Synthesis and biological evaluation of a novel β-D-2′-deoxy-2′-α-fluoro-2′-β-C-(fluoromethyl)uridine phosphoramidate prodrug for the treatment of hepatitis C virus infection

Article abstract of DOI:10.1016/j.ejmech.2017.11.024

A novel β-D-2′-deoxy-2′-α-fluoro-2′-β-C-(fluoromethyl)uridine phosphoramidate prodrug (1) has been synthesized. This compound exhibits submicromolar-level antiviral activity in vitro against HCV genotypes 1b, 1a, 2a, and S282T replicons (EC₅₀ = 0.18–1.13 μM) with low cytotoxicity (CC₅₀ > 1000 μM). Administered orally, prodrug 1 is well tolerated at doses of up to 4 g/kg in mice, and produces a high level of the corresponding triphosphate in rat liver.

Full text of DOI:10.1016/j.ejmech.2017.11.024

Accepted Manuscript
Synthesis and biological evaluation of a novel β-D-2′-deoxy-2′-α-fluoro-2′-β-C-
(fluoromethyl)uridine phosphoramidate prodrug for the treatment of hepatitis C virus
infection
Ertong Li, Yafeng Wang, Wenquan Yu, Zhigang Lv, Youmei Peng, Bingjie Liu,
Shiliang Li, Wenzhe Ho, Qingduan Wang, Honglin Li, Junbiao Chang
PII:
S0223-5234(17)30919-4
10.1016/j.ejmech.2017.11.024
EJMECH 9899
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 September 2017
Revised Date: 1 November 2017
Accepted Date: 8 November 2017
Please cite this article as: E. Li, Y. Wang, W. Yu, Z. Lv, Y. Peng, B. Liu, S. Li, W. Ho, Q. Wang,
H. Li, J. Chang, Synthesis and biological evaluation of a novel β-D-2′-deoxy-2′-α-fluoro-2′-β-C-
(fluoromethyl)uridine phosphoramidate prodrug for the treatment of hepatitis C virus infection, European
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.11.024.
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ACCEPTED MANUSCRIPT
A novel β-D-2'-deoxy-2'-α-fluoro-2'-β-C-(fluoromethyl)uridine phosphoramidate
prodrug has been discovered with submicromolar-level anti-HCV activity and low
toxicity profiles.

ACCEPTED MANUSCRIPT
Synthesis
β-D-2'-Deoxy-2'-α-fluoro-2'-β-C-(fluoromethyl)uridine
Prodrug for the Treatment of Hepatitis C Virus Infection
and
Biological
Evaluation
of
a
Novel
Phosphoramidate
Ertong Li,1, # Yafeng Wang,2, 3, # Wenquan Yu,1, # Zhigang Lv,1 Youmei Peng,4 Bingjie Liu,5 Shiliang
Li,6 Wenzhe Ho,3 Qingduan Wang,4 Honglin Li,6 and Junbiao Chang1, *
1College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, China
2School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
3Department of Pathology and Laboratory Medicine, Temple University School of Medicine, Philadelphia, PA
19140, USA
4Institute of Medical and Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450052, China
5School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang 453007, China
6Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and
Technology, Shanghai 200237, China
#E. Li, Y. Wang and W. Yu contributed equally to this work.
*Corresponding Author. College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou
450001, China. E-mail Address: changjunbiao@zzu.edu.cn
ABSTRACT: A novel β-D-2'-deoxy-2'-α-fluoro-2'-β-C-(fluoromethyl)uridine phosphoramidate
prodrug (1) has been synthesized. This compound exhibits submicromolar-level antiviral activity in
vitro against HCV genotypes 1b, 1a, 2a, and S282T replicons (EC50 = 0.18~1.13 µM) with low
cytotoxicity (CC50 > 1000 µM). Administered orally, prodrug 1 is well tolerated at doses of up to 4
g/kg in mice, and produces a high level of the corresponding triphosphate in rat liver.
Keywords: 2'-α-fluoro-2'-β-C-(fluoromethyl)uridine; phosphoramidate prodrug; anti-HCV;
triphosphate

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1. INTRODUCTION
Hepatitis C is still a major global health problem, estimated to affect 142 million people
worldwide.1 Every year, hepatitis C virus (HCV) infection causes approximately 495,000 deaths
from cirrhosis, hepatocellular carcinoma (HCC), and other liver diseases.2 The potential molecular
targets for anti-HCV therapy include the NS5B protein, an RNA-dependent RNA polymerase
(RdRp)3 which plays a key role in the replication of the viral RNA genome. To date, various kinds of
NS5B RdRp inhibitors have been identified against HCV infection.4-7 In particular, nucleoside or
nucleotide inhibitors (NIs) have attracted considerable attention from medicinal chemists owing to
their potent inhibitory activity, broad genotype coverage, and high genetic barrier to viral resistance.8
However, sofosbuvir (17, Figure 1) is the only NI-type NS5B inhibitor approved by FDA for the
treatment of hepatitis C and consequently, the discovery and development of novel nucleos(t)ide
NS5B inhibitors to treat HCV infection is still of great importance for both monotherapy or
combination HCV therapy.
NIs active against HCV infection are usually developed as the corresponding phosphate or
phosphoramidate prodrugs, which can be conveniently converted into the bioactive nucleoside
triphosphates in cells. The 2′-β-C-methyl9-14 (e.g. INX-08189 (16)) and the
2'-deoxy-2'-α-fluoro-2'-β-C-methyl15-20 (e.g. 17) ribose nucleos(t)ides are two important classes of
NIs. Over the past decade, extensive efforts have been devoted to the modification of the base and
prodrug moieties in such compounds and in recent years, attention has been paid to the structural
exploration of the sugar ring. For example, the anti-HCV drug candidate MK-368221 contains a
2'-α-chloro group instead of the fluoro group. In 2014, Du, et al.22 and Jonckers, et al.23 reported
anti-HCV nucleotide analogues bearing 2′-spirocyclic ether substructures. More recently, Schinazi et
al.24 synthesized a 2′-α-fluoro-2′-β-chloro anti-HCV ribonucleotide prodrug in which the
2′-β-C-methyl substituent is replaced by a chloro group. As a part of our effort to discover novel
fluoronucleos(t)ide analogues for antiviral research, we report in this paper the design and synthesis

