Phthalocyanine-polyamine conjugates as pH-controlled photosensitizers for photodynamic therapy

Article abstract of DOI:10.1002/chem.200903580

A series of aryl hydroxyamines prepared by reductive animation were treated with silicon(IV) phthalocyanine dichloride in the presence of pyridine to give the diaxially substituted phthalocyanine-polyamine conjugates 1-5. The electronic absorption, fluorescence emission, and efficiency at generating reactive oxygen species of these compounds were all sensitive to the pH environment. Under acidic conditions, the fluorescence quantum yields and the singlet oxygen quantum yields of these compounds were greatly enhanced in DMF as a result of protonation of the amino moieties, which inhibited the photoinduced electron-transfer deactivation pathway. The Q band was diminished and broadened, and the fluorescence intensity decreased as the pH increased in citrate buffer solutions. The rate of superoxide radical formation was also reduced in a higher pH environment. Compound 3, containing a terminal 4chlorophenyl group at the axial substituent, showed the most desirable pH-responsive properties, which makes it a promising tumor-selective fluorescence probe and photosensitizer for photodynamic therapy. All of the phthalocyanines 1-5 were highly photocytotoxic against HT29 and HepG2 cells with IC₅₀ values as low as 0.03 μM. Compound 3 was highly selective toward lysosomes, but not mitochondria of HT29 cells.

Full text of DOI:10.1002/chem.200903580

FULL PAPER
DOI: 10.1002/chem.200903580
Phthalocyanine–Polyamine Conjugates as pH-Controlled Photosensitizers
for Photodynamic Therapy
Xiong-Jie Jiang,[a] Pui-Chi Lo,[a] Yee-Man Tsang,[b] Sin-Lui Yeung,[b]
Wing-Ping Fong,[b] and Dennis K. P. Ng*[a]
Abstract: A series of aryl hydroxy-
amines prepared by reductive amina-
pounds were greatly enhanced in DMF
as a result of protonation of the amino
moieties, which inhibited the photoin-
duced electron-transfer deactivation
pathway. The Q band was diminished
and broadened, and the fluorescence
intensity decreased as the pH increased
in citrate buffer solutions. The rate of
superoxide radical formation was also
reduced in a higher pH environment.
Compound 3, containing a terminal 4-
chlorophenyl group at the axial sub-
stituent, showed the most desirable
pH-responsive properties, which makes
it a promising tumor-selective fluores-
cence probe and photosensitizer for
photodynamic therapy. All of the
phthalocyanines 1–5 were highly photo-
cytotoxic against HT29 and HepG2
cells with IC50 values as low as 0.03 mm.
ACHTUNGTRENNUNG
tion were treated with silicon(IV)
phthalocyanine dichloride in the pres-
ence of pyridine to give the diaxially
substituted phthalocyanine–polyamine
conjugates 1–5. The electronic absorp-
tion, fluorescence emission, and effi-
ciency at generating reactive oxygen
species of these compounds were all
sensitive to the pH environment.
Under acidic conditions, the fluores-
cence quantum yields and the singlet
oxygen quantum yields of these com-
Keywords: photodynamic therapy ·
photosensitizers · phthalocyanines ·
polyamines · singlet oxygen
Compound
3 was highly selective
toward lysosomes, but not mitochon-
dria of HT29 cells.
Introduction
cacy of the treatment depends greatly on the behavior of
the photosensitizers, including their selectivity toward dis-
eased tissues and efficiency at generating ROS, considerable
research efforts have been devoted to optimize their photo-
biological and photophysical characteristics.[2] For targeted
delivery of photosensitizers to cancer cells, various ap-
proaches have been explored. These include conjugation of
photosensitizers to tumor-specific vectors, such as antibod-
ies, synthetic peptides, epidermal growth factor, and adeno-
viruses,[3] and encapsulation in colloidal carriers, such as li-
Photodynamic therapy (PDT) is a noninvasive therapeutic
modality for a variety of premalignant and malignant diseas-
es.[1] It utilizes the combined action of three individually
nontoxic components, namely a photosensitizer, light, and
molecular oxygen to cause cellular and tissue damage. Sin-
glet oxygen generated through the photosensitization pro-
cess is believed to be the key cytotoxic reactive oxygen spe-
cies (ROS) responsible for the damage. As the overall effi-
somes, polymeric micelles, and silica nanoparticles.[4] An
ACHUTNGRENpNUG oACHTUNGTRENNUGN
alternative strategy takes advantage of the lower extracellu-
lar pH in tumors (ca. 6.8) relative to normal tissues (ca.
7.3).[5] The different pH environment may change the stabili-
ty, aggregation tendency, lipophilicity, and cellular uptake of
the photosensitizers, thereby offering a new level of selectiv-
ity.
