Synthesis of unsymmetrical phthalocyanine derivatives and their interaction with mammary MCF7 cells

Article abstract of DOI:10.1016/j.dyepig.2013.05.012

We report herein the synthesis and characterization of a group of unsymmetrical phthalocyanines modified with different combinations of hydrophobic and hydrophilic groups. Our results demonstrated that their solubility in polar media varies with the proportion of hydroxylated groups present in each structure and that the cell interactions and generation of reactive oxygen species in aqueous media change according to the hydrophobic to hydrophilic balance in the macrocycle.

Full text of DOI:10.1016/j.dyepig.2013.05.012

Accepted Manuscript
Synthesis of unsymmetrical phthalocyanine derivatives and their interaction with
mammary MCF7 cells
F.B. do Nascimento, T.M. Manieri, G. Cerchiaro, A.O. Ribeiro
PII:
S0143-7208(13)00177-0
10.1016/j.dyepig.2013.05.012
DYPI 3949
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 5 January 2013
Revised Date: 9 May 2013
Accepted Date: 14 May 2013
Please cite this article as: do Nascimento FB, Manieri TM, Cerchiaro G, Ribeiro AO, Synthesis of
unsymmetrical phthalocyanine derivatives and their interaction with mammary MCF7 cells, Dyes and
Pigments (2013), doi: 10.1016/j.dyepig.2013.05.012.
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ACCEPTED MANUSCRIPT
Research Highlights
1. The phthalocyanines water solubility is related to the proportion of free hydroxyl groups.
2. The cell interaction changes according to the hydrophobic to hydrophilic balance.
3. Unprotected derivatives presented a greater tendency to aggregate but higher cell interaction.

