Synthesis of N3,5′-cyclo-4-(β-D-ribofuranosyl)-vic- triazolo[4,5-b]pyridin-5-one, a novel compound with anti-hepatitis C virus activity

Article abstract of DOI:10.1021/jm0401210

A novel anti-hepatitis C virus (HCV) agent, N³,5′-cyclo-4- (β-D-ribofuranosyl)-vic-triazolo(4,5-b]pyridinin-5-one, was identified, and the structure was confirmed by chemical synthesis from 2-hydroxy-5- nitropyridine.

Full text of DOI:10.1021/jm0401210

6100
J. Med. Chem. 2004, 47, 6100-6103
Brief Articles
Synthesis of N3,5′-Cyclo-4-(â-D-ribofuranosyl)-vic-triazolo[4,5-b]pyridin-5-one, a
Novel Compound with Anti-Hepatitis C Virus Activity
Peiyuan Wang,*,† Laurent Hollecker,† Krzysztof W. Pankiewicz,† Steven E. Patterson,† Tony Whitaker,†
Tamara R. McBrayer,† Phillip M. Tharnish,† Robert W. Sidwell,‡ Lieven J. Stuyver,† Michael J. Otto,†
Raymond F. Schinazi,§ and Kyoichi A. Watanabe†
Pharmasset Inc., Tucker, Georgia 30084, Utah State University, Logan, Utah 84322, and Department of Pediatrics,
Emory University, School of Medicine/VA Medical Center, Decatur, Georgia 30033
Received June 25, 2004
A novel anti-hepatitis C virus (HCV) agent, N3,5′-cyclo-4-(â-D-ribofuranosyl)-vic-triazolo[4,5-
b]pyridinin-5-one, was identified, and the structure was confirmed by chemical synthesis from
2-hydroxy-5-nitropyridine.
1
Hepatitis C virus (HCV) is one of the most clinically
important Flaviviridae infections affecting humans and
is the second most common cause of viral hepatitis.
Currently, nearly 2% of the U.S. population, and an
estimated 170 million people worldwide, are HCV
carriers.1 HCV is an RNA virus, which replicates
without involvement of DNA. Presently there is no
universally effective treatment for this infection, and
the only drugs available for treatment of chronic hepa-
titis C are various forms of alpha interferon (IFN-R),
either alone or in combination with ribavirin.2 However,
the therapeutic value of these treatments has been
compromised largely due to adverse effects,2,3 which
highlights the need for development of additional op-
tions for treatment.
Therefore, we embarked upon systematic investiga-
tion of ribonucleosides from our extensive chemical
library of nucleosides in the hope that HCV hits may
provide clues for the development of effective anti-HCV
drugs. One of our programs was to synthesize various
base-modified nucleosides and evaluate them for their
activity in a replicon system.4,5 None of the synthetic
ribonucleosides with modified purine bases tested showed
activity in our evaluation system. However, when we
treated tribenzoylated 1-(â-D-ribofuranosyl)-2-oxo-5-ni-
tropyridine (1, Scheme 1) with sodium azide in DMF,
followed by saponification with NaOMe to a product,
which we expected to be 4-(â-D-ribofuranosyl)-5-oxo-vic-
triazolo[4,5-b]pyridine (3),6 we found weak activity.
Inactive preparations consisted only of 4-(â-D-ribofura-
nosyl)-5-oxo-vic-triazolo[4,5-b]pyridine (3),6 but the ac-
tive preparation was contaminated with a second com-
ponent, which is less polar than 3 on TLC. Therefore,
we focused our efforts on structural determination of
the minor component, which was separated and purified
by repeated silica gel column chromatography. This
compound showed no H-6 signal of pyridine in its H
NMR spectrum, suggesting that it is a derivative of
5-oxo-vic-triazolo[4,5-b]pyridine. However, there were
only two D2O exchangeable doublets for 2′- and 3′-OH
groups and no exchangeable triplet for 5′-OH. The
chemical shifts of H-5′a and H-5′b were at δ 4.83 (dd,
J4′,5′a ) 4 and J5′a,5′b ) 13.6 Hz) and at δ 5.02 (apparent
d, J4′,5′b ) 0, J5′a,5′b ) 13.6 Hz). The large difference in
chemical shifts and geminal coupling of H5′a and H5′b
strongly suggest no free rotation about the C-4′-C-5′
bond. This was further supported by NOE difference
spectra: irradiation of the resonances at 4.83 ppm gave
enhancement of the signals at 4.60 and 5.02 ppm,
whereas irradiation of the resonances at 5.02 ppm gave
enhancement of the signals at 4.83, 4.60, and 3.98 ppm.
