2′-Modified Guanosine Analogs for the Treatment of HCV

Article abstract of DOI:10.1080/15257770.2016.1154968

Novel 2′-modified guanosine nucleosides were synthesized from inexpensive starting materials in 7–10 steps via hydroazidation or hydrocyanation reactions of the corresponding 2′-olefin. The antiviral effectiveness of the guanosine nucleosides was evaluated by converting them to the corresponding 5′-O-triphosphates (compounds 38–44) and testing their biochemical inhibitory activity against the wild-type NS5B polymerase.

Full text of DOI:10.1080/15257770.2016.1154968

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
2′-Modified Guanosine Analogs for the Treatment
of HCV
Vinay Girijavallabhan, Ashok Arasappan, Frank Bennett, Kevin Chen, Qun
Dang, Ying Huang, Angela Kerekes, Latha Nair, Dmitri Pissarnitski, Vishal
Verma, Carmen Alvarez, Ping Chen, David Cole, Sara Esposite, Yuhua Huang,
Qingmei Hong, Zhidan Liu, Weidong Pan, Haiyan Pu, Randall Rossman,
Quang Truong, Bancha Vibulbhan, Jun Wang, Zhiqiang Zhao, David Olsen,
Andrew Stamford, Stephane Bogen & F. George Njoroge
To cite this article: Vinay Girijavallabhan, Ashok Arasappan, Frank Bennett, Kevin Chen, Qun
Dang, Ying Huang, Angela Kerekes, Latha Nair, Dmitri Pissarnitski, Vishal Verma, Carmen
Alvarez, Ping Chen, David Cole, Sara Esposite, Yuhua Huang, Qingmei Hong, Zhidan Liu,
Weidong Pan, Haiyan Pu, Randall Rossman, Quang Truong, Bancha Vibulbhan, Jun Wang,
Zhiqiang Zhao, David Olsen, Andrew Stamford, Stephane Bogen & F. George Njoroge (2016): 2′-
Modified Guanosine Analogs for the Treatment of HCV, Nucleosides, Nucleotides and Nucleic
Acids, DOI: 10.1080/15257770.2016.1154968
To link to this article: http://dx.doi.org/10.1080/15257770.2016.1154968
Published online: 22 Apr 2016.
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Date: 23 April 2016, At: 07:01

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
, VOL. , NO. , –
http://dx.doi.org/./..
ꢀ-Modified Guanosine Analogs for the Treatment of HCV
Vinay Girijavallabhan, Ashok Arasappan, Frank Bennett, Kevin Chen, Qun Dang,
Ying Huang, Angela Kerekes, Latha Nair, Dmitri Pissarnitski, Vishal Verma,
Carmen Alvarez, Ping Chen, David Cole, Sara Esposite, Yuhua Huang,
Qingmei Hong, Zhidan Liu, Weidong Pan, Haiyan Pu, Randall Rossman,
Quang Truong, Bancha Vibulbhan, Jun Wang, Zhiqiang Zhao, David Olsen,
Andrew Stamford, Stephane Bogen, and F. George Njoroge
Merck Research Laboratories, Kenilworth, NJ , USA
ABSTRACT
ARTICLE HISTORY
Received  August 
Accepted  February 
Novel 2ꢀ-modified guanosine nucleosides were synthesized from
inexpensive starting materials in 7–10 steps via hydroazidation
or hydrocyanation reactions of the corresponding 2ꢀ-olefin. The
antiviral effectiveness of the guanosine nucleosides was evalu-
atedbyconvertingthemtothecorresponding5ꢀ-O-triphosphates
(compounds 38–44) and testing their biochemical inhibitory
activity against the wild-type NS5B polymerase.
Introduction
Nucleoside analogues have found widespread therapeutic application for more
than half a century for the treatment of both DNA and RNA viral infec-
tions as well as a variety of cancers.[1] Nucleosides are prodrugs that inhibit
viral replication or cancer cell division by being metabolized intracellularly
into the corresponding 5ꢀ-O-nucleoside triphosphates (NTPs) which are sub-
sequently incorporated into the growing DNA or RNA chain.[2,3] Compound
1 (Figure 1) was synthesized in 1966 and it was later determined that
the introduction of a 2ꢀ-beta methyl group leads to the inhibition of the
HCV NS5b polymerase.[4] Since then numerous inhibitors with the 2ꢀ-beta
methyl scaffold such as NM-1075a (2, Figure 1), PSI-9385a (3, Figure 1) and
most recently sofosbuvir5b (4, Figure 1) have provided proof of the concept
that inhibition of the viral polymerase can be an effective method of HCV
treatment.
Most known effective inhibitors in the literature possessed a 2ꢀ-alpha OH or
isosteres such as 2ꢀ-alpha F, but there was very little known about other modifi-
cations. Therefore, our goal was to probe the tolerance for steric and electronic
modifications at the 2ꢀ-alpha position as well as the need for hydrogen bond
CONTACT Vinay Girijavallabhan
vinay.girijavallabhan@merck.com
ing Hill Road, Kenilworth, NJ , USA.
Merck Research Laboratories,  Gallop-
©  Taylor & Francis Group, LLC

