Synthesis, Photochemical and In Vitro Cytotoxic Evaluation of New Iodinated Aminosquaraines as Potential Sensitizers for Photodynamic Therapy

In this work, several benzothiazole-based aminosquaraine dyes, displaying strong absorption within the so-called phototherapeutic window (650–800 nm), were synthesized. The ability, of all the new dyes, to generate singlet oxygen was assessed by determining the correspondent phosphorescence emission and through the comparison with a standard. The quantum yields of singlet oxygen generation were determined and exhibited to be strongly dependent on the nature of the amino substituents introduced in the squaric ring. The photodynamic activity of the synthesized dyes was tested against four human tumor cell lines: breast (MCF-7), lung (NCI-H460), cervical (HeLa) and hepatocellular (HepG2) carcinomas; and a non-tumor porcine liver primary cell culture (PLP2). All the compounds synthesized were found to be able to inhibit tumor cells growth upon irradiation more than in the dark, in most of the cases, very significantly. Considering the photodynamic activity exhibited and the low toxicity displayed for the non-tumor cells, some of the synthetized dyes can be regarded as potential candidates as photosensitizers for PDT.


Introduction
Photodynamic therapy (PDT) has emerged as a very promising treatment for certain types of cancer and other non-oncologic disorders [1]. Over the years, PDT has received regulatory approval worldwide for several conditions and has now become an established therapeutic modality [2].
The basic principle underlying this therapeutic approach is simple: a light-absorbing molecule, after being administrated to the patient, is activated with light of an appropriate wavelength and gives rise to the production of reactive oxygen species (ROS). This species are responsible to induce damage of the surrounding cells [3]. Following interaction with light, the sensitizer, which is firstly excited to its excited singlet state, may efficiently populate, via intersystem crossing, the longer-lived excited triplet state. The triplet state of the sensitizer can then produce ROS by two different competing mechanisms [4]. In a Type I reaction, the sensitizer in the triplet state can react with organic substrates or solvents to generate free radicals that subsequently interact with oxygen to generate ROS such

Synthesis of Dyes
The molecular design rationale of the aminosquaraines synthesized was driven by the attempt to amend molecule's rigidity, which influences the singlet oxygen production ability, dye's maximum absorption, which depends on the electron-donating characteristics of the amine auxochrome, and the ability to establish intermolecular hydrogen bonds, which may increase water solubility and provide a potential means for interaction with biological substrates. Therefore, the strategy consisted in selecting substituent groups able to generate secondary and tertiary amines, some of which possessing flexible hydroxyethyl arms.
Aminosquaraines 5a-e were prepared by an expeditious synthetic method developed earlier by some of us [37], based on the methylation of one of the oxygen atoms of the squaraine core followed by nucleophilic substitution of the so-formed methoxy group by appropriate amines (Scheme 1).
Molecules 2019, 24 x FOR PEER REVIEW 3 of 13

Synthesis of Dyes
The molecular design rationale of the aminosquaraines synthesized was driven by the attempt to amend molecule's rigidity, which influences the singlet oxygen production ability, dye's maximum absorption, which depends on the electron-donating characteristics of the amine auxochrome, and the ability to establish intermolecular hydrogen bonds, which may increase water solubility and provide a potential means for interaction with biological substrates. Therefore, the strategy consisted in selecting substituent groups able to generate secondary and tertiary amines, some of which possessing flexible hydroxyethyl arms.
Aminosquaraines 5a-e were prepared by an expeditious synthetic method developed earlier by some of us [37], based on the methylation of one of the oxygen atoms of the squaraine core followed by nucleophilic substitution of the so-formed methoxy group by appropriate amines (Scheme 1). Scheme 1. Synthesis of squaraine dyes 5a-e.
Thus, condensation of two molar equivalents of benzothiazolium salt 2, easily prepared by Nalkylation of 6-iodo-2-methylbenzothiazole (1) with iodoethane, with one equivalent of squaric acid in refluxing n-butanol/pyridine, resulted in the starting zwitterionic squaraine 3. Methylation of the latter with methyl trifluoromethanessulfonate produced the crucial O-methyl ether derivative 4 from which the triflate analogues of 5a-e could be obtained by treatment with the appropriate amines. Counter-ion exchange by iodine by treatment of the methanolic solutions of the dyes with excess 14% aqueous KI, produced the final aminosquaraine iodides 5a-e in rather good yields. The counter ion exchange by iodine is carried out to potentially increase singlet oxygen generation through the external heavy atom effect [39]. As previously observed for several aminosquaraine dyes bearing a secondary amino group in the squaric ring [37,38], the two methylene protons of the polymethylene chain of 5b and 5d appear in the 1 H-NMR spectrum as separated signals as consequence of the consequent local magnetic field inhomogeneity, resulting from the intramolecular hydrogen bonding and/or hindered rotation around the C-N bond linking the amino substituent to the squaric ring.

