Synthesis and structure-activity relationships of novel anti-hepatitis C agents: N3,5′-cyclo-4-(β-D-ribofuranosyl)-vic-triazolo[4, 5-b]pyridin-5-one derivatives

Article abstract of DOI:10.1021/jm058223t

Several 6- and 7-monosubstituted N³,5′-cyclo-4-(β-D- ribofuranosyl)-vic-triazolo[4,5-b]pyridin-5-one derivatives as well as the 5-thiono analogue were synthesized, providing structure - anti-hepatitis C virus (HCV) activity relationships for th

Full text of DOI:10.1021/jm058223t

6454
J. Med. Chem. 2005, 48, 6454-6460
Synthesis and Structure-Activity Relationships of Novel Anti-hepatitis C
Agents: N3,5′-Cyclo-4-(â-D-ribofuranosyl)-vic-triazolo[4,5-b]pyridin-5-one
Derivatives
Peiyuan Wang,*,† Jinfa Du,† Suguna Rachakonda,† Byoung-Kwon Chun,† Phillip M. Tharnish,†
Lieven J. Stuyver,† Michael J. Otto,† Raymond F. Schinazi,‡ and Kyoichi A. Watanabe†
Pharmasset Inc., Princeton, New Jersey 08540, and Department of Pediatrics, Emory University, School of Medicine/Veterans
Affairs Medical Center, Decatur, Georgia 30033
Received April 22, 2005
Several 6- and 7-monosubstituted N 3,5′-cyclo-4-(â-D-ribofuranosyl)-vic-triazolo[4,5-b]pyridin-
5-one derivatives as well as the 5-thiono analogue were synthesized, providing structure-
anti-hepatitis C virus (HCV) activity relationships for the series. Among the compounds
synthesized, the 6-bromo, 7-methylamino, and 5-thiono analogues exhibited more potent anti-
HCV activity in an HCV subgenomic replicon cell based assay (EC90 ) 1.9, 7.4, and 10.0 µM,
respectively) than the lead compound N 3,5′-cyclo-4-(â-D-ribofuranosyl)-vic-triazolo[4,5-b]pyridin-
5-one (EC90 ) 79.8 µM).
Introduction
subgenomic RNA replicon system.6 In an effort to
discover better anti-HCV agents, a series of 6- or
7-substituted derivatives were synthesized and their
potency was compared to that of the lead compound, 8.
The effect of substitution on the heterocyclic moiety of
the molecule on the biological activity was explored, in
order to gain some insight into the mode of action. The
facile one-pot synthesis of 8, synthesis of derivatives
thereof, structural determination, synthesis of novel
5-thiono, 6-halo, 7-alkylamino, and 7-methyl analogues
of 8, and anti-HCV and cytotoxicity evaluation of these
compounds are reported.
The hepatitis C virus (HCV) has infected approxi-
mately 2% of the worldwide population.1 It is estimated
that there are up to 230 000 new HCV infections every
year, and in the United States, this infection is respon-
sible for over 8000 deaths annually. Chronic hepatitis
C is predicted to become a major burden on the health
care system as individuals that are currently asymp-
tomatic with relatively mild disease progress to end-
stage liver disease and develop hepatocellular carci-
noma. Furthermore, the number of deaths due to HCV
infection is predicted to triple in the next 10-20 years.2
The HCV is a single-stranded, enveloped, positive-
sense RNA virus in the flaviviridae family. It contains
a genome of RNA with approximately 9600 nucleotides,
a capsid, a matrix, and an envelope. It encodes a single
polyprotein precursor which is processed into three
structural (C, E1, and E2) and into several nonstruc-
tural (NS1, NS2, NS3, NS4, and NS5) viral proteins.3
Currently, the only treatment available for patients
with chronic HCV is R-interferon (IFN-R), either alone
or in combination with ribavirin (RBV).4 The overall
sustained response rate to treatment by the combination
therapy with RBV and IFN-R for 6-12 months is around
40% in patients infected with HCV genotype 1. Response
rates are better in genotype 2 or 3. Anemia is the most
common adverse event associated with RBV, and neu-
ropsychiatric adverse effects due to IFN-R can lead to
premature cessation of therapy in 10-20% of patients.
This treatment is far from satisfactory, as the response
rate is low and adverse side effects are common.4,5
Therefore, it is critical to discover new, more potent anti-
HCV agents.
Results and Discussion
Chemistry. We first attempted to improve the syn-
thesis of 8, which was synthesized from 1-(2,3,5-tri-O-
benzoyl-â-D-ribofuranosyl)-5-nitropyridin-2-one(1,Scheme
1). It is known that 2′,3′-O-isopropylidene pyrimidine
nucleosides undergo cyclization to form 5′,2-anhydro-
pyrimidine much more readily than the corresponding
2′,3′-unprotected nucleosides.7 We, therefore, synthe-
sized 4-(1-â-D-ribofuranosyl)-vic-triazolo[4,5-b]pyridin-
5-one8 (3) in two steps by de-O-benzoylation to 2,
followed by treatment with a mixture of acetone and
2,2-dimethoxypropane in the presence of acid to afford
the 2′,3′-O-isopropylidene derivative 4. Upon treatment
of 4 with diethyl azodicarboxylate and triphenylphos-
phine under Mitsunobu conditions, cyclization occurred
to give the desired 3,5′-cyclonucleoside 5. Acid hydrolysis
of 5 gave the desired product 8 in 83% yield. We found,
however, that this procedure was not suitable for
preparative scale synthesis because three of the five
steps require chromatographic purification and the
overall yield was less than 20%.
Recently, we discovered that the novel molecule N3,5′-
cyclo-4-(â-D-ribofuranosyl)-vic-triazolo[4,5-b]pyridin-5-
one (8) exhibited moderate anti-HCV activity in a
A more efficient two-step procedure was developed.
