Synthesis and antifungal photodynamic activities of a series of novel zinc(II) phthalocyanines substituted with piperazinyl moieties

Article abstract of DOI:10.1016/j.dyepig.2013.04.032

A series of novel zinc(II) phthalocyanines tetra-substituted or mono-substituted with piperazinyl moieties linked by different ethoxy chains has been prepared and characterized. The effects of the N-protecting group of piperazinyl moiety, length of ethoxy chains and number of substitutes on the photophysical, photochemical properties, cellular uptakes and in vitro photodynamic antifungal activities have also been examined. All of these compounds are essentially non-aggregated and good singlet oxygen generators with quantum yields (Φ_Δ) of 0.54-0.77 in N,N-dimethylformamide. The photodynamic activity of these compounds against Candida albicans follows the order: 7 > 5a > 5b > 4a ≈ 4b. The mono-substituted phthalocyanine 7 exhibits the highest photodynamic inactivation of C. albicans with an IC₉₀ value of 9 μM, which can be attributed to its better amphiphilicity and even higher cellular uptake. The results suggest that phthalocyanine 7 is a potential photosensitizer for antifungal photodynamic therapy.

Full text of DOI:10.1016/j.dyepig.2013.04.032

Dyes and Pigments 99 (2013) 185e191
Contents lists available at SciVerse ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Synthesis and antifungal photodynamic activities of a series of novel
zinc(II) phthalocyanines substituted with piperazinyl moieties
Bi-Yuan Zheng, Hong-Peng Zhang, Mei-Rong Ke, Jian-Dong Huang*
College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou 350108, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 March 2013
Received in revised form
A series of novel zinc(II) phthalocyanines tetra-substituted or mono-substituted with piperazinyl moi-
eties linked by different ethoxy chains has been prepared and characterized. The effects of the N-pro-
tecting group of piperazinyl moiety, length of ethoxy chains and number of substitutes on the
photophysical, photochemical properties, cellular uptakes and in vitro photodynamic antifungal activ-
ities have also been examined. All of these compounds are essentially non-aggregated and good singlet
24 April 2013
Accepted 25 April 2013
Available online 9 May 2013
D
oxygen generators with quantum yields (F ) of 0.54e0.77 in N,N-dimethylformamide. The photody-
namic activity of these compounds against Candida albicans follows the order: 7 > 5a > 5b > 4a z 4b.
The mono-substituted phthalocyanine 7 exhibits the highest photodynamic inactivation of C. albicans
with an IC90 value of 9 mM, which can be attributed to its better amphiphilicity and even higher cellular
uptake. The results suggest that phthalocyanine 7 is a potential photosensitizer for antifungal photo-
dynamic therapy.
Keywords:
Zinc(II) phthalocyanine
Photosensitizer
Photodynamic therapy
Single oxygen
Antifungal
Candida albicans
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Moreover, the resistance of C. albicans against traditional antifungal
agents, such as fluconazole, is increasing year by year [14,15]. PDT
Photodynamic therapy (PDT) is a minimally invasive method
that employs a photosensitizer molecule, which produces reactive
oxygen species (ROS) to cause cell and tissue damage by exposure
to lights with a specific wavelength [1]. It is a well-recognized
therapeutic modality for a variety of premalignant and malignant
diseases [2e4]. Recently, PDT utilized for the treatment of patho-
genic fungi has received considerable interest as a result of the
emergence of antibiotic-resistant mycoses and the increase of
fungal infections [5e10]. As a promising therapeutic approach,
antifungal PDT, which inactivates the pathogenic microorganisms
by ROS generated by photosensitizers, presents several advantages,
such as broad spectrum of action, high therapeutic efficacy which is
independent of the antibiotic resistance pattern of the given mi-
crobial strain, and limited damage to the host tissue, in contrast to
the typical antibiotic therapeutics [5e10].
has been employed to inactivate C. albicans and the antifungal ef-
fects of several kinds of photosensitizers (such as phenothiazine
dyes, porphyrins, and 5-aminolevulinic acid) have been briefly re-
ported [12,13,16e19].
Phthalocyanines have found to be highly promising as second-
generation photosensitizers for PDT owing to their desirable
photophysical and photochemical properties [20,21]. However,
the photodynamic activities toward fungi remain little studied for
phthalocyanine-based photosensitizers [22e27]. In order to
rationally design the phthalocyanines especially for the photo-
dynamic treatment of fungi, it is necessary to carefully explore
the structureeactivity relationships via variedly functionalized
phthalocyanines. Very recently, our group has investigated the
photodynamic activities against C. albicans of a series of silico-
n(IV) phthalocyanines axially substituted with different func-
tional groups, and discussed the effect of axial substituents [27].
The results from our [27] and other groups [23,24] suggest that
the photosensitizers bearing cationic or protonable groups such
as tertiary amines generally show higher photocytotoxicities
against pathogenic microorganisms compared with the neutral
and anionic analogs. As an extension of our work, we report
herein a series of novel zinc(II) phthalocyanines tetra-substituted
or mono-substituted with piperazinyl moieties linked by
different ethoxy chains. The synthesis, photophysical and
Candida albicans is the most common fungal pathogen that can
cause both superficial and systemic infections. Owing to the
augmented numbers of immunocompromised or hospitalized pa-
tients who suffer from cancer or AIDS, the infections associated
with C. albicans have increased dramatically in last decades [11e13].
