Synthesis and in vitro antiviral activities of 3′-fluoro (or chloro) and 2′,3′-difluoro 2′,3′-dideoxynucleoside analogs against hepatitis B and C viruses

Article abstract of DOI:10.1016/j.bmc.2010.08.048

Chronic infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) lead to serious liver diseases worldwide. Co-infection with HBV and HCV is very common and is associated with increased risk of liver pathogenesis, liver cancer, and liver failure. Several 5-substituted 3′-fluoro (or chloro) (1-4, 6, 7, 17-19) and 2′,3′-difluoro 2′,3′- dideoxynucleosides (15 and 16) were synthesized and evaluated for in vitro antiviral activities against duck hepatitis B virus (DHBV), human hepatitis B virus, and hepatitis C virus. Of these compounds 4, 7, 17, and 19 demonstrated moderate anti-HBV activity, and 2, 4, 7, 8, and 19 were weak inhibitors of HCV. Although 5-iodo derivative (7) was most inhibitory against HCV, it exhibited a reduction in cellular RNA levels in Huh-7 cells. The 5-hydroxymethyl-3′- fluoro-2′,3′-dideoxyuridine (4) and 1-(3-chloro-2,3-dideoxy-β- d-erythro-pentofuranosyl)-5-fluorouracil (19) provided the most inhibition of both viruses without cytotoxicity.

Full text of DOI:10.1016/j.bmc.2010.08.048

Bioorganic & Medicinal Chemistry 18 (2010) 7542–7547
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro antiviral activities of 30-fluoro (or chloro) and
20,30-difluoro 20,30-dideoxynucleoside analogs against hepatitis B and C viruses
Naveen C. Srivastav a, Neeraj Shakya a, Michelle Mak a, Chao Liang a, D. Lorne J. Tyrrell b,
Babita Agrawal c, Rakesh Kumar a,
⇑
a Department of Laboratory Medicine and Pathology, 1-71 Medical Sciences Building, University of Alberta, Edmonton, AB, Canada T6G 2H7
b Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada T6G 2H7
c Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada T6G 2H7
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2010
Revised 25 August 2010
Accepted 26 August 2010
Available online 23 September 2010
Chronic infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) lead to serious liver diseases
worldwide. Co-infection with HBV and HCV is very common and is associated with increased risk of liver
pathogenesis, liver cancer, and liver failure. Several 5-substituted 30-fluoro (or chloro) (1–4, 6, 7, 17–19)
and 20,30-difluoro 20,30-dideoxynucleosides (15 and 16) were synthesized and evaluated for in vitro anti-
viral activities against duck hepatitis B virus (DHBV), human hepatitis B virus, and hepatitis C virus. Of
these compounds 4, 7, 17, and 19 demonstrated moderate anti-HBV activity, and 2, 4, 7, 8, and 19 were
weak inhibitors of HCV. Although 5-iodo derivative (7) was most inhibitory against HCV, it exhibited a
reduction in cellular RNA levels in Huh-7 cells. The 5-hydroxymethyl-30-fluoro-20,30-dideoxyuridine (4)
Keywords:
Hepatitis B virus
Hepatitis C virus
Pyrimidine nucleosides
and 1-(3-chloro-2,3-dideoxy-b-
D-erythro-pentofuranosyl)-5-fluorouracil (19) provided the most inhibi-
tion of both viruses without cytotoxicity.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
of therapy peg-IFN-
a
and ribavirin is expensive, limited in efficacy
and associated with serious side effects.9,10 Further, this therapy is
only effective in <50% of the people with genotype 1a, the most
prevalent genotype in North America.11
Hepatitis B and C viruses (HBV and HCV) belong to hepadnavir-
idae and flaviviridae families, respectively. The infection with these
two hepatitis viruses is the major cause of serious liver diseases
such as hepatitis, liver failure, cirrhosis, and hepatocellular carci-
noma worldwide.1–3
Approximately 2 billion people have been infected with HBV and
ꢀ400 million people worldwide are chronically infected with HBV
(chronic carriers of HBV).4 Up to 1.2 million deaths occur annually
due to HBV.5 The HBV genome consists of a partly double stranded
DNA, but goes through reverse transcription of an RNA intermediate
to complete genome replication. Current vaccine for HBV, although
useful for preventing new infections, is not useful for thechronic car-
riers. The antiviral therapy with approved nucleoside/nucleotide
agents has limitations due to emergence of resistance, side effects,
and/or viral rebound after the cessation of therapy.6
Worldwide, approximately 170 million people are chronically
infected with HCV.7 Unlike HBV infection where most of the adults
acquiring HBV develop acute infection and clear the virus, 70–85%
of the people acquiring HCV develop chronic infection and later
liver diseases. HCV contains a single stranded RNA genome and
goes through replication via RNA dependent RNA polymerase.8
There is no vaccine available for HCV, and the current standard
People co-infected with both HBV and HCV demonstrate a faster
progression to liver fibrosis, more severe liver disease, increased risk
of developing hepatocellular carcinoma, and markedly increased
risk of liver related death and overall mortality.12,13 Currently, there
is no standard of care treatment for HBV/HCV co-infected patients.