Treatment of 16 with NaN3 at 110-120 °C in dimeth-
ylformamide (DMF) for 3 days afforded the 3,5′-cyclo
product 6 in 60% yield. After saponification of 6,
compound 8 was obtained in good yield (78%). The
overall yield from 1 was 47%. Treatment of 6 with
* Corresponding author. Office: 609-613-4100. Fax: 609-613-4150.
E-mail: peiyuan.wang@pharmasset.com
† Pharmasset Inc.
‡ Emory University.
10.1021/jm058223t CCC: $30.25 © 2005 American Chemical Society
Published on Web 09/14/2005

Novel Anti-hepatitis C Agents
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6455
Scheme 1
Reagents: (a) NH3/MeOH, rt; (b) NaN3, DMF, 110-120 °C, 12 h; (c) DMP/DMF/acetone/p-TsOH, rt; (d) Ph3P/DEAD/DMF, rt; (e) NaN3,
DMF, 110-120 °C, 48 h; (f) i. Lawesson’s reagent/DCE, reflux; ii. NH3/MeOH, rt; (g) 0.5 M NaOMe in MeOH, rt; (h) NCS/AcOH, reflux
to 9a; Br2/H2O to 9b; (i) RNH2 or R2NH, heated; (j) HCl/dioxane, 50 °C, 12 h.
Scheme 2
Reagents: (a) n-BuNH2/MeOH, rt; (b) HCl in Et2O/acetone, rt; (c) MsCl/Pyr/CH2Cl2, rt; (d) NaN3, DMF, 120 °C, 3 days; (e) concentrated
HCl/MeOH, 50 °C, 20 min.
Lawesson’s reagent in toluene afforded the 5-thio de-
rivative, which, without purification, was treated with
MeONa/MeOH at room temperature. The free nucleo-
side 7 that precipitated was crystallized from MeOH.
Bromination with Br2 in water or chlorination with
NCS in acetic acid of 8 afforded the 6-substituted
halides 9a and 9b. Treatment of 9b with various amines
yielded the corresponding 7-substituted products 10a-
f. The structure of 9 can readily be verified by 1H NMR
spectroscopy. In structure 8, 6-H and 7-H appeared at
δ 6.36 and 8.05, respectively, as doublets. On halogena-
tion, the higher field 6-H signal disappeared, and 7-H
stayed at δ 8.54 for 9a and δ 8.69 for 9b as a singlet.
On treatment of 9b with a nucleophile, such as MeNH2,
the 7-H singlet disappeared and the 6-H singlet emerged
at δ 6.18. Apparently, halogenation of 8 occurred at the
R-position of the carbonyl group on the heterocyclic ring
to form 9; Michael addition of an amine on 9 took place,
giving rise to the saturated intermediates 9′ and 9′′, and
from the latter, HX came off, leading to the thermody-
namic product 10.
The 7-methyl analogue 16 was prepared by a different
method from the one used for the synthesis of 8, since,
due to the presence of the 7-methyl group, the nucleo-
philicity of N-3 is apparently reduced, resisting anhydro-
linkage formation. Thus, we prepared the 5′-azido-2′,3′-
O-isopropylidene derivative 14 (Scheme 2) first and then
tried to add the azido group across the 5,6-double bond
with concomitant elimination of the 5-nitro group. We
condensed 4-methyl-5-nitropyridin-2-one with 1-O-acetyl-
tri-O-benzoyl-D-ribofuranose under Vorbru¨ggen’s condi-
tions and obtained 11 in 57% yield. Compound 11 was

6456 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Wang et al.
Table 1. Anti-HCV Activity of Synthesized Compounds in the
Huh-7 Replicon Cells
compound
EC90a (µM)
CC50b (µM)
7
10.0
79.8
11.86
30.6
8
9a
9b
2.1
0.06
1.9
1.5
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
16
29.7
74.2
7.4
4.6
>100
>100
>100
>100
>100
>100
86.1
>100
>100
>100
>100
>100
>100
97.3
>100
15.4
>100
22.6
a EC90 ) effective concentration to reduce the HCV RNA by 90%.
b CC50 ) concentration at which cell number as detected by the
MTS assay was reduced by 50%.
subjected to de-O-benzoylation to give 12, which was
treated with acetone and acid to afford 2′,3′-O-isopro-
pylidene derivative 13, followed by mesylation to 14.
Treatment of 14 with NaN3 afforded the desired triaz-
olopyridine cyclonucleoside 15 in 53% crude overall
yield. Acid hydrolysis of 15 afforded the desired product
16 in 77% yield in crystalline form.
Figure 1. (A) Dose response of compound 7 and dose-
dependent antiviral effect on HCV replicon RNA containing
Huh-7 cells. Cells were seeded at 1000 cells per well in a 96
well plate in the presence of the compound. After 96 h of
incubation, replicon HCV RNA and rRNA levels were quanti-
fied by real-time reverse transcription-PCR. (B) Comparison
of the effects of compound 7 on cell growth and on HCV
replicon dynamics over 7 days.
Anti-HCV Activity. Compounds 7, 8, 9a, 9b, 10a-
j, and 16 were assayed for their ability to inhibit HCV
RNA replication in a subgenomic replicon Huh7 cell line,
as described previously.9 The anti-HCV replicon activity
and cytotoxicity of these compounds are summarized in
Table 1. The 5-thiono (7), 6-chloro (9a), 6-bromo (9b),
7-amino (10a), 7-methylamino (10b), and 7-methyl (16)
derivatives exhibited more potent anti-HCV activity
than 8.6 Among them, the 6-bromo and 6-chloro deriva-
tives, 9b and 9a, exhibited the most potent anti-HCV
activity with EC90 values of 1.9 and 2.1 µM, respectively
(Table 1). The 7-methylamino derivative (10b) was
found to exhibit significant anti-HCV activities with an
EC90 value of 7.4 µM. The 5-thiono and 7-methyl
derivatives, 7 (EC90 ) 10.0 µM) and 16 (EC90 ) 15.4
µM) (part A of Figures 1-3), are the next most active
group, followed by 7-methoxyethylamino derivatives,
i.e., 10i (EC90 ) 86.1 µM). The 7-amino derivative 10a
(EC90 ) 29.7 µM) exhibited more potent anti-HCV
activity than 8. Other 7-alkylamino derivatives 10c-h
were neither active nor cytotoxic up to 100 µM. From
the biological results, the most active compound was the
6-bromo analogue 9b, but unfortunately, it was also
toxic. Among 7-amino substituted derivatives, the most
active compound was the methylamino analogue 10b.
