A zinc(II) phthalocyanine conjugated with an oxaliplatin derivative for dual chemo- and photodynamic therapy

Article abstract of DOI:10.1021/jm300398q

A novel zinc(II) phthalocyanine substituted with an oxaliplatin derivative via a triethylene glycol linker has been synthesized. The two components work in a cooperative manner in the antitumor action. The conjugate shows a cytotoxic effect in the dark due to the cytostatic oxaliplatin moiety and an enhanced cytotoxicity upon illumination due to the photosensitizing phthalocyanine unit against the HT29 human colon adenocarcinoma cells. The IC₅₀ value of the conjugate is as low as 0.11 μM, which is 5-fold lower than that of the reference compound without the platinum complex. The high photodynamic activity of the conjugate can be attributed to its high cellular uptake and efficiency in generating intracellular reactive oxygen species. The conjugate also shows preferential localization in the lysosomes of the cells and induces cell death mainly through apoptosis.

Full text of DOI:10.1021/jm300398q

Article
pubs.acs.org/jmc
A Zinc(II) Phthalocyanine Conjugated with an Oxaliplatin Derivative
for Dual Chemo- and Photodynamic Therapy
Janet T. F. Lau,† Pui-Chi Lo,*,† Wing-Ping Fong,‡ and Dennis K. P. Ng*,†
‡
†Department of Chemistry and School of Life Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China
S
* Supporting Information
ABSTRACT: A novel zinc(II) phthalocyanine substituted with an
oxaliplatin derivative via a triethylene glycol linker has been synthesized.
The two components work in a cooperative manner in the antitumor action.
The conjugate shows a cytotoxic effect in the dark due to the cytostatic
oxaliplatin moiety and an enhanced cytotoxicity upon illumination due to the
photosensitizing phthalocyanine unit against the HT29 human colon
adenocarcinoma cells. The IC50 value of the conjugate is as low as 0.11
μM, which is 5-fold lower than that of the reference compound without the
platinum complex. The high photodynamic activity of the conjugate can be
attributed to its high cellular uptake and efficiency in generating intracellular
reactive oxygen species. The conjugate also shows preferential localization in
the lysosomes of the cells and induces cell death mainly through apoptosis.
enhance the cellular uptake and increase the antitumor activity
of the platinum complexes. Guo et al. prepared another two
conjugates in which a silicon(IV) phthalocyanine core is axially
substituted with two cisplatin derivatives via the 3- or 4-
pyridyloxy group.16 These compounds were also photo-
cytotoxic and potentially useful for DNA-targeting PDT.
Recently, Donzello et al. have reported a series of tetra-2,3-
pyrazinoporphyrazines appended with pyridine rings which can
complex with a PtCl2 moiety. The singlet oxygen generation
efficiency of these conjugates and the interactions of one of
these complexes with a telomeric DNA sequence having a G-
quadruplex structure have also been studied.17,18 Apart from
these covalent conjugates, several noncovalent systems in which
the photosensitizer and the anticancer drug are held either by
host−guest interactions19 or in polymeric nanocarriers20−22
have also been prepared and briefly evaluated for their potential
in dual PDT and chemotherapy.
Over the past decade, we have been interested in the
development of efficient and selective photosensitizers for
PDT. A vast number of novel photosensitizers based on
phthalocyanines23−25 and distyryl boron dipyrromethenes26,27
have been synthesized and evaluated for their in vitro and in
vivo photodynamic activities. In light of the potential
advantages of combined chemo- and photodynamic therapy,
we have recently extended our study to phthalocyanines
substituted with platinum-based anticancer drugs. The latter
have been widely used for the treatment of a variety of cancers
through disruption of DNA in the nucleus.28,29 It is believed
that the combination of these two components can embrace the
advantages of the two very different therapeutic mechanisms
INTRODUCTION
■
Photodynamic therapy (PDT) is a clinically established
treatment modality for a range of cancers and wet age-related
macular degeneration.1−3 It utilizes the combined action of a
photosensitizer, light, and molecular oxygen to generate
reactive oxygen species (ROS), particularly singlet oxygen, to
eradicate cancer cells. The ROS generated through the
photosensitization process react rapidly with biological
substrates, triggering an apoptotic or necrotic response and
eventually leading to oxidative damage of the cells.4 Recently,
there has been considerable interest in combining this modality
with other anticancer therapeutic methods. The combination of
modalities that act on different disease pathways has shown
several advantages, such as enhanced therapeutic efficacy,
reduced side effects, and retarded drug-resistance problem.5 For
dual PDT and chemotherapy, there are generally three
approaches, including sequential administration of a photo-
sensitizer and an anticancer drug, the use of their covalent and
noncovalent conjugates, and coencapsulation of these agents in
a polymeric nanocarrier. A number of anticancer drugs,
including cisplatin,6,7 carboplatin,8,9 and doxorubicin,10,11 have
been evaluated for their outcomes in sequential treatment with
PDT. For most of the cases, additive or synergistic effects have
been observed which can reduce the effective doses of
anticancer drugs. The sequence of the two treatment modalities
has also been found to be crucial.9,11,12
A number of covalent conjugates of photosensitizers and
platinum complexes have been prepared with a view to
combining their cytotoxic effects.13−18 Brunner et al.
synthesized a series of these conjugates, which contain a
porphyrin-based photosensitizing unit and a cisplatin derivative,
and studied their antiproliferative activity toward several types
of cancer cells.13−15 The porphyrin moieties could generally
Received: March 22, 2012
Published: May 31, 2012
© 2012 American Chemical Society
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Scheme 1. Synthesis of Phthalocyanine−Platinum Complex Conjugate 1
and the resulting conjugates can exhibit synergistic anticancer
effects. In this paper, we report the preparation and in vitro
photodynamic activities of such a conjugate, in which an
oxaliplatin derivative is linked to a zinc(II) phthalocyanine core
through a triethylene glycol spacer. The conjugate is highly
potent of which the cytotoxicity is 5-fold higher than that of the
analogue without the platinum complex, demonstrating the
cooperative effects of the two cytotoxic components.
