Design, synthesis and evaluation of novel phenyl propionamide derivatives as non-nucleoside hepatitis B virus inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.12.042

As an ongoing search for potent non-nucleoside anti-HBV agents with novel structures, we described a series of phenyl propionamide derivatives (3a-b, 4a-e, 7a-g, 8a-h and 9a-b) by pharmacophore fusion strategy in the present work. All the compounds exhibited an anti-HBV activity to some extent. Among them, compounds 8d and 9b displayed most potent anti-HBV activity with IC₅₀ values on HBV DNA replication of 0.46 and 0.14 μM, respectively. And the selective index values of 8d and 9b were more than 217.39 and 153.14, suggesting that 8d and 9b exhibited favorable safety profiles. Interestingly, 8d and 9b possessed significantly antiviral activities against lamivudine and entecavir resistant HBV mutants with IC₅₀ values of 0.77 and 0.32 μM. Notably, preliminary anti-HBV action mechanism studies showed that 8d could inhibit intracellular HBV pgRNA and RT activity of the HBV polymerase. Molecular docking studies suggested that compound 8d could fit into the dimer-dimer interface of HBV core protein by hydrophobic interaction. In addition, in silico prediction of physicochemical properties showed that 8d conformed well to the Lipinski's rule of five, suggesting its potential for use as a drug like molecule. Taken together, 8d possessed significantly anti-HBV activity, low toxicity, diverse anti-HBV mechanism and favorable physicochemical properties, and warranted further investigation as a promising non-nucleoside anti-HBV candidate.

Full text of DOI:10.1016/j.ejmech.2017.12.042

Accepted Manuscript
Design, synthesis and evaluation of novel phenyl propionamide derivatives as non-
nucleoside hepatitis B virus inhibitors
Jingying Qiu, Qineng Gong, Jian Gao, Wang Chen, Yinpeng Zhang, Xiaoke Gu,
Daoquan Tang
PII:
S0223-5234(17)31075-9
10.1016/j.ejmech.2017.12.042
EJMECH 10020
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 September 2017
Revised Date: 30 November 2017
Accepted Date: 13 December 2017
Please cite this article as: J. Qiu, Q. Gong, J. Gao, W. Chen, Y. Zhang, X. Gu, D. Tang, Design,
synthesis and evaluation of novel phenyl propionamide derivatives as non-nucleoside hepatitis B virus
inhibitors, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.042.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Design, synthesis and evaluation of novel phenyl propionamide derivatives as
non-nucleoside hepatitis B virus inhibitors
Jingying Qiua, b, #, Qineng gongb, #, Jian Gaoa, Wang Chenb, Yinpeng Zhangb, Xiaoke Gua,
Daoquan Tanga, b,
,
a
Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical
University, Xuzhou 221004, People’s Republic of China
b
Department of Pharmaceutical Analysis, School of Pharmacy, Xuzhou Medical University,
Xuzhou 221004, People’s Republic of China
Graphical Abstract
Compound 8d exhibited significantly anti-HBV activity against wild-type strain as well as
lamivudine and entecavir resistant HBV strain.
Corresponding authors. Tel.: +86-516-83262137; fax: +86-516-83262630; e-mail: gu_xk@163.com (X. G.)
Tel.: +86-516-83262138; fax: +86-516-83262630; e-mail: tdq993@hotmail.com (D. Q.)
# These two authors contributed equally to this work.

ACCEPTED MANUSCRIPT
Design, synthesis and evaluation of novel phenyl propionamide derivatives as
non-nucleoside hepatitis B virus inhibitors
Jingying Qiua, b, #, Qineng gongb, #, Jian Gaoa, Wang Chenb, Yinpeng Zhang b, Xiaoke Gua, ,
Daoquan Tanga, b,
a Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University,
Xuzhou 221004, People’s Republic of China
b
Department of Pharmaceutical Analysis, School of Pharmacy, Xuzhou Medical University, Xuzhou
221004, People’s Republic of China
Abstract
As an ongoing search for potent non-nucleoside anti-HBV agents with novel structures, we described a
series of phenyl propionamide derivatives (3a-b, 4a-e, 7a-g, 8a-h and 9a-b) by pharmacophore fusion
strategy in the present work. All the compounds exhibited an anti-HBV activity to some extent. Among
them, compounds 8d and 9b displayed most potent anti-HBV activity with IC50 values on HBV DNA
replication of 0.46 and 0.14 µM, respectively. And the selective index values of 8d and 9b were more than
217.39 and 153.14, suggesting that 8d and 9b exhibited favorable safety profiles. Interestingly, 8d and 9b
possessed significantly antiviral activities against lamivudine and entecavir resistant HBV mutants with
IC50 values of 0.77 and 0.32 µM. Notably, preliminary anti-HBV action mechanism studies showed that 8d
could inhibit intracellular HBV pgRNA and RT activity of the HBV polymerase. Molecular docking
studies suggested that compound 8d could fit into the dimer-dimer interface of HBV core protein by
hydrophobic interaction. In addition, in silico prediction of physicochemical properties showed that 8d
conformed well to the Lipinski's rule of five, suggesting its potential for use as a drug like molecule. Taken
together, 8d possessed significantly anti-HBV activity, low toxicity, diverse anti-HBV mechanism and
favorable physicochemical properties, and warranted further investigation as a promising non-nucleoside
anti-HBV candidate.
Keywords: Anti-HBV agents; Phenyl propionamide derivatives; Non-nucleoside; Synthesis.
Corresponding authors. Tel.: +86-516-83262137; fax: +86-516-83262630; e-mail: gu_xk@163.com (X. G.)
Tel.: +86-516-83262138; fax: +86-516-83262630; e-mail: tdq993@hotmail.com (D. Q.)
# These two authors contributed equally to this work.
1

ACCEPTED MANUSCRIPT
1. Introduction
Hepatitis B virus (HBV) infection remains a major threat to public health despite the fact that HBV
vaccination programs have been extensively implemented in the past. There are about 350 million chronic
HBV carriers in the world and each year about one million chronic hepatitis patients develop into more
life-threatening diseases such as liver failure, cirrhosis or hepatocellular carcinoma (HCC) [1]. More
depressingly, current treatment options, such as interferons (IFNs) and nucleos(t)ides-based reverse
transcriptase inhibitors, do not offer a satisfactory clinical cure rate for chronic HBV infection [2].
Remarkably, nucleos(t)ide therapies typically require lifetime treatment to prevent viral rebound[3, 4], and
the HBV can easily develop resistance to this kind of drugs [5, 6], while IFNs need parenteral
administration and usually cause adverse effects such as “flu-like” symptoms [7]. Therefore, there is a
tremendous unmet medical requirement to develop novel and safety anti-HBV agents with diverse action
mechanisms [8].
In an ongoing search for potent non-nucleoside HBV inhibitors started a few years ago, we described a
series of Matijing-Su (MTS) derivatives as anti-HBV agents [9-11], which possessed a phenylalanine
dipeptide scaffold. Among them, Y101 was the most potent one (Figure 1) [12]. Notably, these
phenylalanine dipeptide derivatives displayed a different anti-HBV action mechanism from those of
conventional nucleotide drugs, and exhibited little inhibitory effect on HBV DNA polymerase. More
interestingly, Y101 possessed potent antiviral activities against wild-type as well as polymerase drug
resistant HBV strains. Several years ago, a series of phenyl acrylamide derivatives (AT-130) was reported
as novel anti-HBV agents by interfering the assembly of HBV core particles instead of inhibiting HBV
DNA polymerase [13, 14]. Excitingly, like Y101, AT-130 also displayed antiviral activities against
polymerase drug resistant HBV strains. With these in mind, and considering that both Y101 and AT-130
possessed novel anti-HBV action mechanism, a series novel phenyl propionamide derivatives was designed
by fusing the pharmacophore of Y101 and AT-130 (Figure 2), and their anti-HBV activities, including
preliminary anti-HBV action mechanisms were evaluated in the present work.
2

ACCEPTED MANUSCRIPT
Figure 1. Chinese traditional medicine and the structures of the anti-HBV natural product compound MTS and its
derivative Y101.
Figure 2. Structures of lead compounds and the newly designed phenyl propionamide derivatives via fused
pharmacophore strategy.
2. Chemistry
The synthetic procedure of the target compounds 3a-b and 4a-e is illustrated in Scheme 1. Briefly,
L
-Phenylalanine methyl ester hydrochloride or -tyrosine methyl ester hydrochloride was condensed with
L
benzoic acid to give intermediate 1a-b, which was then hydrolyzed to yield intermediate 2a-b as
described previously. Compound 3a-b was obtained by the condensation of 2a-b with piperidine. With
compound 2b as the starting material, and using the procedure described for the preparation of 3a-b, 3b-f
were prepared by changing piperidine into various amine, which was then alkylated with
2-dimethylaminoethyl chloride hydrochloride to provide alkoxyl substituted derivatives 4a-e,
respectively.
3

ACCEPTED MANUSCRIPT
Scheme 1. Reagents and conditions: (a) benzoic acid, CH2Cl2, IBCF, NMM, 0 °C; (b) NaOH, DMF, rt.; (c) piperidine,
CH2Cl2, IBCF, NMM, 0 °C; (d) amine, CH2Cl2, IBCF, NMM, 0 °C; (e) 2-dimethylaminoethyl chloride hydrochloride,
1,4-dioxane, K2CO3, 90 °C.
The synthetic routes of the target compounds 7a-g, 8a-h and 9a-b are depicted in Scheme 2.
Compound 5 was obtained by the acylation reaction with glycine and benzoyl chloride or benzoyl chloride
derivatives as the raw materials, which was then reacted with benzaldehyde or benzaldehyde derivatives in
the presence of acetic anhydride (Ac2O) and sodium acetate (AcONa) to obtain compound 6. Next, the
intermediate mentioned above was reacted with -phenylalaninol to give the target compounds 7a-g.
L
Finally, the crude reaction product was directly brominated with bromine to give the target compounds
8a-h, respectively. Compound 7b or 8b was alkylated with 2-dimethylaminoethyl chloride hydrochloride to
provide alkoxyl substituted derivative 9a or 9b.
4

