Synthesis of 6-arylthio analogs of 2′,3′-dideoxy-3′- fluoroguanosine and their effect against hepatitis B virus replication

Article abstract of DOI:10.1080/15257770600686394

A key compound, 2-amino-6-chloro-9-(2,3-dideoxy-3-fluoro-β-D- erythro-pentofuranosyl)purine, was prepared from 2-amino-6-chloropurine riboside in 5 steps, then subjected to the nucleophilic displacement with benzenethiols to afford 6-arylthio congeners. These compounds showed a similar anti-HBV effect to that of 2′,3′-dideoxy-3′-fluoroguanosine. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770600686394

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Synthesis of 6-Arylthio Analogs of
2′,3′-Dideoxy-3′-Fluoroguanosine and
Their Effect against Hepatitis B Virus
Replication
Takayoshi Torii a , Tomoyuki Onishi a , Kunisuke Izawa a , Tokumi
Maruyama b , Yosuke Demizu b , Johan Neyts c & Erik De Clercq c
a AminoScience Laboratories, Ajinomoto Co., Inc., Kawasaki-ku,
Kawasaki, Kanagawa, Japan
b Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima
Bunri University, Sanuki-City, Kagawa, Japan
c Rega Institute for Medical Research, Katholieke Universiteit
Leuven, Leuven, Belgium
Version of record first published: 07 Feb 2007.
To cite this article: Takayoshi Torii , Tomoyuki Onishi , Kunisuke Izawa , Tokumi Maruyama , Yosuke
Demizu , Johan Neyts & Erik De Clercq (2006): Synthesis of 6-Arylthio Analogs of 2′,3′-Dideoxy-3′-
Fluoroguanosine and Their Effect against Hepatitis B Virus Replication, Nucleosides, Nucleotides and
Nucleic Acids, 25:4-6, 655-665
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Nucleosides, Nucleotides, and Nucleic Acids, 25:655–665, 2006
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770600686394
SYNTHESIS OF 6-ARYLTHIO ANALOGS OF
2’,3’-DIDEOXY-3’-FLUOROGUANOSINE AND THEIR EFFECT
AGAINST HEPATITIS B VIRUS REPLICATION
Takayoshi Torii, Tomoyuki Onishi, and Kunisuke Izawa
AminoScience
2
Laboratories, Ajinomoto Co., Inc., Kawasaki-ku, Kawasaki, Kanagawa, Japan
Tokumi Maruyama and Yosuke Demizu Faculty of Pharmaceutical Sciences at
2
Kagawa Campus, Tokushima Bunri University, Sanuki-City, Kagawa, Japan
Johan Neyts and Erik De Clercq
Rega Institute for Medical Research, Katholieke
2
Universiteit Leuven, Leuven, Belgium
A key compound, 2-amino-6-chloro-9-(2,3-dideoxy-3-fluoro-β-D-erythro-pentofuranosyl)purine,
2
was prepared from 2-amino-6-chloropurine riboside in 5 steps, then subjected to the nucleophilic
displacement with benzenethiols to afford 6-arylthio congeners. These compounds showed a similar
anti-HBV effect to that of 2ꢁ,3ꢁ-dideoxy-3 ꢁ-fluoroguanosine.
Keywords FddG analogs; Benzenethiol; Anti-HBV activity
INTRODUCTION
The hepatitis B virus (HBV), an hepadnavirus, was identified in 1963
by Blumberg et al.[22] as the causative agent of hepatitis B. Today, there
are an estimated 300 million chronic carriers worldwide. There is a par-
ticular high prevalence in Asia and Africa. Treatment of hepatitis B in-
fection is a challenge of modern medicine. There is an effective vaccine
available against HBV.[1] Antiviral agents are, however, needed for those
patients already infected with HBV. Although interferon α is approved for
Received 30 January 2006; accepted 8 February 2006.
