Synthesis of novel octa-cationic and non-ionic 1,2-ethanediamine substituted zinc (II) phthalocyanines and their in vitro anti-cancer activity comparison

Article abstract of DOI:10.1016/j.ejmech.2012.09.038

Novel tetra-substituted zinc phthalocyanines (Pcs) bearing 1,2-ethanediamine group and the quaternized derivatives were synthesized and characterized. The photophysical and cellular properties of these Pcs were investigated. The results indicated that the

Full text of DOI:10.1016/j.ejmech.2012.09.038

European Journal of Medicinal Chemistry 58 (2012) 12e21
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel octa-cationic and non-ionic 1,2-ethanediamine substituted
zinc (Ⅱ) phthalocyanines and their in vitro anti-cancer activity comparison
1
1
*
Ao Wang , Li Zhou , Kelong Fang, Lin Zhou, Yun Lin, Jiahong Zhou, Shaohua Wei
College of Chemistry and Materials Science, Jiangsu Key Laboratory of Biofunctional Materials, Nanjing Normal University, Wenyuan Road No.1, Nanjing 210046, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 May 2012
Received in revised form
12 September 2012
Accepted 13 September 2012
Available online 2 October 2012
Novel tetra-substituted zinc phthalocyanines (Pcs) bearing 1,2-ethanediamine group and the quaternized
derivatives were synthesized and characterized. The photophysical and cellular properties of these Pcs
were investigated. The results indicated that the quaternized ionic effect can greatly improve the water-
solubility of Pcs and reduce their aggregation degree in aqueous solution. Comparative studies with
quaternized phthalocyanine and its unquaternized counterpart have also demonstrated that the
quaternary action on the molecules significantly enhances the fluorescence quantum yields, fluorescence
lifetimes, efficiency of singlet oxygen production and, thereby, the in vitro photodynamic therapy efficacy.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Phthalocyanine
Photodynamic therapy
Photosensitizer
Singlet oxygen
1. Introduction
In order to decrease phthalocyanine aggregation and increase
their water-solubility, several hydrophilic groups (e.g., poly-
Photodynamic therapy (PDT) is a well-established modality for
the treatment of a variety of localized tumors, including skin,
mouth, esophageal, lung and bladder tumors [1,2]. Photodynamic
therapy involves administration of a tumor localizing photo-
sensitizing agent, followed by activation of the agent by light of
a specific wavelength. This therapy results in a sequence of
photochemical and photobiologic processes that cause irreversible
photodamage to tumor tissues [3,4].
Pcs, which were first synthesized by chance in 1907, are
remarkable macrocyclic compounds having magnificent physical
and chemical properties [5,6]. Especially, owing to their high effi-
ciency of singlet oxygen generation ability, extraordinary stability,
and strong absorption in the phototherapeutic window (600e
900 nm), Pc has been emerged as promising photosensitizers for
PDT [7e9]. However, for most Pcs, low solubility and aggregation
phenomena are observed in several organic media and in water,
which have strong influence on their singlet oxygen (1O2)
production efficiency, in vivo distribution and bioavailability. Above
problems finally affect their application in PDT [10,11].
ethylene glycol, carbohydrates) and ionic substituent have been
introduced at the macrocycle periphery [12e16]. Of particular
interest are cationic Pcs, since such molecules have superior water-
solubility, low aggregation degree and high cellular uptake effi-
ciency [17e21]. Besides, According to the literature, the intracel-
lular uptake efficacy and aggregation degree of Pcs are directly
related to the number of positive ions in their structures [1,22,23].
Based on these conceptions, a new octa-cationic zinc phthalo-
cyanine and other two unquaternized counterpart, as control, were
synthesized. The properties of the three Pcs were discussed and
compared. The results showed that octa-cationic zinc phthalocya-
nine not only have good solubility, low aggregation, high fluores-
cence quantum yields, high rate of singlet oxygen generation but
also have high efficiency of intracellular uptake and in vitro anti-
cancer activity. Above results indicated that the new octa-cationic
zinc phthalocyanine offers potential as photosensitizer in PDT.
2. Experiments
2.1. Chemicals and reagents
4-nitrophthalonitrile was used after being recrystallized from
methanol. All other necessary chemicals were of analytical grade
obtained from commercial suppliers and were used without further
purification unless otherwise stated. 1,8-diazabicyclo[5,4,0]-undec-
* Corresponding author. Tel./fax: þ86 (0)25 85891761.
E-mail address: shwei@njnu.edu.cn (S. Wei).
Both authors contributed equally to this work.
1
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.038

A. Wang et al. / European Journal of Medicinal Chemistry 58 (2012) 12e21
13
7-ene (DBU), disodium salt of 9,10-anthracenedipropionic acid
2.7. Cellular uptake of ZnPcs 1e3 in HeLa cells
(ADPA) and CT-DNA were all purchased from established suppliers
(SigmaeAldrich, Acros) and used as received. All organic reagents
were of analytical grade and were purified according to reported
procedures [24] before use. TLC was performed on silica gel GF254
plates. Silica gel (300e400 mesh) was used for preparative column
chromatography. Dulbecco’s modified Eagle’s medium (DMEM)
was form Gibco.