In addition, the UV spectrum in water was similar to
that of 4-methyl-5-oxo-v-triazolo[4,5-b]pyridine,7 and the
pattern did not change at pH 2 and pH 11, indicating
no dissociable proton on the base. All these spectral
characteristics are fully consistent with the novel cyclic
compound 6.7 The elemental analyses (C10H10N4O4) and
mass spectral data (HR-FAB MS obsd: m/z 249.0614,
calcd for C10H10N4O4: m/z 249.0624 M - H-) are also
consistent with structure 6.
We then investigated conditions to produce 6 more
efficiently. The improved procedure that we tried was
to start with 1-(2,3,5-tri-O-benzoyl-â-D-ribofuranosyl)-
5-nitropyridin-2-one (1), which we obtained in 70% yield
by condensation of the trimethylsilyated pyridine with
1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose under the
Vorbru¨ggen’s conditions. The 1H NMR spectrum of the
product was consistent with those that were previously
reported.6 The benzoyl protecting groups were removed
with saturated ammonia in methanol, and after crystal-
lization from water, 1-(â-D-ribofuranosyl)-5-nitropyridin-
2-one (2) was obtained in 81% yield. Treatment of 2 with
sodium azide in DMF at 110-120 °C for 12 h afforded
the vic-triazolopyridine nucleoside 3 in 60% yield.
When 1 was directly treated with NaN3, followed by
de-O-benzoylation with NaOMe in MeOH, a mixture of
3 and 6 was produced. In this reaction, 3 was formed
* Corresponding author. Office: 678-395-0042. Fax: 678-395-0030.
E-mail: pwang@pharmasset.com.
† Pharmasset Inc.
‡ Utah State University.
§ Emory University.
10.1021/jm0401210 CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/22/2004

Brief Articles
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 6101
Scheme 1a
a Reagents and conditions: (a) NH3/MeOH, rt; (b) NaN3, DMF, (b′: at 110-120 °C, 12 h: from 2 to give 3; b′′: at 80-95 °C, 24 h, from
1 to 4; b′′′: at 110-120 °C, 48 h, from 1 to 5); (c) 0.5 M NaOMe in MeOH, rt.
from 4 by solvolysis. Compound 6 could have been
produced by two ways: after formation of 4 during NaN3
treatment of 1, the 5′-benzoyloxy group acted as a
leaving group upon attack of N-3 on C-5′ leading to 5
which was then de-O-benzoylated; or intramolecular
cyclization could have occurred between N-3 and C-5′dur-
ing solvolysis of 4. In the latter case two competing
reactions occurred simultaneously; i.e., attack by solvent
in the presence of base on the carbonyl carbon of the
benzoyl group and intramolecular attack of N-3 on C-5′
with removal of the benzoyloxy group. We, therefore,
studied the course of the reaction to find conditions that
favor intramolecular reaction over solvolysis to convert
1 directly to 5. We found that reaction of 1 with NaN3
in DMF at 90 °C afforded only 4-(2,3,5-tri-O-benzoyl-â-
D-ribofuranosyl)-vic-triazolo[4,5-b]pyridin-5-one (4). How-
ever, we observed that by increasing the reaction
temperature to 110 °C or higher, 4 was produced first
then 5 was formed slowly as judged by TLC. The
amount of 5 increased with concomitant decrease of 4.
Thus, after prolonged heating we were able to obtain 5
in 44% yield. Saponification of 5 with MeONa/MeOH
furnished 6 in 62% yield (Scheme 1).
The antiviral activity of 6 was evaluated in the HCV
subgenomic RNA replicon as previously described,5 and
the results are shown in Figure 1. Compound 6 had an
anti-HCV effect with an EC50 (effective concentration
to reduce the HCV RNA by 50%) of 19.7 µM after 96 h
of compound exposure (Figure 1A). In addition to the
antiviral effect, ribosomal RNA (which is a cellular
Figure 1. Antiviral activity of compound 6. (A) Dose-depend-
ent antiviral effect on HCV replicon RNA containing Huh-7
cells. Cells were seeded at 1000 cells per well in a 96-well plate
marker for potential toxic side-effects) was also reduced
in the presence of compound. After 96 h of incubation, replicon
(Figure 1A). To evaluate the specific antiviral effect of
compound 6 over a longer exposure time, Replicon cells
were kept in culture for 7 days, either in the presence
(100 µM) or in absence of the compound (Figure 1B).
