5174
D. B. Belanger et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5170–5174
9. Sun, C. L.; Liang, C.; Huang, P.; Harris, G. D.; Guan, H. US/2004/0220189; Sun, C.
L.; Liang, C.; Huang, P.; Harris, G. D.; Guan, H. US/2005/009832.
10. 3-Bromo-8-(methylsulfonyl)imidazo[1,2-a]pyrazine 4 is a stable solid that can
be stored cold for months without decomposition.
Aurora A assay was modified and utilized 4 nM enzyme, 100 nM PKAtide, and
100 M ATP. For the Aurora B assay, each reaction consisted of 26 nM enzyme
(Aurora B, Invitrogen), 100 nM Tamra-PKAtide (Molecular Devices, 5TAMRA-
GRTGRRNSICOOH), 50 M ATP, 1 mM DTT, and kinase buffer (10 mM Tris,
l
l
11. The coordinates for compound 1j have been deposited in the RCSB Protein Data
Bank under the Accession Code 3NRM.
10 mM MgCl2, 0.01% Tween 20). Dose–response curves were plotted from
inhibition data generated in duplicate, from eight point serial dilutions of
inhibitory compounds. Concentration of compound was plotted against kinase
activity, calculated by degree of fluorescent polarization. To generate IC50
values, the dose–response curves were then fitted to a standard sigmoidal
curve and IC50 values were derived by nonlinear regression analysis.
Immunofluorescent assays: HCT-116 cells were plated at 15,000 cells per well
12. (a) Nowakowski, J.; Cronin, C. N.; McRee, D. E.; Knuth, M. W.; Nelson, C. G.;
Pavletich, N. P.; Rogers, J.; Sang, B.-C.; Scheibe, D. N.; Swanson, R. V.;
Thompson, D. A. Structure 2002, 10, 1659; (b) Zuccotto, F.; Ardini, E.; Casale,
E.; Angiolini, M. J. Med. Chem. 2009, 53, 2681; (c) Morphy, R. J. Med. Chem. 2010,
53, 1413.
13. Subsequent X-ray data with related imidazo[1,2-a]pyrazine inhibitors revealed
a water mediated hydrogen bond between the enzyme and the 3-(4-pyrazolo)
group. Yu, T.; Tagat, J. R.; Kerekes, A. D.; Doll, R. J.; Zhang, Y.; Xiao, Y.; Esposite,
S.; Belanger, D. B.; Curran, P. J.; Amit K. Mandal; Siddiqui, M. A.; Shih, N-Y.;
Basso, A. D.; Liu, M.; Gray, K.; Tevar, S.; Jones, J.; Lee, S.; Ponery, S.; Smith, E. B.;
Hruza, A.; Voigt, J.; Ramanathan, L.; Prosise, W.; Hu, M. J. Med. Chem. Lett. 2010,
14. The conformational preference of thiazoles in related systems has been
documented. (a) Pierce, A. C.; ter Haar, E.; Binch, H. M.; Kay, D. P.; Patel, S. R.; Li,
P. J. Med. Chem. 2005, 48, 1278; (b) Jung, F. H.; Pasquet, G.; Lambert-van der
Brempt, C.; Lohmann, J. J.; Warin, N.; Renaud, F.; Germain, H.; De Savi, C.;
Roberts, N.; Johnson, T.; Dousson, C.; Hill, G. B.; Mortlock, A. A.; Heron, N.;
Wilkinson, R. W.; Wedge, S. R.; Heaton, S. P.; Odedra, R.; Keen, N. J.; Green, S.;
Brown, E.; Thompson, K.; Brightwell, S. J. Med. Chem. 2006, 49, 955.
15. Biochemical assays: Aurora A and B kinase assays were performed in low
protein binding 384-well plates. Compounds were diluted in 100% DMSO to
the desired concentrations. For the Aurora A assay, each reaction consisted of
8 nM enzyme (Aurora A, Upstate), 100 nM Tamra-PKAtide (Molecular Devices,
in poly-D-lysine coated black micro-clear 384-well tissue culture plates. For the
phos-Histone H3 assay, cells were first treated with 0.4 mg/mL nocodazole.
Sixteen hours later cells were treated in triplicate with compound (0.1% final
DMSO concentration) for 1 h. Cells were fixed with PreferÒ fixation solution
(Anatech) plus 1000 nM Hoechst 33342 dye and incubated for 30 min at room
temperature. The fixation solution was removed and cells were washed with
PBS. Cells were permeabilized with 0.2% Triton-X in PBS and incubated for
10 min. Cells were washed with PBS and incubated with PBS containing 3% FBS
for 30 min. Cells were then stained overnight at 4 °C with Phos-Histone H3
(Ser10)-Alexa Flur 488 Conjugate antibody (Cell Signaling) solution in PBS plus
3% FBS. Cells were washed with PBS and then immunofluorescence images
were captured at 10ꢀ with HT Pathway 855 automated fluorescent microscope
(BD Bioscience). Percent positive cells were quantitated using Hoechst staining
for cell number using Attovision software (BD Bioscience). To generate IC50
values, the dose–response curves were then fitted to a standard sigmoidal
curve and IC50 values were derived by nonlinear regression analysis.
16. DeLano, W. L. The PyMOL Molecular Graphics System; DeLano Scientific: Palo
Alto, CA, 2002.
5TAMRA-GRTGRRNSICOOH), 25
Tris, 10 mM MgCl2, 0.01% Tween 20). For the high throughput screen the
l
M ATP, 1 mM DTT, and kinase buffer (10 mM
17. Sorota, S.; Zhang, X. S.; Margulis, M.; Tucker, K.; Priestley, T. Assay Drug Dev.
Technol. 2005, 3, 47.