Synthesis and in vitro anticancer activity of zinc(II) phthalocyanines conjugated with coumarin derivatives for dual photodynamic and chemotherapy

Article abstract of DOI:10.1002/cmdc.201402401

The combination of photodynamic therapy and chemotherapy is a promising strategy to overcome growing problems in contemporary medicine, such as low therapeutic efficacy and drug resistance. Four zinc(II) phthalocyanine-coumarin conjugates were synthesized and characterized. In these complexes, zinc(II) phthalocyanine was used as the photosensitizing unit, and a coumarin derivative was selected as the cytostatic moiety; the two components were linked via a tri(ethylene glycol) chain. These conjugates exhibit high photocytotoxicity against HepG2 human hepatocarcinoma cells, with low IC₅₀ values in the range of 0.014-0.044 μm. The high photodynamic activities of these conjugates are in accordance with their low aggregation tendency and high cellular uptake. One of these conjugates exhibits high photocytotoxicity and significantly higher chemocytotoxicity. The results clearly show that the two antitumor components in these conjugates work in a cooperative fashion. As shown by confocal microscopy, the conjugates can localize in the mitochondria and lysosomes, and one of the conjugates can also localize in the cell nuclei.

Full text of DOI:10.1002/cmdc.201402401

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DOI: 10.1002/cmdc.201402401
Synthesis and in vitro Anticancer Activity of Zinc(II)
Phthalocyanines Conjugated with Coumarin Derivatives
for Dual Photodynamic and Chemotherapy
Xiao-Qin Zhou, Lu-Bo Meng, Qi Huang, Jun Li, Ke Zheng, Feng-Ling Zhang, Jian-Yong Liu,
[a]
and Jin-Ping Xue*
The combination of photodynamic therapy and chemotherapy
is a promising strategy to overcome growing problems in con-
temporary medicine, such as low therapeutic efficacy and drug
resistance. Four zinc(II) phthalocyanine–coumarin conjugates
were synthesized and characterized. In these complexes,
zinc(II) phthalocyanine was used as the photosensitizing unit,
and a coumarin derivative was selected as the cytostatic
moiety; the two components were linked via a tri(ethylene
glycol) chain. These conjugates exhibit high photocytotoxicity
against HepG2 human hepatocarcinoma cells, with low IC50
values in the range of 0.014–0.044 mm. The high photodynamic
activities of these conjugates are in accordance with their low
aggregation tendency and high cellular uptake. One of these
conjugates exhibits high photocytotoxicity and significantly
higher chemocytotoxicity. The results clearly show that the
two antitumor components in these conjugates work in a co-
operative fashion. As shown by confocal microscopy, the con-
jugates can localize in the mitochondria and lysosomes, and
one of the conjugates can also localize in the cell nuclei.
Introduction
Photodynamic therapy (PDT) is an effective treatment for
a range of cancers as well as age-related macular degenera-
efficacy at lower drug doses, and in some cases, tumor drug
[10–12]
resistance was even overcome.
These advantages suggest
[
1–4]
tion.
PDT has been used in the treatment of cancer for de-
that the combination of chemo- and photodynamic therapies
may provide a simple and highly efficient modality for anti-
cancer treatment.
cades. Cancer cells absorb the drug in question, which is deliv-
ered to the tumor via the bloodstream. Visible light then is ap-
plied to the site, which causes the drug to react with oxygen,
creating a burst of reactive oxygen species (ROS) that kill
Coumarin derivatives are important, given their wide use in
[16,17]
synthetic chemistry and their biological activities.
They
[
5]
tumor cells and destroy malignant tissues. Significant effort
has been recently devoted to combining this modality with
other anticancer therapeutic methods. The combination of mo-
dalities that act on different disease pathways has shown sev-
eral advantages, such as enhanced therapeutic outcome, de-
creased side effects, and diminished problems of drug resist-
show good photochemical and photophysical properties,
making them useful in a variety of applications such as optical
brighteners, laser dyes, nonlinear optical chromophores, solar
energy collectors, as well as fluorescent labels and probes in
biology and medicine (e.g., anticoagulation, antibiotic, antifun-
gal, antipsoriasis, cytotoxic, anti-HIV, and anti-inflammatory
[
6]
[16–28]
ance. There are generally three approaches for dual PDT and
chemotherapy: sequential administration of a photosensitizer
and an anticancer drug, use of their covalent and noncovalent
conjugates, and co-encapsulation of these agents in a polymer-
agents).
The photocytotoxicity of coumarin derivatives has
[29–31]
drawn much attention for decades.
Phthalocyanines are promising second-generation photosen-
sitizers for PDT as a result of their strong absorption in the
[
7]
ic nanocarrier. A number of chemotherapeutic drugs includ-
tissue-penetrating red light wavelength range and high effi-
[
8–13]
[8]
[7]
[32–36]
ing doxorubicin (DOX),
cisplatin, oxaliplatin derivatives,
ciency in generating singlet oxygen.
7-Hydroxycoumarin is
[
10]
[14]
vincristine,
platinum(II) complexes,
and organometallic
a highly effective anticancer agent against malignant melano-
ma, leukemia, renal cell carcinoma, and prostate and breast
cancers; it works through inhibition of cell proliferation by de-
creasing the release of cyclin D1, which is overexpressed in
[
15]
ruthenium(II) complexes have been evaluated in combina-
tion with PDT against several human tumor cell lines. Remarka-
bly, the results of these studies showed enhanced anticancer
[37]
many types of cancer. Coumarin-substituted phthalocyanines
have been synthesized, and their photophysical and photo-
[a] X.-Q. Zhou, L.-B. Meng, Q. Huang, J. Li, K. Zheng, F.-L. Zhang, Prof. J.-Y. Liu,
[38–44]
Prof. J.-P. Xue
chemical properties have been examined.