UV-Visible Absorption Spectroscopy
All synthesized aminosquaraines 5a-e display sharp and strong absorption in the red end of the visible spectrum, within the phototherapeutic window ( Figure 1, Table 1). The absorption of dyes 5a-Scheme 1. Synthesis of squaraine dyes 5a-e.
Thus, condensation of two molar equivalents of benzothiazolium salt 2, easily prepared by N-alkylation of 6-iodo-2-methylbenzothiazole (1) with iodoethane, with one equivalent of squaric acid in refluxing n-butanol/pyridine, resulted in the starting zwitterionic squaraine 3. Methylation of the latter with methyl trifluoromethanessulfonate produced the crucial O-methyl ether derivative 4 from which the triflate analogues of 5a-e could be obtained by treatment with the appropriate amines. Counter-ion exchange by iodine by treatment of the methanolic solutions of the dyes with excess 14% aqueous KI, produced the final aminosquaraine iodides 5a-e in rather good yields. The counter ion exchange by iodine is carried out to potentially increase singlet oxygen generation through the external heavy atom effect [39]. As previously observed for several aminosquaraine dyes bearing a secondary amino group in the squaric ring [37,38], the two methylene protons of the polymethylene chain of 5b and 5d appear in the 1 H-NMR spectrum as separated signals as consequence of the consequent local magnetic field inhomogeneity, resulting from the intramolecular hydrogen bonding and/or hindered rotation around the C-N bond linking the amino substituent to the squaric ring.

UV-Visible Absorption Spectroscopy
All synthesized aminosquaraines 5a-e display sharp and strong absorption in the red end of the visible spectrum, within the phototherapeutic window ( Figure 1, Table 1). The absorption of dyes 5a-e is shifted bathochromically in relation to the non-substituted zwitterionic squaraine precursor 3. The observed shifts ranging from 1 to 22 nm. The extent of the bathochromic shifts depends, in a direct way, on the electron donating ability of the amino auxochrome. This typical donor-acceptor mechanism is an usual feature of the chromophoric system of this class of compounds [37,38,40].
Molecules 2019, 24 x FOR PEER REVIEW 4 of 13 e is shifted bathochromically in relation to the non-substituted zwitterionic squaraine precursor 3. The observed shifts ranging from 1 to 22 nm. The extent of the bathochromic shifts depends, in a direct way, on the electron donating ability of the amino auxochrome. This typical donor-acceptor mechanism is an usual feature of the chromophoric system of this class of compounds [37,38,40].  The evaluation of the photosensitizing efficiency of the aminosquaraine dyes was performed in chloroform, by determining the singlet oxygen formation quantum yields (ΦΔ). Phenazine was used as standard [41,42]. The phosphorescence emission of singlet oxygen generated by the sensitizers at approximately 1270 nm is presented in Figure 2 and the determined quantum yields are summarized in Table 1.  Absorption spectra of unsubstituted squaraine 3 and aminosquaraines 5a-e in MeOH/CH 2 Cl 2 , 99/1 (v/v).

Singlet Oxygen Quantum Yields
The evaluation of the photosensitizing efficiency of the aminosquaraine dyes was performed in chloroform, by determining the singlet oxygen formation quantum yields (Φ ∆ ). Phenazine was used as standard [41,42]. The phosphorescence emission of singlet oxygen generated by the sensitizers at approximately 1270 nm is presented in Figure 2 and the determined quantum yields are summarized in Table 1. e is shifted bathochromically in relation to the non-substituted zwitterionic squaraine precursor 3. The observed shifts ranging from 1 to 22 nm. The extent of the bathochromic shifts depends, in a direct way, on the electron donating ability of the amino auxochrome. This typical donor-acceptor mechanism is an usual feature of the chromophoric system of this class of compounds [37,38,40].

