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

1 New benzo[a]phenoxazines bearing the (4,6-dichloro-1,3,5-triazin-2-yl)amino group: synthesis and photophysical properties B. Rama Raju,a,b Joana F. C. Parente,a Maria Inês P. S. Leitão,a Paulo J. G. Coutinho,b M. Sameiro T. Gonçalvesa* aCentre of Chemistry and bCentre of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal e-mail: [email protected] Abstract: 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. Keywords: Benzo[a]phenoxazinium dyes; Nile Blue; 2,4,6-trichloro-1,3,5-triazine; acid-base equilibrium. 1. Introduction Fluorescent probes such as small organic molecules used for optical imaging purposes have emerged as an important tool in various fields of sciences.1 Among 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.3 In addition, these compounds are medicinally important as antifungal4 and antimalarial agents.5 Phenoxazine core compounds also serve as electron donors for dye sensitized solar cells,6 enhance electro-optic activity in second-order nonlinear optical chrmophores7 and function as efficient sensitizers for photodynamic therapy (PDT).8 2 Owing to the importance and our research activity towards Nile Blue (NB) derivatives,9-11 we herein describe the synthesis of new benzo[a]phenoxazinium chlorides possessing the (4,6-dichloro1,3,5-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. 2. Experimental Typical procedure for the preparation of compounds 1 and 2 (illustrated for 1). To an ice cold solution of 5-(ethylamino)-4-methyl-2-nitrosophenol hydrochloride 4a (50 mg, 0.23 mmol) in ethanol (1 mL), was added concentrated hydrochloric acid (0.6 μL) followed by 4,6-dichloroN-(naphthalen-1-yl)-1,3,5-triazin-2-amine 3 (34 mg, 0.12 mmol) and was refluxed for 14 hrs. The progress of the reaction was monitored by TLC (dicloromethane/methanol: 95:5). After evaporation of the solvent and column chromatography purification on silica gel with dichloromethane and dichloromethane/methanol, mixtures of increasing polarity, as the eluent, N-(5-((4,6-dichloro-1,3,5triazin-2-yl)amino)-10-methyl-9H-benzo[a]phenoxazin-9-ylidene)ethanaminium chloride 1 was obtained as a blue solid (0.027 g, 24%). 1H NMR (CD3OD, 400 MHz): δH = 1.37 (t, J = 7.2 Hz, 3H, NCH2CH3), 2.27 (s, 3H, CH3), 3.47 (q, J = 7.2 Hz, 2H, NCH2CH3), 6.70 (s, 1H, 8-H), 6.75 (s, 1H, 6H), 7.52 (s, 1H, 11-H), 7.76 (broad s, 1H, 2-H), 7.88 (t, J = 7.6 Hz, 1H, 3-H), 8.21 (d, J = 7.2 Hz, 1H, 1-H), 8.78 (d, J = 7.6 Hz, 1H, 4-H) ppm. 13C NMR (CD3OD, 100.6 MHz): δc = 14.14 (NCH2CH3), 17.66 (CH3), 39.70 (NCH2CH3), 94.48 (C-8), 97.17 (C-6), 123.96 (Ar-C), 124.51 (C-1), 125.45 (C-4), 128.38 (C-10), 130.61 (C-2), 131.61 (Ar-C), 132.77 (C-11), 132.94 (Ar-C), 133.18 (C-3), 134.32 (ArC), 149.02 (Ar-C), 151.57 (Ar-C), 152.46 (Ar-C), 156.57 (2×Ar-C), 161.94 (2×Ar-C) ppm. Procedure for the N-alkylation of naphthalen-1-amine with 2,4,6-trichloro-1,3,5-triazine. A solution of 2,4,6-trichloro-1,3,5-triazine(0.500 g, 2.71 mmol) in acetone (3 mL) was added with stirring to a cold solution (0-5 °C) of sodium hydrogenocarbonate (0.600 g) in water (6 mL), in a threenecked flask (250 mL) equipped with a magnetic stirrer. This resulted in the formation of slurry of cyanuric chloride. A solution of 1-naphthylamine (0.777 g, 2.71 mmol) in acetone (1 mL) was added to the cold slurry of cyanuric chloride. After sometime the reaction mixture was neutralized by saturated solution of sodium hydrogenocarbonate. The mixture was stirred for 2 hr at 0-5 °C. The pale violet solid was filtered and recrystallized from ethanol to obtain 4,6-dichloro-N-(naphthalen-1-yl)-1,3,5triazin-2-amine 3 in good yield (0.125 g, 79%). TLC (dichloromethane/methanol, 9.5:0.5): Rf = 0.53. 