Synthesis and anti-HCV activity of N9,5′-cyclo-3-(β- D-ribofuranosyl)-8-azapurin-2-one derivatives

Article abstract of DOI:10.1080/15257770500265802

□ A number of 1- or 6-substituted N⁹,5′-cyclo-3- (β-D-ribofuranosyl)-8-azapurin-2-one derivatives were synthesized in multi-step reactions. Their anti-hepatitis C virus activities were evaluated and some structure-activity relationship is discussed. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770500265802

This article was downloaded by: [Thammasat University Libraries]
On: 08 October 2014, At: 12:01
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,
37-41 Mortimer Street, London W1T 3JH, UK
Nucleosides, Nucleotides and Nucleic Acids
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/lncn20
Synthesis and Anti-Hcv Activity of N 9, 5′-Cyclo-3-(β-D-
Ribofuranosyl)-8-Azapurin-2-One Derivatives
Abdalla E. A. Hassan a , Peiyuan Wang a , Tamara McBrayer a , Philip Tharnish a , Lieven
Stuyver a , Michael J. Otto a , Kyoichi A. Watanabe a & Raymond F. Schinazi b
a Pharmasset, Inc. , Tucker, Georgia, USA
b Department of Pediatrics , Emory University, School of Medicine/VA Medical Center ,
Decaur, Georgia, USA
Published online: 16 Aug 2006.
To cite this article: Abdalla E. A. Hassan , Peiyuan Wang , Tamara McBrayer , Philip Tharnish , Lieven Stuyver , Michael
J. Otto , Kyoichi A. Watanabe & Raymond F. Schinazi (2005) Synthesis and Anti-Hcv Activity of N 9, 5′-Cyclo-3-(β-D-
Ribofuranosyl)-8-Azapurin-2-One Derivatives, Nucleosides, Nucleotides and Nucleic Acids, 24:10-12, 1531-1542, DOI:
10.1080/15257770500265802
To link to this article: http://dx.doi.org/10.1080/15257770500265802
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the
Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and
are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and
should be independently verified with primary sources of information. Taylor and Francis shall not be liable for
any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever
or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of
the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions

Nucleosides, Nucleotides, and Nucleic Acids, 24:1531–1542, 2005
c
Copyright ꢀ Taylor & Francis Group, LLC
ISSN: 1525-7770 print/1532-2335 online
DOI: 10.1080/15257770500265802
SYNTHESIS AND ANTI-HCV ACTIVITY OF N 9,5 -CYCLO-3-
(β-D-RIBOFURANOSYL)-8-AZAPURIN-2-ONE DERIVATIVES
Abdalla E. A. Hassan, Peiyuan Wang, Tamara McBrayer, Philip Tharnish,
ᮀ
Lieven Stuyver, MichaelJ. Otto, and Kyoichi A. Watanabe
Pharmasset,
Inc., Tucker, Georgia, USA
ᮀ
Raymond F. Schinazi
Department of Pediatrics, Emory University, School of
Medicine/VA Medical Center, Decaur, Georgia, USA
A number of 1- or 6-substituted N9,5 -cyclo-3-( -D-ribofuranosyl)-8-azapurin-2-one derivatives
ᮀ
were synthesized in multi-step reactions. Their anti-hepatitis C virus activities were evaluated and
some structure–activity relationship is discussed.
Keywords N 9,5 -Cyclo-3-( -D-ribofuranosyl)-8-azapurine nucleosides; N 9,5 -Anhydro-
isonucleosides; Potential anti-HCV nucleosides
INTRODUCTION
The flaviviridae family of viruses includes the causative agents of dengue,
West Nile virus (WNV), yellow fever, and hepatitis C viruses. Hepatitis C
virus (HCV) is one of the most serious flaviviridae infections in humans and
is responsible for the second most common cause of viral hepatitis. HCV-
induced chronic hepatitis with concomitant cirrhosis and hepatocellular
carcinoma is now the leading cause of liver transplantations. Currently,
nearly 2% of the U.S. population, and an estimated 170 million people
worldwide, are HCV carriers.[1] The only approved therapy for chronic
hepatitis C is alpha interferon (INF-α), either alone or in combination with
ribavirin. However, its therapeutic value has been compromised largely due
to its adverse effects[2,3] and additional new therapies are needed.
This article is dedicated to the memory of Dr. John A. Montgomery.
Received 19 January 2005; accepted 4 April 2005.
Supported in part by NIH grants 1R43 AI-52868 (biology) and 1 R43 AI-056720 (chemistry).
RFS is the principal founder and a major shareholder in Pharmasset Inc. His laboratory received no
funding from Pharmasset Inc. for these studies.
Address correspondence to Kyoichi A. Watanabe, Pharmasset, Inc., 1860 Montreal Road, Tucker,
GA, 30084. Fax: 770-414-4208; E-mail: kyowatanabe@comcast.net
1531

