Design, synthesis, and bioactive screen in vitro of cyclohexyl (E)-4-(Hydroxyimino)-4-phenylbutanoates and their ethers for anti-Hepatitis B Virus agents

Article abstract of DOI:10.3390/molecules24112063

A series of oxime Cyclohexyl (E)-4-(hydroxyimino)-4-phenylbutanoates and their ethers were designed, synthesized, and evaluated for anti-hepatitis B virus (HBV) activities with HepG 2.2.15 cell line in vitro. Most of these compounds possessed anti-HBV activities, and among them, compound 4B-2 showed significant inhibiting effects on the secretion of HBsAg (IC₅₀ = 63.85 ± 6.26 μM, SI = 13.41) and HBeAg (IC₅₀ = 49.39 ± 4.17 μM, SI = 17.34) comparing to lamivudine (3TC) in HBsAg (IC₅₀ = 234.2 ± 17.17 μM, SI = 2.2) and HBeAg (IC50 = 249.9 ± 21.51 μM, SI = 2.07). Docking study of these compounds binding to a protein residue (PDB ID: 3OX8) from HLA-A2 that with the immunodominant HBcAg18-27 epitope (HLA-A2.1- restricted CTL epitope) active site was carried out by using molecular operation environment (MOE) software. Docking results showed that behaviors of these compounds binding to the active site in HLA-A protein residue partly coincided with their behaviors in vitro anti-HBV active screening.

Full text of DOI:10.3390/molecules24112063

molecules
Article
Design, Synthesis, and Bioactive Screen In Vitro of
Cyclohexyl (E)-4-(Hydroxyimino)-4-Phenylbutanoates
and Their Ethers for Anti-Hepatitis B Virus Agents
Xinhua Cui †, Min Zhou †, Jie Tan, Zhuocai Wei, Wanxing Wei * , Peng Luo and Cuiwu Lin
Department of Chemistry, Guangxi University, Nanning 530004, China; cuixinhua123@163.com (X.C.);
leelmm@gxu.edu.cn (M.Z.); yulitanjie@163.com (J.T.); muse00@163.com (Z.W.); lcl40261461@163.com (P.L.);
lincuiwu@gxu.edu.cn (C.L.)
* Correspondence: wxwei@gxu.edu.cn
† These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 6 May 2019; Accepted: 27 May 2019; Published: 30 May 2019
Abstract: A series of oxime Cyclohexyl (E)-4-(hydroxyimino)-4-phenylbutanoates and their ethers
were designed, synthesized, and evaluated for anti-hepatitis B virus (HBV) activities with HepG 2.2.15
cell line in vitro. Most of these compounds possessed anti-HBV activities, and among them, compound
4B-2 showed significant inhibiting effects on the secretion of HBsAg (IC50 = 63.85 ± 6.26 µM
SI = 13.41) and HBeAg (IC50 = 49.39 4.17 M, SI = 17.34) comparing to lamivudine (3TC) in
HBsAg (IC50 = 234.2 17.17 M, SI = 2.2) and HBeAg (IC50 = 249.9 21.51 M, SI = 2.07). Docking
,
±
µ
±
µ
±
µ
study of these compounds binding to a protein residue (PDB ID: 3OX8) from HLA-A2 that with
the immunodominant HBcAg18–27 epitope (HLA-A2.1- restricted CTL epitope) active site was
carried out by using molecular operation environment (MOE) software. Docking results showed that
behaviors of these compounds binding to the active site in HLA-A protein residue partly coincided
with their behaviors in vitro anti-HBV active screening.
Keywords: design; synthesis; anti-HBV activity; structure–activity relationship; molecular docking
1. Introduction
Hepatitis B virus (HBV) remains a serious health problem for leading risks of liver related diseases
for many people in the world. An estimation of 257 million people is chronically infected with HBV in
the world, and among them, about 15–40% develop liver cirrhosis, hepatic failure, and hepatocellular
carcinoma. More than 887,000 people die every year for complications of HBV [
hepatitis B infection currently include methods of direct-acting antivirals (just as with nucleoside
analogs) and host-targeting antivirals (just as with interferon) [ ]. The nucleoside analogs in treatment
of anti-HBV play a role of Troy horse in synthesis of HBV DNA and suppressed replication of HBV, but
they are not effective to eliminate virus completely from patients [ ]. Meanwhile, therapies of HBV
with nucleoside analogs in the long term cause side effects and resistance [ ]. Therefore, it is still a
1,2]. Therapies of
3
4–6
7–9
tremendous challenge to develop new anti-HBV agents for further improvement of anti-HBV therapy.
Many non-nucleoside analog compounds with anti-HBV activities have been found and invented from
synthesized compounds [10
the immunodominant HBcAg18–27 epitope (HLA-A2.1- restricted CTL epitope) binding peptides
of vaccine or of HBcAg initiated specific respond of T cell and resolved cute HBV infection [15 19].
Other peptides specifically bound to the HLA-A2 residue activate CTL responds to prevent infection
and eliminate HBV [20 21]. Therefore, a proposal can be implied that other non-peptide compounds
specifically interact with protein residue in HLA-A2.1 restricted CTL epitope may initiate CTL respond
,11] and natural products [12–14]. Researches showed that HLA-A2 with
–
,
Molecules 2019, 24, 2063; doi:10.3390/molecules24112063
www.mdpi.com/journal/molecules

