© 2017 Elsevier Ltd 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.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.info:eu-repo/semantics/publishedVersio

Calhelha, Ricardo C.

Ferreira, Isabel C.F.R.

Graça, Vânia C.

Magalhães, F.

Santos, Paulo F.

Bioorganic & Medicinal Chemistry Letters

Álvaro F. Magalhães

Vânia C. Graça

Ricardo C. Calhelha

Isabel C.F.R. Ferreira

Paulo F. Santos

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

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Biblioteca Digital do IPB

Bioorganic & Medicinal Chemistry Letters 27 (2017) 4467–4470Contents lists available at ScienceDirectBioorganic & Medicinal Chemistry Lettersjournal homepage: www.elsevier .com/locate /bmclAminosquaraines as potential photodynamic agents: Synthesis andevaluation of in vitro cytotoxicityhttp://dx.doi.org/10.1016/j.bmcl.2017.08.0040960-894X/ 2017 Elsevier Ltd. All rights reserved.⇑ Corresponding authors.E-mail addresses: iferreira@ipb.pt (I.C.F.R. Ferreira), psantos@utad.pt (P.F. Santos).Álvaro F. Magalhães a, Vânia C. Graça a, Ricardo C. Calhelha b, Isabel C.F.R. Ferreira b,⇑, Paulo F. Santos a,⇑aChemistry Dep. and Chemistry Center – Vila Real (CQ-VR), University of Trás-os-Montes and Alto Douro, 5001-801 Vila Real, PortugalbMountain Research Centre (CIMO), Polytechnic Institute of Bragança, Campus de Santa Apolónia, 5300-253 Bragança, Portugala r t i c l e i n f o a b s t r a c tArticle history:Received 4 July 2017Revised 31 July 2017Accepted 2 August 2017Available online 3 August 2017Keywords:Photodynamic therapyCancerPhotosensitizerSquaraine dyesThe synthesis of several aminosquaraine cationic dyes displaying strong absorption within the so-calledphototherapeutic window (650–850 nm) is described. Their cytotoxicity, under dark and illuminatedconditions, was tested against several human tumor cell lines (breast, lung, cervical and hepatocellularcarcinomas) and non-tumor porcine liver primary cells. All compounds showed to inhibit the growthof the tumor cells upon irradiation more than in the absence of light, in more or less extension, clearlyexhibiting photodynamic activity. The photosensitizing ability against some cell lines, together withthe 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. 2017 Elsevier Ltd. All rights reserved.IntroductionPhotodynamic therapy (PDT) is a promising minimally invasivetherapeutic technique that has been shown effective for cancer andother non-oncological conditions [1]. It is based on the use of a sen-sitizer that, after accumulating inside the abnormal cells, is excitedby light of an appropriate wavelength and interacts with oxygen,leading to the production of reactive oxygen species (ROS) whichinduce cell death via oxidative stress mechanisms. Amongst those,singlet oxygen is generally considered to be the main cytotoxicagent responsible for the photodynamic effect [2]. The prerequisiteof concurrence of this triad of components (photosensitizer, oxy-gen and light), which individually are innocuous, for the photody-namic process to take place turns PDT a particularly selectivemodality, with unique advantages compared to conventionaltumor therapies such as surgery, chemotherapy and radiotherapy[3,4].Photofrin, a mixture of hematoporphyrin derivatives, was thefirst photosensitizer to be approved by the regulatory agenciesand still the most extensively used for medical applications ofPDT [5]. This so-called first generation sensitizer presents howeverseveral drawbacks, namely a lack of chemical homogeneity, pro-longed coetaneous photosensitivity and a less than optimal lightabsorption profile (k = 630 nm, e = 3200 M1 cm1) [6], whichentails the use of higher drug dosages, and limits the penetrationdepth of light into tissue.The crucial role of the photosensitizer in the success of PDTturned the development of new sensitizers with better photophys-ical and photobiological properties into an active area of researchin the two last decades and a lot of attention has been given to anumber of different classes of compounds as potential so-calledsecond generation sensitizers [5,7].Squaraines, a class of functional dyes with sharp and intensevisible to NIR absorption which have found wide application inthe domain of photonics [8], have also attracted great interest asa new class of potential photosensitizers for PDT [9] since, in1997, Ramaiah et al. [10] synthesized and investigated the photo-physical properties, including the singlet oxygen generation ability,of some squaraines derived from phloroglucinol. As a result, a lot ofwork has been published on the structural variation of squaraineswhich, however, has been mainly focused on the