304
D. V. Kumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 300–304
compounds are an improvement on a previous series of quinolones
through the replacement of an ester with either a 1,2,4- or a 1,3,4-
oxadiazole. The most potent compounds in this series, 16 and 17,
have replicon EC50s below 250 nM. The continued optimization
of this series will be described in forthcoming papers.
Acknowledgement
The authors thank Dr. Robert Booth (Virobay Inc.) for support of
the research that went into this paper.
References and notes
1. Simmonds, P. J. Gen. Virol. 2004, 85, 3173.
2. Younossi, Z.; Kallman, J.; Kincaid, J. Hepatology 2007, 45, 806.
3. (a) Poordad, F.; McCone, J.; Bacon, B. R.; Bruno, S.; Manns, M. P.; Sulkowski, M.
S.; Jacobson, I. M.; Reddy, K. R.; Goodman, Z. D.; Boparai, N.; DiNubile, M. J.;
Sniukiene, V.; Brass, C. A.; Albrecht, J. K.; Bronowicki, J. P. N. SPRINT-2
Investigators Engl. J. Med. 2011, 364, 1195; (b) Jacobson, I. M.; McHutchison, J.
G.; Dusheiko, G.; Di Bisceglie, A. M.; Reddy, K. R.; Bzowej, N. H.; Marcellin, P.;
Muir, A. J.; Ferenci, P.; Flisiak, R.; George, J.; Rizzetto, M.; Shouval, D.; Sola, R.;
Terg, R. A.; Yoshida, E. M.; Adda, N.; Bengtsson, L.; Sankoh, A. J.; Kieffer, T. L.;
George, S.; Kauffman, R. S.; Zeuzem, S. N. ADVANCE Study Team Engl. J. Med.
2011, 364, 2405.
4. (a) Kumar, D. V.; Rai, R.; Brameld, K. A.; Somoza, J. R.; Rajagopalan, R.; Janc, J.
W.; Xia, Y. M.; Ton, T. L.; Shaghafi, M. B.; Hu, H.; Lehoux, I.; To, N.; Young, W. B.;
Green, M. J. Bioorg Med Chem Lett 2011, 21, 82; (b) While protein binding and
solubility of the lead ester series was not evaluated, the improvement in
replicon potency with the addition of polar moieties at the C7 position is
attributed to reduced protein binding properties.
5. (a) Saunders, J.; Angus, M.; MacLeod, K. M.; Showell, G. A.; Snow, R. J.; Street, L.
J.; Baker, R. J. Chem. Soc., Chem. Commun. 1988, 1618; (b) Kuduk, S. D.; Chang, R.
K.; Ng, C.; Murphy, K. L.; Ransom, R. W.; Tang, C.; Prueksaritanont, T.;
Freidinger, R. M.; Pettibone, D. J.; Bock, M. G. Bioorg. Med. Chem. Lett. 2005, 15,
3925.
6. Smith, D. A.; Waterbeemd, H.; Walker, D. K. In Pharmacokinetics and Metabolism
in Drug Design; Mannhold, R., Kubinyi, H., Folkers, G., Eds.; Wiliey-VCH:
Weinheim, Germany, 2006. second Ed., Vol. 31.
7. Kumar, D. V.; Rai, R.; Young, W. B.; Hu, H, Riggs, J. R.; Ton, T. L.; Green, M. J.;
Hart, B. P.; Brameld, K. A.; Dener, J. M. US 2007/0287699 and WO 2007/130499.
8. The NS5B polymerase IC50 enzyme activity assay determination: As described
in: McKercher, G.; Beaulieu, P. L.; Lamarre, D.; LaPlante, S.; Lefebvre, S.; Pellerin,
C.; Thauvette, L.; Kukolj, G. Nuc. Acids Res. 2004, 32, 422. Scintillation
proximity assays (SPA) were performed at ambient temperature (22 °C) in
96-well plates using 50 nM of enzyme (a C-terminal 21 residue deletion
mutant of NS5b from genotype 1b BK isolate). Reaction components included
20 mM Tris–HCl pH 7.5, 5 mM MgCl2, 1 mM EDTA, 2 mM DTT, 5% DMSO, 0.01%
Figure 3. (a) X-ray structure of 16 bound to NNI-2 site (PDB ID code: 3UDL). (b)
Overlay of compounds 16 (yellow) and 1 (white), showing a high degree of overlap
between the two ligands and very similar protein conformations. Small differences
in Leu 419 and Ile 482 are highlighted.