ACCEPTED MANUSCRIPT
Scheme 2. Reagents and conditions: a) NaOH (aq), HCl; b) AcONa/Ac2O, 100 °C; c) CH3Cl, 0 °C; d) Br2,
CaCO3/CHCl3, 0 °C; e) 2-dimethylaminoethyl chloride hydrochloride, 1,4-dioxane, K2CO3, 90 °C.
3. Results and discussion
3.1. Biological evaluation
All the synthesized compounds were evaluated for the in vitro anti-HBV activity and cytotoxicity in
HepG2 2.2.15 cells using real-time quantitative PCR and MTT methods, respectively. The concentration of
compound required for 50% inhibition of DNA replication was defined as IC50 and the concentration of
compound that induced the death of the HepG2 2.2.15 cells cultures by 50% was defined as CC50.
Selectivity index (SI) was determined as the CC50/IC50 value. Lamivudine was used as the positive control.
3.1.1. Cytotoxicity assays
To evaluate the in vitro cytotoxic effects of the target compounds, HepG2 2.2.15 cells were treated with
various doses of 3a-b, 4a-e, 7a-g, 8a-h and 9a-b for 72 h, and then the viability of the cells was measured
by the MTT assay. All measurements were performed in three replicates, and the results were presented as
a relative percentage of the results of the control group. As shown in Table 1 and Table 2, most of the
5

ACCEPTED MANUSCRIPT
compounds exhibited little (CC50 > 100 µM) or slight (CC50 = 21.44 ~ 66.71 µM) intrinsic cytotoxicity in
vitro, indicating that this kind of compounds possessed a relatively safety profile.
3.1.2. Inhibitory effect on HBV DNA replication
Next, the anti-HBV activity of compounds 3a-b, 4a-e, 7a-g, 8a-h and 9a-b was screened in the HepG2
2.2.15 cell line by determining their inhibitory effect on HBV DNA replication. Lead compound Y101 and
lamivudine (3TC) were selected as the positive controls. After the treatment of the cells with the test
compounds at the set concentration of 20 µM or 4 µM for 6 days, the extra cellar HBV DNA levels were
quantified by real time PCR. The preliminary one dose anti-HBV screening showed that, compounds 3a-b,
4a-e, 7a-g and 9a displayed relative poor inhibitory effect on HBV DNA replication (Table 1 and Table 2),
the inhibition rate of most compounds against HBV DNA replication was less than 50%, except 4a
(51.64%) and 7g (51.36%). Interestingly, analogues 8a-h and 9b exhibited significant anti-HBV activity,
the inhibition rate on HBV DNA replication of all these compounds was higher than that of the positive
control Y101 (with an inhibition rate of 69.17%, Table 2). Clearly, compound 8d and 9b showed the most
potent in vitro anti-HBV DNA activity with an inhibition rate of 87.61% and 99.45%, respectively, which
was comparable with the positive control 3TC.
Table 1. Inhibitory effect of the target compounds 3a-b and 4a-e on HBV DNA levela
Inhibition rate
(%)
CC50 ± SD
Compounds
R1
R2
(µM)
3a
3b
4a
4b
4c
4d
H
>100
>100
>100
>100
>100
>100
42.30 ± 5.44
19.48 ± 3.71
51.64 ± 3.36
47.62 ± 2.12
24.90 ± 4.18
17.47 ± 3.41
4-OH
4-OCH2CH2N(CH3)2
4-OCH2CH2N(CH3)2
4-OCH2CH2N(CH3)2
4-OCH2CH2N(CH3)2
N(CH2CH3)2
6

ACCEPTED MANUSCRIPT
4e
4-OCH2CH2N(CH3)2
89.10 ± 0.117
47.32 ± 4.83
60.61 ± 5.37
69.17 ± 3.68
92.66 ± 2.24
MTS
Y101
3TC
>100
>100
>100
aShown is the mean ± SD of at least three independent experiments with duplicate measurements.
Table 2. Inhibitory effect of the target compounds 7a-g, 8a-h and 9a-b on HBV DNA levela
Inhibition rate ± SD
(%)
CC50 ± SD
Compounds
R1
R3
R4
(µM)
4-OH
H
H
H
H
>100
>100
24.95 ± 4.52
37.55 ± 3.81
7a
9a
7b
7c
7d
7e
7f
4-OCH2CH2N(CH3)2
4-Br
H
H
72.66 ± 0.091
>100
39.05 ± 2.47 (4 µM)
10.18 ± 4.66
4-CN
H
H
3-CH3
H
H
>100
15.80 ± 3.40
2-Cl
H
H
>100
29.75 ± 4.59
2-Br
H
H
>100
26.24 ± 2.11
H
H
2-F
H
>100
51.36 ± 2.93
7g
8a
9b
8b
8c
8d
8e
8f
4-OH
Br
Br
Br
Br
Br
Br
Br
Br
Br
38.95 ± 0.032
21.44 ± 0.059
>100
63.19 ± 4.65 (4 µM)
99.45 ± 0.34 (4 µM)
73.06 ± 2.45
4-OCH2CH2N(CH3)2
H
4-Br
4-CN
3-CH3
2-Cl
2-Br
H
H
H
>100
75.06 ± 3.68
H
>100
87.61 ± 2.13
H
66.71 ± 0.013
>100
80.15 ± 2.63
H
76.64 ± 5.62
2-F
H
>100
76.39 ± 5.91
8g
8h
H
>100
77.64 ± 2.69
7

ACCEPTED MANUSCRIPT
Y101
3TC
>100
>100
69.17 ± 3.68
92.66 ± 2.24
aShown is the mean ± SD of at least three independent experiments with duplicate measurements.
Subsequently, the active compounds 8a-h and 9b were further evaluated in HepG2 2.2.15 cells in a
dose-dependent manner. The anti-HBV activity of each compound was evaluated by the combination of the
IC50 value for inhibition of HBV DNA replication and the selectivity index (SI). As shown in Table 3, all
the tested compounds displayed significant inhibitory effect on HBV DNA replication with IC50 values in
range of 0.14-7.26 µM. Obviously, 8d and 9b were the top two compounds that possessed potent anti-HBV
activity with IC50 values of 0.46 and 0.14 µM, respectively. More importantly, the SI values of 8d and 9b
were more than 217 and 153, suggesting that 8d and 9b exhibited favorable safety profiles.
Table 3. Anti-HBV activity and SI values of target compounds 8a-h and 9ba
DNA replication
Compounds
CC50 ± SD (µM)
IC50 ± SD (µM)
2.00 ± 0.027
0.14 ± 0.025
4.45 ± 0.009
5.50 ± 0.017
0.46 ± 0.033
1.88 ± 0.070
7.26 ± 0.086
2.44 ± 0.051
3.86 ± 0.043
<0.1
SI
38.95 ± 0.032
21.44 ± 0.059
>100
19.48
8a
9b
8b
8c
153.14
>22.47
18.18
>100
>100
>217.39
35.48
8d
8e
66.71 ± 0.013
>100
>13.77
40.98
8f
>100
8g
>100
>25.91
>1000
8h
3TC
>100
aShown is the mean ± SD of at least three independent experiments with duplicate measurements.
From data of Table 1-3, a preliminary structure activity relationship (SAR) conclusions can be drawn as
follows: when the phenylalanine fragment in MTS derivative Y101 was replaced by piperidine (a key
fragment in AT-130) or other amino substituents, such as pyrrolidine, diethylamine, morpholine, the
anti-HBV activity of Y101 was obviously decreased (3a vs. MTS; 4a-e vs. Y101, Table 1). Changing the
phenylalanine scaffold of MTS into phenyl acrylamide, the anti-HBV activity was also significantly
reduced. Notably, introducing a bromine atom into phenyl acrylamide fragment was benefit for the
8

ACCEPTED MANUSCRIPT
anti-HBV activity of phenyl propionamide derivatives (7a-g vs. 8a-g; 9a vs 9b; Y101 vs. 9b, Table 2). In
addition, para substituent in A ring also significantly affect the anti-HBV activity. Generally speaking,
compounds with a dimethylamino ethoxy substituent exhibited higher anti-HBV activity (4a vs. 3a, 9b vs.
8h). When dimethylamino ethoxy substituent was replaced with CN, Br or OH, the anti-HBV activity was
significantly decreased (9b vs. 8a, 8b, 8c). Interestingly, compound 8d, with a meta methyl substituent in A
ring, exhibited significant anti-HBV activity with a inhibition rate of 87.61%, indicating that the
introduction of a meta substituent might be benefit for the anti-HBV activity. However, the precise SAR
remains further investigation when more phenyl propionamide derivatives are available in the near future.
3.1.3. Assessment of activity of compounds 8a, 8d and 9b against polymerase drug resistant HBV strain
Considering that the emergence of drug resistant strains is one of the key limitations associated with the
current nucleotide analogs approved for HBV treatment, it is important to evaluate the activity of new
compound against drug resistant HBV strains. Therefore, the anti-HBV effect on the lamivudine and
entecavir dually resistant mutant (rtL180M + rtM204V + rtT184L) of the active compounds 8a, 8d and 9b
were subsequently evaluated. Lamivudine and entecavir were selected as reference drugs. Briefly, HepG2
cells transiently transfected with lamivudine and entecavir resistant strain (LERS) or wild-type strain were
treated with lamivudine (100 µM), entecavir (10 µM), 8a (1 µM), 8d (1 µM) or 9b (1 µM) respectively for
72 h, followed by examination of the level of HBV DNA replicative intermediate using real time PCR. The
single concentration anti-HBV screening showed that lamivudine and entecavir could significantly suppress
the wild-type HBV replication by 94.28% and 95.67% (Figure 3). As expected, both lamivudine and
entecavir displayed poor anti-HBV activity against lamivudine and entecavir resistant strain, and the
inhibition rate was only 19.37% and 17.90%, respectively. In sharp contrast, compounds 8a, 8d and 9b at
relatively low concentration of 1 µM can significantly inhibit the drug resistant HBV replication with a
potency similar to that observed on wild-type HBV (43.17% vs 34.84%, 65.91% vs 61.36%, 80.12% vs
79.57%), indicating the effectiveness of 8a, 8d and 9b on both strains.
9

