Quinoline- and benzoselenazole-derived unsymmetrical squaraine cyanine dyes: design, synthesis, photophysicochemical features and light-triggerable antiproliferative effects against breast cancer cell lines

Photodynamic therapy is an innovative treatment approach broadly directed towards oncological diseases. Its applicability and efficiency are closely related to the interaction of three main components, namely a photosensitizer, light and molecular triplet oxygen, which should drive cell death. Recently, several studies have demonstrated that squaraine cyanine dyes have a set of photophysical and photochemical properties that have made of these compounds’ potential photosensitizers for this therapeutic modality. In the present research work, we describe the synthesis and characterization of four quinoline- and benzoselenazole-derived unsymmetrical squaraine cyanine dyes. Except for the precursor of aminosquaraine dyes, i.e., O-methylated derivative, all dyes were evaluated for their behavior and absorption capacity in different organic and aqueous solvents, their ability to form singlet oxygen, their light-stability, and in vitro phototherapeutic effects against two human breast cancer cell cultures (BT-474 and MCF-7). Regardless of the nature of the used solvents, the synthesized dyes showed intense absorption in the red and near-infrared spectral regions, despite the formation of aggregates in aqueous media. Dyes showed high light-stability against light exposure. Despite the low ability to produce singlet oxygen, aminosquaraine dyes demonstrated worthy in vitro phototherapeutic activity.This research was funded by the European Investment Funds by FEDER/COMPETE/POCI under projects POCI-01-0145-FEDER-006958 (CITAB) and POCI-01-0145-FEDER-007491 (CICS-UBI) and Funds by FCT—Portuguese Foundation for Science and technology, under the projects UIDB/ 04033/2020 (CITAB) and UIDB/ 00616/2020 (CQ-VR). This work was also supported by funds from the Health Sciences Research Center (CICS-UBI) through National Funds by FCT—Foundation for Science and Technology (UID/Multi/00709/2019).The research at iBB was supported by Project UID/NAN/50024/2019 and M-ERA-NET/0002/2015 from FCT. E.L. was supported by the FCT PhD grant SFRH/BD/147645/2019.info:eu-repo/semantics/publishedVersio

Evaluation of antifungal activity and potential application as fluorescent probes of indolenine and benzo[e]indole-based squarylium dyes

The antifungal performance and the possible use as fluorescent probes of a series of squarylium dyes derived from indolenine and benzo[e]indole previously synthesized was evaluated. Some photophysical properties were performed in ethanol and phosphate buffer, and the type of aggregates form in phosphate buffer was analyzed. Using the 1,3-diphenylisobenzofuran assay, a qualitative assessment of the capacity of dyes to produce singlet oxygen after irradiation was performed. Regarding the antifungal activity, this was studied through a broth microdilution assay using Saccharomyces cerevisiae PYCC 4072 as a biological model. The effect of irradiation of the dyes, with an appropriate light emitting diode system, on the antifungal activity was also evaluated, and it was verified that some of the dyes improve their activity after irradiation. Using fluorescence microscopy techniques, the colocalization of dyes in S. cerevisae cells was investigated and it was possible to verify that some of the squarylium dyes with a barbituric moiety in the four-membered central ring stained and accumulated preferentially in the mitochondrial web and perinuclear membrane of the cells. The possible use as a fluorescent probe for the detection of HSA was also evaluated for one of the dyes of the series, demonstrating a linear variation of the fluorescence intensity accompanied by the increase of the protein concentration.We thank to Fundação para a Ciência e Tecnologia (FCT), Comissão de Coordenação e Desenvolvimento Regional do Norte (CCDR-N) and FEDER (European Fund for Regional Development)-COMPETEQREN-EU for financial support to the research centers CQ/UM (UIDB/00686/2020), CBMA (UID/BIA/04050/2020), CQ/VR (UID/QUI/UI0616/2019) and CICSUBI (POCI-01-0145-FEDER-007491), as well as PhD grants to V.S.D.G. (UMINHO/BD/43/2016) and J.C.C.F. (SFRH/BD/133207/2017)

Squaraine dyes derived from indolenine and benzo[e]indole as potential fluorescent probes for HSA detection and antifungal agents