[a] X.-J. Jiang, Prof. P.-C. Lo, Prof. D. K. P. Ng
Department of Chemistry and Center of
Novel Functional Molecules
The Chinese University of Hong Kong
Shatin, N.T. (Hong Kong)
Fax : (+852)2603-5057
E-mail: dkpn@cuhk.edu.hk
The pH-dependent behavior of several classes of photo-
sensitizers, such as porphyrins,[6] chlorins,[6,7] chalcogeno-
pyrylium dyes,[8] and phenylene vinylenes[9] has been briefly
examined. Recently, OꢀShea et al. have reported a novel
series of amine-containing BF2-chelated azadipyrromethenes
that can switch on and off the singlet-oxygen production in
DMF by changing the pH environment.[10] Upon addition of
[b] Y.-M. Tsang, S.-L. Yeung, Prof. W.-P. Fong
Department of Biochemistry and Center of
Novel Functional Molecules
The Chinese University of Hong Kong
Shatin, N.T. (Hong Kong)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200903580.
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HCl, the singlet oxygen generation rates increase by up to
10-fold. In addition to pH-sensitive photosensitizers, pH-re-
sponsive fluorescence probes[11] and polymeric micelles[12]
have also received much of current attention because of
their potential application in in vitro and in vivo cancer
imaging and targeted delivery of chemotherapeutic agents,
respectively. All of them work on the basis of the slightly
acidic extracellular pH environment of solid tumors.
As part of our continuing efforts in the development of
efficient and selective phthalocyanine-based photosensitizers
for PDT,[13] we report herein a new series of pH-controlled
photosensitizers in which a silicon(IV) phthalocyanine core
is axially substituted with aryl polyamine moieties (com-
pounds 1–5). These substituents can modulate the photo-
physical and photosensitizing properties of the macrocycles
in both organic and aqueous media at different pH values.
An acid-enhanced fluorescence emission effect has also
been observed at the cellular level. These compounds, there-
fore, serve as potential tumor-selective photosensitizers for
PDT. The pH-dependent behavior and the in vitro photody-
namic activity of these compounds is reported herein.
yields. The procedure was similar to that reported earlier for
the preparation of bis(2-pyridylmethyl)amine.[14] These com-
pounds were then treated with silicon(IV) phthalocyanine
dichloride (17) in the presence of pyridine in toluene to give
the disubstituted products 1–5, which were purified by ex-
traction with CH2Cl2 followed by recrystallization from a
mixture of CHCl3 and 1-hexane (1:4 v/v). All the new com-
pounds were characterized with various spectroscopic meth-
ods and elemental analysis (or accurate mass measurement
for hydroxyamines 13–16).
UV/Vis spectra of phthalocyanines 1–5 were measured in
DMF and the data are summarized in Table 1. The spectra
are typical for nonaggregated phthalocyanines, all showing a
Soret band at 354 nm, two vibronic bands at 607 and
644 nm, together with a sharp Q band at 674 nm (see the
spectra of 1, Figure S1 in the Supporting Information, given
as an example). The absorption positions of all these com-
pounds are identical, which suggests that the axial substitu-
ents do not perturb the phthalocyanine p system. Upon ex-
citation at 610 nm, these compounds showed a weak fluores-
cence emission at 675–677 nm with a fluorescence quantum
yield (FF) of 0.01–0.04 relative to unsubstituted zinc(II)
Results and Discussion
Table 1. Electronic absorption and photophysical data for 1–5 in DMF.
Compound lmax [nm] (log e) In the absence of HCl
In the presence of HCl[a]
Scheme 1 shows the synthetic
route used to prepare phthalo-
cyanines 1–5. Treatment of hy-
droxyamines 6–7 with benzalde-
hydes 8–11 and NaBH4 led to
[b]
[c]
[d]
[b]
[c]
[d]
lem
FF
FD
lem
FF
FD
1
2
3
4
5
354 (4.85), 607 (4.59), 644 (4.52), 674 (5.38) 676
354 (4.80), 607 (4.54), 644 (4.47), 674 (5.34) 677
354 (4.74), 607 (4.49), 644 (4.43), 674 (5.30) 676
354 (4.84), 607 (4.56), 644 (4.49), 674 (5.36) 676
354 (4.83), 607 (4.54), 644 (4.49), 674 (5.32) 675
0.04
0.01
0.01
0.01
0.02
0.06
0.03
0.07
0.04
0.06
681
680
681
681
680
0.30
0.28
0.30
0.29
0.26
0.34
0.38
0.65
0.36
0.47
reductive
amination giving
[a] 600 equivalents relative to phthalocyanine. [b] Excited at 610 nm. [c] By using ZnPc in DMF as the refer-
ence (FF =0.28). [d] By using ZnPc as the reference (FD =0.56 in DMF).
compounds 12–16 in good
phthalocyanine (ZnPc) (FF =0.28).[15] The weak fluores-
cence is due to the presence of amino moieties, which
quench the singlet excited state of the phthalocyanine core
by intramolecular photoinduced electron transfer (PET).[16]
Upon addition of HCl (0.6 mm), the emission bands of all
the compounds shifted slightly to the red (by 3–5 nm) and
increased greatly in intensity (see Figure S2 in the Support-
ing Information). The fluorescence quantum yield increased
significantly to 0.26–0.30 (Table 1). The great changes can
be attributed to protonation of the amino moieties under an
acidic environment, which inhibits the PET process.[10,11c]
The absorption and fluorescence spectra of 1–5 were also
measured in citrate buffer solutions with different pH.
