Discovery of Oxime Ethers as Hepatitis B Virus (HBV) Inhibitors by Docking, Screening and In Vitro Investigation

Article abstract of DOI:10.3390/molecules23030637

A series of oxime ethers with C₆-C₄ fragment was designed and virtually bioactively screened by docking with a target, then provided by a Friedel-Crafts reaction, esterification (or amidation), and oximation from p-substituted phenyl derivatives (Methylbenzene, Methoxybenzene, Chlorobenzene). Anti-hepatitis B virus (HBV) activities of all synthesized compounds were evaluated with HepG2.2.15 cells in vitro. Results showed that most of compounds exhibited low cytotoxicity on HepG2.2.15 cells and significant inhibition on the secretion of HBsAg and HBeAg. Among them, compound 5c-1 showed the most potent activity on inhibiting HBsAg secretion (IC₅₀ = 39.93 μM, SI = 28.51). Results of the bioactive screening showed that stronger the compounds bound to target human leukocyte antigen A protein in docking, the more active they were in anti-HBV activities in vitro.

Full text of DOI:10.3390/molecules23030637

molecules
Article
Discovery of Oxime Ethers as Hepatitis B Virus (HBV)
Inhibitors by Docking, Screening and In
Vitro Investigation
Jie Tan 1,†, Min Zhou 1,†, Xinhua Cui 1, Zhuocai Wei 1 and Wanxing Wei 1,2,
*
ID
1
College of Chemistry and Chemical Engineering, Guangxi University, Nanning 53004, China;
yulitanjie@163.com (J.T.); eelmm@gxu.edu.cn (M.Z.); cuixinhua123@163.com (X.C.); liveupto@sina.cn (Z.W.)
Guangxi Colleges and Universities Key Laboratory of Applied Chemistry Technology and Resource
Development, Guangxi University, Nanning 53004, China
2
*
Correspondence: wxwei@gxu.edu.cn; Tel./Fax: +86-771-323-3718
†
These authors contributed equally to the work.
Received: 30 January 2018; Accepted: 9 March 2018; Published: 12 March 2018
Abstract: A series of oxime ethers with C6-C4 fragment was designed and virtually bioactively
screened by docking with a target, then provided by a Friedel–Crafts reaction, esterification (or
amidation), and oximation from p-substituted phenyl derivatives (Methylbenzene, Methoxybenzene,
Chlorobenzene). Anti-hepatitis B virus (HBV) activities of all synthesized compounds were evaluated
with HepG2.2.15 cells in vitro. Results showed that most of compounds exhibited low cytotoxicity
on HepG2.2.15 cells and significant inhibition on the secretion of HBsAg and HBeAg. Among them,
compound 5c-1 showed the most potent activity on inhibiting HBsAg secretion (IC50 = 39.93 µM,
SI = 28.51). Results of the bioactive screening showed that stronger the compounds bound to target
human leukocyte antigen A protein in docking, the more active they were in anti-HBV activities
in vitro.
Keywords: synthesis; oxime ethers derivatives; anti-HBV activity; molecular docking
1. Introduction
Hepatitis B virus (HBV) infection is a serious worldwide health problem, which causes acute and
chronic hepatitis B, cirrhosis, hepatocellular carcinoma, and other hepatic diseases [
World Health Organization, 240 million people in the world suffer chronic infection and develop into
HBV carriers. About 0.78 million people die per year from HBV-related diseases [ ]. The nucleoside
analogs used in treatment of anti-HBV play the role of Trojan horse in the synthesis of HBV DNA and
suppress replication of HBV [ ], but they are not effective in eliminating the virus from patients.
Meanwhile, HBV therapies with nucleoside analogs in the long term cause serious side effects and
resistance [ 12]. For the improvement of HBV therapies, the structural modification of nucleoside
analogs has focused and developed many derivatives [13 16]. Although more and more effective
1]. According to the
2
3–7
8–
–
nucleoside analogs agents for treating HBV have been invented and developed, they only inhibit or
stop replication of HBV DNA, and do not eliminate HBV cccDNA from patients. The cccDNA in
patients will cause HBV DNA replication. Methods to stop and eliminate HBV DNA and cccDNA
from HBV patients are still a great challenge for researchers. In searching for more effective anti-HBV
agents, many significant anti-HBV non-nucleoside analogs from synthesized compounds [17
–23] and
natural products [24 26] have been found. Some were designed according to their interactions with
–
receptor by simulating screen [27]. Researchers showed that HLA-A2 with the immunodominant
HBcAg18–27 epitope (or HLA-A2.1-restricted CTL epitope) binds peptide of vaccine or of HBcAg
to initiate a specific respond of T cell and resolve acute HBV infection [28–30]. Some other peptides
Molecules 2018, 23, 637; doi:10.3390/molecules23030637
www.mdpi.com/journal/molecules

Molecules 2018, 23, 637
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specifically bound to HBcAg18–27 epitope activate CTL response to prevent infection and eliminate
HBV [31 32]. Therefore, this implies that other non-peptide compounds specifically interact with
,
protein residue in HLA-A2.1-restricted CTL epitope may initiate CTL respond to prevent infection
by HBV. A protein residue from HLA-A2.1-restricted CTL epitope (PDB ID 3OX8) with intensive
interaction with non-nucleoside compounds displayed anti-HBV activities in docking investigations,
and this protein residue (3OX8) has been used as a virtual target to design anti-HBV non-nucleoside
compounds in our previous work. A series of oxime-containing compounds with a C6-C3 skeleton
were designed, synthesized, and assayed for anti-HBV activities. Results showed that these designed
compounds possessed significant anti-HBV activities [33–35]. On the basis of these results, we designed
new oximes with a C6-C4 fragment and screened their anti-HBV activities to investigate the structure
of the bioactivity relationship. Results of docking studies of these new oxime derivatives showed that
N in the oxime group (-O-N=C) and O in the carbonyl group (-C=O) of these compounds interacted
with amino acid residue by a hydrogen bond in active site of the HLA-Aprotein (HLA-A*02:03, PDB
ID: 3OX8) [36]. This indicated the roles of oxime and carbonyl group in anti-HBV activity in theory.
2. Results and Discussion
2.1. Molecular Docking Study
Molecular docking studies of the oxime ester derivatives were carried out using MOE 2008.10
as docking software in order to rationalize the biological activity results and understand the various
interactions between ligand and protein in the active site in detail. HLA-A protein (HLA-A*02:03,
PDB ID: 3OX8) [36] was used for docking study. The “Site Finder” tool of this program was used
to search for its active site. Three docking procedures for each ligand were performed and the best
configuration of each of the ligand–receptor complexes was selected based on energetic grounds. The
affinity-scoring function
scores and the hydrogen bonding strength of all the molecules are shown in Table 1. These oxime ester
derivatives had a dock score ranging from 12.0185 to 9.1113, and all of them were involved in at
δG was used to assess and rank the ligand–receptor complexes. The docking
−
−
least one hydrogen-bonding interaction with the active site of 3OX8 protein. Among them, N in the
oxime group (-O-N=C) of 14 compounds interacted with amino acid residues by a hydrogen bond
in the active site, which is similar to the reported works [33]. O in the carbonyl group (-C=O) of
five compounds interacted with amino acid residues by a hydrogen bond in the active sites. This
indicated in theory the roles of the oxime group and carbonyl group in anti-HBV activity. Bioactive
results in vitro showed the compound 5c-1 had the most potent anti-HBV activity with IC50 values
of 39.93
= 94.71
µ
M for HBsAg, the next was 5a-1 (HBsAg IC50 =74.92
µM), followed by 3c-2 (HBsAg IC50
µM, HBeAg IC50 = 93.91 M). Compounds 5c-1 (Figure 1) and 3c-2 (Figure 2) formed two
µ
hydrogen bonds in length 2.50 and 2.44 Å, 2.63 and 2.61 Å with O of O=C in amide group of Tyr27 and
Tyr63, respectively (Figures 1 and 2), and compound 5a-1 formed only one hydrogen bond in length
2.40 Å with N of C=N in oxime group of Tyr26 (Figure 3).
Table 1. Docking results of synthesized compounds with 3OX8.
Compound
S-Score (kcal/mol)
Residue
Site of Actions
Distance (Å)
3a-1
3a-2
3b-1
−9.1113
−9.7265
−10.4709
2.72
2.93
2.94
Tyr27
Tyr27
Tyr26
N of -C=N-
N of -C=N-
N of -C=N-
2.53
2.83
Tyr26
Tyr27
O of -C=O
N of -C=N-
−11.2203
3b-2

