New benzo [ a ] phenoxazines bearing the ( 4 , 6-dichloro-1 , 3 , 5-triazin-2-yl ) amino group : synthesis and photophysical properties

Synthesis of new benzo[a]phenoxazinium chlorides possessing the (4,6-dichloro-1,3,5triazin-2-yl)amino at 5-amino function of the heterocycles is described. The preliminary photophysical properties of these compounds in anhydrous ethanol when acidified with TFA or basified with TEAH is also investigated, as well as their response in aqueous media. These benzo[a]phenoxazinium chlorides exhibited fluorescence with maximum emission wavelengths between 628 and 676 nm, in anhydrous ethanol and water.


Introduction
Fluorescent probes such as small organic molecules used for optical imaging purposes have emerged as an important tool in various fields of sciences. 1Among the fluorescent probes, benzophenoxazinium derivatives are preferred due to their high wavelength of emission maxima, good molar extinction coefficients, photochemical stability and long shelf lives. 2 Due to their interesting optical properties, benzo[a]phenoxazinium chlorides are used as biomarkers for labeling of amino acids, proteins and also utilized for various other analytical applications. 3In addition, these compounds are medicinally important as antifungal 4 and antimalarial agents. 5Phenoxazine core compounds also serve as electron donors for dye sensitized solar cells, 6 enhance electro-optic activity in second-order nonlinear optical chrmophores 7 and function as efficient sensitizers for photodynamic therapy (PDT). 8wing to the importance and our research activity towards Nile Blue (NB) derivatives, [9][10][11] we herein describe the synthesis of new benzo[a]phenoxazinium chlorides possessing the (4,6-dichloro-1,3,5-triazin-2-yl)amino at 5-position of the polycyclic system.The photophysical characterization in anhydrous ethanol and aqueous media along with the addition of acid and base is discussed.

Experimental
Typical procedure for the preparation of compounds 1 and 2 (illustrated for 1).
The 1 H NMR spectra (1 and 2) exhibited a triplet for the terminal methyl groups (δ 1.36 ppm) and methylene protons proximity to the nitrogen atom appeared as quartets at (δ 3.47 to 3.72 ppm).The methyl group of 10-H for the compound 1 showed at a sharp singlet at (δ 2.27 ppm).The aromatic protons of the polycyclic system showed singlets for 6-H (δ 6.75 or 6.92 ppm), and either singlet or doublet for 8-H (δ 6.70 or 6.99 ppm).Similarly, for the other aromatic protons 11-H (δ 7.52 or 7.95 ppm), 2-H (δ 7.76 or 7.87 ppm), 1-H (δ 8.21 or 8.37 ppm) and 3-H (δ 7.88 or 7.99 ppm) appeared in form of singlets doublets, triplets and multiplets.Electronic absorption and emission spectra of 4 × 10 -6 M solutions in anhydrous ethanol and water were measured for synthesized benzo[a]phenoxazinium chlorides (Table 1).The relative fluorescence quantum yields (Φ F ) were determined using Oxazine 1 as a standard (Φ F = 0.11 in ethanol). 13ble 1.Preliminary photophysical studies of compounds 1 and 2 in anhydrous ethanol and water after the addition of TFA.

Dry ethanol + TFA
Water + TFA     In our previous studies, 14,15 it was found that acid-base equilibrium of benzo[a]phenoxazinium chlorides in proton accepting solvents is influenced by the substituents mainly attached at 5-amino position of the heterocyclic system.In ethanol media the emission spectra of the basic form is broad and centered at around 600 nm while the acid form (AH + ) shows a band above 660 nm with a much higher quantum yield. 16The variation in fluorescence quantum yields with change in substitution at 9amino position of the benzophenoxazine is monoalkylated 0.4 and 0.1-0.2 for the dialkylated compounds of similar types. 14 comparison, compound 2 exhibited significant bathochromic shifts both in absorption (13 nm) and emission (24 nm) (Table 1).This is mainly, due to the di-alkylation at the 9-amino position as previously observed.Similarly, compound 1 showed the highest fluorescence efficiency in ethanoic media.On the other hand, greater Stokes shift (40 nm) in aqueous media was observed for compound 2, which clearly indicates the stronger solvent fluorophore interactions.In acidified water, compound 1 shows the highest absorption and compound 2 decreases in its absorption value with a bathochromic shift of 20 nm (Figure 1).The maximum emission was registered by compound 2 in acidic water ( em 676 nm).At 470 nm excitation the basic form is mostly excited with a small fraction of acidic form.
Figure 2 shows the spectra in basified dry ethanol at 470 nm excitation and the excitation spectra were collected at 605 nm emission wavelength.Similarly, figure 3 shows the emission spectra at 575 nm excitation, where only the acidic AH + form is excited.

Conclusion
Benzo[a]phenoxazinium chlorides possessing the (4,6-dichloro-1,3,5-triazin-2-yl)amino at 5position function of the heterocycle were synthesized.The acidic and basic forms were studied in anhydrous ethanol and water by the addition of either strong acid or base.These compounds have tendency towards aggregation in aqueous medium.This type of fluorescent dyes can be used as synthetic intermediates and extension of the studies are in progress.

Figure 1 .
Figure 1.Absorption spectra of compounds 1 and 2 in acidified and basified water.

Figure 2 .
Figure 2. Spectra in basified dry ethanol at 470 nm excitation and the excitation spectra were collected at 605 nm emission wavelength.

Figure 3 .
Figure 3. Emission spectra of compounds 1 and 2 in dry ethanol and aqueous media at 575 nm excitation.