Synthesis of 1,2,3-triazolo-carbanucleoside analogues of ribavirin targeting an HCV in replicon

Article abstract of DOI:10.1016/S0968-0896(03)00349-3

The synthesis of carbocyclic and phosphonocarbocyclic analogues of ribavirin, an anti-HCV inhibitor, are described. Those compounds were evaluated against HCV but also against other important viruses in order to determine their spectrum of antiviral activity. Compounds 6 and 13 displayed a moderate IC₅₀ against HIV-1 of 43.8 and 37 μM, respectively.

Full text of DOI:10.1016/S0968-0896(03)00349-3

Bioorganic & Medicinal Chemistry 11 (2003) 3633–3639
Synthesis of 1,2,3-Triazolo-carbanucleoside Analogues of
Ribavirin Targeting an HCV in Replicon
Yoshio Saito,a Vanessa Escuret,b David Durantel,b Fabien Zoulim,b Raymond
F. Schinazic and Luigi A. Agrofoglioa,*
a
´
Institut de Chimie Organique et Analytique, ICOA UMR 6005, UFR Sciences, BP 6759, 45067 Orleans Cedex 2, France
bInserm U271, 151 Cours Albert Thomas, 69003 Lyon, France
cVeterans Affairs Medical Center and Laboratory of Biochemical Pharmacology, Department of Pediatrics,
Emory University School of Medicine, Atlanta, GA 30033, USA
Received 18 March 2003; accepted 23 May 2003
Abstract—The synthesis ofcarbocyclic and phosphonocarbocyclic analogues ofribavirin, an anti-HCV inhibitor, are described.
Those compounds were evaluated against HCV but also against other important viruses in order to determine their spectrum of
antiviral activity. Compounds 6 and 13 displayed a moderate IC50 against HIV-1 of43.8 and 37 mM, respectively.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
Thus, specific inhibitors ofthis virus-encoded RNA-
dependent RNA polymerase (RdRP) should serve as
potent antiviral agents.
Chronic hepatitis C (HCV) remains a major public
health problem, with an estimated 200–300 million
people infected in the world. Indeed, despite the mass
screening program for early diagnosis and treatment of
HCV infection, and the improvement of antiviral ther-
apy1 relying on the combination pegylated interferon2
and ribavirin3 [1, virazole1, (1-b-d-ribofuranosyl-1,2,4-
triazole-3-carboxamide], 40–50% ofpatients are no
responders to the best available treatments (Fig. 1).
Recently, major knowledge has been gained about the
mechanism ofHCV replication, due to the development
ofHCV inefction study models. The expression of
enzymatically active nonstructural viral proteins
required for HCV genome replication and the develop-
ment ofa replicon system allowing the replication ofan
HCV subgenome in hepatocyte culture should allow us
to study new and specific inhibitors ofHCV replication,
as well as new targets for antiviral therapy. Of several
putative viral enzyme targets, the NS5B polymerase has
been intensively studied because ofits central role in
viral replication.4 Recently Bressanelli et al.5 reported a
first co-crystallization ofHCV NS5B with ribonucleo-
tides, which lead to the identification ofa specific and
unique GTP-binding pocket outside the active site.
The pharmaceutical importance ofcarbocyclic nucleo-
side analogues has prompted the design and synthesis of
many examples ofthese compounds showing activity
against HIV, HBV, and HSV types 1 and 2.6 As part of
our drug discovery program, this paper provides a full
account ofthe synthesis and biological evaluation of
new carbocyclic analogues 4–6 ofthe anti-HCV agent
ribavirin. We have chosen to introduce some 1,2,3-tri-
azole heterocycles, as they have been considered an
interesting component in terms ofbiological activity
and are seen in many drugs.7,8 Because it has been sug-
gested that one ofthe active ofrms ofribavirin is the
monophosphate, which inhibits inosine monophosphate
dehydrogenase (IMPDH), decreasing the intracellular
concentration ofGTP and leading to a decrease ofviral
protein synthesis, we also turn our attention to the
*Corresponding author. Tel.: +33-2-3849-4582; fax: +33-2-3841-
7281; e-mail: luigi.agrofoglio@univ-orleans.fr
Figure 1. Chemical structure ofanti-HCV ribavirin.
0968-0896/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00349-3

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Y. Saito et al. / Bioorg. Med. Chem. 11 (2003) 3633–3639
synthesis ofthe phosphonate carbocyclic analogues of
ribavirin 11, 12, and 14. We also evaluated the newly
synthesized compounds against other important viruses
in order to determine their spectrum ofantiviral activity.
