Use of 2′-spirocyclic ethers in HCV nucleoside design

Article abstract of DOI:10.1021/jm401224y

Conformationally restricted 2′-spironucleosides and their prodrugs were synthesized as potential anti-HCV agents. Although the replicon activity of the new agents containing pyrimidine bases was modest, the triphosphate of a 2′-oxetane cytidine analogue demonstrated potent intrinsic biochemical activity against the NS5B polymerase, with IC₅₀ = 8.48 μM. Activity against NS5B bearing the S282T mutation was reduced. Phosphoramidate prodrugs of a 2′-oxetane 2-amino-6-O-methyl-purine nucleoside demonstrated potent anti-HCV activity in vitro, and the corresponding triphosphate retained similar potent activity against both wild-type and S282T HCV NS5B polymerase.

Full text of DOI:10.1021/jm401224y

Article
pubs.acs.org/jmc
Use of 2′-Spirocyclic Ethers in HCV Nucleoside Design
Jinfa Du,*,† Byoung-Kwon Chun,† Ralph T. Mosley, Shalini Bansal, Haiying Bao, Christine Espiritu,
Angela M. Lam, Eisuke Murakami,† Congrong Niu,† Holly M. Micolochick Steuer,† Phillip A. Furman,
and Michael J. Sofia*,‡
Pharmasset, Inc., 303A College Road East, Princeton, New Jersey 08540, United States
ABSTRACT: Conformationally restricted 2′-spironucleosides and their
prodrugs were synthesized as potential anti-HCV agents. Although the
replicon activity of the new agents containing pyrimidine bases was
modest, the triphosphate of a 2′-oxetane cytidine analogue demonstrated
potent intrinsic biochemical activity against the NS5B polymerase, with
IC50 = 8.48 μM. Activity against NS5B bearing the S282T mutation was
reduced. Phosphoramidate prodrugs of a 2′-oxetane 2-amino-6-O-methyl-
purine nucleoside demonstrated potent anti-HCV activity in vitro, and the
corresponding triphosphate retained similar potent activity against both
wild-type and S282T HCV NS5B polymerase.
INTRODUCTION
■
Approximately 180 million individuals are infected with
hepatitis C virus (HCV) worldwide.1 Options comprising
pegylated interferon-α (PEG-IFN) in combination with
ribavirin (RBV) had been the treatment of choice for HCV
infection for many years; however, this treatment had
demonstrated limited efficacy and generally intolerable side
effects.2,3 Two newly approved drugs, Telaprevir and
Boceprevir, have demonstrated improved efficacy in combina-
Figure 1. Prodrugs of β-D-2′-α-F-2′-β-C-methyl-nuclesides.
tion with PEG-IFN and RBV, but their use is associated with
added side effects including rash, resistance development, and
complex treatment regimens. In addition, these new agents are
only approved for treating genotype 1 HCV-infected patients,
thus excluding a large patient population infected with
genotypes 2 to 6 virus. Therefore, there is still an urgent
need to develop alternative direct acting antiviral agents
(DAAs) that are more efficacious, have an improved safety
profile, a high barrier to resistance, and are pan-genotypic.4,5
The RNA-dependent RNA polymerase (RdRp)6−8 is
essential to viral replication and thus represents a valid target
for therapeutic intervention by design of specific inhibitors.
Nucleoside inhibitors (NIs) of HCV polymerase are attractive
due to their high potency and high generic barrier to
resistance.9 Several classes have been identified as potent
anti-HCV agents in vitro, including the β-D-2′-deoxy-2′-α-F-2′-
β-C-methyl-ribo-nucleosides,10 the β-D-2′-β-C-methyl-ribo-nu-
cleosides,11,12 the 4′-azido-nucleosides,13 and the 2′-O-methyl-
nucleosides.14 These nucleosides or their phosphate prodrugs
have demonstrated broad genotype coverage. Among them,
sofosbuvir (GS-7977)15 demonstrated promising anti-HCV
efficacy in vitro and in humans, and a new drug application has
been filed recently in the U.S. In the same class, I, a prodrug of
2′-α-F-2′-β-C-methyl-guanosine, also showed potent anti-HCV
activity with excellent safety and resistance profiles (Figure
1).16 The superior anti-HCV efficacy, pan-genotypic coverage,
and high genetic barrier to viral resistance of nucleosides makes
them of great interest in the search for novel analogues or their
phosphate prodrugs.
As a part of our effort to identify novel nucleoside NS5B
inhibitors, we explored 2′-spiro analogues (Figure 2). This
investigation was based on the hypothesis that 2′-disubstituted
nucleosides such as 2′-α-F-2′-β-C-methylcytidine (II),10 2′-
methyl cytidine (III),11,12 and the 2′-monosubstituted nucleo-
side 2′-O-methyl-cytidine14 are active as inhibitors of the NS5B
polymerase, and therefore, sufficient space might exist within
the NS5B active-site to accommodate an additional atom
especially if it resides within a constrained ring system. In
addition, the 2′-O-methylcytidine derivative showed that a 2′-α
oriented ether substituent was able to confer NS5B inhibitory
activity. Further support for this hypothesis was obtained when
the low energy conformations of both II and its 2′-spiro
oxetane derivative were compared (Figure 3). Overlay of these
low energy conformations demonstrated that they exhibited
identical ring conformations and afforded good alignment of
their respective 2′-substituents. We herein report the synthesis
Special Issue: HCV Therapies
Received: August 7, 2013
© XXXX American Chemical Society
A
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methyl ester in the presence of triethylamine) in the presence
of N-methylimidazole (NMI) gave the corresponding prodrugs
5a−d as mixtures of diasteromers. A diastereomerically pure
phosphoramidate prodrug 6c was prepared from 4c and the
homochiral phosphorus reagent (X2)18 in the presence of t-
butylmagnesium chloride. Nucleoside triphosphates 7b−c and
8a−d from 2b, 2c, and 4a−d were prepared by reaction with
phosphoryl chloride followed by treatment of the resulting
intermediate with tributylammonium pyrophosphate in 1,3-
dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DTP)
(Scheme 2).
2′-Oxetane 2-amino-6-O-methyl-purine nucleoside 17 was
prepared from 2-C-allyl-1,2,3,5-tetrabenzoyl-riboside (Scheme
3). Coupling of the sugar 917 with 2-amino-6-chloropurine in
the presence of DBN/TMSOTf gave exclusively the β-anomer
10.19 Installation of the desired purine 6-substituent and global
deprotection was achieved in a single step by treatment with
sodium methoxide. Selective protection of 11 followed by
installation of the 2′-oxetane ring17 and deprotection provided
the target nucleoside 17.
Figure 2. Design of 2′-spironucleosides.
