Ultrasonic mediated synthesis of new benzo [ a ] phenoxazinium chlorides and their photophysical studies

Ultrasonic irradiation was demonstrated to be an efficient technique for activating various organic transformations, allowing short reaction times, high yields and sometimes good selectivity. Three new benzo[a]phenoxazinium chlorides possessing isopentylamino, (2-cyclohexylethyl)amino and phenethylamino groups at 5-position of the heterocyclic systems were efficiently synthesized under ultrasound irradiation. Photophysical characterization in ethanol and water, as well as acid-base equilibrium studies of the probes were carried out.


Introduction
Fluorescent chromophores have immense applications in biological sciences, particularly in labeling for diagnosis and analysis. 1,2Numerous reports reveal that a variety of commercially available dyes fluoresce in the visible region of the spectrum.However, long-wavelength fluorophores (600-1000 nm) are preferred for biological applications which can account their minimum interference from absorption scattering and the natural auto-fluorescence of biological molecules. 3,4In this context, oxazine derivatives, such as phenoxazines and benzo[a]phenoxazines have been reported for various spectroscopic research studies in the near-infrared region. 5trasonic irradiation has been considered as a useful and clean protocol in organic synthesis as 2 compared with the traditional methods due to its convenience.Moreover, a large number of organic transformations can be carried out in short reaction times, high yield or milder conditions under ultrasonic irradiation. 6Considering these facts and in continuation of our research interests in the synthesis of organic fluorophores, [7][8][9] we wish to herein report for the first time the use of ultrasound irradiation in the efficient preparation of a new set of benzo[a]phenoxazines. Fundamental photophysical characterization of these compounds was carried out.

Experimental
Typical procedure for the synthesis of 3a-c (described for 3a).
The progress of reaction was monitored by TLC (dichloromethane/methanol, 9.5:0.5).After completion of the reaction, the solvent was evaporated and the mixture was purified by column chromatography on silica using dichloromethane and dichloromethane/methanol (99:1), as the eluent.

Results and discussion
Benzo excellent yields (90-93%, Scheme 1).By using conventional oil bath heating instead of ultrasound irradiation, compound 1a was also synthesized in excellent yield, but the reaction time increased more than five times (from 1.5 to 8 hours).All compounds obtained were fully characterized by the usual analytical techniques.

Scheme 1. Synthesis of benzo[a]phenoxazinium chlorides 1a-c
The 1 H NMR spectra showed the signals of aliphatic protons from the methylenic groups of substituents of positions 5 and 9, directly linked to the nitrogen atom NHCH 2 that appeared as triplets or a broad singlet (1b) (δ 3.72 to 3.91 ppm), as well as groups closed to the same atom, NHCH 2 CH 2 , showed as multiplets (δ 1.70 to 1.90 ppm) or a triplet (1c, δ 3.16 ppm).The CH proton occurred as a multiplet (1a) or broad singlet (1b) (δ 1.53 to 1.90 ppm).The methyl protons of ethyl and isopentyl (1a) groups in amines of positions 5 and 9 of the heterocycle appeared as triplets (δ ~ 1.40 ppm) and doublets (isopentyl group of 1a, δ 1.09 ppm).There was also the presence of protons of the methyl group directly linked to the aromatic ring at position 10, which appeared as singlets (δ 2.30 to 2.36 ppm).In addition, spectra showed the expected aromatic protons of the polycyclic system, in particular H-8 (δ 6.73 to 6.86 ppm), H-6 (δ 6.76 to 6.90 ppm), and H-11 (δ 7.66 to 7.74 ppm), which appeared in the form of singlets.
The 13 C NMR spectra showed the signals of the methylenic groups of substituents of positions 5 and 9, directly linked to the nitrogen atom NHCH 2 (δ 39.72 to 47.0 ppm), as well as groups closed to the same Fundamental photophysical studies of the fluorescent properties of benzo[a]phenoxazinium chlorides 1a and 1b were carried out in ethanol, and distilled water.The longest wavelength of maximum absorption ( abs ) of both compounds in the two solvents was located in the region 621-629 nm.The molar absorptivities of these compounds displayed excellent values in ethanol, and decreased in water.
Maximum emission wavelengths ( em ) and relative fluorescence quantum yields (Φ F ) were obtained and are summarised in Table 1.For the determination of quantum yields, Oxazine 1, used as a standard (Φ F = 0.11 in ethanol), 11 was excited at the wavelength of excitation of each compound.In both solvents, benzo[a]phenoxazinium salts displayed  em in the region of 642-651 nm, and fluorescence quantum yields that are superior in ethanol.
8][9] In the case of compounds 1a and 1b, only the acid form was observed in ethanol and also in water.However, the sensitivity to acid and basic media was studied in both solvents by using trifluoroacetic acid (TFA) and tetraethylammonium hydroxide (TEAH), respectively.The presence of the acid did not affect significantly  abs and  em values, and fluorescence quantum yields are similar or superior (except for 1b in water).In basic medium a hypsochromic shift in the absorption and emission in ethanol and water was observed.The Stokes' shifts were superior (Δ> 100 nm) to those obtained in the absence of TEAH, and as it was expected, the basic form display low fluorescence, being the best Φ F value 0.08 (compound 1b, in ethanol).

Table 1 .
Preliminary photophysical studies of compounds 1a and 1b in ethanol, water and after the addition of TFA and TEAH.