variation of thenature 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 researchfor some years [12–14].Notwithstanding a considerable number of squaraine com-pounds has been designed and synthesized with the purpose ofPDT sensitizers, the in vitro assessment of their photodynamicability has been barely addressed [15–24]. The reports of in vivoevaluation are even scarcer [17,18,21,25–27].In this work we synthesized several squaraine dyes substitutedat the central four member ring with different amino groups andTable 1Visible spectral data for squaraine dyes 3 and 5a–e.Dye R1 R2 kmaxa (nm) (log e)3 – – 650 (5.24)b5a H H 648 (5.41)5b H CH2CH2OH 657 (4.67)5c CH2CH2OH CH2CH2OH 667 (5.21)5d H CH3 658 (5.23)b5e CH3 CH2CH2OH 667 (5.19)a Measured in MeOH/CH2Cl2 (99/1).b From Ref.[12]4468 Á.F. Magalhães et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 4467–4470assessed their cytotoxicity, under dark and illuminated conditions,against several human tumor cell lines (breast, lung, cervical andhepatocellular carcinomas) and non-tumor porcine liver primarycells. These aminosquaraine dyes are envisioned to present someadvantages over the common zwitterionic counterparts as theircationic character may positively influence cellular uptake takingadvantage of the membrane potential, and the presence of aminogroups, besides red-shifting the dye’s absorption, may conve-niently enhance solubility and interaction with the biological med-ium. To the best of our knowledge this is the first report on thein vitro photosensitizing ability of symmetrical aminosquarainedyes.Aminosquaraines 5 were synthesized from the zwitterionic dye3 by an expeditious methodology developed earlier by some of us[12] (Scheme 1). The starting squaraine 3, easily prepared by thecondensation of two molar equivalents of 2-methylbenzothia-zolium salt 2 with squaric acid, in refluxing n-butanol/pyridine,was methylated with methyl triflate to furnish the crucialO-methyl intermediate 4. The later then undergoes nucleophilicsubstitution with appropriate amines to yield the triflate analoguesof 5. Each of the final compounds underwent counter-ion replace-ment by iodine, by treatment of a methanolic solution of the com-pound with excess 14% aqueous KI, in order to take benefit fromthe so-called external heavy atom effect on the potential enhance-ment of singlet oxygen generation [28].All the synthesized dyes exhibit sharp and intense absorption(e > 104 cm1 M1) in the red end of the visible spectrum, closeto the bottom end of the phototherapeutic window (Table 1). Ingeneral, the amino-substituted dyes 5 display absorption at longerwavelengths than the starting non-substituted analogue 3. Thechromophore shows typical donor–acceptor characteristics withincreasing bathochromic shift as the electron donating propertiesof the auxochrome increases. The observed bathochromic shiftsranged from 3 to 22 nm.The cytotoxicity of the compounds synthesized was evaluatedagainst four human tumor cell lines: breast (MCF-7), non-small celllung (NCI-H460), cervical (HeLa) and hepatocellular (HepG2) carci-nomas, and against a porcine liver primary cell culture (PLP2)Scheme 1. Synthesis of amiestablished by some of us, both in the absence of light and underirradiation.As a representative example, Fig. 1 shows the relative growthinhibition ability of dyes 5a–e on the human tumor cell lines stud-ied using 0,1 lM solutions of the dye, a concentration for whichgrowth inhibition in the absence of light was less than 20% in alltests.All compounds showed to inhibit the growth of the tumor cellsupon irradiation more than in the dark, in more or less extension,clearly showing to display photocytotoxic effect. The more suscep-tible cell lines to phototoxicity were HeLa and MCF-7, for which thephotodynamic treatment resulted in more significant differencesin the growth inhibition of the cells in the dark and upon irradia-tion. Controls were set with the cells in the absence of the dyesin 3% DMSO in DMEM and in the growth medium only. The cellswere independently submitted to the photodynamic treatmentand 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 photodynamicaction mechanism of the synthesized dyes is out of the scope ofthis work, it is worthwhile to mention that the majority of thecompounds assayed have shown singlet oxygen generation ability[14,29].The GI50 values (compound concentration resulting in 50% ofgrowth inhibition) determined for squaraine dyes 5a–e arepresented in Table 2.nosquaraine dyes 5a–e.Fig. 1. Growth inhibition of MCF-7, HeLa, HepG2 and NCI-H460 cell lines upon treatment with dyes 5a–e (0.1 lM) in the dark and after irradiation. Results are presented