Design, synthesis, molecular docking studies and anti-HBV activity of phenylpropanoid derivatives

Article abstract of DOI:10.1016/j.cbi.2016.03.011

In this work, a series of phenylpropanoid derivatives were synthesized, and their anti-hepatitis B virus (HBV) activity was evaluated. Most of the synthesized derivatives showed effective anti-HBV activity. And compound 4d-3 showed the most effective anti-HBV activity, performing strong potent inhibitory not only on the secretion of HBsAg (IC₅₀ = 58.28 μM, SI = 23.26) and HBeAg (IC₅₀ = 97.21 μM, SI = 13.95), but also on the HBV DNA replication (IC₅₀ = 42.28 μM, SI = 32.06). The structure-activity relationships (SARs) of the derivatives had been discussed, which were useful for developing phenylpropanoid derivatives as novel anti-HBV agents. Moreover, the docking study of all synthesized compounds inside the HLA-A protein (PDB ID: 3OX8) active site was carried out to explore the molecular interactions and a molecular target for activity and a modified assay method measuring the interaction between our derivatives and HBcAg was investigated, indicating that the HBV core protein might be their potential target for anti-HBV. This study identified a new class of potent non-nucleoside anti-HBV agents.

Full text of DOI:10.1016/j.cbi.2016.03.011

Accepted Manuscript
Design, synthesis, molecular docking studies and Anti-HBV activity of
phenylpropanoid derivatives
Sheng Liu, Yubin Li, Wanxing Wei, Kuiwu Wang, Lisheng Wang, Jianyi Wang
PII:
S0009-2797(16)30072-2
10.1016/j.cbi.2016.03.011
CBI 7613
DOI:
Reference:
To appear in: Chemico-Biological Interactions
Received Date: 13 December 2015
Revised Date: 20 February 2016
Accepted Date: 4 March 2016
Please cite this article as: S. Liu, Y. Li, W. Wei, K. Wang, L. Wang, J. Wang, Design, synthesis,
molecular docking studies and Anti-HBV activity of phenylpropanoid derivatives, Chemico-Biological
Interactions (2016), doi: 10.1016/j.cbi.2016.03.011.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, Synthesis, Molecular Docking Studies and Anti-HBV Activity of
Phenylpropanoid Derivatives
1
,2
1
2
3
2
2
Sheng Liu , Yubin Li , Wanxing Wei * , Kuiwu Wang , Lisheng Wang , Jianyi Wang
1
College of Science, Guangdong Ocean University, Zhanjiang, 524088, P. R. China
2
Department of Chemistry, Guangxi University, Nanning, 530004, P. R. China
3
School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou
3
10035, P. R. China
To whom correspondence should be addressed:
Wanxing Wei
Department of Chemistry, Guangxi University, Nanning, 530004, China
Telephone number: 86-771-3233718, Fax number: 86-771-3272601.
E-mail address: wxwei@gxu.edu.cn
1

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Abstract: In this work, a series of phenylpropanoid derivatives were synthesized, and their
anti-hepatitis B virus (HBV) activity was evaluated. Most of the synthesized derivatives
showed effective anti-HBV activity. And compound 4d-3 showed the most effective anti-HBV
activity, performing strong potent inhibitory not only on the secretion of HBsAg (IC50
=
58.28ꢀM, SI=23.26) and HBeAg (IC = 97.21 ꢀM, SI=13.95), but also on the HBV DNA
50
replication (IC = 42.28 ꢀM, SI = 32.06). The structure-activity relationships (SARs) of the
5
0
derivatives had been discussed, which were useful for developing phenylpropanoid
derivatives as novel anti-HBV agents. Moreover, the docking study of all synthesized
compounds inside the HLA-A protein (PDB ID: 3OX8) active site was carried out to explore
the molecular interactions and a molecular target for activity and a modified assay method
measuring the interaction between our derivatives and HBcAg was investigated, indicating
that the HBV core protein might be their potential target for anti-HBV. This study identified a
new class of potent non-nucleoside anti-HBV agents.
Keywords: Synthesis, Phenylpropanoid derivatives, Anti-HBV activity, Structure-activity
relationships, Molecular docking
2

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1
. Introduction
Hepatitis B virus (HBV), a member of Hepadnaviridae family, is a human pathogen that
causes acute and chronic hepatitisa and may lead to lifelong infection, cirrhosis,
hepatocellular carcinoma, liver failure, or death [1,2]. HBV infects chronically over 350
million people globally with more than 1 million deaths annually [3]. Currently, therapies
including immunomodulator, interferons (interferon-alpha and pegylated interferon), and
nucleoside drugs (lamivudine, adefovir dipivoxil, entecavir, telbivudine and tenofovir) for
treating HBV are still unsatisfactory [4,5]. Interferons is effective only in less than 30% of the
chronic carriers and about 50% of interferons-treated patients experience recurrence of
viremia after cessation of treatment [6,7]. Nucleoside drugs, whose major target is to inhibit
the viral reverse transcriptase (RT)/DNA polymerase, are effective in suppressing the viral
replication for extended periods, while drug resistance remains a critical issue due to the
mutation of DNA polymerase [8,9]. Therefore, new classes of potent antiviral compounds
with different antiviral targets and mechanisms seem highly desirable.
HBV contains a 3.2 kb partially double-stranded DNA genome with four open reading
frames (ORFs) encoding four viral proteins including core protein (Cp), surface protein (S),
polymerase (P), and X protein (X) [10]. Core protein mainly constitutes the nucleocapsid
which harbors the viral DNA and polymerase [11]. It was reported that dominant negative
core protein mutants, which could not support pregenomic RNA packaging and genome
maturation, could effectively inhibit replication HBV [12]. Peptides that could inhibit the
assembly of core proteins and intracellular single chain antibodies against HBV core protein
could also inhibit HBV replication [13,14]. These observations suggest that the assembly
3