Figure 1 shows the UV/Vis spectra of 3 in the buffers given
as an example. It can be seen that the Q band remains sharp
and intense at pH 4–6. However, it becomes weaker and
broadened when the pH increases to 7–8. It is likely that
under acidic conditions, the amino moieties are protonated.
The charged substituents induce mutual repulsion reducing
the aggregation of the phthalocyanine. This results in the oc-
currence of a sharp and intense monomeric Q band. By con-
Scheme 1. Preparation of phthalocyanines 1–5.
trast, the compound remains aggregated under neutral and
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Phthalocyanine–Polyamine Conjugates
FULL PAPER
of the quencher was monitored spectroscopically at 414 nm
with time (see Figure S4 in the Supporting Information),
from which the values of FD could be determined. As
shown in Table 1, all of the phthalocyanines 1–5 can gener-
ate singlet oxygen in DMF, but with a low efficiency. The
values of FD are only 0.03 to 0.07 relative to ZnPc (FD =
0.56).[17] However, in the presence of HCl (600 equiv), their
FD values are greatly enhanced to 0.34–0.65 and follow the
order 1ꢀ2ꢀ4<5<3 (Table 1). The significant enhancement
can again be attributed to the protonation of the amino moi-
eties, which inhibits the PET process and promotes the in-
tersystem crossing and eventually the singlet oxygen forma-
tion.
Figure 1. UV/Vis spectra of 3 (3 mm) in citrate buffer solutions with differ-
ent pH.
To better mimic the biological environment, the ROS
generation efficiency of phthalocyanines 1–5 was also exam-
ined in the buffer solutions by using dihydroethidium
(DHE) as a probe for the superoxide radical (O2·À).[18] It is
generally believed that ethidium is the oxidized product,
which fluoresces strongly at around 600 nm upon excitation.
Figure 3 shows the change in fluorescence intensity of ethid-
slightly alkaline conditions in the buffers leading to a broad
and weak Q band.
All of the phthalocyanines 1–5 showed a similar pH-de-
pendent behavior, but the extent was different for different
compounds. Figure 2 plots the variation of the Q-band ab-
Figure 3. Change in the fluorescence intensity of ethidium with irradia-
tion time (open symbols). The mixture contained phthalocyanine
(4 mm) and dihydroethidium (20 mm) in citrate buffer solution at pH 6.0
3
Figure 2. Change in the Q-band absorbance at 684 nm with pH for 1–5
~
!
&
*
^
(3 mm) in citrate buffer solutions ( : 1, : 2, : 3, : 4, : 5).
~
~
&
(
and &) or 7.4 ( and ) and was irradiated with red light (l>
610 nm). The corresponding data obtained without irradiation are given
as closed symbols as a control.
sorbance at 684 nm with the pH value for all these com-
pounds. Generally, the absorbance decreases as the pH in-
creases. Compound 3 shows the most distinct decrease in ab-
sorbance when the pH increases from 6.0 to 7.4, which can
roughly mimic the environment around tumors and normal
tissues, respectively. The change in fluorescence intensity
with pH for all these compounds also follows a similar trend
as depicted in Figure S3 (Supporting Information). Again,
compound 3 shows the most remarkable change and the
fluorescence intensity decreases by about 10-fold in the
region between pH 6.0 to 7.4. The results show that the
axial substituents of 1–5 can modulate the pH-dependent
absorption and emission properties of the phthalocyanines
in aqueous media, probably through adjustment of the ag-
gregation tendency, and that compound 3 exhibits the most
desirable pH-dependent spectral changes.
ium with time when using compound 3 as the sensitizer at
two different pH environments (6.0 and 7.4). In the absence
of light, no fluorescence could be detected at both pH
values, showing that compound 3 could not generate super-
oxide radicals under these conditions. By contrast, the fluo-
rescence intensity increased steadily upon irradiation and
the rate of enhancement was much faster at pH 6.0 relative
to that at 7.4. The results show that compound 3 works as
an efficient ROS generator, particularly in a low-pH envi-
ronment. All of the compounds 1–5 exhibit a similar behav-
ior and the results are summarized in Table 2. It can be seen
that all of them can generate ROS and are more efficient at
pH 6.0 than 7.4. The difference is most remarkable for com-
pound 3, for which there is a 9.5-fold increase.
To evaluate the photosensitizing efficiency of these com-
pounds, their singlet oxygen quantum yields (FD) were de-
termined by a steady-state method by using 1,3-diphenyliso-
benzofuran (DPBF) as the scavenger.[17] The concentration
The pH-dependent fluorescence emission of 3 at the cellu-
lar level was also examined. In this study, human colon ade-
nocarcinoma HT29 cells were incubated with 3 followed
with the ionophore nigericin at different pH values (6.0, 6.5,
Chem. Eur. J. 2010, 16, 4777 – 4783
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D. K. P. Ng et al.
