Synthesis and photophysics of new benzophenoxazine derivatives fused with julolidine

Phenoxazinium salts are mostly used as markers for various purposes. In this context, we report the synthesis of five new benzophenoxazinium derivatives fused with julolidine moiety. These benzophenoxazinium chlorides were obtained by the condensation of anthracen-1-amine, naphthalen-1amine, N-phenylnaphthalen-1-amine, N-propylnaphthalen-1-amine and 3-(naphthalen-1ylamino)propan-1-ol, with 9-nitroso-8-hydroxyjulolidine hydrochloride as blue solids. The photophysical behavior of these compounds in anhydrous ethanol when acidified with TFA or basified with TEAH was also investigated as well as their response in aqueous media.


Introduction
Fluorescent probes are widely used for imaging in cell biology. 1 Nile Blue (NB), one of the most used benzo[a]phenoxazinium dyes, and their derivatives emit light in the near infrared (NIR) region of the spectrum and are prominent as indicators for fluorimetric and photometric detection. 2 In general, benzo[a]phenoxazinium dyes are well known as markers due to their several interesting spectral properties. These compounds exhibit intense long-wavelength absorption and emission at about 650nm. Hence, they are used as potential candidates for dyeing paper, fiber stuff and laser dyes. 3 Moreover, they are useful as photosensitizers in photodynamic therapy, fluorophor in energy and electron-transfer reactions antitubercolastatic or anticancerogenic agents. 4 In the previous reports by our research group we have synthesised several NB derivatives, [5][6][7][8][9][10] and in the present communication we are interested to explore the properties of a rigid structure in comparison with the unrestricted dye. Herein, we report the synthesis of benzophenoxazinium derivaties fused with julolidine moiety. The photophysical characterization was performed in dry ethanol and aqueous medium. Fluorescence quantum yields and acid-base equilibrium were also examined.

Experimental
Typical procedure for the preparation of compounds 1, 2a-d (illustrated for 1).

Results and discussion
Benzophenoxazinium chlorides 1, 2a-d were synthesised by condensation of 9-nitroso-1,2,3,5,6,7-hexahydropyrido[3,2,1-ij]quinolin-8-ol 3 with anthracen-1-amine, naphthalen-1-amine and N-alkylated naphthalen-1-amines 5a-d in acid media. Intermediates 5b-d were obtained by alkylation of naphthalen-1-amine with chlorobenzene, 1-bromopropane and 3-bromopropan-1-ol in ethanol as the solvent, in low to moderate yields. The required 9-nitroso-1,2,3,5,6,7-hexahydropyrido[3,2,1- Previous studies on this type of compounds showed that the photophysical properties in protonaccepting solvents is influenced by acid-base equilibria mainly located at the 5-amino position. 9,10 In ethanol media the absorption spectra are dominated by an acidic form (AH + ) and a ~100nm blue shifted neutral form (A). 12 The fluorescence of the basic form is broad and centred at around 600nm while the acid form (AH + ) shows a band centred above 660nm with a much higher quantum yield. 12 The later reaches 0.4 when the 9-amino position is mono-alkylated and varies between 0.1 and 0.2 when it is dialkylated. 9,10,12 At 470nm the basic form is mostly excited with a small fraction of acidic form. At higher wavelengths the situation is reversed. In Figures 1 and 2     The above characteristics are confirmed in ethanol media when acidified with trifluoroacetic acid (TFA) or basified with tetraethylammonium hydroxide (TEAH). In contrast, the acid-base behavior is different from what we obtained in our earlier reports for similar compounds but without the julolidine moiety 7,8 as, using the same amount of TEAH, the equilibrium is not completely shifted towards the basic form (Fig.1). For the acid form it seems possible to completely displace the equilibrium with the same amount of TFA previously used, but, particularly for compound 1, the absorption spectra are broader.
The absorption spectra of these type of compounds, but with an amino group at the 9-position, in water usually evidences the presence of non-fluorescent H-aggregates through a ~40nm blue shifted shoulder. 12 The relative amount of that shoulder obviously depends on dye concentration. In the present case of the studied julolidine fused compounds the spectrum at 4×10 -6 M is similar to the ones obtained for compounds without the julolidine moiety at 5×10 -5 M. This clearly indicates the much higher tendency for aggregation of the synthesised benzophenoxazines fused with julolidine. Table 1 shows absorption and emission maxima, Stokes shift and fluorescence quantum yield for the acid form in dried ethanol and in water. Through the stiffening of 9-amino position, it was expected that the fluorescence quantum yield in acidified ethanol should be higher than the values previously obtained for benzophenoxazines without a fused julolidine moiety and di-alkylated compounds (between 0.1 and 0.2). 9,10,12 In fact, the improvement in fluorescence quantum yield was not observed which can be explained by the possible presence of aggregates in ethanolic media that was evidenced in the broader spectrum of the studied compounds in acidified ethanol. Nevertheless, it seems that the 5-amino position (6-or 14-positions in 1 and 2a-d, respectively) is the main pathway of excited state nonradiative desactivation.
Comparing compounds 1 and 2a significant red shifts were observed both in absorption (17nm) and emission (22nm). The origin of this shift is certainly the higher π conjugation system that results from the fusion of anthracene with the phenoxazine moiety instead of naphthalene.
Also worth mentioning is the much lower fluorescence quantum yield in aqueous media. This is easily understandable by the fact that H-aggregates are non-fluorescent and they are very prominent for the studied compounds.

Conclusions
Five benzophenoxazinium chlorides derivatives with fused julolidine bearing different terminal groups at the amino position were synthesised. The photophysics of the acid and basic forms were studied in dried ethanolic media, by adding either a strong acid or a strong base. The acid form was also followed in water. The reported compounds were found to have higher tendency to aggregate than similar compounds without the fused julolidine. This tendency can possibly account for a lower than expected fluorescence quantum yield for the studied compounds.