Synthesis of stable isotope labeled analogs of the anti-hepatitis C virus nucleotide prodrugs PSI-7977 and PSI-352938

Article abstract of DOI:10.1080/15257770.2011.614308

In order to support bioanalytical LC/MS method development and plasma sample analysis in preclinical and clinical studies of the anti-hepatitis C-virus nucleotides, PSI-7977 and PSI-352938, the corresponding stable isotope labeled forms were prepared. These labeled compounds were prepared by addition reaction of the freshly prepared Grignard reagent ¹³CD₃MgI to the corresponding 2′-ketone nucleosides followed by fluorination of the resulting carbinol with DAST. As expected, these 2′-C-(trideuterated- ¹³C-methyl) nucleotide prodrugs showed similar anti-HCV activity to that of the corresponding unlabeled ones. Copyright Pharmasset, Inc..

Full text of DOI:10.1080/15257770.2011.614308

This article was downloaded by: [University of York]
On: 07 May 2013, At: 04:58
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Nucleosides, Nucleotides and Nucleic
Acids
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/lncn20
Synthesis of Stable Isotope Labeled
Analogs of the Anti-Hepatitis C Virus
Nucleotide Prodrugs PSI-7977 and
PSI-352938
Byoung-Kwon Chun a , Jinfa Du a , Hai-Ren Zhang a , Wonsuk Chang a
, Bruce S. Ross a , Ying Jiang a , Donghui Bao a , Christine L. Espiritu
a , Meg Keilman a , Holly M. Micolochick Steuer a , Phillip A. Furman a
& Michael J. Sofia a
a Pharmasset, Inc., Princeton, New Jersey, USA
Published online: 07 Nov 2011.
To cite this article: Byoung-Kwon Chun , Jinfa Du , Hai-Ren Zhang , Wonsuk Chang , Bruce S. Ross ,
Ying Jiang , Donghui Bao , Christine L. Espiritu , Meg Keilman , Holly M. Micolochick Steuer , Phillip A.
Furman & Michael J. Sofia (2011): Synthesis of Stable Isotope Labeled Analogs of the Anti-Hepatitis C
Virus Nucleotide Prodrugs PSI-7977 and PSI-352938, Nucleosides, Nucleotides and Nucleic Acids, 30:11,
886-896
To link to this article: http://dx.doi.org/10.1080/15257770.2011.614308
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation
that the contents will be complete or accurate or up to date. The accuracy of any
instructions, formulae, and drug doses should be independently verified with primary
sources. The publisher shall not be liable for any loss, actions, claims, proceedings,
demand, or costs or damages whatsoever or howsoever caused arising directly or
indirectly in connection with or arising out of the use of this material.

Nucleosides, Nucleotides and Nucleic Acids, 30:886–896, 2011
Copyright ꢀC Pharmasset, Inc.
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2011.614308
SYNTHESIS OF STABLE ISOTOPE LABELED ANALOGS
OF THE ANTI-HEPATITIS C VIRUS NUCLEOTIDE PRODRUGS
PSI-7977 AND PSI-352938
Byoung-Kwon Chun, Jinfa Du, Hai-Ren Zhang, Wonsuk Chang,
Bruce S. Ross, Ying Jiang, Donghui Bao, Christine L. Espiritu, Meg Keilman,
Holly M. Micolochick Steuer, Phillip A. Furman, and Michael J. Sofia
Pharmasset, Inc., Princeton, New Jersey, USA
In order to support bioanalytical LC/MS method development and plasma sample analysis in
2
preclinical and clinical studies of the anti-hepatitis C-virus nucleotides, PSI-7977 and PSI-352938,
the corresponding stable isotope labeled forms were prepared. These labeled compounds were prepared
by addition reaction of the freshly prepared Grignard reagent 13CD3MgI to the corresponding 2 ꢁ-
ketone nucleosides followed by fluorination of the resulting carbinol with DAST. As expected, these
2 ꢁ-C-(trideuterated-13C-methyl) nucleotide prodrugs showed similar anti-HCV activity to that of the
corresponding unlabeled ones.
Keywords PSI-7851; PSI-7977; PSI-352938; stable isotope label; hepatitis C virus; DAST;
fluorination; 13CD3MgI
INTRODUCTION
β-D-2ꢁ-Deoxy-2ꢁ-α-fluoro-2ꢁ-β-C-methyl nucleotides, PSI-7851 (1a),[1,2]
PSI-7977 (1b),[1] and PSI-352938 (2)[3,4] have demonstrated potent anti-
HCV activity. Currently, PSI-7977 (1b), a single diastereomer of PSI-7851
(1a), and PSI-352938 (2) are in Phase II clinical development. In order to
support bioanalytical quantitative LC/MS analysis of plasma samples from
preclinical and clinical studies of these agents, it was necessary to prepare sta-
ble isotope labeled versions to be used as internal standards. These standards
were required to have similar physicochemical characteristics to those of the
nucleotides of interest and have a molecular weight different enough so as
not to overlap with the analytes of interest (PSI-7977 or PSI-352938) in quan-
titative mass spectroscopic analysis. In order to fulfill these requirements,
preparation of nucleotides 3 and 4 each having a 4 amu difference relative
Received 17 June 2011; accepted 10 August 2011.
Address correspondence to Byoung-Kwon Chun, Ph.D. Pharmasset, Inc., 303A College Road East,
Princeton, NJ 08540, USA. E-mail: kwon.chun@pharmasset.com
886