7-ene (DBU) in n-pentanol to give the “3 + 1” unsymmetrical
phthalocyanine 8 in 18% yield. This compound was then
hydrolyzed with NaOH in acetone. After acidic treatment, the
carboxyl derivative 9 was obtained, which was further
complexed with trans-(1R,2R)-1,2-diaminocyclohexanedinitrato
platinum(II) (10)35 in an aqueous NaOH solution to give the
phthalocyanine−platinum complex conjugate 11. This com-
pound was purified by flash column chromatography followed
by recrystallization from a mixture of DMF and ethanol. For
comparison, phthalocyanine 12 and oxaliplatin analogue 13
(Figure 1) were also prepared. The former was synthesized by
RESULTS AND DISCUSSION
■
Molecular Design and Synthesis. In this conjugate, a
zinc(II) phthalocyanine was employed as the photosensitizing
unit due to its near-infrared fluorescence emission, high singlet
oxygen generation efficiency, and high photostability.30−32
Owing to the high therapeutic efficacy of oxaliplatin,28,29,33
a
derivative of this drug was selected as the cytostatic part. The
two components were linked with a triethylene glycol chain,
which can enhance the amphiphilicity and biocompatibility of
the system. Scheme 1 shows the synthetic route used to prepare
this conjugate. Treatment of 1-iodo-8-(tetrahydro-2H-pyran-2-
yloxy)-3,6-dioxaoctane (1)34 with diethyl methylmalonate (2)
and NaH in N,N-dimethylformamide (DMF) led to sub-
stitution giving compound 3. The tetrahydropyranyl protecting
group of 3 was then removed with concentrated sulfuric acid to
give compound 4. Nucleophilic aromatic substitution reaction
of 4-nitrophthalonitrile (5) with alcohol 4 afforded the
substituted phthalonitrile 6. This compound then underwent
mixed cyclization with an excess of phthalonitrile (7) in the
presence of Zn(OAc)2·2H2O and 1,8-diazabicyclo[5.4.0]undec-
Figure 1. Structures of phthalocyanine 12 and oxaliplatin analogue 13.
typical base-promoted mixed cyclization of 4-(3,6,9-
trioxadecyloxy)phthalonitrile36 with an excess of 7 in the
presence of Zn(OAc)2·2H2O, while the latter was prepared by
treating 10 with an aqueous solution of malonic acid and
KOH.35 All the new compounds were fully characterized with
various spectroscopic methods and elemental analysis (for
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phthalocyanines 9, 11, and 12) or accurate mass measurements
(for phthalocyanine 8 and all the precursors).
Electronic Absorption and Photophysical Properties.
The absorption spectrum of 11 in DMF was typical as the
spectra of nonaggregated phthalocyanines (Figure S1 in the
Supporting Information). It showed a B-band at 344 nm, two
vibronic bands at 607 and 639 nm, and an intense and sharp Q-
band at 672 nm, which strictly followed the Lambert−Beer’s
law. Upon excitation at 610 nm, the compound showed a
fluorescence emission at 688 nm with a fluorescence quantum
yield (ΦF) of 0.22 relative to the unsubstituted zinc(II)
phthalocyanine (ZnPc) (ΦF = 0.28).37 The spectral data of the
reference compound 12 were very similar to those of 11, except
its slightly higher value of ΦF (0.25), probably due to the
absence of the heavy platinum complex (Figure S2 and Table
S1 in the Supporting Information).
To evaluate the photosensitizing efficiency of these two
compounds, their singlet oxygen quantum yields (ΦΔ) were
determined in DMF by a steady-state method with 1,3-
diphenylisobenzofuran (DPBF) as the scavenger. The concen-
tration of the quencher was monitored spectroscopically at 411
nm with time of irradiation (Figure S3 in the Supporting
Information), from which the values of ΦΔ could be
determined by the method described previously.38 The results
showed that both compounds are highly efficient singlet oxygen
generators, and the values of ΦΔ were virtually identical to that
of ZnPc (ΦΔ = 0.56) (Table S1 in the Supporting
Information).
Figure 2. (a) Comparison of the cytotoxic effects of 11 (squares), 12
(triangles), 13 (circles), and an equimolar mixture of 12 and 13 (stars)
on HT29 cells 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 standard error of the mean value (SEM) of
three independent experiments, each performed in quadruplicate. (b)
Data for 11 (squares), 12 (triangles), and an equimolar mixture of 12
and 13 (stars) in the range up to 2.0 μM.
In Vitro Photodynamic Activities. The cytotoxic effects
of conjugate 11 in a Cremophor EL emulsion were then
investigated. The surfactant was added to solubilize the
compound in water and reduce its aggregation. Because
oxaliplatin is one of the most important chemotherapeutic
drugs in colorectal cancer treatment,33 HT29 human colon
adenocarcinoma cells were used for these studies. To reveal the
photo- and chemocytotoxic effects of 11, the antiproliferative
properties of the two reference compounds 12 and 13 were
also examined. Figure 2 shows the dose-dependent survival
curves for these compounds. It can be seen in Figure 2a that the
nonplatinum-containing phthalocyanine 12 does not show any
dark toxicity up to 100 μM. However, upon illuminating with
red light generated from a halogen lamp after passing through a
color glass filter cut-on 610 nm, it shows substantial cytotoxicity
with an IC50 value, defined as the dye concentration required to
kill 50% of the cells, of 0.55 μM (Figure 2b, Table 1). The
oxaliplatin analogue 13 exhibits cytostatic activity both in the
absence and presence of light, and the corresponding IC50
values are 10.7 and 5.5 μM, respectively. It is interesting to note
the enhanced cytotoxicity under illumination, although the
actual mechanism remains elusive at this stage. Having a
photosensitizer and a chemotherapeutic drug in the same
molecule, conjugate 11 is highly photocytotoxic. The IC50 value
is as low as 0.11 μM, which is 5-fold lower than that of 12. In
fact, this conjugate is much more potent than the classical
photosensitizer porfimer sodium, which has an IC50 value of 4.5
μg mL−1 under the same experimental conditions (vs 0.12 μg
mL−1 for 11). Its cytotoxicity is also significantly higher than
that of oxaliplatin, of which the IC50 value was found to be ca.