*
Corresponding author. Tel.: þ86 591 22866235; fax: þ86 591 22866227.
E-mail addresses: jdhuang@fzu.edu.cn, 13805009843@163.com (J.-D. Huang).
0143-7208/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.dyepig.2013.04.032

186
B.-Y. Zheng et al. / Dyes and Pigments 99 (2013) 185e191
photochemical properties, cellular uptakes and in vitro photo-
dynamic activities against C. albicans of these compounds are
described in this paper. The effects of the N-protecting group of
piperazinyl moiety, length of ethoxy chains and number of sub-
stitutes on the physicochemical properties and antifungal activ-
ities have also been discussed.
2H); 1.46 (s, 9H). Anal. Calcd for C19
15.72. Found C, 63.81; H, 6.71; N, 15.63.
24 4 3
H N O : C, 64.03; H, 6.79; N,
2.2.2. 3-{2-[2-(4-Boc-piperazine)-1-ethoxy]-ethoxy}phthalonitrile
(3b)
According to the procedure for 3a, a solution of 1-[2-(2-
hydroxyethoxy)ethyl] piperazine (1b) (0.86 ml, 5 mmol) in TFA/
2 2 2
CH Cl (1:5, v/v, 22 ml) was treated with Boc O (1.18 ml, 5 mmol) to
2
. Experimental
give Boc-protected piperazinyl ethoxy ethanol. The obtained Boc-
protected piperazinyl ethoxy ethanol (1.37 g, 5.0 mmol) was then
treated with 3-nitrophthalonitrile (0.87 g, 5.0 mmol) and anhy-
2.1. General
drous K
b (1.13 g, 57%). Rf ¼ 0.59 (ethyl acetate). IR (KBr, cm ): 2226
C^N); 3036 (Ar-H); 2976, 2892, 2803, 1472,1365 (CH , CH ); 1586,
472 (benzene, CaC); 1292, 1176, 1131 (CeOeC, CeN); 1247 (Are
2 3
CO (1.38 g, 10 mmol) in DMF (20 ml) to give a white solid
All the reactions were performed under an atmosphere of ni-
trogen. N,N-dimethylformamide (DMF) and n-pentanol were dried
ꢁ1
3
(
1
3
2
over molecular sieves and further distilled under reduced pressure
ꢀ
before use. Potassium carbonate was activated by muffle at 300 C
þ
1
OeC); 1693 (CaO). MS (ESI): m/z 402.2 [M þ 2H] . H NMR
300 MHz, CDCl , ppm):
¼ 7.62 (t, J ¼ 8.4 Hz, 1H); 7.36 (d,
J ¼ 7.8 Hz, 1H); 7.28 (d, J ¼ 8.7 Hz, 1H); 4.29 (t, J ¼ 4.6 Hz, 2H); 3.89
t, J ¼ 4.6 Hz, 2H); 3.72 (t, J ¼ 5.5 Hz, 2H); 3.42 (t, J ¼ 5.0 Hz, 4H);
.60 (t, J ¼ 5.6 Hz, 2H); 2.43 (t, J ¼ 5.0 Hz, 4H); 1.45 (s, 1H). Anal.
Calcd for C21 : C, 62.98; H, 7.05; N, 13.99. Found C, 63.25; H,
under normal pressure. Cremophor EL and unsubstituted zinc(II)
phthalocyanine (ZnPc) were purchased from SigmaeAldrich.
Chromatographic purifications were performed on silica gel col-
umns (100e200 mesh, Qingdao Haiyang Chemical Co., Ltd, China)
with the indicated eluents. Size-exclusion chromatography was
performed on Bio-Rad Bio-Beads S-X3 beads with the indicated
eluents. All other solvents and reagents were of reagent grade and
used as received.
(
3
d
(
2
28 4 4
H N O
6.80; N, 14.31.
2.2.3. 1,8(11),15(18),22(25)-tetrakis-[2-(4-Boc-piperazine)-1-
1H NMR spectra were recorded on a Bruker DPX 300 spec-
ethoxy] phthalocyanine zinc(II) (4a)
trometer (300 MHz) in CDCl
3
6
or DMSO-d . Chemical shifts were
A suspension of 3a (0.36 g, 1.0 mmol) in n-pentanol (20 ml)
relative to internal SiMe
4
(d
¼ 0 ppm). Mass spectra were recorded
ꢀ
was stirred at 90 C for 10 min, and then anhydrous zinc acetate
on a Finnigan LCQ Deca xpMAX mass spectrometer. IR spectra were
recorded on a PerkineElmer SP2000 FT-IR spectrometer, using KBr
disks. Elemental analyses were performed by Element Vario EL III
equipment. Electronic absorption spectra were measured on a
Shimadzu UV-2450 UVevis spectrophotometer. Fluorescence
spectra were taken on an Edinburgh FL900/FS900 spectrofluo-
(
(
0.10 g, 0.54 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
0.40 ml, 2.6 mmol) were added. The resulting mixture was stirred
ꢀ
at 130 C for 5 h. After removing the volatiles in vacuo, the residue
was purified by a silica gel column chromatography using CHCl
CH OH (18:1, v/v) as eluent. A green band was collected and
concentrated to give a crude product, which was purified by
chromatography again using CHCl /CH OH (15:1, v/v) as eluent.
