Synthesis and evaluation of new phenyl acrylamide derivatives as potent non-nucleoside anti-HBV agents

Article abstract of DOI:10.1016/j.bmc.2020.115892

As a continuation of our previous work, a series of new phenyl acrylamide derivatives (4Aa-g, 4Ba-t, 5 and 6a-c) were designed and synthesized as non-nucleoside anti-HBV agents. Among them, compound 4Bs could potently inhibit HBV DNA replication in wild-type and lamivudine (3TC)/entecavir resistant HBV mutant strains with IC₅₀ values of 0.19 and 0.18 μM, respectively. Notably, the selective index value of 4Bs was above 526, indicating the favorable safety profile. Interestingly, unlike nucleoside analogue 3TC, 4Bs could significantly inhibit 3.5 kb pgRNA expression. Molecular docking study revealed that 4Bs could fit well into the dimer-dimer interface of HBV core protein by hydrophobic, π–π and H-bond interactions. Considering the potent anti-HBV activity, low toxicity and diverse anti-HBV mechanism from that of nucleoside anti-HBV agent 3TC, compound 4Bs might be a promising lead to develop novel non-nucleoside anti-HBV therapeutic agents, and warranted further investigation.

Full text of DOI:10.1016/j.bmc.2020.115892

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Synthesis and evaluation of new phenyl acrylamide derivatives as potent non-
nucleoside anti-HBV agents
Xiaoke Gu, Yinpeng Zhang, Yueting Zou, Xin Li, Mingyu Guan, Qingqing
Zhou, Jingying Qiu
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https://doi.org/10.1016/j.bmc.2020.115892
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Bioorganic & Medicinal Chemistry
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20 November 2020
Please cite this article as: X. Gu, Y. Zhang, Y. Zou, X. Li, M. Guan, Q. Zhou, J. Qiu, Synthesis and evaluation of
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Synthesis and evaluation of new phenyl acrylamide derivatives as potent non-nucleoside
anti-HBV agents
Xiaoke Gua, #, Yinpeng Zhangb, #, Yueting Zoub, Xin Lia, Mingyu Guana, Qingqing Zhoub, Jingying Qiua, 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 a continuation of our previous work, a series of new phenyl acrylamide derivatives (4Aa-g, 4Ba-t, 5
and 6a-c) were designed and synthesized as non-nucleoside anti-HBV agents. Among them, compound 4Bs
could potently inhibit HBV DNA replication in wild-type and lamivudine (3TC)/entecavir resistant HBV
mutant strains with IC50 values of 0.19 and 0.18 μM, respectively. Notably, the selective index value of 4Bs
was above 526, indicating the favorable safety profile. Interestingly, unlike nucleoside analogue 3TC, 4Bs
could significantly inhibit 3.5 kb pgRNA expression. Molecular docking study revealed that 4Bs could fit
well into the dimer-dimer interface of HBV core protein by hydrophobic, π−π and H-bond interactions.
Considering the potent anti-HBV activity, low toxicity and diverse anti-HBV mechanism from that of
nucleoside anti-HBV agent 3TC, compound 4Bs might be a promising lead to develop novel non-nucleoside
anti-HBV therapeutic agents, and warranted further investigation.
Keywords: Anti-HBV agents; Phenyl acrylamide derivatives; Non-nucleoside; Synthesis.
1. Introduction
Hepatitis B virus (HBV) infection is a major public health problem worldwide. Statistically, of the
approximately 5% of the world’s population (350 million people) that is chronic HBV carriers1, 2, and about
20% of them will finally develop into HBV-related cirrhosis or hepatocellular carcinoma (HCC).3-5
 Corresponding author. Tel.: +86-516-83262138; fax: +86-516-83262630; e-mail: jingyqiu@xzhmu.edu.cn (J. Q.)
 These two authors contributed equally to this work.
1

Interferons (IFNs) and nucleos(t)ides-based reverse transcriptase inhibitors are the main treatment options in
clinic, however, both of them do not provide satisfactory clinical outcomes for chronic HBV infection.6-8 For
instance, the long-term administration of nucleos(t)ide analogs always cause an increased viral resistance.9,
10 Therefore, there is an urgent requirement to develop novel non-nucleoside anti-HBV agents.11
Y101, a dipeptide derivative of herbal ingredient, was discovered by our research group.12 As a novel anti-
HBV agent, Y101 is currently in phase I clinical trials for the treatment of HBV infection in China.13 Our
previous work on the modification of Y101 led to novel analogs 8d and 8g, which displayed potent in vitro
anti-HBV activities.14 The IC50 values of 8d and 8g against wild-type HBV DNA replication were 0.46 and
2.44 μM, respectively. Previous structure activity relationship (SAR) study indicated that the 3-bromo-3-
phenylacrylamide moiety was significant for the anti-HBV activity of 8d, and the introduction of a meta
substituent on A ring, such as a methyl substituent, was benefit for the anti-HBV activity. To further
investigate the effect of meta substituents on the anti-HBV activities, we prepared eleven phenylacrylamide
derivatives by introducing various meta substituents into the A ring. In addition, we previously found that
the introduction of an ortho-F substituent on B ring could improve the anti-HBV activities. In order to obtain
more structurally diverse derivatives and further investigate the effect of different substituents on the anti-
HBV activity, we also synthesized twenty new compounds with various substituents on B ring, thus thirty-
one target compounds were obtained in total. Subsequently, their anti-HBV activities against wild and drug
resistant HBV strains, and the preliminary action mechanism were investigated in the present work.
R1
H3C
A
OH
OH
OH
OH
O
O
O
O
N
H
O
Br
N
H
O
Br
N
H
O
Br
N
H
O
SAR
N
HN
HN
HN
HN
O
exploration
F
R2
B
Y101 (Phase I)
IC50: 0.46 M SI: >217.39
8d
IC50: 2.44 M SI: >40.98
8g
Target compounds
Compound
Compound
Figure 1. Chemical structures of Y101, 8d, 8g and the target compounds
2. Results and Discussion
2.1. Chemistry
The synthetic procedures of the target compounds 4Aa-f and 4Ba-t were depicted in Scheme 1. Briefly,
2

compounds 1 were obtained by the acylation reaction with glycine and benzoyl chloride or benzoyl chloride
derivatives as the raw material, which were then reacted with benzaldehyde or benzaldehyde derivatives in
the presence of acetic anhydride (Ac2O) and sodium acetate (AcONa) to obtain compounds 2. Next, the
intermediates 2 were reacted with L-phenylalaninol to give intermediates 3, which were finally brominated
to give the target compounds 4Aa-f and 4Ba-t, respectively.
CHO
O
O
O
R1
H2N
COOH
R1
O
N
COOH
Cl
R2
R2
H
N
a
b
1
2
R2
R1
R1
H2N
OH
OH
O
O
OH
H
N
Br
N
d
H
O
H
HN
R2
HN
O
c
R2
4Aa-f, 4Ba-t
3
Scheme 1. Reagents and conditions: a) NaOH (aq), HCl; b) AcONa/Ac2O, 100 °C; c) CH2Cl2, reflux; d) Br2, CaCO3/CHCl3,
0 °C.
The synthetic route of the target compounds 4Ag, 5 and 6a-c was depicted in Scheme 2. Briefly, compound
1 was reacted with 3-hydroxybenzaldehyde in the presence of acetic anhydride (Ac2O) and sodium acetate
(AcONa) to obtain compound 2. Subsequently, the intermediate 2 was reacted with L-phenylalaninol to give
intermediate 3. Finally, the crude product 3 was directly brominated with bromine to give the target
compound 4Ag. After the hydrolysis of 4Ag, the intermediate 5 was successfully obtained, which was finally
alkylated with 2-dimethylaminoethyl chloride hydrochloride, 2-diethylaminoethyl chloride hydrochloride or
3-dimethylaminopropyl chloride hydrochloride to provide alkoxyl substituted derivatives 6a-c, respectively.
3

O
O
OH
H2N
O
O
HO
CHO
O
O
OH
H
N
O
N
COOH
H
O
HN
OH
H
O
N
a
b
1
2
3
O
HO
R1
O
OH
OH
O
O
O
Br
N
Br
N
Br
N
c
d
e
H
O
H
H
HN
HN
O
HN
O
6a-c
5
4Ag
Scheme 2. Reagents and conditions: a) AcONa/Ac2O, 100 °C; b) CH2Cl2, reflux; c) Br2, CaCO3/CHCl3, 0 °C; d) DMF,
NaOH, rt.; e) 2-dimethylaminoethyl chloride hydrochloride, 2-diethylaminoethyl chloride hydrochloride or 3-
dimethylaminopropyl chloride hydrochloride, 1,4-dioxane, K2CO3, 90 °C.
2.2. Biological evaluation
2.2.1. Cytotoxicity assays
An ideal anti-HBV drug should inhibit HBV DNA replication at a non-toxic concentration. Therefore, we
firstly determined the in vitro intrinsic cytotoxicity of the target compounds against HepG2 2.2.15 cells by
MTT assay. As shown in Table 1, most of the target compounds displayed little (CC50 > 100 μM) or very
low (CC50 = 83.57 ~ 99.51 μM) intrinsic cytotoxicity against HepG2 2.2.15 cells, indicating their relatively
safety profile.
2.2.2. Inhibitory effect on replication of HBV DNA
Next, we investigated the in vitro anti-HBV activity of the target compounds with various meta substituents
on A ring by determining their effect on HBV DNA levels in HepG2 2.2.15 cells using real time PCR assay.15
Lead compound 8d (0.8 μM) and lamivudine (3TC, 0.8 μM) were used as positive controls. As shown in
Table 1, when HepG2 2.2.15 cells were treated with the target compounds at a non-toxic dose (0.8 μM) for
6 days, the extra cellar HBV DNA levels were decreased, with inhibition rates of 14.37-58.38%. Among
them, compounds 6a and 6b displayed the most potent anti-HBV activity with inhibition rates of 58.38% and
56.46%, respectively. Notably, when the meta methyl of compound 8d was replaced with other substituents,
4