0.9 μM.39 Conjugate 11 also exhibits some dark toxicity (IC50 =
78.5 μM), which can be attributed to the oxaliplatin moiety as
12 is nontoxic under these conditions. As a better control, the
cytotoxicity of an equimolar mixture of 12 and 13 was also
studied and compared with that of 11. It can be seen in Figure
Table 1. IC50 Values for 11−13 and an Equimolar Mixture of
12 and 13 against HT29 Cells
IC50 (μM)
compd
in dark
with light
11
12
13
78.5
a
0.11
0.55
5.5
10.7
b
1:1 mixture of 12 and 13
0.42
a
b
Noncytotoxic up to 100 μM. Not determined.
2b and Table 1 that the mixture is relatively more potent than
12 alone (IC50 = 0.42 vs 0.55 μM), probably due to the
additional antitumor effect of the oxaliplatin derivative.
However, its photocytotoxicity is still significantly lower than
that of 11 (IC50 = 0.42 vs 0.11 μM). The results clearly show
that the two antitumor components in conjugate 11 work in a
cooperative fashion.
Considering the fact that the aggregation state of photo-
sensitizers is an important factor relating to their photodynamic
activities,30−32 the aggregation behavior of conjugate 11 and the
reference compound 12, formulated with Cremophor EL in
Dulbecco’s Modified Eagle’s Medium (DMEM), was examined
by absorption and fluorescence spectroscopic methods. As
shown in Figure S4 in the Supporting Information, both
compounds show a relatively sharp and intense Q-band and a
relatively strong fluorescence emission. The results suggest that
both compounds are not significantly aggregated under these
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Figure 3. (a) Confocal fluorescence images of HT29 cells after incubation with 11 or 12 for 2 h (both at 5 μM). The corresponding bright field
images are given in the left column. (b) Comparison of the relative intracellular fluorescence intensity of 11 and 12 (number of cells = 30). (c)
Comparison of the percentage of cellular uptake of 11 and 12 as determined by an extraction method. Data are expressed as mean value standard
deviation (SD) of three independent experiments.
conditions, which seems to be in accord with their high
photocytotoxicity.
To account for the different photocytotoxicity of these two
compounds, their cellular uptake was examined by fluorescence
microscopy and absorption spectroscopy. As shown by the
images captured by confocal laser scanning microscopy (Figure
3a), both compounds could enter into the cells causing
intracellular fluorescence after incubation for 2 h. The average
relative fluorescence intensity per cell of these compounds was
also measured and compared in Figure 3b. It can be seen that
the intracellular fluorescence intensity of 11 is about 5-fold
lower than that of 12. However, to take into account that these
two compounds may have different efficiency in generating
fluorescence inside the cells, an extraction method was also
employed to quantify the cellular uptake. After incubation with
these phthalocyanines, DMF was used to lyse the cells and
extract the dyes. The dye concentrations inside the cells were
quantified by measuring their Q-band absorbance at 672 nm.
The results are depicted in Figure 3c, which shows that the
cellular uptake of 11 is actually about 7-fold higher than that of
12. The presence of the oxaliplatin derivative in 11 may
enhance the amphiphilicity of the overall compound, thereby
promoting the cellular uptake. The higher cellular uptake of 11
is in accord with its higher photocytotoxicity.
In addition, the intracellular production of ROS by these
compounds was also studied using 2′,7′-dichlorodihydrofluor-
escein diacetate (DCFDA) as the quencher.40 As shown in
Figure 4, both 11 and 12 as well as the mixture of 12 and 13
can sensitize the production of ROS upon illumination.
Conjugate 11 is the most efficient compound in producing
intracellular ROS, which may be a result of its higher cellular
uptake. Because the oxaliplatin derivative 13 is not a
photosensitizer, it is expected that it does not produce any
ROS even after illumination. The ROS production efficiencies
of 12 and the mixture of 12 and 13 are therefore comparable to
each other. All these results suggest that the higher photo-
cytotoxicity of conjugate 11 can be attributed to its low
Figure 4. ROS production induced by 11 (squares), 12 (triangles), 13
(circles), and an equimolar mixture of 12 and 13 (stars) in HT29 cells
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.
aggregation tendency and higher cellular uptake and efficiency
in generating intracellular ROS.
The subcellular localization of conjugate 11 was also
investigated. The cells were first incubated with the compound
in the culture medium for 2 h, and then stained with
LysoTracker DND 26, ER-Tracker Green, MitoTracker
Green FM, or SYTO-16 (for 10−20 min), which are specific
dyes for lysosomes, endoplasmic reticulum, mitochondria, and
nucleus, respectively. As shown in Figure 5, the fluorescence
caused by the LysoTracker (excited at 488 nm, monitored at
510−570 nm) can superimpose with the fluorescence caused by
11 (excited at 633 nm, monitored at 650−750 nm). The very
similar fluorescence intensity line profiles of 11 and
LysoTracker traced along the green line in Figure 5a (iv)
also confirms that 11 can target the lysosomes of the cells. By
contrast, the fluorescence images of 11 could not be merged
with those of the ER-Tracker, MitoTracker, and SYTO-16
(excited at 488 nm, monitored at 510−570 nm) (Figure S5 in
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Figure 5. (a) Visualization of (i) the bright field image, intracellular fluorescence of HT29 using filter sets specific for (ii) 11 (in red) and (iii)
LysoTracker (in green), and (iv) the corresponding superimposed image. (b) Fluorescence intensity profiles of 11 (red) and LysoTracker (green)
traced along the green line in (a) (iv).
the Supporting Information), showing that 11 is not localized
in the endoplasmic reticulum, mitochondria, and nucleus of the
cells.
even at 100 μM. The oxaliplatin derivative 13 could induce
apoptotic as well as necrotic cells, both in the absence and
presence of light. The results showed that apoptosis is the
major pathway for the PDT action of 11 and the presence of
the oxaliplatin derivative does not significantly affect the cell
death pathways.
