Design, synthesis, and biological evaluation of new N 4-Substituted 2′-deoxy-2′-fluoro-4′-azido cytidine derivatives as potent anti-HBV agents

Article abstract of DOI:10.1016/j.ejmech.2015.06.030

A series of new 2′-deoxy-2′-β-fluoro-4′-azido-β-d-arabinofuranosyl cytidine derivatives bearing heteroatom-containing N⁴-substituents were designed and synthesized. Antiviral screening in HepG2.2.15 cells identified three analogs (1a, 1d and 1g

Full text of DOI:10.1016/j.ejmech.2015.06.030

European Journal of Medicinal Chemistry 101 (2015) 103e110
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis, and biological evaluation of new N4-Substituted
20-deoxy-20-fluoro-40-azido cytidine derivatives as potent anti-HBV
agents
,
b
Zhigang Lv a, Wu He a, Xianhai Tian a, Jinfeng Kang a, Yangxue Liu a, Youmei Peng a
,
,
, c, *
Liyun Zheng b, Qingduan Wang b, Wenquan Yu a *, Junbiao Chang a
a College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, PR China
b Henan Academy of Medical and Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450052, PR China
c Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, Zhengzhou 450001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A
series of new 20-deoxy-20- -fluoro-40-azido-
b b-D-arabinofuranosyl cytidine derivatives bearing
heteroatom-containing N4-substituents were designed and synthesized. Antiviral screening in
HepG2.2.15 cells identified three analogs (1a, 1d & 1g) with good anti-HBV activity and low cytotoxicty.
Of them, compound 1g exhibited significant inhibitory activity on both HBV antigens secretion (EC50,
Received 16 May 2015
Received in revised form
8 June 2015
Accepted 12 June 2015
Available online 22 June 2015
¼ 9 nM, EC50,
¼ 0.25
m
M) and viral DNA replication (intracellular, EC50 ¼ 0.099
mM; extra-
HBsAg
HBeAg
cellular, EC50 < 0.01 mM).
© 2015 Elsevier Masson SAS. All rights reserved.
Keywords:
b b-D-
20-deoxy-20- -fluoro-40-azido-
arabinofuranosyl cytidines
N4-substituted
Anti-HBV activity
HBV antigens
Viral DNA replication
1. Introduction
polymerase inhibitors. However, due to limited efficacy, side effects
and/or emergence of resistance, the current therapeutic options
still cannot meet the clinic need for the treatment of chronic hep-
atitis B. Thus, it is urgent to develop new and effective anti-HBV
drugs whether for monotherapy or combination therapy.
Although a number of non-nucleoside compounds have been
discovered with good anti-HBV activity [2e8], so far, none of them
has got the FDA approval for clinical use. Compared with inter-
Globally, hepatitis B remains a major infectious disease, which
affects more than 240 million individuals chronically worldwide
and causes as many as 780,000 deaths a year [1]. Currently, there
are only seven antiviral drugs in clinical use against hepatitis B
virus (HBV) infection: two interferon-a (IFN-a) products as immune
system modulators and five nucleoside/nucleotide analogs as viral
feron-a, nucleosides/nucleotides are orally available and effective
in almost all the patients, while causing fewer side effects, which is
still one of the most promising areas for developing new potential
anti-HBV therapeutics.
Abbreviations used: HBV, hepatitis B virus; IFN, interferon; FDA, food and drug
administration; HIV, human immunodeficiency virus; HCV, hepatitis C virus; TPSCl,
2,4,6-triisopropylbenzenesulfonyl chloride; DIPEA, disopropylethylamine; MTT, 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CC50, 50% cytotoxic
concentration; EC50, 50% effective concentration; 3 TC, lamivudine; ETV, entecavir;
PCR, polymerase chain reaction; FQ-PCR, fluorescence quantitative PCR; HBsAg,
HBV surface antigen; HBeAg, HBV e antigen; DNA, deoxyribonucleic acid; ELISA,
enzyme-linked immunosorbent assay; TLC, thin layer chromatography; MEM,
minimum essential medium.
* Corresponding authors. College of Chemistry and Molecular Engineering,
Zhengzhou University, 100 Science Avenue, Zhengzhou, Henan 450001, PR China.
E-mail addresses: wenquan_yu@zzu.edu.cn (W. Yu), changjunbiao@zzu.edu.cn
(J. Chang).
Our research group has been working on the design and dis-
covery of novel nucleosides against human immunodeficiency vi-
rus (HIV) [9e12], HBV [13e16], and hepatitis C virus (HCV) [17].
Previously, we reported the discovery of 20-deoxy-20- -fluoro-40-
b
azido-b-D-arabinofuranosyl cytidine (FNC, 6) with good anti-HBV
activity both in vitro [16] and in vivo [15]. In a recent research
work, it has been demonstrated that N4-alkylation (e.g. compound
7) of nucleoside 6 could significantly reduce the in vivo toxicity of
the compound [14]. However, these N4-alkylated analogs are less
http://dx.doi.org/10.1016/j.ejmech.2015.06.030
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

104
Z. Lv et al. / European Journal of Medicinal Chemistry 101 (2015) 103e110
chloride (TPSCl) [18] to form intermediates 3 or 4. Treatment of
compound 3 (or 4) with substituted amines in the presence of
disopropylethylamine (DIPEA) followed by deprotection with
ammonia in methanol afforded compound 1a¡l in 39e75% yield.
All these new synthesized nucleoside analogs were fully charac-
terized by 1H NMR, 13C NMR and HRMS. Analytical HPLC indicated
that all the tested compounds possess a purity of at least 95%.
2.2. Cytotoxicity
In the HepG2.2.15 cell line, the cytotoxicity of these new cytidine
derivatives (1a¡l) was measured by an MTT assay. As shown in
Table 1, most of these nucleoside analogs (1aꢀc and 1fꢀj) exhibited
further reduced toxicity compared to the parent compound 7.
Among them, two compounds (1a and 1g) are less toxic than the
positive control drug lamivudine (3 TC, CC50 ¼ 859
mM). In partic-
ular, no cytotoxicity was observed for the bis(2-hydroxyethyl)
amino analog 1g up to the highest concentration tested (1250 mM).