ACCEPTED MANUSCRIPT
Synthesis of unsymmetrical phthalocyanine derivatives and their interaction with
mammary MCF7 cells
F. B. do Nascimento, T. M. Manieri, G. Cerchiaro, A. O. Ribeiro*
Center for Natural Sciences and Humanities, Federal University of ABC – UFABC
Avenida dos Estados, 5001, Bloco B, Santo André, SP, Brazil.
*anderson.ribeiro@ufabc.edu.br
RECEIVED DATE (will be automatically inserted after manuscript is accepted).
Abstract
We report herein the synthesis and characterization of a group of unsymmetrical phthalocyanines modified
with different combinations of hydrophobic and hydrophilic groups. Our results demonstrated that their
solubility in polar media varies with the proportion of hydroxylated groups present in each structure and that
the cell interactions and generation of reactive oxygen species in aqueous media change according to the
hydrophobic to hydrophilic balance in the macrocycle.
Keywords: phthalocyanines, synthesis, photodynamic therapy, cell interaction, glycerol
1. Introduction
Photodynamic therapy (PDT) is a technique that combines a photosensitizer, oxygen and light to treat
diseases such as cancer, acne, and psoriasis [1].This treatment uses a photosensitizer that is nontoxic in the
dark to focus the therapeutic action to only those regions irradiated by light [2, 3].
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Of the approved and studied photosensitizers for this treatment [4], phthalocyanine derivatives are the most
promising due to their high molar extinction coefficient in the 600–800 nm region and their high singlet
oxygen quantum yields under irradiation [5, 6]. However, the primary limitation for their use is their low
solubility in water and physiological fluids due to their tendency to aggregate in aqueous media [7].
Recently, various strategies to obtain phthalocyanine derivatives with better solubility in aqueous media have
been reported. These methods primarily involve the incorporation of cationic [8] and anionic [9, 10] groups,
peptides [11], β-cyclodextrins [12, 13] or crown ethers [14] in the phthalocyanine ring to diminish the
aggregation of the compound via either spatial conformation or columbic forces.
Herein, we present a photochemical and photophysical study in polar media of a group of unsymmetrical
phthalocyanines bearing polyhydroxyl moieties, as well as their interaction with mammary MCF7 cells. The
properties of unsymmetrical phthalocyanine structures have been explored in the literature [15], with some
examples of carbohydrate substituted phthalocyanines studied for PDT applications [16]. But only a few
works present the synthesis, characterization and biological studies of phthalocyanines with different
proportions of polyhydroxylated groups in their macrocycle structure [17]. Also, some studies involving
porphyrin and chlorin derivatives have reported that the combination of hydrophobic and hydrophilic groups
within the same molecule results in improved intramolecular polarity, which can facilitate cell membrane
penetration [18]. The primary goal with the phthalocyanine derivatives studied here is to compare the cell
interactions of related macrocycles substituted with different proportions of protected and unprotected
hydroxyl groups (-OH) which promotes change in polarity.
2. Experimental Section.
2.1 General considerations
The 1,2-dicyanobenzene (ii) and 4-methyl-1,2-dicyanobenzene (iii) were purchase from Aldrich and used
without purification. Solketal was synthesized as described in literature [19] and the structure confirmed by
1H NMR. When necessary, solvents were purified following standard literature procedures. Some reactions
1
were carried out under nitrogen atmosphere as specified in the experimental procedures. In H NMR spectra
(500 MHz), CDCl3, DMSO-d6 solventes and tetramethylsilane were used. The chemical shifts are expressed
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in
δ
(ppm). Flash chromatography was carried out by using silica gel (230-400 mesh). Analytical TLC was
carried out on precoated sheets with silica gel (0.2 mm thick). Mass spectra were recorded by HMRS-
MALDI-TOF (Bruker-Daltonics, USA).
2.2. Synthesis of 4-solketalphthalonitrile (i)
Solketal (102 g, 7.75 mmol) and 4-nitrophthalonitrile (1.34 g, 7.75 mmol) were stirred in a three-necked flask
for three days in DMF (6 mL) at room temperature in the presence of an excess of potassium carbonate (7 g).
The mixture was poured in ice water, extracted with dichloromethane (DCM) (3 x 30 mL) and washed with
distilled water (3 x 50 mL). The organic phase was dried over anhydrous magnesium sulfate anhydrous,
filtered and concentrated under reduced pressure. The product was purified by column chromatography (silica
gel: hexane-ethyl acetate 1:1 v / v) resulting in a yellow solid which was finally purified by recrystallization
with methanol, yielding a white solid, 67% yield. FTIR (KBr) umax/cm-1 3072 (Ar–CH, w), 2994, 2062, 2890
(–CH, –CH2, and –CH3, w), 2231 (–C≡N, w), 1600, 1498 (-C=C-, s), 1255, 1033 (C-O-C). 1H NMR (CDCl3):
δ
(ppm) 7.73 (d, J=8.8 Hz, 1H), 7.32 (d, J=2.7 Hz, 1H), 7.25 – 7.22 (dd, J=8.8 Hz and J=2.7 Hz, 1H), 4.50
(m, 1H), 4.19 (m, 1H), 4.09 (m, 2H), 3.89 (m, 1H), 1.45 (s, 3H, CH3), 1.37 (s, 3H, CH3). HRMS-MALDI-
TOF m/z calcd for C14H14N2O3 (M+Na)+ 281.0902, found 281.0896.
2.3. Synthesis of symmetrical phthalocyanines 1 and 2
2.3.1. Synthesis of [tetra(solketal)phthalocyaninate]zinc(II) (1)
In a closed reaction tube, 4-solketalphthalonitrile (i, 800 mg, 3.09 mmol) and zinc acetate (339 mg, 1.54
mmol) were heated under reflux for 10 h in 5 mL of N,N-Dimethylethanolamine , under nitrogen atmosphere,
with constant stirring. After cooling, the reaction mixture was concentrated under reduced pressure and the
dark blue crude product was purified by column chromatography (silica gel: gradient elution, hexane–ethyl
acetate (2:1) to pure ethyl acetate), giving the compound (1) in 72% yield (667 mg; 0.557 mmol). mp > 300
°C. FTIR (KBr) umax/cm-1 3066 (Ar–CH, w), 2983, 2933, 2873 (–CH, –CH2, and –CH3, w), 1733 (–N=C–, s),
1606, 1488 (-C=C-, s), 1236, 1040 (C-O-C). UV-Vis (DMSO), λmax, (log ε): 358 (4.72), 613 (4.36), 682
(5.05). 1H NMR (CDCl3):
δ
7.73 (d, J=8.4 Hz, 4H), 7.31 (d, J=2.2 Hz, 4H), 7.21 (dd, J=8.4 Hz and J=2.2 Hz
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4H), 4.50 – 4.48 (m, 4H), 4.19 – 4.11 (m, 8H), 4.07 – 4.04 (m, 4H), 3.92 – 3.89 (m, 4H), 1.46 (s, 12H, -CH3),
1.41 (s, 12H -CH3). HRMS-MALDI-TOF m/z calcd for C56H56N8O12Zn+ (M)+ 1096.3310, found 1096.3321.
2.3.2. Synthesis of [tetra(glyceryl)phthalocyaninate]zinc(II) (2)
Compound 1 (200 mg, 0.418 mmol) was stirred overnight in excess of acetic acid 80% solution. The reaction
mixture was then evaporated to dryness under reduced pressure and the dark blue powder was washed
successively with ethyl acetate, hexane, dichloromethane and chloroform to yield the compound 2 as a dark
blue powder with 94.,4 % yield (147.7 mg ; 0.157 mmol). mp > 300 °C. FTIR (KBr) υmax/cm-1 3367 (–OH, s)
3068 (Ar–CH, w), 2937, 2881 (–CH and –CH2, w), 1733 (–N=C–, s), 1616, 1490 (-C=C-, s), 1228, 1041 (C-
1
O-C). UV-Vis (DMSO), λmax, (log ε): 358 (4.89), 614 (4.54), 682 (5.23). H NMR (DMSO-d6):
δ 9.13 –
9.04 (m, 3H), 8.73 – 8.62 (m, 3H), 7.73 – 7.66 (m, 4H), 7.35 – 7.28 (m, 2H), 5.33 – 5.18 (m, 3H), 5.10 – 4.88
(m, 5H), 4.65 – 4.58 (m, 3H), 4.56 – 4.45 (m, 3H), 4.21 – 3.98 (m, 6H), 3.85 – 3.68 (m, 8H, -OH). HRMS-
MALDI-TOF m/z calcd for C44H40N8O12Zn+ (M)+ 936.2057, found 936.2052.
2.4. Synthesis of Non symmetrical (A3B) phthalocyanines (3), (4), (5) and (6)
2.4.1. Synthesis of [tris-2,9(10),16(17)-solketal-phthalocyaninate]zinc(II) (3): i (1.81 g, 702 mmol) and ii (