with tosylchloride and the subsequent activation with
sodium iodide provided 7 (77%), which was reacted
with the lithium salt ofdiisopropyl methanephos-
phonate in THF to furnish the phosphono-epoxide 8 in
90% yield.12
Reaction ofphosphono-epoxide 8 with sodium azide in
MeOH–H2O (8:1) gave the azide 9 in 84% yield. The
azido group of 9 was reacted with methylpropiolate and
2-cyanoacetamide to yield the (ꢀ)-1-{4-[2-(diisopro-
poxy-phosphoryl)-ethyl]-2-hydroxy-cyclopentyl}-1H-
[1,2,3]triazole-4-carboxylic acid methyl ester (10) and
(ꢀ)-{2-[3-(5-amino-4-carbamoyl-[1,2,3]triazol-1-yl)-4-
hydroxy-cyclopentyl]-ethyl}-phosphonic acid diisopro-
pyl ester (14), respectively. The final deprotection of 10
and 14 with a solution ofbromotrimethylsilane in
CH2Cl2 afforded the free phosphonic acid final com-
pounds 11 (98%) and 15 (98%), respectively. The amide
analogue 13 was obtained by a first ammonolysis of 10
(97%) and subsequent deprotection (99%), or directly
Chemistry
The preparation ofthe desired ribavirin analogues 4–6,
11, 13, and 15 is illustrated in Schemes 1 and 2. Thus,
starting with the known epoxide (2),9 obtained from the
1-hydroxymethyl-3-cyclopentene,10 new carbocyclic
analogues 4–6 were synthesized through a series ofeffi-
cient chemical transformations (Scheme 1). The key step
for the preparation of 1,2,3-triazoles involves a 1,3-
dipolar cycloaddition11 between azides and alkynes.
Thus, the opening ofthe epoxide ring of 2 by NaN3
yielded 3 (79%), which was reacted with methylpropio-
late to afford directly the (ꢀ)-1-(2-hydroxy-4-hydroxy-
methyl-cyclopentyl)-1H-[1,2,3]triazole-4-carboxylic acid
methyl ester (4) in 66% yield. When using a solution of
2-cyanoacetamide on the azido compound (3), the (ꢀ)-
5-amino-1-(2-hydroxy-4-hydroxymethyl-cyclopentyl)-1H-
[1,2,3]triazole-4-carboxylic acid amide (6) was isolated
in 97% yield. Ammonolysis of( 4) yielded the amide
analogue 5 (97%).
from 11 (in 94%) with a solution ofMeOH/NH
.
3
Compounds have been obtained in good yields and
analyzed by HRMS.
Biological Assay
For preparation ofthe phosphono carbanucleoside
counterparts, 11, 13 and 15, the treatment ofepoxide 2
The synthesized compounds 4–6, 11, 13, and 15, along
with the known antiviral compounds (acyclovir for
Scheme 1. Reagents and conditions: (a) NaN3, MeOH, H2O, rfx, 15 h; (b) methylpropiolate, 50 ꢁC, 16 h; (c) NH3/MeOH, 0 ꢁC, 16 h; (d) 2-cyano-
acetamide, K2CO3, DMSO, 50 ꢁC, 16 h.
Scheme 2. Reagents and conditions: (a) TsCl, pyridine, 0 ꢁC then NaI, acetone; (b) (iPrO)2P(O)CH3, nBuLi, THF, ꢂ78 ꢁC; (c) NaN3, MeOH;
(d) methylpropiolate, 50 ꢁC, 16 h; (e) Me3SiBr, CH2Cl2, rt; (f) MeOH/NH3, 0 ꢁC, 16 h; (g) 2-cyanoacetamide, K2CO3, DMSO, rt.

Y. Saito et al. / Bioorg. Med. Chem. 11 (2003) 3633–3639
3635
Table 1. Evaluation of1,2,3-triazolo-carbanucleoside analogue antiviral activity against human immunodeficiency virus (HIV), herpes simplex
virus (HSV-1), hepatitis B hirus (HBV) and cytotoxicity against PBM, CEM and VERO cells in vitro, expressed in mM
Compd.
Anti-HIV-1 activity in
human PBM cells
HSV-1 plaque
reduction assay
HBV
Toxicity (IC50) in:
EC50
EC90
EC50
EC90
EC90
PBM
CEM
VERO
Acyclovir
>100
0.005
0.016
>100
>100
43.8
>100
37.0
>100
>100
0.002
0.10
>100
>100
89.0
>100
=100
>100
0.11
>100
0.69
>100
>100
0.24ꢀ0.28
>10
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
14.0
>100
>100
>100
>100
>100
>100
>100
>100
29.0
>100
>100
>100
>100
>100
>100
3TC
AZT
4
5
6
11
13
15
>10
>100
>100
>100
>100
>100
>100
>10
>100
>100
>100
>100
>100
>100
>10
>10
>10
>10
>10
>10
Table 2. Evaluation of1,2,3-triazolo-carbanucleoside analogue anti-
viral activity against hepatitis C virus (HCV) Replicon activity,
expressed in mM, except for IFN which is expressed as international
units/mL
antiviral activity in our experimental conditions. It is
worth noting that both ribavirin and MPA, two mole-
cules known for their inhibitory effect on IMPDH, did
not show any specific antiviral activity, suggesting that
this type ofinhibitor might be inactive in this in vitro
system, in contrast with interferon a-2b used as a posi-
tive control. In the absence ofany potent antiviral
activity of 4, 5, and 6 and their phosphonate derivatives
11, 13, and 15, it would be interesting to evaluate the di-
and triphosphonate forms of 4, 5, and 6, to determine
whether they could inhibit the HCV polymerase activity
in this replicon system.
