Synthesis and in vitro photodynamic activity of Oligomeric ethylene glycol-quinoline substituted zinc(II) phthalocyanine derivatives

Article abstract of DOI:10.1021/jm400722d

A new series of zinc(II) phthalocyanine derivatives have been synthesized and characterized. These macrocycles exhibited a sharp absorption band in the red visible region in DMF, which indicated that they were dissolved well and almost did not aggregate i

Full text of DOI:10.1021/jm400722d

Article
pubs.acs.org/jmc
Synthesis and in Vitro Photodynamic Activity of Oligomeric Ethylene
Glycol−Quinoline Substituted Zinc(II) Phthalocyanine Derivatives
Xiao Jia, Feng-Feng Yang, Jun Li, Jian-Yong Liu, and Jin-Ping Xue*
Fujian Engineering Research Center for Drug and Diagnoses, Treat of Photodynamic Therapy, College of Chemistry and Chemical
Engineering, Fuzhou University, Fuzhou, China
S
* Supporting Information
ABSTRACT: A new series of zinc(II) phthalocyanine derivatives
have been synthesized and characterized. These macrocycles
exhibited a sharp absorption band in the red visible region in
DMF, which indicated that they were dissolved well and almost did
not aggregate in this solvent. Compared with the unsubstituted
zinc(II) phthalocyanine, all these phthalocyanines have a red-shifted
Q-band (at 678−699 vs 670 nm) and exhibit a relatively weaker
fluorescence emission and a higher efficiency at generating singlet
oxygen. The monosubstituted photosensitizers also exhibit high
photocytotoxicity toward HepG2 human hepatocarcinoma cells
with IC50 values as low as 0.02−0.05 μM (λ = 670 nm, 80
mW·cm−2, 1.5 J·cm−2). The high photodynamic activities of these compounds are in accordance with their low aggregation
tendency and high cellular uptake. Their structure−activity relationship was assessed by determining the photophysical
properties, cellular uptake, and in vitro photodynamic activities of this series of compounds. As shown by confocal microscopy,
monosubstituted phthalocyanines can target the mitochondria and lysosomes of the cells, and tetrasubstituted phthalocyanines
tend to target the lysosomes of the cells.
being exploited in cancer chemotherapy and a number of them
are in different phases of clinical trials in recent years.8,11,12
Shen et al. demonstrated some 8-hydroxyquinoline derivatives
are potential anticancer drug candidates.13 Vincent and his
colleagues also discovered a new family of bis-8-hydroxyquino-
line substituted benzylamines with proapoptotic activity in
cancer cells.14 It is very interesting that quinoline and its
analogues have recently been examined for their modes of
function in the inhibition of tyrosine kinases, proteasome,
tubulin polymerization, and DNA repair.15,16 Polyethylene
glycols are known to be excellent pharmaceutical intermediates,
which possess excellent biocompatibility and can minimize
nonspecific uptake and enable specific tumor-targeting through
the enhanced permeability and retention (EPR) effect.17
Therefore, these hydrophilic moieties adding to the hydro-
phobic core will enhance the solubility and make the molecules
amphiphilic, which is a desirable characteristic for efficient
photosensitizers. Dennis et al. prepared a series of asymmetric
methylated polyethylene glycol zinc(II) phthalocyanines, which
proved to have high cytotoxic properties despite their water
insolubility.18 Free hydroxyl groups at the end of the
polyoxochain were reported to considerably increase the
water solubility of the phthalocyanine but have been found
the lower photodynamic activity.19 In addition, some photo-
sensitizers such as lutetium texaphyrin involving the use of
INTRODUCTION
■
Photodynamic therapy (PDT) has emerged as a promising and
noninvasive treatment for (against) malignant tumors and wet
age-related macular degeneration. Fundamentally, the treat-
ment utilizes the combination of a photosensitizer, light, and
molecular oxygen to cause cellular and tissue damage, in which
reactive oxygen species (ROS), generated through a series of
photoinduced processes, is believed to be the major cytotoxic
agent. For cancer treatment, PDT has several potential
advantages including its noninvasive nature, tolerance of
repeated doses, and high specificity that can be achieved
through precise application of the light.1−3
Phthalocyanines are promising second-generation photo-
sensitizers for PDT as a result of their strong absorption in
tissue-penetrating red light and high efficiency of generating
singlet oxygen.4−7 However, one important problem related to
phthalocyanine derivatives is their low solubility in several
organic media and water. In addition, aggregation phenomena
are observed and may have a strong influence on the efficiency
of singlet oxygen production and then the bioavailability.8
Quinoline derivatives have displayed a wide range of biological
activity. Luis and his colleagues reported that 2-arylquinolines,
particularly those containing methoxy substituents or no
substituents in the quinoline skeleton, showed antiviral
properties against human immunodeficiency syndrome
(HIV).9 Ridley and Hudson indicated that quinoline exhibited
antimalarial property.10 On the other hand, quinoline related
chemical classes such as quinolines and naphthyridines are
Received: April 2, 2013
Published: June 20, 2013
© 2013 American Chemical Society
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Scheme 1. Synthesis of α-Substituted Phthalocyanine Derivatives
polyethylene glycols derivatives as additive in formulations have
been used in PDT clinical treatment.20
these phthalocyanine derivatives could be purified readily by
silica gel column chromatography in 10−48% yield. All the new
1
In this report, we describe a novel series of zinc(II)
phthalocyanines substituted with oligomeric ethylene glycol
and 8-hydroxyquinoline moieties. The two substituents reduce
their aggregation tendency, thereby promoting the generation
of singlet oxygen and resulting in higher photodynamic
activities. In addition, we report the synthesis, spectroscopic
and photophysical characteristics, and the in vitro photo-
dynamic activities of these compounds.
compounds were characterized with H NMR, HRMS, and IR.
Photophysical and Photochemical Properties. The
electronic absorption spectra of phthalocyanines α-2d, α-3d, α-
4d, β-4d and 4α-2d, 4α-3d, 4α-4d, 4β-3d, 4β-4d in DMF were
recorded. The spectra showed typical features of nonaggregated
phthalocyanines. They displayed a Soret band at 320−350 nm,
an intense and sharp Q-band at 672−699 nm, together with
two vibronic bands at 610−650 nm. For all of these
phthalocyanines, the Q-band strictly followed the Lambert−
Beer law (up to 10 μM), indicating that the aggregation of
these phthalocyanines is not significant in DMF (Figure S1−S8
in the Supporting Information). The electronic absorption
spectra data for all phthalocyanines are summarized in Table 1.
