2566
O. N. Burchak et al. / Bioorg. Med. Chem. 14 (2006) 2559–2568
DCM was added to the mixture. After stirring over-
night, DCM was evaporated, the residue was diluted
with H2O and extracted with ethyl acetate. The organic
phase was washed with 1 M HCl, 1 M NaHCO3, brine,
dried over Na2SO4 and concentrated under reduced
pressure. The residue was subjected to silica gel chroma-
tography with EtOAc/hexane (1:1), EtOAc, MeOH/
4.17. 2-(6-Carbonylmethoxy-3-oxo-3H-xanthen-9-yl)-5-
[3-(9H-fluoren-9-ylmethoxycarbonyl-amino)-propionyl-
amino]-benzoic acid ethyl ester (dye 17)
Dye 17 was obtained from ester 9 (782 mg, 1.0 mmol)
with 97% yield (704 mg). H NMR ((CD3)2SO, ppm):
1
0.85 (t, 3H, J = 7.5 Hz), 2.58 (m, 2H), 3.39 (m, 2H),
3.93 (m, 2H), 4.32 (m, 1H), 4.66 (m, 2H), 4.87 (s, 2H),
6.21 (m, 1H), 6.40 (m, 1H), 6.50–6.59 (m, 3H), 6.69
(m, 1H), 6.70 (m, 1H), 7.15 (m, 1H), 7.26–7.39 (m,
4H), 7.64 (m, 2H), 7.88 (m, 1H), 7.89 (m, 2H), 10.49
(s, 1H). HRMS calcd. for C42H34N2O10 (M)+
726.2214, found 726.2212.
1
DCM (1:20), giving 101 mg of dye 13 (58%). H NMR
((CD3)2SO, ppm): 0.85 (t, 3H, J = 7.5 Hz), 1.42 (s,
9H), 3.11 (m, 2H), 3.19 (m, 2H), 3.92 (m, 2H), 4.08 (s,
2H), 4.16 (m, 1H), 4.21 (s, 2H), 4.28 (m, 2H), 4.86 (s,
2H), 6.24 (m, 1H), 6.37 (m, 1H), 6.80–6.97 (m, 3H),
7.19 (m, 1H), 7.27–7.42 (m, 4H), 7.65 (m, 2H), 7.64
(m, 1H), 7.88 (m, 2H), 7.82 (m, 1H), 8.05 (m, 1H),
10.49 (s, 1H). HRMS calcd. for C49H47N3O12 (M)+
869.3160, found 869.3164.
4.18. 2-(6-Carbonylmethoxy-3-oxo-3H-xanthen-9-yl)-4-
[3-(9H-fluoren-9-ylmethoxycarbonyl-amino)-propionyl-
amino]-benzoic acid ethyl ester (dye 18)
4.13. 2-(6-tert-Butoxycarbonylmethoxy-3-oxo-3H-
xanthen-9-yl)-4-(2-{[2-(9H-fluoren-9-ylmethoxycarbonyl-
amino)-ethylcarbamoyl]-methoxy}-acetylamino]-benzoic
acid ethyl ester (dye 14)
Dye 18 was obtained from ester 10 (782 mg, 1.0 mmol)
with 95% yield (690 mg). H NMR ((CD3)2SO, ppm):
1
0.85 (t, 3H, J = 7.5 Hz), 2.36 (m, 2H), 3.23 (m, 2H),
3.92 (m, 2H), 4.25 (m, 1H), 4.51 (m, 2H), 4.85 (s, 2H),
6.21 (m, 1H), 6.33 (s, 1H), 6.44 (m, 1H), 6.80 (m, 1H),
6.83–6.95 (m, 3H), 7.15 (m, 1H), 7.22 (m, 2H), 7.26–
7.42 (m, 4H), 7.64 (m, 2H), 7.85 (m, 2H), 10.50 (s,
1H). HRMS calcd. for C42H34N2O10 (M)+ 726.2214,
found 726.2211.
Dye 14 was obtained by the same method from acid 12
(121 mg, 0.2 mmol) with 64% yield (111 mg). H NMR
1
((CD3)2SO, ppm): 0.86 (t, 3H, J = 7.5 Hz), 1.42 (s,
9H), 3.07 (m, 2H), 3.15 (m, 2H), 3.91 (m, 2H), 4.04 (s,
2H), 4.16 (m, 1H), 4.19 (s, 2H), 4.26 (m, 2H), 4.85 (s,
2H), 6.22 (m, 1H), 6.48 (m, 1H), 6.82–6.95 (m, 3H),
7.17 (m, 1H), 7.27–7.41 (m, 4H), 7.64 (m, 2H), 7.77
(m, 1H), 7.86 (m, 2H), 8.00 (m, 1H), 8.16 (m, 1H),
10.49 (s, 1H). HRMS calcd. for C49H47N3O12 (M)+
869.3160, found 869.3162.
