Synthesis and antiviral effects of isosteviol-derived analogues against the hepatitis B virus

Article abstract of DOI:10.1016/j.phytochem.2013.12.014

Among several isosteviol-derived analogues, NC-8 (ent-16-oxobeyeran-19-N- methylureido) showed inhibitory potency against the hepatitis B virus (HBV) in HepG2 2.2.15 cells. Its anti-HBV mechanism was then next investigated in a human hepatoma cell culture system. Results showed that it specifically inhibited viral gene expression and reduced the level of encapsidated viral DNA intermediates in Huh7 cells that expressed replicating HBV. It also potently attenuated all viral promoter activity in HBV-expressing Huh7 cells, but not in cells lacking HBV expression. By examining its antiviral mechanism in cellular signaling pathways, NC-8 was found to inhibit the activity of the nuclear factor (NF)-κB element-containing promoter, but only slightly enhanced activities of activator protein (AP)-1- and interferon-sensitive response element (ISRE)-containing promoters in HBV-expressing cells. NC-8 also significantly eliminated NF-κB (p65/p50) and Toll-like receptor (TLR)2 proteins, but increased the IκBα protein level in a dose-dependent manner in HBV-transfected Huh7 cells, while these protein levels were apparently unchanged in non-transfected cells. Meanwhile, NC-8-treated nuclear extracts that co-expressed HBV inhibited the binding of NF-κB to the CS1 site of HBV major surface gene and specifically attenuated CS1-containing promoter activity. Taken together, this study suggests that the antiviral mechanism of NC-8 appears to be mediated by disturbing replication and gene expression of HBV and by inhibiting the host TLR2/NF-κB signaling pathway.

Full text of DOI:10.1016/j.phytochem.2013.12.014

Phytochemistry 99 (2014) 107–114
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Synthesis and antiviral effects of isosteviol-derived analogues against
the hepatitis B virus
Tsurng-Juhn Huang a, Bo-Hon Chou a,b,e, Cheng-Wen Lin c, Jen-Hsien Weng a, Chang-Hung Chou a,
Li-Ming Yang b,d, Shwu-Jiuan Lin b,
⇑
a Department of Biological Science and Technology and Research Center for Biodiversity, China Medical University, Taichung 404, Taiwan
b Department of Pharmaceutical Sciences, School of Pharmacy, Taipei Medical University, Taipei 110, Taiwan
c Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung 404, Taiwan
d Division of Chinese Medicinal Chemistry, National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei 112, Taiwan
e Long Shine Biopharma Co., Tainan 722, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 May 2013
Received in revised form 21 June 2013
Available online 23 January 2014
Among several isosteviol-derived analogues, NC-8 (ent-16-oxobeyeran-19-N-methylureido) showed
inhibitory potency against the hepatitis B virus (HBV) in HepG2 2.2.15 cells. Its anti-HBV mechanism
was then next investigated in a human hepatoma cell culture system. Results showed that it specifically
inhibited viral gene expression and reduced the level of encapsidated viral DNA intermediates in Huh7
cells that expressed replicating HBV. It also potently attenuated all viral promoter activity in HBV-
expressing Huh7 cells, but not in cells lacking HBV expression. By examining its antiviral mechanism
in cellular signaling pathways, NC-8 was found to inhibit the activity of the nuclear factor (NF)-jB ele-
ment-containing promoter, but only slightly enhanced activities of activator protein (AP)-1- and inter-
feron-sensitive response element (ISRE)-containing promoters in HBV-expressing cells. NC-8 also
significantly eliminated NF-jB (p65/p50) and Toll-like receptor (TLR)2 proteins, but increased the IjBa
Keywords:
Isosteviol derivative
Hepatitis B virus
Human hepatoma cells
NF-j
B
TLR2
protein level in a dose-dependent manner in HBV-transfected Huh7 cells, while these protein levels were
apparently unchanged in non-transfected cells. Meanwhile, NC-8-treated nuclear extracts that co-
expressed HBV inhibited the binding of NF-jB to the CS1 site of HBV major surface gene and specifically
attenuated CS1-containing promoter activity. Taken together, this study suggests that the antiviral mech-
anism of NC-8 appears to be mediated by disturbing replication and gene expression of HBV and by inhib-
iting the host TLR2/NF-jB signaling pathway.
Ó 2014 Elsevier Ltd. All rights reserved.
Introduction
and 0.8 kb long, respectively. The 3.5-kb transcript is translated to
produce core, precore, and viral polymerase proteins. It also serves
as a pregenomic RNA template for viral genome synthesis. The 2.4-
and 2.1-kb transcripts encode the small, middle, and large surface
(envelope) proteins; the 0.8-kb transcript is translated to produce
the X protein, a potent transactivator in both viral and host gene
promoters (Chisari, 2000). Determining how to regulate viral gene
expression, instead of viral replication, is a crucial issue for antivi-
Hepatitis B virus (HBV), a member of the Hepadnaviridae, is a
causative agent that frequently leads to acute and chronic infec-
tions in humans (Gitlin, 1997). Approximately 80% of HBV chronic
carriers have varying degrees of liver destruction which can pro-
mote liver cirrhosis and hepatocellular carcinoma (HCC) (Park
et al., 2006). As non-human hosts of HBV are restricted to only a
few species of animals, such as the chimpanzee, Peking duck,
woodchuck, and ground squirrel, the development of efficient ther-
apeutics for HBV infection has been hampered (Chisari et al., 1987).
Despite the development of a recombinant vaccine against HBV
and the use of specific HB immunoglobulin in susceptible popula-
tions, over 400 million people are chronic carriers (Gish, 2005).