Only 10a showed activity at a concentration signifi-
cantly lower than the CC50. It appears that the activity
would be reduced as the size of the substituent on the
amino group at C-7 grows larger. The ribosomal RNA
(a cellular marker for potential toxicity) was also
reduced, but to a lesser extent than the amount of HCV
RNA was reduced (Figures 1-3). To evaluate the
specific antiviral effect of compounds 7, 10b, and 16 over
a longer exposure time, replicon cells were kept in
culture for 7 days, in either the presence (100 µM) or
absence of the compound (part B of Figures 1-3). These
experiments suggest that these compounds caused a
cytostatic effect at 100 µM, since the treated cells did
not proliferate with the same dynamics as the no-
treatment control. Concomitantly with the slower cell
proliferation, a significant decrease in intracellular HCV
RNA was observed, consistent with a previous report
that examined cytostatic agents.9 Other synthesized
compounds also showed similar results (data not shown).
These observations suggest that the anti-HCV activity
may be associated with the cytostatic effects.
This class of compounds did not inhibit purified HCV
RNA-dependent RNA polymerase (NS5B) in vitro when
tested in cell-free systems.6,10 It is not surprising that
they do not appear to inhibit HCV RNA-dependent RNA
polymerase since there is no 5′-OH present in these
compounds. The mode of anti-HCV action is now the
subject of further studies.
In conclusion, a new class of 5-thiono, 6-halo, 7-al-
kylamino, and 7-methyl analogues of N3,5′-cyclo-4-(â-
D-ribofuranosyl)-vic-triazolo[4,5-b]pyridin-5-one was syn-
thesized and evaluated for its biological activity in a
HCV replicon system. Several synthesized compounds
were found to exhibit significant anti-HCV activity. In
view of these interesting preliminary antiviral data,
further chemical synthesis and biological studies are
warranted.
Experimental Section
General. Melting points were determined on an electro-
thermal digital melting point apparatus and are uncorrected.
Nuclear magnetic resonance spectra were recorded on a Varian
Unity Plus 400 spectrometer at room temperature, with Me4-

Novel Anti-hepatitis C Agents
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6457
Figure 3. (A) Dose response of compound 16 and dose-
dependent antiviral effect on HCV replicon RNA containing
Huh-7 cells. Cells were seeded at 1000 cells per well in a 96
well plate in the presence of the compound. After 96 h of
incubation, replicon HCV RNA and rRNA levels were quanti-
fied by real-time reverse transcription-PCR. (B) Comparison
of the effects of compound 16 on cell growth and on HCV
replicon dynamics over 7 days.
Figure 2. (A) Dose response of compound 10b and dose-
dependent antiviral effect on HCV replicon RNA containing
Huh-7 cells. Cells were seeded at 1000 cells per well in a 96
well plate in the presence of the compound. After 96 h of
incubation, replicon HCV RNA and rRNA levels were quanti-
fied by real-time reverse transcription-PCR. (B) Comparison
of the effects of compound 10b on cell growth and on HCV
replicon dynamics over 7 days.
1H), 8.01 (d, J ) 9.6 Hz, 1H). Anal. Calcd for C10H12N4O5: C,
44.78; H, 4.51; N, 20.89. Found: C, 44.60; H, 4.53; N, 20.60.
Si as the internal standard. Chemical shifts (δ) are reported
in parts per million (ppm), and signals as s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of
doublets), or br s (broad singlet). Values given for coupling
constants are first order, and UV spectra were recorded on a
Varian CARY 50 Bio UV-vis spectrophotometer. Fast atom
bombardment (FAB) mass spectroscopic data were obtained
at the Emory University Mass Spectrometry Center. Thin-
layer chromatography (TLC) was performed on Uniplates
(silica gel) from Analtech Co., and column chromatography on
silica gel (60 Å) purchased from Sorbent Technologies, Atlanta,
GA. Elemental analyses were performed by Atlantic Microlab
Inc., Norcross, GA.
4-(2,3-O-Isopropylidene-â-D-ribofuranosyl)-vic-triazolo-
[4,5-b]pyridin-5-one (4). To a solution of 3 (2.68 g, 1.0 mmol)
in a mixture of 2,2-dimethoxypropane (10 mL), DMF (10 mL),
and dry acetone (30 mL) was added p-TsOH‚H2O (100 mg),
and the mixture was stirred overnight at room temperature.
The reaction mixture was neutralized with excess NaHCO3
and filtered, and the solvent was removed in vacuo to give a
pale yellow residue, which was passed through a column of
silica gel eluting with 30% ethyl acetate (EtOAc) in hexane to
give 4 (2.24 g, 72.5%) as a yellow solid. 1H NMR (CDCl3) δ
1.35 (s, 3H), 1.64 (s, 3H), 3.97 (d, J ) 12 Hz, 1H), 4.06 (d, J )
12 Hz, 1H), 4.45 (s, 1H), 5.08 (m, 1H), 5.14 (m, 1H), 5.25 (m,
OH), 6.63 (d, J ) 10 Hz, 1H), 6.72 (d, J ) 4.8 Hz, 1H), 7.81 (s,
J ) 10 Hz, 1H). This product was used directly in the next
step.
