124
P. L. Beaulieu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 119–124
Although the moderate potency of these compounds
and their physicochemical properties may be limiting
their efficacy in a cell-based HCV RNA replication
assay, they do nevertheless, represent an attractive
starting point in our search for more potent molecules
and HCV therapeutics.
7. Kolykhalov, A. A.; Mihalik, K.; Feinstone, S. M.; Rice,
C. M. J. Virol. 2000, 74, 2046.
8
. (a) Behrens, S.-E.; Tomei, L.; De Francesco, R. EMBO J.
996, 15, 12. (b) Lohmann, V.; Korner, F.; Herian, H.;
Bartenschlager, R. J. Virol. 1997, 71, 8416.
1
¨
9
. Full length HCV NS5B was purified as a N-terminal
hexa-histidine fusion protein from baculovirus infected
insect cells. Compounds were tested for inhibitory activity
using a 12 nucleotide RNA oligo-uridylate primer mod-
Acknowledgements
0
ified with biotin at the free 5 C position and a com-
plementary poly-adenylate template of heterogeneous
length (1000–10,000 nucleotides). Polymerase activity was
We wish to acknowledge the contribution of the Boeh-
ringer Ingelheim Pharmaceuticals Inc. (Ridgefield, CT,
USA) screening team, and in particular Drs. Carol
Homon and Paul Kaplita, Gustavo Rodriguez and
George Rogers. The authors also thank Lukxmi Bala-
thasan and Leslie Magtanong for their support running
the polymerase enzymatic assays. We are grateful to
Norman Aubry, Colette Boucher, Sylvain Bordeleau,
Michael Little and Serge Valois for analytical support.
3
measured as incorporation of UMP-[5,6 H] into the
chain elongated from the oligo-U primer using SPA-bead
1
1a
capture and quantification (TopCount).
A more
detailed description of this assay will be published else
where (McKercher, G. et al.).
1
0. Compounds were tested against polio virus RNA-depen-
dent RNA polymerase and a mammalian DNA depen-
dent RNA polymerase II isolated from calf thymus in an
assay format similar to that described for HCV poly-
merase.
1. (a) Austel, V., Beaulieu, P. L., Fazal, G., Gillard, J.,
Kukolj, G. US patents 6,448,281 B1 and 6,479,508 B1,
9
,11a
1
References and notes
2
002, to Boehringer Ingelheim (Canada) Ltd. (b) A recent
patent application from Japan Tobacco Inc. also discloses
derivatives of benzimidazole 5-carboxylic acids as HCV
polymerase inhibitors: Hashimoto, H., Mizutani, K.,
Yishida, A. EP 1 162 196 A1, 2001.
1
. Choo, Q.-L.; Kuo, G.; Weiner, A. J.; Overby, L. R.;
Bradley, D. W.; Houghton, M. Science 1989, 244, 359.
. Wasley, A.; Alter, M. J. Semin. Liver Dis. 2000, 20, 1.
. Berenger, M.; Wright, T. L. Proc. Assoc. Am. Phys. 1998,
2
3
12. The buffer used for NMR experiments consisted of 20
mM Tris-d11 at pH 7.5, 10% glycerol-d , 2 mM DTT-d ,
1
10, 98.
. Di Bisceglie, A. M.; Hoofnagle, J. H. Hepatology 2002,
6 (Suppl. 1), S121.
. (a) Beaulieu, P. L.; Llina
8
10
4
5
1 mM EDTA-d , 300 mM NaCl, 15 mL DMSO-d and
1
2
6
3
2
10% D O (v/v). Data acquisition was performed at
`
s-Brunet, M. Curr. Med. Chem.
600 MHz and 300 K using methods similar to those
reported previously: LaPlante, S. R.; Cameron, D. R.;
Aubry, N.; LeFebvre, S.; Kukolj, G.; Maurice, R.; Thi-
beault, D.; Lamarre, D.; Llinas-Brunet, M. J. Biol. Chem.
1999, 274, 18618. A soluble form of the polymerase,
NS5BÁ21, lacking C-terminal 21 amino acids was used:
Ferrari, E.; Wright-Minogue, J.; Fang, J. W. S.; Baroudy,
B. M.; Lau, J. Y. N.; Hong, Z. J. Virol. 1999, 73, 1649.
Anti-Infective Agents 2002, 1, 163. (b) For recent dis-
closures of other non-nucleoside HCV NS5B polymerase
inhibitors, see: Dhanak, D.; Duffy, K. J.; Johnston, V. K.;
Lin-Goerke, J.; Darcy, M.; Shaw, A. N.; Gu, B.; Silver-
man, C.; Gates, A. T.; Nonnemacher, M. R.; Earnshaw,
D. L.; Casper, D. J.; Kaura, A.; Baker, A.; Greenwood,
C.; Gutshall, L. L.; Maley, D.; DelVecchio, A.; Macar-
ron, R.; Hofmann, G. A.; Alnoah, Z.; Cheng, H.-Y.;
Chan, G.; Khandekar, S.; Keenan, R. M.; Sarisky, R. T.
J. Biol. Chem. 2002, 277, 38322. (c) Chan, L.; Reddy, T. J.;
Proulx, M.; Das, S. K.; Pereira, O.; Wang, W.; Siddiqui,
A.; Yannopoulos, C. G.; Poisson, C.; Turcotte, N.;
Drouin, A.; Alaoui-Ismaili, M. H.; Bethell, R.; Hamel,
M.; L’Heureux, L.; Bilimoria, D.; Nguyen-Ba, N. J. Med.
Chem. 2003, 46, 1283.
¨
13. Lohmann, V.; Korner, F.; Koch, J. O.; Herian, U.;
Theilmann, L.; Bartenschlager, R. Science 1999, 285, 110.
14. (a) Grimmet, M. R.; In Comprehensive Heterocyclic
Chemistry; Katritzky, A. R., Rees, C. W., Eds.; Perga-
mon: Oxford, 1984; Vol. 5, p 457. (b) Mayer, J. P.; Lewis,
G. S.; McGee, C.; Bankaitis-Davis, D. Tetrahedron Lett.
1
1998, 39, 6655. (c) For the use of oxone in the synthesis
of benzimidazoles from aldehydes and 1,2-phenylenedia-
6
. Reed, K. E.; Rice, C. M. Curr. Top. Microbiol. Immunol.
´
mines see: Beaulieu, P. L.; Hache, B.; von Moos, E.
Synthesis 2003, 1683.
2000, 242, 55.