After infection, four viral transcripts are transcribed from four dif-
ferent viral promoters (Core, X, S, and PreS), which are 3.5, 2.4, 2.1,
ral strategies. Several antiviral drugs including interferon (INF)-
a
and nucleoside derivatives are approved for clinical treatment of
HBV (Pramoolsinsup, 2002), yet critical issues remain unresolved,
e.g., low-to-moderate efficacy, adverse side-effects, and resistant
strains (Wakui et al., 2010). In light of these facts, continued devel-
opment of new antiviral agents with novel targets and mecha-
nisms to eradicate HBV in chronic carriers is urgently needed.
Stevioside is a natural sweetener that exists in leaves of Stevia
rebaudiana (Bertoni) Bertoni (Compositae) (Geuns, 2003). Isostevi-
ol, an ent-beyerane diterpenoid that possesses many biological
activities (Chang et al., 2008), can readily be obtained by acid
⇑
Corresponding author. Tel.: +886 2 27361661x6133; fax: +886 2 27390671.
E-mail address: shwu-lin@tmu.edu.tw (S.-J. Lin).
0031-9422/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2013.12.014

108
T.-J. Huang et al. / Phytochemistry 99 (2014) 107–114
Table 1
hydrolysis of stevioside (Avent et al., 1990). Because natural prod-
ucts can provide discovery of molecular targets for developing
drugs, a number of isosteviol analogues obtained from biocatalysis
and/or chemical modification showed bioactive attributes, includ-
Anti-HBV activity of isosteviol derivatives in vitroa
b
Compound
TC50
HBsAgc
HBeAgd
IC50 g/ml)
24.30
e
IC50
(l
g/ml)
SIf
(l
SIf
ing anti-inflammation (Chang et al., 2009), inhibition of a-glucosi-
2 (NC-8)
53.52
7.89
6.78
–
–
2.88
–
4.17
2.20
–
5.06
8.03
–
dase, a cytotoxic effect, and antibacterial activity (Zhu et al., 2013).
Recently, Takasaki et al. (2009) reported that isosteviol displayed a
significant inhibitory effect against early antigen activation of the
Epstein–Barr virus. A literature survey indicated that replacement
of the carboxylic group on triterpenoids by a ureide moiety had
antiviral activity towards the influenza and HSV-1 viruses (Baltina
et al., 2003; Flekhter et al., 2003). Thus, to develop new anti-HBV
agents with novel targets and mechanisms, several isosteviol
derivatives were prepared by replacing the 19-COOH with the ure-
ide moiety, and their inhibitory action against HBV in HepG2 2.2.15
cells were evaluated. Among them, NC-8 (2) (ent-16-oxobeyeran-
19-N-methylureido, Scheme 1) was found to be effective against
HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) secretion.
10
14
70.30
125.86
75.18
6.12
–
–
–
24.83
9.36
16
26.13
49.13
5-FUg
–
–
–
Lamivudineh
205
–
a
Compounds 3–9, 11–13, 15, and 17 were not active against HBV in
HepG2 2.2.15 cells.
b
TC50: 50% cytotoxic concentration in HepG2 2.2.15 cells.
c
HBsAg: HBV surface antigen.
HBeAg: HBV e antigen.
IC50: 50% inhibitory concentration.
SI (selectivity index) = TC50/IC50
d
e
f
.
g
5-FU (fluorouracil): positive control of cytotoxicity.
Lamivudine as the positive control.
h
Its IC50 of inhibition to HBsAg secretion was 7.89
more potent than the positive control lamivudine (49.13
l
g/ml, which was
g/ml).
l
Encouraged by the results, the anti-HBV molecular mechanism of
NC-8 (2) was examined using a human hepatoma cell culture sys-
tem. Herein, described are the synthesis and results of anti-HBV
testing, as well as the mode of antiviral action of NC-8 (2).
Results
Synthesis and screening for anti-HBV activity
The preparation of compounds 2–17 was carried out as outlined
in Scheme 1. Initially, the intermediate of isocyanate 1, which was
previously synthesized (Militsina et al., 2007), was treated with the
appropriate amines in dimethylformamide (DMF) in the presence
of triethylamine at room temperature to afford 2–17 (Scheme 1).
These were structurally identified and elucidated based on NMR
and HRESIMS analyses. Subsequently, the synthesized compounds
were evaluated against hepatitis B virus in HepG2 2.2.15 cells. NC-
8 (2) was effective against HBsAg and HBeAg secretion (Table 1),
prompting further investigation of its effect using a human hepa-
toma cell culture system.
Fig. 1. Effect of NC-8 on Huh7 cytotoxicity. Huh7 cells were transfected with the
pHBV1.2 plasmid for 48 h, and then treated with NC-8 (2) (at doses of 2.5–160.0 lg/
ml) for 3 days. Its cytotoxicity was determined by an MTS assay. (n = 4) (⁄p < 0.05 vs.
untreated cells).
