Synthesis and antiviral activity of 2′-deoxy-2′-fluoro- 2′-C-methyl purine nucleosides as inhibitors of hepatitis C virus RNA replication

Article abstract of DOI:10.1016/j.bmcl.2005.12.002

A series of purine nucleosides containing the 2′-deoxy-2′- fluoro-2′-C-methylribofuranosyl moiety were synthesized and evaluated as potential inhibitors of the hepatitis C virus in vitro. Of the nucleosides that were synthesized, only those possessing a 2

Full text of DOI:10.1016/j.bmcl.2005.12.002

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1712–1715
Synthesis and antiviral activity of 20-deoxy-20-fluoro-20-C-methyl
purine nucleosides as inhibitors of hepatitis C virus RNA replication
Jeremy L. Clark,a,*,à J. Christian Mason,a, Laurent Hollecker,a,§ Lieven J. Stuyver,a,–
Phillip M. Tharnish,a Tamara R. McBrayer,a Michael J. Otto,a Phillip A. Furman,a
Raymond F. Schinazib and Kyoichi A. Watanabea
aPharmasset, Inc.,k 1860 Montreal Road, Tucker, GA 30084, USA
bDepartment of Pediatrics, Emory University, School of Medicine/Veterans Affairs Medical Center, Decatur, GA 30033, USA
Received 30 October 2005; revised 30 November 2005; accepted 1 December 2005
Available online 20 December 2005
Abstract—A series of purine nucleosides containing the 20-deoxy-20-fluoro-20-C-methylribofuranosyl moiety were synthesized and
evaluated as potential inhibitors of the hepatitis C virus in vitro. Of the nucleosides that were synthesized, only those possessing
a 2-amino group on the purine base reduced the levels of HCV RNA in a subgenomic replicon assay.
Ó 2005 Elsevier Ltd. All rights reserved.
The current standard of care for chronic hepatitis C
virus (HCV) infections is combination therapy with al-
pha interferon and ribavirin. Studies have shown that
more patients with hepatitis C respond to pegylated
interferon-a/ribavirin combination therapy than to com-
bination therapy with unpegylated interferon alpha.
Due to the low response rates as well as toxic side effects
and unsustained viral load reductions, these therapies
are inadequate and there is a need for improved thera-
pies for treating chronic HCV infection.1
NH2
N
X
N
N
N
N
N
O
Y
HO
HO
O
O
CH3
CH3
HO
F
HO OH
1: X = OH, Y = NH2
2: X = NH2, Y = H
3
Figure 1. Branched-chain nucleoside HCV polymerase inhibitors.
Several 20-C-methyl purine nucleoside analogs with po-
tent inhibitory activity against the HCV NS5B polymer-
ase have been identified (Fig. 1).2–5 The potent inhibitory
activity against HCV replication of a novel pyrimidine
nucleoside, b-D-20-deoxy-20-fluoro-20-C-methylcytidine
(3), has recently been reported.6 The synthesis and
antiviral activity of several purine analogs containing
the 20-deoxy-20-fluoro-20-C-methylribofuranosyl moiety
are reported herein.
The general synthetic procedure used for 20-deoxy-20-
fluoro-20-C-methyl purine nucleoside analogs (11–14)
described herein is similar to the linear synthesis
described for 20-deoxy-20-fluoro-20-C-methylcytidine
(Scheme 1).6 For the preparation of compounds 6a
and 6b, the 30,50-silyl-protecting groups were removed
with TBAF in THF and replaced with the acetyl groups
in a one-pot desilylation/acetylation reaction. This one-
pot reaction provided a convenient method that avoided
the isolation of the polar 30,50-diol intermediate.
Keywords: Nucleosides; Purines; Hepatitis C; HCV.
*
Corresponding author. Tel.: +1 205 581 2572; fax: +1 440 359
0910; e-mail: j.clark@sri.org
Deceased April 2005.
à
Present address: Southern Research Institute, 2000 Ninth Ave.