3
/
3
rometer. Fluorescence quantum yield (
F
D
F
F
) and singlet oxygen yield
) were determined as described in our previous manuscripts
28].
3
3
(
[
The obtained green solid was further purified by size-exclusion
chromatography by using THF as eluent to give a dark-green
ꢁ
1
solid 4a (0.15 g, 40%). Rf ¼ 0.68 (CH
2929, 2869, 1365 (CH , CH ); 1587, 1488 (benzene, CaC); 1175,
128, 1085 (CeOeC); 1336 (CeN); 1265, 1245 (AreOeC); 1697
3
OH). IR (KBr, cm ): 2970,
2
2
.2. Synthesis
3
2
1
þ
.2.1. 3-[2-(4-Boc-piperazine)-1-ethoxy]phthalonitrile (3a)
solution of 1-(2-hydroxyethyl)piperazine (1a) (1.4 ml,
0 mmol) in triethylamine (TEA)/CH Cl (1:5, v/v, 24 ml) was
stirred at room temperature for 10 min, and then di-tert-butyl
dicarbonate (Boc O) (2.3 ml, 11 mmol) was added slowly over
0 min. The mixture was further stirred for 12 h. The volatiles
were removed under reduced pressure. The oily residue was pu-
rified by a silica gel column chromatography using CH OH as
(CaO); 866, 801, 744 (Ar-H). MS(ESI): m/z 1488.9[M] , 1513.7
þ
1
A
[M þ Na] . H NMR (300 MHz, CDCl
3
, ppm):
d
¼ 8.88e9.03 (m,
1
2
2
4H); 7.69e7.82 (m, 8H); 4.78e4.83 (m, 8H); 3.19e3.28 (m, 16H);
2.67e2.86 (m, 24H); 1.33e1.39 (m, 36H). Anal. Calcd for
2
76 96 16
C H N O12Zn: C, 61.22; H, 6.94; N, 15.03. Found: C, 61.56; H,
6.12; N, 15.42.
1
3
2.2.4. 1,8(11),15(18),22(25)-tetrakis-{2-[2-(4-Boc-piperazine)-1-
ethoxy]-ethoxy} phthalocyanine zinc(II) (4b)
According to the above procedure for 4a, a suspension of 3b
(0.20 g, 0.50 mmol) in n-pentanol (10 ml) was treated with anhy-
drous zinc acetate (0.05 g, 0.27 mmol) and DBU (0.4 ml, 2.6 mmol)
eluent to give Boc-protected piperazinyl ethanol (Boc ¼ tert-
butyloxycarbonyl).
Then, a mixture of Boc-protected piperazinyl ethanol (1.15 g,
5
.0 mmol), 3-nitrophthalonitrile (2) (0.87 g, 5.0 mmol), and anhy-
ꢀ
drous K
2
CO
3
(1.38 g, 10 mmol) in DMF (20 ml) was stirred at 40 C
to give a dark-green solid 4b (0.06 g, 33%). Rf ¼ 0.68 (CH
3
OH). IR
ꢁ
3 2
(KBr, cm ): 2970, 2927, 2867, 1365 (CH , CH ); 1588, 1489 (ben-
1
for 48 h. The reaction mixture was poured into ice water (200 ml) to
give light red precipitate, which was collected by filtration, washed
with water until pH 7 and dried in vacuo. The crude product was
zene, CaC); 1122, 1082 (CeOeC); 1335 (CeN); 1265, 1245 (AreOe
C); 1695 (CaO); 866, 801, 745 (Ar-H). MS (ESI): m/z 1668.7
þ
1
purified by recrystallization with DMF/H
dissolved in CHCl , and followed by chromatography on a silica gel
column using CHCl /CH OH (10:1, v/v) as eluent to give a white
2
O. The oily residue was
[M þ H] . H NMR (300 MHz, CDCl
3
, ppm):
d
¼ 8.99e9.11 (m, 4H);
3
7.77e8.17 (m, 8H); 4.16e4.38 (m, 8H); 3.62e3.77 (m, 16H); 3.08 (br,
16H); 2.34 (br, 16H); 2.23 (br, 8H,); 1.19e1.27 (m, 36H). Anal. Calcd
3
3
ꢁ
1
solid 3a (1.24 g, 70%). Rf ¼ 0.62 (ethyl acetate). IR (KBr, cm ): 2229
for C84
112 16
H N O16Zn: C, 60.51; H, 6.77; N, 13.44. Found: C, 61.02; H,
(
C^N); 3077 (Ar-H); 2971, 2869, 1364 (CH
3
); 2928, 2817 (CH
2
);
7.15; N, 13.89.
