P. L. Beaulieu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3658–3663
3663
Table 4
7. IC50 values were determined as previously described: (a) McKercher, G.;
Beaulieu, P. L.; Lamarre, D.; LaPlante, S.; Lefebvre, S.; Pellerin, C.; Thauvette, L.;
Kukolj, G. Nucleic Acids Res. 2004, 32, 422; EC50 determinations were performed
using Huh-7 cells with a stable subgenomic HCV 1b replicon. The HCV RNA
levels were normalized to total cellular RNA in this 72 h assay. The
quantification of total RNA recovery allowed for an assessment of cellular
homeostasis to eliminate the possibility of antiviral activity due to subtle toxic
effects. For an overview and basic protocols on the use of HCV replicons, see (b)
Lohmann, V. Hepatitis C: Methods and Protocols, 2nd edition In Methods in Mol.
Biol.; Tang, H., Ed.; Humana Press, 2009; Vol. 510, p 145.
RLM metabolic stability and rat oral PK parameters for 24 and 32 at 10 mg/kg oral
dose
Compds
RLM T1/2 (min)a
Cmax
(
lM)
AUC (
lMÁh)
Oral MRT (h)
24
32
9
21
1.23
0.99
0.87
1.18
0.5
3.4
a
T1/2 measured in rat liver microsomes (RLM) at 10 lM initial concentration.
8. (a) Beaulieu, P. L.; Gillard, J.; Bykowski, D.; Brochu, C.; Dansereau, N.; Duceppe,
J.-S.; Haché, B.; Jakalian, A.; Lagacé, L.; LaPlante, S.; McKercher, G.; Moreau, E.;
Perreault, S.; Stammers, T.; Thauvette, L.; Warrington, J.; Kukolj, G. Bioorg. Med.
Chem. Lett. 2006, 16, 4987; (b) Harper, S.; Pacini, B.; Avolio, S.; Di Filippo, M.;
Migliaccio, G.; Laufer, R.; De Francesco, R.; Rowley, M.; Narjes, F. J. Med. Chem.
2005, 48, 1314; (c) Ontoria, J. M.; Martin Hernando, J. I.; Malancona, S.; Attenni,
B.; Stansfield, I.; Conte, I.; Ercolani, C.; Habermann, J.; Ponzi, S.; Di Filippo, M.;
Koch, U.; Rowley, M.; Narjes, F. Bioorg. Med. Chem. Lett. 2006, 16, 4026.
prediction method is based on the algorithm described in Viswanadhan, V. N.;
Ghose, A. K.; Revankar, G. R.; Robins, R. K. J. Chem. Inf. Comput. Sci. 1989, 29, 163.
10. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev.
1997, 23, 3.
Acknowledgments
We are grateful to Dr. Gordon Bolger, Christine Zouki and Josie
De Marte for in vitro ADME data and to Colette Boucher, Michael
Little and Serge Valois for analytical support. We thank Francine
Liard, Manon Rhéaume and Hélène Montpetit for providing rat
in vivo data. Nathalie Dansereau, Lisette Lagacé, Martin Marquis,
Ginette McKercher and Louise Thauvette are acknowledged for
profiling in HCV biological assays.
11. Performed on amorphous TFA salts using the 24 h shaking flask method (pH
7.2 phosphate buffer).
12. Kalgutkar, A. S.; Gardner, I.; Obach, R. S.; Shaffer, C. L.; Callegari, E.; Henne, K.
R.; Mutlib, A. E.; Dalvie, D. K.; Lee, J. S.; Nakai, Y.; O’Donnell, J. P.; Boer, J.;
Harriman, S. P. Curr. Drug Metab. 2005, 6, 651.