IFN-a/ribavirin treatment showed lower response rate in co-
infected patients compared to mono-infected HCV patients in a
clinical trial.14 Due to the limited treatment options available cur-
rently and their limited efficacy, new antiviral agents are urgently
required for the treatment of both HBV and HCV.
Due to common mode of parenteral transmission, HIV-infected
individuals also have a high prevalence of multiple hepatitis co-
infection (HBV and HCV).15 However, limited studies are available
on the virological aspects, clinical impact and therapeutic aspects
of HIV-infected people co-infected with HBV and HCV. In HIV
positive patients, the immunodeficiency may lead to increase in
both HCV and HBV replication with a more aggressive liver disease,
and the use of anti-retroviral agents (some of which also inhibit
HBV replication), may influence the dynamics of HCV/HBV
co-infection.16,17
In earlier studies, 30-fluorinated pyrimidine nucleosides have
been investigated as anti-HIV agents.18 Of these, 30-fluoro-20,30-
dideoxythymidine (FddThd) and 30-fluoro-20,30-dideoxy-5-chloro-
⇑
Corresponding author. Tel.: +1 (780) 492 7545; fax: +1 (780) 492 7521.
E-mail address: rakesh.kumar@ualberta.ca (R. Kumar).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.048

N. C. Srivastav et al. / Bioorg. Med. Chem. 18 (2010) 7542–7547
7543
uridine (FddClUrd) emerged as the most active inhibitors of HIV-1
replication.18 30-Fluorinated nucleosides have also been explored
as anti-HBV agents where 20,30-dideoxyguanosine (FLG) has shown
promise against duck HBV (DHBV) and human HBV (HBV) and has
undergone clinical trials.19 FddThd and FddClUrd were also found
to be strong inhibitors of HBV in vitro using the HepG2 2.2.15 cells.
FddThd was effective against DHBV in vivo.20 Inspite of the potent
anti-HBV activity shown by FddThd, its long-term use as a thera-
peutic anti-HBV drug is limited due to its toxicities.20
In order to gain a better insight into the structural requirements
for potent and selective anti-HBV activity as a part of our ongoing
antiviral research program, a number of 30-fluoro (or chloro)
pyrimidine nucleoside analogs were synthesized and evaluated
to determine their effects against DHBV and HBV. Increasing num-
ber of HBV and HCV co-infections have also prompted us to search
for agents that could be able to block the replication of both
viruses, therefore, these compounds were investigated for their po-
tential against HCV as well.
confluent cultures of primary duck hepatocytes obtained from
chronically infected Pekin ducks.28,29 The anti-HBV activity of the
compounds 1–4, 6–8, 12, and 15–19 was also assessed in confluent
cultures of the human hepatoma cell line 2.2.15 that chronically pro-
duces infectious HBV. 2.2.15 is a stable human HBV-producing hu-
man hepatoblastoma cell line, which carries HBV DNA stably
integrated into the genome of HepG2 cells.30 The antiviral activity
was determined by analysis of intracellular viral DNA, using dot blot
hybridization. The concentrations required to inhibit 50% of HBV
DNA (EC50) are shown in Table 1. Lamivudine (3-TC) was used as a
reference antiviral drug. As shown in Table 1, among the 30-fluori-
nated compounds, 5-hydroxymethyl (4) and 5-iodo (7) exhibited
significant anti-DHBV activity with EC50 values of 1 and 5 lg/mL,
respectively. The 5-unsubstituted (1) and 5-trifluoromethyl (2)
analogs showed weak inhibition and 5-ethyl (3), 5-hydroxy (6),
and 5-fluoro (8 and 12) did not show any activity at 10 lg/mL. In
these studies, 5-hydroxymethyl-30-fluoro-20,30-dideoxyuridine (4)
emerged as the most active inhibitor of DHBV replication that was
20-times less active than the reference drug 3-TC. The 20,30-difluoro
nucleosides (15 and 16) were not found to possess anti-HBV activity.