Compared with ZnPc (670 nm), all these analogues exhibit a
red-shifted Q-band (672−699 nm). The α-substituted
phthalocyanine further shifts its Q-band to 677 or 699 nm,
while the β-substituted one has its Q-band at 672 or 679 nm.
Compared with monosubstituted phthalocyanines, tetrasub-
stituted phthalocyanines have a further red-shift Q-band (at 699
nm vs 677 nm and at 679 nm vs 672 nm) (Figure S9−S18 in
the Supporting Information). For α-2d, α-3d, α-4d, β-4d, 4β-
3d, 4β-4d excited at 610 nm, it shows a fluorescence emission
at 684−691 nm with a quantum yield (ΦF) of 0.26−0.28 in
DMF. For 4α-2d, 4α-3d, 4α-4d excited at 630 nm, the
fluorescence emission was red-shifted to 709−710 nm with an
RESULTS AND DISCUSSION
■
Synthesis and Characterization. The synthesis of
zinc(II) phthalocyanines is shown in Scheme 1 and Scheme
2. First, the alcohols were converted to the corresponding
compounds 2a−4a in triethylamine (TEA) and CH2Cl2
reaction systems. Then treatment of 2a−4a with 8-hydrox-
yquinoline gave the substituted products 2b−4b, which then
underwent nucleophilic substitution with 3-nitrophthalonitrile
or 4-nitrophthalonitrile to give 2c−4c, 3c-1, and 4c-1. Heating
these phthalonitriles in n-pentanol with Zn(OAc)2·2H2O and
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) led to phthalocya-
nines 4α-2d, 4α-3d, 4α-4d, 4β-3d, 4β-4d. Mixed cyclization of
precursors 2c−4c and 4c-1 with an excess of unsubstituted
phthalonitrile (9 equiv) in the presence of Zn(OAc)2·2H2O
and DBU in n-pentanol afforded the corresponding “3 + 1”
unsymmetrical phthalocyanines α-2d, α-3d, α-4d, and β-4d. All
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Scheme 2. Synthesis of β-Substituted Phthalocyanine Derivatives
Table 1. Photophysical/Photochemical Data for All These Phthalocyanines
a
b
abs
max
ex
max
λ
(nm) (log ε)
λ
(nm)
ΦF
ΦΔ
compd
in DMF
in medium
in DMF
in medium
in DMF
in medium
in DMF
c
α-2d
677 (5.34)
677 (5.36)
677 (5.43)
672 (5.41)
699 (5.43)
699 (5.36)
699 (5.34)
679 (5.26)
679 (5.28)
682
681
681
676
707
708
707
687
685
685
684
686
680
709
709
710
691
691
693
690
682
712
717
716
693
691
0.24
0.24
0.24
0.26
0.17
0.17
0.17
0.26
0.28
0.19
0.19
0.21
0.18
0.05
0.06
0.09
0.05
0.06
0.63
0.66
0.68
0.62
0.86
0.86
0.88
0.58
0.75
c
α-3d
c
α-4d
c
β-4d
d
4α-2d
d
4α-3d
d
4α-4d
c
4β-3d
c
4β-4d
690
a
b
c
d
Relative to ZnPc (ΦF = 0.28). Relative to ZnPc (ΦΔ = 0.56). Excited at 610 nm. Excited at 630 nm.
even lower ΦF of 0.17. It is likely that introduction of an
electron-donor alkoxy group at the phthalocyanine macrocycle
makes the ΦF value smaller relative to ZnPc (ΦF = 0.28)21 as
the energy gap decreases between the HOMO and the LUMO.
To evaluate the photosensitizing efficiency of these
phthalocyanines, their singlet oxygen quantum yields (ΦΔ)
were determined in DMF by a steady-state method with 1,3-
diphenylisobenzofuran (DPBF) as the scavenger.22 The
variation of DPBF absorption was monitored using a UV−vis
spectrophotometer. The absorbance at 415 nm gradually
decreased (Figure S19−S24 in the Supporting Information),
while there was no decrease in Q-band λambasx. All the ΦΔ values
are summarized in Table 1. It can be seen that all of these
monosubstituted phthalocyanines are efficient singlet oxygen
generators having comparable quantum yields (ΦΔ = 0.62−
0.68), which is higher than that of ZnPc (ΦΔ = 0.56), and tetra-
α-substituted phthalocyanines exhibit higher ability to generate
singlet oxygen with a singlet oxygen quantum yield of 0.86−
0.88. As shown in Table 1, the ΦΔ data are virtually the same
for the analogues α-2d, α-3d, α-4d, 4α-2d, 4α-3d, and 4α-4d,
showing that the length of the chains does not affect the
photochemical properties of phthalocyanine.
In Vitro Photodynamic Activities. The cytotoxic effects
of these phthalocyanines in Cremophor EL emulsions were
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Figure 1. Cytotoxic effects of title compounds on HepG2 cells in the absence (closed symbols) and presence (open symbols) of light (λ = 670 nm,
80 mW·cm−2, 1.5 J·cm−2). Data are expressed as mean values standard error of the mean value (SEM) of three independent experiments, each
performed in quadruplicate.
investigated against HepG2 human hepatocarcinoma cells.
Figure 1 shows the dose-dependent survival curves for all these
compounds. It can be seen that monosubstituted phthalocya-
nines are essentially noncytotoxic in the absence of light (up to
0.2 μM). However, upon illumination they have significantly
high photocytotoxicity toward HepG2 cells with IC50 values,
defined as the dye concentration required to kill 50% of the
cells, as low as 0.01−0.03 μM (Figure 1a, Table 2) under a
aggregation provides an efficient nonradiative relaxation
pathway, thereby greatly shortening the excited-state lifetime,
and the singlet oxygen quantum yield would decrease,
eventually leading to photodynamic activity greatly reduced
or no photodynamic activity. The aggregation behavior of
phthalocyanines, formulated with Cremophor EL in the RPMI
1640 culture medium, was examined by absorption and
fluorescence spectroscopic methods. As shown in Figure 2,
monosubstituted phthalocyanines show a relatively sharp and
intense Q-band and a relatively strong fluorescence emission.
At the same time, tetra-β-substituted phthalocyanines have a
significantly broadened Q-band and relatively weak fluores-
cence. The results suggest that monosubstituted phthalocya-
nines are not significantly aggregated under these conditions,
which seems to be in accord with their high photocytotoxicity.