Inhibition of HBV HBsAg and HBeAg secretion and viral DNA
intermediates by NC-8
Fig. 2A shows secretion of HBsAg and HBeAg in culture media of
treated Huh7 cells. Production of HBsAg and HBeAg dramatically
dropped with NC-8 (2) treatment in a dose-dependent manner
compared to the vehicle control (p < 0.05). The EC50 of NC-8 (2)
Cytotoxicity of NC-8 to Huh7 cells
Fig. 1 shows that NC-8 (2) caused significant cytotoxic effects
only at doses of >40 lg/ml in HBV-transfected Huh7 cells. How-
ever, it did not exhibit significant cytotoxicity in non-transfected
cells (data not shown) at these concentrations. In the following
experiments, a non-cytotoxic dose (<40
ral treatment of transfected cells.
on HBsAg and HBeAg secretion were 4.8 and 5.9 lg/ml, respec-
tively. To investigate its antiviral activity on HBV DNA production,
Huh7 cells were transiently transfected with the pHBV1.2 viral
genome and treated with NC-8 (2) for 48 h. Cells were harvested,
lg/ml) was used for antivi-
O-Glc-Glc
H+
O
O
RNH2 or R1NH, (C2H5)3N
H
H
H
DMF
H
H
H
CO2-Glc
COOH
isosteviol
NCO
stevioside
1
2: R = CH3 (NC-8)
3: R = C2H5
11: R = p-FC6H4
O
O
12: R = p-ClC6H4
4: R = C3H7
13: R = p-BrC6H4
H
H
H
O
6: R1 = (CH3)2
7: R1 = (C2H5)2
5: R = C4H9
14: R = p-(OH)C6H4
15: R = p-(CH3O)C6H4
16: R = 3,4-di(CH3O)C6H4
17: R = m-(CH3CO)C6H4
8: R = C(CH3)2
9: R = C6H5
H
HN
HN
NHR
NR1
10: R = CH2C6H5
O
Scheme 1. The Synthetic route to compounds 2–17.

T.-J. Huang et al. / Phytochemistry 99 (2014) 107–114
109
Fig. 2. Effects of NC-8 on production of HBV surface antigen (HBsAg), HBV e antigen
(HBeAg), and HBV DNA levels of viral core particles in Huh7 cells. (A) Huh7 cells
were seeded on 6-well plates (3 ꢀ 105 cells/well) in DMEM with 10% FBS and
incubated overnight. Cells were transfected with the pHBV1.2 plasmid for 2 days,
and then treated with NC-8 (2) at non-cytotoxic concentrations (0, 1, 5, and 10 lg/
ml) in medium containing 2% FBS for another 2 days. Culture media were then
collected for HBsAg and HBeAg viral antigen EIA analyses. Data are expressed as the
standard error of the mean. (n = 3) (⁄p < 0.05 vs. untreated cells). (B) Huh7 cells
plated on 60-mm culture dishes (6 ꢀ 105 cells/dish) were transfected and treated
with various concentrations of NC-8 (2) in 2% FBS-containing DMEM for 2 days.
Cells were harvested, and total cellular genomic DNA was extracted and analyzed
Fig. 3. Inhibitory effects of NC-8 on the expression of hepatitis B virus (HBV) genes
and viral promoter activity in Huh7 cells. Huh7 cells were plated on 60-mm dishes.
Transfection and treatment with NC-8 (2) were conducted as described in Fig. 2B.
Treated cells were harvested and subjected to (A) total RNA and (B) protein
isolation. (A) Total RNA from transfected and treated Huh7 cells was subjected to a
Northern blot analysis using HBV DNA as the probe as described in ‘‘Experimental’’.
GAPDH served as an RNA loading control. (B) Total proteins from whole-cell lysates
were separated by SDS–PAGE, followed by a Western blot analysis to detect three
by
a Southern blot analysis using HBV DNA as the probe as described in
‘‘Experimental’’. Data shown are representative of three sets of experiments.
HB surface proteins (LHBs, MHBs, and SHBs) and viral core protein (HBcAg).
a-
and total genomic DNA was isolated. Fig. 2B shows that NC-8 (2)
attenuated the production of HBV viral relaxed circular DNA and
intermediates at doses of >5 lg/ml.
Tubulin served as the protein loading control. Data shown are representative of
three sets of experiments. (C) Huh7 cells were transfected with either a pCore-Luc,
pX-Luc, pS-Luc, or pPreS-Luc HBV gene promoter-luciferase reporter construct
together with a pHBV1.2 HBV whole-genome expression plasmid and pRL-SV40 for
24 h. After transfection, cultures were treated with or without NC-8 (2) (10 lg/ml)
for 2 days. Cells were lysed and subjected to a dual-luciferase assay. Data are
expressed as relative levels of luciferase activity compared to the NC-8 untreated
control. (n = 3) (⁄p < 0.05 vs. untreated cells).
Antiviral activity of NC-8 in HBV-transfected Huh7 cells and viral
promoter activities
The antiviral activity of NC-8 (2) was examined in the HBV-
transfected Huh7 cell line that stably replicated HBV (the adw2
serotype). HBV RNA and viral protein levels were determined by
Northern and Western blot analyses after NC-8 (2) treatment. As
shown in Fig. 3A, expression levels of the 3.5-kb precore/prege-
nomic RNA were slightly reduced during treatment, but the 2.4/
2.1-kb surface antigen RNA was strongly inhibited by NC-8 (2) in
a dose-dependent manner. Consistently, treatment of NC-8 (2) spe-
cifically decreased protein levels of three forms of the surface anti-
gen (LHBs, MHBs, and SHBs) and HBV core protein (HBcAg) in a
dose-dependent manner (Fig. 3B). Furthermore, four viral pro-
moter-reporter plasmids were cloned and constructed for pro-
moter activity assays to understand its molecular mechanism.
These constructs corresponding to the viral core protein (pCore-
Luc), major viral surface antigen (pS-Luc), viral large surface anti-
gen (pPreS-Luc), and viral X protein (pX-Luc) were co-transfected
with the pHBV1.2 plasmid in Huh7 cells. Treatment with NC-8
(2) at a dose of 10 lg/ml strongly suppressed activities of the sur-
face antigen gene promoter (S and PreS) and slightly decreased
those of the core and X promoter (p < 0.05) (Fig. 3C). However,
treatment of HBV non-expressing cells with NC-8 (2) had no effect
on any viral promoter activities (data not shown).
