Peripheral and axial substitution of phthalocyanines with solketal groups: Synthesis and in vitro evaluation for photodynamic therapy

Article abstract of DOI:10.1021/jm061136w

Phthalocyanines (Pcs) are a class of photosensitizers (PSs) with a strong tendency to aggregate in aqueous environment, which has a negative influence on their photosensitizing ability in photodynamic therapy. Pcs with either peripheral or axial solketal substituents, that is, ZnPc(sol)₈ and Si(sol)₂Pc, respectively, were synthesized and their tendency to aggregate as well as their photodynamic properties in 14C and B16F10 cell lines were evaluated. The results were compared to more hydrophilic silicon Pcs, that is, Si(PEG750)₂Pc and Pc4. The order of cellular uptake was Pc4 > ZnPc(sol)₈ > Si(PEG750)₂Pc > Si(sol₂)Pc. In contrast, Si(sol₂)Pc showed the highest photocytotoxicity, while ZnPc(sol)₈ did not show any photocytotoxicity up to a concentration of 10 μM in both cell types. UV/vis spectroscopy showed that Si(sol) ₂Pc is less prone to aggregation than ZnPc(sol)₈, which can explain the lack of photoactivity of the latter. Si(sol)₂Pc was predominantly located in lipid droplets, whereas Si(PEG750)₂Pc was homogeneously distributed in the cytosol, which is probably the main cause of their difference in photoactivity. The very high photodynamic efficacy of Si(sol)₂Pc makes this PS an interesting candidate for future studies.

Full text of DOI:10.1021/jm061136w

J. Med. Chem. 2007, 50, 1485-1494
1485
Peripheral and Axial Substitution of Phthalocyanines with Solketal Groups: Synthesis and In
Vitro Evaluation for Photodynamic Therapy
Jan-Willem Hofman,† Femke van Zeeland,† Selcan Turker,† Herre Talsma,† Saskia A. G. Lambrechts,† Dmitri V. Sakharov,‡
Wim E. Hennink,† and Cornelus F. van Nostrum*,†
Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences (UIPS), Utrecht UniVersity, Post Office Box 80082,
3508 TB Utrecht, The Netherlands, and Department of Biochemistry of Lipids, CBLE, Utrecht UniVersity, Post Office Box 80054,
3508 TB Utrecht, The Netherlands
ReceiVed September 29, 2006
Phthalocyanines (Pcs) are a class of photosensitizers (PSs) with a strong tendency to aggregate in aqueous
environment, which has a negative influence on their photosensitizing ability in photodynamic therapy. Pcs
with either peripheral or axial solketal substituents, that is, ZnPc(sol)8 and Si(sol)2Pc, respectively, were
synthesized and their tendency to aggregate as well as their photodynamic properties in 14C and B16F10
cell lines were evaluated. The results were compared to more hydrophilic silicon Pcs, that is, Si(PEG750)2Pc
and Pc4. The order of cellular uptake was Pc4 > ZnPc(sol)8 > Si(PEG750)2Pc > Si(sol2)Pc. In contrast,
Si(sol2)Pc showed the highest photocytotoxicity, while ZnPc(sol)8 did not show any photocytotoxicity up to
a concentration of 10 µM in both cell types. UV/vis spectroscopy showed that Si(sol)2Pc is less prone to
aggregation than ZnPc(sol)8, which can explain the lack of photoactivity of the latter. Si(sol)2Pc was
predominantly located in lipid droplets, whereas Si(PEG750)2Pc was homogeneously distributed in the cytosol,
which is probably the main cause of their difference in photoactivity. The very high photodynamic efficacy
of Si(sol)2Pc makes this PS an interesting candidate for future studies.
Chart 1. Structural Formula of Pc4
Introduction
Photodynamic therapy (PDT) is an attractive modality for
the treatment of cancer.1 It involves the illumination of a
photosensitive compound with light of an appropriate wave-
length, that is, in the range of 630-800 nm for which tissue
penetration is optimal.2 By directing the light beam to the target
area, PDT can be used to selectively destroy cancer tissue
without harming surrounding cells. The light-activated photo-
sensitizer (PS) can form reactive oxygen species (ROS) via
either a so-called type I reaction or by direct reaction with
molecular oxygen by a so-called type II reaction to form highly
active singlet oxygen.2 ROS are oxidizing agents that react with
cellular components, such as unsaturated lipids, RNA/DNA, or
side chains of certain amino acids, like cystein, methionine,
tyrosine, histidine, and tryptophan.3,4 These oxidative effects
ultimately result in cell death. Singlet oxygen is generally
excepted as the main species responsible for the observed
photodynamic effect.5
Porfimer sodium (Photofrin), a complex mixture of hemato-
porphyrin derivatives, was the first clinically approved PS for
photodynamic treatment of bladder cancer in 1993 in Canada.1,6
Although encouraging results have been obtained, it has some
serious limitations. The molecular absorption coefficient of
porfimer sodium at the clinically used wavelength of 630 nm
is low (ꢀ ) 1170 l‚mol-1‚cm-1).6 More importantly, it causes
skin photosensitivity for a prolonged period of time, up to 30-
90 days after treatment, due to its accumulation and retention
in skin tissues.7 Therefore, much research has been devoted to
developing new photosensitive compounds with improved
photophysical efficiency and fewer side effects. To achieve
optimal efficacy, a PS should meet several requirements,4-6,8
including (i) high absorbance at wavelengths >600 nm, (ii) low
tendency to aggregate intracellularly to achieve a maximal yield
of singlet oxygen, and (iii) favorable intracellular localization
properties, for example, in mitochondrial membranes,4,9 because
the diffusion range of singlet oxygen is small. Phthalocyanines
(Pcs) are very well suited to be used as PSs for PDT due to
their high molecular absorption coefficients at higher wave-
lengths of the visible spectrum (ꢀ ≈ 105 l‚mol-1‚cm-1 at 640-
710 nm), lack of dark toxicity, and chemical stability. Pcs used
for PDT include the zinc-phthalocyanine (ZnPc),10 aluminum-
phthalocyanine (AlPc),11,12 and silicon-phthalocyanine (SiPc)13,14
derivatives. However, these compounds often show aggregation
in aqueous media and have unfavorable intracellular localization.
To prevent stacking of the hydrophobic Pc core and intracellular
aggregation, those compounds can be substituted with bulky or
charged groups at the periphery of the Pc ring.15-17 Furthermore,
incorporation of a tri- or tetravalent metal atom in the Pc core,
like aluminum(III) or silicon(IV), enables axial derivatization,
which can affect the solubility in aqueous media even more by
improved steric shielding of the Pc core.12,14,18 Pc419-22 (Chart
1), an axially substituted Pc that localizes intracellularly in
mitochondria, is known as one of the most potent Pc-based PSs
and has been used in clinical trials.23
* To whom correspondence should be addressed. Phone: +31(0)30-
2537306. Fax: +31(0)30-2517839. E-mail: c.f.vannostrum@pharm.uu.nl.
† Department of Pharmaceutics.