Four squaraine dyes derived from 2,3,3-trimethylindolenine and 1,1,2-trimethyl-1H-benzo[e]indole with different combinations of barbituric groups attach to the central ring, having ester groups and alkyl chains in the nitrogen atoms of heterocyclic rings were synthesized. These dyes were fully characterized and their photophysical behavior was studied in ethanol and phosphate-buffered saline solution. Absorption and emission bands between 631 and 712 nm were detected, with the formation of aggregates in aqueous media, which is typical of this class of dyes. Tests carried out with 1,3-diphenylisobenzofuran allowed us to verify the ability of the dyes to produce singlet oxygen. The interaction of synthesized dyes with human serum albumin (HSA) was also evaluated, being demonstrated a linear correlation between fluorescence intensity and protein concentration. The antifungal potential of the dyes against the yeast Saccharomyces cerevisiae was evaluated using a broth microdilution assay. In order to test the photosensitizing capacity of the synthesized dyes, tests were carried out in the dark and with irradiation, using a custom-built light-emitting diode that emits close to the absorption wavelength of the studied dyes. The results showed that the interaction of dyes with HSA and the antifungal activity depends on the different structural modifications of the dyes.We thanks to Fundação para a Ciência e Tecnologia (FCT), Comissão de Coordenação e Desenvolvimento Regional do Norte (CCDR-N) and FEDER (European Fund for Regional Development)-COMPETEQREN-EU for financial support to the research centers CQ/UM (UIDB/00686/2020), CBMA (UID/BIA/04050/2020), CQ/VR (UID/QUI/UI0616/2019) and CICSUBI (POCI-01-0145-FEDER-007491), as well as PhD grants to V.S.D.G. (UMINHO/BD/43/2016) and J.C.C.F. (SFRH/BD/133207/2017)

Red and near-infrared absorbing dicyanomethylene squaraine cyanine dyes: photophysicochemical properties and anti-tumor photosensitizing effects

Photodynamic therapy is a medical modality developed for the treatment of several diseases of oncological and non-oncological etiology that requires the presence of a photosensitizer, light and molecular oxygen, which combined will trigger physicochemical reactions responsible for reactive oxygen species production. Given the scarcity of photosensitizers that exhibit desirable characteristics for its potential application in this therapeutic strategy, the main aims of this work were the study of the photophysical and photochemical properties and the photobiological activity of several dicyanomethylene squaraine cyanine dyes. Thus, herein, the study of their aggregation character, photobleaching and singlet oxygen production ability, and the further application of the previously synthesized dyes in Caco-2 and HepG2 cancer cell lines, to evaluate their phototherapeutic effects, are described. Dicyanomethylene squaraine dyes exhibited moderate light-stability and, despite the low singlet oxygen quantum yields, were a core of dyes that exhibited relevant in vitro photodynamic activity, as there was an evident increase in the toxicity of some of the tested dyes exclusive to radiation treatments.This research was funded by the European Investment Funds by FEDER/COMPETE/POCI under projects POCI-01-0145-FEDER-006958 (CITAB) and POCI-01-0145-FEDER-007491 (CICS-UBI) and Funds by FCT – Portuguese Foundation for Science and technology, under the projects UIDB/04033/2019 (CITAB) and UIDB/00616/2020 (CQ-VR). This work was also supported by funds from the Health Sciences Research Center (CICS-UBI) through National Funds by FCT—Foundation for Science and Technology (UID/Multi/00709/2019). The research at CQFM was supported by Project UID/NAN/50024/2019 and M-ERA-NET/0002/2015 from FCT. E. L. was supported by the FCT PhD grant SFRH/BD/147645/2019

In Vitro and In Silico Evaluation of Indole-Bearing Squaraine Dyes as Potential Human Serum Albumin Fluorescent Probes

The quantitative determination of proteins is an important parameter in biochemistry, biotechnology and immunodiagnostics, and the importance of serum albumin in clinical diagnosis should be highlighted, given that alterations in its concentration are generally associated with certain diseases. As possible probes for this purpose, squaraine dyes have been arousing the interest of many researchers due to their unique properties, such as absorption in the visible spectra, moderate relative fluorescence quantum yields and increased fluorescence intensity after non-covalent binding to specific ligands. In this work, five squaraine dyes, four of which have never been reported in the literature, were characterized and evaluated in vitro and in silico concerning their potential application as fluorescent probes for human serum albumin detection. After interaction with the protein, the fluorescence intensity increased from 12 to 41 times, depending on the dye under study. High sensitivity (1.0 × 105–5.4 × 105 nM), low detection limits (168–352 nM) and moderate quantitation limits (560–1172 nM) were obtained, proving the efficiency of the method. In addition, moderate-to-excellent selectivity was observed compared to γ-globulin proteins. Molecular docking suggests that the dyes interact more effectively with the Sudlow site I, and binding energies have been markedly higher than those of warfarin, a molecule known to bind to this site specifically

