Synthesis and biological activity of 5′,9-anhydro-3-purine- isonucleosides as potential anti-hepatitis C virus agents

Article abstract of DOI:10.1080/15257770601052307

In order to study structure-activity relationships among the derivatives and congeners of 5′,9-anhydro-3-(β-D-ribofuranosyl)xanthine for anti-hepatitis C virus activity, a series of 5′,9-anhydro-purine- isonucleosides with a substituent (s) at 6- or/and 8

Full text of DOI:10.1080/15257770601052307

Nucleosides, Nucleotides, and Nucleic Acids, 26:83–97, 2007
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770601052307
SYNTHESIS AND BIOLOGICAL ACTIVITY OF
ꢀ
5
,9-ANHYDRO-3-PURINE-ISONUCLEOSIDES AS POTENTIAL
ANTI-HEPATITIS C VIRUS AGENTS
Byoung-Kwon Chun, Peiyuan Wang, Abdalla Hassan, Jinfa Du,
Phillip M. Tharnish, Eisuke Murakami, Lieven Stuyver, and
Michael J. Otto
2
Pharmasset, Inc., Princeton, New Jersey, USA
Raymond F. Schinazi
2
Department of Pediatrics, Emory University School of
Medicine/Veterans Affairs Medical Center, Decatur, Georgia, USA
Kyoichi A. Watanabe Pharmasset, Inc., Princeton, New Jersey, USA
2
2
In order to study structure-activity relationships among the derivatives and congeners of
,9-anhydro-3-(β-D-ribofuranosyl)xanthine for anti-hepatitis C virus activity, a series of 5 ,9-
ꢁ
ꢁ
5
anhydro-purine-isonucleosides with a substituent (s) at 6- or/and 8-position of the purine moiety
were synthesized, and their anti-hepatitis C virus activity and cytotoxicity were evaluated and
discussed.
ꢁ
Keywords 5 ,9-Anhydro-purine-isonucleoside; nucleoside; hepatitis C virus; HCV
INTRODUCTION
Hepatitis C virus (HCV)-induced chronic hepatitis with concomitant
cirrhosis and hepatocellular carcinoma is now the leading cause of liver
transplant in the United States. There are about 3 million HCV carriers (2%
of the population) in the United States and an estimate 170 million people
worldwide. In up to 80% of the infected patients, the virus causes a chronic
infection that can progress to chronic active hepatitis with cirrhosis and/or
hepatocellular carcinoma.[
1,2]
HCV is a 9.6 Kb positive strand RNA virus of the flaviviridae, genus Hep-
acivirus. It contains a single open reading frame coding for a 3,000 amino
acid polyprotein, which is further processed by host and viral proteases
Received 15 February 2006; accepted 15 July 2006.
This work was supported in parts by the NIH grants 1R43 AI-52868 (biology) and 1R43AI-056720
(
chemistry).
Address correspondence to Byoung-Kwon Chun, Pharmasset, Inc. 303A College Road East,
Princeton, New Jersey, 08540. E-mail: kwon.chun@pharmasset.com
8
3

8
4
B. Kwon-Chun et al.
into various structural (core, E1, and E2) and nonstructural (NS2, NS3,
[
3]
NS4A, NS4B, NS5A, and NS5B) viral proteins. The NS3 protease/helicase
and the NS5B RNA dependent RNA polymerase are probably the most
well studied targets for anti-HCV therapy since they are crucial for the
viral replication.[
4–6]
However, the only approved therapies for chronic
hepatitis C are interferon-α (INF-α), or pegylated-interferon-α either alone
or in combination with ribavirin.[ Combination therapy with ribavirin and
pegylated-IFN for 6 to 12 months is currently the treatment of choice for
chronic HCV infection. Although patients infected with HCV genotype 2 or
7]
-3 show high overall sustained response rate to treatment, defined as loss of
HCV from serum 6 months after completion of treatment, those chronically
infected with the genotype1 show only between 46% and 65% of response
rate. Anemia and neuropsychiatric effects are the common adverse events
of the combination therapy, which lead to premature cessation of therapy
in 10–20% of patients.[
7]
In light of these problems, we initiated a search for anti-HCV agents
ꢁ
and discovered anti-HCV activity with 3,5 -cyclo-4-(β-D-ribofuranosyl)-vic-
triazolo[4,5-b]pyridin-5-one (1).[ Compound 1 showed no specific in-
8]
hibitory activity against purified HCV RNA-dependent RNA polymerase
(NS5B), which excluded the notion that the anti-HCV activity may be
derived from the allosteric inhibition of the polymerase like that of non-
nucleoside reverse transcriptase inhibitors (NNRTI) in HIV. Nevertheless, it
was believed that this class of compounds might have a very unique mode
of action worth further exploration. Therefore, as a part of structure-activity
relationship study, the triazolopyridinone moiety of the lead compound 1
was replaced by a purine base, xanthine, which resulted in the discovery of
ꢁ
5
,9-anhydro-isoxanthosine (2) with improved anti-HCV activity in a replicon
[
9]
system. Encouraged by this result, the derivatives with various substitu-
tions on the purine moiety were prepared in search for more potent and
less toxic anti-HCV agents. Herein, we report the synthesis and anti-HCV
ꢁ
activity of 5 ,9-anhydro-purine-isonucleoside derivatives (3–14) (Figure 1).
RESULTS AND DISCUSSION
ꢁ
ꢁ
ꢁ
The starting material for 3–7 was 2 ,3 -O-isopropylidene-5 ,9-anhydro-
[
9]
ꢁ
isoxanthosine (15).
For preparation of 5 ,9-anhydro-2-hydroxy-3-
ꢁ
isoadenosine or 5 ,9-anhydro-isocrotonoside (4), 15 was treated with
Lawesson’s reagent to give a 6-thio derivative 16 in 47% yield. Then 16 was
heated with methanolic ammonia to afford a 6-amino derivative 17 in 63%
yield, which was hydrolyzed with 0.5 N HCl to afford the target compound
4
as the HCl salt in 88% yield.
Alternatively, 17 also was prepared from known tri-O-benzoyl
isocrotonoside 18.[
10,11]
Debenzoylation of 18 with n-butylamine, followed