The work in Leuven is part of the activities of the VIRGIL European Network of Excellence on
Antiviral Drug Resistance supported by a grant (LSHM-CT-2004-503359) from the Priority 1 “Life Sciences,
Genomics and Biotechnology for Health” Programme in the 6th Framework Programme of the EU.
This paper is dedicated to Professor Eiko Ohtsuka on the occasion of her 70th birthday.
Address correspondence to Tokumi Maruyama, Faculty of Pharmaceutical Sciences at Kagawa
Campus, Tokushima Bunri University, 1314-1 Shido, Sanuki City, Kagawa, 769-2193, Japan. E-mail:
maruyama@kph.bunri-u.ac.jp
655

656
T. Torii et al.
the treatment of chronic HBV infection, it is effective in only 10–30% of
treated patients.[2,3] Several nucleoside reverse transcriptase inhibitors are
effective against both HIV and HBV. Lamivudine acts as an inhibitor of HBV
DNA polymerase as well as HIV reverse transcriptase after it has been con-
verted to the 5ꢁ-triphosphate.[4,5] A class of phosphonomethoxyethylpurine
analogs such as 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) and 9-
[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP) show a broad
spectrum of activity against several viruses including HBV.[6] Sekiya et al.
developed a new type of phosphonomethoxyethylpurine analogs, 2-amino-
6-arylthio-9-[2-(phosphonomethoxy)ethyl]purine bis (2,2,2-trifluoroethyl)
esters, as HBV-specific antiviral agents.[7] Recently, 2ꢁ,3ꢁ-dideoxy-3ꢁ-
fluoroguanosine (FddG) has been proved to be a strong inhibitor of duck
hepatitis B virus.[8] We have already reported practical synthetic approaches
to lodenosine (FddA) starting from inosine, in which fluorination of 6-
chloropurine nucleosides has been involved as a key step.[9] In this per-
spective, our current attention was focussed on the synthesis of FddG via
2-amino-6-chloropurine riboside. Also reported here is the antiviral effect of
6-arylthio analogs of FddG against HBV.
SYNTHESIS
Introduction of fluorine at the sugar moiety of nucleosides has been
recognized as important since many fluoronucleosides are biologically ac-
tive and stable by chemical and PNP catalyzed hydrolysis. Three synthetic
methods are classified as follows: (1) Epoxide ring-opening using HF; (2)
nucleophilic displacement by fluoride ion; (3) opening of the anhydro bond
formed between sugar and pyrimidine.[10] However, vigorous conditions are
needed for the reaction due to the relatively poor nucleophilicity of the
fluoride ion. Recently, diethylaminosulfur trifluoride (DAST) has been ex-
tensively used for the introduction of the fluorine into carbohydrate.[11]
For the synthesis of FddG, several approaches have been reported.[12] In
1988, Herdewijn et al. reported the conversion of guanosine to FddG, in
which reaction of 9-(2-deoxy-β-D-threo-pentofuranosyl)guanine with DAST
was involved as a key step.[13] However, the yield was not satisfactory. In our
research to prepare FddA,[9a] introduction of 6-chloro on purine proved
to be efficient to prevent intramolecular nucleophilic attack by 3-nitrogen.
