A. Chris Krueger et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2212–2215
2215
Table 3
Cell culture replicon potency of analogs 54–61
HN
HN
and
S
S
N
N
N
N
R1
N
N
N
OH
RO
N
N
OH
R2
55 - 57
58 - 61
Compd
R
R1
R2
Replicon 1a EC50
(lM)
Replicon 1b EC50
(lM)
MTT (lM)
54
55
56
57
58
59
60
61
see Scheme 3
4.8
>8
7.3
5.3
14.1
22.0
47.6
>50
0.01
>8
12.0
3.5
7.3
2.8
46.9
>8
Methyl
Ethyl
Phenyl
—
—
—
—
—
—
—
—
—
29.7
17.1
>50
>50
>50
>50
Morpholine
Piperidine
Ethyl
H
30.0
>50
—
Methyl
Methyl
acid to provide the ethylsulfanyl analog 54. The ethylsulfanyl
functionality could be displaced with other nucleophiles to provide
the corresponding ether 55–57 and amine 58, 59 analogs directly.
Alternately, the acyclic amine analogs 60 and 61 were generated by
first oxidizing the benzyl mercapto group in 52 to the correspond-
ing benzyl sulfone followed by displacement of this group with the
corresponding amines. Amidine formation and cyclization pro-
vided the desired amine analogs 60 and 61. The ethylsulfanyl-
pyrimidopyrimidine analog 54 displayed excellent 1b replicon
potency (EC50 10 nM), however it was much weaker against the
assessed for inhibitory potency in genotypes 1a and 1b HCV
replicon assays. Overall we observed that the SAR was very sensi-
tive to seemingly small structural modifications and that the most
potent analog displayed submicromolar potency in one cell culture
assay containing 40% human serum.
References and notes
1. Choo, Q. L.; Kuo, G.; Weiner, A. J.; Overby, L. R.; Bradley, D. W.; Houghton, M.
Science 1989, 244, 359.
2. (a) Alter, M. J.; Kruszon-Moran, D.; Nainan, O. V.; McQuillan, G. M.; Gao, F.;
Moyer, L. A.; Kaslow, R. A.; Margolis, H. S. N. Engl. J. Med. 1999, 341, 556; (b)
Hepatology, 1997, 26, pp. 2S–10S.
1a replicon (EC50 4.8 lM, Table 3). Unfortunately the EC50s of all
of the ether 55–57 and amine 58–61 analogs were greater than 2
micromolar in both assays.
3. Simmonds, P. J. Hepatol. 1999, 31, 54.
4. Rice, C. Nature 2011, 474, S8.
The most potent analog, iso-propyl pyrido[2,3-d]pyrimidine 33,
was retested in the replicon assay in the presence of 40% human
serum to access how much potency loss would occur due to unpro-
ductive protein binding. The potency loss was 10-fold in the 1b
replicon assay (16 to 170 nm). We also evaluated the pharmacoki-
netic properties of analog 33 after i.v. and oral administration in
rats (2.5 mg/kg as a solution). Compound 33 displayed high
clearance (1.58 L/h/kg) and a relatively short half-life (3.1 h). Cmax
5. (a) Randolph, J. T.; Zhang, X.; Huang, P. P.; Klein, L. L.; Kurtz, K. A.;
Konstantinidis, A. K.; He, W.; Kati, W. M.; Kempf, D. J. Bioorg. Med. Chem. Lett.
2008, 18, 2745; (b) Krueger, A. C.; Madigan, D. L.; Green, B. E.; Hutchinson, D.
K.; Jiang, W. W.; Kati, W. M.; Liu, Y.; Maring, C. J.; Masse, S. V.; McDaniel, K. F.;
Middleton, T. R.; Mo, H.; Molla, A.; Montgomery, D. A.; Ng, T. I.; Kempf, D. J.
Bioorg. Med. Chem. Lett. 2007, 17, 2289.
6. Ikeda, M.; Yi, M.; Li, K.; Lemon, S. M. J. Virol. 2002, 76, 2997; (b) Blight, K. J.;
Kolykhalov, A.; Rice, C. M. Science 2000, 290, 1972.
7. Betebenner, D. A.; DeGoey, D. A.; Maring, C. L.; Krueger, A. C.; Iwasaki, N.;
Rockway, T. W.; Cooper, C. S.; Anderson, D. D.; Donner, P. L.; Green, B. E.;
Kempf, D. J.; Liu, D.; McDaniel, K. F.; Madigan, D. L.; Motter, C. E.; Pratt, J. K.;
Shanley, J. P.; Tufano, M. D.; Wagner, R.; Zhang, R.; Molla, A.; Mo, H.; Pilot-
Matias, T. J.; Masse, S. VL.; Carrick, R. J.; He, W.; Lu, L.; Grampovnik, D. J. Patent
Application WO 2007/081517, 19 July 2007.
8. Tsou, H-R.; Mamuya, N.; Johnson, B. D.; Reich, M. F.; Gruber, B. C.; Ye, F.;
Nilakantan, R.; Shen, R.; Discafani, C.; DeBlanc, R.; Davis, R.; Koehn, F. E.;
Greenberger, L. M.; Wang, Y-F.; Wissner, A. J. Med. Chem 2001, 44, 2719.
9. Krishnan, P.; Pilot-Matias, T. J. unpublished results.
(0.07 lM), plasma AUC (1.58 lg h/mL) and bioavailability (10%)
after oral administration were low. In a future publication we will
disclose our efforts related to improving upon this pharmacoki-
netic profile. Finally, based on resistance studies which will be
published in the future,9 we believe that the target of these HCV
replicon inhibitors is the HCV NS5A protein which has been shown
to be critical for viral replication.10
10. Nettles, R. E.; Gao, M.; Bifano, M.; Chung, E.; Persson, A.; Marbury, T. C.;
Goldwater, R.; DeMicco, M. P.; Rodriquez-Torres, M.; Vutikullird, A.; Fuentes,
E.; Lawitz, E.; Lopez-Talavera, J. C.; Grasela, D. M. Hepatology 2011, 54, 1956.
In summary, a series of substituted pyrido[2,3-d]pyrimidine
and pyrimido[4,5-d]pyrimidine analogs were synthesized and