Sonochemical synthesis, optical properties and DFT studies on novel (N-arylamino)phenothiazinium dyes suitable for fluorescence cells imaging.

Novel (N-arylamino)phenothiazinium dyes containing meta-substituted-arylamine auxochrome units were successfully obtained by applying a sonochemical protocol designed for a more efficient energy usage in the preparation of methylene blue (MB) analogues. Single crystal X-ray diffraction analysis revealed the spatial arrangement in aggregated crystalline state of (N-(meta-bromoaryl)amino)phenothiazinium dye with minor variances induced by the nature of the halogenide counterion (iodide or chloride). The optical UV-vis properties of the novel (N-arylamino)phenothiazinium dyes were comparable to those of the parent MB, with the longest wavelength absorption maxima situated in the visible range (640-680 nm), large molar extinction coefficients (log ε = 4.5-5.1) and weak solvatochromism in polar solvents. Their fluorescence emission in solid state was evidenced by One Photon Excited Fluorescence Lifetime Imaging (OPE-FLIM) and Two Photon Excited Fluorescence Lifetime Imaging (TPE-FLIM) experiments. Theoretical calculations based on Time Dependent-Density Functional Theory (TD-DFT) at B3PW91 and CAM-B3LYP/def2-SV(P) level of theory predicted absorption and fluorescence emission wavelength maxima in reasonable agreement with experimental data. Computational results suggest that the electronic excitations imply a departure from the planar molecular ground state towards geometrically rearranged excited states disfavoring the vibronic couplings due to a high degree of flexibility induced by the conformational motion of the N-arylamino auxochromes. Preliminary studies regarding the dyes' relevance in biological environment indicated lipophilicity (log P octanol/water 0.5-2.3), no aggregation tendency in diluted solutions in the concentration range 10-50 microM and ability for cytoplasmatic staining of D407 human retinal pigment epithelial cells.

Transient radical species and oxygen colorimetric indicators grounded on phenothiazinium dyes.

This study investigates four phenothiazinium dyes including methylene blue (MB) and three analogues containing auxochrome variation 2-4 as oxygen colorimetric indicators prepared by their incorporation into two types of oxygen permeable materials containing: a) polyvinyl alcohol substrate with additional TiO2 photocatalysts (PhOxIn), and b) carboxymethylcellulose polymer matrix containing glucose and KOH (ChOxIn). In vacuum packages where volumetric concentrations of oxygen were below 0.01 %, the leuco forms of the tested phenothiazinum dyes were readily oxidised, initiating the colour turn from white to blue. The redox processes involved were explored by experimental electron paramagnetic resonance EPR and UV-vis spectroscopic methods and further supported by theoretical quantum chemistry. The EPR experiments showed that the chemical oxidation of the leuco-form of the phenothiazinium dyes 1-4 produced transient aminyl radical species with the single electron mainly located at the nitrogen atom of the heterocyclic core. The formation of these transient aminyl species was also perceived by UV-vis spectroscopy, their absorption maxima situated in the 552-592 nm range being also supported by TD-DFT theoretical calculations.

Novel (Phenothiazinyl)Vinyl-Pyridinium Dyes and Their Potential Applications as Cellular Staining Agents.

We report here the synthesis and structural characterization of novel cationic (phenothiazinyl)vinyl-pyridinium (PVP) dyes, together with optical (absorption/emission) properties and their potential applicability as fluorescent labels. Convective heating, ultrasound irradiation and mechanochemical synthesis were considered as alternative synthetic methodologies proficient for overcoming drawbacks such as long reaction time, nonsatisfactory yields or solvent requirements in the synthesis of novel dye (E)-1-(3-chloropropyl)-4-(2-(10-methyl-10H-phenothiazin-3-yl)vinyl)pyridin-1-ium bromide 3d and its N-alkyl-2-methylpyridinium precursor 1c. The trans geometry of the newly synthesized (E)-4-(2-(7-bromo-10-ethyl-10H-phenothiazin-3-yl)vinyl)-1-methylpyridin-1-ium iodide 3b and (E)-1-methyl-4-(2-(10-methyl-10H-phenothiazin-3-yl)vinyl)pyridin-1-ium tetrafluoroborate 3a' was confirmed by single crystal X-ray diffraction. A negative solvatochromism of the dyes in polar solvents was highlighted by UV-Vis spectroscopy and explanatory insights were supported by molecular modeling which suggested a better stabilization of the lowest unoccupied molecular orbitals (LUMO). The photostability of the dye 3b was investigated by irradiation at 365 nm in different solvents, while the steady-state and time-resolved fluorescence properties of dye 3b and 3a' in solid state were evaluated under one-photon excitation at 485 nm. The in vitro cytotoxicity of the new PVP dyes on B16-F10 melanoma cells was evaluated by WST-1 assay, while their intracellular localization was assessed by epi-fluorescence conventional microscopy imaging as well as one- and two-photon excited confocal fluorescence lifetime imaging microscopy (FLIM). PVP dyes displayed low cytotoxicity, good internalization inside melanoma cells and intense fluorescence emission inside the B16-F10 murine melanoma cells, making them suitable staining agents for imaging applications.

Ultrasound-assisted Strecker synthesis of novel 2-(hetero)aryl-2-(arylamino)acetonitrile derivatives.