Therefore, 2-amino-6-chloropurine riboside (1)[14] was selected as a start-
ing material for the synthesis of 2-amino-6-chloro-9-(2,3-dideoxy-3-fluoro-β-
D-erythro-pentofuranosyl)purine (6),[15] an intermediary compound for the
6-substituted derivative (Scheme 1). At first, 2ꢁ-O-tosyl compound was pre-
pared as follows. The 2ꢁ,3ꢁ-O-di-n-butylstannylene complex is useful interme-
diate for introducing only one tosyl group at the 2ꢁ-OH.[16] Thus, compound
1 has been successively treated with di-n-butyltin (IV) oxide and excess tosyl

6-Arylthio Analogs and Hepatitis B Virus
657
SCHEME 1
chloride in the presence of triethylamine (Et3N) in MeOH to give the 2ꢁ-O-
tosyl congener[17] (2) as white prisms in 85% yield. This product was sub-
jected to a deoxygenative [1,2]-hydride shift rearrangement which converts
cis-diol monotosylates to inverted secondary alcohols.[18] A solution of 2 in
anhydrous dimethyl sulfoxide (DMSO) under nitrogen was treated with 1 M
lithium triethylborohydride (LTBH) in THF. Since 6-chloro group is suscep-
tible to alkaline condition, the reaction was quenched with acetic acid. After
usual workup and separation using silica gel chromatography, the 2-deoxy-
β-D-threo-pentofuranose 3 was obtained as a colorless solid in 83% yield. To
protect the 5ꢁ-OH and 2-NH2 group,[19] 3 was treated with trityl chloride
in the presence of triethylamine and 4-dimethylaminopyridine (DMAP) to
give the ditrityl congener (4) as a foam in 96% yield. Then, 4 was treated
with DAST in CH2Cl2 at 40◦C for 30 min. After work-up of the solution,
3ꢁ-fluoro derivative (5) was isolated as colorless prisms in 60% yield. The
yield of this step was superior to that of guanine nucleoside (35%).[13] Next,

658
T. Torii et al.
SCHEME 2
the trityl group was removed by acid treatment to give a key intermediary
compound (6) in 69% yield. Several 6-modified derivatives were prepared
by nucleophilic displacement or hydrogenation as follows (Scheme 2): at
first 6 was converted to FddG (7)[13] by treatment with 2-mercaptethanol
and NaOMe in MeOH[20] in 67% yield. Next, catalytic hydrogenation of 6
using 5% Pd-C was achieved to give the 2-aminopurine nucleoside (8)[21]
in 63% yield. When 6 was treated with benzenethiol[7] in the presence of
pyridine in DMF, 6-phenylthio derivative (9) was obtained in 90% yield. 6-(4-
Methoxyphenyl)thio congener (10) was also synthesized in a similar manner
to that of 9 using p-methoxybenzenethiol as a nucleophile.
ANTI-HBV ACTIVITY
The antiviral effects against HBV of the 2ꢁ,3ꢁ-dideoxy-3ꢁ-fluororibosides
6-10 were assayed according to the procedures described in the experimental
section and the results are presented in Table 1. Compound 7 (FddG) ex-
hibited moderate anti-HBV activity as was expected from earlier reports.[8,9]
6-Chloro derivative (6) also showed anti-HBV activity to almost the same
degree as that of 7, suggesting 6-chloro had been converted to 6-oxo be-
fore it acts as an inhibitor of HBV DNA polymerase. It is interesting that the

6-Arylthio Analogs and Hepatitis B Virus
659
TABLE 1 Anti-HBV Activity of FddG and
Its 6-Substituted Analogs Prepared by
Nucleophilic Displacement or Catalytic
Hydrogenation of 6
Compound
EC50 (µM) CC50 (µM)
6
7
8
9
10.4
9
>99
9.7
3.6
0.26
>93
>87
>99
>69
>64
>92
10
PMEA
activity of 6-(4-methoxy)phenylthio (10) derivative was superior to that of 7.
Also, 6-phenylthio derivative (9) maintained inhibitory effect against HBV
comparable to that of 7. These results indicate that 2-amino-6-arylthiopurine
is also changeable to guanine in the assay system. One exception to this
rule is 2-aminopurine derivative (8), which showed no anti-HBV activity
even at the concentration of 99 µM, suggesting that 10 had not been con-
verted to an active form. However, antiviral effect of all compounds includ-
ing FddG against HBV was weak compared to that of the positive control,
PMEA. Therefore, we will attempt to optimize in further studies the activity of
compound 7.