such as nitryl (compound 4Aa), trifluoromethyl (compound 4Ab), halogen (compounds 4Ac-e), methoxyl
(compound 4Af) and hydroxyl (compound 5), the in vitro anti-HBV activity was decreased, confirming that
a meta methyl was indeed benefit for the improvement of anti-HBV activity. Interestingly, when meta
hydroxyl group was alkylated with dimethylamino ethyl (compound 6a), diethylamine ethyl (compound 6b)
or dimethylamino propyl (compound 6c), the anti-HBV activity was significantly increased, which was
consistent with our previous findings that such substituents were beneficial for anti-HBV activity.14
Table 1. Inhibitory effect of the target compounds 4Aa-g, 5 and 6a-c (0.8 μM) on HBV DNA levels
R1
H3C
A
A
OH
OH
O
O
Br
N
H
O
Br
N
H
O
HN
HN
B
B
Lead compound 8d
Target compound 4Aa-g, 5 and 6a-c
aCC50
Inhibition rate
CC50
Inhibition rate
(%)
Compounds
R1
Compounds
R1
(μM)
93.72
>100
>100
>100
>100
>100
>100
(%)
(μM)
4Aa
4Ab
4Ac
4Ad
4Ae
4Af
4Ag
NO2
CF3
2.28
5
6a
OH
99.51
94.66
83.57
85.64
>100
>100
29.63
58.38
56.46
37.72
57.31
86.23
23.07
42.61
48.16
14.37
48.90
32.35
OCH2CH2N(CH3)2
F
6b
OCH2CH2N(CH2CH3)2
Cl
6c
OCH2CH2CH2N(CH3)2
Br
8d
/
/
OCH3
OCOCH3
3TC
a CC50: defined as the concentration that induced 50% death of HepG2 2.2.15 cells.
Subsequently, we determined the in vitro anti-HBV activity of the target compounds with various
substituents on B ring (Table 2), and selected lead compound 8g (0.8 μM) and lamivudine (3TC, 0.8 μM) as
positive controls. Data showed that lead compound 8g (with an ortho-F atom on B ring) exhibited moderate
in vitro anti-HBV activity with an inhibition of control of 33.62%. When the ortho-F atom was changed into
ortho-Cl (compound 4Ba) or Br atom (compound 4Bb), the anti-HBV activity of the compound was
decreased. And the introduction of an ortho-electron-donating group (ie, OCH3 or CH3) in B ring also resulted
5

in a dramatic decrease in the inhibition of control, suggesting that ortho-electron-donating group was
detrimental to the in vitro anti-HBV activity. In addition, when a substituent was introduced into the meta
position of B ring (Compounds 4Be-i), the in vitro anti-HBV activity was almost lost. Notably, the
introduction of a para-substituent, especially an electron withdrawing substituent, such as F, Cl or Br atom,
could significantly increase the anti-HBV activity. Clearly, para-F and Cl atom were the best, for the
inhibition rates of compound 4Bj and 4Bk were 74.94% and 70.65%, respectively, which were much higher
than that of lead compound 8g. Interestingly, introduction of a second substituent in the B ring (compounds
4Bo-t) could significantly affect the anti-HBV activity. Among them, compound 4Bs was the most potent
one with an inhibition rate of 87.98%, which was even higher than that of classic anti-HBV drug 3TC
(86.23%).
Table 2. Inhibitory effect of the target compounds 4Ba-t (0.8 μM) on HBV DNA level
A
A
OH
OH
O
O
Br
N
H
O
Br
N
H
O
HN
HN
F
R2
B
B
Lead compound 8g
Target compound 4Ba-t
CC50
Inhibition of
CC50
Inhibition of
control (%)
Compounds
R2
Compounds
R2
(μM)
control (%)
13.93
(μM)
4Ba
4Bb
4Bc
4Bd
4Be
4Bf
4Bg
4Bh
4Bi
2-Cl
2-Br
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
4Bl
4Bm
4Bn
4Bo
4Bp
4Bq
4Br
4Bs
4Bt
8g
4-Br
4-OCH3
4-CH3
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
43.00
6.25
17.19
11.86
5.75
9.07
5.36
2.48
4.89
2.94
74.91
2-OCH3
2-CH3
3-F
47.36
58.08
73.65
56.08
83.22
87.98
83.76
33.62
2-F, 4-F
3-F, 4-F
2-Cl, 4-F
2-CH3, 4-F
2-Cl, 4-Cl
3-Cl, 5-Cl
/
3-Cl
3-Br
3-OCH3
3-CH3
4-F
4Bj
6

4Bk
4-Cl
>100
70.65
3TC
/
>100
86.23
To further confirm the in vitro anti-HBV activity, we subsequently evaluated the IC50 values for the HBV
DNA replication of the active compounds, and also determined their selectivity index (SI). As shown in Table
3, all the tested compounds could remarkably inhibit HBV DNA replication with IC50 values of 0.19-0.48
μM, indicating their potent in vitro anti-HBV activity. Clearly, compound 4Bs was the most prominent one,
with an IC50 value of 0.19 μM. Notably, 4Bs exhibited a high SI value of above 526, indicating that 4Bs
possessed a relatively favorable safety profile.
Table 3. Anti-HBV activity and SI values of target compounds 4Bj, 4Bk, 4Bp and 4Br-t
DNA replication
Compounds
CC50 (μM)
aIC50 (μM)
0.38 ± 0.043
0.43 ± 0.040
0.48 ± 0.047
0.33 ± 0.044
0.19 ± 0.015
0.27 ± 0.05
bSI
4Bj
4Bk
4Bp
4Br
4Bs
4Bt
3TC
>100
>100
>100
>100
>100
>100
>100
>263
>232
>208
>303
>526
>370
>1000
0.031 ± 0.008
a IC50 : defined as the concentration of compound required for 50% inhibition of HBV DNA replication, and expressed as
means ± SD of triplicate experiments. b SI : Selectivity index (SI), calculated by dividing the CC50 value by the IC50 value.
2.2.3. In vitro anti-HBV activity of compounds 4Bj and 4Bs against polymerase drug resistant HBV strain
As mentioned above, during the long-term administration of nucleoside drugs, HBV strains will gradually
become resistant, thus resulting in the treatment failure. Therefore, we next evaluated the anti-HBV effect of
the active compounds 4Bj and 4Bs on 3TC and entecavir dually resistant mutant (rtL180M + rtM204V +
rtT184L). As shown in Figure 2, nucleoside drugs 3TC and entecavir could remarkably inhibit the wild-type
HBV DNA replication with inhibition rates of 90.01% and 96.82%, respectively, while the HBV DNA
replication inhibition rates of 3TC and entecavir resistant strain were significantly decreased to 18.58% and
20.71%, respectively. Notably, compounds 4Bj and 4Bs exhibited potent inhibitory effect on HBV DNA
replication of drug resistant strain with inhibition rates of 72.90% and 87.02%, respectively, which were
7

comparable to that of wild type strain.
Figure 2. Antiviral effect of 4Bj and 4Bs 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), 4Bj (0.8 μM), 4Bs (0.8 μM) respectively. HBV DNA replicative intermediate level
was measured using real time PCR. **P < 0.01 represents significant difference from the WT HBV group.
To further confirm the anti-resistant HBV activity, we investigated whether compounds 4Bj and 4Bs could
dose-dependently inhibit the HBV DNA replication in drug resistant HBV strain. As shown in Table 4, the
IC50 values for HBV DNA replication of 4Bj and 4Bs were 0.43 and 0.18 μM, respectively, validating their
potent inhibitory effect on HBV DNA replication in drug resistant strain. Excitingly, the SI values of
compound 4Bj and 4Bs were above 232 and 556, respectively, further confirming their relatively safety
profiles.
Table 4. Inhibitory activity of compounds 4Bj and 4Bs against lamivudine and entecavir resistant HBV strain
DNA replication
Compounds
CC50 (μM)
a IC50 (μM)
0.43 ± 0.035
0.18 ± 0.021
>100
b SI
>232
>556
/
4Bj
4Bs
3TC
>100
>100
>100
a IC50 : defined as the concentration of compound required for 50% inhibition of HBV DNA replication, and expressed as
means ± SD of triplicate experiments. b SI : Selectivity index (SI), calculated by dividing the CC50 value by the IC50 value.
2.2.4. Inhibitory Effect of compound 4Bs on HBV viral gene expression
Considering that pregenomic RNA (pgRNA) plays a crucial role in HBV lifecycle and is related to HBV
DNA replication, we subsequently determined whether compound 4Bs could inhibit HBV viral gene
8