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of a novel β-D-2'-deoxy-2'-α-fluoro-2'-β-C-(fluoromethyl)uridine (15) by incorporation of a fluorine
atom in the 2′-β-C-methyl group and its phosphoramidate prodrug (1).25, 26 The anti-HCV activity of
compound 1 was evaluated in a cell line-based assay containing different HCV genotype (GT)
replicons. The in vivo toxicity, liver nucleoside triphosphate (NTP) levels, and interaction with NS5B,
assessed by molecular docking, were also investigated.
<Figure 1>
2. RESULTS AND DISCUSSION
2.1.
Chemistry.
The
synthetic
route
for
the
preparation
of
β-D-2'-deoxy-2'α-fluoro-2'β-C-(fluoromethyl)uridine (15) is outlined in Scheme 1. Selective
3',5'-protection of uridine (2) with tetraisopropyl dichlorodisilyloxane (TIPDSCl)27 followed by
oxidation and a Wittig reaction gave the alkene (5). The silyl group in compound 5 was then
removed by tetrabutylammonium fluoride (TBAF), and the 3',5'-dihydroxy moieties were further
protected by methoxymethyl (MOM) ether groups using dimethoxymethane (DMM). OsO4-mediated
dihydroxylation of alkene 7 resulted in a diol (8) as a mixture of diastereomers. Selective protection
of the primary alcohol group with benzoyl chloride followed by treatment with
diethylaminosulfurtrifluoride (DAST) generated a mixture of compounds 10 and 11, which were
both converted to diol (12) by the treatment with 2N NaOH in MeOH. Fluorination of compound 12
by DAST with and without base in sequence produced the desired difluorouridine (14). Deprotection
in 50% formic acid solution afforded the desired 2'-α-fluoro-2'-β-C-(fluoromethyl)uridine (15). To
synthesize
the
SP-phosphoramidate
prodrug
1,
isopropyl
((S)-(perfluorophenoxy)-
(phenoxy)phosphoryl)-L-alaninate was prepared according to a literature procedure.28 Finally, the
reaction of this chiral phosphoramidating reagent with nucleoside 15 in the presence of tert-butyl
magnesium chloride as base formed the expected prodrug 1 as the pure SP-enantiomer. The absolute
configuration of compound 1 was verified by X-ray crystallography (see the Supporting

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Information).
2.2. In Vitro Anti-HCV Activity. The cytotoxicity of 2'-α-fluoro-2'-β-C-(fluoromethyl) uridine (15)
and its prodrug (1) was evaluated in the Huh7 cell line. No cytotoxicity was observed for either
compound up to the highest concentration tested (1000 µM, Table 1). In order to examine the
antiviral activity of compound 1, HCV JFH-1 infected Huh7 cells were cultured in the presence of
the test compound at different concentrations. Quantitative real-time RT-PCR demonstrated that
prodrug 1 significantly suppresses the viral RNA replication in a dose-dependent manner (Figure 2A),
with EC50 and EC90 values that were slightly better than those of the positive control (Table 1).
Moreover, western blotting assay also indicated that, compared to the control group, the level of
HCV core protein was markedly reduced by compound 1 at a concentration of 1 µM (Figure 2B).
<Table 1>
<Figure 2>
In light of these results, the anti-HCV activity of prodrug 1 was further evaluated in different
cell-based HCV replicon assays. The inhibitory effect of 1 on the viral RNA replication was
measured by luciferase activity, which demonstrated that compound 1 possesses submicromolar-level
EC50 values (0.24 ~ 1.13 µM) against the HCV genotype (GT) 1b, 1a, 2a, and S282T replicons. The
test compound also displays EC90 values in the 0.50 to 7.70 µM range (Table 2).
<Table 2>
2.3. Acute Toxicity. A toxicity study of prodrug 1 was conducted in Kunming (KM) mice. Single
oral doses of the test compound at 2 or 4 g/kg were administered to the mice (three males and three
females), which were then observed for 15 days. During the study period, all mice remained alive
with no observed clinical signs of toxicity or body weight loss at either dose (Figure 3). All the
animals were sacrificed on day 15, and no macroscopic pathology of the heart, liver, spleen, lung or