A recent study
Fujian Engineering Research Center for Drugs and
Diagnoses – Photodynamic Therapy, College of Chemistry
Fuzhou University, Fuzhou, Fujian, 350108 (P.R. China)
E-mail: xuejinping66@fzu.edu.cn
indicated that butanoic acid bearing coumarin-substituted
zinc(II) phthalocyanine (ZnPc) complexes and their sodium
salts show excellent solubility in aqueous solution and are
highly efficient singlet oxygen generators, but these coumarin-
substituted ZnPc complexes cannot exhibit cooperative anti-
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402401.
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[
39]
cancer effects.
Herein we report the preparation
and in vitro photodynamic anticancer activities of
four novel ZnPc–coumarin conjugates, in which a cou-
marin moiety is linked to a ZnPc core at peripheral
positions on the Pc scaffold via a tri(ethylene glycol)
spacer. Conjugate 10a, synthesized in this study,
shows high photocytotoxicity and higher chemocyto-
toxicity than other corresponding coumarin com-
pounds, exhibiting dual anticancer effects.
Results and Discussion
Molecular design and synthesis
For these conjugates, zinc(II) phthalocyanine was
used as the photosensitizing unit owing to its strong
near-infrared absorption efficiency, high efficiency in
generating singlet oxygen, and excellent photostabil-
[
45,46]
ity.
7-Hydroxycoumarin or 7-hydroxy-4-trifluoro-
methylcoumarin were selected as the chemothera-
peutic cytostatic portions. The two components were
linked by a tri(ethylene glycol) chain, which enhances
the amphiphilicity and biocompatibility of the
system. Scheme 1 shows the synthetic route used to
prepare the conjugates. First, treatment of tri(ethy-
lene glycol) (1) with 4-toluenesulfonyl chloride (TsCl)
and triethylamine in dichloromethane gave the tolue-
nesulfonate 2, which then underwent nucleophilic
substitution with coumarin derivative 3 in N,N-dime-
thylformamide (DMF) to give the coumarin deriva-
tives 4 or 5. Subsequently, another nucleophilic sub-
stitution between compounds 4 or 5 and 3-nitro-
phthalonitrile (6a) or 4-nitrophthalonitrile (6b) in
DMF afforded the corresponding substituted phthalo-
Scheme 1. Synthesis of zinc(II) phthalocyanine–coumarin conjugates. Reagents and condi-
nitriles 7a, 8a, 7b, and 8b. Finally, mixed cyclization tions: a) TsCl, Et
of compounds 7a, 8a, 7b, or 8b with excess unsub-
stituted phthalonitrile 9 in the presence of Zn(OA-
3
N, CH
methylcoumarin (3b), K
cyano-1-nitrobenzene, DMF, K
pentanol, DBU, 140–1508C, 5 h.
2
Cl
2
, RT, 8–12 h; b) 7-hydroxycoumarin (3a) or 7-hydroxy-4-trifluoro-
CO , DMF, 608C, 8–12 h; c) 2,3-dicyano-1-nitrobenzene or 3,4-di-
CO , 808C, 5 h; d) 1,2-dicyanobenzene, Zn(OAc) ·2H O, n-
2
3
2
3
2
2
c) ·2H O
and
DBU) in n-pentanol gave the
ZnPc–coumarin conjugates 10a,
1a, 10b, and 11 b as blue
1,8-diazabicyclo[5.4.0]undec-7-ene
2
2
(
1
solids in 15, 28, 17, and 25%
yields, respectively. The higher
yields of 11 a and 11 b than
those of 10a and 10b may be
explained by the increased steric
hindrance from the trifluoro-
methyl group, resulting in the fa-
vored formation of 3:1 asymmet-
[
47,48]
Figure 1. Structures of the reference zinc(II) phthalocyanines 12a and 12b.
ric
phthalocyanine.
All
ZnPc–coumarin conjugates were
readily purified by silica gel
Photophysical and photochemical properties
1
column chromatography and were characterized by H NMR,
HRMS, and IR. For comparison purposes, phthalocyanines 12a
and 12b (Figure 1) were also prepared according to published
The UV/Vis absorption spectra of the title conjugates and their
reference phthalocyanines in DMF were recorded. The spectra
showed typical features of non-aggregated phthalocyanines
[
7,49]
methods.
(
Figure S1–S6 in the Supporting Information, Table 1). Com-
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Table 1. Photophysical and photochemical data of conjugates and refer-
ence compounds in DMF.
[
a]
[b]
[c]
Compd
l
max [nm] (loge)
l
em [nm]
F
F
F
D
1
1
0a
0b
677 (5.40)
672 (5.33)
677 (5.45)
672 (5.40)
677 (5.41)
672 (5.30)
684
680
693
686
684
680
0.25
0.27
0.20
0.20
0.26
0.28
0.64
0.57
0.54
0.53
0.61
0.60
11 a
11 b
1
1
2a
2b
[
52]
[a] Excitation at 610 nm. [b] Relative to ZnPc (F
F
=0.28 in DMF). [c] Rel-
ative to ZnPc (F
D
=0.56 in DMF). Data listed are from a single representa-
tive experiment.
pared with unsubstituted ZnPc (670 nm), the a-substituted
complexes exhibit a red-shifted Q-band (677 nm), whereas b-
substituted complexes do not show such a clear red-shifted Q-
band (672 nm). The observed red shifts are typical of Pcs with
substituents at the a positions and have been explained in the
[
45,46,50]
literature.
For 10a, 10b, 12a, and 12b excited at
610 nm, fluorescence emission is observed at 680–693 nm with
a quantum yield (F ) of 0.20–0.27 in DMF. The results show
f
that the introduction of a trifluoromethyl group at the coumar-
in unit causes the fluorescence emission to red-shift (a-substi-
tuted Pc: 693 vs. 684 nm, b-substituted Pc: 686 vs. 680 nm)
and results in a smaller F value (a-substituted Pc: 0.20 vs.
f
0.25, b-substituted Pc: 0.20 vs. 0.27).