expression according to the previous methods.16, 17 Briefly, HepG2 2.2.15 cells were firstly incubated with 1
μM compound 4Bs for 6 days, and then the total cellular RNA was extracted and subsequently detected by
real time PCR. 3TC was used as a nucleoside analog control. As shown in Table 5, 3TC displayed little
inhibitory effect on pgRNA expression. In sharp contrast, compound 4Bs could significantly inhibit 3.5 kb
pgRNA expression with an inhibition of control of 63.13%, suggesting that compound 4Bs may possess a
different anti-HBV action mechanism from that of nucleoside analog 3TC.
Table 5. Effect of compound 4Bs on intracellular HBV pgRNA
Concentration
pgRNA
Inhibition of
control (%)
Compounds
(μM)
(copies/mL)
4Bs
3TC
1
1
-
2.54E+06
6.73E+06
6.89E+06
63.13
0.26
-
Control
2.2.5. Potential effect on HBV core protein
It is well-known that HBV core protein performs multiple roles in HBV life cycle, and is essential for
HBV replication, viral assembly, and cccDNA maintenance.18 Moreover, as a viral protein, HBV core protein
is not present in human cells.19 Therefore, HBV core protein is regarded as a promising target for developing
novel, virus-selective, and efficacious anti-HBV agents.19, 20 Considering the advantages of HBV core protein
as non-nucleoside anti-HBV drug target, we analyzed the binding mode of 4B with HBV core protein by
molecular docking to preliminarily understand the potential effect of 4Bs on HBV core protein.
9

Figure 3. Binding modes of compound 4Bs with HBV capsid protein Y132A (PDB code 5e0i). The binding pocket was at
Chains B and C interface. Chains B and C were shown as cartoon model, and colored in blue and orange, respectively.
Compound 4Bs was highlighted in white stick. The key residues in the active site were shown as blue and orange line. Red
dash lines indicated the hydrogen bonds between compounds and core protein (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article).
As shown in Figure 3, compound 4Bs, as compared to the reference ligand, was flexibly docked into the
dimer-dimer interface of HBV core protein. Generally, 4Bs was stabilized in a well-defined hydrophobic
cavity composed of Phe23, Trp102, Tyr118, Val124, Arg127 Ile139, Leu140 (Chain B) and Pro25, Leu30,
Thr33, Ile105 Trp125,Thr128, Pro134 (Chain C). Meanwhile, several main interactions were predicted
between 4Bs and the HBV core protein, including that π−π interactions were indicated between the benzene
ring in the phenyl acrylamide scaffold of 4Bs with the phenyl ring of Phe23 and Tyr118 of HBV core protein;
another π−π interactions were observed between the benzene ring in the phenyl acrylamide scaffold and 3,4-
dichlorophenyl of 4Bs with Trp102 and Trp 125 of HBV core protein, respectively; two hydrogen bonds
(3.09 and 2.86 Å) occurred between amide carbonyl, amino of phenyl acrylamide scaffold of 4Bs with Thr128
of HBV core protein, respectively; and another hydrogen bond (2.70 Å) was observed between hydroxyl
group of phenylpropanolamine of 4Bs with Trp102. The molecular docking studies indicated that compound
4Bs displayed molecular interactions with HBV core protein, and might be a potential inhibitor of HBV core
protein.
10

3. Conclusions
In summary, thirty-one compounds with phenyl acrylamide scaffold were designed and prepared as non-
nucleoside anti-HBV agents. Among them, compound 4Bs exhibited the most potent inhibitory effect on
HBV DNA replication with an IC50 value of 0.19 μM. In addition, compound 4Bs possessed a high selective
index values (above 526), indicating that the safety profile was relatively favorable. Notably, 4Bs also
displayed a significant antiviral effect on 3TC and entecavir resistant HBV mutants with an IC50 value was
0.18 μM. More interestingly, unlike nucleoside analog 3TC, 4Bs could significantly inhibit 3.5 kb pgRNA
expression. Molecular docking study revealed that compound 4Bs could fit well into the dimer-dimer
interface of HBV core protein by hydrophobic, π−π and H-bond interactions. Considering the potent anti-
HBV activity, low toxicity and diverse anti-HBV mechanism from that of nucleoside anti-HBV drug 3TC,
compound 4Bs might be a promising lead to develop novel non-nucleoside anti-HBV therapeutic agents, and
warranted further investigation.
4. Experimental protocols
4.1. Chemistry
Compounds were purified by column chromatography using silica gel 60 (200–300 mesh) or thin layer
chromatography (TLC) using silica gel 60 F254 plates (250 mm; Qingdao Ocean Chemical Company, China).
1
Melting points were determined with a model YRT-3 apparatus and are uncorrected. H NMR (400 MHz)
and 13C NMR (100 MHz) spectra were run on a JEOL (400 MHz) spectrometer in CDCl3 or DMSO-d6 with
Me4Si (TMS) as an internal standard. Mass detection was performed on an Agilent G6460C triple quadrupole
mass spectrometer. Analytical HPLC was run on the Waters Alliance E2695 HPLC instrument, equipped
with Chiralcel OD-RH column and UV detection at 254 nm.. Individual compounds with a purity of >95%
were used for subsequent experiments. All solvents were reagent grade and were purified and dried by
standard methods when necessary.
4.1.1. General procedure for the synthesis of compounds 4Aa-f and 4Ba-t
Compounds 1 were 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 were 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 2. Next, the intermediates mentioned
11

above were reacted with L-phenylalaninol (0.01 mol) to yield the crude products 3, respectively. Finally, the
crude products 3 (0.01 mol) were directly brominated with bromine (0.015 mol) and anhydrous calcium
carbonate (0.01 mol) in CHCl3 to give the crude products, which were purified by column chromatography
to yield the title compounds 4Aa-f and 4Ba-t, respectively.
4.1.1.1. (S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-1-(3-nitrophenyl)-3-oxoprop-1-en- 2-
yl)benzamide (4Aa)
As a yellow powder, yield: 32.7%, m.p.163.4-164.7 °C. Analytical data for 4Aa: 1H NMR (DMSO, 400
MHz,  ppm): 10.08 (s, 1H, NH), 8.15-8.16 (m, 3H, Ar-H), 8.04 (d, J = 8.4 Hz, 2H, Ar-H), 7.54-7.71 (m,
5H, Ar-H), 7.04-7.18 (m, 5H, Ar-H), 4.14 (t, d = 4.0 Hz, 1H, OH), 3.75 (br, 1H, CH), 2.98-3.20 (m, 2H,
CH2), 2.40-2.62 (m, 2H, CH2); 13C NMR (DMSO, 100 MHz, δ ppm): 165.67, 162.86, 147.69, 139.88, 139.30,
136.07, 135.81, 133.37, 132.79, 130.38, 129.35 (2C), 129.05 (2C), 128.55 (2C), 128.52 (2C), 126.33, 124.30,
124.17, 117.18, 61.96, 53.51, 36.08; ESI-MS: m/z 546.0 [M + Na]+; ESI-HRMS (TOF): m/z [M + H]+ calcd
for C25H23BrN3O5, 524.0821, found 524.0831.
4.1.1.2. (S,Z)-N-(1-bromo-1-(3-bromophenyl)-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxoprop-1-en-
2-yl)benzamide (4Ab)
1
As a white powder, yield: 28.2%, m.p.173.1-174.3 °C. Analytical data for 4Ab: H NMR (DMSO, 400
MHz,  ppm): 9.99 (s, 1H, NH), 7.97-8.01 (m, 3H, Ar-H, NH), 7.48-7.68 (m, 7H, Ar-H), 7.08-7.21 (m, 3H,
Ar-H), 7.00-7.02 (m, 2H, Ar-H), 3.65-3.70 (m, 1H, CH), 2.91-3.10 (m, 2H, CH2), 2.31-2.53 (m, 2H, CH2);
13C NMR (DMSO, 100 MHz, δ ppm): 165.66, 162.88, 139.51, 139.27, 135.35, 133.56, 133.40, 132.75,
2
130.01, 129.46 (2C), 129.23 (d, JCF = 31.8 Hz), 129.03 (2C), 128.61 (2C), 128.50 (2C), 126.36, 126.19,
126.03, 125.75 (d, 1JCF = 270.3 Hz), 118.33, 61.78, 53.42, 36.02; ESI-MS: m/z 569.1 [M + Na]+; ESI-HRMS
(TOF): m/z [M + H]+ calcd for C26H23BrF3N2O3, 547.0844, found 547.0856.
4.1.1.3. (S,Z)-N-(1-bromo-1-(3-fluorophenyl)-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxoprop-1-en-
2-yl)benzamide (4Ac)
1
As a white powder, yield: 32.7%, m.p.164.3-165.8 °C. Analytical data for 4Ac: H NMR (DMSO, 400
MHz,  ppm): 9.94 (s, 1H, NH), 7.99 (d, J = 7.2 Hz, 2H, Ar-H), 7.90 (d, J = 8.4 Hz, 1H, NH), 7.59 (t, J =
7.6 Hz, 1H, Ar-H), 7.50 (t, J = 6.0 Hz, 2H, Ar-H), 7.03-7.22 (m, 9H, Ar-H), 4.37 (t, J = 5.6 Hz, 1H, OH),
3.67-3.75 (m, 1H, CH), 2.94-3.15 (m, 2H, CH2), 2.37-2.57 (m, 2H, CH2); 13C NMR (DMSO, 100 MHz, δ
12

