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

Article abstract of DOI:10.1016/j.tetlet.2006.01.052

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.

Full text of DOI:10.1016/j.tetlet.2006.01.052

Tetrahedron Letters 47 (2006) 1721–1724
New reactive fluorophores in the 1,2,3-triazine series
Richard N. Butler,a,* Aoife M. Fahy,a Anthony Fox,a John C. Stephens,a P. McArdle,a
D. Cunninghama and Alan G. Ryderb
aChemistry Department, National University of Ireland, Galway, Ireland
bNational Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland
Received 29 November 2005; revised 22 December 2005; accepted 12 January 2006
Abstract—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.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Table 1. 2,5-Dihydro-1,2,3-triazines and pyrrolo-triazoline products
Fluorescent molecules with reactive functional groups
are of particular interest because of their potential for
use as real-time sensors in biomolecular systems.1–3
Among the triazine series the 1,2,3-triazine system is
the least studied in comparison with the 1,2,4- and
1,3,5-triazine structures because the ring system is the
least stable of the three and synthetic routes are limited.4
Herein, we describe the first fluorophores of the 1,2,3-
triazine series from an experimentally simple one-pot
reaction developed from our work on azolium ylide
1,3-dipole systems.5
Compd
Yield (%)
Mp (ꢁC)
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6
52
35
45
38
26
33
8
23
27
13
35
25
72
92
122–123
193–194
179–180
146–147
163–164
158–159
186–187
157–158
174–175
164–166
169–170
163–164
142–143
193–196
Solutions of the 1,2,3-triazolium-1-aminide 1,3-dipoles
1 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 of
the fused pyrrolo[2,3-d]-1,2,3-triazolines 4 and 5 (Table
1).6 When the reaction was carried out in undried ace-
tone, small quantities of the products 6 and 7, which
7
were also fluorescent, were encountered from hydro-
lytic degradation of the imine function in 2 and 3 to
Table 2. Fluorescent properties of substituted 2,5-dihydro-1,2,3-triazines
Compd
kabs maxa
Band 1 (nm)
kabs maxa
Band 2 (nm)
Area ratiob
(est)
eabs max
kem maxc
Band 1 (nm)
FWHMd
kem maxc
Band 2 (nm)
FWHMd
(Mꢀ1 cmꢀ1
)
3a
3b
3c
7
307
310
309
322
392
391
391
377
1.5
3.1
1.7
1.1
19,914
25,971
26,540
19,192
483.6
482.6
482.8
478.9
47.5
48.6
48.3
42.3
528.7
528.1
528.1
516.7
75.1
74.1
75.8
57.8
a Steady state excitation data recorded at concentration 10ꢀ6 M in MeOH.
b Estimated approximate area ratios band 1:band 2 from best-fit Gaussian functions at the offset baseline.
c Steady state emission data recorded with 317 nm excitation at concentration 10ꢀ6 M in MeOH.
d FWHM, full width half maximum.
*
Corresponding author. Tel.: +353 91 492478; fax: +353 91 525700; e-mail: r.debuitleir@nuigalway.ie
0040-4039/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.01.052

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R. N. Butler et al. / Tetrahedron Letters 47 (2006) 1721–1724
a formyl group, which was oxidised in situ and
decarboxylated.
The products 6 and 7 could be readily obtained in over
90% yield by separately heating solutions of 2 and 3 in
1.0
Stokes Shift
200 nm
0.8
0.6
0.4
0.2
0
λabs
λem
200
300
400
500
600
700
800
Wavelength (nm)
Figure 1. Excitation spectrum (emission set at 500 nm) and fluorescence emission spectrum (excitation set at 317 nm) for compound 3a.
Ph
N
N
Ph
Ph
N
N
X
H
CO2R
N
Ar
N
Ph
N
N
X
RO2C
Ar
H
1a X = H
b X= Br
c X =Cl
9
93.0
107.8
Ph
Ph
RO2C
H
RO2C
N1
N
N
6
6a
5
X
Ar
N2
N3
N
3a
4
H
N
8.40
N
Ph
Ph
Ar
148.6
105.7
8
X
X
4 R = Et
5 R = Me
159.9
8.51
H
53.7
N
C
CO2R
4
CO2R
Ph
H
N
5
6
aq. EtOH
Ph
Ph
Ph
N1
N3
N
N2
N
X
6 R = Et
2 R = Et
3 R = Me
7 R = Me
Scheme 1. 1H and 13C NMR shifts in CDCl3 for 2a and 4a.