4.19. 2-(6-Carbonylmethoxy-3-oxo-3H-xanthen-9-yl)-5-
(2-{[2-(9H-fluoren-9-ylmethoxycarbonyl-amino)-ethyl-
carbamoyl]-methoxy}- acetylamino]-benzoic acid ethyl
ester (dye 19)
4.14. Removal of Boc-protection (dyes 15–20)
Dye 19 was obtained from ester 13 (87 mg, 0.1 mmol)
with 95% yield (77 mg). 1H NMR ((CD3)2SO, ppm):
0.85 (t, 3H, J = 7.5 Hz), 3.07 (m, 2H), 3.15 (m, 2H),
3.95 (m, 2H), 4.04 (s, 2H), 4.16 (m, 1H), 4.19 (s, 2H),
4.26 (m, 2H), 4.85 (s, 2H), 6.32 (m, 1H), 6.43 (m, 1H),
6.94–7.00 (m, 3H), 7.26 (m, 1H), 7.24–7.36 (m, 4H),
7.64 (m, 2H), 7.78 (m, 1H), 7.86 (m, 2H), 8.01 (m,
1H), 8.28 (m, 1H), 10.49 (s, 1H). HRMS calcd. for
C45H39N3O12 (M)+ 813.2534, found 813.2537.
TFA (2.0 g, 1.3 mL) was added to the solution of ester
in DCM (2.0 g, 1.5 mL) at room temperature. After
3 h, DCM and TFA were evaporated, the residue was
solidified by addition of diethyl ester, washed thorough-
ly with the same solvent, and dried.
4.15. 5-Acetylamino-2-(6-carboxymethoxy-3-oxo-3H-
xanthen-9-yl)-benzoic acid ethyl ester (dye 15)
4.20. 2-(6-Carbonylmethoxy-3-oxo-3H-xanthen-9-yl)-4-
(2-{[2-(9H-fluoren-9-ylmethoxycarbonyl-amino)-ethyl-
carbamoyl]-methoxy}-acetylamino]-benzoic acid ethyl
ester (dye 20)
Dye 15 was obtained by ester 7 (53 mg, 0.1 mmol)
deprotection with 95% yield (45 mg). NMR
((CD3)2SO, ppm): 0.85 (t, 3H, J = 7.5 Hz), 2.12 (s,
3H), 3.96 (m, 2H), 4.87 (s, 2H), 6.22 (m, 1H), 6.39
(m, 1H), 6.84–6.92 (m, 3H), 7.17 (m, 1H), 7.40 (m,
1H), 8.04 (m, 1H), 8.40 (s, 1H), 10.45 (s, 1H).
HRMS calcd. for C26H21NO8 (M)+ 475.1268, found
475.1269.
Dye 20 was obtained from ester14 (87 mg, 0.1 mmol)
with 90% yield (73 mg). 1H NMR ((CD3)2SO, ppm):
0.87 (t, 3H, J = 7.5 Hz), 3.05 (m, 2H), 3.15 (m, 2H),
3.93 (m, 2H), 4.02 (s, 2H), 4.18 (m, 1H), 4.19 (s, 2H),
4.25 (m, 2H), 4.86 (s, 2H), 6.42 (m, 1H), 6.53 (m, 1H),
6.93–6.99 (m, 3H), 7.25 (m, 1H), 7.29–7.40 (m, 4H),
7.64 (m, 2H), 7.80 (m, 1H), 7.85 (m, 2H), 7.99 (m,
1H), 8.18 (m, 1H), 10.51 (s, 1H). HRMS calcd. for
C45H39N3O12 (M)+ 813.2534, found 813.2534.
4.16. 4-Acetylamino-2-(6-carboxymethoxy-3-oxo-3H-
xanthen-9-yl)-benzoic acid ethyl ester (dye 16)
Dye 16 was obtained from ester 8 (53 mg, 0.1 mmol)
with 93% yield (44 mg). 1H NMR ((CD3)2SO, ppm):
0.83 (t, 3H, J = 7.5 Hz), 2.10 (s, 3H), 3.83 (m, 2H),
4.93 (s, 2H), 6.41 (m, 1H), 6.55 (m, 1H), 6.95–7.02 (m,
3H), 7.29 (m, 1H), 7.72 (m, 1H), 7.89 (m, 1H), 8.19
(m, 1H), 10.52 (s, 1H). HRMS calcd. for C26H21NO8
(M)+ 475.1268, found 475.1267.
4.21. Physico-chemical characterization of the dyes
Standard UV–vis and fluorescence spectra in 50 mM
sodium tetraborate buffer, pH 9.8, were recorded at
0.5–10 lM dye concentration. The fluorescence