These experiments showed that 6 caused a cytostatic
effect at high concentration, since the treated cells did
not proliferate with the same dynamics as the no-
compound control. Concomitantly with the slower cell
proliferation, a significant decrease in intracellular HCV
RNA was observed.
In addition, an HCV RNA-dependent RNA poly-
merase (NS5B) assay was designed essentially as
described.8 Compound 6 showed no specific inhibitory
activity against this recombinant NS5B enzyme (data
HCV RNA and rRNA levels were quantified by real time
reverse transcription-PCR. (B) Comparison of the effects of
compound 6 on cell growth and on HCV replicon dynamics over
7 days.
not shown). Since there is no 5′-OH in compound 6, it
is not surprising that it does not appear to inhibit HCV-
RNA-dependent RNA polymerase.
Compound 6 was tested against a range of other RNA
viruses (Table 1). The compound was inactive and gen-
erally nontoxic (Table 2) with the exception of weak ac-
tivity against influenza B virus with an EC50 of 23 µM.
In conclusion, we discovered and synthesized a novel
compound 6 as a good lead for development of anti-HCV

6102 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Brief Articles
Table 1. Antiviral Activity of Compound 6 When Tested
kept at room-temperature overnight, the reaction mixture is
diluted with CH2Cl2, washed with saturated NaHCO3 solution,
and filtered through Celite. The organic layer was separated,
washed with H2O, dried, filtered, and concentrated to give a
residue, which was crystallized from ethanol. The product 1
(15.53 g, 70%) was obtained as a solid. 1H NMR (DMSO-d6) δ
4.71 (m, 1H), 4.90 (m, 1H), 6.04 (m, 2H), 6.43 (d, J ) 2.0 Hz,
1 H), 6.59 (d, J ) 10.0 Hz, 1H), 7.40-8.02 (m, 15 H), 8.19 (dd,
J ) 3.2, 10.0 Hz, 1 H), 9.16 (d, J ) 3.2 Hz, 1 H).6
1-(â-D-Ribofuranosyl)-5-nitropyrine-2(1H)-one (2). A
mixture of 1 (100 mg, 0.17 mmol) and saturated MeOH/NH3
(10 mL) was stirred at room temperature for 12 h. The mixture
was concentrated to dryness, and the residue was triturated
with EtOH to precipitate (2), which was collected and crystal-
lized from water (37 mg, 81%) as a white solid. 1H 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, 1 H), 8.12 (dd, J ) 3.2, 10
Hz, 1H), 9.65 (d, J ) 3.2 Hz, 1 H). This product was used
directly in the next step without further purification.
against Various Viruses in Culture
virus
EC50, µM
EC90, µM
influenza a virus
influenza b virus
respiratory syncytial virus
measles virus
rhinovirus
parainfluenza virus
pinchinde virus
VEE
yellow fever
West Nile
adenovirus type 1
punta toro A
hepatitis B virus
HCV replicon
BVDV
>100
28
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
19.7
>100
79.8
>100
>100
>100
>100
>100
>100
HIV
HSV type 1
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 mixture was concentrated to dryness, and the residue
was purified by silica gel column chromatography with 15%
agents. Compound 6 showed an interesting anti-
hepatitis C effect in the HCV subgenomic RNA replicon.
The mode of anti-HCV action is now the subject of
further studies. In addition, structure-activity relation-
ship studies aimed at optimizing the anti-HCV activity
while reducing the cytostatic effect are underway.
1
MeOH in CH2Cl2 to give 3 (32 mg, 60%) as a solid, H 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, 1 H), 4.79 (m, 1H), 5.04 (d, J ) 5.2 Hz,
1H), 5.17 (d, J ) 6 Hz, 1 H), 6.33 (d, J ) 6 Hz, 1 H), 6.46 (d,
J ) 9.6 Hz, 1 H), 8.01 (d, J ) 9.6 Hz, 1 H). Anal. Calcd for
C10H12N4O5: C, 44.78; H, 4.51; N, 20.89; Found: C, 44.60; H,
4.53; N, 20.60.
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
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. TLC was performed on
Uniplates (silica gel) purchased from Analtech Co., and column
chromatography was performed using silica gel (60 Å) from
Sorbent Technologies, Atlanta, GA. Elemental analyses were
performed by Atlantic Microlab Inc., Norcross, GA.