ACCEPTED MANUSCRIPT
Figure 3. Antiviral effect of 8a, 8d and 9b in lamivudine and entecavir resistant HBV. HepG2 cells were transiently
transfected with the full genome of wide-type or lamivudine and entecavir resistant HBV, followed by 72 h treatment with
lamivudine (100 µM), entecavir (10 µM), 8a (1 µM), 8d (1 µM) or 9b (1 µM), respectively. HBV DNA replicative
intermediate level was measured using real time PCR. **P < 0.01 represents significant difference from the WT HBV group.
And also, compounds 8a, 8d and 9b were further evaluated in a dose-dependent manner and the activity
of each compound were evaluated by the combination of the IC50 value for inhibition of HBV DNA
replication and the selectivity index (SI) (Table 4). As shown in Table, the IC50 values of 8d and 9b were
0.77 and 0.32 µM, indicating that both of them displayed potent inhibitory effect on HBV DNA replication
against drug resistant strain. Notably, SI values of compounds 8d and 9b were more than 129.87 and 89.72,
suggesting their relatively safety profiles.
Table 4. Inhibitory activity of compounds 8a, 8d and 9b against lamivudine and entecavir resistant HBV straina
DNA replication
Compounds
CC50 (µM)
IC50 (µM)
2.97 ± 0.010
0.77 ± 0.013
0.32 ± 0.038
>100
SI
11.55
>129.87
89.72
<1
34.31 ± 0.016
>100
8a
8d
28.71 ± 0.029
>100
9b
3TC
aShown is the mean ± SD of at least three independent experiments with duplicate measurements.
3.2. In silico prediction of physicochemical properties
Furthermore, some key physicochemical parameters of compounds 8a, 8d and 9b were predicted for
their compliance with the Lipinski's rule of five using free online software (http://www.molinspiration.com/)
[8]. As shown in Table 5, 8a and 8d are well accordance with the Lipinski's rule of five, indicating their
10

ACCEPTED MANUSCRIPT
potential for use as drug like molecule. Considering that compound 8d exhibited potent anti-HBV activity
with low toxicity and reasonable physicochemical properties, we selected 8d to explore the anti-HBV
mechanism of the target compounds.
Table 5. Prediction of physicochemical propertiesa of 8a, 8d and 9b
Compounds
nViol
MW
＜500
495.37
493.4
natoms
miLogP
＜5
nON
nOHNH
Nrotb
TPSA
＜140
98.65
78.42
90.90
MV
Range
8a
＜10
＜5
4
＜=10
0
0
1
32
32
37
3.60
6
5
7
8
8
401.40
409.94
481.88
4.51
3
8d
566.50
4.17
3
12
9b
a nViol: number of violations; natoms: number of atoms; miLogP: molinspiration predicted LogP; MW: molecular weight;
nON: number of hydrogen bond acceptors; nOHNH: number of hydrogen bond donors; nrotb: number of rotatable bonds;
TPSA: topological polar surface area; MV: molar volume.
3.3. Anti-HBV mechanism studies of compound 8d
3.3.1. Effect of compound 8d on HBV viral gene expression
It is well known that pregenomic RNA (pgRNA) plays a significant role in HBV lifecycle and is
associated with HBV DNA replication. Briefly, HepG2 2.2.15 cells were treated with compound 8d at the
concentration of 1 µM for 6 days. After the treatment, the total cellular RNA was extracted, and
intracellular HBV RNA was detected by real time PCR. Notably, unlike 3TC, compound 8d could inhibit
3.5 kb pgRNA expression (Table 6), suggesting that compound 8d possessed different anti-HBV action
mechanism than polymerase inhibitor 3TC.
Table 6. Effect of compound 8d on intracellular HBV pgRNA
Concentration
pgRNA
Inhibition rate
(%)
Compounds
(µM)
(copies/mL)
1
1
-
4.13E+06
8.10E+06
8.17E+06
49.42
0.81
-
8d
3TC
Control
3.3.2. Evaluation of the inhibitory activity of compound 8d on the DHBV RT activity
11

ACCEPTED MANUSCRIPT
Considering that compound 8d possessed novel chemical structure different from that of nucleoside
analogues, we finally evaluated whether 8d exhibits the inhibitory effect on RT activity of DHBV
polymerase in a cell-free assay, using lamivudine (3TC) as a positive control, which inhibits virus
replication by acting as a chain terminator of both the RT and DNA polymerase activities of the
enzyme. Interestingly, study on elongation of viral minus strand DNA showed that 8d could inhibit the
incorporation of radiolabelled dNTPs in a dose-dependent manner (Figure 4), suggesting that 8d
exhibited inhibitory effect on RT activity of DHBV polymerase. This result, together with the result
on HBV viral gene expression demonstrated that compound 8d, as a novel anti-HBV agent, possessed
multiple action mechanisms.
Figure 4. In vitro inhibitory activity of 8d on the RT activity of the DHBV polymerase. The DHBV polymerase was
expressed in vitro in a reticulocyte system. The enzymatic activity was studied in the absence or presence of increasing
concentrations of nucleoside analog triphosphates. The inhibition rate of compound 8d correspond to the concentration
required to inhibit the incorporation of [α32P]-dNTP in viral minus strand DNA. *P<0.05, **P<0.01 vs. control group.
3.3.3 Molecular docking
HBV core protein performs multiple essential roles at different stages of HBV virus life cycle, which is
regarded as an excellent target for developing novel, efficacious and safe anti-HBV agents. Recently,
phenyl acrylamide derivatives, such as AT-130, were reported as a HBV core protein assembly effectors
thus exerting anti-HBV activity. With these in mind and considering that 8d shared a similar phenyl
12

ACCEPTED MANUSCRIPT
acrylamide skeleton, we preliminarily examined the interaction of 8d with the interface of HBV core
protein by Molecular docking. Interestingly, compound 8d could be successfully docked into the
dimer-dimer interface of HBV core protein, with the docking score value was of 6.00. As shown in Figure
5A, compound 8d was fitted into a well-defined, largely hydrophobic groove composed of Pro25, Asp29,
Leu30, Thr33, Leu37, Trp102, Ile105, Ser106, Thr109, Phe110, Tyr118, Phe122, Ile139, Leu140, and
Ser141 in one protein subunit. Meanwhile, compound 8d was covered by a “lid” formed by the C-terminal
helix and loop (including residues Val124, Trp125, Arg127, Thr128, Pro129, Ala132, Arg133, and Pro134)
of the other protein subunit (Figure 5B). It is likely that the binding affinity between HBV core protein and
compound 8d was nearly dominated by the hydrophobic interactions.
Figure 5. Binding mode of compound 8d in the dimer-dimer interface of HBV core protein. (A) The ligand 8d
accommodates in the hydrophobic groove formed by the dimer-dimer interface, with the lid removed for clarity. (B) The
ligand 8d binds to the lid, with the groove removed for clarity. The protein and ligand 8d are shown in cartoon and stick
mode, respectively. The key resides and ligand 8d are colored in yellow and green, respectively.
4. Conclusions
Overall, a series of phenyl propionamide derivatives (3a-b, 4a-e, 7a-g, 8a-h and 9a-b) was designed and
synthesized as novel non-nucleoside anti-HBV agents. Among them, compounds 8d and 9b displayed most
potent anti-HBV activity with IC50 values on HBV DNA replication of 0.46 and 0.14 µM, respectively.
And the selective index values of 8d and 9b were more than 217.39 and 153.14, suggesting that 8d and 9b
exhibited favorable safety profiles. Interestingly, 8d and 9b possessed significantly antiviral activities
13

ACCEPTED MANUSCRIPT
against lamivudine and entecavir resistant HBV mutants with IC50 values of 0.77 and 0.32 µM. Notably,
preliminary anti-HBV action mechanism studies showed that 8d could inhibit intracellular HBV pgRNA
and RT activity of the HBV polymerase. Molecular docking studies suggested that compound 8d could fit
into the dimer-dimer interface of HBV core protein by hydrophobic interaction. In addition, in silico
prediction of physicochemical properties showed that compounds 8d conformed well to the Lipinski's rule
of five. Considering the significantly anti-HBV activity, low toxicity, diverse anti-HBV mechanism and
favorable physicochemical properties, 8d might be novel non-nucleoside anti-HBV candidates to warrant
further investigation.
5. Experimental protocols
5.1. Chemical analysis
All of the synthesized compounds were purified by column chromatography on silica gel 60 (200–300
mesh) or thin layer chromatography (TLC) on silica gel 60 F254 plates (250 mm; Qingdao Ocean Chemical
1
Company, China). Melting points were measured on a model YRT-3 apparatus and are uncorrected. H
13
NMR (400 MHz) and C NMR (100 MHz) spectra were recorded on a JEOL (400 MHz) spectrometer in
CDCl3 or DMSO-d6 with Me4Si (TMS) as an internal standard. MS detection was performed using an
Agilent G6460C triple quadrupole mass spectrometer. All solvents were reagent grade and, when necessary,
were purified and dried by standards methods. The purity of all investigated compounds in biological
testing was determined as ≥ 95% unless otherwise stated. Analytical HPLC was run on the Agilent 1260
HPLC instrument, equipped with Agilent SB-C18 column and UV detection at 254 nm. Eluent system was:
80% MeOH in H2O; flow rate = 0.2 mL/min.
5.1.1. General procedure for the synthesis of compounds 3a-b
Isobutyl chloroformate (IBCF) (1.1 mmol) was added dropwise to the mixture of benzoic acid (1.0
mmol), -phenylalanine methyl ester hydrochloride (1.1 mmol) and N-methylmorpholine (NMM) (2.3
L
mmol) in CH2Cl2 (50 mL) at 0 °C within 30 min. The mixture was stirred for 30 min and the bulk of
CH2Cl2 was removed in vacuo. The residue was dissolved in ethyl acetate and washed sequentially with
water, 5% HCl, saturated NaHCO3 solution and brine, dried with anhydrous Na2SO4. Removal of the
solvent gave a residue which was recrystallized from ethyl acetate to afford target compound 1. 2 M NaOH
(1 mL) was added to the solution of compound 1 (1.0 mmol) in DMF (5 mL). After stirring at room
14