South, Birmingham, AL 35255, USA.
§
Present address: Via San Filippo, 19, Monserrato (CA) 09042, Italia.
Present address: Virco BVBA, Mechelen, Belgium.
Present address: Pharmasset Inc: 303A College Rd E., Princeton,
NJ 08540, USA.
–
k
Upon treating 6a and 6b with DAST in toluene, the de-
sired fluorinated products (7a and 7b) were obtained in
approximately 20% isolated yield after silica gel purifica-
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.002

J. L. Clark et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1712–1715
1713
Cl
N
Cl
N
Cl
Cl
N
N
N
N
N
N
N
N
N
N
N
N
N
Y
Y
Y
Y
N
O
RO
AcO
AcO
a
c
O
O
O
O
OH
CH3
CH3
+
TIDPS
R
CH2
O
O
RO
AcO
AcO
4a: Y = NHTr
4b: Y = H
5a: R = TIDPS, Y = NHTr
5b: R = TIDPS, Y = H
7a: R = F, Y = NHTr
7b: R = F, Y = H
8a: R = OH, Y = NHTr
8b: R = OH, Y = H
9a: Y = NHTr
9b: Y = H
b
6a: R = Ac, Y = NHTr
6b: R = Ac, Y = H
Scheme 1. Reagents and conditions: (a) MeLi, ꢀ78 °C; (b) i—1 M TBAF, concd AcOH, rt, ii—Ac2O, TEA; (c) DAST, toluene, 0 °C to rt.
Table 1. 1H NMR data for compounds 7b and 10–14a
Compound
20-CH3
H-10
H-30
H-40
H-50/H-5a0
4.41–4.55 (m)
H-2
H-8
7b
10
1.24 (d, J = 22.8)
1.26 (d, J = 22.5)
6.37 (d, J = 17.6)
6.12 (d, J = 17.7)
5.70 (dd, J = 9.1, 22.1)
5.84 (dd, J = 9.2, 22.8)
8.82 (s)
—
8.45 (s)
7.97 (s)
4.47 (m)
4.34, 4.55 (dd,
J = 2.9, 12.4)
4.25, 4.04 (dd,
J = 2.11, 12.5)
11
12
13
14
1.23 (d, J = 22.4)
1.14 (d, J = 22.3)
1.21 (d, J = 22.8)
1.20 (d, J = 22.3)
6.37 (d, J = 17.4)
6.28 (d, J = 17.4)
6.19 (d, J = 17.3)
6.10 (d, J = 17.8)
4.61 (dd, J = 9.2, 22.3)
4.40 (dd, J = 9.4, 24.3)
4.38 (dd, J = 9.5, 24.5)
4.32 (dd, J = 8.9, 24.3)
4.17 (dd,
J = 1.5, 9.2 Hz)
8.78 (s)
8.52 (s)
—
8.70 (s)
8.20 (s)
8.51 (s)
8.15 (s)
4.03–4.09 (m), 3.87 (dd,
J = 3.0, 12.6 Hz)
4.02–4.07 (m), 3.87 (dd,
J = 3.1, 12.6 Hz)
3.85–4.04 (m)
—
a Spectra were obtained at 400 MHz. Chemical shifts are reported as d (ppm) downfield with respect to an internal standard of tetramethylsilane. J
values are in Hertz. Spectra were obtained in CDCl3 for compounds 7b, 10, 11 and in CD3OD for compounds 12–14.
tion (Scheme 1).10 The structures of the fluorinated
compounds 8a and 8b were confirmed by the transfor-
mation to the known compounds 1 and 2 using standard
methods.4 Compound 8a was converted to 20-C-meth-
ylguanosine (1) by the same methods used to prepare
20-deoxy-20-fluoro-2-C-methyl-guanosine 14 from 7a
(Scheme 2).