No studies to date have been directed to the comparison of
Pcs substituted with the same groups either on the axial or
‡ Department of Biochemistry of Lipids.
10.1021/jm061136w CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/10/2007

1486 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Hofman et al.
Scheme 1. Synthesis Route to the Peripherally Substituted ZnPc(sol)8
Scheme 2. Synthesis Route to the Axially Substituted Si(sol)2Pc and Si(PEG750)2Pc
peripheral positions. In this study, two Pcs either peripherally
or axially substituted with solketal groups were synthesized,
that is, ZnPc(sol)8 (1) and Si(sol)2Pc (2), respectively (Schemes
1 and 2). Solketal, which is a hydrophobic unit that can be
converted, in vivo, by acid-catalyzed hydrolysis to form
hydrophilic glyceryl,24 was chosen with the aim to combine the
favorable tumor uptake of hydrophobic PSs with the expected
rapid clearance, for example, from the skin, once converted to
the hydrophilic counterpart. In this study, the cellular uptake,
intracellular localization, and overall photodynamic efficacy of
the two hydrophobic Pc derivatives were investigated and
compared with two hydrophilic SiPcs, that is, Si(PEG750)2Pc
(3), containing polyethylene glycol (PEG) chains, and Pc4.
which was first brominated using elemental bromine in tetra-
chloromethane.25 The product (4) was used without further
purification for the reaction with allylbromide to form 5. This
compound was isolated as a crystalline solid or as an oil, which
solidified in a few hours. Next, the allyl groups were dihy-
droxylated using osmium tetraoxide as a catalyst in a tert-
butanol/water (1:1) mixture. This reaction was done in the
presence of 4-methylmorpholine-4-oxide, to regenerate the
catalyst, and citric acid, as suggested by Dupau et al.26 Citric
acid was added as pH modifier, and it was hypothesized that it
can also act as a ligand for osmium, stabilizing the catalytically
active Os(IV) species in solution against disproportionation to
Os(VIII) and insoluble Os(IV) species.
The bis(dihydroxylated) product (6) was formed in an
excellent yield (95%), with a high purity (>99%), as was
established by NMR and HPLC. The OH groups of 6 were
reacted with 2,2-dimethoxypropane (DMP) to form 7. Nitrile
Results
Synthesis. The peripherally substituted ZnPc(sol)8 was
synthesized according to Scheme 1, starting from catechol,

Substitution of Phthalocyanines with Solketal Groups
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1487
Figure 2. UV-vis spectra of Si(sol)2Pc (-), ZnPc(sol)8 (- - -), and
Si(PEG750)2Pc (...), 5 µM in DMF.
Figure 1. 1H NMR of Si(PEG750)2Pc in CDCl3. Methylene groups
of the PEG chain closest to the Pc ring have the higest upfield shift.
See Experimental Section for assignment of the peaks.
as solvent, good resolution was obtained in the 1H NMR
spectrum and the core signals were visible in the 13C NMR
spectrum. ZnPc(sol)8 is probably aggregated in CDCl3, yielding
a colloidal solution, at the concentrations used for the NMR
measurements, whereas in DMSO-d6, it is molecularly dissolved.
groups were introduced, according to the Rosenmund-von
Braun reaction, by substitution of the bromine groups of 7 with
CuCN in DMF, which yielded phthalonitrile derivative 8 in a
reasonable yield (59%). The 2,3,9,10,16,17,23,24-octasolketal-
substituted Pc ZnPc(sol)8 (1) was formed in a cyclotetramer-
ization reaction of 8 in dimethylethanolamine as solvent in the
presence of Zn(OAc)2. The crude product was purified by flash
chromatography with gradient elution using a mixture of EtOAc
and hexane as eluents. Shifting the eluent from EtOAc/hexane
(2:1) to EtOAc after removal of side products caused the elution
of the product. The overall yield starting from catechol was
31%.
UV/vis spectroscopy was used to investigate in more detail
the aggregation behavior of Si(sol)2Pc and ZnPc(sol)8 at relevant
concentrations (µM range). Aggregation was induced by titration
of a solution of PS in DMF with H2O. The absorption spectra
of ZnPc(sol)8, Si(sol)2Pc, and Si(PEG750)2Pc in DMF showed
a B (or Soret) band at 330-370 nm and a strong absorption
peak (Q-band) at 674 nm (Figure 2), together with two
vibrational bands at 603 and 639 nm, characteristic of nonag-
gregated Pcs. The change in the absorbance of the Q-band in
the UV-vis spectrum upon addition of water was recorded and
plotted against the dielectric constant of the solvent mixture
(see Figure 3). Figure 3c shows that the critical dielectric
constant of the solvent mixture above which aggregation
occurred is ∼45 and 58 for ZnPc(sol)8 and Si(sol)2Pc, respec-
tively. This demonstrates that, as anticipated, axially substituted
Si(sol)2Pc has a lower tendency for stacking and aggregation
than the peripherally substituted ZnPc(sol)8.
Photocytotoxicity. The concentration-dependent phototox-
icity of ZnPc(sol)8, Si(sol)2Pc, and Si(PEG750)2Pc toward
B16F10 and 14C cells was determined after 6 h of incubation
and subsequent illumination with a light intensity of 3.5
mW‚cm-2 for 10 min (Figure 4). Under these conditions, the
light dose was 2.1 J‚cm-2. None of the PSs showed dark
cytotoxicity (Figure 4). ZnPc(sol)8 did not show photocytotox-
icity up to the highest concentration used (10 µM, not shown
in the figure). The IC50 values (i.e., concentration of PS added
to the medium during incubation, at which illumination resulted
in 50% cell death) of the other PSs are reported in Table 1,
together with the values of Pc4. Si(sol)2Pc is approximately 1
order of magnitude more effective than Si(PEG750)2Pc and two
to three times more effective than Pc4 in both cell lines.
Cellular Uptake. The uptake of ZnPc(sol)8, Si(sol)2Pc, Si-
(PEG750)2Pc, and Pc4 by B16F10 and 14C cells was investi-
gated. The amount of PS that was taken up was determined by
fluorescence spectrometry after cell lysis and expressed relative
to the cellular protein amount (Table 1). For both cell lines, the
amount of ZnPc(sol)8 taken up was about ten times higher than
for Si(sol)2Pc and Si(PEG750)2Pc. Table 1 also shows that the
uptake of Pc4 was about 20 times higher than for Si(sol)2Pc
and Si(PEG750)2Pc. To establish whether an energy-dependent
process is responsible for the uptake of Si(sol)2Pc, ZnPc(sol)8,
and Si(PEG750)2Pc by B16F10 cells, the same experiments were
carried out at 4 °C. At this temperature, energy-dependent
processes are inhibited and compounds can penetrate into the
Si(Cl)2Pc was synthesized starting from phthalonitril (Scheme
2), which was allowed to react with ammonia to form 1,3-
diiminoisoindoline (9), according to Sasa et al.27 Si(Cl)2Pc was
formed by reaction of 9 with SiCl4 in tetraline, according to
Davison et al.,28 in a yield of 70%. The identity of the product
was confirmed by comparison of its FTIR spectrum with that
of the commercial-derived product (Sigma). Si(sol)2Pc (2) was
synthesized by substitution of Si(Cl)2Pc with solketal in toluene.