2
V. GIRIJAVALLABHAN ET AL.
Figure . Examples of base and sugar modified nucleosides active intracellularly as inhibitors of RNA
viruses.
donors/acceptors. Functionalities such as the alpha substituted azide, amino and
cyano groups (5, Figure 2) fit this criteria and also have the potential to be readily
converted to additional functionalities to further elaborate the SAR.
Due to the challenges of nucleoside chemistry it was important to develop
robust methodologies that would allow us to incorporate functionality in an
efficient manner. Our group had previously demonstrated that the hydroazida-
tion/hydrocyanation chemistry developed in the Carreira group could be performed
on complex systems.[6] Thus, we envisioned that a variety of novel substituents
could be obtained from a common intermediate such as the exocyclic alkene (6,
Figure 2).[7]
The other differentiating factor for novel analogs was the nucleoside base. Since
it was unknown how the potency of analogs would compare to 2ꢀ-OH/2ꢀ-F analogs,
it was important to use a base that was highly active in cellular/biochemical assays
which have been used to benchmark compounds in the literature.[5] Even though
guanosine, uridine and cytidine are all found in clinical candidates, literature sug-
gested that guanosines generally have the best intrinsic potency, therefore, they were
the initial focus of our attention.[5]
Synthesis
The chemistry to synthesize the 2ꢀ-alkene was initiated from the readily available
compound 7 which was peracetylated to provide 8 (Scheme 1). Protection of the
guanosine was necessary to avoid functional group incompatibilities during later
parts of the synthesis, therefore, we chose to use both the 6-OMe and 6-OEt groups
that have been shown to be viable guanosine prodrugs motifs in the clinic.[5] Pro-
tection as the 6-OEt/OMe was accomplished using Mitsunobu conditions with
either methanol or ethanol followed by removal of the hydroxy protecting groups to
Figure . ꢀalpha position modified nucleosides via the ꢀ-olefin.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
Scheme . Reagents and conditions. (a) acetic anhydride, pyridine, °C; %; (b) i. DIAD, EtOH or
MeOH, dioxane. ii. Ammonium hydroxide, methanol; 9a: % over two steps, 9b: % over two
steps.; (c) tetraisopropyldisiloxanedichloride pyridine; 10a: %, 10b: %; (d) Dess-Martin periodi-
nane, CHCl; 11a: %, 11b: %; (e) methyltriphenylphosphonium bromide, potassium hexamethyl-
disilazide, THF; 12a: %, 12b: %.
provide 9a and 9b. The 3ꢀ- and 5ꢀ- alcohols were protected as the 3ꢀ-5ꢀ-bis-silyl ether
using 1,3-dichloro-1,1,3,3-tetrakis(2-methylethyl)-disiloxane to provide 10a and
10b. TEMPO mediated oxidation of the 2ꢀ-alcohols to the ketones provided com-
pounds 11a and 11b. These compounds could be converted to the exocyclic alkenes
12a and 12b by treatment with an excess of the potassium salt of methylphospho-
nium bromide in good yield and on a multigram scale.
With compound 12b in hand, it was possible to test the hydroazidation reaction
(Scheme 2).[8] First attempts of the hydroazidation chemistry (Table 1, Entries 1 and
2) were performed by dissolving a catalytic amount of Co catalyst 13 and 1.5 or 3 eq
Scheme . Hydroazidation reaction of 12b.
Table . Reagents and conditions.
Entry
Co (eq)
TsN (eq)
PhSiH (eq)
TsN/EtOHa
Yield%

.
.
.
.
.
.
.





.
.
.
.
.
.b
/
/
/
/
/
/
<
<









aRatio by volume of TsN and ethanol at start of reaction. bPhenylsilane was added dropwise in EtOH (equal to
volume of TsN).

4
V. GIRIJAVALLABHAN ET AL.
of TsN3 in ethanol and adding neat phenylsilane. This provided very small amounts
of the desired product 14 and large amounts of side product 15. Even though the
yield was very low, we isolated only the desired alpha-isomer of the 2ꢀ-azide most
likely due to the steric congestion of the beta face of the ribose. Several attempts were
made to improve the yield of the reaction including modifications to the protecting
groups on the substrate, catalyst (various ligands/alternative metals), solvent, azide
reagent and silane but none of these modifications improved the reaction. Therefore,
it was essential to better understand the reaction by making incremental changes to
solvent and equivalents of reagents. From entries 1 and 2, it was observed that the
reaction was extremely fast (all starting olefin was consumed upon the complete
addition of Ph3SiH), therefore, it was hypothesized that the product ratio could be
improved by having a larger effective concentration of TsN3. Incremental increases
in the equivalents of TsN3 increased the yield of compound 14 by reducing the
amount of side product 15 (entries 3 and 4). The best yield of 45% was achieved
by removing the solvent and using 30 equivalents of TsN3 (entry 5). Due to the fast
reaction rate upon addition of the phenylsilane, it was hypothesized that the addition
rate may also impact the yield of the reaction. Addition of the phenylsilane dropwise
in ethanol further improved the yield of the conversion to 65% which was sufficient
to access the desired targets (entry 6).
With the yield optimized to be able to provide gram quantities of 14, we were able
to obtain the desired 6-OEt nucleoside 16 by deprotection using TBAF followed by
treatment with methanolic ammonia (Scheme 3). The 6-OEt protecting group was
removed using 1M HCl at 50°C to provide the desired 2ꢀ-azido guanosine analog
17. The 2ꢀ-alpha amino analog 18 was synthesized via hydrogenation of the azide
17 in the presence of Pd(OH)2.
Scheme . Synthesis of ꢀ-amino modified nucleosides.
Reagents and conditions. (a) i. TBAF, THF, room temperature, ii. 7M ammonia
in methanol, (98% over two steps); (b) 1M HCl, THF, 50°C, 64%; (c) H2, Pd(OH)2,
MeOH, 91%.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
The hydrocyanation reaction[9] (Scheme 4) proved to require additional opti-
mization since the optimized hydroazidation reaction was run solvent-free and
TsCN is a solid. Therefore, it was important to identify an appropriate solvent for
the reaction that could solubilize the TsCN while maintaining high effective concen-
trations. After screening numerous solvents it was found that the use of 20 equiv-
alents of TsCN dissolved in a minimal amount of dioxane provided a reasonable
yield of desired compound 19 as a single diastereomer. Once again, it was impor-
tant to add the phenylsilane very slowly in ethanol in order to minimize production
of the reduced side product. The target 6-OMe nucleoside 20 was reached via global
deprotection of 19 using TBAF followed by sodium methoxide. Treatment of the
6-OMe nucleoside 20 with 1M HCl at 50°C provided the desired 2ꢀ-CN guanosine
nucleoside 21.
Scheme . Synthesis of ꢀ-CN guanosine analog.
Reagents and conditions. (a) 13, tosyl cyanide, phenylsilane, dioxane, ethanol,
25%; (b) i. TBAF, THF, 98%, ii. NaOMe, MeOH, 67%; (c) 1M HCl, THF, 50°C, 68%.
Scheme . Synthesis of ꢀ-triazolo guanosine nucleoside.
Reagents and conditions. (a) i. Acetic anhydride, DMAP, triethylamine, 66%, ii.
vinyl acetate, 140°C, 90%; (b) 7M ammonia in MeOH, 90%; (c) 1M HCl, THF, 60°C,
76%.
The versatility of the 2ꢀ-azido/amino functionality enabled us to investigate
additional functionality at this position that would probe the steric/electronic