Molecules 2019, 24, 2063
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to prevent infection of HBV. A protein residue from HLA-A2.1-restricted CTL epitope (PDB ID: 3OX8)
interacted intensively with anti-HBV active compounds in docking investigations, was used as a
virtual target to design anti-HBV new compounds in our previous works. Series of oxime-contained
compounds with C6-C3 and C6-C4 skeleton were designed, synthesized, and assayed for anti-HBV
activities. Results showed these compounds possessed significant anti-HBV activities [22–25]. On the
basis of these results, we designed and synthesized a series of another part of novel oximes and their
derivatives with C6-C4 skeletons, and screened for anti-HBV activities in our present works. All
these compounds were evaluated theoretically with a mimic target HLA-A protein (PDB ID 3OX8)
by molecular docking using molecular operating environment (MOE) method. Results showed these
compounds with significant anti-HBV activities in vitro partly coincided with molecular docking.
2. Results and Discussion
2.1. Chemistry
A series of oxime and oxime ether derivatives of (E)-4-(alkyl-imino)-4-(4-substitute-phenyl)
cyclohexylbutanoate were designed for anti-HBV agents according molecular docking conclusions.
General synthesis of these compounds was depicted in Scheme 1. Compounds 2A
by Friedel-Crafts reaction of succinic anhydride with substituted benzene in yields of 95–99%.
Compounds 3A 3D were prepared by reaction of compounds 2A 2D with cyclohexanol catalyzed with
p-CH3C6H4SO3H (TsOH) in yields of 81-89%. Oxime ether derivatives (4A-1 4A-3 4B-1 4B-3 4C-1 4C-3
4D-1 4D-3) were obtained by reaction of substitute-hydroxylamine hydrochloride (hydroxylamine
hydrochloride methoxylamine hydrochloride and benzylhydroxylamine hydrochloride) with
compounds 3A 3D in yields of 75–88%. The structures of the newly synthesized compounds
were characterized by H NMR, 13C NMR and MS. H NMR spectra of the derivatives showed a
singlet at about 8.69–9.23 ppm corresponding to N-O-H proton in oxime compounds 4A-1 4C-1 and
–2D were prepared
–
–
–
,
–
,
–
,
–
–
1
1
,
4D-1 (Compound 4B-1 was deuterated in CH3OD). The signal at 6.92–7.64 ppm of multiplets in the
region attributed to aromatic hydrogens of the phenyl ring, especially 6.92–7.63 and 7.37–7.69 ppm of
two doubles in the region attributed to the phenyl ring of R1. 13C NMR spectrum for the derivatives
showed signals at 155.68–158.24, 171.90–172.35 corresponding to C=N and C=O, respectively.
2.2. Anti-HBV Activities In Vitro
Inhibition of secreting HBsAg and HBeAg of these synthesized compounds were assayed in
HepG 2.2.15 cells with lamivudine (3TC) as a positive control. Results of cytotoxicity and anti-HBV
activities were showed in Table 1 and Figure 1. Many compounds showed more effective in inhabiting
secretion of HBsAg and HBeAg than that of lamivudine, and among them compound 4B-2 showed
inhibition of HBsAg (IC50 = 63.85
toxicity (CC50 = 856.2 M), and compound 4B-3 in inhibiting secretion of HBsAg (IC50 = 78.46
and HBeAg (IC50 = 84.57 M) and low toxicity (CC50 = 859.5 M) with SI values (SIHBsAg = 12.90,
SIHBeAg = 10.89). When R=H, all oximes in the four groups decreased inhibitions of HBsAg in 505.7,
257.4, 489.3, and 352.4 M, respectively, HBeAg in 375.0, 283.7, 404.7, and 373.4 M, respectively, and
elevated cytotoxicity than their relative ethers.
µM, SI = 13.41) and HBeAg (IC50 = 49.39
µM, SI = 17.34), with low
µ
µM,)
µ
µ
µ
µ