Scheme 1. Structure Exploration of the N4-Alkylated FNC Derivatives.
2.3. Anti-HBV activity screening
potent than the parent compound. Herein, we carried out further
structural exploration of the N4-substituents by incorporating ox-
ygen atoms as hydroxyl groups or ether bonds (Scheme 1), aiming
at further increasing the antiviral potency. The biological activity of
all the new cytidine derivatives was evaluated in HepG2.2.15 cell
lines.
The anti-HBV activity of these nucleosides (1a¡l) was firstly
screened in the HepG2.2.15 cell line using entecavir (ETV) as a
positive control. After the treatment of the cells with the test
compounds at the concentration of 1 mM for 9 days, their inhibitory
activity against HBV DNA replication was measured by an FQ-PCR
assay (Table 2). Compared to the parent compound 7, direct N4-
hydroxylation greatly increased the anti-HBV activity of the com-
pound (1a). O-Alkylation of 1a did not improve the biological ac-
tivity, with the ethoxy cytidine 1c being more potent than its
methoxy analog 1b. Further N-methylation of 1b significantly
decreased the biological activity of the compound (1d). Analogs
bearing N4-hydroxyalkyl groups (1eꢀf) maintained the good anti-
viral activity. Incorporation of one more hydroxyethyl group to the
N4-position of 1e led to compound 1g with significant potency
against HBV DNA replication (96.9%). However, the morpholine
analog (1h) completely lost the anti-HBV activity. Introduction of
aromatic rings (e.g. benzene, furan and pyridine rings) to the N4-
2. Results and discussion
2.1. Chemistry
The target nucleosides 1 were synthesized as outlined in
Scheme 2. The key starting material, bisbenzoyl uridine 2 was
prepared from 1,3,5-O-tribenzoyl-2-deoxy-2-fluoro-D-arabinofur-
anoside using a previously reported synthetic route [10]. The 4-
carbonyl group in compound 2 was selectively activated by reac-
tion with 1,2,4-triazole [14] or 2,4,6-triisopropylbenzenesulfonyl
Scheme 2. Synthesis of Compounds 1 from Bisbenzoyl Protected Uridine 2.

Z. Lv et al. / European Journal of Medicinal Chemistry 101 (2015) 103e110
105
Table 1
Cytotoxicity of compounds 1.a
Compound
Structure
CC50
(m
M)
Compound
Structure
CC50 (mM)
7
391
1g
>1250
1a
1b
1c
951
1h
700
418
572
1i
834
722
1j
1d
1e
1f
a
>250
1k
<2
>10
859
<2
1l
668
3 TC
e
CC50: 50% cytotoxic concentration, measured by an MTT assay in the HepG2.2.15 cell line.
side-chain resulted in derivatives (1iꢀl) with poor antiviral activity.
Overall, three compounds (1a, 1d & 1g) exhibited potent inhibitory
activity against HBV DNA replication (97.0%, 93.9% and 96.9%),
which are equally good or slightly better than that of compound 6
and the potent anti-HBV drug entecavir (ETV, 95.7%) at the same
concentration tested.
significant reduction on both HBsAg and HBeAg secretion in a dose-
dependent manner. As illustrated in Table 3, all the three nucleo-
sides displayed nanomolar level inhibitory activity on HBsAg
secretion on Day 9, with the bis(2-hydroxyethyl)amino analog 1g
being the most potent one (EC50 ¼ 9 nM). In addition, compound 1g
also exhibited an EC50 of 0.25
mM on the inhibition of HBeAg
secretion. On Day 9, the positive control drug lamivudine (3 TC) at
the concentration of 25 M only showed 43.4% and 46.0% inhibition
of HBsAg and HBeAg, respectively.
m
2.4. Inhibitory effect on HBsAg and HBeAg secretion
According to the SAR results above, the inhibitory effect of
selected analogs (1a, 1d & 1g) on the secretion of HBsAg and HBeAg
was further evaluated in the HepG2.2.15 cell line. The supernatant
was collected after the cells were treated with these compounds at
various concentrations (1, 0.1 and 0.01 mM), and the levels of HBsAg
and HBeAg in the supernatant were detected by ELISA assays.
Treatment of HepG2.2.15 cells with the test compounds resulted in
2.5. Inhibitory effect of compound 1g on HBV DNA replication
Consistent with the inhibitory activity on HBV antigens secre-
tion, compound 1g also reduced both extracellular and intracellular
HBV DNA levels in HepG2.2.15 cells in a dose-dependent manner.
On Day 9, the average inhibition of viral DNA levels at the dosages

106
Z. Lv et al. / European Journal of Medicinal Chemistry 101 (2015) 103e110
Table 2
Inhibitory effect of compounds 1 on HBV DNA replication.a
Compound
Structure
Inhibition rate (1
98.6%
m
M)
Compound
Structure
Inhibition rate (1
96.9%
mM)
6
1g
7
86.6%
1h
<10%
<10%
<10%
1a
97.0%
1i
1b
1c
1d
1e
1f
a
81.2%
93.9%
34.0%
82.1%
77.6%
1j
1k
1l
53.6%
24.3%
95.7%
ETV
e
HepG2.2.15 cells were treated with the test compounds at the concentration of 1.0 mM and the inhibition of HBV DNA replication was measured by an FQ-PCR assay.
of 1, 0.1 and 0.01
(Fig. 1A, EC50 ¼ 0.099
larly (Fig. 1B, EC50 < 0.01
m
M was 61.9%, 51.8% and 36.4% intracellularly
M) and 76.1%, 62.2% and 53.6% extracellu-
M), respectively.
