A one-pot synthesis of new fluorescent 2,5-dihydro-1,2,3-triazines with reactive functional groups and a large Stokes shift of 200 nm is described

Butler, Richard

Cunningham, D.

Fahy, Aoife

Fox, Anthony

McArdle, P.

Ryder, Alan

Stephens, John C.

English

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New reactive fluorophores in the 1,2,3-trianze series

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Tetrahedron Letters 47 (2006) 1721–1724New reactive fluorophores in the 1,2,3-triazine seriesRichard N. Butler,a,* Aoife M. Fahy,a Anthony Fox,a John C. Stephens,a P. McArdle,aD. Cunninghama and Alan G. RyderbaChemistry Department, National University of Ireland, Galway, IrelandbNational Centre for Biomedical Engineering Science, National University of Ireland, Galway, IrelandReceived 29 November 2005; revised 22 December 2005; accepted 12 January 2006Abstract—A one-pot synthesis of new fluorescent 2,5-dihydro-1,2,3-triazines with reactive functional groups and a large Stokes shiftof 200 nm is described. 2006 Elsevier Ltd. All rights reserved.Table 1. 2,5-Dihydro-1,2,3-triazines and pyrrolo-triazoline productsCompd Yield (%) Mp (C)2a 52 122–1232b 35 193–1942c 45 179–1803a 38 146–1473b 26 163–1643c 33 158–1594a 8 186–1874b 23 157–1584c 27 174–1755a 13 164–1665b 35 169–1705c 25 163–1646 72 142–1437 92 193–196Fluorescent molecules with reactive functional groupsare of particular interest because of their potential foruse as real-time sensors in biomolecular systems.1–3Among the triazine series the 1,2,3-triazine system isthe least studied in comparison with the 1,2,4- and1,3,5-triazine structures because the ring system is theleast stable of the three and synthetic routes are limited.4Herein, we describe the first fluorophores of the 1,2,3-triazine series from an experimentally simple one-potreaction developed from our work on azolium ylide1,3-dipole systems.5Solutions of the 1,2,3-triazolium-1-aminide 1,3-dipoles1 and alkylpropiolates in dry acetone when heated un-der reflux for 24 h produced the new fluorescent 1,2,3-triazine derivatives 2 and 3 along with lesser yields ofthe fused pyrrolo[2,3-d]-1,2,3-triazolines 4 and 5 (Table1).6 When the reaction was carried out in undried ace-tone, small quantities of the products 6 and 7, which0040-4039/$ - see front matter  2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.tetlet.2006.01.052* Corresponding author. Tel.: +353 91 492478; fax: +353 91 525700; e-mail:Table 2. Fluorescent properties of substituted 2,5-dihydro-1,2,3-triazinesCompd kabs maxaBand 1 (nm)kabs maxaBand 2 (nm)Area ratiob(est)eabs max(M1 cm3a 307 392 1.5 19,9143b 310 391 3.1 25,9713c 309 391 1.7 26,5407 322 377 1.1 19,192a Steady state excitation data recorded at concentration 106 M in MeOH.b Estimated approximate area ratios band 1:band 2 from best-fit Gaussian fc Steady state emission data recorded with 317 nm excitation at concentratiod FWHM, full width half maximum.were also fluorescent, were encountered from hydro-lytic degradation of the imine function in 2 