Chlorin derivatives sterically-prevented from self-aggregation with high antitumor activity for photodynamic therapy

Article abstract of DOI:10.1016/j.dyepig.2017.06.011

In this study two new chlorin derivatives sterically prevented from aggregation were synthesised by the Diels-Alder reaction between protoporphyrin IX dimethyl ester and 1-(2-hydroxyethyl)maleimide. The compounds were fully characterised by ¹H NMR, ¹³C NMR, UV-Vis and high-resolution mass spectroscopy (HRMS) and their photochemical properties such as singlet oxygen quantum yield (?₀), fluorescence quantum yield (?_f) and photodegradation were also evaluated. Furthermore, the partition coefficient (log P) revealed that these compounds present amphiphilic properties. Studies of the photodynamic action in tumour cells (HEp-2 and HeLa) and non-tumour cells (Vero) were also performed in order to confirm the photodynamic therapy (PDT) activity that was indicated by the preliminary photophysical studies. Those phototoxicity results were 55–77% higher than the results obtained with the commercial photosensitiser verteporfin. Finally, cytotoxic assays were performed with the new photosensitiser candidates and cell death was determined using fluorescence microscopy, which provided information about the mechanisms of cell death. In general, we have obtained improved and accessible compounds for PDT studies, as highlighted by the research presented here.

Full text of DOI:10.1016/j.dyepig.2017.06.011

Accepted Manuscript
Chlorin derivatives sterically-prevented from self-aggregation with high antitumor
activity for photodynamic therapy
Irwin A.P. Linares, Kleber T. de Oliveira, Janice Rodrigues Perussi
PII:
S0143-7208(17)30665-4
10.1016/j.dyepig.2017.06.011
DYPI 6032
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 30 March 2017
Revised Date: 25 May 2017
Accepted Date: 7 June 2017
Please cite this article as: Linares IAP, de Oliveira KT, Perussi JR, Chlorin derivatives sterically-
prevented from self-aggregation with high antitumor activity for photodynamic therapy, Dyes and
Pigments (2017), doi: 10.1016/j.dyepig.2017.06.011.
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Chlorin derivatives sterically-prevented from self-aggregation with high
antitumor activity for photodynamic therapy
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Irwin A. P. Linaresa,Kleber T. de Oliveirab and Janice Rodrigues Perussia
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aInstituto de Química de São Carlos, Universidade de São Paulo, São Carlos-
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SP, Brazil
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b Departamento de Química, Universidade Federal de São Carlos, São Carlos-
SP, Brazil
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Corresponding author address:
Instituto de Química de São Carlos, Universidade de São Paulo, Av.
Trabalhador São-Carlense, 400. São Carlos-SP, Brazil.
13560-970
*janice@usp.br
Abstract
In this study two new chlorin derivatives sterically prevented from
aggregation were synthesised by the Diels-Alder reaction originated from
protoporphyrin IX dimethyl ester and 1-(2-hydroxyethyl)maleimide. The
compounds were fully characterised by H-NMR, 13C-NMR, UV-Vis and high-
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resolution mass spectroscopy (HRMS) and their photochemical properties such
as singlet oxygen quantum yield (φ0), fluorescence quantum yield (φ ) and
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photodegradation were also evaluated. Furthermore, the partition coefficient
(log P) revealed that these compounds present amphiphilic properties. Studies

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of the photodynamic action in tumour cells (HEp-2 and HeLa) and non-tumour
cells (Vero) were also performed in order to confirm the photodynamic therapy
(PDT) activity that was indicated by the preliminary photophysical studies.
Those phototoxicity results were 55–77% higher than the results obtained with
the commercial photosensitiser verteporfin. Finally, cytotoxic assays were
performed with the new photosensitiser candidates and cell death was
determined using fluorescence microscopy, which provided information about
the mechanisms of cell death. In general, we have obtained improved and
accessible compounds for PDT studies, as highlighted by the research
presented here.
Keywords: Chlorins, aggregation, amphiphilic character, photobleaching,
phototoxicity, mechanism of cell death

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Highlights
New chlorin derivatives, sterically prevented from aggregation, were
synthesised by the Diels-Alder reaction.
Chlorin derivatives exhibit no aggregation in an aqueous medium (phosphate-
buffered saline).
The cytotoxicity of the chlorin derivatives in the tumour cells is higher than
verteporfin.
The mechanism of cell death occurs via an apoptotic process in three cell lines
after irradiation.

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1. Introduction
Photodynamic therapy (PDT) is a non-invasive therapeutic method that
induces cell death of tumour cells by apoptosis or necrosis. The therapy uses
dyes or pigments (photosensitiser) that are capable of absorbing visible light
energy and transferring part of the absorbed energy to adjacent molecules. The
transfer of energy is usually to the oxygen (O2) molecules which are present in
the tissues. This process leads to a series of photochemical reactions that
produce singlet oxygen, which is extremely toxic, and other reactive oxygen
species and free radicals, thus resulting in a strong cytotoxic effect and tumour
cell death [1–5].
Different types of photosensitisers, for example, porphyrin [6–8], chlorin [9–
11] and phthalocyanine [12, 13] derivatives, have been used in PDT studies.
Hematoporphyrin derivative (HpD) and Photofrin® are considered to be part of
the first generation of photosensitizers used in the therapy against cancer.
However, the strong aggregation of these photosensitizers in physiologic
medium, the low singlet oxygen quantum yields, and the short penetration of
red light (typically 630 nm) has resulted in limited use of these light-absorbing
molecules. Those limitations have led to the development of a new generation
of compounds with improved physical, chemical and photobiological properties
[14].The most recent generation of compounds was developed to exhibit
characteristics such as intense absorption bands at longer wavelengths and
shorter skin photosensitivity periods, which would enable the treatments to
reach deeper tissues compared to the first generation of photosensitisers [15].