Si(PEG750)2Pc (3) was synthesized according to Huang et
al.,29 that is, Si(Cl)2Pc was reacted with monomethoxy PEG750
and NaH in toluene. However, work up of the product by
extraction with H2O/EtOAc, to remove excess of free PEG, was
not effective in our hands. After extensive purification with flash
chromatography, the product was obtained as a blue paste.
Characterization. The synthesized Pcs had high purity
(>97%) according to NMR, HPLC, and elemental analysis. The
PEGylated Pc had a good solubility in water (>5 mg/mL),
whereas ZnPc(sol)8 and Si(sol)2Pc did not show detectable
dissolution in water. 1H NMR spectra recorded in CDCl3 clearly
showed the shielding of the substituent protons by the electron
cloud of the Pc ring in the axially substituted SiPcs, that is,
Si(sol)2Pc and Si(PEG750)2Pc. The closer the atoms of the axial
substituents are to the point of attachment to the Pc ring, the
larger the upfield shift of the group. For Si(PEG750)2Pc, this
effect was visible from the closest methylene group (δ -1.95
ppm) up to nine methylene units away (Figure 1), which was
in agreement with previously reported data for this com-
pound.29,30 The same phenomenon was also observed for the
solketal substituents of Si(sol)2Pc, where the methylene unit
1
closest to the ring had a shift of -2.0 ppm. The H NMR
spectrum of ZnPc(sol)8 in CDCl3 showed very broad and
unresolved peaks, as was also shown by Kimura et al.31
Furthermore, the signals from the aromatic Pc ring were missing
in the 13C NMR spectrum. However, when DMSO-d6 was used

1488 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Hofman et al.
Figure 3. Selected absorption spectra of Si(sol)2Pc (a) and ZnPc(sol)8 (b) upon titration of a PS solution (5 µM) in DMF with H2O. (c) Decrease
of Amax of the Q-band of Si(sol)2Pc (squares) and ZnPc(sol)8 (triangles) as a function of the dielectric constant of the solvent mixture. The absorbances
were corrected for the shift in concentration due to addition of H2O.
Figure 4. Relative cell viability after 6 h of incubation with PS at various concentrations in the medium, followed by illumination for 10 min with
3.5 mW (open symbols) and without illumination (closed symbols) of (a) ZnPc(sol)8, (b) Si(sol)2Pc, and (c) Si(PEG750)2Pc toward B16F10 cells
(squares) and 14C cells (circles). Values are expressed as the average of three experiments ( SEM (n ) 3).
cells only by passive diffusion. For Si(sol)2Pc and ZnPc(sol)8,
no significant differences in uptake were found between the
experiment run at 4 °C and the experiment run at 37 °C. The
cellular uptake of Si(PEG750)2Pc at 4 °C was ∼3 times lower
than the uptake at 37 °C.
Intracellular Localization. Confocal fluorescence micros-
copy was used to examine qualitatively the distribution kinetics
and intracellular localization of ZnPc(sol)8, Si(PEG750)2, and
Si(sol)2Pc in both B16F10 and 14C cells. The cells were
incubated with 1 µM solutions of the PSs for 6 h, and the

Substitution of Phthalocyanines with Solketal Groups
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1489
Table 1. Photocytotoxicity and Cell Uptake of Four PSs for B16F10
and 14C Cells
cell uptakea (nmol/mg protein)
IC50b (µM)
sensitizer
B16F10
14C
B16F10
14C
ZnPc(sol)8
Si(sol)2Pc
Si(PEG750)2Pc 0.94 ( 0.10
Pc4
12.5 ( 1.0
10.6 ( 0.3
0.88 ( 0.19
1.17 ( 0.03
n.d.
n.a.c
0.006
0.17
n.a.c
0.003
0.09
0.66 ( 0.11
17.8 ( 1.5
0.019
0.006
a Cells were incubated with PS (10 µM) for 6 h. The dye content was
analyzed as described under Experimental Section. Values are ( SEM (n
) 3). b The IC50 is the concentration of PS added to the medium during 6
h of incubation, at which subsequent illumination (10 min, 3.5 mW) resulted
in 50% reduced cell viability relative to cells without PS added. c Cells did
not show any reduced viability after illumination up to the highest
concentration tested (10 µM).
fluorescence was examined at different time points. After 6 h,
the incubation medium was removed, and the cells were washed
twice with PBS and again visualized after illumination of the
cells (10 min, 3.5 mW/cm2). Cells incubated with ZnPc(sol)8,
at concentrations up to 10 µM, showed no intracellular
fluorescence. For Si(PEG750)2Pc, a more or less homogeneous
distribution in the cytosol of both cell types was observed
(Figure 5a). Illumination did not result in a detectable different
intracellular distribution. Si(sol)2Pc showed a different distribu-
tion pattern in both cell types as compared to Si(PEG750)2Pc,
that is, fluorescence was mainly observed in spots (most clearly
visible in 14C cells; Figure 5b). The fluorescence intensity of
these spots increased in time during incubation. Illumination
after 6 h of incubation caused an increased intensity of the
fluorescence and a more homogeneous distribution pattern
(Figure 5c), indicating redistribution of the PS in the cells. To
investigate the identity of the spots observed in the Si(sol)2Pc
treated cells, the cells were also incubated with several
fluorescent dyes to stain different organelles, that is, mitochon-
dria, lysosomes, endosomes, and lipid droplets. No colocaliza-
tion of Si(sol)2Pc and the fluorescent dye was observed for
MitoTracker green, LysoTracker green, and FITC-dextran,
suggesting that the PS did not localize in mitochondria,
lysosomes, and endosomes, respectively (Figure 5d-f). How-
ever, when B16F10 or 14C cells, preincubated with Si(sol)2Pc,
Figure 6. Colocalization of Si(sol)2Pc and nile red in 14C cells after
6 h of incubation with PS (1 µM): (a) nile red fluorescence; (b)
fluorescence of Si(sol)2Pc; (c) overlay of nile red and Si(sol)2Pc
fluorescence; and (d) DIC photo of the cells.
were stained with nile red, colocalization of the PS and the dye
was observed (Figure 6a-c). The spots were also observed in
differential interference contrast (DIC) mode (Figure 6d).
Greenspan et al.32 showed that nile red preferentially stains lipid
droplets in cells. Therefore, colocalization of Si(sol)2Pc with
the dye indicates that the PS is primarily localized in these
hydrophobic organelles and redistributes upon illumination.