Singlet Oxygen Quantum Yields.
The evaluation of the photosensitizing efficiency of the aminosquaraine dyes was performed in chloroform, by determining the singlet oxygen formation quantum yields (ΦΔ). Phenazine was used as standard [41,42]. The phosphorescence emission of singlet oxygen generated by the sensitizers at approximately 1270 nm is presented in Figure 2 and the determined quantum yields are summarized in Table 1.  These Φ ∆ values were obtained by a simple comparison of the integrated areas determined for the standard and samples, using the same OD at the excitation wavelength. For each determination an average of 100 phosphorescence spectra were used. Dye 5b displays the highest quantum yield when compared to the other squaraine derivatives (Φ ∆ = 0.26). As previously reported [43,44], the amino group in the squaric ring enhances singlet oxygen generation efficiency when compared with the unsubstituted zwitterionic dye 3. The results for Φ ∆ are strongly dependent on the nature of the amino substituents: the highest Φ ∆ values occurred for the smallest substituents, and the lowest values were observed for the hydroxyethylamino substituents, especially the bis(hydroxyethyl)-amino group (dye 5c). This is related with the intramolecular charge transfer (ICT) mechanism which provides a non-radiative deactivation pathway, therefore decreasing the singlet oxygen formation quantum yields ( [44] and references therein). Table 1 also presents the fluorescence quantum yields Φ F for the aminosquaraines 5a-e in CHCl 3 . As expected, due to the covalent link between iodine and the aminosquaraine, a strong heavy atom effect can be observed in all cases. This effect is emphasized in the case of the bis(hydroxyethyl)amino substituent group (dye 5c).

In Vitro Photodynamic Activity
Four human tumor cell lines were used to assess the in vitro cytotoxicity of the synthesized dyes (5a-e): cervical (HeLa), breast (MCF7), hepatocellular (HepG2) and non-small cell lung (NCI-H460) carcinomas.
Although cytotoxicity was found to depend on the cell line, in general, all aminosquaraine dyes exhibited much higher cytotoxicity upon irradiation than in the absence of light. This fact revealed that the dyes are endowed with photodynamic action capacity. The assurance that the photodynamic effect was due, exclusively, to the sensitizing properties of the squaraine dyes, was secured by observing, in independent assays submitted to the photodynamic treatment and maintained in the dark, that, in the absence of the dyes neither DMSO nor light individually, nor the combination of both, were able to induce toxicity on the cells. Figure 3 shows the growth inhibition ability of aminosquaraines 5a-e against the different human tumor cell lines, in the absence of light and upon irradiation, at a dye concentration of 0.3 µM. In this representative example, the growth inhibition in the dark was less than 20% in more than 75% of the assays. These ΦΔ values were obtained by a simple comparison of the integrated areas determined for the standard and samples, using the same OD at the excitation wavelength. For each determination an average of 100 phosphorescence spectra were used. Dye 5b displays the highest quantum yield when compared to the other squaraine derivatives (ΦΔ = 0.26). As previously reported [43,44], the amino group in the squaric ring enhances singlet oxygen generation efficiency when compared with the unsubstituted zwitterionic dye 3. The results for ΦΔ are strongly dependent on the nature of the amino substituents: the highest ΦΔ values occurred for the smallest substituents, and the lowest values were observed for the hydroxyethylamino substituents, especially the bis(hydroxyethyl)amino group (dye 5c). This is related with the intramolecular charge transfer (ICT) mechanism which provides a non-radiative deactivation pathway, therefore decreasing the singlet oxygen formation quantum yields ( [44] and references therein). Table 1 also presents the fluorescence quantum yields ΦF for the aminosquaraines 5a-e in CHCl3. As expected, due to the covalent link between iodine and the aminosquaraine, a strong heavy atom effect can be observed in all cases. This effect is emphasized in the case of the bis(hydroxyethyl)amino substituent group (dye 5c).