3,5′-Cyclo-4-(2,3-O-isopropylidene-â-D-ribofuranosyl)-
vic-triazolo[4,5-b]pyridin-5-one (5). Compound 4 (0.5 g,
1.61 mmol) was dissolved in 15 mL of anhydrous DMF along
with triphenylphosphine (1.27 g, 4.85 mmol) and diethyl
azadicarboxylate (0.84 g, 4.85 mmol), and the mixture was
stirred at room temperature overnight. The mixture was
concentrated to dryness, and the residue was partitioned
between EtOAc and water. The organic layer was washed with
water (2 × 20 mL), dried (Na2SO4), and concentrated in vacuo
to give a yellow residue that was purified by column chroma-
tography using 5-10% MeOH in CH2Cl2 to give 5 (306 mg,
65.4%) as a pale yellow solid. 1H NMR (CDCl3) δ 1.26 (s, 3H),
1.53 (s, 3H), 4.52 (d, J ) 5.2 Hz, 1H), 4.70 (m, 1H), 4.79 (d, J
) 5.6 Hz, 1H), 4.89 (d, J ) 4.0 Hz, 1H), 5.06 (d, J ) 14 Hz,
1H), 6.40 (d, J ) 10 Hz, 1H), 6.83 (1H), 7.89 (d, J ) 10 Hz,
1H). HRMS (FAB) obsd: m/z 291.1100. Calcd for C13H14N4O4
+ H: m/z 291.1093 (M + H)+.
5-Nitro-1-(â-D-ribofuranosyl)pyridin-2-one (2). A mix-
ture of 5-nitro-1-(2,3,5-tri-O-benzoyl-â-D-ribofuranosyl)-2-py-
ridone (1, 100 mg, 0.17 mmol)8 and saturated NH3/MeOH (10
mL) was stirred at room temperature for 12 h. The reaction
mixture was concentrated to dryness, and the residue was
triturated with EtOH until precipitation was completed. The
precipitates were collected and recrystallized with water to
1
give 2 (37 mg, 81%) as a white solid. H NMR (DMSO-d6 +
D2O) δ 3.64 (m, 1H), 3.90 (m, 1H), 4.01 (m, 3H), 5.89 (s, 1H),
6.48 (d, J ) 10.4 Hz, 1H), 8.12 (dd, J ) 3.2, 10 Hz, 1H), 9.65
(d, J ) 3.2 Hz, 1H).
4-(â-D-Ribofuranosyl)-vic-triazolo[4,5-b]pyridin-5-
one (3). A mixture of 2 (54 mg, 0.2 mmol) and NaN3 (20 mg,
0.3 mmol) in DMF (20 mL) was stirred at 110-120 °C for 12
h. The reaction mixture was concentrated to dryness, and the
residue was purified on a silica gel column with 15% MeOH
in CH2Cl2 to give 3 (32 mg, 60%) as a solid. 1H NMR (DMSO-
d6) δ 3.53 (m, 1H), 3.66 (m, 1H), 3.88 (m, 1H), 4.16 (dd, J )
5.2, 9.2 Hz, 1H), 4.79 (m, 1H), 5.04 (d, J ) 5.2 Hz, 1H), 5.17
(d, J ) 6 Hz, 1H), 6.33 (d, J ) 6 Hz, 1H), 6.46 (d, J ) 9.6 Hz,
3,5′-Cyclo-4-(2,3-di-O-benzoyl-â-D-ribofuranosyl)-vic-
triazolo[4,5-b]pyridin-5-one (6). A mixture of 1 (25 g, 42.8

6458 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Wang et al.
mmol), NaN3 (4.17 g, 64.2 mmol), and DMF (700 mL) was
stirred at 110-120° C for 3 days. The mixture was cooled, the
insoluble material was filtered off, and the filtrate was
evaporated in vacuo to give a residue, which was extracted
with EtOAc (3 × 500 mL) in the presence of water (500 mL).
The ethyl acetate solution was back washed with water, dried
(Na2SO4), and evaporated to give a crystalline residue. Re-
crystallization from a mixture of EtOAc and EtOH gave 6 (11.8
precipitated was collected (135 mg): mp 294-296 °C; UV
1
(MeOH) λmax 295 nm; H NMR (DMSO-d6) δ 3.86 (t, J ) 4.8
Hz, 1H), 4.07 (dd, J ) 5.2, 11.6 Hz, 1H), 4.53 (t, J ) 4 Hz,
1H), 4.74 (dd, J ) 4.0, 14.0 Hz, 1H), 4.96 (d, J ) 13.6 Hz, 1H),
5.16 (s, 1H), 5.34 (d, J ) 6.8 Hz, 1H, D2O exchangeable), 5.66
(d, J ) 4.8 Hz, 1H, D2O exchangeable), 6.16 (s, 1H), 6.98 (s,
2H, D2O exchangeable). Anal. Calcd for C10H11N5O4‚H2O: C,
42.40; H, 4.63; N, 24.73. Found: C, 42.17; H, 4.64; N, 24.43.
1
g, 60.2%). H NMR (DMSO-d6) δ 5.00 (dd, J ) 4.4, 14.0 Hz,
3,5′-Cyclo-7-methylamino-4-(â-D-ribofuranosyl)-vic-tri-
azolo[4,5-b]pyridin-5-one (10b). A mixture of 9b (150 mg,
0.456 mmol) and 33% MeNH2 in EtOH (15 mL) was heated at
110 °C in a sealed bottle for 16 h. The mixture was concen-
trated in vacuo, and the residue was triturated with EtOH.
The precipitated product was collected by suction (35 mg,
1H), 5.33 (m, 2H), 5.79 (t, J ) 4.8 Hz, 1H), 5.88 (d, J ) 5.2
Hz, 1H), 6.44 (d, J ) 9.6 Hz, 1H), 6.75 (s, 1H), 7.32-8.08 (m,
10H), 8.14 (d, J ) 9.6, 1H). Anal. Calcd for C24H18N4O3‚
0.5H2O: C, 61.67; H, 4.10; N, 11.99. Found: C, 61.41; H, 4.10;
N, 12.39.