Table 2. Comparison of the rates of ROS generation by using 1–5 as the
photosensitizers at pH 6.0 and 7.4.
fluorescence probe and photosensitizer that can target
tumors on the basis of its remarkable pH-responsive proper-
ties in the region between pH 6.5 and 7.4, which are the gen-
eral pH environments for tumors and normal tissues, respec-
tively.[5]
The photodynamic activities of phthalocyanines 1–5 were
also evaluated against two different cell lines, namely HT29
and human hepatocarcinoma HepG2 cells. Figure 5 shows
Compound Rate of ROS gen- Rate of ROS gen-
eration (pH 6.0)[a] eration (pH 7.4)[a]
Relative rate of
ROS generation[b]
1
2
3
4
5
39
345
331
341
238
13
236
35
220
76
3.0
1.5
9.5
1.6
3.1
[a] Determined as the slope of the best-fitted line plotting the fluores-
cence intensity of ethidium versus irradiation time (as shown in
Figure 3). [b] Ratio of the rate of ROS generation at pH 6.0 to that at
pH 7.4.
7.4, and 8.0). Nigericin is an H+/K+ antiporter, which en-
ACHTUNGTRENNUNG
ables the electroneutral transport of extracellular H+ ions in
exchange for intracellular K+ ions, and can equilibrate
intra- and extracellular pH.[19] The bright field and fluores-
cence images of the cells were then captured with a confocal
microscope (Figure 4a), and the intracellular fluorescence
intensities were determined (Figure 4b). As shown in
Figure 4, the intracellular intensities are much stronger at
pH 6.0 and 6.5 relative to those at pH 7.4 and 8.0. The re-
sults further demonstrate that compound 3 is a promising
&
Figure 5. Cytotoxic effects of 3 on HT29 cells in the absence ( ) and
presence (&) of light (l>610 nm, 40 mWcmÀ2, 48 JcmÀ2). Data are ex-
pressed as the mean Æstandard error of the means of three independent
experiments, each performed in quadruplicate.
the effects of 3 on HT29 cells, which are typical for all these
phthalocyanines. All of them are essentially noncytotoxic in
the absence of light, but exhibit high photocytotoxicity. The
IC50 values, defined as the dye concentration required to kill
50% of the cells, are summarized in Table 3. Compounds 2–
Table 3. Comparison of the IC50 values of phthalocyanines 1–5 against
HT29 and HepG2 cells.
Compound
IC50 [mm]
HT29
HepG2
1
2
3
4
5
0.45
0.03
0.05
0.03
0.03
0.49
0.06
0.06
0.03
0.06
5 are highly potent with IC50 values in the range of 0.03 to
0.06 mm, which are comparable with those of some other sili-
con(IV) phthalocyanine-based photosensitizers reported by
us earlier.[13a,16,20] Compound 1, which only has one amino
group in the axial substituent is significantly less photocyto-
toxic. This can easily be seen in Figure S5 (Supporting Infor-
mation), which compares the effects of 1 and 2 on HepG2
cells. The IC50 values of 1 (0.45–0.49) are roughly 10-fold
higher relative to those of the other analogues (Table 3).
The subcellular localization of 3 in HT29 cells was also in-
vestigated. The cells were stained with 3 together with Lyso-
Tracker Green DND 26 or MitoTracker Green FM, which
are specific fluorescence dyes for lysosomes and mitochon-
dria, respectively. As shown in Figure 6a–c, the fluorescence
Figure 4. a) Visualization of bright field (upper row) and intracellular
fluorescence (lower row) images of HT29 cells after incubation with 3
(0.5 mm) for 30 min, followed by nigericin solutions (25 mm) at i) pH 6.5
and ii) pH 7.4 for 20 min. b) Comparison of the intracellular fluorescence
intensity of 3 in the presence of nigericin at different pH values. Data are
expressed as the mean Æstandard deviation (number of cells=25).
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Phthalocyanine–Polyamine Conjugates
FULL PAPER
of particular interest because of its remarkably different be-
havior in the pH range (ca. 6.5 to 7.4) differentiating the
tumor and normal tissue environments. Phthalocyanines 1–5
also exhibit high photocytotoxicity against HT29 and
HepG2 cells with IC50 values as low as 0.03 mm. As revealed
by confocal microscopy, compound 3 also shows a high se-
lectivity towards the lysosomes of the cells. The results pre-
sented herein show that these phthalocyanines, particularly
compound 3, are promising pH-controlled and tumor-selec-
tive photosensitizers for PDT.
Experimental Section
Materials and methods: All the reactions were performed under an at-
mosphere of nitrogen. THF, DMF, pyridine, and toluene were distilled
from sodium benzophenone ketyl, barium oxide, calcium hydride, and
sodium, respectively. All other solvents and reagents were of reagent
grade and used as received.