To determine the mode of cell death induced by 11, a flow
cytometric assay of annexin V-green fluorescent protein (GFP)
and propidium iodide (PI) double staining was employed.41 To
find out whether the introduction of the oxaliplatin derivative
will alter the cell death mechanism, the results for 12 and 13
were also compared. The cell populations in different phases of
cell death, namely viable (annexin V-GFP−/PI−), early
apoptotic (annexin V-GFP+/PI−), and necrotic or late-stage
apoptotic (annexin V-GFP+/PI+) were examined at the
respective IC50 values of the drugs. The only exception was
for 12 in the dark for which the IC50 value could not be
determined, a dye concentration of 100 μM was used for the
measurements. As shown in Table 2, when the cells were
treated with 11 or 12 in the presence of light, about 50% of
apoptotic cells and less than 10% of necrotic cells were
observed. Compound 11 also exhibited a significant dark
toxicity, showing 41% of apoptotic cells and 9% of necrotic cells
after the treatment due to the oxaliplatin derivative. For 12 in
the dark, more than 95% of the tumor cells remained viable
CONCLUSIONS
■
In summary, we have synthesized and characterized a zinc(II)
phthalocyanine−platinum complex conjugate and evaluated its
in vitro photodynamic activities. This conjugate contains both
photo- and chemotherapeutic agents which are covalently
linked and function in a cooperative manner. The introduction
of the oxaliplatin derivative can enhance the cellular uptake and
intracellular ROS generation efficiency of the phthalocyanine
unit, resulting in a higher cytotoxicity. The IC50 value of the
conjugate is as low as 0.11 μM toward the HT29 cells, which is
5-fold lower than that of the reference compound without the
oxaliplatin derivative. The conjugate also shows preferential
localization in the lysosomes of the cells and induces cell death
mainly through apoptosis. The overall results show that this
conjugate is a highly promising antitumor agent for dual
chemo- and photodynamic therapy.
Table 2. Flow Cytometric Analysis of the Cell Death
Mechanism Induced by 11, 12, and 13 at Their Respective
IC50 Values in the Absence and Presence of Light on HT29
EXPERIMENTAL SECTION
■
General. All the reactions were performed under an atmosphere of
nitrogen. DMF was predried with barium oxide and distilled under
reduced pressure. n-Pentanol was distilled from sodium under reduced
pressure. Chromatographic purifications were performed on silica gel
(Macherey−Nagel, 230−400 mesh) columns with the indicated
eluents. Size exclusion chromatography was carried out on Bio-Rad
Bio-Beads S-X1 beads (200−400 mesh) with THF as the eluent. All
other solvents and reagents were of reagent grade and used as received.
Compounds 1,34 10,35 and 1335 were prepared as described.
a
Cells
cell population (%)
[Drug]
(μM) illumination
viable
apoptotic
3.3 0.5
necrotic
blank
0
−
95.0 1.2
1.2 0.3
1H and 13C{1H} NMR spectra were recorded on a Bruker
AVANCE III 400 spectrometer (1H, 400; 13C, 100.6 MHz) in
CDCl3 unless otherwise stated. Spectra were referenced internally
using the residual solvent (1H: δ 7.26) or solvent (13C: δ 77.2)
resonances relative to SiMe4. 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, China.
The purity of all the new compounds was determined by elemental
11
0.11
78
+
36.5 1.1
48.9 1.5
54.2 2.9
41.0 1.2
8.4 1.6
9.3 0.7
−
12
13
0.55
+
44.9 1.9
95.3 1.8
50.2 1.9
2.2 1.5
4.4 0.7
2.1 1.2
b
100
−
5.5
+
45.2 4.8
53.8 3.5
41.4 1.8
25.7 3.0
13.0 3.1
19.9 0.6
10.7
−
a
1
Data are expressed as mean value
SD of three independent
analysis (for 9, 11, and 12) or H NMR spectroscopy and was found
b
experiments. The IC50 value could not be determined.
to be ≥95%.
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UV−vis and steady-state fluorescence spectra were taken on a Cary
5G UV−vis−NIR spectrophotometer and a Hitachi F-7000 spectro-
fluorometer, respectively. The fluorescence quantum yields of the
recrystallization from a mixture of THF and hexane to give a green
solid (0.19 g, 18%). 1H NMR (CDCl3 with a trace amount of pyridine-
d5): δ = 8.92−9.10 (m, 6 H, Pc-Hα), 8.71 (d, J = 8.0 Hz, 1 H, Pc-Hα),
8.24 (s, 1 H, Pc-Hα), 7.89−8.01 (m, 6 H, Pc-Hβ), 7.42 (d, J = 8.0 Hz, 1
H, Pc-Hβ), 4.57 (virtual t, J = 4.4 Hz, 2 H, CH2), 4.16−4.23 (m, 6 H,
CH2), 3.91 (t, J = 4.4 Hz, 2 H, CH2), 3.74 (t, J = 4.4 Hz, 2 H, CH2),
3.66 (t, J = 6.8 Hz, 2 H, CH2), 2.30 (t, J = 6.8 Hz, 2 H, CH2), 1.50 (s,
3 H, CH3), 1.25 (t, J = 7.2 Hz, 6 H, CH3). 13C{1H} NMR (CDCl3
with a trace amount of pyridine-d5): δ = 172.1, 160.1, 153.0, 152.9,
152.7, 152.6, 152.3, 152.2, 139.8, 138.1, 138.0, 137.9, 137.7, 131.2,
128.6, 128.5, 128.4, 128.3, 123.1, 122.2, 122.1, 122.0, 117.9, 104.8,
70.9, 70.4, 69.9, 67.9, 67.3, 61.2, 52.0, 35.1, 20.0, 14.0 (some of the
aromatic signals were overlapped). MS (ESI): an isotopic cluster
peaking at m/z 881 (80%, [M + H]+). HRMS (ESI): m/z calcd for
C46H41N8O7Zn [M + H]+, 881.2384; found, 881.2391.
