S. Tamura et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2082–2085
2085
Table 3
chemical structures of ACA (1), the analogs, and H2O were fully optimized by
the semiempirical PM3 method. The oxygen atom of H2O was vertically located
to the carbonyl carbon linked to the oxygen at C-4 at a distance of 1.95 Å, then
the potential energy of the stationary state was calculated. According to the
reaction mechanism of hydrolysis of the acetyl group,20 the primary structure
of the transition state was respectively generated. Each transition state was
further optimized by the semiempirical PM3 method to calculate the potential
energy. The activation energy (E1 and E2) was determined by difference of the
potential energy between the stationary state and transition state.
Inhibitory activity of halogenated analogs of 1 for nuclear export of Rev
Compound
Model fusion protein in
HA-Rev in HeLa
cell (IC50, lM)
fission yeast (MIC,
l
M)
ACA (1)
4.3
1.7
2.2
1.7
1.4
0.98
0.48
0.62
0.49
0.13
3-FluoroACA (20a)
3-ChloroACA (20b)
3-BromoACA (20c)
2,3-DifluoroACA (20d)
12. Compound 20a: colorless oil, ½a 2D4 max (KBr) cmꢁ1: 1765,
ꢁ34.2 (c 1.0, EtOH), IR m
ꢃ
1740, 1608, 1H NMR (300 MHz, CDCl3) d: 7.17 (1H, brd, J = 10.9 Hz, 2-H), 7.13
(1H, br d, J = 8.5 Hz, 6-H), 6.98 (1H, dd, J = 8.5, 6.1 Hz, 5-H), 6.23 (1H, d,
J = 6.1 Hz, 10-H), 5.95 (1H, ddd, J = 17.0, 10.4, 6.1 Hz, 20-H), 5.32 (1H, d,
J = 17.0 Hz, 30-Ha), 5.28 (1H, d, J = 10.4 Hz, 30-Hb), 2.33 (3H, s, 4-OAc), 2.12 (3H,
s, 10-OAc), FAB-MS (m/z): 253 [M+H]+, HR FAB-MS (m/z): calcd for
C13H13FO4+H; 253.0876, found 253.0873.
would indicate that the activities share the mechanism of action
involving formation of the quinone methide intermediate ii. Thus,
the logical design by the activation energy E1 would open up a new
avenue to develop new agents using ACA (1) as a scaffold.
Compound 20b: colorless oil, ½a 2D4 max (KBr) cmꢁ1: 1763,
ꢁ40.8 (c 1.0, EtOH), IR m
ꢃ
1740, 1605, 1H NMR (300 MHz, CDCl3) d: 7.44 (1H, d, J = 2.0 Hz, 2-H), 7.27 (1H,
d, J = 8.3, 2.0 Hz, 6-H), 7.12 (1H, d, J = 8.3 Hz, 5-H), 6.22 (1H, br d, J = 5.9 Hz, 10-
H), 5.95 (1H, ddd, J = 17.1, 10.4, 5.9 Hz, 20-H), 5.33 (1H, ddd, J = 17.1, 1.3, 1.1 Hz,
30-Ha), 5.28 (1H, ddd, J = 10.4, 1.3, 1.1 Hz, 30-Hb), 2.36 (3H, s, 4-OAc), 2.13 (3H,
s, 10-OAc), FAB-MS (m/z): 269 [M+H]+, HR FAB-MS (m/z): calcd for
C13H1335ClO4+H; 269.0581, found 269.0586, calcd for C13H1337ClO4+H;
271.0552, found 271.0554.
Acknowledgments
We wish to thank Professor Minoru Yoshida in RIKEN Advanced
Science Institute for giving the fission yeast S. pombe. This work
was supported in part by Grants-in-Aid for Scientific Research
(Grant No. 19590100) from the Ministry of Education, Science, Cul-
ture and Sports. The authors are grateful to the Shorai Foundation
for Science and Technology for financial support.