EXPERIMENTAL
Melting points (mp) were determined using a Yanagimoto micro-melting
point apparatus (hot stage type) and are uncorrected. UV spectra were
recorded with a Shimadzu UV-190 digital spectrometer. Low-resolution mass
spectra were obtained on a FAB-JMS-700 mass spectrometer in the direct-
inlet mode. High-resolution mass spectra were obtained on a JMS-T100-CSI
1
spectrometer in the ESI mode. H-NMR spectra were recorded on JEOL
JMM-ECX400 (400 MHz) in CDCl3 (or dimethyl sulfoxide (DMSO)-d6) with
tetramethylsilane as an internal standard. Merck Art 5554 plates pre-coated
with silica gel 60 containing fluorescent indicator F254 were used for thin-layer
chromatography and silica gel 60 (Merck 7734, 60–200 mesh) was employed
for column chromatography.
2-Amino-6-chloro-9-(2-O-tosyl-β-D-ribofuranosyl)purine ( 2). Dibutyltin (IV)
oxide (2.7 g, 11.6 mmol) was added to the solution of 1 (3.5 g, 11.6 mmol)
in MeOH (200 ml), and refluxed for 1 h. After being cooled to 0◦C, Et3N
(24 ml, 174 mmol) and TsCl (32.5 g, 174 mmol) were added to a solution
and stirred at 4◦C for 12 h, and the solvent was removed. The residue was di-
luted with H2O and extracted with EtOAc, and dried over MgSO4. Removal
of the organic layer afforded an oily residue, which was purified by column

660
T. Torii et al.
chromatography on silica gel (66% EtOAc in hexane) and recrystallization
1
from EtOH to give 2 (4.5 g, 85%) as colorless crystals: mp 194–196◦C. H
NMR (DMSO-d6, RT) δ 8.11 (1H, s, H8), 7.43 (2H, d, J = 8.1 Hz, Ph), 7.05
(2H, d, J = 8.1 Hz, Ph), 6.96 (2H, brs, 2-NH2), 6.04 (1H, brs, 3ꢁ-OH), 5.96
(1H, d, J = 7.3 Hz, CH1ꢁ), 5.45 (1H, dd, J = 5.0, 7.3 Hz, CH2ꢁ), 5.18 (1H, brs,
5ꢁ-OH), 4.34-4.28 (1H, m, CH3ꢁ), 3.98–3.92 (1H, m, CH4ꢁ), 3.70–3.50 (2H,
m, CH25ꢁ), 2.28 (3H, s, CH3). 13C NMR (DMSO-d6, RT) δ 159.90, 153.32,
149.76, 145.21, 141.32, 132.00, 129.79, 127.21, 123.73, 87.17, 84.02, 79.69,
70.16, 61.46, 21.42. ESI-MS m/z: 456 [M+H]+.
2-Amino-6-chloro-9-(2-deoxy-β-D-threo-pentofuranosyl)purine ( 3). To a solu-
tion of 2 (3.42 g, 7.5 mmol) in DMSO (75 ml) was added 1 M lithium triethyl-
borohydride in THF solution (75 ml) and stirred at 35◦C for 14 h. The solu-
tion was diluted with H2O (35 ml) at 0◦C and neutralized with CH3COOH.