R. N. Butler et al. / Tetrahedron Letters 47 (2006) 1721–1724
1723
These are normally stable high melting solids5 when
there is a substitutent at C-5 but in the present first cases
with a H-atom at C-5 they proved to be highly labile and
ring expanded in situ into the fluorescent triazines 2 and
3 thereby providing a convenient one-pot synthesis of
these triazines.7 We envisage that the ring-expansion in-
volves an intermediate of type 8, which can arise from a
cleavage of the C(3a)–N(4) bond in 4.
C(7)
C(28)
C(29)
C(6)
C(8)
C(9)
C(27)
C(30)
C(5)
C(26)
C(25)
N(4)
C(4)
C(3)
C(24)
C(22)
C(2)
N(1)
N(2)
O(1)
C(11)
C(1)
N(3)
C(23)
Acknowledgements
C(12)
O(2)
C(21)
C(16)
C(10)
C(15)
C(20)
A.M.F. acknowledges the financial support of the Irish
Research Council for Science Engineering and Techno-
logy. Alan Ryder acknowledges the support of Science
Foundation Ireland (Grant No. O2/IN.1/M231).
C(13)
C(17)
C(19)
C(18)
C(14)
Figure 2. X-ray crystal structure of 3a.
References and notes
C(10)
C(9)
1. 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.
C(11)
C(12)
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, pp
1–188; Ohsawa, A.; Itoh, T. In Comprehensive Heterocyclic
Chemistry 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.
C(7)
C(8)
N(1)
N(2)
C(14)
O(26)
C(5)
C(6)
C(4)
C(15)
C(25)
C(13)
C(18)
C(16)
C(24)
N(3)
C(28)
O(27)
C(19)
C(17)
C(23)
C(20)
5. Butler, R. N.; Lysaght, R. A.; Burke, L. A. J. Chem. Soc.,
Perkin Trans. 2 1992, 1103–1106.
C(22)
C(21)
6. A suspension of 1,2-bis(phenyl)hydrazone of benzil (1 g,
2.56 mmol) in dichloromethane (25 ml) was treated with
lead dioxide (0.73 g, 3.07 mmol) and stirred for 18 h at
ambient temperature. Insoluble salts were removed, and
washed thoroughly with dichloromethane. Evaporation of
the 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 stirred
under reflux for 24 h after which time the solvent was
removed under reduced pressure. The residue (in 2 cm3 of
methylene chloride) was placed on a silica gel column (230–
400 mesh ASTM). The column was eluted with a gradient
mixture (1:0 to 0:1) (v/v) of petroleum spirit (bp 40–60 ꢁC)/
methylene chloride using a 2.5% (v/v) changing gradient to
give 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-10,
C-20, C-30, C-40, respectively, iminyl-N-phenylring), 145.4,
116.1, 128.9, 123.6 (C-10, C-20, C-30, C-40, respectively, 2-N-
phenyl ring), 160.5 (AN@CH), 170.2 (C@O). Anal. Calcd
for C30H24N4O2: C, 76.3; H, 5.1; N, 11.9. Found: C, 76.3;
H, 4.8; N, 11.6.
Figure 3. X-ray crystal structure of 7.
1:1 (v/v) aqueous ethanol.6 The structures 2, 3, 6 and 7
are new fluorophores, which display a bright green fluo-
rescence (Table 2), and contain reactive functional
groups (ester and imine) with potential for binding as
biomarkers. The UV absorption of these structures
showed a dual absorption at ca. 310 nm with a shoulder
at ca. 390 nm. The fluorescence emission (for excitation
at 317 nm) displayed a dual band at ca. 480 and 528 nm.
Hence, the complex system comprises at least two
ground states and two excited states with the 310 nm
absorption correlating with the 520 nm emission and
the 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 from
microanalyses, IR, proton and 13C NMR spectra.6 Some
representative NMR shifts are shown in Scheme 1.
X-ray crystal structure determinations7,8 on compounds
3a and 7a supported the structures (Figs. 2 and 3).
The products 4 and 5 arise from the 1,3-dipoles 1 by Huis-
gen cycloaddition5 to give the unstable cycloadduct 9,
which undergoes a 1,4-N!C rearrangement9 (Scheme 1).
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) 1721–1724
˚
7.06–7.09 (m, 4H, aromatic), 7.25–7.26 (m, 4H, aromatic),
10.2928(19), b = 16.430(4), c = 14.593(3) A, U = 2466.9(8)
3
˚
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-10, 6a-Ph), 137.6 (C-10, 3a-Ph), 140.6, 127.0, 123.2, 129.1
(C-10, C-20, C-30, C-40, respectively, 2-N-phenyl ring),
139.5, 118.2, 122.9, 128.5 (C-10, C-20, C-30, C-40, 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 for
C30H24N4O2: 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 product
was extracted into dichloromethane (4 · 10 cm3) and dried
over MgSO4. The solvent was evaporated under reduced
pressure and the product 7 was crystallized from (2:1 v/v)
methylene chloride/hexane (92%) ethanol (40 cm3) was
crystallised from (2:1 v/v) methylene chloride/hexane
(92%), mp 193–196 ꢁC (from methylene chloride/hexane
2:1 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-10, C-20, C-30, C-40, 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.
A , T = 298(2) K, space group P21/n, Z = 4, l(Mo-Ka) =
0.082 mmꢀ1
, 10,421 reflections collected, 2665 unique
(Rint = 0.0603), which were used in all calculations. The
final wR(F2) was 0.1251 (all data). Crystallographic data
(excluding structure factors) for the structures in this letter
have been deposited with the Cambridge Crystallographic
Data Centre as supplementary publication numbers CCDC
286736. Copies of the data can be obtained, free of charge,
on application to CCDC, 12 Union Road, Cambridge CB2
1EZ, UK [fax: +44(0)-1223-336033 or e-mail: deposit@
ccdc.cam.ac.uk].
8. Crystal structure determination for structure 7. Crystal
data C23H19N3O2, M = 369.41, orthorhombic, a =
˚
9.9253(13), b = 10.973(7), c = 17.458(4) A, U = 1901.4(14)
A , T = 292(2) K, space group P212121, Z = 4,
3
˚
l(Mo-Ka) = 0.084 mmꢀ1, 7353 reflections measured, 1886
unique (Rint = 0.0796), which were used in all calculations.
The final wR(F2) was 0.1406 (all data). Crystallographic
data (excluding structure factors) for the structures in this
letter have been deposited with the Cambridge Crystallo-
graphic Data Centre as supplementary publication number
CCDC 286737. Copies of the data can be obtained, free
of charge, on application to CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK [fax: +44(0)-1223-336033 or
e-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.
7. Crystal structure determination for structure 3a. Crys-
tal data C30H24N4O2, M = 472.53, Monocyclic, a =