HBV activity, low toxicity and reasonable physicochemical proper-
ties, and thus could be considered as a promising candidate for
further development.
m
m
2.6. In silico prediction of physicochemical properties
3. Conclusions
Furthermore, some physicochemical properties of 1g, its parent
compounds (6e7), and 3 TC, ETV (as control) were predicted using
free online software (http://www.molinspiration.com/) for their
compliance with the Lipinski's rule of five. As shown in Table 4, 1g,
6e7, and reference compounds conformed well to the Lipinski's
rule of five. Overall, compound 1g exhibited potent in vitro anti-
Building upon the structures of cytidine nucleosides 6 and 7, we
have designed and synthesized a series of new analogs bearing
heteroatom-containing N4-substituents. Antiviral screening on
HepG2.2.15 cell line identified three compounds (1a, 1d & 1g) with
potent inhibitory activity against HBV DNA replication. Treatment
of the cells with compound 1g for 9 days resulted in significant

Z. Lv et al. / European Journal of Medicinal Chemistry 101 (2015) 103e110
107
Table 3
These encouraging results justify its further development as a po-
tential anti-HBV agent.
Inhibitory Activity of selected compounds (1a, 1c, and 1g) on HBsAg and HBeAg
secretion.a
Compound
Structure
EC50
0.69
(
m
M, HBsAg)
EC50 (mM, HBeAg)
4. Experimental section
1a
>1
4.1. Chemistry
1H NMR spectra were recorded on a 400 MHz (13C NMR spectra
were recorded on a 100 MHz) spectrometer. Chemical shift values
are given in ppm and referred to the internal standard of TMS
(tetramethylsilane). The peak patterns are indicated as follows: s,
singlet; d, doublet; t, triplet; q, quadruplet; quint, quintet; m,
multiplet and dd, doublet of doublets. The coupling constants (J) are
reported in Hertz (Hz). Flash column chromatography was per-
formed over silica gel 200e300 mesh, and the eluent was a mixture
of MeOH/CH2Cl2 or EtOAc/petroleum ether. High-resolution mass
spectra (HRMS-ESI) were obtained on a Q-TOF Mass Spectrometer.
All the tested compounds possess a purity of at least 95%. Analytical
HPLC was run on the Agilent 1260 HPLC instrument, equipped with
Agilent XDB-C18 column and UV detection at 270 nm. Eluent sys-
tem was: 40% MeOH in H2O; flow rate ¼ 0.8 mL/min.
1c
1g
0.12
>1
0.009
0.25
a
EC50: 50% effective concentration, measured by HBsAg and HBeAg ELISA assays,
respectively, in the HepG2.2.15 cell line.
4.1.1. General Procedure A for the synthesis of nucleoside 1 via
intermediate 3
inhibition on the secretion of HBV antigens (EC50, HBsAg ¼ 9 nM,
The crude intermediate 3 was prepared as a pale yellow solid in
nearly quantitative yield according to our previously reported
procedure [14], which was used directly in the next step without
further purification. Compound 3 (116 mg, 0.2 mmol) was dissolved
in CH2Cl2 (2 mL), and then treated with the corresponding amine
EC50,
¼ 0.25
mM), and reduction of both intracellular
HBeAg
(EC50 ¼ 0.099
levels. In addition, no cytotoxicity was observed for this compound
at the highest concentration tested (1250 M) on the same cell line.
mM) and extracellular (EC50 < 0.01 mM) HBV DVA
m
Fig. 1. Inhibitory effect of compound 1g on HBV DNA level. HepG2.2.15 cells were cultured in the presence of compound 1g at various concentrations (1, 0.1 and 0.01
mM) or 3 TC at
436 M for 9 days, and then intracellular (A) and extracellar (B) HBV DNA levels were quantified by real-time FQ-PCR. The experiments were performed three times, **P < 0.01
m
compared to control.
Table 4
Prediction of physicochemical propertiesa of 1g, 6e7, 3 TC and ETV.
Compound
nViol
natoms
miLogP
MW/Da
nON
nOHNH
Nrotb
TPSA/Å2
MV
Accepted range
1g
6
7
3 TC
ETV
<5
<500
<10
12
10
10
6
<5
4
4
3
3
ꢁ10
8
3
5
2
<140
1
1
0
0
0
26
20
23
15
20
ꢀ1.84
ꢀ1.20
ꢀ0.46
ꢀ1.09
ꢀ1.06
374.33
286.22
326.29
229.26
277.28
178.04
160.37
146.37
90.38
308.05
223.32
264.02
187.07
237.36
8
5
2
130.06
a
nViol: no. of violations; natoms: no. of atoms; miLogP: molinspiration predicted LogP; MW: molecular weight; nON: no. of hydrogen bond acceptors; nOHNH: no. of
hydrogen bond donors; nrotb: no. of rotatable bonds; TPSA: topological polar surface area; MV: molar volume.

108
Z. Lv et al. / European Journal of Medicinal Chemistry 101 (2015) 103e110
(0.5 mmol) and N,N-diisopropylethylamine (DIPEA, 87
m
L,
JC-F ¼ 24.4 Hz), 61.7, 60.5; HRMS (m/z) [M þ H]þ calcd for
0.5 mmol; another 0.5 mmol of DIPEA was added, if the HCl salt of
the amine was used). After stirring at room temperature for 4e12 h
(TLC indicated that the conversion was complete), the reaction was
quenched with 5% citric acid (10 mL) and stirred for 10 min. The
organic layer was separated and the aqueous layer was extracted
with CH2Cl2 (10 mL ꢂ 3). The combined organic layer was washed
with brine (20 mL), dried over anhydrous sodium sulfate, and
concentrated. The resulting residue was purified by silica gel col-
umn chromatography to give the corresponding intermediate 5,
which was stirred in sat. NH3 solution in MeOH (5 mL) until TLC
indicated that the debenzoylation was complete. The solvent was
evaporated, and the resulting residue was purified through silica
gel column chromatography to afford products 1a, 1def and 1hel
in 39e70% yield overall three steps.
C10H14FN6O5 317.1004, found 317.1008.