and 3 tor.debuitleir@nuigalway.ie1)kem maxcBand 1 (nm)FWHMd kem maxcBand 2 (nm)FWHMd483.6 47.5 528.7 75.1482.6 48.6 528.1 74.1482.8 48.3 528.1 75.8478.9 42.3 516.7 57.8unctions at the offset baseline.n 106 M in MeOH.1722 R. N. Butler et al. / Tetrahedron Letters 47 (2006) 1721–1724a formyl group, which was oxidised in situ anddecarboxylated.200 300 400 50WavelenIntensity00.20.40.60.81.0Stokes Shift  200 nmλabsFigure 1. Excitation spectrum (emission set at 500 nm) and fluorescence em1a X = Hb X= Brc X =Cl2 R = EtNNNPhPhNXXN1N2N3CO2RCHNXPhPhX159.953.78.51456NNNArRO2CHNArPhPh8H CO2Raq. EtO3 R = MeScheme 1. 1H and 13C NMR shifts in CDCl3 for 2a and 4a.The products 6 and 7 could be readily obtained in over90% yield by separately heating solutions of 2 and 3 in0 600 700 800gth (nm)λemission spectrum (excitation set at 317 nm) for compound 3a.6 R = Et7 R = MeNNNNArArPhPhRO2CH993.0NNNNHXXRO2C PhPh148.6107.8105.78.401234 3a566aNNNCO2RHPhPh4 R = Et5 R = MeHC(23) O(2)  C(12) C(7)  C(8)  C(6)  C(13) C(11) O(1)  C(22) C(9)  C(5)  C(14) C(4)  C(10) N(1)  C(15) C(3)  N(2)  C(2)  N(3)  C(1)  C(24) C(21) C(16) N(4)  C(20) C(17) C(25) C(26) C(19) C(18) C(27) C(30) C(28) C(29) Figure 2. X-ray crystal structure of 3a.C(22) C(23) C(21) C(24) C(20) C(9)  C(10) C(19) C(8)  C(11) C(4)  C(7)  C(5)  C(12) C(6)  N(3)  O(27) C(25) N(1)  N(2)  O(26) C(28) C(13) C(18) C(14) C(17) C(15) C(16) Figure 3. X-ray crystal structure of 7.R. N. Butler et al. / Tetrahedron Letters 47 (2006) 1721–1724 17231:1 (v/v) aqueous ethanol.6 The structures 2, 3, 6 and 7are new fluorophores, which display a bright green fluo-rescence (Table 2), and contain reactive functionalgroups (ester and imine) with potential for binding asbiomarkers. The UV absorption of these structuresshowed a dual absorption at ca. 310 nm with a shoulderat ca. 390 nm. The fluorescence emission (for excitationat 317 nm) displayed a dual band at ca. 480 and 528 nm.Hence, the complex system comprises at least twoground states and two excited states with the 310 nmabsorption correlating with the 520 nm emission andthe 390 nm absorption correlating with the 480 nm emis-sion. The very significant Stokes shift of ca. 200 nm(Fig. 1) shows the potential of these molecules for bio-logical fluorescent labelling experiments.The structures of the products 2–7 were established frommicroanalyses, IR, proton and 13C NMR spectra.6 Somerepresentative NMR shifts are shown in Scheme 1.X-ray crystal structure determinations7,8 on compounds3a and 7a supported the structures (Figs. 2 and 3).The products 4 and 5 arise from the 1,3-dipoles 1 byHuis-gen cycloaddition5 to give the unstable cycloadduct 9,which undergoes a 1,4-N!C rearrangement9 (Scheme 1).These are normally stable high melting solids5 whenthere is a substitutent at C-5 but in the present first caseswith a H-atom at C-5 they proved to be highly labile andring expanded in situ into the fluorescent triazines 2 and3 thereby providing a convenient one-pot synthesis ofthese triazines.7 We envisage that the ring-expansion in-volves an