300 mg, 2.34 mmol) was heated under reflux in N,N-dimethylethanolamine (6 mL), under a nitrogen
atmosphere, for 12h, in the presence of excess of zinc acetate. After reaction, the solution was cooled and the
product was precipitated and washed with water/methanol (10:1), and dried under vacuum. The compound
was purified by column chromatography (silica gel, toluene/THF gradient 95/5 to 50/50) as a green solid in
8% yield (181 mg; 0.187 mmol). mp > 300 °C. FTIR (KBr)υmax/cm-1 3062 (Ar–CH, w), 2979, 2923, 2852 (–
CH, –CH2 and –CH3, w), 1608, 1488 (-C=C-, s), 1340 (-C=N-), 1226, 1040 (C-O-C). UV-Vis (DMSO),
1
λmax, (log ε): 356 (4.71), 612 (4.38), 680 (5.09). H NMR (CDCl3):
δ 7.78 – 7.70 (m, 4H), 7.27 - 7.22 (m,
9H), 4.57 – 4.50 (m, 3H), 4.23 – 4.13 (m, 6H), 4.11 – 4.05 (m, 3H), 3.96 – 3.89 (m, 3H), 1.48 (s, 9H, -CH3),
1.42 (s, 9 H, -CH3). HRMS-MALDI-TOF m/z calcd for C50H46N8O9Zn+ (M)+ 966.2679, found 966.2679.
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Synthesis of [tris-2,9(10),16(17)-glyceryl-phthalocyaninate]zinc(II) (4): Compound (3) (80 mg, 0.0827 mmol)
was stirred overnight in excess of acetic acid 80% solution. The purification procedure was treated under the
same conditions used for compound (2). Yield 87.5% (181 mg; 0.187 mmol). mp > 300 °C. FTIR
(KBr)υmax/cm-1 3421 (–OH, s), 3063 (Ar–CH, w), 2981, 2931 (–CH and –CH2, w), 1731 (–N=C–, s), 1606,
1486 (-C=C-, s), 1333, 1289 (-C=N-), 1232, 1054 (C-O-C). UV-Vis (DMSO), λmax, (log ε): 357 (4.79), 612
(4.46), 680 (5.17). 1H NMR (DMSO-d6):
δ 7.73- 7.65 (m, 4H), 7.35 - 7.26 (m, 9 H), 5.10 – 4.98 (m, 3H), 4.76
– 4.66 (m, 3H), 4.20 – 4.02 (m, 6H), 3.84 – 3.80 (m, 3H), 3.54 – 3,38 (m, 6H, -OH). HRMS-MALDI-TOF
m/z calcd for C41H34N8O9Zn+ (M)+ 846.1740, found 846.1743.
Synthesis of [tris-2,9(10),16(17) solketal-23(24)-mono(methyl)phthalocyaninate]zinc(II) (5): i (1.63 g, 6.33
mmol) and iii (300 mg, 2.11 mmol) was treated under the same conditions adopted for compound (3). Yield
13% (269 mg ; 0.270 mmol). mp > 300 °C. FTIR (KBr)υmax/cm-1 3062 (Ar–CH, w), 2923, 2857 (–CH, –CH2,
and –CH3, w), 1608, 1465 (-C=C-, s), 1378, 1330 (-C=N-, -C–N=), 1240 (C-O-C). UV-Vis (DMSO), λmax,
(log ε): 356 (4.50), 613 (4.16), 681 (4.87). 1H NMR (CDCl3):
δ 7.78 - 7.69 (m, 4H), 7.56 - 7.47 (m, 1H), 7.33
- 7.22 (m, 4H), 7.21 - 7.13 (m, 3H), 4.53 - 4.45 (m, 3H), 4.20 - 4.15 (m, 3H), 4.13 - 4.08 (m, 3H), 4.07 - 4.02
(m, 3H), 3.96 - 3.82 (m, 3H), 2.52 - 2.42 (m, 3H, Ar-CH3), 1.46 (s, 9H, -CH3), 1.40 (s, 9H, -CH3). HRMS-
MALDI-TOF m/z calcd for C51H48N8O9Zn+ (M)+ 980.2836, found 980.2834.
Synthesis of [tris-2,9(10),16(17)- glyceryl-23(24)-mono(methyl)phthalocyaninate]zinc(II) (6): Compound (5)
(130 mg, 0.132 mmol) was stirred overnight in excess of acetic acid 80% solution. The purification procedure
was the same used for compound (2). Yield 80.7% (141 mg ; 0.164 mmol). mp > 300 °C. FTIR
(KBr)υmax/cm-1 3421 (–OH, s), 3004 (Ar–CH, w), 2917 (–CH, –CH2 and –CH3, w), 1648, 1485 (-C=C-,w),
1
1333 (-C=N-), 1236, 1022 (C-O-C). UV-Vis (DMSO), λmax, (log ε): 356 (4.66), 613 (4.32), 681 (5.03). H
NMR (DMSO-d6):
δ 7.73 – 7.66 (m, 4H), 7.34 - 7.26 (m, 8H), 5.11 - 5.01 (m, 3H), 4.83 - 4.64 (m, 3H), 4.22 -
4.15 (m, 3H), 4.08 - 4.01 (m, 3H), 3.85 - 3.79 (m, 3H), 3.50 - 3.42 (m, 6H, -OH), 2.51 (s, 3H, Ar-CH3).
HRMS-MALDI-TOF m/z calcd for C42H36N8O9Zn+ (M)+ 860.1897, found 860.1905.
2.5. Unsymmetrical (AB3) phthalocyanines (7), (8), (9) and (10)
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2.5.1. Synthesis of [mono(solketal)phthalocyaninate]zinc(II) 7: i (300 mg, 1.16 mmol) and ii (0.446 g, 3.48
mmol) was heated under reflux in N,N-dimethylethanolamine (6 mL), under a nitrogen atmosphere, for 12h,
in the presence of excess of zinc acetate. After that, the solution was cooled and the product was precipitated
and washed with water/methanol (10:1) and dried under vacuum. The compound was purified by column
chromatography (silica gel, toluene/THF) to yield 7 as a green solid in 12% yield (98 mg ; 0.139 mmol). mp >
300 °C. FTIR (KBr)υmax/cm-1 3128, 3068 (Ar–CH, w), 2983, 2933, 2877 (–CH, –CH2, –CH3 w), 1722 (–
N=C–, s), 1608, 1488 (-C=C-, s), 1336, 1124 (-C=N-), 1228, 1040 (C-O-C). UV-Vis (DMSO), λmax, (log ε):
1
350 (4.79), 608 (4.55), 674 (5.32). H NMR (DMSO-d6):
δ 9.22 - 9.14 (m, 3H), 9.12 - 9.08 (m, 2H), 9.07 -
9.01 (m, 2H), 8.78 -8.64 (m, 1H), 8.02 - 8.22 (m, 6H), 7.48 - 7.45 (m, 1H), 4.78 - 4.71 (m, 1H), 4.58 - 4.50
(m, 2H), 4.47 - 4.41 (m, 1H), 4.22 - 4.18 (m, 1H), 1.62 (s, 3 H, -CH3), 1.49 (s, 3 H, -CH3). HRMS-MALDI-
TOF m/z calcd for C38H26N8O3Zn+ (M)+ 706.1422, found 706.1436.
Synthesis of [mono(glyceryl)phthalocyaninate]zinc(II) (8): Compound 7 (49 mg, 0.0073 mmol) was stirred
overnight in excess of acetic acid 80% solution. The purification procedure was the same used for compound
(2). Yield 91.6% (42.3 mg ; 0.0635 mmol). mp > 300 °C. FTIR (KBr)υmax/cm-1 3405 (–OH, s), 3004 (Ar–CH,
w), 2919 (–CH and –CH2, w), 1718 (–N=C–, s), 1608, 1484 (-C=C-, w), 1333, 1231 (-C=N-). UV-Vis
(DMSO), λmax, (log ε): 354 (4.76), 612 (4.45), 679 (5.18). 1H NMR (DMSO-d6):
δ 8.23 - 8.11 (m, 5H), 7.86
- 7.78 (m, 1H), 7.76-7.69 (m, 4H), 7.40 - 7.24 (m, 5H), 5.01 - 4.93 (m, 1H), 4.77- 4.68 (m, 2H), 4.65 - 4.57
(m, 1H), 4.55 - 4.46 (m, 1H), 3.49 - 3.43 (m, 2H, -OH). HRMS-MALDI-TOF m/z calcd for C35H22N8O3Zn+
(M)+ 666.1106, found: 666.1108.
Synthesis of [tris-2,9(10),16(17)-methyl-23(24)-solketal-phthalocyaninate]zinc(II) (9): i (300 mg, 1.16 mmol)
and iii (494 mg, 3.48 mmol) was treated under the same conditions adopted for compound 7. Yield 15% (130
mg ; 0.174 mmol). mp > 300 °C. FTIR (KBr)υmax/cm-1 3128, 3068 (Ar–CH, w), 2983, 2933, 2877 (–CH, –
CH2, –CH3, w), 1703 (–N=C–, w), 1610, 1488 (-C=C-, s) 1328, 1271 (-C=N-), 1236, 1046 (C-O-C). UV-Vis
1
(DMSO), λmax, (log ε): 353 (4.53), 612 (4.22), 679 (5.00). H NMR (DMSO-d6):
δ 7.72 – 7.67 (m, 4 H),
7.65 - 7.59 (m, 8H), 4.80 - 4.73 (m, 1H), 4.60 - 4.52 (m, 2H), 4.44 - 4.36 (m, 1H), 4.19 - 4.11 (m, 1H), 2.51 -
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2.48 (m, 9H, Ar-CH3), 1.62 (s, 3H, -CH3), 1.50 (s, 3H, -CH3). HRMS-MALDI-TOF m/z calcd for
C41H32N8O3Zn+ (M)+ 748.1889, found 748.1901.
Synthesis of [tris-2,9(10),16(17)-methyl-23(24)-glyceryl-phthalocyaninate]zinc(II) (10): Compound 9 (65 mg,
0.0868 mmol) was stirred overnight in excess of acetic acid 80% solution. The purification procedure was the
same used for compound 2. Yield 94% (57.8 mg ; 0.081 mmol). mp > 300 °C. FTIR (KBr)υmax/cm-1 3444 (–
OH, s), 3087 (Ar–CH, w), 2981, 2917, 2865 (–CH, –CH2, and –CH3, w), 1733 (–N=C–, w) 1606, 1488 (-
C=C-,w), 1333, 1271(-C=N-), 1226, 1093 (C-O-C). UV-Vis (DMSO), λmax, (log ε): 353 (4.62), 612 (4.31),
680 (5.06). 1H NMR (DMSO-d6):
δ 7.96 - 7.79 (m, 3H), 7.74 - 7.58 (m, 6H), 7.36 - 7.11 (m, 3H), 4.70 - 4.51
(m, 1H), 4.55 - 4.41 (m, 1H), 4.23 - 4.14 (m, 2H), 4.08 - 3.96 (m, 1H), 2.49 (m, 9H, Ar-CH3). HRMS-
MALDI-TOF m/z calcd for C38H28N8O3Zn+ (M)+ 708.1370, found 708.1371.
2.6. Photophysichal and photochemical studies
2.6.1. Singlet oxygen quantum yields
All emission, excitation and electronic absorption spectra of the phthalocyanines were recorded in 10 mm
path length fluorescence cuvettes in DMSO. Photogeneration quantum yields of singlet oxygen (Φ∆ ) were
obtained by indirect method, using diphenylisobenzofuran (DBPF) [20, 21] as chemical quencher. Typically,
a mixture of the phthalocyanine (absorption ~0.2 in 680 nm) and the DPBF (absorption ~ 1.2 in 418 nm) was
irradiated with a red LED lamp (23 mW) in 20 cycles of 6 seconds each one. The Φ∆ values were determined
using zinc phthalocyanine (ZnPc) (Eq.1):
PcZn
Where Φ∆
is the singlet oxygen quantum yield for the ZnPc (Φ∆= 0.67 in DMSO [20]); W and WPcZn is