samples [ΦF(sample)] were determined by the equation: ΦF(sample)
=
42
(Fsample/Fref)(Aref/Asample)(nsample2/nref2)ΦF(ref)
,
where F, A, and n are
the measured fluorescence (area under the emission peak), the
absorbance at the excitation position, and the refractive index of the
solvent, respectively. ZnPc in DMF was used as the reference [ΦF(ref)
=
0.28].37
Compound 3. Diethyl methylmalonate (2) (4.00 g, 22.96 mmol)
was added to a slurry suspension of NaH (0.83 g, 34.58 mmol) in
DMF (30 mL) at 0 °C under a nitrogen atmosphere. A solution of 1
(7.90 g, 22.95 mmol) in DMF (30 mL) was then added in dropwise to
the reaction mixture. The mixture was heated at 60 °C for 24 h, and
then the solvent was evaporated under reduced pressure. The residue
was then subject to silica gel column chromatography using ethyl
acetate/hexane (9:1 v/v) as the eluent to obtain the product as a
yellow oily liquid (4.83 g, 54%). Rf [ethyl acetate/hexane (9:1 v/v)] =
0.42. 1H NMR: δ = 4.62 (t, J = 4.0 Hz, 1 H, CH), 4.16 (dq, J = 2.0, 7.2
Hz, 4 H, CH2), 3.83−3.89 (m, 2 H, CH2), 3.47−3.67 (m, 10 H, CH2),
2.18 (t, J = 6.4 Hz, 2 H, CH2), 1.67−1.87 (m, 3 H, CH and CH2),
1.48−1.63 (m, 3 H, CH and CH2), 1.43 (s, 3 H, CH3), 1.24 (t, J = 7.2
Hz, 6 H, CH3). 13C{1H} NMR: δ = 172.1, 98.9, 70.5 (two overlapping
signals), 70.2, 67.2, 66.6, 62.2, 61.2, 52.0, 35.0, 30.5, 25.4, 20.0, 19.5,
14.0. MS (ESI): m/z 413 (100%, [M + Na]+). HRMS (ESI): m/z
calcd for C19H34NaO8 [M + Na]+, 413.2146; found, 413.2145.
Compound 4. Concentrated sulfuric acid (5 drops) was added to a
solution of 3 (1.08 g, 2.77 mmol) in ethanol (10 mL). The mixture
was stirred at room temperature for 3 h, and then it was neutralized
with an aqueous solution of NaHCO3. The solvent was evaporated
under reduced pressure, and then the residue was redissolved in
diethyl ether. The insoluble material was removed by filtration. The
Phthalocyanine 9. A mixture of 8 (0.15 g, 0.17 mmol), 5 M
NaOH (0.3 mL, 1.5 mmol), and acetone (20 mL) was heated under
reflux for 2 h. The volatiles were removed under reduced pressure. The
green residue was washed thoroughly with acetone, and then
redissolved in water and acidified with 3 M HCl until pH = 4. The
green precipitate was washed thoroughly with water and ethanol, and
1
then dried in vacuo (0.11 g, 78%). H NMR (DMSO-d6 with a trace
amount of pyridine-d5): δ = 9.25−9.34 (m, 6 H, Pc-Hα), 9.05 (d, J =
8.0 Hz, 1 H, Pc-Hα), 8.65 (d, J = 2.0 Hz, 1 H, Pc-Hα), 8.17−8.23 (m, 6
H, Pc-Hβ), 7.69 (dd, J = 2.0, 8.0 Hz, 1 H, Pc-Hβ), 4.66 (virtual t, J =
4.4 Hz, 2 H, CH2), 4.08 (virtual t, J = 4.4 Hz, 2 H, CH2), 3.80 (t, J =
4.4 Hz, 2 H, CH2), 3.64 (t, J = 4.4 Hz, 2 H, CH2), 3.53 (t, J = 7.2 Hz, 2
H, CH2), 2.08 (t, J = 7.2 Hz, 2 H, CH2), 1.35 (s, 3 H, CH3). 13C{1H}
NMR (DMSO-d6 with a trace amount of pyridine-d5): δ = 174.3,
160.3, 152.8, 152.6, 152.4, 152.2, 152.1, 139.9, 138.0, 137.8, 137.7,
130.9, 129.2, 129.0, 123.2, 122.4, 122.2, 117.9, 105.4, 70.4, 70.0, 69.5,
68.1, 67.4, 51.2, 35.5, 20.9 (some of the aromatic signals were
overlapped). MS (ESI): isotopic clusters peaking at m/z 780 (100%,
[M − CO2]+) and 824 (35%, M+). HRMS (ESI): m/z calcd for
C42H32N8O7Zn [M]+, 824.1680; found, 824.1654. Anal. Calcd for
C42H32N8O7Zn: C, 61.06; H, 3.90; N, 13.56. Found: C, 60.81; H, 3.56;
N, 13.27.
1
filtrate was evaporated to give a pale-yellow oil (0.62 g, 73%). H
NMR: δ = 4.16 (dq, J = 2.8, 7.2 Hz, 4 H, CH2), 3.72 (t, J = 4.0 Hz, 2
H, CH2), 3.52−3.61 (m, 8 H, CH2), 2.19 (t, J = 6.4 Hz, 2 H, CH2),
1.43 (s, 3 H, CH3), 1.23 (t, J = 7.2 Hz, 6 H, CH3). 13C{1H} NMR: δ =
172.2, 72.4, 70.3, 70.2, 67.1, 61.8, 61.3, 51.9, 35.0, 19.9, 14.0. MS
(ESI): m/z 329 (100%, [M + Na]+). HRMS (ESI): m/z calcd for
C14H26NaO7 [M + Na]+, 329.1571; found, 329.1580.