This work describes an efficient, simple, and ecofriendly sonochemical procedure for the preparation of new α-(arylamino)acetonitrile derivatives C-substituted with phenothiazine or ferrocene units. The synthetic protocol is based on the Strecker reaction of a (hetero)aryl aldimine substrate with trimethylsilyl cyanide (TMSCN) in poly(ethylene glycol) (PEG) solution. The advantages of the sonochemical versus the conventional α-(arylamino)acetonitrile synthesis are the significantly shorter reaction time (30 min instead of 72 hours), the higher purity and the easier separation of the product that precipitated from the reaction mixture in crystalline form as depicted by scanning electron microscopy (SEM) analysis. The single crystal X-ray diffraction analysis disclosed the arrangement of the α-(arylamino)acetonitrile molecules in the aggregated crystalline state as a racemic mixture. The mutagenic/antimutagenic potential for three representative derivatives containing phenothiazinyl, ferrocenyl, and phenyl units, respectively, was evaluated by the Ames Salmonella/microsome test using S. typhimurium TA98 and TA100 strains with and without metabolic activation. The preliminary screening results pointed out that the C-(hetero)aryl-α-(arylamino)acetonitrile derivatives can be considered genotoxically safe and possibly antimutagenic.

Ethyne Functionalized Meso-Phenothiazinyl-Phenyl-Porphyrins: Synthesis and Optical Properties of Free Base Versus Protonated Species.

Synthesis, structural characterization and photophysical properties for a series of new trans-A2B2- and A3B-type ethynyl functionalized meso-phenothiazinyl-phenyl porphyrin derivatives are described. The new compounds displayed the characteristic porphyrin absorption spectra slightly modified by weak auxochromic effects of the substituents and fluorescence emission in the range of 651-659 nm with 11-25% quantum yields. The changes recorded in the UV-vis absorption spectra in the presence of trifluoroacetic acid (TFA) are consistent with the protonation of the two internal nitrogen atoms of the free-base porphyrin (19 nm bathochromic shift of the strong Soret band and one long wave absorption maxima situated in the range of 665-695 nm). Protonation of the phenothiazine substituents required increased amounts of TFA and produced a distinct hypsochromic shift of the long wave absorption maxima. The density functional theory (DFT) calculations of a porphyrin dication pointed out a saddle-distorted porphyrin ring as the ground-state geometry.

Novel Phenothiazine-Bridged Porphyrin-(Hetero)aryl dyads: Synthesis, Optical Properties, In Vitro Cytotoxicity and Staining of Human Ovarian Tumor Cell Lines.

We report here the synthetic procedure applied for the preparation of new AB3-type and trans-A2B2 type meso-halogenophenothiazinyl-phenyl-porphyrin derivatives, their metal core complexation and their peripheral modification using Suzuki-Miyaura cross coupling reactions with various (hetero)aryl (phenothiazinyl, 7-formyl-phenothiazinyl, (9-carbazolyl)-phenyl and 4-formyl-phenyl, phenyl) boronic acid derivatives. The meso-phenothiazinyl-phenyl-porphyrin (MPP) dyes family was thus extended by a series of novel phenothiazine-bridged porphyrin-(hetero)aryl dyads characterized by UV-Vis absorption/emission properties typical to the porphyrin chromophore, slightly modulated by increasing the size of peripheral substituents. Three phenothiazine-bridged porphyrin-heteroaryl dyads with fluorescence emission above 655 nm were selected as fluorophores in red spectral region for applications in cellular staining of human ovarian tumors. In vitro experiments of cell metabolic activity displayed a moderate toxicity on human ovarian tumor cell lines (OVCAR-3, cisplatin-sensitive A2780 and cisplatin-resistant A2780cis respectively). Visualization of the stained living cells was performed both by fluorescence microscopy imaging and by fluorescence lifetime imaging under two photon excitation (TPE-FLIM), confirming their cellular uptake and the capability of staining the cell nucleus.

Novel Thiazolo[5,4-b]phenothiazine Derivatives: Synthesis, Structural Characterization, and In Vitro Evaluation of Antiproliferative Activity against Human Leukaemia.

The molecular frame of the reported series of new polyheterocyclic compounds was intended to combine the potent phenothiazine and benzothiazole pharmacophoric units. The synthetic strategy applied was based on oxidative cyclization of N-(phenothiazin-3-yl)-thioamides and it was validated by the preparation of new 2-alkyl- and 2-aryl-thiazolo[5,4-b]phenothiazine derivatives. Optical properties of the series were experimentally emphasized by UV-Vis absorption/emission spectroscopy and structural features were theoretically modelled using density functional theory (DFT). In vitro activity as antileukemic agents of thiazolo[5,4-b]phenothiazine and N-(phenothiazine-3-yl)-thioamides were comparatively evaluated using cultivated HL-60 human promyelocytic and THP-1 human monocytic leukaemia cell lines. Some representatives proved selectivity against tumour cell lines, cytotoxicity, apoptosis induction, and cellular metabolism impairment capacity. 2-Naphthyl-thiazolo[5,4-b]phenothiazine was identified as the most effective of the series by displaying against THP-1 cell lines a cytotoxicity close to cytarabine antineoplastic agent.

Heterocycles 27. Microwave assisted synthesis and antitumour activity of novel phenothiazinyl-thiazolyl-hydrazine derivatives.

A series of new phenothiazinyl-thiazolyl-hydrazine derivatives were synthesized by Hantzsch cyclization of 1-(10-ethyl-10H-phenothiazin-3-yl)-methylidene-thiosemicarbazide with α-halocarbonyl derivatives. Comparison between classical and microwave assisted synthesis emphasizes the great advantages induced by microwaves irradiation which afforded high reaction yields in much shorter reaction time. Structural assignments were based on spectroscopic methods (high resolution NMR, FTIR, MS). The new compounds were tested in vitro for their antiproliferative activity against tumor cell lines using spectrometric methods. Most of the compounds exhibit cytotoxicity against hepatic and colon tumor cells in a dose-dependent mode and a relationship between the structure and their biological activity was observed.