1H and 13C{1H} NMR spectra were recorded on a Bruker DPX 300 spec-
trometer (1H, 300; 13C, 75.4 MHz) in CDCl3. Spectra were referenced in-
ternally by using the residual solvent (1H: d=7.26 ppm) or solvent (13C:
d=77.0 ppm) resonances relative to SiMe4. Fast-atom bombardment
(FAB) and electrospray ionization (ESI) mass spectra were recorded on
a Thermo Finnigan MAT 95 XL mass spectrometer. Elemental analyses
were performed by the Shanghai Institute of Organic Chemistry, Chinese
Academy of Sciences.
UV/Vis and steady-state fluorescence spectra were taken on a Cary 5G
UV/Vis-NIR spectrophotometer and a Hitachi F-7000 spectrofluorome-
ter, respectively. The FF values were determined by the equation:
[21]
FF(sample) =(Fsample/Fref)(Aref/Asample)(ns2ample/ n2ref)FF(ref)
,
in which F, A, and
Figure 6. Visualization of the intracellular fluorescence of HT29 by using
filter sets specific for a) phthalocyanine 3 (in green) and b) LysoTracker
(in red), and c) the corresponding superimposed image. Figure (d) shows
the fluorescence intensity profiles of 3 (green) and LysoTracker (red)
traced along the line in (d).
n are the measured fluorescence (area under the emission peak), the ab-
sorbance at the excitation position (610 nm), and the refractive index of
the solvent, respectively. ZnPc in DMF was used as the reference
(FF(ref) =0.28).[15] To minimize reabsorption of radiation by the ground-
state species, the emission spectra were obtained in very dilute solutions
of which the absorbance at 610 nm was about 0.03. The FD values were
measured in DMF by the method of chemical quenching of DPBF by
using ZnPc as a reference (FD =0.56).[17]
caused by the LysoTracker (excited at 488 nm, monitored at
500–570 nm) is well superimposed with the fluorescence
caused by 3 (excited at 633 nm, monitored at 640–700 nm).
The very similar fluorescence intensity profiles of 3 and Ly-
soTracker traced along the green line in Figure 6c (Fig-
ure 6d) also confirms that compound 3 can target the lyso-
somes of the cells. By contrast, the fluorescence images of 3
and the Mitotracker (excited at 488 nm, monitored at 500–
570 nm) cannot be superimposed (see Figure S6 in the Sup-
porting Information), which indicates that 3 is not localized
in the mitochondria.
ROS measurements for 1–5 were performed in citrate buffer solutions
with pH values at 6.0 and 7.4. A mixture of the phthalocyanine (4 mm)
and dihydroethidium (20 mm) in the buffer (3 mL, at pH 6.0 or 7.4) was
prepared in a quartz cell. Its fluorescence spectra (lex =465 nm, lem
=
500–700 nm due to the oxidized product) were recorded immediately
after irradiation with a red light for every 5 s. The light source consisted
of a 100 W halogen lamp and a color glass filter (Newport) cut-on
610 nm. The rate of oxidation of dihydroethidium, which reflects the
ROS generation efficiency of that phthalocyanine, was monitored for a
total of 30 s irradiation.
Hydroxyamine 12: A solution of benzaldehyde (8) (2.80 g, 26.4 mmol) in
methanol (150 mL) was cooled in an ice bath. 2-Aminoethanol (6)
(1.40 g, 22.9 mmol) was then added, and the mixture was stirred at room
temperature for 8 h. Sodium borohydride (2.00 g, 52.9 mmol) was added
slowly to the reaction mixture, which was maintained at 08C by using an
ice bath. After the addition, the mixture was stirred at room temperature
for 12 h. The reaction was quenched by the addition of HCl (6m) with
cooling until the pH was adjusted to 4. The solvent was removed under
reduced pressure. The residue was dissolved in water (100 mL) and
washed with CH2Cl2 (100 mLꢂ3) to remove the organic impurities. The
aqueous phase was separated and its pH was adjusted to 10 by using
solid Na2CO3. It was then extracted with CH2Cl2 (150 mL). The organic
portion was dried over anhydrous MgSO4 and evaporated in vacuo to
give the product as a colorless oil (3.25 g, 94%). 1H NMR: d=7.27–7.33
(m, 5H; ArH), 3.80 (s, 2H; CH2), 3.65 (t, J=5.1 Hz, 2H; CH2), 2.80 ppm
(t, J=5.1 Hz, 2H; CH2); 13C{1H} NMR: d=138.9, 128.1, 128.0, 126.8,
Conclusion
In summary, we have prepared and characterized a series of
novel silicon(IV) phthalocyanines axially substituted with
aryl polyamine moieties. These compounds show pH-depen-
dent UV/Vis and fluorescence spectroscopic properties in
DMF and citrate buffer solutions. In acidic media, all of
them can generate ROS including singlet oxygen and the su-
peroxide radical effectively as a result of protonation of the
amino moieties that inhibit the PET process. Compound 3 is
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D. K. P. Ng et al.
60.0, 53.0, 50.3 ppm; the NMR spectroscopic data are essentially the
same as those given elsewhere.[22]
C54H52N12O3Si (2·H2O): C 68.62, H 5.55, N 17.78; found: C 68.45, H 5.79,
N 17.33.