To account for the different photocytotoxicity of three
phthalocyanines (α-3d, 4α-3d, and 4β-3d), their cellular uptake
was examined by fluorescence microscopy. As shown by the
images captured by confocal laser scanning microscopy (Figure
3), three compounds could enter into the cells causing
intracellular fluorescence after incubation for 24 h. After
incubation with these phthalocyanines, SDS was used to lyse
the cells and extract the phthalocyanines. The phthalocyanine
concentrations inside the cells were quantified by measuring
their fluorescence at 690 nm by using the fluorescence
microscopy (excitation at 610 nm). The results are depicted
in Figure 3 g, which shows that the cellular uptake of α-3d is
only little higher than that of 4α-3d and actually about 5-fold
higher than that of 4β-3d. The higher cellular uptake of α-3d is
in accord with its higher photocytotoxicity.
Table 2. IC50 for Phthalocyanines against HepG2 Cells (λ =
670 nm, 80 mW·cm−2, 1.5 J·cm−2)
compd
IC50 (μM)
compd
IC50 (μM)
α-2d
α-3d
α-4d
β-4d
0.016
0.019
0.027
0.012
4α-2d
4α-3d
4α-4d
4β-4d
4β-3d
>40
10
8
10
>40
rather low light dose (λ = 670 nm, 80 mW·cm−2, 1.5 J·cm−2).
Tetrasubstituted phthalocyanines show less photodynamic
activity toward HepG2 cells (λ = 670 nm, 80 mW·cm−2, 1.5
J·cm−2). For 4α-2d, 4α-3d, 4β-3d, they are almost non-
cytotoxic without light, but for 4α-4d and 4β-4d, they are
cytotoxic in the absence of light for HepG2 cells (Figure 1b,
Table 2). The IC50 value of α-3d is as low as 0.019 μM toward
the HepG2 cells under a rather low light dose, which is 526-fold
lower than that of 4α-3d. The results show that monosub-
stituted phthalocyanines (α-2d, α-3d, α-4d, and β-4d) have
significantly high photocytotoxicity.
We also investigate the subcellular localization of these
phthalocyanines by confocal laser scanning microscopy. The
The aggregation state of photosensitizers is an important
factor relating to their photodynamic activities.23 Usually,
Figure 2. Electronic absorption spectra and fluorescence spectra of all new phthalocyanines, formulated with Cremophor EL, in the RPMI 1640
culture medium (all phthalocyanines at 8 μM).
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Figure 3. Confocal fluorescence images of HepG2 cells after incubation with α-3d (b) or 4α-3d (d) or 4β-3d (f) for 24 h (at 10 μM). The
corresponding bright field images are given in the left column (a) for α-3d, (c) for 4α-3d, and (e) for 4β-3d. (f) Comparison of the percentage of
cellular uptake of α-3d, 4α-3d, and 4β-3d. Data are expressed as mean value standard deviation (SD) of three independent experiments.
Figure 4. (a) Visualization of the bright field image. (b) Corresponding superimposed image of intracellular fluorescence of HepG2 using filter sets
specific for α-3d (in red) and Mito Tracker (in green). (c) Fluorescence intensity profiles of α-3d (in red) and Mito Tracker (in green). (d)
Visualization of the bright field image. (e) Corresponding superimposed image of intracellular fluorescence of HepG2 using filter sets specific for 4α-
3d (in red) and Lyso Tracker (in green). (f) Fluorescence intensity profiles of 4α-3d (in red) and Lyso Tracker (in green).
cells were first incubated with the compound in the culture
medium for 2 h and then stained with LysoTracker DND 26 or
MitoTracker Green FM (for 10−20 min), which are specific
dyes for lysosomes or mitochondria, respectively. As shown in
Figure 4 and Figure S25 (in the Supporting Information), the
fluorescence caused by the MitoTracker (excited at 488 nm,
monitored at 500−570 nm) can superimpose with the
fluorescence caused by α-3d (excited at 633 nm, monitored
at 640−700 nm). The very similar fluorescence intensity line
profiles of α-3d and MitoTracker traced along the green line in
Figure 4 c also confirms that α-3d can target the mitochondria
of the cells. Meanwhile the fluorescence images of α-3d could
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under an atmosphere of nitrogen for 48 h. The volatiles were removed
in vacuo. Then the residue was mixed with water (100 mL) and
extracted with CH2Cl2 (80 mL × 3). The combined organic extracts
were dried over anhydrous MgSO4. After evaporation under reduced
pressure, the residue was purified by silica gel column chromatography
using CHCl3/EA/CH3OH (20:30:1 v/v/v) as the eluent.
Phthalonitrile 2c. According to the general procedure, 2b (3.09 g,
13.25 mmol) was treated with 3-nitrophthalonitrile (3.45 g, 19.93
mmol) and K2CO3 (5.56 g, 40.29 mmol) in DMF to give 2c as a white
solid (54%). 1H NMR (500 MHz, CDCl3): δ 8.91 (dd, J = 1.5, 4.0 Hz,
1 H, ArH), 8.13 (dd, J = 2.0, 8.5 Hz, 1 H, ArH), 7.48−7.39 (m, 4 H,
ArH), 7.28 (d, J = 9.0 Hz, 1 H, ArH), 7.22 (d, J = 7.5 Hz, 1 H, ArH),
7.09 (d, J = 8.0 Hz, 1 H, ArH), 4.41 (t, J = 4.5 Hz, 2 H, CH2), 4.34 (t, J
= 4.5 Hz, 2 H, CH2), 4.15 (t, J = 4.5 Hz, 2 H, CH2), 4.05 (t, J = 4.5
Hz, 2 H, CH2). HRMS (ESI): m/z calcd for C21H17N3O3 [M + H]+
360.1342, found 360.1364. IR: υ(−CN) 2227 cm−1; υas(−CH2−)
2942 cm−1, υs(−CH2−) 2876 cm−1; υ(Ar) 1581, 1499, 1470 cm−1;
υ(CH) 3085 cm−1; δ(CH) 816 cm−1; υas(C−O−C) 1111 cm−1;
υ(Ar−O−R) 1290 cm−1; υ(CN) 1613 cm−1; υ(H−O−H) 3440
cm−1.
be merged with that of the LysoTracker (excited at 488 nm,
monitored at 500−570 nm) (parts e and f of Figure S25 in the
Supporting Information), showing that α-3d can target the
lysosomes of the cells too. The fluorescence caused by the
LysoTracker (excited at 488 nm, monitored at 500−570 nm) is
superimposed with the fluorescence caused by 4α-3d (or 4β-
3d) (excited at 633 nm, monitored at 640−700 nm) (Figure 4e
or Figure S25k). The very similar fluorescence intensity line
profiles of 4α-3d (or 4β-3d) and LysoTracker traced along the
green line in Figure 4f (or Figure S25l)) also confirms that 4α-
3d (or 4β-3d) can target the lysosomes of the cells. By contrast,
the fluorescence images of 4α-3d (or 4β-3d) could not be
merged with that of the MitoTracker (part c or part i of Figure
S25 in the Supporting Information), showing that 4α-3d (or
4β-3d) is not localized in the mitochondria of the cells. The
difference between mono- and tetrasubstituted phthalocyanine
for subcellular locatization may be explained by the varying
degrees of the protonation of hydroxyquinoline of phthalocya-
nines caused by lower pH in lysosomes and by tetrasubstituted
phthalocyanines becoming water-soluble and having difficulty
crossing the membrane of lysosomes and being detained in
lysosomes of the cells. It may explain the trend of photo-
cytotoxicity observed for this series of compounds.
