4854
C. McGuigan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4850–4854
deaminase16 These data, together with the potent HCV replicon
inhibition data, demonstrate that 4g can be metabolised rapidly
in vitro to the active 20-C-methyl guanosine triphosphate; a direct
inhibitor of the HCV RNA dependent RNA polymerase NS5b.
Given the considerable interest in 4g, the two diastereoisomers
were separated on a preparative scale using a Chiral Pak AD chiral
chromatography column with 1:1 ethanol/hexane as eluant (Chiral
Technologies, West Chester, PA). The absolute configuration of
each of the two diastereomers was determined by Vibrational Cir-
cular Dichroism (VCD) (BioTools, Inc., Jupiter, FL). Replicon data on
the separate isomers are given in Table 5.
Muhammad, J.; Chamberlain, S.; Henson, G. J. Med. Chem. 2010, 53.
9. Blight, K.; Kolykhalov, A. A.; Rice, C. M. Science 2000, 290, 1972.
10. Migliaccio, G.; Tomassini, J. E.; Carroll, S. S.; Tomei, L.; Altamura, S.; Bhat, B.;
Bartholomew, L.; Bosserman, M. R.; Ceccacci, A.; Colwell, L. F.; Cortese, R.; ele
De Francesco, R.; Eldrup, A. B.; Getty, K. L.; Hou, D. S.; LaFemina, R. L.;
Ludmerer, S. W.; MacCoss, M.; McMasters, D. R.; Stahlhut, M. W.; Olsen, D. B.;
Hazuda, D. J.; Flores, O. A. J. Biol. Chem. 2003, 278, 49164.
11. Docking results were scored based on the RMSD value calculated between the
GTP base present in the original model and the base of the docked compounds.
12. Gupta, M.; Nair, V. Collect. Czech. Chem. Commun. 2006, 71, 769.
13. Lambe, C. U.; Averett, D. R.; Paff, M. T.; Reardon, J. E.; Wilson, J. G.; Krenitsky, T.
A. Cancer Res. 1995, 55, 3352.
14. Derudas, M.; Carta, D.; Brancale, A.; Vanpouille, C.; Lisco, A.; Margolis, L.;
Balzarini, J.; McGuigan, C. J. Med. Chem. 2009, 52, 5520.
Interestingly the HCV replicon activities of the two separated
diastereomers are within twofold, suggesting that the initial cleav-
age of the amino acid ester and subsequent elimination of the
naphthol, to give the common amino acid monophosphate metab-
olite, happens at similar rates. A Carboxypeptidase A assay has
proved a useful in vitro model of the initial metabolism of such
ProTides.7 Thus, we carried out such an assay on the separated iso-
mers of 4g. The data shown in Figure 5 is a series of 31P NMRs taken
over a 3.5 h incubation of each diastereomer (10 mM) with Car-
boxypeptidase A in Trizma buffer.
15. Brief synthetic and analytical details for the preparation of 2 and 4g.
To a pre-cooled (0 °C) solution of 2,3,4,5-tetra-O-benzoyl-2-C-methyl-beta-
ribofuranose (10.0 g, 17.22 mmol), 2-amino-6-chloropurine (3.2 g,
D-
18.87 mmol), and 1,8-diazabicycl[5.4.0]undec-7-ene (DBU) (7.7 ml, 51 mmol)
in anhydrous acetonitrile (200 ml) trimethysilyl triflate (12.5 ml, 68.8 mmol)
was added dropwise. The reaction mixture was heated at 65 °C for 4–6 h,
allowed to cool down to room temperature, poured into saturated aqueous
sodium bicarbonate (300 ml), and extracted with dichloromethane
(3 ꢀ 150 ml). The combined organic phase was dried over sodium sulfate and
evaporated under reduced pressure. The residue was precipitated from
dichloromethane and methanol. Precipitate was filtered and washed with
methanol, to give the desired compound (8.5 g, 79%) as a white solid. 1H NMR
(500 MHz, CDCl3) d 8.13 (dd, J = 1.2, 8.3, 2H), 8.02–7.94 (m, 5H), 7.65–7.60 (m,
1H), 7.58–7.45 (m, 4H), 7.35 (q, J = 7.7, 4H), 6.65 (s, 1H), 6.40 (d, J = 6.7, 1H),
5.31 (s, 2H), 5.08 (dd, J = 4.2, 11.6, 1H), 4.79 (dd, J = 6.4, 11.6, 1H), 4.74 (td,
J = 4.2, 6.5, 1H), 1.60 (s, 3H). 13C NMR (126 MHz, CDCl3) d 166.31 (C@O), 165.38
(C@O), 165.32 (C@O), 159.13 (C2), 152.87 (C6), 152.06 (C4), 141.42 (C8),
133.77 (C–H Bn), 133.69 (C–H Bn), 133.28 (C–H Bn), 129.90 (C–H Bn), 129.82
(C–H Bn), 129.78 (C Bn), 129.70 (C–H Bn), 129.41 (C Bn), 128.78 (C Bn), 128.61
(C–H Bn), 128.50 (C–H Bn), 128.41 (C–H Bn), 126.00 (C5), 88.84 (C10), 85.68
(C20), 79.43 (C40), 76.07 (C30), 63.57 (C50), 17.77 (20-Me).