ꢁ
5
,9-Anhydro-3-Purine-Isonucleosides
85
ꢁ
FIGURE 1 5 ,9-Anhydro-3-purine-isonucleosides.
by selective protection using 2,2-dimethoxypropane/TsOH afforded 19,
which was subjected to a Mitsunobu reaction to give 17 in 79% yield
from 18. Treatment of 15 with sodium hydride and LiBr in a mixture
of 1,2-dimethoxyethane and N,N -dimethylformamide, and then with
bromoacetonitrile gave an N 1-cyanomethyl intermediate, which was
deprotected with 0.5 N HCl to afford 3 in 43% yield from 15. The
6
N -methylamino purine derivative 5 was obtained by 6-O-sulfonylation of
15 with 2,4,6-triisopropylbenzenesufonyl chloride followed by treatment
with methylamine and then acidic hydrolysis with 0.5 N HCl. The 6-thio
derivative 6 was obtained simply by acidic hydrolysis of 16 in 48% yield. The
6
S -methyl derivative 7 was obtained in 52% yield by treatment of 16 with
methyl iodide in presence of aqueous NaOH followed by acidic hydrolysis
with 75% trifluoroacetic acid (Scheme 1).
In order to prepare the 8-substiuted anhydro-purine-isonucleosides, two
different strategies were employed (Scheme 2). Cyclization of a diamine
intermediate 20 with an appropriate reagent was employed for preparation
of 8, 9, and 10, while electrophilic substitution on the anhydro-xanthosine
15 and 4 was used for 11, 12, 13, and 14. For preparation of 8, the diamime
2
0 was treated with carbonyl diimidazole in a mixture of dioxane and N ,N -
ꢁ
ꢁ
ꢁ
dimethylformate to give 5 ,9-anhydro-8-hydroxyl-2 ,3 -O-isopropylidene-3-
isoxanthosine (21),[
12]
which was hydrolyzed with aqueous trifluoroacetic
acid to afford the target compound 8 in 50% yield from 20. For the
preparation of 9, compound 20 was treated with triethyl orthoacetate and
ꢁ
ꢁ
ꢁ
p-toluenesulfonic acid in N ,N -dimethylformate to give 5 ,9-anhydro-2 ,3 -
[
13]
O-isopropylidene-8-methyl isoxanthosine (22) in 61% yield,
which was
treated with 0.5 N HCl to afford the target compound 9 in 80% yield. For

8
6
B. Kwon-Chun et al.
◦
SCHEME 1 a) Lawesson’s reagent, 1,2-dichloroethane, reflux, 15 h; b) NH3, MeOH, 90 C,
2
+
◦
◦
4h; c) H 3O; d) NaH, DME-DMF, 0 C, 10 min, then BrCH2CN, LiBr, 65 C, 2 h; e) 2,4,6-
triisopropylbenzenesulfonyl chloride, DMAP, Et3N, CH3CN, rt, 7 h, then aqueous CH3NH2, rt, 15 h;
f) Mel, 0.5 N NaOH, rt, 30 min; g) n- i) DEAD, PPh3, DMF, rt, 30 min.
preparation of 10, compound 20 was treated with benzaldehyde and acetic
acid to generate an imine intermediate, which was then oxidatively cyclized
ꢁ
ꢁ
ꢁ
using thionyl chloride to give 5 ,9-anhydro-2 ,3 -O-isopropylidene-8-phenyl-
[
14]
3
-xanthosine (23) in 53% yield,
which was similarly hydrolyzed to afford
the target compound 10 in 78% yield. To prepare the target compounds,
1
1, 12, and 13, the 8-halo intermediates, 24, 25, and 26 were obtained by
[
15]
treating 15 with a corresponding N -halosuccinimide.
8-Chlorination of
1
5 was effected by N -chlorosuccinimide (NCS) in presence of acetic acid to
ꢁ
ꢁ
ꢁ
give 5 ,9-anhydro-8-chloro-2 ,3 -O-isopropylidene-3-xanthosine (24) in 84%
yield,
[
16]
which was hydrolyzed with aqueous trifluoroacetic acid to afford
target compound 11 in 41% yield.
It is noteworthy that the chlorination of 15 with NCS did not sig-
nificantly progress without acetic acid-catalysis. The 8-bromination of 15
with N -bromosuccinimide (NBS) occurred readily without acid catalyst in
ꢁ
ꢁ
ꢁ
dioxane at room temperature to give a protected 5 ,9-anhydro-8-bromo-2 ,3 -
O-isopropylidene-3-xanthosine (25),[
17]
which was then hydrolyzed with

ꢁ
5
,9-Anhydro-3-Purine-Isonucleosides
87
◦
SCHEME 2 a) CO(Im)2, dioxane-DMF, rt, 15h; b) CH3(OEt)3, TsOH, DMF, 90 C, 1 hr; c) PhCHO,
AcOH, MeOH, rt, 30 min, then SOCl2, rt, 30 min; d) NCS-AcOH, dioxane, rt, 15h; e) NBS, dioxane, rt,
◦ +
h; f) NIS, dioxane, 90 C, 24h; g) NBS, dioxane-H2O, rt, 2h; h) H 3O.
2
trifluoroacetic acid to afford the target compound 12 in 48% yield from
5. Another target compound 13 was obtained similarly from 15 in 26%
1
yield using N -iodosuccinimide (NIS). Finally, the target compound 14 was
prepared by bromination of compound 4 using NBS in 70% yield. The
structure of the intermediates and final compounds were characterized by
1
13
NMR ( H, C, COSY, NOE), UV, and high-resolution mass spectroscopy.
The antiviral activity of compounds 1–14 was evaluated in a HCV
subgenomic RNA replicon system,[ along with cytotoxicity in the replicon
cell and several other cell lines (Table 1). A 6-thiopurine analogue 6, its
18]
6
S -methyl derivative 7, 8-methyl (9), and 8-chloro-(11) analogues exhibited
9
the more potent anti-HCV activity than the lead compound 2 while all the
newly synthesized compounds except 10 were more potent than the original
8
lead 1 . An isoxanthosine analogue 2 and 6-aminopurine analogue 4 are less
active than 6 and 7, but more active than its N 1-cyanomethyl derivative 3
6
and N -methyl-6-aminopurine analogue 5. However, the anti-HCV activity of
these nucleosides paralleled the cytotoxicity (CC ) in the replicon system.
50
To further evaluate the specific antiviral effect over a longer exposure time,
replicon cells were kept in culture for 7 days, either in the presence or in
absence of the compound 1 (at 100 µM), 2 (at 40 µM), 4 (at 100 µM), or
Interferon α (100 IU/ml) (Figure 2).