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process of core protein could serve as a target for anti-HBV agents.
In our previous study, we found that lignans niranthin, nirtetralin, nirtetralin A and
nirtetralin B isolated from Phyllanthus niruri L. exhibited significant anti-HBV activity
[
15-17]. We investigated the potential anti-HBV targets of the anti-HBV lignans with reverse
docking approach using fifteen HBV related proteins and RNA including human leukocyte
antigen HLA-A*02:03 (PDB ID: 3OX8), human leukocyte antigen HLAA*02:06 (PDB ID:
3
OXR), human leukocyte antigen HLA-A*02:07 (PDB ID: 3OXS), hepatitis B virus preS1
protein (PDB ID: 3ZHF), hepatitis B virus preS2 surface antigen (PDB ID: 1WZ4), human
hepatitis B virus surface antigen HzKR127 (PDB ID: 2EH8), human hepatitis B virus
e-antigen (PDB ID: 3V6F, 3V6Z), Hepatitis B X-interacting protein HBXIP (PDB ID: 3MS6,
4
WZR, 4WZW), HBV RNA polymerase (PDB ID: 2HN7), and human hepatitis B virus
encapsidation signal (PDB ID: 2IXY, 2K5Z). The reverse docking results showed that HLA-A
protein (PDB ID: 3OX8) may be the potential target for anti-HBV.
The HBV core 18-27 peptide (HBcAg18-27) was originally described as a human
leukocyte antigen (HLA)-A epitope and detected in more than 90% of human leukocyte
antigen (HLA)-A protein which was associated with severe liver inflammation in Chinese
patients with chronic HBV infection [18]. The crystal structure of HLA-A protein (PDB ID:
3
OX8) was investigated to be a fragment of HBV core protein and could serve as a target for
anti-HBV agents [19].
In order to increase the activity of the lignans, we designed and synthesized series of
analogues based on the reverse docking results and combination principle. HLA-A protein
(
PDB ID: 3OX8) was used as molecular targets for anti-HBV activity of the analogues in a
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moe-docking technique. And the same structural fragments that the anti-HBV active lignans
possess ((E)-3-(3,4-dimethoxyphenyl)acrylic acid, (E)-3-(3,4,5-trimethoxyphenyl)acrylic acid,
(
E)-3-(benzo[d][1,3]dioxol-5-yl)acrylic acid and (E)-3-(7-methoxybenzo[d][1,3]dioxol-5-yl)
acrylic acid) were used as the main scaffold for the design and synthesis of novel compounds
as potent anti-HBV agents. As a part of our continuous search for active anti-HBV leads from
synthetic compounds, some phenyl acryloyl type oxime esters derivatives with heterocycle
have been reported [20]. We found that the lignans were observed to exhibit low cytotoxicity
and effective anti-HBV activity while the cytotoxicity increased after heterocycle were
introduced into the derivatives. Then, the phenyl acryloyl type oxime esters derivatives also
showed anti-HBV activity and our docking study shown that the oxime ester group could
interact with the protein [20]. In the current studies, it was of interest to know how the oxime
ester group maintained the anti-HBV activity by simplifying the structure of the active
lignans with an oxime ester group.
In this paper, we reported the synthesis, SARs, molecular docking study, and anti-HBV
activity of phenylpropanoid derivatives by modification of ester group heterocycle part with
the structural fragment of the active lignans. Thus, the two fragments of the active lignans
was connected and simplified by an oxime ester group. For further study, an assay was
investigated to explore the interactions between the derivatives and the protein.
2
2
. Results and Discussion
.1. Chemistry
General synthesis for the intermediate and target compounds is depicted in Scheme
1
. Substituted benzaldehyde was reacted with hydroxylamine hydrochloride in EtOH
5

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in the presence of sodium acetate to yield oxime 1-3 in a good yield [21]. Intermediates
2
a-d were prepared by Knoevenagel condensation of malonic acid and the aldehyde
group of four benzaldehydes with yields of 80%-90% [22].The final oxime ester derivatives
4a-1~ 4a-3), (4b-1~ 4b-3), (4c-1~ 4c-3), (4d-1~ 4d-3) were obtained by reaction of oxime
(
with cinnamoyl chloride 3a-d in the presence of TEA, which were obtained by reaction of
substituted phenylacrylic acid 2a-d and thionyl chloride in DCM [23].
The structures of the newly synthesized compounds (4a-1~ 4a-3), (4b-1~ 4b-3), (4c-1~
1
13
4
c-3), (4d-1~ 4d-3) were characterized by H NMR, CNMR and MS data and their data are
1
presented in the supplementary material. H NMR spectra of the derivatives showed a singlet
at about 8.35 ppm corresponding to N=CH proton. Two doublets at 6.29-6.47 and 7.57-7.80
ppm with J=15.57-15.93 Hz corresponding to trans hydrogens of CH=CH respectively. The
singlet at 3.89-3.95 ppm attributed to O-CH protons, and at 6.00-6.05 corresponding to
3
OCH O protons. The chemical shifts of aromatic hydrogens of the phenyl ring appeared as
2
1
3
multiplets in the region δ 6.74-7.25. C NMR chemical shifts for title compounds were
1
3
observed in their expected regions. C NMR spectrum for the derivatives showed signals at
5
5.87-56.92, 101.52-102.35, 155.76-156.70 and 164.53-167.66 corresponding to CH , CH ,
3 2
C=N and C=O, respectively.
2
.2. QSAR Study
The 3D structures of all the compounds were generated using the Built Optimum option of
Hyperchem software (version 8.0), and subsequently energy minimized using MM+ force
field. Then, the structures were fully optimized. Molecular descriptors were determined by
6