Compd.
Anti-HCV replicon
activity (IC50
Cell viability
assays (CC50)
)
RNA (ꢂ)
RNA (+)
Interferon a-2b
Ribavirin
79
230
74
170
>1000
110
Mycophenolic acid
4
5
>40
40
7
>320
>320
>320
>320
>320
>320
>320
>320
>320
>320
>320
>320
>320
>320
>320
>320
>320
>320
6
11
13
15
Experimental
Commercially available chemicals and solvents were
reagent grade and used as received. Dry tetra-
hydrofuran, pyridine and dichloromethane were
obtained from distillation over CaH2 or Na, N,N-dime-
thylformamide over BaO. The reactions were monitored
by thin-layer chromatography (TLC) analysis using
silica gel plates (Kieselgel 60 F254, E. Merck). Com-
pounds were visualized by UV irradiation and/or
spraying with 20% H2SO4 in EtOH, followed by char-
ring at 150 ꢁC. Column chromatography was performed
on Silica Gel 60 M (0.040–0.063 mm, E. Merck). Melt-
ing points were recorded on a Buchi (Dr. Tottoli) and
were uncorrected. 1H and 13C NMR spectra were
recorded on a Bruker AVANCE DPX 250 Fourier
Transform spectrometer at 250 MHz for 1H and
62.9 MHz for 13C, respectively, using tetramethylsilane
as the internal standard; signals are reported as s (sing-
let), d (doublet), t (triplet), q (quartet), m (multiplet).
Mass spectra were recorded on a Perkin-Elmer SCIEX
API-300 (heated nebullizer) spectrometer. High-resolu-
tion mass analyses (HRMS) were performed by the
CRMPO, University ofRennes 1, and Fr using the Fast
Atom Bombardment (FAB ) or Electron Spray Ioni-
zation (ESI) mode. The nomenclature ofthe obtained
compounds is in accordance with the IUPAC rules and
was checked with Autonome.13 The numbering and
assignment ofthe chemical shifts for all described com-
pounds are related to the corresponding ribose or
carbanucleosides derivatives.
HSV, IFN for HCV, 3TC for HBV and AZT for HIV),
were tested for their antiviral activities in vitro, and the
results are shown in Table 1. The procedures used were
the same as described previously. Among these nucleo-
side analogues, only compounds (ꢀ)-5-amino-1-(2-
hydroxy-4-hydroxy-methyl-cyclopentyl)-1H-[1,2,3]tri-
azole-4-carboxylic acid amide (6) and the phosphonate
(ꢀ)-{2-[3-(5-amino-4-carbamoyl-[1,2,3]triazol-1-yl)-4-
hydroxy-cyclopentyl]-ethyl}-phosphonic acid diisopro-
pyl ester (14) were found to exhibit moderate anti-HIV
activity, with an EC50 of43.8 and 37.0 mM, respectively.
Both compounds showed no cytotoxicity against PBM,
CEM or VERO cells. The carbocyclic analogues of
ribavirin 4, 5, and 6 and their respective phosphonate
derivatives 11, 13, and 15, were reasoned to be potential
inhibitors ofthe inosine monophosphate dehydrogenase
(IMPDH), an enzyme involved in the metabolism of
guanosine monophosphate (GMP) and a potential tar-
get for anti-HCV strategy. Therefore, these compounds,
along with other anti-HCV compounds (interferon
a-2b, ribavirin and mycophenolic acid [MPA]) were
evaluated for their antiviral activities and toxicity in
vitro using Huh7 cells harboring and replicating sub-
genomic HCV replicons, as described in the experi-
mental section. The results are presented in Table 2.
None ofthe synthesized compounds reached the inhibi-
tory concentration 50 at the highest concentrations tes-
ted (e.g., 320 mM), indicating a lack ofsignificant

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Y. Saito et al. / Bioorg. Med. Chem. 11 (2003) 3633–3639
(ꢀ)-2-Azido-4-hydroxymethyl-cyclopentanol (3). A sus-
pension of 2 (155 mg, 1.36 mmol), NaN3 (176 mg, 2.72
mmol) in MeOH–H2O (8:1, 35 mL) was refluxed over-
night. After filtration, the organics were concentrated
under reduced pressure, and the residue was purified by
silica gel column chromatography (10:1 CH2Cl2–
(ddd, 1H, J=7.0, 7.1, 7.1 Hz, 20-H), 4.53 (ddd, 1H, J=6.8,
7.0, 7.1 Hz, 10-H); 13C NMR (CD3OD) d 32.8 (C50), 36.0
(C30), 37.9 (C40), 65.5 (C60), 66.4 (C20), 76.5 (C10), 122.9
(C5), 146.7 (C6), 166.7 (C¼O); UV lmax (H2O, pH 7) 234
nm (e 8900), 261 (e 8600); HR-FAB-MS m/z 242.1253
calcd for C9H16N5O3 [M+H]+, found m/z 242.1253.