ACCEPTED MANUSCRIPT
temperature for 2 h, the mixture was acidified to pH 6-7 with concentrated hydrochloric acid, and
partitioned between ethyl acetate and water. The organic phase was separated and washed with brine, dried
over anhydrous Na2SO4 and evaporated in vacuo to give compound 2. Compound 3a-b was prepared with a
similar procedure to that described for the synthesis of compound 1, and the crude product was purified by
column chromatography to yield the title compound, respectively.
5.1.1.1. (S)-N-(1-oxo-3-phenyl-1-(piperidin-1-yl)propan-2-yl)benzamide (3a)
As a yellow oil, yield: 41.2%. Analytical data for 3a: 1H NMR (CDCl3, 400 MHz, δ ppm): 7.78-7.80 (d,
J = 8.0 Hz, 2H, Ar-H), 7.47-7.51 (t, 1H, Ar-H), 7.40-7.43 (t, 2H, Ar-H), 7.21-7.30 (m, 5H, Ar-H), 5.36-5.41
(m, 1H, CH), 3.46-3.57 (m, 2H, CH2), 3.04-3.30 (m, 4H, 2×CH2), 0.83-1.60 (m, 6H, 3×CH2); 13C NMR
(CDCl3, 100 MHz, δ ppm): 169.39, 166.54, 136.32, 134.12, 131.66, 129.76 (2C), 128.58 (2C), 128.55 (2C),
127.15 (2C), 127.09, 50.24, 46.71, 43.24, 39.86, 25.98, 25.47, 24.33; ESI-MS: m/z 359.1 [M+Na]+,
HRESIMS: calcd for C21H25N2O2 [M+H]+ 337.1910, found 337.1900. Purity: 99.8%.
5.1.1.2. (S)-N-(3-(4-hydroxyphenyl)-1-oxo-1-(piperidin-1-yl)propan-2-yl)benzamide (3b)
As a yellow oil, yield: 43.7%. Analytical data for 3b: 1H NMR (CDCl3, 400 MHz, δ ppm): 7.78-7.80 (d,
2H, J = 8.0 Hz, Ar-H), 7.48-7.52 (t, 1H, Ar-H), 7.41-7.43 (t, 2H, Ar-H), 7.00 (d, J = 8.0 Hz, 2H, H-5, 9),
6.62 (d, J = 8.0Hz, 2H, H-6, 8), 5.34-5.39 (m, 1H, CH), 3.46-3.57 (m, 2H, CH2), 3.03-3.29 (m, 4H, 2×CH2),
13
0.81-1.60 (m, 6H, 3×CH2); C NMR (CDCl3, 100 MHz, δ ppm): 169.39, 166.54, 157.09, 134.21, 131.64,
130.66 (2C), 128.61, 128.57 (2C), 127.14 (2C), 114.64 (2C), 51.45, 46.70, 46.65, 38.91, 26.05, 26.03,
24.34; ESI-MS: m/z 353.2 [M+H]+, HRESIMS: calcd for C21H25N2O3 [M+H]+ 353.1860, found 353.1852.
Purity: 92.9%.
5.1.2. General procedure for the synthesis of compounds 4a-e
Compound 3b (0.5 mmol) was followed by the treatment with 2-dimethylaminoethyl chloride
hydrochloride (0.6 mmol) and K2CO3 (3.5 mmol) in dioxane (15 mL) at 90 °C for 3 h. The crude product
was purified by column chromatography to yield the title compound, respectively.
5.1.2.1. (S)-N-(3-(4-(2-(dimethylamino)ethoxy)phenyl)-1-oxo-1-(piperidin-1-yl)propan-2-yl)benzamide (4a)
As a yellow oil, yield: 36.4%. Analytical data for 4a: 1H NMR (CDCl3, 400 MHz, δ ppm): 7.77-7.80 (d,
2H, J = 8.0 Hz, Ar-H), 7.47-7.51 (t, 1H, Ar-H), 7.40-7.44 (t, 2H, Ar-H), 7.10-7.12 (d, J = 8.0 Hz, 2H, Ar-H),
6.82-6.84 (d, J = 8.0 Hz, 2H, Ar-H), 5.31-5.36 (m, 1H, CH), 4.03-4.06 (t, 2H, OCH2), 3.22-3.34 (m, 4H,
15

ACCEPTED MANUSCRIPT
2×CH2), 2.92-3.13 (m, 2H, CH2), 2.72-2.75 (t, 2H, NCH2), 2.35 (s. 6H, 2×CH3), 0.90-1.92 (m, 6H, 3×CH2);
13C NMR (CDCl3, 100 MHz, δ ppm): 169.44, 166.50, 157.93, 134.21, 131.64, 130.66 (2C), 128.61, 128.57
(2C), 127.14 (2C), 114.64 (2C), 65.97, 58.25, 51.45, 46.71, 46.65, 45.82 (2C), 38.92, 26.06, 26.04, 24.35;
ESI-MS: m/z 424.2 [M+H]+, HRESIMS: calcd for C21H25N2O2 [M+H]+ 424.2595, found 424.2593. Purity:
90.6%.
5.1.2.2.
(S)-N-(3-(4-(2-(dimethylamino)ethoxy)phenyl)-1-oxo-1-(pyrrolidin-1-yl)propan-2-yl)benzamide
(4b)
As a yellow oil, yield: 38.1%. Analytical data for 4b: 1H NMR (CDCl3, 400 MHz, δ ppm): 7.78-7.80 (d,
J = 8.0 Hz, 2H, Ar-H), 7.47-7.51 (t, 1H, Ar-H), 7.40-7.44 (t, 2H, Ar-H), 7.09-7.12 (d, J = 8.0 Hz, 2H, Ar-H),
6.80-6.85 (d, J = 8.0 Hz, 2H, Ar-H), 5.05-5.11 (m, 1H, CH), 4.03-4.06 (t, 2H, OCH2), 3.27-3.49 (m, 4H,
2×CH2), 2.91-3.13 (m, 2H, CH2), 2.72-2.75 (t, 2H, NCH2), 2.35 (s. 6H, 2×CH3), 1.58-1.92 (m, 4H, 2×CH2);
13C NMR (CDCl3, 100 MHz, δ ppm): 169.79, 166.61, 157.95, 134.12, 131.67, 130.56 (2C), 128.60, 128.58
(2C), 127.16 (2C), 114.62 (2C), 66.09, 58.33, 52.96, 46.50, 46.39, 45.79 (2C), 38.92, 25.91, 25.88; ESI-MS:
m/z 410.2 [M+H]+, HRESIMS: calcd for C24H32N3O3 [M+H]+ 410.2438, found 410.2434. Purity: 92.2%.
5.1.2.3. (S)-N-(3-(4-(2-(dimethylamino)ethoxy)phenyl)-1-morpholino-1-oxopropan-2-yl)benzamide (4c)
As a yellow oil, yield: 37.2%. Analytical data for 4c: 1H NMR (CDCl3, 400 MHz, δ ppm): 7.79-7.81 (d,
J = 8.0 Hz, 2H, Ar-H), 7.49-7.53 (t, 1H, Ar-H), 7.41-7.45 (t, 2H, Ar-H), 7.13-7.15 (d, J = 8.0 Hz, 2H, Ar-H),
6.85-6.87 (d, J = 8.0 Hz, 2H, Ar-H), 5.27-5.33 (m, 1H, CH), 4.03-4.06 (t, 2H, OCH2), 2.86-3.63 (m, 5H,
4×CH2, CH), 2.73-2.76 (t, 2H, NCH2), 2.35 (s. 6H, 2×CH3); 13C NMR (CDCl3, 100 MHz, δ ppm): 170.14,
166.62, 158.15, 133.95, 131.83, 130.67 (2C), 130.58, 128.66 (2C), 127.14 (2C), 114.79 (2C), 66.57, 66.23,
65.99, 58.26, 50.20, 45.88 (2C), 39.25; ESI-MS: m/z 426.2 [M+H]+, HRESIMS: calcd for C24H32N3O4
[M+H]+ 426.2387, found 426.2373. Purity: 94.0%.
5.1.2.4. (S)-N-(1-(diethylamino)-3-(4-(2-(dimethylamino)ethoxy)phenyl)-1-oxopropan-2-yl)benzamide (4d)
As a yellow oil, yield: 40.9%. Analytical data for 4d: 1H NMR (CDCl3, 400 MHz, δ ppm): 7.78-7.80 (d,
J = 8.0 Hz, 2H, Ar-H), 7.47-7.51 (t, 1H, Ar-H), 7.40-7.43 (t, 2H, Ar-H), 7.12-7.14 (d, J = 8.0 Hz, 2H, Ar-H),
6.81-6.83 (d, J = 8.0 Hz, 2H, Ar-H), 5.20-5.26 (m, 1H, CH), 4.02-4.05 (t, 2H, OCH2), 3.49-3.86 (m, 2H,
CH2), 2.92-3.13 (m, 4H, 4×CH2), 2.71-2.74 (t, 2H, NCH2), 2.34 (s. 6H, 2×CH3), 0.97-1.09 (m, 6H, 2×CH3);
13C NMR (CDCl3, 100 MHz, δ ppm): 170.63, 166.42, 157.95, 143.13, 131.66, 130.68 (2C), 130.59, 128.67
16