Effects of NC-8 on host cellular signaling pathways
To determine how NC-8 (2) inhibits the effect of HBV on host
cellular signaling pathways, three luciferase reporters containing

110
T.-J. Huang et al. / Phytochemistry 99 (2014) 107–114
binding elements of either NF-jB, AP-1, or the ISRE were co-trans-
fected with the pHBV1.2 plasmid or a control vector into Huh7
cells. After treatment with NC-8 (2) at a dose of 10 g/ml, cells
were harvested and subjected to promoter activity assay.
Fig. 4A shows that NC-8 (2) specifically inhibited NF- B-containing
l
a
j
promoter activity but enhanced AP-1- and ISRE-containing (Fig. 4B,
C, respectively) promoter activity (p < 0.05) when the HBV was ex-
pressed in cells. To our surprise, treatment of HBV-negative Huh7
cells with NC-8 (2) had the same effect on promoter activities.
The latter activities of NC-8 (2)-treated HBV non-expressing cells
were lower in the NF-
jB reporter, but higher in the AP-1 and ISRE
reporters compared to untreated control.
Effects of NC-8 on NF-
jB, IjB, and TLR2 protein expressions in
HBV-transfected or non-transfected Huh7 cells
In view of the key role of the NF-jB signaling pathway in HBV
gene regulation and viral genome replication in HBV infection
(Doria et al., 1995; Hildt et al., 1996; Kwon and Rho, 2002; Lin
et al., 2009; Meyer et al., 1992), Huh7 cells-transfected with or
without the pHBV1.2 plasmid were treated with NC-8 (2) followed
by Western blot analysis. Fig. 5A shows that HBV non-transfected
Huh7 cells expressed a low level of NF-
(10 g/ml) did not affect its expression. In contrast, expressions of
the two forms of the NF- B protein (p65/p50) significantly in-
jB, and NC-8 (2) treatment
l
j
creased in Huh7 cells transfected with the HBV genome. Nonethe-
less, increasing the treated dose of NC-8 (2) of HBV-expressing
Huh7 cells greatly reduced NF-
pare correspondence with the expression of NF-
inhibitor of the I protein was also detected by an immunoblot
analysis. Results showed that treatment of HBV-negative Huh7
cells with NC-8 (2) slightly decreased I protein expression.
However, transfection of HBV into cells dramatically reduced I
protein levels. In addition, treatment of HBV-expressing cells with
NC-8 (2) significantly increased expression of I in a dose-
jB protein levels (Fig. 5A). To com-
jB, the NF-
jB
jBa
jBa
jBa
jBa
dependent manner (Fig. 5A). By contrast, TLR2 was previously de-
tected in Huh7 cells (Preiss et al., 2008), which was speculated to
be an activator of NF-jB in the cellular innate immune response
during HBV infection (Thompson et al., 2009). Thus, the expression
of TLR2 was examined by immunoblotting to determine the poten-
tial factors of the NF-jB signaling pathway regulated by NC-8 (2).
Fig. 5B shows that expression levels of TLR2 protein were not al-
tered by NC-8 (2) in HBV non-transfected Huh7 cells. In cells trans-
fected with the HBV genome, expression of the TLR2 protein
significantly increased, whereas NC-8 (2) treatment dramatically
reduced its levels in a dose-dependent manner.
Effect of NC-8 on DNA-binding activity of NF-
gene
jB in the HBV surface
Several putative NF-
genome (Lin et al., 2009). Among them, a specific NF-
j
B-binding sites were identified in the HBV
B-binding
j
site had a positive regulatory role in HBV gene expression. The site
is located in the HBV genome at nt207 to 216 (CS1) which corre-
sponds to the viral surface gene coding region. To examine
whether NC-8 (2) regulated viral gene expression by NF-jB as
mediated through the CS1 site, nuclear extracts were prepared
from cells with and without HBV transfection and were tested
for the presence of NF-
The results showed that transfection of the HBV genome signifi-
cantly increased the binding activity of NF- B to the HBV CS1 site.
However, NC-8 (2) slightly decreased NF- B DNA-binding activity
in non-HBV-transfected nuclear extracts. In contrast, NC-8 (2) dra-
matically reduced the DNA-binding activity of NF- B to the CS1
jB to CS1 site-binding activity by an EMSA.
Fig. 4. Cellular signaling pathways were modulated by NC-8. Huh7 cells were
transfected with either (A) pNF- B-Luc, (B) pAP1-Luc, or (C) pISRE-Luc promoter–
reporter constructs together with or without the pHBV1.2 plasmid and treated with
or without NC-8 (2) as described in Fig. 3. Data are expressed as relative levels of
luciferase activity compared to the NC-8 (2) untreated control. (n = 3) (⁄p < 0.05).
j
j
j
j
was constructed and transfected into Huh7 cells with or without
HBV expression. The transfections were then treated with or
site (Fig. 6A). To further examine the possibility of an effect of
NC-8 (2) on HBV CS1-containing promoter activity, p5CS1-Luc

T.-J. Huang et al. / Phytochemistry 99 (2014) 107–114
111
Fig. 5. Effects of NC-8 on NF-jB p65/p50, IjBa, and TLR2 protein expressions in
Huh7 cells. Huh7 cells were seeded in 60-mm culture dishes and then transfected
with or without the pHBV1.2 plasmid for 2 days. After treatment with or without
NC-8 (2) (1–10
were isolated for a Western blot analysis to determine expression levels of (A) NF-
B p65/p50, I , or (B) TLR2 proteins. -Tubulin protein expression served as the
lg/ml) for another 2 days, cells were harvested, and total proteins
j
j
Ba
a
loading control. Data shown are representative of three sets of experiments.
without NC-8 (2) for 2 days and tested for luciferase activity. Re-
sults showed that CS1-containing promoter activity increased in
HBV-expressing cells, but not in HBV-negative cells. Furthermore,
treatment with NC-8 (2) significantly decreased CS1-containing
promoter activity in HBV-expressing cells, but not in HBV-negative
cells (Fig. 6B).