1532
A. E. A. Hassan et al.
FIGURE 1 Structures of potential anti-HCV nucleosides, N 3,5 -cyclo-4-(β-D-ribofuranosyl)-v-triazo-
lo[4,5-b]-pyridine-2-one (1) and N 9,5 -cyclo-3-(β-D-ribofuranosyl)-8-azaxanthine (2).
In a previous report[4,5] we described the chemical synthesis of N3,5ꢁ-
cyclo-4-(β-D-ribofuranosyl)-v-triazolo[4,5-b]pyridin-2-one (1), which inhibits
the HCV-RNA in the HCV-subgenomic replicon cell line (Huh 7 cells) with
an EC90 of 79.8 µM. As a part of our program for discovery of more potent
anti-HCV agents, we have synthesized the xanthine analogue 2 (Figure 1)
and several related purine derivatives[6] and found some of the compounds
to be rather active in the replicon system. We report herein the chemical
synthesis of the analogues and structure-activity relationship studies from
anti-HCV screening test results.
RESULTS AND DISCUSSION
Chemistry
5-Bromo-2ꢁ,3-O-isopropylidene nucleoside derivative 3 was prepared
from uridine by reported procedures.[7] Mesylation of 3 to 4a (Scheme 1),
followed by sodium azide treatment in DMF afforded the 5ꢁ-azido-5ꢁ-deoxy
derivative 5, which was obtained in 86% yield from 3 after crystallization
from ethanol. Upon heating 5 in DMF at 110–120◦C for 30 h, [2,3]-
dipolar addition of the 5ꢁ-azido group to the 5,6-double bond occurred
with concomitant elimination of HBr from the adduct 5ꢁ resulting in
the formation of 9,5ꢁ-cyclo-nucleoside 6, which was obtained as colorless
crystals in 76% yield after recrystallization from methanol and ethyl acetate.
Compound 3 was acylated with benzoyl chloride or p-fluorobenzoyl chloride
in pyridine at 0◦C to give 4b and 4c in 84–87% yield. Upon treatment of
4b or 4c with sodium azide in DMF at 110–120◦C for 3–5 days, the cyclo-
nucleoside 6 was obtained directly in 75–80% yield. Acid hydrolysis of 6
in a 1:1 mixture of tetrahydrofuran and 1M hydrochloric acid at 90◦C
afforded 9,5ꢁ-cyclo-3-(β-D-ribofuranosyl)-8-azaxanthine (2) in quantitative
yield as colorless crystals (Scheme 1).

Anti-HCV Derivatives
1533
SCHEME 1 Reagent: (a) NaN3/DMF, 80 C, from 4a to 5; (b) DMF, 110–120 C, 30 h; (c) 1N HCl/THF,
90 C; (d) MeI/K2CO3/DMF for 7a; 60% NaH/LiBr/BrCH2CN/DMF for 7b; (e) NaN3/DMF, 110–
120 C, from 4b or 4c.
Treatment of 6 with MeI and K2CO3 in DMF afforded the N-1 methyl
derivative 7a in high yield. The N-1 cyanomethyl derivative 7b was obtained
in quantitative yield by treatment of 6 with NaH and bromoacetonitrile in
a 1:1 mixture of DME and DMF. Upon acid hydrolysis of 7a and 7b, the
corresponding 2ꢁ,3ꢁ-diols 8a and 8b were obtained in high yield.
Compound 6 was converted to the 6-imidazolyl derivative 9 in 81% yield
by treatment with triphenyl phosphine, imidazole, N,N-diisopropylethyl-
amine and I2 in toluene at 95–100◦C for 2 h (Scheme 2). When 9 was
treated with NH4OH for 24 h at room temperature, the 8-aza-isoguanine
derivative 10a was obtained. Various 6-(substituted amino) derivatives 10b-e
were prepared directly from 6 by treatment with triisopropylbenzenesulfonyl
chloride and DAMP, followed by reaction with the corresponding amines.
Compounds 10a-e were then deprotected to the corresponding diols (11a-e)
by acid hydrolysis. Some of the mono-substituted amino derivatives of 10 and
1
11 exist as a mixture of amino-imino tautomers as evidenced by H N MR
showing a pair of signals, which merged to simple spectra when heated.

1534
A. E. A. Hassan et al.
SCHEME 2 Reagent: (a) Ph3P/imidazole/iPr2NEt/I2/toluene; (b) Et3N/DMAP/TIPSCI/amine; (c)
1NHCl/THF, 90 C; (d) Lawesson’s reagent/DCE, reflux; (e) CF3CO2H/H2O, 50–55 C.
Compound 6 was treated with Lowesson’s reagent in dichloroethane at
reflux to obtain the 6-thio derivative 12 in 67.5% as a pale yellow solid. Acid
treatment of 12 afforded 63% yield of 9,5ꢁ-cyclo-(3-β-D-ribofuranosyl)-8-aza-
6-thioxanthine 13.
Anti-HCV Activity
Compounds 2, 8a-b, 11a-e, and 13 were assayed for their ability to inhibit
HCV RNA replication in a subgenomic replicon Huh7 cell line as described
previously.[8] Table 1 summarizes the HCV replicon activity and cytotoxicity
of these compounds. As shown, 6-amino (11a), 6-methylamino (11b) and
6-methoxyamino (11e) derivatives were found to exhibit interesting anti-
HCV activities with the EC90 values of 10.9, <6.25, and 9.7 µM, respectively.
But they were also toxic with the CC50 values of 11.0, 6.7, and 1.9 µM,
respectively. The 8-aza-xanthine 2 and 8-aza-6-thio-xanthine 13 showed
moderate anti-HCV activities with the EC90 values of 23.7 and 43.2 µM,
respectively. Methyl substitution on N-1 position intensifies the activity (e.g.,
8a, EC90 = 1.5 µM), but causes toxicity. Cyanomethyl substitution on N-1
(e.g., 8b) did not show significant anti-HCV activity.

