A R T I C L E S
Ueno et al.
chromatography. The product was recrystallized from methanol/CH
2
-
The organic layer was dried over Na
2
SO
4
and concentrated in vacuo
Cl
2
to afford fluorescein methyl ester derivatives (1b, 3b-5b).
-[1-(2,4,5-Trimethoxycarbonyl)phenyl]-6-hydroxy-3H-xanthen-
to afford a crude product. The crude product (100 mg) was dissolved
in DMF (5 mL); then iodomethane (42 mg, 0.30 mmol) and cesium
carbonate (97 mg, 0.3 mmol) were added. The mixture was stirred at
room temperature for 12 h, water (50 mL) was added, and the reaction
mixture was extracted with AcOEt (3 × 60 mL). The combined organic
layers were dried over anhydrous Na SO and concentrated in vacuo.
9
1
3
-one (2,4,5-triCOOMePh X, 1b). H NMR (300 MHz, CDCl
3
): δ
3
.66 (s, 3H); 3.91 (s, 3H); 3.99 (s, 3H); 6.67 (br, 4H); 6.93 (d, 2H, J
9.3 Hz); 7.78 (s, 1H); 8.61 (s, 1H). 1 C NMR (75 Hz, CDCl
3
+
)
CD
3
3
OD): δ 53.3, 53.6, 78.6, 104.4, 115.5, 130.8, 131.6, 132.5, 133.4,
2
4
+
1
33.7, 136.6, 138.3, 164.9, 167.2, 167.4. HRMS (ESI ): calcd for [M
The crude product was purified by silica gel chromatography to afford
1d in 28% yield in two steps.
+
+
Na] , 458.0848; found, 458.0819. Mp: 270 °C (dec). Anal. Calcd
for C25
H, 3.92.
-[1-(3,4-Dichloro-2-methoxycarbonyl)phenyl]-6-hydroxy-3H-
H
18
O
9
‚CH
3
OH: N, 0; C, 63.16; H, 4.48. Found: N, 0; C, 64.94;
9
-[1-(2,4,5-Triethoxycarbonyl)phenyl]-6-methoxy-3H-xanthen-3-
1
one (1d). H NMR (300 MHz, CDCl
3
): δ 0.88 (t, 3H, J ) 3.7 Hz);
9
1
(
.37 (t, 3H, J ) 3.7 Hz); 1.44 (t, 3H, J ) 3.7 Hz); 3.93 (s, 3H, b); 4.09
m, 2H); 4.09 (q, 2H, J ) 3.7 Hz); 4.40 (q, 2H, J ) 3.7); 6.45 (d, 1H,
J ) 0.9 Hz); 6.55 (dd, 1H, J ) 1.1, 4.5 Hz); 6.81 (d, 1H, J ) 4.5 Hz);
.83 (d, 1H, J ) 4.5 Hz); 6.96 (d, 1H, J ) 1.1 Hz); 8.02 (s, 1H); 8.56
s, 1H). C NMR (75 MHz, CDCl
2.3, 62.4, 100.4, 105.9, 113.5, 114.1, 117.6, 128.5, 129.7, 130.2, 130.8,
31.6, 133.0, 135.6, 136.8, 147.7, 154.1, 158.5, 163.8, 164.3, 165.7,
65.9, 185.5. HRMS (ESI ): calcd for [M + Na] , 541.1474; found,
43.1436. absmax ) 460, 483 nm. emmax ) 527 nm. Φfl ) 0.148.
1
xanthen-3-one (4.5-diCl 2-COOMePh X, 3b). H NMR (300 MHz,
DMSO-d ): δ 3.59 (s, 3H); 6.52 (br, 4H); 6.90 (d, 2H, J ) 9.4 Hz);
6
13
7
.91 (s, 1H); 8.33 (s,1H); 11.07 (br, 1H). C NMR (300 MHz, DMSO-
6
(
6
1
1
5
d
6
): δ 53.8, 104.6, 105.1, 108.1, 131.1, 131.5, 133.3, 134.3, 165.3.
13
3
): δ 13.5, 13.9, 14.0, 56.0, 62.0,
+
+
HRMS (ESI ): calcd for M + 1, 421.0559; found, 421.0528. Mp:
30 °C (dec). Anal. Calcd for C21 ‚0.25H O: N, 0; C, 60.09;
H, 3.00. Found: N, 0; C, 60.21; H, 3.21.
