# Synthesis, Characterization, and In Vitro Photodynamic Activity of Novel Amphiphilic Zinc(II) Phthalocyanines  Bearing Oxyethylene-Rich Substituents

Authors: Jian-Yong Liu; Xiong-Jie Jiang; Wing-Ping Fong; Dennis K. P. Ng
Journal: Metal-Based Drugs
 (2008)

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## Abstract

Three novel zinc(II) phthalocyanines substituted with one or two 3,4,5-tris(3,6,9-trioxadecoxy)benzoxy group(s) have been prepared and spectroscopically characterized.  These compounds are highly soluble and remain nonaggregated inN,N-dimethylformamide.  Upon excitation, they exhibit a relatively weak fluorescence emission and high efficiency to generate singlet oxygen compared with the unsubstituted zinc(II) phthalocyanine.  These amphiphilic photosensitizers formulated with Cremophor EL are highly photocytotoxic against HT29 human colon adenocarcinoma and HepG2 human hepatocarcinoma cells. The mono-α-substituted analogue 4 is particularly potent with IC50 values as low as 0.02 μM.
The higher photodynamic activity of this compound can be attributed to its lower aggregation tendency in the culture media as shown by absorption spectroscopy and higher cellular uptake as suggested by the stronger intracellular fluorescence, resulting in a higher efficiency to generate reactive oxygen species inside the cells.


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## Body

## 1. Introduction

Photodynamic therapy (PDT) has emerged as a promising
modality for the treatment of malignant tumors and wet age-related macular
degeneration [1–3]. It is a binary therapy that involves the combination of visible light
and a photosensitizer. Each component is
harmless by itself, but in combination with molecular oxygen, they result in
the generation of reactive oxygen species (ROS) causing oxidative cellular and
tissue damage. This treatment has
several potential advantages including its minimally invasive nature, tolerance
of repeated doses, and high specificity that can be achieved through precise
application of the light with modern fiber-optic systems and various types of
endoscopy [3]. Currently, only a few
porphyrin derivatives including porfimer sodium, temoporfin, and verteporfin
are clinically approved for systemic administration. These compounds, though giving a positive
response in a high percentage of patients, still have various deficiencies that
demand a further development of better candidates [4]. Owing to the desirable electronic absorption
and photophysical properties, phthalocyanines are one of the most promising
classes of candidates for this application [5]. Over the last few years, we have been interested in rational
modification of this class of functional dyes with the goal of enhancing their
PDT efficiency. Several new series of silicon(IV) and zinc(II) phthalocyanines have been synthesized
and evaluated for their photo-physical and biological properties; see [6] and the references. As the amphiphilicity of photosensitizers is
believed to have a beneficial effect on their photodynamic activity [7],
amphiphilic phthalocyanines have been our targets. In this paper, we report the synthesis, photophysical properties, and in vitro photodynamic
activity of three novel zinc(II)
phthalocyanines bearing one or two 3,4,5-tris(3,6,9-trioxadecoxy)benzoxy
substituent(s). Having three or six triethylene glycol chains on one side
of the macrocycle, these compounds are amphiphilic in nature, exhibiting a high in vitro photocytotoxicity.


## 2. Materials and Methods

### 2.1. General

All the reactions were performed under an atmosphere of
nitrogen. Tetrahydrofuran (THF),n-pentanol, dichloromethane, and N,N-dimethylformamide
(DMF) were distilled from sodium benzophenone ketyl, sodium, calcium
hydride, and barium oxide, respectively. Chromatographic purifications were performed on silica gel (Macherey-Nagel, 70–230 mesh) columns with the
indicated eluents. Size exclusion chromatography
was carried out on Bio-Rad Bio-Beads S-X1 beads (200–400 mesh). All other solvents and reagents were of
reagent grade and used as received. Compounds 1 and 6 were prepared as described
[8].1H and 13C{1H} NMR spectra were
recorded on a Bruker DPX 300 spectrometer (1H, 300; 13C,
75.4 MHz) in CDCl3 or DMSO-d6. Spectra were referenced internally using the
residual solvent [1H: CDCl3 (δ 7.26); DMSO-d6 (δ
2.50)] or solvent [13C: CDCl3 (δ 77.0);
DMSO-d6 (δ 39.7)] resonances relative to SiMe4. Electrospray ionization (ESI) mass spectra
were measured on a Thermo Finnigan MAT 95 XL mass spectrometer.UV-Vis and
steady-state fluorescence spectra were taken on a Cary 5G UV-Vis-NIR
spectrophotometer and a Hitachi F-4500 spectrofluorometer, respectively. Fluorescence quantum yields(ΦF) were determined by the equation: ΦF(sample)=(Fsample/Fref)(Aref/Asample)(nsample2/nref2)ΦF(ref) [9], where F,A, and n are the measured
fluorescence (area under the emission peak), the absorbance at the excitation
position (610 nm), and the refractive index of the solvent, respectively. The unsubstituted zinc(II) phthalocyanine (ZnPc)
in DMF was used as the reference [ΦF(ref)=0.28] [10]. To minimize reabsorption of radiation by the ground-state species, the
emission spectra were obtained in very dilute solutions where the absorbance at
610 nm was less than 0.03. Singlet oxygen
quantum yields (ΦΔ) were measured in DMF by
the method of chemical quenching of 1,3-diphenylisobenzofuran (DPBF) using ZnPc
as reference (ΦΔ=0.56) [11].


### 2.2. Syntheses

#### 2.2.1. Preparation of 3-[3,4,5-tris(3,6,9-Trioxadecoxy)Benzoxy]Phthalonitrile (2)

To a mixture of 3-nitrophthalonitrile
(1.73 g, 10 mmol) and compound1 (2.97 g, 5 mmol) in DMF (30 mL) was added anhydrous K2CO3 (6.90 g, 50 mmol). The resulting mixture was
stirred at 80°C for 4 days. The solvent was then evaporated under reduced pressure and the residue
was mixed with CHCl3 (60 mL) and water (60 mL). The aqueous layer was separated and extracted
with CHCl3 (60 mL ×3). The
combined organic layers was dried over anhydrous MgSO4, then
evaporated to dryness. The residue was
purified by silica-gel column chromatography using CHCl3/MeOH (60 : 1 v/v) as eluent to give compound 2 as
a colorless liquid (1.41 g, 39%). 1H
NMR: δ 7.61 (vt, J = 8.7 Hz, 1 H, ArH), 7.37 (d, J = 7.5 Hz, 1 H, ArH), 7.25 (d, J =
7.5 Hz, 1 H, ArH), 6.66 (s, 2 H, ArH), 5.17 (s, 2 H, ArCH2), 4.13–4.18
(m, 6 H, CH2),  3.85 (t, J = 4.8 Hz, 4 H,
CH2), 3.79 (t, J = 5.1 Hz, 2 H, CH2), 3.70–3.74 (m, 6 H, CH2), 3.62–3.68 (m, 12 H, CH2),
3.53–3.57 (m, 6 H, CH2), 3.37 (two partially overlapping s, 9 H, CH3); 13C{1H} NMR (DMSO-d6): δ 160.9, 152.4, 137.7,
136.0, 130.7, 126.2, 119.3, 116.0, 115.6, 113.9, 107.1, 103.6, 72.0, 71.5,
71.2, 70.2, 70.0, 69.9, 69.8, 69.1, 68.5, 58.2 (some of the CH2 signals are overlapped); MS (ESI): an isotopic cluster peaking at m/z 743 {100%, [M + Na]+}; HRMS (ESI): m/z calcd for C36H52N2NaO13[M + Na]+: 743.3362, found 743.3365.


#### 2.2.2. Preparation of 4-[3,4,5-tris(3,6,9-Trioxadecoxy)Benzoxy]Phthalonitrile (3)

According to the above procedure
using 4- instead of 3-nitrophthalonitrile as a starting material, compound3 was obtained as a colorless liquid
(1.71 g, 47%). 1H NMR (CDCl3): δ 7.70
(d, J = 8.7 Hz, 1 H, ArH), 7.31 (d, J = 2.4 Hz, 1 H, ArH), 7.22 (dd, J = 2.4, 8.7 Hz, 1 H, ArH), 6.60 (s, 2 H, ArH), 5.02
(s, 2 H, ArCH2), 4.11–4.15 (m, 6 H, CH2), 3.82 (t, J = 4.8 Hz, 4 H, CH2), 3.76 (t, J = 5.1 Hz, 2 H, CH2), 3.68–3.71 (m, 6 H,
CH2), 3.60–3.65 (m, 12 H, CH2), 3.50–3.53 (m, 6 H, CH2),
3.34 (s, 9 H, CH3); 13C{1H} NMR (CDCl3):
δ 161.5, 152.9, 138.7, 135.2, 129.7, 120.0, 119.6, 117.3, 115.5, 115.1, 107.5,
107.2, 72.2, 71.8, 71.0, 70.7, 70.6, 70.4, 69.6, 68.9, 58.9 (some of the CH2 signals are overlapped); MS (ESI): an isotopic cluster peaking at m/z 743 {100%, [M + Na]+}; HRMS (ESI): m/z calcd for C36H52N2NaO13[M + Na]+: 743.3362, found 743.3361.