To evaluate the photosensitizing efficiency of these Pc con-
jugates, their singlet oxygen quantum yields (F ) were also ex-
D
amined by a steady-state method with 1,3-diphenylisobenzo-
furan (DPBF) as the singlet oxygen quencher and ZnPc as the
Figure 2. a) Cytotoxic effects of 10a, 10b, 12a, and 12b toward HepG2 cells
upon irradiation (l=670 nm, 80 mWcm , 1.5 Jcm ) (closed symbols) and
in the dark (open symbols). b) Dark cytotoxic effects of 10a,b, 12a,b, and
À2
À2
[
51]
reference in DMF.
The variation of DPBF absorption was
monitored by UV/Vis spectroscopy. The absorbance at 415 nm
gradually decreased (Figure S7 in the Supporting Information).
3
a as a function of concentration up to 10 mm. Data are expressed as mean
values ÆSEM of three independent experiments, each performed in quadru-
All F values are summarized in Table 1. It can be seen that all
plicate.
D
of these conjugates are efficient singlet oxygen generators,
having similar quantum yields (F =0.53–0.64) to that of ZnPc
D
(
F =0.56).
D
Table 2. IC50 values for compounds 10a–12b and 3a toward HepG2
cells.
[
a]
Compd
IC50 [mm]
In vitro biological activities
Light
Dark
4.43
The cytotoxic effects of conjugates in a Cremophor EL emul-
sion were investigated against HepG2 human hepatocarcino-
ma cells. To reveal the chemo- and photocytotoxic effects of
these conjugates, the antiproliferative activities of the refer-
ence phthalocyanines and 7-hydroxycoumarin (3a) were also
investigated. Figure 2 and Figure S8 (Supporting Information)
show the dose-dependent survival curves for these com-
pounds. It is apparent that reference phthalocyanines 12a and
1
10b
11 a
0a
0.044Æ0.0087
0.018Æ0.0025
0.014Æ0.0021
0.021Æ0.0026
0.027Æ0.0075
0.063Æ0.0084
>10
>10
>10
>10
>10
>10
11 b
1
1
2a
2b
[
b]
3a
ND
À2
À2
[
a] l=670 nm, 80 mWcm , 1.5 Jcm . [b] Not determined. Data are the
mean ÆSEM of three independent experiments, each performed in quad-
12b do not show dark toxicity up to 0.5 mm. However, upon il-
ruplicate.
lumination at 670 nm, they show substantial cytotoxicity, with
respective IC50 values of 0.027 and 0.063 mm (Figure 2a,
Table 2). Coumarin-containing conjugates 10b, 11 a, and 11 b
do not show dark toxicity up to 0.5 mm. Upon illumination at
dual chemo- and photodynamic activities, the test concentra-
tion was increased to 10 mm. Figure 2b shows that among
these compounds, coumarin derivative 3a does not show che-
motoxicity up to 10 mm, reference phthalocyanine 12a exhibits
slight dark toxicity under the same conditions, whereas the
6
0
70 nm, they show substantial cytotoxicity, with IC50 values of
.018, 0.014, and 0.021 mm (Figure 2a, Table 2). To determine
whether the ZnPc–coumarin conjugates show the expected
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coumarin-containing phthalocyanine conjugate 10a exhibits
clear chemotoxicity, with an IC50 value of 4.43 mm (Table 2).
Having both a photosensitizer and a chemotherapeutic agent,
conjugate 10a is highly photocytotoxic, with an IC50 value as
low as 0.044 mm toward HepG2 cells under a rather low light
À2
À2
dose (l=670 nm, 80 mWcm , 1.5 Jcm ). It also shows signifi-
cantly improved chemocytotoxicity, with an IC50 value of
4.43 mm compared with that of coumarin 3a. The results clear-
ly show that the two antitumor components in conjugate 10a
work in a cooperative fashion.
The aggregation state of photosensitizers in the culture
medium is an important factor in their photodynamic activi-
[
53]
ties. Aggregation usually provides an efficient non-radiative
relaxation pathway, thereby greatly shorting the excited-state
lifetime. This effect decreases the singlet oxygen quantum
yield, eventually leading to greatly diminished or even com-
pletely suppressed photodynamic activity. The aggregation be-
havior of compounds, formulated with Cremophor EL in RPMI
Figure 4. Cellular uptake of compounds by HepG2 cells. Values are ex-
pressed as means ÆSD of three independent experiments, each performed
in sextuple; statistical significance: **p<0.01, *p<0.05.
1640 culture medium, was examined by spectroscopic absorp-
tion. As shown in Figure 3, all phthalocyanines show a relatively
uptake of a-substituted Pc is much higher than that of corre-
sponding b-substituted Pc (p<0.01). It is also clear that the
uptake of conjugates 11 a and 11 b is significantly higher than
that of 10a or 10b (p<0.01). The presence of the trifluoro-
methyl unit in 11 a or 11 b may enhance the overall amphiphi-
licity of the compound, thereby promoting the cellular uptake.
The greater uptake of 11 a, 11 b, and 12a is in agreement with
the higher photocytotoxicity observed for these three com-
pounds.
The subcellular localization of these compounds was also in-
vestigated by confocal laser scanning microscopy. HepG2 cells
were first incubated with test compounds in the culture
medium for 24 h and then stained with LysoTracker DND 26
(
30 min), MitoTracker Green FM (30 min), or DAPI (5 min),
which are specific dyes for lysosomes, mitochondria, and
nuclei, respectively. As shown in Figure 5, the fluorescence
caused by DAPI (l : 405 nm, monitored at 425–475 nm) is par-
ex
tially superimposed with that caused by 10a (l : 633 nm,
ex
monitored at 640–700 nm). The fluorescence intensity line pro-
files of 10a and DAPI traced along the red line in Figure 5 also
confirm that 10a can partly localize in the cell nuclei. The re-
sults are accord with the higher chemocytotoxicity of 10a
than other phthalocyanines. In contrast, the fluorescence
images of 10b, 11 a, 11 b, 12a, and 12b could not be merged
with that of DAPI (Figure 5 and Figures S11–S13 in the Sup-
porting Information), showing that 10b, 11 a, 11 b, 12a, or
12b cannot localize in the cell nuclei. Meanwhile, the fluores-
Figure 3. Electronic absorption spectra of compounds 10a–12b (all at
10 mm), formulated with Cremophor EL, in RPMI 1640 cell culture medium.
sharp and intense Q-band. The results suggest that these
phthalocyanines are not significantly aggregated in culture
medium.