Stable Isotope Labeled Anti-HCV Nucleotide Prodrugs
887
FIGURE 1 Structures of the nucleotide prodrugs and their labeled versions.
to their parent drugs, PSI-7977 and PSI-352938, was undertaken. We de-
cided to label the 2ꢁ-methyl group using Grignard reaction with 13CD3MgBr
because of the easy synthesis using the same chemistry developed for the un-
labeled versions.[5,6] Otherwise, the labeling would have involved additional
difficult multistep reactions to introduce 4 amu as shown in the reported ex-
amples.[7–10] Accordingly, compounds 3 and 4 each replaced the 2ꢁ-C-methyl
group of the parent prodrugs 1a and 2 with a 13CD3 group.
RESULTS AND DISCUSSION
In order to prepare the target nucleotide prodrug 3, 3ꢁ,5ꢁ-TIPDS-
protected uridine (5) was readily prepared from uridine and then subjected
to Swern oxidation to afford 2ꢁ-ketone nucleoside 6 in 97% yield.[5] The
labeled Grignard reagent was prepared by reacting commercially available
13CD3I with magnesium metal in dry ether. The freshly prepared 13CD3MgI
was then reacted with ketone 6 at low temperature to give carbinol 7 as
the major product in 46% yield. A large excess (ca 5 eq) of 13CD3MgI
was required to force the reaction to completion. Compound 7 was then
deprotected with ammonium fluoride and reprotected with acetic anhy-
dride to give diacetate 8 in 68% yield. Fluorination of compound 8 was
effected by treating with DAST[5] to afford 2-α- fluorinated compound 9 in
74% yield, which was then deacetylated with n-BuNH2 to give compound
10 in 85% yield. Finally, stable isotope labeled phosphoramidate 3 was

888
B.-K. Chun et al.
SCHEME 1 Synthesis of the labeled phosphoramidate nucleotide prodrug.
prepared as a Sp/Rp diastereomeric mixture by reacting 10 with (2S)-
isopropyl-[(chloro(phenoxy)phosphoryl)amino]-propanoate in the pres-
ence of N -methylimidazole in 52% yield (Scheme 1).[1] Mass spectroscopic
analysis of compound 3 showed greater than 99% isotopic purity and molec-
ular weight increase by 4 amu as expected.
In order to prepare labeled purine nucleotide 4, protected purine nucle-
oside 11[6] was further protected at the 2-amino group through a transient
protection of the 2ꢁ-hydroxyl with TMSCl followed by acetylation with Ac2O,
and then selective hydrolysis of the 2ꢁ-O-TMS by treatment with TsOH to pro-
vide the N 2-acetyl-nucleoside 13 in 57% yield. The oxidation of compound
13 was best accomplished by CrO3/Py/Ac2O to give 2ꢁ-ketone nucleoside
14 in 85% yield. In order to prepare the labeled carbinol intermediate, ke-
tone 14 was treated with 13CD3MgI to give the addition product in low yield.
However, treatment with 13CD3MgI/AlCl3 gave an improved yield, probably
due to in situ production of more reactive Al(13CD3)3.[11] Desilylation of
the carbinol intermediate with TBAF followed by reprotection with Ac2O
gave compound 15 in 30% yield. Fluorination of 15 with DAST afforded
2ꢁ-a-fluorinated compound 16 in 31% yield,[5,6] which, in turn, was treated
with NaOEt to give labeled 6-OEt-nucleoside 17 in 81% yield. Treatment of
compound 17 with 1-chloro-N,N,N’,N’-tetraisopropyl-phosphinediamine in
the presence of 4,5-dicyanoimidazole formed a cyclic phosphite triester that