Anti-HCV Derivatives
1535
TABLE 1 Anti-HCV Activity of Synthesized
Compounds in the Huh 7 Replicon Cells
Compounds
EC90 (µM)
CC50 (µM)
1
2
8a
79.8
23.7
1.5
30.6
24.4
1.8
8b
>100
10.9
<6.25
>100
>100
9.7
>100
11.0
6.7
>100
>100
1.9
11a
11b
11c
11d
11e
13
43.2
32
From the biological results, the most active compound among C-6-
amino substituted derivatives was the methylamino analogue (11b). Methyl
substitution on N-1 position intensifies the activity, but unfortunately it
was also toxic. It seems that the activity would be reduced as the size of
substituent on the amino group at C-6 or alkyl group at N-1 grows larger.
This class of compounds did not inhibit purified the HCV RNA-depend-
ent RNA polymerase (NS5B) in vitro when tested in cell-free systems.[4] It
is not surprising that they do not appear to inhibit HCV-RNA-dependent
RNA polymerase since there is no 5ꢁ-OH in these compounds. The mode of
anti-HCV action is now the subject of further studies.
It should be noted that the difference between antiviral activity and
cell toxicity (IC90 and CC50 values) are rather close in most cases. Roughly,
the concentration of drug required to inhibit cell growth by 50% reduces
90% of viral RNA production. Thus, it is unclear whether the extent of
apparent reduction of HCV RNA is derived from antiviral activity of the
test compounds or is due to reduction of viable cells. Nevertheless, these
data still warrant further chemical synthesis and biological studies of this
class of molecules to explore the possibility of discovering compounds with
reduced cytotoxicity and retained or enhanced anti-HCV activity.
EXPERIMENTAL
ChemicalSynthesis
General. Melting points were determined on an Electrothermal digi-
tal melting point apparatus and are uncorrected. Nuclear magnetic reso-
nance spectra were recorded on a Varian Unity Plus 400 spectrometer at
room temperature, with tetramethylsilane as an internal standard. Chemi-
cal shifts (δ) are reported in parts per million (ppm), and signals are re-
ported as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet),

1536
A. E. A. Hassan et al.
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 spectropho-
tometer. Fast atom bombardment mass spectroscopy was performed by the
Emory University Mass Spectrometry Center. TLC was performed on Uni-
plates (silica gel) purchased from Analtech Co., and column chromatog-
raphy was performed using silica gel (60 Å) from Sorbent Technologies,
Atlanta, Georgia. Elemental analyses were performed by Atlantic Microlab
Inc., Norcross, Georgia.
2 ,3 -O -Isopropylidine-5-bromouridine (3). 5-Bromouridine (130 g,
0.403 mol) was suspended in acetone (1 L) and treated with 1M HCl in
Et2O (25 mL) for 48 h at room temperature with stirring. The mixture was
neutralized with 1N NH4OH to pH = 7 and the solvent was evaporated in
vacuo. The residue was crystallized from EtOH to give (137 g, 94%) as a
1
white solid. H-NMR (DMSO-d6) δ 1.28 (3H, s, CH3), 1.48 (3H, s, CH3),
3.59 (2H, m, H-5ꢁa and H-5ꢁb), 4.12 (1H, brq, H-4ꢁ), 4.75 (1H, dd, H-3ꢁ, J =
3.6, J = 6.4 Hz), 4.92 (1H, dd, H-2ꢁ, J = 2.4, J = 6.0 Hz), 5.32 (1H, t, J =
5.2 Hz), 5.83 (1H, d, H-1ꢁ, J = 2.8 Hz), 8.38 (1H, s, H-6), 11.92 (1H, s, NH).
5 -O-Benzoyl-2 ,3 -O-isopropylidine-5-bromouridine (4b). Benzoyl chlo-
ride (7.7 mL, 66.1 mmol) was added dropwise to a solution of 3 (12 g, 33.0
mmol) in pyridine (50 mL) at 0◦C. The mixture was stirred at room temper-
ature for 3 h then the reaction was quenched with ice-H2O and stirred for
further 30 min. The solvent was evaporated and co-evaporated with toluene.
The residue was partitioned between EtOAc and H2O. The organic phase
was washed with sat. NaHCO3, H2O, dried (MgSO4), and the solvent was
evaporated to give 4b as a white solid (13.0 g, 84%), which was crystallized,
1
from MeOH. H-NMR (CDCl3) δ 8.70 (1H, br s, NH), 8.01 (2H, m, Bz),
7.65–7.45 (3H, m, Bz), 5.74 (1H, d, H-1ꢁ), 4.99 (1H, dd, 5ꢁa), 4.91 (1H, dd,
5ꢁb), 4.65–4.53 (3H, m, H2ꢁ, H-3ꢁ and H-4ꢁ), 1.60 (3H, s, CH3), 1.39 (3H, s,
CH3).
5 -O-(4-Fluorobenzoyl)-2 ,3 -O-isopropylidine-5-bromouridine (4c). p-
Fluoro-benzoyl chloride (0.9 mL, 7.6 mmol) was added dropwise to a
solution of 3 (2.5 g, 6.88 mmol) in pyridine (20 mL) at 0◦C and the mixture
was stirred at room temperature for 1 h. The reaction was quenched with
ice-H2O and stirred for further 30 min. The solvent was evaporated and
co-evaporated with toluene. The residue was partitioned between EtOAc
and H2O. The organic phase was washed with sat. NaHCO3, H2O, dried
(MgSO4), and evaporated, and the residue was purified by a flash silica gel
column (eluate; 20% EtOAc in hexanes to give 4c as a white solid (2.91 g,
1
87%). H-NMR (DMSO-d6) δ 11.93 (1H, br s, NH), 8.14 (1H, s, H-6), 8.03
(2H, dd, F-Bz), 7.35 (2H, dd like t, F-Bz), 5.79 (1H, d, H-1ꢁ), 5.14 (1H, d,