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Chlorin derivatives, when compared with porphyrins, have a reduced double
bond in one β-position of one tetrapyrrolic ring [16, 17], resulting in a red-shifted
absorption band (640 to 680 nm) and allowing deeper light penetration in the
tissue [18–20]. Several chlorins have been approved for clinical use: Foscan®,
talaporfin (NPe6) and verteporfin (Visudyne®) [21, 22]. Visudyne® is derived
from the natural protoporphyrin IX (PpIX) (Figure 1) [23–25]. It is used in the
treatment of age-related macular degeneration, choroidal neovascularization
due to secondary pathological myopia, and suspected ocular histoplasmosis
[26, 27]. In the last 15 years, this photosensitizer has been used as an efficient
compound in PDT studies and in clinical treatments for macular degeneration
[28].
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Fig. 1. Protoporphyrin IX analogues.
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Several approved photosensitisers, for example, Photofrin® [29, 30], Foscan
[31, 32], Laserphyrin [33, 34] and SnET2 [35, 36], are limited in their ability to be
applied to PDT because of skin photosensitivity caused by the slow clearance
of photosensitisers from the organism, low solubility, penetration of light and low
tumour-targeting efficacy [37]. Therefore, there are great challenges for

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synthetic organic chemistry researchers to improve photosensitisers for PDT
treatments.
Different approaches have been used for the synthesis of chlorins derived
from PpIX dimethyl ester, which has resulted in better solubility and non-
aggregation in physiological medium [38–41]. Recently, chlorin derivatives
synthesised by the Diels-Alder reaction between diene-appended porphyrins
and maleimides yielded L-shape chlorin derivatives. These L-shape structures
prevent the aggregation caused by the strong attraction between the π-systems
of the polyaromatic heterocycles, thus resulting in molecules with high singlet
oxygen quantum yields [39, 41]. Therefore, in this study, we carried out the
synthesis of a new chlorin derivatives by the Diels-Alder reaction between the
PpIX dimethyl ester and different maleimides. We characterised the new
photosensitiser derivatives and investigated their photodynamic activity,
cytotoxicity, phototoxicity, photobleaching, cell death and accumulation in cells.
2. Materials and Methods
2.1 Reagents and measurements
All reagents were of analytical grade and were purchased from Aldrich or
national suppliers. Solvents and reagents that were used for the synthesis were
only used after purification according to standard procedures [42]. Ultrasound
irradiation was used to deoxygenate the toluene for the Diels-Alder reactions.
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1H and C-NMR spectra were recorded on a Bruker Avance 400 spectrometer
at 400.13 MHz and 100.13 MHz, respectively, using CDCl3 as the solvent and

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tetramethylsilane (TMS) as the internal reference. The chemical shifts were
expressed in parts per million (ppm) and the coupling constants (J) in hertz
(Hz). High-resolution mass spectrometry (HRMS) analyses were obtained using
the ESI-LTQ orbitrap (Thermo Scientific) and Xcalibur 2.1 analysis processing
software. The UV-Vis absorption spectra analyses of the chlorin derivatives
were performed using a Perkin Elmer Lambda 25 spectrophotometer.
Preparative purifications were carried out by using column chromatography
and silica gel 200 and 400 mesh, and preparative thin-layer chromatography
(TLC) and analytical TLC were conducted on TLC Silica gel 60 aluminium
sheets (1mm thick) (Merck). The light-source for photobleaching was a 660 nm
diode laser (FTC 500, OPTO, Sao Carlos-SP, Brazil) in the PDT/iPDT mode.
The partition coefficients and fluorescence measurements of the chlorin
derivatives were performed using a spectrophotometer (HITACHI U-2800,
Japan) and a spectrofluorometer (HITACHI F-4500, Japan), respectively, with
10 mm path length quartz cuvettes. The light-source used for the photodynamic
treatment was an irradiation table called Biotable that contained a series of 660
± 10 nm LEDs with a fluence rate of 27.6 mWcm−2 (LAT, IFSC-USP). The
absorbance measurements of the formazan dye after the phototoxic assays
were read in a Biotek-Synergy HT spectrophotometer. To visualise cell death, a
fluorescence microscope (Olympus BX41) was used with the following
parameters: 20X magnification, excitation filter of 460/90 nm, dichromatic mirror
of 50 nm and barrier filter of 520 nm. Images were captured with an Olympus
DP72 digital camera.