Stable Isotope Labeled Anti-HCV Nucleotide Prodrugs
889
SCHEME 2 Synthesis of the labeled cyclic phosphate nucleotide prodrug.
was then directly oxidized with iodine to afford cyclic phosphate triester tar-
get compound 4 in 34% yield as a single Rp diastereomer as determined by
31P- and 1H-NMR comparison with the unlabeled parent structure (Scheme
2).[3,12] Mass spectroscopic analysis of compound 4 showed greater than 99%
isotopic purity and molecular weight increase by 4 amu as expected.
Although it was not a main objective to evaluate labeled compounds 3
and 4 for antiviral activity, we were interested in evaluating whether labeling
affects anti-HCV activity of the 2ꢁ-fluoro-C-methyl class of nucleosides. In fact,
the anti-HCV activity of labeled nucleotides 3 and 4 was not significantly
different in an in vitro replicon assay[2] from that of their corresponding
unlabeled clinical agents 1a and 2 (Table 1).
In summary, we successfully prepared stable isotope labeled nucleotides
3 and 4 through 2ꢁ-ketone nucleoside intermediates using freshly prepared
Grignard reagent 13CD3MgI and evaluated their anti-HCV activity. These
products were used as internal standards to support the quantitative mass
TABLE 1 Anti-HCV activitya: unlabeled versus labeled
Phosphoramidate
Cyclic phosphate
Unlabeled
Labeled
Unlabeled
Labeled
Compound
la
3
2
4
EC50
EC90
CC50
0.17 µM
0.73 µM
>100 µM
0.14 µM
0.27 µM
>100 µM
0.07 µM
0.26 µM
>100 µM
0.03 µM
0.22 µM
>50 µM
aAntiviral activity was indirectly measured through luciferase activity in a replicon system and shown as
an average value of three tests for the unlabeled compounds and a result of a single test for the labeled.[2]

890
B.-K. Chun et al.
spectroscopic analysis of samples for preclinical and clinical studies of PSI-
7851 (1a), PSI-7977 (1b), and PSI-352938 (2).
EXPERIMENTAL
General
Nuclear magnetic resonance spectra were recorded on a Varian AS 400
spectrometer at room temperature with tetramethylsilane as an internal
standard. Chemical shifts (δ) are reported in parts per million (ppm), and
signals are reported as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), or br s (broad singlet). Values given for coupling constants
are first order. UV spectra were recorded on a Spectronic Genesis 10 Bio
from Thermo Eclectron Corporation. Electron Spray Mass Spectrometry
was recorded on Acquity Ultra Performance LC from Waters. Flash silica gel
chromatography was performed on Intelli Flash 280 from AnaLogix.
[3-13C]-1-((2R,3S,4R,5R)-3,4-Dihydroxy-5-(Hydroxymethyl)-3-
Trideu-teromethyltetrahydrofuran-2-yl)Pyrimidine-2,4(1H,3H)-
Dione (7)
To a dry three-necked round flask were added anhydrous CH2Cl2
(600 mL) and DMSO (30.82 g, 394.5 mmol). The solution was cooled to
−78◦C under an atmosphere of nitrogen. Trifluoroacetic anhydride (77.7 g,
369.8 mmol) was added via a syringe over 40 minutes to give a cloudy mix-
ture. A solution of the protected uridine (5) (38.30 g, 78.70 mmol) in CH2Cl2
(600 mL) was added dropwise over 75 minutes at −78◦C via an addition fun-
nel. The resulting heterogeneous mixture was stirred for 2 hours at −78 to
−65◦C and then dry triethylamine (92 mL) was added via a syringe quickly
to form a clear light yellow solution. After 1 hour at that temperature, the
cooling bath was removed and the reaction mixture was warmed up slowly
to room temperature over 1 hour. The reaction was quenched by addition
of saturated NH4Cl (180 mL). Water (200 mL) was added and the organic
layer was separated. The aqueous layer was extracted with CH2Cl2 (300 mL).
The combined organic layer was washed with water (3 × 400 mL), brine
(150 mL), and dried over Na2SO4. Removal of solvent afforded a sticky
brown residue. The crude oil residue was stored overnight in the freezer.
After overnight storage, some crystal solid was observed in the oil. The oil was
dissolved in 500 mL hexanes at room temperature. The solution was stored
in the freezer for 24 hours, resulting in the formation of a solid. Solid (6)
was collected via filtration and rinsed with cold 10% CH2Cl2 (1 L) in hex-
anes to remove most of the orange color. The filtrate was concentrated and
the residue was purified by flash silica gel column chromatography (EtOAc
10%–70% in hexanes) to afford an additional amount of compound 6 as a