Compound 20c: colorless oil, ½a 2D4 max (KBr) cmꢁ1: 1766,
ꢁ39.9 (c 1.0, EtOH), IR m
ꢃ
1738, 1610, 1H NMR (300 MHz, CDCl3) d: 7.60 (1H, d, J = 2.0 Hz, 2-H), 7.31 (1H,
dd, J = 8.3, 2.0 Hz, 6-H), 7.12 (1H, d, J = 8.3 Hz, 5-H), 6.22 (1H, d, J = 6.0 Hz, 10-H),
5.95 (1H, ddd, J = 16.9, 10.2, 6.0 Hz, 20-H), 5.33 (1H, d, J = 16.9 Hz, 30-Ha), 5.28
(1H, d, J = 10.2 Hz, 30-Hb), 2.36 (3H, s, 4-OAc), 2.13 (3H, s, 10-OAc), FAB-MS (m/
z): 313 [M+H]+, HR FAB-MS (m/z): calcd for C13H1379BrO4+H; 313.0075, found
313.0071, calcd for C13H1381BrO4+H; 315.0056, found 315.0053.
Compound 20d: colorless oil, ½a 2D4 max (KBr) cmꢁ1: 1763,
ꢁ21.6 (c 1.0, EtOH), IR m
ꢃ
1736, 1610, 1H NMR (300 MHz, CDCl3) d: 7.13 (1H, ddd, J = 8.9, 6.9, 2.3 Hz, 5-H),
6.92 (1H, ddd, J = 8.9, 6.6, 2.1 Hz, 6-H), 6.48 (1H, br d, J = 6.0 Hz, 10-H), 5.99 (1H,
ddd, J = 17.2, 10.4, 6.0 Hz, 20-H), 5.32 (1H, dd, J = 17.2, 1.1 Hz, 30-Ha), 5.29 (1H,
ddd, J = 10.4, 1.1, 1.1 Hz, 30-Hb), 2.35 (3H, s, 4-OAc), 2.12 (3H, s, 10-OAc), FAB-
MS (m/z): 271 [M+H]+, HR FAB-MS (m/z): calcd for C13H12F2O4+H; 271.0782,
found 271.0785.
References and notes
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fusion protein for 24 h at 37 °C. Then the cells were seeded in 96-well
microplates along with the test samples in the medium containing 1% DMSO
and incubated at 37 °C for further 3 h. Distribution of the GST-NLS-GFP-
HA-tagged Rev protein) and pCRRE/DRev (plasmid encoding Gag protein)
plasmids into HeLa cells were performed using PolyFect transfection reagent
kit (QIAGEN) for 16 h according to the manufacturer’s instructions. After the
cells were washed, each solution of tested sample at an appropriate
concentration in the medium containing 1% DMSO was inoculated and the
whole was incubated at 37 °C for further 12 h. Cells were rinsed with cold D-
PBS (ꢁ) twice and fixed with 4% formaldehyde/D-PBS (ꢁ) for 20 min. Then the
cells were defatted with MeOH under shaking for 10 min and washed with cold
D-PBS (ꢁ) thrice. After treatment with 10% FBS in Dulbecco’s MEM medium for
30 min, the samples were incubated with anti-HA antibody (Roche) for 45 min
followed by incubation with FITC-labeled anti-mouse IgG antibody (Vector) for
45 min. Localization of the HA-tagged Rev protein in the cells was examined
with a fluorescence microscope, then image analysis was conducted by Scion
image software (Scion) to determine Rev-export inhibitory activity. In the
depicted pictures, several cells free from transfection displayed disperse weak
fluorescence due to nonspecific binding of the antibodies.
RevNES-fused protein was monitored with
determine MIC values.
a fluorescence microscope to
8. Each sample (20
lL of 0.2 mM EtOH solution) was treated with RPMI1640
medium containing 10% fetal bovine serum (2 mL) and the whole was
incubated at 37 °C for 0, 30, 60, 180 min, respectively. After extraction of the
treated mixture with EtOAc (2 mL), each extract was concentrated under
reduced pressure. The residue was dissolved with 100
aliquot (20 L) of the solution was analyzed by reversed-phase HPLC [column:
Mightysil RP-18 (5
lL of CH3CN, then an
l
l
m) 4.6 ꢂ 250 mm, mobile phase: CH3CN–H2O (1:9?2:8,
liner gradient, 5 min; 2:8, 5 min; 2:8?8:2, liner gradient, 20 min), flow
rate = 1.0 mL/min, detection: UV 220 nm] to determine the remaining
amount of the tested sample at the indicated times in triplicate. In this
experiment, fetal bovine serum was diluted with RPMI1640 medium to
modulate enzymatic activity of esterase.
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11. The activation energy for the E1 and E2 steps was calculated by MO calculation
using SPARTAN ’02 (Wavefunction) in the following protocol. At first, the