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2.2 Synthesis of 4,10-Dioxatricyclo[5.2.1.0]dec-8-ene-3,5-dione (3)
The synthesis of 4,10-Dioxatricyclo[5.2.1.0]dec-8-ene-3,5-dione (3) was
carried out following methods reported in a previous publication [43]. In a 100
mL round-bottom flask, maleic anhydride (1) (10.00 g, 102 mmol), toluene (50
mL), and furan (2) (7.5 mL, 103.1 mmol) were stirred at room temperature
under an inert atmosphere for 24 h. The product was obtained after filtration
and washing of the precipitate with cold diethyl ether (80% yield, 13.57 g, 81.73
mmol). 1H-NMR (400 MHz, DMSO-d6) δ 6.56 (s, 2H), 5.32 (s, 2H), 3.29 (s, 2H);
13C-NMR (100 MHz, DMSO-d6) δ 172.0, 137.3, 82.1, 49.5.
2.3 Synthesis of 4-(2-Hydroxy-ethyl)-10-oxa-4-aza-tricyclo [5.2.1.0]-dec-8-ene-
3,5-dione (4)
The synthesis of 4-(2-Hydroxy-ethyl)-10-oxa-4-aza-tricyclo [5.2.1.0]-dec-8-
ene-3,5-dione (4) was carried out following methods reported in a previous
publication [44]. A 50 mL two-neck round-bottom flask was adapted with a reflux
condenser, the intermediate 3 (5.0 g, 30.1 mmol) was combined with dry
methanol (15 mL). After stabilizing the magnetic stirring and the reaction
temperature at 0°C, ethanolamine was added dropwise (1.84 g, 30.12mmol).
The mixture was refluxed for 24 h. The methanol was then distilled off under
reduced pressure, and the product was recrystallised from diethyl ether at -
20°C. The crystals were collected by filtration and washed with cold diethyl
ether in 61% yield. (3.81 g, 18.21 mmol). 1H-NMR (400 MHz, DMSO-d6) δ 6.53

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(s, 2H), 5.10 (s, 2H), 3.39 (s, 4H), 2.90 (s, 2H); 13C-NMR (100 MHz, DMSO-d6)
δ 176.9, 136.9, 80.7, 57.7, 47.6, 41.0.
2.4 Synthesis of 1-(2-Hydroxyethyl)-1H-pyrrole-2,5-dione (5)
The synthesis of 1-(2-Hydroxyethyl)-1H-pyrrole-2,5-dione (5)was carried out
following methods reported in a previous publication [43]. In a50 mL round-
bottom flask adapted with a flux condenser and magnetic stirring, the
intermediate 4 (2.30 g, 10.99 mmol) was combined with toluene (30 mL). The
reaction was refluxed for 7 h. The resulting mixture was immediately filtered,
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and the product crystallised from toluene in 81% yield(1.25 g, 8.87 mmol). H-
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NMR(400 MHz, DMSO-d6) δ7.01(s, 2H), 4.78 (br, 1H), 3.46 (br, 4H); C-NMR
(100 MHz, DMSO-d6) δ171.1, 134.5, 57.9, 39.9.
2.5 Synthesis of protoporphyrin IX dimethyl ester (7)
The synthesis of protoporphyrin IX dimethyl ester (7) was carried out
following methods reported in a previous publication [39]in75% (1.59 g, 2.70
mmol).1H-NMR (CDCl3, 400 MHz) δppm: -3.69 (s, 2H), 3.28 (t, 4H, J = 7.7 Hz);
3.56 (s, 3H); 3.61 (s, 3H); 3.62 (s, 6H), 3.65 (s, 3H); 3.70 (s, 3H); 4.34 (t, 4H, J
=7.7 Hz); 6.16 (d, 2H, J =11.5 Hz); 6.35 (d, 2H, J =17.8 Hz), 8.27 (dd, 2H, J
=17.9 Hz, J =11.3 Hz); 9.92 (s, 3H); 9.93 (s, 1H); 10.05 (s, 3H); 10.06 (s, 3H).