1
products were determined by analyzing the H and 13C
NMR multiplicities arising from the 20-fluorine coupling
and by nuclear Overhauser effect (NOE) H NMR dif-
1
ference spectroscopy (Table 1). Upon irradiation of
the doublet at d 1.26 (20-CH3) in compound 10, NOEs
were observed with the doublet of doublets at d 5.84
(H-30) and the anomeric doublet at d 6.12 (Fig. 2).
Similar NOEs were observed with the 6-chloropurine
analog 7b.
The N2 trityl group in 7a was removed using CHCl3-
TFA (9:1) to provide compound 10. Treating com-
pounds 7b and 10 with methanolic ammonia at room
temperature provided 11 and 13, respectively; perform-
ing the aminolysis at 80 °C in a sealed tube for 24–
36 h provided 12 from 7b. 20-deoxy-20-fluoro-20-C-meth-
ylguanosine (14) was prepared from 11 using an excess
of mercaptoethanol and sodium methoxide.7
The DAST fluorination of compounds 6a and 6b also
provided two additional corresponding products that
were isolated by silica gel chromatography and identi-
fied as the exomethylene products 9a and 9b, and the
protected 20-C-methyl nucleoside products 8a and 8b.
Analysis of the 1H NMR spectra of compounds 9a
and 9b revealed two doublets (J = 3.9 Hz) at around d
5.6 and 5.3, indicating the presence of an exomethylene
containing diastereotopic protons. The structures of
Although the yields of fluorinated products were essen-
tially the same for both compounds 6a and 6b, the yields
Cl
X
N
N
N
N
N
N
Cl
Y
Y
N
N
AcO
HO
O
O
b
N
N
CH3
CH3
AcO
F
HO
F
N
Y
AcO
N
CH
2 O
7a: Y = NHTr
10: Y = NH2
7b: Y = H
11: X = Cl, Y = H
12: X = NH2, Y = H
13: X = Cl, Y = NH2
14: X = OH, Y = NH2
a
H3C
H
H
c
AcO
F
7b: Y = H
10: Y = NH2
Scheme 2. Reagents and conditions: (a) CHCl3-TFA (9:1), rt;
(b) MeOH/NH3, rt for 10 ! 13 and 7b ! 11, 80 °C for 7b ! 12;
(c) mercaptoethanol, NaOMe, reflux.
Figure 2. 1D NOE correlations for compounds 7b and 10.

1714
J. L. Clark et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1712–1715
Table 2. Anti-HCV activity and cellular toxicity of compounds 11–14,
20-C-methylguanosine (1) and 20-C-methyladenosine (2)
Acknowledgments
a
b
Dr. R. F. Schinazi is the principal founder and former
consultant of Pharmasset Inc. His laboratory received
no funding for this work.
Compound
HCV EC90 (lM)
Cytotoxicity CC50 (lM)
11
12
13
14
1
88.2 3.7
>100
27.1 13.0
>100
>100
62.4 22.6
56.0 31.7
10.8
>100
>100
References and notes
2
1.40 0.5
15.0 1.6
a Average of at least two experiments.
1. Davis, G. L. Gastroenterology 2000, 118, S104.
2. Carroll, S. S.; Tomassini, J. E.; Bosserman, M.; Getty, K.;
Stahlhut, M. W.; Eldrup, A. B.; Bhat, B.; Hall, D.;
Simcoe, A. L.; LaFemina, R.; Rutkowski, C. A.; Wolan-
ski, B.; Yang, Z.; Migliaccio, G.; De Francesco, R.; Kuo,
L. C.; MacCoss, M.; Olsen, D. B. J. Biol. Chem. 2003, 278,
11979.
3. Stuyver, L. J.; McBrayer, T. R.; Tharnish, P. M.; Hassan,
A. E.; Chu, C. K.; Pankiewicz, K. W.; Watanabe, K. A.;
Schinazi, R. F.; Otto, M. J. J. Virol. 2003, 77, 10689.