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Table 1. Cont.
3c-1
3c-2
−9.1497
3.09
Tyr63
N of -C=N-
2.63
2.16
Tyr27
Tyr63
O of -C=O
O of -C=O
−10.0032
4a-1
4a-2
4b-1
4b-2
4c-1
−9.9143
−11.0692
−10.4782
−10.8746
−10.0261
3.01
2.94
2.82
2.82
3.01
Tyr26
Tyr63
Tyr26
Tyr63
Tyr27
N of -C=N-
O of -C=O
N of -C=N-
N of -C=N-
N of -C=N-
2.81
2.73
Tyr27
Tyr63
N of -C=N-
N of -C=N-
−10.3516
−9.9471
−10.3332
4c-2
5a-1
2.40
Tyr26
N of -C=N-
1.96
2.52
Asp30
Tyr63
H of -N-H
N of -C=N-
5a-2
5b-1
5b-2
−10.2883
−12.0185
2.75
2.66
Tyr63
Tyr63
N of -C=N-
O of -C=O
2.50
2.44
Tyr27
Tyr63
O of -C=O
O of -C=O
−9.9406
5c-1
5c-2
−11.2609
2.92
Tyr63
N of -C=N-
Figure 1. Binding mode of compound 5c-1 with 3OX8. The hydrogen bond formed is colored in green.
Figure 2. Binding mode of compound 3c-2 with 3OX8. The hydrogen bond formed is colored in green.

Molecules 2018, 23, 637
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Figure 3. Binding mode of compound 5a-1 with 3OX8. The hydrogen bond formed is colored in green.
2.2. Chemistry
General synthesis of the intermediate and target compounds is depicted in Scheme 1. In the
initial step, 3-aroylpropionic acids (2a 2b and 2c) were prepared by condensing substituted benzenes
with succinic anhydride under Friedel–Crafts acylation reaction conditions [37]. Treatment of
3-aroylpropionic acids (2a 2b and 2c) with ethanol in the presence of p-toluenesulfonic acid provided
ethyl 4-(4-substituted benzoyl)-4-oxobutanoate (3a 3b and 3c) in 94~96% yield. Compounds 4a 4b
4c 5a 5b and 5c were formed by the reaction of 3-aroylpropionic acids (2a 2b and 2c) with furfuryl
alcohol or aniline in the presence of N,N-dicyclohexylcarbodiimide and 4-dimethylamioprdine in THF
with yields of 88~96% [38 43]. The intermediate compounds (3a 3b 3c 4a 4b 4c 5a 5b and 5c
finally reacted with methoxylamine or hydroxylamine hydrochloride in pyridine and DCM to afford
oxime ethers derivatives (3a-1 3a-2 3a-3 3b-1 3b-2 3b-3 3c-1 3c-2 3c-3 4a-1 4a-2 4a-3 4b-1 4b-2
4b-3 4c-1 4b-2 4b-3 5a-1 5a-2 5a-3 5b-1 5b-2 5b-3; and 5c-1 5c-2 5c-3) as mixture of isomers.
,
,
,
,
,
,
,
,
–
,
,
,
,
,
,
,
)
,
,
;
,
,
;
,
,
;
,
,
;
,
,
;
,
,
;
,
,
;
,
,
,
,
These isomers were easily separated by column chromatography. The major isomer of oxime ether
compounds is presumed to have the E configuration [44]. The structures of the synthesized compounds
were characterized by 1H NMR, 13C NMR and MS data and their data are presented in the experimental
section. Signals at 2.28~2.35, 3.77~3.84, 3.91~3.98 and 5.17~5.26 ppm in 1H NMR were related to CH3,
OCH3, N-OCH3, N-OCH2 protons, respectively. The chemical shifts of aromatic hydrogens of the
phenyl ring appeared in the region δ
7.71~6.83. 13C NMR spectra of the derivatives showed signals
at about 155.61~157.93 and 170.16~172.80 ppm related to carbons in C=N and C=O respectively. The
signals at 60.91~62.36 ppm attributed to carbons in N-OCH3, and 75.74~76.51 ppm to carbons in
N-OCH2.
2.3. Anti-Hepatitis B Virus (HBV) Activity
All synthesized derivatives were assayed for their anti-HBV activities in vitro, which included
inhibiting the secretion of HBsAg and HBeAg in HepG2.2.15 cells with lamivudine (3TC, a clinically
popular anti-HBV agent) as a positive control. The anti-HBV activities of the compounds were
expressed as the concentration of compound that achieved 50% inhibition (IC50) to the secretion of
HBsAg and HBeAg. Some compounds in which the inhibition rate of the secretion of HBsAg or HBeAg
did not reach 50% after 9d would not have the IC50 value calculated (Figure 4A,B). The cytotoxicity
of compounds was expressed as the concentration of compound required to kill 50% (TC50) of the
HepG2.2.15 cells. Parts of compounds were shown to be more active for inhibiting the secretion of
HBsAg and had lower cytotoxicity than lamivudine. Eleven among the 18 derivatives displayed higher
inhibitory activity against the secretion of HBsAg than lamivudine (Figure 5), and five among those
derivatives demonstrated better inhibitory effect in the secretion of HBeAg than lamivudine (Figure 6).
Among them, compounds 5c-1 and 5a-1 had the most potential as anti-HBV agents.

Molecules 2018, 23, 637
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OR2
N
R
O
O
R1
R
3a-1 R=OEt, R1= Me; 4a-1 R=OEt, R1= OMe;
5a-1 R=OEt, R1= Cl
O
R1
O
O
O
O
3b-1 R=
, R1=Me; 4b-1 R=
, R1=Cl
, R1=OMe;
3a R=OEt, R1= Me
4a R=OEt, R1= OMe
5a R=OEt, R1= Cl
b
O
O
5b-1 R=
O
NH
3c-1 R= NH
5c-1 R= NH
, R1=Me; 4c-1 R=
, R1=Cl
, R1=OMe;
O
O
e
OH
3b R=
4b R=
5b R=
3c R=
4c R=
5c R=
, R1=Me
, R1=OMe
, R1=Cl
a
R2=Et
c
O
O
O
R1
R1
R1=Me, OMe, Cl
O
O
2a, R1=Me
2b, R1=OMe
2c, R1=Cl
NH
f
3a-2 R=OEt, R1= Me; 4a-2 R=OEt, R1= OMe;
5a-2 R=OEt, R1= Cl
, R1=Me
, R1=OMe
, R1=Cl
R2=Bn
NH
d
O
O
O
O
3b-2 R=
, R1=Me; 4b-2 R=
, R1=Cl
, R1=OMe;
, R1=OMe;
NH
O
O
5b-2 R=
NH
3c-2 R= NH
, R1=Me; 4c-2 R=
, R1=Cl
NH
5c-2 R=
Scheme 1. Synthetic route of the series of compounds. Reagents and conditions: (a) succinic anhydride,
AlCl3/DCM, overnight, r.t.; (b) TsOH/EtOH, refiux, 6–8 h; (c) Furfuryl alcohol, DMAP, DCC/THF, 0.5 h
◦
◦
0 C; 6–8 h, r.t.; (d) Aniline, DMAP, DCC/THF, 0.5 h 0 C; 6–8 h, r.t.; (e) NH2OCH3 HCl, Pyridine/DCM,
·
◦
◦
70 C, 6–7 h; (f) NH2OCH2C6H5·HCl, Pyridine/DCM, 70 C, 6–7.
Figure 4. Inhibitory effects of the compounds on secretion of HBsAg (A) and HBeAg (B) in the HepG
2.2.15 cell line. Date were expressed as mean ± S.D. (n = 3).