the DPBF photobleaching rates in the presence of the phthalocyanine and ZnPc, respectively (this value was
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PcZn
obtained by the first exponential curves using the Origin® program); Iabs and Iabs
are the light absorption
rates by the phthalocyanines and ZnPc, respectively.
2.6.2. Fluorescence quantum yields
Fluorescence quantum yields (ΦF) of all compounds were determined by comparative method using the
following equation (Eq.2):
.
.
=
(
)
.
.
Were F and FZnPc are the areas under the fluorescence emission curves of the pthalocyanines (1 – 10) and the
standard, respectively. A and AZnPc are the relative absorbance of the sample and standard at the excitation
wavelength, respectively. η2 and η2
are the refractive indices of the solvents for the sample and standard,
ZnPc
respectively. The unsubstituted ZnPc (ΦF(ZnPc)=0.20) [21] was employed as the standard. The solutions of
the compounds were prepared in DMSO with absorbance ranged between 0.28 and 0.35 at the excitation
wavelength.
2.7. Determination of partition coefficients (PO/W
)
Five milliliters of 10 µM solution from each phthalocyanine derivative was prepared in n-octanol. The UV-vis
spectrum of solution was sampled. Then water (5 mL) was added to solution and the container was stirred for
30 minutes. The centrifugation (5 minutes at 5000 rpm) enabled a phase separation and the organic phase was
sampled again. The partition coefficient was obtained from the difference in the phthalocyanine absorption
intensity (in 650 nm region) in both stages. At least three independent measures were performed and the
corresponding PO/W value was taken as the overall average.
2.8. Cell interaction studies
Cell culture – Cell culture was manipulated using sterile disposable non-pyrogenic plastic ware and were
maintained at 37° C in an atmosphere of 5% CO2 in air at a relative humidity of 80%. Human breast epithelial
cells MCF-7 (adenocarcinoma, estrogen receptor +; ATCC) were incubated in RPMI-1640 medium (Sigma)
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supplemented with 10% fetal bovine serum (Gibco), 10.0 µg/mL insulin purified from bovine pancreas
(Sigma), 100 U/mL penicillin and 10.0 µg/mL streptomycin (Sigma). Cells were routinely trypsinized and
reinoculated onto plates at a density of 4 x 104 cells/cm2.
2.9. Fluorescence microscopy
Phthalocyanines formulated in DMSO solution were diluted in fresh cell medium to give a 45.0 µM
concentration and incubated with MCF-7 cells. MCF-7 cells were grown on chamber slides at a density of 4 x
104 cells/cm2 and then treated with 300 nM DAPI solution (4’,6-diamidino-2-phenylindole, Invitrogen) at 37
°C for 30 minutes. The cells were then washed with phosphate buffered saline (PBS: 137 mM NaCl and 2.7
mM KCl in 10 mM phosphate buffer at pH 7.4), and incubated with 45 µM phthalocyanines in dark. After 1-h
treatment, the cells were pre-fixed with 30% and 50% ethanol diluted in cells medium (v/v) followed by a
100% ethanol post-fixation. The cells were then washed with phosphate buffered saline (PBS). The
fluorescence images were produced by 340 nm excitation, using a 580 nm emission filter in a AXIO –
observer A1 fluorescence microscope, Zeiss. DAPI was used due their interaction with cell nuclei and its blue
fluorescence, allowing following the positions of studied phthalocyanine (red fluorescence) related to the
nuclei.
2.10. Flow cytometry Assay
The percentage of phthalocyanines entrance was determined quantitatively by flow cytometry. MCF-7 cells
were seeded in 12-well plates and incubated with 1 h with 45.0 µM phtalocyanines. The cells were harvested
and washed with ice-cold PBS buffer for 3 times. The cell pellet was resuspended in 200 µL of PBS buffer
and the flow cytometric analysis (Cytometer FC 500 MPL – Beckman Coulter) was performed to determine
the percentage of phthalocyanines entrance in each sample using a 365 nm arc lamp.
3. Results and discussion
3.1. Preparation and structural characterization
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Phthalocyanine 1 (Fig. 1) was synthesized by heating 4-solketalphthalonitrile in the presence of zinc acetate
and a solvent (N,N-dimethylethanolamine ) [22]. To remove the acetal group, compound 1 was stirred for 24
h in acetic acid (80% solution) at room temperature, which resulted in the precipitation and isolation of 2 in
68% yield. These two compounds were synthesized to support the comparative study of unsymmetrical
phthalocyanines with similar moieties.
The new unsymmetrical phthalocyanines 3–10 were prepared via the statistical cyclotetramerization of a
mixture of two different phthalonitriles. We used a 3-fold excess of 4-solketalphhtalonitrile versus 1,2-
dicyanobenzene or 4-methylphthalonitrile to obtain A3B-phthalocyanines 3 and 5. The AB3-phthalocyanines 7
and 9 were synthesized using the reverse molar excess combination of the same phthalonitriles. The applied
conditions led to a mixture of different macrocycles that were purified via silica-gel column chromatography.
Phthalocyanines with free hydroxyl groups were obtained by stirring the macrocycles in an acetic acid
solution.
<Fig. 1>
1
All of the synthesized structures were confirmed by H NMR and HRMS. The presence (and integration) of
1
signals in the region between 4.0-5.0 ppm and 1.40-1.62 ppm in all of the H NMR spectra confirmed the
solketal derivative proportions in the structure (Fig 2). Disappearance of the acetal signals for (2), (4), (6), (8)
and (10) in the NMR spectrum in the region between 1.40-1.62 ppm confirmed the efficiency of the
deprotection reaction conditions used to convert of the solketal groups into glycerol moieties. It should be
noted that each phthalocyanine derivative presented in this study is a mixture of the expected constitutional
isomers.
<Fig. 2>
3.2. Photophysical and photochemical properties
The UV/Vis absorption spectra of compounds 1 -10 presented behavior typical of nonaggregated
phthalocyanines in DMSO as confirmed by the intense, narrow Q-band in the red visible region between 674
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and 682 nm (Fig. 3) (Table 1). Additionally, by comparing the absorbance spectra, it is possible to observe a
slight red shift in the maximum absorption in the Q band region for compounds with three or more
hydroxylated derivatives. This result is in accordance with theoretical studies [7] that show the linear
combination of atomic orbitals (LCAO) is lower when phthalocyanines are modified in the β peripheral
position.
<Fig. 3>
<Table 1>
Phthalocyanines have great tendency to aggregate in aqueous media, which decreases their efficient
generation of reactive oxygen species [5]. Such aggregation is explained by strong π−π electron interactions
between the planar phthalocyanine rings and can be diminished by introducing peripheral substituents with
distinct characteristics.
For macrocycles studied here with at least three peripheral glyceryl groups, we observed a predominance of
the monomeric species in solution until a 4:6 Water:DMSO mixture, which can be verified by following the
relative intensities of the Q-band in the absorption spectra (Fig.4).
<Fig. 4>
However, even phthalocyanine derivatives with three unprotected groups presented a higher tendency to
aggregation in increased aqueous conditions, i.e. 6:4 Water:DMSO media (observed by Q-band relatively
intensities). This behavior can be associated to the fact that free hydroxyl groups can both promote better
macrocycle solvation in water and an enhancement in hydrogen bonding interactions, which increases the
11