The 31P NMR traces show the parent phosphoramidate near
4.2 ppm at time zero, is rapidly cleaved by Carboxypeptidase A to
the free amino acid carboxylate (peak 2/20) with a 31P NMR shift
near 5 ppm, and then to the aminoacyl phosphate intermediate
(peak 3/30) with a 31P NMR shift near 7.0 ppm. The formation of
this intermediate is considered essential for the activity of Pro-
Tides.6,7 Consistent with the replicon assay data, the estimated
T1/2 values of 4g–1 and 4g–2 are similar, 20 and 17 min, respec-
tively. Further work is underway on 4g–1 and 4g–2 that may dis-
tinguish the two diastereomers, and will be reported elsewhere.
Based on the exceptional HCV antiviral activity of 4g (INX-
08189), along with other advantageous properties, such as cell per-
meability, ease of synthesis, and bioavailability, it was advanced
into in vivo studies supporting its selection as a clinical candidate
for HCV. Full details on the DMPK studies used to support the
selection of 4g will be reported elsewhere. The agent has now pro-
gressed into human clinical trials for HCV.
Synthesis of 2-amino-6-methoxy-9-(2-C-methyl-b-
D-ribofuranosyl) purine
To a suspension of 2-amino-6-chloro-9-(2-C-methyl-2,3,5-tri-O-benzoyl-b-
D-
ribofuranosyl)purine (3.0 g, 4.78 mmol) in anhydrous methanol (36 ml) at 0 °C
NaOMe in methanol (5.4 ml, 25% w/w) was added. The mixture was stirred at
room temperature for 24 h then quenched by addition of amberlite (H+). The
mixture was then filtrated and solvent was removed under reduced pressure.
The resultant residue was dissolved in water (50 ml) and extracted with
hexane (50 ml). The organic layer was then extracted with water (50 ml), and
the combined water fractions were concentrated under reduced pressure. The
residue was purified by silica gel chromatography (CHCl3/MeOH 85:15) to give
the pure compound (1.125 g, 76%) as a white solid. 1H NMR (500 MHz, MeOD)
d 8.26 (s, 1H, H8), 5.99 (s, 1H), 4.24 (d, J = 9.1, 1H), 4.08 (s, 3H), 4.04 (ddd, J = 2.3,
5.7, 8.6, 2H), 3.87 (dd, J = 3.0, 12.4, 1H), 0.96 (s, 3H). 13C NMR (126 MHz, MeOD)
d 162.75 (C6), 161.86 (C2), 154.50 (C4), 139.35 (C8), 115.36 (C5), 93.00 (C10),
84.15 (C40), 80.34 (C20), 73.57 (C30), 61.17 (C50), 54.25 (6-OMe), 20.35 (20-Me).
HPLC: tR = 9.00 min. (solvent gradient needed O?100% ACN in H2O 30 mins).
HRMS (ESI): calcd for C12H17N5O5+Na+ 334.1127, found 334.1125. Calcd for
Acknowledgements
C
12H17N5O5ꢂ0.75 H2O: C, 44.37; H, 5.74; N, 21.56. Found: C, 44.24; H, 5.49; N,
This study was supported by a grant from Inhibitex, Inc. to C.M.
C.M. is a board member and shareholder of Inhibitex, Inc. All authors
from Inhibitex, Inc. own options and are shareholders. We would
like to thank CiVentiChem, RTP, NC for scaling up INX-08189 for
separation of the diastereomers and Dr. Mark Gumbleton and Mr.
Mathew Smith, Cardiff University for initial Caco-2 studies. We
would like to thank Helen Murphy for secretarial assistance.
20.83.
Synthesis of 2-amino-6-methoxy-9-(2-C-methyl-b-D
-ribofuranosyl) purine 50-O-
[
a
-naphthyl-(2,2-dimethylpropoxy-
L
-alaninyl)] phosphate 4g
-ribofuranosyl) purine (1.35 g,
To 2-amino-6-methoxy-9-(2-C-methyl-b-
D
4.34 mmol) in THF (35 ml), phosphorochloridate (10.83 mmol, 1 M solution
in THF)) in THF was added, followed by addition of N-methyl-imidazole
(1.71 ml, 21.7 mmol). The mixture was stirred overnight and the solvent was
removed under reduced pressure. To remove the N-methyl-imidazole, the
phosphoramidate was dissolved in chloroform and washed
3 times with
hydrochloric acid (HCl 0.5 N). The organic layer was then dried over sodium
sulfate and evaporated under reduce pressure. The residue was then purified
on silica gel using CHCl3 to CHCl3/MeOH 95:5 as an eluent, to give the pure
phosphoramidate as a white solid (510 mg, 18%). 31P NMR (202 MHz, CDCl3) d
4.33, 4.29. 1H NMR (500 MHz, MeOD) d 8.19–8.15 (m, 1H, H8-naph), 7.98, 7.96
(2 ꢀ s, 1H, H8), 7.86–7.82 (m, 1H, H5-napht), 7.68, 7.65 (2 ꢀ d, J= 7.0 Hz, 1H, H4-
napht), 7.53–7.44 (m, 3H, H2, H7, H6-napht), 7.39, 7.37 (2 ꢀ t, J= 8.0 Hz, 1H, H3-