Phthalonitrile 3c. According to the general procedure, 3b (1.59 g,
5.72 mmol) was treated with 3-nitrophthalonitrile (1.48 g, 8.50 mmol)
and K2CO3 (4.26 g, 30.65 mmol) in DMF to give 3c as a white solid
(47%). 1H NMR (500 MHz, CDCl3): δ 8.92 (dd, J = 1.5, 4.5 Hz, 1 H,
ArH), 8.13 (dd, J = 1.5, 8.5 Hz, 1 H, ArH), 7.54 (t, J = 8.5 Hz, 1 H,
ArH), 7.47−7.39 (m, 3 H, ArH), 7.27 (vd, J = 8.5 Hz, 2 H, ArH), 7.12
(d, J = 7.5 Hz, 1 H, ArH), 4.42 (t, J = 5.0 Hz, 2 H, CH2), 4.27 (d, J =
4.5 Hz, 2 H, CH2), 4.07 (t, J = 5.0 Hz, 2 H, CH2), 3.93 (t, J = 5.0 Hz, 2
H, CH2), 3.78 (s, 4 H, CH2). HRMS (ESI): m/z calcd for
C23H21N3O4 [M + H]+ 404.1604, found 404.1614. IR: υ(−CN)
2229 cm−1; υas(−CH2−) 2904 cm−1; υs(−CH2−) 2872 cm−1; υ(Ar)
1584, 1502, 1473 cm−1; υas(C−O−C) 1103 cm−1; υ(Ar−O−R) 1285
cm−1; υ(H−O−H) 3567 cm−1.
CONCLUSIONS
■
We have prepared and characterized a series of novel zinc(II)
phthalocyanines substituted with quinolin-8-yloxy-poly-ethoxy
at the peripheral positions and evaluated their in vitro
photodynamic activities. All these monosubstituted phthalo-
cyanines are less aggregated in the culture medium, and upon
illumination they are highly photodynamic active toward
HepG2 cells with IC50 values as low as about 0.02 μM,
which is 450-fold lower than that of the tetrasubstituted
phthalocyanines. As shown by confocal microscopy, mono-
substituted phthalocyanines can target the mitochondria and
lysosomes of the cells and tetrasubstituted phthalocyanines
tend to target the lysosomes of the cells. The overall results
show that these monosubstituted phthalocyanines are highly
promising antitumor agents for photodynamic therapy.
Phthalonitrile 4c. According to the general procedure, 4b (6.23 g,
19.37 mmol) was treated with 3-nitrophthalonitrile (4.65 g, 26.86
mmol) and K2CO3 (8.93 g, 64.71 mmol) in DMF to give 4c as a pale
1
yellow oil (46%). H NMR (500 MHz, CDCl3): δ 8.91 (dd, J = 1.5,
4.0 Hz, 1 H, ArH), 8.12 (dd, J = 1.5, 8.5 Hz, 1 H, ArH), 7.56 (vt, J =
8.5 Hz, 1H, ArH), 7.46−7.37 (m, 3 H, ArH), 7.28−7.25 (m, 2 H,
ArH), 7.09 (dd, J = 1.5, 8.0 Hz, 1 H, ArH), 4.39 (t, J = 5.0 Hz, 2 H,
CH2), 4.26 (t, J = 4.5 Hz, 2 H, CH2), 4.06 (t, J = 5.0 Hz, 2 H, CH2),
3.89 (t, J = 5.0 Hz, 2 H, CH2), 3.78−3.76 (m, 2 H, CH2), 3.73−3.71
(m, 2 H, CH2), 3.70−3.68 (m, 2 H, CH2), 3.67−3.65 (m, 2 H, CH2).
HRMS (ESI): m/z calcd for C25H25N3O5 [M + H]+ 448.1866, found
448.1905. IR: υ(−CN) 2230 cm−1; υas(−CH2−) 2917 cm−1
;
υs(−CH2−) 2876 cm−1; υ(Ar) 1583, 1503, 1472 cm−1; υ(CH)
3086 cm−1; δ(CH) 732−824 cm−1; υas(C−O−C) 1109 cm−1;
υ(Ar−O−R) 1294 cm−1; υ(CN) 1616 cm−1; υ(H−O−H) 3430
cm−1.
EXPERIMENTAL SECTION
■
Materials and Methods. All reactions were carried out under an
atmosphere of nitrogen. n-Pentanol was distilled over sodium, and all
other solvents and reagents were reagent grade and used as received.
Compounds 2a,24 3a,24 4a,24 2b,25 3b,26 and 4b27 were prepared as
previously described. 1H NMR spectra were recorded on an AVANCE
III 500 MHz spectrometer, in CDCl3 unless otherwise stated. Spectra
were referenced internally using the residual solvent (1H: δ 7.26)
resonances relative to SiMe4. Electronic adsorption spectra were
measured on a TU-1901 spectrometer, and fluorescence spectra were
obtained on a Varian Cary eclipse spectrometer. IR spectra were
measured on a Perkin-Elmer SP2000. HRMS spectra were obtained on
ion trap mass spectrometry DECAX-30000 LCQ Deca XP mass
spectrometer. The fluorescence quantum yields of the samples
Phthalonitrile 3c-1. According to the general procedure, 3b (2.45
g, 8.83 mmol) was treated with 4-nitrophthalonitrile (1.6 g, 9.24
mmol) and K2CO31 (3.6 g, 26.09 mmol) in DMF to give 3c-1 as a
white solid (84%). H NMR (500 MHz, CDCl3): δ 8.90 (dd, J = 1.5,
4.5 Hz, 1 H, ArH), 8.14 (dd, J = 1.5, 8.0 Hz, 1 H, ArH), 7.58 (d, J =
9.0 Hz, 1 H, ArH), 7.47−7.40 (m, 3 H, ArH), 7.22 (d, J = 2.5 Hz, 1 H,
ArH), 7.14 (dd, J = 3.0, 9.0 Hz, 1 H, ArH), 7.08 (d, J = 7.5 Hz, 1 H,
ArH), 4.39 (t, J = 5.0 Hz, 2 H, CH2), 4.17 (t, J = 4.5 Hz, 2 H, CH2),
4.06 (t, J = 5.0 Hz, 2 H, CH2), 3.88 (t, J = 4.5 Hz, 2 H, CH2), 3.81−
3.79 (m, 2 H, CH2), 3.76−3.74 (m, 2 H, CH2). HRMS (ESI): m/z
calcd for C23H21N3O4 [M + H]+ 404.1605, found 404.1621. IR:
υ(−CN) 2226 cm−1; υas(−CH2−) 2929 cm−1; υs(−CH2−) 2881
cm−1; υ(Ar) 1596, 1574 cm−1; 1503 cm−1; 1474 cm−1; υas(C−O−C)
1126 cm−1; υ(Ar−O−R) 1259 cm−1; υ(H−O−H) 3401 cm−1.