Anti-HCV Derivatives
1537
H-2ꢁ), 4.92 (1H, br dd, H-5ꢁa), 4.52 (1H, dd, 5ꢁb, J = 3.6, J = 11.6 Hz),
4.46–4.39 (2H, m, H-3ꢁ, and H-4ꢁ), 1.50 (3H, s, CH3), 1.31 (3H, s, CH3).
5 -Azido-5 -deoxy-2 ,3 -O-isopropylidine-5-bromouridine (5). Method A.
To a solution of 3 (3 g, 8.3 mmol) in dry pyridine (20 mL) was added
MsCl (0.7 mL, 9.1 mmol) at 0◦C. The mixture was stirred for 1 h at 0◦C,
then another (0.12 mL) MsCl was charged, and the stirring continued
for 1 h. The solvent was evaporated and coevaporated with toluene 2
times, and the residue was partitioned between CH2Cl2 and H2O. The
organic layer was separated, dried (MgSO4), and the solvent was removed
in vacuo to give 4a as a pale yellow foam, which, without purification was
dissolved in DMF (20 mL). NaN3 (2.15 g, 33.2 mmol) was added to the
solution, and the mixture was heated for 2 h at 80◦C. After cooling to room
temperature, the mixture was concentrated in vacuo, and the residue was
flash chromatographed on a silica gel column (eluate: 5% MeOH in CHCl3)
to give 5 (2.78 g, 86%) as a colorless solid, which was crystallized, from EtOH
in hexanes. 1H-NMR (DMSO-d6) δ 11.96 (1H, s, NH), 8.27 (1H, s, H-6), 5.81
(1H, s, H-1ꢁ), 5.13 (1H, br dd, H-2ꢁ), 4.76 (1H, dd, H-3ꢁ, J = 4.4, J = 6.0
Hz), 4.15 (1H, q, H-4ꢁ), 3.60 (2H, br d, H-5ꢁa and H-5ꢁb), 1.49 (3H, s, CH3),
1.29 (3H, s, CH3).
Method B. A mixture of 5ꢁ-azido-5ꢁ-deoxy-2ꢁ,3ꢁ-O-isopropylideneuridine
(5, 2.30 g, 7.44 mmol) and ammonium cerium nitrate (6.52 g, 11.89 mmol),
and LiBr (768 mg, 8.84 mmol) in anhydrous acetonitrile (120 mL) was
heated at 80◦C for 30 min, neutralized by triethylamine, and concentrated.
The residue was partitioned between H2O and CHCl3. The aqueous layer
is extracted with CHCl3, and the combined extracts were concentrated and
the residue is purified on a silica gel column (CHCl3:MeOH = 30:1) to give
1
5 (2.23 g, 77%). H NMR (CDCl3) δ 8.80 (bs, 1H), 7.67 (s, 1H), 5.66 (d,
1H, J = 2.0 Hz), 4.86 (m, 1H), 4.73 (m, 1H), 4.21 (m, 1H), 3.59 (m, 2H),
1.51 (s, 3H), 1.29 (m, 3H).
9,5 -Cyclo-3-(2,3-O-isopropylidine-β-D-ribofuranosyl)-8-azaxanthine (6).
Method A. A solution of 5 (1 g, 2.58 mmol) in DMF (10 mL) was heated for
30 h at 110–120◦C. The solvent was removed in vacuo and the residue was
partitioned between EtOAc and H2O. The organic layer was dried (MgSO4),
and evaporated to leave a pale crystalline residue. Recrystallization from
1
MeOH and EtOAc afforded 6 (602 mg, 76%) as colorless solid. H-NMR
(DMSO-d6) δ 11.67 (1H, s, NH), 6.30 (1H, s, H-1ꢁ), 5.21 (1H, d, H-5ꢁa),
4.95–4.89 (3H, m, H-2ꢁ, H-3ꢁ, and H-4ꢁ), 4.61 (1H, d, H-5ꢁb), 1.45 (3H, s,
CH3), 1.24 (3H, s, CH3).
Method B. A mixture of 4b (3.82 g, 8.22 mmol) and NaN3 (3.2 g, 49.2
mmol) in DMF (30 mL) was heated for 3 days at 110–120◦C. The mixture
was cooled to room temperature. Insoluble material precipitated and was