6
V. GIRIJAVALLABHAN ET AL.
environment. As shown in Scheme 5, it was possible to convert the azido guanosine
intermediate 16 to the corresponding triazole 22 by protecting it as the bis-acetyl
followed by stirring at high temperature with vinyl acetate.[10] Subsequent depro-
tection using methanolic ammonia provided the 6-OEt guanosine analog 23. The
2ꢀ-triazolo guanosine nucleoside 24 was accessed by treatment of 23 with 1M HCl
at 50°C.
Scheme . Synthesis of ꢀ-NHMe, NHEt, and NMe guanosine analogs

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
Reagents and conditions. (a) H2, Pd(OH)2, methanol 61%; (b) formaldehyde,
sodium triacetoxyborohydride, triethylamine; 43%; (c) i. TBAF, THF, ii. 7M ammo-
nia, methanol, (58% over two steps); (d) 1M HCl, dioxane, 74%; (e) H2, 10% Pd/C,
methanol, 81%; (f) acetaldehyde, sodium triacetoxyborohydride, triethylamine,
100%; (g) TBAF, THF, 99%; (h) 7M ammonia, methanol, 99%; (i) 1M HCl, diox-
ane; 77%; (j) formaldehyde, sodium triacetoxyborohydride, triethylamine, 34%; (k)
i. TBAF, THF, ii. 7M ammonia, methanol, (78% over 2 steps); (l) 1M HCl, dioxane,
82%.
The 2ꢀ-amino guanosine was an excellent starting point for the synthesis of
N-alkyl analogs. The azido intermediate 14 could be reduced to the correspond-
ing amine 25 (Scheme 6). Mono alkylation of the amine to the corresponding
NHMe (26) was accomplished using reductive amination chemistry. The corre-
sponding nucleoside 27 was synthesized via global deprotection using TBAF fol-
lowed by treatment with methanolic ammonia. Treatment of 27 with 1M HCl at
50°C provided the desired guanosine analog 28. The corresponding NHEt com-
pound 34 was formed using similar chemistry from compound 29 (29 was syn-
thesized from 12a in 60% yield). The synthesis of the NMe2 guanosine analog
was initiated by the reductive amination of compound 25 in the presence of
excess formaldehyde to provide compound 35. Global deprotection using TBAF
followed by methanolic ammonia gave access to compound 36. Treatment of the
6-OMe nucleoside 36 with 1M HCl at 50°C provided the desired NMe2 guanosine
analog 37.
Results and discussion
With these 2ꢀ-diversified nucleosides in hand, the next step was to evaluate their
HCV inhibitory activity in the 1b genotype replicon assay.[11] If the nucleosides were
successfully converted to the nucleoside triphosphate within the replicon cell, there
could be viral inhibition. While none of the nucleosides demonstrated potency in
the cellular assay (Table 2), it was encouraging that none of the them were cyto-
toxic. The lack of potency in the replicon assay can be explained by the fact that the
Table . Anti-HCV and cytotoxicity of nucleosides.
Nuc.
Base
R
EC (µM)
CC (µM)













-OEt guanosine
-OMe guanosine
guanosine
guanosine
guanosine
-OEt guanosine
-OMe guanosine
-OEt guanosine
-OEt guanosine
guanosine
guanosine
guanosine
N
CN
N
NH
CN
NHMe
NHEt
NMe
triazole
NHMe
NHEt
NMe
triazole
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
guanosine

8
V. GIRIJAVALLABHAN ET AL.
Table . Activities of the corresponding NTPs against HCV NSB polymerase.
Compound
NTPa
R
IC (µM)







guanosine
guanosine
guanosine
guanosine
guanosine
guanosine
guanosine
N
NH
CN
NHMe
NHEt
NMe
triazole
.
.
.
.

