F. Ruebsam et al. / Bioorg. Med. Chem. Lett. 19 (2009) 451–458
457
these assessments will be reported in the future. Importantly, the
enantiopure inhibitor 4ad showed a good combination of replicon
potency and in vitro/in vivo DMPK properties and exhibited plasma
levels in monkeys that significantly exceeded its replicon (1b) EC50
value for at least 12 h following oral dosing (Fig. 3). The other com-
pounds described in Table 4 did not exhibit similarly high C12h/EC50
values, either due to poorer PK properties or weaker antiviral activ-
ity (or both).
100000
10000
1000
100
In summary, we describe a novel series of non-nucleoside
inhibitors of genotype 1b HCV NS5B polymerase (4) that incorpo-
rate an aliphatic, fused 5,6-dihydro-1H-pyridin-2-one moiety.
Extensive SAR studies identified a number of very potent com-
pounds in both biochemical and replicon assays. This work led to
the discovery of the promising inhibitor 4ad which exhibited po-
tent antiviral activity and significantly improved oral bioavailabil-
ity compared to our previously reported NS5B inhibitors. Our
ongoing efforts to further improve the PK properties of the benzo-
thiadiazine-containing NS5B inhibitors will be reported in a future
communication.
10
EC50 (1b) = 8.5 ng/mL
1
0
6
12
18
24
Time (h)
Figure 3. Plasma concentrations of compound 4ad in cynomolgus monkeys at
various times after iv (solid symbols) and po (open symbols) administration of a
single 1 mg/kg dose.
meta-positions (4al) of the R1 benzyl group resulted in significant
loss of activity. Compound 4am, bearing a smaller 3-methylbenzyl
R1 substituent, displayed improved activities compared to 4al.
Potencies in the enzymatic and replicon assays were also restored
by introducing 3,4-disubstituted benzyl groups containing a fluo-
rine atom in the 4-position as illustrated by compounds 4an–4ap.
Table 4 details the in vitro and in vivo DMPK parameters for
selected 5-membered (4ad, 4ae, 4p, 4h, and 4ao), 6-membered
(4af, 4ag, 4ab, and 4i), and 7-membered (4f) compounds.21 All
pyridinones in Table 4 exhibited good solubility in the biochemical
Acknowledgement
The authors thank Drs. Devron Averett and Steve Worland for
their support and helpful discussions during the course of this
work.
References and notes
1. Choo, Q. L.; Kuo, G.; Weiner, A. J.; Overby, L. R.; Bradley, D. W.; Houghton, M.
Science 1989, 244, 359.
assay (>100 lM) and generally displayed low to moderate in vivo
clearance. Most compounds bearing aliphatic R1 moieties (4p, 4h,
4ab, and 4i) displayed low stability toward monkey liver micro-
somes (MLM), while all inhibitors containing benzylic R1 substitu-
ents (4ad, 4ae, 4ao, 4af, 4ag, and 4f) were stable in such
assessments. Generally, MLM stability correlated loosely with the
corresponding in vivo clearance data suggesting that the clearance
of less stable compounds bearing aliphatic R1 substituents is likely
to be mediated via oxidative biotransformation. Encouragingly,
Caco-2 data indicated improved permeability for the new inhibi-
tors described in Table 4 relative to the previously studied com-
pounds 2 and 3. The bioavailabilities and AUCs of compounds 4
observed following oral dosing were also improved relative to
those exhibited by compounds 2 and 3, suggesting that the perme-
ability gains, when combined with good microsomal stability
(MLM t1/2 > 60 min), translated into increased in vivo exposures.
Interestingly, comparison of enantiomers 4af and 4ag revealed that
these compounds showed distinct in vivo DMPK parameters, with
the (4aR,7aS)-isomer 4af being superior. This difference was even
more pronounced in the 5-membered enantiomeric pair 4ad and
4ae, again with the (4aR,7aS)-isomer (4ad) favorably standing
out. However, these differences in the in vivo parameters were
not easily predicted from the corresponding in vitro DMPK data
(MLM, Caco-2), possibly suggesting differential recognition of each
enantiomer pair by biological systems.22 Thus, while we originally
envisioned that the lower PSA values associated with molecules 4
containing the 5,6-dihydro-1H-pyridin-2-one motif would afford
improved permeabilities relative to inhibitors such as 2 and 3,
we currently suspect that the superior in vivo performance of the
former compounds may partially result from more favorable inter-
actions with one or more biological systems.23 We hypothesize
that the incorporation of two sp3-hybridized centers into the de-
sign of 4 significantly altered the shape of the resulting molecules
relative to those we previously studied and thereby contributed to
the in vivo DMPK improvements. Additional experiments are
underway to better characterize the pharmacokinetic properties
of the dihydropyridinone-containing molecules, and results from
2. Shepard, C. W.; Finelle, L.; Alter, M. J. Lancet Infect. Dis. 2005, 5, 558.
4. (a) Sidwell, R. W.; Huffman, J. H.; Khare, G. P.; Allen, L. B.; Witkowski, J. T.;
Robins, R. K. Science 1972, 177, 705; (b) Smith, R. A.; Kirkpatrick, W.. In Ribavirin
a Broad Spectrum Antiviral Agent; Academic Press: New York, 1980; Vol. 3, p
237; (c) De Clercq, E. Adv. Virus Res. 1994, 42, 1.