-[2-(3-Methoxycarbonyl)naphthyl]-6-hydroxy-3H-xanthen-3-
3
H12Cl
2
O
5
2
+
+
9
1
one (2-COOMeNaph X, 4b). H NMR (300 MHz, DMSO-d
6
): δ 3.63
Synthesis and Characterization of MTM-CF. 6-CF-DA (150 mg,
0.32 mmol) was dissolved in DMF (5 mL). To this solution, chloro-
methyl methyl thioether (81 mg, 0.86 mmol) and cesium carbonate
(
(
1
s, 3H); 6.30 (br, 4H); 6.84 (m, 4H); 7.76 (m, 2H); 8.06 (m, 2H); 8.30
+
+
m, 1H); 8.89 (s, 1H); 11.04 (br, 1H). HRMS (ESI ): calcd for M +
, 397.107; found, 397.105. Mp: 325 °C (dec). Anal. Calcd for
‚0.75H O: N, 0; C, 73.25; H, 4.30. Found: N, 0; C, 73.43;
(
309 mg, 0.65 mmol) were added. The mixture was stirred for 6 h at
C
25
H O
18 6
2
room temperature, water was added, and the whole mixture was
extracted with 3 × 50 mL of AcOEt. The organic layer was dried and
concentrated in vacuo. The crude product was purified by silica gel
chromatography to afford MTM-CF DA (49 mg, yield 30%). MTM-
CF DA (10 mg, 0.02 mmol) was dissolved in 0.1 M sodium phosphate
buffer (50 mL) (2% DMF cosolvent). To this solution porcine liver
esterase (10 mg) was added. The mixture was incubated for 25 min at
H, 4.37.
9-[1-(2-Methoxycarbonyl)phenyl]-6-hydroxy-3H-xanthen-3-one-
1
(2-COOMePh X, 5b). H NMR (300 MHz, DMSO-d
6
): δ 3.63 (s,
3
2
H); 6.23-6.33 (br, 2H); 6.70-6.87 (m, 4H); 7.77 (m, 2H); 8.06 (m,
1
3
H); 8.31 (m, 1H); 8.89 (s, 1H); 11.04 (br, 1H). C NMR (75 MHz,
+ CD OD): δ 52.6, 103.0, 103.9, 115.6, 129.8, 130.7, 131.0,
31.3, 131.5, 133.6, 134.9, 153.7, 166.1. HRMS (ESI ): calcd for M
DMSO-d
1
6
3
+
+
2
5 °C. After the reaction, reaction mixture was diluted with 1 M HCl
aq, 50 mL) and whole mixture was extracted with AcOEt (3 × 50
mL). The organic layer was dried over anhydrous Na SO and
+
1, 347.0919; found, 347.0889. Mp: 285 °C (dec). Anal. Calcd for
: N, 0; C, 72.83; H, 4.07. Found: N, 0; C, 72.58; H, 4.33.
(
C
21
14
H O
5
2
4
Synthesis of Compound 2c. 6-Carboxyfluorescein diacetate (6-CF-
DA)31 was prepared according to the literature. 6-CF-DA (50 mg, 0.11
concentrated in vacuo. The crude product was purified by silica gel
chromatography to afford MTM-CF (1.3 mg, yield 13%).
mmol) was dissolved in DMF (5 mL); iodomethane (19 mg, 0.13 mmol)
and cesium carbonate (43 mg, 0.13 mmol) were added. The mixture
was stirred at room temperature for 2 h, water was added, and reaction
mixture was extracted with AcOEt (3 × 30 mL). The organic layer
1
MTM-CF-DA. H NMR (300 MHz, CDCl
3
): δ 2.27 (s, 3H); 2,32
(s, 6H); 5.38 (s, 2H); 6.80 (d, 2H, J ) 4.3 Hz); 6.84 (dd, 2H, J ) 1.8,
4.3 Hz); 7.12 (d, 2H, J ) 1.8 Hz); 7.83 (d, 1H, J ) 1.3 Hz); 8.12 (d,
1H, J ) 8.1 Hz); 8.33 (dd, 1H, J ) 1.3, 8.1 Hz). 13C NMR (75 Hz,
2 4
was dried over anhydrous Na SO and concentrated in vacuo. The crude
product was dissolved in methanol (10 mL). To this solution sodium
methoxide (47 mg, 0.87 mmol) was added. The mixture was stirred at
CDCl ): δ 15.7, 21.0, 69.9, 81.9, 110.4, 115.5, 117.8, 125.2, 125.3,
3
128.7, 129.7, 131.4, 136.2, 151.4, 152.1, 152.8, 164.4, 167.8, 168.6.