8
8
B. Kwon-Chun et al.
TABLE 1 Anti-HCV activity and cytotoxicity of the prepared compounds in the HCV replicon system
EC90(µM)1
CC50(µM)2
IC50(µM)3
Replicon
(Huh7)
Replicon
(Huh7)
Compounds
Huh7
HepG2
PBM
CEM
Vero
1
2
3
4
5
6
7
8
9
79.8
ND
49.6
57.4
42.2
28.0
22.3
ND
66.7
ND
ND
ND
ND
ND
ND
ND
>81
49.2
>100
>100
16.1
ND
33.5
>100
ND
ND
50.2
ND
ND
ND
>100
6.8
13.8
0.33
13.5
4.4
>100
>100
>100
3.5
ND
ND
ND
ND
ND
ND
13.0
36.7
11.9
41.8
3.5
4.0
67.3
8.4
>100
8.7
52.3
74.6
24.8
19.5
50.5
14.5
35.5
3.3
3.7
>100
2.9
>100
9.4
37.5
37.1
22.4
72.1
22.1
4
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
10
11
12
13
14
ND
ND
ND
ND
1EC90(µM) is a concentration that reduces 90% of the viral RNA in the replicon system cell.
2
50
CC (µM) is a concentration that reduces 50% of cellular mRNA in the replicon system cell.
3
4
50
IC (µM) is a concentration that reduces 50% of cellular growth.
Not determined.
FIGURE 2 Comparison of the effect of compounds 1, 2, 4, and interferon on HCV RNA and cellular
growth in a HCV replicon system over 7 days.

ꢁ
5
,9-Anhydro-3-Purine-Isonucleosides
89
Compounds 2 achieved more than 1 log reduction in HCV RNA in the
replicon system which sustained for 7 days while interferon α also achieved
a similar viral RNA reduction but failed to sustain the suppression. However,
this experiment also showed that the compounds tested were cytostatic
at the given concentration, which is less apparent for interferon. Similar
results were obtained with other synthesized compounds (data not shown).
These observations suggested that the apparent anti-HCV activity might be
associated with the cytostatic effects of these compounds in the replicon
system. Most of the compounds showed certain degree of cytotoxicity in
other cell lines (Table 1).
ꢁ
In conclusion, we synthesized 5 ,9-anhydro-purine-isonucleosides
(
3–14), and evaluated their anti-HCV activity and cytotoxicity in a replicon
cell system and other cell lines. It appears that the reduction of the HCV
RNA in the replicon system by the synthesized compounds (2–14) may be
attributed to their cytostatic effects.
EXPERIMENTAL
General
Nuclear magnetic resonance spectra were recorded on a Varian Unity
Plus 400 spectrometer (Palo Alto, CA, USA) at room temperature, with
tetramethylsilane as an internal standard. Chemical shifts (δ) are reported
in parts per million (ppm), and signals are reported as s (singlet), d
(
doublet), t (triplet), q (quartet), m (multiplet), or br s (broad singlet).
Values given for coupling constants are first order. UV spectra were
recorded on a Varian CARY 50 Bio UV-visible spectrophotometer. Fast atom
bombardment mass spectroscopy was performed by the Emory University
Mass Spectrometry Center (Palo Alto, CA, USA). TLC was performed on
Uniplates (silica gel) purchased from Analtech Co. (Newark, DE, USA),
and column chromatography was performed using silica gel (60 A˚) from
Sorbent Technologies (Atlanta, GA, USA).
ꢁ
6
-Amino-5 ,9-anhydro-3-(2,3-O-isopropylidene-β-D-ribofuranosyl)-9H-
purine-2(3H)-one (17) Method 1: A mixture of 15 (5.0 g, 16.33 mmol) and
Lawesson’s reagent (10.0 g, 24.72 mmol) in anhydrous 1,2-dichloroethane
(250 mL) was refluxed for 15 hours and concentrated to dryness. The
residue was purified by silica gel column chromatography (CHCl :MeOH =
3
3
0:1 to 10:1) to give 16 (2.5 g, 47%), which was dissolved in methanolic
◦
ammonia (50 mL), heated at 90 C in a sealed reaction flask for 24 hours
and concentrated to dryness. The residue was purified by silica gel column
chromatography (CHCl :MeOH = 10:1) to give 17 (1.5 g, 63% from 16).
3
11
Method 2: A mixture of 18 (3.7 g, 6.21 mmol) and n-butylamine (10 mL)
in methanol (30 mL) was stirred at rt for 2 days and concentrated to dryness.
The residue was stirred in ethyl acetate (30 mL) for 30 minutes, filtered,

