2′-Modified Guanosine Analogs for the Treatment of HCV

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 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 evalu-

atedbyconvertingthemtothecorresponding5^ꢀ-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 5^ꢀ-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 2^ꢀ-beta methyl group leads to the inhibition of the

HCV NS5b polymerase.^[4]Since then numerous inhibitors with the 2^ꢀ-beta

methyl scaﬀold such as NM-107^5a(2, Figure 1), PSI-938^5a(3, Figure 1) and

most recently sofosbuvir^5b(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 2^ꢀ-alpha OH or

isosteres such as 2^ꢀ-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 2^ꢀ-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-

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 2^ꢀ-OH/2^ꢀ-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 2^ꢀ-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 3^ꢀ- and 5^ꢀ- alcohols were protected as the 3^ꢀ-5^ꢀ-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 2^ꢀ-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 (T able 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_/EtOH^a

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 TsN₃in 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 2^ꢀ-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 TsN₃increased 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 2^ꢀ-azido guanosine analog

17. The 2^ꢀ-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 2^ꢀ-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 2^ꢀ-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

2^ꢀ-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 2^ꢀ-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 NMe₂guanosine 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 NMe₂guanosine

analog 37.

Results and discussion

With these 2^ꢀ-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 (T able 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

-OEt guanosine

-OMe guanosine

-OEt 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

NTP^a

R

IC_(µM)















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 2^ꢀ-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 (T able 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 2^ꢀ-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 2^ꢀ-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 2^ꢀ-alpha posi-

tion were synthesized from the 2^ꢀ-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 2^ꢀ-alpha position. The 2^ꢀ-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 2^ꢀ-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

6,6^ꢀ-((1E,1^ꢀE)-((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+) C₃₆H₅₄CoN₂O₂requires: 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+) C₁₄H₁₉N₅O₆requires: 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+) C₂₆H₄₅N₅O₇Si₂requires: 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+) C₂₆H₄₃N₅O₇Si₂requires: 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+) C₂₇H₄₅N₅O₆Si₂requires:

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+) C₂₇H₄₆N₈O₆Si₂requires: 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+) C₁₃H₁₈N₈O₄requires: 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+) C₁₁H₁₄N₈O₄requires: 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 H₂and the

ﬂask was ﬁlled and purged 5×, then allowed to stir under H₂for 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+) C₁₁H₁₆N₆O₄requires: 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+) C₂₇H₄₄N₆O₆Si₂requires: 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+) C₁₉H₂₄N₈O₆requires: 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+)

C₂₇H₄₈N₆O₆Si₂requires: 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+) C₂₈H₅₀N₆O₆Si₂requires: 622.3, found: 623.4 (M+H)⁺.

Compound 35: MS (ES+) C₂₉H₅₂N₆O₆Si₂requires: 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+) C₁₄H₂₂N₆O₄requires: 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+)

C₁₂H₁₈N₆O₄requires: 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+) C₂₆H₄₆N₆O₆Si₂requires: 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+) C₂₈H₅₀N₆O₆Si₂requires: 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+) C₁₆H₂₄N₆O₅requires: 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+) C₁₄H₂₂N₆O₄requires: 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+) C₁₃H₂₀N₆O₄requires: 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+) C₁₃H₂₀N₆O₄requires: 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. EC₅₀values 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. CC₅₀is 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 IC₅₀values can then be calculated from experiments with

10 serial 3-fold dilutions of the inhibitor in duplicate.

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Article Doi

2′-Modified Guanosine Analogs for the Treatment of HCV

DOI: 10.1080/15257770.2016.1154968

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