Post‐modification of phthalocyanines via isocyanide-based multicomponent reactions: Highly dispersible peptidomimetic metallophthalocyanines as potent photosensitizers

Article abstract of DOI:10.1016/j.dyepig.2019.03.018

In this work, one-step synthesis of highly dispersible phthalocyanines containing carboxamide moieties via four-component Ugi reaction was demonstrated for the first time. These pseudopeptide-modified-phthalocyanines were characterized by means of IR, NMR, CHN and UV–Vis spectroscopy. Additionally, the purity of the pseudopeptide phthalocyanines was confirmed by capillary zone electrophoresis. Most of the synthesized phthalocyanines showed good dispersity in common organic solvents and especially in water. The in vitro photodynamic activities of water-soluble phthalocyanine 5k upon MCF-7 cancer cells were investigated and revealed that the cell viability was high in dark conditions, while the compound was phototoxic at visible light and the intensity of damage was directly related to its concentration. Also, phthalocyanine 5k showed high cellular uptake and more killing cells after the modification with Ugi-4CR. Since, many potent phthalocyanine photosensitizers are hydrophobic and poorly water-soluble, which limit their therapeutic applications, the novel phthalocyanines which modified through Ugi approach could be introduced as potent hydrophilic, nonaggregated and biocompatible sensitizers which can able to improve the effectiveness of photodynamic therapy.

Full text of DOI:10.1016/j.dyepig.2019.03.018

Accepted Manuscript
Post‐modification of phthalocyanines via isocyanide-based multicomponent reactions:
Highly dispersible peptidomimetic metallophthalocyanines as potent photosensitizers
Ronak Afshari, Vahid Ghasemi, Shabnam Shaabani, Ahmad Shaabani, Zolfaghar
Aladaghlo, Ali Reza Fakhari
PII:
S0143-7208(18)31490-6
https://doi.org/10.1016/j.dyepig.2019.03.018
DYPI 7407
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 6 July 2018
Revised Date: 18 January 2019
Accepted Date: 8 March 2019
Please cite this article as: Afshari R, Ghasemi V, Shaabani S, Shaabani A, Aladaghlo Z, Fakhari AR,
Post‐modification of phthalocyanines via isocyanide-based multicomponent reactions: Highly dispersible
peptidomimetic metallophthalocyanines as potent photosensitizers, Dyes and Pigments (2019), doi:
https://doi.org/10.1016/j.dyepig.2019.03.018.
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ACCEPTED MANUSCRIPT
Post‐modification of phthalocyanines via isocyanide-based multicomponent
reactions: Highly dispersible peptidomimetic metallophthalocyanines as potent
photosensitizers
Ronak Afshari, Vahid Ghasemi, Shabnam Shaabani, Ahmad Shaabani , Zolfaghar Aladaghlo, and Ali Reza
Fakhari
Faculty of Chemistry, Shahid Beheshti University, G. C., P. O. Box 19396-4716, Tehran, Iran
Corresponding author. Tel.: +982129902800; e-mail: a-shaabani@sbu.ac.ir
Abstract: In this work, one-step synthesis of highly dispersible phthalocyanines containing carboxamide
moieties via four-component Ugi reaction was demonstrated for the first time. These pseudopeptide-modified-
phthalocyanines were characterized by means of IR, NMR, CHN and UV-Vis spectroscopy. Additionally, the
purity of the pseudopeptide phthalocyanines was confirmed by capillary zone electrophoresis. Most of the
synthesized phthalocyanines showed good dispersity in common organic solvents and especially in water. The in
vitro photodynamic activities of water-soluble phthalocyanine 5k upon MCF-7 cancer cells were investigated and
revealed that the cell viability was high in dark conditions, while the compound was phototoxic at visible light
and the intensity of damage was directly related to its concentration. Also, phthalocyanine 5k showed high
cellular uptake and more killing cells after the modification with Ugi-4CR. Since, many potent phthalocyanine
photosensitizers are hydrophobic and poorly water-soluble, which limit their therapeutic applications, the novel
phthalocyanines which modified through Ugi approach could be introduced as potent hydrophilic, nonaggregated
and biocompatible sensitizers which can able to improve the effectiveness of photodynamic therapy.
Keywords: Metallophthalocyanine, Functionalization, Ugi four-component reaction, Post-modification,
Photosensitizer
1.
Introduction
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Metallopththalocyanines have gained widespread attention mainly due to being analogs of nature’s porphyrins.