The inactivity of 20,30-difluoro-20,30-deoxythymidine (16) was sur-
prising. Although 30 fluoro-20,30-deoxythymidine (FddThd) is known
to be an excellent anti-DHBV and anti-HBV agent, replacement of
the 20-hydrogen with a fluorine atom in FddThd (as in compound
16) completely abrogated the anti-HBV activity despite the fact that
the fluorine atom is similar to a hydrogen atom in terms of van der
waal’s radii. The 30-chloro analog of 20,30-dideoxythymidine (17)
2. Chemistry
The 4-thio-30-fluoro-20,30-dideoxyuridine (1), 5-trifluoromethyl-
30-fluoro-20,30-dideoxyuridine (2), 5-ethyl-30-fluoro-20,30-dideoxy-
uridine (3), and 5-hydroxymethyl-30-fluoro-20,30-dideoxyuridine
(4) were prepared according to the published procedures.21,22 The
target 5-hydroxy pyrimidine nucleoside (6) was synthesized by
the treatment of 30-fluoro-20,30-dideoxyuridine (5)23 with bro-
mine–water followed by addition of pyridine at room temperature
in 37% yield. (Scheme 1). Previously published methods of Balzarini
et al.24 were used to synthesize 5-iodo-30-fluoro-20,30-dideoxyuri-
dine (7) and 5-fluoro-30-fluoro-20,30-dideoxyuridine (8). 30,5-Di-
fluoro-4-thio-20,30-dideoxyuridine (12) was prepared as illustrated
in Scheme 2. Thus, reaction of 9 with trityl chloride and 4-(dimeth-
ylamino)pyridine in dry pyridine provided 50-O-tritylated derivative
10 in 81% yield which upon thiation with P2S5 in pyridine gave the 4-
thio nucleoside 11. Compound 11 after detritylation with 80% aque-
ous AcOH at room temperature afforded 12 in 71% yield. 1-(2,3-
was found to exhibit moderate anti-HBV activity (EC50 = 10 lg/
mL) suggesting that substitutionof 30-fluoro by a chloro group is det-
rimental to anti-HBV activity. This observation was further sup-
ported by the fact that a 30-chloro derivative of 5-fluoro-20,30-
dideoxyurdine (18) was also devoid of anti-HBV activity. However,
it is interesting to note that when the 30-chloro substituent was in-
verted in the ‘up’ configuration (as in compound 19), it leads to sig-
nificant anti-DHBV activity (EC50 = 10 lg/mL). In our next studies, it
would be interesting to investigate the anti-HBV activity of the 20,30-
dideoxythymidine analog possessing a 30-chloro substituent in ery-
thro configuration.
Dideoxy-2,3-difluoro-b-
D-ribofuranosyl)uracil (15) was synthe-
Similar SARs were made when the compounds1–4, 6–8, 12, and
15–9 were screened against human HBV in 2.2.15 cells,31 except
that the antiviral activity of compound 4 was reduced compared
to that against DHBV. In this system, compounds 4, 7, 17, and 19
showed the best inhibition of HBV (Table 1).
sized in 37% yield by the reaction of 1-(3-fluoro-3-deoxy-b-
D
-arabi-
nofuranosyl)uracil (13) with diethylaminosulfur trifluoride in
benzene at ꢁ5 °C followed by subsequent detritylation of the ob-
tained product 14 using 80% aqueous AcOH (Scheme 3). Pyrimidine
nucleoside analogs, 1-(2,3-dideoxy-2,3-difluoro-b-
D-ribofurano-
The anti-HCV activity of synthesized compounds was evaluated
in the HCV RNA subgenomic replicon system in Huh-7 cells.32
Pyrimidine nucleosides 1–4, 6–8, 12, and 15–19 were initially
screened at 20 lg/mL concentration (Table 2). In these studies,
we observed that compounds 2, 4, 7, 8, and 19 exhibited apprecia-
ble inhibition of HCV replicon. The iodo derivative (7) exhibited
40% inhibition of HCV at 20 lg/mL, however, a concomitant de-
syl)thymine (16) and 1-(3-chloro-2,3-dideoxy-b- -erythro-pento-
D
furanosyl)thymine (17) were prepared according to the method
reported by Huang et al.25 and Verheyden et al.26 The 1-(3-chloro-
2,3-dideoxy-b-
D-threo-pentofuranosyl)-5-fluorouracil (18) and
1-(3-chloro-2,3-dideoxy-b-
D-erythro-pentofuranosyl)-5-fluoroura-
cil (19) were synthesized according to the procedures reported by us
earlier.27
crease in cellular RNA levels was also observed (data not shown).
The exact mechanism of action of compounds inhibiting both
HBV and HCV replication is not clear, however, it is possible that
compounds 2, 4, 7, and 19 exhibiting both anti-HBV and anti-
HCV activities undergo phosphorylation by cellular kinases and
their triphosphates can inhibit both HBV and HCV polymerases,
and/or due to lack of 20- and 30-OH groups, they may also lead to
viral DNA and RNA chain termination by acting as substrates of
both viral polymerases. Although HBV and HCV belong to two dif-
ferent virus families and have DNA and RNA as their genomes, the
replication of viral genome involves reverse transcription and DNA
dependent DNA synthesis, and RNA dependent RNA synthesis,
respectively. By the nature of general mechanism of action of mod-
ified nucleosides, it is possible to find analogs which can inhibit
different enzymes found in these viruses and provide both anti-
HBV and anti-HCV activities.