1-(2,3,5-Tri-O-benzoyl-â-D-ribofuranosyl)-5-nitropyri-
dine-2(1H)-one (1). 2-Hydroxy-5-nitropyridine (5.6 g, 40
mmol) in hexamethyldisilazane (80 mL) is refluxed with a
catalytic amount of ammonium sulfate for 6 h in an argon
atmosphere. Excess solvent is removed in vacuo and the
residue dissolved in 1,2-dichloroethane (100 mL). To this
solution are added 1-acetyl-2,3,5-tri-O-benzoyl-â-D-ribofura-
nose (19 g, 37.6 mmol) in anhydrous 1,2-dichloroethane (100
mL) and 1 N solution of tin(IV) chloride in CH2Cl2 (7.68 mL),
and the mixture was heated under reflux for 8 h. After being
4-(2,3,5-Tri-O-benzoyl-â-D-ribofuranosyl)-vic-triazolo-
[4,5-b]pyridin-5-one (4). To a solution of 1 (3.82 g, 6.5 mmol)
in DMF (33 mL) was added NaN3 (1.55 g, 23.8 mmol) under
argon. The suspension was stirred at 80-95 °C for 24 h. The
solvent was removed in vacuo, and 2 N HCl (12 mL) was added
the residue. The mixture was extracted with CH2Cl2, dried
(Na2SO4), and concentrated in vacuo. The oily residue was
purified on a silica gel column with a stepwise gradient of
EtOAc (0-20%)in CH2Cl2 to give 3.19 g (84%) of a pale yellow
1
solid. H NMR (DMSO-d6) δ 4.57 (dd, J ) 5.2, 12.4 Hz, 1H),
4.71 (dd, J ) 3.6, 12.0 Hz, 1H), 4.79 (m, 1H), 6.23 (t, J ) 8.0
Hz, 1 H), 6.33 (m, 1H), 6.60 (d, J ) 10.0 Hz, 1H), 6.70 (s, 1H),
7.38-7.94 (m, 15 H), 8.10 (d, J ) 10 Hz, 1 H).6
3,5′-Cyclo-3-(2,3-di-O-benzoyl-â-D-ribofuranosyl)-vic-
triazolo[4,5-b]pyridin-5-one (5). A mixture of 1 (1 g, 1.71
mmol), NaN3 (167 mg, 2.56 mmol), and DMF (28 mL) was
stirred and heated at 110-120° C for 48 h. The solvent was
removed in vacuo, and the residue purified by silica gel column
chromatography with 20% EtOAc in hexanes to give 5 as a
solid (343 mg, 44%). mp 232-233 °C (after crystallization from
EtOAc/EtOH). 1H NMR (DMSO-d6) δ 5.00 (dd, J ) 4.4 and 14
Hz, 1 H), 5.33 (m, 2 H), 5.79 (t, J ) 4.8 Hz, 1 H), 5.88 (d, J )
5.2 Hz, 1 H), 6.44 (d, J ) 9.6 Hz,1H), 6.75 (s, 1 H), 7.32-8.08
(m, 10 H), 8.14 (d, 1H, J ) 9.6). HR-FAB MS Obsd; m/z
459.1321. Calcd for C24H19N4O6: m/z 459.1305 (M + H)+. Anal.
Table 2. Cytotoxicity Results for Compound 6 in Various Cell Lines.
cell line
HepG2
Huh-7
PBM
Vero
cell origination
CC50 (µM), MTSa
CC50 (µM), neutral red
>100
human hepatocellular carcinoma
human hepatocellular carcinoma
primary human peripheral blood mononuclear cells
African green monkey kidney
human malignant melanoma
human T-cell lymphoma
74.4
49.6
>100
>100
>100
13.8
SK-MEL-28
CEM
B-SC-1
MDCK
A-549
African green monkey kidney
Madin Darby canine kidney
>100
>100
>100
>100
>100
>100
>100
human lung carcinoma
CV-1
African green monkey kidney
African green monkey kidney
rhesus monkey kidney
MA-104
LLC-MK2
KB
human epidermoid carcinoma of the nasopharnyx
a MTS dye: Cell titer 96-cell proliferation assay (Promega, Madison, WI).

Brief Articles
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 6103
Calcd for C24H18N4O3‚0.5 H2O: C, 61.67; H, 4.10; N, 11.99;
Found: C, 61.41; H, 4.10; N, 12.39.