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QSAR study, including logP, molar refractivity, surface area, volume, hydration energy and
polarizability. And the results showed that all molecules had drug like properties (Table 1). All
the compounds have the molecular weight ranging from 350 to 450 Da. The log P values of
these compounds are superior to act as drug which is -0.14 to -3.39 and the molar refractivity
is in the range of 101-121.
2
.3. Molecular Docking
Molecular docking studies of phenylpropanoid derivatives were carried out using MOE
008.10 as the docking software in order to rationalize the biological activity results and
2
understand the various interactions between ligand and protein in the active site in detail. The
crystal structure of HLA-A protein (PDB ID: 3OX8) was used for docking study and the ‘Site
Finder’ tool of the program was used to search for its active site. We performed three docking
procedures for each ligand and the best configuration of each of the ligand-receptor
complexes was selected based on energetic grounds. The affinity scoring function δG was
used to assess and rank the receptor-ligand complexes. The docking scores and the hydrogen
bonding strength of all the molecules were shown in Table 2.
The synthesized series derivatives had dock score ranging from -14.6098 to -19.5089.
Compound 4d-3 was showing the best least docking score of -19.5089 and the next best least
docking score was found with 4b-3 followed by 4a-3. Four hydrogen bonds were present in
the derivative 4b-3 and 4d-3, which was the highest among the series. Compound 4d-3 is
found to be forming four hydrogen bonds of lengths 1.48, 1.55, 2.85 and 3.03 Å each with
1
5-OCH , 14-OCH , 13-OCH in benzene ring of Asp53 and O-N in oxime ester group of
3
3
3
Tyr27 respectively (Fig. 1). Compound 4b-3 also formed four hydrogen bonds of lengths 1.41,
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1
.65, 2.95 and 2.98 Å with 13, 14, 15-OCH in benzene ring of Asp53 and O-N in oxime
3
ester group of Tyr27 (Fig. 2). Compound 4a-3 formed three hydrogen bonds. In 4a-3, the 14,
5-OCH3 in benzene ring of Asp53 and O-N in oxime ester group of Tyr27 were with bond
1
length of 2.61, 2.16 and 2.02 Å respectively (Fig. 3). The compounds 4d-3, 4b-3 and 4a-3
exhibited the best least docking score had good in vitro anti-HBV activity as well.
2
.4. Anti-HBV Activity
All the newly synthesized derivatives were tested for their anti-HBV activity, namely
inhibiting the secretion of HBsAg, and HBeAg in HepG 2.2.15 cells using lamivudine (3TC, a
clinically popular anti-HBV agent) as a positive control. The anti-HBV activity of each
compound was expressed as the concentration of compound that achieved 50% inhibition
(
IC ) to the secretion of HBsAg and HBeAg. And the cytotoxicity of each compound was
50
expressed as the concentration of compound required to kill 50% (CC ) of the HepG 2.2.15
5
0
cells. The selectivity index (SI), a major pharmaceutical parameter that estimates possible
future clinical development, was determined as the ratio of CC to IC . The results of their
5
0
50
anti-HBV activity and cytotoxicity were listed in Table 3.
The treatment of HBV-transfected HepG2.2.15 cells with various concentrations of drugs
for 9 days exhibited a time-and dose-dependent inhibitory effect on the secretion of HBsAg
and HBeAg (Fig. 4). In synthesized derivatives, all compounds showed better activity
inhibiting the secretion of HBsAg than that of lamivudine. And eleven of twelve derivatives,
with higher inhibitory activity against the secretion of HBeAg than lamivudine were obtained
except for 4a-1. Moreover, all the twelve derivatives were observed analogical activities, but
lower cytotoxicity than those with heterocycle [20]. Compound 4d-3 showed the most potent
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anti-HBV activity, demonstrating potent inhibitory effect on the secretion of HBsAg (IC50
=
58.28ꢀM, SI=23.26) and HBeAg (IC50 = 97.21 ꢀM, SI=13.95). Compound 4c-2 showed
inhibitory potency to the secretion of HBsAg (IC =55.51 ꢀM) and HBeAg (IC =109.34 ꢀM),
5
0
50
but appeared toxic (CC =381.35ꢀM), which led to relatively low SI values (SIHBsAg=6.87,
5
0
SIHBeAg=3.49). Compared to compound 4c-2, compound 4b-3 relatively low inhibitory
potency to the secretion of HBsAg (IC =76.39 ꢀM) and HBeAg (IC =145.774 ꢀM), but
5
0
50
weak toxic (CC =1554.70 ꢀM) led to higher SI values (SIHBsAg=20.35, SIHBeAg=10.67).
5
0
Importantly, the most active compounds 4a-3, 4b-3, 4c-2, 4c-3 and 4d-3 with high activities
against HBsAg and HBeAg were selected to investigate inhibition of HBV DNA replication
using lamivudine as the reference drug. Compounds 4a-3, 4b-3, and 4d-3 exhibited anti-HBV
activity with their IC values against HBV DNA replication of 151.57, 100.62, 42.28 ꢀM,
5
0
respectively. Compounds 4a-3, 4b-3, and 4d-3 displayed inhibiting not only HBsAg and
HBeAg secretion but also HBV DNA replication, however, 3TC showed significantly activity
against HBV DNA replication (IC = 6.86) while showed little inhibitory on HBsAg and
5
0
HBeAg secretion.
2
.5. Structure-Activity Relationship
The start reactants substituted benzaldehyde 1a-d, intermediates 2a-d and oximes
1
-3 showed low suppressant properties on the HBV while most of the derivatives
showed high potency activity against of the secretion of HBsAg and HBeAg as shown
in Table 3. In the docking study, we also found that the O of O-N in the oxime ester
group interacted with Tyr27 by hydrogen bond.
The phenylpropanoid part of the derivatives showed the similar structure-activity
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relationship as our previous study [20]. The results also shown that the introduction of
methoxy group to 5-C could enhance the anti-HBV activity and decrease cytotoxicity along
with the high SI values, and the substituent of 3,4-dimethoxy by 3,4-methylenedioxy could
increase activity and cytotoxicity along with relatively low SI values.
Derivatives 4a-1~3, 4b-1~3, 4c-1~3 and 4d-1~3 contained the same phenylpropanoid part
respectively. Compound 4a-3 showed the best anti-HBsAg activity (IC =85.00 ꢀM) and the
50
next was 4a-1 (IC =128.86 ꢀM), followed by 4a-2 (IC =222.19 ꢀM), while the most potent
5
0
50
anti-HBeAg activity was observed with 4a-3 (IC =146.24 ꢀM), followed by 4a-2
5
0
(
IC =196.15 ꢀM), then 4a-1 (IC =290.28 ꢀM). The order also applied to 4b-1~3, 4c-1~3 and
50 50
4
d-1~3 except for 4c-2, which showed more effective on inhibiting HBsAg and HBeAg
secretion than that of 4c-3. It suggested that the introduction of methoxy group to 13-C could
enhance the anti-HBV activity, and the substituent of 13,14-dimethoxy by
1
3,14-methylenedioxy could decrease inhibition to the secretion of HBsAg and HBeAg.
Compound 4a-1 showed cytotoxicity with CC values of 546.41 ꢀM. The cytotoxicity of
5
0
derivatives 4a-3 (CC =601.46 ꢀM) decreased with methoxy group at 13-C. Compound 4a-2
5
0
(
CC =582.83 ꢀM) was observed with stronger cytotoxicity than 4a-3 with
50
1
3,14-methylenedioxy. Actually, all the derivatives obtained from oxime 3 did show weakest
cytotoxicity, followed by that from oxime 2. It indicated that the introduction of methoxy
group to 13-C could enhance the anti-HBV activity and decrease the cytotoxicity, and the
substituent of 13,14-dimethoxy by 13,14-methylenedioxy could decrease the anti-HBV
activity but increase cytotoxicity, leading to a low SI values.
According to the results mentioned above, SARs were summarized as followed: (1)
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0