Their unique spectroscopic and electrochemical properties, stronger absorption in the red visible region and
higher performance at creating singlet oxygen [1], makes them convenient candidates for semiconductors [2],
photodynamic therapy (PDT) [3], chemosensors [4] and catalysis [5]. However, phthalocyanines have some
disadvantages like low solubility in water or polar solvents and their aggregation tendency which reduces their
photosensitizing activity [6]. By rationally tuning the metal center as well as modification and functionalization
of substrates, important electrical and optical properties, intermolecular interactions, solubility and dispersity of
phthalocyanines can be fine-tuned that can play important roles in the plethora of applications [7]. As a
consequence, various types of functionalized substrates which involve organic scaffolds with diverse chemical
nature as peripheral substituents, have been introduced for the construction of functionalized phthalocyanines
with tetra- and octa- groups [8], water-soluble anionic sulfo groups [9], nitroxide segments [10], tetra-substituted
with 3,4-(dimethoxyphenylthio) moieties [11] and pyrazole scaffolds [12]. The focus of most of these procedures
is on the development of new synthetic strategies for the synthesis of substituted phthalocyanines which can be
synthesized step-by-step from versatile precursor compounds. In the rare but praiseworthy cases, phthalocyanines
with potential organic functional groups such as amine, acid or acetylene have been prepared that can further
participate in the in-situ post-modification processes [13].
Multicomponent reactions (MCRs) as diversity-oriented syntheses are superior synthetic procedures which
provide highly valuable scaffolds with a minimum synthesis and purification steps [14]. In the MCRs, through
one-pot procedures, three or more components combined to form a skeletal diverse product that incorporates
essentially all the atoms of the starting materials [15]. Among the various synthetic routes, performing MCRs is
an ideal strategy for the environmentally friendly modification of supramolecules and materials [16-18]. On the
other hand, MCRs are one of the applicable organic chemist’s toolbox for the synthesis of functional organic
chromophores with unique photophysical and electrochemical features [19-21]. These innovative strategies have
the benefit of operational simplicity, substrate diversity, improve E-factor and mass intensity and in particular,
pot, atom and step economic feature which are important aspects of green chemistry [22]. Meanwhile, Ugi-4CR
as a most known isocyanide-based MCR (I-MCR), has been frequently utilized as an efficient synthetic tool for
the synthesis of macrocycles [23], functionalization of applicable materials [24-28], and construction of spin
labeled compounds [29]. As we reported previously, the modification of carbonaceous materials through Ugi-
4CR approach conducted to the formation of highly dispersed materials which served as a nanocarrier [30] and
catalyst [31]. Also, very recently, efficient utilization of Ugi-4CR approach for the construction of the
fluorescently labeled fullerenes was demonstrated [32].
Our ongoing investigations focus on the functionalization of phthalocyanines via Ugi four-component reactions
(Ugi-4CR), in a one-pot manner for the first time. For this purpose, tetra-peripherally acid and amine substituted
phthalocyanine derivatives was participated in a one-pot Ugi-4CR for the synthesis of pseudopeptide-
phthalocyanines without using any catalysts. In order to assess the potential of the Ugi-4CR approach for
modification of phthalocyanines, their dispersities were investigated in detail. It is worth mentioning that in the
course of new post-modification, highly soluble and dispersible metallophthalocyanines were prepared in which
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some of them are stable for a month or more. Additionally, the photodynamic activity of water-dispersible
phthalocyanine (5k) was examined and the results highlight the potential of Ugi-4CR for the synthesis of efficient
photosensitizers for cancer therapy.
2.
Experimental Section
2.1.
Materials and methods
All of the materials including phthalic acid analogous, metal salts and etc. were purchased from Merck, Fluka and
Sigma-Aldrich and used without further purification. Fourier transform infrared (FT-IR) spectra were made in
KBr pellets on a Shimadzu IR470 spectrometer. Microwave irradiation experiment was carried out in a
Microsynth Milestone SRL microwave apparatus with MW output power of 1000 W. UV–vis spectra were
recorded with UV–vis Shimadzu 2100. Fluorescence measurements were performed on a PerkinElmer
spectrofluorimeter. The apillary zone electrophoresis (CZE) experiments with UV detection were carried out on
C
a Lumex Capel 105 capillary electrophoresis system (Ohio lumex, Twinsburg, Russia). Acquisition of
electropherograms was computer-controlled by Chrom & Spec software version 1.5. The elemental analyses were
investigated with an Elementar Analysensysteme GmbH Vario EL. 1H NMR and 13C NMR spectra were recorded
on BRUKER DRX-300 AVANCE spectrometer at 300.13 and Bruker DRX-500 Avance instrument using TMS
as internal standard and CDCl3 as applied solvent at 125.7 MHz, respectively.
2.2.
Synthesis of metallophthalocyanine tetracarboxylic acid
Metallophthalocyanine tetracarboxylic acids were synthesized based on previous work [33]. In a ceramic mortar,
trimellitic anhydride (0.192 g, 1.00 mmol), cobalt acetate tetrahydrate (0.112 g, 0.58 mmol), ammonium
molybdate (0.0180 g, 0.016 mmol), ammonium chloride (0.0380 g, 0.72 mmol) and urea (0.496 g, 6.39 mmol)
were mixed and grinded into powders. Then, the produced powders were transferred to a flask and irradiated in a
microwave oven for 5 min at 140 °C. After reaction completion, tetra carboxamide metallophthalocyanine was
hydrolyzed by heating at 100 °C in an aqueous KOH solution (10 g, 90 ml) for 24 h. The prepared
metallophthalocyanine tetracarboxylic acid precipitated through the addition of HCl (37 wt %) and separated by
filtration. The obtained yields for cobalt, zinc, nickel, iron and copper-phthalocyanine tetracarboxylic acid were
73, 55, 70, 76, 60% respectively.