>

aPrepared and tested as triethylamine salts.
initial phosphorylation during the step-wise transformation to the NTP is rate lim-
iting as observed with previous 2ꢀ-F guanosines analogs.[12] Therefore, the antiviral
effectiveness of the guanosine nucleosides was evaluated by converting them to the
corresponding NTPs (compounds 38–44) and testing their biochemical inhibitory
activity against the wild-type NS5B polymerase (Table 3).
Several of the guanosine NTPs demonstrated excellent activity suggesting that
there was indeed a rate limiting phosphorylation step. It was also apparent from
these data that sterics and electronics both play a role in the tolerance of substitu-
tion at the 2ꢀ-alpha position of HCV nucleosides. The NTP potency of the azido and
CN analogs (38, 40) suggest that hydrogen bond acceptors/donors are not necessary
at the 2ꢀ-alpha position for inhibitory effect. The NTP data for the amines (39, 41–
43) and triazole (44) also suggest that the success of an inhibitor is driven by ster-
ics. The amine (39) and NHMe (41) appear to be tolerated but dialkylation (43) or
conversion to NHEt (42) leads to a significant loss in potency. The data trend for
this series of analogs will assist in the discovery of novel HCV nucleoside inhibitors.
Conclusions
In summary, a series of nucleosides bearing novel substitutions at the 2ꢀ-alpha posi-
tion were synthesized from the 2ꢀ-exocyclic alkene of the ribose. These nucleosides
demonstrate that a variety of sterically unhindered functionalities with significantly
different electronic properties as well as hydrogen bonding potential are tolerated
at the 2ꢀ-alpha position. The 2ꢀ-azido, amino,cyano and NHMe guanosine nucleo-
sides (17, 18, 21 and 28) are of particular interest due to the intrinsic biochemi-
cal potency of their corresponding NTPs. Although the parent nucleosides did not
demonstrate antiviral activity in the cellular assays, it is possible that monophos-
phate prodrugs such as the one demonstrated in sofosbuvir can be used to bypass
the inefficient initial kinase step. Application of this as well as the use of alterna-
tive pyrimidine and purine bases and other novel 2ꢀ-modifications will be described
elsewhere.
Experimental section
1H-NMR spectra were obtained on 400 MHz Bruker spectrometers. MS analyses
were performed on a Waters micromass ZQ instrument equipped with a Waters

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
Acquity UPLC system. The solvent system used was MeCN/H2O with 0.1% formic
acid. All reagents and solvents were obtained from commercial sources.
Synthesis of compound 13
6,6ꢀ-((1E,1ꢀE)-((2,3-dimethylbutane-2,3-diyl)bis(azanylylidene)) bis(methanylyli-
dene))bis(2,4-di-tert-butylphenol) (733 mg, 1.33 mmol) was taken up in ethanol
(10 mL) and the resulting suspension was heated to 80°C and allowed to stir at this
temperature for 5 minutes. Cobalt (II) acetate (236 mg, 1.33 mmol) was then added
and the resulting reaction was allowed to stir at 80°C for an additional 2 hours.
The reaction was cooled to room temperature using an ice bath and was filtered.
The collected red solid was dried under vacuum to provide compound 13 (579 mg,
72%). MS (ES+) C36H54CoN2O2 requires: 605.3, found: 606.2 (M+H)+. See the
supplementary information section of reference 9 for additional characterization
information.
Synthesis of compound 8
Guanosine hydrate (15 g, 53 mmol) was dissolved in pyridine (120 mL) and acetic
anhydride (60 mL). To the resulting solution was added 4-dimethylamino pyridine
(6.46 g, 53 mmol) and the reaction was heated to 70°C and allowed to stir at this tem-
perature for 3 hours. The reaction was cooled in an ice bath and treated dropwise
with methanol (60 mL). Half of the reaction volume was removed in vacuo and the
reaction was diluted with dichloromethane and washed with 0.2 M potassium dihy-
drogensulfate, water, and saturated sodium bicarbonate. The organic layer was dried
over sodium sulfate, filtered and concentrated in vacuo and the residue obtained
was purified using flash column chromatography on silica gel (5% methanol in
dichloromethane) to provide 22 g of compound 8 (92%). MS (ES+) C18H21N5O9
requires: 451.1, found: 452.2 (M+H)+
Synthesis of compound 9b
Compound 8 (3.2 g, 7.09 mmol) was dissolved in dioxane (50 mL) and treated with
diisopropylazodicarboxylate (1.65 mL, 8.5 mmol) and ethanol (391 mg, 8.5 mmol).
The resulting reaction was allowed to stir for fifteen minutes and then concentrated
in vacuo. The residue obtained was dissolved in methanol (15 mL) and ammonium
hydroxide (15 mL) and stirred for 3 hours to provide a suspension, which was filtered
to provide 1.4 g of compound 9b. The remaining solution was concentrated in vacuo
and the residue obtained was triturated with chloroform to provide an additional
0.8 g of compound 9b (86%). MS (ES+) C14H19N5O6 requires: 353.1, found: 354.2
(M+H)+
Synthesis of compound 10b
Compound 9b (1.46 g, 4.13 mmol) was azeotroped with pyridine (2 × 20 mL)
and then suspended in pyridine (30 mL). The resulting solution was treated with