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kidney was observed. These results demonstrated that orally administered compound 1 is
well-tolerated in the course of treatment and during the post-treatment period in KM mice at a
dosage of up to 4 g/kg.
<Figure 3>
2.4. Stability Assessment of Prodrug 1 at in SGF, SIF, Human Plasma, and Human Liver S9
Fraction. To investigate if this prodrug (1) can survive the exposure in the gastrointestinal tract and
preferentially release the phosphate in hepatocytes. Compound 1 was further evaluated for
gastrointestinal stability utilizing simulated gastric fluid (SGF) and simulated intestinal fluid (SIF).
Its stability in human plasma and human liver S9 fraction was also examined, respectively. As shown
in Table 3, prodrug 1 exhibited good stability (t1/2 > 17 h) in SGF, SIF, and human plasma but a short
half-life in liver S9, which indicates that 1 could rapidly release in the liver.
<Table 3>
2.5. Exposure of Liver Nucleoside Triphosphate (NTP) in Sprague-Dawley Rats. NTP levels
are critical to predictions of in vivo potency of antivirus nucleos(t)ide analogues. The distribution of
the difluorouridine (15), the biologically active metabolite of prodrug 1, and its triphosphate (15-TP),
was evaluated in Sprague-Dawley rats after administration by intragastric gavage (i.g.) of 50 mg/kg
of 1 (Table 4 and Figure 4). The mean Cmax and AUC0–t were 1895 ± 312 ng/g and 14540 ± 2448
ng†h/g respectively, indicating the test compound produces high concentration of 15-TP in the liver.
The mean t1/2 was 3.73 ± 1.06 h, implying 15 has a moderate residence time in the liver. These
results demonstrate that prodrug 1 is able to traverse the gastrointestinal tract, be absorbed effectively
into systemic circulation, transport intact to the target organ, and finally produce high exposure of the
bioactive metabolite (15-TP) in the liver.
<Table 4>

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<Figure 4>
2.6. Binding Mode Analysis. Molecular docking was conducted to further study the action
mechanism of the bioactive form, 15-TP. The enzyme-RNA-nucleotide ternary complex structure of
NS5B (PDB ID: 4WTG), which binds the diphosphate metabolite of sofosbuvir, was adopted for
protein preparation and molecular docking. The predicted binding mode shows that 15-TP binds at
the nucleotide binding site of NS5B and forms a beneficial Watson-Crick interaction with the pairing
base of the template strand of RNA (Figure 5A). Specifically, the 4’-carbonyl of the uracil moiety
appears to form a hydrogen bond with K141 of the protein (Figure 5B). In the ribose ring of 15-TP,
the 2'-fluorine-2’-fluoromethyl moiety can form polar interactions with residues S282 and N291, and
the 3'-hydroxyl interacts with D225 by forming a hydrogen bond with the backbone nitrogen atom of
D225. In addition, the α and β phosphates of 15-TP are coordinated with the two catalytic Mn2+ ions
and the conserved catalytic triad of D220, D318, and D319, at which the two metal ions and the
catalytic triad are perfectly oriented to coordinate each other. Opposite to the two Mn2+ ions, three
conserved basic residues R48, R158 and H223 are engaged in coordinating mainly the β and γ
phosphates. Thus, the triphosphate is appropriately accommodated in the catalytic site. In the
simulated NS5B ternary complex, we also observed that the 3'-hydroxyl of the primer strand that
coordinates with one catalytic Mn2+ ion is located adjacent to the scissile bond of the α phosphate of
15-TP in an in-line conformation, which is important for the common polymerase mechanism.29
Collectively, the favorable hydrogen bonding interactions, combined with the strong complementary
base-pairing interactions to the template strand, helps elucidate the structural basis for the successful
recognition of 15-TP as a ribonucleotide substrate that can be incorporated into the growing RNA
strand.
<Figure 5>

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3. CONCLUSIONS
In summary,
we
have
designed
and
synthesized
a
novel
β-D-2'-deoxy-2'-α-fluoro-2'-β-C-(fluoromethyl)uridine (15) from uridine via a 12-step synthetic
route. Its phosphoramidate prodrug (1) was obtained by the reaction of the nucleoside 15 with
isopropyl ((S)-(perfluorophenoxy)(phenoxy)phosphoryl)-L-alaninate in the presence of t-BuMgCl. In
cell-based HCV replicon assays, the prodrug (1) displays submicromolar level antiviral activity
(EC50 = 0.18 ~ 1.13 µM) against the HCV genotype (GT) 1b, 1a, 2a, and S282T replicons, with no
cytotoxicity observed at the highest concentration tested (1000 µM). Orally administered, compound
1 is well tolerated at doses up to 4 g/kg in KM mice, and produces a high level of the corresponding
uridine triphosphate in the liver of Sprague-Dawley rats. A molecular docking study indicates that
the biologically active metabolite of this prodrug (15-TP) could be successfully recognized as a
ribonucleotide substrate for incorporation into the growing RNA strand. These encouraging results
make phosphoramidate 1 a prospective candidate for further development as a new anti-HCV agent.
4. EXPERIMENTAL SECTION
1
13
13
4.1. Chemistry. H NMR and C NMR spectra were recorded on a 400 MHz (100 MHz for C
NMR) spectrometer. Chemical shift values are given in parts per million (ppm) with
tetramethylsilane (TMS) as an internal standard. The peak patterns are indicated as follows: s, singlet;
d, doublet; t, triplet; q, quadruplet; m, multiplet; dd, doublet of doublets and dt, doublet of triplets.
The coupling constants (J) are reported in Hertz (Hz). Flash column chromatography was performed
over 200-300 mesh silica gel. High-resolution mass spectra (HRMS-ESI) were obtained on a Q-TOF
mass spectrometer. The compounds tested possess a purity of at least 95%. Analytical HPLC was run
on an Agilent 1260 HPLC System, equipped with a Phenomenex Luna column (150 mm × 4.6 mm, 5
µm) and UV detection at 260 nm. The mobile phase consisted of linear gradients of A (MeOH) and B
(H2O): 20% to 68% A (9 min), 68% A (4 min), 68-90% A (0.1 min), 90% A (3.9 min), 90% to 20% A