1
3
ppm): 165.63, 163.18 (d, JCF = 232.8 Hz), 162.96, 140.65 (d, JCF = 232.8 Hz), 139.37, 134.88, 133.43,
3
132.71, 130.74 (d, JCF = 7.5 Hz), 129.49 (2C), 129.01 (2C), 128.61 (2C), 128.48 (2C), 126.36, 125.75,
118.70, 116.63 (d, 2JCF = 20.8 Hz), 116.56 (d, 2JCF = 22.6 Hz), 61.96, 53.41, 36.09; ESI-MS: m/z 519.1 [M
+ Na]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C25H23BrFN2O3, 497.0876, found 497.0884.
4.1.1.4. (S,Z)-N-(1-bromo-1-(3-chlorophenyl)-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxoprop-1-en-
2-yl)benzamide (4Ad)
1
As a white powder, yield: 28.4%, m.p.162.6-163.7 °C. Analytical data for 4Ad: H NMR (DMSO, 400
MHz,  ppm): 9.94 (s, 1H, NH), 7.98 (d, J = 7.2 Hz, 2H, Ar-H), 7.93 (d, J = 8.4 Hz, 1H, NH), 7.59 (t, J =
7.2 Hz, 1H, Ar-H), 7.50 (t, J = 7.2 Hz, 2H, Ar-H), 7.35-7.38 (m, 2H, Ar-H), 7.27 (t, J = 7.6 Hz, 1H, Ar-H),
7.03-7.21 (m, 6H, Ar-H), 4.37 (t, J = 7.2 Hz, 1H, OH), 3.69-3.74 (m, 1H, CH), 2.95-3.16 (m, 2H, CH2), 2.37-
2.57 (m, 2H, CH2); 13C NMR (DMSO, 100 MHz, δ ppm): 165.62, 162.94, 140.42, 139.36, 135.01, 133.43,
133.15, 132.71, 130.59, 129.50 (2C), 129.24, 129.01 (2C), 128.62 (2C), 128.50 (2C), 128.32, 118.51, 61.91,
53.45, 36.09; ESI-MS: m/z 535.0 [M + Na]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C25H23BrClN2O3,
513.0581, found 513.0585.
4.1.1.5. (S,Z)-N-(1-bromo-1-(3-bromophenyl)-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxoprop-1-en-
2-yl)benzamide (4Ae)
As a yellow powder, yield: 26.2%, m.p.185.8-186.6 °C. Analytical data for 4Ae: 1H NMR (DMSO, 400
MHz,  ppm): 9.95 (s, 1H, NH), 7.98 (d, J = 7.2 Hz, 2H, Ar-H), 7.95 (d, J = 8.4 Hz, 1H, NH), 7.58 (t, J =
7.6 Hz, 1H, Ar-H), 7.48-7.52 (m, 4H, Ar-H), 7.11-7.26 (m, 5H, Ar-H), 7.05 (d, J = 8.0 Hz, 2H, Ar-H), 4.38
(br, 1H, OH), 3.69-3.74 (m, 1H, CH), 2.97-3.16 (m, 2H, CH2), 2.38-2.56 (m, 2H, CH2); 13C NMR (DMSO,
100 MHz, δ ppm): 165.62, 162.93, 140.64, 139.35, 135.01, 133.43, 132.72, 132.33, 132.02, 130.84, 129.51
(2C), 129.02 (2C), 128.69, 128.62 (2C), 128.49 (2C), 126.38, 121.62, 118.44, 61.89, 53.45, 36.10; ESI-MS:
m/z 559.0 [M + Na]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C25H23Br2N2O3, 557.0075, found 557.0079.
4.1.1.6. (S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-1-(3-methoxyphenyl)-3-oxoprop-1-
en-2-yl)benzamide (4Af)
1
As a yellow powder, 26.3%, m.p.86.4-87.8 °C. Analytical data for 4Af: H NMR (DMSO, 400 MHz, 
ppm): 9.92 (s, 1H, NH), 8.00 (d, J = 8.0 Hz, 2H, Ar-H), 7.76 (d, J = 8.4 Hz, 1H, NH), 7.58 (t, J = 7.6 Hz,
1H, Ar-H), 7.50 (t, J = 6.8 Hz, 2H, Ar-H), 7.08-7.21 (m, 4H, Ar-H), 7.04 (d, J = 8.0 Hz, 2H, Ar-H), 6.86-
13

6.92 (m, 3H, Ar-H), 4.34 (t, J = 6.8 Hz, 1H, OH), 3.67-3.74 (m, 4H, CH, OCH3), 2.93-3.14 (m, 2H, CH2),
2.36-2.53 (m, 2H, CH2); 13C NMR (DMSO, 100 MHz, δ ppm): 165.59, 163.16, 159.26, 139.61, 139.36,
134.19, 133.53, 132.63, 129.79, 129.51 (2C), 129.00 (2C), 128.60 (2C), 128.43 (2C), 126.34, 121.93, 120.58,
115.40, 115.15, 61.85, 55.69, 53.35, 36.10; ESI-MS: m/z 531.1 [M + Na]+; ESI-HRMS (TOF): m/z [M + H]+
calcd for C26H26BrN2O4, 509.1076, found 509.1047.
4.1.1.7.
(S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en-2-yl)-2-
chlorobenzamide (4Ba)
1
As a white powder, yield: 36.4%, m.p.167.8-168.6 °C. Analytical data for 4Ba: H NMR (DMSO, 400
MHz,  ppm): 10.13 (s, 1H, NH), 7.81 (d, J = 8.4 Hz, 1H, NH), 7.40-7.56 (m, 4H, Ar-H), 7.26-7.29 (m, 5H,
Ar-H), 7.04-7.21 (m, 5H, Ar-H), 4.34 (br, 1H, OH), 3.70-3.75 (m, 1H, CH), 2.93-3.12 (m, 2H, CH2), 2.35-
2.56 (m, 2H, CH2); 13C NMR (DMSO, 100 MHz, δ ppm): 165.49, 162.74, 139.44, 138.35, 135.70, 133.66,
132.00, 130.79, 130.29, 130.01, 129.60, 129.53 (4C), 128.66 (4C), 127.54, 126.37, 119.86, 61.94, 53.25,
36.09; ESI-MS: m/z 535.4 [M + Na]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C25H23BrClN2O3, 513.0581,
found 513.0578.
4.1.1.8. (S,Z)-2-bromo-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en -
2- yl)benzamide (4Bb)
1
As a white powder, yield: 29.6%, m.p.174.4-175.8 °C. Analytical data for 4Bb: H NMR (DMSO, 400
MHz,  ppm): 10.16 (s, 1H, NH), 7.82 (d, J = 8.4 Hz, 1H, NH), 7.72 (d, J = 8.0 Hz, 1H, Ar-H), 7.57 (dd, J =
7.6 Hz, 1.6 Hz, 1H, Ar-H), 7.50 (t, J = 7.6 Hz, 1H, Ar-H), 7.43 (t, J = 7.6 Hz, 1H, Ar-H), 7.15-7.35 (m, 8H,
Ar-H), 7.09 (d, J = 6.8 Hz, 2H, Ar-H), 3.72-3.78 (m, 1H, CH), 2.97-3.17 (m, 2H, CH2), 2.40-2.60 (m, 2H,
CH2); 13C NMR (DMSO, 100 MHz, δ ppm): 166.37, 162.73, 139.44, 138.37, 137.86, 133.66, 133.40, 132.06,
129.99, 129.59 (3C), 129.52 (2C), 128.66 (2C), 128.64 (2C), 127.97, 126.37, 120.02, 119.62, 61.96, 53.27,
36.12; ESI-MS: m/z 557.0 [M + H]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C25H23Br2N2O3, 557.0075,
found 557.0069.
4.1.1.9.
(S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en-2-yl)-2-
methoxybenzamide (4Bc)
1
As a white powder, yield: 38.5%, m.p. 127.3-128.9 °C. Analytical data for 4Bc: H NMR (DMSO, 400
MHz,  ppm): 10.12 (s, 1H, NH), 8.00 (d, J = 8.4 Hz, 1H, Ar-H), 7.95 (dd, J = 8.0, 2.0 Hz, 1H, NH), 7.60 (t,
14

