J. Kohno et al. / Tetrahedron 57 (2001) 1731±1735
1735
1
10.1), 4.10 (N±OCH3, 3H, s), 4.00 (H-11ax, 1H, d, 13), 3.35
(OCH3, 3H, s), 2.46 (H-9eq, 1H, dd, 14, 4.2), 2.70 (H-8, 1H,
m), 2.0±2.2 (H-9ax and H-17, 2H, m), 1.32 (H-18, 2H, m),
1.01 (H-21, 3H, d, 6.8), 0.85 (H-19, 3H, t, 7.3), 0.82 (H-20,
3H, d, 6.6).
C29H31NO7F3 562.2053); H NMR (CDCl3, 400 MHz) d
7.503 (H-6, 1H, s), 7.4±7.5 (Ph£2, 10H, m), 5.234 (H-10,
1H, m), 4.514 (H-7, 1H, d, 11), 4.145 (H-11eq, 1H, ddd,
10.5, 5.0, 2.0), 4.098 (N±OCH3, 3H, s), 3.557 (OCH3, 3H,
s), 3.362 (OCH3, 3H, s), 3.348 (H-11ax, 1H, t, 10.5), 2.833
(H-8, 1H, m), 2.370 (H-9eq, 1H, m), 1.468 (H-9ax, q, 12),
0.843 (H-20, 3H, d, 6.6).
2.1.5. Preparation of 6 from 4. Dry O3 was bubbled into
the solution of 4 (80 mg, 0.18 mmol) in MeOH (5 mL) at
2788C, until the starting material was disappeared on TLC
(SiO2, EtOAc/n-hexane (1:1)). To the reaction mixture,
Me2S was added (200 mL, 2.7 mmol) and then the mixture
was stirred at room temperature for 1 h. To the resulting
mixture, NaBH4 was added (27 mg, 0.72 mmol) and then
the mixture was stirred at 08C for 30 min. After adding
acetone (1 mL) to the reaction mixture, water was added
and the mixture was extracted with CH2Cl2. The organic
phase was washed with water, saturated NaCl, and dried
over anhydrous Na2SO4. The solvent was concentrated in
vacuo and the residual oil was puri®ed on preparative TLC
(SiO2, 6% MeOH±CHCl3) to yield a colorless oil of 6
2.2. In vitro cytotoxic activity
The cells used for assay were cultured in the following
medium; HCT-116: complete McCoy's 5A supplemented
with 10% fetal bovine serum, B16: complete D-MEM
supplemented with 10% fetal bovine serum, P388D1:
complete RPMI-1640 supplemented with 5% fetal bovine
serum.
In vitro cytotoxic activity was tested in 96-well microtiter
plates of which well containing 1£104 each cell lines in
135 mL medium. The test samples were dissolved in 10%
DMSO. The serially diluted DMSO solution (15 mL) was
added to each well of plates. After addition, the cells were
incubated at 378C for 72 h in a humidi®ed 5% CO2
atmosphere. In vitro cytotoxic activity was evaluated by
the microculture tetrazolium (MTT) assay method for
each cell and by the colorimetrical determination method
at 540 nm.
1
(9 mg, 22% yield). ESI-MS m/z 346 (M1H)1; H NMR
(CDCl3, 300 MHz) d 7.51 (H-6, 1H, s), 7.35±7.45 (Ph,
5H, m), 4.46 (H-7, 1H, d, 10), 4.15 (H-11eq, 1H, ddd,
10.3, 5, 2), 4.10 (N±OCH3, 3H, s), 3.95 (H-10, 1H, m),
3.36 (OCH3, 3H, s), 3.24 (H-11ax, 1H, t, 10.3), 2.75 (H-8,
1H, m), 2.24 (H-9eq, 1H, m), 1.25 (H-9ax, 1H, q, 12), 0.82
(H-20, 3H, d, 6.8).
2.1.6. (S)-(2)-MTPA ester of 6. To a stirred solution of 6
(2.3 mg, 0.0067 mmol) in dry CH2Cl2 (0.5 mL), (R)-(2)- a-
methoxy-a-tri¯uoromethylphenylacetic chloride (MTPACl;
12 mg, 0.05 mmol), triethylamine (12 mg, 0.1 mmol) and
4-dimethylaminopyridine (DMAP; catalytic amount) was
added and the mixture was stirred at room temperature for
7 h. The reaction mixture was directly puri®ed on prepara-
tive TLC (SiO2, 5% MeOH±CHCl3) to yield 2 mg of
(S)-(2)-MTPA ester, 7 (53% yield). FAB-MS m/z 562
(M1H)1; HRFAB-MS m/z 562.2050 (M1H)1, (calcd for
Acknowledgements
We wish to thank Ms Naoko Fukui for NMR measurements,
and Dr Noriko Ohashi and Ms Sonoko Shiina for mass
measurements.
References
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1
C29H31NO7F3 562.2053); H NMR (CDCl3, 400 MHz) d
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