In Vitro Photodynamic Activity
Four human tumor cell lines were used to assess the in vitro cytotoxicity of the synthesized dyes (5a-e): cervical (HeLa), breast (MCF7), hepatocellular (HepG2) and non-small cell lung (NCI-H460) carcinomas.
Although cytotoxicity was found to depend on the cell line, in general, all aminosquaraine dyes exhibited much higher cytotoxicity upon irradiation than in the absence of light. This fact revealed that the dyes are endowed with photodynamic action capacity. The assurance that the photodynamic effect was due, exclusively, to the sensitizing properties of the squaraine dyes, was secured by observing, in independent assays submitted to the photodynamic treatment and maintained in the dark, that, in the absence of the dyes neither DMSO nor light individually, nor the combination of both, were able to induce toxicity on the cells. Figure 3 shows the growth inhibition ability of aminosquaraines 5a-e against the different human tumor cell lines, in the absence of light and upon irradiation, at a dye concentration of 0.3 μM. In this representative example, the growth inhibition in the dark was less than 20 % in more than 75% of the assays.  Whatever the cell line, dyes 5a and 5b exhibited, invariably, the larger differences between the growth inhibition percentages upon illumination and in the dark (~ 46-68% and ~ 43-73%, respectively). In case of dye 5a the more pronounced differences in growth inhibition under the two conditions was found against the MCF-7 cell line, closely followed by the NCI-H460 cell line, while for dye 5b the larger difference was observed for the MCF-7 cell line. Dye 5c displayed the weaker photocytotoxic capability, presenting the smaller differences of growth inhibition in the presence and in the absence of light, for the HepG2, MCF-7 and NCI-H460 cell lines. For the HeLa cells dye 5d showed to be the less efficient sensitizer. It is worthwhile to mention that, though determined in CHCl3, dye 5c exhibited the lowest singlet oxygen generation quantum yield among all dyes tested.
The GI50 values (concentration causing 50% cell growth inhibition) for aminosquaraines 5a-e were also determined and are presented in Table 2. The differences found between the values determined for the assays in the dark and upon irradiation, clearly demonstrated once more the photocytotoxic ability of the dyes. Among all compounds, 5a and 5b consistently presented the lowest GI50 values upon illumination for all the cell lines tested, ranging from 0.004 μM to 0.19 μM. In the absence of light the GI50 values increased ca. 80-1500 times for 5a and ca. 45-250 times for 5b, depending on the cell line. With exception of the HeLa cells, dye 5d showed to be inactive in the dark (GI50 > 25 μM) against all the cells lines within the range of the concentrations used. Whatever the cell line, dyes 5a and 5b exhibited, invariably, the larger differences between the growth inhibition percentages upon illumination and in the dark (~46-68% and~43-73%, respectively). In case of dye 5a the more pronounced differences in growth inhibition under the two conditions was found against the MCF-7 cell line, closely followed by the NCI-H460 cell line, while for dye 5b the larger difference was observed for the MCF-7 cell line. Dye 5c displayed the weaker photocytotoxic capability, presenting the smaller differences of growth inhibition in the presence and in the absence of light, for the HepG2, MCF-7 and NCI-H460 cell lines. For the HeLa cells dye 5d showed to be the less efficient sensitizer. It is worthwhile to mention that, though determined in CHCl 3 , dye 5c exhibited the lowest singlet oxygen generation quantum yield among all dyes tested.
The GI 50 values (concentration causing 50% cell growth inhibition) for aminosquaraines 5a-e were also determined and are presented in Table 2. The differences found between the values determined for the assays in the dark and upon irradiation, clearly demonstrated once more the photocytotoxic ability of the dyes. Among all compounds, 5a and 5b consistently presented the lowest GI 50 values upon illumination for all the cell lines tested, ranging from 0.004 µM to 0.19 µM. In the absence of light the GI 50 values increased ca. 80-1500 times for 5a and ca. 45-250 times for 5b, depending on the cell line. With exception of the HeLa cells, dye 5d showed to be inactive in the dark (GI 50 > 25 µM) against all the cells lines within the range of the concentrations used.