2.3.
Synthesis of carboxamide-modified-metallophthalocyanines (5a-5k)
In a typical procedure, aldehydes (4 mmol) and amines (4 mmol) were dissolved in DMSO (25 mL). The
solution was stirred at room temperature for 30 min. The appropriate isocyanides (4 mmol) and
metallophthalocyanine tetracarboxylic acids (1 mmol) were added and the resulting mixture was stirred in a flask
for 3 days at 60 °C. On completion, carboxamide-functionalized metallophthalocyanine was purified by washing
with distilled water and acetone followed by centrifuge [30].
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2.4.
Synthesis of 4-nitrophthalimide
Nitric acid (48 mL; d. 1.50) was added to a solution of 280 mL of concentrated sulfuric acid and phthalimide (20
g) followed by stirring at a temperature range of 10-15 °C. The reaction mixture was then allowed to warm
slowly to room temperature. After 16 h, the crude product which precipitated was filtered, washed four times
with ice water, and purified by crystallization from ethanol [34].
2.5.
Deep eutectic solvent preparation
Deep eutectic solvents were prepared according to the literature [35], and used without further purification. For
this purpose, choline chloride was mixed by urea with a mole ratio of (1:2) then heated at 80 °C in air with
stirring until a clear liquid was obtained.
2.6.
Synthesis of tetra-nitro cobalt (II) phthalocyanine
Tetra-nitro cobalt (II) phthalocyanine was synthesized based on our previous work [35]. A mixture of 4-
nitrophthalimide (0.192 g, 1 mmol), cobalt (II) acetate tetrahydrate (0.044 g, 0.25 mmol), ammonium molybdate
(0.001 g, 0.01 mmol), ammonium chloride (0.053 g, 1 mmol) and urea (0.240 g, 4 mmol) in DES (2 mL) was
heated at 150 °C for 60 minutes. As the reaction proceeded, a dark-green solid gradually appeared. After
completion of the reaction, 10 mL of distilled water was added. The insoluble crude product was filtered and
washed with water, acetone and then was dried. Next, the product was dissolved in the concentrated H2SO4 (5
mL), precipitated from distilled water (150 mL), filtrated off and washed with water dried in an oven (80 °C) to
give the corresponding product with the 62% yield.
2.7.
Synthesis of tetra-amino cobalt (II) phtalocyanine
Tetra-amino cobalt (II) phthalocyanine was synthesized by adding sodium sulfide nonahydrate (0.72 g, 3.00
mmol) to tetra-nitro cobalt (II) phthalocyanine (0.19 g, 0.25 mmol) in DMF (10 mL) in the flask and heated at
70 °C for 24h. After completion of the reaction, the mixture was cooled and collected by centrifugation. The
product was washed with distilled water (20 mL), ethanol (20 mL) and washed two times with acetone followed
by centrifugation. Then, the mixture was dried to obtain a dark-blue solid (Yield=54%) [36].
2.8.
Synthesis of carboxamide-functionalized Co-phthalocyanine (8a, 8b)
For the synthesis of carboxamide-functionalized Co-phthalocyanine, tetra-amino cobalt (II) phthalocyanine (0.63
g, 1 mmol) and aldehydes (4 mmol) were dissolved in DMSO (25 mL). The solution was stirred at room
temperature for 60 min. Then the isocyanide (4 mmol) and carboxylic acid (4 mmol) were added and the
resulting mixture was stirred in a flask for 3 days at 60 °C. On completion, the product was easily purified by
washing with distilled water and acetone followed by centrifuge [30].
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2.9.
Capillary Zone Electrophoresis (CZE) analysis
In order to prove the purity of modified phthalocyanines, the CZE analysis was performed in an
uncoated fused-silica capillary (Ohio lumex, Twinsburg, Russia) 60 cm × 50 µm I.D. (50 cm effective
length). The background electrolyte solution was 100 mM phosphate buffer adjusted to pH 7. The
separation was carried out at 20 kV, generating a current level in the range of 140-160 µA. The sample
solutions were injected by hydrodynamic injection at 60 mbar for 7 s. Detection of the analytes was
accomplished at 334 nm.
2.10. Cytotoxicity study
The MTT assay was used for the investigation of cell cytotoxicity of carboxamide-modified-phthalocyanine.
