4320
C. McGuigan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4316–4320
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McBrayer, T. R.; Schinazi, R. F.; Watanabe, K. A.; Otto, M. J.; Furman, P. A.; Stec,
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7. (a) McGuigan, C.; Cahard, D.; Sheeka, H. M.; Le Clercq, E.; Balzarini, J. J. Med.
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contrast the t-butyl ester is only ca. 24% hydrolysed after 13 h, with
an estimated half life of 33 h. Thus the (active) benzyl ester is
processed ca. 700-fold more efficiently to the key amino acyl
metabolite in this assay than is the (poorly active) t-butyl ester.
Secondly, it is notable that the intermediate (24) observed in
Figure 7, corresponding to the ester cleaved ProTide, is not
observed in Figure 9; presumably the ester cleavage is greatly rate
limiting in the case of (23) and aryl loss is much more rapid, so
intermediate (24) never builds up to detectable levels.
In conclusion, we have reported the synthesis of 1 and 2 and a
small series of their corresponding phosphoramidates. The most
potent compounds were the 1-naphthyl derivatives with benzyl
(20), ethyl (21) or methyl (22) ester moieties with 84-fold increase
in activity against HCV compared to the parent nucleoside (2). The
application of phosphoramidate approach to 1 did not significantly
improve the activity against HCV compared to the parent nucleo-
side (1). We also note the valuable predictive power of a P-31
NMR based enzyme metabolic assay for the activity of these Pro-
Tides versus HCV in vitro.
8. Harry-O0kuru, R. E.; Smith, J. M.; Wolfe, M. S. J. Org. Chem. 1997, 62, 1754.
9. Li, N.-S.; Piccirilli, J. A. J. Org. Chem. 2006, 71, 4018.
10. Perrone, P.; Daverio, F.; Valente, R.; Rajyaguru, S.; Martin, J. A.; Lévêque, V.; Le
Pogam, S.; Najera, I.; Klumpp, K.; Smith, D. B.; McGuigan, C. J. Med Chem. 2007,
50, 5463.
11. Perrone, P.; Luoni, G. M.; Kelleher, M. R.; Daverio, F.; Angell, A.; Mulready, S.;
Congiatu, C.; Rajyaguru, S.; Martin, J. A.; Lévêque, V.; Le Pogam, S.; Najera, I.;
Klumpp, K.; Smith, D. B.; McGuigan, C. J. Med Chem. 2007, 50, 1840.
12. Uchiyama, M.; Aso, Y.; Noyori, R.; Hayakawa, Y. J. Org. Chem. 1993, 58, 373.
13. Standard procedure for the synthesis of 20,30-protected phosphoramidates:
tBuMgCl (2.0 mol equiv, 1 M solution in dry THF) and the appropriate
nucleoside (11 or 12, 1.0 mol equiv) were dissolved in dry THF
(31 mol equiv) and stirred for 15 min. Then a 1 M solution of the appropriate
phosphorochloridate (2.0 mol equiv, 13 or 19) in dry THF was added dropwise,
then stirred for 14 h. A saturated solution of NH4Cl was added and the solvent
was removed under reduced pressure to give a yellow solid, which was
purified by column chromatography using CHCl3/MeOH (from 95/5) as eluent.
The appropriate fractions were collected and the solvent was removed under
reduced pressure to give a white solid.
Acknowledgments
14. Standard
procedure
for
the
the
appropriate
deprotection
of
b-20-methyladenosine
phosphoramidates:
20,30-O,O-cyclopentylidene-b-20-
We would like to thank Helen Murphy for secretarial assistance
and Katrien Geerts for excellent technical assistance. This work is
supported by a grant of the Flemish Fund of Scientific Research
(FWO) and VIRGIL, the European Network of Excellence on Antivi-
ral Drug Resistance (Grant LSHM-CT-2004-503359 from the Prior-
ity 1 ‘Life Sciences, Genomics and Biotechnology’).
methyladenosine phosphoramidate (1.0 mol equiv) was added to a solution
of formic acid (80% v/v solution in water). The reaction was stirred at rt for 6 h.
The solvent was removed under reduced pressure and the obtained yellow oil
was subsequently purified by column chromatography using CHCl3/MeOH
(95:5) as eluent followed by a semipreparative HPLC to give a white solid.
15. Standard
procedure
for
the
deprotection
of
b-20-methylguanosine
phosphoramidates: the appropriate 20,30-isopropylidene-b-20-methyladenosine
phosphoramidates (1.0 mol equiv) was added to a solution of 60% v/v of acetic
acid in water at 90 °C for 15 h. The solvent was removed under reduced
pressure and the obtained yellow oil was subsequently purified by column
Supplementary data
chromatography using CHCl3/MeOH (95:5) as eluent followed by
a
semipreparative HPLC to give a white solid. Key spectroscopic data on (20)
(NMR assignments confirmed by 2D spectra): dP (CH3OH-d4): 4.25, 4.14; dH
(CH3OH-d4): 8.17 (1H, m, H8-guanosine), 7.88 (1H, m, CH-naphthyl), 7.79 (1H,
m, CH-naphthyl), 7.53 (2H, m, CH-naphthyl, CH-benzyl), 7.42–7.40 (1H, m, CH-
naphthyl), 7.36–7.21 (7H, m, CH-naphthyl, CH-benzyl), 6.05 (1H, d, H10-
guanosine, J= 8.4 Hz), 5.15–4.90 (2H, m, CH2-benzyl), 4.58–4.49 (2H, d, H30-
guanosine, H40-guanosine), 4.44–4.34 (2H, m, H50-guanosine), 4.17–4.11 (1H,
Supplementary data (additional spectroscopic and analytical
data on the target compounds) associated with this article can be
m, CHa
), 1.37 (3H, m, CH3-alanine), 1.00 (3H, s, CH3-20-guanosine). MS (ES) m/
References and notes
e: 687.2 (MNa+, 100%); accurate mass: C31H33N6O9NaP required 687.1954,
found 687.1944. Spectroscopic and Analytical data on other analogues are
listed in SI.
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17. Carboxypeptidase
suspended in 200
Y
l
(Sigma CAS No. 9046-67-7, EC 3.4.16.1, 0.5 mg) was
L Trizma buffer and added to the ProTide (5 mg) in 200
L of Trizma, and the reaction followed by 31P NMR.
lL
of acetone-d6 and 400
l
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