ACCEPTED MANUSCRIPT
tendency of the ring to aggregate. Kimura et al. [23] have already presented results showing the self-
organization of some phthalocyanines derivatives containing one glyceryl group in the structure due to the
intermolecular diol hydrogen bonding.
To complement, our UV-Vis absorbance related studies have shown that, at same conditions, the macrocycles
with only one glyceryl unprotected moiety presented higher aggregation tendency than macrocycles with
three unprotect moieties around the ring. This fact demonstrates the influence of the balance between the
water solvation and the hydrogen bonding interactions on the equilibrium between monomers, dimers and
oligomers in solution.
3.3. In vitro photodynamic activities
The interaction of phthalocyanines with cells was evaluated using fluorescence microscopy and flow
cytometry. In the fluorescence microscopy experiments, the nuclei of MCF-7 tumor cells were labeled with
DAPI, which fluoresces blue, to evaluate the localization of phthalocyanine via red fluorescence [Fig.5].
<Fig. 5>
We performed flow cytometry analysis to accurately measure the rate of interaction between each macrocycle
and the MCF7 cells. In this technique, cells were incubated with the photosensitizer and their fluorescence
emissions were analyzed to determine the percentage of cells with increased emission intensities (due to the
presence of the photosensitizer). We observed that symmetrical macrocycle 1 with protect hydroxyl groups
showed higher cellular uptake than their unprotected counterpart, 2. This result can be related to the highest
polar character of macrocycle 2 and to the free hydroxyl group hydrogen bonds, which increase the
macrocycle tendency to form aggregates and decrease their interaction with the target cells.
Almost all of the unsymmetrical phthalocyanines synthesized interacted well with the target cells. The results
for unsymmetrical macrocycles (4), (6) and (10), with unprotected hydroxyl groups, indicated greater
interaction with the target cells compared with their respective counterpart with protected hydroxyl groups
(compounds (3), (5) and (9), respectively). The compounds (7) and (8) presented similar results of cell
interaction. These observations indicate the importance of the hydrophobic/hydrophilic balance in the
12