Human breast cancer like cell line (MCF-7) was obtained from Iran Pasteur Institute (ATCC, CCL-121). In
summary, the MCF-7 cells line were seeded onto four 96-well plates at a density of 104 cells/well in DMEM
medium supplemented with 100 U/mL penicillin, 0.1 mg/mL streptomycin and 10 % fetal bovine serum and kept
in CO2 incubator under standard culturing conditions. Then, 100 µL of culture medium containing different
concentration of compound 5k (5, 10, 25, 50 and 100 µg/mL) was added to two plates. From the two plates
prepared for each series of test, one is selected for the determination of cytotoxicity (dark cytotoxicity), and one
for the determination of photocytotoxicity which were irradiated with visible light source whereas the control
plates are kept in the dark (Light source: 667 nm diode laser, Shenzhen Taiyongxin Technology, China. Output
power was measured before exposure by utilization of the FieldMate Laser Power Meter for calculating the
exposure time. Irradiation power: 52 mW, power density: 5.73 mW cm-2 and energy density: 20 J cm-2 for 56 min
exposure. The exposure time for the dose-dependent experiment was: 14, 29, 43 and 56 min to obtain an energy
density of 5, 10, 15 and 20 J cm-2, respectively). The samples were irradiated at room temperature for the desired
minutes. Non-irradiated plates kept at room temperature in a dark box at the light exposure time. After incubation
at 37 °C for 24 h, the medium was replaced with 100 µL of MTT solution with 0.5 mg/mL concentration in each
well. For the investigation of comparative cytotoxicity of acid and carboxamide-functionalized phthalocyanines,
100 µL of culture medium containing Co-phthalocyanine tetracarboxylic acid were added to two plates and the
other plates were filled with 100 µL of culture medium containing compound 5k with 25, 50 and100 µg/mL
concentration of each sample. Then prepared the same as mentioned above. Finally, the optical density (OD) of
samples was measured with an ELISA plate reader (Stat Fax 2100, USA) after continuous incubation for 4 h. The
culturing experiments were repeated in triplicate for each experiment.
2.11. Detection of singlet oxygen
The efficiency of compound 5k to generate singlet oxygen was monitored over time by measuring
photobleaching of the radical quencher ADPA [37]. In brief, 1.5ꢀmL of ADPA (1ꢀ×ꢀ10−4ꢀM) was mixed with
1.5ꢀmL of compound 5k (1ꢀ×ꢀ10−5ꢀM) suspension. The control solution was prepared which contains ADPA alone.
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The samples were irradiated with the visible light source and their optical densities at 378ꢀnm were recorded
every 100ꢀseconds in a spectrophotometer.
2.12. Time-dependent cellular uptake
Time-dependent cellular uptake efficiency of Co-phthalocyanine tetracarboxylic acid and compound 5k were
investigated. MCF-7 cells with Co-phthalocyanine tetracarboxylic acid and compound 5k were incubated under
the same experimental conditions as mentioned above for certain time (2, 4, 6 and 24 h) in the dark. After then,
the upper drug-containing medium was transferred in a 1 cm cuvette to measure the absorption spectra. The
cellular uptake amounts were calculated according to the standard curves.
3.
Results and Discussion
In view of our interests in design and synthesis of neoteric functionalized materials through MCRs [30, 31], and
following our numerous works on the synthesis and applications of phthalocyanines [35, 38-40], herein we
demonstrated the novel corporation of functionalized phthalocyanines in Ugi-4CR. First, the phthalocyanines
were functionalized by acid or amine moieties which caused to regioisomers. It is well known that template
synthesis of substituted phthalocyanines could give mixtures of regioisomers that the isomers portion depends on
the phthalic acid derivatives and reaction conditions [41]. Importantly, isolation of individual isomers is often a
very difficult challenge [42]. Since our main purpose was the functionalization of phthalocyanines and we were
unsuccessful in separating the isomers by normal phase chromatography we used the mixture of regioisomers for
participation as one part of the Ugi-4CR. The carboxamide-modified-phthalocyanines were obtained from readily
available starting materials such as various amines, aromatic aldehydes, and isocyanides in DMSO at 60 °C
within 3 days in moderate yields without the need of any catalysts or additives.
In order to optimize the reaction conditions, we investigated the effect of various solvents for the synthesis of
phthalocyanine 5a as a model system. Since most of the phthalocyanines have a low dispersity in common
solvents, the use of organic solvents such as AcOH, MeOH, EtOH and CHCl3 caused to inferior results in yield.
The best results obtained in DMSO, therefore, we choose DMSO as a solvent for this procedure. In continuation,
the effect of temperature (room temperature, 60 and 100 °C) was investigated and the best yield was found at
60 °C.
With optimized conditions in hand, we explored the scope and limitations of Ugi-4CR, by extending the
procedure to the various metallophthalocyanine tetracarboxylic acids, aniline derivatives, aromatic aldehydes
containing electron-donating and –withdrawing functional groups, and aliphatic and aromatic isocyanides. As
indicated in Table 1, the reactions proceeded very efficiently in DMSO and led to the formation of novel
carboxamide-modified-metallophthalocyanines (5a-k) in moderate to good yields.
In a typical procedure, metallophthalocyanine tetracarboxylic acids 1 (1 mmol), benzaldehydes 2 (4 mmol),
aniline derivatives 3 (4 mmol) and isocyanides 4 (4 mmol) were dissolved in DMSO (25 mL). Then, the
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resulting mixture was stirred in a flask for 3 days at 60 °C to yield carboxamide-modified-metallophthalocyanine
5 (Table 1).