ACCEPTED MANUSCRIPT
(2C), 127.15 (2C), 114.60 (2C), 65.98, 58.36, 50.76, 45.94 (2C), 41.76, 40.58, 39.25, 14.29, 12.91; ESI-MS:
m/z 412.2 [M+H]+, HRESIMS: calcd for C24H33N3O3Na [M+Na]+ 434.2414, found 434.2419. Purity:
93.8%.
5.1.2.5.
N-((S)-3-(4-(2-(dimethylamino)ethoxy)phenyl)-1-(((S)-1-hydroxy-3-methylbutan-2-yl)amino)-1-oxopropan-
2-yl)benzamide (4e)
As a yellow oil, yield: 36.9%. Analytical data for 4e: 1H NMR (CDCl3, 400 MHz, δ ppm): 7.74-7.76 (d,
J = 8.0 Hz, 2H, Ar-H), 7.48-7.51 (t, 1H, Ar-H), 7.38-7.42 (t, 2H, Ar-H), 7.19-7.21 (d, J = 8.0 Hz, 2H, Ar-H),
6.83-6.85 (d, J = 8.0 Hz, 2H, Ar-H), 4.85-4.90 (m, 1H, CH), 4.06-4.07 (t, 2H, OCH2), 3.42-3.53 (m, 2H,
CH2), 3.05-3.23 (m, 3H, CH2, CH), 2.79-2.81 (t, 2H, NCH2), 2.39 (s. 6H, 2×CH3), 0.78-0.85 (m, 6H,
2×CH3); 13C NMR (CDCl3, 100 MHz, δ ppm): 171.59, 167.47, 157.74, 133.79, 131.91, 130.52 (2C),
129.17, 128.67 (2C), 127.22 (2C), 114.87 (2C), 65.55, 63.06, 58.02, 57.23, 55.65, 45.58 (2C), 37.94, 28.87,
19.45, 18.78; ESI-MS: m/z 442.2 [M+H]+, HRESIMS: calcd for C25H35N3O4Na [M+Na]+ 464.2510, found
464.2527. Purity: 98.4%.
5.1.3. General procedure for the synthesis of compounds 7a-g [15]
Compounds 5 was obtained by the acylation reaction with glycine (0.01 mol) and benzoyl chloride or
benzoyl chloride derivatives (0.02 mol) as the raw materials in sodium hydroxide solution, which was then
reacted with benzaldehyde or benzaldehyde derivatives (0.01 mol) in the presence of acetic anhydride
(Ac2O) (10 mL) and sodium acetate (AcONa) (0.01 mol) to obtain compounds 6. Next, the intermediate
mentioned above was reacted with -phenylalaninol (0.01 mol). The crude product was purified by column
L
chromatography to yield the title compound 7a-g, respectively.
5.1.3.1.
(S,E)-N-(3-((1-hydroxy-3-phenylpropan-2-yl)amino)-1-(4-hydroxyphenyl)-3-oxoprop-1-en-2-yl)benzamide
(7a)
As a white powder, yield: 39.3%; mp: 82.5-83.6 °C. Analytical data for 7a: 1H NMR (DMSO, 400 MHz,
δ ppm): 9.92 (s, 1H, OH), 7.98-8.00 (d, 2H, J = 8.0 Hz, Ar-H), 7.83-7.85 (d, J = 8.0 Hz, 1H, NH),
7.52-7.58 (m, 5H, Ar-H), 7.17-7.25 (m, 5H, Ar-H), 7.11-7.13 (d, J = 8.0 Hz, 2H, Ar-H), 7.06 (s, 1H, CH=),
4.75-4.77 (t, 1H, OH), 4.02-4.07 (m, 1H, CH), 3.30-3.44 (m, 2H, CH2), 2.75-2.90 (m, 2H, CH2); 13C NMR
17

ACCEPTED MANUSCRIPT
(DMSO, 100 MHz, δ ppm): 166.42, 165.41, 150.87, 139.75, 134.15, 132.49, 132.29, 131.01, 130.91 (2C),
129.75 (2C), 128.90 (2C), 128.66 (2C), 128.42 (2C), 127.97, 126.43, 122.48 (2C), 62.82, 53.66, 36.84;
ESI-MS: m/z 417.2 [M+H]+, HRESIMS: calcd for C22H25N2O4 [M+H]+ 417.1809, found 417.1821. Purity:
98.9%.
5.1.3.2.
(S,E)-N-(1-(4-bromophenyl)-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxoprop-1-en-2-yl)benzamide
(7b)
As a white powder, yield: 48.1%; mp: 85.5-86.4°C. Analytical data for 7b: 1H NMR (DMSO, 400 MHz,
δ ppm): 9.90 (s, 1H, NH), 7.98-8.00 (d, J = 8.0 Hz, 1H, Ar-H), 7.89-7.91 (d, J = 8.8 Hz, 1H, NH),
7.46-7.62 (m, 7H, Ar-H), 7.16-7.27 (m, 5H, Ar-H), 6.99 (s, 1H, CH=), 4.74-4.77 (t, 1H, OH), 3.99-4.08 (m,
1H, CH), 3.32-3.49 (m, 2H, CH2), 2.76-2.92 (m, 2H, CH2); 13C NMR (DMSO, 100 MHz, δ ppm): 166.36,
165.38, 139.68, 134.19, 134.03, 132.36, 131.99 (2C), 131.73, 131.62 (2C), 129.73 (2C), 128.94 (2C),
128.68 (2C), 128.39 (2C), 127.33, 126.49, 122.12, 62.84, 53.67, 36.83; ESI-MS: m/z 479.01 [M+H]+,
HRESIMS: calcd for C25H24BrN2O3 [M+H]+ 479.0965, found 479.0952. Purity: 99.9%.
5.1.3.3.
(S,E)-N-(1-(4-cyanophenyl)-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxoprop-1-en-2-yl)benzamide
(7c)
As a white powder, yield: 50.6%; mp: 94.5-95.3°C. Analytical data for 7c: 1H NMR (DMSO, 400 MHz,
δ ppm): 10.01 (s, 1H, NH), 8.02-8.04 (d, J = 8.0 Hz, 1H, NH), 7.97-7.99 (d, J = 8.0 Hz, 2H, Ar-H),
7.81-7.83 (d, J = 8.0 Hz, 2H, Ar-H), 7.66-7.69 (d, J = 8.0 Hz, 2H, Ar-H), 7.59-7.63 (t, 1H, Ar-H), 7.51-7.55
(t, 2H, Ar-H), 7.17-7.28 (m, 5H, Ar-H), 7.00 (s, 1H, CH=), 4.75-4.77 (t, 1H, OH), 4.01-4.07 (m, 1H, CH),
3.32-3.50 (m, 2H, CH2), 2.76-2.93 (m, 2H, CH2); 13C NMR (DMSO, 100 MHz, δ ppm): 166.35, 165.18,
139.95, 139.69, 133.90, 133.79, 132.85 (2C), 132.46, 130.25 (2C), 129.73 (2C), 128.95 (2C), 128.69 (2C),
128.43 (2C), 126.50, 126.07, 119.26, 110.82, 62.86, 53.78, 36.83; ESI-MS: m/z 426.2 [M+H]+, HRESIMS:
calcd for C26H24N3O3 [M+H]+ 426.1812, found 426.1798. Purity: 99.6%.
5.1.3.4. (S,E)-N-(3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-(m-tolyl)prop-1-en-2-yl)benzamide
(7d)
As a white powder, yield: 47.2%; mp: 70.9-71.6°C. Analytical data for 7d: 1H NMR (DMSO, 400 MHz,
18

ACCEPTED MANUSCRIPT
δ ppm): 9.87 (s, 1H, NH), 7.98-8.80 (d, J = 7.8 Hz, 2H, Ar-H), 7.79-7.81 (d, J = 8.0 Hz, 1H, NH),
7.58-7.61 (t, 1H, Ar-H), 7.51-7.55 (t, 2H, Ar-H), 7.10-7.35 (m, 9H, Ar-H), 7.06 (s, 1H, CH=), 4.73-4.76 (t,
1H, OH), 3.99-4.07 (m, 1H, CH), 3.31-3.47 (m, 2H, CH2), 2.76-2.91 (m, 2H, CH2); 13C NMR (DMSO, 100
MHz, δ ppm): 166.49, 165.44, 139.70, 137.99, 134.71, 134.26, 132.26. 130.79, 130.53, 129.76 (2C),
129.25, 128.92, 128.90 (2C), 128.67 (2C), 128.32 (2C), 126.88, 126.46, 62.81, 53.58, 36.82, 21.50;
ESI-MS: m/z 415.2 [M+H]+, HRESIMS: calcd for C26H27N2O3 [M+H]+ 415.2016, found 415.2007. Purity:
100.0%.
5.1.3.5.
(S,E)-N-(1-(2-chlorophenyl)-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxoprop-1-en-2-yl)benzamide
(7e)
1
As a white powder, yield: 43.9%; mp: 107.7-108.5°C. Analytical data for 7e: H NMR (DMSO, 400
MHz, δ ppm): 9.83 (s, 1H, NH), 8.00-8.02 (d, J = 8.0 Hz, 1H, NH), 7.93-7.94 (d, J = 7.8 Hz, 2H, Ar-H),
7.49-7.61 (m, 5H, Ar-H), 7.18-7.34 (m, 7H, Ar-H), 7.11 (s, 1H, CH=), 4.76-4.79 (t, 1H, OH), 4.00-4.08 (m,
1H, CH), 3.33-3.52 (m, 2H, CH2), 2.77-2.95 (m, 2H, CH2); 13C NMR (DMSO, 100 MHz, δ ppm): 166.49,
165.18, 139.73, 133.79, 133.67, 133.35, 133.22, 132.23, 130.37, 130.10, 130.01, 129.73 (2C), 128.86 (2C),
128.68 (2C), 128.38 (2C), 127.57, 126.46, 123.88, 62.94, 53.77, 36.80, ESI-MS: m/z 435.2 [M+H]+,
HRESIMS: calcd for C25H24ClN2O3 [M+H]+435.1470, found 435.1461. Purity: 99.8%.
5.1.3.6.
(S,E)-N-(1-(2-bromophenyl)-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxoprop-1-en-2-yl)benzamide
(7f)
As a white powder, yield: 41.5%; mp: 99.7-100.4°C. Analytical data for 7f: 1H NMR (DMSO, 400 MHz,
δ ppm): 9.83 (s, 1H, NH), 7.98-8.00 (d, J = 8.0 Hz, 1H, NH), 7.90-7.92 (d, J = 8.8 Hz, 2H, Ar-H),
7.68-7.70 (d, J = 8.0 Hz, 1H, Ar-H), 7.47-7.59 (m, 4H, Ar-H), 7.17-7.29 (m, 7H, Ar-H), 7.05 (s, 1H, CH=),
4.74-4.77 (t, 1H, OH), 3.98-4.07 (m, 1H, CH), 3.31-3.50 (m, 2H, CH2), 2.75-2.93 (m, 2H, CH2); 13C NMR
(DMSO, 100 MHz, δ ppm): 166.54, 165.16, 139.71, 135.11, 133.97, 133.21, 132.99, 132.33, 130.56,
130.25, 129.73 (2C), 128.84 (2C), 128.72 (2C), 128.38 (2C), 128.09, 126.46, 126.34, 124.53, 62.91, 53.77,
36.78; ESI-MS: m/z 479.1 [M+H]+, HRESIMS: calcd for C25H24BrN2O3 [M+H]+ 479.0965, found
479.0952. Purity: 97.5%.
19

