(m, 12H, CH3), 1.02–1.63(m, 56H, γ-CH2), 1.88–2.61(m, 8H, β-CH2), 4.26–4.50(m, 4H, α-CH2), 7.37(m, 4H, Arom),
8.22(m, 4H, Py):
IR(ν KBr/cm−1)
3480(νO–H), 3050, 2960(νC–H), 1600, 1460, 1400(νC=C), 1350, 1150(νS=O), 1250, 920, 770(δC–H), 580(δC−S); DMS yield 25%,   (δ 400 MHz, C6H6-d6/ppm) 0.90(m, 12H,
CH3), 0.95–1.45(m, 56H, γ-CH2), 1.60–2.41(m, 8H, β-CH2), 4.05(s, 6H, CH3), 4.25–4.42(m, 4H, α-CH2), 7.45(m, 4H, Arom),
8.02(m, 6H, Py): IR(ν KBr/cm−1)
3070, 2980(νC–H), 1500, 1400(νC=C), 1250, 1100, 950, 810,
660(δC–H).


## 3.5. Cell Culture

IU-002 cells were maintained in MEM medium supplemented 5% fetal calf
serum.Cells seeded into 96-well tissue culture plates and incubated to allow
attachment to the plates. The
sensitizer was added to the medium at concentration ranging from 0 to 2 mgcm3. Cells were
incubated for 3 hours. The medium was removed, the cells were washed with
phosphate-buffered saline (PBS), and fresh medium was added.  Cells were exposed halogen light for 10
minutes. Appearance of cells was observed used a fluorescence microscope.



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*Source: 392090-2008-02-26.xml*