Table 1. Synthesis of carboxamide-modified-metallophthalocyanines through Ugi-4CR approach
Entry Metal
R1
R2
R3
Product Yield(%)a
1
2
3
Co
Co
Co
4-nitrobenzaldehyde
p-toluidine
p-toluidine
p-toluidine
cyclohexyl
cyclohexyl
cyclohexyl
67
60
63
5a
5b
5c
2,4-dimethoxybenzaldehyde
4-(benzyloxy)benzaldehyde
3,4-
4
Co
benzaldehyde
tert-butyl
55
5d
dimethylaniline
5
6
7
8
9
Co
Cu
Fe
Ni
Zn
4-chlorobenzaldehyde
2,4-dimethoxybenzaldehyde
2,4-dimethoxybenzaldehyde
2,4-dimethoxybenzaldehyde
2,4-dimethoxybenzaldehyde
4-methoxyaniline
p-toluidine
tert-butyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
60
40
30
45
15
5e
5f
p-toluidine
5g
5h
5i
p-toluidine
p-toluidine
Tosylmeth
yl
10
Co
2,4-dimethoxybenzaldehyde
p-toluidine
25
5j
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2 6-
11
Co
2,4-dimethoxybenzaldehyde
p-toluidine
dimethylph
enyl
65
5k
a Isolated yield
The structures of products 5 were deduced from their IR, UV-vis and CHN analysis data. All the studied
phthalocyanines showed well-defined Q and Soret bands and un-split Q bands (Supporting Information).
Importantly, IR, NMR and titration method was used for confirming the tetra-decoration of phthalocyanines
based on the absence of the acidic group. As can be seen in SI there is no peak for acidic proton higher than 10
ppm in NMR spectrums which are in agreement with IR data. Additionally, the acidic proton titrations were
carried out with an automatic titrator and the results revealed no evidence of acidic group in synthesized
compounds. The resulting carboxamide-modified-metallophthalocyanines (5a, b, c, f, h, j, k) are well soluble in
organic solvents, which allowed 1H NMR spectroscopic characterization. The 1H NMR spectra of compounds 5d,
5g, showed broadened signals, a feature that may due to the inherent paramagnetic characteristic of the products
and intermolecular aggregation. Additionally, it was impossible to obtain acceptable NMR spectrum for product
1
5e, because of low solubility and product 5i, despite its good dispersity in DMSO and CHCl3. The H NMR
spectrum of 5c consisted of a multiplet for the methylene protons of the cyclohexyl ring (δ=1.1-1.98 ppm,40H), a
broad singlet for the NH−CH cyclohexyl (δ=3.68 ppm, 4H), a singlet for the methyl group of aniline (δ= 2.34
ppm, 12H), a singlet for benzyl group of the 4-(benzyloxy)benzaldehyde (δ= 5.18 ppm, 8H), a singlet for CH
(δ=6.28 ppm, 4H), a multiplet for aromatic protons (7.01-7.85 ppm, 60H) and a singlet for NH of amide group (δ
= 8.42 ppm, 4H). Also, the 13C NMR spectrum of 5c indicated the presence of methyl group of aniline,
cyclohexyl ring, eight amide carbonyls and sp3 methine signals of Ugi product which confirmed the formation of
carboxamide-modified-Co-phthalocyanine. The FT-IR spectra showed the bands at 2923 and 2854 cm-1 assigned
to the asymmetric and symmetric methylene vibrations of cyclohexyl moiety. Also, N-H stretching of the amide
observed around 3200–3400 cm-1 and the C=O stretching amide bond at around 1686 cm-1. Four absorption peaks
at 283, 331, 604 and 669 cm-1 in DMSO were observed. In addition, the chemical composition of the product has
been accurately evaluated by CHN analysis data.
Continuing the work and for investigation of the substrate diversity of this post-modification approach, we used
tetra-amino cobalt (II) phthalocyanine as an amine part in Ugi-4CR. For this purpose, first of all, tetra-nitro cobalt
(II) phthalocyanine was synthesized in a deep eutectic solvent (DES) as green and robust multipurpose solvent
caused to a dark-green solid [35]. Then, tetra-amino cobalt (II) phthalocyanine which is a dark-blue solid were
synthesized through the reduction reaction. In the second step, the reaction of tetra-amino cobalt (II)
phthalocyanine 6 (1 mmol), aldehyde 2 (4 mmol), carboxylic acid 7 (4 mmol) and isocyanide 4 (4 mmol) under
1
the previously mentioned conditions were investigated (Table 2). The IR, H NMR, UV-vis and CHN analytical
data obtained on the final modified-Co-phthalocyanines 8 confirmed the efficiency of Ugi-4CR approach for
modification of amine-phthalocyanines with acceptable yields.