To explain the varied cytotoxicity of these conjugates (10a,
10b, 11a, and 11 b), reference ZnPcs 12a and 12b, and 7-hy-
cence images of LysoTracker (l : 488 nm, monitored at 500–
ex
droxycoumarin (3a), the cellular uptake of these compounds
was examined by fluorescence spectroscopy. After incubation
with these compounds for 24 h, sodium dodecylsulfate (SDS)
was used to lyse the cells and extract the phthalocyanines. The
intracellular Pc concentrations were determined by measuring
their fluorescence at 670 nm (excitation at 610 nm). The intra-
cellular coumarin concentrations were determined by measur-
ing their fluorescence at 460 nm (excitation at 329 nm). The re-
sults are depicted in Figure 4, which shows that the cellular
570 nm) and MitoTracker (l : 488 nm, monitored at 510–
ex
570 nm) could be superimposed well with those of 10a–12a
(Figure S9 in the Supporting Information), 10b–12b (Figur-
es S11–S13 in the Supporting Information), and 10a–12a (Fig-
ure S10 in the Supporting Information), 10b–12b (Figur-
es S11–13 in the Supporting Information), correspondingly.
These results show that these conjugates and reference phtha-
locyanines can localize in the lysosomes and mitochondria of
the cells.
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Figure 5. Corresponding superimposed images of intracellular fluorescence in HepG2 cells using filter sets specific for phthalocyanine (red) and DAPI (blue).
Fluorescence intensity profiles of phthalocyanine (c) and DAPI (c) traced along the red lines in the merged images. Magnification: 60ꢁ; scale bars:
30 mm.
Conclusions
Experimental Section
Chemistry
In summary, we have synthesized and characterized a series of
zinc(II) phthalocyanine–coumarin conjugates and evaluated
their in vitro chemo- and photodynamic activities. These conju-
gates contain both photo- and chemotherapeutic agents cova-
lently linked by a tri(ethylene glycol) spacer. The introduction
of the coumarin derivative did not significantly affect the pho-
tocytotoxicity of the zinc(II) phthalocyanines, and all conju-
gates maintained the high phototoxicity of the phthalocyanine
core (IC : 0.014–0.044 mm). Among these conjugates, coumar-
in-containing phthalocyanine 10a exhibits significantly im-
proved chemocytotoxicity, with an IC50 value of 4.43 mm com-
pared with coumarin 3a. The results clearly show that the two
antitumor components in conjugate 10a work in a cooperative
manner. The conjugates show preferential localization in lyso-
somes and mitochondria, and in particular conjugate 10a can
also localize in cell nuclei. Overall, these results indicate conju-
gate 10a as a highly promising antitumor agent for dual
chemo- and photodynamic therapies.
General: All reactions were carried out under an atmosphere of ni-
trogen. n-Pentanol was distilled over sodium, and all other solvents
and reagents were of reagent grade and used as received. Com-
[54]
1
pound 2 was prepared as described. H NMR spectra were re-
corded on Avance III spectrometers (400 and 500 MHz) in CDCl
3
unless otherwise stated. Spectra were referenced internally using
the residual solvent ( H: d=7.26 ppm) resonances relative to
1
SiMe . Electronic adsorption spectra were measured on a TU-1901
4
50
spectrometer, fluorescence spectra were obtained on a Varian Cary
eclipse spectrometer, and IR spectra were measured on a Perki-
nElmer SP2000 instrument. HRMS data were collected on an Ion
Trap Mass Spectrometry DECAX-30000 LCQ Deca XP mass spec-
trometer. The fluorescence quantum yields of the samples
[FF(sample)] were determined by the equation: FF(sample) =(Fsample
/
F )(A /Asample)(nsample2/nref2)FF(ref), for which F, A, and n are the mea-
ref ref
sured 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 [FF(ref) =0.28].
Singlet oxygen quantum yields (F ) were measured using 1,3-di-
D
phenylisobenzofuran (DPBF) as the scavenger. Laser light (670 nm,
À2
8
0 mWcm ) was used as the light source, and ZnPc was selected
as the reference (F =0.56 in DMF). The purity of all new com-
D
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pounds was determined by HPLC analysis and was found to be
30 mmol) and 4 (2.94 g, 10 mmol) were mixed in anhydrous DMF
1
ꢀ
95%.
to give 7b (1.30 g, 31%). H NMR (500 MHz, CDCl ): d=7.70 (d, J=
3
8
1
.5 Hz, 1H, ArH), 7.66 (d, J=9.5 Hz, 1H, ArH), 7.40 (d, J=8.5 Hz,
H, ArH), 7.31 (vd, J=2.5 Hz, 1H, ArH), 7.24 (dd, J=2.5, 9.5 Hz, 1H,
General procedure for the preparation of compounds 4 and 5:
Toluene-4-sulfonic acid 2-[2-(2-hydroxyethoxy)ethoxy]ethyl ester (2)
ArH), 6.88–6.86 (m, 2H, ArH), 6.28 (d, J=9.5 Hz, 1H, ArH), 4.25 (t,
J=4.5 Hz, 2H, CH ), 4.21 (t, J=4.5 Hz, 2H, CH ), 3.93–3.90 (m, 4H,
(
1 equiv), coumarin derivative (3a or 3b) (1.1~1.5 equiv), and
2
2
K CO (3–5 equiv) in DMF (15–20 mL) were stirred at 608C for 8–
2
3
CH ), 3.77 ppm (s, 4H, CH ); HRMS (ESI): m/z calcd for C H N O
6
2
2
23 21
2
1
2 h under nitrogen. At the end of reaction, the solvent was re-
+
[
M+H] : 421.1399, found: 421.1462; IR (KBr pellet): n˜ =3435 (HÀ
moved in vacuo. Water (90 mL) was added to the residue and ex-
tracted with CH Cl (3ꢁ100 mL). The combined organic extracts
OÀH), 2945 (ÀCH À), 2231 (CꢁN), 1706 (C=O), 1616 (C=C), 1500,
2
2
2
1
481, 1444, 1402 (Ar), 1353, 1325 (ArÀOÀR), 1117, 1089, 1051 (CÀ
were dried with saturated NaHCO solution, filtered and concen-
À1
3
OÀC), 983, 948, 886, 839 cm (ArÀH).
trated. The yellow oil product was purified over a silica gel column
using PE/EtOAc (1:1 v/v) as eluent.