1
27.5%): mp 264-266 °C; UV (MeOH) λmax 275 nm; H NMR
3,5′-Cyclo-4-(â-D-ribofuranosyl)-vic-triazolo[4,5-b]pyri-
din-5-one (8). Compound 6 (2.2 g, 4.8 mmol) was treated with
0.5 M MeONa/MeOH (44 mL), and the mixture was stirred at
room temperature for 3 h. After neutralization with HOAc,
the mixture was concentrated to dryness, and the residue was
chromatographed on a silica gel column with 5% MeOH in CH2-
Cl2 to give a solid which was triturated with EtOH to afford
pure 8 (0.93 g, 77.5%). 1H NMR (DMSO-d6 + D2O) δ 3.98 (t, J
) 4.4 Hz, 1H), 4.13 (m, 1H), 4.61 (t, J ) 4.2 Hz, 1H), 4.83 (dd,
J ) 4, 13.6 Hz, 1H), 5.02 (d, J ) 13.6 Hz, 1H), 5.37 (d, J ) 7.2
Hz, 1H), 5.76 (d, J ) 4.8 Hz, 1H), 6.21 (s, 1H), 6.36 (d, J ) 9.6
Hz, 1H), 8.05 (d, J ) 9.6 Hz, 1H). Anal. Calcd for C10H10N4O4‚
0.2H2O: C, 47.32; H, 4.13; N, 22.07. Found: C, 47.10; H, 4.09;
N, 21.97.
3,5′-Cyclo-4-(â-D-ribofuranosyl)-vic-triazolo[4,5-b]pyri-
din-5-thione (7). A mixture of 6 (2.0 g, 4.36 mmol) and
Lawesson’s reagent (2.0 g) in anhydrous dichloroethane (85
mL) was heated under reflux for 16 h, after which it was
concentrated to dryness. The residue was treated with satu-
rated NH3/MeOH (80 mL), and the mixture was stirred at room
temperature for 12 h. Yellowish precipitates were collected by
suction and washed with MeOH. Recrystallization of the
precipitate from hot water gave 7 (910 mg, 78%) as a yellowish
solid. 1H NMR (DMSO-d6 ) δ 4.13 (t, J ) 4.4 Hz, 1H), 4.23 (m,
1H), 4.70 (t, J ) 4.8 Hz, 1H), 4.97 (dd, J ) 4.4, 13.6 Hz, 1H),
5.11 (d, J ) 14.0 Hz, 1H), 5.43 (d, J ) 7.2 Hz, 1H), 5.84 (d, J
) 5.2 Hz, 1H), 6.88 (s, 1H), 7.31 (d, J ) 8.8 Hz, 1H), 7.96 (d,
J ) 9.6 Hz, 1H). Anal. Calcd for C10H10N4O3S‚0.2H2O: C,
44.50; H, 3.88; N, 20.76. Found: C, 44.25; H, 3.94; N, 20.58.
3,5′-Cyclo-6-chloro-4-(â-D-ribofuranosyl)-vic-triazolo-
[4,5-b]pyridin-5-one (9a). A mixture of 8 (94 mg, 0.38 mmol)
and N-chlorosuccinimide (69 mg) in 3.5 mL of glacial HOAc
was refluxed for 30 min. The mixture was concentrated to
dryness, and the residue was purified by silica gel column
chromatography with 1-2% MeOH in CH2Cl2 to give 9a (43
mg, 40.2%) as a solid: mp 192-195 °C; UV (MeOH) λmax 319.9
nm; 1H NMR (DMSO-d6) δ 4.06 (t, J ) 5 Hz, 1H), 4.15 (m,
1H), 4.62 (t, J ) 4.2 Hz, 1H), 4.89 (dd, J ) 4, 13.6 Hz, 1H),
5.05 (d, J ) 14.0 Hz, 1H), 5.42 (d, J ) 7.6 Hz, 1H, D2O
exchangeable), 5.84 (d, J ) 4.8 Hz, 1H, D2O exchangeable),
6.23 (s, 1H), 8.54 (s, 1H). HRMS (FAB) Obsd: m/z 283.0222.
Calcd for C10H8N4O4Cl: m/z 283.0234 (M - H)-.
3,5′-Cyclo-6-bromo-4-(â-D-ribofuranosyl)-vic-triazolo-
[4,5-b]pyridin-5-one (9b). To a stirred solution of 8 (3 g, 11.99
mmol) in H2O (60 mL) was added Br2 (0.9 mL) dropwise at
room temperature. The product 9b that precipitated while
stirring at room temperature was collected by suction and
washed with MeOH to give 9b (3.35 g, 85.1%): mp 199-201
°C; UV (MeOH) λmax 325 nm; 1H NMR (DMSO-d6 + D2O) δ
4.04 (d, J ) 5.2 Hz, 1H), 4.15 (t, J ) 4.8 Hz, 1H), 4.62 (t, J )
4.4 Hz, 1H), 4.87 (dd, J ) 4, 14 Hz, 1H), 5.04 (d, J ) 14.0 Hz,
1H), 6.22 (s, 1H), 8.69 (s, 1H). Anal. Calcd for C10H9N4O4Br‚
0.75H2O: C, 35.06; H, 3.09; N, 16.35. Found: C, 34.71; H, 3.13;
N, 16.03.
(DMSO-d6) δ 2.77 (d, J ) 4.4 Hz, 3H), 3.87 (t, J ) 4.8 Hz, 1H),
4.07 (dd, J ) 5.2, 12 Hz, 1H), 4.54 (t, J ) 4.4 Hz, 1H), 4.75
(dd, J ) 3.6, 13.6 Hz, 1H), 4.96 (d, J ) 13.6 Hz, 1H), 5.05 (s,
1H), 5.34 (d, J ) 7.6 Hz, 1H, D2O exchangeable), 5.68 (d, J )
4.8 Hz, 1H, D2O exchangeable), 6.18 (s, 1H), 7.56 (s, 1H, D2O
exchangeable). Anal. Calcd for C11H13N5O4‚0.5H2O: C, 45.83;
H, 4.89; N, 24.29. Found: C, 45.56; H, 4.96; N, 23.97.