One of the desirable features of any new sensitizer is the lack of toxicity for non-tumor cells.
For that reason aminosquaraines 5a-e were also tested against a non-tumor porcine liver primary cell culture (PLP2) established by some of us, both in the absence of light and under irradiation. Dye 5a presented a GI 50 value under illumination considerably superior to those determined for the tumor cell lines. Dyes 5b-d displayed GI 50 values against PLP2 cells, both in the dark and under irradiation, not appreciably different than those obtained for the tumor cell lines used.
Having recently demonstrated the photocytotoxic properties of several aminosquaraine dyes derived from benzothiazole [36], we anticipated that the introduction of iodine atoms in the terminal aromatic nucleus of the dyes could increase singlet oxygen generation through the heavy atom effect and, ultimately, enhance their photocytotoxicity. In fact, the GI 50 values obtained for dyes 5a-e under irradiation were in general smaller, in some cases substantially, than those determined previously for their non-halogenated analogues. The GI 50 values in the dark, on the other hand, were higher, making aminosquaraines 5a-e safer for application.

General Information
All reagents were obtained commercially and used as received. Solvents were of analytical grade. Anhydrous solvents were dried [45] and freshly distilled. All reactions were monitored by TLC using 0.20 mm aluminum-backed silica-gel plates (SIL G UV 254 , Macherey-Nagel, Düren, Germany). Melting points were measured in a melting-point apparatus equipped with a binocular microscope (Rotoquímica, Maia, Portugal) and are uncorrected. IR spectra were recorded on a Unicam Research Series FT-IR spectrophotometer (Mattson Instruments Inc., Madison, WI, USA); ν max in cm −1 . Vis spectra were performed on a Lambda 25 instrument (Perkin-Elmer, Singapore); λ max in nm. 1 H-(400.13 MHz) and 13 C-NMR spectra (100.61 MHz) were recorded on an ARX 400 spectrometer (Brüker, Germany); δ in ppm relative to residual solvent signals, J in Hz. High resolution electrospray ionization time-of-flight mass spectra (HRMS ESI-TOF) were determined on Apex-Q FT-ICR and micrOTOF mass spectrometers (Bruker, Germany). 6-Iodo-2-methylbenzothiazole (1) was prepared as previously described [40].

Singlet Oxygen Quantum Yield Determinations
The singlet oxygen measurement set-up was assembled in our laboratory as previously described [43]. As an excitation source we used a nitrogen laser, excitation wavelength = 337 nm. The detector was an InGaAs CCD (model i-Dus from Andor Technology Limited, Belfast, UK) working at low temperature (−60 • C) coupled to a fixed spectrograph, model Shamrock 163i, also from Andor. Phenazine (standard) was used at O.D. = 0.6 in chloroform. The Φ ∆ values were obtained by comparing the total area of the emission spectra for the reference and for each dye under study in the same solvent, with the same optical density at the excitation wavelength. Phenazine (standard) was used at OD = 0.60 in CHCl 3 .

Preparation of the Solutions of Aminosquaraine Dyes
Stock solutions of the aminosquaraines 5a-e with different concentrations (0.0375 µM, 0.15 µM, 0.6 µM, 6 µM, 12 µM, 25 µM, 50 µM, 500 µM) were prepared using a solution of 3% DMSO in DMEM and kept at−20 • C. Prior to the in vitro assays diluted solutions were prepared with the same solvent to obtain solutions with final concentrations of 0.03 µM, 0.3 µM, 0.6 µM, 1.25 µM, 2.5 µM and 25 µM. For the HeLa MCF-7 cell lines solutions with final concentrations of 0.0075 µM and 0.001875 µM were additionally prepared once the inhibition percentage for the 0.03 µM solution was already superior to 50%.

Photodynamic Treatment
The cytotoxicity of the squaraine dyes was tested in the dark and under irradiation. For irradiation of the cells a halogen/tungsten lamp (24 V and 250 W, Osram, Carnaxide, Portugal) was used with a fluence rate of 23-24 µW/cm 2 (measured with an ILT 1400-A radiometer equipped with a SEL033 detector, ILT, Peabody, MA, USA). The cells were irradiated continuously for 30 min. A 3% aqueous solution of K 2 Cr 2 O 7 was placed between the lamp and the cells as a liquid cut off filter to remove light of wavelength shorter than~500 nm. The temperature to which the cells were exposed was carefully monitored to guarantee cell viability.