Phthalonitrile 4c-1. According to the general procedure, 4b (7.42
g, 23.09 mmol) was treated with 4-nitrophthalonitrile (5.97 g, 34.48
mmol) and K2CO3 (6.45 g, 46.74 mmol) in DMF to give 4c-1 as a
pale yellow oil (45%). 1H NMR (500 MHz, CDCl3): δ 8.92−8.91 (m,
1 H, ArH), 8.13 (dd, J = 1.5, 8.5 Hz, 1 H, ArH), 7.62 (d, J = 9.0 Hz, 1
H, ArH), 7.46−7.39 (m, 3 H, ArH), 7.23 (d, J = 2.5 Hz, 1 H, ArH),
7.15 (dd, J = 2.5, 8.5 Hz, 1 H, ArH), 7.11−7.10 (m, 1 H, ArH), 4.41 (t,
[ΦF(sample)] were determined by the equation ΦF(sample) = (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].21
The purity of all the new compounds was determined by HPLC
analysis and was found to be ≥95%.
General Procedure for the Preparation of 2c, 3c, 4c, 3c-1,
and 4c-1. A mixture of compounds 2b−4b (1 equiv), 3-nitro-
phthalonitrile or 4-nitrophthalonitrile (1.1−1.5 equiv), and K2CO3
(3−5 equiv) in DMF (20−30 mL) was stirred at room temperature
5802
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Journal of Medicinal Chemistry
Article
J = 5.0 Hz, 2 H, CH2), 4.16 (t, J = 4.5 Hz, 2 H, CH2), 4.06 (t, J = 5.0
Hz, 2 H, CH2), 3.86 (t, J = 4.5 Hz, 2 H, CH2), 3.79−3.77 (m, 2 H,
CH2), 3.70−3.68 (m, 6 H, CH2). HRMS(ESI): m/z calcd for
C25H25N3O5 [M + H]+ 448.1866, found 448.1809. IR: υ(−CN) 2230
cm−1; υas(−CH2−) 2917 cm−1; υs(−CH2−) 2876 cm−1; υ(Ar) 1598,
1503, 1472 cm−1; υ(CH) 3086 cm−1; δ(CH) 732−816 cm−1;
υas(C−O−C) 1107 cm−1; υ(Ar−O−R) 1257 cm−1; υ(H−O−H) 3436
cm−1.
General Procedure for the Preparation of Phthalocyanines
α-2d, α-3d, α-4d, and β-4d. A mixture of phthalonitriles 4a−c and
4c-1 (1 equiv), phthalonitrile (9 equiv), and Zn(OAc)2·2H2O (5
equiv) in n-pentanol (15 mL) was heated to 100 °C. Then a small
amount of DBU (0.5 mL) was added. The mixture was stirred at 150
°C for 12 h. After a brief cooling, the volatiles were removed under
reduced pressure. The residue was purified by silica gel column
chromatography using CHCl3/CH3OH (30:1 v/v) as the eluent. The
crude product was further purified by recrystallization from a mixture
of THF and hexane to give the product as a blue solid.
Phthalocyanine α-2d. According to the above procedure,
phthalonitrile 2c (0.18 g, 0.50 mmol) was treated with unsubstituted
phthalonitrile (0.58 g, 4.52 mmol) and Zn(OAc)2·2H2O (0.88 g, 4.00
mmol) to give α-2d as a blue solid (0.04 g, 10%). 1H NMR (500 MHz,
DMSO-d6): δ 9.33−9.24 (m, 6 H, Pc-Hα), 8.88 (d, J = 8.0 Hz, 1 H, Pc-
Hα), 8.75 (s, 1 H, ArH), 8.22−8.02 (m, 8 H, Pc-Hβ), 7.70 (d, J = 8.0
Hz, 1 H, ArH), 7.43−7.40 (m, 1 H, ArH), 7.32−7.26 (m, 2 H, ArH),
7.15 (d, J = 7.5 Hz, 1 H, ArH), 4.92 (s, 2 H, CH2), 4.54 (t, J = 4.5 Hz,
2 H, CH2), 4.50 (t, J = 4.5 Hz, 2 H, CH2), 4.43 (t, J = 4.5 Hz, 2 H,
CH2). HRMS (ESI): m/z calcd for C45H29N9O3Zn [M + H]+
808.1757, found 808.1671. IR: υ(Pc) 1648, 1588, 1488, 1452 cm−1;
736; υas(−CH2−) 2921 cm−1; υs(−CH2−) 2852 cm−1; υas(C−O−C)
1114 cm−1; υs(Ar−O−R) 1265 cm−1; υ(C−N) 1332 cm−1; υ(H−O−
H) 3437 cm−1.