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(0.1 min); 20% A (4.9 min). The flow rate was set at 0.5 mL/min, and the column temperature was
maintained at 30 °C.
1-((6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadis
ilocin-8-yl)pyrimidine-2,4(1H,3H) -dione (3). A solution of uridine (2, 9.8 g, 40 mmol) in dry
pyridine (50 mL) was treated with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPDSCl, 12.6 mL,
40.4 mmol) and stirred at 0 °C overnight. The reaction mixture was diluted with EtOAc (100 mL)
and washed with 1 M HCl (50 mL x 3). The combined aqueous phase was extracted with EtOAc (50
ml x 3). All the organic layers were combined, dried over anhydrous Na2SO4, concentrated, and then
purified through silica gel column chromatography [EtOAc/petroleum ether (PE) 25:75] to give
1
compound 3 (17.7 g, 91%) as a colorless solid: mp 75-76 °C; H NMR (400 MHz, CDCl3) δ 9.88
(br, s, 1H), 7.80 (d, J = 8.4 Hz, 1H), 5.75 (s, 1H), 5.71 (dd, J = 8.0, 2.0 Hz, 1H), 4.32-4.29 (m, 1H),
4.24-4.16 (m, 3H), 4.00 (dd, J = 13.2, 2.4 Hz, 1H), 1.11-1.00 (m, 28H) ; 13C NMR (100 MHz, CDCl3)
δ 163.7, 150.3, 139.9, 102.0, 91.0, 81.9, 75.1, 68.6, 60.1, 17.5, 17.4, 17.3, 17.2, 17.04, 16.97, 16.9,
16.8, 13.4, 13.0, 12.9, 12.4; HRMS (m/z) [M + Na]+ calcd for C21H38N2NaO7Si2, 509.2110, found
509.2101.
1-((6aR,8R,9aS)-2,2,4,4-tetraisopropyl-9-methylenetetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisil
ocin-8-yl)pyrimidine-2,4(1H,3H) -dione (5). To a stirred suspension of CrO3 (18.2 g, 182 mmol) in
CH2Cl2 (200 mL) at 0 °C under nitrogen atmosphere was carefully added pyridine (24.9 mL, 309.4
mmol) and Ac2O (17.2 mL, 182 mmol) in sequence. The resulting mixture was stirred at 0 °C until
homogeneous (1 h), and then treated with a solution of compound 3 (17.7 g, 36.4 mmol) in CH2Cl2
(50 mL). After stirred at room temperature for 3 h, it was poured into cold EtOAc (200 mL) and
stirred for 10 min. The mixture was concentrated and subjected to flash silica gel chromatography
(EtOAc/PE 30:70) to give intermediate 4 (16.9 g, crude) as a white solid which was used directly in
the following Wittig reaction.
To a stirred suspension of MePh3PBr (39.1 g, 109.2 mmol) in dry THF (200 mL) was added

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n-BuLi (1.6 M in n-hexane; 54.6 mL, 87.4 mmol) at -78 °C under a nitrogen atmosphere. After
stirring for 2 h, it was treated with a solution of 4 (above) in dry THF (50 mL). The reaction was
allowed to warm to room temperature for 24 h, quenched with sat. NH4Cl (100 mL), and then
extracted with EtOAc (100 mL x 3). The combined organic layer was dried over anhydrous Na2SO4,
concentrated, and purified through silica gel column chromatography (EtOAc/PE 25:75) to give
1
olefin 5 (11.4 g, 65% from 3) as a white solid: mp 118-120 °C; H NMR (400 MHz, CDCl3) δ 9.75
(br s, 1H), 7.46 (d, J = 8.0 Hz, 1H), 6.54 (s, 1H), 5.72 (dd, J = 8.0, 1.6 Hz, 1H), 5.56 (s, 1H), 5.46 (s,
1H), 4.82 (dd, J = 8.8, 1.2 Hz, 1H), 4.17-4.02 (m, 2H), 3.69 (d, J = 8.8 Hz, 1H), 1.11-1.05 (m, 28H);
13C NMR (100 MHz, CDCl3) δ 163.6, 150.7, 146.6, 139.7, 112.1, 102.7, 83.8, 83.0, 69.7, 60.3, 17.4,
17.34, 17.27, 17.01, 16.98, 16.9, 16.8, 13.5, 13.0, 12.8, 12.5; HRMS (m/z) [M + Na]+ calcd for
C22H38N2NaO6Si2, 505.2161, found 505.2149.
1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-3-methylenetetrahydrofuran-2-yl)pyrimidine-2,4(1H,3
H)-dione (6). A solution of compound 5 (11.4 g, 23.5 mmol) in dry THF (100 mL) was treated with
TBAF (1 M in THF; 70.5 mL, 70.5 mmol) and stirred at room temperature for 2 h. It was then
concentrated and purified through silica gel column chromatography (MeOH/CH2Cl2 5:95) to give
nucleoside 6 (5.6 g, 99%) as a colorless solid: mp 169-170 °C; 1H NMR (400 MHz, CD3OD) δ 7.63
(d, J = 8.0 Hz, 1H), 6.60 (d, J = 2.0 Hz, 1H), 5.71 (d, J = 8.0 Hz, 1H), 5.54 (t, J = 2.4 Hz, 1H), 5.38
13
(t, J = 2.4 Hz, 1H), 4.66-4.64 (m, 1H), 3.92-3.88 (m, 1H), 3.79-3.74 (m, 2H); C NMR (100 MHz,
CD3OD) δ 164.7, 151.2, 149.5, 142.0, 112.1, 101.9, 84.8, 84.4, 69.9, 60.5; HRMS (m/z) [M + Na]+
calcd for C10H12N2NaO5, 263.0638, found 263.0638.
1-((2R,4S,5R)-4-(methoxymethoxy)-5-((methoxymethoxy)methyl)-3-methylenetetrahydrofuran-2-yl
)pyrimidine-2,4(1H,3H)-dione (7). A solution of compound 6 (5.6 g, 23.3 mmol) in CH2Cl2 (280
mL)/dimethoxymethane (DMM, 110 mL) was treated with TfOH (0.7 mL), stirred at room
temperature for 6 h, and then quenched with sat. NaHCO3 (100 mL). The organic layer was separated,
and the aqueous phase was extracted with CH2Cl2 (60 mL x 3). The combined organic layer was