Phthalocyanine 11. Phthalocyanine 9 (0.25 g, 0.30 mmol) was
dissolved in water (50 mL) containing 5 M NaOH (0.15 mL, 0.75
mmol). It was then added to a solution of 10 (0.13 g, 0.30 mmol) in
water (30 mL). The mixture was heated at 70 °C for 48 h. The
resulting blue precipitate was filtered and washed thoroughly with
water and ethanol. The crude product was then subject to flash silica
gel column chromatography using DMF as the eluent. The product
was further purified by recrystallization from a mixture of DMF and
Phthalonitrile 6. A mixture of 4 (0.58 g, 1.89 mmol), 4-
nitrophthalonitrile (5) (0.16 g, 0.92 mmol), and K2CO3 (0.38 g, 2.75
mmol) in DMF (20 mL) was stirred at 110 °C under nitrogen for 24
h. The solvent was removed at 60 °C under reduced pressure. The
residue was mixed with H2O (50 mL), and then it was extracted with
CHCl3 (50 mL × 3). The combined organic layers was dried over
anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude
product was subject to silica gel column chromatography using
hexane/ethyl acetate (1:1 v/v) as the eluent to afford the product as a
pale-yellow oil (0.21 g, 53%). Rf [hexane/ethyl acetate (1:1 v/v)] =
0.29. 1H NMR: δ = 7.70 (d, J = 8.8 Hz, 1 H, ArH), 7.31 (d, J = 2.8 Hz,
1 H, ArH), 7.23 (dd, J = 2.8, 8.8 Hz, 1 H, ArH), 4.22 (t, J = 4.8 Hz, 2
H, CH2), 4.16 (dq, J = 2.4, 7.2 Hz, 4 H, CH2), 3.85−3.89 (m, 2 H,
CH2), 3.64−3.69 (m, 2 H, CH2), 3.47−3.59 (m, 4 H, CH2), 2.18 (t, J
= 6.4 Hz, 2 H), 1.42 (s, 3 H, CH3), 1.23 (t, J = 7.2 Hz, 6 H, CH3).
13C{1H} NMR: δ = 172.1, 162.0, 135.2, 119.8, 119.5, 117.4, 115.6,
115.2, 107.4, 70.9, 70.2, 69.1, 68.6, 67.3, 61.2, 51.9, 35.0, 20.0, 14.0.
MS (ESI): m/z 455 (100%, [M + Na]+). HRMS (ESI): m/z calcd for
C22H28N2NaO7 [M + Na]+, 455.1789; found, 455.1800.
Phthalocyanine 8. A mixture of 6 (0.53 g, 1.23 mmol),
phthalonitrile (7) (1.42 g, 11.08 mmol), and Zn(OAc)2·2H2O (0.81
g, 3.69 mmol) in n-pentanol (25 mL) was heated to 100 °C, and then
a small amount of DBU (1 mL) was added. The mixture was stirred at
140−150 °C for 24 h. After cooling, the volatiles were removed under
reduced pressure. The residue was dissolved in CHCl3 (150 mL), and
then part of the ZnPc formed was removed by filtration. The filtrate
was collected and evaporated to dryness in vacuo. The residue was
purified by silica gel column chromatography using CHCl3 and then
CHCl3/MeOH (100:1 v/v) as the eluents. The crude product was
purified by size exclusion chromatography with Bio-Beads S-X1 beads
using THF as the eluent. The product was then further purified by
1
ethanol to afford a blue solid (0.12 g, 35%). Rf (DMF) = 0.65. H
NMR (DMSO-d6 with a trace amount of pyridine-d5): δ = 9.38−9.44
(m, 6 H, Pc-Hα), 9.24 (d, J = 8.4 Hz, 1 H, Pc-Hα), 8.87 (d, J = 2.0 Hz,
1 H, Pc-Hα), 8.21−8.24 (m, 6 H, Pc-Hβ), 7.77 (dd, J = 2.0, 8.4 Hz, 1
H, Pc-Hβ), 4.67 (virtual t, J = 4.4 Hz, 2 H, CH2), 4.04 (virtual t, J = 4.4
Hz, 2 H, CH2), 3.75 (t, J = 4.4 Hz, 2 H, CH2), 3.61 (t, J = 4.4 Hz, 2 H,
CH2), 3.53 (t, J = 7.2 Hz, 2 H, CH2), 2.34 (br s, 2 H, CH2), 1.88−1.97
(m, 4 H, CH2), 1.49 (br s, 2 H, CH2), 1.16−1.37 (m, 5 H, CH2 and
CH3), 0.98−1.05 (m, 2 H, CH2). The 13C{1H} NMR spectrum could
not be obtained due to its low solubility in common organic solvents.
MS (ESI): an isotopic cluster peaking at m/z 1133 (38%, [M + H]+).
HRMS (ESI): m/z calcd for C48H45N10O7PtZn [M + H]+, 1133.2404;
found, 1133.2415. Anal. Calcd for C48H44N10O7PtZn: C, 50.87; H,
3.91; N, 12.35. Found: C, 50.50; H, 3.63; N, 11.95.