#### 2.2.3. Preparation of Phthalocyanine (4)

A mixture of phthalonitrile2 (0.26 g, 0.36 mmol), unsubstituted phthalonitrile (0.46 g, 3.59 mmol), and Zn(OAc)2· 2H2O
(0.22 g, 1.00 mmol) in n-pentanol (15 mL) was heated to 100°C, then a small
amount of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.5 mL) was added. The mixture was stirred at 140–150°C
for 24 hours  After a brief cooling, the
volatiles were removed under reduced pressure.
The residue was dissolved in CHCl3 (120 mL), then filtered to
remove part of the unsubstituted zinc(II) phthalocyanine formed. The filtrate was collected and evaporated to
dryness in vacuo. The residue was
purified by silica-gel column chromatography using CHCl3/CH3OH
(30 : 1 v/v) as eluent, followed by size exclusion chromatography using THF as
eluent. The crude product was further
purified by recrystallization from a mixture of THF and hexane (0.11 g, 26%). 1H
NMR (CDCl3 with a trace amount of
pyridine-d5): δ 9.41–9.46 (m, 5 H, Pc-Hα), 9.16 (d, J = 7.5 Hz, 1 H, Pc-Hα), 9.13
(d, J = 6.9 Hz, 1 H, Pc-Hα),
8.07–8.15 (m, 7 H, Pc-Hβ), 7.69 (d, J = 7.8 Hz, 1 H, Pc-Hβ), 7.30 (s, 2 H, ArH), 5.80 (s, 2 H, ArCH2), 4.31 (t, J = 5.1 Hz, 2 H, CH2), 4.19 (t, J = 5.1 Hz, 4 H,
CH2), 3.90 (t, J = 5.1 Hz, 2 H, CH2), 3.78–3.82 (m, 2H, CH2), 3.66–3.75 (m, 8 H, CH2),
3.55–3.59 (m, 6 H, CH2), 3.47–3.51 (m, 8 H, CH2), 3.38–3.41
(m, 7 H, CH2 and CH3), 3.24 (s, 6 H, CH3); 13C{1H}
NMR (DMSO-d6): δ 155.6, 152.7, 152.6, 152.3, 152.2, 140.3, 138.4,
137.9, 137.7, 137.6, 133.5, 130.6, 129.1, 125.3, 122.5, 122.4, 122.1, 115.3,
114.0, 107.8, 72.3, 71.6, 71.5, 71.0, 70.2, 70.0, 69.9, 69.8, 69.3, 68.8, 58.3,
58.2 (some of the signals are overlapped); MS (ESI):
an isotopic cluster peaking at m/z
1191 {100%, [M + Na]+}; HRMS (ESI): m/z calcd for C60H64N8NaO13Zn
[M + Na]+: 1191.3777, found 1191.3783.


#### 2.2.4. Preparation of Phthalocyanine (5)

According to the above procedure, phthalonitrile3 (0.26 g, 0.36 mmol) was treated with
unsubstituted phthalonitrile (0.46 g, 3.59 mmol) and Zn(OAc)2·2H2O (0.22 g, 1.00 mmol) to give phthalocyanine 5 as a blue solid (0.09 g, 21%). 1H
NMR (CDCl3 with a trace amount of
pyridine-d5): δ 9.20–9.35 (m, 6 H, Pc-Hα), 9.05 (d, J = 7.8 Hz, 1 H, Pc-Hα), 8.66
(s, 1 H, Pc-Hα), 8.06–8.12 (m, 6 H, Pc-Hβ), 7.62 (d, J = 8.4 Hz, 1 H, Pc-Hβ), 6.99 (s, 2  H, ArH), 5.46 (s, 2 H, ArCH2), 4.33 (t, J = 4.8 Hz, 4 H, CH2), 4.24 (t, J = 5.1 Hz, 2 H, CH2), 3.94
(t, J = 4.8 Hz, 4 H, CH2),
3.86 (t, J = 4.8 Hz, 2 H, CH2), 3.76–3.80 (m, 6 H, CH2),
3.62–3.71 (m, 12 H, CH2), 3.53–3.57 (m, 6 H, CH2), 3.38
(s, 3 H, CH3), 3.36 (s, 6 H, CH3); 13C{1H}
NMR (DMSO-d6): δ 159.9, 152.6, 152.4, 152.3, 152.2, 152.0, 151.7,
151.6, 139.5, 137.8, 137.5, 132.7, 130.7, 129.0, 128.9, 123.0, 122.2, 117.8, 107.2,
105.6, 72.1, 71.5, 70.3, 70.1, 70.0, 69.9, 69.4, 68.8, 58.3, 55.1 (some of the
signals are overlapped); MS (ESI): an isotopic
cluster peaking at m/z 1191 {95%,
[M + Na]+}; HRMS (ESI): m/z calcd for C60H64N8NaO13Zn
[M + Na]+: 1191.3777, found 1191.3771.


#### 2.2.5. Preparation of 3,6-bis[3,4,5-tris(3,6,9-Trioxadecoxy)Benzoxy]Phthalonitrile (7)

A mixture of compound6 (2.06 g, 3.36 mmol), 2,3-dicyanohydroquinone (0.27 g, 1.69 mmol), and K2CO3 (1.17 g, 8.47 mmol) in DMF (10 mL) was stirred at 100°C
for 24 hours The volatiles were then removed under reduced
pressure. The residue was mixed with
water (50 mL) and the mixture was extracted with CHCl3 (50 mL ×   3). The combined organic extracts was
dried over anhydrous MgSO4 and evaporated under reduced pressure.
The residue was then purified by silica-gel column chromatography using CHCl3/MeOH
(20:1 v/v) as eluent to give the product as a pale yellow transparent liquid
(1.95 g, 88%).1H NMR (CDCl3): δ 7.13 (s, 2 H, ArH), 6.64 (s, 4 H,
ArH), 5.07 (s, 4 H, ArCH2), 4.12–4.17 (m, 12 H, CH2),
3.84 (t, J = 4.8 Hz, 8 H, CH2), 3.78 (t, J = 5.1 Hz, 4 H, CH2), 3.71–3.74 (m, 12 H,
CH2), 3.62–3.67 (m, 24 H, CH2), 3.52–3.57 (m, 12 H, CH2),
3.37 (two partially overlapping s, 18 H, CH3); 13C{1H}
NMR (CDCl3): δ 154.7, 152.8, 138.2, 130.3, 119.4, 112.9, 106.4,
105.7, 72.2, 71.7, 71.6, 70.6, 70.5, 70.3, 69.5, 68.7, 58.8 (some of the CH2 signals are overlapped); MS (ESI): an isotopic cluster peaking at m/z 1336 {100%, [M + Na]+}; HRMS (ESI): m/z calcd for C64H100N2NaO26 [M + Na]+: 1335.6457, found 1335.6462.


#### 2.2.6. Preparation of Phthalocyanine (8)

According to the procedure described for4, phthalonitrile 7 (0.50 g, 0.38 mmol) was treated with unsubstituted
phthalonitrile (0.49 g, 3.82 mmol) and Zn(OAc)2·2H2O
(0.23 g, 1.05 mmol) to give phthalocyanine 8 as a blue-green oil (54 mg, 8%). 1H
NMR (CDCl3): δ 9.43–9.47 (m, 4 H, Pc-Hα),
9.20 (d, J = 7.5 Hz, 2 H, Pc-Hα), 8.03–8.15 (m, 6 H, Pc-Hβ), 7.68 (s, 2 H, Pc-Hβ), 7.37 (s, 4 H, ArH), 5.90 (s, 4 H, ArCH2), 4.05 (t, J = 5.1 Hz, 4 H, CH2), 3.80 (t, J = 5.1 Hz, 8 H, CH2), 3.70 (t, J = 5.1 Hz, 4 H, CH2), 3.61–3.65 (m, 4 H,
CH2), 3.55–3.58 (m, 8 H, CH2),
3.44–3.48 (m, 12 H, CH2), 3.34–3.37 (m,
12 H, CH2), 3.30 (s, 6 H, CH3), 3.18–3.21 (m, 8 H, CH2),
3.12 (s, 12 H, CH2), 2.90 (s, 12 H, CH3); 13C{1H}
NMR (CDCl3): 153.6, 153.4, 153.3, 152.5, 152.2, 150.2, 138.6, 138.3,
138.1, 137.5, 133.6, 129.0, 128.8, 128.6, 128.5, 122.6, 122.3, 122.1, 116.5,
107.0, 72.3, 72.2, 71.8, 71.3, 70.5, 70.4, 69.9, 69.7, 69.2, 68.1, 58.8, 58.4
(some of the CH2 signals are overlapped); MS
(ESI): an isotopic cluster peaking at m/z 1784 {20%, [M + Na]+}; HRMS
(ESI): m/z calcd for C88H112N8NaO26Zn
[M + Na]+: 1783.6871, found 1783.6862.


### 2.3. In Vitro Studies

#### 2.3.1. Cell Lines and Culture Conditions

The HT29 human colorectal carcinoma
cells (from ATCC, no.
HTB-38) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, cat no. 10313-021) supplemented with
fetal calf serum (10%), penicillin-streptomycin (100 units mL-1 and
100 mgmL-1, resp.), L-glutamine (2 mM), and transferrin (10 mgmL-1). The HepG2 human hepatocarcinoma cells (from
ATCC, no. HB-8065) were maintained in RPMI medium 1640 (Invitrogen, cat no.
23400-021) supplemented with fetal calf serum (10%) and penicillin-streptomycin
(100 units mL-1 and 100 mgmL-1, resp.). Approximately 3×104 (for HT29)
or 4×104 (for HepG2) cells per well in these media were inoculated
in 96-multiwell plates and incubated overnight at 37°C in a humidified 5% CO2 atmosphere.