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dried over anhydrous Na2SO4, concentrated, and then purified through silica gel column
chromatography (EtOAc/PE 60:40) to give compound 7 (5.0 g, 65%) as a white solid: mp 79-80 °C;
1H NMR (400 MHz, CDCl3) δ 10.02 (br, s, 1H), 7.47 (d, J = 8.4 Hz, 1H), 6.72 (s, 1H), 5.74 (d, J =
8.4 Hz, 1H), 5.57 (s, 1H), 5.36 (s, 1H), 4.82 (d, J = 7.2 Hz, 1H), 4.74 (d, J = 7.2 Hz, 1H), 4.68-4.67
13
(m, 3H), 4.15-4.13 (m, 1H), 3.88-3.77 (m, 2H), 3.43 (s, 3H), 3.38 (s, 3H); C NMR (100 MHz,
CDCl3) δ 163.6, 151.0, 145.4, 141.0, 115.3, 103.0, 96.6, 95.7, 84.3, 82.5, 76.4, 66.8, 55.8, 55.5;
HRMS (m/z) [M + Na]+ calcd for C14H20N2NaO7, 351.1163, found 351.1165.
1-((2R,4R,5R)-3-hydroxy-3-(hydroxymethyl)-4-(methoxymethoxy)-5-((methoxymethoxy)methyl)tetr
ahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (8). To a solution of compound 7 (5.0 g, 15.2 mmol)
in THF (20 mL)/t-butanol (20 mL)/water (8 mL) was added N-methylmorpholine-N-oxide (50% in
water, 3.6 mL, 15.2 mmol) and OsO4 (4% in t-butanol, 5.7 mL, 0.9 mmol) in sequence. The reaction
was stirred at room temperature for 24 h and then quenched with 5% Na2S2O3 (50 mL) and extracted
with EtOAc (50 mL x 3). The combined organic layer was dried over anhydrous Na2SO4,
concentrated, and then purified through silica gel column chromatography (MeOH/EtOAc 5:95) to
give diol 8 (5.3 g, 96%) as a syrup: HRMS (m/z) [M + Na]+ calcd for C14H22N2NaO9, 385.1218,
found 385.1205.
1-((2R,3S,4R,5R)-3-hydroxy-3-(hydroxymethyl)-4-(methoxymethoxy)-5-((methoxymethoxy)methyl)t
etrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (12). A solution of diol 8 (5.3 g, 14.6 mmol) in
pyridine (16 mL) and dry CH2Cl2 (50 mL) was treated with benzoyl chloride (3.4 mL, 29.5 mmol)
slowly at 0 ºC and then stirred at room temperature for 0.5 h. The reaction was quenched with water
(50 mL) and then extracted with CH2Cl2 (50 mL x 3). The combined organic layer was dried over
anhydrous Na2SO4, concentrated, and then purified through silica gel column chromatography
(EtOAc/PE 60:40) to give compound 9 (5.3 g, 96%) as a syrup: HRMS (m/z) [M + Na]+ calcd for
C21H26N2NaO10, 489.1480, found 489.1474.
To a mixture of 9 (5.3 g, 14.6 mmol) in NEt3 (10.2 mL, 73.2 mmol)/CH2Cl2 (50 mL) was added