1
MeOH) to give 3 (79%, 170 g) as a colorless oil. H
NMR (CD3OD–D2O) d 1.35–1.42 (m, 2H, 3-Ha, 5-Ha),
1.69 (m, 1H, 4-H), 2.25–2.34 (m, 2H, 3-Hb, 5-Hb), 3.44
(d, 2H, J=6.5 Hz, 6-H), 3.67 (dd, 1H J=5.4, 12.5 Hz,
2-H), 3.95 (dd, 1H, J=5.9, 13.8 Hz, 1-H). 13C NMR
(CDCl3) d 31.9 (C5), 34.9 (C3), 37.0 (C4), 66.1 (C6),
68.6 (C2), 76.5 (C1); HR-FAB-MS m/z 158.1739 calcd
for C6H12N3O2 [M+H]+, found m/z 158.1736.
(ꢀ)-3-Iodomethyl-6-oxabicyclo[3.1.0]hexane (7). To a
solution of 2 (1.02 g, 8.95 mmol) in anhydrous pyridine
(10 mL) at 0 ꢁC was added TsCl (2.21 g, 11.60 mmol).
After being stirred for 2 h, the reaction mixture was fil-
tered, washed with brine, dried over MgSO4 then con-
centrated in vacuo. The residue was purified by silica gel
column chromatography (2:1 hexanes–EtOAc) to give
the tosylate intermediate (90%, 2.16 g) as a yellow oil.
1H NMR (CDCl3) d 2.01–2.10 (m, 4H, 3-H, 5-H), 2.46
(s, 3H, Me), 2.62 (m, 1H, 4-H), 3.94 (d, 2H, J=7.2 Hz,
6-H), 5.62 (s, 2H, 1-H, 2-H), 7.37 (d, 2H, J=8.1 Hz, H-
aryl), 7.82 (d, 2H, J=8.1 Hz, H-aryl). This compound
was directly engaged into the next reaction. Thus, a
suspension oftosylate intermediate (2.16 g, 8.07 mmol),
NaI (1.57 g, 10.50 mmol) in anhydrous acetone (15 mL)
was refluxed under Ar for 3 h. After filtration, the
organics were concentrated under reduced pressure, and
the residue was purified by silica gel column chromato-
graphy (2:1 hexanes–EtOAc) to give 7 (85%, 1.53 g) as a
(ꢀ)-1-(2-Hydroxy-4-hydroxymethyl-cyclopentyl)-1H-
[1,2,3]triazole-4-carboxylic acid methyl ester (4). The
mixture of 3 (101 mg, 0.64 mmol) and methylpropiolate
(300 mL) was heated at 50 ꢁC for 16 h. The reaction
mixture was then cooled to ambient temperature and
concentrated in vacuo. The residue was purified by silica
gel column chromatography (10:1 CH2Cl2-MeOH) to
give 4 (66%, 102.6 mg) as a colorless oil: 1H NMR
(CD3OD) d 1.86 (m, 1H, 40-H), 1.99 (m, 2H, 30-H), 2.47
(m, 2H, 50-H), 3.55 (d, 2H, J=5.9 Hz, 60-H), 3.90 (s, 3H,
OMe), 4.42 (ddd, 1H, J=7.1, 7.3, 7.5 Hz, 20-H), 4.75
(ddd, 1H, J=7.3, 7.3, 7.3 Hz, 10-H), 8.61 (s, 1H, 5-H);
13C NMR (CD3OD) d 34.3 (C50), 35.9 (C30), 37.8 (C40),
52.5 (OMe), 66.5 (C60), 70.1 (C20), 77.4 (C10), 128.9
(C5), 140.3 (C6), 162.4 (C¼O); UV lmax (MeOH) 214
nm (e 11000); HR-FAB-MS m/z 242.1141 calcd for
C10H16N3O4 [M+H]+, found m/z 242.1143.
1
colorless oil. H NMR (CDCl3) d 1.35–1.98 (m, 4H, 3-
H, 5-H), 2.34 (m, 1H, 4-H), 3.26 (d, 2H, J=6.3 Hz, 6-
H), 3.50 (s, 2H, 1-H, 2-H); 13C NMR (CDCl3) d 31.8
(C4), 33.6 (C3, C5), 56.7 (C1, C2), 58.2 (C6); HR-FAB-
MS m/z 225.0395 calcd for C6H10IO [M+H]+, found
m/z 225.0391.
