Aminosquaraines as potential photodynamic agents: Synthesis and evaluation of in vitro cytotoxicity

Article abstract of DOI:10.1016/j.bmcl.2017.08.004

The synthesis of several aminosquaraine cationic dyes displaying strong absorption within the so-called phototherapeutic window (650–850 nm) is described. Their cytotoxicity, under dark and illuminated conditions, was tested against several human tumor cell lines (breast, lung, cervical and hepatocellular carcinomas) and non-tumor porcine liver primary cells. All compounds showed to inhibit the growth of the tumor cells upon irradiation more than in the absence of light, in more or less extension, clearly exhibiting photodynamic activity. The photosensitizing ability against some cell lines, together with the low toxicity for the non-tumor primary PLP2 cells displayed by some of the compounds synthetized, turns them into potential candidates as photosensitizers for PDT.

Full text of DOI:10.1016/j.bmcl.2017.08.004

Accepted Manuscript
Aminosquaraines as potential photodynamic agents: synthesis and evaluation
of in vitro cytotoxicity
Álvaro Magalhães, Vânia C. Graça, Ricardo C. Calhelha, Isabel C.F.R. Ferreira,
P.F. Santos
PII:
S0960-894X(17)30791-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.08.004
BMCL 25198
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
4 July 2017
31 July 2017
2 August 2017
Please cite this article as: Magalhães, A., Graça, V.C., Calhelha, R.C., C.F.R. Ferreira, I., Santos, P.F.,
Aminosquaraines as potential photodynamic agents: synthesis and evaluation of in vitro cytotoxicity, Bioorganic
& Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.08.004
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Aminosquaraines as potential photodynamic agents: synthesis and evaluation of in vitro
cytotoxicity
Álvaro Magalhãesa, Vânia C. Graçaa, Ricardo C. Calhelhab, Isabel C.F.R. Ferreirab,*, P. F.
Santosa,*
aChemistry Dep. and Chemistry Center - Vila Real (CQ-VR), University of Trás-os-Montes and
Alto Douro, 5001-801 Vila Real, Portugal.
bMountain Research Centre (CIMO), Polytechnic Institute of Bragança, Campus de Santa
Apolónia, 5300-253 Bragança, Portugal.
* Authors to whom correspondence should be addressed: psantos@utad.pt telephone +351-259-
350276; fax +351-259-350480 and iferreira@ipb.pt; Tel.: +351-273-303219; Fax: +351-273-
325405)

Abstract
The synthesis of several aminosquaraine cationic dyes displaying strong absorption within the
so-called phototherapeutic window (650-850 nm) is described. Their cytotoxicity, under dark
and illuminated conditions, was tested against several human tumor cell lines (breast, lung,
cervical and hepatocellular carcinomas) and non-tumor porcine liver primary cells. All
compounds showed to inhibit the growth of the tumor cells upon irradiation more than in the
absence of light, in more or less extension, clearly exhibiting photodynamic activity.
The photosensitizing ability against some cell lines, together with the low toxicity for the non-
tumor primary PLP2 cells displayed by some of the compounds synthetized, turns them into
potential candidates as photosensitizers for PDT.
Keywords: Photodynamic therapy; Cancer; Photosensitizer; Squaraine dyes

Photodynamic therapy (PDT) is a promising minimally invasive therapeutic technique that
has been shown effective for cancer and other non-oncological conditions [1]. It is based on the
use of a sensitizer that, after accumulating inside the abnormal cells, is excited by light of an
appropriate wavelength and interacts with oxygen, leading to the production of reactive oxygen
species (ROS) which induce cell death via oxidative stress mechanisms. Amongst those, singlet
oxygen is generally considered to be the main cytotoxic agent responsible for the photodynamic
effect [2]. The prerequisite of concurrence of this triad of components (photosensitizer, oxygen
and light), which individually are innocuous, for the photodynamic process to take place turns
PDT a particularly selective modality, with unique advantages compared to conventional tumor
therapies such as surgery, chemotherapy, and radiotherapy [3, 4].
Photofrin® a mixture of hemotoporphyrin derivatives, was the first photosensitizer to be
approved by the regulatory agencies and still the most extensively used for medical applications
of PDT [5]. This so-called first generation sensitizer presents however several drawbacks,
namely a lack of chemical homogeneity, prolonged coetaneous photosensitivity and a less than
optimal light absorption profile ( = 630 nm,  = 3200 M-1 cm-1) [6], which entails the use of
higher drug dosages, and limits the penetration depth of light into tissue.
The crucial role of the photosensitizer in the success of PDT turned the development of new
sensitizers with better photophysical and photobiological properties into an active area of
research in the two last decades and a lot of attention has been given to a number of different
classes of compounds as potential so-called second generation sensitizers [5, 7].
Squaraines, a class of functional dyes with sharp and intense visible to NIR absorption which
have found wide application in the domain of photonics [8], have also attracted great interest as a
new class of potential photosensitizers for PDT [9] since, in 1997, Ramaiah et al. [10]
synthesized and investigated the photophysical properties, including the singlet oxygen
generation ability, of some squaraines derived from phloroglucinol. As a result, a lot of work has
been published on the structural variation of squaraines which, however, has been mainly
focused on the variation of the nature and substitution of the heterocyclic terminal groups [11].
The derivatization of the central squaric core, on the other hand, has been poorly addressed and
has been the focus of our research for some years [12-14].
Notwithstanding a considerable number of squaraine compounds has been designed and
synthesized with the purpose of PDT sensitizers, the in vitro assessment of their photodynamic