ACCEPTED MANUSCRIPT
5.1.3.7.
(S,E)-2-fluoro-N-(3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en-2-yl)benzamide
(7g)
1
As a white powder, yield: 45.7%; mp: 105.7-106.2°C. Analytical data for 7g: H NMR (DMSO, 400
MHz, δ ppm): 9.80 (s, 1H, NH), 7.78-7.80 (d, J = 8.0 Hz, 1H, NH), 7.67-7.71 (t, 1H, Ar-H), 7.58-7.62 (m,
3H, Ar-H), 7.17-7.41 (m, 10H, Ar-H), 7.05 (s, 1H, CH=), 4.79-4.81 (t, 1H, OH), 4.00-4.08 (m, 1H, CH),
3.38-3.48 (m, 2H, CH2), 2.75-2.93 (m, 2H, CH2); 13C NMR (DMSO, 100 MHz, δ ppm): 166.15, 163.86,
158.68, 139.63, 134.58, 130.82, 130.79. 130.11, 129.83 (2C), 129.73 (2C), 129.20, 129.01 (2C), 128.96,
128.69 (2C), 126.51, 125.08, 124.00, 116.87, 62.76, 53.58, 36.92; ESI-MS: m/z 419.2 [M+H]+, HRESIMS:
calcd for C25H24FN2O3 [M+H]+ 419.1766, found 419.1757. Purity: 99.3%.
5.1.4. General procedure for the synthesis of compounds 8a-h [15]
Finally, the crude reaction product (7a-h) (0.01 mol) was directly brominated with bromine (0.015 mol)
and anhydrous calcium carbonate (0.01 mol) in CHCl3. The crude product was purified by column
chromatography to yield the title compound 8a-h, respectively.
5.1.4.1.
(S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-1-(4-hydroxyphenyl)-3-oxoprop-1-en-2-yl)be
nzamide (8a)
1
As a yellow powder, yield: 18.0%, m.p.223.7-224.6 °C. Analytical data for 8a: H NMR (DMSO, 400
MHz, δ ppm): 9.87 (s, 1H, OH), 9.80 (br, 1H, NH), 7.99-8.01 (d, J = 8.0 Hz, 2H, Ar-H), 7.64-7.66 (d, J =
8.0 Hz, 1H, NH), 7.59-7.63 (t, 1H, Ar-H), 7.51-7.55 (t, 2H, Ar-H), 7.08-7.23 (m, 7H, Ar-H), 6.68-6.70 (d, J
= 8.0 Hz, 2H, Ar-H), 4.19-4.27 (m, 1H, OH), 3.73-3.81 (m, 1H, CH), 3.01-3.21 (m, 2H, CH2), 2.44-2.62 (m,
13
2H, CH2); C NMR (DMSO, 100 MHz, δ ppm): 166.65, 163.47, 158.80, 139.47, 133.65, 132.42, 131.21,
129.58 (4C), 128.97 (2C), 128.87, 128.61 (2C), 128.40 (2C), 126.35, 122.69, 115.44 (2C), 62.16, 53.36,
36.25; ESI-MS: m/z 495.1 [M+H]+, HRESIMS: calcd for C25H24BrN2O4 [M+H]+ 495.0914, found
495.1089. Purity: 98.5%.
5.1.4.2.
(S,Z)-N-(1-bromo-1-(4-bromophenyl)-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxoprop-1-en-2-yl)benz
amide (8b)
20

ACCEPTED MANUSCRIPT
1
As a white powder, yield: 15.4%, m.p.99.7-101.1 °C. Analytical data for 8b: H NMR (DMSO, 400
MHz, δ ppm): 9.90 (s, 1H, NH), 7.96-7.98 (d, J = 8.0 Hz, 2H, Ar-H), 7.84-7.87 (d, J = 8.0 Hz, 1H, NH),
7.57-7.61 (t, 1H, Ar-H), 7.49-7.52 (t, 2H, Ar-H), 7.43-7.45 (d, J = 8.0 Hz, 2H, Ar-H), 7.13-7.28 (m, 5H,
Ar-H), 7.03-7.05 (d, J = 8.0 Hz, 2H, Ar-H), 3.70-3.77 (m, 1H, CH), 3.01-3.21 (m, 2H, CH2), 2.39-2.62 (m,
13
2H, CH2); C NMR (DMSO, 100 MHz, δ ppm): 165.58, 162.98, 139.47, 137.68, 134.43, 133.47, 132.31,
131.64 (2C), 129.50 (2C), 129.09 (2C), 128.99 (2C), 128.61 (2C), 128.46 (2C), 126.34, 122.85, 119.54,
62.23, 53.46, 36.17; ESI-MS: m/z 558 [M+H]+, HRESIMS: calcd for C25H23Br2N2O3 [M+H]+ 557.0070,
found 557.0065. Purity: 94.2%.
5.1.4.3.
(S,Z)-N-(1-bromo-1-(4-cyanophenyl)-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxoprop-1-en-2-yl)benz
amide (8c)
As a white powder, yield: 15.9%, m.p.98.8-99.4 °C. Analytical data for 8c: 1H NMR (DMSO, 400 MHz,
δ ppm): 9.96 (s, 1H, NH), 7.97-7.99 (m, 2H, Ar-H), 7.66-7.68 (d, J = 8.0 Hz, 2H, Ar-H), 7.57-7.61 (t, 1H,
Ar-H), 7.49-7.53 (t, 2H, Ar-H), 7.38-7.40 (d, J = 8.0 Hz, 2H, Ar-H), 7.14-7.19 (m, 3H, Ar-H), 7.02-7.04 (d,
J = 8.0 Hz, 2H, Ar-H), 4.20 (br, 1H, OH), 3.71-3.79 (m, 1H, CH), 3.00-3.21 (m, 2H, CH2), 2.38-2.63 (m,
13
2H, CH2); C NMR (DMSO, 100 MHz, δ ppm): 165.73, 162.82, 143.04, 139.41, 135.58, 133.36, 132.79,
132.54 (2C), 130.41 (2C), 129.43 (2C), 129.04 (2C), 128.62 (2C), 128.52 (2C), 126.35, 119.01, 118.00,
62.21, 53.46, 36.10; ESI-MS: m/z 504 [M+H]+, HRESIMS: calcd for C26H23BrN3O3 [M+H]+ 504.0917,
found 504.0906. Purity: 97.1%.
5.1.4.4.
(S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-(m-tolyl)prop-1-en-2-yl)benzamide
(8d)
1
As a white powder, yield: 14.3%, m.p.98.9-100.6 °C. Analytical data for 8d: H NMR (DMSO, 400
MHz, δ ppm): 9.91 (s, 1H, NH), 8.01-8.03 (d, J = 8.0 Hz, 2H, Ar-H), 7.67-7.69 (d, J = 8.0 Hz, 1H, NH),
7.60-7.62 (t, 1H, Ar-H), 7.52-7.56 (t, 2H, Ar-H), 7.06-7.21 (m, 9H, Ar-H), 3.73-3.75 (m, 1H, CH),
2.99-3.17 (m, 2H, CH2), 2.41-2.58 (m, 2H, CH2), 2.28 (s, 3H, CH3); 13C NMR (DMSO, 100 MHz, δ ppm):
165.62, 163.18, 139.38, 138.29, 137.78, 133.82, 133.54, 132.63, 130.05, 129.54 (2C), 129.00 (2C), 128.99,
128.61 (2C), 128.45 (2C), 127.98, 126.80, 126.36, 121.52, 61.87, 53.34, 36.08, 31.43; ESI-MS: m/z 493
21

