Synthesis and spectral characterisation of naphtho [ 2 , 3-a ] phenoxazinium salts

A series of new functionalised naphtho[2,3-a]phenoxazinium chlorides was synthesised by condensation of 5-ethylamino-2-nitrosophenol hydrochloride with 1-aminoanthracene and its Nsubstituted derivatives. These cationic dyes showed maximum absorption in the range between 590 and 647 nm and strong fluorescence with maximum emission wavelengths between 630 and 674 nm, as well as high fluorescent quantum yields in ethanol and water at physiological pH.


Introduction
[3] Oxazine derivatives are included in this category of dyes and they have been used in various biomedical applications, 4,5 as biomarkers for nucleic acid detection 6 and protein labelling. 7though the large amount of fluorescent reagents has been reported, development of new longwavelength fluorophores still continues to be a subject of great interest.
Bearing this in mind, and following our previous research in the synthesis and application of benzo[a]phenoxazine dyes, [8][9][10] we decided to extend our previous work to the synthesis of new fluorochromophores, such us naphtho[2,3-a]phenoxazinium salts.These cationic compounds were obtained in low to excellent yields and evaluation of their absorption and emission properties was carried out in ethanol and water (pH 7.4).

Results and Discussion
Naphtho[2,3-a]phenoxazinium chlorides 1a-d were prepared by the reaction of 5-ethylamino-2nitrosophenol hydrochloride 2, with 1-aminoanthracene or its N-substituted-derivatives 3a-d in an acidic medium (Scheme 1).The required nitrosophenol 2 was synthesised using the usual procedure involving treatment of the corresponding 3-ethylamino-4-methylphenol with sodium nitrite in an acid solution.Compounds 3a-d were prepared by alkylation of 1-aminoanthracene with the appropriate bromo reagent in yields of about 53% (3c and 3d; 3b was not isolated).
The cationic dyes 1a-d were obtained as blue solids in yields ranging from 16 to 98% (Table 1) and were fully characterised by high resolution mass spectrometry, IR and NMR ( 1 H and 13 C) and visible spectroscopy.The visible absorption spectra of 10 -6 M solutions of compounds 1a-d in degassed absolute ethanol showed absorption peaks between 615 nm (1a) and 647 nm (1b) with ε values ranging from 4781 (1d) to 62112 (1a) (Table 1).
The fluorescent properties of these polycyclic heterocycles measured in the same solvent, using Oxazine 1 as a standard, 11 are summarised in Table 2.All compounds exhibited high levels of fluorescence, with quantum yields (Φ F ) between 0.20 (1b) and 0.37 (1c) and showed moderate to good Stokes' shifts (44 -99 nm).
The photophysical properties of compounds 1a and 1c were also studied in water at physiological pH (Tables 1 and 2).Their maximum absorption (λ max ) and emission (λ em ) wavelengths in water and ethanol were equal for compound 1a (615 nm), whereas for compound 1c a hypsochromic shift of 50 nm (λ max ) or 42 nm (λ em ) occurred from ethanol to water.When compared to ethanol, Φ F in water was superior for compound 1a (0.47 in water, 0.34 in ethanol) and inferior for compound 1c (0.37 in ethanol < 0.1 in water).synthesised in low to excellent yields.The photophysical properties of these long-wavelength polycyclic heterocycles in ethanol and water at physiological pH, strongly justifies further studies with them as fluorescent probes in biological applications.

Table 2 .
Fluorescence data for compounds 1a-d in ethanol and water (pH 7.4).Units of λ exc , λ em and Stokes' shift: nm. a Spectra measured in ethanol.