ability has been barely addressed [15-23]. The reports of in vivo evaluation are even scarcer [17,
18, 21, 24-26].
In this work we synthesized several squaraine dyes substituted at the central four member ring
with different amino groups and assessed their cytotoxicity, under dark and illuminated
conditions, against several human tumor cell lines (breast, lung, cervical and hepatocellular
carcinomas) and non-tumor porcine liver primary cells. These aminosquaraine dyes are
envisioned to present some advantages over the common zwiterionic counterparts as their
cationic character may positively influence cellular uptake taking advantage of the membrane
potential, and the presence of amino groups, besides red-shifting the dye’s absorption, may
conveniently enhance solubility and interaction with the biological medium. To the best of our
knowledge this is the first report on the in vitro photosensitizing ability of aminosquaraine dyes.
Aminosquaraines 5 were synthesized from the zwitterionic dye 3 by an expeditious
methodology developed earlier by some of us [12] (Scheme 1). The starting squaraine 3, easily
prepared by the condensation of two molar equivalents of 2-methylbenzothiazolium salt 2 with
squaric acid, in refluxing n-butanol/pyridine, was methylated with methyl triflate to furnish the
crucial O-methyl intermediate 4. The later then undergoes nucleophilic substitution with
appropriate amines to yield the triflate analogues of 5. Each of the final compounds underwent
counter-ion replacement by iodine, by treatment of a methanolic solution of the compound with
excess 14 % aqueous KI, in order to take benefit from the so-called external heavy atom effect
on the potential enhancement of singlet oxygen generation [27].

Scheme 1. Synthesis of aminosquaraine dyes 5a-e.
All the synthesized dyes exhibit sharp and intense absorption ( 104 cm-1 M-1) in the red end
of the visible spectrum, close to the bottom end of the phototherapeutic window (Table 1). In
general, the amino-substituted dyes 5 display absorption at longer wavelengths than the starting
non-substituted analogue 3. The chromophore shows typical donor–acceptor characteristics with
increasing bathochromic shift as the electron donating properties of the auxochrome increases.
The observed bathochromic shifts ranged from 3 to 22 nm.
Table 1
Visible spectral data for squaraine dyes 3 and 5a-e.
Dye
R1
R2
maxa (nm) (log )
-
-
650 (5.24)b
648 (5.41)
657 (4.67)
3
H
H
H
5a
5b
CH2CH2OH

CH2CH2OH
CH2CH2OH
CH3
667 (5.21)
658 (5.23)b
667 (5.19)
5c
5d
5e
H
CH3
CH2CH2OH
a Measured in MeOH/CH2Cl2 (99/1). b From ref. [12]
The cytotoxicity of the compounds synthesized was evaluated against four human tumor cell
lines: breast (MCF7), non-small cell lung (NCI-H460), cervical (HeLa) and hepatocellular
(HepG2) carcinomas, and against a porcine liver primary cell culture (PLP2) established by some
of us, both in the absence of light and under irradiation
As a representative example, Fig. 1 shows the relative growth inhibition ability of dyes 5a-e
on the human tumor cell lines studied using 0,1 M solutions of the dye, a concentration for
which growth inhibition in the absence of light was less than 20% in all tests