General procedure for the preparation of compounds 8a and
8
b: A mixture of compounds 5 (1 equiv), 3-nitrophthalonitrile (6a)
Compound 4: According to the general procedure, 2 (3.04 g,
or 4-nitrophthalonitrile (6b) (1.1~1.5 equiv) and K CO (3–5 equiv)
in DMF (15–20 mL) was stirred in 12 mL CH CN at 808C for ~5 h
under nitrogen atmosphere. At the end of the reaction, the solvent
was removed in vacuo. Saturated NaCl in water (100 mL) was
added to the residue and extracted with CH Cl (3ꢁ100 mL). The
combined organic extracts were dried over saturated NaHCO solu-
tion, filtered and concentrated. The white solid product was puri-
fied over a silica gel column using CH Cl /CH OH (30:1 v/v) as
eluent.
2
3
1
0 mmol), was treated with 7-hydroxycoumarin 3a (1.78 g,
3
1
4
7
6
4
1 mmol) and K CO (4.56 g, 33 mmol) in DMF to give 4 (1.38 g,
2
3
1
7%). H NMR (500 MHz, CDCl ): d=7.62 (d, J=9.5 Hz, 1H, ArH),
3
.35 (d, J=8.5 Hz, 1H, ArH), 6.87 (dd, J=2.0 Hz, 8.5 Hz, 1H, ArH),
.82 (d, J=2.0 Hz, 1H, ArH), 6.24 (d, J=9.5 Hz, 1H, ArH), 4.18 (t, J=
.5 Hz, 2H, CH ), 3.88 (t, J=4.5 Hz, 2H, CH ), 3.73–3.71 (m, 4H,
2
2
3
2
2
CH ), 3.70–3.68 (m, 2H, CH ), 3.61 (t, J=4.5 Hz, 2H, CH ), 2.23 ppm
2
2
2
+
2
2
3
(
s, 1H, OH); HRMS (ESI): m/z calcd for C H NaO [M+Na] :
15 18 6
3
17.1001, found: 317.1091; IR (KBr pellet): n˜ =3408 (HÀOÀH), 3084
(
ArÀH), 2881 (ÀCH À), 1727 (C=O), 1613, 1556, 1508, 1453 (C=C),
Phthalonitrile 8a: According to the above procedure, 5 (1 g,
2.75 mmol), 3-nitrophthalonitrile (0.51 g, 2.95 mmol), and anhy-
drous K CO (1.52 g, 11.01 mmol) were mixed in anhydrous DMF to
2
À1
1
230 (ArÀOÀR), 1123, 1050 (CÀOÀC), 893, 840 cm (ArÀH).
2
3
Compound 5: According to the general procedure, 2 (0.52 g,
.71 mmol) was treated with anhydrous K CO (0.90 g, 6.52 mmol)
1
give 8a (0.80 g, 59%). H NMR (400 MHz, CDCl ): d=7.65 (t, J=
3
1
2
3
1
6
0.5 Hz, 2H, ArH), 7.38 (d, J=9.5 Hz, 1H, ArH), 7.31 (s, 1H, ArH),
.98 (d, J=9.2 Hz, 1H, ArH), 6.92 (s, 1H, ArH), 6.64 (s, 1H, ArH), 4.32
and 7-hydroxy-4-trifluoromethylcoumarin (3b) (0.45 g, 1.96 mmol)
1
in DMF to give 5 (0.52 g, 73%). H NMR (400 MHz, CDCl ): d=7.63–
3
(vt, J=4.4 Hz, 2H, CH ), 4.25 (vt, J=4 Hz, 2H, CH ), 3.98 (vt, J=
2
2
7
1
4
4
3
.66 (d, J=8.8 Hz, ArH), 6.97–7.00 (m, 1H, ArH), 6.93 (d, J=2.4 Hz,
5
Hz, 2H, CH ), 3.94 (vt, J=6 Hz, 2H, CH ), 3.83 (d, J=5.5 Hz, 2H,
2 2
H, ArH), 6.65 (s, 1H, ArH), 4.24 (t, J=4.8 Hz, 2H, CH ), 3.93 (t, J=
2
CH ), 3.79–3.78 ppm (m, 2H, CH ); HRMS (ESI): m/z calcd for
C H F N O [M+H] : 489.1267, found: 489.1216; IR (KBr pellet):
2
2
.8 Hz, 2H, CH ), 3.74–3.76 (m, 7H, CH +OH), 3.646 ppm (t, J=
+
2
2
+
24 19
3
2
6
.8 Hz, 2H, CH ); HRMS (ESI): m/z calcd for C H F O [M+H] :
2
16 17
3
6
n˜ =3082 (ArÀH), 2226 (CꢁN), 1741 (C=O), 1138, 1064 (CÀOÀC),
63.1050, found: 363.1091; IR (KBr pellet): n˜ =3466 (HÀOÀH), 1744
1
285 (ArÀOÀR), 2871 (ÀCH À), 2909 (ÀCH À), 1472, 1447, 1408 (À
2
2
(
C=O), 1614 (C=C), 1007 (CÀOÀC), 1279 (ArÀOÀR), 1145, 1195 (CF ),
3
CH À), 1612 (C=C), 1516, 1582 (Ar), 795, 810, 866, 823 (ArÀH), 1198,
À1
2
3
091 (ArÀH), 1557, 1516 (C=C), 819, 870 cm (ArÀH).