#### 2.3.2. Photocytotoxicity Assay

Phthalocyanines4, 5, and 8 were first dissolved in DMF to give 1.5 mM solutions, which
were diluted to 80 μM with an aqueous solution of Cremophor EL (Sigma, 0.47 g in
100 mL of water). The solutions were
filtered with a 0.2 μm filter, then diluted with the culture medium to appropriate
concentrations (two-fold dilutions from 8 μM). The cells, after being rinsed with phosphate buffered saline (PBS), were
incubated with 100 μL of these phthalocyanine solutions for 2 hours at 37°C under
5% CO2. The cells were then
rinsed again with PBS and refed with 100 μL of the culture medium before being
illuminated at ambient temperature. The
light source consisted of a 300 W halogen lamp, a water tank for cooling, and a
color glass filter (Newport) cut-on 610 nm. The fluence rate (λ>610 nm) was 40 mW cm-2. An illumination of 20 minutes led to a total
fluence of 48 J cm-2.Cell viability was
determined by means of the colorimetric MTT assay [12]. After illumination, the cells were
incubated at 37°C under 5% CO2 overnight. An MTT (Sigma) solution in PBS (3 mgmL-1,
50 μL) was added to each well followed by incubation for 2 hours under the same
environment. A solution of sodium
dodecyl sulfate (SDS, Sigma) (10% by weight, 50 μL) was then added to each
well. The plate was incubated in an oven
at 60°C for 30 minutes, then 80 μL of iso-propanol was added to each
well. The plate was agitated on a
Bio-Rad microplate reader at ambient temperature for 10 sec before the
absorbance at 540 nm at each well was taken.
The average absorbance of the blank wells, which
did not contain the cells, was subtracted from the readings of the other
wells. The cell viability was then
determined by the equation: % Viability = [Σ(Ai/Acontrol×100)]/n,
where Ai is the absorbance of the ith data (i=1,2,…,n), Acontrol is the average absorbance of the control
wells, in which the phthalocyanine was absent, and n (= 4) is the number of the data points.


#### 2.3.3. Fluorescence Microscopic Studies

For the detection of the intracellular
fluorescence intensity of compounds4, 5, and 8, approximately
1.2×105 HT29 cells in the culture medium (2 mL) were seeded on a
coverslip (diameter = 25 mm) and incubated overnight at 37°C under 5% CO2. The medium was removed, then the cells were
incubated with 2 mL of an 8 μM phthalocyanine dilution in the medium for 2 h
under the same conditions. The cells
were then rinsed with PBS and viewed with an Olympus IX 70 inverted
microscope. The excitation light source
(at 630 nm) was provided by a multiwavelength illuminator (Polychrome IV, TILL
Photonics). The emitted fluorescence
(>660 nm) was collected using a digital cooled CCD camera (Quantix,
Photometrics). Images were digitalized
and analyzed using MetaFluor V.4.6 (Universal Imaging).


## 2.1. General

All the reactions were performed under an atmosphere of
nitrogen. Tetrahydrofuran (THF),n-pentanol, dichloromethane, and N,N-dimethylformamide
(DMF) were distilled from sodium benzophenone ketyl, sodium, calcium
hydride, and barium oxide, respectively. Chromatographic purifications were performed on silica gel (Macherey-Nagel, 70–230 mesh) columns with the
indicated eluents. Size exclusion chromatography
was carried out on Bio-Rad Bio-Beads S-X1 beads (200–400 mesh). All other solvents and reagents were of
reagent grade and used as received. Compounds 1 and 6 were prepared as described
[8].1H and 13C{1H} NMR spectra were
recorded on a Bruker DPX 300 spectrometer (1H, 300; 13C,
75.4 MHz) in CDCl3 or DMSO-d6. Spectra were referenced internally using the
residual solvent [1H: CDCl3 (δ 7.26); DMSO-d6 (δ
2.50)] or solvent [13C: CDCl3 (δ 77.0);
DMSO-d6 (δ 39.7)] resonances relative to SiMe4. Electrospray ionization (ESI) mass spectra
were measured on a Thermo Finnigan MAT 95 XL mass spectrometer.UV-Vis and
steady-state fluorescence spectra were taken on a Cary 5G UV-Vis-NIR
spectrophotometer and a Hitachi F-4500 spectrofluorometer, respectively. Fluorescence quantum yields(ΦF) were determined by the equation: ΦF(sample)=(Fsample/Fref)(Aref/Asample)(nsample2/nref2)ΦF(ref) [9], where F,A, and n are the measured
fluorescence (area under the emission peak), the absorbance at the excitation
position (610 nm), and the refractive index of the solvent, respectively. The unsubstituted zinc(II) phthalocyanine (ZnPc)
in DMF was used as the reference [ΦF(ref)=0.28] [10]. To minimize reabsorption of radiation by the ground-state species, the
emission spectra were obtained in very dilute solutions where the absorbance at
610 nm was less than 0.03. Singlet oxygen
quantum yields (ΦΔ) were measured in DMF by
the method of chemical quenching of 1,3-diphenylisobenzofuran (DPBF) using ZnPc
as reference (ΦΔ=0.56) [11].


## 2.2. Syntheses

### 2.2.1. Preparation of 3-[3,4,5-tris(3,6,9-Trioxadecoxy)Benzoxy]Phthalonitrile (2)

To a mixture of 3-nitrophthalonitrile
(1.73 g, 10 mmol) and compound1 (2.97 g, 5 mmol) in DMF (30 mL) was added anhydrous K2CO3 (6.90 g, 50 mmol). The resulting mixture was
stirred at 80°C for 4 days. The solvent was then evaporated under reduced pressure and the residue
was mixed with CHCl3 (60 mL) and water (60 mL). The aqueous layer was separated and extracted
with CHCl3 (60 mL ×3). The
combined organic layers was dried over anhydrous MgSO4, then
evaporated to dryness. The residue was
purified by silica-gel column chromatography using CHCl3/MeOH (60 : 1 v/v) as eluent to give compound 2 as
a colorless liquid (1.41 g, 39%). 1H
NMR: δ 7.61 (vt, J = 8.7 Hz, 1 H, ArH), 7.37 (d, J = 7.5 Hz, 1 H, ArH), 7.25 (d, J =
7.5 Hz, 1 H, ArH), 6.66 (s, 2 H, ArH), 5.17 (s, 2 H, ArCH2), 4.13–4.18
(m, 6 H, CH2),  3.85 (t, J = 4.8 Hz, 4 H,
CH2), 3.79 (t, J = 5.1 Hz, 2 H, CH2), 3.70–3.74 (m, 6 H, CH2), 3.62–3.68 (m, 12 H, CH2),
3.53–3.57 (m, 6 H, CH2), 3.37 (two partially overlapping s, 9 H, CH3); 13C{1H} NMR (DMSO-d6): δ 160.9, 152.4, 137.7,
136.0, 130.7, 126.2, 119.3, 116.0, 115.6, 113.9, 107.1, 103.6, 72.0, 71.5,
71.2, 70.2, 70.0, 69.9, 69.8, 69.1, 68.5, 58.2 (some of the CH2 signals are overlapped); MS (ESI): an isotopic cluster peaking at m/z 743 {100%, [M + Na]+}; HRMS (ESI): m/z calcd for C36H52N2NaO13[M + Na]+: 743.3362, found 743.3365.


### 2.2.2. Preparation of 4-[3,4,5-tris(3,6,9-Trioxadecoxy)Benzoxy]Phthalonitrile (3)

According to the above procedure
using 4- instead of 3-nitrophthalonitrile as a starting material, compound3 was obtained as a colorless liquid
(1.71 g, 47%). 1H NMR (CDCl3): δ 7.70
(d, J = 8.7 Hz, 1 H, ArH), 7.31 (d, J = 2.4 Hz, 1 H, ArH), 7.22 (dd, J = 2.4, 8.7 Hz, 1 H, ArH), 6.60 (s, 2 H, ArH), 5.02
(s, 2 H, ArCH2), 4.11–4.15 (m, 6 H, CH2), 3.82 (t, J = 4.8 Hz, 4 H, CH2), 3.76 (t, J = 5.1 Hz, 2 H, CH2), 3.68–3.71 (m, 6 H,
CH2), 3.60–3.65 (m, 12 H, CH2), 3.50–3.53 (m, 6 H, CH2),
3.34 (s, 9 H, CH3); 13C{1H} NMR (CDCl3):
δ 161.5, 152.9, 138.7, 135.2, 129.7, 120.0, 119.6, 117.3, 115.5, 115.1, 107.5,
107.2, 72.2, 71.8, 71.0, 70.7, 70.6, 70.4, 69.6, 68.9, 58.9 (some of the CH2 signals are overlapped); MS (ESI): an isotopic cluster peaking at m/z 743 {100%, [M + Na]+}; HRMS (ESI): m/z calcd for C36H52N2NaO13[M + Na]+: 743.3362, found 743.3361.


### 2.2.3. Preparation of Phthalocyanine (4)

A mixture of phthalonitrile2 (0.26 g, 0.36 mmol), unsubstituted phthalonitrile (0.46 g, 3.59 mmol), and Zn(OAc)2· 2H2O
(0.22 g, 1.00 mmol) in n-pentanol (15 mL) was heated to 100°C, then a small
amount of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.5 mL) was added. The mixture was stirred at 140–150°C
for 24 hours  After a brief cooling, the
volatiles were removed under reduced pressure.
The residue was dissolved in CHCl3 (120 mL), then filtered to
remove part of the unsubstituted zinc(II) phthalocyanine formed. The filtrate was collected and evaporated to
dryness in vacuo. The residue was
purified by silica-gel column chromatography using CHCl3/CH3OH
(30 : 1 v/v) as eluent, followed by size exclusion chromatography using THF as
eluent. The crude product was further
purified by recrystallization from a mixture of THF and hexane (0.11 g, 26%). 1H
NMR (CDCl3 with a trace amount of
pyridine-d5): δ 9.41–9.46 (m, 5 H, Pc-Hα), 9.16 (d, J = 7.5 Hz, 1 H, Pc-Hα), 9.13
(d, J = 6.9 Hz, 1 H, Pc-Hα),
8.07–8.15 (m, 7 H, Pc-Hβ), 7.69 (d, J = 7.8 Hz, 1 H, Pc-Hβ), 7.30 (s, 2 H, ArH), 5.80 (s, 2 H, ArCH2), 4.31 (t, J = 5.1 Hz, 2 H, CH2), 4.19 (t, J = 5.1 Hz, 4 H,
CH2), 3.90 (t, J = 5.1 Hz, 2 H, CH2), 3.78–3.82 (m, 2H, CH2), 3.66–3.75 (m, 8 H, CH2),
3.55–3.59 (m, 6 H, CH2), 3.47–3.51 (m, 8 H, CH2), 3.38–3.41
(m, 7 H, CH2 and CH3), 3.24 (s, 6 H, CH3); 13C{1H}
NMR (DMSO-d6): δ 155.6, 152.7, 152.6, 152.3, 152.2, 140.3, 138.4,
137.9, 137.7, 137.6, 133.5, 130.6, 129.1, 125.3, 122.5, 122.4, 122.1, 115.3,
114.0, 107.8, 72.3, 71.6, 71.5, 71.0, 70.2, 70.0, 69.9, 69.8, 69.3, 68.8, 58.3,
58.2 (some of the signals are overlapped); MS (ESI):
an isotopic cluster peaking at m/z
1191 {100%, [M + Na]+}; HRMS (ESI): m/z calcd for C60H64N8NaO13Zn
[M + Na]+: 1191.3777, found 1191.3783.