References and notes
1. Bostan, N.; Mahmood, T. Crit. Rev. Microbiol. 2010, 36, 91.
2. Hoofnagle, J. H.; Seef, L. B. N. Engl. J. Med. 2006, 355, 2444.
3. Soriano, V.; Madejon, A.; Vispo, E.; Labarga, P.; Garcia-Samaniego, J.; Martin-
Carbonero, L.; Sheldon, J.; Bottecchia, M.; Tuma, P.; Barreiro, P. Expert Opin.
Emerg. Drugs 2008, 13, 1.
4. Eldrup, A. B.; Prhavc, Ml; Brooks, J.; Bhat, B.; Prakash, T. P.; Song, Q.; Bera, S.;
Bhat, N.; Dande, P.; Cook, P. D.; Bennett, C. F.; Carroll, S. S.; Ball, R. G.;
Bosserman, M.; Burlein, C.; Colwell, L. F.; Fay, J. F.; Flores, O. A.; Getty, K.;
LaFemina, R. L.; Leone, J.; MacCoss, M.; McMasters, D. R.; Tomassini, J. E.;
Langen, D. V.; Wolanski, B.; Olsen, D. B. J. Med. Chem. 2004, 47, 5284.
5. Eldrup, A. B.; Allerson, C. R.; Bennett, C. F.; Bhat, S. B. B.; Bhat, N.; Bosserman, M.
R.; Brooks, J.; Burlein, C.; Carroll, S. S.; Cook, P. D.; Getty, K. L.; MacCoss, M.;
McMasters, D. R.; Olsen, D. B.; Prakash, T. P.; Prhavc, M.; Song, Q.; Tomassini, J.
E.; Xia, J. J. Med. Chem. 2004, 47, 2283.
0
0
0
napht), 6.01, 6.00 (2 ꢀ s, 1H, H1 ), 4.67–4.64 (m, 1H, H5 ), 4.63- 4.59 (m, 1H, H3
,
0
H
4 ), 4.09–4.05 (m, 1H, Ha), 4.04 (s, 3H, 6OCH3), 3.75, 3.72, 3.64, 3.58 (2 ꢀ AB,
JAB = 10.5 Hz, 2H, CH2 ester), 1.33 (d, J = 7.5 Hz, 3H, CH3 Ala), 0.98, 0.96 (2 ꢀ s,
3H, 20CCH3), 0.85, 0.84 (2 ꢀ s, 9H, 3 ꢀ CH3 ester). 13C NMR (126 MHz, MeOD) d
3
175.06, 174.80 (2 ꢀ d, JC–C–N–P = 5.0 Hz, C@O ester), 162.73 (C6), 161.86 (C2),
2
154.55, 154.51 (C4), 148.00, 147.95 (d, JC–O–P = 3.8 Hz, ipso naph), 139.36,
139.08 (CH8), 136.27, 136.25 (C10-naph), 128.86, 128.80 (CH-naph), 127.88,
3
127.73 (2 ꢀ d, JC–C–O–P = 6.3 Hz, C9-naph), 127.48, 126.53, 126.49, 125.97,
122.81, 122.76 (CH-naph), 116.24, 116.22 (C2-naph), 116.19, 115.63 (C5),
3
93.34, 93.18 (C10), 82.32, 82.16 (2 ꢀ d, JC–C–O–P = 8.8 Hz, C40), 79.99, 79.95
2
(C20), 75.52, 75.37 (CH2 ester), 74.95, 74.70 (C30), 68.11, 67.62 (2 ꢀ d, JC–O–
P = 5.0 Hz, C50), 54.28, 54.07 (6OCH3)51.79, 51.71 (Ca Ala), 32.26, 32.22 (C
6. Cahard, D.; McGuigan, C.; Balzarini, J. Mini-Rev. Med. Chem. 2004, 4, 371.
7. McGuigan, C.; Perrone, P.; Madela, K.; Neyts, J. Bioorg. Med. Chem. Lett. 2009, 19,
431.
8. McGuigan, C.; Gilles, A.; Madela, K.; Aljarah, M.; Holl, S.; Jones, S.; Vernachio, J.;
Hutchins, J.; Bryant, D.; Gorovits, E.; Ganguly, B.; Hall, A.; Kolykhalov, A.;
3
ester), 26.74, 26.71 (3 ꢀ CH3 ester), 20.89, 20.69 (2 ꢀ d, JC–C–N–P = 6.3 Hz, CH3
Ala), 20.39, 20.35 (20CCH3). HPLC Rt = 20.95, 21.48 min.
16. Faletto, M. B.; Miller, W. H.; Garvey, E. P.; St. Clair, M. H.; Daluge, S. M.; Good, S.
S. Antimicrobial Agents Chemother. 1997, 41, 1099.