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2.6 Diels-Alder reaction between protoporphyrin IX dimethyl ester (7) and 1-(2-
Hydroxyethyl)-1H-pyrrole-2,5-dione (5)
The syntheses were carried out following the modification of a procedure
that has been previously published [39]. In a glass pressure tube, were added 7
(60 mg, 0.1 mmol), dry toluene (5 mL) and 5 (282 mg, 2 mmol). The reaction
was stirred at 125oC for 24 h. The reaction was monitored by TLC and UV-Vis
spectroscopy in order to verify the consumption of the starting material. The
reaction products were purified by flash column in silica gel, and preparative
TLC chromatography using chloroform, ethyl acetate and methanol in a 5:4:1
ratio as eluent. Two chlorin derivatives, 8-a and 8-b, were crystallised from
CH2Cl2 and hexane in 33 and 34%, respectively.
21,22[N,N-dicarbonil-N-4-(2-hydroxyethyl)phenyl]-13,17-bis[2-
(methoxycarbonyl)ethyl]-2,7,12,18-tetramethyl-8-vinyl-2,21,22,23-
tetrahydrobenzo[b]porphyrin (8a): 1H-NMR (CDCl3, 400 MHz) δ (ppm): -2.49 (s,
2H,); 2.01 (s, 3H); 3.13 - 3.17 and 3.20 - 3.24 (m, 9H); 3.40 (s, 3H); 3.42 - 3.45
(m, 2H); 3.50 (s, 3H); 3.54 (s, 3H); 3.66 and 3.69 (s, 6H); 3.75 - 3.79 (m, 1H);
4.18 (t, 2H, J=7.8 Hz); 4.31 (t, 2H, J=7.7 Hz), 4.51 (d, 1H, J=8.4 Hz); 6.15 (dd,
1H, J=1.4 Hz, J=11.5 Hz); 6.34 (dd, H, J=1.4 Hz and J=17.8 Hz); 7.35 (t, 1H,
J=5.3 Hz); 8.17 (dd, 1H, J=17.8 Hz and 11.5 Hz); 9.07 (s, 1H); 9.30 (s, 1H);
9.70 (s, 1H), 9.86 (s, 1H); 13C-NMR (CDCl3, 100 MHz) δ (ppm): 11.4, 11.6; 12.3,
21.5,21.9, 25.6, 26.5,36.6, 37.0, 38.5, 50.1, 51.7, 51.8, 52.3, 90.4, 93.4, 97.9,
99.8, 115.6, 121.4, 129.2, 129.8, 130.9, 132.6, 133.7, 133.9, 136.2, 136.5,
138.3, 139.6, 149.5, 151.0, 151.3, 152.2, 165.9, 173,4, 173.8,174.8,178.5;

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HRMS (ESI-LTQ orbitrap velos): m/z calculated for [M+H]+ 732.33716, found
732.33918.
21,22[N,N-dicarbonil-N-4-(2-hydroxyethyl)phenyl]-8,12-bis[2-
(methoxycarbonyl)ethyl]-2,7,12,18-tetramethyl-8-vinyl-2,21,22,23-
tetrahydrobenzo[b]porphyrin (8b): 1H-NMR (CDCl3, 400 MHz) δ (ppm): -2.45 (s,
2H,); 2.01 (s, 3H); 3.13 - 3.18 and 3.20 - 3.25 (m, 9H); 3.41 (s, 3H); 3.41 (s,
3H);3.45 (s, 3H); 3.50 - 3.57 (m, 2H); 3.62 (s, 3H); 3.64 and 3.65 (s, 6H); 3.74 e
3.79 (m, 1H); 4.17 (t, 2H, J=7.8 Hz); 4.30 (t, 2H, J=7.7 Hz), 4.50 (d, 1H, J=8.4
Hz); 6.12 (dd, 1H, J=1.4 Hz, J=11.5 Hz); 6.34 (dd, H, J=1.4 Hz, J=17.8 Hz);
7.34 (t, 1H, J=5.4 Hz); 8.13 (dd, 1H, J=6.3 Hz and 17.8 Hz); 9.23 (s, 1H); 9.24
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(s, 2H), 9.69 (s, 1H), 9.76 (s, 1H); C-NMR (CDCl3, 100 MHz) δ (ppm): 11.2,
11.7, 12.4, 21.6, 21.9, 25.6, 26.6, 36.7, 37.1, 38.6, 50.1, 51.7, 51.8, 52.2, 89.9,
94.1, 98.4, 99.4, 116.0, 120.9, 129.9, 130,4, 131.2, 133.3, 133.8, 135.9, 136.8,
137.3, 138.0, 139.7, 149.6, 150.6, 151.4, 152.8, 165.6, 173.4, 173.8, 174.7,
178.5; HRMS (ESI-LTQ orbitrap velos): m/z calculated for [M+H]+ 732.33698,
found 732.33918.
2.7 Singlet oxygen quantum-yield measurement
The singlet oxygen quantum yields (Φ∆) of 8-a and 8-b were determined
from the intensity of the phosphorescence decay at 1270 nm [45]. The data
were recorded with a time-resolved NIR fluorometer (Edinburgh Analytical
Instruments) at the Departamento de Bioquímica, Instituto de Química,
Universidade de São Paulo. The NIR fluorimeter was equipped with an Nd:YAG

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LASER (Quantel) and a system optical parameter oscillator (OPO) allowing to
choose the desired wavelength in which the samples were excited at 420 nm (5
ns pulse). The emitted light was passed through silicon, an interference filter
and a monochromator before detection with an NIR photomultiplier
(Hammamatsu Co. R5509). The singlet oxygen lifetime was determined by
applying first-order exponential fittings to the phosphorescence decay curve.
The Φ∆ values were obtained using verteporfin as a reference (Φ∆=0.77) [46].
This procedure was performed in triplicate.
2.8 Fluorescence quantum yield measurements
The fluorescence quantum yields were determined using Rhodamine B in
ethanol as a reference (Φf = 0.65) [47]. The solutions of the chlorin derivatives,
8a and 8-b, were prepared in DMSO from stock solutions. The absorbance was
maintained at approximately 0.04 and 0.05 to avoid the inner filter effect.
Excitation of both chlorins was performed at 510 nm, and the emission spectra
were recorded. This procedure was performed in triplicate.
2.9 Photobleaching studies
A solution of 8-a or 8-b was prepared in CH2Cl2 with an absorbance value
of approximately 1. The solution was irradiated at 661 nm for 10 minutes, with 1
min intervals, using a laser potency of 50 mW. The absorbance spectra were
monitored after each irradiation interval in order to observe whether
photobleaching of the chlorin derivatives occurred [17].