+
+
0
°C for 1 h, then poured into 1 N HCl (aq), and extracted with AcOEt
SO and
HRMS (ESI ): calcd for [M + Na] , 543.07257; found, 543.07687.
(3 × 50 mL). The organic layer was dried over anhydrous Na
2
4
1
MTM-CF. H NMR (CD
3
OD): δ 2.29 (s, 3H); 5.38 (s, 2H); 6.53
evaporated. The crude product was purified by silica gel chromatog-
(dd, 2H, J ) 2.3, 8.6 Hz); 6.60 (d, 2H, J ) 8.6 Hz); 6.70 (d, 2H, J )
raphy to afford 2c (7 mg, yield 16%).
2
1
.3 Hz); 7.76 (d, 1H, J ) 1.5 Hz); 8.11 (d, 1H, J ) 7.9 Hz); 8.33 (dd,
H, J ) 1.5, 7.9 Hz). HRMS (ESI ): calcd for [M - 1] , 435.05385;
9
-[1-(5-Methoxycarbonyl 2-carboxy)phenyl]-6-hydroxy-3H-xan-
-
-
1
3
then-3-one (5-COOMe 2-COOHPh X, 2c). H NMR (300 MHz, CD -
found, 435.05384.
OD): δ 3.86 (s, 3H); 6.52 (dd, J ) 2.2, 8.6 Hz, 2H); 6.56 (d, J ) 8.6
Hz, 2H); 6.69 (d, J ) 2.2 Hz, 2H); 7.75 (d, J ) 1.3 Hz, 1H); 8.09 (d,
Preparation of Benzene Moieties of Fluorescein Derivatives. All
the benzene moieties of methylated fluorescein derivatives, i.e.,
substituted methyl benzoates, are commercially available except 3,4-
dichlorobenzoic acid methyl ester.
1
3
J ) 7.9 Hz, 1H); 8.30 (dd, J ) 1.3, 7.9 Hz, 1H). C NMR (75 MHz,
CD OD): 53.1, 103.6, 110.8, 113.8, 126.3, 130.2, 132.0, 137.8, 154.1,
3
+
+
1
3
66.9, 170.3. HRMS (ESI ): calcd for M + 1, 391.0817; found,
91.0795. Anal. Calcd for C22 ‚H O: N, 0; C, 64.71; O, 3.95.
3,4-Dichlorobenzoic Acid Methyl Ester. 3,4-Dichlorobenzoic acid
H O
14 7
2
(
(
100 mg, 1.05 mmol) was dissolved in DMF (10 mL), and iodomethane
177 mg, 1.26 mmol) and cesium carbonate (408 mg, 1.26 mmol) were
Found: N, 0; C, 64.81; H, 3.85.
Synthesis and Characterization of 1d and 5d. 9-[1-(2-Methoxy-
carbonyl)phenyl]-6-methoxy-3H-xanthen-3-one (5d) was prepared ac-
added. The mixture was stirred at room temperature for 12 h, water
(50 mL) was added, and reaction mixture was extracted with AcOEt
9
cording to the literature. 9-[1-(2,4,5-Triethoxycarbonyl)phenyl]-6-
(
3 × 60 mL). The organic layers were dried over anhydrous Na
2 4
SO
hydroxy-3H-xanthen-3-one (1d) was prepared via the following
procedure: to a suspension of 1a (500 mg, 1.19 mmol) in ethanol (500
mL) was added a few drops of sulfuric acid. The reaction mixture was
refluxed for 20 h and then concentrated in vacuo. The residue was
poured onto ice, and the whole mixture was extracted with AcOEt.
and concentrated in vacuo. The crude product was purified by silica
gel chromatography and recrystallized from methanol to afford 3,4-
dichlorobenzoic acid methyl ester (145 mg, yield 68%). H NMR (300
1
MHz, CDCl
2
3
): 3.93 (s, 3H); 7.52 (d, J ) 8.4 Hz, 1H); 7.86 (dd, J )
.0, 8.4 Hz, 1H); 8.12 (d, J ) 2.0 Hz, 1H). 13C NMR (75 MHz,
(31) Mattingly, P. G. Bioconjugate Chem. 1992, 9, 430-431.
3
CDCl ): 52.4, 128.5, 129.8, 130.4, 131.4, 132.8, 137.4, 165.0. HRMS
1
4084 J. AM. CHEM. SOC. VOL. 126, NO. 43, 2004
9