4. Eldrup, A. B.; Allerson, C. R.; Bennett, C. F.; Bera, S.;
Bhat, 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.
b MTS CC50 was determined in a 96 h assay using the Celltiter 96
nonradioactive cell proliferation assay from Promega (Madison,
WI).
of the by-products (8a and 8b) were sensitive to the pur-
ine ring substituents. The DAST fluorination of 6a pro-
vided the protected 20-C-methylpurine (8a) in 72%
isolated yield, suggesting that the N3 moiety, made more
nucleophilic by the exocyclic N2 amino substituent, is in-
volved in this epimerization.8 In contrast, fluorination of
compound 6b, that contains no 2-amino group, consis-
tently afforded a modest 20% yield of the C-20 epimer-
ized compound 8b.
5. 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.
6. Clark, J. L.; Hollecker, L.; Mason, J. C.; Stuyver, L. J.;
Tharnish, P. M.; Lostia, S.; McBrayer, T. R.; Schinazi, R.
F.; Watanabe, K. A.; Otto, M. J.; Furman, P. A.; Stec, W.
J.; Patterson, S. E.; Pankiewicz, K. W. J. Med. Chem.
2005, 48, 5504.
Compounds 11–14 were evaluated as inhibitors of the
HCV in a subgenomic replicon assay as previously de-
scribed.9 The anti-HCV activity of 20-C-methylguano-
sine (1) and 20-C-methyladenosine (2), as obtained
from the DAST fluorination, is shown for comparison.
As indicated in Table 2, the 20-deoxy-20-fluoro-20-C-
methylpurine analogs 13 and 14 demonstrate modest
inhibition against HCV replication. 20-Deoxy-20-fluo-
ro-20-C-methylguanosine (14) was ꢁ5 times less potent
than the corresponding 20-C-methylguanosine (1),
while 20-deoxy-20-fluoro-20-C-methyladenosine (12)
showed no activity or cytotoxicity when tested up to
100 lM.
7. Tong, G. L.; Ryan, K. J.; Lee, W. W.; Acton, E. M.;
Goodman, L. J. Org. Chem. 1967, 32, 859.
8. For references discussing the participation of the N3
during related DAST fluorinations of purines, see: (a)
Pankiewicz, K. W.; Krzeminski, J.; Ciszewski, L. A.; Ren,
W.-Y.; Watanabe, K. A. J. Org. Chem. 1992, 57, 553; (b)
Izawa, K.; Takamatsu, S.; Katayama, S.; Hirose, N.;
Kozai, S.; Maruyama, T. Nucleosides Nucleotides Nucleic
Acids 2003, 22, 507.
The DAST fluorination of purine nucleosides such as 6a
and 6b provides modest yields of the desired fluorinated
nucleosides 7a and 7b. However, this synthetic route
provides a direct method for the preparation of the cor-
responding 20-C-methylnucleosides and thereby a con-
cise method for determining the effect of fluorine
substitution with respect to in vitro HCV inhibition.
While the reason for the lack of potency of compound
12 is unclear, the modest activity of compounds 13
and 14 suggests that both the 2-amino and the 20-b-
methyl groups are important substituents for selective
HCV inhibition in these molecules. The weak inhibition
of compounds 13 and 14 in the cell-based replicon assay
may be due to inefficient uptake and/or metabolism to
the active 50-triphosphate. The lack of efficient phos-
phorylation or uptake of 20-C-methylguanosine (1) in
a cell-based replicon assay has been suggested. Eldrup
et al. reported a remarkable anti-HCV potency of the
50-triphosphate of compound 1 in an NS5B enzyme as-
say even though the free nucleoside lacked the same level
of potency in a cell-based replicon system.4 The latter as-
say requires penetration of the parent nucleoside into
the cells followed by metabolism to the active triphos-
phate species.