3. Results and discussion
The synthesized 30-halo or 20,30-dihalo nucleosides (1–4, 6–8, 12,
and 15–19) were screened against duck hepatitis B virus (DHBV) in
O
O
OH
HN
HN
(i)
O
N
O
N
O
O
HO
HO
F
F
6
5
Scheme 1. Reagents and conditions: (i) Br2–H2O, H2O, room temperature; pyridine,
room temperature, 22 h, 37%.

7544
N. C. Srivastav et al. / Bioorg. Med. Chem. 18 (2010) 7542–7547
O
N
O
N
S
N
S
N
F
F
F
F
HN
HN
HN
HN
(i)
(ii)
(iii)
O
O
O
O
O
O
O
O
HO
TrO
TrO
HO
F
F
F
F
10
11
12
9
Scheme 2. Reagents and conditions: (i) trityl chloride, 4-(dimethylamino)pyridine, dry pyridine, 80 °C, 8 h, 81% (for 10); (ii) P2S5, pyridine–H2O, reflux, 8 h, 27% (for 11); (iii)
80% AcOH, room temperature, 72 h, 71% (for 12).
O
N
O
N
O
N
mined for samples in Me2SO-d6 or CDCl3 on a Bruker AM 300 spec-
trometer. Chemical shifts are given in ppm relative to TMS as an
internal standard and signals are described as s (singlet), d (dou-
blet), t (triplet), br s (broad signal), m (multiplet), dm (doublet of
multiplet) and dd (doublet of doublets). The assignment of all
exchangeable protons (OH, NH) was confirmed by the addition of
the D2O. Microanalysis results were within 0.4% of theoretical val-
ues for all elements listed, unless otherwise indicated. Silica gel
column chromatography was carried out using Merck 7734 silica
HN
HN
HN
(i)
(ii)
O
O
O
O
O
O
HO
TrO
TrO
HO
F
F
F
F
F
13
14
15
Scheme 3. Reagents and conditions: (i) diethylaminosulfur trifluoride, anhydrous
benzene, ꢁ5 °C to room temperature, 3 h, 39% (for 14); (ii) 80% aq acetic acid, 90 °C,
30 min, 37% (for 15).
gel (100–200
was performed with Machery-Nagel Alugram SiL G/UV silica gel
slides (20 M thickness).
lM particle size). Thin-layer chromatography (TLC)
l
In conclusion, from these studies, 5-hydroxymethyl-30-fluoro-
4.1. 5-Hydroxy-30-fluoro-20,30-dideoxyuridine (6)
20,30-dideoxyuridine (4) and 1-(3-chloro-2,3-dideoxy-b-
D-erythro-
pentofuranosyl)-5-fluorouracil (19) emerged as interesting com-
pounds that displayed significant anti-HBV activity and notable
inhibition of hepatitis C replicon without causing cytotoxicity to
host cells.
A freshly prepared solution of bromine in water was added
drop-wise to a solution of compound 5 (0.50 g, 2.17 mmol) in
water (40 mL) at 25 °C with stirring until a yellow color persisted.
TLC showed that the starting material was consumed. Excess bro-
mine was evaporated through aeration. Pyridine (15 mL) was
added slowly to the colorless reaction mixture, so that the temper-
ature did not exceed 25 °C. The reaction mixture was stirred at
room temperature for 22 h. Solvent was evaporated in vacuo fol-
lowed by co-evaporation with ethanol. The residue thus obtained
4. Experimental
Melting points were determined on Electrothermal melting
point apparatus and are uncorrected. 1H NMR spectra were deter-
Table 1
Antiviral activities of pyrimidine nucleosides against duck HBV and human HBV
X
O
R
F
HN
HN
O
N
O
N
O
O
Cl
HO
HO
R1
R2
b
b
No.
R
R1
R2
X
DHBV primary duck hepatocytes
EC50
(
lg/mL)
2.2.15 HBV % inhibitiona
EC50
g/mL)
% inhibitiona (concd
lg/mL)
(concd
l
g/mL)
(l
1
2
3
4
6
7
8
H
CF3
C2H5
CH2OH
OH
I
F
F
H
CH3
CH3
F
—
—
F
F
F
F
F
F
F
F
F
F
Cl
Cl
—
—
H
H
H
H
H
H
H
H
F
S
25 (10)
20 (10)
0
75 (10), 50 (1)
0
63 (10), 50 (5)
0
0
0
0
20 (10)
20 (10)
0
58 (10), 50 (5)
0
60 (10), 50 (5)
0
0
0
O
O
O
O
O
O
S
O
O
O
O
—
—
1
5
5–10
5
12
15
16
17
18
19
3-TCc
F
0
H
H
—
55 (10)
0
50 (10)
96 (0.5–1.0)
10
10
56 (10)
0
50 (10)
88 (0.5–1)
10
10
0.05
0.2
a
b
c
The data is expressed as percent inhibition of viral DNA in the presence of 10
The drug concentration ( g/mL) required to reduce the viral DNA in infected cells to 50% of untreated infected controls.