4.1.2.3. 1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydrox-
ymethyl)tetrahydro-furan-2-yl)-4-(ethoxyamino)pyrimidin-2(1H)-
one (1c). The product was obtained according to General Procedure
B, as a white solid (75%); 1H NMR (400 MHz, CD3OD)
d 6.99 (dd,
J ¼ 8.4, 1.2 Hz, 1H), 6.39 (dd, J ¼ 11.2, 5.6 Hz, 1H), 5.57 (d, J ¼ 8.4 Hz,
1H), 5.13 (dt, J ¼ 54.4, 5.6 Hz,1H), 4.47 (dd, J ¼ 22.8, 5.2 Hz, 1H), 4.01
(q, J ¼ 7.2 Hz, 2H), 3.85e3.77 (m, 2H), 1.25 (t, J ¼ 7.2 Hz, 3H); 13C
NMR (100 MHz, CD3OD)
d
149.4, 144.2, 131.4 (d, JC-F ¼ 2.7 Hz), 97.1,
96.3 (d, JC-F ¼ 8.9 Hz), 94.9 (d, JC-F ¼ 192.7 Hz), 81.4 (d, JC-
¼ 16.9 Hz), 74.9 (d, JC-F ¼ 24.4 Hz), 68.9, 61.7, 13.4; HRMS (m/z)
F
[M þ H]þ calcd for C11H16FN6O5 331.1161, found 331.1167.
4.1.2.4. 1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydrox-
ymethyl)tetrahydro-furan-2-yl)-4-(methoxy(methyl)amino)pyr-
imidin-2(1H)-one (1d). The product was obtained according to
General Procedure A, as a white solid (70%); 1H NMR (400 MHz,
4.1.2. General Procedure B for the synthesis of nucleoside 1 via
intermediate 4
4-Dimethylaminopyridine (DMAP, 122 mg, 1 mmol) and DIPEA
(6.97 mL, 40 mmol) were added sequentially to a suspension of
compound 2 (2.65 g, 5 mmol) in anhydrous CH2Cl2 (30 mL). After
compound 2 was completely dissolved, the resulting mixture was
treated with 2,4,6-triisopropylbenzenesulfonyl chloride (TPSCl,
3.03 g, 10 mmol). After stirring at room temperature for 24 h, the
reaction mixture was concentrated and purified through silica gel
column chromatography to give intermediate 4 as a white solid
(3.90 g, 86%). Compound 4 (239 mg, 0.3 mmol) was dissolved in
CH2Cl2 (3 mL), and then treated with the corresponding amine
CD3OD)
d
7.88 (dd, J ¼ 7.6, 1.2 Hz, 1H), 6.46 (dd, J ¼ 12.4, 5.2 Hz, 1H),
6.31 (d, J ¼ 7.6 Hz, 1H), 5.20 (dt, J ¼ 53.6, 4.8 Hz, 1H), 4.47 (dd,
J ¼ 21.6, 4.4 Hz, 1H), 3.83 (s, 2H), 3.75 (s, 3H), 3.36 (s, 3H); 13C NMR
(100 MHz, CD3OD)
d
164.8, 155.9, 142.3 (d, JC-F ¼ 1.7 Hz), 97.3 (d, JC-
¼ 7.5 Hz), 94.6 (d, JC-F ¼ 192.5 Hz), 91.4, 83.5 (d, JC-F ¼ 17.1 Hz), 75.0
F
(d, JC-F ¼ 25.1 Hz), 61.9, 60.2, 32.9; HRMS (m/z) [M þ H]þ calcd for
C11H16FN6O5 331.1161, found 331.1166.
4.1.2.5. 1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydrox-
ymethyl)tetrahydro-furan-2-yl)-4-((2-hydroxyethyl)amino)pyr-
imidin-2(1H)-one (1e). The product was obtained according to
General Procedure A, as a white solid (39%). 1H NMR (400 MHz,
(0.9 mmol), DIPEA (157 mL, 0.9 mmol; another 0.9 mmol of DIPEA
was added, if the HCl salt of the amine was used) and DMAP (7 mg,
0.06 mmol). After stirring at room temperature for 4e12 h (TLC
indicated that the conversion was complete), the reaction was
quenched with 5% citric acid (10 mL) and stirred for 10 min. The
organic layer was separated and the aqueous layer was extracted
with CH2Cl2 (10 mL ꢂ 3). The combined organic layer was washed
with brine (20 mL), dried over anhydrous sodium sulfate, and
concentrated. The resulting residue was purified by silica gel col-
umn chromatography to give the corresponding intermediate 5,
which was stirred in sat. NH3 solution in MeOH (5 mL) until TLC
indicated that the debenzoylation was complete. The solvent was
evaporated, and the resulting residue was purified through silica
gel column chromatography to afford products 1b, 1c and 1g in
68e75% yield overall three steps.
CD3OD)
d
7.64 (dd, J ¼ 7.6, 1.2 Hz, 1H), 6.47 (dd, J ¼ 12.4, 5.2 Hz, 1H),
5.89 (d, J ¼ 7.6 Hz, 1H), 5.17 (dt, J ¼ 54.0, 4.8 Hz, 1H), 4.45 (dd,
J ¼ 22.0, 4.8 Hz, 1H), 3.82 (s, 2H), 3.67 (t, J ¼ 5.6 Hz, 2H), 3.49 (t,
J ¼ 5.6 Hz, 2H); 13C NMR (100 MHz, CD3OD)
d 164.3, 156.7, 140.0 (d,
JC-F ¼ 1.7 Hz), 97.0 (d, JC-F ¼ 7.7 Hz), 95.6, 94.7 (d, JC-F ¼ 192.6 Hz),
83.2 (d, JC-F ¼ 17.0 Hz), 75.0 (d, JC-F ¼ 25.0 Hz), 61.9, 59.9, 42.8;
HRMS (m/z) [M þ H]þ calcd for C11H16FN6O5 331.1161, found
331.1179.