Phthalocyanine 12. A mixture of phthalocyanine (7) (2.06 g,
16.08 mmol), 4-(3,6,9-trioxadecyloxy)phthalonitrile36 (0.52 g, 1.79
mmol), and Zn(OAc)2·2H2O (1.18 g, 5.38 mmol) in n-pentanol (20
mL) was heated to 100 °C, and then a small amount of DBU (1 mL)
was added. The mixture was stirred at 140−150 °C for 24 h. After a
brief cooling, the volatiles were removed under reduced pressure. The
residue was dissolved in CHCl3 (150 mL) and then filtered to remove
part of the ZnPc formed. The filtrate was collected and evaporated to
dryness in vacuo. The residue was purified by silica gel column
chromatography using CHCl3 and then CHCl3/MeOH (100:1 v/v) as
the eluents. The crude product was purified by size exclusion
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chromatography using THF as the eluent followed by recrystallization
from a mixture of CHCl3 and hexane to give a green solid (0.32 g,
24%). H NMR (CDCl3 with a trace amount of pyridine-d5): δ =
seeded on a coverslip and incubated overnight at 37 °C under 5%
CO2. The medium was removed, and then the cells were incubated
with 11 or 12 in the medium (5 μM, 2 mL) for 2 h under the same
conditions. The cells were then rinsed with PBS twice and viewed with
a Leica SP5 confocal laser-scanning microscope equipped with a 633
nm helium−neon laser. The emission signals in 650−750 nm were
collected, and the images were digitized and analyzed by Leica
Application Suite Advanced Fluorescence Software. The intracellular
fluorescence intensities (a total of 30 cells in each sample) were also
determined.
1
9.05−9.10 (m, 3 H, Pc-Hα), 9.01 (d, J = 7.2 Hz, 1 H, Pc-Hα), 8.95 (d, J
= 6.4 Hz, 1 H, Pc-Hα), 8.90 (d, J = 6.8 Hz, 1 H, Pc-Hα), 8.68 (d, J =
8.0 Hz, 1 H, Pc-Hα), 8.20 (d, J = 2.0 Hz, 1 H, Pc-Hα), 7.88−8.01 (m, 6
H, Pc-Hβ), 7.39 (dd, J = 2.0, 8.0 Hz, 1 H, Pc-Hβ), 4.57 (virtual t, J =
4.4 Hz, 2 H, CH2), 4.19 (virtual t, J = 4.4 Hz, 2 H, CH2), 3.97−3.99
(m, 2 H, CH2), 3.85−3.87 (m, 2 H, CH2), 3.77−3.79 (m, 2 H, CH2),
3.64−3.66 (m, 2 H, CH2), 3.42 (s, 3 H, CH3). 13C{1H} NMR (CDCl3
with a trace amount of pyridine-d5): δ = 160.2, 153.2, 152.9, 152.8,
152.5, 139.9, 138.1, 138.0, 137.8, 131.3, 128.7, 128.5, 123.2, 122.3,
118.1, 105.1, 71.9, 71.0, 70.8, 70.6, 70.0, 68.0, 59.0 (some of the
aromatic signals were overlapped). MS (ESI): an isotopic cluster
peaking at m/z 738 (100%, M+). HRMS (ESI): m/z calcd for
C39H30N8O4Zn [M]+, 738.1676; found, 738.1674. Anal. Calcd for
C39H30N8O4Zn: C, 63.29; H, 4.09; N, 15.13. Found: C, 63.52; H, 4.35;
N, 14.75.
Cell Lines and Culture Conditions. The HT29 human colon
adenocarcinoma cells (from ATCC, no. HTB-38) were maintained in
DMEM (Invitrogen, cat no. 10313−021) supplemented with fetal calf
serum (10%), penicillin−streptomycin (100 units mL−1 and 100 μg
mL−1 respectively), L-glutamine (2 mM), and transferrin (10 μg
mL−1).
Cellular Uptake (Determined by Extraction). About 2.0 × 106
HT29 cells in the culture medium (2 mL) were seeded on a Petri dish
(diameter = 35 mm) and incubated overnight at 37 °C under 5% CO2.
The medium was removed, and the cells were rinsed with PBS (2 mL).
The cells were then incubated with a solution of 11 or 12 in the
medium (5 μM, 2 mL) for 2 h under the same conditions. The
solution was then removed and the cells were rinsed with PBS (2 mL)
and then harvested by 0.25% trypsin-EDTA (Invitrogen, 500 μL),
followed by quenching the trypsin with medium (500 μL). The
solution was transferred to 1.5 mL centrifuge tubes and centrifuged at
2400 rpm for 3 min. The pellet was then washed with PBS (1 mL),
and the suspension was centrifuged again. After removing the PBS, the
cells were lysed with DMF (1 mL). The mixture was sonicated for 20
min and then centrifuged again. The supernatants were transferred for
UV−vis spectroscopic measurements. The absorbance at 672 nm for
both phthalocyanines was compared with the respective calibration
curves to give the uptake concentrations. Each experiment was
repeated three times.
Photocytotoxicity Assay. Approximately 3 × 104 HT29 cells per
well in the culture medium were inoculated in 96-multiwell plates and
incubated overnight at 37 °C in a humidified 5% CO2 atmosphere.
Compounds 11−13 were first dissolved in DMF to give 10 mM
solutions, which were diluted to 1 mM with the culture medium in the
presence of 0.5% Cremophor EL. These served as the stock solutions
for the following in vitro studies. For cytotoxicity studies, the solutions
were further diluted with the culture medium. The cells, after being
rinsed with phosphate buffered saline (PBS), were incubated with 100
μL of the diluted phthalocyanine solutions for 2 h 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. Illumination of
20 min led to a totally fluence of 48 J cm−2.
Cell viability was determined by means of the colormetric MTT
assay.43 After illumination, the cells were incubated at 37 °C under 5%
CO2 overnight. An MTT (Sigma) solution in PBS (3 mg mL−1, 50
μL) 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 μL) was then added to each well. The plate was
incubated in an oven at 60 °C for 30 min, and then 80 μL of isopropyl
alcohol was added to each well. The plate was agitated on a Bio-Rad
microplate reader at ambient temperature for 10 s 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 = [∑(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 data points.
Subcellular Localization Studies. About 3 × 104 HT29 cells in
the culture medium (2 mL) were seeded on a coverslip and incubated
overnight at 37 °C under 5% CO2. The medium was then removed.