### 2.2.4. Preparation of Phthalocyanine (5)

According to the above procedure, phthalonitrile3 (0.26 g, 0.36 mmol) was treated with
unsubstituted phthalonitrile (0.46 g, 3.59 mmol) and Zn(OAc)2·2H2O (0.22 g, 1.00 mmol) to give phthalocyanine 5 as a blue solid (0.09 g, 21%). 1H
NMR (CDCl3 with a trace amount of
pyridine-d5): δ 9.20–9.35 (m, 6 H, Pc-Hα), 9.05 (d, J = 7.8 Hz, 1 H, Pc-Hα), 8.66
(s, 1 H, Pc-Hα), 8.06–8.12 (m, 6 H, Pc-Hβ), 7.62 (d, J = 8.4 Hz, 1 H, Pc-Hβ), 6.99 (s, 2  H, ArH), 5.46 (s, 2 H, ArCH2), 4.33 (t, J = 4.8 Hz, 4 H, CH2), 4.24 (t, J = 5.1 Hz, 2 H, CH2), 3.94
(t, J = 4.8 Hz, 4 H, CH2),
3.86 (t, J = 4.8 Hz, 2 H, CH2), 3.76–3.80 (m, 6 H, CH2),
3.62–3.71 (m, 12 H, CH2), 3.53–3.57 (m, 6 H, CH2), 3.38
(s, 3 H, CH3), 3.36 (s, 6 H, CH3); 13C{1H}
NMR (DMSO-d6): δ 159.9, 152.6, 152.4, 152.3, 152.2, 152.0, 151.7,
151.6, 139.5, 137.8, 137.5, 132.7, 130.7, 129.0, 128.9, 123.0, 122.2, 117.8, 107.2,
105.6, 72.1, 71.5, 70.3, 70.1, 70.0, 69.9, 69.4, 68.8, 58.3, 55.1 (some of the
signals are overlapped); MS (ESI): an isotopic
cluster peaking at m/z 1191 {95%,
[M + Na]+}; HRMS (ESI): m/z calcd for C60H64N8NaO13Zn
[M + Na]+: 1191.3777, found 1191.3771.


### 2.2.5. Preparation of 3,6-bis[3,4,5-tris(3,6,9-Trioxadecoxy)Benzoxy]Phthalonitrile (7)

A mixture of compound6 (2.06 g, 3.36 mmol), 2,3-dicyanohydroquinone (0.27 g, 1.69 mmol), and K2CO3 (1.17 g, 8.47 mmol) in DMF (10 mL) was stirred at 100°C
for 24 hours The volatiles were then removed under reduced
pressure. The residue was mixed with
water (50 mL) and the mixture was extracted with CHCl3 (50 mL ×   3). The combined organic extracts was
dried over anhydrous MgSO4 and evaporated under reduced pressure.
The residue was then purified by silica-gel column chromatography using CHCl3/MeOH
(20:1 v/v) as eluent to give the product as a pale yellow transparent liquid
(1.95 g, 88%).1H NMR (CDCl3): δ 7.13 (s, 2 H, ArH), 6.64 (s, 4 H,
ArH), 5.07 (s, 4 H, ArCH2), 4.12–4.17 (m, 12 H, CH2),
3.84 (t, J = 4.8 Hz, 8 H, CH2), 3.78 (t, J = 5.1 Hz, 4 H, CH2), 3.71–3.74 (m, 12 H,
CH2), 3.62–3.67 (m, 24 H, CH2), 3.52–3.57 (m, 12 H, CH2),
3.37 (two partially overlapping s, 18 H, CH3); 13C{1H}
NMR (CDCl3): δ 154.7, 152.8, 138.2, 130.3, 119.4, 112.9, 106.4,
105.7, 72.2, 71.7, 71.6, 70.6, 70.5, 70.3, 69.5, 68.7, 58.8 (some of the CH2 signals are overlapped); MS (ESI): an isotopic cluster peaking at m/z 1336 {100%, [M + Na]+}; HRMS (ESI): m/z calcd for C64H100N2NaO26 [M + Na]+: 1335.6457, found 1335.6462.


### 2.2.6. Preparation of Phthalocyanine (8)

According to the procedure described for4, phthalonitrile 7 (0.50 g, 0.38 mmol) was treated with unsubstituted
phthalonitrile (0.49 g, 3.82 mmol) and Zn(OAc)2·2H2O
(0.23 g, 1.05 mmol) to give phthalocyanine 8 as a blue-green oil (54 mg, 8%). 1H
NMR (CDCl3): δ 9.43–9.47 (m, 4 H, Pc-Hα),
9.20 (d, J = 7.5 Hz, 2 H, Pc-Hα), 8.03–8.15 (m, 6 H, Pc-Hβ), 7.68 (s, 2 H, Pc-Hβ), 7.37 (s, 4 H, ArH), 5.90 (s, 4 H, ArCH2), 4.05 (t, J = 5.1 Hz, 4 H, CH2), 3.80 (t, J = 5.1 Hz, 8 H, CH2), 3.70 (t, J = 5.1 Hz, 4 H, CH2), 3.61–3.65 (m, 4 H,
CH2), 3.55–3.58 (m, 8 H, CH2),
3.44–3.48 (m, 12 H, CH2), 3.34–3.37 (m,
12 H, CH2), 3.30 (s, 6 H, CH3), 3.18–3.21 (m, 8 H, CH2),
3.12 (s, 12 H, CH2), 2.90 (s, 12 H, CH3); 13C{1H}
NMR (CDCl3): 153.6, 153.4, 153.3, 152.5, 152.2, 150.2, 138.6, 138.3,
138.1, 137.5, 133.6, 129.0, 128.8, 128.6, 128.5, 122.6, 122.3, 122.1, 116.5,
107.0, 72.3, 72.2, 71.8, 71.3, 70.5, 70.4, 69.9, 69.7, 69.2, 68.1, 58.8, 58.4
(some of the CH2 signals are overlapped); MS
(ESI): an isotopic cluster peaking at m/z 1784 {20%, [M + Na]+}; HRMS
(ESI): m/z calcd for C88H112N8NaO26Zn
[M + Na]+: 1783.6871, found 1783.6862.


## 2.2.1. Preparation of 3-[3,4,5-tris(3,6,9-Trioxadecoxy)Benzoxy]Phthalonitrile (2)

To a mixture of 3-nitrophthalonitrile
(1.73 g, 10 mmol) and compound1 (2.97 g, 5 mmol) in DMF (30 mL) was added anhydrous K2CO3 (6.90 g, 50 mmol). The resulting mixture was
stirred at 80°C for 4 days. The solvent was then evaporated under reduced pressure and the residue
was mixed with CHCl3 (60 mL) and water (60 mL). The aqueous layer was separated and extracted
with CHCl3 (60 mL ×3). The
combined organic layers was dried over anhydrous MgSO4, then
evaporated to dryness. The residue was
purified by silica-gel column chromatography using CHCl3/MeOH (60 : 1 v/v) as eluent to give compound 2 as
a colorless liquid (1.41 g, 39%). 1H
NMR: δ 7.61 (vt, J = 8.7 Hz, 1 H, ArH), 7.37 (d, J = 7.5 Hz, 1 H, ArH), 7.25 (d, J =
7.5 Hz, 1 H, ArH), 6.66 (s, 2 H, ArH), 5.17 (s, 2 H, ArCH2), 4.13–4.18
(m, 6 H, CH2),  3.85 (t, J = 4.8 Hz, 4 H,
CH2), 3.79 (t, J = 5.1 Hz, 2 H, CH2), 3.70–3.74 (m, 6 H, CH2), 3.62–3.68 (m, 12 H, CH2),
3.53–3.57 (m, 6 H, CH2), 3.37 (two partially overlapping s, 9 H, CH3); 13C{1H} NMR (DMSO-d6): δ 160.9, 152.4, 137.7,
136.0, 130.7, 126.2, 119.3, 116.0, 115.6, 113.9, 107.1, 103.6, 72.0, 71.5,
71.2, 70.2, 70.0, 69.9, 69.8, 69.1, 68.5, 58.2 (some of the CH2 signals are overlapped); MS (ESI): an isotopic cluster peaking at m/z 743 {100%, [M + Na]+}; HRMS (ESI): m/z calcd for C36H52N2NaO13[M + Na]+: 743.3362, found 743.3365.