Discussion
The lack of water solubility of ZnPc(sol)8 and Si(sol)2Pc
emphasizes the hydrophobicity of these compounds. In contrast,
the hydrophilic PEG chains of Si(PEG750)2Pc are very effective
in shielding the hydrophobic Pc core, making the PS much more
Figure 5. Intracellular localization of Si(PEG750)2Pc (a) and Si(sol)2Pc (b-f) in 14C cells after 6 h of incubation with 1 µM PS: (c) illuminated
after incubation; (d) incubated with MitoTracker green; (e) incubated with LysoTracker green; and (f) incubated with FITC-dextran. PSs and dyes
are presented in blue and green, respectively.

1490 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Hofman et al.
hydrophilic and highly soluble in water. Aggregation, due to
π-π stacking of the aromatic Pc rings, is frequently observed
for Pcs.25,33 Singlet oxygen quantum yields have been shown
to be directly related to the aggregation state of the PS, that is,
the quantum yield is low when the sensitizer is in an aggregated
form.34,35 Also, the fluorescence of the PS will be reduced
accordingly. Substitution of the macrocycle with bulky groups
can reduce aggregation tendency by shielding of the Pc core.16,36
Moreover, axial substitution may be even more effective in
reducing the formation of these stacks.13,18 Indeed, Figure 3
shows that ZnPc(sol)8 has a stronger tendency to aggregate as
compared to Si(sol)2Pc, demonstrating the higher efficiency of
axial substitution in reducing the aggregation behavior. The
strong aggregation tendency of ZnPc(sol)8 can explain why it
could not be visualized intracellularly by LSCFM, despite its
high uptake (Table 1), and why it did not show any photocy-
totoxicity upon illumination. Unlike ZnPc(sol)8, both Si(sol)2Pc
and Si(PEG750)2Pc could be visualized by LSCFM and were
photoactive, indicating that these PSs are not in an aggregated
state inside the cell and that the axial substituents are effective
in preventing stacking of the Pc rings.
specific location of the PS inside the cell in order to be able to
induce cell death. Localization of PSs in membranes of
organelles like lysosomes or mitochondria have been reported
to be most effective.9,42 Pc4 mainly localizes in mitochondrial
membranes,43 which is one of the main factors contributing to
its high photodynamic efficacy. Si(sol)2Pc was found to be
localized in lipid droplets. These compartments have been
reported to be involved in PS accumulation only on very few
occasions.44,45 However, those PSs are chlorin derivatives, and
Pc-based compounds have not been explicitly mentioned before
in this context. Interestingly, Lee et al.14 also observed a spotted
pattern in HepG2 cells, much resembling our microscopic
images, for a similar SiPc containing axial isopropylidene-
protected galactose substituents. By incubation with MitoTracker
green, they showed only minor colocalization with mitochondria.
It is therefore very well possible that localization of the PS in
lipid droplets was largely responsible for the pattern that they
observed. In mammalian cells, lipid droplets are cell organelles
that consist of neutral lipids surrounded by a monolayer of
phospholipids and associated proteins.46,47 Among the membrane
proteins found in lipid droplets are caveolin-1 and caveolin-
2.48 These proteins are normally found as structural components
of caveolae,49 a class of clathrin-independent vesicles involved
in receptor-mediated endocytosis,50 which may suggest that Si-
(sol)2Pc is actively taken up by the cells. However, as no
difference in cellular uptake was observed between 4 °C and
37 °C, it is unlikely that the Si(sol)2Pc was taken up by active
transport. Time-dependent uptake studies have shown that it
took 6 h of incubation at 37 °C to reach equilibrium, which
was the standard incubation period in our experiments (results
not shown). If possible alterations in membrane properties
(increased membrane rigidity, for example) would have played
a role in the diffusion-driven uptake rate at 4 °C, one would
expect that equilibrium is obtained more slowly at the lower
temperature. This was likely not the case, because we observed
similar uptake after 6 h at 4 °C and at 37 °C. Probably,
intracellular complexation of the PS with certain proteins or
other lipid droplet constituents is responsible for the observed
localization of the PS in lipid droplets.
When evaluating the IC50 values of the tested PSs, Si(sol)2Pc
appears about 25 times more effective in inducing cell death
upon illumination than Si(PEG750)2Pc in both B16F10 and 14C
cells. The photocytotoxicity of Si(PEG750)2Pc is comparable
to values reported previously for axially substituted SiPcs.13,14,37
For example, Huang et al.29 tested the same compound in HepG2
cells and found an IC50 concentration of 0.75 µM, which is about
the same IC50 value we found for this compound in B16F10
and 14C cells. Interestingly, when Si(sol)2Pc and Pc4 are
compared, they showed similar photodynamic activity (IC50
values were comparable). Because the site of PS activity is
supposed to be located intracellularly, as mentioned in the
Introduction section, intracellular concentrations are more
relevant as a measure for phototoxicity than the concentrations
of PS added to the medium in the cell cultures (i.e., the IC50
values). However, the intracellular concentrations at the amounts
of PS used in the phototoxicity studies are so low that it is
impossible to measure intracellular concentrations. Therefore,
just to get an indication about the cellular uptake, we measured
fluorescence of cell lysates at much higher concentrations of
added PS (i.e., 10 µM). The uptake of Si(sol)2Pc and Si-
(PEG750)2Pc by B16F10 and 14C cells was similar to the values
reported by Cauchon et al.38 for sulfonated ZnPcs with alkyl
substituents of various chain lengths. The cellular uptake of the
latter compounds by EMT-6 cells was between 0.05 and 0.97
ng/mg protein after incubation with 10 µM PS for 1 or 24 h.
Although Si(sol)2Pc and Si(PEG750)2Pc were taken up to the
same extent, Si(sol)2Pc was the most phototoxic one. More
importantly, while Pc4 and Si(sol)2Pc showed similar photo-
toxicity at equal amounts of PS added to the medium, the
amount of PS taken up by B16F10 cells was about 25 times
lower for Si(sol)2Pc.
The difference in photodynamic activity between Si(sol)2Pc
and Si(PEG750)2Pc is most likely due to their different
intracellular distribution, that is, the latter PS is not specifically
located in membrane containing cellular compartments, but is
homogeneously distributed inside the cell, owing to its greater
hydrophilicity. In view of the proposed preferred localization
site of PSs, for example, in mitochondrial membranes, the
similar photodynamic efficacy of Si(sol)2Pc and Pc4 is remark-
able, especially because the former PS is present at lower
intracellular concentrations. This would suggest that lipid
droplets are very effective targets for PDT. On the other hand,
it is also possible that the localization of Si(sol)2Pc in lipid
droplets is not the prime reason for its high phototoxicity,
because redistribution of this compound over the cytosol was
observed upon illumination (Figure 5c). Similar behavior has
been found by Liu et al.51 who observed that PSs, that is, ZnPc
derivatives containing carboxylate groups accumulated in ly-
sosomes and redistributed upon brief light exposure. Redistribu-
tion of Si(sol)2Pc may be due to the destruction of the lipid
droplets by ROS. On the other hand, it has been shown
previously that acetals and ketals can be photochemically
deprotected by a catalytic amount of PS.52,53 Therefore, the
redistribution of Si(sol)2Pc could also be the result of the
removal of the ketal functionality under the illumination
conditions, leading to the formation of a more hydrophilic Pc
The above results indicate that the intracellular concentration
of PS needed to affect cell death upon illumination was much
less for Si(sol)2Pc than for Pc4, and that Si(sol)2Pc is more
efficient than Si(PEG750)2Pc at equal intracellular concentration.