Fig. 6. Effects of NC-8 on (A) DNA-binding activity of NF-
CS1 site-containing promoter activity. (A) Huh7 cells were transfected with or
j
B to the CS1 site and (B)
without the pHBV1.2 plasmid for 2 days. After transfection, cells were treated with
or without NC-8 (2) (10 lg/ml) for another 2 days. Cells were harvested, and
Discussion
nuclear extracts were prepared for a binding assay. (B) Huh7 cells were transfected
with either the p5CS1-Luc promoter–reporter constructs together with or without
the pHBV1.2 plasmid for 24 h and treated with or without NC-8 (2) for 2 days. Data
are expressed as relative levels of luciferase activity compared to the NC-8
untreated control or pHBV1.2 non-transfected control. (n = 3) (⁄p < 0.05).
Developing anti-HBV agents with novel modes of action to alle-
viate problems associated with currently approved drugs is a major
challenge (Wakui et al., 2010). The current study found that NC-8
(2) exhibited unique anti-HBV activity by decreasing expressions
of HBV DNA, RNA, and protein in Huh7 cells that expressed the
HBV viral genome. Among them, four HBV viral transcripts de-
creased (Fig. 3A). NC-8 (2) potently eliminated expressions of three
forms of the viral surface antigen and core protein in Huh7 cells
(Fig. 3B). In addition, a Southern blot analysis indicated that NC-
8 (2) significantly inhibited the production of encapsidated DNA
intermediates of HBV particles (Fig. 2B). Therefore, it can be spec-
ulated that NC-8 (2) might suppress viral gene expression by mod-
ulating viral promoter activity. Further investigation demonstrated
that activities of the four viral gene promoters (Core, X, S, and PreS)
were abolished by NC-8 (2) in Huh7 cells that expressed the repli-
cating HBV genome (Fig. 3C); in particular, viral S promoter activity
was dramatically reduced. In contrast, this compound did not alter
the viral promoter activity in HBV non-transfected cells (data not
shown). These results suggest that HBV viral proteins may serve
as auto-regulators of viral gene expression itself and disturb this
action. In addition, NC-8 (2) seems to have a potential role in viral
surface gene regulation. Previous studies showed that HBcAg and
HBx were essential to HBV gene regulation (Kwon and Rho,
2002; Tang et al., 2005). To examine the possible mechanism by
which NC-8 (2) affects the function of HBV in host cellular signal-
ing pathways, transcriptional activities of three major signaling
promoter-reporter assay. After transfection, NC-8 (2) specifically
decreased NF-jB promoter activity, while somehow slightly
increasing AP-1 and ISRE promoter activities in HBV-transfected
and non-transfected cells (Fig. 4), suggesting that HBV infection
might be required for the actions of these transcription factors
on promoter activity-related genes and that NC-8 (2) could inter-
fere this regulation. Previous reports showed that several DNA-
binding elements for NF-jB are essential for HBV viral gene regu-
lation. Even so, a study by Kwon and Rho (2002) clearly demon-
strated that the HBC core protein (HBc) upregulates the HBV
enhancer II/pregenomic promoter through the NF-
located upstream of the promoter. Therefore, the importance of
NF- B in HBV gene regulation is the main issue we addressed. To
address the role of NF- B in NC-8 (2)-regulated HBV viral promoter
activity, the specific NF- B activation inhibitor, BAY 11-7085, was
jB binding site
j
j
j
used to examine this possibility. When Bay11-7085 was co-treated
with NC-8 (2) in HBV-expressing cells after transfection, inhibitory
effect on viral surface gene promoter activity was reversed to a
non-treatment level (unpublished data), thus suggesting that the
action of NC-8 (2) on HBV viral surface gene regulation might be
mediated by NF-jB’s activation.
Prior studies showed that the innate immune response serves
pathways, i.e., NF-jB, AP-1, and ISRE, were analyzed using a
as the first line of host defense against exogenous pathogen

112
T.-J. Huang et al. / Phytochemistry 99 (2014) 107–114
infections, which is via recognition of pathogen-associated molec-
ular patterns. In addition, TLRs are the primary pathogen pattern-
recognition receptors involved in innate immunity. Activation of
the TLR signaling pathway is induced by dimerization of receptors
on the surfaces of cell membranes, which in turn triggers MyD88
activation, leading to the nuclear translocation and activation of
Conclusions
In summary, it’s hypothesized that NC-8 (2) initially inhibits
HBV RNA production by decreasing transcription of viral RNAs
through downregulating levels of host TLR2 and NF-jB signaling
factors required for transcription of viral genes. It was noted that
downregulation of viral gene expression by NC-8 (2), especially of
the HBV Core and X genes, disrupted feedback activation of HBV
gene expression and further decreased viral RNA transcription. In-
deed, this process is required for viral Core and X proteins to en-
hance the initiation of pregenomic 3.5-kb RNA (Hildt et al., 1996;
Kwon and Rho, 2002). However, interactions of viral proteins with
NF-jB, and this regulates cellular gene expression and immune re-
sponses (Takeda and Akira, 2005). Among TLR members, TLR2 was
found to be involved in the inhibitory effects of interleukin (IL)-1
on HBV replication (Thompson et al., 2009) that was shown to be
expressed by Huh7 and HepG2 cells (Preiss et al., 2008). Thus, host
TLR2/NF-
tigated in an HBV-dependent manner. Results indicated that NC-8
(2) specifically reduced expressions of NF- B and TLR2 proteins in
jB signaling pathways inhibited by NC-8 (2) were inves-
NF-jB and the contribution to S gene promoter activation are still
j
unknown. The current study indicated that the mode of antiviral ac-
tion of NC-8 (2) is distinct from that of typical HBV reverse-trans-
criptase/polymerase inhibitors and other inhibitors that activate
HBV-expressing Huh7 cells, but not in HBV-negative Huh7 cells
(Fig. 5). This implies that an inhibitory mode of NC-8 (2) on the
NF-jB signaling pathway is through modulation of HBV gene
the TLR2/NF-
8 (2) on HBV infectious disease is not only via a unique signaling
pathway (TLR2/NF- B) but also other pathways like AP-1 and ISRE
transcription factors. Mechanisms of AP-1 and ISRE exerted by NC-8
(2) to regulate HBV gene expression therefore warrant future inves-
tigation. To our knowledge, this is the first report of an isosteviol
analog exerting an inhibitory effect on the hepatitis B virus.