1
582, 1473, 1455 (benzene, CaC); 1294, 1283, 1246 (CeOeC, CeN);
þ
1
1
123 (AreOeC); 1682.6 (CaO). MS (ESI): m/z 357.1 [M þ H] . H
2.2.5. 1,8(11),15(18),22(25)-tetrakis-(2-piperazine-1-ethoxy)
phthalocyanine zinc(II) (5a)
A solution of phthalocyanine 4a (0.150 g, 0.10 mmol) in CH OH
3
(40 ml) was added with TFA (2.0 ml), and then the mixture was
NMR (300 MHz, CDCl
J ¼ 7.68 Hz, 1H); 7.25 (d, J ¼ 10.4 Hz, 1H); 4.26 (t, J ¼ 5.48 Hz, 2H);
.43 (t, J ¼ 4.88 Hz, 2H); 2.90 (t, J ¼ 5.46 Hz, 2H); 2.56 (t, J ¼ 4.92 Hz,
3
, ppm):
d
¼ 7.64 (t, J ¼ 8.4 Hz, 1H); 7.36 (d,
3

B.-Y. Zheng et al. / Dyes and Pigments 99 (2013) 185e191
187
stirred at room temperature for 30 min. The volatiles were
removed in vacuo, and the oily residue was purified by size-
exclusion chromatography using THF as eluent to give a dark-
performed and the corresponding PO/W value was taken as the
overall average.
ꢁ
1
green solid 5a (0.058 g, 53%). IR (KBr, cm ): 2928, 1365 (CH
487, 1457 (benzene, CaC); 1200, 1131 (CeOeC); 1333 (CeN);
253, 1268 (AreOeC); 832, 800, 746 (Ar-H). MS (ESI): m/z 1113.4
2
);
2.4. Biological studies
1
1
2.4.1. Photosensitizer
þ
1
[M þ Na] . H NMR (300 MHz, DMSO-d
6
, ppm):
H); 7.71e7.78 (m, 8H); 4.90e4.94 (m, 8H); 4.65e4.70 (m, 8H);
.78e3.82 (m, 16H); 3.53e3.58 (m, 16H). Anal. Calcd for
Zn (5a$H O): C, 60.67; H, 6.00; N, 20.22. Found: C,
0.20; H, 6.34; N, 19.88.
d
¼ 8.91e9.00 (m,
Stock solutions of the studied zinc phthalocyanines (1 mM)
ꢀ
4
3
C
6
were prepared in N,N-dimethylformamide (DMF) and stored at 4 C
in the dark. The solution was then diluted to appropriate concen-
tration with PBS (0.01 M, pH 7.4) containing 1% Cremophor EL (1 g,
in 100 ml) before use.
H
56 66
N
16
O
5
2
2.2.6. 1,8(11),15(18),22(25)-tetrakis-[2-(2-piperazine-1-ethoxy)-
2.4.2. Organisms and growth conditions
ethoxy]phthalocyanine zinc(II) (5b)
Candida albicans strain CMCC(F)C1a was purchased from Nanj-
ing Institute of Dermatology, Chinese Academy of Sciences. Candida
cells were plated on Sabouraud dextrose agar and incubated
According to the procedure for 5a, phthalocyanine 4b (0.17 g,
0
.10 mmol) in CH
3
OH (40 ml) was treated with TFA (2.0 ml) at room
ꢀ
temperature for 30 min to give a dark-green solid 5b (80 mg, 63%).
aerobically at 37 C. After 48 h of incubation, a sample of colonies
ꢁ
1
IR (KBr, cm ): 2924 (CH
2
); 1487,1477 (benzene, CaC); 1271 (CeOe
was removed from the surface of the agar plate and suspended in
sterile PBS (0.01 M, pH 7.4). Cells were harvested by centrifugation,
and washed with PBS, and re-suspended in PBS to appropriate cell
density prior to the experiment.
C); 1326 (CeN); 1271 (AreOeC); 3268, 832, 800, 746 (Ar-H); 3424
ꢁ
1
(
tertiary amine). MS (ESI): m/z 1302.3 [M þ Cl] . H NMR (300 MHz,
DMSO-d , ppm):
¼ 8.99e9.11 (m, 4H); 7.77e8.17 (m, 8H); 4.16e
.38 (m, 8H); 3.62e3.77 (m,16H); 3.08 (br,16H); 2.34 (br,16H); 2.23
br, 8H); 1.19e1.27(m, 4H). Anal. Calcd for Zn
5b$H O): C, 59.83; H, 6.43; N, 17.44. Found: C, 59.87; H, 6.60; N,
6
d
4
(
(
C
64
H
82
N
16
O
9
2.4.3. Photosensitizer uptake by Candida cells
7
2
Suspensions of C. albicans (0.8 ml, about 2.5 ꢂ 10 cells/ml) in
ꢀ
17.92.
PBS were incubated in dark at 37 C with 0.1 mM of phthalocya-
nines for 3 h. The cultures were centrifuged (8000 rpm for 10 min)
and the cell pellets were washed with twice PBS, and then re-
suspended in 0.8 ml DMF and sonicated for 10 min. The absorp-
tion spectrum of the supernatant was measured. The photosensi-
tizer concentration was calculated from the Q-band absorbance in
this diluted DMF reference to the corresponding molar absorptivity
(see Table 1). Each experiment was repeated 3 times.