9
0
B. Kwon-Chun et al.
and the obtained solid refluxed in 2,2-dimethoxypropane (10 mL)-DMF
50 mL) in the presence of catalytic amount of TsOH for 1 hour. After
(
cooling and concentration in vacuo, the residue was purified by silica gel
column chromatography (CHCl :MeOH = 10:1 to 5:1) to give 19 (2.0 g,
3
9
9%, crude). To a solution of compound 19 (2.0 g, 6.19 mmol) and
triphenylphosphine (2.4 g, 9.15 mmol) in anhydrous DMF (10 mL) was
added diethyl azodicarboxylate (1.5 mL, 9.28 mmol) slowly. The reaction
mixture was stirred at rt for 30 minutes, concentrated after the reaction
was quenched with water (0.5 mL), and purified by silica gel column
chromatography (CHCl :MeOH = 10:1) to give 17 (1.5 g, 79% from 18):
3
1
H NMR (DMSO-d ) δ 7.71 (s, 1H, H-8), 7.56 (d, 2H, J = 6.8 Hz, H N),
6
2
ꢁ
ꢁ
ꢁ
6
2
.42 (s, 1H, H-1 ), 4.84 (d, 1H, J = 6.0 Hz, H-2 or 3 ), 4.76 (d, 1H, J =
ꢁ
ꢁ
.8 Hz, H-4 ), 4.71 (d, 1H, J = 14.0 Hz, H-5 ), 4.56 (d, 1H, J = 5.6 Hz,
ꢁ
ꢁ
ꢁꢁ
H-2 or 3 ), 4.19 (dd, 1H, J = 3.6, 13.6 Hz, H-5 ), 1.45 (s, 3H, CH ), 1.24
3
(
s, 3H, CH ).
3
ꢁ
6
-Amino-5 ,9-anhydro-3-(β-D-ribofuranosyl)-9H-purine-2(3H)-one-HCl
salt (4) 17 (1.5 g, 4.91 mmol) was dissolved in 0.5 N HCl (20 mL), stirred at
rt for 15 hours, concentrated, and co-evaporated with toluene to dryness.
The obtained solid was triturated with methanol and the crystalline
compound 4 precipitated was collected by filtration and dried under high
vacuum (1.3 g, 88%): UV λ
245 (shoulder), 287 nm (peak) (MeOH);
max
1
H NMR (DMSO-d ) δ 13.00 (br s, 1H, HN), 9.77 (s, 1H, HN), 8.68 (s, 1H,
6
ꢁ
ꢁ
HN), 8.04 (s, 1H, H-8), 6.00 (s, 1H, H-1 ), 5.77 (br s, 1H, HO-2 ), 5.43 (br s,
ꢁ
ꢁ
ꢁ
1
4
(
1
H, HO-3 ), 4.73 (d, 1H, J = 14.0 Hz, H-5 ), 4.55 (t, 1H, J = 4.0 Hz, H-4 ),
ꢁ
ꢁ
ꢁ
.48 (dd, 1H, J = 3.6, 13.6 Hz, H-5 ), 4.14 (t, 1H, J = 4.8 Hz, H-3 ), 4.11
ꢁ
13
d, 1H, J = 4.8 Hz, H-2 ); C NMR (DMSO-d ) δ 152.25, 146.52, 141.53,
6
41.25, 111.55, 94.08, 82.52, 73.86, 70.96, 52.81; HRFABMS estimated
−
3
00.0500 for C H N O Cl (M-H) , observed 300.0487.
10
12
5
4
ꢁ
5
,9-Anhydro-1-cyanomethyl-3-(β-D-ribofuranosyl)-9H-purine-2,6(1H,
3
H)-dione (3) To a solution of 15 (140 mg, 0.46 mmol) in DME (4 mL)-DMF
◦
(4 mL) was added NaH (28 mg, 0.69 mmol) at 0 C. The mixture was
◦
stirred at 0 C for 10 minutes. Then LiBr (80 mg, 0.92 mmol) and
bromoacetonitrile (64 µL, 0.92 mmol) were added in 10-minute interval.
◦
The resulting mixture was then heated at 65 C for 2 hours and concentrated
to dryness. The residue was purified by silica gel column chromatography
(
CHCl :MeOH = 30:1) to give a N 1-cyanomethyl intermediate as a white
3
solid, which was dissolved in 0.5 N HCl (3 mL), stirred for 2 hours,
concentrated, and co-evaporated with toluene to dryness. Upon triturating
the residue with MeOH, the crystalline compound 3 precipitated was
collected by filtration and dried under high vacuum (60 mg, 43%): UV λ
1
2
36, 266 nm (MeOH); H NMR (DMSO-d ) δ 7.84 (s, 1H, H-8), 6.08 (s,
max
6
ꢁ
ꢁ
ꢁ
1
4
1
H, H-1 ), 5.73 (d, 1H, J = 4.4 Hz, HO-2 ), 5.36 (d, 1H, J = 7.2 Hz, HO-3 ),
ꢁ
.83 (d, 2H, J = 2.0 Hz, CH CN), 4.67 (d, 1H, J = 14.0 Hz, H-5 ), 4.53 (t,
2
ꢁ
ꢁꢁ
H, J = 3.6 Hz, H-4 ), 4.42 (dd, 1H, J = 3.6, 13.6 Hz, H-5 ), 4.16–4.10 (m,