Molecules 2018, 23, 637
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TC50
IC50 (μM, HBsAg)
1600
1400
1200
1000
800
600
400
200
0
Figure 5. Cytotoxicity and inhibiting HBsAg activity of compounds in 9 days.
TC50
IC50 (μM, HBeAg)
1600
1400
1200
1000
800
600
400
200
0
Figure 6. Cytotoxicity and inhibiting HBeAg activity of compounds in 9 days.
2.4. Structure–Activity Relationship (SAR)
Most of these compounds showed low cytotoxicity to HepG2.2.15 cells lines except the derivatives
3c-2 and 4b-1. The inhibition ratio of part of the derivatives on HBeAg was less than 50% in the test
concentration range. The anti-HBV activity of the derivatives was evaluated from their inhibition on
the secretion of HBsAg. Compounds 5a-1
different oxime ether groups showed different anti-HBV activity (Table 2). Compound 5a-1, with IC50
values of 74.92 M and 273.87 M for HBsAg and HBeAg, respectively, was shown to be more effective
at inhibiting HBsAg secretion but weaker at HBeAg secretion than that of compound 5a-2 (HBsAg
IC50 = 156.27 M, HBeAg IC50 = 220.09 M). It was similar to the compounds 5b-1 5b-2 5c-1 and
5c-1. Compound 5c-1 (HBeAg IC50 = 245.96 M) was shown to be more effective at inhibiting HBeAg
, 5a-2, 5b-1, 5b-2, 5c-1 and 5c-1 with similar structures but
µ
µ
µ
µ
,
,
µ

Molecules 2018, 23, 637
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secretion than that of 5c-1 (HBeAg IC50 = non). A series of derivative 4 also had similar behavior
at inhibiting HBsAg secretion. With an electronic withdrawing group Cl, compound 5a-1 showed a
better inhibitory effect on the secretion of HBsAg (IC50 = 74.92
(IC50 = 292.73, 154.50 M). Similar behaviors were shown in compounds 3a-2
non, 156.27 M) and 3c-1 4c-1 and 5c-1(IC50 =224.82, non, 39.93 M), respectively. This revealed the
µM) than compound 3a-1 and 4a-1
µ
,
4a-2 and 5a-2 (IC50 = non,
µ
,
µ
formation of an amide fragment increased bioactivity. Compared to compound 3b-1, whose inhibitory
activity could be neglected, compound 5b-1 had relatively high inhibitory potency to the secretion
of HBsAg (IC50 = 207.63
value of 127.68 M for HBsAg) was shown to be more effective at inhibiting HBsAg secretion than
compound 5b-1, but appeared toxic (TC50 = 347.55 M, SI = 2.72), but the Me group in 3b-1 presented
less bioactivity. As amides, in compounds 3c-1 4c-1 and 5c-1 an electronic withdrawing group Cl in
5c-1 presented a more significant inhibiting effect than compounds 3c-1 and 4c-1. For compounds
3b-2 4b-2 and 5b-2, a more electronic donating group MeO in 4b-2 showed less bioactivity, and
compound 5b-2 showed better inhibition of HBsAg secretion (IC50 = 101.19 M) than compound 4b-2
(IC50 = 233.60 M), but weaker than 3b-2 (IC50 = 86.85 M). For amide compounds 3c-2 4c-2 and 5c-1,
µM). An electronic donating group MeO in compound 4b-1 (with an IC50
µ
µ
,
,
µ
µ
µ
,
only a mild electronic donating group Me in 3c-2 showed more bioactivity. In general, most of the
compounds with R2 = OMe were more bioactive than ther compounds with R2 = Bn.
Table 2. Inhibition on the secretion of HBsAg, HBeAg and cytotoxicity of compounds in 9 days.
HBeAg c
HBsAg b
a
Compound
TC50 (µM)
SI e
SI e
d
d
IC50 (µM)
IC50 (µM)
f
3a-1
3a-2
3b-1
3b-2
3c-1
3c-2
4a-1
4a-2
4b-1
4b-2
4c-1
4c-2
5a-1
5a-2
5b-1
5b-2
5c-1
5c-1
3TC g
>1500
>1500
1383.74
429.88
450.67
127.10
>1500
>1500
347.55
1153.49
>1500
>1500
>1500
>1500
473.25
>1500
1138.45
>1500
517.40
292.73
-
>5.12
-
-
-
-
-
-
-
-
-
-
-
-
86.85
224.82
94.71
154.50
-
127.68
233.60
-
4.95
2.00
1.34
>9.71
-
2.72
4.94
-
-
93.91
-
-
-
-
-
-
273.87
220.09
-
191.58
245.96
-
1.34
-
-
-
-
-
-
>5.48
>6.82
-
>7.83
4.63
-
-
-
74.92
156.27
207.63
101.19
39.93
-
>20.02
>9.60
2.28
>14.82
28.51
-
290.73
1.78
358.59
1.44
a
b
c
TC50 is 50% cytotoxicity concentration in HepG 2.2. 15 cell; HBsAg: hepatitis B surface antigen; HBeAg:
hepatitis B e antigen; d IC50 is 50% inhibitory concentration; e SI (selectivity index) = TC50/IC50
;
The inhibition
f
ratio less than 50% in the test concentration range; g Lamivudine (3TC) as the positive control.
Most N-phenyl amide derivatives among all the compounds exhibited an inhibitory effect on
HBeAg secretion. These results suggested that the introduction of N-phenyl amide shows relatively
greater anti-HBV activity than the introduction of furan-2-ylmethyl or ethyl.
According to the results mentioned above, the SARs were summarized. The N-phenyl
amide-substituted methoxy oxime ether derivatives possessed higher anti-HBV activity than
other analogs.

Molecules 2018, 23, 637
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3. Methods
3.1. Molecular Docking
The docking study was performed using the MOE 2008.10 to understand the ligand–receptor
interactions in detail. The crystal structure of human leukocyte antigen (HLA-A) protein (PDB ID:
3OX8) [36] was retrieved from the Protein Data Bank (http://www.rcsb.org/pdb/home/home.do).
Conformation of all the compounds was constructed in ChemDraw Ultra 7.0 and optimized in
HyperChem 8.0.7 Software, and included hydrogen addition, 3D structure conversion, force-field
optimization, and geometry optimization. The optimized ligands were built using the builder interface
of the MOE program. The crystal structure was imported into MOE and chain A was considered
for the docking process as the protein is a dimer consisting of A and B chains. The structure was
protonated, unbound waters were deleted, polar hydrogens were added, and energy minimization
was carried out. The active site was correlated with the ‘Site Finder’ module of MOE to define the
docking site for the ligands [33–35]. The docking procedure was followed using the standard protocol
implemented in MOE 2008.10 and the geometry of the resulting complexes was studied using the
MOE’s Pose Viewer utility.
3.2. Synthesis Methods
3.2.1. Materials and Methods
Melting points were determined using a fully automatic melting point apparatus MP420
(Jinan, China) and were uncorrected. Mass spectrometry (MS) spectra ware recorded on Thermo
Scientific ITQ 1100 instrument (Thermo Fisher Scientific, Waltham, MA, USA), Varian CP 3800+ Satum
2200 instrument (Agilent Technologies, Santa Clara, CA, USA). NMR spectra were recorded on a
Bruker AV III HD 600 MHz (1H/13C, 600 MHz/150 MHz) spectrometer (Brucker, Corp, MA, USA) by
using CDCl3, CD3OH, or DMSO as solvent and TMS as an internal standard. All the chemicals were
obtained from local suppliers and were used without further purification.
3.2.2. Chemistry
The general procedure (Scheme 1) for the preparation of compounds (2a, 2b and 2c): succinic
anhydride (1 equiv, 10 mmol) was reacted with an appropriate aromatic compound (substituted
benzene, 1 equiv, 10 mmol) in DCM (20 mL) in the presence of anhydrous aluminium chloride
(1.5 equiv, 15 mmol). The reaction mixture was stirred under anhydrous conditions overnight at room
temperature and then ice-cold diluted hydrochloric acid solution was added dropwise. A solid mass
separated out which was filtered and purified by recrystallization to give 2a, 2b and 2c [44].
The general procedure for the preparation of compounds (3a, 3b and 3c): a mixture of the
appropriate acid 2a-c (1 equiv, 10 mmol) and p-toluenesulfonic acid (0.4 equiv, 4 mmol) in EtOH
(20 mL) was refluxed for 6–8 h and evaporated to remove EtOH. The residue was suspended in H2O
(30 mL) and extracted with EtOAc (2
on silica gel eluting with AcOEt–petroleum ether (1:4, v/v) to get 3a-c as crystals. The general
procedure for the preparation of compounds (4a-c 5a-c): an appropriate acid 2a-b (1 equiv, 10 mmol),
×
50 mL) which was further purified by column chromatography
–
and 4-dimethylamioprdine (0.02 equiv, 0.2 mmol) was added to a solution of the furfuryl ◦alcohol
or aniline (1 equiv, 10 mmol) in THF (20 mL). The mixture was stirred and cooled to 0 C and
then N,N-dicyclohexylcarbodiimide (DCC) (1.1 equiv, 11 mmol) was added over a 5-min period and
the reaction was stirred under anhydrous conditions for 6–8 h at room temperature. The mixture
was filtered and the filtrate was evaporated to yield a crude product which was finally purified by
recrystallization to give 4a, 4b, 4c, 5a, 5b and 5c as crystals [38–43].
3-(4-Methylbenzoyl)propionic acid (2a). Yield 95%; m.p. 93.7~94.9 ◦C. 1H NMR (600 MHz, MeOD)
δ:
7.91–7.86 (m, 2H, H-6, 10), 7.29 (d, J = 8.0 Hz, 2H, H-7, 9), 3.30–3.25 (m, 2H, H-3), 2.70–2.66 (m, 2H,
H-2), 2.39 (s, 3H, H-11).