10
V. GIRIJAVALLABHAN ET AL.
tetraisopropyldisiloxanedichloride (1.43 g, 4.43 mmol) dropwise over fifteen min-
utes and allowed to stir at room temperature for 3 hours. The reaction was diluted
with water (1 mL) and concentrated in vacuo. The residue obtained was diluted
with water (50 mL) and ethyl acetate (50 mL). The organic layer was washed with
brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue
obtained was azeotroped with toluene (2 × 50 mL) and purified using flash column
chromatography on silica gel (5% methanol in dichloromethane) to provide 2.02 g
of product 10b as a white solid (82%). MS (ES+) C26H45N5O7Si2 requires: 595.3,
found: 596.2 (M+H)+.
Synthesis of compound 11b
Compound 10b (0.4 g, 0.671 mmol) was dissolved in dichloromethane (100 mL)
and the resulting solution was cooled in an ice bath and treated with Dess-Martin
Periodinane (0.569 g, 1.34 mmol). The reaction was stirred for 15 hours and then
filtered through a pad of silica and sodium sulfate. The solution was diluted with
diethyl ether (400 mL) and washed with a mixture of saturated sodium bicarbonate
and 10% sodium thiosulfate (1:1). The organic layer was dried over sodium sulfate,
filtered and concentrated in vacuo. The resulting residue was purified using flash
column chromatography on silica gel (hexanes/ethyl acetate 0% →100%) to pro-
vide 310 mg of 11b (78%). MS (ES+) C26H43N5O7Si2 requires: 593.3, found: 594.2
(M+H)+.
Synthesis of compound 12b
Methyltriphenylphosphonium bromide (626 mg, 1.75 mmol) was dissolved in
tetrahydrofuran (15 mL) and the resulting suspension was treated with KHMDS
(0.5 M in tetrahydrofuran, 3.5 mL, 1.75 mmol). The reaction was allowed to stir at
room temperature for 20 minutes and then was cooled in an ice bath. A solution of
compound 11b (260 mg, 0.438 mmol) in tetrahydrofuran (5 mL) was added drop-
wise and the reaction was allowed to warm to room temperature over 4 hours. Satu-
rated ammonium chloride (30 mL) was added and resulting solution was extracted
with ethyl acetate (3 × 20 mL). The organic extracts were washed with brine, dried
over sodium sulfate, filtered and concentrated in vacuo. The residue obtained was
purified using flash column chromatography on silica gel (2:1 hexanes/ethyl acetate)
to provide 170 mg of compound 12b (66%). MS (ES+) C27H45N5O6Si2 requires:
591.3, found: 592.2 (M+H)+.
Synthesis of compound 14
Compound 12b (637 mg, 1.07 mmol) and Compound 13 (19 mg, 0.032 mmol)
were dissolved in tosyl azide (6.0 g, 30.4 mmol) and stirred for 5 minutes at
room temperature. A solution of phenylsilane (151 mg, 1.39 mmol) in ethanol
(3 mL) was added dropwise over 20 minutes and the reaction was stirred for

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
an additional 30 minutes. The reaction was quenched with brine (50 mL) and
extracted with ethyl acetate (2 × 50 mL). The combined organic layers were
washed with brine, dried over sodium sulfate and concentrated in vacuo. Sil-
ica gel column chromatography (3:1 hexanes/ethyl acetate) provided 440 mg of
compound 14 (65%). MS (ES+) C27H46N8O6Si2 requires: 634.3, found: 635.2
(M+H)+.
Synthesis of compound 16
To a solution of compound 14 (80 mg, 0.126 mmol) in tetrahydrofuran (1 mL) was
added tetrabutylammonium fluoride (1.0 M, 0.252 mL). The reaction was allowed
to stir for 2 hours and was then concentrated in vacuo. The resulting residue was
dissolved in 7M ammonia in methanol (3 mL) and allowed to stir at 100°C in a
pressure tube for 15 hours. The reaction mixture was cooled to room tempera-
ture and concentrated in vacuo. The resulting residue was purified using column
chromatography (dichloromethane/methanol 0% to 5%) to provide 43 mg of com-
pound 16 (98%). MS (ES+) C13H18N8O4 requires: 350.1, found: 373.2 (M+Na).
1H-NMR (400 MHz, CD3OD): δ:8.3 (s, 1H), 5.86 (s, 1H), 4.52 (q, 2H, J = 7.1 Hz),
4.49 (d, 1H, J = 9.21 Hz), 4.0 (m, 2H), 3.84 (m, 1H), 1.42 (t, 3H, J = 7.09 Hz),
1.15 (s, 3H).
Synthesis of compound 17
A solution of compound 16 (5 mg, 10.4 µmol) and 1M HCl (0.5 mL) in tetrahy-
drofuran (0.5 mL) was heated to 50°C and allowed to stir at this temperature for
24 hours. The reaction mixture was then concentrated in vacuo and the residue
obtained was purified using flash column chromatography on silica gel (0 to
20% dichloromethane/methanol) to provide 2.1 mg of compound 17 (64%). MS
(ES+) C11H14N8O4 requires: 322.1, found: 345.2 (M+Na)+. 1H-NMR (400 MHz,
CD3OD): δ:8.22 (s, 1H), 5.81 (s, 1H), 4.34 (m, 1H), 4.0 (m, 2H), 3.81 (m, 1H), 1.17
(s, 3H).
Synthesis of compound 18
The azide 17 (30 mg, 0.09 mmol) was dissolved in MeOH (2 mL) and a small portion
of Pd(OH)2 (∼15 mg) was added. To the flask was affixed a balloon of H2 and the
flask was filled and purged 5×, then allowed to stir under H2 for 1 hour. The solution
was filtered over celite and washed with MeOH. The combined MeOH solution was
concentrated in vacuo to give the pure amine product 18 as a white solid (24 mg,
1
91%). MS (ES+) C11H16N6O4 requires: 296.1, found: 319.2 (M+Na)+. H NMR
(400 MHz, CD3OD) δ 8.05 (s, 1H), 6.03 (s, 1H), 4.30 (d, 1H, J = 7.2 Hz), 3.99 (ddd,
1H, J = 7.2, 3.3, 2.7 Hz), 3.92 (dd, 1H, J = 12.5, 2.5 Hz), 3.77 (dd, 1H, J = 12.7,
3.5 Hz), 1.09 (s, 3H).