1538
A. E. A. Hassan et al.
removed by filtration. The filtrate was concentrated to dryness and the
residue was worked up as above to give (2.0 g, 79%) of 6.
Method C. A mixture of 4c (23 g, 47.4 mmol) and NaN3 (4.6 g, 71.1
mmol) in DMF (500 mL) was heated for 5 days at 110–120◦C. The mixture
was cooled to room temperature and the insoluble material was removed
by filtration. The filtrate was concentrated to dryness and the residue was
partitioned between EtOAc and H2O. The organic layer was evaporated and
the residue was crystallized from MeOH and EtOAc to give 6 (9.04 g, 62.2%).
The aqueous layer was evaporated to dryness and the residue was combined
to that obtained from evaporation of the mother liquor of the organic layer
and purified by flash silica gel column chromatography (hexanes:EtOAc,
1:1.5) to give 2.1 g (14.5%) of 6.
9,5 -Cyclo-3-(β-D-ribofuranosyl)-8-azaxanthine (2). A solution of 6 (4.1
g, 13.35 mmol) in a 1:1 mixture of THF and 1N HCl (1:1, 40 mL) was heated
at 90◦C for 4 h. Upon cooling the mixture to room temperature, white
crystals separated, which were collected, washed with cold water, and dried
in vacuo to give 3.5 g (98%) of 2. 1H-NMR (DMSO-d6) δ 11.59 (1H, s, NH),
5.98 (1H, s, H-1ꢁ), 5.74 (1H, d, 2ꢁ-OH, J = 4.8 Hz), 5.39 (1H, d, 3ꢁ-OH, J =
7.2 Hz), 4.98 (1H, d, H5ꢁa, J = 13.6 Hz), 4.80 (1H, dd, H5ꢁb, J = 13.6, J =
4.0 Hz), 4.56 (1H, dd like t, H-2), 4.16–4.06 (2H, m, H-3ꢁ and H-4ꢁ).
9,5 -Cyclo-1-methyl-3-(2,3- O-isopropylidine- β-D-ribofuranosyl)-8-aza-
xanthine (7a). To a mixture of 6 (152 mg, 0.5 mmol) and K2CO3 (172
mg, 1.25 mmol) in DMF (5 mL) was added MeI (62 mL, 1 mmol), and the
mixture was stirred at room temperature for 20 h. The solvent was removed
in vacuo, and the residue was partitioned between EtOAc and H2O. The
organic phase was dried (MgSO4) and evaporated. The residue was purified
on a silica gel column (eluate; EtOAc:hexanes 2:1) to give (150 mg, 93%) of
1
7a as a white solid which was crystallized from EtOAc in hexanes: H-NMR
(CDCl3) δ 6.68 (1H, s, H-1ꢁ), 5.05 (1H, d, H5ꢁa, J = 14 Hz), 4.92 (2H, m,
H-2ꢁ, and H-3ꢁ), 4.74 (1H, d, H-4ꢁ, J = 5.6 Hz), 4.67 (1H, dd, H-5ꢁb, J = 14,
J = 4 Hz), 3.41 (3H, s, N-CH3), 1.58 (3H, s, CH3), 1.34 (3H, s, CH3).
1-Cyanomethyl-9,5 -cyclo-3-(2,3- O-isopropylidine-β-D-ribofuranosyl)-8-
azaxanthine (7b). Sodium hydride dispersion in mineral oil (68 mg, 1.71
mmol) was added to a solution of 9,5ꢁ-cyclo-3-(2,3-O-isopropylidine-β-D-
ribofuranosyl)-8-azaxanthine (500 mg, 1.63 mmol) in DME:DMF (4:1, 10
mL) at 0◦C. After stirring for 10 min at 0◦C, LiBr (283 mg, 3.26 mmol)
was added and the suspension was stirred at room temperature for 15
min. BrCH2CN(0.23 mL, 3.26 mmol) was added and the mixture was
heated for 2 h at 65◦C. The solvent was evaporated and the residue
was partitioned between EtOAc and H2O. The organic phase was dried

Anti-HCV Derivatives
1539
(MgSO4) and evaporated. The residue was purified by a flash silica gel
column chromatography (eluate; 2% MeOH in CHCl3) to give (560 mg,
1
99%) as a white solid. H-NMR (DMSO-d6) δ 6.47 (1H, s, H-1ꢁ), 5.27 (1H,
d, H5ꢁa, J = 14.4 Hz), 5.01–4.87 (5H, m, H-2ꢁ, H-3ꢁ, and N-CH2CN), 4.67
(1H, dd, H-5ꢁb, J = 14.4, J = 4.0 Hz), 1.47 (3H, s, CH3), 1.25 (3H, s, CH3).
9,5 -Cyclo-1-methyl-3-( β-D-ribofuranosyl)-8-azaxanthine (8a). A solu-
tion of 7a (99 mg, 0.31 mmol) in a 1:1 mixture of THF and 1N HCl (10 mL)
was heated at 90◦C for 3 h. The solvent was evaporated and the residue was
purified by a flash silica gel column chromatography (eluate 12% MeOH
1
in CHCl3) to give 8a (78 mg, 89%) as a white solid: H-NMR (DMSO-d6) δ
6.03 (1H, s, H-1ꢁ), 5.77 (1H, d, 2ꢁ-OH, J = 4.8 Hz), 5.42 (1H, d, 3ꢁ-OH, J =
6.8 Hz), 5.03 (1H, br d, H5ꢁa, J = 15.2 Hz), 4.81 (1H, dd, H-5ꢁb, J = 14,
J = 4 Hz), 4.58 (1H, br dd, H-4ꢁ), 4.10 (2H, m, H-2ꢁ, and H-3ꢁ), 3.20 (3H,
s, N-CH3).
In a similar manner, from 300 mg (0.87 mmol) of 7b, 260 mg (98%)
of 1-cyanomethyl-9,5ꢁ-cyclo-3-(β-D-ribofuranosyl)-8-azaxanthine (8b) was ob-
1
tained after crystallization from MeOH. H-NMR (DMSO-d6+D2O) δ 6.04
(1H, s, H-1ꢁ), 5.04 (1H, d, H-5ꢁa, J = 13.6 Hz), 4.91–4.81 (2H, m, H-5ꢁb,
and H-4ꢁ), 4.62 (1H, m, H-3ꢁ or H-2ꢁ), 4.38 (1H, m, H-3ꢁ or H-2ꢁ), 4.20-4.06
(2H, m, N-CH2CN).
9,5 -Cyclo-3-(2,3- O -isopropylidine- β-D-ribofuranosyl)-6-imidazolyl-8-
azapurine-2-one (9). A mixture of 6 (565 mg, 1.84 mmol), PPh3 (1.16 g,
4.42 mmol), imidazol (0.44 g, 6.51 mmol), diisopropylethylamine (1.6 mL,
9.2 mmol), and iodine (1 g, 3.81 mmol) in toluene (15 mL) was heated
for 2 h at 95–100◦C. After cooling to room temperature, the mixture was
concentrated in vacuo, and the residue was mixed with EtOAc. Insoluble
materials were filtered through a Celite pad, the filtrate was washed with
water, brine, dried (MgSO4), and concentrated in vacuo. The residue was
purified on a flash silica gel column (eluate; 7:3 EtOAc:hexanes) to give
(0.536 g, 81%) of 9 as a pale yellow solid which was crystallized from EtOH
in hexanes. 1H-NMR (DMSO-d6) δ 7.64 (1H, s, 6-Im), 7.11 (1H, br s, 6-Im),
6.92 (1H, br s, 6-Im), 6.37 (1H, s, H-1ꢁ), 5.23 (1H, d, H5ꢁa, J = 14 Hz),
4.92–4.81 (3H, m, H-2ꢁ, H-3ꢁ, and H4ꢁ), 4.66 (1H, dd, H-5ꢁb, J = 4.4, J =
14 Hz), 1.47 (3H, s, CH3), 1.26 (3H, s, CH3).
9,5 -Cyclo-3-(2,3- O -isopropylidine- β-D-ribofuranosyl)-6-amino-8-aza-
purine-2-one (10a). Triethylamine (0.35 mL, 2.48 mmol) was added to
a mixture of 6 (152 mg, 0.5 mmol), DMAP (150 mg, 1.23 mmol), and
triisopropylbenzenesulfonyl chloride (373 mg, 1.23 mmol) in CH3CN(10
mL) at ca. 0◦C, and the mixture was stirred at room temperature for 24 h.
To the mixture was added conc. NH4OH (20 mL), and stirring continued