Molecules 2018, 23, 637
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3-(4-Methoxybenzoyl)propionic acid (2b). Yield 98%; m.p. 148.9~150.2 ◦C. 1H NMR (600 MHz, MeOD)
δ:
7.90–7.85 (m, 2H, H-6, 10), 6.92–6.87 (m, 2H, H-7, 9), 3.76 (s, 3H, H-11), 3.18–3.14 (m, 2H, H-3), 2.60–2.56
(m, 2H, H-2).
3-(4-Chlorobenzoyl)propionic acid (2c). Yield 90%; m.p. 130.2~131.5 ◦C. 1H NMR (600 MHz, DMSO)
δ:
12.16 (s, 1H, H-OH), 7.99 (dd, J = 8.5, 1.7 Hz, 2H, H-6, 10), 7.62–7.58 (m, 2H, H-7, 9), 3.26–3.22 (m, 2H,
H-3), 2.58 (t, J = 6.2 Hz, 2H H-2).
◦
Ethyl 4-(4-methylbenzoyl)-4-oxobutanoate (3a). Yield 96%; m.p. 66.6~68.0 C. 1H NMR (600 MHz, MeOD)
δ
: 7.88 (dd, J = 8.2, 1.7 Hz, 2H, H-6, 10), 7.29 (d, J = 6.8 Hz, 2H, H-7, 9), 4.11 (q, J = 7.1 Hz, 2H, H-100),
3.29 (td, J = 6.5, 2.3 Hz, 2H, H-3), 2.70–2.66 (m, 2H, H-2), 2.39 (s, 3H, H-11), 1.23 (t, J = 7.1 Hz, 3H, H-2 ).
◦
Ethyl 4-(4-methoxyphenyl)-4-oxobutanoate (3b). Yield 98%; m.p. 55.9~57.1 C. 1H NMR (600 MHz, MeOD)
δ
: 7.90–7.85 (m, 2H, H-6, 10), 6.92–6.87 (m, 2H, H-7, 9), 4.02 (q, J = 7.1 Hz, 2H, H-10), 3.76 (s, 3H, H-11),
3.19–3.16 (m, 2H, H-3), 2.58 (dd, J = 7.3, 5.4 Hz, 2H, H-2), 1.13 (t, J = 7.1 Hz, 3H, H-20).
Ethyl 4-(4-chlorophenyl)-4-oxobutanoate (3c). Yield 94%; m.p. 55.5~56.5 ◦C. 1H NMR (600 MHz, CDCl3)
δ
: 7.93 (t, J = 5.4 Hz, 2H, H-6, 10), 7.44 (t, J = 5.4 Hz, 2H, H-7, 9), 4.16 (q, J = 7.1 Hz, 2H, H-10), 3.27
(t, J = 6.6 Hz, 2H, H-3), 2.75 (t, J = 6.6 Hz, 2H, H-2), 1.27 (t, J = 7.1 Hz, 3H, H-20).
Furan-2-ylmethyl-4-(4-methylbenzoyl)-4- oxobutanoate (4a). Yield 95%, m.p. 79.6~80.5 C; H NMR
(600 MHz, CDCl3)
: 7.87 (d, J = 8.2 Hz, 2H, H-6, 10), 7.41 (d, J = 1.1 Hz, 1H, H-50), 7.25 (d, J = 8.1 Hz,
2H, H-7, 9), 6.40 (d, J = 3.2 Hz, 1H, H-30), 6.36 (dd, J = 3.1, 1.9 Hz, 1H, H-40), 5.09 (s, 2H, H-10), 3.29
◦
1
δ
(t, J = 6.7 Hz, 2H, H-3), 2.78 (t, J = 6.7 Hz, 2H, H-2), 2.41 (s, 3H, H-11); 13C NMR (151 MHz, CDCl3)
δ:
197.56 (C-4), 172.66 (0C-1), 149.45 (C-20), 144.03 0(C-8), 143.24 (C-50), 134.07 (C-5), 129.29 (C-7, 9), 128.16
0
(C-6, 10), 110.63 (C-4 ), 110.56 (C-3 ), 58.28 (C-1 ), 33.19 (C-3), 28.19 (C-2), 21.65 (C-11); ESIMS: m/z 273
[M + H]+, 290 [M + NH4]+, 295 [M + Na]+, 567 [2M + Na]+, calc. for C16H16O4 (272.2958).
◦
1
Furan-2-ylmethyl 4-(4-methoxyphenyl)-4-oxobutanoate (4b). Yield 96%, m.p. 61.0~61.7 C; H NMR
(600 MHz, CDCl3)
: 7.97–7.94 (m, 2H, H-6, 10), 7.43–7.40 (m, 1H, H-50), 6.96–6.91 (m, 2H, H-7, 9), 6.40
(d, J = 3.2 Hz, 1H, H-30), 6.36 (dd, J = 3.1, 1.9 Hz, 1H, H-40), 5.09 (s, 2H, H-10), 3.86 (s, 3H, H-11), 3.27
δ
(t, J = 6.7 Hz, 2H, H-3), 2.78 (t, J = 6.7 Hz, 2H, H-2); 13C NMR (151 MHz, CDCl3)
δ: 196.44 (C-4), 172.72
(C-1), 163.59 (C-8), 149.46 (C-20), 143.24 (C-50), 130.31 (C-6, 10), 129.65 (C-5), 113.75 (C-7, 9), 110.62
(C-40), 110.57 (C-30), 58.27 (C-10), 55.47 (C-11), 32.93 (C-3), 28.25 (C-2); ESIMS: m/z 289 [M + H]+, 311
[M + Na]+, calc. for C16H16O5 (288.2952).
Furan-2-ylmethyl 4-(4-chlorophenyl)-4-oxobutanoate (4c). Yield 90%, m.p. 64.2~65.1 ◦C; 1H NMR (600
MHz, CDCl3)
δ
: 7.91 (d, J = 8.5 Hz, 2H, H-6, 10), 7.44 (d, J = 8.5 Hz, 2H, H-7, 9), 7.42 (s, 1H, H-50), 6.41
(d, J = 3.2 Hz, 1H, H-30), 6.36 (d, J = 1.8 Hz, 1H, H-40), 5.09 (s, 2H, H-10), 3.28 (t, J = 6.6 Hz, 2H, H-3),
2.80 (t, J = 6.6 Hz, 2H, H-2); 13C NMR (151 MHz, CDCl3) : 196.76 (C-4), 172.43 (C-1), 149.34 (C-20),
δ
143.29 (C-50), 139.70 (C-8), 134.86 (C-5), 129.47 (C-6, 10), 128.95 (C-7, 9), 110.70 (C-40), 110.58 (C-30),
58.35 (C-10), 33.26 (C-3), 28.08 (C-2); ESIMS: m/z 293 [M + H]+, 310 [M + NH4]+, 315 [M + Na]+, calc.
for C15H13ClO4 (292.7143).
◦
4-(4-Methylbenzoyl)-4-oxo-N-phenylbutanamide (5a). Yield 90%, m.p. 147.5~148.5 C; 1H NMR (600 MHz,
DMSO)
δ
: 10.04 (s, 1H, H-NH), 7.91 (d, J = 8.2 Hz,02H, H-6, 10), 7.60 (t, J = 8.4 Hz, 2H, H-20, 60), 7.34
0
0
(d, J = 7.1 Hz, 2H, H-7, 9), 7.29 (t, J = 7.9 Hz, 2H, H-3 , 5 ), 7.02 (t, J = 7.4 Hz, 1H, H-4 ), 3.31 (t, J = 6.4 Hz,
2H, H-3), 2.72 (t, J = 6.3 Hz, 2H, H-2), 2.38 (s, 3H, H-11); 13C NMR (151 MHz, DMSO)
δ: 198.80 (C-4),
170.84 (C-1), 143.94 (C-8), 139.83 (C-10), 134.55 (C-5), 129.70 (C-7, 9), 129.11 (C-30, 50), 128.43 (C-6, 10),
123.33 (C-40), 119.35 (C-20, 60), 33.46 (C-3), 30.76 (C-2), 21.61 (C-11); ESIMS: m/z 268 [M + H]+, calc. for
C17H17NO2 (267.3224).
◦
4-(4-Methoxyphenyl)-4-oxo-N-phenylbutanamide (5b). Yield 93%, m.p. 113.3~114.2 C; 1H NMR (600 MHz,
DMSO)
: 10.01 (s, 1H, H-NH), 8.00–7.96 (m, 2H, H-6, 10), 7.59 (d, J = 7.6 Hz, 2H, H-20, 60), 7.30–7.26
(m, 2H, H-30, 50), 7.07–7.03 (m, 2H, H-7, 9), 7.01 (t, J = 7.4 Hz, 1H, H-40), 3.85 (s, 3H, H-11), 3.28 (t,
δ