The free guanosine nucleoside 18 was prepared from 17 by
treatment with adenosine deaminase (ADA) in water.
Compound 18 was then converted to its 5′-triphosphate 19
by the method used for the preparation of compound 7b.
Phosphoramidate prodrugs of 17 were synthesized as
diastereomeric mixtures by the method used for the preparation
of compounds 5a−d. A series of diastereomerically pure
phosphoramidate prodrugs (20b−o) were obtained by SFC
separation.18 For the assignment of stereochemistry at
phosphorus, the stereospecific method used for the preparation
of 6c was applied with X2 of known absolute stereochemistry18
to generate the single Sp isomer 20c. The 31P NMR resonance
frequency of 20c appeared at higher field (4.16 ppm) than that
(4.74 ppm) of its Rp diastereomer 20d, which is consistent with
similar assignments in the literature.20 The absolute stereo-
chemistry at phosphorus in other diastereomers were assigned
by analogy.
Figure 3. Superposition of the structures of the crystallographically
determined compound II (green C’s) and modeled 2′-oxetane cytidine
(cyan C’s).
BIOLOGICAL ACTIVITY
■
The anti-HCV activities of the uridine 2a−d and cytidine
analogues 4a−d were evaluated in the genotype 1b replicon
assay (Table 1).21 Among them, compounds 2b and 4c
displayed weak activity, with the rest inactive when tested up to
100 μM concentration.
and biological evaluation of 2′-spironucleosides and their
phosphate prodrugs as anti-HCV agents.
CHEMISTRY
Nucleosides must be phosphorylated in cells to their
corresponding nucleoside triphosphates by kinases to exert
their biological activity. Phosphoramidate prodrugs of nucleo-
sides have been shown to deliver the nucleoside mono-
phosphate directly into tissues, particularly into the liver,
bypassing the rate-limiting first step of phosphorylation.15 The
phosphoramidate prodrugs 5a−d and 6c of the nucleosides
2a−d and 4c were prepared but resulted in modest levels of
cellular activity (Table 2), suggesting that inefficient initial
phosphorylation in the first step is not the primary reason for
the weak activity of these pyrimidine nucleosides and their
prodrugs.
The triphosphates of a range of the parent nucleosides were
also prepared and their intrinsic activity against wild-type and
S282T NS5B polymerase compared to that of II (Table 3). The
2′-oxetane uridine triphosphate 7c demonstrated measurable
inhibition of the wild-type enzyme. The intrinsic activity of the
triphosphates (8a−8d) of each of the possible 4- and 5-
membered spirocyclic cytidine variants 4a−d was also
■
2′-Spirouridine analogues 2a−d were synthesized from the
corresponding protected intermediates 1a−d prepared accord-
ing to literature methods.17 The ara-configuration of 1a and 1b
was established stereoselectively by reaction of allylmagnesium
chloride with 3′,5′-TIPS-protected 2′-oxouridine, while 1c and
1d having the ribo-configuration were prepared by coupling of
2-β-allyl-1,2,3,5-tetrabenzoyl-D-ribofuranoside with silylated
uracil in the presence of SnCl4. 2′-Spirocytidine analogues
4a−d were also prepared from 1a−d by treatment with TsCl/
Et3N in the presence of 4-dimethylaminopyridine (DMAP),
followed by amination with ammonium hydroxide and
desilylation with TBAF (Scheme 1).
The preparation of nucleotide phosphoramidate derivatives is
depicted in Scheme 2 and relied upon coupling with reagents
already bearing the desired amino acid ester and phenol
substituents. Reaction of uridine analogues 2a−d with X1 (as a
diastereomeric mixture at P, prepared in situ by the reaction of
phenyl phosphorus dichloride with the requisite amino acid
B
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a
Scheme 1. Preparation of Pyrimidine Nucleosides
a
Reaction and conditions: (i) (1) TsCl/Et3N/DMAP, (2) NH4OH; (ii) TBAF.
tetrahydrofuran 8d and was comparable to that of the 2′-α-F-
2′-C-methyl cytidine derivative II. Both triphosphates 8c and
8d showed a similar resistance profile to the triphosphate of II,
in that significant potency was lost against the S282T mutant
enzyme.10
Scheme 2. Preparation of Phosphoramidates and
Triphosphates
a
A good resistance profile is desirable for the next generation
of nucleoside or nucleotide anti-HCV agents. Because
phosphate prodrugs of 2′-α-F-2′-β-C-methylguanosine ana-
logues such as compound I (PSI-353661) and PSI-352938 have
demonstrated excellent resistant profiles,16 we next turned to
explore 2′-oxetane guanosine analogues. Although the 2′-
oxetane-2-amino-6-O-methyl-purine 17 and 2′-oxetane guano-
sine (18) nucleosides were inactive in the whole cell replicon
assay up to 100 μM (data not shown), it was encouraging that
the triphosphate 19 of guanosine analogue 18 demonstrated
comparable potencies against wild-type NS5B polymerase and
its S282T mutant, like 2′-F-2′-β-C-methylguanosine (I, Table
4).
To optimize cellular potency in this series, a variety of
prodrugs were prepared (Table 5). Most of the prodrugs
demonstrated moderate activity without significant cytotoxicity
up to 20 μM. The n-butyl ester 20e demonstrated the best anti-
HCV potency, with EC50 of 0.27 μM. Generally, as in the GS-
7977 series, prodrugs with Sp-chirality on the phosphorus
demonstrated greater anti-HCV potency than those with Rp,15
but the activities of both isomers of the cyclopentyl esters (20i
and 20j) and benzyl esters (20n and 20o) were similar. An
enhancement in activity was observed when the phenyl group
in 20k was replaced with naphthyl group in 20p. Nevertheless,
even the most active compound in the series was still
significantly less potent than compound I.
a
Reaction and conditions: (i) X1/NMI for 5a−d and X2/t-BuMgCl
for 6C; (ii) POCl3/Bu3N/Bu3N·pyrophosphate/DTP.