HeLa cells were incubated under the same experimental
conditions with ZnPc1 (5 M), ZnPc2 (5 M) and ZnPc3 (5 M) for
24 h in the dark. After 24 h incubation, the drug concentration
remaining in the medium was detected and calculated. All cellular
uptake amounts were calculated according to the standard curves.
m
m
m
2.8. Intracellular ROSs detection by DCFH-DA
2.2. Characteristics
Intracellular generation of reactive oxygen species was
measured by using an oxidation-sensitive fluorescent probe, 2070-
dichlorofluorescin diacetate (DCFH-DA), whose oxidized form (2070-
dichlofluorescein, DCF) is highly fluorescent [26,27]. HeLa cells
were seeded in 6 well plates at 1 ꢀ 105 mLꢁ1 density. After incu-
The water-soluble Pcs were dissolved in water and in the case of
Pcs insoluble in water, the stock solution was prepared in dimethyl
sulfoxide (DMSO) and diluted with water to the final concentration
for all experiments. Infrared spectra were measured in KBr pellets
on IR-Spectrometer Nicolet Nexus 670. 1H NMR and 13C NMR
spectra were recorded using a Bruker Advance 400 MHz NMR
spectrometer. Elemental analyses were taken with Vario MICRO,
Elementar. UVevis spectra were recorded on spectrophotometer
Cary 5000, Varian. Fluorescence spectra were carried out using
Perkin Elmer LS 50B fluorescence spectrophotometer. Fluorescence
quantum yields and lifetimes were recorded on FM-4P-TCSPC,
Horiba Jobin Yvon. Cell morphology changes were observed
under a Zeiss Observer fluorescence microscope. A 665 nm LED was
used as light source.
bation with ZnPcs (5 mM) for 24 h, cells were treated with 10 mM
DCFH-DA. The control experiment used cells incubated in serum-
free DMEM and DCFH-DA without ZnPcs. After 60 min incubation,
cells were washed twice with PBS and then exposed to light for
5 min. After light exposure, cells were digested with pancreatic
enzymes and dilute with 1 mL DMEM immediately, and the fluo-
rescence of DCF was detected (Ex ¼ 488 nm; Em ¼ 530 nm).
2.9. Dark cytotoxicity
For determination of dark cytotoxicity human embryonic kidney
293 cells (HEK 293) and HeLa cells were seeded into 96 well plates
at a density of 5 ꢀ 105 cells/cm2 and incubated for 24 h in growth
medium to allow for attachment. After 24 h, cellular survival was
measured by using the classical [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] (MTT) colorimetric assay [28].
2.3. Photostability assaying
The photostability of the three Pcs were investigated in water.
And the decrease in the Q-band absorption of the phthalocyanine
was monitored every 30 s using UVevis spectrophotometer.
2.10. In vitro anti-cancer activity studies
2.4. Singlet oxygen generation detection
For photo-induced anti-cancer experiments HeLa cells were
incubated as described above, 24 h later, the old medium was
replaced by fresh medium (without FBS) with ZnPc1, ZnPc2 or
ZnPc3, separately. Following 24 h incubation, the culture medium
was replaced by FBS-free medium and rinsed for three times by PBS
to remove adhered drugs. The cells were immediately exposed to
light and after 24 h incubation, cells viability was measured as
described above.
The singlet oxygen ability of ZnPcs was carried out using diso-
dium salt of 9,10-anthracenedipropionic acid (ADPA) as probe. Pcs
(10 ꢀ 10ꢁ6 M) and ADPA (5.5 ꢀ 10ꢁ6 M) were mixed and irradiated.
The reaction was monitored spectrophotometrically by measuring
the decrease in optical density every 1 min at an absorbance
maximum of 378 nm of ADPA.
2.11. Statistical analysis
2.5. Cell morphology
Student’s t-test was used to analyze the differences in photo-
toxicity and mitochondrial membrane potential assay following
treatment with free or HA-Tf. Experimental results are given as
means ꢃ SD of the indicated number of experiments. P < 0.05 was
considered necessary for statistical significance.
The cervical carcinoma cell line (HeLa) was maintained in Dul-
becco’s modified eagle’s medium (DMEM) containing glucose
supplemented with
L-glutamine, pyridoxine hydrochloride,
110 mg Lꢁ1 sodium pyruvate, 0.1 mg mLꢁ1 penicillin, 0.1 mg mLꢁ1
streptomycin and 10% (v/v) fetal bovine serum (FBS), in a humidi-
fied atmosphere at 37 ꢂC and 5.0% CO2. After treatment with ZnPcs
overnight and irradiating by light, cells were washed with phos-
phate buffered saline (PBS) three times and the cell morphology
changes were observed under a fluorescence microscope.
3. Results and discussion
The synthetic procedure followed is outlined in Schemes 1 and 2.
3.1. Synthesis
2.6. Hoechst 33342 staining
3.1.1. N-tritylethane-1,2-diamine (1, Scheme 1)
Chromatin condensation was detected by nuclear staining with
Hoechst 33342 [25]. After treatment with ZnPcs overnight and
irradiating by light, cells were washed with phosphate buffered
A mixture of anhydrous ethylenediamine (1.5 g, 24.96 mmol)
and finely ground anhydrous K2CO3 (3.45 g, 24.96 mmol) in CH2Cl2
(40 mL) was stirred under nitrogen at room temperature. Then,
(chloromethanetriyl) tribenzene (3.48 g, 12.48 mmol) in CH2Cl2
(40 mL) was added dropwise to the solution over a period of 4 h,
and stirring was continued overnight after the titration was
completed. The formed solid material and K2CO3 were filtered off
saline (PBS) three times and treated with 25
m
g mLꢁ1 Hoechst
33342 at 37 ꢂC with 5% CO2 in the dark for 30 min. Nuclear
morphology change was observed under
microscope.