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J = 6.5 Hz, 2H, H-3), 2.70 (t, J = 6.5 Hz, 2H, H-2); 13C NMR (151 MHz, DMSO)
δ
: 197.64 (C-4), 170.90
(C-1), 163.56 (C-8), 139.87 (C-10), 130.64 (C-6, 10), 130.00 (C-5), 129.12 (C-30, 50), 123.31 (C-40), 119.34
(C-20, 60), 114.34 (C-7, 9), 55.99 (C-11), 33.22 (C-3), 30.83 (C-2); ESIMS: m/z 284 [M + H]+, calc. for
C17H17NO3 (283.3218).
4-(4-Chlorophenyl)-4-oxo-N-phenylbutanamide (5c). Yield 88%, m.p. 140.2~141.0 ◦C; 1H NMR (600 MHz,
DMSO)
7, 9), 7.28 (t, J = 7.9 Hz, 2H, H-30, 50), 7.02 (t, J = 7.4 Hz, 1H, H-40), 3.33 (t, J = 6.4 Hz, 2H, H-3), 2.73
(t, J = 6.4 Hz, 2H, H-2); 13C NMR (151 MHz, MeOD)
: 198.08 (C-4), 171.77 (C-1), 139.13 (C-8), 138.58
δ
: 10.04 (s, 1H, H-NH), 8.03–8.00 (m, 2H, H-6, 10), 7.60 (dd, J = 12.5, 5.5 Hz, 4H, H-20, 60,
δ
0
0
0
0
0
0
(C-1 ), 135.26 (C-5), 129.41 (C-6, 10), 128.56 (C-3 , 5 ), 128.37 (C-7, 9), 123.62 (C-4 ), 119.76 (C-2 , 6 ), 33.17
(C-3), 30.05 (C-2); ESIMS: m/z 288 [M + H]+, calc. for C16H14ClNO2 (287.7409).
Ethyl (E)-4-(4-methylbenzoyl)-4-[(methoxy)imino]butanoate (3a-1). Yield 78%; 1H NMR (600 MHz, MeOD)
δ
: 7.52 (dd, J = 4.9, 3.3 Hz, 2H, H-6, 10), 7.22–7.17 (m, 2H, H-7, 9), 4.07 (dd, J = 12.7, 5.5 Hz, 2H, H-10),
3.95 (s, 3H, H-1”), 3.04–2.98 (m, 2H, H-3), 2.50 (ddd, J = 7.8, 4.1, 1.5 Hz, 2H, H-2), 2.34 (s, 3H, H-11), 1.20
(t, J = 7.1 Hz, 3H, H-20); 13C NMR (151 MHz, MeOD)
δ: 172.80 (C-1), 156.95 (C-4), 139.20 (C-8), 132.18
(C-5), 128.81 (C-7, 9), 125.99 (C-6, 10), 60.91 (C-1”), 60.34 (C-10), 30.59 (C-2), 21.83 (C-3), 19.93 (C-11),
13.09 (C-20); ESIMS: m/z 250.1442 [M + H]+, 272.1261 [M + Na]+, calc. for C14H19NO3 (249.3056).
Ethyl (E)-4-(4-methylbenzoyl)-4-[(benzyloxy)imino]butanoate (3a-2). Yield 76%; 1H NMR (600 MHz, CDCl3)
δ
: 7.52 (d, J = 8.1 Hz, 2H, H-6, 10), 7.40 (d, J = 7.3 Hz, 2H, H-3”, 7”), 7.35 (t, J = 7.5 Hz, 2H, H-4”, 6”),
7.30 (t, J = 7.2 Hz, 1H, H-5”), 7.16 (d, J = 8.0 Hz, 2H, H-7, 9), 5.22 (s, 2H, H-1”), 4.08 (q, J = 7.1 Hz, 2H,
H-10), 3.06 (dd, J = 10.5, 5.8 Hz, 2H, H-3), 2.57–2.52 (m, 2H, H-2), 2.35 (s, 3H, H-11), 1.20 (t, J = 7.1 Hz,
3H, H-20); 13C NMR (151 MHz, MeOD)
δ: 172.77 (C-1), 157.28 (C-4), 139.20 (C-8), 138.02 (C-2”), 132.20
(C-5), 128.80 (C-7, 9), 128.03 (C-3”, 7”), 127.84 (C-4”, 6”), 127.48 (C-5”), 126.04 (C-6, 10), 75.86 (C-1”),
0
0
60.34 (C-1 ), 30.58 (C-2), 22.02 (C-3), 20.01 (C-11), 13.08 (C-2 ); ESIMS: m/z 326.1766 [M + H]+, 348.1579
[M + Na]+, calc. for C20H23NO3 (325.4015).
Ethyl (E)-4-(4-methoxyphenyl)-4-[(methoxy)imino]butanoate (3b-1). Yield 76%; 1H NMR (600 MHz, CDCl3)
δ
: 7.60–7.57 (m, 2H, H-6, 10), 6.91–6.87 (m, 2H, H-7, 9), 4.11 (q, J = 7.1 Hz, 2H, H-10), 3.96 (s, 3H,
H-1”), 3.82 (s, 3H, H-11), 3.03–2.99 (m, 2H, H-3), 2.56–2.52 (m, 2H, H-2), 1.23 (t, J = 7.1 Hz, 3H, H-20);
13C NMR (151 MHz, CDCl3)
δ: 172.73 (C-1), 160.52 (C-8), 156.46 (C-4), 127.67 (C-5), 127.64 (C-6, 10),
113.94 (C-7, 9), 61.91 (C-1”), 60.59 (C-10), 55.32 (C-11), 30.99 (C-2), 22.28 (C-3), 14.18 (C-20); ESIMS: m/z
266.1389 [M + H]+, 288.1199 [M + Na]+, calc. for C14H19NO4 (265.3050).
1
Ethyl (E)-4-(4-methoxyphenyl)-4-[(benzyloxy)imino]butanoate (3b-2). Yield 78%; H NMR (600 MHz,
CDCl3) δ: 7.66–7.62 (m, 2H, H-6, 10), 7.45 (d, J = 7.3 Hz, 2H, H-3”, 7”), 7.40 (t, J = 7.5 Hz, 2H, H-4”, 6”),
7.34 (t, J = 7.3 Hz, 1H, H-5”), 6.94–6.90 (m, 2H, H-7, 9), 5.26 (s, 2H, H-1”), 4.13 (q, J = 7.1 Hz, 2H, H-10),
3.84 (s, 3H, H-11), 3.14–3.07 (m, 2H, H-3), 2.63–2.57 (m, 2H, H-2), 1.25 (t, J = 7.2 Hz, 3H, H-20); 13C
NMR (151 MHz, CDCl3)
δ
: 172.72 (C-1), 160.57 (C-8), 156.76 (C-4), 138.11 (C-2”), 128.38 (C-3”, 7”),
0
128.11 (C-4”, 6”), 127.76 (C-5”), 127.72 (C-6), 127.68 (C-5), 113.93 (C-7, 9), 76.22 (C-1”), 60.60 (C-1 ), 55.32
(C-11), 30.99 (C-2), 22.40 (C-3), 14.19 (C-20); ESIMS: m/z 342.1694 [M + H]+, 364.1506 [M + Na]+, calc.
for C20H23NO4 (341.4009).
Ethyl (E)-4-(4-chlorophenyl)-4-[(methoxy)imino]butanoate (3c-1). Yield 75%; 1H NMR (600 MHz, CDCl3)
7.60–7.56 (m, 2H, H-6, 10), 7.35–7.31 (m, 2H, H-7, 9), 4.10 (q, J = 7.2 Hz, 2H, H-10), 3.98 (s, 3H, H-1”),
δ:
0
3.02–2.98 (m, 2H, H-3), 2.55–2.52 (m, 2H, H-2), 1.22 (t, J = 7.2 Hz, 3H, H-2 ); 13C NMR (151 MHz, CDCl3)
δ
: 172.46 (C-1), 155.74 (C-4), 135.21 (C-8), 133.66 (C-5), 128.71 (C-7, 9), 127.55 (C-6, 10), 62.16 (C-1”),
60.65 (C-10), 30.77 (C-2), 22.12 (C-3), 14.15 (C-20); ESIMS: m/z 270.0901 [M + H]+, 292.0720 [M + Na]+,
calc. for C13H16ClNO3 (269.7240).
Ethyl (E)-4-(4-chlorophenyl)-4-[(benzyloxy)imino]butanoate (3c-2). Yield 77%; 1H NMR (600 MHz, CDCl3)
δ
: 7.59–7.56 (m, 2H, H-6, 10), 7.41–7.34 (m, 4H, H-3”, 7”, 4”, 6”), 7.34–7.29 (m, 3H, H-7, 9, 5”), 5.22 (s,
2H, H-1”), 4.08 (q, J = 7.1 Hz, 2H, H-10), 3.06–3.02 (m, 2H, H-3), 2.56–2.52 (m, 2H, H-2), 1.20 (t, J = 7.1