CONCLUSION
■
evaluated, with the ribo-analogues 8c and 8d exhibiting greater
activity than their xylo-counterparts. The activity of the 2′-
oxetane analogue 8c was greater than that of the 2′-
We have synthesized and evaluated 2′-spironucleosides and
their prodrugs as potential anti-HCV agents. The triphosphate
of 2′-oxetane cytidine demonstrated comparable intrinsic
C
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a
Scheme 3. Preparation of 2′-Oxetane 2-Amino-6-O-methyl-purine Nucleoside
a
Reaction and conditions: (i) 2-amino-6-chloropurine/DBN/TMSOTf/ACN; (ii) NaOMe/MeOH; (iii) TIPSCl2/Pyr; (iv) BzCl/Pyr; (v) (1)
OsO4/NMO/tBuOH/H2O, (2) NaIO4/THF, (3) NaBH4/EtOH/EtOAc; (vi) (1) MsCl/Pyr, (2) NaH/THF; (vii) NH4F/MeOH; (viii) NaOMe/
MeOH.
a
Scheme 4. Preparation of 2′-Oxetane Guanosine and its Prodrugs
a
Reagents and conditions: (i) phosphorus agent/NMI; (ii) ADA/H2O; (iii) POCl3/Bu3N/Bu3N·pyrophosphate/DTP.
activity against NS5B polymerase to that of compound II. For
the 2′-oxetane cytidine analogues activity against NS5B bearing
the S282T amino acid change was reduced, as was observed for
other pyrimidine anti-HCV nucleosides or their phosphate
prodrugs. However, the triphosphate of 2′-oxetane-guanosine,
like those of the 2′-α-F-2′-β-C-methylguanosine series, retained
comparable activity against both S282T and wild-type NS5B.
Although 2′-oxetane guanosine analogues did not show
significant replicon activity, their phosphoramidate prodrugs
demonstrated potent anti-HCV activity in vitro and an excellent
resistance profile.
D
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column (3.0 mm × 30 mm, 3.0 mm i.d.), eluting with 0.0375% TFA in
water (solvent A) and 0.01875% TFA in acetonitrile solvent B). High-
resolution mass spectra were obtained on a Agilent G1969a
spectrometer.
Table 1. Anti-HCV Activity and Cytotoxicity of Pyrimidine
Nucleosides
General Procedure for the Preparation of Compounds 2a−
d. To a solution of compound 1a17 (300 mg, 0.59 mmol) in
anhydrous THF (10 L) was added Et3N·3HF (0.15 mL), and the
reaction mixture was stirred at room temperature for 2 h. The mixture
was concentrated to dryness under reduced pressure, and the residue
was purified by flash silica gel column chromatography (0−15%
MeOH in CH2Cl2) to give compound 2a (61.26 mg, 38.8%).
2a: δH (DMSO-d6): 11.42 (s, 1H), 7.68 (d, J = 8.0 Hz, 1H), 6.08 (s,
1H), 5.87 (d, J = 5.2 Hz, 1H), 5.60 (d, J = 8.0 Hz, 1H), 5.04−5.06 (m,
1H), 4.30−4.35 (m, 1H), 4.19−4.24 (m, 1H), 3.95−3.98 (m, 1H),
3.50−3.61 (m, 3H), 3.01−3.08 (m, 1H), 2.39−2.45 (m, 1H). HRMS
(TOF-ESI): calcd for C11H15N2O6, 271.0925; found, 271.0917.
2b: δH (DMSO-d6): 11.32 (s, 1H), 7.80 (d, J = 8.0 Hz, 1H), 5.83 (s,
1H), 5.63 (d, J = 4.2 Hz, 1H), 5.59 (d, J = 8.0 Hz, 1H), 5.03−5.05 (m,
1H), 3.83−3.86 (m, 1H), 3.64−3.70 (m, 3H), 3.47−3.60 (m, 2H),
2.27−2.29 (m, 1H), 1.74−1.81 (m, 3H). HRMS (TOF-ESI): calcd for
C12H17N2O6, 285.1087; found, 285.1070.
2c: δH (CD3OD): 7.93 (d, J = 8.0 Hz, 1H), 6.17 (s, 1H), 5.67 (d, J
= 8.0 Hz, 1H), 4.53 (m, 2H), 3.95 (m, 2H), 3.72 (m, 2H), 2.60 (m,
2H). LC-MS (ESI): 270 [M + H]+.
2d: δH (CD3OD): 8.09 (d, J = 8.4 Hz, 1H), 5.91 (s, 1H), 5.68 (d, J
= 8.4 Hz, 1H), 3.90 (m, 6H), 1.95 (m, 4H). LC-MS (ESI): 284 [M +
H]+.
General Procedure for the Preparation of Cytidine
Analogues 4a−d. To a solution of compound 1d (0.08 g, 0.14
mmol) in MeCN (10 mL) was added DMAP (0.02 g, 0.14 mmol) and
Et3N (0.07 g, 0.71 mmol) followed by addition of TsCl (0.08 g, 0.43
mmol). The solution was stirred at room temperature for 1 h. To the
solution was added NH4OH (30%, 2 mL), and the mixture was stirred
at room temperature for 1 h. Solvent was evaporated to dryness, and
the residue was coevaporated with toluene twice to give crude cytosine
analogue, which was dissolved in CH2Cl2 (10 mL) and pyridine (1
mL). To the solution was added BzCl (0.1 mL, 0.86 mmol), and the
solution was stirred at room temperature for 2 h. Water (5 mL) was
added, and the mixture was evaporated to dryness under reduced
pressure. The residue was dissolved in EtOAc (100 mL), and the
solution was washed with water and brine and dried over Na2SO4.
Solvent was evaporated, and the residue was purified by flash silica gel
column chromatography (0−60% EtOAc in hexanes) to give N-
benzoylcytosine analogue, which was dissolved in THF (10 mL). To
the solution was added TBAF (0.12 g, 0.48 mmol), and the solution
was stirred at room temperature for 1 h. Solvent was evaporated and
the residue was purified by flash silica gel column chromatography (0−
8% MeOH in CH2Cl2) to give N-benzoyl nucleoside, which was
dissolved in 7N NH3 in MeOH (8 mL). The solution was stirred at
room temperature for 20 h. Solvent was evaporated and the residue
was purified by flash silica gel column chromatography (0−30%
MeOH in CH2Cl2) to give product 4d (0.02 g, 56% from 1d).
4a: δH (DMSO-d6): 7.55 (d, J = 7.2 Hz, 1H), 7.12−7.20 (m, 2H),
6.16 (s, 1H), 5.76 (d, J = 5.2 Hz, 1H), 5.68 (d, J = 8.0 Hz, 1H), 4.91−
4.94 (m, 1H), 4.24−4.29 (m, 1H), 4.06−4.11 (m, 1H), 3.93−3.96 (m,
1H), 3.46−3.63 (m, 3H), 2.87−2.94 (m, 1H), 2.42−2.47 (m, 1H).
LC-MS(ESI): m/z 269.9 [M + 1]+. HRMS (TOF-ESI): calcd For
C11H16N3O5, 270.1084; found, 270.1081.
a
Effective concentration at which 50% inhibition occurs as determined
by luciferase based genotype 1b replicon assays in Lunet cells.