2
.2.7. 1-[2-piperazine-1-ethoxy] phthalocyanine zinc(II) (7)
A mixture of phthalonitrile 3a (0.18 g, 0.50 mmol) and unsub-
stituted phthalonitrile (6) (0.32 g, 2.5 mmol) in n-pentanol (20 ml)
was stirred at 70 C for 10 min, and then anhydrous zinc acetate
ꢀ
(
0.30 g, 1.66 mmol) and DBU (0.4 ml, 2.6 mmol) were added. The
ꢀ
resulting mixture was stirred at 130 C for 12 h. After removing the
3
volatiles in vacuo, the residue was dissolved in CH OH (30 ml), and
then treated with TFA (30 ml) at room temperature for 30 min. The
reaction mixture was poured into ice water (200 ml) to give green
precipitate, which was collected by filtration, washed with water
until pH 7 and dried in vacuo. The oily residue was purified by a
2.4.4. In vitro photodynamic inaction of Candida cells in PBS
Candida cellular suspensions were incubated with PBS solution
containing different concentrations of photosensitizer in a 96-well
6
plate (0.2 ml/well, 2 ꢂ 10 cells/ml), in the dark for 3 h at room
silica gel column chromatography using CHCl
3
/CH
3
OH (5:1, v/v) as
temperature. After that, the cultures were exposed to visible light
for 30 min. The light source consisted of a 500 W halogen lamp, a
eluent to give a blueegreen crude, which was further purified by
size-exclusion chromatography using THF as eluent to give a bluee
water tank for cooling and a color glass filter cut-on 610 nm. The
ꢁ
1
ꢁ2
green solid 7 (0.019 g, 5%). IR (KBr, cm ): 2957, 2925, 2856, 1365
CH ); 1608, 1588 (benzene, CaC); 1165, 1092 (CeOeC); 1333 (Ce
N); 1259 (AreOeC); 3056, 885, 800, 772, 751, 736 (Ar-H). MS (ESI):
fluence rate (
l
> 610 nm) was 15 mW cm . An illumination of
ꢁ
2
(
2
30 min led to a total fluence of 27 J cm . In parallel, three control
experiments were designed as follows: (1) with photosensitizer,
but non-illuminated, (2) without photosensitizer, but illuminated,
(3) no photosensitizer, non-illuminated. Control and irradiated cell
suspensions were serially diluted (100-fold) with PBS, and each
solution was plated in triplicate onto Sabouraud dextrose agar.
ꢁ
1
m/z 703.1 [M ꢁ H] . H NMR (300 MHz, DMSO-d
6
, ppm):
.41 (m, 6H); 9.01e9.03 (m, 1H); 8.23e8.25 (m, 8H); 6.68 (s, 1H);
.31e5.33 (m, 2H); 4.68e4.69 (m, 2H); 3.32e3.43 (m, 8H). Anal.
10OZn: C, 64.64; H, 4.00; N, 19.84. Found: C, 64.19;
d
¼ 9.39e
9
5
Calcd for C38
28
H N
ꢀ
H, 4.42; N, 20.21.
After incubation of the plates at 37 C for 48 h, the number of
colonies on each plate was counted. The survival percentage was
determined by the comparing the number of colony-forming units
with the control (no photosensitizer, non-illuminated). Each
experiment was repeated separately 3 times.
2
.3. Oil/water partition coefficients
The oil/water partition coefficients were determined according
to the procedure reported by Dei et al. with minor modification
[
(
23]. The n-octanol (0.5 ml) and phosphate buffered saline (PBS)
0.5 ml) was mixed with vPBS/voctanol (1:1) in a tube, and then about
2.4.5. Growth delay of C. albicans
The growth delay of C. albicans caused by photodynamic actions
was determined according to the literature procedure [18]. Sus-
20 ml of phthalocyanine solution (1 mM in DMF) was added into it.
6
The tube was shaken for 10 min. After that, the separation of the
two phases was enabled by centrifugation and the aqueous solution
was sampled by using a procedure that minimizes the risk of
including traces of n-octanol. The phthalocyanine concentration
was calculated from the Q-band absorbance in a diluted DMF so-
lution with reference to the corresponding molar absorptivity (see
Table 1). The distribution coefficient was then determined by the
ratio of phthalocyanine concentration in octanol phase to that in
PBS phase. At least three independent measurements were
pensions of C. albicans (1 ml, about 2 ꢂ 10 cells/ml) in PBS was
diluted to 20 ml of fresh Sabouraud broth medium. The suspension
was homogenized and aliquots of 0.2 ml were incubated with
ꢀ
25
m
M of phthalocyanine 7 in flasks at 37 C. The flasks were then
ꢁ
2
irradiated with red light (
l
> 610 nm, 15 mW cm ) for 30 min, as
described above. The culture grown was measured by turbidity at
492 nm using a MK3 multiskan ascent (Thermo). This wavelength
was chosen to avoid the interference of the absorption of phtha-
locyanines in the range of 300e400 nm and 610e700 nm. In all

188
B.-Y. Zheng et al. / Dyes and Pigments 99 (2013) 185e191
cases, control experiments were carried out without illumination in
the absence and in the presence of sensitizers. Each experiment
was repeated separately 3 times.
followed by N-Boc-deprotection to afford the targeted phthalo-
cyanine 7.