jB signaling pathway. Meanwhile, the action of NC-
expression. Previous studies showed that transcriptional activators
like HBx (Doria et al., 1995), HBcAg (Kwon and Rho, 2002), a C-ter-
minally truncated form of the middle-sized HBV surface antigen
(MHBst) (Meyer et al., 1992), and HB large surface antigens (LHBs)
j
(Hildt et al., 1996) are capable of activating NF-
clearly suggests that NC-8 (2) specifically downregulates TLR2
and NF- B expressions in HBV-infected hepatoma cells in addition
to viral gene regulation. Lin et al. (2009) indicated that one NF-
jB. Thus, this
j
jB
Experimental
binding site (CS1, nt207 to 216) was identified in the HBV surface
gene open reading frame (ORF) and proposed that this site can be
Chemistry
upregulated by NF-
jB. Thus, to examine the possible role of NC-8
(2) in binding NF- B to the CS1 site, its inhibitory effect on the sur-
face gene promoter mediated through this element was examined
j
General
The instrumental analysis was described previously (Chang
et al., 2008, 2009).
and analyzed by an EMSA. Results indicated that NC-8 (2) signifi-
cantly decreased binding activity of NF-jB to the CS1 site in nucle-
ar extracts of HBV-expressing cells (Fig. 6A). Furthermore,
transfection of HBV into cells obviously increased CS1-containing
promoter activity, and NC-8 (2) reduced this promoter activity
(Fig. 6B). These results suggest that viral proteins can transactivate
surface genes and are inhibited by NC-8 (2) through binding of NF-
Synthesis of NC-8 (2) and anti-HBV assay
Isosteviol was obtained by acid hydrolysis of stevioside
(Scheme 1) as described previously (Avent et al., 1990). Isocyanate
(1) was synthesized as described previously (Militsina et al., 2007)
and characterized by NMR and HRESIMS analyses. Subsequently,
methylamine (4 mmole) and Et3N were added to a solution of 1
(0.3 mmole) in DMF (5 ml). The reaction mixture was stirred over-
night at room temperature. After addition of 1 N HCl, the resulting
white precipitate was collected and crystallized from H2O–MeOH
to afford NC-8 (2) in 85% yield.
The same experimental procedure was adopted for the prepara-
tion of compounds 3–17. The spectroscopic data of these com-
pounds were given in Supplementary data. The anti-HBV activity
was performed following procedures described previously (Cheng
et al., 2011).
jB to the CS1 site. This study results thus suggested that HBV can
utilize the TLR2/NF- B signaling pathways as a major factor in viral
j
gene regulation and virus propagation, whereas a discrepancy of
anti-HBV activity on IL-1 arose in a comparative study by Preiss
et al. (2008). They proposed that the inhibitory effect of IL-1 on
HBV replication was mediated through activation of TLR2. How-
ever, elevated expressions of TLR2 and NF-jB in hepatocytes in-
fected with HBV seemed to be critical to HBV gene upregulation
and viral replication in our study. A possible explanation is that
the early phase of an immediate HBV infection will trigger an in-
nate immune response including TLR2 activation, lead to nuclear
translocation of preexisting NF-jB, and inhibit HBV replication.
4-Isocyanato-19-nor-ent-16-oxobeyeran (1). 1H NMR (500 MHz,
C5D5N): d 2.66 (dd, J = 18.5, 3.6 Hz, 1H), 1.79 (d, J = 18.5 Hz, 1H),
1.52–1.61 (m, 5H), 1.41–1.46 (m, 3H), 1.17–1.38 (m, 7H), 1.24 (s,
3H), 1.01 (s, 3H), 0.98 (d, J = 4.1 MHz, 1H), 0.89 (s, 3H), 0.84 (d,
J = 11.6 MHz, 1H), 0.69 (td, J = 11.4, 3.3 MHz, 1H); 13C NMR
(125 MHz, C5D5N): d 220.5, 122.5, 59.4, 54.7, 54.4, 54.3, 48.9,
48.5, 41.3, 40.2, 39.2, 38.3, 37.5, 37.2, 31.6, 20.2, 20.1, 20.0, 18.2,
14.0; HRESIMS m/z 316.2272 [M+H]+ (calcd for C20H30NO2,
316.2277).