Molecules 2018, 23, 637
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Hz, 3H, H-20); 13C NMR (151 MHz, CDCl3)
δ
: 172.47 (C-1), 156.11 (C-4), 137.74 (C-2”), 135.26 (C-8),
133.65 (C-5), 128.69 (C-7, 9), 128.40 (C-3”, 7”), 128.12 (C-4”, 6”), 127.87 (C-5”), 127.62 (C-6), 76.49 (C-1”),
60.66 (C-10), 30.75 (C-2), 22.26 (C-3), 14.14 (C-20); ESIMS: m/z 346.1204 [M + H]+, 368.1020 [M + Na]+,
calc. for C19H20ClNO3 (345.8200).
Furan-2-ylmethyl (E)-4-(4-methylbenzoyl)-4-[(methoxy)imino] butanoate (4a-1). Yield 76%; 1H NMR
(600 MHz, CDCl3) δ
: 7.50 (d, J = 8.1 Hz, 2H, H-6, 10), 7.41 (dd, J = 1.8, 0.8 Hz, 1H, H-50), 7.16
(d, J = 8.0 Hz, 2H, H-7, 9), 6.39 (d, J = 3.1 Hz, 1H, H-30), 6.35 (dd, J = 3.2, 1.9 Hz, 1H, H-40), 5.04 (s, 2H,
H-10), 3.95 (s, 3H, H-1”), 3.05–3.00 (m, 2H, H-3), 2.60–2.55 (m, 2H, H-2), 2.35 (s, 3H, H-11); 13C NMR
(151 MHz, CDCl3) δ
: 172.29 (C-1), 156.63 (C-4), 149.39 (C-20), 143.27 (C-50), 139.27 (C-8), 132.25 (C-5),
129.25 (C-7, 9), 126.16 (C-6, 10), 110.68 (C-40), 110.57 (C-30), 61.96 (C-1”), 58.18 (C-10), 30.72 (C-2), 22.22
(C-3), 21.25 (C-11); ESIMS: m/z 302 [M + H]+, calc. for C17H19NO4 (301.3371).
Furan-2-ylmethyl (E)-4-(4-methylbenzoyl)-4-[(benzyloxy)imino]butanoate (4a-2). Yield 77%; 1H NMR
(600 MHz, CDCl3) δ
: 7.50 (d, J = 8.2 Hz, 2H, H-6, 10), 7.41–7.40 (m, 1H, H-50), 7.38 (d, J = 7.3 Hz, 2H,
H-3”, 7”), 7.34 (t, J = 7.4 Hz, 2H, H-4”, 6”), 7.31–7.27 (m, 1H, H-5”), 7.15 (d, J = 8.0 Hz, 2H, H-7, 9),
6.36 (d, J = 3.2 Hz, 1H, H-30), 6.35 (dd, J = 3.2, 1.9 Hz, 1H, H-40), 5.20 (s, 2H, H-1”), 5.01 (s, 2H, H-10),
3.09–3.04 (m, 2H, H-3), 2.60–2.55 (m, 2H, H-2), 2.34 (s, 3H, H-11); 13C NMR (151 MHz, CDCl3)
δ: 172.28
(C-1), 156.94 (C-4), 149.38 (C-20), 143.25 (C-50), 139.31 (C-8), 137.97 (C-2”), 132.24 (C-5), 129.22 (C-7, 9),
128.35 (C-3”, 7”), 128.10 (C-4”, 6”), 127.74 (C-5”), 126.22 (C-6, 10), 110.66 (C-40), 110.55 (C-30), 76.25
0
(C-1”), 58.17 (C-1 ), 30.69 (C-2), 22.32 (C-3), 21.25 (C-11); ESIMS: m/z 378 [M + H]+, 400 [M + Na]+, calc.
for C23H23NO4 (377.4330).
Furan-2-ylmethyl (E)-4-(4-methoxyphenyl)-4-[(methoxy)imino]butanoate (4b-1). Yield 77%; 1H NMR
(600 MHz, CDCl3) δ
: 7.60–7.54 (m, 2H, H-6, 10), 7.41 (dd, J = 1.8, 0.8 Hz, 1H, H-50), 6.90–6.85 (m,
2H, H-7, 9), 6.39 (d, J = 3.1 Hz, 1H, H-30), 6.36 (dd, J = 3.2, 1.9 Hz, 1H, H-40), 5.04 (s, 2H, H-10), 3.94
(s, 3H, H-1”), 3.81 (s, 3H, H-11), 3.04–2.98 (m, 2H, H-3), 2.60–2.56 (m, 2H, H-2); 13C NMR (151 MHz,
CDCl3) δ
: 172.33 (C-1), 160.51 (C-8), 156.29 (C-4), 149.39 (C-20), 143.28 (C-50), 127.63 (C-6, 10), 127.58
(C-5), 113.94 (C-7, 9), 110.70 (C-40), 110.57 (C-30), 61.91 (C-1”), 58.20 (C-10), 55.31 (C-11), 30.76 (C-2),
22.19 (C-3); ESIMS: m/z 318 [M + H]+, calc. for C17H19NO5 (317.3365).
1
Furan-2-ylmethyl (E)-4-(4-methoxyphenyl)-4-[(benzyloxy)imino]butanoate (4b-2). Yield 78%; H NMR
(600 MHz, CDCl3) δ
: 7.61–7.53 (m, 2H, H-6, 10), 7.40 (s, 1H, H-50), 7.38 (d, J = 7.7 Hz, 2H, H-3”, 7”),
7.34 (dd, J = 10.9, 4.0 Hz, 2H, H-4”, 6”), 7.29 (t, J = 7.1 Hz, 1H, H-5”), 6.91–6.83 (m, 2H, H-7, 9), 6.37 (d,
J = 2.8 Hz, 1H, H-30), 6.36–6.31 (m, 1H, H-40), 5.19 (s, 2H, H-1”), 5.01 (s, 2H, H-10), 3.80 (s, 3H, H-11),
3.11–3.00 (m, 2H, H-3), 2.63–2.53 (m, 2H, H-2); 13C NMR (151 MHz, CDCl3)
δ
: 172.30 (C-1), 160.53 (C-8),
0
0
156.56 (C-4), 149.37 (C-2 ), 143.26 (C-5 ), 138.03 (C-2”), 128.34 (C-3”, 7”), 128.08 (C-4”, 6”), 127.72 (C-5”),
0
0
0
127.69 (C-6, 10), 127.57 (C-5), 113.90 (C-7, 9), 110.67 (C-4 ), 110.56 (C-3 ), 76.19 (C-1”), 58.18 (C-1 ), 55.30
(C-11), 30.73 (C-2), 22.27 (C-3); ESIMS: m/z 394 [M + H]+, calc. for C23H23NO5 (393.4324).
Furan-2-ylmethyl (E)-4-(4-chlorophenyl)-4-[(methoxy)imino]butanoate (4c-1). Yield 75%; 1H NMR (600 MHz,
CDCl3)
δ
: 7.58–7.54 (m, 2H, H-6, 10), 7.41 (dd, J = 1.7, 0.6 Hz, 1H, H-50), 7.34–7.30 (m, 2H, H-7, 9),
6.38 (d, J = 3.2 Hz, 1H, H-30), 6.36 (dd, J = 3.2, 1.9 Hz, 1H, H-40), 5.04 (s, 2H, H-10), 3.96 (s, 3H, H-1”),
3.03–2.99 (m, 2H, H-3), 2.60–2.56 (m, 2H, H-2); 13C NMR (151 MHz, CDCl3)
δ: 172.11 (C-1), 155.61
(C-4), 149.28 (C-20), 143.32 (C-50), 135.24 (C-8), 133.58 (C-5), 128.73 (C-7, 9), 127.55 (C-6, 10), 110.77
(C-40), 110.58 (C-30), 62.18 (C-1”), 58.24 (C-10), 30.59 (C-2), 22.07 (C-3); ESIMS: m/z 322 [M + H]+, calc.
for C16H16ClNO4 (321.7555).
Furan-2-ylmethyl (E)-4-(4-chlorophenyl)-4-[(benzyloxy)imino]butanoate (4c-2). Yield 76%; 1H NMR
(600 MHz, CDCl3) δ
: 7.55 (dt, J = 4.3, 1.8 Hz, 2H, H-6, 10), 7.41 (s, 1H, H-50), 7.40–7.37 (m, 2H,
H-3”, 7”), 7.37–7.33 (m, 2H, H-4”, 6”), 7.33–7.28 (m, 3H, H-7, 9, 5”), 6.36 (s, 1H, H-30), 6.35 (dd, J = 3.2,
1.7 Hz, 1H, H-40), 5.21 (s, 2H, H-1”), 5.01 (s, 2H, H-10), 3.04 (dd, J = 11.7, 4.2 Hz, 2H, H-3), 2.61–2.56
(m, 2H, H-2); 13C NMR (151 MHz, CDCl3) : 172.09 (C-1), 155.95 (C-4), 149.26 (C-20), 143.30 (C-50),
δ
137.69 (C-2”), 135.27 (C-8), 133.57 (C-5), 128.70 (C-7, 9), 128.41 (C-3”, 7”), 128.14 (C-4”, 6”), 127.88 (C-5”),