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Table 2. Post-modification of tetra-amino cobalt (II) phtalocyanine via Ugi-4CR
Entry
R1
R2
R3
Product
Yield(%)a
1
2
benzoic acid
cyclohexyl
60
52
8a
8b
2,4-dimethoxybenzaldehyde
4-nitrobenzaldehyde
4-methylbenzoic
acid
2 6-dimethylphenyl
a Isolated yields
During the purification process of modified-phthalocyanines, we observed that some carboxamide-modified-
metallophthalocyanines showed high dispersity in solvents such as acetone, water and chloroform. It is important
to mention that solubility of phthalocyanines in common organic solvents is important for certain modern
technological applications, therefore, we decided to check the solubility and dispersity of modified-
phthalocyanines in different solvents over the period of time. The results demonstrated that all of the products
show high dispersity in DMSO. In most of the cases, modified-phthalocyanines exhibited high dispersity and
long-term stability in acetone and chloroform for a week or more, while tetra acid or amine-functionalized
metallophthalocyanines have poor solubility in these solvents. For example, compounds 5g and 5i showed high
dispersity in both DMSO and CHCl3, even after a month, although the water solubility was low and the
aggregation happened (Fig. S1). Also, the compounds 8a and 8b are well dispersed in DMSO for a long period of
time, while water-dispersity were not observed. Interestingly, some of the products showed unprecedented
dispersity and stability in the water (Fig. S2). For example, the product 5k exhibited high dispersity in water
leading to a slow aggregation even after four months. The homogeneous blue color in the vial indicates the stable
suspension while there were no sign of dispersity in the case of chloroform and the product was aggregated (Fig.
S3).
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Noteworthy, aggregation is not desired in metallophthalocyanines bioapplications due to the photodynamic
inactivity of aggregates which is a crucial factor for designing ideal photosensitizer. This issue is more serious in
polar media such as water, which cause to self-associate and repel the hydrophobic ᴨ systems to form aggregates
[43]. We used UV-vis absorption spectroscopy to monitor the aggregation of some compounds. The aggregation
and electronic absorption properties of compound 5h and 5i before and after functionalization were studied in
DMSO. As shown in Figure 1a, both of the Ni phthalocyanines showed absorption band at 340 nm (B or Soret
band) and clear Q-bands with a slight shift to longer wavelength with the increase of the intensity of the Q bands.
Notably, in the case of compound 5h highly resolved Q-band, at 618 and 675 nm, may be assigned to the
monomeric species. Highly resolved Q-bands in Figure 1b, confirmed that compound 5i exists mainly in
monomer type which confirmed the good dispersity of these compounds in DMSO after Ugi-functionalization.
Figure 1. The comparative electronic absorption spectra of (a) NiPc and (b) ZnPc derivatives in DMSO
(Concentration:1×10-5 M)
In continuation, the concentration effect on the aggregation properties of the carboxamide-functionalized copper
phthalocyanine (5f) was also studied at various concentrations ranging from 45 to 1.4 µg/mL in DMSO. As can
be seen in Figure 2, compound 5f exhibited B- and Q-band absorptions typical of phthalocyanine species at
approximately 343, 628 and 683 nm. Compound 5f was obeyed with the Lambert-Beer law at the studied
concentration range, consequently, aggregation was not observed (Fig. 2a). The UV-Vis (Fig. 2b) and
fluorescence (Fig. 2c) spectra of the 5f were also performed in various solvents. The 5f compound exhibited
analogous and nonaggregated UV-spectra in DMSO while it showed negligible fluorescence emission. The
electronic spectrum of 5f in ethanol, water and CH2Cl2 showed some differences from that in DMSO as a result of
differing solvent. First, the B-bands are slightly shifted to a shorter wavelength. Second, the intensity of the Q-
bands is much lower. In UV spectrum of ethanol, the broad shoulder at around 620 nm was observed due to little
aggregation along with a sharp emission band at 680 nm. The shape of the UV-curve indicates that 5f exists
predominantly in the form of dimers in a distilled water with a maximum emission. Also, compound 5f showed
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obvious aggregation and low-stability in CH2Cl2 as concluded from the broadened Q-band in the absorbance
spectrum.
Figure 2. Absorbance changes of 5f in DMSO at different concentrations (µg/mL) (Inset: Plot of absorbance
versus concentration) (a), absorbance (b) and emission (c) spectra of 5f in different solvents (Concentration:10-5
M, λexc =338 nm).
Since compound 5k showed very high stability in water even after four months we decided to check its
dissolution and dispersity behavior in different modes. For this purpose, first of all the the UV analysis of 5k at
different concentrations ranging from 45 to 1.4 µg/mL in DMSO were studied and the typical absorbance of
phthalocyanines were observed at 334 nm along with highly resolved Q-band at 667 nm, assigned to the
monomeric species, accompanied with transition into excited vibration state at 608 nm which confirmed that
compound 5k exists mainly in monomer type (Fig. 3a). Additionally, the calibration curve showed that 5k
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followed the Lambert-Beer law at the studied concentration range which confirmed the high dispersity and non-
aggregation of 5k (Fig. 3b).