It has been shown that the intracellular distribution of a PS is
very important for the overall photodynamic effect.4 Singlet
oxygen has a very short life span of about 0.03 - 0.18 µs.39
During that short time, its diffusion inside the cell was between
10 and 70 nm.40,41 These diffusion distances are very small
compared to the dimensions of eukaryotic cells, which are in
the order of 10-100 µm.23 This stresses the importance of the

Substitution of Phthalocyanines with Solketal Groups
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1491
(ESI) mass spectrometry was carried out on a Shimadzu LCMS
QP-800 single quadrupole benchtop spectrometer, coupled to a QP-
800 data system. Melting points were determined using a TA
Intruments DSC Q1000 machine. Scans were taken from 20 to 200
°C at a heating rate of 10 °C/min. Elemental analysis of the end
products was done by H. Kolbe, Mikroanalytisches Laboratorium,
Mu¨lheim an der Ruhr, Germany.
containing two axial 1,2-diols, which could dissolve in the
cytosol or other organelles. The possibility of light-induced
conversion of the solketal groups in close proximity of the Pc
ring is highly interesting, and this will be the subject of future
investigations. Thus, it remains elusive whether redistribution,
for example, to mitochondria, is essential for the high efficacy
of the Si(sol)2Pc or if it is the result of disruption of the lipid
droplet integrity, which itself could be responsible for inducing
the observed photodynamic effect. Investigating the site of ROS
formation54-56 or determination of the activity of marker
enzymes for specific organelles57,58 after illumination of cells
incubated with Si(sol)2Pc could be a valuable tool to elucidate
this phenomenon.
3,4-Dibromocatechol (4). Compound 4 was synthesized accord-
ing to van Nostrum et al.25 Catechol was recrystallized from toluene
before use. TLC (MTBE/hexane 1:1): Rf ) 0.23; mp 117 °C (lit.
1
119 °C25). H NMR (CDCl3): δ 7.12 (s, 2H, CHAr), 5.23 (s, 2H,
OH). 13C NMR (CDCl3): δ 143.46 (CAr-OH), 119.88 (CHAr),
114.83 (CAr-Br).
1,2-Bis(allyloxy)-4,5-dibromobenzene (5). 3,4-Dibromocatechol
(4; 5.00 g, 18.66 mmol), Na2CO3 (4.35 g, 41.06 mmol), and
allylbromide (4.97 g, 41.06 mmol) were suspended in acetone (25
mL). The reaction mixture was refluxed for 44 h, until TLC showed
no monosubstituted product. After cooling to rt, H2O (100 mL)
was added, and the product was extracted with Et2O (3 × 100 mL).
The combined organic layers were washed with brine (100 mL)
and dried over Na2SO4. Removal of the solvent in vacuo afforded
5 (6.24 g, 96%) as a light yellow crystalline solid, which was pure
according to TLC and NMR. TLC (MTBE/hexane 1:1): Rf ) 0.58;
mp 41 °C. 1H NMR (CDCl3): δ 7.07 (s, 2H, CHAr), 6.02 (M, 2H,
-CHdCH2), 5.35 (m, 4H, dCH2), 4.54 (m, 4H, OsCH2). 13C
NMR (APT, CDCl3): δ 148.28 (qC, CArsO), 132.39 (CHdCH2),
118.34 (qC, CArsBr), 118.32 (CHAr), 115.00 (CH2d), 70.15 (O-
CH2). HPLC: Rt ) 38.6 min, 100%.
Conclusions
This study shows that the axially substituted Si(sol)2Pc has a
much higher photocytotoxicity and a much lower tendency to
aggregate than the peripherally substituted ZnPc(sol)8. This can
be attributed to the effect of the axial substituents, which prevent
stacking of the aromatic Pc rings. Despite its relatively low
uptake compared to a known PS (Pc4), Si(sol)2Pc had a high
phototoxic effect, with IC50 values comparable to those of Pc4.
Si(sol)2Pc accumulated intracellularly in lipid droplets and
redistributed upon illumination, which can be the reason for
the high photodynamic activity. Increasing the cellular uptake
by formulation of this PS in, for example, targeted micelles or
liposomes might result in an even higher intracellular concentra-
tion and a concomitant increased photodynamic effect.
3,3′-(4,5-Dibromo-1,2-phenylene)bis(oxy)dipropane-1,2-diol
(6). Citric acid (11.04 g, 57.47 mmol), 2 (10.00 g, 28.73 mmol),
and OsO4 (0.709 mL of a 4 w-% solution in H2O, 0.4 mol-%) were
added to H2O/t-BuOH (1:1, 60 mL), yielding a green suspension.
4-Methylmorpholine-4-N-oxide (NMO, 14.81 g of a 50 w-%
solution in H2O, 61.2 mmol) was added, and the reaction was stirred
at rt for 2 h. After removal of t-BuOH in vacuo, a HCl solution
(1M, 100 mL) was added, followed by extraction with EtOAc (3
× 100 mL). The combined organic layers were dried over Na2-
SO4. The solvent was removed in vacuo, yielding 6 as a white solid
(11.30 g, 95%). TLC (CH2Cl2/MeOH 5:1): Rf ) 0.23; mp 94 °C.
1H NMR + COSY (CD3OD): δ 7.26 (s, 2H, CHAr), 4.06 (m, 2H,
-CH2-CH), 3.96 (m, 4H, -CH-OH, CH2′-CH), 3.67 (m, 4H,
CH2-OH). 13C NMR (APT, CD3OD): δ 150.42 (qC, CAr-O),
119.77 (CHAr), 116.10 (qC, CAr-Br), 72.16 (O-CH2-), 71.59
(-CH-OH), 63.96 (CH2-OH). HPLC: Rt ) 17.3 min, 100%.
4,4′-(4,5-Dibromo-1,2-phenylene)bis(oxy)bis(methylene)bis-
(2,2-dimethyl-1,3-dioxolane) (7). Compound 6 (500 mg, 1.20
mmol) was suspended in DMP (10 mL). Upon addition of p-toluene
sulfonic acid (50 mg), the starting materials dissolved almost
completely. The solution was stirred at rt for 30 min. NaHCO3 (aq;
5 w-%, 20 mL) was added, and the product was extracted with
CH2Cl2 (2 × 50 mL). The combined organic layers were washed
with H2O (3 × 100 mL) and dried over Na2SO4. Removal of the
solvent in vacuo yielded an oil, which solidified upon storage to
give 7 as a white product (0.56 g, 94%). TLC (MTBE/ Hex 1:1):
Experimental Section
Materials and Equipment for Synthesis. Chemicals were
obtained from commercial sources and used without purification,
unless stated otherwise. 1,3-Diiminoisoindoline was synthesized
according to Sasa et al.,27 with minor modifications in the workup
procedure, that is, the reaction solvent was removed in vacuo, and
the product was resuspended in Et2O before filtration. The product
was used to prepare Si(Cl)2Pc according to a literature procedure.28
Pc459 was kindly donated by Prof. Dr. Nancy Oleinick, Case
Western Reserve University School of Medicine, Cleveland, Ohio.