12
V. GIRIJAVALLABHAN ET AL.
Synthesis of compound 19
Tosyl cyanide (20.3 g, 112 mmol) was added to a solution of compound 12a (2.16 g,
3.74 mmol) and compound 13 (113 mg, 0.19 mmol) in dioxane (7 mL) and the
reaction was stirred for 5 minutes at 20°C. A solution of phenylsilane (485 mg,
4.49 mmol) in ethanol (14 mL) was added dropwise over 30 minutes. The reaction
was stirred for an additional 1 hour and then quenched with brine (100 mL) and
extracted with ethyl acetate (3 × 100 mL). The solids were filtered and ethyl acetate
(200 mL) was added. The combined organic layers were washed with water, brine,
dried over sodium sulfate and concentrated in vacuo. Silica gel column chromatog-
raphy (4:1 hexanes/ethyl acetate → 1:1 hexanes ethyl acetate) provided 560 mg
of compound 19 (25%). MS (ES+) C27H44N6O6Si2 requires: 604.3, found: 605.2
(M+H)+.
Synthesis of compound 20
To a stirred solution of compound 19 (495 mg, 0.82 mmol) in THF (5 mL) was
added 1M TBAF in THF (2.5 mL). The reaction was stirred for 1 hour and then
concentrated under reduced pressure to provide a yellow oil. Silica gel column chro-
matography (5% methanol/DCM → 10% methanol/DCM) provided 290 mg of the
free diol (98%). The material (290 mg, 0.8 mmol) was dissolved in MeOH (5 mL)
and treated with sodium methoxide (0.43 g, 0.8 mmol). The reaction was stirred
at 65 ºC for 1 hour and then concentrated in vacuo. The residue was dissolved in
water (20 mL) and ethyl acetate (20 mL). The aqueous layer was extracted with ethyl
acetate (3 × 20 mL) and the combined organic layers were dried over sodium sulfate
and concentrated to provide 172 mg of compound20 (67%). MS (ES+) C13H16N6O4
1
requires: 320.1, found: 321.1 (M+H)+. H-NMR (400 MHz, CD3OD): δ:8.32 (s,
1H), 6.46(s, 1H), 4.40 (m, 1H), 4.05 (s, 3H), 4.04 (m, 2H), 3.87 (m, 1H), 1.14 (s,
3H).
Synthesis of compound 21
To a stirred solution of compound 20 (110 mg, 0.343 mmol) in THF (6 mL)
was added 1N HCl (6 mL). The solution was heated at 50 ºC for 48 hours and
then the solvent was removed in vacuo. The residue was purified by HPLC (5%
water/acetonitrile) to provide compound 21. (71 mg, 68%). MS (ES+) C12H14N6O4
1
requires: 306.1, found: 307.2 (M+H)+. H-NMR (400 MHz, CD3OD): δ:8.48 (s,
1H), 6.41(s, 1H), 4.33 (m, 1H), 4.05–4.0 (m, 2H), 3.85 (m, 1H), 1.19 (s, 3H).
Synthesis of compound 22
Compound 16 (150 mg, 0.428 mmol), DMAP (5.2 mg, 0.043 mmol), triethylamine
(0.13 mL, 0.94 mmol), and acetic anhydride (0.089 mL, 0.94 mmol) were all dis-
solved in acetonitrile (2 mL) and stirred for 12 hours at room temperature. The reac-
tion was quenched with water (20 mL) and extracted with ethyl acetate (3 × 20 mL).

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
The combined organic layers were dried over sodium sulfate and concentrated in
vacuo. The residue was purified by silica gel column chromatography (0–5% methy-
lene chloride methanol) to provide the di-acetate (171 mg, 66%). The intermediate
was dissolved in vinyl acetate (0.5 mL) and the reaction was stirred at 140°C in a
microwave reactor for 4 hours. The solvent was removed in vacuo and the residue
was purified by silica gel chromatography (methylene chloride/methanol 0–10%)
to provide compound 22 (117 mg, 90%). MS (ES+) C19H24N8O6 requires: 460.2,
found: 461.2 (M+H)+.
Synthesis of compound 23
Compound 22 (38 mg, 0.083 mmol) was dissolved in 7M ammonia in methanol
(2 mL) and stirred at room temperature for 2 hours. The solvent was removed
in vacuo and the residue was purified by silica gel chromatography (0–15%
methanol/DCM) to provide compound 23 (28 mg, 90%). MS (ES+) C15H20N8O4
requires: 376.1, found: 376.98 (M+H)+. 1H-NMR (400 MHz, CD3OD): δ:8.48 (s,
1H), 8.24 (s, 1H), 7.85 (s, 1H), 7.05 (s, 1H), 4.64 (q, 2H, J = 7.0 Hz), 4.52 (d, 1H,
J = 9.1 Hz), 4.0 (m, 2H), 3.84 (m, 1H), 1.42 (t, 3H, J = 7.15 Hz), 1.2 (s, 3H).
Synthesis of compound 24
Compound 23 (50 mg, 0.13 mmol) was dissolved in THF (2 mL) and 1 M HCl (2 mL)
and stirred at 60°C for 24 hours. The solvent was removed in vacuo and the residue
was purified by silica gel chromatography (0–25% methanol/methylene chloride)
to provide compound 24 as a white solid (35 mg, 76%). MS (ES+) C13H16N8O4
1
requires: 348.1, found: 349.2 (M+H)+. H-NMR (400 MHz, DMSO): δ:10.65 (s,
1H), 8.24 (s, 1H), 7.9 (s, 1H), 7.05 (s, 1H), 6.8 (s, 1H). 6.5 (bs, 1H), 5.9 (bs, 1H), 4.44
(m, 1H), 3.85 (m, 1H), 3.71 (m, 1H). 3.65 (m, 1H), 1.2 (s, 3H).
Synthesis of compound 25
Compound 14 (1.2 g, 1.89 mmol) was dissolved in MeOH (10 mL) and
Pd(OH)2(2.0 g) was added. The reaction was stirred for 2.5 hours under an atmo-
sphere of hydrogen. The reaction was filtered over celite and concentrated in vacuo.
The residue was purified by silica gel column chromatography (0 to 10% to 15%
MeOH/DCM) to provide the desired compound 25 (700 mg, 61%). MS (ES+)
C27H48N6O6Si2 requires: 608.3, found: 609.2 (M+H)+.
Synthesis of compounds 26 and 35
Compound 25 (121 mg, 0.2 mmol), triethyl amine (0.11 mL, 0.8 mmol), and
paraformaldehyde (179 mg, 5.96 mmol) were dissolved in dichloroethane (3 mL)
and stirred for 30 minutes. Sodium triacetoxyborohydride (211 mg, 0.99 mmol)
was added and the reaction was stirred for 12 hours. The reaction was quenched