EXPERIMENTAL SECTION
■
Nuclear magnetic resonance (NMR) spectra were recorded on a
Bruker advance II 400 MHz and a Varian Unity Plus 400 MHz
spectrometers 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). Purities of the final
compounds were determined by HPLC and were greater 95%. HPLC
conditions to assess purity were as follows: Shimadzu HPLC 20AB
Sepax HP-C18 4.6 mm × 50 mm (5 μm); flow rate, 3.0 mL/min;
acquisition time, 6 min; wavelength, UV 220 nm; oven temperature,
40 °C. Chiral HPLC was conducted on Shimadzu HPLC 20A. The
preparative HPLC system includes two sets of Gilson 306 pumps, a
Gilson 156UV/vis detector, and a Gilson 215 injector and fraction
collector, with Unipoint control software. A YMC 25 mm × 30 mm ×
2 mm column was used. The mobile phase was HPLC grade water (A)
and HPLC grade acetonitrile (B) system. SFC was conducted on
Berger Multi-Gram SFC from Mettler Toledo Co, Ltd. LC/MS was
conducted on Shimadzu LCMS 2010EV using electrospray positive
[ES + ve to give MH+] equipped with a Shim-pack XR-ODS 2.2 μm
4b: δH (DMSO-d6): 7.65 (d, J = 7.2 Hz, 1H), 7.05−7.19 (m, 2H),
5.98 (s, 1H), 5.68 (d, J = 7.2 Hz, 1H), 5.57 (d, J = 5.6 Hz, 1H), 4.86−
4.92 (m, 1H), 3.74−3.77 (m, 1H), 3.54−3.70 (m, 4H), 3.35−3.38 (m,
1H), 2.17−2.24 (m, 1H), 1.66−1.85 (m, 3H). LC-MS (ESI): m/z
283.9 [M + 1]+. HRMS(TOF-ESI): calcd for C12H18N3O5, 284.1241;
found, 285.1235.
4c: δH (CD3OD): 8.04 (d, J = 7.6 Hz, 1H), 6.26 (s, 1H), 5.87 (d, J
= 7.6 Hz, 1H), 4.55 (m, 2H), 3.96 (m, 2H), 3.74 (m, 2H), 2.54 (m,
2H). LC-MS (ESI): 270 [M + H]+.
4d: δH (CD3OD): 8.09 (d, J = 7.6 Hz, 1H), 5.99 (s, 1H), 5.87 (d, J
= 7.6 Hz, 1H), 3.95 (m, 6H), 2.80 (m, 4H). LC-MS (ESI): 284 [M +
H]+.
E
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Table 2. Anti-HCV Activity and Cytotoxicity of Prodrugs of Pyrimidine Nucleosides
General Procedure for the Preparation of Prodrugs As a
Mixtures of Diastereomers. To a precooled solution (−78 °C) of
phenyl dichlorophosphate (2.1 g, 9.96 mmol) in CH2Cl2 (40 mL) was
added L-alanine methyl ester hydrochloride (1.39 g, 9.96 mmol)
followed by addition of Et3N (2.02 g, 19.92 mmol) in CH2Cl2 (5 mL)
slowly, and the mixture was stirred at −78 °C for 1 h then at room
temperature for 16 h. Solvent was evaporated, and the residue was
filtered with Et2O (20 mL). Solvent was evaporated to dryness to give
crude monochlorophosphate reagent, which was dissolved in CH2Cl2
(10 mL, 10.0 M) for the next reaction. To a mixture of compound 2a
(20.0 mg, 0.07 mmol) in CH2Cl2 (15 mL) were added N-
methylimidazole (0.2 mL) and a solution of above monochlorophos-
phate reagent (0.5 mL, 0.50 mmol), and the resulting mixture was
stirred at room temperature for 3 h. EtOAc (100 mL) was added and
the mixture was washed with water, 1N HCl, aqueous NaHCO3, and
brine, sequentially. Organic solution was dried over Na2SO4 and
evaporated, and the residue was purified by flash silica gel column
chromatography (0−8% MeOH in CH2Cl2) to give compound 5a (22
mg, 58.0%).
5a: δH (CDCl3): 8.18 (s, 1H), 7.30 (m, 6H), 6.12 (ss, 1H), 5.62 (m,
1H), 4.07, 4.11, 3.80 (m, 8H), 3.74, 3.72 (ss, 3H), 3.17 (m, 1H), 2.60
(m, 1H), 1.37 (d, J = 7.2 Hz, 3H). 31P NMR (162 MHz): 4.84, 4.26.
LC-MS (ESI): 512 [M + H]+.
5b: δH (CDCl3): 8.75 (s, 1H), 7.60, 7.52 (dd, J = 8.0 Hz, 1H), 7.24
(m, 5H), 6.05, 6.04 (ss, 1H), 5.65, 5.58 (d, J = 8.0, 1H), 4.35 (m, 2H),
4.00 (m, 4H), 3.80 (m, 4H), 3.72, 3.70 (ss, 3H), 2.39 (m, 1H), 1.90
(m, 2H), 1.72 (m, 1H), 1.36 (m, 3H). 31P NMR (162 MHz): 4.32,
4.01. LC-MS (ESI): 526 [M + H]+.
3.73, 3.72 (ss, 3H), 2.83 (m, 1H), 1.95 (m, 2H), 1.69 (m, 1H), 1.37
(m, 3H). 31P NMR (162 MHz): 4.20, 3.79. LC-MS (ESI): 526 [M +
H]+.
Preparation of Prodrug 6c. To a suspension of nucleoside 4c (50
mg, 0.45 mmol) and corresponding diastereomerically pure
phosphoramidate reagent18 (0.15 g, 0.33 mmol) in THF (5 mL)
was added a solution of t-butylmagnesium chloride (1M, 0.45 mL, 0.45
mmol) in THF at 0 °C slowly. The reaction mixture was stirred at 0
°C for 3 h. EtOAc (100 mL) was added, and the solution was washed
with aqueous NH4Cl and brine and dried over Na2SO4. Solvent was
removed and the residue was purified by flash silica gel column
chromatography (0−5% MeOH in CH2Cl2) to give compound 6c (40
mg, 40%). δH (CDCl3): 7.11−7.43 (m, 6H), 6.18 (s, 1H), 5.76 (d, J =
7.6 Hz, 1H), 4.96 (m, 1H), 4.63 (m, 1H), 4.33−4.50 (m, 4H), 4.04
(m, 1H), 3.94 (m, 1H), 3.79 (m, 1H), 2.63, 2.43 (mm, 2H), 1.33 (d, J
= 7.2 Hz, 3H), 1.24 (d, J = 6.4 Hz, 6H). 31P NMR (162 MHz): 4.03.
LC-MS (ESI): 539 [M + H]+.
Preparation of Nucleoside Triphosphates. Triphosphates (7b,
7c and 8a−d and others) of the nucleosides were prepared by
Nublocks, LLC, San Diego, CA.