3 Mp 149-151 oC. 1H NMR (CD3OD, 400 MHz): δH = 7.51-7.63 (m, 2H, 2-H and 3-H), 7.65-7.72 (m, 2H, 6-H and 7-H), 7.94-8.02 (m, 2H, 4-H and 5-H), 8.05-8.14 (m, 1H, 8-H) ppm. 13C NMR (CD3OD, 100.6 MHz): δc = 123.46 (C-4), 125.39 (Ar-C), 126.39 (Cnap-2), 127.04 (Cnap-6), 127.64 (Cnap-3), 127.92 (Cnap-7), 129.04 (Cnap-5), 129.50 (Cnap-8), 135.71 (Ar-C), 148.61 (Ar-C), 164.49 (Ar-C), 164.95 (Ar-C), 166.96 (Ar-C) ppm. IR (KBr 1%, cm-1): ν = 3126, 3063, 2921, 2788, 2615, 1667, 1631, 1588, 1543, 1509, 1392, 1373, 1346, 1313, 1267, 1236, 1171, 1092, 1055, 1020, 995, 957, 886, 846 cm-1. 3. Results and discussion Benzophenoxazinium chlorides 1 and 2 were synthesized by condensation of nitroso derivatives, namely 5-(ethylamino)-4-methyl-2-nitrosophenol hydrochloride 4a and 5-(diethylamino)- 2-nitrosophenol hydrochloride 4b with 4,6-dichloro-N-(naphthalen-1-yl)-1,3,5-triazin-2-amine 3 in acidic conditions. 4,6-Dichloro-N-(naphthalen-1-yl)-1,3,5-triazin-2-amine 3 was obtained by Nalkylation of naphthalen-1-amine with 2,4,6-trichloro-1,3,5-triazine in acetone under ice cold conditions. The other intermediates such as 5-(ethylamino)-4-methyl-2-nitrosophenol hydrochloride 4a, 5-(diethylamino)-2-nitrosophenol hydrochloride 4b were obtained by the nitrosation of 3-(ethylamino)- 4-methylphenol and 3-(diethylamino)phenol with sodium nitrite in the presence of hydrochloric acid, in a mixture of ethanol-water as the solvent under ice cold conditions.12 Scheme 1. Synthesis of benzo[a]phenoxazinium chlorides 1 and 2. 4 The condensation reaction of nitroso derivatives 5-(ethylamino)-4-methyl-2-nitrosophenol hydrochloride 4a, 5-(diethylamino)-2-nitrosophenol hydrochloride 4b with 4,6-dichloro-N-(naphthalen1-yl)-1,3,5-triazin-2-amine 3 was carried out in the presence of concentrated hydrochloric acid under reflux conditions. The progress of the reaction was checked with TLC and upon completion; the crude reaction mixture was purified with dichloromethane and mixtures of dichloromethane/methanol as eluents. The benzo[a]phenoxazinium chlorides 1 and 2 were obtained as blue solids (Scheme 1). All compounds obtained were fully characterized by the usual analytical techniques. The 1H 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. The 13C NMR spectra showed the carbons of the methyl groups (1 and 2) (δ 12.86 to 14.14 ppm) and methylene carbons adjacent to the nitrogen (39.70 to 46.97 ppm). The methyl group attached to C-10 for the compound 1 at (δ 17.66 ppm). The aromatic signals for the carbons C-6 and C-8 (δ 97.17 or 94.48 ppm), C-2 and C-11 (δ 130.61 to 134.29 ppm) and C-3 (δ 133.18 or 133.50 ppm) were exhibited. 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 (ΦF) were determined using Oxazine 1 as a standard (ΦF = 0.11 in ethanol).13 Table 1. Preliminary photophysical studies of compounds 1 and 2 in anhydrous ethanol and water after the addition of TFA. Cpd Dry ethanol + TFA Water + TFA abs (nm)  (M-1cm-1)em (nm)   (nm)  F  abs (nm)  (M-1cm-1)em (nm)  (nm)  F 1 615 97750 639 24 0.73 616 59250 644 28 0.29 2 628 74400 663 35 0.33 636 35025 676 40 0.06 5 300 400 500 600 700 0 1x10 4 2x10 4 3x10 4 4x10 4 5x10 4 6x10 4 Molar absorptivity (M -1 cm -1 ) Wavelength (nm) 1-Acidic 2-Acidic 1-Basic 2-Basic Figure 1. Absorption spectra of compounds 1 and 2 in acidified and basified water. 