3,5′-Cyclo-7-(ethylamino)-4-(â-D-ribofuranosyl)-vic-tri-
azolo[4,5-b]pyridin-5-one (10c). In a similar manner but
using 75% EtNH2 instead of 33% MeNH2, the corresponding
7-ethylamino product 10c was obtained in 36% yield from 9b:
1
mp 265-267 °C; UV (MeOH) λmax 275 nm; H NMR (DMSO-
d6) δ 1.16 (t, J ) 4.4 Hz, 3H), 3.20 (brs, 2H), 3.87 (t, J ) 4.4
Hz, 1H), 4.08 (dd, J ) 5.2, 12.8 Hz, 1H), 4.54 (t, J ) 4.0 Hz,
1H), 4.74 (dd, J ) 4.0, 13.6 Hz, 1H), 4.96 (d, J ) 13.6 Hz, 1H),
5.11 (s, 1H), 5.33 (d, J ) 7.2 Hz, 1H, D2O exchangeable), 5.66
(d, J ) 4.8 Hz, 1H, D2O exchangeable), 6.18 (s, 1H), 7.52 (t, J
) 5.2 Hz, 1H, D2O exchangeable). Anal. Calcd for C12H15N5O4‚
0.25H2O: C, 48.40; H, 5.25; N, 23.51. Found: C, 48.17; H, 5.31;
N, 23.33. Similarly, the following compounds were synthesized.
3,5′-Cyclo-7-propylamino-4-(â-D-ribofuranosyl)-vic-tri-
azolo[4,5-b]pyridin-5-one (10d) in 32% yield: mp 269-272
°C; UV (MeOH) λmax 275 nm; 1H NMR (DMSO-d6) δ 0.89 (t, J
) 7.2 Hz, 3H), 1.57 (dd, J ) 8.0, 14.8 Hz, 2H), 3.12 (m, 2H),
3.87 (t, J ) 4.4 Hz, 1H), 4.08 (dd, J ) 5.2, 12 Hz, 1H), 4.53 (t,
J ) 4 Hz, 1H), 4.74 (dd, J ) 4.0, 14.0 Hz, 1H), 4.96 (d, J )
13.2 Hz, 1H), 5.11 (s, 1H), 5.33 (d, J ) 7.2 Hz, 1H, D2O
exchangeable), 5.67 (d, J ) 4.8 Hz, 1H, D2O exchangeable),
6.17 (s, 1H), 7.56 (s, 1H, D2O exchangeable). Anal. Calcd for
C13H17N5O4: C, 50.81; H, 5.58; N, 22.79. Found: C, 50.87; H,
5.74; N, 22.69.
3,5′-Cyclo-7-(2-hydroxyethylamino)-4-(â-D-ribofurano-
syl)-vic-triazolo[4,5-b]-pyridin-5-one (10e) in 41% yield: mp
253-256 °C; UV (MeOH) λmax 275 nm; 1H NMR (DMSO-d6) δ
3.23 (brs, 2H), 3.56 (dd, J ) 6.4, 12.0 Hz, 2H), 3.87 (t, J ) 4.8
Hz, 1H), 4.07 (dd, J ) 5.2, 12 Hz, 1H), 4.54 (t, J ) 4.0 Hz,
1H), 4.75 (dd, J ) 4.0, 14.0 Hz, 1H), 4.79 (t, J ) 6.0 Hz, 1H,
D2O exchangeable), 4.97 (d, J )14.0 Hz, 1H), 5.17 (s, 1H), 5.34
(d, J ) 7.6 Hz, 1H, D2O exchangeable), 5.68 (d, J ) 4.4 Hz,
1H, D2O exchangeable), 6.18 (s, 1H), 7.32 (t, J ) 6 Hz, 1H,
D2O exchangeable). Anal. Calcd for C12H15N5O5: C, 46.60; H,
4.89; N, 22.64. Found: C, 46.49; H, 5.05; N, 22.48.
3,5′-Cyclo-7-hydroxypropylamino-4-(â-D-ribofuranosyl)-
vic-triazolo[4,5-b]pyridin-5-one (10f) in 39.7% yield: mp
279-281 °C; UV (MeOH) λmax 275 nm; 1H NMR (DMSO-d6) δ
1.73 (m, 2H), 3.24 (m, 2H), 3.48 (dd, J ) 6.0, 11.6 Hz, 2H),
3.89 (t, J ) 4.8 Hz, 1H), 4.09 (m, 1H), 4.56 (m, 2H), 4.76 (dd,
J ) 3.6, 13.6 Hz, 1H), 4.97 (d, J ) 14.0 Hz, 1H), 5.14 (s, 1H),
5.35 (d, J ) 7.2 Hz, 1H), 5.69 (d, J ) 4.4 Hz, 1H), 6.19 (s, 1H),
7.53 (t, J ) 5.6 Hz, 1H). Anal. Calcd for C13H17N5O5: C, 48.29;
H, 5.30; N, 21.66. Found: C, 48.44; H, 5.49; N, 21.56.