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2.10 Determination of the partition coefficients in a solution of 1-octanol and
PBS
The partition coefficients were determined at room temperature by a spread
shake-flask method [48].The phosphate-buffered saline (PBS) and 1-octanol
were mixed for five days to promote solvent saturation in both phases.
Compounds 8-a or 8-b were dissolved in the organic phase. Subsequently,
equal amounts of PBS, 1-octanol and photosensitisers at 1.0×105 mol L-1 were
mixed for 3 h under magnetic stirring and protection from light. The phase
separation was followed by 10 min of centrifugation at 1000 rpm. The UV-Vis
absorption spectra were measured before and after partitioning, and the
differences were measured at the Soret band.
2.11 Aggregation study
Tetrapyrrolic molecules, such as chlorin derivatives, frequently aggregate in
an aqueous medium due to the strong attractive interactions between the π-
systems of the polyaromatic compound and the low interaction with the solvent.
To evaluate the aggregation process, compounds 8-a or 8-b and verteporfin,
were evaluated in different concentrations. Each sample solution was prepared
in DMSO and PBS in order to determine the presence or absence of
aggregation in solution. UV-Vis analyses were monitored for changes in the λmax
band of the spectra.

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2.12 Phototoxicity assay
In this study, two cell lines were used: HeLa cell line (CCL-2TM, ATCC,
USA), a human cervical carcinoma cell, and HEp-2 (CCL-23TM), an epithelial
tumour-cell line. These cell lines were grown in Iscove’s Modified Dulbecco’s
media (Sigma-Aldrich) and were supplemented with 10% foetal bovine serum
(FBS, Cultilab) and 0.01% antibiotics (penicillin and streptomycin, Cultilab) in 75
cm2 cell culture flasks incubated at 37°C and 5% CO2. The cells were seeded
(5x104 cells mL-1) in 96-well plates and underwent photodynamic treatments.
Cell viability was evaluated using the MTT (3,4,5-dimethylthiazol-2,5-diphenyl
tetrazolium bromide) assay [49]. This method is based on the ability of viable
cells with active metabolisms to use mitochondrial dehydrogenase enzymes to
cleave the tetrazolium rings of the yellow MTT and form an insoluble precipitate
(formazan crystals) inside of the cells [50]. Cells were incubated for 2 h in the
absence of light with the photosensitisers and different concentrations of 8-a
and 8-b. Subsequently, the cells were irradiated at 660 ± 10 nm using the
Biotable at two time intervals in order to obtain two different light doses: 3.0 and
6.0 J cm-2.
At 48 h post-irradiation, the medium was removed and the cells were
incubated for 3 h with an MTT solution in culture medium added to each well of
the plate. Following the incubation period, the solution that contained MTT was
removed and the cells washed with 100 µL PBS. The formazan crystals were
solubilised in 100 µL of absolute ethanol and 100 µL of isopropyl alcohol. The
absorbance was read at 570 nm. The cell survival rate (%) was assessed in
relation to the photosensitiser concentration. The average inhibitory

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concentrations (IC50) of the photosensitisers were determined. We also carried
out an analysis of controls with no treatment, cells treated only with
photosensitisers (not irradiated), and cells treated only with irradiation (without
photosensitiser).
2.13 Cell death detection by acridine orange/ethidium bromide (AO/EB)
staining
HeLa and Hep-2 cells were seeded (5x104 cells mL-1) in sterile chamber
slides with culture medium. After 24 h the cells were incubated with different
concentration of 8-a or 8-b for 2 h. After the incubation period, the cells were
washed with PBS, maintained in culture medium, and then irradiated with a light
dose of 3 or 6 J cm-2. After 48 h, the cells were stained with a mixture of
ethidium bromide and acridine orange (100 µg mL-1). The labelled cells were
visualised by fluorescence microscopy [51].
2.14 Statistical Analysis
The data from the biological assays were analysed by one-way analysis of
variance (ANOVA) followed by Student-Newman-Keuls multiple-comparisons
test (Graph Pad InStat software). Differences were considered significant when
p ≤ 0.05.

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3. Results and discussion
3.1 Chlorin derivatives syntheses
The preparation of protoporphyrin IX dimethyl ester (PpIX) (7), described in
scheme 1, began with a process of elimination of two hydroxyl (-OH) groups of
hematoporphyrin (Hp) (6). Then, an esterification in the presence of methanol
and sulfuric acid (H2SO4) (5%) was performed maintaining the protection from
light. The final product 7 was obtained after solvent evaporation and a simple
crystallization from CHCl3:MeOH (1:3). Compound 7 was isolated in 79% yield
1
and characterised by H-NMR and UV-Vis in order to confirm its identity and
purity.
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Scheme 1. Synthesis of protoporphyrin IX dimethyl ester from hematoporphyrin.
After obtaining 7, the synthesis of the dienophile maleimide 5 was
performed, as described in scheme 2. For this purpose, the Diels-Alder adduct,
3, was obtained from maleic anhydride (1) and furan (2) (80% yield) [43]. Next,
ethanolamine was added to compound 3, yielding the maleimide 4 (61%) [44].
Compound 5 was obtained after a retro-Diels-Alder reaction in 81%yield [43].
The Diels-Alder reaction between 5 and 7 was performed in glass-pressure