(ꢀ)-1-(2-Hydroxy-4-hydroxymethyl-cyclopentyl)-1H-
[1,2,3]triazole-4-carboxylic acid amide (5). A solution of
4 (42.6 mg, 0.18 mmol) in MeOH (2.5 mL) saturated
with NH3 was stirred at 0 ꢁC for 16 h. After evaporation
ofthe volatiles, the residue was purified by silica gel
column chromatography (10:1 CH2Cl2-MeOH) to give
trans-[2-(6-Oxa-bicyclo[3.1.0]hex-3-yl)-ethyl]-phosphonic
acid diisopropyl ester (8). To a solution ofdiisopro-
pylmethyl phosphonate (479 mg, 2.66 mmol) in THF
(5.0 mL) at ꢂ78 ꢁC was added dropwise 1.6 M nBuLi
(1.66 mL, 2.66 mmol) in hexane under an argon atmos-
phere. The mixture was stirred at the same temperature
for 1 h. After the addition of THF solution (4.0 mL) of
7 (542 mg, 2.42 mmol), the stirring was continued for
another 2 h. The reaction mixture was poured into satd.
aq NH4Cl (10 mL), and extracted with CH2Cl2 (50
mLꢃ3). The combined organic layers were washed with
brine (50 mL), dried (MgSO4), then concentrated in
vacuo. The residue was purified by silica gel column
chromatography (12:1 CH2Cl2-MeOH) to give 8 (90%,
1
5 (97%, 38.6 mg) as a colorless waxy solid: H NMR
(CD3OD) d 1.88 (m, 1H, 40-H), 2.00 (m, 2H, 30-H), 2.47
(m, 2H, 50-H), 3.55 (d, 2H, J=5.7 Hz, 60-H), 4.41 (ddd,
1H, J=7.1, 7.3, 7.3 Hz, 20-H), 4.74 (ddd, 1H, J=7.1,
7.3, 7.3 Hz, 10-H), 8.44 (s, 1H, 5-H); 13C NMR
(CD3OD) d 34.3 (C50), 36.0 (C30), 37.8 (C40), 66.5 (C60),
70.0 (C20), 77.4 (C10), 126.8 (C5), 143.5 (C6), 164.8
(C¼O); UV lmax (MeOH) 210 nm (e 12135); HR-FAB-
MS m/z 227.1144 calcd for C9H15N4O3 [M+H]+,
found m/z 227.1145.
1
601 mg) as a colorless oil: H NMR (CDCl3) d 1.34 (d,
12H, J=6.2 Hz, 4ꢃMe), 1.15–2.07 (m, 7H, 3-H, 4-H, 5-H,
6-H), 2.26 (m, 2H, CH2P), 3.47 (s, 2H, 1-H, 2-H), 4.76 (m,
2H, 2 X CH(Me)2); 13C NMR (CDCl3) d 24.0 (d, J=2.9
Hz, (Me)2CH), 27.1 (d, J=143 Hz, CH2P), 27.6 (C4), 33.4
(d, J=18 Hz, C6), 31.8 (C3, C5), 56.7 (C1, C2), 68.7 (d,
J=6.5 Hz, (Me)2CH); HR-FAB-MS m/z 276.2931 calcd
for C13H25O4P [M+H]+, found m/z 276.2928.
(ꢀ)-5-Amino-1-(2-hydroxy-4-hydroxymethyl-cyclopentyl)-
1H-[1,2,3]triazole-4-carboxylic acid amide (6). To a
solution of2-cyanoacetamide (80.2 mg, 0.95 mmol) in
DMSO (1.0 mL) at rt was added K2CO3 (131.8 mg, 0.95
mmol) under an argon atmosphere. The mixture was
stirred at the same temperature for 1 h. After the addi-
tion ofDMSO solution (1.0 mL) of 3 (50.0 mg, 0.32
mmol), the stirring was continued for 16 h at 50 ꢁC.
After evaporation of the solvent, the residue was purified
by silica gel column chromatography (5:1 CH2Cl2–
(ꢀ)-[2-(3-Azido-4-hydroxy-cyclopentyl)-ethyl]-phosphonic
acid diisopropyl ester (9). To a solution of 8 (228 mg,
0.83 mmol) in MeOH (24 mL) and H2O (3.0 mL) was
added NaN3 (102 mg). After being stirred at refluxing
temperature for 14 h, the reaction mixture was cooled to
ambient temperature and concentrated in vacuo. The
residue was diluted with CH2Cl2 (25 mL), and the
1
MeOH) to give 6 (97%, 38.6 mg) as a colorless solid: H
NMR (CD3OD) d 1.86 (m, 1H, 40-H), 1.98 (m, 2H, 30-H),
2.44 (m, 2H, 50-H), 3.56 (d, 2H, J=6.2 Hz, 60-H), 4.42

Y. Saito et al. / Bioorg. Med. Chem. 11 (2003) 3633–3639
3637
organic phase was washed with brine (30 mL), dried
(MgSO4), then evaporated. The residue was purified by
silica gel column chromatography (10:1 CH2Cl2–
purified by silica gel column chromatography (10:1
CH2Cl2–MeOH) to give 12 (97%, 38.6 mg) as a color-
less waxy solid: 1H NMR (CDCl3) d 1.31 (d, 12H,
J=5.5 Hz, 4ꢃMe), 1.62–1.98 (complex, 7H, 30-H, 40-H,
50-H, 60-H), 2.33 (m, 2H, CH2P), 4.46 (m, 1H, 20-H),
4.60–4.73 (complex, 3H, 10-H, 2ꢃCH(Me)2), 8.39 (s,
1H, 5-H); 13C NMR (CDCl3) d 24.0 (d, J=2.8 Hz,
(Me)2CH), 25.4 (d, J=143 Hz, CH2P), 28.8 (C40), 35.3
(d, J=17.5 Hz, C60), 36.5 (C50), 38.2 (C30), 68.9 (C20),
70.2 (d, J=6.7 Hz, (Me)2CH), 77.5 (C10), 125.9 (C5),
142.2 (C6), 162.5 (C¼O); HR-FAB-MS m/z 389.1954
calcd for C16H30N4O5P [M+H]+, found m/z 389.1958.