À1
1168 cm (CF ).
3
General procedure for the preparation of compounds 7a and
b: A mixture of compound 4 (1 equiv), 3-nitrophthalonitrile (6a)
or 4-nitrophthalonitrile (6b) (1.1~1.5 equiv) and K CO (3–5 equiv)
in DMF (15–20 mL) was stirred at 608C for 8–12 h under nitrogen.
The reaction mixture was poured into 500 mL cold water; the pre-
cipitated crude product was filtered off. The brown solid product
was purified over a silica gel column using PE/EtOAc (1:1 v/v) as
eluent.
Phthalonitrile 8b: According to the above procedure, 5 (1 g,
7
2
.75 mmol), 3-nitrophthalonitrile (0.51 g, 2.95 mmol), and anhy-
2
3
drous K CO (1.52 g, 11.01 mmol) were mixed in anhydrous DMF to
2
3
1
give 8b (1.00 g, 75%). H NMR (400 MHz, CDCl ): d=8.03 (d, J=
3
8
.8 Hz, 1H, ArH), 7.76 (d, J=2.4 Hz, 1H, ArH), 7.61–7.58 (m, 1H,
ArH), 7.46–7.43 (m, 1H, ArH), 7.15 (d, J=2.8 Hz, 1H, ArH), 7.06–7.04
(
m, 1H, ArH), 6.85 (s, 1H, ArH), 4.27 (vt, J=4.4 Hz, 2H, CH ), 4.24
2
(
vt, J=4.4 Hz, 2H, CH ), 3.79–3.78 (m, 4H, CH ), 3.62 ppm (s, 4H,
2
2
+
Phthalonitrile 7a: According to the above procedure, 3-nitro-
phthalonitrile (6a) (2.08 g, 12 mmol) and 4 (2.94 g, 10 mmol) were
mixed in anhydrous DMF (15 mL) under nitrogen atmosphere, then
anhydrous K CO (4.14 g, 30 mmol) was added to give 7a (1.56 g,
CH
2
); HRMS (ESI): m/z calcd for C24H F N NaO [M+Na] : 511.1107,
18 3 2 6
found: 511.1060; IR (KBr pellet): n˜ =3085 (ArÀH), 2230 (CꢁN), 1743
(C=O), 1138, 1044 (CÀOÀC), 1278 (ArÀOÀR), 2903 (ÀCH À), 1612
2
(C=C), 1557, 1513 (Ar), 713, 792, 837, 863 (ArÀH), 1130, 1174,
2
3
1
À1
3
7
7%). H NMR (500 MHz, CDCl ): d=7.66 (d, J=9.0 Hz, 1H, ArH),
.63 (d, J=9.5 Hz, 1H, ArH), 7.39 (d, J=9.0 Hz, 1H, ArH), 7.36 (d,
1197 cm (CF
).
3
3
General procedure for the preparation of compounds 10a, 10b,
1a, and 11b: A mixture of phthalonitrile 7a,b, 8a or 8b (1 equiv),
phthalonitrile (9) (9 equiv), and Zn(OAc) ·2H O (5 equiv) in n-penta-
J=9.0 Hz, 1H, ArH), 7.32 (d, J=8.5 Hz, 1H, ArH), 6.89 (dd, J=
1
2
.0 Hz, 8.5 Hz, 1H, ArH), 6.84 (d, J=2.0 Hz, 1H, ArH), 6.26 (d, J=
2
2
9
.5 Hz, 1H, ArH), 4.29 (t, J=4.5 Hz, 2H, CH ), 4.18 (t, J=4.5 Hz, 2H,
2
nol (15 mL) was heated at 1008C, and then a small amount of DBU
~1 mL) was added. The mixture was stirred at 140–1508C for 5 h.
CH ), 3.94 (t, J=4.5 Hz, 2H, CH ), 3.89 (t, J=4.5 Hz, 2H, CH ), 3.79–
2
2
2
(
3
.77 (m, 2H, CH ), 3.75–3.73 ppm (m, 2H, CH ); HRMS (ESI): m/z
2 2
After cooling, the volatiles were removed under reduced pressure.
The reaction mixture was dissolved by a small amount of CH Cl
+
calcd for C H N O [M+H] : 421.1399, found: 421.1458; IR (KBr
2
3
21
2
6
2
2
pellet): n˜ =3078 (ArÀH), 2942, 2881 (ÀCH À), 2229 (CꢁN), 1725 (C=
2
and purified over a silica gel column using CH Cl /CH OH (20:1 v/v)
2
2
3
O), 1618 (C=C), 1584, 1477, 1456 (Ar), 1230 (ArÀOÀR), 1140, 1022
as eluent, followed by size-exclusion chromatography (Bio-Beads S-
X1 beads) with THF as eluent. The crude product was further puri-
fied by re-crystallization from a mixture of THF and petroleum
ether to give phthalocyanine as a blue solid.
À1
(
CÀOÀC), 866, 799 cm (ArÀH).
Phthalonitrile 7b: According to the above procedure, 4-nitro-
phthalonitrile (6b) (2.08 g, 12 mmol), anhydrous K CO3 (4.14 g,
2
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Phthalocyanine 10a: According to the general procedure, a mix-
ture of 7a (0.25 g, 0.59 mmol), phthalonitrile 9 (0.69 g, 5.35 mmol),
and Zn(OAc) ·2H O (0.65 g, 2.97 mmol) were added to give 10a
Biological assays
Cell culture: HepG2 cells (from ATCC) were maintained in RPMI
1640 medium (Invitrogen) supplemented with 10% fetal bovine
serum (FBS, Invitrogen) and Primocin antibiotic (Invitrogen). The
cells were incubated at 378C in a humidified 5% CO 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.