In the long run, the production of viral proteins (i.e., Core and X
proteins) is essential for upregulation of viral genes (Kwon and
Rho, 2002; Tang et al., 2005). Unidentified cellular signaling factors
are transactivated by viral proteins, induce novel synthesis of TLR2
and NF-jB, and then provide feedback that augments viral gene
expression and viral replication. This suggests that HBV may take
over host signaling pathways and switch this regulatory machinery
toward its own gene expression and genome replication after
infection, despite inhibitory effects of the TLR2/NF-jB signaling
pathway on HBV in early stages of infection. Several issues still re-
main to be elucidated in the future, including how HBV regulates
cellular c-Jun/c-Fos and Jak-Stat signaling pathways and finally
ent-16-Oxobeyeran-19-N-methylureido (NC-8) (2)
26
White crystals; m.p. 251–253 °C; [
a
]
-77.5 (c 0.55, CH3OH);
D
activates AP-1 and ISRE in addition to NF-
j
B; how NF-
j
B is in-
1H NMR (500 MHz, C5D5N): d 6.75 (br s, 1H), 4.77 (s, 1H), 3.29
(br d, J = 13.4 MHz, 1H), 2.85 (d, J = 4.7 MHz, 3H), 2.47 (dd,
J = 18.4, 3.5 MHz, 1H), 1.27–1.75 (m, 13H), 1.59 (s, 3H), 1.00–1.15
(m, 3H), 1.00 (s, 3H), 0.90 (d, J = 12.2 MHz, 1H), 0.87 (s, 3H), 0.77
(m, 1H); 13C NMR (125 MHz, C5D5N): d 220.9, 159.0, 56.7, 54.9,
volved in regulating viral gene promoter activity other than the S
gene; and how HBV switches the regulatory machinery and takes
over cellular signaling pathways in the early or late phase of
infection.

T.-J. Huang et al. / Phytochemistry 99 (2014) 107–114
113
54.9, 54.2, 48.7, 48.6, 41.0, 39.5, 39.3, 37.7, 37.3, 37.2, 28.3, 26.8,
20.2, 20.1, 19.6, 18.2, 15.1; HRESIMS m/z 347.2658 [M+H]+ (calcd
for C21H35N2O2, 347.2699).
RIPA (10 mM Tris–HCl, 150 mM NaCl, 5 nM EDTA, 0.1% SDS, 1.0%
Nonidet P-40, and 0.25% deoxycholate) buffer containing 1 mM
phenylmethylsulfonyl fluoride (PMSF), 25 ng/ml aprotinin, and
5 ng/ml leupeptin protease inhibitors. Suspensions were subjected
to shearing several times using a syringe with a 25-gauge needle,
and then centrifuged at 14,000 rpm for 20 min at 4 °C. Cellular pro-
tein concentrations were determined by a bicinchoninic acid (BCA)
protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA), and
Bioassays
Materials
Dulbecco’s modified Eagle’s medium (DMEM), OPTI-MEM, and
fetal bovine serum (FBS) were purchased from GIBCO/BRL (Gaithers-
burg, MD, USA). Enzyme immunoassay (EIA) kits for HBsAg and
HBeAg were obtained from Johnson and Johnson (Skillman, NJ,
USA). The anti-HBsAg and TLR2 antibodies for the immunoblot anal-
ysis were purchased from GeneTex (San Antonio, TX, USA). The anti-
25 lg of protein from each sample was applied to SDS–PAGE. Pro-
teins were transferred onto polyvinylidene difluoride (PVDF) mem-
branes (Amersham, GE Healthcare, Buckinghamshire, UK) by
electroblotting, and then blocked for 1 h with PBST (PBS buffer
with 0.05% Tween-20) containing 4% nonfat milk. The immunoblot
was incubated with a primary antibody followed by a horseradish
peroxidase (HRP)-conjugated secondary antibody. The same mem-
HBcAg, anti-NF-jB (p65/p50), and anti-IjBa antibodies were pur-
chased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The
anti-preS2 protein antibody of HBV was purchased from Chemicon
(Millipore, Billerica, MA, USA). The DIG high prime DNA labeling
and detection starter kit was purchased from Roche (Mannheim,
Germany). Power SYBR Green PCR master mix was purchased from
Applied Biosystem (Foster City, CA, USA). A dual-Luciferase reporter
assay kit was purchased from Promega (Madison, WI, USA). The Tri-
zolÒ total RNA isolation solution and Lipofectamine 2000 transfec-
tion reagent were purchased from Invitrogen (Carlsbad, CA, USA).
The QIAquick Gel extraction kit was purchased from Qiagen (Valen-
branes were then stripped and reprobed with an anti-
antibody (Sigma) as the loading control.
a-tubulin
Analysis of intracellular HBV-RNA by Northern blotting
Total RNA from treated cells was extracted using the TrizolÒ iso-
lation buffer (Invitrogen) according to the manufacturer’s protocol.
Total RNA (5 lg) was denatured, separated on a 1.0% agarose gel
(Amresco), and transferred to a positively charged Hybond-N+ ny-
lon membrane (Amersham Biosciences, Bucks, UK). After UV cross-
linking, the membrane was hybridized with a DIG-labeled full-
length HBV genome probe, washed, and exposed to X-ray film.