5. (a) Hayashi, N.; Takehara, T. J. Gastroenterol. 2006, 41, 17; (b) Hoofnagle, J. H.;
Seeff, L. B. N. Engl. J. Med. 2007, 355, 2444.
6. Kolykhalov, A. A.; Agapov, E. V.; Blight, K. J.; Mihalik, K.; Feinstone, S. M.; Rice,
C. M. Science 1997, 277, 570.
7. Koch, U.; Narjes, F. Curr. Top. Med. Chem. 2007, 7, 1302.
8. (a) Slater, M. J.; Amphlett, E. M.; Andrews, D. M.; Bravi, G.; Burton, G.; Cheasty,
A. G.; Corfield, J. A.; Ellis, M. R.; Fenwick, R. H.; Fernandes, S.; Guidetti, R.; Haigh,
D.; Hartley, C. D.; Howes, P. D.; Jackson, D. L.; Jarvest, R. L.; Lovegrove, V. L. H.;
Medhurst, K. J.; Parry, N. R.; Price, H.; Shah, P.; Singh, O. M. P.; Stocker, R.;
Thommes, P.; Wilkinson, C.; Wonacott, A. J. Med. Chem. 2007, 50, 897; (b)
Dhanak, D.; Duffy, K. J.; Johnston, V. K.; Lin-Goerke, J.; Darcy, M.; Shaw, A. N.;
Gu, B.; Silverman, 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.; Macarron, 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) Evans, K. A.;
Chai, D.; Graybill, T. L.; Burton, G.; Sarisky, R. T.; Lin-Goerke, J.; Johnston, V. K.;
Rivero, R. A. Bioorg. Med. Chem. Lett. 2006, 16, 2205; (d) Blake, J. F.; Fell, J. B.;
Fischer, J. P.; Hendricks, R. T.; Spencer, S. R.; Stengel, P. J. WO2006117306,
2006.; (e) Pratt, J. K.; Donner, P.; McDaniel, K. F.; Maring, C. J.; Kati, W. M.; Mo,
H.; Middleton, T.; Liu, Y.; Ng, T.; Xie, Q.; Zhang, R.; Montgomery, D.; Molla, A.;
Kempf, D. J.; Kohlbrenner, W. Bioorg. Med. Chem. Lett. 2005, 15, 1577; (f)
Hutchinson, D. K., et al. U.S. Patent US2005107364, 2005.; (g) Bosse, T. D.;
Larson, D. P.; Wagner, R.; Hutchinson, D. K.; Rockway, T. W.; Kati, W. M.; Liu, Y.;
Masse, S.; Middleton, T.; Mo, H.; Montgomery, D.; Jiang, W.; Koev, G.; Kempf, D.
J.; Molla, A. Bioorg. Med. Chem. Lett. 2008, 18, 568.
9. (a) Zhou, Y.; Webber, S. E.; Murphy, D. E.; Li, L.-S.; Dragovich, P. S.; Tran, C. V.;
Sun, Z.; Ruebsam, F.; Shah, A.; Tsan, M.; Showalter, R.; Patel, R.; Li, B.; Zhao, Q.;
Han, Q.; Hermann, T.; Kissinger, C.; LeBrun, L.; Sergeeva, M. V.; Kirkovsky, L.
Bioorg. Med. Chem. Lett. 2008, 18, 1413; (b) Zhou, Y.; Li, L.-S.; Dragovich, P. S.;
Murphy, D. E.; Tran, C. V.; Ruebsam, F.; Webber, S. E.; Shah, A.; Tsan, M.; Averill,
A.; Showalter, R.; Patel, R.; Han, Q.; Zhao, Q.; Hermann, T.; Kissinger, C.; LeBrun,
L.; Sergeeva, M. V. Bioorg. Med. Chem. Lett. 2008, 18, 1419; (c) Li, L.-S.; Zhou, Y.;
Murphy, D. E.; Stankovic, N.; Zhao, J.; Dragovich, P. S.; Bertolini, T.; Sun, Z.;
Ayida, B.; Tran, C. V.; Ruebsam, F.; Webber, S. E.; Shah, A. M.; Tsan, M.;
Showalter, R. E.; Patel, R.; LeBrun, L. A.; Bartkowski, D. M.; Nolan, T. G.; Norris,
D. A.; Kamran, R.; Brooks, J.; Sergeeva, M. V.; Kirkovsky, L.; Zhao, Q.; Kissinger,
C. R. Bioorg. Med. Chem. Lett. 2008, 18, 3446; (d) Sergeeva, M. V.; Zhou, Y.;
Bartkowski, D. M.; Nolan, T. G.; Norris, D. A.; Okamoto, E.; Kirkovsky, L.;
Kamran, R.; LeBrun, L. A.; Tsan, M.; Patel, R.; Shah, A. M.; Lardy, M.; Gobbi, A.; Li,
L.-S.; Zhao, J.; Bertolini, T.; Stankovic, N.; Sun, Z.; Murphy, D. E.; Webber, S. E.;
Dragovich, P. S. Bioorg. Med. Chem. Lett. 2008, 18, 3421.