1540
A. E. A. Hassan et al.
for further 12 h. The solvent was concentrated in vacuo, and the residue
was chromatographed on a silica gel column (eluate: EtOAc:hexanes 3:1)
to give 10a (130 mg, 86%) as a pale yellow solid, which was used directly in
the next step without further purification.
9,5 -Cyclo-3-(β-D-ribofuranosyl)-6-amino-8-azapurine-2-one Hydrochlo-
ride (11a, HCl salt). A solution of 10a (120 mg, 0.39 mmol) in a 1:1 mixture
of THF and 1N HCl (1:1, 10 mL) was heated at 90◦C for 1 h and cooled
to room temperature. The solvent was evaporated and coevaporated with
EtOH several times. The solid residue was crystallized from EtOH and H2O
1
to give (92 mg, 89%) of the HCl salt of 11a. H-NMR of the major isomer
(DMSO-d6 + D2O) δ 5.99 (1H, s, H-1ꢁ), 5.09 (1H, d, H5ꢁa, J = 13.6 Hz),
4.90 (1H, dd, H5ꢁb, J = 13.6, J = 4.4 Hz), 4.61 (1H, t, H-4ꢁ, J = 4.4 Hz),
1
4.14–4.06 (2H, m, H-2ꢁ and H-3ꢁ); H-NMR of the minor isomer (DMSO-
d6 + D2O) δ 5.97 (1H, s, H-1ꢁ), 4.98 (1H, d, H5ꢁa, J = 14.0 Hz), 4.87 (1H,
dd, H5ꢁb, J = 14.0, J = 3.6 Hz), 4.56 (1H, t, H-4ꢁ, J = 4.4 Hz), 4.14–4.06
(2H, m, H-2ꢁ and H-3ꢁ).
N 9,5 -Cyclo-3-(2,3-O-isopropylidine-β-D-ribofuranosyl)-6-methylamino-8-
azapurine-2-one (10b). Triethylamine (0.4 mL, 2.83 mmol) was added to
a mixture of 6 (200 mg, 0.65 mmol), DMAP (200 mg, 1.64 mmol), and
triisopropylbenzenesulfonyl chloride (400 mg, 1.32 mmol) in CH3CN(10
mL) at ca. 0◦C, and the mixture was stirred at room temperature for 12 h.
To the mixture was CH3NH2 (2 mL), and the stirring continued for 2 h. The
solvent was removed in vacuo, and the residue purified on a silica gel column
(eluate; EtOAc:hexanes 3:1) to give (181 mg, 91%, white solid) of 10b as a
mixture of tautomers (6-MeNH−: 6-MeN=; 20:1 at room temperature). 1H-
NMR (DMSO-d6) δ 8.93 (1H, q, 6-NH, J = 4.8 Hz), 6.34 (1H, s, H-1ꢁ), 5.15
(1H, d, H-5ꢁa, J = 14.0 Hz), 4.87–4.84 (2H, dd, H-4ꢁ, and H-3ꢁ, J = 4.4, J =
5.6 Hz), 4.70 (1H, d, H-2ꢁ, J = 6.0 Hz), 4.59 (1H, dd, H-5ꢁb, J = 4.4, J =
14.0 Hz), 2.90 (3H, d, N-CH3, J = 4.8 Hz), 1.46 (3H, s, CH3), 1.24 (3H, s,
CH3).
In a similar manner, 10c-10e were prepared.
10c obtained in 84% yield as a white powder: 1H NMR (DMSO-d6+D2O)
δ 6.05 (1H, s, H-1ꢁ), 4.96 (1H, d, H-5ꢁa, J = 14.0 Hz), 4.76 (1H, dd, H-5ꢁb,
J = 4.4, J = 14.0 Hz), 4.57 (1H, dd like t, H-4ꢁ, J = 4.0 Hz), 4.11 (1H, t,
H-3ꢁ, J = 5.2 Hz), 3.96 (1H, t, H-2ꢁ, J = 5.2 Hz), 3.75 (3H, s, N-CH3), 3.71
(3H, s, N-CH3).
10d (95%, white powder), 1H-NMR (DMSO-d6) δ 9.00 (1H, t, 6-NH, J =
5.6 Hz), 6.33 (1H, s, H-1ꢁ), 5.16 (1H, d, H-5ꢁa, J = 14.0 Hz), 4.88–4.84 (2H,
dd, H-4ꢁ, and H-3ꢁ, J = 4.0, J = 6.0 Hz), 4.71 (1H, d, H-2ꢁ, J = 6.0 Hz), 4.60
(1H, dd, H-5ꢁb, J = 4.0, J = 14.0 Hz), 3.41 (2H, m, N-Bu), 1.59–1.52 (2H,
m, N-Bu), 1.46 (3H, s, CH3), 1.37–1.28 (2H, m, N-Bu), 1.24 (3H, s, CH3),
0.90 (3H, t, N-Bu, J = 5.6 Hz).