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interaction with the target cells. Further analyses are underway to understand the influence of these groups in
each structure and to model their behavior and interactions with target cells.
4. Conclusions
A series of unsymmetrical phthalocyanines bearing polyhydroxylated moieties were synthesized and
characterized and then investigated as photosensitizers in photodynamic therapy. The compounds have
different tendencies to form aggregates in polar media, according to the number of attached moieties.
In vitro results showed that the presence of different proportions of hydroxylated moieties in the
phthalocyanine macrocycle promoted different balances of hydrophilic and hydrophobic characteristics,
which promoted distinct interactions with the target cells. These in vitro findings indicate these molecules
offer good potential as photosensitizers in photodynamic therapy.
Acknowledgments.
The authors thank FAPESP (2011/22561-9 and 2007/50765-2), CNPq and CAPES for their financial support
of this research. Thanks are also given to Professor Paolo Di Mascio, Dr. Fernanda M. Prado and Alexandre
Sardelli for their contributions.
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their analogs. J. Porphyrins Phthalocyanines 2010; 14: 1-40.
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[6] Villemin D, Hammadi M, Hachemi M, Bar N. Applications of Microwave in Organic Synthesis: An Improved
One-step Synthesis of Metallophthalocyanines and a New Modified Microwave Oven for Dry Reactions. Molecules
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[7] Alexey Lyubimtsev ZI, Crucius G, Syrbu S, Taraymovich ES, Ziegler T, Hanack M. Aggregation behavior and
UV-vis spectra of tetra- and octaglycosylated zinc phthalocyanines. J. Porphyrins Phthalocyanines 2011; 15: 39-46.
[8] Arslanoğlu Y, Nyokong T. Synthesis and photophysical studies of monocarboxy phthalocyanines containing
quaternizable groups. Polyhedron 2011; 30: 2733-2739.
[9] de Oliveira K T, de Assis FF, Ribeiro AO, et al. Synthesis of Phthalocyanines−ALA Conjugates: Water-Soluble
Compounds with Low Aggregation J. Org. Chem 2009; 74: 7962-7965.
[10] Opris DM, Nüesch F, Löwe C, Molberg M, Nagel M. Synthesis, Characterization, and Dielectric Properties of
Phthalocyanines with Ester and Carboxylic Acid Functionalities. Chem. Mater 2008; 20: 6889-6896.
[11] Bıyıklıoğlu Z, Kantekin H. Synthesis and spectroscopic properties of a series of octacationic water-soluble
phthalocyanines. Synth. Met 2011; 161: 943-948.
[12] Ribeiro AO, Tomé JPC, Tomé AC, et al. First phthalocyanine–β-cyclodextrin dyads. Tetrahedron Lett 2006;
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[13] Silva ARA, Simioni AR, Tedesco AC. Photophysical and Complexation Studies of Chloro-Aluminum
Phthalocyanine with Beta-Cyclodextrin and Hydroxypropyl-Beta-Cyclodextrin. J. Nanosci. Nanotechnol 2011; 11:
4046-4055.
[14] Durmuş M, Yaman H, et al. Water-soluble quaternized mercaptopyridine-substituted zinc-phthalocyanines:
Synthesis, photophysical, photochemical and bovine serum albumin binding properties. Dyes Pigm 2011; 91: 153-
163.
[15] Mack J, Kobayashi N, Low Symmetry Phthalocyanines and Their Analogues, Chem. Rev. 2011; 111: 281–
321.
[16] Zorlu Y, Ermeydan MA, et al. Glycerol and galactose substituted zinc phthalocyanines. Synthesis and
photodynamic activity. Photochem. Photobiol. Sci 2009; 8: 312-318
[17] Dumoulin F, Durmus M¸ Ahsen V, Tebello N. Synthetic pathways to water-soluble phthalocyanines and close
analogs. Coord. Chem. Rev 2007; 254: 2792-2847
[18] Bronshtein I, Aulova S, et al. In Vitro and In Vivo Photosensitization by Protoporphyrins Possessing Different
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Optically Active Phthalocyanine Dimers. Langmuir 2003; 19: 4825-4830.
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Figure Captions
Fig. 1. The route of new phthalocyanines derivatives
Fig 2. 1H NMR (500 MHz) spectrum of phthalocyanine 3 in CDCl3.
Fig 3. UV-vis spectra of all phthalocyanine derivatives synthesized 1 (A), 3 (B) and 7 (C) in different
concentrations (in DMSO).
Fig. 4. UV-vis spectra of phthalocyanines 5 (left) and 6 (right) in different proportions of H2O and DMSO
(each one at same concentration in all analysis, 7.6 µmol.L-1).
Fig.5. Fluorescence images of the interaction between phthalocyanine 4 (45.0 µM final concentration) and
MCF7 cells (grown on chamber slides at a density of 4 x 104 cells/cm2 and treated with 300 nM DAPI
solution). The images were produced by 340 nm excitation, using a 580 nm emission filter. DAPI was used to
following the studied phthalocyanine (red fluorescence) according to the nuclei position.
16