ꢁ
5
,9-Anhydro-3-Purine-Isonucleosides
91
ꢁ
ꢁ
13
2
H, H-2 , 3 ); C NMR (DMSO-d ) δ 155.75, 148.08, 139.11 (2C), 116.67,
6
1
16.24, 94.40, 82.74, 74.28, 71.09, 52.34, 28.78.
ꢁ
5
,9-Anhydro-6-methylamino-3-(β-D-ribofuranosyl)-9H-purine-2(3H)-
one-HCl salt (5) To a solution of 15 (250 mg, 0.82 mmol), 4-(dimethyl-
amino)pyridine (200 mg, 1.64 mmol), and Et N (2 mL) in anhydrous
3
CH CN (4 mL) was added 2,4,6-triisopropylbenzenesulfonyl chloride
3
(223 mg, 0.74 mmol) at rt. The reaction mixture was stirred at rt for 7
hours and then 40% aqueous CH NH (2 mL) was added. The resulting
3
2
mixture was stirred at rt for 15 hours, concentrated, and co-evaporated with
toluene to dryness. The obtained residue was purified by silica gel column
chromatography (CHCl :MeOH = 30:1 containing 1% Et N) to give a
3
3
6
N -methyl derivative, which was dissolved in 0.5 N HCl (2 mL), stirred at
rt for 15 hours, concentrated, and co-evaporated with toluene to dryness.
Upon triturating the residue with methanol, the crystalline compound
5
precipitated was collected by filtration and dried under high vacuum
1
(82 mg, 32% from 15): UV λ max 290 nm (MeOH); H NMR (DMSO-d )
6
ꢁ
δ 9.87 (s, 1H, HN), 7.59 (s, 1H, H-8), 5.98 (s, 1H, H-1 ), 5.58 (d, 1H, J =
ꢁ
ꢁ
4
.4 Hz, HO-2 ), 5.31 (d, 1H, J = 6.8 Hz, HO-3 ), 4.57 (d, 1H, J = 13.6 Hz,
ꢁ
ꢁ
ꢁꢁ
H-5 ), 4.44 (m, 1H, H-4 ), 4.29 (dd, 1H, J = 3.2, 13.2 Hz, H-5 ), 4.11 (m,
ꢁ
ꢁ
13
1
1
2
3
H, H-3 ), 3.98 (m, 1H, H-2 ), 3.60 (s, 3H, CH ); C NMR (DMSO-d ) δ
3 6
51.06, 147.11, 140.48, 139.32, 111.81, 93.92, 82.44, 73.89, 70.88, 52.54,
+
9.16; HRFABMS estimated 316.1900 for C H N O Cl (M+H) , observed
11
15
5
4
16.2051.
ꢁ
5
,9-Anhydro-3-(β-D-ribofuranosyl)-6-thio-9H-purine-2(3H)-one (6) 16
(34 mg, 0.11 mmol) was dissolved in 0.5 N HCl (1 mL), stirred at rt for
2
hours, concentrated, and co-evaporated with toluene to dryness. Upon
triturating the residue with MeOH, the crystalline product 6 precipitated
was collected by filtration and dried under high vacuum (15 mg, 48%): UV
1
λ
256, 341 nm (MeOH); H NMR (DMSO-d ) δ 12.39 (s, 1H, HN or
max
6
ꢁ
ꢁ
HS), 7.76 (s, 1H, H-8), 6.01 (s, 1H, H-1 ), 5.68 (br s, 1H, HO-2 ), 5.35 (br s,
ꢁ
ꢁ
ꢁ
1
1
H, HO-3 ), 4.58 (d, 1H, J = 13.6 Hz, H-5 ), 4.48 (m, 1H, H-4 ), 4.40 (dd,
ꢁ
ꢁ
ꢁ
H, J = 3.6, 14.0 Hz, H-5 ), 4.15 (t, 1H, J = 4.8 Hz, H-3 ), 4.04 (d, 1H,
ꢁ
13
J = 5.2 Hz, H-2 ); C NMR (DMSO-d ) δ 157.33, 148.73, 139.33, 138.28,
6
1
17.51, 93.12, 82.60, 74.41, 71.06, 52.05; HRFABMS estimated 289.0583 for
+
C H N O SLi (M+Li) , observed 289.0595.
10
10
4
4
ꢁ
5
,9-Anhydro-6-methylthio-3-(β-D-ribofuranosyl)-9H-purine-2(3H)-one
(
7) To a mixture of 16 (100 mg, 0.31 mmol) and 0.5 N NaOH (1 mL) was
added MeI (0.1 mL) at rt. The resulting mixture was stirred at rt for 30
minutes and concentrated to dryness. The residue was purified by silica
6
gel column chromatography (CHCl :MeOH = 30:1) to give a S -methyl
3
1
thioxanthine derivative (54 mg, 52%): UV λ
271, 316 nm (MeOH); H
max
ꢁ
NMR (DMSO-d ) δ 7.86 (s, 1H, H-8), 6.43 (s, 1H, H-1 ), 4.86 (d, 1H, J =
6
ꢁ
ꢁ
ꢁ
6
.4 Hz, H-2 or 3 ), 4.84 (d, 1H, J = 3.6 Hz, H-4 ), 4.77 (d, 1H, J = 14.0 Hz,
ꢁ
ꢁ
ꢁ
H-5 ), 4.74 (d, 1H, J = 6.0 Hz, H-2 or 3 ), 4.28 (dd, 1H, J = 4.0, 14.0 Hz,

9
2
B. Kwon-Chun et al.
H-5 ), 2.51 (s, 3H, CH S), 1.47 (s, 3H, CH ), 1.24 (s, 3H, CH ); 13C NMR
ꢁ
ꢁ
3
3
3
(
DMSO-d ) δ 169.00, 151.54, 139.98, 139.90, 121.93, 112.49, 91.69, 84.96,
6
84.50, 81.21, 51.92, 26.14, 24.59, 11.35. The intermediate was dissolved
in 75% trifluoroacetic acid (2 mL) and stirred at rt for 24 hours. Upon
concentration, the residue was co-evaporated with toluene and purified by
silica gel column chromatography (CHCl :MeOH = 10:1) to give 7 (37 mg,
3
1
4
0% from 16): UV λ
223, 270, 316 nm (MeOH); H NMR (DMSO-d )
max
6
ꢁ
ꢁ
δ 7.87 (s, 1H, H-8), 6.05 (s, 1H, H-1 ), 5.75 (d, 1H, J = 4.8Hz, HO-2 ),
ꢁ
ꢁ
5
1
.30 (d, 1H, J = 7.6 Hz, HO-3 ), 4.63 (d, 1H, J = 13.6 Hz, H-5 ), 4.49 (t,
ꢁ
ꢁ
ꢁꢁ
H, J = 4.0 Hz, H-4 ), 4.42 (dd, 1H, J = 4.0, 13.6 Hz, H-5 ), 4.12 (dt, 1H,
ꢁ
J = 4.8, 7.2 Hz, H-3 ), 4.00 (d, 1H, J = 4.4 Hz, H-2 ), 2.48 (s, 3H, CH S);
3
13
C NMR (DMSO-d ) δ 168.19, 151.32, 139.38 (2C), 121.74, 93.90, 82.33,
6
7
3.95, 71.08, 52.02, 11.09; HRFABMS estimated 297.0658 for C H N O S
11
13
4
4
+
(
M+H) , observed 297.0643.
ꢁ
5
,9-Anhydro-8-hydroxy-3-(β-D-ribofuranosyl)xanthine (8) To a suspen-
sion of compound 20 (50 mg, 0.17 mmol) in anhydrous dioxane (1 mL)-
DMF (0.5 mL) was added carbonyl diimidazole (100 mg, 0.62 mmol) at
rt. The mixture was stirred for 1 hour and additional carbonyl diimidazole
(100 mg, 0.62 mmol) added. Then the mixture was stirred for 15 hours at
rt, concentrated, and co-evaporated with toluene. Th residue was washed
with methanol to remove imidazole and dried to give compound 21 as a
white solid. Compound 21 was dissolved in 70% aqueous trifluoroacetic
acid (1 mL), stirred for 15 hours, concentrated, co-evaporated with toluene,
and the residue was triturated with methanol. The precipitated crystals (8)
were collected by filtration and dried to give in vacuo (24 mg, 50%): UV
1
λ
289 nm (MeOH); H NMR (DMSO-d ) δ 11.27 (s, 1H, NH or OH),
max
6
ꢁ
ꢁ
1
(
1
1.19 (s, 1H, NH or OH), 5.99 (s, 1H, H-1 ), 5.59 (s, 1H, HO-2 ), 5.33
ꢁ
ꢁ
ꢁ
s, 1H, HO-3 ), 4.44 (s, 1H, H-4 ), 4.18 (m, 1H, H-3 ), 4.06 (d, 1H, J =
ꢁ
ꢁ
4.0 Hz, H-5 ), 4.01 (d, 1H, J = 4.4 Hz, H-2 ), 3.78 (d, 1H, J = 14.0 Hz,
ꢁ
ꢁ
13
H-5 ); C NMR (DMSO-d ) δ 152.89, 151.47, 148.18, 135.64, 127.87, 93.49,
6
8
2.58, 74.46, 71.08, 50.06. HRFABMS estimated 283.0679 for C H N O
10 11 4 6
+
(
M+H) , found 283.0678.
ꢁ
5
,9-Anhydro-8-methyl-3-(β-D-ribofuranosyl)xanthine (9) A mixture of
compound 20 (300 mg, 1.01 mmol), p-toluenesulfonic acid monohydrate
(
30 mg, 0.16 mmol), and triethyl orthoformate (3 mL, 17.91 mmol) in N,N -
◦
dimethylformamide (6 mL) was heated at 90 C for 1 hour. After cooling
to rt, the reaction mixture was treated with sodium bicarbonate powder
(30 mg), concentrated, and the residue was purified by silica gel column
chromatography (CH Cl :MeOH = 10:1) to give compound 22 (197 mg,
2
2
6
1%). 22 was then treated with 0.5 N HCl (2 mL), stirred for 15 hours,
concentrated in vacuo, and the residue was co-evaporated with toluene
to give a solid. The solid was triturated with methanol, filtered, and the
collected crystal dried under high vacuum to give compound 9 (138 mg,
1
8
0% from 22): UV λmax 237, 272 nm (H O); H NMR (DMSO-d ) δ 11.14
2
6