(ꢁ)-b-
-20,30-Dideoxy-30-thiacytidine (Lamivudine, 3-TC).
lg/mL of the test compounds as compared to untreated infected controls.
l
L

N. C. Srivastav et al. / Bioorg. Med. Chem. 18 (2010) 7542–7547
7545
Table 2
Anti-hepatitis C virus (HCV) activity of pyrimidine nucleoside analogs in the stably HCV RNA replicon expressing Huh7 cell line containing HCV 1a subtype
X
O
R
F
HN
HN
O
N
O
N
O
O
Cl
HO
HO
R1
R2
Compd
R
R1
R2
X
Anti-HCV activity HCV 1a
Viability (XTT assay)
CC50 (lg/mL)
b
% inhibitiona
(l
g/mL)
1
2
3
4
6
7
8
12
15
16
17
18
19
Ribavirin
H
CF3
C2H5
CH2OH
OH
I
F
F
H
F
F
F
F
F
F
F
F
F
F
Cl
Cl
—
H
H
H
H
H
H
H
H
F
S
0 (20)
25 (20)
0 (20)
30 (20)
0 (20)
40 (20)
28 (20)
0 (20)
0 (20)
0 (20)
0 (20)
0 (20)
>100
>100
>200
>200
>100
<100
>100
>100
>200
>100
>200
>200
>200
NDc
O
O
O
O
O
O
S
O
O
O
O
—
CH3
CH3
F
F
H
H
—
—
30 (20)
50 (20)
a
b
c
Anti-HCV activity was determined at concentrations 20
Concentration required to reduce Huh-7 cell viability by 50%.
ND = not determined.
l
g/mL.
was purified on a silica gel column using EtOAc as eluent to give 6
(450 mg) as an impure solid, which was crystallized with MeOH/
acetone to yield pure 6 (200 mg, 37%) as solid; mp 198–200 °C
organic layer was dried over anhydrous Na2SO4 and concentrated
to give the crude product which was purified on silica gel column
using CHCl3 as eluent to give 11 (0.50 g, 27%) as a sirup; 1H NMR
(DMSO-d6) d 2.38–2.68 (m, 2H, H-20), 3.21–3.42 (m, 2H, H-50),
(dec); [a]
ꢁ9.8 (c 0.40, DMSO); 1H NMR (DMSO-d6) d 2.13–2.47
D
(m, 2H, H-20), 3.52–3.66 (m, 2H, H-50), 4.14 (dm, JH-4 ,3 -F
=
4.27 (dm, JH-4 ,3 -F = 27.17 Hz, 1H, H-40), 5.33 (dm, JH-3 ,3 -
F = 52.49 Hz, 1H, H-30), 6.06–6.11 (m, 1H, H-10), 7.26–7.47 (m,
15H, 50-O-trityl), 7.89 (d, J = 4.58 Hz, 1H, H-6), 13.16 (s, 1H, NH);
ES-MS (+ve mode) = 529.1 (M+Na)+; ES-MS (ꢁve mode) = 505.1
(Mꢁ1)+.
0
0
0
0
0
0
25.02 Hz, 1H, H-40), 5.21 (t, J = 4.58 Hz, 1H, 50-OH), 5.29 (dd,
JH-3 ,3 -F = 53.10 Hz, 4.27 Hz, 1H, H-30), 6.19–6.25 (m, 1H, H-10),
0
0
7.37 (s, 1H, H-6), 8.75 (s, 1H, 5-OH), 11.52 (s, 1H, NH); 13C NMR
(DMSO-d6)
d
36.66 (d, C-20, J = 20.12 Hz), 60.93 (d, C-50,
J = 11.16 Hz), 83.73 (C-10), 84.64 (d, C-40, J = 22.63 Hz), 94.95 (d,
C-30, J = 173.60 Hz), 119.03 (C-6), 132.68 (C-5), 149.07 (C-2),
160.41 (C-4). Anal. Calcd for C9H11FN2O5 + 0.9H2O: C, 41.20; H,
4.92; N, 10.68. Found: C, 41.48; H, 5.01; N, 10.33.