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tubes (toluene at 125oC, 24h), and catalytic amounts of BHT were used to avoid
polymerised by-products (Scheme 3).
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Scheme 2. Synthesis of N-(hydroxyethyl)maleimide.
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The chlorin derivatives 8-a and 8-b were separated by preparative TLC over
silica at a 1:1 ratio, resulting in 68% overall yield. These compounds were fully
characterised by 1H-NMR, 13C-NMR, HRMS and UV-Vis.
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Scheme 3. Synthesis of chlorins 8-a and 8-b.
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3.2 Singlet Oxygen Quantum Yields (Φ∆) measurements
The singlet oxygen quantum yields of 8-a, 8-b and verteporfin (standard)
were determined from equation (1). This term is used to describe the
measurement of the efficiency in which molecules are able to absorb light and
convert molecular oxygen (3O2) to singlet oxygen (1O2) [52].
ꢇ
ꢈ
409
Ф∆ = ꢀꢁꢂꢃꢄꢅ ꢆꢃ ꢃꢄꢅ Ф∆ꢉꢊꢋ (1)
ꢀꢁꢂ ꢇ ꢈ
ꢆꢃ ꢃꢄꢅ ꢆꢃ
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Where (Φ∆) represents the singlet oxygen quantum yield of the sample and
(Ф∆ꢉꢊꢋ) is the standard (Verteporfin) singlet oxygen quantum yield. AbsPS and
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AbsStd are the absorbance values of the photosensitiser samples and the
ꢉꢊꢋ
standard. ꢌꢍꢁꢂ and ꢌ
are the maxima intensity of phosphorescence decay
ꢍꢁꢂ
curve for the sample and the standard. ꢎꢏꢉ and ꢎꢉꢊꢋ are the single-oxygen
lifetime of the sample and the standard, respectively. The solutions of 8-a, 8-b
and verteporfin were solubilised in DMSO. The chlorins 8-a and 8-b showed
similar singlet oxygen quantum yields when compared to verteporfin (Table 1).
Table 1. Quantum yield values of singlet oxygen formation (Φ∆) and
fluorescence (Φf) of verteporfin and of chlorins 8-a and 8-b. Data are expressed
as average ± SD.
(Ф∆)
(Фf)
Verteporfin
0.77 ± 0.02[46]
0.75 ± 0.02
0.73 ± 0.03
0.0085 ± 0.0006 α
0.0096 ± 0.0003
0.0110 ± 0.0006*, α
8-a
8-b
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Experiment was performed in triplicate at 20oC
α Statistically significant difference from CHL-OH-A (p<0.05)
* Statistically significant difference from Verteporfin (p<0.01)

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3.3 Fluorescence Quantum yields (Φf) measurements
The fluorescence quantum yield is defined as the number of emitted
photons relative to the number of absorbed photons and was determined using
equation (2) [53].
ꢓ
ꢑ .ꢀ
.ꢒ
ꢃꢄꢅ
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Фꢐ = Фꢐꢉꢊꢋ
(2)
ꢓ
ꢑ
ꢃꢄꢅ
.ꢀ .ꢒ
ꢃꢄꢅ
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Where (Фꢔꢉꢊꢋ) represent the fluorescence quantum yield of the standard. F and
FStd represent the areas under the fluorescence emission curves for the sample
and standard, respectively. A and AStd are the absorbance of the chlorin
derivatives and the standard acquired from the same amount of solution used to
measure the fluorescence emission spectra of both. The parameter, n and nStd
are the refractive indices for the used solvents. Solutions of each compound, 8-
a, 8-b, were prepared in DMSO and exhibited emissions at 676 nm with
quantum yield of fluorescence values comparable to the verteporfin (standard)
(table 1).
3.4 Photobleaching studies
Photostability is a physicochemical property important to photosensitisers in
general, since during the irradiation time, the molecule may lose its
photodynamic activity due to chemical changes in its structure resulting in small
fragments (photo-products) that have no appreciable absorption in the visible
region [54]. The synthesised compounds 8-a and 8-b showed a slight

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photobleaching effect, depending on the irradiation time. A reduction of
approximately 5% in the absorbance band at both 404 nm (Soret band) and 668
nm (Q-band) was observed, thus allowing to consider these new chlorin
derivatives as photo-stable under visible light irradiation (Figure 2).
Irradiation
time
1,0
1.0
0.8
0.6
0.4
0.2
0.0
0 min
1 min
2 min
3 min
4 min
5 min
6 min
7 min
8 min
9 min
10 min
0,9
0,8
0,7
0,6
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wavelength (nm)
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700
800
wavelength (nm)
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Fig. 2. Photobleaching study of the chlorin 8-a (inset: expansion of Soret band).
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3.5 Partition coefficient in 1-octanol/PBS (Log P)
The lipophilicity of the synthesised chlorin derivatives and verteporfin was
determined from the partition coefficient. The log P is defined as the ratio of
concentrations of the photosensitiser at equilibrium between the organic and
aqueous phases [55, 56]. This feature is important since it allows the
incorporation of molecules within the cell. Compounds 8-a, 8-b and verteporfin
were solubilised in 1-octanol relative to the aqueous phase (PBS). The log P for
each compound was calculated according to an adaptation of the method first
proposed by Pooler and Valenzeno [57], because the studied photosensitisers
are sparingly soluble in the aqueous phase (Equation 3).