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DAST (3.9 mL, 29.5 mmol) dropwise at 0 °C under a nitrogen atmosphere. After stirring at room
temperature for 4 h, the reaction was quenched with sat. NaHCO3 (50 mL), added slowly. The
organic layer was separated, and the aqueous phase was extracted with CH2Cl2 (50 mL x 3). The
combined organic layer was dried over anhydrous Na2SO4, concentrated to give a mixture of
compounds 10 and 11 as a faint yellow solid, which was used directly for the next step without
further purification.
A solution of the above 10 and 11 in MeOH (50 mL) was treated with 2 N NaOH (29.2 mL, 58.4
mmol) slowly and stirred at room temperature for 24 h. Then the reaction was neutralized with acetic
acid and concentrated. The resulting residue was suspended in MeOH/EtOAc (2:98, 50 mL), then
filtered through a silica gel pad, and washed with MeOH/EtOAc (2:98, 50 mL). The combined
filtrate was concentrated and purified through silica gel column chromatography (MeOH/EtOAc
1
1:99) to give diol 12 (3.2 g, 60% from 9) as a white solid: mp 111-112 °C; H NMR (400 MHz,
CD3OD) δ 7.82 (d, J = 8.4 Hz, 1H), 6.11 (s, 1H), 5.67 (d, J = 8.0 Hz, 1H), 4.81 (d, J = 6.8 Hz, 1H),
4.71-4.70 (m, 3H), 4.17-4.12 (m, 2H), 3.84-3.82 (m, 2H), 3.77 (d, J = 7.2 Hz, 1H), 3.68 (d, J = 7.2
Hz, 1H), 3.40 (s, 3H), 3.38 (s, 3H); 13C NMR (100 MHz, CD3OD) δ 164.8, 151.0, 143.0, 99.9, 96.3,
96.2, 86.3, 81.6, 81.3, 80.8, 66.6, 61.9, 54.8, 54.4; HRMS (m/z) [M + Na]+ calcd for C14H22N2NaO9,
385.1218, found 385.1215.
1-((2R,3S,4R,5R)-3-(fluoromethyl)-3-hydroxy-4-(methoxymethoxy)-5-((methoxymethoxy)methyl)tet
rahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (13). To a mixture of diol 12 (3.2 g, 8.8 mmol) and
Na2CO3 (1.1 g, 10.4 mmol) in dry CH2Cl2 (40 mL) was added DAST (2.3 mL, 17.4 mmol) dropwise
at 0 °C under nitrogen atmosphere. The reaction was stirred at room temperature for 24 h, quenched
with sat. NaHCO3 (50 mL) slowly, and extracted with CH2Cl2 (40 mL x 3). The combined organic
layer was dried over anhydrous Na2SO4, concentrated, and then purified through silica gel column
chromatography (MeOH/EtOAc/PE 2:49:49) to give compound 13 (2.4 g, 75%) as a syrup. 19F NMR
(376 MHz, CDCl3) δ -233.3; 1H NMR (400 MHz, CDCl3) δ 10.12 (br, s, 1H), 7.70 (d, J = 84 Hz, 1H),

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6.12 (s, 1H), 5.67 (d, J = 8.4 Hz, 1H), 4.88-4.52 (m, 7H), 4.22 (s, 2H), 3.91-3.81 (m, 2H), 3.42 (s,
13
6H); C NMR (100 MHz, CDCl3) δ 164.3, 150.7, 142.0, 101.2, 96.7, 96.5, 85.9 (d, JC−F = 7.1 Hz),
83.3, 81.6, 81.1, 80.6 (d, JC−F = 18.0 Hz), 66.5, 56.1, 55.8; HRMS (m/z) [M + Na]+ calcd for
C14H21FN2NaO8, 387.1174, found 387.1161.
1-((2R,3R,4R,5R)-3-fluoro-3-(fluoromethyl)-4-(methoxymethoxy)-5-((methoxymethoxy)methyl)tetrah
ydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (14). To a solution of compound 13 (2.4 g, 6.6 mmol)
in dry CH2Cl2 (30 mL) was added DAST (1.7 mL, 12.9 mmol) dropwise at 0 °C under a nitrogen
atmosphere. The reaction was then stirred at room temperature for 6 h, quenched with sat. NaHCO3
(40 mL) slowly, and extracted with CH2Cl2 (30 mL x 3). The combined organic layer was dried over
anhydrous Na2SO4, concentrated, and then purified through silica gel column chromatography
19
(MeOH/EtOAc/PE 2:49:49) to give compound 14 (967 mg, 40%) as a syrup. F NMR (376 MHz,
CDCl3) δ -123.5, -233.0; 1H NMR (400 MHz, CDCl3) δ 9.79 (s, 1H), 7.78 (d, J = 8.0 Hz, 1H), 6.26
(d, J = 18.8 Hz, 1H), 5.73 (d, J = 8.4 Hz, 1H), 4.82-4.51 (m, 6H), 4.45-4.37 (m, 1H), 4.29-4.27 (m,
13
1H), 4.04 (dd, J = 11.6, 2.0 Hz, 1H), 3.85 (dd, J = 11.6, 2.8 Hz, 1H), 3.44 (s, 3H), 3.40 (s, 3H); C
NMR (100 MHz, CDCl3) δ 163.5, 150.6, 140.3, 102.5, 98.1 (dd, JC−F = 186.4, 19.1 Hz), 97.6, 96.7,
88.7, 79.3, 78.7 (d, JC−F = 172.2 Hz), 73.4 (d, JC−F = 13.3 Hz), 64.5, 56.1, 55.6; HRMS (m/z)
[M+Na]+ calcd for C14H20F2N2NaO7, 389.1131, found 389.1129.
1-((2R,3R,4R,5R)-3-fluoro-3-(fluoromethyl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyr
imidine-2,4(1H,3H)-dione (15). A solution of compound 14 (967 mg, 2.6 mmol) in 50% formic acid
(20 mL) was stirred at 80 °C for 6 h, concentrated, and then purified through silica gel column
chromatography (MeOH/EtOAc 1:99) to give the difluoro uridine 15 (334 mg, 46%) as a white solid.
19F NMR (376 MHz, CD3OD) δ -179.3, -237.3; 1H NMR (400 MHz, CD3OD) δ 7.99 (d, J = 8.0 Hz,
1H), 6.23 (d, J = 19.2 Hz, 1H), 5.69 (d, J = 8.4 Hz, 1H), 4.78-4.47 (m, 2H), 4.31 (dd, J = 24.0, 9.6
Hz, 1H), 4.04-3.98 (m, 2H), 3.83-3.79 (m, 1H); 13C NMR (100 MHz, CD3OD) δ 164.5, 150.7, 141.0,
101.4, 99.0 (dd, JC−F = 183.8, 18.3 Hz), 88.3, 81.6, 79.2 (dd, JC−F = 172.7, 34.0 Hz), 67.0 (d, JC−F
=