## 2.2.2. Preparation of 4-[3,4,5-tris(3,6,9-Trioxadecoxy)Benzoxy]Phthalonitrile (3)

According to the above procedure
using 4- instead of 3-nitrophthalonitrile as a starting material, compound3 was obtained as a colorless liquid
(1.71 g, 47%). 1H NMR (CDCl3): δ 7.70
(d, J = 8.7 Hz, 1 H, ArH), 7.31 (d, J = 2.4 Hz, 1 H, ArH), 7.22 (dd, J = 2.4, 8.7 Hz, 1 H, ArH), 6.60 (s, 2 H, ArH), 5.02
(s, 2 H, ArCH2), 4.11–4.15 (m, 6 H, CH2), 3.82 (t, J = 4.8 Hz, 4 H, CH2), 3.76 (t, J = 5.1 Hz, 2 H, CH2), 3.68–3.71 (m, 6 H,
CH2), 3.60–3.65 (m, 12 H, CH2), 3.50–3.53 (m, 6 H, CH2),
3.34 (s, 9 H, CH3); 13C{1H} NMR (CDCl3):
δ 161.5, 152.9, 138.7, 135.2, 129.7, 120.0, 119.6, 117.3, 115.5, 115.1, 107.5,
107.2, 72.2, 71.8, 71.0, 70.7, 70.6, 70.4, 69.6, 68.9, 58.9 (some of the CH2 signals are overlapped); MS (ESI): an isotopic cluster peaking at m/z 743 {100%, [M + Na]+}; HRMS (ESI): m/z calcd for C36H52N2NaO13[M + Na]+: 743.3362, found 743.3361.


## 2.2.3. Preparation of Phthalocyanine (4)

A mixture of phthalonitrile2 (0.26 g, 0.36 mmol), unsubstituted phthalonitrile (0.46 g, 3.59 mmol), and Zn(OAc)2· 2H2O
(0.22 g, 1.00 mmol) in n-pentanol (15 mL) was heated to 100°C, then a small
amount of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.5 mL) was added. The mixture was stirred at 140–150°C
for 24 hours  After a brief cooling, the
volatiles were removed under reduced pressure.
The residue was dissolved in CHCl3 (120 mL), then filtered to
remove part of the unsubstituted zinc(II) phthalocyanine formed. The filtrate was collected and evaporated to
dryness in vacuo. The residue was
purified by silica-gel column chromatography using CHCl3/CH3OH
(30 : 1 v/v) as eluent, followed by size exclusion chromatography using THF as
eluent. The crude product was further
purified by recrystallization from a mixture of THF and hexane (0.11 g, 26%). 1H
NMR (CDCl3 with a trace amount of
pyridine-d5): δ 9.41–9.46 (m, 5 H, Pc-Hα), 9.16 (d, J = 7.5 Hz, 1 H, Pc-Hα), 9.13
(d, J = 6.9 Hz, 1 H, Pc-Hα),
8.07–8.15 (m, 7 H, Pc-Hβ), 7.69 (d, J = 7.8 Hz, 1 H, Pc-Hβ), 7.30 (s, 2 H, ArH), 5.80 (s, 2 H, ArCH2), 4.31 (t, J = 5.1 Hz, 2 H, CH2), 4.19 (t, J = 5.1 Hz, 4 H,
CH2), 3.90 (t, J = 5.1 Hz, 2 H, CH2), 3.78–3.82 (m, 2H, CH2), 3.66–3.75 (m, 8 H, CH2),
3.55–3.59 (m, 6 H, CH2), 3.47–3.51 (m, 8 H, CH2), 3.38–3.41
(m, 7 H, CH2 and CH3), 3.24 (s, 6 H, CH3); 13C{1H}
NMR (DMSO-d6): δ 155.6, 152.7, 152.6, 152.3, 152.2, 140.3, 138.4,
137.9, 137.7, 137.6, 133.5, 130.6, 129.1, 125.3, 122.5, 122.4, 122.1, 115.3,
114.0, 107.8, 72.3, 71.6, 71.5, 71.0, 70.2, 70.0, 69.9, 69.8, 69.3, 68.8, 58.3,
58.2 (some of the signals are overlapped); MS (ESI):
an isotopic cluster peaking at m/z
1191 {100%, [M + Na]+}; HRMS (ESI): m/z calcd for C60H64N8NaO13Zn
[M + Na]+: 1191.3777, found 1191.3783.


## 2.2.4. Preparation of Phthalocyanine (5)

According to the above procedure, phthalonitrile3 (0.26 g, 0.36 mmol) was treated with
unsubstituted phthalonitrile (0.46 g, 3.59 mmol) and Zn(OAc)2·2H2O (0.22 g, 1.00 mmol) to give phthalocyanine 5 as a blue solid (0.09 g, 21%). 1H
NMR (CDCl3 with a trace amount of
pyridine-d5): δ 9.20–9.35 (m, 6 H, Pc-Hα), 9.05 (d, J = 7.8 Hz, 1 H, Pc-Hα), 8.66
(s, 1 H, Pc-Hα), 8.06–8.12 (m, 6 H, Pc-Hβ), 7.62 (d, J = 8.4 Hz, 1 H, Pc-Hβ), 6.99 (s, 2  H, ArH), 5.46 (s, 2 H, ArCH2), 4.33 (t, J = 4.8 Hz, 4 H, CH2), 4.24 (t, J = 5.1 Hz, 2 H, CH2), 3.94
(t, J = 4.8 Hz, 4 H, CH2),
3.86 (t, J = 4.8 Hz, 2 H, CH2), 3.76–3.80 (m, 6 H, CH2),
3.62–3.71 (m, 12 H, CH2), 3.53–3.57 (m, 6 H, CH2), 3.38
(s, 3 H, CH3), 3.36 (s, 6 H, CH3); 13C{1H}
NMR (DMSO-d6): δ 159.9, 152.6, 152.4, 152.3, 152.2, 152.0, 151.7,
151.6, 139.5, 137.8, 137.5, 132.7, 130.7, 129.0, 128.9, 123.0, 122.2, 117.8, 107.2,
105.6, 72.1, 71.5, 70.3, 70.1, 70.0, 69.9, 69.4, 68.8, 58.3, 55.1 (some of the
signals are overlapped); MS (ESI): an isotopic
cluster peaking at m/z 1191 {95%,
[M + Na]+}; HRMS (ESI): m/z calcd for C60H64N8NaO13Zn
[M + Na]+: 1191.3777, found 1191.3771.


## 2.2.5. Preparation of 3,6-bis[3,4,5-tris(3,6,9-Trioxadecoxy)Benzoxy]Phthalonitrile (7)

A mixture of compound6 (2.06 g, 3.36 mmol), 2,3-dicyanohydroquinone (0.27 g, 1.69 mmol), and K2CO3 (1.17 g, 8.47 mmol) in DMF (10 mL) was stirred at 100°C
for 24 hours The volatiles were then removed under reduced
pressure. The residue was mixed with
water (50 mL) and the mixture was extracted with CHCl3 (50 mL ×   3). The combined organic extracts was
dried over anhydrous MgSO4 and evaporated under reduced pressure.
The residue was then purified by silica-gel column chromatography using CHCl3/MeOH
(20:1 v/v) as eluent to give the product as a pale yellow transparent liquid
(1.95 g, 88%).1H NMR (CDCl3): δ 7.13 (s, 2 H, ArH), 6.64 (s, 4 H,
ArH), 5.07 (s, 4 H, ArCH2), 4.12–4.17 (m, 12 H, CH2),
3.84 (t, J = 4.8 Hz, 8 H, CH2), 3.78 (t, J = 5.1 Hz, 4 H, CH2), 3.71–3.74 (m, 12 H,
CH2), 3.62–3.67 (m, 24 H, CH2), 3.52–3.57 (m, 12 H, CH2),
3.37 (two partially overlapping s, 18 H, CH3); 13C{1H}
NMR (CDCl3): δ 154.7, 152.8, 138.2, 130.3, 119.4, 112.9, 106.4,
105.7, 72.2, 71.7, 71.6, 70.6, 70.5, 70.3, 69.5, 68.7, 58.8 (some of the CH2 signals are overlapped); MS (ESI): an isotopic cluster peaking at m/z 1336 {100%, [M + Na]+}; HRMS (ESI): m/z calcd for C64H100N2NaO26 [M + Na]+: 1335.6457, found 1335.6462.


## 2.2.6. Preparation of Phthalocyanine (8)

According to the procedure described for4, phthalonitrile 7 (0.50 g, 0.38 mmol) was treated with unsubstituted
phthalonitrile (0.49 g, 3.82 mmol) and Zn(OAc)2·2H2O
(0.23 g, 1.05 mmol) to give phthalocyanine 8 as a blue-green oil (54 mg, 8%). 1H
NMR (CDCl3): δ 9.43–9.47 (m, 4 H, Pc-Hα),
9.20 (d, J = 7.5 Hz, 2 H, Pc-Hα), 8.03–8.15 (m, 6 H, Pc-Hβ), 7.68 (s, 2 H, Pc-Hβ), 7.37 (s, 4 H, ArH), 5.90 (s, 4 H, ArCH2), 4.05 (t, J = 5.1 Hz, 4 H, CH2), 3.80 (t, J = 5.1 Hz, 8 H, CH2), 3.70 (t, J = 5.1 Hz, 4 H, CH2), 3.61–3.65 (m, 4 H,
CH2), 3.55–3.58 (m, 8 H, CH2),
3.44–3.48 (m, 12 H, CH2), 3.34–3.37 (m,
12 H, CH2), 3.30 (s, 6 H, CH3), 3.18–3.21 (m, 8 H, CH2),
3.12 (s, 12 H, CH2), 2.90 (s, 12 H, CH3); 13C{1H}
NMR (CDCl3): 153.6, 153.4, 153.3, 152.5, 152.2, 150.2, 138.6, 138.3,
138.1, 137.5, 133.6, 129.0, 128.8, 128.6, 128.5, 122.6, 122.3, 122.1, 116.5,
107.0, 72.3, 72.2, 71.8, 71.3, 70.5, 70.4, 69.9, 69.7, 69.2, 68.1, 58.8, 58.4
(some of the CH2 signals are overlapped); MS
(ESI): an isotopic cluster peaking at m/z 1784 {20%, [M + Na]+}; HRMS
(ESI): m/z calcd for C88H112N8NaO26Zn
[M + Na]+: 1783.6871, found 1783.6862.