Phthalocyanine 3: According to the procedure described for 1, sili-
AHCTUNGERTGcNNUN on(IV) phthalocyanine dichloride (17) (0.20 g, 0.33 mmol) was treated
Hydroxyamine 13: According to the procedure described for 12, com-
pound 13 was prepared as a colorless oil by treating benzaldehyde (8)
(2.12 g, 20.0 mmol) with 2-(2-aminoethylamino)ethanol (7) (2.08 g,
20.0 mmol), followed by reduction with sodium borohydride (1.52 g,
with hydroxyamine 14 (0.23 g, 1.0 mmol) and pyridine (0.5 mL) in tolu-
ene (30 mL) to give 3 as a blue solid (0.22 g, 67%). 1H NMR: d=9.57–
9.61 (m, 8H; Pc-Ha), 8.30–8.34 (m, 8H; Pc-Hb), 7.19 (d, J=8.4 Hz, 4H;
ArH), 6.89 (d, J=8.4 Hz, 4H; ArH), 3.21 (s, 4H; CH2), 1.55 (t, J=
5.7 Hz, 4H; CH2), 0.89 (t, J=5.7 Hz, 4H; CH2), À0.34 (t, J=5.1 Hz, 4H;
CH2), À1.97 ppm (t, J=5.1 Hz, 4H; CH2); 13C{1H} NMR: d=149.2,
138.9, 135.9, 132.2, 131.0, 129.1, 128.2, 123.6, 53.8, 52.5, 47.9, 47.7,
46.9 ppm; HRMS (FAB): m/z: calcd for C54H49Cl2N12O2Si: 995.3242
[M+H]+; found: 995.3214; elemental analysis calcd (%) for
C54H48Cl2N12O2Si: C 65.12, H 4.86, N 16.87; found: C 64.75, H 4.62, N
16.56.
1
40.2 mmol) (3.38 g, 87%). H NMR: d=7.27–7.33 (m, 5H; ArH), 3.78 (s,
2H; CH2), 3.62 (t, J=5.1 Hz, 2H; CH2), 2.71–2.74 ppm (m, 6H; CH2);
13C{1H} NMR: d=139.9, 128.2, 128.0, 126.8, 60.4, 53.7, 51.2, 48.7,
48.4 ppm; MS (FAB): m/z (%): 195 [M+H]+ (100); HRMS (FAB): m/z:
calcd for C11H19N2O: 195.1492 [M+H]+; found: 195.1490.
Hydroxyamine 14: According to the procedure described for 12, com-
pound 14 was synthesized by treating 4-chlorobenzaldehyde (9) (1.97 g,
14.0 mmol) with 2-(2-aminoethylamino)ethanol (7) (1.46 g, 14.0 mmol),
followed by reduction with sodium borohydride (1.06 g, 28.0 mmol). The
product was isolated as a brown oil (2.72 g, 85%). 1H NMR: d=7.22–
7.29 (m, 4H; ArH), 3.73 (s, 2H; CH2), 3.61 (t, J=5.4 Hz, 2H; CH2),
2.69–2.72 ppm (m, 6H; CH2); 13C{1H} NMR: d=138.5, 132.5, 129.4,
128.4, 60.6, 53.0, 51.2, 48.7, 48.5 ppm; MS (FAB): m/z (%): 229 [M]+
(100); HRMS (FAB): m/z: calcd for C11H17ClN2O: 229.1102 [M]+; found:
229.1094.
Phthalocyanine 4: According to the procedure described for 1, sili-
AHCTUNGERTGcNNUN on(IV) phthalocyanine dichloride (17) (0.20 g, 0.33 mmol) was treated
with hydroxyamine 15 (0.22 g, 1.0 mmol) and pyridine (0.5 mL) in tolu-
ene (30 mL) to give 4 as a blue solid (0.23 g, 71%). 1H NMR: d=9.59–
9.62 (m, 8H; Pc-Ha), 8.30–8.33 (m, 8H; Pc-Hb), 6.91 (d, J=8.4 Hz, 4H;
ArH), 6.76 (d, J=8.4 Hz, 4H; ArH), 3.78 (s, 6H; CH3), 3.19 (s, 4H;
CH2), 1.56 (t, J=6.0 Hz, 4H; CH2), 0.88 (t, J=6.0 Hz, 4H; CH2), À0.36
(t, J=5.4 Hz, 4H; CH2), À1.98 ppm (t, J=5.4 Hz, 4H; CH2);
13C{1H} NMR: d=158.3, 149.2, 135.9, 132.5, 130.9, 129.0, 123.7, 113.5,
55.2, 53.8, 52.7, 47.9, 47.7, 46.9 ppm; HRMS (FAB): m/z: calcd for
C56H55N12O4Si: 987.4233 [M+H]+; found: 987.4196; elemental analysis
calcd (%) for C56H56N12O5Si (4·H2O): C 66.91, H 5.62, N 16.72; found: C
67.36, H 5.53, N 16.45.