4.4. 30,5-Difluoro-4-thio-20,30-dideoxyuridine (12)
The compound 11 (0.50 g, 0.99 mmol) in 80% acetic acid
(20 mL) was stirred at room temperature for 72 h. Solvent was re-
moved in vacuo and the obtained crude product was purified on
silica gel column using EtOAc/hexane (40:60, v/v) as eluent to give
12 (0.185 g, 71%) as sirup that could not be crystallized out using
4.2. 30,5-Difluoro-50-O-trityl-20,30-dideoxyuridine (10)
A
mixture of 30,5-difluoro-20,30-dideoxyuridine (9, 2.0 g,
8.06 mmol), trityl chloride (3.37 g, 12.09 mmol), and 4-(dimethyl-
amino)pyridine (0.1 g, 0.82 mmol) in dry pyridine (40 mL) was
heated at 80 °C for 8 h. The solvent was removed in vacuo and
the crude product thus obtained was purified on silica gel column
using MeOH/CHCl3 (4:96, v/v) as eluent to give 10 (3.20 g, 81%) as a
sirup; 1H NMR (CDCl3) d 2.18–2.41 (m, 1H, H-20), 2.73–2.87 (m, 1H,
ethanol; [
a]
+65.04 (c 0.36, MeOH); 1H NMR (DMSO-d6) d 2.20–
D
2.61 (m, 2H, H-20), 3.58–3.70 (m, 2H, H-50), 4.23 (dm, JH-4 ,3 -
0
0
F = 27.16 Hz, 1H, H-40), 5.29 (dm, JH-3 ,3 -F = 53.40 Hz, 1H, H-30),
5.33 (t, J = 4.89 Hz, 1H, 50-OH), 6.09–6.16 (m, 1H, H-10), 8.18 (d,
J = 4.58 Hz, 1H, H-6), 13.13 (s, 1H, NH); 13C NMR (CD3OD) d 39.65
(d, C-20, J = 20.52 Hz), 62.58 (d, C-50, J = 11.06 Hz), 87.24 (C-10),
87.45 (d, C-40, J = 23.94 Hz), 95.99 (d, C-30, J = 175.61 Hz), 121.69
(d, C-6, J = 41.64 Hz), 148.68 (C-2), 148.86 (d, C-5, J = 222.18 Hz),
182.77 (d, C-4, J = 30.88 Hz). Anal. Calcd for C9H10F2N2O3S +
0.3C2H5OH: C, 41.47; H, 4.28; N, 10.07; S, 11.53. Found: C, 41.61;
H, 4.00; N, 10.38; S, 11.13.
0
0
H-200), 3.40–3.57 (m, 2H, H-50), 4.38 (dm, JH-4 ,3 -F = 27.16 Hz, 1H,
0
0
H-40), 5.32 (dm, JH-3 ,3 -F = 53.71 Hz, 1H, H-30), 6.34–6.41 (m, 1H,
H-10), 7.28–7.50 (m, 15H, 50-O-trityl), 7.81 (d, J = 6.10 Hz, 1H, H-
6), 8.96 (br s, 1H, NH); ES-MS (+ve mode) = 513.1 (M+Na)+; ES-
MS (ꢁve mode) = 489.2 (Mꢁ1)+.
0
0
4.3. 30,5-Difluoro-4-thio-50-O-trityl-20,30-dideoxyuridine (11)
4.5. 1-(2,3-Dideoxy-2,3-difluoro-5-O-trityl-b-D-
ribofuranosyl)uracil (14)
A mixture of 10 (1.80 g, 3.67 mmol), phosphorus pentasulfide
(2.93 g, 6.59 mmol), pyridine (100 mL), and water (0.1 mL) was re-
fluxed for 14 h and then cooled to room temperature. The solvent
was removed in vacuo and ice-water mixture was added to the res-
idue. The product was extracted with CHCl3 (3 ꢂ 50 mL). Combined
Diethylaminosulfur trifluoride (DAST) (0.95 g, 5.9 mmol) was
added to a solution of 13 (0.41 g, 0.82 mmol) in benzene (10 mL)
at ꢁ5 °C. The reaction mixture was stirred at room temperature for
24 h and poured into 5% aqueous NaHCO3 (w/v, 10 mL) dropwise.

7546
N. C. Srivastav et al. / Bioorg. Med. Chem. 18 (2010) 7542–7547
The aqueous portion was extracted with EtOAc (3 ꢂ 50 mL). The or-
ganic layer was dried over Na2SO4 and concentrated in vacuo. The
residue was purified on a silica gel column using MeOH/CHCl3
(2:98, v/v) as eluent to yield 14 (0.16 g, 39%) as a sirup; 1H NMR
(DMSO-d6) d 3.32 (m, 2H, H-50), 4.23–4.39 (m, 1H, H-40), 5.31–5.50
(m, 1H, H-30), 5.52 (dd, J = 7.93 Hz and 1.88 Hz, 1H, H-5), 5.54–5.67
(m, 1H, H-20), 5.96 (d, J = 18 Hz, 1H, H-1,), 7.16–7.47 (m, 15H, trityl),
7.72 (d, J = 8.54 Hz, 1H, H-6), 11.53 (s, 1H, NH); ES-MS (+ve
mode) = 513.1 (M+Na)+; ES-MS (ꢁve mode) = 489.2 (Mꢁ1)+.