Stable Isotope Labeled Anti-HCV Nucleotide Prodrugs
891
light orange solid. The combined solid was dried under vacuum at 50◦C for
15 hours before use in the next reaction (37 g, 97%).
In order to prepare the Grignard reagent, magnesium metal (3.53 g,
160.45 mmol) was washed with d-HCl and dried in a hot oven, put into an
argon-filled two-neck round-bottomed flask equipped with a magnetic stirrer
and a condenser; then anhydrous ether (80 mL) was added. To the magne-
sium in ether was added trideuterated [13C]-methyliodide (15.06 g, 103.16
mmol) slowly, which generated an exothermic reaction. After the reaction
mixture cooled, the supernatant was transferred to a solution of compound
6 (10.0 g, 20.63 mmol) in anhydrous THF (1 L) at −50◦C over 20 minutes.
The temperature was allowed to rise to −20◦C and the mixture was stirred
at −20◦C for 4 hours. Upon completion of the reaction, the mixture was
diluted with EtOAc (1 L) and brine (300 mL) was added slowly with stirring.
Then, the organic layer was separated, washed with ammonium chloride
solution (300 mL × 2), and dried with sodium sulfate. After filtration and
concentration, the residue was dissolved in MeOH (250 mL) to give a so-
lution to which ammonium fluoride (12 g, 323.97 mmol) and TBAF (400
mg, 1.53 mmol) were added. The resulting mixture was stirred at 90◦C for
7 hours and concentrated in vacuo and purified by silica gel column chro-
matography (MeOH:CH2Cl2 = 1:20 to 1:10) to give compound 7 (5 g, 46%)
1
as a white solid. H NMR (DMSO-d6) δ (ppm) 11.26 (s, 1H, NH), 7.65 (d,
1H, J = 8.4 Hz), 5.77 (d, 1H, J = 2.4 Hz,), 5.57 (d, 1H, J = 8.0 Hz), 5.46 (d,
1H, J = 5.2 Hz), 5.24 (d, 1H, J = 2.4 Hz), 5.14 (t, 1H, J = 5.6 Hz), 3.74–3.56
(m, 4H).
[4-13C]-((2R,3R,4R,5R)-3-Acetoxy-5-(2,4-Dioxo-3,4-Dihydropyrimidin-
1(2H)-yl)-4-Fluoro-4-Trideuteromethyltetrahydrofuran-2-yl)Methyl
Acetate (9)
To a solution of compound 7 (5.0 g, 19.07 mmol) in anhydrous pyridine
(100 mL) was added acetic anhydride (3 mL) at room temperature. The
resulting mixture was stirred at room temperature for 15 hours, diluted with
EtOAc (250 mL), washed with water (50 mL × 3), and dried with sodium
sulfate. After filtration and concentration, the residue was purified by flash
column chromatography (MeOH 0%–5% in CH2Cl2) to give compound 8
(4.0 g, 68%) as a gray solid. To a solution of compound 8 (2.33 g, 6.73 mmol)
in anhydrous CH2Cl2 (60 mL) was added DAST (1.33 mL, 10.09 mmol) at
−78◦C slowly. The resulting mixture was stirred for 30 minutes after being
exposed to room temperature, diluted with CH2Cl2 (100 mL), washed with
ice water (30 mL × 2) and then cold aqueous NaHCO3 solution (30 mL ×
2), and finally, dried with sodium sulfate. Upon filtration and concentration,
the residue was purified by flash silica gel column chromatography (EtOAc
0%–50% in hexanes) to give compound 9 (580 mg, 24%) as a white solid.
1H NMR (CDCl3) δ (ppm) 8.27 (s, 1H), 7.55 (d, 1H, J = 8.4 Hz), 6.17 (d,

892
B.-K. Chun et al.
1H, J = 18.8 Hz), 5.78 (dd, 1H, J = 1.2, 8.4 Hz), 5.12 (dd, 1H, J = 9.6, 21.6
Hz), 4.40–4.31 (m, 3H), 2.19 (s, 3H), 2.15 (s, 3H).
[3-13C]-1-((2R,3R,4R,5R)-3-Fluoro-4-Hydroxy-5-(Hydroxymethyl)-3-
Trideuteromethyltetrahydrofuran-2-yl)Pyrimidine-2,4(1H,3H)-
Dione (10)
To a solution of compound 9 (2.0 g, 5.74 mmol) in methanol (20 mL)
was added n-butylamine (6 mL). The resulting mixture was stirred at room
temperature for 15 hours and concentrated in vacuo. The obtained residue
was purified by flash silica gel column chromatography (MeOH 0%–10%
1
in CH2Cl2) to give compound 10 (1.3 g, 85%) as a white solid. H NMR
(CD3OD) δ (ppm) 8.08 (d, 1H, J = 8.0 Hz), 6.13 (d, 1H, J = 18.4 Hz), 5.70
(d, 1H, J = 8.0 Hz), 3.99 (d, 1H, J = 13.6 Hz), 3.97–3.91 (m, 2H), 3.80 (dd,
1H, J = 2.0, 12.8 Hz), LRMS (ESI) [M + H] calculated for C913CH11D3FN2O5
265, found 265.
[4-13C]-(2S)-Isopropyl 2-(((((2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-
Dihydropyrimidin-1(2H)-yl)-4-Fluoro-3-Hydroxy-4-
Trideuteromethyltetrahydrofuran-2-yl)Methoxy)
(Phenoxy)Phosphoryl)Amino)Propanoate (3)
To a solution of compound 10 (207 mg, 0.783 mmol) and N -
methylimidazole (0.4 mL, 5.0 mmol) in THF (4 mL) was added dropwise
(2S)-isopropyl-[(chloro(phenoxy)phosphoryl)amino]-propanoate in THF
(1.0 M, 2.35 ml, 2.35 mmol) at 0◦C. The reaction was slowly warmed to
room temperature over 1 hour, and then, water (1 mL) and EtOAc (5
mL) were added. The organic solution was washed with saturated aqueous
sodium citrate mono basic (2 × 2 mL), saturated aqueous NaHCO3 (1 ×
2 mL), dried over magnesium sulfate, and concentrated in vacuo. The
residue obtained was purified by flash silica gel column chromatography
(iPrOH 0–5% in CH2Cl2) to give compound 3 (216 mg, 52%, 1:1 mixture
1
of P-diastereomers) as a white solid: H NMR (DMSO-d6) δ 11.54 (s, 1H),
7.56 (d, 1H, J = 6.8 Hz), 7.40–7.35 (m, 2H), 7.23–7.18 (m, 3 H), 6.14–5.96
(m, 2H), 5.89 (dd, 1H, J = 5.6, 25.6 Hz), 5.55 (t, 1H, J = 8.4 Hz), 4.85 (dq,
1H, J = 1.6, 6.0 Hz), 4.44–4.32 (m, 1H), 4.25 (m, 1H), 4.06–3.98 (m, 1H),
3.86–3.70 (m, 2H), 1.30–1.08 (m, 9H); 13C NMR (CDCl3) δ 173.3, 163.1,
150.6, 139.5, 130.2, 125.6, 120.0, 103.2, 100.6, 89.5, 80.0, 77.5, 71.8, 70.0,
64.2, 50.1, 21.8, 21.0, 15.9; 31P NMR (DMSO-d6) δ 4.90, 4.77; HRMS (ESI)
[M + H]+ calculated for C2113CH27D3FN3O9P 534.1925, found 534.1924.