350 400 450 500 550 600 650 700 750 0.0 5.0x105 1.0x106 1.5x106 2.0x106 2.5x106 3.0x106 3.5x106 0 1x107 2x107 3x107 4x107 5x107 Fluorescence Intensity (a.u.) Fluorescence Intensity (a.u.) Wavelength (nm) 1 2 1 2 Figure 2. Spectra in basified dry ethanol at 470 nm excitation and the excitation spectra were collected at 605 nm emission wavelength. 6 580 600 620 640 660 680 700 720 740 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 Normalized intensity Normalized intensity Wavelength (nm) 1 Eth 2 Eth 1 Wat 2 Wat Figure 3. Emission spectra of compounds 1 and 2 in dry ethanol and aqueous media at 575 nm excitation. 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.16 The 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 In 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. 7 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. 4. 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. Acknowledgements Thanks are due to the Fundação para a Ciência e Tecnologia (FCT, Portugal) for financial support to the NMR portuguese network (PTNMR, Bruker Avance III 400-Univ. Minho), FCT and FEDER (European Fund for Regional Development)-COMPETE-QREN-EU for financial support to the Research Centres CFUM [PEst-C/FIS/UI0607/2011 (F-COMP-01-0124-FEDER-022711)] and CQ/UM [PEst-C/QUI/UI0686/2013(FCOMP-01-0124-FEDER-022716)]. A post-doctoral grant to B. R. Raju (SFRH/BPD/62881/2009) is also acknowledged to FCT, POPH-QREN, FSE. References 1. Yu, L.; Wang, S.; Huang, K.; Liu, Z.; Gao, F.; Zeng, W. Tetrahedron 2015, 71, 4679. 2. Tong, H.; Lou, K.; Wang, W. Acta Pharm. Sin. B 2015, 5, 25. 3. Raju, B. R.; Naik, S.; Coutinho, P. J. G.; Gonçalves, M. S. T. Dyes Pigm. 2014, 220. 4. Frade, V. H. J.; Sousa, M. J.; Moura, J. C. V. P.; Gonçalves, M. S. T. Bioorg. Med. Chem. 2008, 16, 3274. 5. Mizukawa, Y.; Ge, J-F.; Md, A. B.; Itoh, I.; Scheurer, C.; Wittlin, S.; Brun, R.; Matsuoka, H.; Ihara M. Bioorg. Med. Chem. 2014, 22, 3749. 6. Li, J.; Yang, X.; Cheng, M.;Wang, M.; Sun, L. Dyes Pigm. 2015, 116, 58. 8 7. Liu, F.; Wang, H.; Yang, Y.; Xu, H.; Yang, D.; Bo, S.; Liu, J.; Zhen, Z.; Liu, X.; Qiu, L. Dyes Pigm. 2015, 114, 196. 8. Lopes, M.; Alves, C. T.; Raju, B. R.; Gonçalves, M. S. T.; Coutinho, P. J. G.; Henriques, M.; Belo, I. J. Photochem. Photobiol: B 2014, 141, 93. 9. Raju, B. R.; Garcia, A. M. F.; Costa, A. L. S.; Coutinho, P. J. G.; Gonçalves, M. S. T. Dyes Pigm. 2014, 203. 10. Raju, B. R.; Sampaio, D. M. F.; Silva, M. M.; Coutinho, P. J. G.; Gonçalves, M. S. T. Ultrason. Sonochem. 2014, 360. 11. Raju, B. R.; Firmino, D. G.; Costa, A. L. S.; Coutinho, P. J. G.; Gonçalves, M. S. T. Tetrahedron 2013, 69, 2451. 12. Crossley, M. L.; Turner, R. J.; Hofmann, C. M.; Dreisbach, P. F.; Parker, R. P. J. Am. Chem. Soc. 1952, 74, 578. 13. Sens, R.; Drexhage, K. H. J. Lumin. 1981, 24, 709. 14. Naik, S.; Alves, C. M. A.; Coutinho, P. J. G.; Gonçalves, M. S. T. Eur. J. Org. Chem. 2011, 13, 2491. 15. Alves, C. M. A.; Naik, S.; Coutinho, P. J. G.; Gonçalves, M. S. T. Tetrahedron 2009, 65, 10441. 16. Frade, V. H. J.; Gonçalves, M. S. T.; Coutinho, P. J. G.; Moura, J. C. V. P. J. Photochem. Photobiol. 2007, 185, 220.