Figure 3. Absorbance changes of 5k in DMSO at different concentrations (µg/mL) (a) Plot of absorbance versus
concentration)(b)
In continuation, the comparative absorbance and emission spectrums in DMSO and water were provided. Figure
4a shows the electronic absorption spectra of compound 5k in DMSO and H2O. As can be seen accordingly,
phthalocyanine 5k shows major absorption band at 334 nm and highly resolved Q-band at 608 and 667 nm. In the
case of water, the shape of the UV-curve observed that compound 5k exists predominantly in the form of dimers
[8]. But, their fluorescence emission properties are different. Furthermore, compound 5k exhibited sharp
fluorescent emission band in water at 677 nm stronger than emission peak for DMSO (excitation at 334 or 610
nm) although fluorescence has been rarely observed for phthalocyanines in aqueous media (Fig. 4b). In addition
to the aggregation study, the fluorescence emission of the compound 5k in water for a sequence of concentrations
in the range of 0.22×10-5-7.2×10-5 M was examined (Fig. 4c). Here one can notice that by increasing the
concentration, the emission of the compound 5k also increased. Notably, functionalization with acid moieties and
then post-modification through Ugi-4CR approach caused to decrease in fluorescence emission intensity that may
due to the quenching effect by the presence of peptide scaffolds (Fig. 4d). Additionally, the electronic absorbance
and emission changes of 5k with the concentration of 10-5 M in water on different days were studied. As can be
seen in Figure 4e, there is no changing in the electronic absorption of 5k even after 3 months. According to the
beer-lambert law, in the same conditions, the absorbance is directly related to the molar absorptivity coefficient
(ɛ) which related to the nature of the solvent. The compound 5k had a sharp emission band at 699 nm in 7 days
but the fluorescence emissions showed an obvious decrease in day 14 although it was constant for 3 months (Fig.
4f).
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Figure 4. The electronic absorption spectra (a) and fluorescence emission spectra (b) of phthalocyanine 5k in
DMSO and water, emission changes of 5k in water at different concentrations (M) (c), fluorescence emission
comparison of Co-phthalocyanine after functionalization and post-modification in DMSO (d), absorbance (e) and
emission (f) changes of 5k in water for the period of 90 days (Concentration:10-5 M
, λexc = 334 nm)
Since the purity of the biomaterials for biological application is the vital factor, to gain more confidence in the
purity of the pseudopeptide phthalocyanines, we profited from the capillary zone electrophoresis (CZE)
technique. CZE is an applicable part of the bioanalytical chemist toolbox for purity assay of proteins and peptides
based on their charge and size [44-47]. As can be shown in Figure 5, only one peak was found in each of the
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electropherograms for phthalocyanines 5e, 5f, 5k and 8a achieved by the CZE analysis that confirmed each the
synthesized phthalocyanines is pure.
Figure 5. The electropherograms of compounds 5e, 5f, 5k and 8a (Concentration:8×10-5 M
, λ= 334 nm)
Also to confirm that the observed peaks in the CZE electropherograms are related to the target compounds, the
analysis of the compound 5k was repeated at several concentrations and the results showed that the surface area
of the peak increased with increasing concentrations (Figure 6).
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Figure 6. The electropherograms of compound 5k at the different concentration (λ= 334 nm)
High water-dispersity and stability of compound 5k, also knowing the role of pseudopeptide scaffolds with amide
functionality in biocompatibility and bioactivities of organic structures [30, 48] encouraged us to study the
cytotoxicity effect and photodynamic activity of compound 5k. First of all, since an acceptable photosensitizer
for biomedical applications must be non-toxic or low-toxic in a normal situation, the concentration-dependent
dark cytotoxicity of compound 5k was investigated. The MTT assay after 24 h in dark revealed that compound
5k caused low dark cytotoxicity even at concentrations as high as 100 µg/mL (Fig. 7). On the other hand,
compound 5k was found to be phototoxic toward MCF-7 cells, upon exposure to light. As can be seen in Figure
7, concentrations higher than 10 µg/mL were found to have a pronounced effect on cell viability. In fact, even at
100 µg/mL concentration in the dark, we found more than 85% cell viability, whereas upon exposure to light the
viability decreased to 26 %.
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Figure 7. Toxicity of compound 5k toward MCF-7 cells in the absence and presence of light exposure
In the next step, the effect of the Ugi-4CR modification on the cell viability before and after the functionalization
of compound 5k were examined. In the dark conditions, no obvious cytotoxicity by MTT assay was detected for
Co-phthalocyanine tetracarboxylic acid and compound 5k after 24 h of incubation. More viability of cells for
compound 5k might be due to the presence of the pseudopeptide scaffolds and agglomeration of tetra-acid
compound which rise from their low solubility. The results for irradiated samples revealed that the photodynamic
effect was higher for compound 5k and it is more toxic to cells in comparison with the attributed tetra-acid
derivative which showed lower cytotoxic effect after exposure to light. The comparative viability of the cell in
100 µg/mL concentrations showed higher differences (26% for compound 5k and 52% for tetra-acid compound)
(Fig. 8). The results confirmed that the photosensitizing efficiency of phthalocyanines was increased through the
modification via Ugi-4CR.