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16.0 Hz), 58.6; HRMS (m/z) [M + Na]+ calcd for C10H12F2N2NaO5, 301.0606, found 301.0609.
(S)-isopropyl
2-(((S)-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-4-(fluoromethyl)-3-hy
droxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino) propanoate (1). A mixture of
uridine
15
(835
mg,
3
mmol)
and
isopropyl
((S)-(perfluorophenoxy)(phenoxy)phosphoryl)-L-alaninate (1.63 g, 3.6 mmol) in anhydrous THF (50
mL) was treated with t-BuMgCl (1 M in THF, 6.3 mL, 6.3 mmol) at 0 °C under a nitrogen
atmosphere. After the addition, it was stirred at 0 °C for 10 min, and then at room temperature for 6 h.
The reaction was quenched with sat. NH4Cl (50 mL) and extracted with EtOAc (50 mL x 3). The
combined organic layer was dried over anhydrous Na2SO4, concentrated, and then purified through
silica gel chromatography (MeOH/CH2Cl2 2:98) to afford prodrug 1 (953 mg, 58%) as a white solid.
19
1
31P NMR (162 MHz, CDCl3) δ 3.35; F NMR (376 MHz, CDCl3) δ -178.3, -234.7; H NMR (400
MHz, CDCl3) δ 9.66 (s, br, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.33 (t, J = 8.0 Hz, 2H), 7.22-7.16 (m, 3H),
6.19 (m, 1H), 5.67 (dd, J = 8.0, 2.0 Hz, 1H), 5.03-4.97 (m, 1H), 4.72-4.33 (m, 7H), 4.19-4.17 (m,
13
1H), 3.99-3.89 (m, 1H), 1.34 (d, J = 7.2 Hz, 3H), 1.22 (d, J = 6.4 Hz, 6H); C NMR (100 MHz,
CDCl3) δ 173.4 (d, JC−P = 6.1 Hz), 163.6, 150.45, 150.38, 140.2, 129.9, 125.3, 119.9 (d, JC−P = 5.0
Hz), 102.6, 98.4 (d, JC−F = 159.0 Hz), 88.1, 79.4, 69.6, 67.5, 64.1, 53.5, 50.3, 21.7 (d, JC−P = 8.9 Hz),
20.7 (d, JC−P = 5.8 Hz); HRMS (m/z) [M + Na]+ calcd for C22H28F2N3NaO9P, 570.1423, found
570.1422.
ACKNOWLEDGMENT
We acknowledge the National Natural Science Foundation of China (No. 81330075) and
Outstanding Young Talent Research Fund of Zhengzhou University (No. 1521316004) for financial
support. We also thank Professor Ren Guobin of East China University of Science and Technology
for the help in X-ray structure determination.

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ABBREVIATIONS USED
HCV, hepatitis C virus; HCC, hepatocellular carcinoma; RNA, ribonucleic acid; RdRp,
RNA-dependent RNA polymerase; NI, nucleos(t)ide inhibitor; TIPDSCl, tetraisopropyl
dichlorodisilyloxane; TBAF, tetrabutylammonium fluoride; MOM, methoxymethyl; DMM,
dimethoxymethane; DAST, diethylaminosulfurtrifluoride; CC50, 50% cytotoxic concentration; EC50,
50% effective concentration; EC90, 90% effective concentration; SGF, simulated gastric fluid; SIF,
simulated intestinal fluid; NTP, nucleoside triphosphate; TP, triphosphate; AUC, area under the
plasma concentration-time curve; t1/2, elimination half-life; Tmax, time to reach Cmax; Cmax, maximum
plasma concentration.
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Figure Captions
Figure 1. Representative 2'-C-methyl substituted nucleotide prodrugs with potent anti-HCV
activity.
Figure 2. Inhibitory effect of prodrug 1 on HCV RNA replication (A) and core protein
expression (B). ÈP < 0.05; ÈÈÈP < 0.001 vs control.
Figure 3. Mean body weight change of KM mice from day 1 to 15 after oral administration of
1 at a single dose of 2 and 4 g/kg.
Figure 4. Time course of NTP levels in rat livers after i.g. administration of prodrug 1 at a
dose of 50 mg/kg.
Figure 5. (A) Close-up view of the active site of NS5B. The protein is represented by cartoon
and surface, and is colored gray. The self-complementary RNA is displayed with the template
strand in pink and the primer strand in green. 15-TP is shown as a ball-and-stick model with
carbon atoms in gray. Two catalytic Mn2+ ions are shown as brown spheres. (B) Close-up view
of the predicted binding mode of 15-TP. Substrates are colored as in (A), and key protein
residues are represented as thin sticks with carbon atoms colored light purple. Protein-ligand
hydrogen bonds are displayed as gray dashed lines, whereas base-pair hydrogen bonds are
displayed as red ones.