Stable Isotope Labeled Anti-HCV Nucleotide Prodrugs
893
N-(6-Chloro-9-((6aR,8R,9R,9aR)-2,2,4,4-Tetraisopropyl-9-
((Trimethylsilyl)Oxy)Tetrahydro-6H-Furo[3,2-f][1,3,5,2,4]
Trioxadisilocin-8-yl)-9H-Purin-2-yl)Acetamide (12)
To a solution of compound 11 (5.70 g, 10.5 mmol) in pyridine (10 mL)
and CH2Cl2 (90 mL) was added TMSCl (3.42 g, 31.5 mmol) at 0◦C. The
reaction mixture was stirred at 0◦C for 30 minutes and Ac2O (3.21 g, 31.5
mmol) was added. The resulting mixture was then stirred at 0◦C for 1 hour
and then at room temperature for 2 hours. EtOAc (200 mL) was added
and the mixture was washed with brine and dried over Na2SO4. Solvent was
evaporated in vacuo and the residue was purified by flash silica gel column
chromatography (EtOAc 10%–60% in hexanes) to give compound 12 (5.0 g,
72%). 1H NMR (CDCl3) δ 8.45 (s, 1H), 7.95 (s, 1H), 5.93 (s, 1H), 4.00–4.41
(m, 5H), 2.60 (s, 3H), 0.91–1.11 (m, 28H), 0.21 (s, 9H).
N-(6-Chloro-9-((6aR,8R,9R,9aS)-9-Hydroxy-2,2,4,4-
Tetraisopropyltetrahydro-6H-Furo[3,2-f][1,3,5,2,4]
Trioxadisilocin-8-yl)-9H-Purin-2-yl)Acetamide (13)
To a solution of 12 (5.0 g, 7.6 mmol) in THF (100 mL) was added
TsOH.H2O (1 g) and the solution was stirred at room temperature for
3 hours and neutralized with triethylamine. Solvent was evaporated and
the residue was purified by flash silica gel column chromatography (EtOAc
10%–80% in hexanes) to give product 13 (3.5 g, 79%). 1H NMR (CDCl3) δ
8.21 (d, 1H, J = 1.2 Hz), 8.00, 8.02 (ds, 1H), 6.00 (s, 1H), 4.68 (t, 1H, J =
7.2 Hz), 4.45 (d, 1H, J = 4.8 Hz), 4.05–4.19 (m, 3H), 3.17 (s, 1H), 2.54 (s,
3H), 1.03–1.10 (m, 28H).
N-(6-Chloro-9-((6aR,8R,9aR)-2,2,4,4-Tetraisopropyl-9-
Oxotetrahydro-6H-Furo[3,2-f][1,3,5,2,4]
Trioxadisilocin-8-yl)-9H-Purin-2-yl)Acetamide (14)
To a mixture of CrO3 (2.2 g, 21.99 mmol) in CH2Cl2 (30 mL) were
sequentially added pyridine (1.74 g, 22.0 mmol), Ac2O (2.24 g, 22 mmol),
and then a solution of 13 (4.3 g, 7.33 mmol) in CH2Cl2 (10 mL). The
resulting mixture was stirred at room temperature for 30 minutes and EtOAc
(200 mL) was added slowly. After stirring at room temperature for 1 hour,
the solid generated was filtered off through a silica gel pad. The filtrate was
concentrated to dryness and the residue was co-evaporated with toluene.
EtOAc (100 mL) was added to the residue, which was then filtered through
a silica gel pad. The filtrate was evaporated and the residue co-evaporated
with toluene to give a white solid. The residue was purified by flash silica gel
column chromatography (EtOAc 5 to 80% in hexane) to give compound
14 as a syrup, which was co-evaporated with toluene (2 × 50 mL) and dried
under vacuum overnight before use (3.65 g, 85%). 1H NMR (CDCl3) δ 8.00