Preparation of 6-Chloropurine Nucleoside 10. To a precooled
solution (0 °C) of compound 917 (4.00 g, 6.59 mmol) and 6-
chloroguanine (1.68 g, 9.89 mmol) in MeCN (80 mL) were added
DBN (2.46 g, 19.78 mmol) then TMSOTf (5.86 g, 26.38 mmol), and
the solution was heated at 65 °C for 5 h then room temperature for 16
h. The solution was poured into a mixture of EtOAc (300 mL) and
excess NaHCO3 with ice. Organic solution was washed with NaHCO3
and brine and dried over Na2SO4. Solvent was evaporated and the
residue was purified by flash silica gel column chromatography (5−
60% EtOAc in hexanes) to give compound 10 (3.20 g, 74%). δH
(CD3OD): 8.18−7.25 (m, 16 Hz), 6.73 (s, 1H), 5.40 (m, 3H), 5.12
(m, 2H), 4.82 (m, 1H), 4.74 (m, 3H), 3.04 (m, 1H), 2.52 (m, 1H).
LC-MS (ESI): 654 [M + H]+.
5c: δH (CDCl3): 9.15, 9.07 (ss, 1H), 7.26 (m, 7H), 6.19, 6.15 (ss,
1H), 5.65 (m, 1H), 4.50 (m, 4H), 3.95 (m, 4H), 3.72, 3.70 (ss, 3H),
3.42 (s, 1H), 2.75 (m, 1H), 2.46 (m, 1H), 1.35 (m, 3H). 31P NMR
(162 MHz): 4.21, 3.83. LC-MS (ESI): 512 [M + H]+.
5d: δH (CDCl3): 8.51, 8.40 (ss, 1H), 7.48, 7.42 (d, 8.0 Hz, 1H),
7.29 (m, 5H), 5.98 (s, 1H), 5.62 (m, 1H), 4.48 (m, 2H), 3.95 (m, 6H),
F
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(s, 1H), 5.93 (s, 1H), 5.66 (m, 1H), 4.88 (m, 1H), 4.82 (s, 2H), 4.73
(d, J = 7.6 Hz, 1H), 4.64 (m, 1H), 4.20 (m, 1H), 4.08 (m, 7H), 2.20
(m, 2H), 1.07 (m, 28H). LC-MS (ESI): 450 [M + H]+.
Table 3. Anti-HCV Activity of the Nucleoside Triphosphates
against NS5B Polymerase and Its S282T Mutant
To a solution of the above intermediate in pyridine (10 mL) and
CH2Cl2 (20 mL) was added benzoyl chloride (0.63 g, 4.48 mmol), and
the solution was stirred at room temperature for 5 h. Water (10 mL)
was added and the solution was evaporated to give a residue which was
dissolved in EtOAc (200 mL). Organic solution was washed with brine
and dried over Na2SO4. Solvent was evaporated and the residue was
purified by flash silica gel column chromatography (0−5% MeOH in
CH2Cl2) to give compound 13 (1.50 g, 76%) as foam. δH (CD3OD):
8.46 (s, 1H), 7.78 (m, 6H), 5.98 (s, 1H), 5.72 (m, 1H), 5.00 (d, J = 8.0
Hz, 1H), 4.83 (d, J = 10.4 Hz, 1H), 4.50 (m, 1H), 4.35 (m, 1H), 4.10
(m, 5H), 2.32 (m,1H), 2.20 (m, 1H), 1.05 (m, 28H). LC-MS (ESI):
684 [M + H]+.
Preparation of Compound 14. To a mixture of compound 13
(0.30 g. 0.44 mmol) in THF (6 mL), t-butanol (6 mL) and water (1
mL) was added OsO4 in t-butanol (0.25% 0.5 mL), followed by
addition of 50% N-methylmorpholine N-oxide (NMO, 0.2 mL, 0.85
mmol). The solution was stirred at room temperature for 16 h. Solvent
was evaporated and the residue was coevaporated with toluene twice
to give diol as a mixture of diastereomers which was dissolved in THF
(10 mL). To the solution was added water (1 mL) followed by
addition of NaIO4 (excess) portionwise until starting material
disappeared at room temperature for 3 h. EtOAc (100 mL) was
added, and the solution was washed with brine and dried over Na2SO4.
Solvent was evaporated, and the residue was dissolved in a solution of
EtOAc (10 mL) and EtOH (10 mL). To the precooled solution of the
resulting aldehyde at 0 °C was added NaBH4 (50.16 mg, 1.32 mmol),
and the mixture was stirred at 0 °C for 1 h. EtOAc (100 mL) was
added, and the organic solution was washed with brine and dried over
Na2SO4. The solvent was evaporated to dryness, and the residue was
purified by flash silica gel column chromatography (0−10% MeOH in
CH2Cl2) to give compound 14 (0.14 g, 43% from 3). δH (CDCl3):
8.57 (s, 1H), 8.48 (s, 1H), 7.70 (m, 5H), 6.26 (s, 1H), 4.15 (m, 9H),
1.28 (m, 2H), 1.15 (m, 28H). LC-MS (ESI): 688 [M + H]+.
Preparation of Compound 17. To a solution of compound 14
(0.33 g, 0.47 mmol) in CH2Cl2 (30 mL) and pyridine (3 mL) was
added MsCl (0.11 g, 0.94 mmol), and the solution was stirred at room
temperature for 3 h. Water (10 mL) was added, and the mixture was
extracted with EtOAc (100 mL). The organic solution was washed
with brine and dried over Na2SO4. Solvent was evaporated and the
residue was purified by flash silica gel column chromatography (0−
100% EtOAc in hexanes) to give a mesylate (0.30 g, 82%). δH
(CDCl3): 8.53 (s, 1H), 8.21 (s, 1H), 7.65 (m, 5), 6.14 (s, 1H), 4.80
(s, 1H), 4.54 (m, 2H), 4.33 (m, 1H), 4.16 (s, 3H), 4.10 (m, 3H), 2.95
(s, 3H), 2.05 (m, 2H), 1.05 (m, 28H). LC-MS (ESI): 766 [M + H]+.
To a solution of the mesylate (0.20 g, 0.26 mmol) in THF (10 mL)
was added NaH (60% mineral oil, 110 mg, 2.75 mmol), and the
mixture was stirred at room temperature for 1 h. The mixture was
poured into EtOAc (100 mL), and the solution was washed with brine
and dried over Na2SO4. Solvent was evaporated and the residue was
purified by flash silica gel column chromatography (0−80% EtOAc in
hexanes) to give 2′-oxetanyl intermediate 15 (0.15 g, 84%). δH
(CDCl3): 8.47 (s, 1H), 8.23 (s, 1H), 7.65 (m, 5H), 6.38 (s, 1H),
7.74 (m, 1H), 4.59 (m, 1H), 4.46 (d, J = 9.2 Hz, 1H), 4.26 (d, J = 13.2
Hz, 1H), 4.15 (s, 3H), 4.00 (m, 2H), 2.56 (m, 2H), 1.09 (m, 28H).