## 2.3. In Vitro Studies

### 2.3.1. Cell Lines and Culture Conditions

The HT29 human colorectal carcinoma
cells (from ATCC, no.
HTB-38) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, cat no. 10313-021) supplemented with
fetal calf serum (10%), penicillin-streptomycin (100 units mL-1 and
100 mgmL-1, resp.), L-glutamine (2 mM), and transferrin (10 mgmL-1). The HepG2 human hepatocarcinoma cells (from
ATCC, no. HB-8065) were maintained in RPMI medium 1640 (Invitrogen, cat no.
23400-021) supplemented with fetal calf serum (10%) and penicillin-streptomycin
(100 units mL-1 and 100 mgmL-1, resp.). Approximately 3×104 (for HT29)
or 4×104 (for HepG2) cells per well in these media were inoculated
in 96-multiwell plates and incubated overnight at 37°C in a humidified 5% CO2 atmosphere.


### 2.3.2. Photocytotoxicity Assay

Phthalocyanines4, 5, and 8 were first dissolved in DMF to give 1.5 mM solutions, which
were diluted to 80 μM with an aqueous solution of Cremophor EL (Sigma, 0.47 g in
100 mL of water). The solutions were
filtered with a 0.2 μm filter, then diluted with the culture medium to appropriate
concentrations (two-fold dilutions from 8 μM). The cells, after being rinsed with phosphate buffered saline (PBS), were
incubated with 100 μL of these phthalocyanine solutions for 2 hours at 37°C under
5% CO2. The cells were then
rinsed again with PBS and refed with 100 μL of the culture medium before being
illuminated at ambient temperature. The
light source consisted of a 300 W halogen lamp, a water tank for cooling, and a
color glass filter (Newport) cut-on 610 nm. The fluence rate (λ>610 nm) was 40 mW cm-2. An illumination of 20 minutes led to a total
fluence of 48 J cm-2.Cell viability was
determined by means of the colorimetric MTT assay [12]. After illumination, the cells were
incubated at 37°C under 5% CO2 overnight. An MTT (Sigma) solution in PBS (3 mgmL-1,
50 μL) was added to each well followed by incubation for 2 hours under the same
environment. A solution of sodium
dodecyl sulfate (SDS, Sigma) (10% by weight, 50 μL) was then added to each
well. The plate was incubated in an oven
at 60°C for 30 minutes, then 80 μL of iso-propanol was added to each
well. The plate was agitated on a
Bio-Rad microplate reader at ambient temperature for 10 sec before the
absorbance at 540 nm at each well was taken.
The average absorbance of the blank wells, which
did not contain the cells, was subtracted from the readings of the other
wells. The cell viability was then
determined by the equation: % Viability = [Σ(Ai/Acontrol×100)]/n,
where Ai is the absorbance of the ith data (i=1,2,…,n), Acontrol is the average absorbance of the control
wells, in which the phthalocyanine was absent, and n (= 4) is the number of the data points.


### 2.3.3. Fluorescence Microscopic Studies

For the detection of the intracellular
fluorescence intensity of compounds4, 5, and 8, approximately
1.2×105 HT29 cells in the culture medium (2 mL) were seeded on a
coverslip (diameter = 25 mm) and incubated overnight at 37°C under 5% CO2. The medium was removed, then the cells were
incubated with 2 mL of an 8 μM phthalocyanine dilution in the medium for 2 h
under the same conditions. The cells
were then rinsed with PBS and viewed with an Olympus IX 70 inverted
microscope. The excitation light source
(at 630 nm) was provided by a multiwavelength illuminator (Polychrome IV, TILL
Photonics). The emitted fluorescence
(>660 nm) was collected using a digital cooled CCD camera (Quantix,
Photometrics). Images were digitalized
and analyzed using MetaFluor V.4.6 (Universal Imaging).


## 2.3.1. Cell Lines and Culture Conditions

The HT29 human colorectal carcinoma
cells (from ATCC, no.
HTB-38) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, cat no. 10313-021) supplemented with
fetal calf serum (10%), penicillin-streptomycin (100 units mL-1 and
100 mgmL-1, resp.), L-glutamine (2 mM), and transferrin (10 mgmL-1). The HepG2 human hepatocarcinoma cells (from
ATCC, no. HB-8065) were maintained in RPMI medium 1640 (Invitrogen, cat no.
23400-021) supplemented with fetal calf serum (10%) and penicillin-streptomycin
(100 units mL-1 and 100 mgmL-1, resp.). Approximately 3×104 (for HT29)
or 4×104 (for HepG2) cells per well in these media were inoculated
in 96-multiwell plates and incubated overnight at 37°C in a humidified 5% CO2 atmosphere.


## 2.3.2. Photocytotoxicity Assay

Phthalocyanines4, 5, and 8 were first dissolved in DMF to give 1.5 mM solutions, which
were diluted to 80 μM with an aqueous solution of Cremophor EL (Sigma, 0.47 g in
100 mL of water). The solutions were
filtered with a 0.2 μm filter, then diluted with the culture medium to appropriate
concentrations (two-fold dilutions from 8 μM). The cells, after being rinsed with phosphate buffered saline (PBS), were
incubated with 100 μL of these phthalocyanine solutions for 2 hours at 37°C under
5% CO2. The cells were then
rinsed again with PBS and refed with 100 μL of the culture medium before being
illuminated at ambient temperature. The
light source consisted of a 300 W halogen lamp, a water tank for cooling, and a
color glass filter (Newport) cut-on 610 nm. The fluence rate (λ>610 nm) was 40 mW cm-2. An illumination of 20 minutes led to a total
fluence of 48 J cm-2.Cell viability was
determined by means of the colorimetric MTT assay [12]. After illumination, the cells were
incubated at 37°C under 5% CO2 overnight. An MTT (Sigma) solution in PBS (3 mgmL-1,
50 μL) was added to each well followed by incubation for 2 hours under the same
environment. A solution of sodium
dodecyl sulfate (SDS, Sigma) (10% by weight, 50 μL) was then added to each
well. The plate was incubated in an oven
at 60°C for 30 minutes, then 80 μL of iso-propanol was added to each
well. The plate was agitated on a
Bio-Rad microplate reader at ambient temperature for 10 sec before the
absorbance at 540 nm at each well was taken.
The average absorbance of the blank wells, which
did not contain the cells, was subtracted from the readings of the other
wells. The cell viability was then
determined by the equation: % Viability = [Σ(Ai/Acontrol×100)]/n,
where Ai is the absorbance of the ith data (i=1,2,…,n), Acontrol is the average absorbance of the control
wells, in which the phthalocyanine was absent, and n (= 4) is the number of the data points.


## 2.3.3. Fluorescence Microscopic Studies

For the detection of the intracellular
fluorescence intensity of compounds4, 5, and 8, approximately
1.2×105 HT29 cells in the culture medium (2 mL) were seeded on a
coverslip (diameter = 25 mm) and incubated overnight at 37°C under 5% CO2. The medium was removed, then the cells were
incubated with 2 mL of an 8 μM phthalocyanine dilution in the medium for 2 h
under the same conditions. The cells
were then rinsed with PBS and viewed with an Olympus IX 70 inverted
microscope. The excitation light source
(at 630 nm) was provided by a multiwavelength illuminator (Polychrome IV, TILL
Photonics). The emitted fluorescence
(>660 nm) was collected using a digital cooled CCD camera (Quantix,
Photometrics). Images were digitalized
and analyzed using MetaFluor V.4.6 (Universal Imaging).


## 3. Results and Discussion

### 3.1. Molecular Design and Chemical Synthesis

Zinc(II) phthalocyanines are good candidates
for PDT application. In addition to
their relatively high stability, the closed-shell zinc(II) center imparts desirable photophysical characteristics to the
macrocycles [13]. Introduction of substituents
at the peripheral positions can also tailor the properties of the macrocycles such as their
solubility in biological media, aggregation behavior, and targeting properties. As a
result, zinc(II) phthalocyanines have received considerable attention as
efficient photosensitizers [5]. We
describe herein three novel zinc(II) phthalocyanines (compounds 4, 5, and 8) which contain the
bulky and hydrophilic 3,4,5-tris(3,6,9-trioxadecoxy)benzoxy
moiety. Having one or two of these substituents, the π-π stacking tendency is reduced and the macrocycles
become amphiphilic in nature. These
properties should be advantageous for singlet oxygen generation and cellular
uptake, by which the photodynamic activity can be enhanced. The relatively rare 1,4-disubstituted phthalocyanine 8 also has a longer Q-band maximum
compared with the α-
and β-monosubstituted counterparts, which is also an advantage that can
increase the light penetration depth [14].Scheme1
shows the synthetic route used to prepare the monosubstituted phthalocyanines 4 and 5. Reaction of benzyl alcohol 1 with 3- or 4-nitrophthalonitrile in the presence of K2CO3 in DMF gave the substituted phthalonitrile 2 or 3, respectively. These
compounds then underwent a mixed cyclization with an excess of the
unsubstituted phthalonitrile (10 equiv.) in the presence of Zn(OAc)2·2H2O
and DBU in n-pentanol to afford the
corresponding “3+1” products 4 and 5. These reactions also produced the unsubstituted ZnPc as a major
side-product, which could be separated readily by filtration and chromatography
as a result of its lower solubility and slower mobility in the silica gel
column. During the chromatographic
purification, a trace amount of some other blue products was also separated,
but no attempt was made to characterize these minor side-products. Similarly, treatment of 2,3-dicyanohydroquinone with benzyl chloride 6 and K2CO3 afforded dinitrile 7,
which was then cyclized with the unsubstituted phthalonitrile in the presence of Zn(OAc)2·2H2O
to give 8 (Scheme 2). All these zinc(II)
phthalocyanines were soluble in common organic solvents and possessed high
stability, which facilitated the purification by silica-gel column chromatography, size exclusion
chromatography, followed by recrystallization.Scheme 1Scheme 2