9. Stuyver, L. J.; Whitaker, T.; McBrayer, T. R.; Hernandez-
Santiago, B. I.; Lostia, S.; Tharnish, P. M.; Ramesh, M.;
Chu, C. K.; Jordan, R.; Shi, J.; Rachakonda, S.; Watan-
abe, K. A.; Otto, M. J.; Schinazi, R. F. Antimicrob. Agents
Chemother. 2003, 47, 244–254.
10. Reaction of 6a with DAST: To a stirred solution of 6a
(0.830 g, 1.29 mmol) in anhydrous toluene (30.0 mL) was
added DAST (0.280 mL, 2.12 mmol) at 0 °C under argon.
After the addition was complete, the cooling bath was
removed and stirring was continued for 1 h. The reaction
mixture was poured into satd NaHCO3 (30.0 mL) and
washed until gas evolution ceased. The organic phase was
dried (Na2SO4), concentrated to dryness, and purified by
silica gel chromatography washing with 1:1 Et2O–petro-
leum ether and eluting with 1:7:7 EtOAc–Et2O–petroleum
ether to afford 7a (0.171 g, 20.5%) as a syrup, followed by
9a (0.062 g, 7.8%). Further elution afforded 8a (0.598 g,
72.0%) as a syrup. The same procedure, starting from 6b,
was used to obtain 7b, 8b, and 9b. 1H, 19F, and 13C NMR
spectra were obtained with a Varian Unity Plus 400
1
spectrometer at 400, 376, and 100 MHz, respectively. H
and 13C NMR chemical shifts are reported as d (ppm)

J. L. Clark et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1712–1715
1715
downfield with respect to an internal standard of tetra-
methylsilane, while 19F chemical shifts are reported
downfield from an external standard of hexafluoroben-
3H), 2.14 (s, 3H), 2.17 (s, 3H), 4.25–4.37 (m, 3H), 5.04 (d,
1H, J = 7.7 Hz), 5.38 (s, 1H), 6.76 (s, 1H), 7.14–7.35 (m,
15H), 8.01 (s, 1H). 13C NMR (CDCl3): d 20.3, 20.7, 20.9,
61.9, 71.0, 73.4, 78.8, 90.6, 124.7, 125.4, 127.0, 127.9,
128.3, 129.07, 129.1, 138.8, 144.8, 151.0, 152.1, 157.5,
169.7, 170.4; 9a: 1H NMR (CDCl3): d 2.10 (s, 3H), 2.16 (s,
3H), 4.29 (d, 2H, J = 3.5 Hz), 4.79 (bs, 1H), 5.31 (bs, 1H),
5.32 (d, 1H, J = 3.5 Hz), 5.65 (d, 1H), 6.00 (bs, 1H), 6.69
(s, 1H), 7.21–7.34 (m, 15H), 7.80 (s, 1H). 13C NMR
(CDCl3): d 20.9, 21.0, 63.3, 71.2, 72.2, 80.5, 83.2, 116.9,
126.9, 127.9, 128.0, 129.0, 129.1, 139.4, 143.4, 144.9, 157.8,
170.2, 170.4.
1
zene. 7a: H NMR (CDCl3): d 0.67 (d, 3 H, J = 22.5 Hz),
2.16 (s, 3H), 2.19 (s, 3H), 4.25–4.40 (m, 3H), 5.19 (dd, 1H,
J = 8.5, 22.4 Hz), 5.52 (d, 1H, J = 17.3 Hz), 6.76 (s, 1H),
7.19–7.34 (m, 15H), 8.01 (s, 1H). 13C NMR (CDCl3): d
16.6 (d, J = 24.6 Hz), 20.6, 20.9, 61.1, 70.8 (d,
J = 15.9 Hz), 71.0, 88.3 (d, J = 38.6 Hz), 99.9 (d,
J = 186.0 Hz), 124.6, 127.0, 128.0, 129.0, 129.1, 138.4,
144.7, 151.1, 152.1, 157.7, 170.0, 170.3. 19F NMR
(CDCl3): d 5.10 (m); 8a: 1H NMR (CDCl3): d 0.63 (s,