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5
-OCH -substituted compounds with methylenedioxy at 13,14-C could provide higher
3
anti-HBV activity than other analogues. (2) 13,14,15-OCH -substituted compounds could
3
provide higher anti-HBV activity than that with H-substituted or methylenedioxy-substituted.
2
.6. The Interaction Between Derivatives and HBcAg
A modified enzyme-linked assay method using 96-well plates was used to explore the
interactions between the derivatives and HBcAg. The most active compounds 4a-3, 4b-3, 4c-2,
c-3 and 4d-3 were chosen as models to explore the interactions and the results were provided
4
–
in Fig. 5. It was evident that there was a reduction of the OD values in a dose dependent way,
which indicated that there would be an interaction between the compounds and HBcAg
blocking the binding of HBcAg. Among them, compound 4c-2 showed the lowest IC value
5
0
(
(
17.25 ꢀM) maybe for its cytotoxicity as mentioned above, and the next was compound 4d-3
22.50 ꢀM). It was compared to that of docking and in vitro anti-HBV activity results, and
was analogous.
3
. Conclusion
In conclusion, synthesis of phenylpropanoid derivatives and their potential anti-HBV
activities was evaluated. Results observed in the present study clearly demonstrated that most
of derivatives of the oximes ester phenylpropanoid family could exert interesting anti-HBV
activities with the SIHBsAg values from 2.14 to 23.26 and SIHBeAg values from 1.88 to 13.95.
Moreover, all the twelve derivatives were observed analogical activities, but lower
cytotoxicity than those with heterocycle. Interestingly, compounds 4c-1, 4d-1, and 4d-2
displayed inhibiting not only HBsAg and HBeAg secretion but also HBV DNA replication,
however, 3TC showed significantly activity against HBV DNA replication while showed little
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inhibitory on HBsAg and HBeAg secretion. In addition, the docking study of all synthesized
compounds inside the HLA-A protein (PDB ID: 3OX8) active site were carried out to explore
the molecular interactions and a molecular target for activity, and indicated that the HBV core
protein might be the potential target for anti-HBV. It was further proved by a modified assay
method measuring the interaction between our derivatives and HBcAg. The results of the
biological activities screened were consistent with the docking results and measurement of the
interaction, indicating that the anti-HBV effect of the prepared compounds may exert its
anti-HBV activity by inhibiting HBcAg. This study identified a new class of potent anti-HBV
agents and offered valuable information for seeking non-nucleoside anti-HBV drug
candidates.
4
4
. Materials and methods
.1. General
Melting points were determined using electrothermal melting point apparatus WRX-4
(
Shanghai, China) and were uncorrected. MS spectra were run on a Finnigan LCQ Deca XP
MAX mass spectrometer (Thermo Fisher, San Jose, CA, USA) equipped with an ESI source
and an ion trap analyzer in the positive ion mode/in the negative ion. NMR spectra were
1
13
recorded on Bruker AM 400 MHz ( H/ C, 400 MHz/100 MHz) or Bruker DRX 500 MHz
1
13
(
H/ C, 500 MHz/125 MHz) spectrometer (Bruker, Bremerhaven, Germany) and chemical
shifts were quoted in δ as parts per million (ppm) downfield with tetramethylsilane (TMS) as
internal reference. Coupling constants, J, are expressed in hertz (Hz). Column
chromatography (CC): silica gel (200 - 300 mesh; Qingdao Makall Group Co., Ltd; Qingdao;
China). All reactions were monitored using thinlayer chromatography (TLC) on silica gel
1
2