a
fluorescence

14
A. Wang et al. / European Journal of Medicinal Chemistry 58 (2012) 12e21
H2N
K2CO3
CH2Cl2
HO
CHO
H2N
+
Tr-Cl
NH2
HN Tr
1
C2H5OH
OH
CN
CN
O2N
NaBH4
NC
O
H
N
Tr
C2H5OH
DMF,K2CO3
NC
N
H
H
N
Tr
N
H
3
2
Tr=
Scheme 1. Synthetic route to 4-(4-((2-(tritylamino)ethylamino)methyl)phenoxy)phthalonitrile.
and the filtrate was washed with water (3 ꢀ 100 mL). The solution
was dried over anhydrous magnesium sulfate. After filtered, the
solvent was evaporated in vacuum; the residue was purified by
column chromatography with silica gel as column material and
methanol/ethyl acetate (1:3) solvent system as elution. Yield: 3.39 g
(90.0%). M.P. 83 ꢂC. IR (KBr, cmꢁ1): 3420 (NH2), 3370 (NH), 3290,
3090e3030 (PheH), 2930, 2850(CH2), 1590, 1490, 1440, 756, 702.
3.1.3. 4-(4-((2-(tritylamino)ethylamino)methyl)phenoxy)
phthalonitrile (3, Scheme 1)
Under nitrogen atmosphere, a mixture of 4-nitrophthalonitrile
(0.94 g, 5.40 mmol) and finely ground anhydrous K2CO3 (1.24 g,
9.01 mmol) in dry DMF (10 mL) was stirred at room temperature.
Then, 4-((2-(tritylamino)ethylamino)methyl)phenol (2) (1.84 g,
4.50 mmol) was added to the solution at once. The reaction mixture
was heated to 40 ꢂC, and left to stir for 5 h. During the whole
process, the reaction was monitored by TLC using ethyl acetate/
petroleum ether (1:2) solvent system for elution. After cooling to
room temperature, the solution was filtered off. The filtrate was
poured into CH2Cl2 (60 mL), then washed with distilled water
(4 ꢀ 50 mL) and saturated brine (2 ꢀ 50 mL), and dried over
anhydrous magnesium sulfate. The solvent was removed under
vacuum and the crude product was purified by column chro-
matographic separations with ethyl acetate/petroleum ether (1:2)
solvent system as elution. Yield: 2.20 g (91.3%). M.P. 52 ꢂC. IR (KBr,
cmꢁ1): 3425, 3320 (NH), 3050 (AreH), 2920, 2840 (CH2), 2230 (CN),
1H NMR (400 MHz, DMSO-d6):
d
(ppm) 7.41 (d, 6H, J ¼ 7.66 Hz, Ar),
7.29 (t, 6H, J ¼ 7.31 Hz, Ar), 7.16e7.2 (m, 3H, Ar), 2.63 (t, 2H,
J ¼ 6.04 Hz, CH2), 2.45 (t, 2H, J ¼ 5.64 Hz, CH2). 13C NMR (100 MHz,
DMSO-d6):
d (ppm) 146.7, 128.9, 128.1, 126.4, 70.6, 46.7, 42.3. Anal.
Calcd. For C21H22N2: C, 83.40; H, 7.33; N, 9.27. Found: C, 83.37; H,
7.37; N, 9.26.
3.1.2. 4-((2-(tritylamino)ethylamino)methyl)phenol (2, Scheme 1)
4-hydroxybenzaldehyde (1.87 g, 15.28 mmol) was slowly added
to the solution of N-tritylethane-1,2-diamine (1) (4.20 g,
13.89 mmol) in dry C2H5OH (35 mL) at room temperature. About
10 min later, white precipitate appeared, and the reaction mixture
was left stirred for another 3 h under ambient conditions. The
reaction was monitored by TLC using methanol/ethyl acetate (1:3)
solvent system for elution. The reaction mixture was poured into
water (50 mL). The white solid material formed was filtered off and
washed with absolute alcohol. Without further processing, the
resulting precipitate was dissolved with dry C2H5OH (40 mL) and
thereafter, NaBH4 (0.58 g, 15.33 mmol) was added portion-wise
over a period of 30 min. Then, the reaction mixture left to stir for
a further 3 h at room temperature. At the end of this period, the
resulting precipitate was filtered off and purified by column chro-
matographic separations with silica gel as column material and
petroleum ether/ethyl acetate (1:3) solvent system as elution.
Yield: 4.43 g (78.0%). M.P. 145 ꢂC. IR (KBr, cmꢁ1): 3440 (OH), 3320
(NH), 3270, 3010e3070 (PheH), 2930, 2840 (CH2),1620,1590,1520,
1590, 1510, 1490, 1250, 708. 1H NMR (400 MHz, DMSO-d6):
d (ppm)
8.09 (d, 1H, 8.70, Ar), 7.75 (d, 1H, 2.18 Hz, Ar), 7.4 (t, 8H, 8.90 Hz, Ar),
7.35e7.27 (m, 7H, Ar), 7.20e7.12 (m, 5H, Ar), 3.6 (s, 2H, CH2), 2.65 (t,
2H, 5.44 Hz, CH2), 2.11 (s, 2H, CH2). 13C NMR (100 MHz, DMSO-d6):
d
(ppm) 161.8, 152.6, 146.7, 139.2, 136.7, 130.4, 128.9, 128.1, 126.5,
122.9, 122.1, 120.5, 117.1, 116.4, 115.8, 108.4, 70.7, 52.4, 49.3, 43.4.
