Benzo[a]phenoxazinium chlorides functionalized with chloride atoms and/or ester groups

With the aim of contributing to the development of fluorescent near-infrared (NIR) probes with applications in biomedicine, our research group is committed to the development of new water-soluble benzo[a]phenoxazine derivatives and the evaluation of their photophysical and biological potential. Herein we report the photophysical behaviour in anhydrous ethanol of four synthesised benzo[a]phenoxazinium chlorides, possessing the (3-chloropropyl)amino and/or (4-ethoxy-4oxobutyl)amino groups at 5and 9-positions of the polycyclic system.


Introduction
Fluorescent probes are recognised tools in the various fields, including in studies of biochemical processes, diagnosis of disease, and detection of hazardous compounds, among others.[3][4] Molecules possessing an oxazine ring as a part of a tetracyclic aromatic structure are called benzophenoxazines, and their derivatives having adequate substituents, namely the most well-known, Nile Blue, are associated with an absorbance and a fluorescence emission at longer wavelengths, belonging to the group of near-infrared fluorescence probes. 5esearch in the design, synthesis and evaluation of the potential applications of compounds that can belong to this class of probes is required and still a very actual subject due to the challenging of developing more specific molecules and follow the advances of fluorescent techniques.As a result, several studies with different families of heterocyclic systems have been reported, and all are necessary and extremely important.
2][3] These two thiol-based compounds are present in biological systems with both being detected at the same time by the NIR dyes. 2 On the other hand, Wang et al. achieved the enhancement of the fluorescence intensity of the NIR probe in presence of cysteine, while showing little fluorescence by interacting with glutathione. 3In 2015, Chai et al. reported a series of phosphorussubstituted rhodamines presenting phosphorous groups instead of the oxygen atom in traditional rhodamines.These new NIR probes enter animal cells both during in vitro and in vivo studies. 4A localized fluorescence signal was still detected 48 hours after the intramuscular injection in mice of one of these compounds, despite the decay in the fluorescence intensity.
As a continuation of the investigation of our research group, [6][7][8][9][10] in this proceeding we intend to give a short contribution to the development of NIR probes, presenting the photophysical properties of four synthesized benzo[a]phenoxazinium chlorides functionalized with chloride atoms and/or ester groups.The experiments were performed in dry ethanol, in this medium acidified with trifluoroacetic acid (TFA) and basified with tetraethylammonium hydroxide (TEAH).Electronic absorption and emission spectra of 4 × 10 -6 M solutions in anhydrous ethanol were measured for synthesized benzo[a]phenoxazinium chlorides 1-4 (Table 1).The relative fluorescence quantum yields (ΦF) were determined using Oxazine 1 as a standard (ΦF = 0.11 in ethanol).In our earlier investigation with similar type of compounds in reagent grade ethanol showed inconsistence and irreproducible data due to variations of water concentrations in different batches of ethanol.Hence, we prefer to use anhydrous ethanol following the drying procedure in literature. 13evious studies on benzo[a]phenoxazinium chlorides showed that the photophysical properties in proton-accepting solvents is influenced by acid-base equilibria mainly located at the 5-amino position. 14,4 15 In ethanol media the absorption spectra are dominated by an acidic form (AH + ) and a 100 nm blue shifted neutral form (A). 16 The fluorescence of the basic form is broad and centred at around 600 nm while the acid form (AH + ) shows a band centred above 660 nm with a much higher quantum yield. 16[16] Table 1 shows that compound 4 has the greatest absorption and emission maxima.This is mainly due to the result of di-alkylation at 9-amino position of the heterocycle as previously observed. 16,17 pounds 1 and 3 showed the highest fluorescence efficiency in ethanolic media and greater Stokes shift of 34 nm was registered for compound 3 (Figure 1).Additionally, the behaviour for the three compounds with a mono-alkylated amine at 9-position is in essence the same, being shown only one band for the emission and for the absorbance of dye 1 (Figures 4 and 5) which are representative of those obtained for compounds 2 and 3. Figure 4 shows the absorption and emission spectra excited at 575 nm of compounds 1 and 4. At 575 nm excitation only the acidic AH + form is excited as clearly evident.In acidified dry ethanol media the emission maxima is 645 nm and 676 nm for fluorophores 1 and 4, respectively, with a bathochromic shift of 31 nm.Moreover, in these conditions a small bump around 550 nm indicates aggregation in solution.To conclude, Figure 5 presents the normalized absorbance and emission spectra at 470 nm excitation of compounds 1 and 4. At 470 nm excitation, the basic form is excited and it is seen that by the addition of TEAH it is possible to shift the equilibrium towards the basic form.Comparing compounds 1 and 4 in basified dry ethanol significant bathochromic shifts were observed in absorption (15 nm) and emission (17 nm).In particular the emission spectrum is broad for the dye 1 in comparison with the other fluorophore.Furthermore, in a basic medium the Stoke's shift is largely increased to ca. 100 nm, the only exception being dye 2 whose main absorption band in plain ethanol is already the correspondent to the basic form and so already there displays a Stoke's shift of a comparable magnitude.

Conclusions
Four new benzo[a]phenoxazinium chlorides were efficiently synthesized and preliminary photophysics of the acidic and basic forms were studied in dried ethanolic media, by the addition of either a strong acid or a strong base.Overall, the highest absorbance and emission was observed for compound 4, which may due to the effect of di-alkylation at 9-amino position of the polycyclic system.Compound 2 possessing two chloride atoms as terminals at 5-and 9-postion behaved the reverse way in comparison with 1 and 3.

Figure 2 4 ,
Figure2shows the absorption spectra for compounds 1-4 (C = 4 × 10 -6 M) in dry ethanol.It was observed that dyes 1 and 3 display mostly acidic form with less basic form.On the other hand, the benzo[a]phenoxazinium chloride 2 possessing chloride atoms as terminals at 5-and 9-amino position presents mostly the basic form, twice the absorbance value in comparison with the acidic form.In case of

Figure 3
Figure3displays the normalised emission spectra of compounds 1-4 (C = 4 × 10 -6 M) excited at 470 nm and 575 nm in anhydrous ethanol.At 470 nm excitation, the basic form is mostly excited with little acidic form.It is evident from the emission spectra of compounds 3 and 4 the presence of a blue shoulder around 600 nm confirming the basic form.In contrast, dye 2 shows mostly the basic form with a hypsochromic shift in comparison to others with little acidic form at 470 nm excitation wavelength.

Figure 5 .
Figure 5. Normalized absorbance and emission spectra at 470 nm excitation of compounds 1 and 4 in basified dry ethanol. 12