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(ꢀ
)
ꢕꢅ
470
log P = log (ꢀ
(3)
)
ꢗꢀ
ꢕꢖ
ꢕꢅ
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Where Aλo is the absorption solution before the partition and Aλd is the
absorption solution after the partition at wavelength 404 nm. Positive log P
values mean that the compound exhibit lipophilic characteristics (log P > 0) and
negative log P values mean that it shows a hydrophilic character (log P < 0)
(Table 2). The photosensitiser candidates 8-a and 8-b were less lipophilic than
verteporfin when the absorption spectrum before and after partition was
considered. This means that a number of molecules of chlorin (8-a or 8-b) were
transferred toward the aqueous phase, resulting in stronger amphiphilic
characteristics and displaying a stronger interaction with the polar environment
[58, 59]. This affinity allows the compounds to diffuse through the aqueous
medium and easily across the cell membrane.
Table 2: Partition coefficients using a mixture of 1-octanol and phosphate-buffer
saline (PBS). Data are expressed as average ± SD.
Photosensitiser
Partition Coefficient (Log P)
Verteporfin
8-a
1.61 ± 0.10
1.20 ± 0.04*
1.17 ± 0.03*
8-b
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* Statistically significant difference from Verteporfin (p<0.001)
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The chlorins showed amphiphilic characteristics and log-P values that were
approximately 25% lower than verteporfin (which presented lipophilic
characteristics). This result shows the importance of hydrophilic groups
attached to the chlorin structure, which increases the solubility in an aqueous
medium without changing the penetration power through the plasmatic

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membrane of cells. Once verteporfin has no hydrophilic groups in its structure, it
becomes more lipophilic and less soluble in aqueous medium. This result is
also consistent with the determined Φ∆ values.
3.6 Aggregation study
The concentrations of 8-a, 8-b and verteporfin analysed by UV-Vis
spectroscopy were on the same scale of those used in the cytotoxic assays.
Chlorin derivatives 8-a and 8-b exhibited no aggregation in an aqueous medium
(PBS) of up to 10 µmol L-1because the “L shape” of the molecules prevented
some π-stacking interactions (Figure 4A and 4B). Verteporfin showed a shift of
20 nm in the Soret band, which was caused when the strong interactions of the
π-orbitals of the aromatic ring induced aggregation (Figure 3) [40]. Once again,
the synthesised photosensitiser candidates appeared to be more adequate
options for PDT.

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Concentration
λ = 672
y = 15151.515x + 0.0012
R2 = 0.9962
0.20
0.16
0.12
0.08
0.04
0.00
Concentration
0.6
0.6
0.5
0.4
0.3
0.2
0.1
0.0
(uM)
1
λ
= 672
B
A
0.24
0.20
0.16
0.12
0.08
0.04
0.00
uM
y = 16490.91x - 0.0112
R2 = 0.9854
1
2
3
4
5
6
7
8
9
10
2
3
4
5
6
7
8
9
0.5
0.4
0.3
0.2
0.1
0.0
2.0x10-6 4.0x10-6 6.0x10-6 8.0x10-6 1.0x10-5
0.0
2.0x10-6 4.0x10-6 6.0x10-6 8.0x10-6 1.0x10-5
0.0
Concentration (uM)
Concentration (uM)
10
300
400
500
600
700
800
300
400
500
600
700
800
Wavelength (nm)
Wavelength (nm)
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Concentration
uM
λ = 690
0.25
0.20
0.15
0.10
0.05
0.00
0.6
C
y = 9868.06x - 0.0056
R2 = 0.9983
1
2
3
4
5
6
7
8
0.5
0.4
0.3
0.2
0.1
5.0x10-6 1.0x10-5 1.5x10-5 2.0x10-5
0.0
Concentration (uM)
9
10
15
20
0.0
300
400
500
600
700
800
Wavelength (nm)
Fig. 3. Aggregation study of (A) Chlorin 8-a (B) Chlorin 8-b and (C) Verteporfin in PBS
by UV-Vis spectroscopy.
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3.7 Phototoxicity assay
The phototoxic assays of 8a, 8-b and verteporfin were performed at two
light doses (3 and 6 J cm-2) with a light intensity of 27.6 mW cm-2 in the tumour
cell lines HEp-2 and HeLa and in the non-tumour cells Vero. These assays
were important for the determination of the medium inhibitory concentrations
(IC50) of each molecule using the MTT colorimetric method.