4.1.2.6. 1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydrox-
ymethyl)tetrahydro-furan-2-yl)-4-((3-hydroxypropyl)amino)pyr-
imidin-2(1H)-one (1f). The product was obtained according to
General Procedure A, as a pale yellow semisolid (65%). 1H NMR
4.1.2.1. 1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydrox-
ymethyl)tetrahydro-furan-2-yl)-4-(hydroxyamino)pyrimidin-2(1H)-
one (1a). The product was obtained according to General Proce-
(400 MHz, CD3OD)
d
7.63 (dd, J ¼ 7.6, 1.2 Hz, 1H), 6.47 (dd, J ¼ 12.4,
5.2 Hz,1H), 5.85 (d, J ¼ 7.6 Hz,1H), 5.17 (dt, J ¼ 53.6, 4.8 Hz,1H), 4.45
(dd, J ¼ 21.6, 4.4 Hz, 1H), 3.82 (s, 2H), 3.59 (t, J ¼ 6.4 Hz, 2H), 3.46 (t,
J ¼ 6.8 Hz, 2H), 1.77 (quint, J ¼ 6.8 Hz, 2H); 13C NMR (100 MHz,
dure A, as a white solid (53%); 1H NMR (400 MHz, CD3OD)
d 6.95
(dd, J ¼ 8.4, 1.6 Hz, 1H), 6.39 (dd, J ¼ 11.2, 5.6 Hz, 1H), 5.57 (d,
CD3OD)
d
164.2, 156.7, 139.9 (d, JC-F ¼ 1.7 Hz), 97.0 (d, JC-F ¼ 7.7 Hz),
J ¼ 8.4 Hz, 1H), 5.13 (dt, J ¼ 54.0, 5.6 Hz, 1H), 4.47 (dd, J ¼ 22.8,
95.5, 94.7 (d, JC-F ¼ 192.3 Hz), 83.2 (d, JC-F ¼ 17.1 Hz), 75.0 (d, JC-
5.2 Hz, 1H), 3.82e3.80 (m, 2H); 13C NMR (100 MHz, CD3OD)
d
149.7,
¼ 25.0 Hz), 61.9, 58.7, 37.1, 31.5; HRMS (m/z) [M þ H]þ calcd for
F
144.4, 130.9 (d, JC-F ¼ 2.7 Hz), 97.5, 96.3 (d, JC-F ¼ 9.0 Hz), 94.9 (d, JC-
C12H18FN6O5 345.1317, found 345.1324.
¼ 192.7 Hz), 81.4 (d, JC-F ¼ 16.9 Hz), 74.9 (d, JC-F ¼ 24.4 Hz), 61.7;
F
HRMS (m/z) [M þ H]þ calcd for C9H12FN6O5 303.0848, found
4.1.2.7. 1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydrox-
ymethyl)tetrahydro-furan-2-yl)-4-(bis(2-hydroxyethyl)amino)pyr-
imidin-2(1H)-one (1g). The product was obtained according to
General Procedure B, as a white solid (68%); 1H NMR (400 MHz,
303.0851.
4.1.2.2. 1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydrox-
ymethyl)tetrahydro-furan-2-yl)-4-(methoxyamino)pyrimidin-2(1H)-
one (1b). The product was obtained according to General Proce-
dure B, as a pale yellow solid (73%); 1H NMR (400 MHz, CD3OD)
CD3OD)
d
7.78 (dd, J ¼ 8.0, 1.2 Hz, 1H), 6.47 (dd, J ¼ 12.4, 5.2 Hz, 1H),
6.24 (d, J ¼ 7.6 Hz, 1H), 5.19 (dt, J ¼ 54.0, 4.8 Hz, 1H), 4.47 (dd,
J ¼ 21.6, 4.4 Hz, 1H), 3.82 (s, 2H), 3.79 (s, 4H), 3.73 (t, J ¼ 5.6 Hz, 2H),
d
7.00 (dd, J ¼ 8.4, 2.0 Hz, 1H), 6.38 (dd, J ¼ 11.2, 5.6 Hz, 1H), 5.55 (d,
3.64 (t, J ¼ 5.6 Hz, 2H); 13C NMR (100 MHz, CD3OD)
d 163.9, 156.0,
J ¼ 8.4 Hz, 1H), 5.13 (dt, J ¼ 54.0 Hz, 5.6 Hz, 1H), 4.46 (dd, J ¼ 22.8,
141.0 (d, JC-F ¼ 1.6 Hz), 97.1 (d, JC-F ¼ 7.7 Hz), 94.6 (d, JC-F ¼ 192.4 Hz),
93.2, 83.3 (d, JC-F ¼ 17.0 Hz), 75.0 (d, JC-F ¼ 25.0 Hz), 61.9, 59.4, 59.2,
52.1, 51.1; HRMS (m/z) [M þ H]þ calcd for C13H20FN6O6 375.1423,
found 375.1425.
5.2 Hz, 1H), 3.82e3.80 (m, 2H), 3.77 (s, 3H); 13C NMR (100 MHz,
CD3OD)
d
149.4, 144.3, 131.6 (d, JC-F ¼ 2.8 Hz), 96.9, 96.3 (d, JC-
¼ 8.8 Hz), 94.9 (d, JC-F ¼ 192.7 Hz), 81.4 (d, JC-F ¼ 17.0 Hz), 74.9 (d,
F

Z. Lv et al. / European Journal of Medicinal Chemistry 101 (2015) 103e110
109
4.1.2.8. 1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydrox-
4.2. Cytotoxicity assay
ymethyl)tetrahydro-furan-2-yl)-4-morpholinopyrimidin-2(1H)-one
(1h). The product was obtained according to General Procedure A,
To determine the cell viability, an MTT-based assay was per-
formed [19]. Briefly, HepG2.2.15 cells were seeded at a density of
2 ꢂ 104 cells per well on 96-well plates. After incubation for 24 h,
the cells were treated with medium containing the test compound
as a white solid (69%); 1H NMR (400 MHz, CD3OD)
d 7.83 (d,
J ¼ 7.6 Hz, 1H), 6.48 (dd, J ¼ 12.4, 5.2 Hz, 1H), 6.24 (d, J ¼ 8.0 Hz, 1H),
5.20 (dt, J ¼ 53.6, 4.8 Hz, 1H), 4.47 (dd, J ¼ 21.6, 4.4 Hz, 1H), 3.77 (m,
10H); 13C NMR (100 MHz, CD3OD)
d
163.0, 156.2, 141.7 (d, JC-
of different concentrations (2, 10, 50, 250, 1250
medium was replaced with a fresh one on day 3 or day 6 during the
9-day experiment. On day 9, 20 L of MTT solution (5 mg/mL) was
added to each well. After incubating routinely for 4 h, the super-
natants were discarded and 180 L of DMSO was added into each
mM). The culture
¼ 1.2 Hz), 97.2 (d, JC-F ¼ 7.8 Hz), 94.7 (d, JC-F ¼ 192.4 Hz), 91.9, 83.4
F
(d, JC-F ¼ 17.0 Hz), 75.0 (d, JC-F ¼ 25.0 Hz), 66.1, 61.9; HRMS (m/z)
m
[M þ H]þ calcd for C13H18FN6O5 357.1317, found 357.1323.