13. Part 2: Beaulieu, P. L. et al. (accompanying paper).
14. Rorrer, L. C.; Hopkins, S. D.; Connors, M. K.; Lee, D. W., III; Smith, M. V.; Rhodes,
H. J.; Uffelman, E. S. Org. Lett. 1999, 1, 1157.
15. All inhibitors in this study were purified to >95% homogeneity by reversed-
phase HPLC and isolated as TFA salts. All compounds were characterized by
mass spectrometry and gave 1H NMR spectra consistent with expected
structures.
16. Beaulieu, P. L.; Bös, M.; Bousquet, Y.; Fazal, G.; Gauthier, J.; Gillard, J.; Goulet, S.;
LaPlante, S.; Poupart, M.-A.; Lefebvre, S.; McKercher, G.; Pellerin, C.; Austel, V.;
Kukolj, G. Bioorg. Med. Chem. Lett. 2004, 14, 119.
17. LaPlante, S. R.; Jakalian, A.; Aubry, N.; Bousquet, Y.; Ferland, J.-M.; Gillard, J.;
Lefebvre, S.; Poirier, M.; Tsantrizos, Y. S.; Kukolj, G.; Beaulieu, P. L. Angew Chem.,
Int. Ed. 2004, 43, 4306.
18. ACD/pKa DB Version 11; Advanced Chemistry Development, Inc: Toronto,
Canada, 2007.
References and notes
1. (a) Choo, Q.-L.; Kuo, G.; Weiner, A. J.; Overby, L. R.; Bradley, D. W.; Houghton, M.
Science 1989, 244, 359; (b) Lavanchy, D. Liver Int. 2009, 29, 74.
2. For reviews on NS5B inhibitors see (a) Beaulieu, P. L. Expert Opin. Ther. Pat.
2009, 19, 145; (b) Li, H.; Shi, S. T. Future Med. Chem. 2010, 2, 121; (c) Watkins,
W. J.; Ray, A. S.; Chong, L. S. Curr. Opin. Drug Discov. Devel. 2010, 13, 441; For a
review on inhibition of other HCV targets, see (a) Manns, M. P.; Foster, G. R.;
Rockstroh, J. K.; Zeuzem, S.; Zoulim, F.; Houghton, M. Nat. Rev. Drug Disc. 2007,
6, 991; (b) Lange, C. M.; Sarrazin, C.; Zeuzem, S. Aliment. Pharmacol. Ther. 2010,
32, 14.
3. Farnik, H.; Mihm, U.; Zeuzem, S. Liver Int. 2009, 29, 23.
4. (a) Beaulieu, P. L. Curr. Opin. Drug Discov. Devel. 2006, 9, 618; (b) Rigat, K.;
Wang, Y.; Hudyma, T. W.; Ding, M.; Zheng, X.; Gentles, R. G.; Beno, B. R.; Gao,
M.; Roberts, S. B. Antiviral Res. 2010, 88, 197.
5. (a) Goulet, S.; Poupart, M.-A.; Gillard, J.; Poirier, M.; Kukolj, G.; Beaulieu, P. L.
Bioorg. Med. Chem. Lett. 2010, 20, 196; (b) Beaulieu, P. L.; Dansereau, N.; Duan,
J.; Garneau, M.; Gillard, J.; McKercher, G.; LaPlante, S.; Lagacé, L.; Thauvette, L.;
Kukolj, G. Bioorg. Med. Chem. Lett. 2010, 20, 1825.
6. LaPlante, S. R.; Gillard, J. R.; Jakalian, A.; Aubry, N.; Coulombe, R.; Brochu, C.;
Tsantrizos, Y. S.; Poirier, M.; Kukolj, G.; Beaulieu, P. L. J. Am. Chem. Soc. 2010,
132, 15204.
19. Male Sprague–Dawley rats were fasted overnight and dosed by oral gavage at
10 mg/kg using 0.5% methocel and 0.3% Tween-80 as vehicle. Plasma samples
from three animals were pooled at each time points (0–8 h) for analysis.
Compound detection in plasma samples was performed following liquid–liquid
extraction and HPLC analysis using UV detection.