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Y. Takaoka et al. / Tetrahedron 73 (2017) 3079e3085
5% citric acid aq., and then extracted with EtOAc three times, and
then the organic layer was washed with distilled water and brine.
The organic layer was dried over Na2SO4. The solvent was evapo-
rated and the residue was purified by column chromatography
(silica, CH2Cl2:hexane ¼ 1:1 to 2:1, and then 1:0) to afford 4e2 as a
AcOEt:hexane ¼ 1:2 to AcOEt:CHCl3 ¼ 1:1 (1% MeOH)) to afford
4e6 as a yellow amorphous compound (108 mg, 0.15 mmol, 53%
(2steps)). 1H NMR (CDCl3, 400 MHz, r.t.):
d/ppm ¼ 1.14e1.18 (m,
12H), 1.43 (s, 9H), 3.42 (s, 2H), 3.52 (b, 2H), 4.01e4.09 (m, 12H), 4.14
(s, 4H), 4.27 (m, 4H), 6.23 (dd, J ¼ 2.4, 8.4, 1H), 6.31 (d, J ¼ 2.4, 1H),
clear oil (764 mg, 79%). 1H NMR (CDCl3, TMS, 400 MHz, r.t.):
d/
6.76e6.79 (m, 4H). 13C NMR (CDCl3, 400 MHz, r.t.):
28.0, 42.2, 53.5, 53.9, 60.5, 60.7, 67.1, 67.1, 80.7, 102.1, 107.7, 114.1,
119.0, 121.6, 122.0, 128.63, 131.42, 138.1, 142.5, 150.2, 152.0, 171.0,
171.6, 171.7.
d
/ppm ¼ 14.0,
ppm ¼ 1.46 (s, 9H), 3.62 (s, 2H), 4.54 (m, 4H), 6.98(dd, J ¼ 1.6, 8.4,
1H), 7.06e7.10 (m, 1H), 7.16 (d, J ¼ 2.0, 1H), 7.25 (dd, J ¼ 1.2, 8.4, 1H),
7.55e7.59 (m, 1H), 7.81e7.84 (m, 2H). 13C NMR (CDCl3, 400 MHz,
r.t.):
d
/ppm ¼ 28.0, 42.4, 68.6, 68.6, 81.7, 115.8, 116.8, 121.3, 122.2,
125.5, 125.7, 134.3, 138.8, 140.3, 142.0, 151.8, 152.0, 169.4. IR (film)
2979, 2933, 1729, 1607, 1523, 1352, 1254, 1280, 1145, 1092. HR-ESI-
MS: Calcd. for [M(C20H22N2O8)þNa]þ: m/z ¼ 441.1274; Found:
441.1287.
4.3.6. Synthesis of 4e8
To a stirred solution of compound 4e6 (108 mg, 0.15 mmol) in
CH2Cl2 (1 mL) was added TFA. The mixture was stirred at r.t. for 2 h.
Excess TFA was removed via azeotrope with CH3CN in toluene,
yielding a crude product (178 mg). Then, to a stirred solution of the
above crude product in dry DMF (2 mL) was added propargylamine
(50 mg, 0.9 mmol, 6 eq), WSC$HCl (35 mg, 0.18 mmol, 1.2eq),
4.3.3. Synthesis of 4e3
To a stirred solution of 4e2 (500 mg, 1.20 mmol) in MeOH
(2.5 mL) and CH2Cl2 (2.5 mL) was added Pd/C (100 mg). The mixture
was stirred under H2 at r.t for 3 h. After the reaction, the suspension
was filtrated through celite. The solvent was evaporated to afford
4e3 as a white solid (479 mg, quant.). 1H NMR (CDCl3, TMS,
HOBt$H2O (27 mg, 0.18 mmol, 1.2eq), and DIPEA (78 mL, 0.45 mmol,
3eq). The mixture was stirred at r.t. for 16 h. The solvent was
evaporated. The residue was diluted with EtOAc and washed with
sat. NaHCO3, distilled water and brine. The organic layer was dried
over Na2SO4. The solvent was evaporated and the residue was pu-
rified by column chromatography (silica, CHCl3:MeOH ¼ 30:1) to
afford compound 4e7 as a brown solid (62 mg, 59% in two steps)
without any further purification. Finally, to a stirred solution of the
above crude compound 4e7 in dry DMF (2 mL) was added 5(6)-
400 MHz, r.t.):
4.37 (m, 4H), 6.66e6.74 (m, 4H), 6.79e6.87 (m, 3H).
d
/ppm ¼ 1.43 (s, 9H), 3.41 (s, 2H), 3.78e3.83 (b, 2H),
4.3.4. Synthesis of 4-4
To a stirred solution of 4e3 (479 mg, 1.20 mmol) in dry CH3CN
(20 mL) was added DIPEA (2025 mL, 12.0 mmol, 10eq) and ethyl
carboxy-2,7-dichlorofluorescein (47 mg, 89
mmol, 1 eq), HATU
bromoacetate (1.06 mL, 9.6 mmol, 8eq). The mixture was stirred
and refluxed for 5 h, and ethyl bromoacetate (1 mL, 9.6 mmol, total
16 eq) was added. The mixture was stirred and refluxed over night.