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plates. Yields refer to isolated pure products and were not maximized. On the basis of NMR
and HPLC (Thermo Fisher UItiMate 3000, USA) data, all final compounds reported in the
manuscript were >95% pure.
4
.2. Chemistry (Scheme 1)
The chemical synthesis and further characterization were available in the supplementary
material.
4
.3. Molecular Docking Studies
The ligand study was carried out by HyperChem software, a sophisticated molecular
modeling environment that uniting with quantum chemical calculations, dynamics, and
molecular mechanics [24].Three-dimensional structures were constructed and optimized for
all the molecules, and then QSAR descriptors were studied, which is a powerful lead
optimization tool that can quantitatively relate variations in biological activity to changes in
molecular properties.
The molecular docking study was performed using MOE 2008.10 to understand the
ligand-protein interactions in detail. The target compounds were built using the builder
interface of the MOE program and subjected to energy minimization. The crystal structure of
human leukocyte antigen (HLA-A) protein (PDB ID: 3OX8) was retrieved from Protein Data
Bank (http://www.rcsb.org/pdb/home/home.do) [25].The edited crystal structure after
removing water molecules was imported into MOE and chain A was considered for docking
process as the protein is a dimer consisting of A and B chains. The structure is protonated,
1
3

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polar hydrogens were added and energy minimization was carried out till the gradient
convergence 0.05 kcal/mol was reached to get the stabilized conformation. The active site was
correlated with ‘Site Finder’ module of MOE to define the docking site for the ligands.
Docking procedure was followed using the standard protocol implemented in MOE 2008.10
and the geometry of resulting complexes was studied using the MOE’s Pose Viewer utility.
4
4
.4. Pharmacology
.4.1. Cells and Cell culture
HepG2.2.15 (clonal cells derived fromhuman hepatoma cell line G2) cells were provided
by the Chinese Academy of Medical Sciences (P.R. China) and maintained in MEM medium
supplemented with 10% fetal bovine serum and 380 µg/mLof G418, 50 u/mL of kanamycin,
o
and 0.03% L-glutamine at 37 C in a 5% CO atmosphere with 100% humidity.
2
4
.4.2. Drug Treatment
5
HepG 2.2.15 cells were seeded at a density of 1×10 cells/mL (200 µL/well) in 96-well
o
plates and maintained at 37 C for 24 h prior to extract addition, followed by treatment with
various concentrations of drugs. Lamivudine (3TC) was served as the positive control. Cells
were refed with drug-containing fresh medium every 3 d for up to 9 d in time-dependent
experiment. Medium was taken at third day of treatment (T3), the sixth day of treatment (T6)
o
and the ninth day of treatment (T9), and stored at -20 C until analysis. The IC and selected
5
0
index (SI) of each compound were calculated, respectively.
1
4

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4
.4.3. Cell Toxicity
5
Logarithmically growing cells were seeded in 96-well culture plates at a density of 1×10
cells/mL (200 µL/well). They were cultured for 24 h and then treated with various
concentrations of drugs. OD values were read at 450 nm after 9days and the percent of cell
death was calculated and the cells were refed with drug-containing fresh medium every 3 d
for up to 9 d. After drug treatment, the cytotoxicity was measured using the MTT assay
[
26,27].
4
.4.4. Determination of HBsAg and HBeAg
The levels of HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) were
simultaneously detected using ELISA kits (Rongsheng Biotechnology Co. Ltd, Shanghai,
China) according to the manufacturer’s instructions.
4
.4.5. Determination of HBV replication
Inhibitory activity against HBV was determined by a real-time fluorescence quantitative
PCR (FQ-PCR) according to our previous description [12]. Briefly, 2.0 µL of HBV DNA was
amplified in a 25 mL of mixture containing 12.5 µL 2 × SYBR Green Master (ROX) and 2
primers specific for HBV: a forward primer (5′-AAC CAT TGAAGC AAT CAC TAG AC-3′)
and a reverse primer (5′- ATC TAT GGT GGC TGC TCG AAC TA -3′). The thermal program
o
comprised of an initial denaturation at 95 C for 10 min followed by 40 amplification cycles
o
o
with each of the two following steps: 95 C for 15 s and 60 C for 1 min.
1
5