m
well to solubilize the formazan. The absorbance (A) at 490 nm was
measured by using an automatic plate reader (BIO-RAD, 168-
1000XC, USA). The survival rate of HepG2.2.15 cells (%) was calcu-
lated as [1 ꢀ (A490 of experimental group/A490 of negative
control) ꢂ 100%] [20].
4.1.2.9. 1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydrox-
ymethyl)tetrahydro-furan-2-yl)-4-((4-hydroxyphenethyl)amino)pyr-
imidin-2(1H)-one (1i). The product was obtained according to
General Procedure A, as a white solid (65%); 1H NMR (400 MHz,
CD3OD)
d
7.61 (d, J ¼ 7.6 Hz, 1H), 7.03 (d, J ¼ 8.4 Hz, 2H), 6.69 (d,
J ¼ 8.8 Hz, 2H), 6.47 (dd, J ¼ 12.4, 5.2 Hz, 1H), 5.81 (d, J ¼ 7.6 Hz, 1H),
5.18 (dt, J ¼ 53.6, 4.8 Hz, 1H), 4.45 (dd, J ¼ 21.6, 4.0 Hz, 1H), 3.81 (s,
2H), 3.55 (t, J ¼ 7.6 Hz, 2H), 2.76 (t, J ¼ 7.6 Hz, 2H); 13C NMR
4.3. Assessment of Anti-HBV activity screening
The HepG2.2.15 cells were plated on 24-well plates at a density
of 1 ꢂ 105 cells per well and routinely cultured for 24 h. Then, the
cells were treated with freshly prepared medium containing the
(100 MHz, CD3OD)
d 163.9, 156.8, 155.5, 139.8, 129.7, 129.3, 114.8,
97.0 (d, JC-F ¼ 7.7 Hz), 95.6, 94.7 (d, JC-F ¼ 192.3 Hz), 83.3 (d, JC-
¼ 17.2 Hz), 75.1 (d, JC-F ¼ 25.1 Hz), 61.9, 42.0, 33.8; HRMS (m/z)
F
test compound at the concentration of 1
mM or ETV as a positive
[M þ H]þ calcd for C17H19FN6O5 407.1474, found 407.1475.
control at 1 M. Normal control cells were treated with minimum
m
essential medium (MEM) only. Media were changed every 3 days,
and the supernatants were collected on day 9. HBV DNA was
extracted by a viral DNA extraction kit according to the manufac-
turer's instructions and the viral DNA levels were detected by an
FQ- PCR detection kit according to the manufacturer's protocol.
Amplification and detection were performed on ABI7500 fluores-
cent quantitative PCR instrument. The program was optimized as
follows: 37 ꢃC for 5 min, at 94 ꢃC for 2 min (denaturation), followed
by 40 cycles of amplification (at 94 ꢃC for 20 s, 55 ꢃC for 30 s, 72 ꢃC
for 10 s). Inhibition rate of HBV DNA (%) ¼ [1 ꢀ (HBV DNA con-
centration of experimental group/HBV DNA concentration of
negative control) ꢂ 100%].
4.1.2.10. 1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydrox-
ymethyl)tetrahydro-furan-2-yl)-4-((2,4-dimethoxyphenyl)amino)
pyrimidin-2(1H)-one (1j). The product was obtained according to
General Procedure A, as a white solid (56%); 1H NMR (400 MHz,
DMSO-d6)
d
9.02 (s, 1H), 7.76 (s, 1H), 7.64 (d, J ¼ 7.2 Hz, 1H), 6.62 (d,
J ¼ 2.0 Hz, 1H), 6.50 (dd, J ¼ 8.8, 2.4 Hz, 1H), 6.42e6.35 (m, 2H), 6.19
(s, 1H), 5.68 (s,1H), 5.17 (dt, J ¼ 54.0, 5.2 Hz,1H), 4.42 (d, J ¼ 22.4 Hz,
1H), 3.78e3.72 (m, 8H); 13C NMR (100 MHz, DMSO-d6)
d 162.5,
157.4, 154.4, 152.5, 141.1, 125.8, 119.7, 104.1, 98.8, 96.9 (d, JC-
¼ 8.4 Hz), 95.7, 94.5 (d, JC-F ¼ 191.2 Hz), 82.2 (d, JC-F ¼ 16.8 Hz), 74.9
F
(d, JC-F ¼ 24.1 Hz), 62.3, 55.7, 55.4; HRMS (m/z) [M þ H]þ calcd for
C
17H19FN6O6 423.1423, found 423.1426.