Anti-HCV Derivatives
1541
10e (86%, white powder), 1H-NMR (DMSO-d6) δ 10.53 (1H, s, NH), 6.24
(1H, s, H-1ꢁ), 5.14 (1H, d, H5ꢁa, J = 14.4 Hz), 4.92–4.83 (3H, m, H-2ꢁ, H-3ꢁ,
and H4ꢁ), 4.55 (1H, dd, H-5ꢁb, J = 4.0, J = 14.4 Hz), 3.78 (3H, s, NOCH3),
1.45 (3H, s, CH3), 1.24 (3H, s, CH3).
N 9,5 -Cyclo-3-(β-D-ribofuranosyl)-6-methylamino-8-azapurine-2-one
(11b). A solution of 10b (130 mg, 0.41 mmol) in THF:1N HCl (1:1, 5 mL)
was heated at 90◦C for 4 h. The reaction mixture was concentrated in vacuo,
and the residue was dried by several azeotropic distillations with EtOH. The
solid residue was recrystallized from EtOH and H2O to give (97 mg, 82%)
1
11b. H-NMR of the major isomer (DMSO-d6 + D2O) δ 6.03 (1H, s, H-1ꢁ),
5.04 (1H, d, H5ꢁa, J = 13.6 Hz), 4.86 (1H, dd, H-5ꢁb, J = 3.6 Hz, J = 14
Hz), 4.61 (1H, dd like t, H-4ꢁ, J = 4.8, J = 4 Hz), 4.15 (1H, t, H-3ꢁ, J = 4.8
Hz), 4.06 (1H, d, H-2ꢁ, J = 5.2 Hz), 3.74 (3H, s, N-CH3).
In a similar manner, 11c-e were prepared from 10c-e.
1
11c, 83% yield, white powder, (recrystallized from EtOH). H-NMR
(DMSO-d6 + D2O) δ 6.05 (1H, s, H-1ꢁ), 4.96 (1H, d, H-5ꢁa, J = 14.0 Hz),
4.76 (1H, dd, H-5ꢁb, J = 4.4, J = 14.0 Hz), 4.57 (1H, dd like t, H-4ꢁ, J =
4.0 Hz), 4.11 (1H, t, H-3ꢁ, J = 5.2 Hz), 3.96 (1H, t, H-2ꢁ, J = 5.2 Hz), 3.75
(3H, s, N-CH3), 3.71 (3H, s, N-CH3).
1
11d (85%, colorless powder), H-NMR (DMSO-d6 + D2O) δ 6.00 (1H,
s, H-1ꢁ), 4.94 (1H, d, H-5ꢁa, J = 13.6 Hz), 4.76 (1H, dd, H-5ꢁb, J = 4.0, J =
13.6 Hz), 4.54 (1H, dd like t, H-4ꢁ, J = 3.6), 4.12 (1H, dd like t, H-3ꢁ, J =
5.2 Hz), 3.96 (1H, d, H-2ꢁ, J = 5.2 Hz), 3.40 (2H, m, N-Bu), 1.59-1.52 (2H,
m, N-Bu), 1.37-1.28 (2H, m, N-Bu), 0.90 (3H, t, N-Bu, J = 7.2 Hz).
11e (60%, white solid), 1H-NMR (DMSO-d6 + D2O) δ 5.95 (1H, s, H-1ꢁ),
4.92 (1H, d, H5ꢁa, J = 14.0 Hz), 4.74 (1H, dd, H-5ꢁb, J = 4.0, J = 14.0 Hz),
4.61 (1H, dd like t, H-4ꢁ, J = 4.4 Hz), 4.13 (1H, t, H-3ꢁ, J = 4.8 Hz), 4.03
(1H, d, H-2ꢁ, J = 5.2 Hz), 3.78 (3H, s, N-OMe).
9,5 -Cyclo-3-(2,3- O-isopropylidene-β-D-ribofuranosyl)-8-aza-6-thioxan-
thine (12). A mixture of compound 6 (2.5 g, 8.1 mmol) and Lawesson’s
reagent (4.94 g) in anhydrous dichloroethane (160 mL) was heated under
reflux for 12 h. The reaction mixture was concentrated to dryness and
the residue was purified by silica gel column chromatography with 10–20%
1
EtOAc in hexanes to give 12 (1.78 g, 67.5%) as a yellowish solid. H N MR
(DMSO-d6) δ 1.23 (s, 3H, CH3), 1.45 (s, 3H, CH3), 4.63 (m, 1H, H-5ꢁa), 4.90
and 5.00 (m, 3H, 2ꢁ,3ꢁ,4ꢁ-H), 5.20 (d, 1H, J = 14 Hz, H-5ꢁb), 6.28 (s, 1H,
H-1ꢁ), 12.86 (s, 1H, NH).
9,5 -Cyclo-3-β-D-ribofuranosyl-8-aza-6-thioxanthine (13). A mixture of
compound 12 (55 mg, 0.17 mmol) in 4 mL of CF3CO2H/H2O (2:1, v/v)
solution was stirred at 50–55◦C for 3 h. A precipitate appears. The mixture