Anal. Calcd. For C36H30N4O: C, 80.87; H, 5.66; N, 10.48. Found: C,
80.86; H, 5.67; N, 10.46.
3.1.4. 2(3), 9(10), 16(17), 23(24)-tetra-(((2-(tritylamino)
ethylamino)methyl)phenoxy)phthalocyaninato-zinc (Ⅱ) (ZnPc1,
Scheme 2)
A mixture of anhydrous zinc acetate (0.09 g, 0.49 mmol), 4-(4-
((2-(tritylamino)ethylamino)methyl)phenoxy)phthalonitrile
(0.42 g, 0.79 mmol) and DBU (0.4 g, 2.63 mmol) was stirred and
heated at 140 ꢂC in dry n-pentanol (10 mL) for 24 h under nitrogen
atmosphere. After cooling to room temperature, the reaction
solution was poured in to CH3OH (60 mL). The deep blue solid
product was precipitated and collected by filtration, then washed
with H2O and methanol till the filtrate was colorless. The green
crude product was purified by passing through a silica gel column
with firstly petroleum ether/ethyl acetate (1:3), then methanol/
(3)
1490, 1250, 750, 710. 1H NMR (400 MHz, DMSO-d6):
d (ppm) 9.18 (s,
1H, eOH), 7.40 (d, 6H, 7.92 Hz, Ar), 7.28 (t, 6H, 7.63 Hz, Ar), 7.17 (t,
3H, 7.24 Hz, Ar), 7.04 (d, 2H, 8.26 Hz, Ar), 6.67 (d, 2H, 8.31, Ar), 3.43
(s, 2H, ArCH2), 2.59 (t, 2H, 5.85, CH2), 2.09 (t, 2H, 9.33 Hz, CH2). 13
C
NMR (100 MHz, DMSO-d6): d (ppm) 156.4, 146.7, 131.4, 129.5, 128.8,
128.1, 126.5, 115.3, 70.7, 52.7, 49.0, 43.3. Anal. Calcd. For C28H28N2O:
C, 82.32; H, 6.91; N, 6.86. Found: C, 82.30; H, 6.92; N, 6.90.

A. Wang et al. / European Journal of Medicinal Chemistry 58 (2012) 12e21
15
R1
R1
N
N
N
N
Zn(CH3COO)2
3
N
N
Zn
DBU,1400C,n-pentanol
N
Tr
N
NH
HN
R1
R1
O
R1=
ZnPc1
R2
R2
N
N
N
N
N
N
Zn
CF3COOH
CH2Cl2
ZnPc1
N
N
R2
R2
NH2
HN
ZnPc2
O
R2=
R3
R3
N
N
N
K2CO3,CH3I
CF3COOH
N
ZnPc1
N
Zn
N
N
I
I
N
N
N
O
R3=
R3
R3
ZnPc3
Scheme 2. Synthetic route to tetra-substituted zinc Pcs (ZnPc1, ZnPc2 and ZnPc3).
CH2Cl2 (1:40) as elution. The blue fraction was collected, evapo-
rated under vacuum, and vacuum-dried at 50 ꢂC for 12 h to afford
the product as a dark green solid. Yield: 0.27 g (62.36%). M.P.
>200 ꢂC. IR (KBr, cmꢁ1): 3450, 3060, 2930, 2840, 1710, 1600, 1480,
3.1.5. 2(3), 9(10), 16(17), 23(24)-tetra-(((2-aminoethylamino)
methyl)phenoxy)phthalocyaninato-zinc (Ⅱ) (ZnPc2, Scheme 2)
Under the condition of ice-water bath, ZnPc1 (0.48 g, 0.22 mmol)
and excess trifluoroacetic acid (TFA) (0.5 mL) were dissolved in
CH2Cl2 (10 mL) and stirred for 1 h. Then, the reaction mixture was
heated to room temperature and left to stir for another 2 h. The crude
product was collected by filtration and washed successively with
CH2Cl2. Thereafter, the green solid was dissolved in water and
precipitated by adjusted pH to 9e10. The residue product collected
by filtration was washed successively with water and methanol. The
product was vacuum-dried at 50 ꢂC for 12 h to afford the final
1230, 945, 708. 1H NMR (400 MHz, DMSO-d6)
d (ppm): 8.75e8.55
(br, 4H, PceH), 8.32 (s, 4H, PceH), 7.13e7.43 (br, 80H, PceH, Ar),
3.62 (m, 8H, CH2), 2.71 (s, 8H, CH2), 2.02 (s, 8H, CH2). 13C NMR
(100 MHz, CDCl3):
d (ppm) 146.2, 129.9, 129.8, 128.7, 128.1, 127.8,
127.6, 126.2, 120.3 (br), 70.8, 53.2, 52.9, 49.5, 46.2, 46.1, 43.1, 31.9,
29.7, 29.6, 29.4, 22.7, 14.1, 11.3. Anal. Calcd. For C144H120N16O4Zn: C,
78.47; H, 5.49; N, 10.17. Found: C, 77.67; H, 5.86; N, 9.80.

16
A. Wang et al. / European Journal of Medicinal Chemistry 58 (2012) 12e21
Table 1
0.6
0.4
0.2
0.0
ZnPc3
ZnPc2
ZnPc1
The monomer and aggregation electronic absorption data of ZnPc3 in water.
