P. L. Beaulieu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 857–861
861
6. (a) 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; (b) Beaulieu, P. L.;
Bös, M.; Bousquet, Y.; DeRoy, P.; Fazal, G.; Gauthier, J.; Gillard, J.; Goulet, S.;
McKercher, G.; Poupart, M.-A.; Valois, S.; Kukolj, G. Bioorg. Med. Chem. Lett.
2004, 14, 967; Similar lead structures were also discovered by a Japan Tobacco
Ltd research group: (c) Hirashima, S.; Suzuki, T.; Ishida, T.; Noji, S.; Yata, S.;
Ando, I.; Komatsu, M.; Ikeda, S.; Hashimoto, H. J. Med. Chem. 2006, 49, 4721.
7. (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.
lent metabolic stability (RLM T1/2 = 288 min) and low potential for
CYP-mediated drug–drug interactions. At an oral dose of 4 mg/kg,
plasma concentration at the 1 h time point reached 1
lM. Several
piperazine analogs (50–52, 54) were also identified that provided
promising plasma exposures in the 0.3–0.6
dosing.
lM range, 1 h post-
In conclusion, we described how alkylation of the nitrogen atom
of indole-based allosteric inhibitors of HCV NS5B polymerase with a
variety of acetamide side chains led to the discovery of compounds
with generally good cell culture potency in subgenomic HCV RNA
replicon assays. The SAR patterns suggested that the acetamide sub-
stituent is solvent-exposed and allows for the incorporation of
chemical diversity and the modulation of the molecule’s physico-
chemical properties. Many analogs showed improved aqueous solu-
bility and promising ADME-PK profiles in rats and in human in vitro
assays over previously reported series. Further optimization of this
class of compounds will be the focus of future studies.
8. Beaulieu, P. L.; Bousquet, Y.; Gauthier, J.; Gillard, J.; Marquis, M.; McKercher, G.;
Pellerin, C.; Valois, S.; Kukolj, G. J. Med. Chem. 2004, 47, 6884.
9. (a) The HT-NS5B
D21 polymerase scintillation proximity assay was performed
as described in Ref. 5 with the following modifications: 12 nM of the soluble
truncated enzyme was used but the reaction had 0.01% w/v BSA and lacked the
previously supplemented 0.33% dodecyl-b-
determinations were performed in duplicates (or more) using Huh-7 cells with
stable subgenomic HCV 1b replicon that encodes modified luciferase
D-maltoside, 0.01% IGEPAL. (b) EC50
a
a
reporter gene as previously described in: Tsantrizos, Y. S.; Chabot, C.; Beaulieu,
P. L.; Brochu, C.; Poirier, M.; Stammers, T.; Thavonekham, B.; Rancourt, J. WO
05/080388, 2005. Compounds were devoid of cytotoxicity (TC50 >10 lM) at
Acknowledgments
inhibitory concentrations. A sub-set of inhibitors were also tested in a 72 h
subgenomic replicon assay where HCV RNA levels were normalized to total
cellular RNA. Similar EC50 values were determined in both assays and the
quantification of total RNA recovery allowed for an alternative 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 Lohmann, V. In Methods in Mol. Biol., 2nd ed.; Tang, H., Ed.;
‘Hepatitis C: Methods and Protocols’; Humana Press; 2009, Vol. 510, p 145.
10. (a) Beaulieu, P. L.; Brochu, C.; Chabot, C.; Jolicoeur, E.; Kawai, S.; Poupart, M.-A.;
Tsantrizos, Y. WO 04/065367, 2004.; Similar findings were recently reported by
another group: (b) Harper, S.; Avolio, S.; Pacini, B.; Di Filippo, M.; Altamura, S.;
Tomei, L.; Paonessa, G.; Di Marco, S.; Carfi, A.; Giuliano, C.; Padron, J.; Bonelli,
F.; Migliaccio, G.; De Francesco, R.; Laufer, R.; Rowley, M.; Narjes, F. J. Med.
Chem. 2005, 48, 4547.
11. All final inhibitors were purified by C-18 reversed-phase HPLC to >97%
homogeneity and gave 1H NMR, 13C NMR and MS data consistent with their
assigned structure.
12. Solubility data was acquired with amorphous solids after incubation for 24 h in
pH 7.2 buffer using the shaking flask method.
13. A similar derivative was profiled by the Merck group: Giuliano, C.; Fiore, F.; Di
Marco, A.; Padron Velazquez, J.; Bishop, A.; Bonelli, F.; Gonzalez-Paz, O.;
Marcucci, I.; Harper, S.; Narjes, F.; Pacini, B.; Monteagudo, E.; Migliaccio, G.;
Rowley, M.; Laufer, R. Xenobiotica 2005, 35, 1035.
We thank Serge Valois, Norman Aubry, Colette Boucher and Mi-
chael Little for analytical support and Dr. Ma’an Amad, Josie De
Marte, Martin Jutras Hélène Montpetit, François Otis and Manon
Rhéaume for DMPK profiling.
References and notes
1. Shepard, C. W.; Finelli, L.; Alter, M. J. Lancet Infect. Dis. 2005, 5, 558.
2. Beaulieu, P. L. Expert. Opin. Ther. Patents 2009, 19, 145. and references cited
therein.
3. NM283 (Idenix) is the first NI to enter clinical trial: (a) O’Brien, C.; Godofsky, E.;
Rodriguez-Torres, M.; Afdhal, N.; Pappas, S. C.; Pockros, P.; Lawitz, E.; Bzowej,
N.; Rustgi, V.; Sulkowski, M.; Sherman, K.; Jacobson, I.; Chao, G.; Knox, S.;
Pietropaolo, K.; Brown, N. A. Hepatology 2005, 42, 234A; (b) Brown, N. Expert.
Opin. Invest. Drugs 2009, 18, 709.
4. Available clinical data for HCV-796 (ViroPharma/Wyeth, first disclosure on
clinical efficacy for a NNI) has been reviewed in: Huang, Z.; Murray, M. G.;
Secrist, J. A., III Antiviral Res. 2006, 71, 351.
5. McKercher, G.; Beaulieu, P. L.; Lamarre, D.; LaPlante, S.; Lefebvre, S.; Pellerin, C.;
Thauvette, L.; Kukolj, G. Nucleic Acids Res. 2004, 32, 422.