NaCl plus 0.3 M sodium citrate, pH 7.0)-0.1% SDS at 65 °C for
30 min and 0.1 SSC, 0.15 DS at room temperature for 30 min. After
an autoradiographic image was obtained, the filters were exposed
in a phosphoimaging screen for 1–2 h, samples were quantitated
by a Fujix BAS1000, and the percentage density of phosphoimaging
units were calculated.20 Lamivudine was used as the reference
compound. Tests were repeated two to three times and the data
for each test compound were compared with a positive and nega-
tive control performed at the same time under identical conditions.
For the compounds where the EC50 obtained from two to three
experiments was within 10% standard deviation, average values
are shown, otherwise a range of EC50 values are shown. Percent
inhibition was calculated by using the formula:
4.6. 1-(2,3-Dideoxy-2,3-difluoro-b-D-ribofuranosyl)uracil (15)
A solution of 14 (0.15 g, 0.31 mmol) in 80% aqueous acetic acid
(10 mL) was heated at 90 °C for 30 min. The solvent was removed
in vacuo. The residue was purified on a silica gel column using
MeOH/CHCl3 (5:95, v/v) as eluent to yield 15 (0.028 g, 37%) as a
Untreated positive control ꢁ treated test sample
%Inhibition ¼
Untreated positive control
ꢁ20.87 (c 0.23, MeOH); 1H NMR (DMSO-d6) d 3.62 (m,
ꢂ 100
sirup; [
a
]
D
1H, H-50), 4.27 (dm, J = 22.58 Hz, 1H, H-40), 5.27 (dm, J = 53.10 Hz,
1H, H-30), 5.38 (m, 1H, 50-OH), 5.39 (dm, J = 51.26 Hz, 1H, H-20),
5.71 (d, 1H, H-5, J = 7.93 Hz), 6.06 (dd, J = 15.2 Hz and 4.88 Hz,
1H, H-10), 7.85 (d, J = 7.93 Hz, 1H, H-6), 11.50 (s, 1H, NH); 13C
NVR CD3OD: d 61.61 (C-50), 84.12 (C-40, J = 22.7 Hz), 88.25 (C-10,
J = 32.6 Hz), 90.17 (C-30, J = 163.90 Hz and 14.4 Hz), 92.07 (C-20,
J = 169.60 Hz and 14.4 Hz), 103.40 (C-5), 142.51 (C-6), 152.19
(C-2), 165.90 (C-4). Anal. Calcd for C9H10F2N2O4: C, 43.55; H,
4.06. Found: C, 43.95; H, 4.48.
4.8. In vitro antiviral assay (human hepatitis B virus, 2.2.15
cells)
The human HBV transfected 2.2.15 cells were obtained from
Dr. M.A. Sells and were used to determine the anti-HBV activity
of test compounds, as reported previously.30,31 These cells were de-
rived from HepG2 cells that were transfected with a plasmid vector
containing G418-resistant sequences and two head to tail dimers
of the HBV genome. The cell culture medium consisted of MEM
(Sigma), supplemented with 10% heat inactivated FBS (Gibco),
4.7. In vitro antiviral assay (duck hepatitis B virus, DHBV)
geneticin/G148 sulfate (380
bonate, and -glutamine (2 mM). Initially, the test compounds
were screened at a 10 g/mL final concentration. Inhibition of
HBV replication at 10 g/mL was calculated as the average of trip-
lg/mL), 5–7 mL of 7.5% sodium bicar-
Primary hepatocyte cultures obtained from congenitally in-
fected ducks were used to determine the anti-DHBV activity of test
compounds, as reported previously.28,29 Pekin duck eggs were ob-
tained from a duck colony maintained at the University of Alberta
farm and were stored in a 37 °C egg incubator until hatching oc-
curred. Livers from congenitally DHBV infected ducks were used.