14
V. GIRIJAVALLABHAN ET AL.
with methanol (0.5 mL) and was filtered over celite and concentrated in vacuo. The
residue was purified by silica gel column chromatography (0 → 100% hexanes/ethyl
acetate) to provide compound 26 (53 mg, 43%) and compound 35 (43 mg, 34%).
Compound 26: MS (ES+) C28H50N6O6Si2 requires: 622.3, found: 623.4 (M+H)+.
Compound 35: MS (ES+) C29H52N6O6Si2 requires: 636.3, found: 637.5 (M+H)+.
Synthesis of compound 27
Compound 26 (53.6 mg, 0.086 mmol) was dissolved in THF (1.5 mL) and treated
with 1M TBAF (0.17 mL, 0.17 mmol) and the reaction was stirred for 2 hours. The
solvent was removed in vacuo and the crude residue was treated with 7M ammonia
in methanol (2 mL) and stirred at 80°C in a sealed tube reactor. The solvent was
removed in vacuo and the residue was purified by silica gel column chromatography
(0 15% methanol in DCM) to provide compound 27 as a white solid (17 mg, 58%).
MS (ES+) C14H22N6O4 requires: 338.3, found: 339.2 (M+H)+. 1H-NMR (400 MHz,
CD3OD): δ:8.32 (s, 1H), 6.08 (s, 1H), 4.55 (q, 2H, J = 7.0 Hz), 4.25 (d, 1H, J =
9.1 Hz), 4.0 (m, 2H), 3.84 (m, 1H), 2.5 (s, 3H). 1.42 (t, 3H, J = 7.15 Hz), 0.85 (s, 3H).
Synthesis of compound 28
Compound 27 (15.2 mg, 0.045 mmol) was dissolved in THF (0.5 mL) and 1M HCl
(0.5 mL) and stirred at 60°C for 24 hours. The solvent was removed in vacuo and
the residue was purified by silica gel column chromatography (0 → 35% methanol
in DCM) to provide compound 28 as a white solid (10.3 mg, 74%). MS (ES+)
C12H18N6O4 requires: 310.3, found: 311.3 (M+H)+. 1H-NMR (400 MHz, CD3OD):
δ:8.22 (s, 1H), 6.02 (s, 1H), 4.2 (m, 1H), 4.05–3.95 (m, 2 H), 3.84 (m, 1H), 2.5 (s, 3H).
0.85 (s, 3H).
Synthesis of compound 29
Compound 29 was synthesized from 12a in 60% yield using the same chemistry
outlined for compound 14. The 6-OMe compound 12a was synthesized using the
chemistry outlined for compound 12b except MeOH was used in the Mitsunobu
reaction rather than EtOH.
Synthesis of compound 30
Compound 29 (2.05 g, 3.31 mmol) and 10% Pd/C (410 mg) were suspended in
MeOH (33 mL) and stirred under a hydrogen atmosphere for 12 hours. The reaction
mixture was filtered through Celite and concentrated in vacuo. The residue was puri-
fied by silica gel column chromatography (EtOAc) to provide compound 30 (1.59 g,
81%). MS (ES+) C26H46N6O6Si2 requires: 594.3, found: 595.5 (M+H)+.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
15
Synthesis of compound 31
Compound 30 (100 mg, 0.168 mmol), triethyl amine (0.023 mL, 0.168 mmol), and
acetaldehyde (0.014 mL, 0.252 mmol) were dissolved in dichloroethane (3 mL) and
stirred for 30 minutes. Sodium triacetoxyborohydride (43 mg, 0.2 mmol) was added
the reaction was stirred for 12 hours. The reaction was quenched with methanol
(0.5 mL) and was filtered over celite and concentrated in vacuo. The residue was
purified by silica gel column chromatography (0 → 100% hexanes/ethyl acetate) to
provide compound 31 (105 mg, 100%). MS (ES+) C28H50N6O6Si2 requires: 622.4,
found: 623.6 (M+H)+.
Synthesis of compound 32
Compound 31 (105 mg, 0.169 mmol) was dissolved in THF (3 mL) and treated with
1M TBAF (0.17 mL, 0.17 mmol). The reaction was stirred for 4 hours and then the
solvent was removed in vacuo and the crude residue was purified by silica gel column
chromatography (0 → 15% methanol in DCM) to provide compound 32 as a white
solid (64 mg, 99%). MS (ES+) C16H24N6O5 requires: 380.3, found: 381.3 (M+H)+.
Synthesis of compound 33
Compound 32 (64 mg, 0.168 mmol) was treated with 7M ammonia in methanol
(2 mL) and stirred at 80°C in a sealed tube reactor. The solvent was removed in
vacuo and the residue was purified by silica gel column chromatography (0 → 15%
methanol in DCM) to provide compound 33 as a white solid (56.4 mg, 99%). MS
1
(ES+) C14H22N6O4 requires: 338.3, found: 339.2 (M+H)+. H-NMR (400 MHz,
CD3OD): δ:8.32 (s, 1H), 6.1 (s, 1H), 4.23 (m, 1H), 4.1 (m, 1H), 4.05 (s, 3H), 4.0 (m,
1H), 3.84 (m, 1H), 2.9–2.8 (m, 2 H), 1.2 (t, 3H, J = 7.0 Hz), 0.85 (s, 3H).
Synthesis of compound 34
Compound 33 (53.3 mg, 0.158 mmol) was dissolved in THF (2 mL) and 1M HCl
(2 mL) and stirred at 60°C for 24 hours. The solvent was removed in vacuo and the
residue was purified by silica gel column chromatography (0 → 35% 2N ammonia
methanol in DCM) to provide compound 34 as a white solid (39.3 mg, 77%). MS
1
(ES+) C13H20N6O4 requires: 324.3, found: 325.2 (M+H)+. H-NMR (400 MHz,
CD3OD): δ:8.2 (s, 1H), 6.1 (s, 1H), 4.23 (m, 1H), 4.1 (m, 1 H), 3.95 (m, 1H), 3.8 (m,
1H), 3.05–2.95 (m, 2H). 1.25 (t, 3H, J = 7.0 Hz), 1.0 (s, 3H).
Synthesis of compound 36
Compound 35 (43.6 mg, 0.068 mmol) was dissolved in THF (1.5 mL) and
treated with 1M TBAF (0.17 mL, 0.17 mmol) and the reaction was stirred
for 2 hours. The solvent was removed in vacuo and the crude residue was