### 3.2. Spectroscopic Characterization and Photophysical Properties

All
the new compounds were fully
characterized with various spectroscopic methods. The NMR signals for the
phthalocyanine ring protons of4, 5, and 8 are very distinct in CDCl3 (with a trace amount of
pyridine-d5 for the former two complexes to reduce their
aggregation), which provide a useful means for characterization. As shown in Figure 1, the 1H NMR spectrum of the α-substituted phthalocyanine 4 shows a multiplet at δ 9.41–9.46 (5 H) and two doublets at δ 9.16 (1 H) and 9.13 (1 H) for the 7 phthalocyanine α protons. The 8 β protons resonate as a multiplet at δ 8.07–8.15 (7 H) and a doublet at δ 7.69 (1 H). For the β-analogue 5, a multiplet at δ 9.20–9.35 (6 H), a doublet at δ 9.05 (1 H), and a
singlet at δ 8.66 (1 H) are seen for the 8 phthalocyanine α protons, while the
signals for the 7 β protons appear as a multiplet at δ 8.06–8.12 (6 H) and a doublet at δ 7.62 (1 H). Phthalocyanine 8 has a
C2v symmetry. The doublet at δ 9.20 can be assigned
to the two phthalocyanine α ring protons close to the benzoxy groups,
while the multiplet at δ 9.43–9.47 is due to the remaining four α protons. The singlet at δ 7.68 can be readily
assigned to the two β protons adjacent to the benzoxy groups, while
the multiplet at δ 8.03–8.15 is attributed to the remaining six β protons.Figure 1
The aromatic region of the1H NMR spectra of 4, 5, and 8 in CDCl3; * indicates the
trace amount of pyridine-d5 added for the former two complexes.The13C NMR data of these compounds were
also in accord with the structures though some of the phthalocyanine ring
carbon signals (for 4 and 5) and the chain CH2 signals
were overlapped. For compound 8, a total of 20 signals were observed
in the aromatic region (δ 107.0–153.6) for the 16 phthalocyanine
and 4 benzene ring carbons, which is consistent with the C2v symmetry.The ESI mass
spectra of these phthalocyanines were also recorded. The molecular ion {[M + Na]+} isotopic cluster could be detected
in all the cases. The isotopic
distribution was in good agreement with the corresponding simulated pattern. The identity of these species was also
confirmed by accurate mass measurements.The electronic absorption and basic photophysical
data of phthalocyanines4, 5, and 8 were measured in DMF and are summarized in Table 1. All the three compounds gave very similar
UV-Vis spectra, which are  typical for nonaggregated phthalocyanines. The spectrum of compound 5, for example, showed the B-band at 344 nm, a vibronic band at 606 nm, and an intense and sharp Q-band at 672 nm, which strictly followed the
Lambert Beer's law (Figure 2). Upon
excitation at 610 nm, the compound is emitted at 677 nm with a fluorescence
quantum yield of 0.19. Substitution at the α position
(compound 4) slightly shifted the Q-band and
fluorescence emission to the red by 4-5 nm. Introduction of an additional α-substituent (compound 8) further shifted the Q-band to 690 nm
and the fluorescence emission to 696 nm.Table 1
Electronic absorption and photophysical data for4, 5, and 8 in DMF.
Compoundλmax (nm) (log ε)λem (nm)aΦFbΦΔc4334 (4.69), 611 (4.58), 677 (5.40)6810.200.605344 (4.79), 606 (4.62), 672 (5.39)6770.190.628337 (4.73), 621 (4.55), 690 (5.31)6960.140.84aExcited at 610 nm.bUsing unsubstituted zinc(II) phthalocyanine (ZnPc) in DMF as the reference (ΦF=0.28).cUsing ZnPc as
the reference (ΦΔ = 0.56 in
DMF).Figure 2
UV-Vis spectra of5 in DMF. The inset plots
the Q-band absorbance versus the concentration of 5.The singlet oxygen quantum yields (ΦΔ) of these
compounds  were also determined using 1,3-diphenylisobe-nzofuran (DPBF) as the
scavenger. The concentration of the quencher was
monitored spectroscopically at 411 nm along with time, from which the values of
ΦΔ could be determined by the method described previously [11]. These data are also summarized in Table 1. Figure 3 compares the rates of decay
of DPBF using these compounds and ZnPc as the photosensitizers. It can be seen that all these phthalocyanines
are efficient singlet oxygen generators, particularly the 1,4-disubstituted
analogue 8, of which the value of ΦΔ (0.84) is significantly higher than that of ZnPc
(0.56), which was used as the reference.Figure 3
Comparison of the rates of decay of DPBF in DMF, as
monitored spectroscopically at 411 nm, using phthalocyanines4, 5,
and 8 as the photosensitizers and
ZnPc as the reference.


### 3.3. In Vitro Photodynamic Activity

The in vitro
photodynamic activity of photosensitizers4, 5, and 8 in Cremophor EL emulsions was investigated against two different
cell lines, namely, HT29 human colorectal carcinoma
and HepG2 human hepatocarcinoma cells. As shown in Figure 4, the three compounds are essentially
noncytotoxic in the absence of light, but exhibit a very high
photocytotoxicity. The corresponding IC50 values are summarized in Table 2. It can
be seen that all these compounds are highly potent and the
effects on HT29 are greater than those on HepG2. The phthalocyanine 4 is particularly potent with the IC50 values down to 0.02 μM. About 1 μM of the dye is sufficient to kill 90% of the cells.Table 2
Comparison of the IC50 valuesa of phthalocyanines 4, 5, and 8 against HT29 and HepG2.
CompoundFor HT29 (μM)For HepG2 (μM)40.020.0350.360.3980.150.49aDefined as the dye concentration
required to kill 50% of the cells.Effects of4 (triangles), 5 (stars),
and 8 (squares) on (a) HT29 and (b)
HepG2 in the absence (closed symbols) and presence (open symbols) of light (λ > 610 nm, 40 mW cm-2, 48 J cm-2). Data are expressed as mean value ± SEM
of three independent experiments, each performed in quadruplicate.
(a)(b)It
is worth noting that although phthalocyanine4 exhibits a relatively lower singlet oxygen quantum yield than the
other two analogues in DMF (Table 1), its photocytotoxicity is the highest
among the three photosensitizers (Table 2). To account for the results, the absorption spectra of these
compounds in the culture media were recorded. As shown in Figure 5, the Q-band for compound 4 in the DMEM medium (for HT29) remains very sharp and intense,
while those for 5 and 8 are weaker and broadened. Very similar results were obtained in the
RPMI medium (for HepG2). This is a strong
indication that compound 4 is
significantly less aggregated in these media, which should lead to a higher
photosensitizing efficiency.Figure 5
Electronic absorption spectra of4 (solid line), 5 (dashed line), and
8 (dotted line), formulated with Cremophor EL, in the DMEM culture
medium (all at 8 μM).To further explain
the photocytotoxicity results, fluorescence microscopic studies were also
carried out to shed light on the cellular uptake of  photosensitizers4, 5,
and 8. After incubation with these compounds
(formulated with Cremophor EL) for 2 hours and upon excitation at 630 nm, the HT29
cells showed intracellular fluorescence throughout
the cytoplasm as shown in Figure 6,
indicating that there
was a substantial uptake of the dyes. The qualitative fluorescence intensity
follows the order 4 > 5 > 8, suggesting
that the α-substituted
phthalocyanine 4 also has the highest uptake among the three
photosensitizers. This may also account
for the highest photocytotoxicity of this compound.Fluorescence microscopic images of HT29 tumor cells
after incubation with (a)4, (b) 5, and (c) 8 at a concentration of 8 μM for 2 hours.
(a)(b)(c)In conclusion, we
have prepared and characterized three novel “3+1” zinc(II) phthalocyanines
substituted with one or two 3,4,5-tris(3,6,9-trioxadecoxy)benzoxy group(s). These compounds exhibit a high
photocytotoxicity against HT29 and HepG2 cells with IC50 values down
to 0.02 μM. The mono-α-substituted phthalocyanine 4 is more potent than the other two analogues, which can be partly
explained by its lower aggregation tendency in the culture media and higher
cellular uptake as shown by absorption spectroscopy and fluorescence
microscopy, respectively.