1
MeOH) to give 9 (84%, 220 mg) as a colorless oil: H
NMR (CDCl3) d 1.20–1.36 (complex, 12H, 4ꢃMe),
1.60–1.93 (complex, 9H, 3-H, 4-H, 5-H, 6-H, CH2P),
2.90 (brs, D2O exchg, 1H, OH), 4.09 (ddd, 1H, J=5.8,
6.0, 7.5 Hz, 2-H), 3.71 (ddd, 1H, J=5.8, 6.0, 7.5 Hz, 1-
H), 4.70 (dt, 2H, J=6.1, 12.5 Hz, 2 X CH(Me)2); 13C
NMR (CDCl3) d 24.0 (d, J=4.2 Hz, (Me)2CH), 25.5 (d,
J=143 Hz, CH2P), 29.1 (C4), 35.4 (C3), 35.6 (J=3.6
Hz, C6), 38.4 (C5), 68.5 (C2), 70.1 (d, J=6.7 Hz,
(Me)2CH), 76.8 (C1); IR (film) nmax 2101 (azide) cmꢂ1
;
HR-FAB-MS m/z 320.1739 calcd for C13H27N3O4P
(ꢀ)-{2-[3-(4-Carbamoyl-[1,2,3]triazol-1-yl)-4-hydroxy-
cyclopentyl]-ethyl}-phosphonic acid (13). Method A. A
solution of 11 (22.1 mg, 0.06 mmol) in CH2Cl2 (3.0 mL)
was treated with bromotrimethylsilane (166.4 mg, 1.09
mmol) under argon atmosphere. The resulting solution
was protected from light and refluxed for 14 h. The
reaction mixture was concentrated to dryness and the
residue was added to MeOH (2 mL) and coevaporated.
After evaporation of the solvent, the residue was tritu-
rated with Et2O and dried to afford 13 (94%, 15.5 mg)
as a waxy solid.
[M+H]+, found m/z 320.1736.
(ꢀ)-1-{4-[2-(Diisopropoxy-phosphoryl)-ethyl]-2-hydroxy-
cyclopentyl}-1H-[1,2,3]triazole-4-carboxylic acid methyl
ester (10). The mixture of 9 (245.6 mg, 0.77 mmol) and
methylpropiolate (2.5 mL) was heated at 80 ꢁC for 13 h.
The reaction mixture was then cooled to ambient tem-
perature and concentrated in vacuo. The residue was
purified by silica gel column chromatography (10:1
CH2Cl2-MeOH) to give 10 (78%, 242.3 mg) as a color-
less waxy solid. 1H NMR (CDCl3) d 1.31 (d, 12H,
J=6.2 Hz, 4ꢃMe), 1.64–1.98 (complex, 7H, 30-H, 40-H,
50-H, 60-H), 2.35–2.56 (m, 2H, CH2P), 3.93 (s, 3H,
OMe), 4.51 (m, 1H, 20-H), 4.59–4.72 (complex, 3H, 10-
H, 2ꢃCH(Me)2), 8.22 (s, 1H, 5-H); 13C NMR (CDCl3)
d 24.0 (d, J=2.9 Hz, (Me)2CH), 25.4 (d, J=143 Hz,
CH2P), 28.9 (C40), 35.3 (d, J=17.0 Hz, C60), 36.5 (C50),
38.2 (C30), 52.2 (OMe), 68.7 (C20), 70.1 (d, J=6.8 Hz,
(Me)2CH), 127.2 (C5), 141.4 (C6), 161.1 (C¼O); HR-
FAB-MS m/z 404.1951 calcd for C17H31N3O6P
[M+H]+, found m/z 404.1944.
Method B. A solution of 12 (25.1 mg 0.08 mmol) in
MeOH (2.5 mL) saturated with NH3 was stirred at 0 ꢁC
for 14 h. After evaporation of the solvent, the residue
was triturated with Et2O and dried to afford 13 (99%,
1
23.7 mg) as a waxy solid: H NMR (CD3OD) d 1.53–
1.89 (complex, 7H, 30-H, 40-H, 50-H, 60-H), 2.28-(m, 2H,
CH2P), 4.38 (ddd, 1H, J=6.6, 6.8, 7.1 Hz, 20-H), 4.71
(ddd, 1H, J=6.8, 7.1, 7.1 Hz, 10-H), 8.43 (s, 1H, 5-H); 13
C
NMR (CD3OD) d 28.6 (d, J=133 Hz, CH2P), 31.7 (C40),
37.1 (d, J=16.9 Hz, C60), 38.2 (C50), 39.5 (C30), 70.4 (C20),
77.7 (C10), 126.8 (C5), 143.4 (C6), 164.8 (C¼O); UV lmax
(MeOH) 210 nm (e 11935); HRESIMS m/z 305.1015 calcd
for C10H18N4O5P [M+H]+, found 305.1018.
