K. Ohgane et al. / Bioorg. Med. Chem. 18 (2010) 7022–7028
7027
DMEM containing 5% FBS (DMSO final concentration; 0.1%). After
4.11. TB4Es: 4-formylbenzyl 4-tert-butylbenzoate (TB4Es)
20 h incubation, the cells were fixed, blocked as described above
and incubated with a 1:7000 dilution of HRP-conjugated anti-HA
polyclonal antibody (A190-108P; Bethyl Laboratories, Inc., CanGet-
Signal immunoreaction enhancer solution; Toyobo Co., Ltd) for
1.5 h at room temperature. After extensive washing of the cells
1H NMR (CDCl3): d 10.03 (1H, s), 8.03 (2H, d, J = 8.5 Hz), 7.90
(2H, d, J = 8.5 Hz), 7.60 (2H, d, J = 8.5 Hz), 7.48 (2H, d, J = 8.5 Hz),
5.44 (2H, s), 1.34 (9H, s); MS (FAB): m/z 297 (M+H)+; HR-MS calcd
for C19H21O3 (M+H)+: 297.1491. Found 297.1496; mp: 33–35 °C.
with PBS containing 0.05% Tween 20, 50 lL of chemiluminescent
HRP substrate (SuperSignal ELISA Pico chemiluminescent sub-
strate; Pierce Biotechnology, Inc.) was added. After shaking at
room temperature for 10 min, the chemiluminescence was
measured with a Perkin Elmer Wallac 1420 multilabel counter.
The relative luminescence was defined as (compound-treated
well ꢁ average of background wells)/(average of vehicle-treated
wells ꢁ average of background wells).
4.12. TB35Es: 4-formylbenzyl 3,5-di-tert-butylbenzoate
(TB35Es)
1H NMR (CDCl3): d 10.03 (1H, s), 7.94 (2H, d, J = 1.8 Hz), 7.91
(2H, d, J = 7.9 Hz), 7.66 (1H, t, J = 1.8 Hz), 7.61 (2H, d, J = 7.9 Hz),
5.46 (2H, s), 1.35 (18H, s); MS (FAB): m/z 353 (M+H)+, 351
(MꢁH)+; HR-MS calcd for C23H29O3 (M+H)+: 353.2117. Found
353.2147; mp: 51–53 °C.
4.6. WST-1 viability assay
4.13. CF35Es: 4-formylbenzyl 3,5-bis(trifluoromethyl)benzoate
(CF35Es)
HEK293 cells were seeded into poly-Lys-coated 96-well plates
(1 ꢀ 104 cells/well), and incubated for 16–20 h. The cells were then
treated with prediluted compounds in DMEM containing 5% FBS
(DMSO final concentration; 0.1%). After 20–22 h incubation,
1H NMR (CDCl3): d 10.05 (1H, s), 8.52 (2H, s), 8.09 (1H, s), 7.94
(2H, d, J = 7.9 Hz), 7.62 (2H, d, J = 7.9 Hz), 5.51 (2H, s); MS (FAB): m/
z 377 (M+H)+, 375 (MꢁH)+; HR-MS calcd for C17H11F6O3 (M+H):
377.0612. Found 377.0652. Calcd for C17H9F6O3 (MꢁH):
375.0456. Found 375.0473; mp: 83–85 °C.
10 lL of WST-1 reagent (WST-1 5 mM, 1-methoxy-5-methylphen-
azinium methylsulfate 100 mM, HEPES 20 mM, pH 7.4) were
added. After 2 h incubation the absorbance was read at 405 nm
using a Perkin Elmer Wallac 1420 multilabel counter. The viability
was defined as Abs405 (treated)/Abs405 (vehicle) after subtraction of
background absorbance (from wells without cells). WST-1 reagent
was purchased from Dojindo.
4.14. CF35EsB: 4-((3,5-bis(trifluoromethyl)benzoyloxy)methyl)-
phenylboronic acid (CF35EsB)
1H NMR (CDCl3): d 8.50 (2H, s), 8.07 (1H, s), 7.80 (2H, d,
J = 7.9 Hz), 7.50 (2H, d, J = 7.9 Hz), 5.46 (2H, s); mp: 194–197 °C.
4.7. N-(4-Formylbenzyl)-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-
naphthalene-2-carboxamide (TMAm)
4.15. CF35EsC: 4-((3,5-bis(trifluoromethyl)benzoyloxy)methyl)-
benzoic acid (CF35EsC)
1H NMR (CDCl3): d 10.00 (s, 1H), 7.86 (d, J = 8.0 Hz, 2H), 7.83 (d,
J = 1.8 Hz, 1H), 7.52 (d, J = 8.0 Hz, 2H), 7.49 (dd, J = 8.3, 1.8 Hz, 1H),
7.37 (d, J = 8.32 Hz, 1H), 6.49 (br s, 1H), 4.74 (d, J = 6.1 Hz, 2H), 1.70
(s, 4H), 1.31 (s, 6H), 1.29 (s, 6H); MS (FAB): m/z 350 (M+H)+; HR-
MS calcd for C23H28NO2 (M+H)+: 350.2120. Found 350.2095; mp:
158–160 °C.