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A)
8-a
8-b
verteporfin
500
400
300
200
100
0
α
∆
*,
*
*
*
*
Hela
Hep-2
Vero
B)
8-a
8-b
verteporfin
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300
200
100
0
β
*,
,∆
*
*
*
*
Hela
Hep-2
Vero
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Fig. 4. Medium inhibitory concentrations (IC50) of the chlorins 8-a, 8-b and verteporfin
in HeLa, HEp-2 and Vero cells. The cells were incubated for 2 h, following by red
irradiation (660 nm) and light doses at (A) 3 and (B) 6 J cm-2. The MTT assay was
performed to determine the cell viability after 48 h of irradiation. The results are
expressed as means ± standard deviation (SD). *, α: statistically significant difference
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from Vero cells (p<0.001; p<0.05). ∆, β: statistically significant difference from HeLa
cells (p<0.01; p<0.001).

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Compounds 8-a, 8-b and verteporfin showed a lower cytotoxicity in normal cell
lines (Vero) for the two tested light doses (Fig. 4), indicating a good selectivity
for the tumour cell. This can be due to various factors such as an increase in
the permeability of tumour cells, which has been observed in many experiments
using animal models [60]. In addition, the location of photosensitisers within the
tumour cell may be different compared to normal cells because of the selective
retention of each cell such as those with low pH [61], over-expression of low-
density lipoprotein receptors [62] (LDL, apoB/E), and the over-expression of
some tumour associated macrophages [63]. The chlorin 8-a showed the lowest
IC50 value, which means the highest cytotoxicity in HeLa tumour cells (26.9 ±
1.9) at a dose 6 J cm-2, and they were 57% more cytotoxic than verteporfin; the
chlorin 8-b exhibited the lowest value of IC50 in the HEp-2 cells (9.2 ± 1.1), and
they were 77% more cytotoxic than verteporfin at the same light dose.
Furthermore, the tests were carried out in the dark for the three compounds (8-
a, 8-b and verteporfin) which demonstrated a very low cytotoxicity in the studied
cells. These achievements highlight the important phototoxic effects of newly
synthesised photosensitiser candidates and the promising possibility of using
these chlorins in PDT.
3.8 Cell death detection by acridine orange/ethidium bromide (AO/EB) staining
In order to determine the mechanism of cell death, cells were labelled with a
mixture of acridine orange and ethidium bromide after 48h irradiation. Cell
differentiation was analysed with a 20X objective and treated with 1x10-8 mol L-1
of chlorin 8-a or 8-b. The three cell lines (HeLa, HEp-2 and Vero) that were

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treated with the shorter irradiation time (3 J cm-2) showed a low percentage of
cell death (30%). However, those with the largest irradiation time (6 J cm-2)
showed a late apoptotic process (condensation and fragmentation of chromatin
stained in orange) and a few cells in a necrotic state (red nuclei stained) caused
by the loss of the integrity of the cytoplasm membrane (Figure 5B [a-c]).
Explanations for this difference include the time length of light incidence in
order to obtain different light doses using the same light source on molecules
photosensitiser (in ground state), leading to different number of molecules in the
excited triplet state (PS*) and to generation of reactive oxygen species and the
higher oxidative stress in tumour cells [64]. These species play an important
role in the intrinsic pathway of apoptosis signalling because mitochondrial DNA
damage is responsible for respiratory chain electron [65]. Morphological
information observed by differential staining with AO/EB suggests that 8-a and
8-b induce cell death by apoptosis in all the studied cell lines. However, the
route of induction of apoptosis is not completely understood.

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B
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Fig. 5. Fluorescence microscopy images of three cell lines incubated with 1x10-8 mol L-
1 chlorin 8-b followed by red light irradiation (660 nm). (A) Untreated (control) cells: (a)
HeLa (b) HEp-2 and (c) Vero. (B) Apoptotic cells after 48h of treatment (6 J cm-2): (a)
HeLa (b) HEp-2 and (c) Vero, respectively.
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4. Conclusion
In this study, we synthesised two chlorin derivatives, 8-a and 8-b, which
have an L-shaped structure and are self-prevented from aggregation in
aqueous mediums. The photophysical and photochemical properties - for
example, singlet oxygen quantum yields, low photobleaching and partition
coefficient (log P) made 8-a and 8-b better photosensitiser candidates for PDT
compared to verteporfin. The cytotoxicity of the chlorin derivatives in the tumour
cells was 60% higher than verteporfin, which allowed lower concentrations to be
used to have a targeted effect. Fluorescence microscopy showed that the
mechanism of cell death occurs by a highly apoptotic process in the three cell
lines after irradiation. Therefore, we suggest that the new chlorin derivatives 8-a
and 8-b exhibit strong potential as photosensitizer, and they should be
evaluated further in animal models and other PDT studies.
Acknowledgements
The authors wish to thank the CNPq, FAPESP (2015/21110-4), CEPOF
(2013/07276-1) and CAPES for financial support and fellowships, as well as
Prof. Ilce Mara de Syllos Cólus for kindly proving the HeLa cell line. We are very
grateful to Dr. Mauricio S. Baptista for allow us to do the measurements of
singlet oxygen quantum yield in his lab and to Dr. Helena Junqueira for
performing the experiments.

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