ꢁ
5
,9-Anhydro-3-Purine-Isonucleosides
93
ꢁ
ꢁ
ꢁ
(
4
1
s, 1H, NH), 6.02 (s, 1H, H-1 ), 5.71 (br s, 1H, HO-2 ), 5.31 (br s, 1H, HO-3 ),
ꢁ
ꢁ
.49 (m, 1H, H-4 ), 4.43 (d, 1H, J = 13.2 Hz, H-5 ), 4.30 (dd, 1H, J = 3.6,
ꢁ
ꢁ
ꢁ
ꢁ
3.2Hz, H-5 ), 4.19 (m, 1H, H-3 ), 4.02 (d, 1H, J = 4.8, H-2 ), 2.36 (s, 3H,
13
CH ); C NMR (DMSO-d ) δ 157.12, 148.69, 145.76, 139.76, 115.63, 93.47,
3
6
8
2.27, 74.18, 71.34, 51.79, 14.02.
ꢁ
5
,9-Anhydro-3-(β-D-2,3-O-isopropylidene ribofuranosyl)-8-phenylxant-
hine (23) A mixture of compound 20 (100 mg, 0.34 mmol), benzaldehyde
(
(
50 µL, 0.50 mmol), and acetic acid (30 µL, 0.524 mmol) in methanol
5 mL) was stirred at rt for 30 minutes and concentrated to dryness.
The residue was purified by silica gel column chromatography
CH Cl :MeOH = 10:1) to give an imine intermediate, which was
(
2
2
then dissolved in thionyl chloride (3 mL). The solution was stirred for
3
2
0 minutes and concentrated to dryness. The residue was dissolved in
,2-dimethoxypropane (3 mL), heated at 90 C for 1 hour for reprotection,
◦
and cooled to rt. The precipitated crystals (23) were collected by filtration
1
(
69 mg, 53%): UV λmax 273 nm (MeOH); H NMR (DMSO-d ) δ 11.35 (s,
6
ꢁ
1
5
4
1
H, NH), 7.66 (m, 2H, phenyl), 7.52 (m, 3H, phenyl), 6.37 (s, 1H, H-1 ),
ꢁ
ꢁ
ꢁ
ꢁ
.04 (d, 1H, J = 6.0 Hz, H-2 or 3 ), 4.87 (d, 1H, J = 6.0 Hz, H-2 or 3 ),
ꢁ
ꢁ
.74 (m, 1H, H-4 ), 4.54 (d, 1H, J = 14.4 Hz, H-5 ), 4.13 (dd, 1H, J = 2.8,
ꢁ
ꢁ
13
4.4 Hz, H-5 ), 1.43 (s, 3H, CH ), 1.25 (s, 3H, CH ); C NMR (DMSO-d )
3
3
6
δ 157.09, 149.09, 148.19, 141.67, 129.71, 129.49, 129.18, 128.68, 117.22,
1
11.83, 90.56, 84.56, 83.33, 80.99, 52.96, 25.82, 24.26; HRFABMS estimated
+
383.1355 for C H N O (M+H) , found 383.1360.
1
9
19
4
5
ꢁ
5
,9-Anhydro-8-phenyl-3-(β-D-ribofuranosyl)xanthine (10) To a solution
of compound 23 (60 mg, 0.157 mmol) in methanol (5 mL) was added c-HCl
30 µL). The resulting solution was evaporated under reduced pressure.
(
The residue was dissolved methanol (5 mL) and the resulting solution was
concentrated under reduced pressure, which was repeated additional two
times. The resulting residue was triturated with methylene chloride to give
a precipitated crystals (10), which were collected by filtration and dried in
1
vacuo (42 mg, 78%): UV λmax 272 nm (MeOH); H NMR (DMSO-d ) δ 11.33
6
(
1
s, 1H, NH), 7.71–7.69 (m, 2H, phenyl), 7.55–7.53 (m, 3H, phenyl), 6.11 (s,
ꢁ
ꢁ
ꢁ
H, H-1 ), 4.55 (dd, 1H, J = 3.6, 13.6 Hz, H-5 ), 4.47 (m, 1H, H-4 ), 4.33
ꢁ
ꢁ
ꢁ
(
5
1
d, 1H, J = 13.2Hz, H-5 ), 4.24 (t, 1H, J = 4.8 Hz, H-3 ), 4.09 (d, 1H, J =
ꢁ
13
.2 Hz, H-2 ); C NMR (DMSO-d ) δ 157.16, 148.74, 146.98, 140.50, 129.69,
6
29.49, 129.45, 128.61, 117.01, 93.27, 82.64, 74.47, 70.67, 53.48; HRFABMS
+
estimated 343.1042 for C H N O (M+H) , found 343.1055.
16
15
4
5
ꢁ
5
,9-Anhydro-8-chloro-3-(β -D-2,3-O-isopropylidene ribofuranosyl)
xanthine (24) A mixture of compound 15 (100 mg, 0.33 mmol), N -
chlorosuccinimide (44 mg, 0.33 mmol), and acetic acid (2 mL) in dioxane
(10 mL) was stirred at rt for 15 hours, concentrated, co-evaporated with
toluene, and the residue was purified by silica gel column chromatography
(
chloroform:methanol = 30:1) to give compound 24 (93 mg, 84%) as a
1
white solid: UV λ
230, 268 nm (MeOH); H NMR (DMSO-d ) δ 11.09
max
6