1H NMR (CDCl3): d 8.52 (2H, s), 8.14 (2H, d, J = 7.9 Hz), 8.09 (1H,
s), 7.56 (2H, d, J = 7.9 Hz), 5.50 (2H, s); HR-MS calcd for C17H11F6O3
(M+H): 393.0561. Found 393.0538; mp: 160–163 °C.
4.16. CF35EsA: 4-carbamoylbenzyl 3,5-bis(trifluoromethyl)-
benzoate (CF35EsA)
4.8. 4-Formylbenzyl 5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-
naphthalene-2-carboxylate (TMEs)
1H NMR (CDCl3): d 8.51 (2H, s), 8.09 (1H, s), 7.87 (2H, d,
J = 8.0 Hz), 7.55 (2H, d, J = 8.0 Hz), 6.06 (1H, br s), 5.60 (1H, br s),
5.48 (2H, s); HR-MS Calcd for C17H11F6O3 (M+H): 392.0721. Found
392.0568; mp: 169–172 °C.
1H NMR (CDCl3): d 10.03 (s, 1H), 8.05 (d, J = 1.8 Hz, 1H), 7.90 (d,
J = 8.5 Hz, 2H), 7.82 (dd, J = 8.5, 1.8 Hz, 1H), 7.59 (d, J = 7.9 Hz, 2H),
7.39 (d, J = 7.9 Hz, 1H), 5.43 (s, 2H), 1.70 (s, 4H), 1.31 (s, 6H), 1.30
(6H); MS (FAB): m/z351 (M+H)+; HR-MS calcd for C23H27O3
(M+H): 351.1960. Found 351.1966.
Acknowledgments
The work described in this paper was partially supported by
Grants-in-Aid for Scientific Research from The Ministry of Educa-
tion, Culture, Sports, Science and Technology, Japan, and the Japan
Society for the Promotion of Science.
4.9. 4-Formylbenzyl 5,6,7,8-tetrahydronaphthalene-2-
carboxylate (HNEs)
1H NMR (CDCl3): d 10.03 (1H, s), 7.90 (2H, d, J = 7.9 Hz), 7.79
(1H, s), 7.79 (1H, d, J = 8.5 Hz), 7.59 (2H, d, J = 7.9 Hz), 7.14 (1H,
d, J = 8.5 Hz), 5.42 (2H, s), 2.82 (4H, br s), 1.84–1.80 (4H, br m);
MS (FAB): m/z 295 (M+H)+; HR-MS calcd for C19H19O3 (M+H)+:
295.1334. Found 295.1368.
References and notes
1. Rattner, A.; Sun, H.; Nathans, J. Annu. Rev. Genet. 1999, 33, 89.
2. Chapple, J. P.; Grayson, C.; Hardcastle, A. J.; Saliba, R. S.; van der Spuy, J.;
Cheetham, M. E. Trends Mol. Med. 2001, 7, 414.
3. Kennan, A.; Aherne, A.; Humphries, P. Trends Genet. 2005, 21, 103.
4. Hartong, D.; Berson, E. L.; Dryja, T. P. Lancet 2006, 368, 1795.
5. Dryja, T. P.; McGee, T. L.; Reichel, E.; Hahn, L. B.; Cowley, G. S.; Yandell, D. W.;
Sandberg, M. A.; Berson, E. L. Nature 1990, 343, 364.
6. Sung, C. H.; Schneider, B. G.; Agarwal, N.; Papermaster, D. S.; Nathans, J. Proc.
Natl. Acad. Sci. U.S.A. 1991, 88, 8840.
7. Sung, C. H.; Davenport, C. M.; Nathans, J. J. Biol. Chem. 1993, 268, 26645.
8. Kaushal, S.; Khorana, H. G. Biochemistry 1994, 33, 6121.
9. Illing, M. E.; Rajan, R. S.; Bence, N. F.; Kopito, R. R. J. Biol. Chem. 2002, 277, 34150.
10. Mendes, H. F.; Cheetham, M. E. Human Mol. Genet. 2008, 17, 3043.
4.10. 4-Formylbenzyl 3-tert-butylbenzoate (TB3Es)
1H NMR (CDCl3): d 10.03 (1H, s), 8.13 (1H, t, J = 2.1 Hz), 7.92–
7.90 (3H, m), 7.63–7.61 (3H, m), 7.40 (1H, t, J = 7.6 Hz), 5.45 (2H,
s), 1.35 (9H, s); MS (FAB): m/z 297 (M+H)+, 295 (MꢁH)+; HR-MS
calcd for C19H21O3 (M+H)+: 297.1491. Found 297.1462; mp: 53–
55 °C.