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4
.5. Measurement of the interaction between the derivatives and HBV core protein
HBcAg protein was purified by ultracentrifugation and gel filtration chromatography as
described [28]. The interaction between the derivatives and HBV core protein was measured
by ELISA with some modifications [28,29]. Briefly, 100 µL of HBcAg in 50mM sodium
o
phosphate (pH 8.0) was incubated in 96-well plates overnight at 4 C. After blocking the plate
surface with 2% bovine serum albumin (Sigma, USA) (w/v) in PBS (50mM sodium
phosphate, 0.15MNaCl, pH 7.4), various concentrations of derivatives were added and
incubated for 20 min. The amount of HBcAg bound on the plate was measured using
anti-HBcAb (Rongsheng Biotechnology Co. Ltd, Shanghai, China).
Acknowledgements
This work was financially supported by the Foundation of High level innovation team
and outstanding scholar program in Guangxi collages, department of education of Guangxi
Zhuang autonomous region (2015), National Natural Science Foundation of China (No.
8
1060261), Natural Science Foundation of Guangxi Province (2012GXNSFAA053021), and
Department of Health of Guangxi Zhuang Autonomous Region (No. GZBZ14-19).
References
[
[
[
1] C. Seeger, W.S. Mason, Hepatitis B virus biology. Microbiol. Mol. Biol. Rev. 2000, 64,
1-68.
2] M. Rizzetto, A. Ciancio, Chronic HBV-related liver disease. Mol. Aspects Med. 2008, 29,
2-84.
3] D. Lavanchy, Hepatitis B virus epidemiology, disease burden, treatment, and current and
5
7
1
6

ACCEPTED MANUSCRIPT
emerging prevention and control measures. J. Viral Hepat. 2004, 11, 97-107.
4] K. Sato, M. Mori, Current and Novel Therapies for Hepatitis B Virus Infection. Mini-Rev.
Med. Chem. 2010, 10, 20-31.
[
[
[
[
[
5] G. Fattovich, L. Brollo, A. Alberti, P. Pontisso, G. Giustina, G. Realdi, Hepatology. 1998, 8,
1
651.
6] G. Fattovich, L. Brollo, A. Alberti, P. Pontisso, G. Giustina, G. Realdi, Long-term follow
up anti-Hbe-positive chronic active hepatitis B. Hepatology. 1988, 8, 1651-1654.
7] J.H. Hoofnagle, A.M. di Bisceglie, The treatment of chronic viral hepatitis. N. Engl. J.
Med. 1997, 336, 347-356.
8] P. Marcellin, T. Chang, S.G. Lim, M.J. Tong, W. Sievert, M.L. Shiffman, L. Jeffers, Z.
Goodman, M.S. Wulfsohn, S. Xiong, J. Fry, C.L. Brosgart, Adefovir dipivoxil for the
treatment of hepatitis B e antigen-positive chronic hepatitis B. N. Engl. J. Med. 2003, 348,
8
08-816.
[
9] S. Pol, P. Lampertico, First-line treatment of chronic hepatitis B with entecavir or
tenofovir in 'real-life' settings: from clinical trials to clinical practice. J. Viral Hepat. 2012,
1
9, 377-386.
10] M. Nassal, Hepatitis B viruses: reverse transcription a different way. Virus Res. 2008,
34, 235ꢀ249.
[
[
1
11] R.A. Crowther, N.A. Kiselev, B. Bottcher, J.A. Berriman, G.P. Borisova, V. Ose, P.
Pumpens, Three-dimensional structure of hepatitis B virus core particles determined by
electron cryomicroscopy. Cell. 1994, 77, 943-950.
[
12] P. Scaglioni, M. Melegari, M. Takahashi, J.R. Chowdhury, J. Wands, Use of dominant
1
7

ACCEPTED MANUSCRIPT
negative mutants of the hepadnaviral core protein as antiviral agents. Hepatology. 1996,
2
4, 1010-1017.
[
13] M. Yamamoto, N. Hayashi, T. Takehara, K. Ueda, E. Mita, T. Tatsumi, Y. Sasaki, A.
Kasahara, M. Hori, Intracellular singlechain antibody against hepatitis B virus core
protein inhibits the replication of hepatitis B virus in cultured cells. Hepatology. 1999, 30,
3
00-307.
[
14] K. Butz, C. Denk, B. Fitscher, I. Crnkovic-Mertens, A. Ullmann, C.H. Schroder, F.
Hoppe-Seyler, Peptide aptamers targeting the hepatitis B virus core protein: a new class of
molecules with antiviral activity. Oncogene. 2001, 20, 6579-6586.
[
[
15] W.X. Wei, X.R. Li, K.W. Wang, Z.W. Zheng, M. Zhou, Lignans with Anti-Hepatitis B
Virus Activities from Phyllanthus niruri L. Phytother. Res. 2012, 26, 964-968.
16] S. Liu, W.X. Wei, K.C. Shi, X. Cao, M. Zhou, Z.P. Liu, In vitro and in vivo anti-hepatitis
B virus activities of the lignan niranthin isolated from Phyllanthus niruri L. J.
Ethnopharmacol. 2014, 155, 1061-1067.
[
[
[
17] S. Liu, W.X. Wei, Y.B. Li, X. Lin, K.C. Shi, X. Cao, M. Zhou, In vitro and in vivo
anti-hepatitis B virus activities of the lignan nirtetralin B isolated from Phyllanthus niruri
L. J. Ethnopharmacol. 2014, 157, 62-68.
18] A. Sette, A. Vitiello, B. Reherman, P. Fowler, R. Nayersina, W.M. Kast, C.J. Melief, The
relationship between class I binding affinity and immunogenicity of potential cytotoxic T
cell epitopes. J. Immunol. 1994, 153, 5586–5592.
19] J.X. Liu, K.Y. Chen, E.C. Ren, Structural insights into the binding of hepatitis B virus
core peptide to HLA-A2 alleles: Towards designing better vaccines. Eur. J. Immunol.
1
8