All the phthalocyanines have good solubility in common organic
solvents, such as DMF, tetrahydrofuran (THF), ethyl acetate, and
1
ethanol. These new compounds were characterized by MS, H NMR,
3
. Results and discussion
and FT-IR spectroscopic methods together with elemental analysis.
3.1. Synthesis
3.2. Photophysical and photochemical properties
Scheme 1 shows the synthetic pathway of tetra-substituted
phthalocyanines 4aeb and 5aeb. Firstly, the secondary amines
on the piperazinyl group of compounds 1a and 1b were protected
with tert-butoxycarbonyl (Boc) group by treating the compound 1a
or 1b with di-tert-butyl dicarbonate in the presence of TEA in
The spectroscopic properties of the phthalocyanines 4a, 4b, 5a,
5b, and 7 were measured in DMF, and the data are summarized in
Table 1. The absorption spectra of all compounds in DMF are typical
for non-aggregated phthalocyanines, exhibiting an intense and
sharp Q-band at 678e700 nm. Fig. S1 (in supporting information)
shows the UVevis spectra of 7 in DMF at various concentrations
given as an example. The Q-band strictly followed the Lamberte
Beer law, indicating that it is essentially free from aggregation in
DMF. Compared with unsubstituted ZnPc, both of the mono- and
tetra-substituted phthalocyanines have red-shift Q-band (670 nm
vs. 678e700 nm). Furthermore, the Q-band absorption of tetra-
substituted phthalocyanines 4a, 4b, 5a, and 5b (699e700 nm)
shows obviously red-shifted compared with that of mono-
substituted analog 7 (678 nm). It has been reported that substitu-
2 2
CH Cl . The N-Boc-protected compounds then underwent nucleo-
philic substitution with 3-nitrophthalonitrile under alkaline con-
dition to give the corresponding 3-substituted phthalonitriles 3a
and 3b. The cyclotetramerization of 3a or 3b in the presence of zinc
acetate and organic base DBU in n-pentanol afforded the N-Boc-
protected tetra-substituted phthalocyanines 4a and 4b in 40% and
3
3% yield, respectively. Subsequently, N-Boc-deprotection of these
compounds using TFA gave phthalocyanines 5a and 5b,
respectively.
To demonstrate the effect of the number of substitutes, the
mono-substituted zinc(II) phthalocyanine 7 was also prepared
using a synthetic route as shown in Scheme 2. The mixed cycli-
zation of N-Boc-protected phthalonitrile 3a with an excess of
unsubstituted phthalonitrile (6) in the presence of zinc acetate and
DBU afforded the N-Boc-protected phthalocyanine, which was
tion at
a position can lead to reduction of the HOMOeLUMO gap,
which is consistent with the result of red-shift of these
a
substituted compounds [29,30]. Upon excitation at 610 nm, the
tetra-substituted compounds showed fluorescence emission at
707e709 nm with a Stokes shift of ca. 9 nm. Their fluorescence
Scheme 1. Synthesis of tetra-substituted zinc(II) phthalocyanines 4e5a and 4e5b.
Scheme 2. Synthesis of mono-substituted phthalocyanine 7.

B.-Y. Zheng et al. / Dyes and Pigments 99 (2013) 185e191
189
Table 1
Photophysical and photochemical data for phthalocyanines in DMF.
0
.6
[
7] (µM)
5
b
c
0.6
Phthalocyanines
l
max
l
em
Stokes
shift (nm)
ε ꢂ 10
F
F
F
D
a
ꢁ1
ꢁ1
)
0.4
0.2
0.0
6
5
4
3
2
1
(
nm)
(nm)
(M cm
4
4
5
5
7
a
b
a
b
700
699
700
700
678
709
707
709
708
680
9
8
9
8
2
1.73
1.76
1.08
1.14
1.77
0.12
0.13
0.11
0.11
0.27
0.64
0.77
0.56
0.69
0.54
0.4
0
2
4
6
[7] (µM)
0.2
a
Excited at 610 nm.
b
Using unsubstituted zinc(II) phthalocyanine (ZnPc) in DMF as the reference
¼ 0.28) [31].
(F
F
0.0
300
c
Determined using DPBF as chemical quencher, and using ZnPc in DMF as the
400
500
600
700
800
reference (
F
D
¼ 0.56) [32,33].
Wavelength (nm)
Fig. 2. UVevis spectra of phthalocyanine 7 at different concentrations in H
2
O con-
taining 1% Cremophor EL. The inset shows the plot of the Q-band absorbance against
the concentration.
quantum yields (
ZnPc (
¼ 0.28) [31]. For the mono-substituted analog 7, the
fluorescence emission was at 680 nm with a comparable fluores-
cence quantum yield (
¼ 0.27) with that of ZnPc. This is in accord
with the general observation that the lower the energy of the Q-
band, the smaller the value [29,30].
The singlet oxygen quantum yields (
were also determined in DMF by a steady-state method by using
,3-diphenylisobenzofuran (DPBF) as the scavenger [32,33]. As
shown in Table 1, all the compounds are efficient singlet oxygen
generators with of 0.54e0.77, which suggests that the singlet
F
F
¼ 0.11e0.13) are obviously lower than that of
F
F
formulation vehicle of hydrophobic drugs [35], the Q-band is
basically intense and sharp similar to that in DMF. Similar results
were observed for the other counterparts 4a, 4b, and 5b. Further-
more, the absorbance at different concentrations was also
measured in the aqueous (Fig. 2, 7 is given as an example). The
absorption well obeys the LamberteBeer law, suggesting that ag-
gregation is not significant for these phthalocyanines when
formulated with Cremophor EL in the aqueous solution.