7-n-Butylamino-3,5′-cyclo-4-(â-D-ribofuranosyl)-vic-tri-
azolo[4,5-b]pyridin-5-one (10g) in 32.8% yield: mp 228-232
°C; UV (MeOH) λmax 275 nm; 1H NMR (DMSO-d6) δ 0.89 (t, J
) 7.2 Hz, 3H), 1.33 (m, 2H), 1.53 (m, 2H), 3.15 (brs, 2H), 3.87
(t, J ) 4 Hz, 1H), 4.08 (dd, J ) 5.2, 11.6 Hz, 1H), 4.53 (t, J )
3.6 Hz, 1H), 4.74 (dd, J ) 4, 14 Hz, 1H), 4.96 (d, J ) 13.6 Hz,
1H), 5.10 (s, 1H), 5.34 (d, J ) 7.2 Hz, 1H, D2O exchangeable),
5.67 (d, J ) 4.4 Hz, 1H, D2O exchangeable), 6.17 (s, 1H), 7.55
7-Amino-3,5′-cyclo-4-(â-D-ribofuranosyl)-vic-triazolo-
[4,5-b]pyridin-5-one (10a). A mixture of 9b (150 mg, 0.456
mmol) and liquid ammonia (10 mL) was heated at 70 °C in a
sealed vessel for 15 h. The vessel was cooled and opened at
-78 °C, and 5 mL of MeOH was added. The mixture was
warmed to room temperature, and the amount of 10a that

Novel Anti-hepatitis C Agents
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6459
(br s, 1H, D2O exchangeable). Anal. Calcd for C14H19N5O4‚
0.4MeOH: C, 51.76; H, 6.21; N, 20.96. Found: C, 51.51; H,
6.53; N, 20.63.
added pyridine (5 mL), CH2Cl2 (5 mL), and MsCl (0.5 mL, 4.5
mmol), and the resulting solution was stirred at room tem-
perature for 16 h. CH2Cl2 (50 mL) was added, and the solution
was washed with brine and dried (Na2SO4). Solvent was
removed to give mesylate 14 as a foam [1H NMR (CDCl3) δ
1.36 (s, 3H), 1.60 (s, 3H), 2.55 (s, 3H), 3.09 (s, 3H), 4.53 (m,
3H), 4.94 (m, 2H), 5.92 (d, J ) 1.2 Hz, 1H), 6.37 (s, 1H), 8.78
(s, 1H)], which was dissolved in DMF (10 mL). To the solution
was added NaN3 (390 mg, 6 mmol), and the mixture was
heated at 120 °C for 3 days. The solvent was removed under
reduced pressure, and the residue was purified by silica gel
chromatography (50% EtOAc in hexanes) to give 2′,3′-O-
isopropylidene-5′,9-anhydronucleoside (15, 320 mg, 52.6%) as
a white solid: mp 247-249 °C; 1H NMR (CDCl3) δ 1.31 (s, 3H),
1.58 (s, 3H), 2.53 (s, 3H), 4.53 (d, J ) 5.6 Hz, 1H), 4.67 (dd, J
) 5.6, 14.0 Hz, 1H), 4.78 (d, J ) 5.6 Hz, 1H), 4.91 (d, J ) 4.4
Hz, 1H), 5.10 (d, J ) 14.0 Hz, 1H), 6.25 (d, J ) 0.8 Hz, 1H),
6.88 (s, 1H). HRMS (FAB) Obsd: m/z 305.1263. Calcd for
C14H16N4O4 + H: m/z 305.1250 (M + H)+.
3,5′-Cyclo-7-methyl-4-(â-D-ribofuranosyl)-vic-triazolo-
[4,5-b]-pyridin-5-one (16). To a solution of 15 (300 mg, 0.99
mmol) in MeOH (10 mL) was added concentrated HCl (2 mL),
and the solution was heated at 50 °C for 20 min. The solvent
was removed in vacuo to give a solid which was washed with
MeOH to provide pure 16 (200 mg, 76.6%): mp 252-253.5 °C;
1H NMR (DMSO-d6) δ 2.42 (s, 3H), 3.94 (t, J ) 4.8 Hz, 1H),
4.11 (m, 1H), 4.68 (t, J ) 4.8 Hz, 1H), 4.81 (dd, J ) 4.4, 14.0
Hz, 1H), 5.00 (d, J ) 14.0 Hz, 1H), 5.37 (d, J ) 7.6 Hz, 1H,
D2O exchangeable), 5.76 (d, J ) 4.4 Hz, 1H, D2O exchange-
able), 6.18 (s, 1H), 6.20 (s, 1H). Anal. Calcd for C11H12N4O4‚
0.25H2O: C, 49.12; H, 4.65; N, 20.84. Found: C, 48.85; H, 4.67;
N, 20.58. HRMS (FAB) Obsd: m/z 265.0977. Calcd for
C11H12N4O4 + H: m/z 265.0937 (M + H)+.
3,5′-Cyclo-7-cyclopropylamino-4-(â-D-ribofuranosyl)-
vic-triazolo[4,5-b]pyridin-5-one (10h) in 45.8% yield: mp
258-260 °C; UV (MeOH) λmax 275 nm; 1H NMR (DMSO-d6) δ
0.56 (m, 2H), 0.73 (m, 2H), 3.26 (m, 1H), 3.88 (t, J ) 4.8 Hz,
1H), 4.07 (dd, J ) 5.2, 12.0 Hz, 1H), 4.54 (t, J ) 4.0 Hz, 1H),
4.74 (dd, J ) 4.0, 14.0 Hz, 1H), 4.96 (d, J ) 13.6 Hz, 1H), 5.34
(d, J ) 7.2 Hz, 1H, D2O exchangeable), 5.37 (s, 1H), 5.69 (d, J
) 4.8 Hz, 1H, D2O exchangeable), 6.19 (s, 1H), 7.87 (s, 1H,
D2O exchangeable). Anal. Calcd for C12H15N5O4‚0.9H2O: C,
48.57; H, 5.26; N, 21.78. Found: C, 48.37; H, 5.22; N, 21.65.
3,5′-Cyclo-7-(dimethylamino)-4-(â-D-ribofuranosyl)-vic-
triazolo[4,5-b]pyridin-5-one (10i) in 35% yield: mp 292-
294 °C (dec); UV (MeOH) λmax 275 nm; 1H NMR (DMSO-d6) δ
2.48 and 2.49 (2 s, 2 × 3H), 3.86 (t, J ) 4.8 Hz, 1H), 4.07 (m,
J ) 1H), 4.55 (t, J ) 4.0 Hz, 1H), 4.74 (dd, J ) 4.0, 13.6 Hz,
1H), 4.99 (d, J ) 14.0 Hz, 1H), 5.10 (s, 1H), 5.35 (d, J ) 7.2
Hz, 1H, D2O exchangeable), 5.68 (d, J ) 4.8 Hz, 1H, D2O
exchangeable), 6.22 (s, 1H). Anal. Calcd for C12H15N5O4‚
0.9H2O: C, 46.57; H, 5.47; N, 22.63. Found: C, 46.80; H, 5.22;
N, 22.71.