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Tables
Table 1. Cytotoxicity and Antiviral Activity against HCV JFH-1 Infection
compound CC50 (µM) EC50 (µM) EC90 (µM)
−
−
15
1
＞1000
＞1000
＞1000
0.18 ± 0.05 0.74 ± 0.02
0.27 ± 0.06 0.83 ± 0.02
17
Table 2. EC50 and EC90 Values of Prodrug 1 against HCV GT 1b, 1a, 2a, and S282T Replicons
HCV GT 1b HCV GT 1a HCV GT 2a HCV GT S282T
EC50 (µM) 0.24 ± 0.03 0.31 ± 0.06 0.54 ± 0.00 1.13 ± 0.06
EC90 (µM) 0.50 ± 0.04 1.26 ± 0.11 3.05 ± 0.07 7.70 ± 0.04
Table 3. Stability Assessment of Prodrug 1 at in SGF, SIF, Human Plasma, and Human Liver
S9 Fraction
stability t1/2 (h)
compound
SGFa
> 24
SIFb
> 24
human plasma
17.3
human S9
0.49
1
aSGF = simulated gastric fluid (pH 1.2). bSIF=simulated intestinal fluid (pH 7.4).

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Table 4. The Mean Pharmacokinetic Parameters of the NTP Level in Rat Livers after an Oral
Dose of Prodrug 1 at 50 mg/kg
AUC0-t
(ng·h/g)
AUC0-∞
(ng·h/g)
compound
t
1/2 (h)
Tmax (h)
Cmax (ng/g)
14540 ± 2448 15655 ± 2455 3.73 ± 1.06 5.83 ± 1.32 1895 ± 312
1

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Figures
Figure 1. Representative 2'-C-methyl substituted nucleotide prodrugs with potent anti-HCV
activity.
Figure 2. Inhibitory effect of prodrug 1 on HCV RNA replication (A) and core protein
expression (B). ÈP < 0.05; ÈÈÈP < 0.001 vs control.
Figure 3. Mean body weight change of KM mice from day 1 to 15 after oral administration of

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1 at a single dose of 2 and 4 g/kg.
Figure 4. Time course of NTP levels in rat livers after i.g. administration of prodrug 1 at a
dose of 50 mg/kg.
Figure 5. (A) Close-up view of the active site of NS5B. The protein is represented by cartoon
and surface, and is colored gray. The self-complementary RNA is displayed with the template
strand in pink and the primer strand in green. 15-TP is shown as a ball-and-stick model with
carbon atoms in gray. Two catalytic Mn2+ ions are shown as brown spheres. (B) Close-up view
of the predicted binding mode of 15-TP. Substrates are colored as in (A), and key protein
residues are represented as thin sticks with carbon atoms colored light purple. Protein-ligand
hydrogen bonds are displayed as gray dashed lines, whereas base-pair hydrogen bonds are
displayed as red ones.

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Schemes
Scheme 1. Synthesis of β-D-2'-Deoxy-2'-α-fluoro-2'-β-C-(fluoromethyl)uridine (15) and its
Phosphoramidate Prodrug 1a
aReagents and conditions: (a) TIPDSCl, Py, 0 °C, 91%; (b) CrO3, Py, Ac2O, CH2Cl2, 0 °C ~ rt; (c)
MePh3PBr, n-BuLi, THF, -78 °C ~ rt, 65% from 3; (d) TBAF, THF, rt, 99%; (e) DMM, TfOH,
CH2Cl2, rt, 65%; (f) OsO4, NMO, THF/t-BuOH/H2O, rt, 96%; (g) BzCl, Py, CH2Cl2, 96%; (h) DAST,
NEt3, CH2Cl2, 0 °C ~ rt; (i) 2 N NaOH, MeOH, rt, 60% from 9; (j) DAST, Na2CO3, CH2Cl2, 0 °C ~ rt,
75%; (k) DAST, CH2Cl2, 0 °C ~ rt, 40%; (l) 50% HCO2H (aq), 80 °C, 46%; (m) isopropyl
((S)-(perfluorophenoxy)(phenoxy)phosphoryl)-L-alaninate, t-BuMgCl/THF, then sat. NH4Cl, 58%.

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Highlights
°
A
novel 2'-α-fluoro-2'-β-C-(fluoromethyl)uridine phosphoramidate was
synthesized.
°
This prodrug (1) exhibits submicromolar-level anti-HCV activity with low
cytotoxicity.
°
°
Compound 1 is well tolerated up to 4 g/kg in mice via oral administration.
Prodrug 1 produces a high level of the corresponding triphosphate in rat liver.