Reactions were monitored by thin layer chromatography (TLC) on
Merck precoated silica gel 60 F254 (0.25 mm) plates or Merck
precoated aluminum oxide 150 F254 neutral plates. Flash column
chromatography was performed on Acros organics silica gel,
0.035-0.070 mm, pore diameter about 6 nm, or dried (150 °C)
neutral alumina (activity 1, ICN Biomedicals B.V., The Nether-
lands). NMR spectra were recorded on a Varian Gemini 300 (1H
NMR at 300 MHz, 13C NMR at 75.4 MHz) or a Varian Inova-500
(1H NMR at 500 MHz). 13C NMR spectra were recorded using the
attached proton test (APT) sequence. Chemical shifts are reported
in ppm, relative to the solvent peak, and are given downfield from
TMS. HPLC analyses were carried out using a Waters 2965
AllianceXC with a Waters 2487 dual wavelength UV detector and
a Waters 2414 RI dectector. The oven temperature was set to 30
°C. A Waters XTerra MS C18, 5 µm, 4.6 × 250 mm column was
used for intermediates; an eluent gradient from H2O/MeCN 95:5
to H2O/MeCN 5:95 in 60 min at 1 mL/min was run. Pcs were run
on a Waters, sunfire C18, 5 µm, 4.6 × 150 mm column; eluent
H2O/THF 95:5 to H2O/THF 5:95 in 60 min, 1 mL/min. GPC
analysis of Si(PEG750)2Pc was done on a GPC system containing
a Waters 600E gradient pump with a Waters 717 autosampler,
connected to a Waters 486 UV detector, set at 600 nm, and a Waters
410 RI detector. The used column was an Oligopore, 300 × 7.5
mm, Polymer Laboratories, with a guard column, oligopore guard,
50 × 7.5 mm, Polymer Laboratories. PEG standards were used for
calibration. Samples were run in CHCl3 at 1 mL/min. Absorpion
spectra were recorded on a Perkin-Elmer Lambda 2 UV-vis
spectrometer. A Fluorolog, FL3-21, fluorescence spectrophotometer
was used for fluorescence measurements. Electrospray ionization
1
Rf ) 0.45; mp 81 °C. H NMR (CDCl3): δ 7.10 (s, 2H, CHAr),
4.43, 4.13, 4.05, 3.91 (4 m, 2H, 2H, 2H, 4H, Ar-OCH2, CH-O,
CH-CH2-O), 1.43 (s, 6H, CH3), 1.37 (s, 6H, CH3′). 13C NMR
(APT, CDCl3): δ 148.51(qC, CAr-O), 118.97 (CHAr), 115.64 (qC,
CAr-Br), 109.73 (qC, O-C-O), 73.76 (CH-O), 70.10 (Ar-O-
CH2), 66.55 (O-CH2), 26.66 (CH3), 25.35 (CH3′). HPLC: Rt )
36.8 min, 99.3%.
4,5-Bis[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]phthaloni-
trile (8). A 3-neck flask (250 mL) with a cooler and oil lock were
dried at 150 °C and allowed to cool to rt under a N2 flow. The
flask was charged with freshly distilled DMF (100 mL) and 7 (5.00
g, 10.08 mmol). The suspension was stirred under a N2 flow until
all the starting material was dissolved. CuCN (2.70 g, 30.23 mmol)
and pyridine (2.45 mL, 30.23 mmol) were added followed by
refluxing overnight. The suspension was poured into a beaker
containing 28-30% ammonia (50 mL). Another 50 mL of ammonia
was used to rinse the reaction flask. Air was bubbled through for

1492 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Hofman et al.
2.5 h, upon which the color changed from green to blue. The product
was extracted with CH2Cl2 (4 × 150 mL). The combined organic
layers were washed with H2O (4 × 150 mL) and dried over Na2-
SO4, and the solvent was removed in vacuo, yielding the crude
product as a dark green solid (99%). The product was purified by
flash chromatography (MTBE/hexane 2:1) to afford 8 (2.31 g, 59%).
TLC (MTBE/hexane 2:1): Rf ) 0.38; mp 124 °C. 1H NMR
(CDCl3): δ 7.19 (m, 2H, CHAr), 4.47, 4.17-4.12, 3.92 (3 m, 2H,
6H, 2H, Ar-O-CH2, CH-O, CH-CH2-O), 1.42 (s, 6H, CH3),
1.37 (s, 6H, CH3′). 13C NMR (APT, CDCl3): δ 151.86 (qC, CAr-
O), 116.65 (CHAr), 115.52 (qC, CN), 109.94 (qC, O-C-O), 108.95
(qC, CAr-CN), 73.41 (CH-O), 69.60 (Ar-O-CH2), 66.06 (O-
CH2), 26.47 (CH3), 25.22 (CH3′). ESI-MS: m/z 406.65 (100%, [M
+ NH4]+); 389.15 (7%, [M + H]+). HPLC: Rt ) 29.3 min, 98.7%.
ZnPc(sol)8 (1). A Schlenk flask (dried at 150 °C and cooled to
rt under an Ar flow), charged with 8 (232 mg, 0.60 mmol) and
Zn(OAc)2 (24 mg, 0.13 mmol) was evacuated and refilled with N2
(3×). N,N-Dimethylaminoethanol (3 mL) was added through a
septum, and the suspension was refluxed for 24 h. A clear solution
was obtained upon heating, which turned green within 1 h. The
solvent was removed in vacuo, and the crude product was purified
by gradient flash chromatography (EtOAc/hexane 2:1 to EtOAc),
yielding ZnPc(sol)8 (164 mg, 68%) as a green powder. TLC (CH2-
PEG), 3.43 (m, 4H, SisOs(CH2CH2O)3sCH2), 3.35 (s, 6H, CH3),
3
3.19 (t, 4H, SisOs(CH2CH2O)2sCH2CH2, J ) 4.88 Hz), 2.94
(t, 4H, SisOs(CH2CH2O)2-CH2, 3J ) 4.88 Hz), 2.42 (t, 4H, Sis
OsCH2CH2OsCH2CH2, 3J ) 4.88 Hz), 1.64 (t, 4H, SisOsCH2-
3
3
CH2OsCH2, J ) 4.88 Hz), 0.44 (t, 4H, SisOsCH2CH2, J )
3
5.86, 5.37 Hz), -1.94 (t, 4H, SisOsCH2, J ) 5.37, 5.86 Hz).