Phthalocyanine α-3d. According to the above procedure,
phthalonitrile 3c (0.20 g, 0.50 mmol) was treated with unsubstituted
phthalonitrile (0.58 g, 4.49 mmol) and Zn(OAc)2·2H2O (0.54 g, 2.45
mmol) to give α-3d as a blue solid (0.04 g, 10%). 1H NMR (500 MHz,
DMSO-d6): δ 9.21−9.08 (m, 3 H, Pc-Hα), 9.02−8.98 (m, 2 H, Pc-Hα),
8.97 (d, J = 2.0 Hz, 1 H, Pc-Hα), 8.69−8.68 (m, 1 H, Pc-Hα), 8.63 (d, J
= 7.0 Hz, 1 H, Pc-Hβ), 8.16−8.13 (m, 5 H, Pc-Hβ), 8.10−8.03 (m, 2
H, Pc-Hβ), 7.87 (t, J = 8.0 Hz, 1 H, ArH), 7.47 (d, J = 8.0 Hz, 1 H,
ArH), 7.37−7.34 (m, 1 H, ArH), 7.30−7.24 (m, 2 H, ArH), 6.91 (dd, J
= 1.5 Hz, 7.5 Hz, 1 H, ArH), 4.75 (br s, 2 H, CH2), 4.34 (t, J = 4.0 Hz,
2 H, CH2), 4.10−4.07 (m, 4 H, CH2), 3.88 (t, J = 4.5 Hz, 2 H, CH2),
3.85 (t, J = 4.5 Hz, 2 H, CH2). HRMS (ESI): m/z calcd for
C47H33N9O4Zn [M + H]+ 852.2019, found 852.2002. IR: υ(Pc)1599,
1488, 1456, 736 cm−1; υas(−CH2−) 2924 cm−1; υs(−CH2−) 2868
cm−1; υas(C−O−C) 1115 cm−1; υs(Ar−O−R) 1257 cm−1; υ(C−N)
1332 cm−1; υ(H−O−H) 3430 cm−1.
Phthalocyanine α-4d. According to the above procedure,
phthalonitrile 4c (0.36 g, 0.80 mmol) was treated with unsubstituted
phthalonitrile (0.92 g, 7.19 mmol) and Zn(OAc)2·2H2O (0.88 g, 3.99
mmol) to give α-4d as a blue solid (0.08 g, 11%). 1H NMR (500 MHz,
DMSO-d6): δ 9.40−9.38 (m, 6 H, Pc-Hα), 9.00 (d, J = 7.0 Hz, 1 H, Pc-
Hα), 8.71−8.70 (m, 1 H, ArH), 8.24−8.22 (m, 6 H, Pc-Hβ), 8.14−8.11
(m, 2 H, Pc-Hβ), 7.73 (d, J = 8.0 Hz, 1 H, ArH), 7.39−7.36 (m, 1 H,
ArH), 7.30 (d, J = 8.0 Hz, 1 H, ArH), 7.24 (t, J = 8.0 Hz, 1 H, ArH),
6.87 (d, J = 8.0 Hz, 1 H, ArH), 4.87 (vt, J = 4.0 Hz, 2 H, CH2), 4.39 (t,
J = 4.5 Hz, 2 H, CH2), 4.07 (t, J = 4.5 Hz, 2 H, CH2), 4.00 (t, J = 4.5
Hz, 2 H, CH2), 3.76 (t, J = 4.5 Hz, 2 H, CH2), 3.68 (t, J = 4.5 Hz, 2 H,
CH2), 3.60−3.58 (m, 2 H, CH2), 3.55−3.53(m, 2 H, CH2). HRMS
(ESI): m/z calcd for C49H37N9O5Zn [M + H]+ 896.2281, found
896.2210. IR: υ(Pc) 1642, 1589, 1488, 1456, 745 cm−1; υas(−CH2−)
2917 cm−1; υs(−CH2−) 2870 cm−1; υas(C−O−C) 1095 cm−1; υs(Ar−
O−R) 1256 cm−1; υ(C−N) 1331 cm−1; υ(H−O−H) 3426 cm−1.
Phthalocyanine β-4d. According to the above procedure,
phthalonitrile 4c-1 (0.51 g, 1.14 mmol) was treated with unsubstituted
phthalonitrile (1.15 g, 8.98 mmol) and Zn(OAc)2·2H2O (1.09 g, 4.97
mmol) to give β-4d as a blue solid (0.15 g, 15%). 1H NMR (500 MHz,
DMSO-d6): δ 9.22−9.16 (m, 3 H, Pc-Hα), 9.11 (d, J = 7.0 Hz, 1 H, Pc-
Hα), 9.06 (d, J = 7.0 Hz, 1 H, Pc-Hα), 9.03 (d, J = 7.0 Hz, 1 H, Pc-Hα),
8.8−8.79 (m, 1 H, Pc-Hα), 8.76 (d, J = 8.5 Hz, 1 H, Pc-Hα), 8.34 (s, 1
H, ArH), 8.21−8.04 (m, 7 H, Pc-Hβ), 7.52−7.50 (m, 1 H, ArH),
7.45−7.42 (m, 1 H, ArH), 7.38−7.12 (m, 2 H, ArH), 7.11−7.09 (m, 1
H, ArH), 4.57 (s, 2 H, CH2), 4.23 (t, J = 4.5 Hz, 2 H, CH2), 4.07 (t, J
= 4.5 Hz, 2 H, CH2), 3.88 (t, J = 4.5 Hz, 2 H, CH2), 3.83−3.81 (m, 2
H, CH2), 3.74−3.72 (m, 4 H, CH2), 3.70−3.68 3.83−3.81 (m, 2 H,
CH2). HRMS(ESI): m/z calcd for C49H37N9O5Zn [M + H]+
896.2281, found 896.2211. IR: υ(Pc) 1503, 1486 cm−1; υas(−CH2−)
2920 cm−1; υs(−CH2−) 2867 cm−1; υas(C−O−C) 1060 cm−1; υs(Ar−
O−R) 1240 cm−1; υ(C−N) 1334 cm−1; υ(H−O−H) 3443 cm−1.
General Procedure for the Preparation of Phthalocyanines
4α-2d, 4α-3d, 4α-4d, 4β-3d, 4β-4d. A mixture of phthalonitriles
4a−c, 3c-1, and 4c-1 (1 equiv) and Zn(OAc)2·2H2O (2 equiv) in n-
pentanol (15 mL) was heated to 100 °C. Then a small amount of
DBU (0.5 mL) was added. The mixture was stirred at 150 °C for 12 h.
After a brief cooling, the volatiles were removed under reduced
pressure. The residue was purified by silica gel column chromatog-
raphy using CHCl3/CH3OH (15:1 v/v) as the eluent. The crude
product was further purified by recrystallization from a mixture of
CHCl3 and hexane to give the product as a green solid.