LC-MS (ESI): 686 [M + H]+.
Table 4. Intrinsic Anti-HCV Activity of Triphosphate of
Synthesized Guanosine Analogue against Wild-Type NS5B
Polymerase and Its S282T Mutant
triphosphate
IC50 (wild type) (μM)
IC50 (S282T) (μM)
19
9.5
4.2
10.4
5.31
I-TP
Preparation of 6-O-Methylnucleoside 11. To a mixture of
compound 10 (3.20 g, 4.89 mmol) in MeOH (80 mL) was added 25%
NaOMe in MeOH (1.86 g, 48.92 mmol), and the solution was stirred
at room temperature for 24 h. Solvent was evaporated, and the residue
was purified by flash silica gel column chromatography (0−15%
MeOH in CH2Cl2) to give compound 11 (1.33 g, 57%) as white solid.
δH (CD3OD): 8.09, 8.13 (s, 1H), 5.97 (s, 1H), 5.67 (m, 1H), 4.77 (m,
1H), 4.56 (m, 1H), 4.45 (d, J = 8.8 Hz, 1H), 4.13−3.83 (m, 6H), 2.25
(m, 1H), 2.05 (m, 1H). LC-MS (ESI): 338 [M + H]+.
Preparation of Compound 13. To a solution of compound 11
(1.33 g, 3.94 mmol) in pyridine (20 mL) was added TIPSCl (1.37 g,
4.34 mmol) slowly, and the solution was stirred at room temperature
for 16 h. Solvent was evaporated, and the residue was dissolved in
EtOAc (400 mL). The organic solution was washed with brine and
dried over Na2SO4. Solvent was evaporated and the residue was
purified by flash silica gel column chromatography (0−5% MeOH in
CH2Cl2) to give intermediate 12 (1.30 g, 73%). δH (CD3OD): 7.757
To the solution of the 2′-oxetanyl intermediate in MeOH (10 mL)
was added NH4F (1.3 mmol, 46.8 mg), and the mixture was heated at
60 °C for 5 h. Solvent was evaporated and the residue was purified by
flash silica gel column chromatography (0−10% MeOH in CH2Cl2) to
give compound 16 (0.05 g, 43% from 14) as white solid. LC-MS
(ESI): 428 [M + H]+.
A solution of compound 16 (0.20 g, 0.45 mmol) in MeOH (10 mL)
was added NaOMe (4.8 M, 0.8 mL), and the solution was stirred at
room temperature for 20 h. Solvent was evaporated and the residue
was purified by flash silica gel column chromatography (0−15%
MeOH in CH2Cl2) to give compound 17 (0.10 g, 69%). δH
(CD3OD): 815 (s, 1H), 6.26 (s, 1H), 4.50 (m, 3H), 4.05 (s, 3H),
G
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Table 5. Anti-HCV Activity and Cytotoxicity of Prodrugs of 6-O-Methylguanosine Analogues
compd
Ar
R
Sp/Rp
alogP
EC50 (1b) (μM)
CC50 (μM)
20a
20b
20c
20d
20e
20f
20g
20h
20i
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Me
Et
Sp/Rp = 1/1
0.27
0.62
1.0
7.16
0.86
1.49
4.52
0.27
3.1
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>100
Sp
iPr
iPr
Sp
Rp
1.0
n-Bu
Sp
1.6
n-Bu
Rp
1.6
i-Bu
Sp
1.46
1.46
1.67
1.67
1.74
1.74
1.85
1.85
2.64
2.0
0.57
4.42
0.45
0.67
1.02
9.31
8.65
11.7
0.31
<0.0045
i-Bu
Rp
c-pentyl
c-pentyl
neopentyl
neopentyl
Bn
Sp
20j
Rp
20k
20m
20n
20o
20p
I
Sp
Rp
Sp
Bn
Rp
naphthyl
Ph
neopentyl
iPr
Sp/Rp = 1/1
Sp
3.96 (m, 1H), 3.80 (m, 2H), 2.57 (m, 1H), 2.27 (m, 1H). LC-MS
(ESI): 324 [M + H]+.
1H), 4.15−4.09 (m, 5H), 4.00−3.91 (m, 2H), 3.77 (dd, 1H, J = 9.6,
11.2 Hz), 3.36 (d, 1H, J = 10.4 Hz), 2.67 (m, 1H), 2.18 (m, 1h), 1.31
(d, 3H, J = 7.2 Hz), 1.20 (t, 3H, J = 7.2 Hz). 31P NMR (162 MHz):
4.08. LC-MS (ESI): 579 [M + H]+.
20d: δH (CDCl3): 7.62 (s, 1H), 7.32−7.12 (m, 5H), 6.20 (s, 1H),
5.11 (s, 2H), 4.97 (m, 1H), 4.6−4.53 (m, 4H), 4.38 (m, 1H), 4.07 (s,
3H), 3.99−3.92 (m, 2H), 3.85 (t, 1H, J = 11.2 Hz), 3.61 (br s, 1H),
2.62 (m, 1H), 2.15 (m, 1H), 1.98 (bs, 1H), 1.29 (d, 3H, J = 6.8 Hz),
1.20 (t, 3H, J = 6.4 Hz). 31P NMR (162 MHz): 4.74. LC-MS (ESI):
593 [M + H]+.
20e: δH (CDCl3): 7.70 (s, 1H), 7.32−7.12 (m, 5H), 6.17 (s, 1H),
5.11 (s, 2H), 4.71 (t, 1H, J = 6.8 Hz), 4.59−4.52 (m, 3H), 4.35 (m,
1H), 4.11−3.91 (m, 7H), 3.81 (dd, 1H, J = 9.6, 11.6 Hz), 3.41 (d, 1H,
J = 10.0 Hz), 2.67 (m, 1H), 2.17 (m, 1H), 1.54 (m, 2H), 1.36−1.27
(m, 5H), 0.87 (t, 3H, J = 7.6 Hz). 31P NMR (162 MHz): 4.12. LC-MS
(ESI): 607 [M + H]+.
Preparation of Compound 18. To a solution of compound 17
(0.04 g, 0.12 mmol) in MOPS buffer (0.1 M, 10 mL) was added
adenosine deaminase (2.0 mg), and the solution was kept at 37 °C for
2 days. Solvent was evaporated and the residue was purified by flash
silica gel column chromatography (0−30% MeOH in CH2Cl2) to give
a crude compound 18 containing MOPS. Crystalline MOPS was
removed by filtration after heating the crude 18 in MeOH. The
mother liquor containing 18 was concentrated to dryness and the
residue was purified by flash silica gel column chromatography (0−
30% MeOH in CH2Cl2) to give compound 18 as white solid (0.02 g,
53%). δH (CD3OD): 8.04 (s, 1H), 6.21 (s, 1H), 4.54 (m, 2H), 4.36 (d,
J = 8.8 Hz, 1H), 4.94 (m, 1H), 3,78 (m, 2H), 2.60 (m, 1H), 2,33 (m,
1H). LC-MS (ESI): 310 [M + H]+.