After reaction, the solvent was evaporated. The residue was diluted
with distilled water and extracted with EtOAc, and the combined
organic layer was washed with brine. The organic layer was dried
over Na2SO4. The solvent was evaporated and the residue was pu-
rified by column chromatography (silica, AcOEt:hexane ¼ 1:3) to
afford 4e4 as a yellow solid (663 mg, 79%). 1H NMR (CDCl3, TMS,
(41 mg, 0.11 mmol, 1.2eq), and DIPEA (31 L, 0.18 mmol, 2eq). The
m
mixture was stirred at r.t. for 8 h. The solvent was evaporated. The
residue was purified by column chromatography (silica,
CHCl3:MeOH ¼ 30:1). The obtained solid was suspended in diiso-
propylether, filtered, and dried in vacuo to yield the 5,6-mixture of
compound 4e8 (72 mg, 67%). The mixture of 5-, and 6-isomers of
4e8 was then purified by HPLC with a linear gradient (ODS, CH3CN
(0.05% TFA):H2O (0.05% TFA) ¼ 75:25 (5min) to 90:10 (55 min). By
using the authentic samples of the 6- or 5-isomer of compound 4e8
prepared separately, the retention time of the 6-isomer was iden-
tified as 22 min and that of the 5-isomer was 18 min 1H NMR of 4e9
400 MHz, r.t.):
3.00e4.07 (m, 8H), 4.13e4.15 (m, 8H), 4.27 (m, 4H), 6.73e6.92 (m,
7H). 13C NMR (CDCl3, 400 MHz, r.t.):
d
/ppm ¼ 1.12e1.16 (m, 12H), 1.43 (s, 9H), 3.42 (s, 2H),
d
/ppm ¼ 13.9, 13.9, 28.0, 42.2,
(6-isomer) (CDCl3:CD3OD ¼ 1:1, 400 MHz, r.t.): /ppm ¼ 1.12e1.17
d
53.4, 53.4, 60.7, 60.7, 66.9, 80.6, 113.0, 113.7, 118.7, 118.8, 121.4, 121.9,
122.0, 128.5, 138.0, 139.3, 150.1, 150.2, 170.8, 171.5. IR (film) 2981,
2933, 1747, 1508, 1369, 1246, 1178, 1027. HR-ESI-MS: calcd. for
[M(C36H50N2O12)þNa]þ: m/z ¼ 725.3261; found: 725.3249.
(m, 12H), 2.36e2.39 (m, 7H), 3.44 (s, 2H), 3.92e3.94 (m, 2H),
4.00e4.06 (m, 16H), 4.28 (m, 4H), 6.76e6.82 (m, 3H), 6.87 (m, 1H),
6.95 (s, 2H), 7.18 (d, J ¼ 1.6 Hz, 1H), 7.28 (s, 2H), 7.32e7.39 (m, 2H),
7.81 (m, 1H), 8.20 (d, J ¼ 8.0 Hz, 1H), 8.30 (dd, J ¼ 1.2 Hz, 8.0 Hz, 1H).
IR (film) 3299, 2927, 2850, 1776, 1733, 1672, 1605, 1516, 1411, 1371,
1259, 1200, 1167, 1026, 758. HRMS (ESI, positive) m/z [MþH]þ calcd
for C60H59N4O19Cl2 1209.3145, found 1209.3144.
4.3.5. Synthesis of 4e6
To a stirred solution of 4e4 (200 mg, 0.285 mmol) in AcOH
(1 mL) was added 70% HNO3 aq. (19 mL, 0.314 mmol, 1.1eq) and
NaNO3 (2.0 mg, 0.029 mmol, 0.1eq). After stirring for 30 min, the
mixture was poured into distilled water, and extracted with EtOAc
(three times), and combined organic layer was washed with water
(three times) and brine (twice). The organic layer was dried over
Na2SO4. Excess AcOH was removed via azeotrope in toluene, to
yield the crude product 4e5 a yellow oil (220 mg) was isolated. This
compound was used for the next step without any purification.
4.3.7. Synthesis of 4a
To a stirred solution of compound 4e9 (6-isomer, 0.25 mg,
0.21 mmol) in MeOH (34 mL) was added 1N KOH aq. (21 mL). The
mixture was stirred at r.t. for overnight. The solution was neutral-
ized with 1N HCl aq. to pH 7, and lyophilized to afford 4a (6-isomer)
as a red solid (quant.) containing the inorganic salt. The amount of
4a was calculated from the UVevis absorption spectrum with the
molar extinction coefficient of 2,7-dichlorofluorescein.23
4e5: 1H NMR (CDCl3, TMS, 400 MHz, r.t.):
d
/ppm ¼ 1.12e1.16 (m,
12H),1.43 (s, 9H), 3.42 (s, 2H), 4.02e4.09 (m, 8H), 4.13e4.15 (m, 8H),
4.28e4.32 (m, 4H), 6.68 (d, J ¼ 9.2 Hz, 1H), 6.76 (m, 3H), 7.73 (d,
J ¼ 2.8 Hz, 1H), 7.84 (DMSO-d6dd, J ¼ 9.2, 2.8 Hz, 1H). IR (film) 3382,
2981, 2933, 1743, 1588, 1517, 1331, 1249, 1181, 1026. ESI-MS: calcd.
for [MþNa]þ: m/z ¼ 770.311; found: 770.284.
Acknowledgement
To a stirred solution of 4e5 (213 mg, 0.285 mmol) in MeOH
(3 mL) and CH2Cl2 (2 mL) was added Pd/C (20 mg). The mixture was
stirred under H2 at r.t for 3 h. After reaction, the suspension was
filtrated through celite. The solvent was evaporated and the residue
We thank Prof. H. Isobe and Dr. S. Sota (Univ. of Tokyo) for the
use of DLS. This work was supported in part by a Grant-in-Aid for
Scientific Research (No. 26282207 to M.U. and No. 25708026, No.
16K13093 to Y.T.) on Innovative Areas “Chemical Biology of Natural
Products” from MEXT, Japan, and by JST (No. JPMJPR16Q4 to Y.T.).
was
purified
by
column
chromatography
(silica,