Persistently infected ducks were identified by detection of DHBV
DNA in sera by dot hybridization.28,29 Primary cultures of duck
hepatocytes were prepared from 9 to 14 day old DHBV-infected
ducklings using a modified method.28,29 Cells were cultured in 6-
well plates in 3 mL L-15 medium containing 5% fetal bovine serum,
L
l
l
licate wells. Standard deviations were within 10% of the average
values. After initial testing, the compounds were serially diluted
to determine more precise anti-HBV EC50 values. The 2.2.15 cells
were grown in MEM media in a humidified 37 °C incubator with
a 5% CO2 atmosphere and seeded into 6-well plates overnight to
obtain a confluent monolayer. The compounds were tested in trip-
licate for each concentration. The drug solutions were added at a
volume of 3 mL per well, replacing the prior media. The media con-
taining test compounds was replaced every second day, for four to
five treatments in total. On the day subsequent to the last treat-
ments, the cells were harvested for the analysis of intracellular
HBV DNA. The method for isolated intracellular DNA and dot-blot
hybridization were essentially similar to that for DHBV, except hu-
man HBV specific radioactive probe was used. Percent inhibition
was calculated by using the formula: (Untreated positive con-
trol ꢁ treated test sample) ꢂ 100/Untreated positive control. Tests
were repeated two to three times and the data for each test com-
pound were compared with a positive and negative control per-
formed at the same time under identical conditions. For the
compounds where the EC50 obtained from three experiments was
within 10% standard deviation, average values are shown, other-
wise a range of EC50 values are shown.
penicillin G sodium (10 IU/mL), and streptomycin sulfate (10 lg/
mL). The test compounds were added in triplicate to the hepato-
cyte cultures on day 2, and were maintained in culture with media
changed every second day until day 16. Cells were harvested at day
17. Initially, the test compounds were screened at a 10
lg/mL final
concentration. Inhibition in DHBV replication at 10 g/mL was cal-
l
culated as the average of triplicate wells. Standard deviations were
within 10% of the average values. After initial testing, the com-
pounds were serially diluted to determine more precise anti-DHBV
EC50 values. The hepatocytes were lysed with 1.0 mL lysis buffer
containing 10 mM Tris–HCl and 1% SDS. The lysate was digested
with 0.2 mg proteinase K and extracted with an equal volume of
phenol saturated with Tris–HCl EDTA and 0.1% 8-hydroxyquino-
line, followed by extraction with chloroform. Concentrated NaCl
(5 M) was added to the aqueous phase to yield a final concentra-
tion of 0.5 M NaCl, and the DNA was precipitated with two
volumes of 95% ethanol. The DNA pellet was washed with 70% eth-
4.9. In vitro antiviral assay for HCV
anol and dried. The dried DNA was dissolved in 50 lL of a solution
Anti-HCV activity was determined in HCV 1a subgenomic repli-
con cells as described previously.32 Briefly, 1 ꢂ 105 replicon cells
per well were plated in 24-well plates. On the next day, replicon cells
containing Tris–HCl EDTA. DNA samples were applied to a nylon
filter (Hybond-N, Amersham) using a Bio-Dot (Bio-Rad Laborato-
ries) microfiltration apparatus. DNA on the filter was denatured
with NaOH/NaCl at room temperature for 30 min and neutralized
in Tris–HCl/NaCl. The filters were exposed to ultraviolet irradiation
for 3 min. DNA hybridization was initiated by adding a recently
prepared DHBV (32P) DNA probe at 106 CPM/mL and incubating
overnight. Filters were washed twice in 1 ꢂ SSC (20 ꢂ SSC = 3 M
were incubated at 37 °C with test compounds at 20 lg/mL. The cells
were treated with fresh mediacontainingtest compoundsevery sec-
ond day for a total of three to four treatments. Total cellular RNA was
extracted using an RNAeasy-96 kit (Qiagen, Valencia, CA), cDNA was
synthesized using iScript cDNA synthesis kit (BioRAD, CA) and the

N. C. Srivastav et al. / Bioorg. Med. Chem. 18 (2010) 7542–7547
7547
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TCG CAA GCA CCC TAT CA-30, b-actin F: 50-CGA TGC AGA AGG AGA
TCA CTG-30, b-actin R: 50-CGA TCC ACA CGG AGT ACT TG-30. The %
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4.10. Cell cytotoxicity assay
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XTT assay. Cell viability was measured using the cell proliferation
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centrations of 200, 100, 50, 10, and 1
cluded as control. Plates were incubated for 2 days at 37 °C. The
color reaction involved adding 50 L XTT reagents per well and
lg/mL. DMSO was also in-
l
incubating for 4 h at 37 °C. Plates were read on an ELISA plate read-
er (Abs 450–500 nm).
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Acknowledgments
We are grateful to the Canadian Institutes of Health Research
(CIHR) for an operating Grant (MOP-36393) for the financial sup-
port of this research. B.A. is thankful to the Alberta Innovates
Health Solutions (Alberta Heritage Foundation for Medical Re-
search, AHFMR) for a Senior Scholar Award.
28. Lee, B.; Luo, W.; Suzuki, S.; Robins, M. J.; Tyrrell, D. L. J. Antimicrob. Agents
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