4132
A. Cho et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4127–4132
19. Olsen, D. B.; Eldrup, A. B.; Bartholomew, L.; Bhat, B.; Bosserman, M. R.; Ceccacci,
A.; Colwell, L. F.; Fay, J. F.; Flores, O. A.; Getty, K. L.; Grobler, J. A.; LaFemina, R.
L.; Markel, E. J.; Migliaccio, G.; Prhavc, M.; Stahlhut, M. W.; Tomassini, J. E.;
MacCoss, M.; Hazuda, D. J.; Carroll, S. S. Antimicrob. Agents Chemother. 2004, 48,
3944.
20. Carroll, S. S.; Ludmerer, S.; Handt, L.; Koeplinger, K.; Zhnag, N. R.; Graham, D.;
Davies, M.-E.; MacCoss, M.; Hazuda, D.; Olsen, D. B. Antimicrob. Agents
Chemother. 2009, 53, 926.
corresponding N-nucleoside (Fig. 4). Consistent with the high oral
bioavailability observed in dogs for 2, high nucleoside levels were
observed in hamster plasma and, relative to a separate intravenous
study, the oral bioavailability in rats was 50% (data not shown).
In summary, characterization of a series of 20-C-Me C-nucleo-
sides has identified nucleoside analogs with potent anti-HCV activ-
ity. While the pyrimidine C-nucleosides (4 and 5) lacked replicon
activity due to poor enzymatic potency and, despite excellent
activity as its triphosphate, the guanosine C-nucleoside (3) and
its nucleotide prodrug (26) were inefficiently metabolized, adeno-
sine C-nucleosides (1 and 2) were found to have selective replicon
activity and potent inhibition of NS5B as their triphosphates. Fur-
thermore, 2 was found to have a favorable pharmacokinetic profile
and in vivo potential for enhanced potency over the corresponding
N-nucleoside 20-C-Me 7-deaza A. These results have prompted the
subsequent further characterization of the toxicological profile of
2, which will be published in due course. C-Nucleosides may hold
promise as potential future therapies for HCV infection.
21. Nucleosides (10 lM) were incubated at 37 °C with 0.0125 U/mL bovine
adenosine deaminase (Sigma A5168) in 0.01 M potassium phosphate buffer
pH 6.0 and samples collected over a 1 hour time course. The reactions were
stopped by adding 2.5 volumes of 0.2% formic acid in ice cold methanol.
Nucleoside levels were then measured using liquid chromatography (0.2%
formic acid in water and an acetonitrile gradient) coupled to triple quadrapole
mass spectrometry running in positive ion multiple reaction monitoring mode.
22. Groups of 3 non-naïve male beagle dogs were administered a single dose of 2
formulated as
a
solution in 5/5/5/85 ethanol/polypropylene glycol/
polyethylene glycol/aqueous 50 mM citrate buffer (pH 3.3) either by
intravenous infusion at 0.5 mg/kg or orally at 1 mg/kg. Blood samples were
collected over a 24 h period postdose into VacutainerTM tubes containing
EDTA-K3 (BD Biosciences) and plasma was isolated by the manufacturers
suggested protocol. Plasma samples were processed via protein precipitation
by adding acetonitrile to a final concentration of 60%. Following filtration to
remove precipitated proteins, samples were dried and reconstituted in 20%
acetonitrile in water. Samples were then analyzed by reversed phase liquid
chromatography coupled to triple quadrapole mass spectrometry (LC/MS/MS)
in positive ion and multiple reaction monitoring mode.
References and notes
23. Replicon cells were maintained in Dulbecco0s modified eagle medium
containing glutamax supplemented with 10% heat inactivated fetal bovine
serum, penicillin–streptomycin, and G418 disulphate salt solution. Cells were
transferred to 12-well tissue culture treated plates by trypsonization and
grown to confluency (0.88 ꢂ 106 cells/well). Cells were treated for 24 h with a
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A. B.; Bhat, B.; Hall, D.; Simcoe, A. L.; LaFemina, R.; Rutkowski, C. A.; Wolanski,
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Bartholomew, L.; Bosserman, M. R.; Ceccacci, A.; Colwell, L. F.; Cortese, R.;
Francesco, R. D.; Eldrup, A. B.; Getty, K. L.; Hou, Z. S.; LaFemina, R. L.; Ludmerer,
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test compound (10 lM). Following 24 hours, cells were washed twice with
2.0 mL ice cold 0.9% sodium chloride saline. Cells were then scraped into
0.5 mL 70% methanol and frozen overnight to facilitate the extraction of
nucleotide metabolites. Extracted cell material in 70% methanol was
transferred into tubes and dried. After drying, samples were re-suspended in
1 mM ammonium phosphate pH 8.5. TP levels were quantified using liquid
chromatography coupled to triple quadrapole mass spectrometry by methods
similar to those previously reported in Durand-Gasselin, L.; Van Rompay, K.K.;
Vela, J.E.; Henne, I.N.; Lee, W.A.; Rhodes, G.R. Mol. Pharm. 2009, 6, 1145.
24. Primary human hepatocytes were maintained in Williams
containing Cellz Direct0s proprietary supplement cocktail. Cells were
purchased from Cellz Direct as twelve well plate format grown to
confluency (0.88 ꢂ 106 cells/well). Cells were treated for 1 h with test
E medium
6. Butora, G.; Olsen, D. B.; Carroll, S. S.; McMasters, D. R.; Schmitt, C.; Leone, J. F.;
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a
a
compound (10 M) followed by a 23 h treatment with non-drug containing
media. Cells were collected at 0, 0.5, and 1 h during compound incubation and
at 0, 0.5, 1, 3, 6, 8 and 23 h following compound removal. Cells were collected
and intracellular metabolites were analyzed as described for replicon cells.
25. Groups of 12 Golden Syrian hamsters or Sprague–Dawley rats were
administered nucleosides formulated in water at respective doses. At 1, 4, 8
and 12 h, livers were harvested under isoflurane anesthesia. Collected livers
were wrapped in aluminum foil and snap-frozen in liquid nitrogen
immediately following removal to avoid sample dephosphorylation. Livers
were processed by sectioning into smaller pieces with a razor blade and
collecting into pre-weighed 15 mL conical tubes kept on dry ice. Four volumes
of ice-cold extraction buffer (0.1% KOH and 67 mM EDTA in 70% MeOH,
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containing 0.25 lM Cl-ATP) were added and samples were promptly
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homogenized using an Omni-Tip THTM with disposable, hard tissue
homogenizer probes (Omni International). Aliquots of homogenate were then
centrifuged (20,000ꢂg at 4 °C for 10 min). Aliquots of supernatant were
transferred to clean tubes, evaporated to dryness in a heated centrifugal
evaporator, and reconstituted with an equal volume of 1 mM ammonium
phosphate (pH 7.0). Intracellular triphosphate levels were measured by LC/MS/
MS as described for samples generated in vitro. Levels of endogenous
adenosine triphosphate were also determined to assure the sample stability.
Nucleoside levels in rat plasma were determined by the same methods
described for dogs.
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