F
F
F
F
D
F ) of these compounds
1
F
D
oxygen generation efficiency of zinc(II) phthalocyanines are not
quenched significantly by the amine of the piperazinyl moiety.
However, it has been reported that, for silicon(IV) phthalocyanines,
both their fluorescence and singlet oxygen generation efficiency are
badly quenched by amino moieties through photoinduced electron
effect (PET), which leads to the obvious reduction of photodynamic
activities of these compounds [27,34]. On the other hand, the
phthalocyanines 4b and 5b containing longer ethoxy chain be-
tween macrocycle and piperazinyl moiety can generate singlet
oxygen more efficiently than their counterparts 4a and 5a,
3.3. In vitro antifungal studies
The in vitro photodynamic antifungal activities of these com-
pounds 4a, 4b, 5a, 5b, and 7 formulated with Cremophor EL were
firstly investigated against C. albicans. The cells were incubated with
these photosensitizers at different concentrations in the dark at
room temperature for 3 h. After that, the cells were irradiated with
red light, and then the viable cells were determined. Fig. 3 shows the
dose-dependent survival curves in the absence and presence of
light. It is clear that all the compounds are essentially noncytotoxic
in dark. Their curves in dark are similar with that of the control, in
which the culture was incubated without sensitizer just illuminated
with the red light. In the presence of light, the photocytotoxicities of
these compounds follow the trend: 7 > 5a > 5b > 4a z 4b. The N-
Boc-protected 4a and 4b are basically noncytotoxic against the cells
respectively (
for 5a).
F
D
¼ 0.77 for 4b vs.0.64 for 4a, and 0.69 for 5b vs. 0.56
Aggregation behavior of photosensitizers will reduce their
photosensitizing efficiencies. Therefore, the aggregation trends of
these compounds were also examined in aqueous solutions by
absorption spectroscopic method. Fig. 1 shows the UVevis spectra
of tetra-substituted 5a and mono-substituted 7 as examples. They
show very broad and weak Q-band at approximately 705 nm and
until 100
mM upon illumination (Fig. 3a). For the tetra-substituted 5a
and 5b, the compound 5a containing shorter ethoxy chain shows
higher photocytotoxicity than the analog 5b. For all the compounds,
the mono-substituted 7 presents the highest photocytotoxicity with
6
85 nm in water with 0.5% DMF, which means they are significantly
aggregated in water. However, in the presence of a tiny amount of
Cremophor EL, which is a non-ionic surfactant usually used as a
an IC90 value as low as 9 mM.
(a)
0
0
0
0
.6
.4
.2
.0
(b) 0
1
% CEL
1% CEL
DMF
.6
DMF
H O
H O
0
.4
.2
0
0.0
300
3
00
400
500
600
700
800
400
500
600
700
800
Wavelength(nm)
Wavelength (nm)
Fig. 1. UVevis spectra of (a) 5a and (b) 7 in 1% CEL (—), DMF (- -), and water (with 0.5% DMF) (e) (All at 4
mM). 1% CEL: 1% Cremophor EL in water (1 g in 100 mL of water).

190
B.-Y. Zheng et al. / Dyes and Pigments 99 (2013) 185e191
(a) 100
(b) 100
1
0
Control
4
awith light
5awith light
5ain dark
5bwith light
5bin dark
1
10
4ain dark
4
bwith light
bin dark
4
0.1
.01
7
with light
in dark
7
0
1
1
1
E-3
E-4
0.1
0
20
40
60
80
100
0
20
[
40
60
80
100
[Photosensitizer] (µM)
Photosensitizer] (µM)
Fig. 3. Photocytotoxic effects of (a) 4aeb, and (b) 5aeb and 7 on C. albicans in the absence and presence of red light (
l
> 610 nm) at a dose of 27 J cmꢁ2. Control means the culture
was incubated without sensitizer and kept illumination as above. Values represent as the mean ꢃ standard deviation of three separate experiments. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
a much higher photocytotoxicity against C. albicans than MB, sug-
gesting that 7 is a potential photosensitizer for antifungal photo-
dynamic therapy.
The photodynamic antifungal activity of 7 can also be observed
from Fig. 4. The C. albicans cells were virtually killed for the dish
incubated with 7 (50 mM) for 3 h upon illumination, while for the
dish without 7, a large amount of C. albicans was found.
To ensure that photodynamic inactivation of fungal cells is still
maintained when the cultures were not under starvation condi-
tions, growth delay of C. albicans sensitized by phthalocyanine 7
was further carried out in Sabouraud medium. The cell growth of
the C. albicans was examined under four conditions, containing in
the absence or presence of phthalocyanine 7 (25 mM), and with or
without illumination. As shown in Fig. 5, when the C. albicans
cultures were treated with phthalocyanine 7 upon illumination, cell
growth of the C. albicans can be suppressed efficiently in the
following 16 h. On the other hand, the C. albicans cells incubated
with phthalocyanine 7 in dark shows no significant growth delay,
which is comparable with the controls. The result indicates that the
observed growth delay can be ascribed to the photodynamic effect
of the sensitizers on the cells.