BSA and 25 mM KCl. 80
0.5 Ci of [3H]UTP, 1
M UTP, 250 nM 50-biotinylated oligo(rU12) and 0.8
ml poly(rA) to assay wells containing enzyme preincubated with inhibitor.
Reactions were terminated after 120 min by the addition of 20 l of stop
solution containing 150 g/ml tRNA and 10 mg/ml streptavidin-coated SPA
beads (Amersham) in 20 mM Tris–HCl pH 7.5, 25 mM KCl, 0.5 M EDTA and
0.025% sodium azide. After incubation for 30 min, 65 l of 5 M cesium chloride
l
l reactions were initiated by adding a mixture of
l
l
l
g/
l
l
l
is difficult to assess the validity of either of these hypotheses.
However, this observation suggests that the interactions made by
the aromatic portion of the benzyl substituent in this region of
the binding pocket are weak, non-specific, and easily influenced
by subtle chemical changes.
Scheme 1 describes the conversion of a C3 carboxylic acid to
various ester isosteric heterocycles. Activation of the acid as the
acid chloride affords an intermediate that reacts with a hydroxami-
dine, 1,2-amino alcohol or hydrazide to afford a 1,2,4-oxadiazole
(3, 6), 1,3-oxazoline (4), and a 1,3,4 oxadiazole respectively (7, 8,
12, 19). Scheme 2 describes the conversion of the C3 quinolone
nitrile to the C3 oxazole and tetrazole derivatives. Reaction of the
nitrile with TMSN3 affords the intermediate tetrazole that is benzy-
lated to afford compound 9.11 Oxazole 8 is obtained by reacting the
nitrile with 1-diazo-3-phenylpropan-2-one.
were added to the wells and further incubated for 1 h before top-counting in a
Microbeta Trilux plate reader.
9. The cell-based Replicon EC50 determination: HCV replicon-containing cells
(Huh7/Clone A, genotype 1b) were maintained in growth medium (DMEM
medium, Invitrogen), supplemented with 10% Fetal Bovine Serum, non
essential amino acids and 1 mg/mL G418) (Blight K. J., Kolykhalov A. A. and
Rice C. M. (2000) Science 290, 1972–1974). For the HCV replicon assay, Huh7/
Clone A cells were trypsinized from culture flasks, seeded in 1 ml of Clone A
growth medium without G418 at 40,000 cells per well in 24-well plates and
incubated at 37 °C in a humidified CO2 (5%) incubator overnight. Following
overnight incubation, test compound was serially diluted in DMSO and added
to the test system such that the final concentration of DMSO was 0.5% in each
well. For IC50 determinations, compounds were tested at 7 concentrations in
triplicates. Plates were incubated at 37 °C for 48 h. After incubation, cells were
harvested, transferred to 96-well plates, and subjected to total RNA extraction
using the RNA Isolation Kit (RNeasy 96, Qiagen). TaqMan quantitative PCR (RT-
qPCR) was used to quantify the amount of HCV replicon RNA in each sample.
The samples without compound treatment served as a control and the HCV
replicon RNA level from untreated cells was defined as 100%. Compound
inhibitory activity was determined as the ratio of the normalized HCV RNA
amount in treated samples relative to the untreated control. Compound EC50’s
were calculated using a standard 4 parameter curve fit model. The cytotoxicity
of compounds, the GI50,was assayed in parallel in which qPCR of the cellular
host protein GAPDH was used as an indicator of viable cells.
Scheme 3 describes the conversion of quinolone C3 nitriles to
the intermediate hydroxyamidine through reaction with hydroxyl
amine. The resulting hydroxyamidine intermediates are converted
to a variety of 1,2,4-oxadiazole target compounds by reacting with
appropriate carboxylic acids.
10. In general, the 7-position piperazine substituted compounds had low stability
in human liver microsomes with 20–50% remaining after a 1 h incubation.
In conclusion, we have described a new series of allosteric-site
(NNI-2) inhibitors of the HCV NS5B polymerase enzyme. These