Concentrations
(M)
Absorbance
(680 nm)
Absorbance
(640 nm)
Proportion
A680/A640
1 ꢀ 10ꢁ6
5 ꢀ 10ꢁ6
9 ꢀ 10ꢁ6
13 ꢀ 10ꢁ6
17 ꢀ 10ꢁ6
0.1407
0.5757
0.9439
1.1695
1.4608
0.0591
0.2905
0.5651
0.9005
1.1939
2.38
1.98
1.67
1.29
1.22
PceH), 4.76 (d, 8H, 3.22 Hz, CH2), 4.10 (s, 8H, CH2), 4.01 (s, 8H, CH2),
3.15e3.25 (br, 60H, CH3). 13C NMR (100 MHz, DMSO-d6):
(ppm)
d
300
400
500
600
700
800
159.9, 159.7, 157.6 (br), 152.9 (br), 140.4, 135.9, 135.8, 134.5, 124.8,
122.9,122.7, 122.1, 119.4, 119.2, 119.1, 113.6, 113.1, 67.1, 57.5, 56.3, 53.7,
50.1, 19.0. Anal. Calcd. For C88H111I8N16O4Zn: C, 41.65; H, 4.41; I,
40.01, N, 8.83. Found: C, 41.23; H, 4.76; N, 8.18.
Wavelength(nm)
Fig. 1. Absorption spectra of the three Pcs (ZnPc1, ZnPc2 and ZnPc3) in water.
Concentration ¼ 5 ꢀ 10ꢁ6 M.
compound as a dark green solid. Yield: 0.21 g (78.07%). M.P. >200 ꢂC.
3.2. UVevis spectral characterization
IR (KBr, cmꢁ1): 3370, 2940, 2730,1600,1470,1240,1040, 945, 820.1H
The typical UVevis spectra of the zinc Pcs usually exhibited
characteristic monomer absorption band at w680 nm while
aggregate absorption band at 630e640 nm [31]. For PDT applica-
tion, aggregation effect of Pcs always reduce their 1O2 generation
and, thereby, the in vitro anti-cancer activity. The UVevis absorp-
tion spectra for ZnPc1, ZnPc2 and ZnPc3 are gathered in Fig. 1.
Comparing studies indicated that only ZnPc3 has strong monomer
band at 680 nm in aqueous solution, which implies its strong
photodynamic activity.
NMR (400 MHz, DMSO-d6) d (ppm): 9.25 (d, 4H,17.4 Hz, PceH), 8.83
(s, 4H, PceH), 8.1 (s, 4H, PceH), 7.68e7.85 (m, 8H, Ar), 7.48 (d, 8H,
10.14 Hz, Ar) 4.33 (d, 8H,10.52 Hz, CH2), 3.31 (d, 8H, 5.2 Hz, CH2), 3.23
(s, 8H, CH2). 13C NMR (100 MHz, D2O):
d (ppm) 157.7 (br), 150.9 (br),
137.8 (br), 132.3, 131.3, 126.1, 124.3, 121.5, 120.0, 112.1 (br), 57.5, 51.2,
43.9, 35.7,16.9,16.8. Anal. Calcd. ForC68H64N16O4Zn:C, 66.15; H, 5.22;
N, 18.15. Found: C, 65.81; H, 5.56; N, 17.68.
3.1.6. Quaternized 2(3), 9(10),16(17), 23(24)-tetra-(((2-aminoethyl-
amino)methyl)phenoxy)phthalocyaninato-zinc (Ⅱ) (ZnPc3, Scheme
2) [29,30]
8000000
ZnPc3
A mixture of ZnPc1 (0.43 g, 0.19 mmol) and excess trifluoroacetic
acid (TFA) (0.5 mL) in CH2Cl2 was stirred at room temperature for
3 h. The solid was filtered and successively washed with CH2Cl2. The
green solid product and K2CO3 (0.53 g, 3.80 mmol) were dissolved in
CH3OH (10 mL) and heated to boiling point of the solvent under
nitrogen atmosphere. Then, excess CH3I (1 mL, 16.00 mmol) were
added, and the reaction mixture was left to stir for 24 h at refluxing.
The crude solid green product was collected by filtration while hot,
then washed by methanol (3 ꢀ 10 mL) and ethanol (5 ꢀ 10 mL)
successively. The green product was dissolve in dry DMF (5 mL) and
purified by filtering again to remove residual K2CO3. The solvent was
removed under reduced pressure. The product was vacuum-dried at
50 ꢂC for 24 h. The title product was obtained as a dark green solid.
Yield: 0.40 g (80.8%). M.P. >200 ꢂC. IR (KBr, cmꢁ1): 3427, 3011,
2586,1601,1475,1234,1069, 934, 751. 1H NMR (400 MHz, DMSO-d6)
ZnPc2
7000000
ZnPc1
6000000
5000000
4000000
3000000
2000000
1000000
0
640 660 680 700 720 740 760 780 800
Wavelength(nm)
Fig. 3. Fluorescence spectra of the three Pcs (ZnPc1, ZnPc2 and ZnPc3) in water.