Hydroxyamine 15: According to the procedure described for 12, 4-me-
thoxybenzaldehyde (10) (1.91 g, 14.0 mmol) was treated with 2-(2-amino-
ethylamino)ethanol (7) (1.46 g, 14.0 mmol) and sodium borohydride
1
(1.06 g, 28.0 mmol) to give 15 as a brown oil (2.61 g, 83%). H NMR: d=
7.24 (d, J=8.7 Hz, 2H; ArH), 6.87 (d, J=8.7 Hz, 2H; ArH), 3.80 (s, 3H;
CH3), 3.74 (s, 2H; CH2), 3.64 (t, J=5.4 Hz, 2H; CH2), 2.75–2.79 ppm (m,
6H; CH2); 13C{1H} NMR: d=158.7, 131.7, 129.5, 113.8, 60.5, 55.2, 53.1,
51.0, 48.4, 48.1 ppm; MS (FAB): m/z (%): 121 [C6H4(OCH3)CH2]+ (100),
225 [M+H]+ (54); HRMS (FAB): m/z: calcd for C12H21N2O2: 225.1598
[M+H]+; found: 225.1605.
Phthalocyanine 5: According to the procedure described for 1, sili-
AHCTUNGERTGcNNUN on(IV) phthalocyanine dichloride (17) (0.20 g, 0.33 mmol) was treated
with hydroxyamine 16 (0.28 g, 1.0 mmol) and pyridine (0.5 mL) in tolu-
ene (30 mL) to give 5 as a blue solid (0.27 g, 74%). 1H NMR: d=9.59–
9.62 (m, 8H; Pc-Ha), 8.31–8.34 (m, 8H; Pc-Hb), 6.20 (s, 4H; ArH), 3.78
(s, 6H; CH3), 3.72 (s, 12H; CH3), 3.19 (s, 4H; CH2), 1.59 (t, J=6.0 Hz,
4H; CH2), 0.91 (t, J=6.0 Hz, 4H; CH2), À0.34 (t, J=5.1 Hz, 4H; CH2),
À1.96 ppm (t, J=5.1 Hz, 4H; CH2); 13C{1H} NMR: d=152.9, 149.2,
136.5, 136.1, 135.9, 131.0, 123.6, 104.5, 60.8, 55.9, 53.7, 53.5, 47.9, 47.7,
46.9 ppm; HRMS (FAB): m/z: calcd for C60H63N12O8Si: 1107.4656
[M+H]+; found: 1107.4604; elemental analysis calcd (%) for
C60H64N12O9Si: (5·H2O): C 64.04, H 5.73, N 14.94; found: C 64.21, H 5.91,
N 14.79.
Hydroxyamine 16: According to the procedure described for 12, 3,4,5-tri-
methoxybenzaldehyde (11) (2.75 g, 14.0 mmol) was reacted with 2-(2-
aminoethylamino)ethanol (7) (1.46 g, 14.0 mmol) and sodium borohy-
dride (1.06 g, 28.0 mmol) to give 16 as a brown oil (3.11 g, 78%).
1H NMR: d=6.56 (s, 2H; ArH), 3.87 (s, 6H; CH3), 3.83 (s, 3H; CH3),
3.74 (s, 2H; CH2), 3.64 (t, J=5.1 Hz, 2H; CH2), 2.75–2.78 ppm (m, 6H;
CH2); 13C{1H} NMR: d=153.2, 136.8, 135.6, 104.9, 60.8, 60.7, 56.1, 54.1,
51.0, 48.5, 48.4 ppm; MS (FAB): m/z (%): 181 [C6H2(OCH3)3CH2]+
(100), 285 [M+H]+ (13); HRMS (FAB): m/z: calcd for C14H25N2O4:
285.1809 [M+H]+; found: 285.1806.
Cell lines and culture conditions: The HT29 human colorectal carcinoma
cells (from ATCC, no. HTB-38) were maintained in Dulbeccoꢀs modified
Eagleꢀs medium (DMEM; Invitrogen, no.10313–021) supplemented with
fetal calf serum (10%), penicillin-streptomycin (100 unitsmlÀ1 and
Phthalocyanine 1: A mixture of silicon(IV) phthalocyanine dichloride
(17) (0.20 g, 0.33 mmol), 2-benzylaminoethanol (12) (0.15 g, 1.0 mmol),
and pyridine (0.5 mL) in toluene (30 mL) was refluxed for 4 h. After
evaporating the solvent in vacuo, the residue was dissolved in CH2Cl2
(100 mL) and then washed with water (100 mLꢂ3). The organic layer
was collected and evaporated under reduced pressure. The crude product
was recrystallized from CHCl3/1-hexane (1:4 v/v) to give the product as a
blue solid (0.21 g, 76%). 1H NMR: d=9.60–9.64 (m, 8H; Pc-Ha), 8.32–
8.36 (m, 8H; Pc-Hb), 6.94 (t, J=7.2 Hz, 2H; ArH), 6.84 (t, J=7.2 Hz,
4H; ArH), 6.09 (d, J=7.2 Hz, 4H; ArH), 2.01 (s, 4H; CH2), À0.27 (t, J=
5.4 Hz, 4H; CH2), À1.93 ppm (t, J=5.4 Hz, 4H; CH2); 13C{1H} NMR:
d=149.2, 139.5, 135.9, 130.9, 127.7, 126.9, 126.0, 123.7, 53.7, 51.3,
47.6 ppm; HRMS (ESI): m/z: calcd for C50H40N10NaO2Si: 863.2997
[M+Na]+; found: 863.2999; elemental analysis calcd (%) for
C50H40N10O2Si: C 71.41, H 4.79, N 16.65; found: C 71.56, H 5.06, N 16.36.