Removal of the solvent afforded an oily residue, which was dissolved EtOH
(50 ml) and filtered through Celite. After removal of the filtrate, the residue
was purified by column chromatography on silica gel (15% EtOH in EtOAc)
and recrystallization from EtOH-H2O to give 3 (1.77 g, 83%) as colorless
1
crystals: mp 126–127◦C; H NMR (DMSO-d6, RT): δ 8.28 (1H, s, H8), 6.91
(2H, brs, 2-NH2), 6.12 (1H, d, J = 7.8 Hz, 3ꢁ-OH), 5.34 (1H, d, J = 4.2 Hz,
CH1ꢁ), 4.64 (1H, t, J = 5.8 Hz, 5ꢁ-OH), 4.32–4.31 (1H, m, CH3ꢁ), 3.89–3.88
(1H, m, CH4ꢁ), 3.69 (1H, ddd, J = 5.5, 11.2, 17.0 Hz, CH25ꢁ-one), 3.58 (1H,
ddd, J = 6.0, 11.7, 17.5 Hz, CH25ꢁ-one), 2.66 (1H, ddd, J = 5.1, 8.3, 14.2
Hz, CH22ꢁ-one), 2.21 (1H, d, J = 14.7 Hz, CH22ꢁ-one); UV λmax (MeOH)
nm: 248, 310; ESI-MS m/z: 308 [M + Na]+; elemental analysis calcd (%) for
C10H12ClN5O3 · H2O: C 39.55, H 4.65, N 23.06; found C 39.57, H 4.34, N
23.05.
6-Chloro-9-(5-O-trityl-2-deoxy-β-D-threo-pentofuranosyl)-2-tritylaminopurine ( 4).
Et3N (0.95 ml, 6.8 mmol), DMAP (85 mg, 0.7 mmol), and TrCl (1.67 g,
6.0 mmol) were added to a solution of 3 (500 mg, 1.8 mmol) in DMF (15
ml) and stirred at 45◦C for 24 h. MeOH (5 ml) was added to the solution
and stirred at RT for 30 min and the solvent was removed. The residue
was diluted with toluene and washed with H2O, and dried over MgSO4.
Removal of the organic layer afforded an oily residue, which was purified
by column chromatography on silica gel (50% EtOAc in hexane) to give
1
4 (1.30 g, 96%) as a colorless foam: mp 140–142◦C; H NMR (CDCl3, RT,
TMS): δ 8.00 (1H, s, H8), 7.42–7.21 (30H, m, Ph), 6.51 (1H, brs, 2-NH),
5.69–5.61 (1H, m, CH1ꢁ), 4.39–4.38 (1H, m, CH3ꢁ), 4.05 (1H, ddd, J = 3.2,
5.9, 9.1 Hz, CH4ꢁ), 3.58 (1H, dd, J = 5.9, 10.1 Hz, CH25ꢁ-one), 3.35 (1H, dd,
J = 6.4, 9.6 Hz, CH25ꢁ-one), 2.28-2.20 (1H, m, CH22ꢁ-one), 2.00–1.95 (1H, m,
CH22ꢁ-one); UV λmax (MeOH) nm: 312; ESI-MS m/z: 792 [M+Na]+;
elemental analysis calcd (%) for C48H40ClN5O3 · 0.5 H2O: C 73.79, H 5.16,
N 8.96; found C 73.74, H 5.36, N 8.65.
6-Chloro-9-(5-O-trityl-2,3-didexy-3-fluoro-β-D-erythro-pentofuranosyl)-2-trityl-
aminopurine ( 5). Diethylaminosulfur trifluoride (DAST) (14 µl, 0.104

6-Arylthio Analogs and Hepatitis B Virus
661
mmol) was added to a solution of 4 (50 mg, 0.065 mmol) in CH2Cl2 (2ml)
at 0◦C. After being stirred at 40◦C for 30 min, the solution was added to 5%
aqueous NaHCO3 at 0◦C and extracted with CH2Cl2, and dried over MgSO4.
Removal of the organic layer afforded a white solid residue, which was
purified by column chromatography on silica gel (33% EtOAc in hexane)
and recrystallization from EtOAc-hexane gave 5 (30 mg, 60%) as colorless
1
crystals: mp 232–234◦C; H NMR (CDCl3, RT, TMS): δ 7.79 (1H, s, H8),
7.33–7.19 (30 H, m, Ph), 6.62 (1H, brs, 2-NH), 5.72–5.62 (1H, m, CH1ꢁ),
4.98 (1H, d, J = 54.5 Hz, CH3ꢁ), 4.28 (1H, d, J = 25.6 Hz, CH4ꢁ), 3.27 (2H,
d, J = 4.2 Hz, CH25ꢁ), 2.20–2.15 (2H, m, CH22ꢁ); UV λmax (MeOH) nm:
313, 259; ESI-MS m/z: 794 [M + Na]+; elemental analysis calcd (%) for
C48H39ClFN5O2 · 0.5 H2O: C 73.79, H 5.16, N 8.96; found C 74.04, H 5.05,
N 8.93.