J = 7.2 Hz, 1H, Ar-H), 7.04-7.28 (m, 12H, Ar-H), 4.32 (t, J = 6.0 Hz, 1H, OH), 4.05 (s, 3H, OCH3), 3.69-
3.74 (m, 1H, CH), 2.96-3.19 (m, 2H, CH2), 2.35-2.59 (m, 2H, CH2); 13C NMR (CDCl3, 100 MHz, δ ppm):
163.50, 163.25, 157.89, 137.04, 136.93, 134.32, 133.64, 133.07, 129.61, 129.45 (2C), 129.25 (2C), 128.61
(2C), 128.59 (2C), 126.59, 121.63, 119.70, 113.63, 111.48, 63.03, 56.32, 52.86, 36.78; ESI-MS: m/z 531.0
[M + Na]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C26H25BrN2O4, 509.1076, found 509.1066.
4.1.1.10. (S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en-2-yl)-2-
methylbenzamide (4Bd)
1
As a white powder, yield: 34.6%, m.p. 137.7-138.6 °C. Analytical data for 4Bd: H NMR (DMSO, 400
MHz,  ppm): 9.84 (s, 1H, NH), 7.77 (d, J = 8.0 Hz, 1H, NH), 7.49 (d, J = 7.2 Hz, 2H, Ar-H), 7.36 (d, J =
6.4 Hz, 1H, Ar-H), 7.23-7.30 (m, 7H, Ar-H), 7.13-7.20 (m, 3H, Ar-H), 7.05 (d, J = 6.8 Hz, 2H, Ar-H), 4.33
(t, J = 6.0 Hz, 1H, OH), 3.70-3.75 (m, 1H, CH), 2.91-3.14 (m, 2H, CH2), 2.35-2.55 (m, 5H, CH2, CH3); 13
C
NMR (DMSO, 100 MHz, δ ppm): 168.40, 163.04, 139.46, 138.38, 136.56, 135.77, 134.02, 131.12, 130.56,
129.61 (2C), 129.52 (3C), 128.64 (4C), 128.14, 126.35, 126.00, 120.30, 62.05, 53.23, 36.11, 20.08; ESI-MS:
m/z 515.1 [M + Na]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C26H26BrN2O3, 493.1127, found 493.1119.
4.1.1.11. (S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en-2-yl)-3-
fluorobenzamide(4Be)
1
As a white powder, yield: 32.4%, m.p. 157.1-158.7 °C. Analytical data for 4Be: H NMR (DMSO, 400
MHz,  ppm): 10.07 (s, 1H, NH), 7.77-7.84 (m, 3H, Ar-H, NH), 7.54-7.59 (m, 1H, Ar-H), 7.45 (t, J = 8.4
Hz, 1H, Ar-H), 6.99-7.29 (m, 10H, Ar-H), 4.35 (br, 1H, OH), 3.70 (br, 1H, CH), 2.94-3.13 (m, 2H, CH2),
2.36-2.56 (m, 2H, CH2); 13C NMR (DMSO, 100 MHz, δ ppm): 164.37, 163.67 (d, 1JCF = 243.2 Hz), 162.91,
139.44, 138.37, 135.88 (d, 3JCF = 6.8 Hz), 133.70, 131.28 (d, 3JCF = 8.0 Hz), 129.56 (5C), 128.68 (2C), 128.60
(2C), 126.35, 124.71 (d, 4JCF = 2.1 Hz), 121.86, 119.66 (d, 2JCF = 21.3 Hz), 115.38 (d, 2JCF = 22.9 Hz), 62.05,
53.32, 36.11; ESI-MS: m/z 497.1 [M + H]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C25H23BrFN2O3,
497.0876, found 497.0866.
4.1.1.12. (S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en-2-yl)-3-
chlorobenzamide (4Bf)
1
As a white powder, yield: 33.4%, m.p. 140.7-141.9 °C. Analytical data for 4Bf: H NMR (DMSO, 400
MHz,  ppm): 10.12 (s, 1H, NH), 8.04 (t, J = 5.6 Hz, 1H, Ar-H), 7.93 (d, J = 8.0 Hz, 1H, Ar-H), 7.80 (d, J =
15

8.4 Hz, 1H, NH), 7.68 (d, J = 8.0 Hz, 1H, Ar-H), 7.54 (t, J = 8.0 Hz,1H, Ar-H), 7.03-7.30 (m, 10H, Ar-H),
3.68-3.73 (m, 1H, CH), 2.92-3.12 (m, 2H, CH2), 2.35-2.57 (m, 2H, CH2); 13C NMR (DMSO, 100 MHz, δ
ppm): 164.39, 162.88, 139.54, 138.39, 135.56, 133.80, 133.69, 132.43, 131.03, 129.56 (5C), 128.67 (2C),
128.60 (2C), 128.31, 127.24, 126.34, 121.92, 62.08, 53.33, 36.12; ESI-MS: m/z 535.1 [M + Na]+; ESI-HRMS
(TOF): m/z [M + H]+ calcd for C25H23BrClN2O3, 513.0581, found 513.0573.
4.1.1.13. (S,Z)-3-bromo-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en-
2-yl)benzamide (4Bg)
1
As a white powder, yield: 32.5%, m.p. 103.9-104.4 °C. Analytical data for 4Bg: H NMR (DMSO, 400
MHz,  ppm): 10.11 (s, 1H, NH), 8.18 (s, 1H, Ar-H), 7.96 (d, J = 7.6 Hz, 1H, Ar-H), 7.77-7.80 (m, 2H, Ar-
H, NH), 7.47 (t, J = 8.0 Hz, 1H, Ar-H), 7.03-7.27 (m, 10H, Ar-H), 3.68-3.71 (br, 1H, CH), 2.92-3.13 (m, 2H,
CH2), 2.36-2.57 (m, 2H, CH2); 13C NMR (DMSO, 100 MHz, δ ppm): 164.32, 162.88, 139.46, 138.40, 135.73,
135.33, 133.68, 131.27, 131.18, 129.56 (5C), 128.67 (2C), 128.60 (2C), 127.62, 126.35, 122.25, 121.93,
62.10, 53.33, 36.11; ESI-MS: m/z 579.0 [M + H]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C25H23Br2N2O3,
557.0075, found 557.0060.
4.1.1.14. (S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en-2-yl)-3-
methoxybenzamide (4Bh)
1
As a white powder, yield: 32.6%, m.p. 180.3-181.6 °C. Analytical data for 4Bh: H NMR (DMSO, 400
MHz,  ppm): 9.92 (s, 1H, NH), 7.75 (d, J = 8.4 Hz, 1H, NH), 7.55-7.58 (m, 2H, Ar-H), 7.41 (t, J = 8.0 Hz,
1H, Ar-H), 7.02-7.29 (m, 10H, Ar-H), 4.43 (br, 1H, OH), 3.80 (s, 3H, OCH3), 3.70-3.75 (m, 1H, CH), 2.94-
3.13 (m, 2H, CH2), 2.35-2.54 (m, 2H, CH2); 13C NMR (DMSO, 100 MHz, δ ppm): 165.39, 163.08, 159.73,
139.41, 138.41, 134.90, 133.98, 130.18, 129.60 (2C), 129.56 (3C), 128.68 (2C), 128.63 (2C), 126.36, 121.21,
120.74, 118.61, 113.42, 62.00, 55.92, 53.31, 36.09; ESI-MS: m/z 531.0 [M + Na]+; ESI-HRMS (TOF): m/z
[M + H]+ calcd for C26H26BrN2O4, 509.1076, found 509.1065.
4.1.1.15. (S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en-2-yl)-3-
methylbenzamide (4Bi)
1
As a white powder, yield: 29.4%, m.p. 149.3-150.6 °C. Analytical data for 4Bi: H NMR (DMSO, 400
MHz,  ppm): 9.85 (s, 1H, NH), 7.72-7.80 (m, 3H, Ar-H, NH), 7.38-7.39 (m, 2H, Ar-H), 7.28 (br, 5H, Ar-
H), 7.12-7.18 (m, 3H, Ar-H), 7.02-7.04 (m, 2H, Ar-H), 3.66-3.74 (m, 1H, CH), 2.92-3.12 (m, 2H, CH2), 2.34-
16

2.58 (m, 5H, CH2, CH3); 13C NMR (DMSO, 100 MHz, δ ppm): 165.45, 162.66, 139.47, 138.23, 133.70,
131.27, 131.18, 129.59 (2C), 129.49 (2C), 128.97, 128.65 (2C), 128.63 (2C), 127.34, 126.95, 126.34, 120.12,
61.93, 53.32, 36.05; ESI-MS: m/z 515.1 [M + H]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C26H26BrN2O3,
493.1127, found 493.1116.
4.1.1.16. (S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en-2-yl)-4-
fluorobenzamide (4Bj)
1
As a white powder, yield: 31.6%, m.p. 160.3-161.9 °C. Analytical data for 4Bj: H NMR (DMSO, 400
MHz,  ppm): 9.99 (s, 1H, NH), 8.03-8.08 (m, 2H, Ar-H), 7.77 (d, J = 8.4 Hz, 1H, NH), 7.03-7.38 (m, 12H,
Ar-H), 3.68-3.73 (m, 1H, CH), 2.91-3.11 (m, 2H, CH2), 2.34-2.55 (m, 2H, CH2); 13C NMR (DMSO, 100
MHz, δ ppm): 166.16 (d, 1JCF = 208.2 Hz), 164.61, 163.04, 139.44, 138.39, 133.90, 131.32 (d, 3JCF = 9.2 Hz,
4
2C), 130.05 (d, JCF = 2.0 Hz), 129.56 (4C), 128.67 (2C), 128.61 (2C), 126.35, 121.43, 121.43, 116.11 (d,
2JCF = 21.5 Hz, 2C), 62.04, 53.32, 36.09; ESI-MS: m/z 519.0 [M + Na]+; ESI-HRMS (TOF): m/z [M + H]+
calcd for C25H23BrFN2O3, 497.0876, found 497.0872.
4.1.1.17. (S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en-2-yl)-4-
chlorobenzamide (4Bk)
1
As a white powder, yield: 29.7%, m.p. 166.6-167.1 °C. Analytical data for 4Bk: H NMR (DMSO, 400
MHz,  ppm): 10.04 (s, 1H, NH), 8.00 (d, J = 8.4 Hz, 2H, Ar-H), 7.76 (d, J = 8.4 Hz, 1H, NH), 7.60 (d, J =
8.4 Hz, 2H, Ar-H), 7.02-7.27 (m, 10H, Ar-H), 3.67-3.72 (m, 1H, CH), 2.91-3.12 (m, 2H, CH2), 2.34-2.55 (m,
2H, CH2); 13C NMR (DMSO, 100 MHz, δ ppm): 164.68, 162.98, 139.43, 138.37, 137.49, 133.79, 132.32,
130.42 (2C), 129.56 (5C), 129.11 (2C), 128.67 (2C), 128.61 (2C), 126.35, 121.59, 62.03, 53.31, 36.09; ESI-
MS: m/z 535.3 [M + Na]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C25H23BrClN2O3, 513.0581, found
513.0576.
4.1.1.18. (S,Z)-4-bromo-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en-
2-yl)benzamide (4Bl)
As a white powder, yield:32.6%, m.p. 103.9-104.5°C. Analytical data for 4Bl: 1H NMR (DMSO, 400 MHz,
 ppm): 10.04 (s, 1H, NH), 7.92 (d, J = 8.8 Hz, 2H, Ar-H), 7.71-7.76 (m, 3H, NH, Ar-H), 7.27 (br, 5H, Ar-
H), 7.02-7.16 (m, 5H, Ar-H), 3.67-3.72 (m, 1H, CH), 2.90-3.11 (m, 2H, CH2), 2.34-2.55 (m, 2H, CH2); 13
C
NMR (DMSO, 100 MHz, δ ppm): 164.82, 162.97, 139.43, 138.36, 133.78, 132.68, 132.05 (2C), 130.57 (2C),
17