Fig. 1. Growth inhibition of MCF7, HeLa, HepG2 and NCI-H460 cell lines upon treatment with
dyes 5a-e (0.1 M) in the dark and after irradiation. Results are presented as mean values ±
standard deviation (SD).
. All compounds showed to inhibit the growth of the tumor cells upon irradiation more than in
the dark, in more or less extension, clearly showing to display photocytotoxic effect. The more
susceptible cell lines to phototoxicity were HeLa and MCF-7, for which the photodynamic
treatment resulted in more significant differences in the growth inhibition of the cells in the dark
and upon irradiation. Controls were set with the cells in the absence of the dyes in 3% DMSO in
DMEM and in the growth medium only. The cells were independently submitted to the
photodynamic treatment and maintained in the dark. Neither DMSO alone, nor light alone, nor
the combination of both was found to induce toxicity on the cells.
Although the study of the exact nature of the photodynamic action mechanism of the
synthesized dyes is out of the scope of this work, it is worthwhile to mention that the majority of
the compounds assayed have shown singlet oxygen generation ability [14, 28].
The GI50 values (compound concentration resulting in 50 % of growth inhibition) determined
for squaraine dyes 5a-e are presented in Table 2.
Table 2
Cytotoxicity (GI50 values, µM) of compounds 5a-e (mean  SD).
Compound
Condition
MCF7
>10
NCI-H460
HepG2
4.571 ± 0.379
HeLa
>10
PLP2
>10
Dark
Irradiated
t-Students test
p-value
>10
0.918 ± 0.095 8.347 ± 0.363 0.665 ± 0.049 0.288 ± 0.017 4.981 ± 0.254
5a
-
-
<0.001
-
-
Dark
7.361 ± 0.764 8.820 ± 0.698 7.829 ± 0.694
>10
3.727 ± 0.579
Irradiated
t-Students test
p-value
0.224± 0.015 0.440 ± 0.024 0.110 ± 0.011 0.097 ± 0.001 0.385 ± 0.006
5b
5c
<0.001
<0.001
<0.001
-
<0.001
Dark
5.337 ± 0.413 0.940 ± 0.085 4.493 ± 0.391
>10
0.621 ± 0.005
Irradiated
t-Students test
p-value
0.257 ± 0.026 0.404 ± 0.034 0.722 ± 0.077 0.088 ± 0.006 0.340 ± 0.017
<0.001 <0.001 <0.001 <0.001
-
Dark
0.576 ± 0.043 0.507 ± 0.064 7.083 ± 0.767 2.223 ± 0.138 0.410 ± 0.050
0.054 ± 0.004 0.171 ± 0.023 0.078 ± 0.005 0.071 ± 0.009 0.126 ± 0.048
Irradiated
t-Students test
p-value
5d
5e
<0.001
<0.001
<0.001
<0.001
0.001
Dark
0.737 ± 0.039 0.767 ± 0.045 7.535 ± 0.818 2.636 ± 0.062 0.717 ± 0.115

Irradiated
t-Students test
p-value
0.239 ± 0.034 0.455 ± 0.012 0.271 ± 0.014 0.273 ± 0.007 0.471 ± 0.044
<0.001 <0.001 <0.001 <0.001 0.008
Regarding the tests conducted in the absence of light, in the range of tested concentrations,
squaraine 5a only showed inhibitory activity against the HepG2 cell line, and yet with a
relatively high GI50 value. The remaining compounds presented inhibitory activity for all the cell
lines tested, though in variable extension, except dyes 5b and 5c which were inactive against the
HeLa cell line in the range of the concentrations used.
Upon irradiation the GI50 values decreased, in some cases very appreciably, showing
significant photodynamic effect of the dyes. For compounds 5a-d, the lowest GI50 values were
obtained consistently against the HeLa cell line, while for dye 5e the lowest GI50 value was
obtained against the MCF7 cell line. The largest differences between the GI50 values obtained in
the dark and upon irradiation, which are very relevant in terms of the usefulness of the
photodynamic effect, were observed precisely for the HeLa cell line, with dyes 5a-c which were
shown to be inactive against this cell line in the dark, in the range of the concentrations used. The
HepG2 cell line turned out to be also very susceptible to the photodynamic treatment using dyes
5b, 5d and 5e. Compound 5d presented the lowest GI50 value of all, whatever the tested cell line;
nevertheless, it was not the compound that presented the highest differences between the dark
and irradiated assays.
The evaluation of the cytotoxicity for PLP2 (non-tumor cells) is very important since
mammalian hepatocytes still represent a mandatory step in the evaluation of toxic compounds
that lead to the production of various metabolites, which are the ultimate cause of toxicity [29].
Apart from dye 5a which, in the range of the tested concentrations, was inactive in the absence
of light and presented a GI50 value of 4.981 ± 0.254 M upon irradiation, all compounds showed
also some cytotoxicity against PLP2 cells. Nevertheless, the GI50 values obtained for the PLP2
cells were consistently higher, either in the dark or under irradiation, than those obtained against
the tumor cell lines. It is worth mentioning that the locoregional nature of the photodynamic
effect can diminish or even surmount the importance of the cytotoxicity observed in the non-
tumor cells.
In conclusion, the aminosquaraine dyes synthesized, displaying strong absorption within the
phototerapeutic window, have shown to able to inhibit the growth of the human tumor cell lines

tested (MCF-7, NCI-H460, HeLa, and HepG2) upon irradiation more than in the dark, in some
cases very substantially, clearly showing to display photocytotoxic activity.
The differences observed for some of the dyes between the GI50 values obtained in the
absence of light and upon irradiation, turn them into potential candidates as photosensitizers for
PDT, in particular compound 5a which showed the lowest toxicity for the non-tumor primary
PLP2 cells.
Acknowledgements
The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and
FEDER for CIMO (UID/AGR/00690/2013) and CQ-VR (UID/QUI/00616/2013) financial
support.
A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at
http://dx.doi.org/
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