Evaluation of Cytotoxicity in Human Tumor Cell Lines
Four human tumor cell lines were used: MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung carcinoma), HeLa (cervical carcinoma) and HepG2 (hepatocellular carcinoma) from DSMZ (Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mönchengladbach, Germany). Cells were routinely maintained as adherent cell cultures in RPMI-1640 medium containing 10% heat-inactivated FBS and 2 mM glutamine at 3 • C, in a humidified air incubator containing 5% CO 2 . Each cell line was plated at an appropriate density (7.5 × 10 3 cells/well for MCF-7 and NCI-H460, and 1.0 × 10 4 cells/well for HeLa and HepG2) in 96-well plates, allowed to attach for 24 h and then treated with different concentrations of each squaraine dye. Controls were set with the cells in the absence of the dyes in 3% DMSO in DMEM and in the growth medium only. After the incubation step (24 h), the cells were submitted to the photodynamic treatment for 30 min. Controls in the absence of light were performed on every test. Neither DMSO alone, nor light alone, nor the combination of both induced toxicity on the cells. Then, the growth medium was changed and the cells were incubated for further 24 h. Following this incubation period, the adherent cells were fixed by adding cold 10% TCA (100 µL) and incubated for 60 min at 4 • C. Plates were then washed with deionized water and subsequently dried. SRB solution (0.1% in 1% acetic acid, 100 µL) was then added to each plate well and incubated for 30 min at room temperature. Unbound SRB was removed by washing with 1% acetic acid. Plates were air dried, the bound SRB was solubilized with 10 mM Tris (200 µL) and the absorbance was measured at 540 nm in an ELX800 Microplate Reader (Bio-Tek Instruments Inc., Winooski, VT, USA) [46]. The results were expressed in GI 50 values, which correspond to the concentration of dye that causes 50% inhibition of cell growth. All the assays were performed in duplicate. Ellipticine was used as standard.

Evaluation of Cytotoxicity in a Porcine Liver Primary Cell Culture
A cell culture was prepared from a freshly harvested porcine liver obtained from a local slaughter house, and it was designed as PLP2. Briefly, the liver tissues were rinsed in Hank's balanced salt solution containing 100 U/mL penicillin and 100 µg/mL streptomycin and divided into 1×1 mm 3 explants to ensure that all liver cells were able to access the nutrients provided by the medium. Some of these explants were placed in 25 cm 2 tissue flasks in DMEM medium supplemented with 10% fetal bovine serum, 2 mM non-essential amino acids and 100 U/mL penicillin, 100 mg/mL streptomycin and incubated at 37 • C with a humidified atmosphere containing 5% CO 2 . The medium was changed every two days. Cultivation of the cells was continued with direct monitoring every two or three days using a phase contrast microscope. Before confluence was reached, cells were subcultured and plated in 96-well plates at a density of 1.0 × 10 4 cells/well, and cultivated in DMEM medium with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin [47]. The PLP2 cells were incubated with the squaraine dyes and submitted to the two independent processes-dark and irradiation-as described earlier for the human tumor cell lines. The SRB assay was performed according to the procedure previously described. The results were expressed in GI 50 values. All the assays were performed in duplicate. Ellipticine was used as standard.

Statistical Analysis
All the assays were carried out in duplicate. The results are expressed as mean values and standard deviation. The results were analysed using a Student´s t-test to determine the significant difference among the different samples, with α = 0.05. This treatment was carried out using SPSS v. 22.0 program (IBM Corp., Armonk, NY, USA).

Conclusions
In conclusion, the synthesized aminosquaraine dyes, with strong absorption within the phototherapeutic window, showed photodynamic activity against the human tumor cell lines studied (MCF-7, NCI-H460, HeLa, and HepG2), inhibiting cell growth upon irradiation more than in the absence of light, in some cases substantially. When compared to the non-halogenated analogues dyes 5a-e exhibited, in general, lower GI 50 values under irradiation, which is probably related to the enhancement of singlet oxygen production through the so-called heavy atom effect. The difference of cytotoxicity observed in the dark and upon irradiation, turns some of the dyes potential candidates as photosensitizers for PDT, in particular compound 5a, which showed the lowest toxicity for the non-tumor primary PLP2 cells.