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127.61 (C-6, 10), 110.75 (C-40), 110.57 (C-30), 76.51 (C-1”), 58.23 (C-10), 30.57 (C-2), 22.17 (C-3); ESIMS:
m/z 398 [M + H]+, calc. for C22H20ClNO4 (397.8515).
◦
(E)-4-(4-Methylbenzoyl)-4-[(methoxy)imino]-N-phenylbutanamide (5a-1). Yield 78%, m.p. 94.4~95.3 C; 1H
NMR (600 MHz, DMSO)
: 9.91 (s, 1H, H-NH), 7.58 (t, J = 8.4 Hz, 4H, H-6, 10, 20, 60), 7.31–7.26 (m,
2H, H-30, 50), 7.22 (d, J = 8.0 Hz, 2H, H-7, 9), 7.02 (t, J = 7.4 Hz, 1H, H-40), 3.91 (s, 3H, H-1”), 3.01–2.96
δ
(m, 2H, H-3), 2.52–2.48 (m, 2H, H-2), 2.32 (s, 3H, H-11); 13C NMR (151 MHz, DMSO)
δ: 170.26 (C-1),
157.46 (C-4), 139.62 (C-10), 139.30 (C-8), 132.45 (C-5), 129.58 (C-7, 9), 129.12 (C-30, 50), 126.56 (C-6, 10),
123.53 (C-40), 119.56 (C-20, 60), 62.12 (C-1”), 33.40 (C-2), 22.43 (C-3), 21.27 (C-11); ESIMS: m/z 297.1607
[M + H]+, 319.1411 [M + Na]+, calc. for C18H20N2O2 (296.3636).
◦
(E)-4-(4-Methylbenzoyl)-4-[(benzyloxy)imino]-N-phenylbutan amide (5a-2). Yield 79%, m.p. 119.5~120.5 C;
1H NMR (600 MHz, DMSO) : 9.95 (s, 1H, H-NH), 7.53 (dd, J = 7.5, 5.1 Hz, 4H, H-6, 10, 20, 60), 7.38
δ
(d, J = 7.4 Hz, 2H, H-3”, 7”), 7.34 (t, J = 7.4 Hz, 2H, H-4”, 6”), 7.30–7.25 (m, 3H, H-30, 50, 5”), 7.19
(d, J = 7.9 Hz, 2H, H-7, 9), 7.03 (t, J = 7.4 Hz, 1H, H-40), 5.17 (s, 2H, H-1”), 3.06–2.98 (m, 2H, H-3),
2.51 (dd, J = 8.1, 6.6 Hz, 2H, H-2), 2.28 (s, 3H, H-11); 13C NMR (151 MHz, DMSO)
δ: 170.46 (C-1),
157.93 (C-4), 139.50 (C-8), 139.36 (C-10), 138.47 (C-2”), 132.34 (C-5), 129.60 (C-7, 9), 129.15 (C-30, 50),
128.79 (C-3”, 7”), 128.27 (C-4”,6”), 128.14 (C-5”), 126.56 (C-6, 10), 123.77 (C-40), 119.72 (C-20, 60), 75.80
(C-1”), 33.26 (C-2), 22.52 (C-3), 21.21 (C-11); ESIMS: m/z 373.2 [M + H]+, 395.1 [M + Na]+, calc. for
C24H24N2O2 (372.4596).
(E)-4-(4-Methoxyphenyl)-4-[(methoxy)imino]-N-phenylbutanamide (5b-1). Yield 78%, m.p. 125.6~126.5 ◦C;
1H NMR (600 MHz, DMSO) δ: 9.95 (s, 01H, H-NH), 7.68 (d, J = 8.6 Hz,02H, H-6, 10), 7.61 (d, J = 8.0 Hz,
0
0
0
2H, H-2 , 6 ), 7.32 (t, J = 7.7 Hz, 2H, H-3 , 5 ), 7.06 (t, J = 7.3 Hz, 1H, H-4 ), 7.00 (d, J = 8.6 Hz, 2H, H-7, 9),
3.94 (s, 3H, H-1”), 3.82 (s, 3H, H-11), 3.04–2.99 (m, 2H, H-3), 2.54 (t, J = 8.0 Hz, 2H, H-2); 13C NMR (151
MHz, DMSO) δ
: 170.32 (C-1), 160.62 (C-8), 157.12 (C-4), 139.61 (C-10), 129.12 (C-30, 50), 128.05 (C-6, 10),
127.57 (C-5), 123.54 (C-40), 119.58 (C-20, 60), 114.39 (C-7, 9), 62.04 (C-1”), 55.67 (C-11), 33.45 (C-2), 22.38
(C-3); ESIMS: m/z 313.1551 [M + H]+, 355.1367 [M + Na]+, calc. for C18H20N2O3 (312.3630).
◦
(E)-4-(4-Methoxyphenyl)-4-[(benzyloxy)imino]-N-phenylbutanamide (5b-2). Yield 79%, m.p. 119.0~119.6 C;
1H NMR (600 MHz, DMSO)
δ: 9.92 (s, 1H, H-NH), 7.66–7.60 (m, 2H, H-6, 10), 7.57 (d, J = 7.6 Hz, 2H,
H-20, 60), 7.41 (d, J = 7.2 Hz, 2H, H-3”, 7”), 7.39–7.33 (m, 2H, H-4”, 6”), 7.32–7.26 (m, 3H, H-30, 50, 5”),
7.03 (t, J = 7.4 Hz, 1H, H-40), 6.98–6.94 (m, 2H, H-7, 9), 5.19 (s, 2H, H-1”), 3.77 (s, 3H, H-11), 3.06–3.00
(m, 2H, H-3), 2.55–2.51 (m, 2H, H-2); 13C NMR (151 MHz, DMSO)
δ: 170.30 (C-1), 160.66 (C-8), 157.55
(C-4), 139.59 (C-10), 138.64 (C-2”), 1290.13 (C-30, 50), 128.78 (C-3”, 7”), 128.31 (C-4”, 6”), 128.11 (C-6, 10),
0
0
128.10 (C-5”), 127.57 (C-5), 123.56 (C-4 ), 119.58 (C-2 , 6 ), 114.41 (C-7, 9), 75.74 (C-1”), 55.69 (C-11), 33.37
(C-2), 22.46 (C-3); ESIMS: m/z 389.1870 [M + H]+, 411.1676 [M + Na]+, calc. for C24H24N2O3 (388.4590).
(E)-4-(4-Chlorophenyl)-4-[(methoxy)imino]-N-phenylbutanamide (5c-1). Yield 76%, m.p. 151.3~152.4 ◦C;
1H NMR (600 MHz, DMSO) δ: 9.91 (s, 1H, H-NH), 7.71 (d, J = 8.5 Hz, 2H, H-6, 10), 7.55 (d, J = 7.6 Hz,
2H, H-20, 60), 7.48 (dd, J = 8.4, 1.4 Hz, 2H, H-7, 9), 7.28 (t, J = 7.8 Hz, 2H, H-30, 50), 7.03 (t, J = 7.3 Hz,
1H, H-40), 3.93 (s, 3H, H-1”), 2.99 (t, J = 8.0 Hz, 2H, H-3), 2.54–2.47 (m, 2H, H-2); 13C NMR (151 MHz,
DMSO) δ
: 170.17 (C-1), 156.75 (C-4), 139.54 (C-10), 134.46 (C-8), 134.13 (C-5), 129.13 (C-30, 50), 129.03
(C-7, 9), 128.47 (C-6, 10), 123.57 (C-40), 119.57 (C-20, 60), 62.36 (C-1”), 33.21 (C-2), 22.35 (C-3); ESIMS:
m/z 317.1048 [M + H]+, 339.0866 [M + Na]+, calc. for C17H17ClN2O2 (316.7821).
(E)-4-(4-Chlorophenyl)-4-[(benzyloxy)imino]-N-phenylbutanamide (5c-1). Yield 78%, m.p. 116.0~117.9 ◦C;
1H NMR (600 MHz, DMSO)
δ: 9.95 (s, 1H, H-NH), 7.70 (t, J = 8.3 Hz, 2H, H-6, 10), 7.56 (dd, J = 22.3,
8.0 Hz, 2H, H-20, 60), 7.47 (d, J = 7.2 Hz, 2H, H-7, 9), 7.42 (t, J = 6.9 Hz, 2H, H-3”, 7”), 7.36 (dd, J = 6.4,
5.3 Hz, 2H, H-4”, 6”), 7.33–7.25 (m, 3H, H-30, 50, 5”), 7.03 (dd, J = 12.2, 6.3 Hz, 1H, H-40), 5.22 (s, 2H,
H-1”), 3.13–2.98 (m, 2H, H-3), 2.55 0(dt, J = 16.4, 8.2 Hz, 2H, H-2); 13C NMR (151 MHz, DMSO)
δ:
0
0
170.16 (C-1), 157.17 (C-4), 139.58 (C-1 ), 138.36 (C-2”), 134.54 (C-8), 134.14 (C-5), 129.12 (C-3 , 5 ), 129.04
(C-7, 9), 128.81 (C-3”, 7”), 128.50 (C-6, 10), 128.34 (C-4”, 6”), 128.19 (C-5”), 123.57 (C-40), 119.59 (C-20,