ACCEPTED MANUSCRIPT
2
011. 41, 2097-2106
[
[
[
[
[
[
20] S. Liu, W.X. Wei, Y.B. Li, X. Liu, X.J. Cao, K.C. Lei, M. Zhou. Design, synthesis,
biological evaluation and molecular docking studies of phenylpropanoid derivatives as
potent anti-hepatitis B virus agents. Eur. J. Med. Chem. 2015, 95, 473-482.
21] V.V. Quan, C. Trenerry, S. Rochfort, J. Wadeson, C. Leyton, A,B. Hughes, Synthesis and
anti-inflammatory activity of aromatic glucosinolates. Bioorgan. Med. Chem. 2013, 21,
5
945-5954.
22] H.B. Zou, H. Wu, X.N. Zhang, Y. Zhao, S. Joachim, Y.J. Lou, Y.P. Yu, Synthesis,
biological evaluation, and structure-activity relationship study of novel cytotoxic
aza-caffeic acid derivatives. Bioorgan. Med. Chem. 2010, 18, 6351-6359.
23] A. Karakurt, A.A.B.S. Mehmet,Ü. Çalıs, S.Dalkara, Synthesis of some novel
1
-(2-naphthyl)-2-(imidazol-1-yl)ethanone oxime ester derivatives and evaluation of their
anticonvulsant activity. Eur. J. Med. Chem. 2012, 57, 275-282.
24] S. Dastmalchi, M. Hamzeh-Mivehroud, T. Ghafourian, H. Hamzeiy, Molecular modeling
of histamine H3 receptor and QSAR studies on arylbenzofuran derived H3 antagonists.
J.Mol. Graph. Model. 2008, 26, 834-844.
25] J.X. Liu, Y. Kenneth, E.C.R. Chen, Structural insights into the binding of hepatitis B
virus core peptide to HLA-A2 alleles: Towards designing better vaccines. Eur. J. Immunol.
2
011, 41, 2097-2106.
[
[
26] M. Ferrari, M.C. Fornasiero, A.M. Isetta, MTT colorimetric assay for testing macrophage
cytotoxic activity in vitro. J. Immunol. Methods. 1990, 131, 165-172.
27] Y.Q. Han, Z.M. Huang, X.B. Yang, H.Z. Liu, G.X. Wu, In vivo and in vitro anti-hepatitis
1
9

ACCEPTED MANUSCRIPT
B virus activity of total phenolics from Oenanthe javanica. J. Ethnopharmacol. 2008, 118,
1
48-153.
[
[
28] K.J. Choi, C.W. Lim, M.Y. Yoon, B.Y. Ahn, Y.G. Yu, Quantitative analysis of the
interaction between the envelope protein domains and the core protein of human hepatitis
B virus. Biochem. Biophys. Res. Commun. 2004, 319, 959-966.
29] M. Asif-Ullah, K.J. Choi, K. Choi, Y.J. Jeong, Y.G. Yu, Identification of compounds that
inhibit the interaction between core and surface protein of hepatitis B virus. Antivir. Res.
2
006, 70, 85-90.
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Tables
Table 1. Molecular descriptors of derivatives from QSAR study.
Molar
Surface
area
Hydration
energy
(Kcal/mol)
48.20
Molecular
Volume
Polarizability
Ligand
LogP refractivity
3
3
weight (Da)
(Å )
(Å )
3
2
(
Å )
(Å )
3TC
4a-1
4a-2
4a-3
4b-1
4b-2
4b-3
4c-1
4c-2
4c-3
4d-1
4d-2
4d-3
229.25
371.38
385.37
401.41
401.41
415.39
431.44
355.34
369.32
385.37
385.37
399.35
415.39
-0.55
-1.40
-2.21
-2.40
-2.40
-3.20
-3.39
-1.21
-2.02
-2.21
-2.21
-3.01
-3.20
55.14
109.06
108.27
115.43
115.43
114.65
121.81
101.90
101.11
108.27
108.27
107.49
114.65
525.55 606.87
852.16 1033.59
899.85 1036.63
906.26 1094.60
905.72 1081.70
953.39 1084.21
959.82 1142.71
862.83 957.70
910.52 961.08
917.00 1019.74
909.16 1012.94
956.84 1016.01
963.46 1075.15
21.71
38.79
38.65
41.26
41.26
41.12
43.73
36.18
36.04
38.65
38.65
38.51
41.12
45.35
39.22
43.26
43.13
39.03
43.04
41.00
36.87
40.91
39.92
35.83
39.83
2
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Table 2. Docking score and bond interactions of synthesized compounds.
Distance
S-score
No. of
Amino acid
involved
Ligand
Molecular structure
(
kcal/mol)
H-bonds
(Å)
3
TC
a-1
-10.7074
-16.1132
2
3
2.03
TYR 27
TYR 27
TYR 27
ASP 53
ASP 53
TYR 27
ASP 53
ASP 53
TYR 27
ASP 53
ASP 53
TYR 27
ASP 53
ASP 53
TYR 27
ASP 53
ASP 53
TYR 27
ASP 53
ASP 53
ASP 53
TYR 27
ASP 53
ASP 53
TYR 27
ASP 53
ASP 53
TYR 27
ASP 53
ASP 53
TYR 27
ASP 53
ASP 53
TYR 27
ASP 53
ASP 53
TYR 27
ASP 53
ASP 53
ASP 53
O of -OH
O of -COC-
2.29
4
2.02
O of -ON
2
.61
.16
O of 14 -OCH3
O of 15 -OCH3
O of -ON
2
4a-2
4a-3
4b-1
4b-2
4b-3
-16.0508
-18.5917
-17.6575
-17.9895
-18.9798
3
3
3
3
4
2.03
2
.72
.14
O of 14 -OCH2-
O of 15 -OCH3
O of -ON
2
2.02
2
2
.61
.16
O of 14 -OCH3
O of 15 -OCH3
O of -ON
2.99
1
.65
.39
O of 14 -OCH3
O of 15 -OCH3
O of -ON
1
2.99
1
.75
.39
O of 14 -OCH3
O of 15 -OCH3
O of -ON
1
2.98
2.95
1.65
1.41
O of 13 -OCH3
O of 14 -OCH3
O of 15 -OCH3
O of -ON
4c-1
4c-2
4c-3
4d-1
4d-2
4d-3
-14.6098
-14.5949
-17.2444
-16.1088
-17.1885
-19.5089
3
3
3
3
3
4
2.02
2
2
.55
.21
O of 14 -OCH3
O of 15 -OCH3
O of -ON
2.00
2
2
.70
.18
O of 14 -OCH3
O of 15 -OCH3
O of -ON
2.03
2
2
.54
.17
O of 14 -OCH3
O of 15 -OCH3
O of -ON
3.02
1
.56
.50
O of 14 -OCH3
O of 15 -OCH3
O of -ON
1
2.97
1
.72
.46
O of 14 -OCH3
O of 15 -OCH3
O of -ON
1
3.03
2.85
1.55
1.48
O of 13 -OCH3
O of 14 -OCH3
O of 15 -OCH3
2
2

