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for 4 h, saturated aqueous sodium bicarbonate was
added. The aqueous layer was extracted with DCM
twice. The combined organic layers were washed with
1 M citric acid, 1 M sodium bicarbonate, water, and
brine, dried over sodium sulfate, and concentrated to
give 1-[(4-methoxyphenyl)methyl]-2-oxo-3-{1-[(trifluoro-
methyl)-sulfonyl]benzimidazol-2-yl}-6-chloro-4-hydroquin-
olyl(trifluoromethyl)sulfonate, which was used for the
next reaction without further purification. A solution of
the bis-triflate prepared above, (S)-3-aminoquinuclidine
6. Koniaras, K.; Cuddihy, A. R.; Christopoulos, H.; Hogg,
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11. Agouron: Reich, S.H.; Belcman, T. M; Kephart, S. E.;
Romines, W. H.; Wallaces, M. B. WO0153268, 2001.
12. For CHK-1 inhibitor, ICP-1, see: Eastman, A.; Kohn, E.
A.; Brown, M. K.; Rathman, J.; Livingstone, M.; Blank,
D. H.; Gribble, G. W. Mol. Cancer Therap. 2002, 1, 1067.
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3118.
(1.2 equiv), and Hunig’s base (4 equiv) in acetonitrile
¨
was heated at 80 ꢁC for 20 h. After cooling down to
room temperature, the reaction mixture was diluted with
ethyl acetate, washed with saturated aqueous sodium
bicarbonate, water, and brine, dried over sodium sulfate,
and concentrated to give
a
a solid. The solid was
dissolved in mixture of trifluoroacetic acid and
concentrated HCl (7:1), and heated at 90 ꢁC for 20 h.
The reaction was diluted with water and ethyl acetate,
and slowly basified to pH 9 with saturated aqueous
sodium bicarbonate. The aqueous layer was extracted
with ethyl acetate. The combined organic layers were
washed with water and brine, and dried over sodium
sulfate, and concentrated. The product was purified with
preparative HPLC to give the desired product 11.
18. The CHK1 complex with compound 11 has been assigned
the PDB ID code: 2GDO.
19. Le, V. et al. manuscript in preparation.
20. Chiron: Barsanti, P.; Bussiere, D.; Harrison, S.; Heise, C.;
Jazan, E.; Machajewski, T.; McBride, C.; McCrea, W.;
Ng, S.; Jansen, H.; Ni, Z.-J.; Pecchi, S.; Pfister, K.;
Ramurthy, S.; Renhowe, P.; Shafer, C.; Wagman, A.;
Wiesmann, M. WO0418419, 2004.
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Aardalen, K.;Embry, M.; Ma, S.; Moler, E.; Ni, Z.-J.;
Menezes, D.; Hibner, B.; Gesner, T. G.; Schwartz, G. K.
Cancer Res. manuscript submitted.
22. Cell Synergy Assay: MDA-MB-435 cells (5· 103)
were plated onto
a 96-well plate in 100 lL cell
14. Lin, N.-H.; Xia, P.; Kovar, P.; Park, C.; Chen, Z.; Zhang,
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Potter, A.; Robertson, A. G. S.; Surgenor, A. E. J. Med.
Chem. 2005, 48, 4332.
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Capiris, T.; Kesten, S. J.; Herzig, D. J. J. Med. Chem.
1981, 24, 735; (b) Hardtmann, G, E.; Koletar, G.; Pfister,
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17. General procedure for the preparation of 4-[((3S)-
quinuclidin-3-yl)amino]-3-benzimidazol-2-yl-6-chloro-hydro-
quinolin-2- one (11): 3-(1H-benzimidazole-2yl)-4-hydrox-
yl-1-(4-methoxybenzyl)-6-chloro-1H- quinolinone (1 equiv)
was suspended in dichloromethane (DCM) in the
presence of pyridine (20 equiv). The mixture was
warmed to dissolve the solid. The mixture was cooled
to ꢀ5 ꢁC and triflic anhydride (8 equiv) in DCM was
added dropwise. After the reaction was stirred at ꢀ5 ꢁC
culture medium and incubated for 4 h at 37 ꢁC
under 5% CO2. DMSO (vehicle control), CPT alone,
compound 11 alone, and compound 11 + CPT, all in
100 lL cell culture medium, were added, resulting in
the final concentrations indicated. The cells were
incubated further for 48 h. MTS was added to the
cultures, permitted to develop for 4 h at 37 ꢁC under
5% CO2, then read on
a microplate reader at
490 nm. The ODs were corrected for background
absorbance by subtracting the values obtained for
wells containing tissue culture medium alone. Percent
viable cells were calculated by: {[A490 (DMSO-treated
cells) ꢀ A490
(test
sample)]/A490
(DMSO-treated
cells)} · 100%. Synergy was defined as the effect
observed by the combination of the two compounds
being greater than the sum of the effect of each
compound alone.
23. Detailed isobologram analysis of the synergistic effect will
be published elsewhere (see Ref. 21).