13C NMR (CDCl3, APT): δ 149.18 (qC, NsCdN); 135.97 (CArs
HR or CArsHâ), 130.81 (qC, AAr), 70.54, 69.78, 69.33, 68.56 (CH2
PEG), 59.01 (CH3 PEG). UV-vis (DMF): λmax (log ꢀ) 674 (5.31),
644 (4.46), 606 (4.52), 355 (4.81) nm. Fluorescence (DMF): λex,max
674, λem,max 678 nm. Anal. (C102H158N8O36Si) C, H, N, Si (see
Supporting Information). GPC: Mp ) 2023 g‚mol-1; Mn ) 1900,
Mw ) 1950 g‚mol-1
.
Aggregation Behavior. Increasing amounts of H2O were added
to PS solutions (5 µM) of Si(sol)2Pc and ZnPc(sol)8 in DMF, and
absorbance spectra were recorded immediately after the addition
of H2O. The maximal absorbance (Amax), corrected for dilution, of
the Q-band was plotted against the dielectric constant (ꢀ) of the
solvent mixture, which was calculated according to the formula ꢀ
) (ꢀwater × H2O (v-%) + ꢀDMF × DMF (v-%))/100, with ꢀwater
)
78.54 (25 °C) and ꢀDMF ) 37.52 (25 °C).
Materials and Equipment for Cell Culture. The murine
B16F10 cell line was kindly provided by Prof. Dr. Ernst Wagner
(Pharmaceutical Biology-Biotechology, Department of Pharmacy,
Ludwig-Maximilans-Universita¨t, Munich, Germany) and was orig-
inally obtained from I. J. Fidler (Texas Medical Center, Houston,
U.S.A.). B16F10 cells were cultured in DMEM (Dulbecco’s
modification of Eagle’s Medium, with 3.7 g/L sodium bicarbonate,
1 g/L glucose, Gibco BRL, Breda, The Netherlands), completed
with antibiotics/antimycotics (100 IU penicilillin G sodium/mL, 100
µg streptomycin sulfate/mL and 0.25 µg amphotericin B/mL, Gibco
BRL, Breda, The Netherlands), L-glutamine (2 mM, Gibco BRL,
Breda, The Netherlands), and 10% Foetal Bovine Serum (FBS,
Integro, Zaandam, The Netherlands).
1
Cl2/MeOH 10:1): Rf ) 0.26. H NMR + COSY (DMSO-d6): δ
8.80 (br s, 8H, H-Pc), 4.73 (m, 8H, Ar-O-CH2), 4.64 (m, 16H,
Ar-O-CH2′, CH-CH2-O), 4.33 (m, 8H, CH-O), 4.13 (m, 8H,
CH-CH2′-O), 1.58 (s, 24H, CH3), 1.45 (s, 24H, CH3′). 13C NMR
(APT, DMSO-d6): δ 151.64, 150.40 (2 × qC, CAr-O, C-N),
131.72 (qC, C-C-N), 109.10 (qC, O-C-O), 106.06 (CHAr), 74.22
(CH-O), 70.10 (Ar-O-CH2), 66.02 (O-CH2), 26.70 (CH3), 25.70
(CH3′). UV-vis (DMF): λmax (log ꢀ) 673 (5.65), 644 (4.81), 607
(4.84), 359 (5.23) nm. Fluorescence (DMF): λex,max 673, λem,max
678 nm. Anal. (C80H96N8O24Zn) C, H, N, Zn (see Supporting
Information).
Si(sol)2Pc (2). A flask and cooler were dried at 150 °C and cooled
under an Ar flow. Si(Cl)2Pc (2.00 g, 3.27 mmol) and NaH (432
mg, 18.0 mmol) were added, and the set up was evacuated and
refilled with Ar (3×). Toluene (160 mL, dried on mol. sieves (4
Å)) was added through a septum, followed by the addition of
solketal (895 µL, 7.20 mmol). After stirring for 15 min, the reaction
mixture was refluxed for 3 days under a N2 atmosphere. The
resulting green reaction mixture was allowed to cool to rt, and the
solvent was removed in vacuo. The crude product was purified by
flash chromatography (CH2Cl2/EtOAc 10:1) with predried silica
(150 °C) to afford Si(sol)2Pc as a blue solid (1.10 g, 42%). TLC
(CH2Cl2/ EtOAc 10:1): Rf ) 0.54. 1H NMR + COSY (CDCl3): δ
9.61 (m, 8H, HRsPc), 8.32 (m, 8H, HâsPc), 1.70 (m, 2H, CHs
CH2sO), 0.93 (m, 2H, CHsO), 0.44 (m, 2H, CHsCH2′sO), 0.29
(s, 6H, CH3), -0.03 (s, 6H, CH3′), -2.00 (m, 4H, SisOsCH2).
13C NMR (APT, CDCl3): δ 149.25 (qC, NsCdN), 135.90 (qC,
AAr), 130.91, 123.71 (CArsHR, CArsHâ), 107.20 (qC, OsCsO),
73.07 (CHsO), 64.89 (OsCH2), 56.32 (ArsOsCH2), 24.99 (CH3),
24.57 (CH3′). UV-vis (DMF): λmax (log ꢀ) 674 (5.45), 645 (4.56),
606 (4.63), 355 (4.93) nm. Fluorescence (DMF): λex,max 674; λem,max
678 nm. Anal. (C44H38N8O6Si) C, H, N, Si (see Supporting
Information).
The human head and neck squamous cell carcinoma cell line
UM-SCC-14C (developed by Dr. T.E. Carey, Ann Arbor, MI, and
further abbreviated as 14C) was kindly provided by Prof. Dr.
G.A.M.S. van Dongen (Department of Otolaryngology/Head and
Neck Surgery, VU University Medical Center, Amsterdam, The
Netherlands). This cell line was cultured in DMEM with 3.7 g/L
sodium bicarbonate, 4.5 g/L glucose, Gibco BRL, Breda, The
Netherlands), completed with antibiotics/antimycotics (100 IU
penicillin G sodium/mL, 100 µg streptomycin sulfate/mL, and 0.25
µg amphotericin B/mL, Gibco BRL, Breda, The Netherlands), and
5% foetal bovine serum (FBS, Integro, Zaandam, The Netherlands).
For illumination of cells, a homemade device consisting of 96
LED lamps (670 ( 10 nm, 1 LED/well) was used, connected to a
water bath thermostated at 37 °C. The well plate was placed on
top of the instrument. The space between the plate and the lamps
was used for water circulation. This water layer did not only give
a constant temperature, but also a more homogeneous illumination
area. Illumination time and intensity could be adjusted by the
controller. The emitted light intensity was measured at the height
of the well plate by an Orion Laser power/energy monitor (Ophir
Optronics LTD, Jerusalem, Israel).