(ꢀ)-1-[2-Hydroxy-4-(2-phosphono-ethyl)-cyclopentyl]-1H-
[1,2,3]triazole-4-carboxylic acid methyl ester (11): gen-
eral procedure for deprotection. A solution of 10 (54.6
mg, 0.14 mmol) in CH2Cl2 (3.0 mL) was treated with
bromotrimethylsilane (413.0 mg, 2.70 mmol) under
argon atmosphere. The resulting solution was protected
from light and stirred at room temperature for 72 h. The
reaction mixture was concentrated to dryness and the
residue was added MeOH (2 mL) and coevaporated.
After evaporation of the solvent, the residue was tritu-
rated with Et2O and dried to afford 11 (98%, 42.1 mg)
(ꢀ)-{2-[3-(5-Amino-4-carbamoyl-[1,2,3]triazol-1-yl)-4-
hydroxy-cyclopentyl]-ethyl}-phosphonic acid diisopropyl
ester (14). To a solution of2-cyanoacetamide (27.1 mg,
0.32 mmol) in DMSO (1.0 mL) at rt was added K2CO3
(44.4 mg, 0.32 mmol) under an argon atmosphere. The
mixture was stirred at the same temperature for 30 min.
After the addition of DMSO solution (1.0 mL) of 9
(51.4 mg, 0.16 mmol), the stirring was continued for 5 h
at 50 ꢁC. After evaporation of the solvent, the residue
was purified by silica gel column chromatography (15:1
CH2Cl2–MeOH) to give 14 (71%, 46.1 mg) as a color-
less solid: 1H NMR (CDCl3) d 1.33 (d, 12H, J=6.2 Hz,
4ꢃMe), 1.60–1.93 (complex, 7H, 30-H, 40-H, 50-H, 60-H),
2.40–2.48 (complex, 2H, CH2P), 4.36–4.55 (complex,
2H, 10-H, 20-H), 4.65 (complex, 2H, 2ꢃCH(Me)2); 13C
NMR (CDCl3) d 23.8 (d, J=2.7 Hz, (Me)2CH), 25.6 (d,
J=142 Hz, CH2P), 28.7 (C40), 35.8 (d, J=17.5 Hz, C60),
36.7 (C50), 37.9 (C30), 69.2 (C20), 70.3 (d, J=6.8 Hz,
(Me)2CH), 71.4 (C10), 118.8 (C5), 142.2 (C6), 163.8
(C¼O); HR-FAB-MS m/z 404.2063 calcd for
C16H31N5O5P [M+H]+, found m/z 404.2065.
1
as a waxy solid: H NMR (CD3OD) d 1.70–1.95 (com-
plex, 7H, 30-H, 40-H, 50-H, 60-H), 2.36 (m, 2H, CH2P),
3.91 (s, 3H, OMe), 4.45 (ddd, 1H, J=6.0, 6.1, 7.9 Hz,
20-H), 4.77 (ddd, 1H, J=6.1, 6.8, 7.0 Hz, 10-H), 8.63 (s,
1H, 5-H); 13C NMR (CD3OD) d 28.6 (d, J=133 Hz,
CH2P) 31.7 (C40), 37.1 (d, J=16.9 Hz, C60), 37.8 (C50),
39.2 (C30), 52.6 (OMe), 70.5 (C20), 77.5 (C10), 129.2
(C5), 140.0 (C6), 162.1 (C¼O); UV lmax (MeOH) 215
nm (e 10,985); HR-ESI-MS m/z 341.0878 calcd for
C12H19N2O6NaP [M+Na]+, found 341.0885.
(ꢀ)-{2-[3-(4-Carbamoyl-[1,2,3]triazol-1-yl)-4-hydroxy-
cyclopentyl]-ethyl}-phosphonic acid diisopropyl ester
(12). A solution of 10 (32.6 mg, 0.08 mmol) in MeOH
(2.5 mL) saturated with NH3 was stirred at 0 ꢁC for 16
h. After evaporation of the volatiles, the residue was
(ꢀ)-{2-[3-(5-Amino-4-carbamoyl-[1,2,3]triazol-1-yl)-4-
hydroxy-cyclopentyl]-ethyl}-phosphonic acid (15).
A

3638
Y. Saito et al. / Bioorg. Med. Chem. 11 (2003) 3633–3639
solution of 14 (21.4 mg, 0.05 mmol) in CH2Cl2 (3.5 mL)
was treated with bromotrimethylsilane (163.3 mg, 1.06
mmol) under argon atmosphere. The resulting solution
was protected from light and refluxed for 14 h. The
reaction mixture was concentrated to dryness and the
residue was added to MeOH (3 mL) and coevaporated.