## 3.1. Molecular Design and Chemical Synthesis

Zinc(II) phthalocyanines are good candidates
for PDT application. In addition to
their relatively high stability, the closed-shell zinc(II) center imparts desirable photophysical characteristics to the
macrocycles [13]. Introduction of substituents
at the peripheral positions can also tailor the properties of the macrocycles such as their
solubility in biological media, aggregation behavior, and targeting properties. As a
result, zinc(II) phthalocyanines have received considerable attention as
efficient photosensitizers [5]. We
describe herein three novel zinc(II) phthalocyanines (compounds 4, 5, and 8) which contain the
bulky and hydrophilic 3,4,5-tris(3,6,9-trioxadecoxy)benzoxy
moiety. Having one or two of these substituents, the π-π stacking tendency is reduced and the macrocycles
become amphiphilic in nature. These
properties should be advantageous for singlet oxygen generation and cellular
uptake, by which the photodynamic activity can be enhanced. The relatively rare 1,4-disubstituted phthalocyanine 8 also has a longer Q-band maximum
compared with the α-
and β-monosubstituted counterparts, which is also an advantage that can
increase the light penetration depth [14].Scheme1
shows the synthetic route used to prepare the monosubstituted phthalocyanines 4 and 5. Reaction of benzyl alcohol 1 with 3- or 4-nitrophthalonitrile in the presence of K2CO3 in DMF gave the substituted phthalonitrile 2 or 3, respectively. These
compounds then underwent a mixed cyclization with an excess of the
unsubstituted phthalonitrile (10 equiv.) in the presence of Zn(OAc)2·2H2O
and DBU in n-pentanol to afford the
corresponding “3+1” products 4 and 5. These reactions also produced the unsubstituted ZnPc as a major
side-product, which could be separated readily by filtration and chromatography
as a result of its lower solubility and slower mobility in the silica gel
column. During the chromatographic
purification, a trace amount of some other blue products was also separated,
but no attempt was made to characterize these minor side-products. Similarly, treatment of 2,3-dicyanohydroquinone with benzyl chloride 6 and K2CO3 afforded dinitrile 7,
which was then cyclized with the unsubstituted phthalonitrile in the presence of Zn(OAc)2·2H2O
to give 8 (Scheme 2). All these zinc(II)
phthalocyanines were soluble in common organic solvents and possessed high
stability, which facilitated the purification by silica-gel column chromatography, size exclusion
chromatography, followed by recrystallization.Scheme 1Scheme 2


## 3.2. Spectroscopic Characterization and Photophysical Properties

All
the new compounds were fully
characterized with various spectroscopic methods. The NMR signals for the
phthalocyanine ring protons of4, 5, and 8 are very distinct in CDCl3 (with a trace amount of
pyridine-d5 for the former two complexes to reduce their
aggregation), which provide a useful means for characterization. As shown in Figure 1, the 1H NMR spectrum of the α-substituted phthalocyanine 4 shows a multiplet at δ 9.41–9.46 (5 H) and two doublets at δ 9.16 (1 H) and 9.13 (1 H) for the 7 phthalocyanine α protons. The 8 β protons resonate as a multiplet at δ 8.07–8.15 (7 H) and a doublet at δ 7.69 (1 H). For the β-analogue 5, a multiplet at δ 9.20–9.35 (6 H), a doublet at δ 9.05 (1 H), and a
singlet at δ 8.66 (1 H) are seen for the 8 phthalocyanine α protons, while the
signals for the 7 β protons appear as a multiplet at δ 8.06–8.12 (6 H) and a doublet at δ 7.62 (1 H). Phthalocyanine 8 has a
C2v symmetry. The doublet at δ 9.20 can be assigned
to the two phthalocyanine α ring protons close to the benzoxy groups,
while the multiplet at δ 9.43–9.47 is due to the remaining four α protons. The singlet at δ 7.68 can be readily
assigned to the two β protons adjacent to the benzoxy groups, while
the multiplet at δ 8.03–8.15 is attributed to the remaining six β protons.Figure 1
The aromatic region of the1H NMR spectra of 4, 5, and 8 in CDCl3; * indicates the
trace amount of pyridine-d5 added for the former two complexes.The13C NMR data of these compounds were
also in accord with the structures though some of the phthalocyanine ring
carbon signals (for 4 and 5) and the chain CH2 signals
were overlapped. For compound 8, a total of 20 signals were observed
in the aromatic region (δ 107.0–153.6) for the 16 phthalocyanine
and 4 benzene ring carbons, which is consistent with the C2v symmetry.The ESI mass
spectra of these phthalocyanines were also recorded. The molecular ion {[M + Na]+} isotopic cluster could be detected
in all the cases. The isotopic
distribution was in good agreement with the corresponding simulated pattern. The identity of these species was also
confirmed by accurate mass measurements.The electronic absorption and basic photophysical
data of phthalocyanines4, 5, and 8 were measured in DMF and are summarized in Table 1. All the three compounds gave very similar
UV-Vis spectra, which are  typical for nonaggregated phthalocyanines. The spectrum of compound 5, for example, showed the B-band at 344 nm, a vibronic band at 606 nm, and an intense and sharp Q-band at 672 nm, which strictly followed the
Lambert Beer's law (Figure 2). Upon
excitation at 610 nm, the compound is emitted at 677 nm with a fluorescence
quantum yield of 0.19. Substitution at the α position
(compound 4) slightly shifted the Q-band and
fluorescence emission to the red by 4-5 nm. Introduction of an additional α-substituent (compound 8) further shifted the Q-band to 690 nm
and the fluorescence emission to 696 nm.Table 1
Electronic absorption and photophysical data for4, 5, and 8 in DMF.
Compoundλmax (nm) (log ε)λem (nm)aΦFbΦΔc4334 (4.69), 611 (4.58), 677 (5.40)6810.200.605344 (4.79), 606 (4.62), 672 (5.39)6770.190.628337 (4.73), 621 (4.55), 690 (5.31)6960.140.84aExcited at 610 nm.bUsing unsubstituted zinc(II) phthalocyanine (ZnPc) in DMF as the reference (ΦF=0.28).cUsing ZnPc as
the reference (ΦΔ = 0.56 in
DMF).Figure 2
UV-Vis spectra of5 in DMF. The inset plots
the Q-band absorbance versus the concentration of 5.The singlet oxygen quantum yields (ΦΔ) of these
compounds  were also determined using 1,3-diphenylisobe-nzofuran (DPBF) as the
scavenger. The concentration of the quencher was
monitored spectroscopically at 411 nm along with time, from which the values of
ΦΔ could be determined by the method described previously [11]. These data are also summarized in Table 1. Figure 3 compares the rates of decay
of DPBF using these compounds and ZnPc as the photosensitizers. It can be seen that all these phthalocyanines
are efficient singlet oxygen generators, particularly the 1,4-disubstituted
analogue 8, of which the value of ΦΔ (0.84) is significantly higher than that of ZnPc
(0.56), which was used as the reference.Figure 3
Comparison of the rates of decay of DPBF in DMF, as
monitored spectroscopically at 411 nm, using phthalocyanines4, 5,
and 8 as the photosensitizers and
ZnPc as the reference.


## 3.3. In Vitro Photodynamic Activity

The in vitro
photodynamic activity of photosensitizers4, 5, and 8 in Cremophor EL emulsions was investigated against two different
cell lines, namely, HT29 human colorectal carcinoma
and HepG2 human hepatocarcinoma cells. As shown in Figure 4, the three compounds are essentially
noncytotoxic in the absence of light, but exhibit a very high
photocytotoxicity. The corresponding IC50 values are summarized in Table 2. It can
be seen that all these compounds are highly potent and the
effects on HT29 are greater than those on HepG2. The phthalocyanine 4 is particularly potent with the IC50 values down to 0.02 μM. About 1 μM of the dye is sufficient to kill 90% of the cells.Table 2
Comparison of the IC50 valuesa of phthalocyanines 4, 5, and 8 against HT29 and HepG2.
CompoundFor HT29 (μM)For HepG2 (μM)40.020.0350.360.3980.150.49aDefined as the dye concentration
required to kill 50% of the cells.Effects of4 (triangles), 5 (stars),
and 8 (squares) on (a) HT29 and (b)
HepG2 in the absence (closed symbols) and presence (open symbols) of light (λ > 610 nm, 40 mW cm-2, 48 J cm-2). Data are expressed as mean value ± SEM
of three independent experiments, each performed in quadruplicate.
(a)(b)It
is worth noting that although phthalocyanine4 exhibits a relatively lower singlet oxygen quantum yield than the
other two analogues in DMF (Table 1), its photocytotoxicity is the highest
among the three photosensitizers (Table 2). To account for the results, the absorption spectra of these
compounds in the culture media were recorded. As shown in Figure 5, the Q-band for compound 4 in the DMEM medium (for HT29) remains very sharp and intense,
while those for 5 and 8 are weaker and broadened. Very similar results were obtained in the
RPMI medium (for HepG2). This is a strong
indication that compound 4 is
significantly less aggregated in these media, which should lead to a higher
photosensitizing efficiency.Figure 5
Electronic absorption spectra of4 (solid line), 5 (dashed line), and
8 (dotted line), formulated with Cremophor EL, in the DMEM culture
medium (all at 8 μM).To further explain
the photocytotoxicity results, fluorescence microscopic studies were also
carried out to shed light on the cellular uptake of  photosensitizers4, 5,
and 8. After incubation with these compounds
(formulated with Cremophor EL) for 2 hours and upon excitation at 630 nm, the HT29
cells showed intracellular fluorescence throughout
the cytoplasm as shown in Figure 6,
indicating that there
was a substantial uptake of the dyes. The qualitative fluorescence intensity
follows the order 4 > 5 > 8, suggesting
that the α-substituted
phthalocyanine 4 also has the highest uptake among the three
photosensitizers. This may also account
for the highest photocytotoxicity of this compound.Fluorescence microscopic images of HT29 tumor cells
after incubation with (a)4, (b) 5, and (c) 8 at a concentration of 8 μM for 2 hours.
(a)(b)(c)In conclusion, we
have prepared and characterized three novel “3+1” zinc(II) phthalocyanines
substituted with one or two 3,4,5-tris(3,6,9-trioxadecoxy)benzoxy group(s). These compounds exhibit a high
photocytotoxicity against HT29 and HepG2 cells with IC50 values down
to 0.02 μM. The mono-α-substituted phthalocyanine 4 is more potent than the other two analogues, which can be partly
explained by its lower aggregation tendency in the culture media and higher
cellular uptake as shown by absorption spectroscopy and fluorescence
microscopy, respectively.



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*Source: 284691-2007-12-02.xml*