2-Amino-6-chloro-9-(2,3-didexy-3-fluoro-β-D-erythro-pentofuranosyl)-purine ( 6).
To a solution of 5 (250 mg, 0.32 mmol) in CH2Cl2 (15 ml) and MeOH
(15 ml) was added 35% aqueous HCl (0.1 ml) dropwise. After being stirred
at 30◦C for 1.5 h, Et3N (0.2 ml) was added to the solution at 0◦C, and the
solvent was removed. The residue was diluted with H2O and extracted with
CH2Cl2, and dried over MgSO4. Removal of the organic layer afforded an
oily residue, which was purified by column chromatography on silica gel
(EtOAc) and recrystallization from EtOH gave 6 (63 mg, 69%) as colorless
1
crystals: mp 169–171◦C. H NMR (DMSO-d6, RT) δ 8.36 (1H, s, H8), 7.00
(2H, brs, 2-NH2), 6.27 (1H, dd, J = 5.8, 9.0 Hz, CH1ꢁ), 5.42 (1H, dd, J = 4.4,
53.6 Hz, CH3ꢁ), 5.13 (1H, t, J = 5.4 Hz, OH), 4.20 (1H, dt, J = 5.0, 26.5
Hz, CH4ꢁ), 4.16 (2H, t, J = 5.4 Hz, CH25ꢁ), 2.90 (1H, ddd, J = 5.0, 9.3, 39.2
Hz, CH22ꢁ-one), 2.71–2.60 (1H, m, CH22ꢁ-one). 13C NMR (DMSO-d6, RT)
δ 159.38, 153.37, 149.18, 140.66, 123.14, 94.40 (J = 173 Hz), 84.97 (J = 22
Hz), 82.65, 60.45 (J = 11 Hz), 36.12 (J = 20 Hz). ESI-MS m/z: 288 [M+H]+.
9-(2,3-Didexy-3-fluoro-β-D-erythro-pentofuranosyl)guanine (FddG, 7). To a
solution of 6 (50 mg, 0.174 mmol) in MeOH (5 ml) was added 2-
mercaptoethanol (0.03 ml) and 28% NaOMe (0.058 ml) and the solution
was kept at 50◦C for 2 days, then neutralized by addition of acetic acid. After
concentration of the solvent, the residue was applied to a column of silica
gel. The main fraction was evaporated to give a solid, which was crystallized
1
from EtOH to give 7 as a pale yellowish powder (31.6 mg, 67%). H NMR
(DMSO-d6, RT) δ 10.54 (1H, brs, NH), 7.92 (1H, s, H8), 6.49 (2 H, brs, 2-
NH2), 6.15 (1H, dd, J = 5.6, 9.4 Hz, CH1ꢁ), 5.38 (1H, dd, J = 4.3, 53.6 Hz,
CH3ꢁ), 5.15 (1H, t, J = 5.4 Hz, OH), 4.16 (1H, dt, J = 4.9, 27.0 Hz, CH4ꢁ),
3.60–3.50 (2 H, m, CH25ꢁ), 2.90–2.70 (1H, m, CH22ꢁ-one), 2.67–2.50 (1H,
m, CH22ꢁ-one). 13C NMR (DMSO-d6, RT) δ 156.26, 153.30, 150.60, 134.88,
116.34, 94.94 (J = 173 Hz), 85.17 (J = 22 Hz), 82.64, 60.98 (J = 11 Hz), 36.87
(J = 20 Hz). ESI-MS m/z: 270 [M+H]+.