Bis-thiobarbiturates as Promising Xanthine Oxidase Inhibitors: Synthesis and Biological Evaluation

Xanthine oxidase (XO) is the enzyme responsible for the conversion of endogenous purines into uric acid. Therefore, this enzyme has been associated with pathological conditions caused by hyperuricemia, such as the disease commonly known as gout. Barbiturates and their congeners thiobarbiturates represent a class of heterocyclic drugs capable of influencing neurotransmission. However, in recent years a very large group of potential pharmaceutical and medicinal applications have been related to their structure. This great diversity of biological activities is directly linked to the enormous opportunities found for chemical change off the back of these findings. With this in mind, sixteen bis-thiobarbiturates were synthesized in moderate to excellent reactional yields, and their antioxidant, anti-proliferative, and XO inhibitory activity were evaluated. In general, all bis-thiobarbiturates present a good antioxidant performance and an excellent ability to inhibit XO at a concentration of 30 µM, eight of them are superior to those observed with the reference drug allopurinol (Allo), nevertheless they were not as effective as febuxostat. The most powerful bis-thiobarbiturate within this set showed in vitro IC50 of 1.79 μM, which was about ten-fold better than Allo inhibition, together with suitable low cytotoxicity. In silico molecular properties such as drug-likeness, pharmacokinetics, and toxicity of this promising barbiturate were also analyzed and herein discussed

An Insight into Symmetrical Cyanine Dyes as Promising Selective Antiproliferative Agents in Caco-2 Colorectal Cancer Cells

Cancer remains one of the diseases with the highest worldwide incidence. Several cytotoxic approaches have been used over the years to overcome this public health threat, such as chemotherapy, radiotherapy, and photodynamic therapy (PDT). Cyanine dyes are a class of compounds that have been extensively studied as PDT sensitisers; nevertheless, their antiproliferative potential in the absence of a light source has been scarcely explored. Herein, the synthesis of eighteen symmetric mono-, tri-, and heptamethine cyanine dyes and their evaluation as potential anticancer agents is described. The influences of the heterocyclic nature, counterion, and methine chain length on the antiproliferative effects and selectivities were analysed, and relevant structure–activity relationship data were gathered. The impact of light on the cytotoxic activity of the most promising dye was also assessed and discussed. Most of the monomethine and trimethine cyanine dyes under study demonstrated a high antiproliferative effect on human tumour cell lines of colorectal (Caco-2), breast (MCF-7), and prostate (PC-3) cancer at the initial screening (10 µM). However, concentration–viability curves showed higher potency and selectivity for the Caco-2 cell line. A monomethine cyanine dye derived from benzoxazole was the most promising compound (IC50 for Caco-2 = 0.67 µM and a selectivity index of 20.9 for Caco-2 versus normal human dermal fibroblasts (NHDF)) and led to Caco-2 cell cycle arrest at the G0/G1 phase. Complementary in silico studies predicted good intestinal absorption and oral bioavailability for this cyanine dye

Squaraine dyes as serum albumins probes: synthesis, photophysical experiments and molecular docking studies

Three barbiturate squaraine dyes derived from indolenine or benzothiazole, with different barbituric acid derivatives were prepared, characterized and photophysically evaluated by standard spectroscopic methods. As expectable for squaraines, these dyes showed narrow and intense absorption and emission bands in the Vis/NIR region. The interaction of synthesized dyes with bovine and human serum albumins (BSA and HSA) was also evaluated in phosphate buffer (PB). The results revealed that upon the addition of BSA or HSA the complex dye-protein emit more fluorescence, and the emission intensity is directly proportional to the concentration of protein used (0–3.5 µM). The titration tests allowed to calculate the binding constants, in an order of magnitude of 104-106 M, as well as the limits of detection and quantification in the nanomolar tens range. All dyes showed a good response to the interaction with both proteins, but the most pronounced envisioning their use as protein labeling was observed for the squaraine dye derived from the indolenine with a 1,3-dimethylbarbituric acid moiety. The molecular docking studies revealed the existence of a binding between the compounds and four sites on the HSA molecule, where one of these four locations is a new binding site with which this series of dye interacts.We acknowledge to Portuguese Foundation for Science and Technology and FEDER (European Fund for Regional Development) -COMPETEQREN-EU for financial support to the research centres CQ/UM (UIDB/00686/2020) , CQ/VR (UID/QUI/UI0616/2019) and CIC-SUBI (POCI-01-0145-FEDER-007491) , as well as PhD grants to Vanessa S. D. Gomes (UMINHO/BD/43/2016)