2
2
1
(
76 mg, 18%). H NMR (500 MHz, [D ]DMSO): d=9.24–9.22 (m, 3H,
6
TM
Pc-H ), 9.18–9.15 (m, 2H, Pc-H ), 9.05 (d, J=7.0 Hz, 1H, Pc-H ), 8.69
a
a
a
2
(
d, J=7.0 Hz, 1H, Pc-H ), 8.19–8.08 (m, 6H, Pc-H ), 7.92 (t, J=
a b
7
.5 Hz, 1H, Pc-H ), 7.69 (d, J=9.5 Hz, 1H, ArH), 7.53 (d, J=7.5 Hz,
b
1
H, Pc-H ), 7.18 (d, J=8.5 Hz, 1H, ArH), 6.65 (vd, J=2.5 Hz, 1H,
b
ArH), 6.52 (dd, J=2.5 Hz, 8.5 Hz, 1H, ArH), 6.10 (d, J=9.5 Hz, 1H,
ArH), 4.77 (brs, 2H, CH ), 4.35 (vt, J=4.5 Hz, 2H, CH ), 4.07 (vt, J=
2
2
Cytotoxicity assays: To assess the cytotoxic effect of the phthalocya-
nines, HepG2 cells (~1.0ꢁ10 per well) in culture medium were
seeded in 96-multiwell plates and incubated at 378C for 24 h in
a humidified 5% CO atmosphere. Phthalocyanines were first dis-
solved in DMSO to give 5 mm solutions in the presence of 5% Cre-
mophor EL. These served as the stock solutions for the following in
vitro studies. For cytotoxicity studies, the solutions were further di-
luted with culture medium. The cells, after being rinsed with phos-
phate-buffered saline (PBS), were incubated with 100 mL of the di-
4
4
.5 Hz, 2H, CH ), 3.96 (vt, J=4.5 Hz, 2H, CH ), 3.80–3.76 (m, 4H,
2 2
+
CH ); HRMS (ESI): m/z calcd for C H N O Zn [M+H] : 869.1809,
2
47 33
8
6
found: 869.1866; IR (KBr pellet): n˜ =3436 (HÀOÀH), 2922 (ÀCH À),
2
2
1
1
729, 1705 (C=O), 1610, 1556, 1488, 1454, 1403 (Pc), 1332 (CꢁN),
115, 1090, 1058 (CÀOÀC), 986, 887, 836 cmÀ1 (ArÀH).
Phthalocyanine 10b: According to the above procedure, phthalo-
nitrile 7b (0.25 g, 0.59 mmol) was treated with unsubstituted
phthalonitrile 9 (0.69 g, 5.35 mmol) and Zn(OAc) ·2H O (0.65 g,
2
2
1
luted phthalocyanine solutions for 24 h at 378C under 5% CO . For
2
2
.97 mmol) to give 10b (81 mg, 16%). H NMR (500 MHz,
dark cytotoxicity, 24 h later, the added compounds were removed
by replacement with fresh medium, and plates were incubated for
another 24 h. The cell survival was assessed by MTT assay. For light
cytotoxicity, after incubation with phthalocyanines for 24 h, cells
were exposed to light (l=670 nm) at a dose of 1.5 Jcm and
then incubated again for 24 h. Finally the MTT cell viability assay
was performed, and each experiment was performed in triplicate.
[
D ]DMSO): d=9.21–9.17 (m, 3H, Pc-H ), 9.12 (d, J=7.0 Hz, 1H, Pc-
6
a
H ), 9.06 (d, J=7.0 Hz, 1H, Pc-H ), 9.03 (d, J=7.0 Hz, 1H, Pc-H ),
a
a
a
8
.77 (d, J=7.0 Hz, 1H, Pc-H ), 8.35 (s, 1H, Pc-H ), 8.19–8.05 (m, 6H,
a a
Pc-H ), 7.79 (d, J=9.5 Hz, 1H, ArH), 7.52 (dd, J=2.0 Hz, 8.5 Hz, 1H,
b
À2
Pc-H ), 7.44 (d, J=8.5 Hz, 1H, ArH), 6.93 (d, J=2.0 Hz, 1H, ArH),
b
6
4
2
.87 (dd, J=2.0 Hz, 8.5 Hz, 1H, ArH), 6.13 (d, J=9.5 Hz, 1H, CH2),
.60 (brs, 2H, CH ), 4.23 (vt, J=4.5 Hz, 2H, CH ), 4.10 (vt, J=4.5 Hz,
2
2
5
H, CH ), 3.89–3.86 (m, 4H, CH ), 3.80 ppm (vt, J=4.5 Hz, 2H, CH );
2
2
2
Intracellular localization: Approximately 1.0ꢁ10 HepG2 cells in cul-
ture medium (1 mL) were plated on a culture dish and incubated
for 24 h. The medium was then replaced by fresh medium with
+
HRMS (ESI): m/z calcd for C H N O Zn [M+] : 869.1809, found:
47
33
8
6
8
69.1856; IR (KBr pellet): n˜ =3435 (HÀOÀH), 2919 (ÀCH À), 1269
2
(
ArÀOÀR), 1730 (C=O), 1610, 1555, 1485, 752, 735, 720 (Pc), 1333
1
0 mm phthalocyanines and incubated for another 24 h. After incu-
À1
(
CꢁN), 1090, 1058 (CÀOÀC), 888, 831 cm (ArÀH).
bation, the cells were rinsed with PBS and incubated with 1 mL Mi-
toTracker Green (40 nm final) or LysoTracker (75 nm final) for
Phthalocyanine 11a: According to the above procedure, unsubsti-
tuted phthalonitrile 9 (0.59 g, 4.6 mmol), phthalonitrile 8a (0.25 g,
3
0 min. The cells were then rinsed with PBS again. The subcellular
localization of these phthalocyanines was revealed by comparing
the intracellular fluorescence images generated from MitoTracker
Green (or LysoTracker) and these compounds. Both MitoTracker
and LysoTracker were excited at 488 nm and monitored at 500–
0
.51 mmol), and anhydrous Zn(OAc)2 (0.56 g, 2.55 mmol) were
1
added to give 11 a (60 mg, 18%). H NMR (400 MHz, [D ]DMSO):
6
d=9.24–9.22 (m, 3H, Pc-H ), 9.18–9.15 (m, 2H, Pc-H ), 9.07 (d, J=
a
a
7
.2 Hz, 1H, Pc-H ), 8.70 (d, J=7.6 Hz, 1H, Pc-H ), 8.19–8.10 (m, 6H,
a a
5
70 nm, whereas phthalocyanines were excited at 633 nm and
Pc-H ), 7.93 (t, J=8 Hz, 1H, Pc-H ), 7.54 (t, J=8 Hz, 1H, ArH), 7.12
b
b
monitored at 640–700 nm.