2-Amino-9-(2,3-didexy-3-fluoro-β-D-erythro-pentofuranosyl) purine ( 8). A mix-
ture of 6 (10 mg, 0.035 mmol), CH3COONa (3.0 mg, 0.035 mmol) and 5%

662
T. Torii et al.
Pd-C (5 mg) in MeOH (2 ml) was vigorously stirred under an H2 atmosphere
for 6 h. The Pd-catalyst was filtered off, and the filtrate was concentrated in
vacuo to leave a white solid residue, which was purified by column chro-
matography on silica gel (15% EtOH in EtOAc) and recrystallization from
EtOH to give 8 (5.5 mg, 63%) as colorless crystals: mp 148–149◦C; 1H NMR
(CD3OD, RT):δ 8.57 (1H, s, H6), 8.27 (1H, s, H8), 6.41 (1H, dd, J = 5.6,
9.2 Hz, CH1ꢁ), 5.41 (1H, dd, J = 4.8, 53.6 Hz, CH3ꢁ), 4.34 (1H, dt, J = 3.6,
26.8 Hz, CH4ꢁ), 3.85-3.77 (2H, m, CH25ꢁ), 2.96 (1H, dddd, J = 4.8, 9.2, 14.4,
39.6 Hz, CH22ꢁ-one), 2.67 (1H, ddd, J = 6.0, 14.4, 21.2 Hz, CH22ꢁ-one); 13
C
NMR (CD3OD, RT): δ 161.7, 154.0, 150.4, 143.5, 128.7, 96.2 (J = 174 Hz),
87.5 (J = 23 Hz), 86.3, 63.1 (J = 11 Hz), 38.9 (J = 21 Hz); UV λmax (MeOH)
nm: 310, 221; FAB-MS m/z: 254 [M + H]+.
2-Amino-9-(2,3-didexy-3-fluoro-β-D-erythro-pentofuranosyl)-6-phenyl-thiopurine
( 9). Benzenethiol (27 µl, 0.26 mmol) and pyridine (69 µl, 0.85 mmol)
were added to a solution of 6 (50 mg, 0.17 mmol) in 1.5 ml of DMF and
stirred at 80◦C for 12 h. Removal of the solvent afforded an oily residue,
which was purified by column chromatography on silica gel (30% EtOAc
in hexane) and recrystallization from EtOH to give 9 (55 mg, 90%) as
1
colorless crystals: mp 192–193◦C; H NMR (DMSO-d6, RT): δ 8.19 (1H, s,
H8), 7.58–7.56 (2H, dd, J = 3.0, 6.6 Hz, Ph), 7.42 (3H, t, J = 3.2 Hz, Ph),
6.39 (2H, brs, 2-NH2), 6.22 (1H, dd, J = 6.0, 9.2 Hz, CH1ꢁ), 5.38 (1H, dd,
J = 4.1, 53.6 Hz, CH3ꢁ), 5.11 (1H, t, J = 4.6 Hz, OH), 4.20–4.11 (1H, dt,
J = 5.0, 27.1 Hz, CH4ꢁ), 3.53 (2H, t, J = 5.1 Hz, CH25ꢁ), 2.89 (1H, dddd,
J = 4.6, 8.7, 14.2, 39.4 Hz, CH22ꢁ-one), 2.59 (1H, ddd, J = 5.5, 14.6, 20.6 Hz,
CH22ꢁ-one); UV λmax (MeOH) nm: 312, 246; ESI-MS m/z: 384 [M + Na]+;
elemental analysis calcd (%) for C16H16FN5O2S: C, 53.17; H, 4.46; N, 19.38;
found C 53.22, H 4.33, N 19.25.