9
4
B. Kwon-Chun et al.
ꢁ
ꢁ
ꢁ
(
(
br s, 1H, NH), 6.32 (s, 1H, H-1 ), 4.94 (d, 1H, J = 6.0 Hz, H-2 or 3 ), 4.84
ꢁ
ꢁ
ꢁ
d, 1H, J = 6.0 Hz, H-2 or 3 ), 4.82 (d, 1H, J = 2.8 Hz, H-4 ), 4.63 (d, 1H,
ꢁ
ꢁꢁ
J = 13.6 Hz, H-5 ), 4.18 (dd, 1H, J = 3.6, 14.0 Hz, H-5 ), 1.44 (s, 3H, CH ),
3
13
1
.24 (s, 3H, CH ); C NMR (DMSO-d ) δ 156.23, 148.70, 140.75, 133, 50,
3 6
1
15.78, 112.23, 91.08, 84.41, 83.47, 81.23, 52.83, 25.97, 24.43; HRFABMS
+
estimated 347.0728 for C H N O ClLi (M+Li) , found 347.0735.
1
3
13
4
5
ꢁ
5
,9-Anhydro-8-chloro-3-(β-D-ribofuranosyl)xanthine (11) Compound 24
(70 mg, 0.21 mmol) was treated with 80% trifluoroacetic acid at rt for 3
hours, concentrated, and co-evaporated with toluene to give a white solid,
which was then triturated with methanol. The precipitated crystals (11) were
1
collected by filtration and dried under high vacuum (25 mg, 41%): H NMR
ꢁ
ꢁ
(
(
1
DMSO-d ) δ 11.38 (s, 1H, NH), 6.00 (s, 1H, H-1 ), 4.50 (m, 1H, H-4 ), 4.43
6
ꢁ
ꢁꢁ
d, 1H, J = 13.6 Hz, H-5 ), 4.34 (dd, 1H, J = 3.2, 13.6 Hz, H-5 ), 4.25 (t,
ꢁ
ꢁ
H, J = 5.2 Hz, H-3 ), 4.04 (d, 1H, J = 5.2 Hz, H-2 ), 3.80 (br s, 2H, 2HO);
1
3
C NMR (DMSO-d ) δ 156.37, 148.29, 139.98, 132.47, 115.66, 93.55, 81.83,
6
7
3.97, 71.03, 53.19.
ꢁ
5
,9-Anhydro-8-bromo-3-(β-D-ribofuranosyl)xanthine (12) A mixture of
compound 15 (36 mg, 0.12 mmol) and NBS (32 mg, 0.18 mmol) in dioxane
2 mL) was stirred at rt for 2 hours, concentrated, and the residue purified
(
by silica gel column chromatography (chloroform:methanol = 30:1) to give
1
compound 25: H NMR (DMSO-d ) δ 11.42 (s, 1H, NH), 6.33 (s, 1H, H-
6
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
1
3
(
), 4.92 (d, 1H, J = 5.6 Hz, H-2 or 3 ), 4.84 (d, 1H, J = 6.0 Hz, H-2 or
ꢁ
ꢁ
), 4.81 (d, 1H, J = 2.4 Hz, H-4 ), 4.62 (d, 1H, J = 14.0 Hz, H-5 ), 4.17
ꢁ
ꢁ
13
dd, 1H, J = 3.6, 14.0 Hz, H-5 ), 1.44 (s, 3H, CH ), 1.23 (s, 3H, CH );
C
3
3
NMR (DMSO-d ) δ 156.09, 148.60, 140.89, 122.44, 117.58, 111.95, 90.88,
6
8
4.26, 83.26, 81.09, 53.74, 25.81, 24.27; HRFABMS estimated 391.0229 for
+
C H N O BrLi (M+Li) , observed 391.0245.
13
13
4
5
Then compound 25 was dissolved in 70% trifluoroacetic acid (1 mL),
stirred for 15 hours, and concentrated to dryness. The residue was co-
evaporated with toluene and triturated with methanol. The precipitated
crystals (12) were collected by filtration and dried in vacuo (20 mg, 48%
1
from 15). UVλmax 244, 268 nm (MeOH); H NMR (DMSO-d ) δ 11.36
6
ꢁ
ꢁ
(
s, 1H, NH), 6.00 (s, 1H, H-1 ), 5.68 (br s, 1H, HO-2 ), 5.35 (br s, 1H,
ꢁ
ꢁ
ꢁ
HO-3 ), 4.50 (t, 1H, J = 3.6 Hz, H-4 ), 4.42 (d, 1H, J = 13.2 Hz, H-5 ),
ꢁ
ꢁ
ꢁ
4
(
.33 (dd, 1H, J = 3.6, 13.2 Hz, H-5 ), 4.23 (t, 1H, J = 4.4 Hz, H-3 ), 4.04
ꢁ
13
d, 1H, J = 4.8 Hz, H-2 ); C NMR (DMSO-d ) δ 156.30, 148.35, 140.30,
6
1
3
21.55, 117.57, 93.53, 81.83, 73.99, 71.02, 34.30; HRFABMS estimated
+
50.9916 for C H N O BrLi (M+Li) , observed 350.9916 (the same as
10
9
4
5
estimated).
ꢁ
5
,9-Anhydro-8-iodo-3-(β-D -2,3-O-isopropylidene ribofuranosyl)xanth-
ine (26) A mixture of compound 15 (200 mg, 0.65 mmol) and NIS (300 mg,
◦
1
.27 mmol) in anhydrous dioxane (20 mL) was heated at 90 C for 24 hours,
concentrated, and the residue purified by silica gel column chromatography
chloroform:methanol = 30:1) to give compound 26 (140 mg, 50%): UV
(