3,5′-Cyclo-7-methoxyethylamino-4-(â-D-ribofuranosyl)-
vic-triazolo[4,5-b]pyridin-5-one (10j) in 29.5% yield: mp
227-228 °C; UV (MeOH) λmax 275 nm; 1H NMR (DMSO-d6) δ
3.25 (s, 3H), 3.34 (m, 2H), 3.50 (t, J ) 5.6 Hz, 1H), 3.87 (t, J
) 4.8 Hz, 1H), 4.08 (m, 1H), 4.54 (t, J ) 3.6 Hz, 1H), 4.75 (dd,
J ) 3.6, 13.6 Hz, 1H), 4.97 (d, J ) 14.0 Hz, 1H), 5.18 (s, 1H),
5.35 (d, J ) 7.2 Hz, 1H), 5.69 (d, J ) 4.8 Hz, 1H), 6.17 (s, 1H),
7.41 (t, J ) 5.6 Hz, 1H). Anal. Calcd for C13H17N5O5‚0.85H2O:
C, 46.11; H, 5.57; N, 20.68. Found: C, 45.85; H, 5.64; N, 20.47.
4-Methyl-5-nitro-1-(2,3,5-tri-O-benzoyl-â-D-ribofurano-
syl)pyridin-2-one (11). A mixture of 2-hydroxy-4-methyl-5-
nitropyridine (3.08 g, 20 mmol) and (NH4)2SO4 (100 mg) in
HMDS (20 mL) was refluxed for 2 h, and the clear solution
was concentrated to dryness under reduced pressure. The
residue was dissolved in CH2Cl2 (60 mL). To the solution were
added 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose (7.56 g, 15
mmol) and TMSOTf (3.99 g, 18 mmol), and the solution was
stirred at room temperature for 16 h. To the solution were
added NaHCO3 (2.27 g, 27 mmol) and then H2O (15 mL)
slowly, and the mixture was stirred at room temperature for
30 min. The aqueous layer was extracted with EtOAc (3 × 50
mL). The combined organic solutions were washed with brine
and dried (Na2SO4). The solvent was removed, and the
resulting solid was recrystallized from EtOAc-hexanes to give
compound 11 (6.80 g, 56.8%): mp 170-171.5 °C; 1H NMR
(CDCl3) δ 8.82 (s, 1H), 8.10-7.34 (m, 15H), 6.49 (d, J ) 4.8
Hz, 1H), 6.37 (s, 1H), 5.90 (t, J ) 5.2 Hz, 1H), 5.84 (t, J ) 5.6
Hz, 1H), 4.84 (m, 3H), 2.52 (s, 3H). Anal. Calcd for C32H26N2O10:
C, 64.21; H, 4.38; N, 4.68. Found: C, 64.37; H, 4.41; N, 4.65.
Antiviral Assay. Briefly, HCV replicon cells (Clone A cells;
Apath LLC, St. Louis, MO) in log phase growth were exposed
to various concentrations of the test compounds for 3 days;
after this time, the HCV RNA was extracted, and the amount
produced was quantified by real-time PCR. The potency of the
compounds against HCV replicon is expressed as EC90 (effec-
tive concentration to reduce the HCV RNA by 90%).6,11 MTS
was utilized to determine the associated potential toxicity
(CC50) as described previously.12,13
Acknowledgment. This work was supported in part
by NIH Grant Nos. 1R43 AI-52868 (biology) and 1R43
AI-056720 (chemistry). R.F.S. is the principal founder
and a former director of Pharmasset, Inc. His laboratory
did not receive any funding from Pharmasset, Inc. for
this work.
4-Methyl-5-nitro-1-(â-D-ribofuranosyl)pyridin-2-one (12).
A mixture of 11 (2.99 g, 5 mmol) and excess n-BuNH2 (10 mL)
in MeOH (100 mL) was stirred at room temperature for 48 h,
and the solvent was removed under reduced pressure. The
residue was triturated with CH2Cl2, and the resulting solid
was collected by filtration to give nucleoside 12 (1.28 g,
89.5%): mp 136-137.5 °C; 1H NMR (DMSO-d6) δ 2.42 (s, 3H),
3.60 (dd, J ) 1.6, 12.0 Hz, 1H), 3.80 (dd, J ) 2.4, 12.4 Hz,
1H), 3.98 (m, 3H), 5.08 (d, J ) 6.4 Hz, 1H, D2O exchangeable),
5.33 (t, J ) 4.2 Hz, 1H, D2O exchangeable), 5.62 (d, J ) 4.8
Hz, 1H, D2O exchangeable), 5.88 (d, J ) 1.6 Hz, 1H), 6.38 (s,
1H), 9.48 (s, 1H). Anal. Calcd for C11H14N2O7: C, 46.16; H,
4.93; N, 9.79. Found: C, 46.19; H, 4.95; N, 9.68. HRMS (FAB)
Obsd: m/z 287.0883. Calcd for C11H14N2O7 + H: m/z 286.0879
(M + H)+.
3,5′-Cyclo-7-methyl-4-(2,3-O-isopropylidene-â-D-ribo-
furanosyl)-vic-triazolo[4,5-b]-pyridin-5-one (15). To a mix-
ture of compound 12 (572 mg, 2 mmol) in acetone (20 mL) was
added HCl (2 M in Et2O, 2 mL, 4 mmol), and the resulting
mixture was stirred at room temperature for 3 h. Pyridine (2
mL) was added, and the resulting solution was concentrated
to dryness under reduced pressure to give crude 13. To 13 were
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