Concentration ¼ 5 ꢀ 10ꢁ6 M; Excitation wavelengths: 600 nm.
d
(ppm): 9.43 (t, 4H, 7.84 Hz, PceH), 9.07e9.01 (m, 4H, PceH), 7.93e
8.00 (m, 4H, PceH), 7.82 (t, 8H, 9.87 Hz, PceH), 7.48e7.56 (m, 8H,
80
monomer
0.8
aggregate
1.4
0.6
E
60
0.4
1.2
1.0
0.2
40
0.0
600
700
0.8
Wavelength(nm)
A
B
0.6
20
C
0.4
0.2
0.0
D
E
A
0
0
40
80
120
160
200
300
400
500
600
700
800
Irradiation Time(s)
Wavelength(nm)
Fig. 4. Photo-bleaching of monomer (-) and aggregate ( ) of ZnPc3 measured by the
Fig. 2. Ground state electronic absorption spectra of ZnPc3 at various concentrations:
1 ꢀ 10ꢁ6 (A), 5 ꢀ 10ꢁ6 (B), 9 ꢀ 10ꢁ6 (C), 13 ꢀ 10ꢁ6 (D), 17 ꢀ 10ꢁ6 (E) M in water.
decrease of the absorbance as the function of irradiation time (Inset: absorption
spectra changes of ZnPc3 irradiated by light in water;
Ot ¼ 30 s).

A. Wang et al. / European Journal of Medicinal Chemistry 58 (2012) 12e21
17
Gabrio Roncucci et al. [33], as concentration was raised, the
absorption at 680 nm and the band at 640 nm both became higher
without new bands observed. The proportion of monomer/aggre-
gation was shown in Table 1. The result indicated that although the
eight positively charged quaternary ammonium groups give the
dye a high solubility in water, increasing concentrations also
enlarge the tendency of ZnPc3 to form aggregates.
1.0
0.8
0.6
0.4
0.2
0.0
A
0s
60s
120s
180s
240s
300s
360s
3.3. Fluorescence spectra and properties
In vivo and in vitro fluorescence detection, fluorescence usually
employed to obtain information about photosensitizer localization
and distribution as well as release from tissues [34].
300
350
400
450
Fluorescence emission spectra of the three zinc phthalocyanines
were observed and collected in Fig. 3. In aqueous system, when
excited at 600 nm, ZnPc1 and ZnPc2 showed very weak fluores-
cence while ZnPc3 showed strong emission peak at 693 nm, same
as other zinc Pcs [35]. This absence of fluorescence of the ZnPc1 and
ZnPc2 dispersed in water can be due to either drug solvent inter-
action promoting nonradiative decay or quenching derived from
self-aggregation of ZnPc1 and ZnPc2. Aggregated metal Pcs are not
known to fluorescence since aggregation lowers the fluorescence
intensity of molecules through dissipation of energy [5]. But in the
case of ZnPc3, the drug molecules can be existed as monomer, and
therefore prevent a complete loss of fluorescence.
Wavelength(nm)
B
0.0
-0.2
-0.4
-0.6
-0.8
ZnPc3
ZnPc1
ZnPc2
3.4. Photostability studies
0
1
2
3
4
5
6
Time(min)
Most photosensitizers will degrade during the process of PDT by
photo-bleaching. In this process, the intensity of the absorption will
decrease [36]. Thus, the stability of Pcs intended for use in PDT is
especiallyimportant. InordertostudythestabilityofthethreeZnPcs,
the photo-bleaching experiments were carried out and the Q-band
absorption decreasing degrees were recorded. After irradiation, all
the Pcs were bleached bylight. Insert panel of Fig. 4 shows the photo-
bleaching process of ZnPc3 in water. Comparing results indicated
that the aggregate is more stable than monomer under the irradia-
tion (Fig. 4). Subsequent experiments prove that when the concen-
tration increase, the photostability of ZnPc3 also become better
because many Pcs molecular were aggregate at high concentration
condition.ForZnPc1, similar resultthat aggregateismorestablethan
monomer under the irradiation was got. The stability of monomer
and aggregate of ZnPc2 were not compared because there was
Fig. 5. (A) UVevis spectrum for the determination of singlet oxygen quantum yields of
ZnPc3 in water use ADPA as quencher at the concentration of 10 ꢀ 10ꢁ6 M. (B) First
order plots of ADPA absorbance versus time of the three Pcs.
Aggregation is usually depicted as a coplanar association of rings
progressing from monomer to dimer and higher order complexes. It
is dependent on the concentration, nature of the substituents, et al.
[32]. In this study, the aggregation behavior of ZnPc3 was investi-
gated at different concentrations in water (Fig. 2). Due to the high
value of the Q-band absorption, we were unable to obtain quanti-
tative data for more concentrated solutions, however we found
high monomer up to 1.7 ꢀ 10ꢁ5 M. Thus, it seems evident that the
presence of eight positive charges efficiently prevents the aggre-
gation of this phthalocyanine. Unlike to compound reported by
Fig. 6. The fluorescence lifetime of ZnPc3 in H2O at the concentration of 7 ꢀ 10ꢁ6 M (21.94787 ns ¼ 1 channel).

18
A. Wang et al. / European Journal of Medicinal Chemistry 58 (2012) 12e21
Table 2
almost no monomer for ZnPc2 in water. Besides, the stability of
ZnPc3 in comparison to ZnPc1 and ZnPc2 was also compared. The
result showed that ZnPc2 showed the best while ZnPc3 the worse
stability inwaterunder irradiation (see Supporting information). The
result can be attributed to the structure and exist states of Pcs
(monomer or aggregate). It is also believed that photo-bleaching of
Pcs is a singlet oxygen mediated process, since singlet oxygen is
highly reactive and it can react with Pcs [31,32], which implies high
singlet oxygen generation ability of ZnPc3.
Cellular uptake percent of ZnPc2 and ZnPc3 with prolonged incubation time.