Photocytotoxicity. Cells were seeded in 96-well tissue culture
plates (Greiner) at 2 × 104 cells per well (100 µL cell suspension)
and incubated overnight at 37 °C under 5% CO2 to reattach. Freshly
prepared stock solutions of the PSs in DMF or THF (0.2 or 2 mg/
mL) were diluted in medium. The concentration of organic solvent
was never higher than 0.5%: a concentration at which no effect
on the cell viability was observed. The medium was removed from
the wells and 100 µL of fresh medium containing different
concentration of PS were added to the wells, and the plates were
incubated at 37 °C in 5% CO2 for 6 h. For every plate, another
identical plate was prepared, which was kept in the dark. After
incubation, the medium was removed and the cells were washed
twice with 100 µL PBS, and 100 µL of fresh medium was added.
The plates were illuminated for 10 min with 3.5 mW/cm2 light
intensity. Similar to previously reported procedures,38,51 the cells
were incubated overnight at 37 °C in 5% CO2 atmosphere, and the
Si(PEG750)2Pc (3). The synthesis of Si(PEG750)2Pc was
essentially carried out according to Huang et al.29 Monomethoxy
PEG (Mn ) 750 g‚mol-1, Aldrich) was dried as a solution in toluene
(0.1 mmol/ mL) over mol. sieves (4 Å). A 3-neck flask (500 mL)
and cooler were dried at 150 °C and cooled under a N2 flow. Si(Cl)2-
Pc (2.00 g, 3.27 mmol) and NaH (432 mg, 18.0 mmol) were added,
and the setup was evacuated and refilled with Ar (3×). The dried
solution of PEG in toluene (72 mL, 7.20 mmol) was added followed
by additional toluene (92 mL, dried on mol. sieves 4 Å). After
stirring for 3 days at reflux and cooling to rt, the solvent was
removed in vacuo, and the crude green product was purified twice
by flash chromatography (CH2Cl2/EtOAc 60:1) with Al2O3 to afford
Si(PEG750)2Pc as a blue paste (1.31 g, 20%). TLC (neutral Al2O,
1
CH2Cl2/EtOH 60:1): Rf ) 0.54. H NMR (CDCl3): δ 9.61 (m,
8H, HRsPc), 8.32 (m, 8H, HâsPc), 3.62-3.48 (m, app. 136H, CH2

Substitution of Phthalocyanines with Solketal Groups
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1493
(3) Bonnett, R.; Martinez, G. Photobleaching of sensitisers used in
photodynamic therapy. Tetrahedron 2001, 57, 9513-9547.
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cell survival, relative to cells without added PS, was then determined
by a colorimetric assay, using the tetrazolium salt XTT (sodium
2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carbox-
anilide60 (Sigma-Aldrich), according to the manufacturers instruc-
tions. With this assay the mitochondrial activity is evaluated, which
is a measure of cell viability because the cells were incubated for
an extended period of time (16 h) after illumination.
Cellular Uptake. B16F10 cells and 14C cells were seeded in a
24-well plate at 15 × 104 cell per well (1000 µL cell suspension),
and the plates were incubated overnight at 37 °C in a 5% CO2
atmosphere. The medium was removed, and 500 µL of fresh
medium, containing 10 µM of PS, was added, and the cells were
incubated for 6 h at 37 °C and 5% CO2. Medium was removed,
and the cells were washed twice with 500 µL of PBS. To lyse the
cells, 500 µL of lysis buffer (50 mM tris/HCl (pH 8), 150 mM
NaCl, and 1% Triton X-100) was added, followed by incubation
on ice for 20 min. The concentration of PS in the cell lysate was
determined by dilution of 100 µL of the cell lysate with 900 µL of
DMF and measuring the fluorescence of the samples (λex ) 355
nm, λem ) 679 nm). Calibration curves were prepared by dilution
of PS stock solutions in DMF with cell lysate to a final lysate
concentration of 10% (v/v). An aliquot (20 µL) of each lysate was
used for determination of the cellular protein content, with the Micro
BCA protein assay61 (Pierce, Rockford, U.S.A.), according to the
instructions of the supplier. The uptake of PS was calculated as
nmol Pc per mg of cellular protein. For evaluation of the cellular
uptake at 4 °C, medium, PS stock, and cells were cooled on ice
before use. Operations were carried out on ice and incubations were
done at 4 °C. The amount of PS taken up was determined according
to the procedure described above.
Intracellular Localization. To promote cell adhesion on the
plates, 35 mm glass bottom microwell dishes (MatTek Corporation,
Ashland, U.S.A.) were precoated with gelatin (Sigma) solution (1:
10 dilution in PBS) by incubation for 4 h at 37 °C. Cell suspensions
of 14C or B16F10 cells (2 × 104 cells in 1.5 mL) were added to
the plates, and they were incubated overnight at 37 °C, 5% CO2.
The medium was removed, and PS solution (1.5 mL, 1 µM) diluted
in medium and supplemented with FBS was added. The cells were
incubated for 6 h, and the plates were examined with a confocal
microscope in regular time intervals during the incubation. To
visualize different organelles, cells preincubated for 6 h with or
without PS were incubated with a number of tracker dyes, that is,
Mytotracer green (Invitrogen, Breda, The Netherlands, 130 nM,
from 0.1 mM stock in DMSO), Lysotracker green (Invitrogen, 130
nM, from 0.1 mM stock in DMSO), Fluorescein isothiocyanate-
dextran (Sigma-Aldrich, FITC-dextran, 40 kDa, Sigma, 130 nM,
from 0.1 mM stock in DMSO), and Nile red (Invitrogen, 160 nM,
from 1 mg/mL stock in DMSO). All dyes were incubated for 5
min at 37 °C, except for FITC-dextran, which was incubated for
10 min. The cells were examined with a laser scanning confocal
fluorescence microscope (Nikon Eclipse TE2000-U microscope),
equipped with a C1 scanning unit, three exitation lasers, that is,
argon-ion laser (488 nm), He-Ne laser (543 nm), and He-Ne
laser (633 nm), two bandpass emission filters, that is, 515/30 nm
and 585/30 nm, and one long-pass emission filter (665 nm).
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Plusquellec, D.; Rollin, P. Original synthesis of linear, branched and
cyclic oligoglycerol standards. Eur. J. Org. Chem. 2001, 875-896.
(11) Chan, W. S.; Brasseur, N.; La Madeleine, C.; Ouellet, R.; van Lier,
J. E. Efficacy and mechanism of aluminium phthalocyanine and its
sulphonated derivatives mediated photodynamic therapy on murine
tumours. Eur. J. Cancer 1997, 33, 1855-1859.
(12) Brasseur, N.; Ouellet, R.; La Madeleine, C.; van Lier, J. E. Water-
soluble aluminium phthalocyanine-polymer conjugates for PDT:
Photodynamic activities and pharmacokinetics in tumour-bearing
mice. Br. J. Cancer 1999, 80, 1533-1541.
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Acknowledgment. This research was financially supported
by NWO-CW/STW (Grant 790.36.110).
Supporting Information Available: A table with combustion
analysis data of ZnPc(sol)8, Si(sol)2Pc, and Si(PEG750)2Pc and a
table with HPLC purity data of compounds 5-8 are presented. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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