129.56 (5C), 128.68 (2C), 128.61 (2C), 126.50, 126.36, 121.55, 62.03, 53.31, 36.08; ESI-MS: m/z 578.9 [M
+ Na]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C25H23Br2N2O3, 557.0075, found 557.0066.
4.1.1.19. (S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en-2-yl)-4-
methoxybenzamide (4Bm)
As a white powder, yield: 30.8%, m.p. 146.9-147.3 °C. Analytical data for 4Bm: 1H NMR (DMSO, 400
MHz,  ppm): 9.75 (s, 1H, NH), 7.99 (d, J = 6.8 Hz, 2H, Ar-H), 7.73 (d, J = 8.4 Hz, 1H, NH), 7.27 (br, 5H,
Ar-H), 7.02-7.19 (m, 7H, Ar-H), 4.31 (t, J = 5.6 Hz, 1H, OH), 3.81 (s, 3H, OCH3), 3.67-3.76 (m, 1H, CH),
2.92-3.11 (m, 2H, CH2), 2.33-2.52 (m, 2H, CH2); 13C NMR (DMSO, 100 MHz, δ ppm): 165.05, 163.20,
162.82, 139.41, 138.46, 134.21, 130.47 (2C), 129.87, 129.61 (2C), 129.56 (2C), 129.48, 128.62 (4C), 126.35,
125.65, 120.49, 114.23 (2C), 113.90, 62.04, 56.02, 53.30, 36.12; ESI-MS: m/z 531.1 [M + Na]+; ESI-HRMS
(TOF): m/z [M + H]+ calcd for C26H26BrN2O4, 509.1076, found 509.1074.
4.1.1.20. (S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en-2-yl)-4-
methylbenzamide (4Bn)
1
As a white powder, yield: 31.4%, m.p. 128.8-129.4 °C. Analytical data for 4Bn: H NMR (DMSO, 400
MHz,  ppm): 9.79 (s, 1H, NH), 7.89 (d, J = 8.4 Hz, 2H, Ar-H), 7.70 (d, J = 8.8 Hz, 1H, NH), 7.28-7.31 (m,
7H, Ar-H), 7.12-7.18 (m, 3H, Ar-H), 7.04 (d, J = 8.0 Hz, 2H, Ar-H), 3.68-3.73 (m, 1H, CH), 2.92-3.12 (m,
2H, CH2), 2.34-2.53 (m, 5H, CH2, CH3); 13C NMR (DMSO, 100 MHz, δ ppm): 165.49, 163.14, 142.73,
139.40, 138.42, 134.09, 130.75, 129.61 (2C), 129.55 (2C), 129.52 (2C), 128.65 (2C), 128.62 (3C), 128.49
(2C), 126.35, 120.75, 62.02, 53.30, 36.12, 21.61; ESI-MS: m/z 515.0 [M + Na]+; ESI-HRMS (TOF): m/z [M
+ H]+ calcd for C26H26BrN2O3, 493.1127, found 493.1127.
4.1.1.21. (S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en-2-yl)-2,4-
difluorobenzamide (4Bo)
1
As a white powder, yield: 31.7%, m.p. 139.9-140.4 °C. Analytical data for 4Bo: H NMR (DMSO, 400
MHz,  ppm): 9.76 (d, J = 3.6 Hz, 1H, NH), 7.78-7.83 (m, 2H, Ar-H, NH), 7.03-7.44 (m, 12H, Ar-H), 4.34
(br, 1H, OH), 3.68-73 (br, 1H, CH), 2.91-3.12 (m, 2H, CH2), 2.34-2.55 (m, 2H, CH2); 13C NMR (CDCl3, 100
MHz, δ ppm): 164.42 (dd, 1JCF = 256.5 Hz, 3JCF = 13.1 Hz), 162.86, 162.75 (dd, 1JCF = 249.8 Hz, 3JCF = 12.5
Hz), 160.26, 136.82, 136.66, 138.16, 134.70 (dd, 3JCF = 20.4 Hz, 3JCF = 2.7 Hz), 132.64, 129.95, 129.39 (2C),
129.26 (2C), 128.74 (2C), 128.63 (2C), 126.67, 115.84 (dd, 2JCF = 10.6 Hz, 4JCF = 3.7 Hz), 113.10 (dd, 2JCF
18

4
2
2
= 20.3 Hz, JCF = 2.7 Hz), 104.96 (dd, JCF = 26.3 Hz, JCF = 26.2 Hz), 62.05, 52.33, 36.60; ESI-MS: m/z
537.0 [M + Na]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C25H22BrF2N2O3, 515.0782, found 515.0784.
4.1.1.22. (S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en-2-yl)-3,4-
difluorobenzamide (4Bp)
1
As a white powder, yield: 32.8%, m.p. 186.6-187.1 °C. Analytical data for 4Bp: H NMR (DMSO, 400
MHz,  ppm): 10.08 (s, 1H, NH), 8.05 (br, 1H, Ar-H), 7.87 (br, 1H, Ar-H), 7.79 (d, J = 8.0 Hz, 1H, NH),
7.62 (d, J = 9.2 Hz, 1H, Ar-H), 7.05-7.27 (m, 10H, Ar-H), 4.35 (br, 1H, OH), 3.70 (br, 1H, CH), 2.90-3.11
(m, 2H, CH2), 2.35-2.57 (m, 2H, CH2); 13C NMR (DMSO, 100 MHz, δ ppm): 163.56, 162.87, 151.73 (dd,
2
1
2
1JCF = 250.3 Hz, JCF = 12.4 Hz), 150.98 (dd, JCF = 245.4 Hz, JCF = 13.0 Hz), 139.43, 138.35, 133.66,
130.98, 129.61, 129.55 (4C), 128.67 (2C), 128.59 (2C), 126.34, 126.22 (2C), 121.95, 118.42 (dd, 2JCF = 37.8
Hz, 3JCF = 17.6 Hz), 62.04, 53.33, 36.11; ESI-MS: m/z 537.1 [M + Na]+; ESI-HRMS (TOF): m/z [M + H]+
calcd for C25H22BrF2N2O3, 515.0782, found 515.0783.
4.1.1.23. (S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en-2-yl)-3,4-
dichlorobenzamide (4Bq)
1
As a white powder, yield: 27.4%, m.p. 195.2-196.1 °C. Analytical data for 4Bq: H NMR (DMSO, 400
MHz,  ppm): 10.13 (s, 1H, NH), 7.81 (d, J = 8.8 Hz, 1H, Ar-H), 7.59-7.63 (m, 1H, Ar-H), 7.56 (dd, J = 9.2,
2.4 Hz, 1H, Ar-H), 7.03-7.35 (m, 11H, Ar-H), 4.33 (br, 1H, OH), 3.67-3.73 (m, 1H, CH), 2.91-3.13 (m, 2H,
CH2), 2.35-2.52 (m, 2H, CH2); 13C NMR (DMSO, 100 MHz, δ ppm): 164.72, 164.23 (d, 1JCF = 248.3 Hz),
162.66, 139.44, 138.31, 133.61, 132.41 (d, 3JCF = 10.1 Hz), 131.96 (d, 3JCF = 10.4 Hz), 129.58 (2C), 129.52
2
2
(3C), 128.65 (5C), 126.37, 119.85, 117.88 (d, JCF = 26.4 Hz), 114.94 (d, JCF = 21.5 Hz), 61.94, 53.25,
36.08; ESI-MS: m/z 553.0 [M + Na]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C25H22BrClFN2O3,
531.0486, found 531.0485.
4.1.1.24. (S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en-2-yl)-4-
fluoro-2-methylbenzamide (4Br)
1
As a white powder, yield: 32.0%, m.p. 180.9-182.2 °C. Analytical data for 4Br: H NMR (DMSO, 400
MHz,  ppm): 9.92 (s, 1H, NH), 7.78 (br, 1H, NH), 7.55 (br, 1H, Ar-H), 7.05-7.26 (m, 12H, Ar-H), 4.35 (br,
1H, OH), 3.72 (br, 1H, CH), 2.94-3.10 (m, 2H, CH2), 2.40-2.52 (m, 5H, CH2, CH3); 13C NMR (DMSO, 100
MHz, δ ppm): 167.50, 164.42 (d, 1JCF = 235.7 Hz), 162.97, 140.36 (d, 3JCF = 8.8 Hz), 139.48, 138.35, 133.93,
19