Briefly, the full-length HBV probe was produced by restriction
enzymatic digestion of the pHBV2 plasmid with EcoRI (Will et al.,
1985), purified, labeled with a DIG high prime DNA labeling kit
(Roche, Mannheim, Germany). The total RNA amount was normal-
ized by hybridization of the membrane with a glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) probe, which was produced
by Pst I digestion of the pGAPDH plasmid (provided by Dr. Hsiao-
Sheng Liu from the Graduate Institute of Microbiology and Immu-
nology, National Cheng-Kung University, Tainan, Taiwan).
cia, CA, USA). pNF-jB-Luc, p-activator protein (AP)-1-Luc, and p-
interferon-sensitive response element (ISRE)-Luc promoter-lucifer-
ase reporter constructs from Stratagene (La Jolla, CA, USA) were pro-
vided by Dr. Cheng-Wen Lin. Other reagents were purchased from
GIBCO/BRL (Invitrogen, Carlsbad, CA, USA), Sigma (St. Louis, MO,
USA), Bio-Rad (Hercules, CA, USA), Merck (Darmstadt, Germany),
J.T. Baker (Mansfield, MA, USA), and Amresco (Solon, OH, USA).
NC-8 was pre-dissolved in dimethyl sulfoxide (DMSO).
Cell culture and bioactivity assays
Huh7 cells were maintained in DMEM supplemented with heat
inactivated 10% (v/v) FBS (GIBCO/BRL, Invitrogen) and 1% antibiotics
at 37 °C in a humidified 5% CO2 incubator as described previously
(Lu et al., 1995). Initially, cells seeded at a density of 4 ꢀ 104/ml
and grown in various cultural dishes at 80% of confluence were used
in all experiments. In routine compound treatments, cells were trea-
ted with serially diluted concentrations of NC-8 (2) for 2–3 days.
After treatment, levels of the viral surface antigen (HBsAg) and e
antigen (HBeAg) in the culture media were measured by an EIA kit
(Johnson and Johnson) according to the manufacturer’s instruc-
tions. In another set of antiviral experiments, Huh7 cells were plated
in 60-mm culture dishes, transfected with the pHBV1.2 plasmid,
and treated with NC-8 (2) in 2% FBS for 48 h. Treated cells were sub-
jected to total RNA, DNA, and protein extraction.
Southern blot analysis of intracellular HBV-DNA synthesis
Encapsidated viral DNA was extracted from intracellular core
particles as described by Pugh et al. (1988), fractionated on 1.0%
agarose gels and transferred onto a Hybond N+ membrane (GE
Healthcare). HBV DNA was detected by a Southern blot analysis
using the DIG-labeled full-length HBV probe.
Plasmid construction
All of the constructs were produced by standard recombinant
DNA techniques (Sambrook et al., 1989). The pHBV1.2 plasmid
containing a head-to-tail dimer of the HBV adw2 serotype was
cloned into the EcoRI site of pGEM-7Zf (+) (Promega, Madison,
WI, USA) (Blum et al., 1991), which was kindly provided by Dr.
Cheng-Chan Lu (Department of Pathology, National Cheng-Kung
University). To construct the four viral promoter-reporter plas-
mids, HBV genomic fragments corresponding to the Core
Cell viability assay
The cytotoxic effect of NC-8 (2) was determined by a CellTiter 96Ò
AQueous one solution cell proliferation assay kit (MTS) (Promega,
Madison, WI, USA) to pinpoint the non-toxic test compound concen-
tration in Huh7 cells. Briefly, Huh7 cells transfected with or without
the pHBV1.2 plasmid for 48 h were treated with serial dilutions of
(nt1636–1851),
S (nt3114–220), PreS (nt2438–2855), and X
(nt1071–1357) gene promoter regions were amplified by a poly-
merase chain reaction (PCR) using the pHBV1.2 plasmid as a tem-
plate and were subsequently inserted into the SacI/XhoI sites of the
pGL4.17 luciferase-reporter expression vector (Promega). The re-
porter plasmid, p5CS1-Luc, which contains five copies of the CS1
site (nt207–216) of the HBV adw serotype genome, was con-
structed from the pGL4.17 expression vector. All of the DNA se-
quences were verified with appropriate restriction enzyme
digestion and direct sequencing.
NC-8 (2) ranging 2.5–160 lg/ml, and the toxicity of cells was mea-
sured according to the manufacturer’s protocol. All measurements
were performed in four replicates, and results are presented as rela-
tive percentages over that of the control group. Non-cytotoxic con-
centrations were used in the antiviral activity assays.
Sodium dodecylsulfate (SDS)–polyacrylamide gel electrophoresis
(PAGE) and Western blot analyses of intracellular viral antigens and
cellular signaling proteins
Transient transfection and luciferase assay
Whole-cell lysates of Huh7 were prepared by washing cells in
phosphate-buffered saline (PBS) three times and suspending in
Huh7 cells were transfected with various plasmids using the
Lipofectamine 2000 transfection reagent (Invitrogen). Briefly, cells

114
T.-J. Huang et al. / Phytochemistry 99 (2014) 107–114
(at 4 ꢀ 104/ml) were plated in a 24-well culture plate and transfec-
ted in DMEM supplemented with 10% FBS for 24 h, washed with
1ꢀ PBS, incubated in DMEM supplemented with 2% FBS, and then
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This work was funded by grants from the Committee on Chi-
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Yuan, Taiwan (CCMP101-RD-015), China Medical University
(CMU99-N2-03-2 and CMU101-S-19), and the National Science
Council of Taiwan (NSC98-2320-B038-011-MY3). The authors ex-
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pHBV2 plasmids, and to Dr. Hsiao-Sheng Liu for the pGAPDH
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Appendix A. Supplementary data
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A., Visvanathan, K., Locarnini, S., 2009. Stimulation of the interleukin-1 receptor
and Toll-like receptor 2 inhibits hepatitis B virus replication in hepatoma cell
lines in vitro. Antivir. Ther. 14, 797–808.
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.phytochem.
2013.12.014.
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