Preparation of Triphosphate 19 Was Accomplished As for
7b and 8a−d. Diastereomerically pure 20c was prepared by method
used for compound 6c. 20c: δH (CDCl3) 7.69 (s, 1H), 7.32−7.12 (m,
5H), 6.16 (s, 1H), 5.09 (s, 2H), 4.96 (m, 1H), 4.73 (dd, 1H, J = 8.8,
10.4 Hz), 4.61−4.53 (m, 3H), 4.33 (m, 1H), 4.06 (s, 3H), 3.98−3.90
(m, 2H), 3.70 (dd, 1H, J = 9.2, 11.2 Hz), 3.24 (d, 1H, J = 10.0 Hz),
2.68 (m, 1H), 2.17 (m, 1H), 1.30 (d, 3H, J = 7.2 Hz), 1.18 (2d, 6H, J =
6.4 Hz). 31P NMR (162 MHz): 4.16. LC-MS (ESI): 593 [M + H]+.
Phosphoramidate prodrugs (20a and 20p) were prepared as
diastereomeric mixtures from the corresponding amino acid esters
according to method used for the preparation of compounds 5a−d.
Diastereomerically pure prodrugs (20b−o) were obtained by SFC
separation of the mixtures. Samples of diastereomeric mixtures were
subjected to SFC using a chiral column (2 × 15 cm) and eluted with
35% isopropyl alcohol in carbon dioxide at 100 bar. An injection
loading of 4 mL of sample at a concentration of 17 mg/mL in
methanol was used. According to the deastereomerically pure 20c, the
Rp isomer eluted first. The appropriate fractions of the multiple runs
were combined and concentrated under reduced pressure to give
product.
20f: δH (CDCl3): 7.62 (s, 1H), 7.33−7.13 (m, 5H), 6.20 (s, 1H),
5.07 (s, 2H), 4.65−4.52 (m, 4H), 4.37 (m, 1H), 4.12−3.88 (m, 6H),
3.93 (m, 1H), 3.79 (t, 1H, J = 11.2 Hz), 3.43 (br s, 1H), 2.63 (m, 1H),
2.15 (m, 1H), 1.57 (m, 2H), 1.38−1.29 (m, 5H), 0.96 (t, 3H, J = 7.2
Hz). 31P NMR (162 MHz): 4.70. LC-MS (ESI): 607 [M + H]+.
20g: δH (CDCl3): 7.71 (s, 1H), 7.31−7.12 (m, 5H), 6.18 (s, 1H),
5.14 (s, 2H), 4.7 (t, 1H, J = 8.8 Hz), 4.60−4.53 (m, 3H), 4.35 (m,
1H), 4.05 (s, 3H), 4.05−3.85 (m, 4H), 3.78 (dd, 1H, J = 6.8, 10.4 Hz),
3.53 (d, 1H, J = 9.6 Hz), 2.66 (m, 1H), 2.17 (m, 1H), 1.87 (m, 1H),
1.33 (d, 3H, J = 6.8 Hz), 0.87 (d, 6H, J = 6.4 Hz). 31P NMR (162
MHz): 4.15. LC-MS (ESI): 607 [M + H]+.
20h δH (CDCl3): 7.61 (s, 1H), 7.34−7.13 (m, 5H), 6.19 (s, 1H),
5.05 (s, 2H), 4.68−4.52 (m, 4H), 4.37 (m, 1H), 4.09−3.98 (m, 4H),
3.93−3.82 (m, 3H), 3.74 (t, 1H, J = 9.2 Hz), 3.34 (d, 1H, J = 9.6 Hz),
2.62 (m, 1H), 2.15 (m, 1H), 1.90 (m, 1H), 1.32 (d, 3H, J = 7.2 Hz),
0.89 (d, 6H, J = 6.4 Hz). 31P NMR (162 MHz): 4.74. LC-MS (ESI):
607 [M + H]+.
20i: δH (CDCl3): 7.69 (s, 1H), 7.32−7.13 (m, 5H), 6.16 (s, 1H),
5.12 (m, 1H), 5.07 (s, 2H), 4.73 (m, 1H), 4.60−4.53 (m, 3H), 4.34
(m, 1H), 4.06 (s, 3H), 3.98−3.90 (m, 2H), 3.66 (dd, 1H, J = 9.6, 11.2
Hz), 3.17 (d, 1H), 2.68 (m, 1H), 2.17 (m, 1H), 1.80 (m, 2H), 1.68−
1.52 (m, 6H), 1.29 (d, 3H, J = 6.8 Hz). 31P NMR (162 MHz): 4.18.
LC-MS (ESI): 619 [M + H]+.
20a: δH (CDCl3): 7.69, 7.61 (ss, 1H), 7.25 (m, 5H), 6.18 (ss, 1H),
5.08 (ss, 2H), 4.60 (m, 3H), 4.35 (m,1H), 4.06 (ss, 3H), 3.90 (m, 3H),
3.60 (ss, 3H), 3.35 (m, 1H), 2.66 (m, 1H), 2.18 (m, 1H), 1.32 (m,
3H). 31P NMR (162 MHz): 4.61, 3.97. LC-MS (ESI): 565 [M + H]+.
20b: δH (CDCl3): 7.70 (s, 1H), 7.32−7.14 (m, 5H), 6.17 (s, 1H),
5.10 (s, 2m), 4.72 (t, 1H, J = 9.2 Hz), 4.59−4.54 (m, 3H), 4.35 (m,
20j: δH (CDCl3): 7.62 (s, 1H), 7.33−7.13 (m, 5H), 6.20 (s, 1H),
5.16−5.13 (m, 3H), 4.64−4.54 (m, 4H), 4.38 (m, 1H), 4.07 (s, 3H),
H
dx.doi.org/10.1021/jm401224y | J. Med. Chem. XXXX, XXX, XXX−XXX

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3.99−3.91 (m, 2H), 3.83 (dd, 1H, J = 9.6, 11.6 Hz), 2.63 (m, 1H),
2.15 (m, 1H), 1.80 (m, 2H), 1.71−1.55 (m, 6H), 1.28 (d, 3H, J = 6.8
Hz). 31P NMR (162 MHz): 4.76. LC-MS (ESI): 619 [M + H]+.