Phthalocyanine 4α-2d. According to the above procedure,
phthalonitrile 2c (0.86 g, 2.4 mmol) was treated with Zn(OAc)2·2H2O
1
(0.26 g, 1.21 mmol) to give 4α-2d as a green solid (0.33 g, 37%). H
NMR (500 MHz, DMSO-d6): δ 8.94−8.80 (m, 4 H, Pc-Hα), 8.79−
8.66 (m, 4 H, Pc- Hβ), 8.19−8.18 (m, 2 H, Pc-Hβ), 8.09 (t, J = 7.0 Hz,
2 H, Pc-Hβ), 8.06 (br s, 2 H, ArH), 7.92 (br s, 2 H, ArH), 7.69−7.68
(m, 4 H, ArH), 7.46−7.43 (m, 2 H, ArH), 7.36−7.26 (m, 8 H, ArH),
7.24−7.14 (m, 4 H, ArH), 6.94 (vt, J = 6.0 Hz, 2 H, ArH), 5.19 (br s, 4
H, CH2), 4.89 (br s, 4 H, CH2), 4.52−4.48 (m, 8 H, CH2), 4.42−4.39
(m, 4 H, CH2), 4.28 (br s, 4 H, CH2), 4.23 (br s, 4 H, CH2), 4.04 (br
s, 4 H, CH2). HRMS (ESI): m/z calcd for C84H68N12O12Zn [M + H]+
1501.4449, found 1501.3867. IR: υ(Pc) 1586, 1501, 1448, 739 cm−1;
υas(−CH2−) 2921 cm−1; υs(−CH2−) 2868 cm−1; υas(C−O−C) 1080
cm−1; υs(Ar−O−R) 1263 cm−1; υ(C−N) 1334 cm−1; υ(H−O−H)
3435 cm−1.
Phthalocyanine 4α-3d. According to the above procedure,
phthalonitrile 3c (1.02 g, 2.53 mmol) was treated with Zn-
(OAc)2·2H2O (0.26 g, 1.17 mmol) to give 4α-3d as a green solid
(0.40 g, 38%). 1H NMR (500 MHz, DMSO-d6): δ 9.05−9.03 (m, 2 H,
Pc-Hα), 8.96−8.92 (m, 2 H, Pc-Hα), 8.71−8.68 (m, 4 H, ArH), 8.13−
8.06 (m, 8 H, Pc-Hβ), 7.78−7.70 (m, 2 H, ArH), 7.65−7.56 (m, 2 H,
ArH), 7.38−7.24 (m, 12 H, ArH), 6.91−6.83 (m, 4 H, ArH), 5.15−
5.10 (m, 4 H, CH2), 4.84−4.80 (m, 4 H, CH2), 4.39 (br s, 4 H, CH2),
4.18−4.15 (m, 4 H, CH2), 4.09 (br s, 8 H, CH2), 3.96 (br s, 4 H,
CH2), 3.87 (vt, J = 3.5 Hz, 8 H, CH2), 3.76−3.75 (t, J = 4.0 Hz, 4 H,
CH2), 3.71 (br s, 4 H, CH2), 3.62 (br s, 4 H, CH2). HRMS (ESI): m/z
calcd for C92H84N12O16Zn [M + H]+ 1677.5492, found 1677.5356. IR:
υ(Pc) 1588, 1501, 1489, 1450 cm−1; υas(−CH2−) 2920 cm−1;
υs(−CH2−) 2868 cm−1; υas(C−O−C) 1096 cm−1; υs(Ar−O−R) 1264
cm−1; υ(C−N) 1335 cm−1; υ(H−O−H) 3435 cm−1.
Phthalocyanine 4α-4d. According to the above procedure,
phthalonitrile 4c (1.13 g, 2.53 mmol) was treated with Zn-
(OAc)2·2H2O (0.29 g, 1.31 mmol) to give 4α-4d as a green solid
(0.46 g, 39%). 1H NMR (500 MHz, DMSO-d6): δ 9.03 (br s, 2 H, Pc-
Hα), 8.91 (br s, 2 H, Pc-Hα), 8.71 (s, 4 H, Pc-Hβ), 8.15 (d, J = 8.0 Hz,
4 H, Pc-Hβ), 8.07 (s, 4 H, ArH), 7.79−7.74 (m, 2 H, ArH), 7.68−7.67
(m, 2 H, ArH), 7.41−7.38 (m, 4 H, ArH), 7.33−7.26 (m, 8 H, ArH),
6.91 (d, J = 7.0 Hz, 4 H, ArH), 5.11 (br s, 4 H, CH2), 4.81−4.78 (m, 4
H, CH2), 4.35 (s, 4 H, CH2), 4.13 (s, 4 H, CH2), 4.03−3.99 (m, 12 H,
CH2), 3.74−3.70 (m, 12 H, CH2), 3.64 (d, J = 4.0 Hz, 4 H, CH2),
3.63−3.54(m, 8 H, CH2), 3.51(s, 4 H, CH2), 3.43(s, 8 H, CH2).
HRMS (ESI): m/z calcd for C100H100N12O20Zn [M + H]+ 1853.6541,
found 1853.6202. IR: υ(Pc) 1641, 1586, 1501, 1488, 1448, 745 cm−1;
υas(−CH2−) 2917 cm−1; υs(−CH2−) 2867 cm−1; υas(C−O−C) 1081
cm−1; υs(Ar−O−R) 1263 cm−1; υ(C−N) 1333 cm−1; υ(H−O−H)
3427 cm−1.
Phthalocyanine 4β-3d. According to the above procedure,
phthalonitrile 3c-1 (0.85 g, 2.11 mmol) was treated with Zn-
(OAc)2·2H2O (0.23 g, 1.04 mmol) to give 4β-3d as a green solid
(0.31 g, 35%). 1H NMR (500 MHz, DMSO-d6): δ 8.98−8.92 (m, 4 H,
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Journal of Medicinal Chemistry
Article
Pc-Hα), 8.85−8.84 (m, 4 H, Pc-Hα), 8.59−8.52 (m, 4 H, Pc-Hβ),
8.22−8.20 (m, 4 H, ArH), 7.65−7.56 (m, 4 H, ArH), 7.54−7.43 (m, 4
H, ArH), 7.42−7.37 (m, 8 H, ArH), 7.16−7.13 (m, 4 H, ArH), 4.61
(br s, 4 H, CH2), 4.54(br s, 4 H, CH2), 4.32−4.29 (m, 8 H, CH2), 4.08
(br s, 4 H, CH2), 4.01−3.93 (m, 12 H, CH2), 3.84 (s, 8 H, CH2), 3.79
(s, 8 H, CH2). HRMS (ESI): m/z calcd for C92H84N12O16Zn [M +
H]+ 1677.5492, found 1677.5499. IR: υ(Pc) 1606, 1571, 1488, 1450
cm−1; υas(−CH2−) 2917 cm−1; υs(−CH2−) 2869 cm−1; υas(C−O−C)
1096 cm−1; υs(Ar−O−R) 1263 cm−1; υ(C−N) 1317 cm−1; υ(H−O−
H) 3418 cm−1.