(
d, J=10 Hz, 1H, ArH), 6.58 (d, J=2.4 Hz, 1H, ArH), 6.55 (s, 1H,
6
ArH), 6.52 (m, 1H, ArH), 4.78–4.77 (m, 2H, CH ), 4.35 (t, J=4 Hz,
2
Cellular uptake: Approximately 3.0ꢁ10 HepG2 cells in culture
medium (2 mL) were seeded on a Petri dish (1=35 mm) and incu-
bated overnight at 378C under 5% CO . The medium was re-
2
4
H, CH ), 4.06 (t, 2H, CH ), 3.88 (vt, J=4 Hz, 2H, CH ), 3.77 (vt, J=
2 2 2
.8 Hz, 2H, CH ), 3.73 ppm (vt, J=4.8 Hz, 2H, CH ); HRMS (ESI): m/z
2
2
2
+
calcd for C H F N O Zn [M+H] : 937.1683, found: 937.1685; IR
4
8
31
3
8
6
moved, and the cells were rinsed with PBS (2 mL). The cells were
then incubated with a solution of phthalocyanine 10a,b, 11 a,b,
12a,b, or compound 3a in the medium (10 mm, 2 mL) for 24 h
under the same conditions. The solution was then removed, and
the cells were rinsed with PBS three times and then lysed by 1%
SDS to give a homogenous solution. The fluorescence of phthalo-
cyanine in the cell extract was measured on a fluorescence spec-
trometer at 670 nm (excitation at 610 nm); for coumarin excitation
at 329 nm and monitoring at 460 nm. Each experiment was repeat-
ed three times.
(
KBr pellet): n˜ =2226 (CꢁN), 1727 (C=O), 1057 (CÀOÀC), 1279 (ArÀ
À1
OÀR), 2923 (ÀCH À), 2855 (ÀCH À), 1609 (Pc), 1138, 1113, 1197 cm
2
2
(
CF ).
3
Phthalocyanine 11b: According to the above procedure, Com-
pound 9 (0.59 g, 4.61 mmol), compound 8b (0.25 g, 0.51 mmol),
and anhydrous Zn(OAc) (0.56 g, 2.56 mmol) were added to give
2
1
1
1 b (60 mg, 20%). H NMR (400 MHz, [D ]DMSO): d=9.21–9.17 (m,
6
3
8
H, Pc-H ), 9.12 (d, J=7.2 Hz, 1H, Pc-H ), 9.07–9.03 (m, 2H, Pc-H ),
a
a
a
.78 (d, J=8.0 Hz, 1H, Pc-H ), 8.37 (s, 1H, Pc-H ), 8.19–8.14 (m, 4H,
a
a
Pc-H ), 8.12–8.06 (m, 2H, Pc-H ), 7.52 (d, J=8.4 Hz, 1H, Pc-H ), 7.40
b
b
b
(
1
d, J=8.4 Hz, 1H, ArH), 6.99 (d, J=2.4 Hz, 1H, ArH), 6.96–6.93 (m,
H, ArH), 6.64 (s, 1H, ArH), 4.61 (s, 2H, CH ), 4.237 (t, J=4 Hz, 2H,
2
Acknowledgements
CH ), 4.105 (t, J=3.6 Hz, 2H, CH ), 3.90–3.86 (m, 4H, CH ), 3.82–
2
2
2
3
.80 ppm (m, 2H, CH ); HRMS (ESI): m/z calcd for C H F N O Zn
2 48 31 3 8 6
+
[
M+H] : 937.1683, found: 937.1602; IR (KBr pellet): n˜ =3053 (ArÀ
The authors are grateful to the Technology Development Founda-
tion of Fuzhou University (Project Nos. 2011-XY-8 and 2011-XY-6),
the Natural Science Foundation of Fujian Province (Project No.
H), 1335 (CꢁN), 2912 (ÀCH À), 1731 (C=O), 1060 (CÀOÀC), 1279
2
À1
(
ArÀOÀR), 1609, 1487, 1429 (Pc), 1117, 1142 cm (CF ).
3
2012J05021), the National Natural Science Foundation of China
(
Project Nos. 21101028 and 21201035), and the Major Project of
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 9
&
7
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
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the State Ministry of Science and Technology of China (Project
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Published online on && &&, 0000
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 9
&
8
&
ÞÞ
These are not the final page numbers!

FULL PAPERS
Light and chemistry combined: A
series of zinc(II) phthalocyanine–cou-
marin conjugates were synthesized and
characterized. These compounds exhibit
high photocytotoxicity against HepG2
human hepatocarcinoma cells. Among
these conjugates, 10a (shown) is a par-
ticularly promising antitumor agent for
dual chemo- and photodynamic thera-
pies.
X.-Q. Zhou, L.-B. Meng, Q. Huang, J. Li,
K. Zheng, F.-L. Zhang, J.-Y. Liu, J.-P. Xue*
&& – &&
Synthesis and in vitro Anticancer
Activity of Zinc(II) Phthalocyanines
Conjugated with Coumarin Derivatives
for Dual Photodynamic and
Chemotherapy
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 9
&
9
&
ÞÞ
These are not the final page numbers!