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Figure 1
O
HO
HO
O
O
O
CN
CN
HO
O
N
N
N
N
OH
N
Zn
N
O
N
Zn
N
CH3COOH 80%
24 h
Zn(AcO)2 . 5 H2
O
O
O
O
N
N
N
O
N
DMAE, 160º C
O
O
N
N
N
O
N
OH
O
HO
O
O
OH
O
OH
O
O
4-solketalphthalonitrile
(i)
(1)
(2)
HO
O
HO
O
O
O
N
N
N
N
R
N
N
N
Zn
N
O
N
Zn
N
Zn(AcO)2 .5 H2O
R
CH3COOH 80%
DMAE, 160º C
CN
Phthalocyanines
A3
O
N
N
N
N
B
OH
24 h
HO
N
N
O
O
R
CN
OH
O
OH
O
(ii) R = H
(iii) R = CH3
O
O
(3) R= H
(5) R= CH3
(4) R= H
(6)
R= CH3
R
R
HO
O
N
N
N
N
Zn(AcO)2 . 5 H2O
DMAE, 160º C
N
N
OH
O
Phthalocyanines
AB3
O
CH3COOH 80%
24 h
O
R
N
Zn
N
N
R
N
Zn
N
CN
N
N
N
N
N
R
CN
(ii) R = H
(iii) R = CH3
R
R
(ii)
(iii)
1,2-Dicyanobenzene
R = 4-Methylphthalonitrile
(8) R= H
(7)
(9)
R= H
R= CH3
(10)
R= CH3
17