ACCEPTED MANUSCRIPT
[M+H]+, HRESIMS: calcd for C26H25BrN2O3Na [M+Na]+ 515.0941, found 515.0942. Purity: 97.4%.
5.1.4.5.
(S,Z)-N-(1-bromo-1-(2-chlorophenyl)-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxoprop-1-en-2-yl)benz
amide (8e)
As a white powder, yield: 19.7%, m.p. 79.3-82 °C. Analytical data for 8e: 1H NMR (DMSO, 400 MHz, δ
ppm): 9.64 (d, J = 8.0 Hz, 1H, NH), 7.73-8.00 (m, 3H, NH, Ar-H), 7.57-7.60 (t, 1H, Ar-H), 7.46-7.53 (m,
3H, Ar-H), 7.02-7.41 (m, 8H, Ar-H), 4.54-4.61 (t, 1H, OH), 3.65-3.70 (m, 1H, CH), 2.97-3.16 (m, 2H,
CH2), 2.35-2.63 (m, 2H, CH2); 13C NMR (DMSO, 100 MHz, δ ppm): 166.09, 162.11, 139.20, 137.48,
135.77, 133.54, 132.97, 132.79, 131.63, 130.13, 129.79, 129.52 (2C), 128.97 (2C), 128.63 (2C), 128.58
(2C), 127.66, 126.41, 62.10, 51.98, 36.19; ESI-MS: m/z 513.1 [M+H]+, HRESIMS: calcd for
C25H23BrClN2O3 [M+H]+ 513.0575, found 513.0569. Purity: 95.5%.
5.1.4.6.
(S,Z)-N-(1-bromo-1-(2-bromophenyl)-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxoprop-1-en-2-yl)benz
amide (8f)
As a white powder, yield: 17.1%, m.p.58.8-59.9 °C. Analytical data for 8f: 1H NMR (DMSO, 400 MHz,
δ ppm): 9.94 (s, 1H, NH), 7.99-8.01 (d, J = 8.0 Hz, 2H, Ar-H), 7.63-7.65 (d, J = 8.0 Hz, 1H, NH),7.57-7.61
(t, 1H, Ar-H), 7.49-7.53 (t, 2H, Ar-H), 7.35-7.36 (d, J = 4.0 Hz, 1H, Ar-H), 7.10-7.30 (m, 6H, Ar-H),
7.02-7.04 (d, J = 8.0 Hz, 2H, Ar-H), 4.40-4.60 (t, 1H, OH), 3.63-3.70 (m, 1H, CH), 2.94-3.13 (m, 2H, CH2),
2.33-2.63 (m, 2H, CH2); 13C NMR (DMSO, 100 MHz, δ ppm): 166.03, 162.01, 139.42, 139.13, 135.52,
133.56, 133.34, 131.64, 131.39, 131.13, 129.54 (2C), 128.96 (2C), 128.64 (2C), 128.59 (2C), 128.23,
126.42, 123.29, 121.09, 61.98, 51.92, 36.17; ESI-MS: m/z 559 [M+H]+, HRESIMS: calcd for
C25H23Br2N2O3 [M+H]+ 557.0070, found 557.0064. Purity: 96.8%.
5.1.4.7.
(S,E)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en-2-yl)-2-fluorobenz
amide (8g)
1
As a white powder, yield: 16.3%, m.p.124.6-125.4°C. Analytical data for 8g: H NMR (DMSO, 400
MHz, δ ppm): 9.91 (s, 1H, NH), 8.01-8.03 (d, J = 8.0 Hz, 2H, Ar-H), 7.67-7.69 (d, J = 8.0 Hz, 1H, NH),
7.60-7.64 (t, 1H, Ar-H), 7.52-7.56 (t, 2H, Ar-H), 7.06-7.27 (m, 9H, Ar-H), 3.73-3.75 (m, 1H, CH),
22

ACCEPTED MANUSCRIPT
2.98-3.18 (m, 2H, CH2), 2.41-2.58 (m, 2H, CH2); 13C NMR (DMSO, 100 MHz, δ ppm): 166.38, 166.02,
162.54, 139.37, 138.13, 137.82, 134.05, 133.00, 129.88 (2C), 128.55 (2C), 129.16, 129.11, 128.98 (2C),
128.86, 128.63 (2C), 127.19, 126.37, 116.99, 62.38, 53.26, 36.12; ESI-MS: m/z 497 [M+H]+, HRESIMS:
calcd for C25H22BrFN2O3Na[M+Na]+ 519.0690, found 519.0686. Purity: 95.1%.
5.1.4.8.
(S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en-2-yl)benzamide
(8h)
1
As a yellow powder, yield: 29.4%, m.p. 89.7-90.5°C. Analytical data for 8h: H NMR (DMSO, 400
MHz, δ ppm): 9.95 (s, 1H, NH), 8.01-8.03 (d, J = 8.0 Hz, 2H, Ar-H), 7.76-7.78 (d, J = 8.0 Hz, 1H, NH),
7.60-7.65 (t, 1H, Ar-H), 7.52-7.57 (t, 2H, Ar-H), 7.07-7.32 (m, 10H, Ar-H), 3.70-3.80 (m, 1H, CH),
2.97-3.17 (m, 2H, CH2), 2.39-2.59 (m, 2H, CH2); 13C NMR (DMSO, 100 MHz, δ ppm): 166.6, 163.11,
139.40, 138.39, 133.93, 133.50, 129.91, 129.59 (2C), 129.56 (2C), 129.01 (2C), 128.99, 128.67 (2C),
128.63 (2C), 128.45 (2C), 126.27, 121.21, 62.05, 53.31, 36.10; ESI-MS: m/z 479 [M+H]+, HRESIMS:
calcd for C25H23BrN2O3Na [M+Na]+ 501.0784, found 501.0781. Purity: 98.6%.
5.1.5. General procedure for the synthesis of compounds 9a-b
Compound 7a or 8a (0.01 mol) was alkylated with 2-dimethylaminoethyl chloride hydrochloride (0.012
mol) to provide alkoxyl substituted derivatives 9a or 9b. The crude product was purified by column
chromatography to yield the title compound, respectively.
5.1.5.1.
(S,E)-N-(1-(4-(2-(dimethylamino)ethoxy)phenyl)-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxoprop-1-e
n-2-yl)benzamide (9a)
The title compound was obtained starting from 7a and 2-dimethylaminoethyl chloride hydrochloride. As
a white powder, yield: 24.9%, m.p. 114.1-114.9 °C. Analytical data for 9a: 1H NMR (DMSO, 400 MHz, δ
ppm): 9.77 (s, 1H, NH), 7.97-7.99 (d, J = 8.0 Hz, 2H, Ar-H), 7.90-7.93 (d, J = 8.0 Hz, 1H, NH),7.45-7.58
(m, 5H, Ar-H), 7.16-7.25 (m, 5H, Ar-H), 7.00 (s, 1H, CH=), 6.88-6.90 (d, J = 8.0 Hz, 2H, Ar-H), 4.24-4.31
(m, 1H, CH2), 3.97-4.02 (m, 4H, 2×CH2), 2.76-2.85 (8, 2H, CH2), 2.54-2.57 (t, 2H, CH2), 2.15 (s, 6H, 2×
CH3); 13C NMR (DMSO, 100 MHz, δ ppm): 166.26, 165.84, 159.32, 138.83, 134.26, 132.20, 131.58 (2C),
129.70 (2C), 129.26, 128.87 (2C), 128.76 (2C), 128.73, 128.40 (2C), 127.12, 126.71, 115.04 (2C), 66.36,
23

ACCEPTED MANUSCRIPT
65.25, 58.12, 50.15, 46.03 (2C), 37.14; ESI-MS: m/z 488.2 [M+H]+, HRESIMS: calcd for C29H34N3O4
[M+H]+ 488.2544, found 488.2561. Purity: 99.0%.
5.1.5.2.
(S,Z)-N-(1-bromo-1-(4-(2-(dimethylamino)ethoxy)phenyl)-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-ox
oprop-1-en-2-yl)benzamide (9b)
The title compound was obtained starting from 8a and 2-dimethylaminoethyl chloride hydrochloride. As
a white powder, yield: 13.6%, m.p.183.0-183.8 °C. Analytical data for 9b: 1H NMR (DMSO, 400 MHz, δ
ppm): 8.27-8.30 (d, J = 8.0 Hz, 2H, Ar-H), 7.12-7.15 (dd, 2H, J = 8.0 Hz, 8.0 Hz, Ar-H), 8.04-8.02 (d, J =
8.0 Hz, 1H, NH), 7.59-7.61 (m, 3H, Ar-H), 7.16-7.31 (m, 5H, Ar-H), 7.06-7.08 (d, J = 8.0 Hz, 2H, Ar-H),
4.98-5.01 (t, 1H, OH), 4.20-4.26 (m, 1H, CH), 4.12-4.15 (t, 2H, CH2), 3.49-3.55 (m, 2H, CH2), 2.91-2.96
(m, 2H, CH2), 2.64-2.67 (t, 2H, CH2), 2.23 (s, 6H, 2×CH3); 13C NMR (DMSO, 100 MHz, δ ppm): 161.01,
160.25, 157.84, 139.54, 131.67, 130.14, 129.75, 129.73, 129.68 (4C), 128.75 (4C), 126.88, 126.57, 126.51,
119.87, 114.90 (2C), 66.48, 62.57, 58.13, 52.85, 46.08 (2C), 37.16; ESI-MS: m/z 566.2 [M+H]+,
HRESIMS: calcd for C29H33BrN3O4 [M+H]+ 566.1649, found 566.1654. Purity: 95.0%.
5.2. Biological assays
HepG2 cell lines were obtained from 302 military hospital of china. The cell lines were cultivated in
DMEM medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL
streptomycin, and 0.38 mg/mL of G418. Cytotoxicity, anti-HBV activities of compounds and effect on
HBV viral gene expression were evaluated on the HepG2 2.2.15 cell line (stably transfected cell lines
containing HBV genome on a plasmid). Determination of antiviral efficacy against nucleoside
analogue-resistant HBV was evaluated on the HepG2 cell line which were transiently transfected
respectively with plasmids containing drug-resistant mutant (rtL180M + rtM204V + rtT184L).
5.2.1. Cytotoxicity assays
Cytotoxicity induced by the target compounds in cultures of HepG2 2.2.15 cells was also determined.
Briefly, HepG2 2.2.15 cells were grown to confluence in 96-well culture plates, and then treated with test
compounds (in 0.2 mL culture medium/well), respectively. Untreated control cultures were maintained on
each
96-well
plate.
The
cytotoxicity
was
determined
by
measuring
MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dye uptake, as determined from the
24