ꢁ
5
,9-Anhydro-3-Purine-Isonucleosides
95
1
λ
245, 270 nm (MeOH); H NMR (DMSO-d ) δ 11.34 (s, 1H, NH),
max
6
ꢁ
ꢁ
ꢁ
ꢁ
6
.31 (s, 1H, H-1 ), 4.84 (d, 1H, J = 5.6 Hz, H-2 or 3 ), 4.80 (m, 2H, H-4 ,
ꢁ
ꢁ
ꢁ
2
or 3 ), 4.54 (d, 1H, J = 14.0 Hz, H-5 ), 4.11 (dd, 1H, J = 3.6, 14.0 Hz,
ꢁ
ꢁ
13
H-5 ), 1.42 (s, 3H, CH ), 1.21 (s, 3H, CH ); C NMR (DMSO-d ) δ 155.61,
3
3
6
1
2
4
48.27, 140.29, 120.25, 111.56, 96.70, 90.46, 83.93, 82.87, 80.76, 55.15, 25.45,
+
3.89; HRFABMS estimated 439.0091 for C H N O ILi (M+Li) , found
13
13
4
5
39.0098.
ꢁ
5
,9-Anhydro-8-iodo-3-(β-D-ribofuranosyl)xanthine (13) Compound 26
(
140 mg, 0.32 mmol) was dissolved in 80% trifluoroacetic acid (1 mL),
stirred for 15 hours, and concentrated to dryness. The resulting residue was
co-evaporated with toluene and triturated with methanol. The precipitated
crystals (13) were collected by filtration and dried in vacuo (35 mg, 28%).
The another crop (30 mg, 24%) was obtained from mother liquor: UV λ
max
1
2
46, 270 nm (MeOH); H NMR (DMSO-d ) δ 11.29 (s, 1H, NH), 6.01 (s, 1H,
6
ꢁ
ꢁ
ꢁ
H-1 ), 4.50 (t, 1H, H-4 ), 4.37 (d, 1H, J = 13.2 Hz, H-5 ), 4.28 (dd, 1H, J =
ꢁ
ꢁ
ꢁ
4.0, 13.2 Hz, H-5 ), 4.18 (t, 1H, J = 4.80 Hz, H-3 ), 4.03 (d, 1H, J = 5.2 Hz,
ꢁ
13
H-2 ); C NMR (DMSO-d ) δ 156.16, 148.34, 140.05, 120.50, 96.27, 93.45,
6
8
1.88, 74.02, 71.02, 56.10; HRFABMS estimated 398.9778 for C H N O ILi
10 9 4 5
+
(
M + Li) , found 398.9785.
ꢁ
5
,9-Anhydro-8-bromo-3-(β-D-ribofuranosyl)-2-oxoadenine (14) A mix-
ture of compound 4 (100 mg, 0.33 mmol) and NBS (89 mg, 0.498 mmol)
in dioxane-water (10:4 mL) was stirred at rt for 2 hours, concentrated, and
co-evaporated with toluene (5 mL × 2). The residue was triturated with
chloroform-methanol (30:1) and filtered through filter paper. The white
crystal was washed with chloroform-methanol (10:1), filtered, and dried
in vacuo to afford compound 14 (67 mg, 53%). An additional amount
of compound 14 (21 mg, 17%) was recovered from the filtrate: UV λ
max
1
2
5
4
5
9
87 nm (MeOH); NMR (DMSO-d ) δ 9.87 (s, 1H, NH), 8.71 (s, 1H, NH),
6
ꢁ
ꢁ
ꢁ
.99 (s, 1H, H-1 ), 4.54 (m, 1H, H-4 ), 4.53 (dd, 1H, J = 5.6, 13.6 Hz, H-5 ),
ꢁ
ꢁ
ꢁ
.43 (dd, 1H, J = 4.4, 13.6 Hz, H-5 ), 4.24 (m, 1H, H-3 ), 4.08 (d, 1H, J =
ꢁ
13
.2 Hz, H-2 ); C NMR (DMSO-d ) δ 146.33, 142.05, 136.84, 125.26, 109.86,
6
4.50, 81.83, 73.50, 70.93, 53.95.
Anti-HCV evaluation. HCV-replicon RNA-containing Huh7 cells (Clone
A cells; Apath, LLC, St. Louis, MO, USA) were kept in exponential growth
in DMEM media (high glucose, no pyruvate) containing 10% fetal bovine
serum, 1X nonessential amino acids, penicillin-streptomycin-glutamine
(
100 units/L, 100 g/L, and 2.92 mg/L, respectively) and G418 (500–1000
19
g/mL). Antiviral assays were performed in the same media without G418.
Cells were seeded in a 96-well plate at 1000 cells per well and test compounds
were added immediately after seeding. After 96 hrs of incubation, total
cellular RNA was isolated (Rneasy 96 kit, Qiagen, Valencia, CA, USA). HCV
RNA and an internal control (TaqMan Ribosomal RNA control Reagents,
Applied Biosystems, Foster City, CA, USA) were amplified in a single-step

9
6
B. Kwon-Chun et al.
multiplex RT-PCR protocol, as recommended by the manufacturer. The
[
12]
HCV primers and probe used have been described previously.
To express the antiviral effectiveness of a compound, the threshold RT-
PCR cycle of the test compound was subtracted from the average threshold
ꢁ
RT-PCR cycle of the ‘no drug control and the concentrate generating a 1-log
reduction (i.e., EC ) in replicon RNA levels was calculated. The cytotoxicity
90
of the test compound was also determined by calculating the effect on
[
20]
ribosomal RNA levels.
Cytotoxicity testing using MTS was performed as
described previously.[
21,22]
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