4.4. Assessment of inhibitory activity on the HBsAg and HBeAg
secretion
4.1.2.11. 1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydrox-
ymethyl)tetrahydro-furan-2-yl)-4-((furan-2-ylmethyl)amino)pyr-
imidin-2(1H)-one (1k). The product was obtained according to
General Procedure A, as a pale yellow solid (67%); 1H NMR
The HepG2.2.15 cells were seeded at a density of 2 ꢂ 104 per well
on 24-well plates and routinely cultured for 24 h. Then, the culture
medium was replaced with freshly prepared medium containing
(400 MHz, CD3OD)
d
7.67 (dd, J ¼ 7.6, 0.8 Hz, 1H), 7.41 (d, J ¼ 0.8 Hz,
the test compound at various concentrations (1, 0.1, 0.01
mM) or
1H), 6.48 (dd, J ¼ 12.4, 5.2 Hz, 1H), 6.34e6.30 (m, 2H), 5.88 (d,
J ¼ 7.6 Hz, 1H), 5.18 (dt, J ¼ 53.6, 4.8 Hz, 1H), 4.56 (s, 2H), 4.46 (dd,
J ¼ 21.6, 4.4 Hz, 1H), 3.82 (s, 2H); 13C NMR (100 MHz, CD3OD)
lamivudine (3 TC, 25 M as positive control) in triplicate. Negative
m
control groups were treated with RPMI 1640 only. Media were
changed on day 3, 6 and 9, and the supernatants were collected. The
levels of HBsAg in the supernatants were detected by the ELISA kit
(Kehua Bio-engineering Corporation, Shanghai, PR China) accord-
ing to the manufacturer's protocol. The absorbance (A) at 450/
630 nm was measured by using an automatic plate reader. Inhibi-
tion rate of HBsAg (%) ¼ [1-(A450/630 of experimental group/A450/
630 of negative control) ꢂ 100%] [13].
d
163.8, 156.7, 151.0, 142.1, 140.3 (d, JC-F ¼ 1.6 Hz), 110.0, 107.3, 97.1
(d, JC-F ¼ 7.7 Hz), 95.4, 94.7 (d, JC-F ¼ 192.4 Hz), 83.3 (d, JC-
¼ 17.0 Hz), 75.0 (d, JC-F ¼ 25.0 Hz), 61.9, 36.9; HRMS (m/z)
F
[M þ H]þ calcd for C14H15FN6O5 367.1161, found 367.1164.
4.1.2.12. 1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydrox-
ymethyl)tetrahydro-furan-2-yl)-4-((pyridin-3-ylmethyl)amino)pyr-
imidin-2(1H)-one (1l). The product was obtained according to
General Procedure A, as a white solid (68%); 1H NMR (400 MHz,
4.5. Assessment of inhibitory effect on HBV DNA replication
CD3OD)
d
8.52 (s, 1H), 8.41 (d, J ¼ 4.8 Hz,1H), 7.83 (d, J ¼ 8.0 Hz, 1H),
The HepG2.2.15 cells were plated at a density of 2 ꢂ 104 cells per
well on 24-well plates and routinely cultured for 24 h. Then, the
cells were treated with freshly prepared medium containing com-
7.71 (d, J ¼ 7.6 Hz, 1H), 7.40e7.36 (m, 1H), 6.47 (dd, J ¼ 12.4, 5.2 Hz,
1H), 5.91 (dd, J ¼ 7.6, 1.6 Hz, 1H), 5.18 (dt, J ¼ 53.6, 4.8 Hz, 1H), 4.63
(s, 2H), 4.46 (dd, J ¼ 21.6, 4.4 Hz, 1H), 3.82 (s, 2H); 13C NMR
pound 1g (1, 0.1, 0.01 mM) or lamivudine (3 TC, 436 mM as positive
(100 MHz, CD3OD)
d
164.1, 156.7, 148.2,147.5, 140.6 (d, JC-F ¼ 1.5 Hz),
control). Normal control cells were treated with RPMI 1640 only.
Media were changed every 3 days, and both the supernatants and
cells were collected. The extracellular and intracellular HBV DNA
was extracted by viral DNA extraction kit according to the manu-
facturer's instructions. The concentration of extracted HBV DNA
136.5, 134.9, 123.8, 97.1 (d, JC-F ¼ 7.5 Hz), 95.3, 94.7 (d, JC-
¼ 192.3 Hz), 83.4 (d, JC-F ¼ 16.9 Hz), 75.0 (d, JC-F ¼ 25.0 Hz), 61.9,
F
41.1; HRMS (m/z) [M þ H]þ calcd for C15H16FN7O4 378.1321, found
378.1322.

110
Z. Lv et al. / European Journal of Medicinal Chemistry 101 (2015) 103e110
as novel non-nucleoside HBV inhibitors, Mini. Rev. Med. Chem. 10 (2010)
162e171.
[8] F. Zhang, G. Wang, A review of non-nucleoside anti-hepatitis B virus agents,
Eur. J. Med. Chem. 75 (2014) 267e281.
[9] J. Chang 2ʹ-Fluoro-4ʹ-substituted nucleosides, the preparation and use. Eur.
Patent 2177527, December 18, 2013.
[10] Q. Wang, W. Hu, S. Wang, Z. Pan, L. Tao, X. Guo, K. Qian, C.-H. Chen, K.-H. Lee,
J. Chang, Synthesis of new 2ʹ-deoxy-2ʹ-fluoro-4ʹ-azido nucleoside analogues
as potent anti-HIV agents, Eur. J. Med. Chem. 46 (2011) 4178e4183.
[11] Q. Wang, Y. Li, C. Song, K. Qian, C.-H. Chen, K.-H. Lee, J. Chang, Synthesis and
anti-HIV activity of 2ʹ-deoxy-2ʹ-fluoro-4ʹ-C-ethynyl nucleoside analogs, Bio-
org. Med. Chem. Lett. 20 (2010) 4053e4056.
was determined by real-time FQ-PCR, performed in Light-Cycler 1.5
(Roche, Mannheim, Germany) using the HBV fluorescent quanti-
tative PCR detection kit (Piji Biotechnology Development, Shenz-
hen, PR China) according to the manufacturer's protocol. Inhibition
rate of HBV DNA (%) ¼ [1 ꢀ (HBV DNA concentration of experi-
mental group/HBV DNA concentration of negative control) ꢂ 100%]
[21,22].
Acknowledgments
[12] J. Wu, W. Yu, L. Fu, W. He, Y. Wang, B. Chai, C. Song, J. Chang, Design, synthesis,
and biological evaluation of new 2ʹ-deoxy-2ʹ-fluoro-4ʹ-triazole cytidine nu-
cleosides as potent antiviral agents, Eur. J. Med. Chem. 63 (2013) 739e745.