The cells were incubated with a solution of 11 in the medium (5 μM, 2
mL) for 2 h under the same conditions. For the study using
LysoTracker, LysoTracker Green DND-26 (Molecular Probes, 2 μM
in culture medium) was then added and the cells were incubated under
these conditions for a further 10 min. For the study using
MitoTracker, the cells were incubated with MitoTracker Green FM
(Invitrogen, 0.25 μM in culture medium) for 10 min. For the study
using ER-Tracker, the cells were incubated with ER-Tracker Green
(Invitrogen, 0.2 μM in culture medium) for 20 min. For the study
using SYTO-16, the cells were incubated with SYTO-16 (Invitrogen,
0.5 μM in culture medium) for 10 min. For all the cases, the cells were
then rinsed with PBS and viewed with a Leica SP5 confocal laser-
scanning microscope equipped with a 488 nm argon laser and a 633
nm helium−neon laser. All the Trackers were excited at 488 nm and
monitored at 510−570 nm, while compound 11 was excited at 633 nm
and monitored at 650−750 nm. Images were digitized and analyzed by
Leica Application Suite Advanced Fluorescence Software. The
subcellular localization of 11 was revealed by comparing the
intracellular fluorescence images caused by the Trackers and this dye.
Flow Cytometric Studies. Approximately 6 × 105 HT29 cells in
the medium (2 mL) were seeded on a 35 mm dish and incubated for
24 h at 37 °C under 5% CO2. The cells were then treated with 11, 12,
or 13 at their respective concentrations as indicated in Table 2 and
incubated under the same conditions for 2 h. The cells were then
rinsed thrice with PBS and refilled with the culture medium (2 mL)
before being illuminated at ambient temperature using a halogen lamp
light source as described above. After 24 h of incubation, the cells were
rinsed with PBS and then harvested by 0.25% trypsin-EDTA
(Invitrogen, 0.5 mL), followed by centrifugation at 2400 rpm for 3
min at room temperature. The pellet was then washed again by PBS
and then subject to centrifugation. The cells were suspended in 1 mL
of binding buffer (10 mM HEPES, 140 mM NaCl, 25 mM CaCl2, pH
= 7.4) containing annexin V-GFP (5 μL) and PI (2 μg mL−1). After
incubation in darkness for 15 min at room temperature, the signals of
annexin V-GFP and PI were measured by a BD FACSCanto flow
cytometer (Becton Dickinson) with 104 cells counted in each sample.
Both annexin V-GFP and PI were excited by a 488 nm argon laser.
The emitted fluorescence was monitored by at 500−560 nm for
ROS Measurements. ROS production was determined by using
DCFDA (Molecular Probes) as the quencher.40 Approximately 3 ×
104 HT29 cells per well in the culture medium was inoculated in a 96-
multiwell plate for 24 h before photodynamic treatment. After the cells
were incubated with different concentrations of the drugs in the
medium (2-fold dilution from 2 μM, 100 μL) for 2 h in darkness, the
cells were rinsed with PBS before 100 μL of DCFDA (10 μM in PBS)
was added to each well. The mixture was incubated at 37 °C under 5%
CO2 for 30 min in darkness, followed by illumination for 20 min.
Fluorescence measurements were made in a fluorescence plate reader
(TECAN Polarion) with a 485 nm excitation filter and a 535 nm
emission filter set at a gain of 60.
Cellular Uptake (Determined by Confocal Microscopy).
About 6 × 104 HT29 cells in the culture medium (2 mL) were
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annexin V-GFP and at >670 nm for PI. The data collected were
analyzed by using WinMDI 2.9.
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of photodynamic therapeutic agent silicon(IV) phthalocyanine and
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(17) Donzello, M. P.; Vittori, D.; Viola, E.; Manet, I.; Mannina, L.;
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ASSOCIATED CONTENT
* Supporting Information
■
S
Absorption and fluorescence spectra/data for 11 and 12 in
DMF or DMEM, comparison of the rate of decay of DPBF
sensitized by 11, 12, or ZnPc in DMF, results of subcellular
1
localization studies, and H and 13C{1H} NMR spectra of all
the new compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*For P.-C.L.: phone, (852) 3943 1326; fax, (852) 2603 5057;
E-mail, pclo@cuhk.edu.hk. For D.K.P.N.: phone, (852) 3943
6375; fax, (852) 2603 5057; E-mail, dkpn@cuhk.edu.hk.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Sin-Lui Yeung for technical support in the in vitro
studies. This work was supported by a grant from the Research
Grant Council of the Hong Kong Special Administrative
Region, China (project no. 402211).
ABBREVIATIONS USED
■
DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DCFDA, 2′,7′-di-
chlorodihydrofluorescein diacetate; DMEM, Dulbecco’s Modi-
fied Eagle’s Medium; DMF, N,N-dimethylformamide; DPBF,
1,3-diphenylisobenzofuran; ESI, electrospray ionization; GFP,
green fluorescent protein; PBS, phosphate buffered saline;
PDT, photodynamic therapy; PI, propidium iodide; ROS,
reactive oxygen species; SD, standard deviation; SEM, standard
error of the mean; ZnPc, zinc(II) phthalocyanine; ΦF,
fluorescence quantum yield; ΦΔ, singlet oxygen quantum
yield; IC50, dye concentration required to kill 50% of the cells
(18) Manet, I.; Manoli, F.; Donzello, M. P.; Ercolani, C.; Vittori, D.;
Cellai, L.; Masi, A.; Monti, S. Tetra-2,3-pyrazinoporphyrazines with
externally appended pyridine rings. 10. A water-soluble bimetallic
(ZnII/PtII) porphyrazine hexacation as potential plurimodal agent for
cancer therapy: exploring the behavior as ligand of telomeric DNA G-
quadruplex structures. Inorg. Chem. 2011, 50, 7403−7411.
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