894
B.-K. Chun et al.
(s, 1H), 7.86 (s, 1H), 5.77 (s, 1H), 5.25 (d, 1H, J = 9.2 Hz), 4.07–4.28 (m,
3H), 2.41 (s, 3H), 1.06–1.16 (m, 28H).
[4-13C]-(2R,3R,4S,5R)-5-(2-Acetamido-6-Chloro-9H-Purin-9-yl)-2-
(Acetoxymethyl)-4-Hydroxy-4-Trideuteromethyltetrahydrofuran-
3-yl Acetate (15)
To a mixture of magnesium metal (1.68 g, 69.14 mmol) in dry ethyl ether
(40 mL) was added 1/3 of 13CD3I (8.70 g, 60.00 mmol) and the mixture was
stirred at room temperature until reflux. To the mixture was added the rest
of 13CD3I at the rate that kept the reaction under gentle reflux. After being
cooled down to room temperature, the solution was transferred to another
flask in an ice bath, to which was added AlCl3 (2.67 g, 20.00 mmol). The
resulting mixture was stirred at 0◦C for 1 hour and concentrated at room
temperature. The obtained syrup was then dissolved in CH2Cl2 (100 mL) to
give a solution, to which was added a solution of compound 14 (5.84 g, 10.00
mmol) in CH2Cl2 (30 mL) slowly at 0◦C. The mixture was stirred at 0◦C for
3 hours and after adding saturated NH4Cl (10 mL) and EtOAc (300 mL),
the mixture was stirred at room temperature for 10 minutes. After removal
of solid by filtration, the filtrate was washed with brine, dried over Na2SO4,
and concentrated in vacuo. The residue obtained was dissolved in THF
(100 mL) and 1 M TBAF in THF (30 mL, 30 mmol) was added. The resulting
mixture was stirred at room temperature for 2 hours, concentrated in vacuo,
and purified by flash silica gel column chromatography (MeOH 0%–15%
in CH2Cl2) to give a labeled nucleoside intermediate, which was dissolved
in pyridine–CH2Cl2 (30:100 mL). After addition of Ac2O (3 mL, excess),
the resulting mixture was stirred at 0◦C for 16 hours. After adding water
(10 mL), the mixture was stirred at room temperature for 10 minutes, diluted
with EtOAc (200 mL), washed with water and brine, and dried over Na2SO4.
Solvent was evaporated in vacuo and the residue was purified by flash silica
gel column chromatography (MeOH 0%–8% in CH2Cl2) to give compound
15 as a white solid (1.33 g, 29.9%). 1H NMR (CDCl3) δ 8.28 (s, 1H), 8.10 (s,
1H), 6.03 (d, 1H, J = 2.4 Hz), 5.22 (d, 1H, J = 3.2 Hz), 4.50 (m, 2H), 4.24
(m, 1H), 4.00 (s, 1H), 2.45 (s, 3H), 2.20 (s, 3H), 2.12 (s, 3H).
[4-13C]-(2R,3R,4R,5R)-5-(2-Acetamido-6-Chloro-9H-Purin-9-yl)-2-
(Acetoxymethyl)-4-Fluoro-4-Trideuteromethyltetrahydrofuran-3-yl
Acetate (16)
To a solution of 15 (0.68 g, 1.53 mmol) in CH2Cl2 (68 mL) precooled
at −78◦C was added DAST (0.74 g, 4.59 mmol) over 20 minutes and the
resulting mixture was stirred at the same temperature for 30 minutes and
then at room temperature for 1 hour. EtOAc (200 mL) was added and the
mixture was washed with aq. NaHCO3 and brine, and dried over Na2SO4.