Compound
Cellular uptake percent (%)
2 h
4 h
24 h
ZnPc2
ZnPc3
32.5
52.9
51.5
61.2
71.3
86.5
2.5
2.0
1.5
1.0
0.5
0.0
3.5. Singlet oxygen generation ability
∗∗
The singlet oxygen generation ability is an important indicator
for the potential of photosensitizers in PDT [37]. Singlet oxygen can
be detected using a chemical method by the photo-oxidation of
ADPA to its endoperoxide derivative.
∗
For all of the three ZnPcs, the absorption intensity of ADPA
(
l
¼ 378 nm) continuously decreased as the irradiation time
increasing. The rate of singlet oxygen generation is calculated by
the following equation described by Wei Tang et al. [38].
À
Á
ZnPc1
ZnPc2
ZnPc3
ln ½ADPAꢄt=½ADPAꢄ0 ¼ ꢁkt
Fig. 7. Effect of Pcs ZnPc1, ZnPc2 and ZnPc3 on ROSs generation in HeLa cells
(*P < 0.05, **P < 0.01 vs. Control; Control: cells were exposed to light without ZnPcs).
where [ADPA]t and [ADPA]0 are the concentrations of ADPA after
and prior irradiation, respectively. Values of k are the rate of singlet
oxygen generation and t is the time of irradiation.
Fig. 8. Photograph showing morphology of normal HeLa cells (A) control untreated with photosensitizer and irradiated for 5 min; (B) treated with 5
with LED light (lex ¼ 665 nm); (C) Treated with 5 M ZnPc2 irradiated for 5 min with LED light (lex ¼ 665 nm); (B) Treated with 5 M ZnPc3 irradiated for 5 min with LED light
lex ¼ 665 nm); 40 microscope objective. Bar ¼ 100 m.
mM ZnPc1 irradiated for 5 min
m
m
(
m

A. Wang et al. / European Journal of Medicinal Chemistry 58 (2012) 12e21
19
In Fig. 5, higher singlet oxygen generation rate is observed for
ZnPc3 (kZnPc3 ¼ 0.1185) comparing with ZnPc1 (kZnPc1 ¼ 0.0253)
and ZnPc2 (kZnPc2 ¼ 0.0245) indicated that the quaternarized ionic
significantly increases the ability of ZnPcs to generate singlet
oxygen. It is due to that quaternary increases the solubility of Pcs in
the water and reduces their aggregation level at the same time.
However, the rates of singlet oxygen generation of ZnPc1 and
ZnPc2 are much lower than ZnPc3, which is in agreement with
those previously reported for similar Pcs [23].
of 7 ꢀ 10ꢁ6 M (ZnPc3 as an example). The average fluorescence
lifetimes of ZnPc2 and ZnPc1 were 2.87 ns and 2.40 ns, separately.
Besides, our results showed different from the literature which
assert the fluorescence lifetime (sf) is directly related to that of
fluorescence quantum yields (FF); i.e. the longer the lifetime, the
higher the quantum yield of fluorescence [37,6]. In our study, there
are no positive correlation relationships between fluorescence life-
times and fluorescence quantum yields. This may be attributed to
the combined effects of structures, nature and environment of
fluorescent groups, and so on, which are all of the crucial influencing
factors of fluorescence lifetime and fluorescence quantum yield.
3.6. Fluorescence quantum yields (FF) and lifetimes (sf
)
3.7. Cellular uptake of ZnPc1, ZnPc2 and ZnPc3 in HeLa cells
The fluorescence quantum yield and lifetime are both important
parameters for practical applications of fluorescence and reactive
oxygen generation process. In our study, different from the
common method [39], the fluorescence quantum yields of us ob-
tained are absolute value rather than measured by comparison to
a standard. ZnPc1 has no fluorescence quantum yields, which can
be attributed to fluorescence quenching of aggregation discussed
above and the solvent effect. Higher fluorescence quantum yields
were obtained for the quaternized ZnPc3 (16.7%) in water
compared to its unquaternized ZnPc2 due to higher aggregation
tendencies of ZnPc2 in aqueous media.
The relative uptake was quantified by comparison between the
two water-soluble Pcs, ZnPc2 and ZnPc3. Significant difference in
cellular uptake efficiency was obtained from ZnPc2 and ZnPc3.
Taking into consideration that ZnPc2 and ZnPc3 both easily dis-
solved in water; these results possibly because of that the intra-
cellular uptake efficiency of the cationic Pc (ZnPc3) was higher than
non-ionic Pc (ZnPc2) due to the electronegative property of cancer
cells (Table 2).
3.8. Intracellular production of reactive oxygen species (ROS)
The fluorescence lifetimes of the Pcs are obtained by the method
of time-correlated single-photon counting (TCSPC). Fig. 6 shows the
average fluorescence lifetime of ZnPc3 in H2O at the concentration
It is well known that, upon illumination, photosensitizer is
excited from the ground state to an excited state, generating free
Fig. 9. Fluorescence micrographs of HeLa cells stained with Hoechst 33342. (A) Normal cells; (B) Cells treated with ZnPc1 (5
ZnPc2 (5 M) and 5 min of irradiation; (D) Cells treated with ZnPc3 (5 m.