intermediate of type 8, which can arise from acleavage of the C(3a)–N(4) bond in 4.AcknowledgementsA.M.F. acknowledges the financial support of the IrishResearch Council for Science Engineering and Techno-logy. Alan Ryder acknowledges the support of ScienceFoundation Ireland (Grant No. O2/IN.1/M231).References and notes1. Wiskur, S. L.; Ait-Haddou, H.; Lavigne, J. J.; Anslyn, E. V.Acc. Chem. Res. 2001, 34, 963–972.2. Weiss, A. Science 1999, 283, 1676–1683.3. Willets, K. A.; Ostroverkhova, O.; He, M.; Twieg, R. J.;Moerner, W. E. J. Am. Chem. Soc. 2003, 125, 1174–1175.4. Neunhoeffer, H.; Wiley, P. F. In Chemistry of 1,2,3-Triazines and 1,2,4-Triazines, Tetrazines and Pentazines;Weissberger, A., Taylor, E. C., Eds.; Heterocyclic Com-pounds; John Wiley & Sons: New York, 1978; Vol. 33, pp1–188; Ohsawa, A.; Itoh, T. In Comprehensive HeterocyclicChemistry II; Katritzky, A. R., Rees, C. W., Scriver, E. F.V., Eds.; Pergamon: Oxford, 1996; Vol. 6 (Boulton, A. J.,vol. ed.) pp 483–505; Ma¨ttner, M.; Neunhoeffer, H.Synthesis 2003, 413–425.5. Butler, R. N.; Lysaght, R. A.; Burke, L. A. J. Chem. Soc.,Perkin Trans. 2 1992, 1103–1106.6. A suspension of 1,2-bis(phenyl)hydrazone of benzil (1 g,2.56 mmol) in dichloromethane (25 ml) was treated withlead dioxide (0.73 g, 3.07 mmol) and stirred for 18 h atambient temperature. Insoluble salts were removed, andwashed thoroughly with dichloromethane. Evaporation ofthe combined mother liquor and washings gave 1a (80%),mp 178–179 C (from toluene–petroleum spirit bp 60–80 C). A suspension of 2,4,5-triphenyl-1,2,3-triazolium-1-phenyl aminide 1a (0.3 g, 0.77 mmol) in dry acetone(10 cm3) was treated with an excess of methyl propiolate(0.14 cm3, 1.57 mmol). The reaction mixture was stirredunder reflux for 24 h after which time the solvent wasremoved under reduced pressure. The residue (in 2 cm3 ofmethylene chloride) was placed on a silica gel column (230–400 mesh ASTM). The column was eluted with a gradientmixture (1:0 to 0:1) (v/v) of petroleum spirit (bp 40–60 C)/methylene chloride using a 2.5% (v/v) changing gradient togive product 3a as a yellow solid (38%), mp 146–147 C(from ethanol); dH (400 MHz, CDCl3) 3.54 (s, 3H, OCH3),6.77–6.79 (m, 2H, 2-N-phenylring, Hortho), 7.11–7.42 (m,12H, aromatic), 7.76–7.92 (m, 6H, aromatic), 8.47 (s, 1H,AN@CH); dC (400 MHz, CDCl3) 53.6 (–OCH3), 53.7 (C-5),134.9 (C-6), 135.3 (C-4), 150.0, 120.3, 129.5, 126.3 (C-1 0,C-2 0, C-3 0, C-4 0, respectively, iminyl-N-phenylring), 145.4,116.1, 128.9, 123.6 (C-1 0, C-2 0, C-3 0, C-4 0, respectively, 2-N-phenyl ring), 160.5 (AN@CH), 170.2 (C@O). Anal. Calcdfor C30H24N4O2: C, 76.3; H, 5.1; N, 11.9. Found: C, 76.3;H, 4.8; N, 11.6.A second product (5a) was isolated as a yellow solid (13%),mp 164–166 C (from ethanol); dH NMR (400 MHz,CDCl3) 3.6 (s, 3H, CO2CH3), 6.89–7.0 (m, 10H, aromatic),1724 R. N. Butler et al. / Tetrahedron Letters 47 (2006) 1721–17247.06–7.09 (m, 4H, aromatic), 7.25–7.26 (m, 4H, aromatic),7.66–7.68 (m, 2H, 2-N-phenylring, Hortho), 8.40 (s, 1H,5-CH, a-enamine). dC NMR (400 MHz, CDCl3):51.2 (OCH3), 92.9 (C-6a), 105.8 (C-3a), 107.4 (C-6), 135.9(C-1 0, 6a-Ph), 137.6 (C-1 0, 3a-Ph), 140.6, 127.0, 123.2, 129.1(C-1 0, C-2 0, C-3 0, C-4 0, respectively, 2-N-phenyl ring),139.5, 118.2, 122.9, 128.5 (C-1 0, C-2 0, C-30, C-4 0, respec-tively, 4-N-phenyl ring), 132.0, 129.2, 127.7, 127.6 (remain-ing aromatics), 149.1 (C-5), 165.5 (C@O). Anal. Calcd forC30H24N4O2: C, 76.3; H, 5.1; N, 11.9. Found: C, 76.1; H,4.88; N, 11.8.A solution of 5-methoxycarbonyl-5-(H-N-phenylformimi-doyl)-2,4,6-triphenyl-2,5-dihydro-1,2,3-triazine 3a (0.15 g,0.32 mmol) in a (1:1 v/v) mixture of aqueous ethanol(40 cm3) was stirred under reflux for 7 days. The productwas extracted into dichloromethane (4 · 10 cm3) and driedover MgSO4. The solvent was evaporated under reducedpressure and the product 7 was crystallized from (2:1 v/v)methylene chloride/hexane (92%) ethanol (40 cm3) wascrystallised from (2:1 v/v) methylene chloride/hexane(92%), mp 193–196 C (from methylene chloride/hexane2:1 v/v). dC NMR (400 MHz, CDCl3) 38.4 (C-5), 53.2 (–OCH3), 134.7 (C-6), 134.8 (C-4), 145.7, 116.2, 128.9, 123.5(C-1 0, C-2 0, C-3 0, C-4 0, respectively, 2-N-phenylring), 126.6,128.7, 129.8, 133.3 (remaining overlapping aromatics),167.8 (C@O). Anal. Calcd for C23H19N3O2: C, 74.8; H,5.2; N, 11.4. Found: C, 74.3; H, 5.1; N, 11.2.7. Crystal structure determination for structure 3a. Crys-tal data C30H24N4O2, M = 472.53, Monocyclic, a =10.2928(19), b = 16.430(4), c = 14.593(3) A˚, U = 2466.9(8)A˚3, T = 298(2) K, space group P21/n, Z = 4, l(Mo-Ka) =0.082 mm1, 10,421 reflections collected, 2665 unique(Rint = 0.0603), which were used in all calculations. Thefinal wR(F2) was 0.1251 (all data). Crystallographic data(excluding structure factors) for the structures in this letterhave been deposited with the Cambridge CrystallographicData Centre as supplementary publication numbers CCDC286736. Copies of the data can be obtained, free of charge,on application to CCDC, 12 Union Road, Cambridge CB21EZ, UK [fax: +44(0)-1223-336033 or e-mail: deposit@ccdc.cam.ac.uk].8. Crystal structure determination for structure 7. Crystaldata C23H19N3O2, M = 369.41, orthorhombic, a =9.9253(13), b = 10.973(7), c = 17.458(4) A˚, U = 1901.4(14)A˚3, T = 292(2) K, space group P212121, Z = 4,l(Mo-Ka) = 0.084 mm1, 7353 reflections measured, 1886unique (Rint = 0.0796), which were used in all calculations.The final wR(F2) was 0.1406 (all data). Crystallographicdata (excluding structure factors) for the structures in thisletter have been deposited with the Cambridge Crystallo-graphic Data Centre as supplementary publication numberCCDC 286737. Copies of the data can be obtained, freeof charge, on application to CCDC, 12 Union Road,Cambridge CB2 1EZ, UK [fax: +44(0)-1223-336033 ore-mail: deposit@ccdc.cam.ac.uk].9. Butler, R. N.; Coyne, A. C.; Cunningham, W. J.; Moloney,E. M.; Burke, L. A. Helv. Chem. Acta 2005, 88, 1626–1628.

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New reactive fluorophores in the 1,2,3-trianze series

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