Stable Isotope Labeled Anti-HCV Nucleotide Prodrugs
895
Solvent was evaporated and the residue was purified by flash silica gel column
chromatography (EtOAc 0%–90% in hexanes) to give compound 16 (0.21
g, 30.6%) as a white solid. 1H NMR (CDCl3) δ 8.26 (s, 1H), 8.11 (s, 1H), 6.23
(d, 1H, J = 17.2 Hz), 5.75 (m, 1H), 4.39, 4.56 (m, 3H), 2.49 (s, 3H), 2.21 (s,
3H), 2.17 (s, 3H).
[3-13C]-N-(6-Ethoxy-9-((2R,3R,4R,5R)-3-Fluoro-4-Hydroxy-5-
(Hydroxymethyl)-3-Trideuteromethyltetrahydrofuran-2-yl)-
9H-Purin-2-yl)Acetamide (17)
To the solution of 16 (0.35 g, 0.78 mmol) in EtOH (36 mL) was added
21% NaOEt in EtOH (3.1 mL, 7.83 mmol). The resulting mixture was stirred
at room temperature for 20 hours and neutralized with AcOH. Solvent was
evaporated and the residue was purified by flash silica gel column chro-
matography (MeOH 0%–10% in CH2Cl2) to give compound 17 (0.21 g,
1
81%). H NMR (CD3OD) δ 8.22 (s, 1H), 6.15 (d, 1H, J = 18.0 Hz), 4.52
(q, 2H, J = 6.8 Hz), 4.38 (dd, 1H, J = 5.8, 23.6 Hz), 4.03 (m, 2H), 3.86
(m, 1H), 1.42 (t, 3H, J = 7.20 Hz). LRMS (ESI) [M + H+] calculated for
C1213CH16D3FN5O4 332, found 332.
[7-13C]-(4aR,6R,7R,7aR)-6-(2-Amino-6-Ethoxy-9H-Purin-9-yl)-7-
Fluoro-2-Isopropoxy-7-Trideuteromethyltetrahydro-4H-
Furo[3,2-d][1,3,2]Dioxaphosphinine-2-Oxide (4)
To a dry 50 mL round-bottomed flask was charged compound 17
(205 mg, 0.62 mmol). Anhydrous aceotonitrile (4 mL) was added and the
suspension was cooled in an ice bath. To the mixture were added 4,5-
dicyanoimidazole (183 mg, 1.52 mmol) and isopropyl tetraisopropylphos-
phorodiamidite (209 mg, 0.72 mmol). The resulting clear solution was then
heated at 45◦C for 6 hours, concentrated, and dissolved in ethyl acetate
(20 mL). White solid was removed by filtration and the filtrate was concen-
trated and cooled in an ice bath. A solution of iodine (ca. 0.1 M in a mixture
of THF (2 mL), pyridine (2 mL), and water (4 mL)) was added and the
brown solution was stirred at room temperature for 5 minutes. An aqueous
solution of sodium thiosulfate (10%) was added dropwise until a light brown
solution was formed. After removal of solvents, the residue was dissolved in
ethyl acetate (20 mL) and solid was removed by filtration. The filtrate was
washed with saturated sodium bicarbonate (2 × 15 mL) and brine (5 mL),
and dried over Na2SO4. Solvent was removed in vacuo and the residue ob-
tained was purified by flash silica gel column chromatography (40% EtOAc
1
in hexanes) to afford product 4 (98.7 mg, 34%) as a white solid. H NMR
(CDCl3) δ 7.59 (s, 1 H), 6.01 (d, 1 H, J = 18.0 Hz), 5.41 (br, 1H), 4.88
(s, 2 H), 4.85 (m, 1H), 4.64–4.46 (m, 4H), 4.38–4.32 (m, 1H), 1.45–1.41
(m, 9 H); 13C NMR (CDCl3) δ 161.8, 159.2, 152.6, 138.0, 117.3, 92.6, 79.8,