R43 AI-056720 (chemistry). We would like to acknowl-
edge the contribution of D.L. Barnard, J. D. Morrey, and
D.F. Smee at the Institute for Antiviral Research at
Utah State University for their testing of RNA viruses
under NIH Contract No. N01-A1-85348. Dr. Raymond
F. Schinazi is supported in part by the Department of
Veterans Affairs. He is a founder and consultant of
Pharmasset Inc., and his protocols have been reviewed
by Emory University’s Conflict of Interest Committee.
Dr. Schinazi and his group received no funding from
Pharmasset.
3,5′-Cyclo-1-(â-D-ribofuranosyl)-vic-triazolo[4,5-b]pyri-
din-5-one (6). Compound 5 (160 mg, 0.35 mmol) was treated
with 0.5 M NaOMe/MeOH at room temperature for 1 h. The
mixture was neutralized with acetic acid, concentrated in
vacuo to dryness, and the residue was purified by silica gel
column chromatography with 5% MeOH in CH2Cl2 to give 6
(54 mg, 62%) as a white solid, mp 248-251 °C. 1H NMR
(DMSO-d6) δ 3.98 (t, J ) 4.8 Hz, 1 H, 2′-H), 4.14 (dd, J ) 5.2
and 12.4 Hz, 1 H, 3′-H), 4.60 (t, J ) 4.4 Hz, 1 H, 4′-H), 4.83
(dd, J ) 4 and 13.6 Hz, 1 H, 5′-Ha), 5.02 (d, J ) 13.6 Hz, 1 H,
5′-Hb), 5.39 (d, J ) 7.2 Hz, 1 H, OH, D2O exchangeable), 5.79
(d, J ) 5.2 Hz, 1 H, OH, D2O exchangeable), 6.21 (s, 1 H, 1′-
H), 6.36 (d, J ) 9.6 Hz, 1 H, 6-H), 8.05 (d, J ) 9.6 Hz, 1 H,
7-H). 13C NMR ((DMSO-d6) δ 159.98, 136.55, 131.96, 129.64,
116.89, 93.22, 81.85, 74.02, 71.31, 54.90; HR-FAB MS Obsd;
m/z 249.0614. Calcd for C10H9N4O4: m/z 249.0624 (M-H)-.
Anal. Calcd for C10H10N4O4: C, 48.00; H, 4.03; N, 22.39;
Found: C, 48.10; H, 4.06; N, 22.45.
Antiviral Testing. HCV-replicon RNA-containing Huh7
cells (Clone A cells; Apath, LLC, St. Louis, MO) were kept in
exponential growth in DMEM media (high glucose, no pyru-
vate) containing 10% fetal bovine serum, 1X nonessential
amino acids, penicillin-streptomycin-glutamine (100 units/
L, 100 µg/L, and 2.92 mg/L, respectively) and G418 (500 to
1000 µg/mL).4 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 im-
mediately after seeding. After 96 h of incubation, total cellular
RNA was isolated (Rneasy 96 kit, Qiagen, CA). Replicon RNA
and an internal control (TaqMan Ribosomal RNA control
Reagents, Applied Biosystems, CA) were amplified in a single-
step multiplex RT-PCR protocol, as recommended by the
manufacturer. The HCV primers and probe used have been
described previously.5
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threshold RT-PCR cycle of the test compound was subtracted
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control (∆CtHCV). A ∆Ct of 3.3 equals a 1-log reduction (i.e.,
EC90) in replicon RNA levels. The cytotoxicity of the test
compound was also determined by calculating the ∆CtrRNA
values. The ∆∆Ct specificity parameter could then be intro-
duced (∆CtHCV - ∆CtrRNA), in which the levels of HCV RNA
were normalized for the rRNA levels and are calibrated against
the no drug control. Recombinant interferon alpha-2a (INF-
R-2a; Roferon-A, Hoffman La Roche Inc, NJ) served as a
positive control.
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L.; Xie, M.-W.; Hart, G. C.; Smith, G. A.; Hahn, E. F. Activity of
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The neutral red and CPE (cytopathic effect) reduction assays
for determining the antiviral activity and cytotoxicity were
performed as described previously.9 Cytotoxicty testing using
MTS was performed as described previously.10,11
Acknowledgment. This work was supported in
parts by the NIH grants 1R43 AI-52868 (biology) and 1
JM0401210