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Figure 2
O
O
O
N
N
N
N
O
N
Zn
N
N
N
O
O
O
O
O
(3)
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
Chemical Shift (ppm)
18

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Figure 3
1,0
0,8
0,6
0,4
0,2
0,0
(A)
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
y = 114487x + 0,0035
R² = 0,9998
-6
-6
1,0x10-6 2,0x10-6 3,0x10-6 4,0x10-6 5,0x10 6,0x10-6 7,0x10 8,0x10-6
Concentration, µM
500
600
700
λ (nm)
1,2
1,0
0,8
0,6
0,4
0,2
0,0
(B)
1,2
1,0
0,8
0,6
0,4
0,2
y = 124459x - 0,0034
R² = 0,9997
1,40x10-6 2,80x10-6 4,20x10-6 5,60x10-6 7,00x10-6 8,40x10-6
Concentration, µM
500
600
700
λ (nm)
(C)
1,0
0,8
0,6
0,4
0,2
0,0
1,0
y = 209969x - 0,0068
R² = 0,9989
0,8
0,6
0,4
0,2
8,0x10-7
1,6x10-6
2,4x10-6
3,2x10-6
4,0x10-6
4,8x10-6
Concentration, µM
500
600
700
λ (nm)
19

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Figure 4
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0,0
Phtalocyanine 5
% H2O in DMSO
0%
20%
40%
60%
80%
500
550
600
650
700
750
800
λ (nm)
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0,0
% H2O in DMSO
0%
Phtalocyanine 6
20%
40%
60%
80%
500
550
600
650
700
750
800
λ (nm)
20

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Figure 5
21

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Table 1: Values for the absorbance maximum and quantum yield of singlet oxygen and fluorescence for
phthalocyanines 1-10. All data were recorded in DMSO.
UV-Vis
λmax (nm)
Cell interaction
(%)*
Pc
Log P
log ε
ΦF
Φ∆
358, 613, 682
358, 614, 682
356, 612, 680
357, 612, 680
356, 613, 681
356, 613, 681
350, 608, 674
354, 612, 679
353, 611, 679
353, 612, 680
4.72, 4.36, 5.05 1,23 ± 0,06 0.13 0.72
4.89, 4.54, 5.23 0,58 ± 0,07 0.13 0.65
4.71, 4.38, 5.09 1,48 ± 0,08 0.13 0.67
4.79, 4.46, 5.17 1,19 ± 0,08 0.13 0.44
4.50, 4.16, 4.87 1,58 ± 0,07 0.13 0.71
4.66, 4.32, 5.03 1,46 ± 0,06 0.13 0.56
4.79, 4.55, 5.32 1,47 ± 0,08 0.15 0.55
4.76, 4.45, 5.18 1,35 ± 0,08 0.14 0.49
4.53, 4.22, 5.00 1,67 ± 0,09 0.14 0.72
4.62, 4.31, 5.06 1,61 ± 0,08 0.14 0.62
80 ± 1,2
0
1
2
3
4
5
6
7
8
9
62 ± 0,1
84 ± 0,9
0
52 ± 0,2
52 ± 0,1
48 ± 0,6
0
81 ± 1,8
10
* The percentage of phthalocyanines entrance was determined quantitatively in MCF-7 cells, seeded in 12-
well plates, incubated with 45.0 µM phthalocyanines for 1h, and performing the flow cytometric analysis.
22

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Graphical abstract
R1 or R4
N
N
N
Zn
N
R4 or R1
N
N
R2 or R3
N
N
CN
CN
CN
R4 or R1
OH
R4 =
O
OH
R1
CN
R3 or R2 , R1
R2 or R3
O
N
N
N
R1 =
R =
R =
CH3
3
H
2
O
O
R3 or R2
N
N
Zn
N
N
R1 or R4
N
R3 or R2
23

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Research Highlights
1. The aggregation and water solubility of the phthalocyanines derivatives is related to the proportion of free
hydroxyl groups present in the structure.
2. The interaction between MCF7 mammary cell and the phthalocyanine derivatives in aqueous media
changes according to the hydrophobic to hydrophilic balance in the macrocycle structure.
3. Unsymmetrical phthalocyanines with unprotected hydroxyl derivatives had a greater tendency to aggregate
in aqueous media but higher interaction with MCF7 mammary cells.
1