16
V. GIRIJAVALLABHAN ET AL.
treated with 7M ammonia in methanol (2 mL) and stirred at 80°C in a sealed
tube reactor. The solvent was removed in vacuo and the residue was purified
by silica gel column chromatography (0 → 15% methanol in DCM) to pro-
vide compound 36 as a white solid (18.8 mg, 77%). MS (ES+) C15H24N6O4
1
requires: 352.3, found: 353.2 (M+H)+. H-NMR (400 MHz, CD3OD): δ:8.2 (s,
1H), 6.22 (s, 1H), 4.55 (q, 2H, J = 7.2 Hz), 4.15 (d, 1H, J = 9.1 Hz), 4.05
(m, 1H), 3.9 (m, 1H), 3.8 (m, 1H), 2.5 (s, 6H), 1.2 (t, 3H, J = 7.0 Hz), 0.85
(s, 3H).
Synthesis of compound 37
Compound 36 (16.3 mg, 0.046 mmol) was dissolved in THF (0.5 mL) and 1M HCl
(0.5 mL) and stirred at 60°C for 24 hours. The solvent was removed in vacuo and the
residue was purified by silica gel column chromatography (0 → 35% 2N ammonia
methanol in DCM) to provide compound 37 as a white solid (12.3 mg, 82%). MS
1
(ES+) C13H20N6O4 requires: 324.3, found: 325.3 (M+H)+. H-NMR (400 MHz,
CD3OD): δ:8.15 (s, 1H), 6.25 (s, 1H), 4.25 (m, 1H), 4.08 (m, 1 H), 3.95 (m, 1H), 3.82
(m, 1H), 2.8 (s, 6H), 1.4 (s, 3H).
Synthesis of nucleoside triphosphates 38–44
Nucleoside triphosphates 38–44 were synthesized as triethylamine salts by Trilink
Biotechnologies using general synthetic methods.[13] All compounds were isolated
as 10 mM solution in water and purity of >95% was confirmed with MS and AX-
HPLC.
Cell-based anti-HCV activity
Replicon cells (1b-Con1) are seeded at 5000 cells/well in 96-well plates one day prior
to inhibitor treatment. Various concentrations of an inhibitor in DMSO are added
to the replicon cells, with the final concentration of DMSO at 0.5% and fetal bovine
serum at 10% in the assay media. Cells are harvested three days post dosing. The
replicon RNA level is determined using real-time RT-PCR (Taqman assay) with
GAPDH RNA as endogenous control. EC50 values are calculated from experiments
with 10 serial twofold dilutions of the inhibitor in triplicate.
To measure cytotoxicity in replicon cells of an inhibitor, an MTS assay is per-
formed according to the manufacturer’s protocol for CellTiter 96 Aqueous One
Solution Cell Proliferation Assay (Promega, Cat # G3580) three days post dosing
on cells treated identically as in replicon activity assays. CC50 is the concentration
of inhibitor that yields 50% inhibition compared to vehicle-treated cells.
Inhibition of HCV NS5B polymerase by nucleoside triphosphate analogs
This assay is a modified version of the assay described in International Publica-
tion No. WO2002/057287. Briefly, 50 µL reactions containing 20 mM HEPES (pH

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
17
7.3); 7.5 mM DTT; 20 units/ml RNasIN; 1 µM each of ATP, GTP, UTP and CTP;
20 µCi/mL [33P]-CTP; 10 mM MgCl; 60 mM NaCl; 100 µg/ml BSA; 0.021 µM
DCoH heteropolymer RNA template; and 5 nM NS5B (1b-BKꢀ55) enzyme are
incubated at room temperature for 1 hour. The assay is then terminated by the addi-
tion of 500 mM EDTA (50 µL). The reaction mixture is transferred to a Millipore
DE81 filter plate and the incorporation of labeled CTP is determined using Packard
TopCount. Compound IC50 values can then be calculated from experiments with
10 serial 3-fold dilutions of the inhibitor in duplicate.
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