2-Amino-9-(2,3-didexy-3-fluoro-β-D-erythro-pentofuranosyl)-6-(4-methoxyphenyl)-
thiopurine ( 10). Compound 9 was prepared from p-methoxybenzenethiol
and 6 in a manner similar to that described for the preparation of 9: mp
184–185◦C; 1H NMR (DMSO-d6, RT): δ 8.17 (1H, s, H8), 7.47 (2H, d, J = 8.7
Hz, Ph), 6.98 (2H, d, J = 9.1 Hz, Ph), 6.33 (2H, brs, 2-NH2), 6.21 (1H, dd,
J = 5.5, 9.2 Hz, CH1ꢁ), 5.38 (1H, dd, J = 4.4, 53.6 Hz, CH3ꢁ), 5.12 (1H, t,
J = 5.5 Hz, 5ꢁ-OH), 4.15 (dt, J = 5.0, 26.9 Hz, CH4ꢁ), 3.77 (3H, s, p-OCH3),
3.53 (2H, t, J = 5.3 Hz, CH25ꢁ), 2.87 (1H, dddd, J = 5.0, 9.1, 14.2, 39.8 Hz,
CH22ꢁ-one), 2.58 (1H, ddd, J = 6.0, 14.7, 21.1 Hz, CH22ꢁ-one); UV λmax
(MeOH) nm: 314; ESI-MS m/z: 414 [M+Na]+; elemental analysis calcd (%)
for C17H18FN5O3S0.2H2O: C 51.69, H 4.69, N 17.73; found C 51.37, H 4.32,
N 17.46.
Anti-HBV Assay
The tetracycline-responsive cell lines HepAD38 was used. These are hep-
atoma cells that have been stably transfected with a cDNA copy of the

6-Arylthio Analogs and Hepatitis B Virus
663
pregenomic RNA of wild-type virus. Withdrawal of tetracycline from the
culture medium results in the initiation of viral replication. Cells were cul-
tured at 37◦C in a humidified 5% CO2/air atmosphere in seeding medium,
DMEM/Ham’s F12 (50/50) supplemented with 10% (v/v) heat-inactivated
fetal calf serum, 100 IU/ml penicillin/50 µg/ml streptomycin mix,
400 µg/ml G418, and 0.3 µg/ml tetracycline. When the assay was started,
the cells were seeded in 48-well plates at a density of 1.5 × 105/well. After
2–3 days the cultures were induced for viral production by washing with
pre-warmed PBS and were fed with 200-µl assay medium (seeding medium
without tetracycline and G418) with or without the antiviral compounds.
The medium was changed after 3 days. The antiviral effect was quantified by
measuring levels of viral DNA [isolated (Qiagen) from the cell cultures] at
day 4 post-induction, by a real-time quantitative PCR (Q-PCR). The Q-PCR
was performed in a reaction volume of 25 µl using the TaqMan Universal
PCR Master Mix (Applied Biosystems, Branchburg, NJ) with forward primer
(5ꢁ-CCG TCT GTG CCT TCT CAT CTG-3ꢁ; final concentration: 600 nM), re-
versed primer (5ꢁ-AGT CCA AGA CTY CTC TTA TRY AAG ACC TT-3ꢁ; final
concentration 600 nM), and Taqman probe (6-FAM-CCG TGT GCA CTT
CGC TTC ACC TCT GC-TAMRA; final concentration 150 nM). The reac-
tion was analyzed using a SDS 7000 (Applied Biosystems, Foster City, CA).
A plasmid containing the full-length insert of the HBV genome was used to
prepare the standard curve. The amount of viral DNA produced in treated
cultures was expressed as a percentage of the mock treated samples. The cy-
tostatic effect of the various compounds was assessed employing the parent
hepatoma cell line HepG2. The effect of the compounds on exponentially
growing HepG2 cells was evaluated by means of the MTS method (Promega,
Leiden, The Netherlands). Briefly, cells were seeded at a density of 3000/well
(96 well plate) and were allowed to proliferate for 3 days in the absence or
presence of compounds after which time cell density was determined.
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