Phthalocyanine 4β-4d. According to the above procedure,
phthalonitrile 4c-1 (1.40 g, 3.13 mmol) was treated with Zn-
(OAc)2·2H2O (0.35 g, 1.59 mmol) to give 4β-4d as a green solid
(0.61 g, 42%). 1H NMR (500 MHz, DMSO-d6): δ 8.98−8.93 (m, 4 H,
Pc-Hα), 8.81−8.80 (m, 4 H, Pc-Hα), 8.57−8.51 (m, 4 H, Pc-Hβ),
8.20−8.18 (m, 4 H, ArH), 7.65−7.59 (m, 4 H, ArH), 7.57−7.56 (m, 4
H, ArH), 7.45−7.09 (m, 8 H, ArH), 7.09−7.08 (m, 4 H, ArH), 4.60
(br s, 4 H, CH2), 4.53 (br s, 4 H, CH2), 4.22 (d, J = 3.5 Hz, 8 H, CH2),
4.05 (br s, 4 H, CH2), 3.98 (br s, 4 H, CH2), 3.87−3.84(m, 8 H, CH2),
3.79−3.78(m, 4 H, CH2), 3.73−3.62(m, 28 H, CH2). HRMS (ESI):
m/z calcd for C100H100N12O20Zn [M + H]+ 1853.6541, found
1853.6550. IR: υ(Pc) 1605, 1570, 1488, 1449 cm−1; υas(−CH2−)
2917 cm−1; υs(−CH2−) 2867 cm−1; υas(C−O−C) 1093 cm−1; υs(Ar−
O−R) 1234 cm−1; υ(C−N) 1317 cm−1; υ(H−O−H) 3435 cm−1.
Singlet Oxygen Quantum Yields. Singlet oxygen quantum yield
(ΦΔ) values were determined by comparative method using 1,3-
diphenylisobenzofuran (DPBF) as singlet oxygen chemical quencher
in DMF28 (eq 1 in air)
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 following 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.
Intracellular Localization. Approximately 1.0 × 105 HepG2 cells
in the culture medium (1 mL) were plated on a culture dish and
incubated for 24 h. Then the medium was replaced by fresh medium
with 10 μM phthalocyanines and incubated for another 24 h. After
incubation, the cells were rinsed with physiological saline and
incubated with 1 mL MitoTracker Green or LysoTracker (40 nM
for MitoTracker Green and 75 nm for LysoTracker in the medium)
for 20 or 60 min. Then the cells were rinsed with physiological saline
again. The subcellular localization of these phthalocyanines was
revealed by comparing the intracellular fluorescence images caused by
the MitoTracker Green (or LysoTracker) and these compounds. Both
MitroTracker and LysoTracker were excited at 488 nm and monitored
at 500−570 nm, while phthalocyanines were excited at 633 nm and
monitored at 640−700 nm.
Cellular Uptake. About 3.0 × 106 HepG2 cells in the culture
medium (2 mL) were seeded on a Petri dish (diameter of 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 phthalocyanine α-3d or 4α-3d or 4β-
3d in the medium (10 μM, 2 mL) for 24 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 of 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 removal of 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 684, 709,
and 691 nm for phthalocyanines α-3d, 4α-3d, and 4β-3d, respectively,
was compared with the respective calibration curves to give the uptake
concentrations. Each experiment was repeated three times.
std
kI
abs
Φ = ΦΔstd
Δ
kstdIabs
(1)
where ΦsΔtd is the singlet oxygen quantum yield for the ZnPc standard29
(ΦsΔtd = 0.56 in DMF); k and kstd are the DPBF photobleaching rates in
the presence of compound 4 and reference, respectively; and Iabs and
std
abs
I
are the rates of light absorption by synthetic phthalocyanines and
reference substance. The degradation of the solutions was monitored
at 415 nm, and DPBF concentrations were reduced to 0.1 mmol·L−1.
Cell Culture. HepG2 cells (from ATCC) were maintained in
RPMI medium 1640 (Invitrogen) supplemented with 10% fetal bovine
serum (FBS, Invitrogen) and Primocin antibiotic (Invitrogen). The
cells were incubated at 37 °C in a humidified 5% CO2 atmosphere. All
the compounds required the preparation of a concentrated DMSO
stock solution (5% CEL), which were then diluted with medium, and
the final DMSO concentration was 1% in medium.
Cytotoxicity Assay. To assess the cytotoxic effect of the
phthalocyanines, about 1.0 × 104 HepG2 cells per well in the culture
medium were seeded in 96-multiwell plates and incubated at 37 °C for
24 h in a humidified 5% CO2 atmosphere. Phthalocyanines 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. For dark
cytotoxicity, phthalocyanines were diluted and added to tetraplicate
wells. After 24 h, the added compounds were removed by fresh
medium and were incubated for another 24 h. The cell survival was
assessed using the MTT assay. For light cytotoxicity, after incubation
with phthalocyanines for about 24 h, the cells were exposed to light (λ
= 670 nm) at a dose of 1.5 J·cm−2 and then incubated again for 24 h,
and finally the MTT cell viability assay was performed and each
experiment was performed in triplicate.
ASSOCIATED CONTENT
* Supporting Information
■
S
Absorption and fluorescence spectra/data for zinc(II) phthalo-
cyanines synthesized in this paper in DMF or DMEM,
comparison of the rate of decay of DPBF sensitized by
phthalocyanines or ZnPc in DMF, results of subcellular
1
localization studies, and H NMR and HRMS 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
*Phone: +86-591-83758209. Fax: +86-591-87892632. E-mail:
xuejinping66@fzu.edu.cn.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors are grateful to the Technology Development
Foundation of Fuzhou University (Project Nos. 2011-XY-8 and
2011-XY-6), the Natural Science Foundation of Fujian
Province (Project No. 2012J05021), The National Natural
Cell viability was determined by means of the colormetric MTT
assay.30 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
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Journal of Medicinal Chemistry
Article
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Science Foundation of China (Project Nos. 21101028 and
21201035), and The Major Project of the State Ministry of
Science and Technology of China (Project No. 2011ZX09101-
001-04).
ABBREVIATIONS USED
■
CEL, Cremophor EL series; DPBF, 1,3-diphenylisobenzofuran;
EPR, enhanced permeability and retention; FBS, fetal bovine
serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide; PDT, photodynamic therapy; TEA, triethyl-
amine; RPMI, Roswell Park Memorial Institute; SD, standard
deviation; ref, reference; SEM, standard error of the mean
value; ZnPc, zinc(II) phthalocyanine; ΦF, fluorescence
quantum yield; ΦΔ, singlet oxygen quantum yield
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