132.17, 130.64, 129.59 (2C), 129.52 (3C), 128.63 (4C), 126.35, 120.47, 117.86 (d, 2JCF = 21.1 Hz), 112.89
(d, 2JCF = 21.4 Hz), 61.06, 53.24, 36.09, 20.13; ESI-MS: m/z 533.0 [M + Na]+; ESI-HRMS (TOF): m/z [M +
H]+ calcd for C26H25BrFN2O3, 511.1033, found 511.1016.
4.1.1.25. (S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en-2-yl)-2,4-
dichlorobenzamide (4Bs)
1
As a white powder, yield: 35.3%, m.p. 169.3-170.4 °C. Analytical data for 4Bs: H NMR (DMSO, 400
MHz,  ppm): 10.20 (s, 1H, NH), 7.84 (br, 1H, NH), 7.72 (br, 1H, Ar-H), 7.55 (br, 2H, Ar-H), 7.05-7.27 (m,
10H, Ar-H), 4.36 (br, 1H, OH), 3.71 (br, 1H, CH), 2.93-3.09 (m, 2H, CH2), 2.39-2.52 (m, 2H, CH2); 13C
NMR (DMSO, 100 MHz, δ ppm): 164.64, 162.62, 139.44, 138.27, 133.73, 134.59, 133.49, 132.09, 131.37,
129.82, 129.78, 129.57 (2C), 129.52 (2C), 128.65 (4C), 127.78, 126.37, 119.94, 61.93, 53.26, 36.07; ESI-
MS: m/z 569.0 [M + Na]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C25H22BrCl2N2O3, 547.0191, found
547.0169.
4.1.1.26. (S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxo-1-phenylprop-1-en-2-yl)-3,5-
dichlorobenzamide (4Bt)
1
As a white powder, yield: 28.5%, m.p. 196.6-197.1 °C. Analytical data for 4Bt: H NMR (DMSO, 400
MHz,  ppm): 10.20 (s, 1H, NH), 7.83 (br, 1H, NH), 7.72 (br, 1H, Ar-H), 7.55 (br, 2H, Ar-H), 7.05-7.27 (m,
10H, Ar-H), 4.35 (br, 1H, OH), 3.71 (br, 1H, CH), 2.94-3.09 (m, 2H, CH2), 2.40-2.52 (m, 2H, CH2); 13C
NMR (DMSO, 100 MHz, δ ppm): 164.64, 162.62, 139.44, 138.28, 135.72, 134.59, 133.50, 132.09, 131.37,
129.83, 129.57 (2C), 129.52 (2C), 128.65 (5C), 127.78, 126.37, 119.92, 61.94, 53.26, 36.07; ESI-MS: m/z
569.1 [M + Na]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C25H22BrCl2N2O3, 547.0191, found 547.0163.
4.1.2. General procedure for the synthesis of compounds 4Ag, 5 and 6a-c
Compound 1 was reacted with 3-hydroxybenzaldehyde (0.01 mol) in the presence of acetic anhydride
(Ac2O) (10 mL) and sodium acetate (AcONa) (0.01 mol) to obtain compound 2. Next, the intermediate
mentioned above was reacted with L-phenylalaninol (0.01 mol) to give the crude product 3. Finally, the
product 3 (0.01 mol) was directly brominated with bromine (0.015 mol) and anhydrous calcium carbonate
(0.01 mol) in CHCl3 to give the target compound 4Ag. The target compound 4Ag (0.01 mol) was then
hydrolyzed to prepare the compound 5. Finally, compound 5 was alkylated with 2-dimethylaminoethyl
chloride hydrochloride, 2-diethylaminoethyl chloride hydrochloride or 3-dimethylaminopropyl chloride
20

hydrochloride (0.012 mol) in the presence of 1, 4-dioxane and K2CO3 to provide the crude product of alkoxyl
substituted derivatives, respectively, which was purified by column chromatography to yield the title
compounds 6a-c, respectively.
4.1.2.1. (S,Z)-3-(2-benzamido-1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-3-oxoprop-1-en-1-
yl)phenyl acetate (4Ag)
As a yellow powder, yield: 32.1%, m.p.140.3-141.5 °C. Analytical data for 4Ag: 1H NMR (DMSO, 400
MHz,  ppm): 9.94 (s, 1H, NH), 7.98 (d, J = 7.2 Hz, 2H, Ar-H), 7.84 (d, J = 8.8 Hz, 1H, NH), 7.59 (t, J =
8.0 Hz, 1H, Ar-H), 7.50 (t, J = 8.0 Hz, 2H, Ar-H), 7.29 (t, J = 8.0 Hz, 1H, Ar-H), 7.04-7.18 (m, 9H, Ar-H),
4.34 (t, J = 5.6 Hz, 1H, OH), 3.68-3.73 (m, 1H, CH), 2.94-3.14 (m, 2H, CH2), 2.37-2.56 (m, 2H, CH2), 2.24
(s, 3H, CH3); 13C NMR (DMSO, 100 MHz, δ ppm): 169.56, 165.63, 162.96, 150.50, 139.68, 139.40, 134.59,
133.46, 132.69, 129.74, 129.57 (2C), 129.01 (2C), 128.61 (2C), 128.48 (2C), 126.98, 126.36, 123,17, 122.92,
119.48, 61.90, 53.37, 36.08, 21.41; ESI-MS: m/z 559.5 [M + Na]+; ESI-HRMS (TOF): m/z [M + H]+ calcd
for C27H26BrN2O5, 537.1025, found 537.1025.
4.1.2.2. (S,Z)-N-(1-bromo-3-((1-hydroxy-3-phenylpropan-2-yl)amino)-1-(3-hydroxyphenyl)-3-oxoprop-1-en
-2-yl)benzamide (5)
As a white powder, yield: 29.3%, m.p.180.4-181.2 °C. Analytical data for 5: 1H NMR (DMSO, 400 MHz,
 ppm): 9.89 (s, 1H, NH), 9.59 (s, 1H, Ar-OH), 7.98 (d, J = 7.2 Hz, 2H, Ar-H), 7.66 (d, J = 8.8 Hz, 1H, NH),
7.58 (t, J = 7.2 Hz, 1H, Ar-H), 7.50 (t, J = 7.2 Hz, 2H, Ar-H), 7.02-7.18 (m, 6H, Ar-H), 6.82 (s, 1H, Ar-H),
6.69-6.73 (m, 2H, Ar-H), 4.32 (br, 1H, OH), 3.68-3.73 (m, 1H, CH), 2.92-3.14 (m, 2H, CH2), 2.37-2.51 (m,
2H, CH2); 13C NMR (DMSO, 100 MHz, δ ppm): 165.55, 163.20, 157.48, 139.42, 139.37, 133.79, 133.54,
132.61, 129.66, 129.58 (2C), 128.99 (2C), 128.63 (2C), 128.41 (2C), 126.36, 121.17, 120.38, 116.73, 116.56,
61.77, 53.30, 36.05; ESI-MS: m/z 517.5 [M + Na]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C25H24BrN2O4,
495.0919, found 495.0924.
4.1.2.3.
(S,Z)-N-(1-bromo-1-(3-(2-(dimethylamino)ethoxy)phenyl)-3-((1-hydroxy-3-phenylpropan-2-yl)
amino)-3-oxoprop-1-en-2-yl)benzamide (6a)
As a white powder, yield: 26.5%, m.p.89.5-91.4 °C. Analytical data for 6a: 1H NMR (DMSO, 400 MHz,
 ppm): 9.91 (s, 1H, NH), 7.98 (d, J = 7.2 Hz, 2H, Ar-H), 7.75 (d, J = 8.8 Hz, 1H, NH), 7.58 (t, J = 7.2 Hz,
1H, Ar-H), 7.50 (t, J = 7.6 Hz, 2H, Ar-H), 7.09-7.20 (m, 4H, Ar-H), 7.03 (d, J = 8.0 Hz, 2H, Ar-H), 6.84-
21