896
B.-K. Chun et al.
77.5, 73.8, 70.5, 69.5, 63.1, 23.9, 23.5, 15.3, 14.5; 31P NMR (CDCl3): δ −5.96;
HRMS (ESI) [M + H]+ calculated for C1513CH21D3FN5O6P 436.1670, found
436.1666.
REFERENCES
1. Sofia, M.J.; Bao, D.; Chang, W.; Du, J.; Nagarathnam, D.; Rachakonda, S.; Reddy, P.G.; Ross, B.S.;
Wang, P.; Zhang, H.R.; Bansal, S.; Espiritu, C.; Keilman, M.; Lam, A.M.; Steuer, H.M.; Niu, C.; Otto,
M.J.; Furman, P.A. Discovery of a β-D-2ꢁ-deoxy-2ꢁ-α-fluoro-2ꢁ-β-C-methyluridine nucleotide prodrug
(PSI-7977) for the treatment of hepatitis C virus. J. Med. Chem. 2010, 53(19), 7202–7218.
2. Lam, A.M.; Murakami, E.; Espiritu, C.; Steuer, H.M.; Niu, C.; Keilman, M.; Bao, H.; Zennou, V.;
Bourne, N.; Julander, J.G.; Morrey, J.D.; Smee, D.F.; Frick, D.N.; Heck, J.A.; Wang, P.; Nagarathnam,
D.; Ross, B.S.; Sofia, M.J.; Otto, M.J.; Furman, P.A. PSI-7851, a pronucleotide of β-D-2ꢁ-deoxy-2ꢁ-
fluoro-2ꢁ-C-methyluridine monophosphate, is a potent and pan-genotype inhibitor of hepatitis C
virus replication. Antimicro. Agents Chemother. 2010, 52(8), 3187–3196.
3. Reddy, P.G.; Bao, D.; Chang, W.; Chun, B.-K.; Du, J.; Nagarathnam, D.; Rachakonda, S.; Ross, B.S.;
Zhang, H.-R.; Bansal, S.; Espiritu, C.L.; Keilman, M.; Lam, A.M.; Niu, C.; Steuer, H.M.; Furman, P.A.;
Otto, M.J.; Sofia, M.J. 2ꢁ-Deoxy-2ꢁ-α-fluoro-2ꢁ-β-C-methyl 3ꢁ,5ꢁ-cyclic phosphate nucleotide prodrug
analogs as inhibitors of HCV NS5B polymerase: discovery of PSI-352938. Bioorg. Med. Chem. Lett. 2010,
20(24), 7376–7380.
4. Lam, A.M.; Espiritu, C.; Murakami, E.; Zennou, V.; Bansal, S.; Micolochick Steuer, H.M.; Niu, C.;
Keilman, M.; Bao, H.; Bourne, N.; Veselenak, R.L.; Reddy, P.G.; Chang, W.; Du, J.; Nagarathnam, D.;
Sofia, M.J.; Otto, M.J.; Furman, P.A. Inhibition of hepatitis C Virus replicon RNA synthesis by PSI-
352938, a cyclic phosphate prodrug of β-D-2ꢁ-deoxy-2ꢁ-α-fluoro-2ꢁ-β-C-methylguanosine. Antimicro.
Agents Chemother. 2011, 55(6), 2566–2575.
5. Clark, J.L.; Hollecker, L.; Mason, J.C.; Stuyver, L.J.; Tharnish, P.M.; Lostia, S.; McBrayer, T.R.;
Schinazi, R.F.; Watanabe, K.A.; Otto, M.J.; Furman, P.A.; Stec, W.J.; Patterson, S.E.; Pankiewicz, K.W.
Design, synthesis, and antiviral activity of 2ꢁ-deoxy-2ꢁ-fluoro-2ꢁ-C-methylcytidine, a potent inhibitor
of hepatitis C virus replication. J. Med. Chem. 2005, 48(17), 5504–5508.
6. Clark, J.L.; Mason, J.C.; Hollecker, L.; Stuyver, L.J.; Tharnish, P.M.; McBrayer, T.R.; Otto, M.J.;
Furman, P.A.; Schinazi, R.F.; Watanabe, K.A. Synthesis and antiviral activity of 2ꢁ-deoxy-2ꢁ-fluoro-2ꢁ-
C-methyl purine nucleosides as inhibitors of hepatitis C virus RNA replication. Bioorg. Med. Chem.
Lett. 2006, 16(6), 1712–1715.
7. Saito, Y.; Nyilas, A.; Zevaco, T.A.: Agrofoglio, L.A. Chemical synthesis of 13C labeled anti-HIV nucle-
osides as mass-internal standards. Tetrahedron 2002, 58(47), 9593–9603.
8. Fo¨ldesi, A.; Trifonova, A.; Kundu, M.K.; Chattopadhyaya, J. The synthesis of deuterionucleosides.
Nucleos. Nucleot. Nucleic Acids 2000, 19(10–12), 1615–1656.
9. Ouwerkerk, N.; Steenweg, M.; de Ruijter, M.; Brouwer, J.; van Boom, J.H.; Lugtenburg, J.; Raap,
J. One-pot two-step enzymatic coupling of pyrimidine bases to 2-deoxy-d-ribose-5-phosphate. A
new strategy in the synthesis of stable isotope labeled deoxynucleosides. J. Org. Chem. 2002, 67(5),
1480–1489.
10. Shallop, A.J.; Gaffney, B.L.; Jones, R.A. Use of 13C as an indirect tag in 15N specifically labeled
nucleosides. Syntheses of [8-13C-1,7-NH2-15N3]adenosine, -guanosine, and their deoxy analogues. J.
Org. Chem. 2003, 68(22), 8657–8661.
11. Li, N.-S.; Lu, J.; Piccirilli, J.A. Synthesis of 2ꢁ-C-β-methyl-2ꢁ-deoxyguanosine. J. Org. Chem. 2009, 74(5),
2227–2230.
12. Chun, B.-K.; Du, J.; Rachakonda, S.; Ross, B.S.; Sofia, M.J.; Pamulapati, G.R.; Chang, W.; Zhang,
H.-R.; Nagarathnam, D. Synthesis of purine nucleosides. WO2010075554.