After evaporation of the solvent, the residue was tritu-
rated with Et2O and dried to afford 15 (98%, 16.6 mg,
0.05 mmol) as a waxy solid : 1H NMR (CD3OD) d 1.75–
1.94 (complex, 7H, 30-H, 40-H, 50-H, 60-H), 2.36–2.52
(m, 2H, CH2P), 4.40–4.56 (complex, 2H, 10-H, 20-H);
13C NMR (CD3OD) d 28.8 (d, J=132 Hz, CH2P), 31.4
(C40), 36.8 (d, J=16.9 Hz, C60), 38.3 (C50), 39.2 (C30),
70.7 (C20), 77.4 (C10), 118.2 (C5), 142.9 (C6), 164.5
(C¼O); UV lmax (H2O, pH 7) 235 nm (e 9100), 260 (e
9150); HR-ESI-MS m/z 320.1124 calcd for
C10H19N5O5P [M+H]+, found 320.1112.
thiocyanate–phenol–chloroform. Northern Blot analy-
sis was then performed using the NorthernMaxTM-Gly
(Ambion) kit, following manufacturer’s instruction. Ten
micrograms oftRNA was denatured in glyoxal buffer at
50 ꢁC for 30 min and separated by agarose gel electro-
phoresis, then transferred for 12 h onto a charged nylon
membrane (Biodyne B, Merck Eurolab). Hybridisation
was carried out with three different [32P]CTP-labelled
riboprobes obtained by in vitro transcription (Pro-
mega). These probes were complementary to the NS5A
region ofthe HCV genome, and to the cellular gene
GAPDH, respectively. First, the blot was hybridized
with two riboprobes directed against the negative strand
ofHCV RNA and the GAPDH mRNA, respectively.
After one night of hybridization at 68 ꢁC, the membrane
was washed then exposed to X-ray film and a phosphor
screen for quantitative analysis. The amount of
GAPDH mRNA was used as an internal loading con-
trol to standardise the amount ofHCV RNA detected.
The same membrane was subsequently hybridized with
a negative-sense riboprobe to determine the level of
HCV-positive strand RNA using the same approach.
Antiviral and cytotoxicity assays for HIV-1 and HBV
Antiviral and cytotoxicity assays were conducted as
described recently by Stuyver et al.14 HIV and HBV
assays were performed in activated primary human
peripheral blood mononuclear cells (PBM) and AD38
cells (derived from 2.2.15HepG2 cells), respectively.
Antiviral and cytotoxicity assays for HSV. The newly
synthesized nucleosides were evaluated for activity
against HSV-1 (strain F) by plaque reduction assay in
Vero cells, using methodologies described previously.16
Cytotoxicity assays were conducted in rapidly dividing
Vero cells, as previously described.16 The median effective
concentration was determined by the median effect
method.17
Antiviral and cytotoxicity assays for HCV
Cell culture system for HCV replication. Huh-7 cells
harbouring the subgenomic HCV replicon BM4-5 were
kindly provided by Dr. C. Seeger.15 Cells were main-
tained in Dulbecco0s modified Eagle0s medium
(DMEM) (Life Technologies) supplemented with 10%
fetal bovine serum, 1% l-glutamine, 1% l-pyruvate,
1% penicillin and 1% streptomycin supplemented with
500 mg/mL G418 (Geneticin, Invitrogen). Cells were
passaged every 4 days.
Acknowledgements
The authors would like to acknowledge the Region
Centre, CNRS and University ofOrle ans for funding
and Dr P. Guenot ofthe CRMPO, University of
Rennes 1, France for HRMS spectroscopy data. The
ANRS is also acknowledged for its support. RFS is
supported by NIH grant 1RO37-AI-41980, the Emory
University Center for AIDS and the Department of
Veterans Affairs.
Cytotoxicity assays for HCV. Huh-7 cells were respec-
tively seeded at a density of3 ꢃ104 cells/well in 96-well
plates for the cell-viability assay, or at a density of
6ꢃ105 cells/well in six-well plates for the antiviral assay.
Sixteen hours post seeding, cells were treated with the
compounds ofinterest ( 4, 5, 6, 11, 13, 15) at various
concentrations (0; 0.312; 0.625; 1.25; 2.5; 5; 10; 20; 40;
80; 160; 320 mM) for 3 days. The administration of each
drug was renewed each day. The medium used during
the treatment phase did not contain the selective drug
G418, as the latter could interfere with the assay. Other
drugs, including ribavirin (ICN Pharmaceuticals, USA),
mycophenolic acid (Sigma, USA), and interferon alpha-
2b (IntronA) were used in the same conditions as posi-
tive controls. The concentrations used for these drugs
were respectively: 0; 0.5; 1; 2; 4; 8; 16; 32; 64; 128; 256,
and 512 mM for ribavirin, 0; 2.5; 5; 10; 20; 40 mM f or
mycophenolic acid, and 0; 0.1; 1; 10; 100 and 1000 UI/
mL for IFN a-2b. At the end oftreatment, cell viability
assays were performed with the 96-well plates using
Neutral Red assay (Sigma).
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