ACCEPTED MANUSCRIPT
absorbance at 490 nm relative to untreated cells, at 24 hours following 72 h of treatment.
5.2.2. Inhibitory effect on HBV DNA replication
For detection of extracellular replication intermediates, HepG2 2.2.15 cells were seeded in 24 well
culture plates at a density of 2×105 cells per well and allowed to recover for 24 h. Culture medium was
replaced by assay medium containing the compound that is being tested or 3TC. Every 2 days, medium was
changed. After 6 days, intracellular HBV DNA was extracted using method reported before[16], and the
levels were quantified by real time PCR.
5.2.3. Determination of antiviral efficacy against nucleoside analogue-resistant HBV [17]
HepG2 cells per well (2×105) were plated into 24-well plates. After overnight incubation, the cells were
transiently transfected respectively with plasmids containing drug-resistant mutant (rtL180M + rtM204V +
rtT184L) for 24 h. The supernatant was discarded, followed by PBS wash. Then, the cells were cultivated
with fresh medium containing lamivudine (100 µM), Entecavir (10 µM), 8a (1 µM), 8d (1 µM) or 9b (10
µM). After 72 h treatment, HBV DNA replicative intermediate was extracted using the method mentioned
above [16], and the level was quantified using real time PCR.
5.3. Anti-HBV action mechanism of compound 8d
5.3.1. Effect of compound 8d on HBV viral gene expression [18, 19]
HepG2 2.2.15 cells were cultured in 6-well plates at a density of 6×105 cells, and were treated with
compounds for 6 days. Total RNA was extracted from HepG2 2.2.15 cells by the use of TRIzol reagent
(Invitrogen), and then detected by quantitative real time PCR.
5.3.2. Evaluation of the Inhibitory Activity of Compounds 8d on the DHBV RT Activity
The DHBV polymerases were obtained from Beijing Weijian Jiye Institute of Biotechnology (302
military hospital of china). The expression of the DHBV polymerases and the analysis of elongation of
viral minus strand DNA were performed as described in detail [20]. The reaction mixture included
[α32P]-dNTP (0.165 µmol/L; 3,000 Ci/mmol) of which the inhibitor is the analog, whereas the 3 other cold
dNTPs were used at 100 µmol/L each.
5.3.3. Molecular docking
For the purpose of investigating the possible binding interactions between HBV core protein and
compound 8d, Surflex docking module in Sybyl X-2.1 was used for molecular docking studies [21]. The
25

ACCEPTED MANUSCRIPT
crystal structure of HBV core protein complex with NVR-010-001-E2 (PDB ID: 5EOI) was obtained from
the RCSB Protein Data Bank [22]. The space where ligand NVR-010-001-E2 placed was selected as the
active pocket, and all water molecules were removed. The missing hydrogen atoms were added by
biopolymer module during protein preparation, the docking parameters were kept as default values.
Acknowledgements
This work was supported by the grants from the Natural Science Foundation of Jiangsu province (No.
BK20140223, BK20140225), and the National Natural Science Foundation of China (NSFC No. 81703359,
81402789).
References and notes
[1] M.Y. Zhu, W. Li, Y. Lu, X. Dong, B. Lin, Y. Chen, X.E. Zhang, J.L. Guo, M.S. Li, HBx drives alpha
fetoprotein expression to promote initiation of liver cancer stem cells through activating PI3K/AKT
signal pathway, Int. J. Cancer, 140 (2017) 1346-1355.
[2] B.Y. Winer, T. Huang, B.E. Low, C. Avery, M.A. Pais, G. Hrebikova, E. Siu, L. Chiriboga, M.V. Wiles,
A. Ploss, Recapitulation of treatment response patterns in a novel humanized mouse model for chronic
hepatitis B virus infection, Virology, 502 (2017) 63-72.
[3] J. Wang, T. Shen, X.B. Huang, G.R. Kumar, X.M. Chen, Z.Z. Zeng, R.Y. Zhang, R. Chen, T. Li, T.Y.
Zhang, Q. Yuan, P.C. Li, Q. Huang, R. Colonno, J.D. Jia, J.L. Hou, M.A. McCrae, Z.L. Gao, H. Ren,
N.S. Xia, H. Zhuang, F.M. Lu, Serum hepatitis B virus RNA is encapsidated pregenome RNA that may
be associated with persistence of viral infection and rebound, J. Hepatol., 65 (2016) 700-710.
[4] Y.J. Wang, L. Yang, J.P. Zuo, Recent developments in antivirals against hepatitis B virus, Virus Res.,
213 (2016) 205-213.
[5] G.Q. Yin, B. Zhong, Efficacy of interferon for chronic hepatitis B in patients with nucleoside and
nucleotide combination therapy failure, J. Gastroen. Hepatol., 31 (2016) 248-255.
[6] S. Manzoor, M. Saalim, M. Imran, S. Resham, J. Ashraf, Hepatitis B virus therapy: What's the future
holding for us?, World J. Gastroentero, 21 (2015) 12558-12575.
[7] J. Liu, A. Kosinska, M. Lu, M. Roggendorf, New therapeutic vaccination strategies for the treatment of
chronic hepatitis B, Virol Sin, 29 (2014) 10-16.
26

ACCEPTED MANUSCRIPT
[8] H. Jia, F. Bai, N. Liu, X. Liang, P. Zhan, C. Ma, X. Jiang, X. Liu, Design, synthesis and evaluation of
pyrazole derivatives as non-nucleoside hepatitis B virus inhibitors, Eur. J. Med. Chem., 123 (2016)
202-210.
[9] B.X. Xu, Z.M. Huang, C.X. Liu, Z.G. Cai, W.D. Pan, P.X. Cao, X.J. Hao, G.Y. Liang, Synthesis and
anti-hepatitis B virus activities of Matijing-Su derivatives, Bioorg. Med. Chem., 17 (2009) 3118-3125.
[10] J.Y. Qiu, B.X. Xu, Z.M. Huang, W.D. Pan, P.X. Cao, C.X. Liu, X.J. Hao, B.A. Song, G.Y. Liang,
Synthesis and biological evaluation of Matijing-Su derivatives as potent anti-HBV agents, Bioorg. Med.
Chem., 19 (2011) 5352-5360.
[11] J.Y. Qiu, B.X. Xu, Q.N. Gong, W.D. Pan, C.X. Liu, Z.M. Huang, X.K. Gu, G.Y. Liang, Synthesis and
Biological Evaluation of Matijin-Su Derivatives as Potential Antihepatitis B Virus and Anticancer
Agents, Chem. Biodivers., 13 (2016) 1584-1592.
[12] Z.X. Hu, Y.G. Zhang, Q. An, B.X. Xu, W.D. Pan, P.X. Cao, C.X. Liu, Z.M. Huang, W. Xia, J.Y. Qiu,
G.Y. Liang, Development of a practical and scalable synthesis of anti-HBV drug Y101, Tetrahedron, 70
(2014) 9592-9600.
[13] S.P. Katen, Z.N. Tan, S.R. Chirapu, M.G. Finn, A. Zlotnick, Assembly-Directed Antivirals
Differentially Bind Quasiequivalent Pockets to Modify Hepatitis B Virus Capsid Tertiary and
Quaternary Structure, Structure, 21 (2013) 1406-1416.
[14] L.C. Li, S.R. Chirapu, M.G. Finn, A. Zlotnick, Phase Diagrams Map the Properties of Antiviral Agents
Directed against Hepatitis B Virus Core Assembly, Antimicrob. Agents. Ch, 57 (2013) 1505-1508.
[15] P.Y. Wang, D. Naduthambi, R.T. Mosley, C.R. Niu, P.A. Furman, M.J. Otto, M.J. Sofia,
Phenylpropenamide derivatives: Anti-hepatitis B virus activity of the Z isomer, SAR and the search for
novel analogs, Bioorg. Med. Chem. Lett, 21 (2011) 4642-4647.
[16] W.E. DELANEY, H.C. ISOM. Hepatitis B Virus Replication in Human HepG2 Cells Mediated by
Hepatitis B Virus Recombinant Baculovirus, Hepatology, (28) 1998 1134-1146.
[17] L.M. Gao, Y.X. Han, Y.P. Wang, Y.H. Li, Y.Q. Shan, X. Li, Z.G. Peng, C.W. Bi, T.A. Zhang, N.N. Du,
J.D. Jiang, D.Q. Song, Design and Synthesis of Oxymatrine Analogues Overcoming Drug Resistance in
Hepatitis B Virus through Targeting Host Heat Stress Cognate 70, J. Med. Chem., 54 (2011) 869-876.
[18] Y.B. Xu, L. Yang, G.F. Wang, X.K. Tong, Y.J. Wang, Y. Yu, J.F. Jing, C.L. Feng, P.L. He, W. Lu, W.
27

ACCEPTED MANUSCRIPT
Tang, J.P. Zuo, Benzimidazole derivative, BM601, a novel inhibitor of hepatitis B virus and HBsAg
secretion, Antivir. Res., 107 (2014) 6-15.
[19] Y.J. Wang, D. Lu, Y.B. Xu, W.Q. Xing, X.K. Tong, G.F. Wang, C.L. Feng, P.L. He, L. Yang, W. Tang,
Y.H. Hu, J.P. Zuo, A Novel Pyridazinone Derivative Inhibits Hepatitis B Virus Replication by Inducing
Genome-Free Capsid Formation, Antimicrob. Agents. Ch, 59 (2015) 7061-7072.
[20] B. Seigneres, C. Pichoud, P. Martin, P. Furman, C. Trepo, F. Zoulim, Inhibitory activity of dioxolane
purine analogs on wild-type and lamivudine-resistant mutants of hepadnaviruses, Hepatology., 36
(2002) 710-722.
[21] SYBYL_X2.1 is availabe from Tripos Associates Inc., S. H. R., St. Louis, MO 631444, USA.
[22] K. Klumpp, A.M. Lam, C. Lukacs, R. Vogel, S.P. Ren, C. Espiritu, R. Baydo, K. Atkins, J. Abendroth,
G.C. Liao, A. Efimov, G. Hartman, O.A. Flores, High-resolution crystal structure of a hepatitis B virus
replication inhibitor bound to the viral core protein, P. Natl. Acad. Sci. USA., 112 (2015) 15196-15201.
28