To account for the different photocytotoxic results of these
compounds, the cellular uptake was also investigated by an
extraction method. As shown in Table 2, the cellular uptake of these
compounds follows the trend 7 > 5a z 5b > 4a z 4b. The cellular
uptake of 7 is significantly higher than all the other compounds.
The compounds 5a and 5b present higher cellular uptake compared
to the corresponding N-Boc-protected 4a and 4b. Therefore, the
trend in cellular uptake of these phthalocyanines is generally in
accord with the observed result of photoactivity toward Candida
cells (7 > 5a > 5b > 4a z 4b). This indicates that the different
photodynamic activity against C. albicans of these compounds is
largely attributed to the differences in cellular uptake.
Fig. 4. The photodynamic activity of phthalocyanine 7 against C. albicans. (a) control
(
(
without 7) (b) incubation with 7 (50 mM) for 3 h, followed by irradiation with red light
ꢁ
2
l
> 610 nm) at a dose of 27 J cm
.
Methylene Blue (MB), a known antifungal photosensitizer
11,12], has been selected as a positive control in this assay. Under
the same experimental condition, the IC90 value of MB against
C. albicans cells was found to be ca. 500 M, which is about 50 times
higher than that of phthalocyanine 7. Clearly, compound 7 exhibits
[
m
0
0
0
0
0
.8
.6
.4
.2
.0
Control with light
with light
Control in dark
in dark
7
7
To understand further the different cellular uptakes of these
compounds, we examined their hydrophilicehydrophobic charac-
teristics by measuring the octanol/PBS partition coefficient (PO/W).
TheLog PO/W values weresummarizedinTable 2. TheLog PO/W values
follow the order: 4a > 4b > 7 > 5a > 5b. Compared to Boc-protected
0
2
4
6
8
10
12
14
16
18
Table 2
Partition coefficients and cellular uptakes of phthalocyanines.
Time (h)
Phthalocyanine 4a
4b
5a
5b
7
Fig. 5. Growth delay curves of C. albicans cells incubated with phthalocyanine 7
Log PO/W
Uptake (%)
2.05
1.81
1.34
0.68
1.77
ꢁ
2
(
25
mM) upon illumination with red light (>610 nm) at a dose of 27 J cm . Control
a
0.78 ꢃ 0.03 0.94 ꢃ 0.03 1.47 ꢃ 0.16 1.85 ꢃ 0.28 4.54 ꢃ 0.14
cultures were incubated without sensitizer in the absence or presence of light. Values
represent as the mean ꢃ standard deviation of three independent experiments. (For
interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
a
Percentages of cellular uptake of phthalocyanines by C. albicans after 3 h incu-
bation. Values represent as the mean ꢃ standard deviation of three independent
experiments.

B.-Y. Zheng et al. / Dyes and Pigments 99 (2013) 185e191
191
compounds 4a and 4b, the corresponding Boc-deprotected analogs
a and 5b show lower Log PO/W values, which can be due to the
[10] Jori G, Fabris C, Soncin M, Ferro S, Coppellotti O, Dei D, et al. Photodynamic
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5
lipophilic t-butyl moieties of the Boc groups on the 4a and 4b. The
phthalocyanines 4b and 5b substituted with longer ethoxy chains
have lower Log PO/W value relative to their analogs 4a and 5a,
respectively, which indicates that the elongation of ethoxy chain can
enhance the hydrophilicity. The Log PO/W value of the mono-
substituted phthalocyanine 7 is higher than the corresponding
tetra-substituted analog 5a. Compared with the other compounds,
the mono-substituted phthalocyanine 7 shows a better amphiphi-
licity. This may account for its higher cellular uptake by C. albicans
than the other compounds.
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We have prepared and characterized a series of zinc(II) phtha-
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The structureeactivity relationship of these phthalocyanines has
been discussed by combination of their photophysical and photo-
chemical properties, cellular uptakes and in vitro photodynamic
antifungal activities. The mono-substituted phthalocyanine 7 pre-
sents the highest photodynamic activity against C. albicans as a
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value of 7 is down to 9 mM. The results indicate that the mono-
substituted zinc(II) phthalocyanine 7 is a promising photosensi-
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sensitisers. Anticancer Agents Med Chem 2008;8(3):280e91.
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Acknowledgments
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4829e35.
This work was supported by the Natural Science Foundation of
China (Grant Nos. 20872016, 21172037), the Natural Science
Foundation of Fujian, China (Grant No. 2011J01040), and Special-
ized Research Fund for the Doctoral Program of Higher Education
[25] So CW, Tsang PW, Lo PC, Seneviratne CJ, Samaranayake LP, Fong WP. Photo-
(
Grant No. 201135141001).
dynamic inactivation of Candida albicans by BAM-SiPc. Mycoses 2009;53:
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Appendix A. Supplementary data
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therapy with Pc4 induces apoptosis of Candida albicans. Photochem Photobiol
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Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.dyepig.2013.04.032.
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