ACCEPTED MANUSCRIPT
a
Table 3. Anti-HBV activity and cytotoxicity of the phenylpropanoid derivatives in vitro .
c
d
HBV DNA replication
HBsAg
HBeAg
b
Compd
CC50 (ꢀM)
e
f
e
f
e
f
IC50 (ꢀM)
SI
IC50 (ꢀM)
SI
IC50 (ꢀM)
SI
g
h
1
a
ꢁ1500
ꢁ1500
1289.31
1075.75
534.66
834.13
503.46
667.25
402.02
475.21
562.19
546.41
582.83
601.46
599.10
759.73
1554.70
255.99
381.35
532.67
567.04
626.14
1355.64
568.25
-
-
-
-
-
-
-
-
-
-
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3.97
ND
ND
15.45
ND
-
1
b
-
-
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
151.57
ND
ND
100.62
ND
-
1
c
-
-
1
d
-
-
2
a
ꢁ600
ꢁ600
433.15
410.96
387.17
479.80
353.56
128.86
222.19
85.00
124.50
194.11
76.39
119.53
55.51
69.22
112.61
167.47
58.28
234.70
ꢂ0.89
ꢂ1.39
1.16
1.62
1.04
1.01
1.59
4.24
2.62
7.08
4.81
3.91
20.35
2.14
6.87
7.70
5.04
3.74
23.26
2.42
ꢁ600
ꢁ600
526.25
476.75
469.11
519.47
431.23
290.28
196.15
146.24
243.86
171.89
145.77
210.97
109.34
125.91
201.90
166.03
97.21
267.16
ꢂ0.89
ꢂ1.39
0.96
1.40
0.86
0.91
1.30
1.88
2.97
4.11
2
b
2
c
2
d
1
2
3
4
4
4
a-1
a-2
a-3
4
4
4
b-1
b-2
b-3
2.47
4.42
10.67
1.21
3.49
4.23
2.81
3.77
13.95
2.13
4
4
4
c-1
c-2
c-3
-
-
4
4
4
d-1
d-2
d-3
ND
ND
42.28
6.86
ND
ND
32.06
82.84
i
3
TC
a. Values are means determined from at least two experiments.
b. CC is 50% cytotoxicity concentration in HepG2 2.2.15 cells.
50
c. HBsAg: hepatitis B surface antigen.
d. HBeAg, hepatitis B e antigen.
e. IC is 50% inhibitory concentration.
5
0
f. SI (selectivity index) = CC /IC .
5
0
50
g. Not active.
h. ND, Not determined.
i. Lamivudine (3TC) as the positive control.
2
3

ACCEPTED MANUSCRIPT
Legend of figures
Scheme 1. Synthetic route to the series of compounds. Reagents and conditions: (a)
CH (COOH) , piperidine, C H N, reflux, 4h, 80-90%; (b) SOCl , CH Cl , reflux, 5h, 95%; (c)
2
2
5
5
2
2
2
o
H NOH-HCl, AcONa, EtOH, 60 C, 1h, 98%; (d) Et N, CH Cl , rt, 12h, 50-70%.
2
3
2
2
2
4

ACCEPTED MANUSCRIPT
Figure 1. Ligand interaction and the binding mode of compound 4d-3 with HLA-A receptor.
The hydrogen bond formed colored in green.
Figure 2. Ligand interaction and the binding mode of compound 4b-3 with HLA-A receptor.
The hydrogen bond formed colored in green.
2
5

ACCEPTED MANUSCRIPT
Figure 3. Ligand interaction and the binding mode of compound 4a-3 with HLA-A receptor.
The hydrogen bond formed colored in green.
Figure 4. Inhibitory effect of the phenylpropanoid derivatives on secretion of HBsAg (A) and
HBeAg (B) in the HepG2.2.15 cell line. Data were expressed as mean ± S.E. (n = 3).
2
6

ACCEPTED MANUSCRIPT
Figure 5. Results of the interaction between the derivatives selected from primary screening
and HBcAg with the ELISA method.
2
7

ACCEPTED MANUSCRIPT
Highlights
ꢀ
ꢀ
ꢀ
Phenylpropanoid derivatives demonstrated potent anti-HBV activity.
Phenylpropanoid derivatives had interaction with protein 3OX8.
The anti-HBV effect of the phenylpropanoid derivatives may exert its anti-HBV
activity by inhibiting HBcAg.