1542
A. E. A. Hassan et al.
was filtered and the solid was washed with EtOH to give 13 (30 mg, 63%)
as a yellowish solid. 1H NMR (DMSO-d6) δ 4.11 and 4.16 (m, 2H, H-2ꢁ, 3ꢁ),
4.55 (m, 1H, H-4ꢁ), 4.80 (dd, 1H, J = 4.4, 14 Hz, H-5ꢁa), 4.97 (d, 1H, J =
14 Hz, H-5ꢁb), 5.96 (s, 1H, H-1ꢁ).
AntiviralAssay
Briefly, HCV-replicon cells (Clone A cells; Apath LLC, St. Louis, Mis-
souri) in log phase growth were exposed to various concentrations of the
test compounds for 3 days, after which time the HCV RNA was extracted
and the amount produced quantified by real-time PCR. The potency of the
compounds against HCV replicon is expressed as EC90 (effective concentra-
tion to reduce the HCV RNA by 90%). MTS was utilized to determine the
associated potential toxic side-effects (CC50) as described previously.[9]
REFERENCES
1. Alter, M.J.; Kruszon-Moran, D.; Nainan, O.V.; McQuillan, G.M.; Gao F.; Moyer, L.A.; Kaslow, R.A.;
Margolis, H.S. The prevalence of hepatitis C virus infection in the United States, 1988 through
1994. N. Engl. J. Med. 1999, 341, 556–562.
2. Collier, J.; Chapman, R. Combination therapy with interferon-alpha and ribavirin for hepatitis C:
Practical treatment issues. BioDrugs 2001, 15, 225–238.
3. Di Bisceglie, A.M.; McHutchison, M.J.; Rice, C.M. New therapeutic strategies for hepatitis C.
Hepatology 2002, 285, 224–231.
4. Wang, P.; Hollecker, L.; Pankiewicz, K.W.; Patterson, S.E.; Whitaker, T.; McBrayer, T.R.; Tharnish,
P.M.; Sidwell, R.W.; Stuyver, L.; Otto, M.J.; Schinazi, R.F.; Watanabe, K.A. Synthesis of N3,5 -
cyclo-4-(β-D-ribofuranosyl)-vic-triazolo[4,5-b]pyridin-5-one: A novel compound with potential anti-
hepatitis C virus activity. J. Med. Chem. 2004, 47, 6100–6103.
5. Wang, P.; Hollecker, L.; Pankiewicz, K.W.; Patterson, S.E.; Whitaker, T.; McBrayer, T.R.; Thar-
nish, P.M.; Stuyver, L.; Schinazi, R.F.; Otto, M.J.; Watanabe, K.A. Synthesis of N3,5 -cyclo-4-
(β-D-ribofuranosyl)-vic-triazolo[4,5-b]pyridin-5-one and its 3 -deoxy analogue as potential anti-
hepatitis C virus agents. Nucleosides, Nucleotides & Nucleic Acids 2005, 24, 957–960.
6. Hassan, A.E.A.; Wang, P.; McBrayer, T.R.; Tharnish, P.M.; Stuyver, L.; Schinazi, R.F.; Otto, M.J.;
Watanabe, K.A. Synthesis and some SAR of anti-HCV nucleoside derivatives of N3,5 -anhydro-4-(β-
D-ribofuranosyl)-8-azapurin-2-ones. Nucleosides, Nucleotides & Nucleic Acids 2005, 24, 961–964.
7. Sasaki, T.; Minamoto, K.; Kino, M.; Mizuno, T. Reactions of the derivatives of 5-bromopyrimidine
nucleosides with sodium azide. J. Org. Chem. 1976, 41, 1100–1104.
8. Stuyver, L.J.; McBrayer, T.R.; Tharnish, P.M.; Hassan, A.E.A.; Chu, C.K.; Pankiewicz, K.W.;
Watanabe, K.A.; Schinazi, R.F.; Otto, M.J. Dynamics of subgenomic hepatitis C virus replicon
RNA levels in Huh cells after exposure to nucleotide anti-metabolites. J. Virol. 2003, 77, 10689–
10694.
9. Schinazi, R.F.; Sommadossi, J.P.; Saalmann, V.; Cannon, D.L.; Xie, M.-W.; Hart, G.C.; Smith,
G.A.; Hahn, E.F. Activity of 3 -azido-3 -deoxythymidine nucleotide dimers in primary lymphocytes
infected with human immunodeficiency virus type 1. Antimicrob. Agents Chemother. 1990, 34(6),
1061–1067.