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60), 76.08 (C-1”), 33.18 (C-2), 22.46 (C-3); ESIMS: m/z 393.1370 [M + H]+, 415.1164 [M + Na]+, calc. for
C23H21ClN2O2 (392.8780).
3.3. Bio-Evaluation Methods
3.3.1. Cells and Cell Culture
HepG2.2.15 (clonal cells derived from human hepatoma cell line G2) cells were provided by the
Chinese Academy of Medical Sciences (P.R. China) and maintained in minimal essential medium
(MEM) supplemented with 10% fetal bovine serum and 380 µg/mL of G418, 50 ug/mL of kanamycin,
and 0.03% L-glutamine at 37 ◦C in a 5% CO2 atmosphere with 100% humidity [26,33].
3.3.2. Drug Treatment
HepG2.2.15 cells were seeded at a density of 1
×
105 cells/ml (200 mL/well) in 96-well plates
◦
and maintained at 37 C for 24 h prior to extract addition, followed by treatment with various
concentrations of drugs. Lamivudine (3TC) was served as the positive control. Cells were treated with
drug-containing fresh medium every 3 d for up to 9 d in a time-dependent experiment. Medium was
taken at the 9th day of the treatment and stored at
(SI) of each compound were calculated, respectively.
−
20 ◦C until analysis. The IC50 and selected index
3.3.3. Cell Toxicity
Logarithmically growing cells were seeded in 96-well culture plates at a density of
105 cells/mL (200 mL/well). They were cultured for 24 h and then treated with various
concentrations of drugs. Optical density (OD) values were read at 450 nm after 9 days and the
percentage of cell death was calculated; the cells were treated with drug-containing fresh medium every
3 d for up to 9 d. After drug treatment, the cytotoxicity was measured using the MTT assay [45,46].
1
×
3.3.4. Method for HBsAg and HBeAg Inhibition Assays
The levels of HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) in the supernatant
of the HepG2.2.15 cell were simultaneously detected using enzyme-linked immunosorbent assay
(ELISA) kits (Rongsheng Biotechnology Co. Ltd., Shanghai, China) according to the manufacturer’s
instructions.The synthesized derivatives were expressed as the concentration of compound that
achieved 50% inhibition (IC50) to the secretion of HBsAg and HBeAg. The selectivity index (SI) was
determined as the ratio of CC50 to IC50, which is a major pharmaceutical parameter of estimates
possible for future clinical application.
4. Conclusions
In a summary, most of the compounds in the series of oxime ethers with a C6-C4 fragment, which
were designed and virtually bioactively screened by docking with a target protein, showed anti-HBV
activities in an in vitro assay. Among them, compound 5c-1 showed the most potent activity inhibiting
HBsAg secretion (IC50 = 39.93 µM, SI = 28.51). The results of bioactive screening showed that the
stronger the compounds bound to a target protein (human leukocyte antigen A protein) in docking,
the more active they were for anti-HBV in vitro.
Acknowledgments: This work was financially supported by the National Natural Science Foundation of China
(No. 81760635), the Foundation of High-Level Innovation Team and Outstanding Scholar program in Guangxi
college, the Department of Education of Guangxi Zhuang autonomous region (2015), and the Science and
Technology Research Foundation for Traditional Chinese Medicine, Health Department of Guangxi province
(gzbz14-19). We thank Jun Xu from the School of Chemical and Chemical Engineering, Sun Yat Sen University, for
providing generous help in MOE docking.
Author Contributions: Wanxing Wei conceived experiments; Jie Tan and Min Zhou designed and performed
the docking study, synthesis and analysis of bioactive assy data; Xinhua Cui and Zhuocai Wei preformed assay
experiments; Jie Tan and Min Zhou wrote the paper.

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Conflicts of Interest: The authors declare no conflicts of interest.
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Sample Availability: Samples of the all compounds are available from the authors.
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