100 mgmLÀ1
,
respectively), l-glutamine (2 mm), and transferrin
(10 mgmLÀ1). The HepG2 human hepatocarcinoma cells (from ATCC,
no. HB-8065) were maintained in RPMI medium 1640 (Invitrogen, no.
23400–021) supplemented with fetal calf serum (10%) and penicillin-
streptomycin (100 unitsmlÀ1 and 100 mgmLÀ1, respectively). Approxi-
mately 3ꢂ104 (for HT29) or 4ꢂ104 (for HepG2) cells per well in these
media were inoculated in 96-multiwell plates and incubated overnight at
378C in a humidified 5% CO2 atmosphere.
pH-dependent intracellular fluorescence studies: About 1.2ꢂ105 HT29
cells in the growth medium (2 mL) were seeded on a coverslip and incu-
bated overnight at 378C under 5% CO2. The medium was removed, then
the cells were incubated with a solution of phthalocyanine 3 in the
medium (0.5 mm, 2 mL) for 30 min under the same conditions. The cells
were then rinsed with phosphate buffered saline (PBS) and incubated
with nigericin (Sigma) in PBS (25 mm, 2 mL) at different pH values (6.0,
6.5, 7.4, and 8.0) for a further 20 min. The cells were viewed with a Leica
SP5 confocal microscope equipped with a 633 nm helium neon laser.
Emission signals from 640–700 nm (gain=750 V) were collected and the
images were digitized and analyzed by Leica Application Suite Advanced
Fluorescence. The intracellular fluorescence intensities (total 25 cells for
each pH solution) were also determined.
Phthalocyanine 2: According to the procedure described for 1, sili-
ACHTUNGTRENNUNGcon(IV) phthalocyanine dichloride (17) (0.20 g, 0.33 mmol) was treated
with hydroxyamine 13 (0.19 g, 1.0 mmol) and pyridine (0.5 mL) in tolu-
ene (30 mL) to give 2 as a blue solid (0.19 g, 62%). 1H NMR: d=9.58–
9.62 (m, 8H; Pc-Ha), 8.29–8.33 (m, 8H; Pc-Hb), 7.20–7.25 (m, 6H; ArH),
7.00 (d, J=7.5 Hz, 4H; ArH), 3.26 (s, 4H; CH2), 1.58 (t, J=6.0 Hz, 4H;
CH2), 0.88 (t, J=6.0 Hz, 4H; CH2), À0.35 (t, J=5.4 Hz, 4H; CH2),
À1.97 ppm (t, J=5.4 Hz, 4H; CH2); 13C{1H} NMR: d=149.2, 140.3,
135.9, 131.0, 128.1, 127.9, 126.6, 123.6, 53.7, 53.3, 47.8 (two overlapping
signals), 46.9 ppm; HRMS (ESI): m/z: calcd for C54H51N12O2Si: 927.4022
[M+H]+; found: 927.4029; elemental analysis calcd (%) for
Photocytotoxicity assay: Phthalocyanines 1–5 were first dissolved in THF
to give 1.6 mm solutions, which were diluted to appropriate concentra-
4782
www.chemeurj.org
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 4777 – 4783

Phthalocyanine–Polyamine Conjugates
FULL PAPER
tions with the culture medium. The cells, after being rinsed with PBS,
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378C under 5% CO2. The cells were then rinsed again with PBS and re-
filled with 100 mL of the culture medium before being illuminated at am-
bient 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.
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. An illumination of
20 min led to a total fluence of 48 JcmÀ2
.
Cell viability was determined by means of the colorimetric MTT assay.[23]
After illumination, the cells were incubated at 378C under 5% CO2 over-
night. An MTT (Sigma) solution in PBS (3 mgmLÀ1, 50 mL) was added to
each well followed by incubation for 2 h under the same environment. A
solution of sodium dodecyl sulfate (SDS, Sigma; 10% by weight, 50 mL)
was then added to each well. The plate was incubated in an oven at 608C
for 30 min, then 80 mL of iso-propanol was added to each well. The plate
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10 s before the absorbance at 540 nm at each well was taken. The average
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n, in which Ai is the absorbance of the ith data (i=1, 2, .., n), Acontrol is
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Acknowledgements
This work was supported by a grant from the Research Grants Council
of the Hong Kong Special Administrative Region, China (project no.
402608) and a strategic investments scheme administered by The Chinese
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Received: December 30, 2009
Published online: March 22, 2010
Chem. Eur. J. 2010, 16, 4777 – 4783
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
4783