20k: δH (CDCl3): 7.69 (s, 1H), 7.32−7.12 (m, 5H), 6.16 (s, 1H),
5.07 (s, 2H), 4.72 (t, 1H, J = 10.0 Hz), 4.60−4.52 (m, 3H), 4.34 (m,
1H), 4.07−4.01 (m, 4H), 3.92 (m, 1H), 3.82 (d, 1H, J = 10.4 Hz),
3.70 (m, 1H), 3.15 (d, 1H, J = 10.0 Hz), 2.68 (m, 1H), 2.17 (m, 1H),
1.35 (d, 3H, J = 6.8 Hz), 0.89 (s, 9H). 31P NMR (162 MHz): 4.11.
LC-MS (ESI): 621 [M + H]+.
AUTHOR INFORMATION
Corresponding Authors
*For J.D.: phone, 650-372-7251; fax, 650-522-5899; E-mail,
Jinfa.Du@gilead.com; address, Chemistry Department, Gilead
Sciences, 333 Lakeside Drive, Foster City, California 94404,
United States.
■
*For M.J.S.: phone, 860-919-0289; E-mail, mjsofia58@gmail.
com.
20m: δH (CDCl3): 7.62 (s, 1H), 7.34−7.13 (m, 5H), 6.19 (s, 1H),
5.07 (s, 2H), 4.66−4.52 (m, 4H), 4.38 (m, 1H), 4.11−4.03 (m, 4H),
3.93 (m, 1H), 3.85−3.72 (m, 3H), 3.42 (br s, 1H), 2.63 (m, 1H), 2.15
(m, 1H), 1.34 (d, 3H, J = 7.2 Hz), 0.91 (s, 9H). 31P NMR (162 MHz):
4.76. LC-MS (ESI): 621 [M + H]+.
20n: δH (CDCl3): 7.73 (s, 1H), 7.37−7.11 (m, 10H), 6.17 (s, 1H),
5.09 (s, 2H), 5.07 (s, 2H), 4.75 (m, 1H), 4.58 (m, 3H), 4.40 (m, 1H),
4.09−4.01 (m, 4H), 3.92 (m, 1H), 3.74 (t, 1H, J = 2.6 Hz), 3.36 (br s,
1H), 2.68 (m, 1H), 2.19 (m, 1H), 1.28 (d, 3H, J = 7.2 Hz). 31P NMR
(162 MHz): 4.27. LC-MS (ESI): 641 [M + H]+.
Present Addresses
†J.D., B.-K.C., E.M., C.N., and H.M.M.S.: Chemistry Depart-
ment, Gilead Sciences, 333 Lakeside Drive, Foster City,
California 94404, United States
‡M.J.S.: OnCore Biopharma, 3805 Old Easton Road, Doyles-
town, Pennsylvania 18902, United States.
Notes
The authors declare no competing financial interest.
Pharmasset was acquired by Gilead Sciences in January, 2012.
20o: δH (CDCl3): 7.60 (s, 1H), 7.36−7.12 (m, 10H), 6.17 (s, 1H),
5.10 (d, 2H, J = 1.2 Hz), 5.05 (s, 2H), 4.66 (m, 1H), 4.62−4.51 (m,
3H), 4.34 (m, 1H), 4.13−4.03 (m, 4H), 3.89 (m, 1H), 3.74 (t, 1H J =
11.2 Hz), 3.31 (d, 1H, J = 7.6 Hz), 2.63 (m, 1H), 2.14 (m, 1H), 1.31
(d, 3H, J = 7.2 Hz). 31P NMR (162 MHz): 4.64. LC-MS (ESI): 641
[M + H]+.
ABBREVIATIONS USED
■
TMSOTf, trimethylsilyl trifluoromethanesulfonate; SFC, super-
critical fluid chromatography; MOPS, 3-(N-morpholino)-
propanesulfonic acid
20p: δH (CDCl3): 8.10 (m, 1H), 7.82 (m, 1H), 7.69−7.63 (m, 2H),
7.49 (m, 3H), 7.36 (m, 1H), 6.15 (ds, 1H), 5.08 and 5.04 (s, 2H),
4.84−4.60 (m, 2H), 4.54 (t, 2H, J = 7.6 Hz), 4.40 (m, 1H), 4.11 (m,
1H), 4.04 (s, 3H), 3.93 (m, 2H), 3.79 (dd, 1H, J = 1.6, 10.8 Hz), 3.64
(dd, 1H, J = 6.4, 10.0 Hz), 3.37 (m, 1H), 2.66 (m, 1H), 2.16 (m, 1H),
1.31 and 1.28 (ss, 3H), 0.86 (s, 9H). 31P NMR (162 MHz): 5.041,
4.47. LC-MS (ESI): 671 [M + H]+.
Molecular Modeling. Initial models for a number of the 2′-spiro
fused nucleosides were energy minimized to a convergence gradient of
0.001 using MMFFs22 with a distance dependent dielectric model of
2r. Conformers were generated for each of these using torsional
sampling (MCMM)23 followed by energy minimization. An RMSD of
0.25 and a 20 kcal/mol energy window were used as criterion to limit
the number of conformers output with an upper limit of 1000 being
possible. None of the compounds resulted in more than 100
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LLC, New York, NY, 2009).
Biological Assays. HCV Replicon Assays. HCV replicon assays
using ET-Lunet cells were performed as described previously.21 Briefly,
replicon-containing cells (3000 cells/well in a 96-well plate) were
incubated for 4 days with serially diluted test compounds. Inhibition of
HCV RNA replication was determined by measuring the levels of
luminescence expressed via the firefly luciferase reporter gene using
Bright-Glo reagents (Promega, Madison, WI). Effect of test
compounds on the proliferation of ET-Lunet cells after 4 days
incubation was determined by using Cell Titer-Glo (Promega,
Madison, WI). The EC50 and CC50 values, the concentrations at
which 50% inhibition were achieved, were determined using GraphPad
Prism software (San Diego, CA).
NS5B Polymerase Assays. IC50 values were determined using
recombinant HCV NS5B from wild-type or S282T genotype 1b NS5B.
All reactions were performed in a 20 μL mixture containing varying
concentrations of the test compound, 5 μM of the four natural
ribonucleotides, [α-32P]UTP, 20 ng/μL of genotype 1b (−) IRES
RNA template, 1 unit/μL of SUPERase•In (Ambion, Austin, TX), 40
ng/μL of NS5B, 5 mM MgCl2, and 2 mM DTT in 50 mM Hepes
buffer (pH 7.5). The reaction was quenched by adding 80 μL of stock
solution (12.5 mM EDTA, 2.25 M NaCl, and 225 mM sodium citrate)
after incubating at 27 °C for 30 min. The radioactive RNA products
were quantified as described previously.16 The IC50 values were
calculated using GraphFit program version 5 (Erithacus Software,
Horley, Surrey, UK).
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