6.92 (m, 3H, Ar-H), 4.32 (t, J = 5.6 Hz, 1H, OH), 4.00 (t, J = 6.0 Hz, 2H, OCH2), 3.65-3.73 (m, 1H, CH),
2.91-3.13 (m, 2H, CH2), 2.59 (t, J = 5.6 Hz, 2H, NCH2), 2.33-2.51 (m, 2H, CH2), 2.17 (s, 6H, N(CH3)2); 13C
NMR (DMSO, 100 MHz, δ ppm): 165.58, 163.19, 158.46, 139.59, 139.34, 134.20, 133.50, 132.65, 129.82,
129.51 (2C), 129.01 (2C), 128.61 (2C), 128.44 (2C), 126.35, 121.97, 120.42, 115.91, 116.73, 66.28, 61.80,
58.02, 53.35, 45.95 (2C), 36.09; ESI-MS: m/z 566.1 [M + H]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for
C29H33BrN3O4, 566.1654, found 566.1635.
4.1.2.4.
(S,Z)-N-(1-bromo-1-(3-(2-(diethylamino)ethoxy)phenyl)-3-((1-hydroxy-3-phenylpropan-2-yl)
amino)-3-oxoprop-1-en-2-yl)benzamide (6b)
As a white powder, yield: 26.7%, m.p.93.4-94.2 °C. Analytical data for 6b: 1H NMR (DMSO, 400 MHz,
 ppm): 9.89 (s, 1H, NH), 7.98 (d, J = 7.2 Hz, 2H, Ar-H), 7.74 (d, J = 8.4 Hz, 1H, NH), 7.58 (t, J = 7.2 Hz,
1H, Ar-H), 7.50 (t, J=7.6 Hz, 2H, Ar-H), 7.10-7.21 (m, 4H, Ar-H), 7.03 (d, J = 6.8 Hz, 2H, Ar-H), 6.86-6.93
(m, 3H, Ar-H), 4.04 (t, J = 5.6 Hz, 2H, OCH2), 3.66-3.72 (m, 1H, CH), 2.94-3.13 (m, 2H, CH2), 2.87 (br,
2H, NCH2), 2.63 (br, 4H, N(CH2)2), 2.35-2.51 (m, 2H, CH2), 0.97 (t, J = 7.2 Hz, 6H, N(CH2CH3)2); 13C
NMR (DMSO, 100 MHz, δ ppm): 165.58, 163.16, 158.28, 139.63, 139.36, 134.21, 133.48, 132.66, 129.84,
129.52 (2C), 129.01 (2C), 128.61 (2C), 128.45 (2C), 126.35, 122.08, 120.44, 115.92, 115.75, 65.98, 61.78,
53.38, 51.39, 47.48 (2C), 36.07, 11.60; ESI-MS: m/z 594.1 [M + H]+; ESI-HRMS (TOF): m/z [M + H]+ calcd
for C31H37BrN3O4, 594.1967, found 594.1979.
4.1.2.5.
(S,Z)-N-(1-bromo-1-(3-(3-(dimethylamino)propoxy)phenyl)-3-((1-hydroxy-3-phenylpropan-2-yl)
amino)-3-oxoprop-1-en-2-yl)benzamide (6c)
As a white powder, yield: 25.8%, m.p.88.7-89.5 °C. Analytical data for 6c: 1H NMR (DMSO, 400 MHz,
 ppm): 9.89 (s, 1H, NH), 7.98 (d, J = 7.2 Hz, 2H, Ar-H), 7.73 (d, J = 8.4 Hz, 1H, NH), 7.58 (t, J = 7.6 Hz,
1H, Ar-H), 7.50 (t, J = 6.8 Hz, 2H, Ar-H), 7.08-7.20 (m, 4H, Ar-H), 7.03 (d, J = 6.8 Hz, 2H, Ar-H), 6.85-
6.91 (m, 3H, Ar-H), 3.95 (t, J = 6.4 Hz, 2H, OCH2), 3.65-3.73 (m, 1H, CH), 2.94-3.13 (m, 2H, CH2), 2.35-
2.52 (m, 4H, 2×CH2), 2.20 (s, 6H, N(CH3)2), 1.82-1.89 (m, 2H, CH2); 13C NMR (DMSO, 100 MHz, δ ppm):
165.58, 163.17, 158.49, 139.59, 139.37, 134.15, 133.49, 132.66, 129.82, 129.53 (2C), 129.01 (2C), 128.61
(2C), 128.45 (2C), 126.35, 121.97, 120.58, 115.86, 115.64, 66.10, 61.80, 55.70, 53.37, 44.81 (2C), 36.07,
26.47; ESI-MS: m/z 580.2 [M + H]+; ESI-HRMS (TOF): m/z [M + H]+ calcd for C30H35BrN3O4, 580.1811,
found 580.1820.
22

4.2. Biological assays
HepG2 2.2.15 cells, stably transfected with HBV genome using a plasmid (302 military hospital of china),
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. The cytotoxicity, effect of the target compounds on HBV
DNA replication and viral gene expression were evaluated on HepG2 2.2.15 cells. Antiviral efficacy against
nucleoside analogue-resistant HBV stains was evaluated on HepG2 cell line transiently transfected with
plasmids containing drug-resistant mutant (rtL180M + rtM204V + rtT184L).
4.2.1. Cytotoxicity assays
Intrinsic cytotoxicity of the target compounds against HepG2 2.2.15 cells were measured by MTT assay.
Briefly, cells were seeded in 96-well culture plates and cultured for 24 h. Then, cells were continue to
incubate with the target compounds (in 0.2 mL culture medium/well) for 72 h. Untreated control cultures
were maintained on each 96-well plate. Cell viability was finally determined by measuring MTT absorbance
at 490 nm, and calculated from the absorbance relative to untreated cells.
4.2.2. Inhibitory effect on replication of HBV DNA
HepG2 2.2.15 cells (2 × 105/well) were seeded in 24 well culture plates and recovered for 24 h. Culture
medium was replaced by assay medium containing the tested compound or 3TC at the indicated dose.
Medium was then changed every 2 days. After 6 days incubation, the intracellular HBV DNA was extracted
and quantified by real time PCR according to the previous method.21
4.2.3. Antiviral activity against nucleoside analogue-resistant HBV stains
HepG2 cells (2 × 105/well) were plated into 24-well plates. After 24 h incubation, the cells were transiently
transfected with plasmids containing drug-resistant mutant (rtL180M + rtM204V + rtT184L) for 24 h. Then
the supernatant was removed followed by washing with PBS. Cells were subsequently treated with fresh
medium containing lamivudine (100 μM), Entecavir (10 μM), 4Bj (0.8 μM) or 4Bs (0.8 μM). After 72 h
incubation, HBV DNA replicative intermediate was extracted and quantified using real time PCR using the
method mentioned above.22
4.2.4. Effect of compound 4Bs on HBV viral gene expression 16, 17
HepG2 2.2.15 cells (6 × 105/well) were cultured in 6-well plates, and treated with tested compound for 6
23

days. Total RNA of the treated HepG2 2.2.15 cells was extracted using TRIzol reagent (Invitrogen), and
detected by quantitative real time PCR.
4.2.5. Molecular docking
The crystal structure of HBV core protein complex (PDB code: 5gmz) was obtained from the Protein Data
Bank, which contained a single point mutation at position Y132A in the N-terminal assembly domain.
Molecular docking was performed by the Sybyl X-2.1 software according to previous methods.23, 24 Briefly,
all the receptor and ligands were prepared using the Sybyl X-2.1Tools module. During the protein preparation,
the missing hydrogen atoms were added by biopolymer module, and all water molecules were abandoned.
The amino acids around the ligand in the crystal structure of HBV core protein were selected and defined as
the active pocket. Docking parameters were kept at default settings. The pose with the highest binding score
was used for further docking analysis.
Acknowledgements
This work was supported by the grants from the National Natural Science Foundation of China (NSFC
No. 81703359), the Natural Science Foundation of Jiangsu province (No. BK20181151 and BK20171179),
the Six Talent Peaks Project in Jiangsu Province (Grant No. 2017-YY-039) and Jiangsu Provincial 333 High-
level Talents Cultivation Project (BRA201927) and the Postgraduate Research & Practice Innovation
Program of Jiangsu Province (No. KYCX19_2251). We thank to Public Experimental Research Center of
Xuzhou Medical University for providing chemical structure analysis.
Appendix A. Supplementary data
Supplementary data to this article can be found online.
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26

Synthesis and evaluation of new phenyl acrylamide derivatives as potent non-nucleoside
anti-HBV agents
Xiaoke Gua, #, Yinpeng Zhangb, #, Yueting Zoub, Xin Lia, Mingyu Guana, Qingqing Zhoub, Jingying Qiua, 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
H3C
OH
O
OH
OH
O
O
SAR exploration
Br
N
H
O
Br
N
H
O
Br
N
H
O
HN
HN
HN
Cl
F
Cl
IC50: 0.19 M SI: >526 wild HBV stain
IC50: 0.18 M SI: >556 resistant HBV stain
IC50: 0.46 M SI: >217.39
8d
IC50: 2.44 M SI: >40.98
8g
Compound
Compound
4Bs
Target compound
Compound 4Bs exhibited potent anti-HBV activity against wild and drug (lamivudine and entecavir)
resistant HBV strains, and possessed a favorable safety profile with a SI value of above 526.
 Corresponding author. Tel.: +86-516-83262138; fax: +86-516-83262630; e-mail: jingyqiu@xzhmu.edu.cn (J. Q.)
 These two authors contributed equally to this work.
27

Highlights
1. Phenyl acrylamide derivatives were synthesized and evaluated as novel anti-HBV agents.
2. 4Bs exhibited potent anti-HBV activity against wild and resistant HBV strains.
3. 4Bs possessed a favorable safety profile with a SI value of above 526.
4. 4Bs inhibited 3.5kb pgRNA expression and fitted well into the interface of HBV core protein.
28

Declaration of Interest Statement
The authors declared that there was no conflicts of interest to this work. We declare that we do not have
any commercial or associative interest that represents a conflict of interest in connection with the work
submitted.
29