[13] Q. Yang, X. Zhao, L. Zang, X. Fang, J. Zhao, X. Yang, Q. Wang, L. Zheng, J. Chang,
We thank the National Natural Science Foundation of China
(Nos. 81302637 and 81330075) and the China Postdoctoral Science
Foundation (Nos. 2013M530341 and 2014T70690) for financial
support.
Anti-hepatitis
B
virus activities of
a
-DDBeFNC,
a
novel nucleosi-
deebiphenyldicarboxylate compound in cells and ducks, and its anti-
immunological liver injury effect in mice, Antivir. Res. 96 (2012) 333e339.
[14] Q. Yang, J. Kang, L. Zheng, X.-J. Wang, N. Wan, J. Wu, Y. Qiao, P. Niu, S.-
Q. Wang, Y. Peng, Q. Wang, W. Yu, J. Chang, Synthesis and biological evalu-
ation of 4-substituted fluoronucleoside analogs for the treatment of hepatitis
B virus infection, J. Med. Chem. 58 (2015) 3693e3703.
References
[1] World Health Organization Hepatitis B; Fact Sheet No. 204; Updated March
2015. http://www.who.int/mediacentre/factsheets/fs204/en/(accessed on
5.12.15).
[15] L. Zheng, Q. Wang, X. Yang, X. Guo, L. Chen, L. Tao, L. Dong, Y. Li, H. An, X. Yu,
Q. Wang, J. Chang, Antiviral activity of FNC, 2ʹ-deoxy-2ʹ-b-fluoro-4ʹ-azidocy-
tidine, against human and duck HBV replication, Antivir. Ther. 17 (2012)
679e687.
[16] Y. Zhou, Y. Zhang, X. Yang, J. Zhao, L. Zheng, C. Sun, J. Jiang, Q. Yang, Q. Wang,
J. Chang, Novel nucleoside analogue FNC is effective against both wild-type
and lamivudine-resistant HBV clinical isolates, Antivir. Ther. 17 (2012)
1593e1599.
[17] W. Hu, P. Wang, C. Song, Z. Pan, Q. Wang, X. Guo, X. Yu, Z. Shen, S. Wang,
J. Chang, Synthesis and anti-HCV activity of a new 2ʹ-deoxy-2ʹ-fluoro-2ʹ-C-
methyl nucleoside analogue, Bioorg. Med. Chem. Lett. 20 (2010) 7297e7298.
[18] Y. Ding, Q. Habib, S.Z. Shaw, D.Y. Li, J.W. Abt, Z. Hong, H. An, Synthesis of new
3ʹ-, 5-, and N4-modified 2ʹ-O-methylcytidine libraries on solid support,
J. Comb. Chem. 5 (2003) 851e859.
[19] M. Ferrari, M.C. Fornasiero, A.M. Isetta, MTT colorimetric assay for testing
macrophage cytotoxic activity in vitro, J. Immunol. Methods 131 (1990)
165e172.
[20] M. Lin, L.Y. Yang, W.Y. Li, Y.P. Peng, J.K. Zheng, Inhibition of the replication of
hepatitis B virus in vitro by oxymatrine, J. Int. Med. Res. 37 (2009) 1411e1419.
[21] L.L. Gao, X.Y. Wang, J.S. Lin, Y.H. Zhang, Y. Li, Efficacies of beta-L-D4A against
hepatitis B virus in 2.2.15 cells, World J. Gastroentero. 14 (2008) 1263e1267.
[22] T.G. Kim, S.Y. Kang, K.K. Jung, J.H. Kang, E. Lee, H.M. Han, S.H. Kim, Antiviral
activities of extracts isolated from terminalis chebula Retz., Sanguisorba
officinalis L., Rubus coreanus Miq. and Rheum palmatum L. against hepatitis B
virus, Phytother. Res. 15 (2001) 718e720.
[2] T.M. Block, X. Lu, F.M. Platt, G.R. Foster, W.H. Gerlich, B.S. Blumberg, R.A. Dwek,
Secretion of human hepatitis B virus is inhibited by the imino sugar N-
butyldeoxynojirimycin, Proc. Natl. Acad. Sci. U S A 91 (1994) 2235e2239.
[3] A. Mehta, S. Ouzounov, R. Jordan, E. Simsek, X. Lu, R.M. Moriarty, G. Jacob,
R.A. Dwek, T.M. Block, Imino sugars that are less toxic but more potent as
antivirals, in vitro, compared with N-n-nonyl DNJ, Antivir. Chem. Chemother.
13 (2002) 299e304.
[4] W.E.I. Delaney, R. Edwards, D. Colledge, T. Shaw, P. Furman, G. Painter,
S. Locarnini, Phenylpropenamide derivatives AT-61 and AT-130 inhibit repli-
cation of wild-type and lamivudine-resistant strains of hepatitis
B virus
in vitro, Antimicrob. Agents Chemother. 46 (2002) 3057e3060.
€
[5] K. Deres, C.H. Schroder, A. Paessens, S. Goldmann, H.J. Hacker, O. Weber,
€
€
T. Kramer, U. Niewohner, U. Pleiss, J. Stoltefuss, E. Graef, D. Koletzki,
R.N.A. Masantschek, A. Reimann, R. Jaeger, R. Gro B. Beckermann,
b
,
K. Schlemmer, D. Haebich, H. Rübsamen-Waigmann, Inhibition of hepatitis B
virus replication by drug-induced depletion of nucleocapsids, Science 299
(2003) 893e896.
[6] W. Yu, C. Goddard, E. Clearfield, C. Mills, T. Xiao, H. Guo, J.D. Morrey,
N.E. Motter, K. Zhao, T.M. Block, A. Cuconati, X. Xu, Design, synthesis, and
biological evaluation of triazolo-pyrimidine derivatives as novel inhibitors of
hepatitis B virus surface antigen (HBsAg) secretion, J. Med. Chem. 54 (2011)
5660e5670.
[7] P. Zhan, X. Jiang, X. Liu, Naturally occurring and synthetic bioactive molecules