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Figure 8. Cytotoxicity of Co-phthalocyanine tetracarboxylic acid (Acid Pc) and compound 5k in dark and after 1
h light irradiation and photosensitization at different concentrations
In continuation, the effect of energy density was studied to identify the appropriate laser fluency on cell death of
MCF-7 cell-lines. The sample that containing 100 µg/mL of compound 5k were choose and incubated for 24 h
then, the dose-response was explored. As can be seen in Figure 9, when compound 5k irradiated with light doses
ranging from 5 to 20 J cm-2, a decrease of cell viability from 44% to 26% was observed which confirmed the
dose-dependent pattern.
Figure 9. Cell viability of MCF-7 cells treated with phthalocyanine 5k and exposed to visible light by different
irradiation dose (Concentration: 100 µg/mL)
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It is well known that reactive oxygen species (ROS), oxidize tissue biomolecules, creating oxidative stress which
1
caused to kill the tumor cells and O2 has been proven to be the main cytotoxic agent of PDT. As we know,
phthalocyanine derivatives show high molar extinction coefficient in the 600–700 nm region of the visible
spectrum besides a long lifetime of triplet excited state to efficiently produce singlet oxygen [49]. Additionally,
most of the phthalocyanines researches consider the singlet oxygen species as the major cytotoxic species
involved in the therapy [50]. Thus, we decided to investigate the singlet oxygen generation of compound 5k by
an indirect method with utilization of anthracene-9,10-dipropionicacid (ADPA) as a molecular scavenger. The
reaction of ADPA with 1O2 directed to an endoperoxide form, which causes a decrease of the optical densities. A
time-dependent production of singlet oxygen was found for compound 5k (Fig. 10). As can be inferred from the
curve, the optical density of ADPA decreased over the light irradiation, indicating the generation of singlet
oxygen. On the other hand, recent studies confirmed that electrons are transferred through proteins or peptides so
they could act as electron donors [51]. This fact could be another reason for more excitation of oxygen that
occurred during the irradiation of psuodopeptide-modified-phthalocyanines.
Figure 10. Change of optical intensity of ADPA’s absorption peak at 378ꢀnm over time when the mixture
containing ADPA and compound 5k was irradiated by light (5k concentration: 10−5ꢀM), Neat ADPA water
solution was used as a control
Since amino acids and proteins show enhanced cellular uptake efficiency and tumor targeting ability [52], we
decided to examine the time-dependent cellular uptakes of Co-phthalocyanine tetracarboxylic acid and compound
5k by MCF-7 cells. As can be seen in Figure 11, the uptakes increase gradually with the increase of time.
Notably, after the incubation for 24 h, the cellular uptake of compound 5k is significantly higher than tetra-acid
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derivative (97%). This result may be attributed to the presence of pseudopeptide scaffolds decorated the
phthalocyanines that can interact with cell membrane phospholipids.
Figure 11. Cellular uptake of Co-phthalocyanine tetracarboxylic acid and compound 5k by MCF-7 cells versus
incubation times
Based on the above results and discussions, concluded that carboxamide scaffolds with amide bonds synthesized
as a phthalocyanine substituent through Ugi-4CR modification tend to reduce aggregation and increase
monomerization of the phthalocyanines which rise from the high stability of carboxamide-modified
phthalocyanines in water and the other solvents. This feature caused to the better direction to cells, energy
transfer to the oxygen molecule on an excited singlet state and facilitation of the singlet oxygen species
production which results in greater cellular uptake and greater efficiency of killing the MCF-7 cells. Although
further investigation will be needed for clearance of the exact biological and photodynamic activity of the
phthalocyanines which modified through Ugi-4CR, we believe that carboxamide-modified-
metallophthalocyanines have the ability to serve as potential photosensitizers in cancer therapy.
4.
Conclusions
A series of carboxamide-modified-metallophthalocyanines by utilization of Ugi four-component reaction were
synthesized and characterized. Using Ugi-4CR approach opens up a new avenue to the realm of synthetic routes
for one-pot modification of phthalocyanines. Results revealed that this post-modification cause unexpected high
stability and dispersity of metallophthalocyanines in common solvents and especially in water, which opens a
new window to the high value-added applications of metallophthalocyanines. Additionally, the hydrophilic and
nonaggregated carboxamide-modified-phthalocyanine 5k showed good ability to photosensitize the species in the
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visible light and high cellular uptake which demonstrates the potential of these novel pseudopeptide-modified-
phthalocyanines as a photosensitizer for photodynamic therapy of cancer disease. These results highlight the
potential of using multicomponent reactions to make functional phthalocyanines with unique properties which
can be utilized as promising carriers and sensors in medicinal applications.
Acknowledgements
We gratefully acknowledge financial support from the Shahid Beheshti University and the Iran National Elites
Foundation (INEF).
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Table of Contents
One-step synthesis of highly dispersible phthalocyanines containing carboxamide moieties via
four-component Ugi reaction as a potent photosensitizer was demonstrated for the first time.
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Highlight
•
•
•
One-step synthesis of phthalocyanines containing carboxamide moieties via Ugi four-component
reaction was demonstrated for the first time.
Modified metallophthalocyanines showed high stability and dispersity in common solvents and
especially in water.
Novel pseudopeptide-modified-phthalocyanines showed potential activity as a photosensitizer for
photodynamic therapy of cancer disease.