M) and 5 min of irradiation. Bar ¼ 100
mM) and 5 min of irradiation; (C) Cells treated with
m
m
m

20
A. Wang et al. / European Journal of Medicinal Chemistry 58 (2012) 12e21
radicals and reactive oxygen species (ROSs), which are responsible
for oxidative damage and cell death [40]. In order to evaluate the
intracellular formation of ROSs after irradiation, HeLa cells pre-
treated with ZnPc1, ZnPc2 and ZnPc3, were loaded with the
probe 2070-dichlorofluorescin-diacetate (DCFH-DA). After diffusing
into cells, DCFH-DA is deacetylated by esterase and is then oxidized
to the fluorescent 2070-dichlorfluorescein (DCF) in the presence of
ROSs. As shown in Fig. 7, results are expressed as the ratio between
DCF fluorescence, and represent the mean ꢃ SD of three different
experiments. The ROSs generation ability of ZnPc3 was higher than
ZnPc2 and ZnPc1, which indicated that the in vitro photodynamic
anti-cancer activity of ZnPc3 should stronger than ZnPc2 and
ZnPc1.
120
100
80
60
40
20
0
ZnPc2
ZnPc1
ZnPc3
∗
∗
∗∗
∗∗∗
∗∗
∗∗∗
∗∗∗
∗∗∗
0
1
2
3
4
5
Irradiation Time(min)
3.9. Cell morphology
Fig. 11. Cell-viability assays with different light doses of ZnPc1, ZnPc2 and ZnPc3
(*P < 0.05, **P < 0.01 vs. Control; Control: cells were treated with ZnPcs but no
irradiation).
The morphology of HeLa cells was observed by microscopy after
incubation with ZnPcs (5
mM) and irradiation for 5 min. Repre-
sentative results are shown in Fig. 8. Microscopic observations
showed that the HeLa cells changed their normal shape after
photodynamic treatment. These results provided evidence about
membrane deformation and an increase in the volume of cells
indicating a higher fragility after the photodynamic action.
Hoechst can bind to DNA, so, it was used to assess changes in
nuclear morphology. PDT process will damage tumor cells and
induce DNA destruction in nuclear of cancer calls by activating
photosensitizers by light. The results showed that there is no
significant change in cell nuclear morphology, when the cells
treated only with irradiation and the fluorescence of chromatin
stained dimly and occupied the majority of the cell (Fig. 9A). In
contrast, the cells treated with ZnPc1 (Fig. 9B), ZnPc2 (Fig. 9C) and
ZnPc3 (Fig. 9D) and irradiated by light showed obvious nuclear
morphology changes, such as nuclear shrinkage, chromatin
condensation, and fragmentation.
3.10. Hoechst 33342 staining
DNA damage is a very important index in PDT. The Hoechst
33342 is one part of a family of blue fluorescent dyes. Because the
120
ZnPc1
A
3.11. Dark cytotoxicity
ZnPc2
100
∗
ZnPc3
∗
∗
∗∗∗
∗
ZnPcs without illumination may have a cytotoxic effect, collec-
tively referred to as dark cytotoxicity in the following. Fig. 10 shows
the dark cytotoxic of ZnPc1, ZnPc2 and ZnPc3 in HeLa and HEK 293
cells. For ZnPc2 and ZnPc3 the cell-viability remains at about 95%
of untreated controls up to a concentration of 5
remains at about 89%. At concentrations higher than 20
viability decreases to lower than 65% (Fig. 10A) and 45% (Fig. 10B)
for ZnPc1. The cell survival of ZnPc2- and ZnPc3-incubated cells
remains higher than 80% up to a final concentration of 20
Because of this, we settled on one concentration of 5
photodynamic reaction.
∗
∗∗
80
60
40
20
0
∗∗
m
M while ZnPc1
M the cell-
m
mM.
mM for the
0
5
10
15
20
Concentration of ZnPc(μmol/L)
120
ZnPc2
ZnPc1
ZnPc3
120
100
80
60
40
20
0
ZnPc1
ZnPc2
B
100
80
60
40
20
0
∗
∗
∗∗
∗
∗
ZnPc3
∗∗∗
∗
∗∗∗
∗∗
∗∗
∗∗∗
∗
∗∗
∗∗
∗∗
∗
∗∗∗
∗∗
∗
∗
∗∗∗
∗∗∗
∗∗∗
∗∗∗
∗∗∗
0
5
10
15
20
0
1
2
3
4
5
Concentration of ZnPc(μmol/L)
Concentration of ZnPc (μmol/L)
Fig. 10. Dark cell-viability assays with different drug doses of ZnPc1, ZnPc2 and ZnPc3
to HeLa (A) and HEK 293 (B) cells (*P < 0.05, **P < 0.01, ***P < 0.001 vs. Control;
Control: cells were treated without ZnPcs).
Fig. 12. Cell-viability assays with different drug doses of ZnPc1, ZnPc2 and ZnPc3
(*P < 0.05, **P < 0.01 vs. Control; Control: cells were exposed to light without ZnPcs).

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and 21.5% respectively.
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We have prepared and characterized a series of novel zinc Pcs,
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Comparative studies with quaternized and unqutaternized Pcs have
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significantly enhances the water-solubility, the monomer percent,
fluorescence intensity, efficiency of 1O2 generation, intracellular
uptake and, thereby, the in vitro PDT efficacy of the Pcs. Above
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Acknowledgements
This work was supported by the National Natural Science
Foundation of China (20673058, 20973093), the Scientific Research
Foundation of Nanjing Normal University (No. 2011103XG0249),
and the Priority Academic Program Development of Jiangsu Higher
Education Institutions (PAPD).
Appendix A. Supplementary data
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Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.09.038.
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