asmean values ± standard deviation (SD).Table 2Cytotoxicity (GI50 values, mM) of compounds 5a–e (mean ± SD).Compound Condition MCF-7 NCI-H460 HepG2 HeLa PLP25a Dark >10 >10 4.571 ± 0.379 >10 >10Irradiated 0.918 ± 0.095 8.347 ± 0.363 0.665 ± 0.049 0.288 ± 0.017 4.981 ± 0.254t-Students test p-value – – <0.001 – –5b Dark 7.361 ± 0.764 8.820 ± 0.698 7.829 ± 0.694 >10 3.727 ± 0.579Irradiated 0.224 ± 0.015 0.440 ± 0.024 0.110 ± 0.011 0.097 ± 0.001 0.385 ± 0.006t-Students test p-value <0.001 <0.001 <0.001 – <0.0015c Dark 5.337 ± 0.413 0.940 ± 0.085 4.493 ± 0.391 >10 0.621 ± 0.005Irradiated 0.257 ± 0.026 0.404 ± 0.034 0.722 ± 0.077 0.088 ± 0.006 0.340 ± 0.017t-Students test p-value <0.001 <0.001 <0.001 – <0.0015d Dark 0.576 ± 0.043 0.507 ± 0.064 7.083 ± 0.767 2.223 ± 0.138 0.410 ± 0.050Irradiated 0.054 ± 0.004 0.171 ± 0.023 0.078 ± 0.005 0.071 ± 0.009 0.126 ± 0.048t-Students test p-value <0.001 <0.001 <0.001 <0.001 0.0015e Dark 0.737 ± 0.039 0.767 ± 0.045 7.535 ± 0.818 2.636 ± 0.062 0.717 ± 0.115Irradiated 0.239 ± 0.034 0.455 ± 0.012 0.271 ± 0.014 0.273 ± 0.007 0.471 ± 0.044t-Students test p-value <0.001 <0.001 <0.001 <0.001 0.008Á.F. Magalhães et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 4467–4470 4469Regarding the tests conducted in the absence of light, in therange of tested concentrations, squaraine 5a only showed inhibi-tory activity against the HepG2 cell line, and yet with a relativelyhigh GI50 value. The remaining compounds presented inhibitoryactivity for all the cell lines tested, though in variable extension,except dyes 5b and 5c which were inactive against the HeLa cellline in the range of the concentrations used.Upon irradiation the GI50 values decreased, in some cases veryappreciably, showing significant photodynamic effect of the dyes.For compounds 5a–d, the lowest GI50 values were obtained consis-tently against the HeLa cell line, while for dye 5e the lowest GI50value was obtained against the MCF-7 cell line. The largestdifferences between the GI50 values obtained in the dark and uponirradiation, which are very relevant in terms of the usefulness ofthe photodynamic effect, were observed precisely for the HeLa cellline, with dyes 5a–c, which were shown to be inactive against thiscell line in the dark, in the range of the concentrations used. TheHepG2 cell line turned out to be also very susceptible to thephotodynamic treatment using dyes 5b, 5d and 5e. Compound5d presented the lowest GI50 value of all, whatever the tested cellline; nevertheless, it was not the compound that presented thehighest differences between the dark and irradiated assays.The evaluation of the cytotoxicity for PLP2 (non-tumor cells) isvery important since mammalian hepatocytes still represent a4470 Á.F. Magalhães et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 4467–4470mandatory step in the evaluation of toxic compounds that lead tothe production of various metabolites, which are the ultimatecause of toxicity [30].Apart from dye 5a which, in the range of the tested concentra-tions, was inactive in the absence of light and presented a GI50value of 4.981 ± 0.254 lM upon irradiation, all compounds showedalso some cytotoxicity against PLP2 cells. Nevertheless, the GI50values obtained for the PLP2 cells were consistently higher, eitherin the dark or under irradiation, than those obtained against thetumor cell lines. It is worth mentioning that the locoregional nat-ure of the photodynamic effect can diminish or even surmountthe importance of the cytotoxicity observed in the non-tumor cells.In conclusion, the aminosquaraine dyes synthesized, displayingstrong absorption within the phototerapeutic window, have shownto able to inhibit the growth of the human tumor cell lines tested(MCF-7, NCI-H460, HeLa, and HepG2) upon irradiation more thanin the dark, in some cases very substantially, clearly showing todisplay photocytotoxic activity.The differences observed for some of the dyes between the GI50values obtained in the absence of light and upon irradiation, turnthem into potential candidates as photosensitizers for PDT, in par-ticular compound 5a which showed the lowest toxicity for thenon-tumor primary PLP2 cells.AcknowledgementsThe authors are grateful to the Foundation for Science and Tech-nology (FCT, Portugal) and FEDER for CIMO (UID/AGR/00690/2013)and CQ-VR (UID/QUI/00616/2013) financial support.Supplementary dataSupplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmcl.2017.08.004.References1. 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Aminosquaraines as potential photodynamic agents: Synthesis and evaluation of in vitro cytotoxicity

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