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Table 1. In Vitro Anti-HBV Activity and Cytotoxicity of the Target Compounds.
HBeAg c
IC50 (µM)
HBsAg b
a
Compounds
CC50 (µM)
SI e
SI
d
IC50 (µM)
4A-1
4A-2
4A-3
4B-1
4B-2
4B-3
4C-1
4C-2
4C-3
4D-1
4D-2
4D-3
3TCf
527.9
659.6
974.3
443.8
856.2
859.5
481.1
629.5
678.1
396.5
581.3
604.3
516.4
505.7 ± 28.64 *
109.3 ± 16.05
113.5 ± 6.15
257.4 ± 16.48 *
63.85 ± 6.26
78.46 ± 9.87
489.3 ± 11.21
106.0 ± 8.67
111.5 ± 7.25
352.4 ± 11.03
67.65 ± 8.81
92.60 ± 4.94
234.2 ± 17.17
1.04
6.03
8.58
1.72
13.41
10.95
0.98
5.94
6.08
1.13
8.59
6.53
2.20
375.0 ± 36.20 *
118.9 ± 13.22
129.4 ± 13.64
283.7 ± 26.73 **
49.39 ± 4.17
1.41
5.55
7.53
1.56
17.34
10.16
1.19
4.92
5.20
1.06
6.83
6.25
2.07
c
84.57 ± 6.60
404.7 ± 4.80
127.9 ± 34.57 *
130.4 ± 21.48 *
373.4 ± 16.86
85.10 ± 11.92
96.76 ± 5.13
249.9 ± 21.51 *
a
b
CC50 is 50% cytotoxicity concentration in HepG 2.2.15 cells. HBsAg: hepatitis B surface antigen. HBeAg,
hepatitis B e antigen. IC50 is 50% inhibitory concentration. SI (selectivity index) = CC50/IC50
d
e
f
. Lamivudine (3TC)
as the positive control. Data are expressed as the mean S.E.M. in each group (n = 3). * Compared with the control
±
group index: p < 0.05. ** Compared with the control group index: p < 0.01.
2.3. Molecular Docking Study
The docking software MOE 2008.10 was used for molecular docking of oxime derivatives and a
protein residue in HLA-A2.1-restricted CTL epitope (PDB ID: 3OX8) was selected for docking study.
Molecular docking results clearly revealed interactions between ligands and protein in the active site.
Affinity scoring function
Table 2. Significant interactions with protein residue in docking were compound 4A-3 and 4D-3 for
22.8858 kcal/mol and 21.2040 kcal/mol, respectively. Two hydrogen bonds of lengths 2.29 and 2.58 Å
∆G of the protein-ligand complexes and other docking results were shown in
−
−
for N of O-N in the oxime group with Tyr27 and Tyr63, respectively, was found for compound 4A-3
(Figures 2 and 3).
Table 2. Docking score and bond interaction of synthesized compounds.
Amino Acid
Involved d
Atoms in Ligands
Involved e
H-Bonds b
Distance (Å) c
Ligands
∆G (kcal/mol) a
2.54
2.94
2.99
2.29
2.58
2.48
2.23
2.47
2.24
2.59
1.75
2.44
2.93
2.70
1.76
2.69
2.70
Tyr27
Tyr63
Ser52
Tyr27
Tyr63
Tyr27
Tyr27
Tyr63
Tyr26
Asp29
Tyr63
Tyr27
Tyr63
Tyr27
Tyr27
Tyr63
Gln32
d
O of -ON
O of -ON
O of –C=O
N of -ON
N of -ON
O of -ON
O of –C=O
O of –C=O
O of –C=O
O of -ON
O of –C=O
O of –OCH3
O of –OCH3
N of -ON
N of -ON
N of -ON
N of -ON
e
4A-1
4A-2
4A-3
4B-1
4B-2
−18.4244
−18.7876
−22.8858
−18.5535
−19.3046
−21.0658
−19.7639
−20.5549
−20.4506
−17.6096
−21.0504
2
1
2
1
2
4B-3
4C-1
4C-2
1
1
1
4C-3
4D-1
4D-2
2
1
2
4D-3
a
−21.2040
1
b
c
Affinity energy. Number of hydrogen bonds. Length of hydrogen bond. Receptor of amino acid. Atoms in
ligands which forming H-bonds with amino acid residues.

Molecules 2019, 24, 2063
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2.4. Structure-Activity Relationship (SAR)
Oximes (4A-1, 4B-1, 4C-1 and 4D-1) which R=H showed weaker anti-HBsAg and anti-HBeAg
activities, weaker affinities to the selected target protein in docking than those compounds which
R=Me and benzyl. When these oximes were etherified, anti-HBV activities of their ethers increased,
meanwhile cytotoxicity of these ethers decreased by comparing to their relative oximes. Methyl ethers
4A-2
,
4B-2
,
4C-2 and 4D-2 showed more significant anti-HBV active in their relative groups (Figure 4).
4C and 4D) increased anti-HBV
Results also revealed that substitute groups in phenyl (groups 4B
,
activities by comparing to relative compounds in 4A group. Docking results showed that N or O atoms
in ON fragment and O atom in C=O groups in the oxime derivatives interacted with ammonic acid
residues by hydrogen bond, and their docking affinity partly coincided with their anti-HBV activities
in vitro. Similar structure-activity relationships were found in our previous works [23].
1000
900
800
700
600
500
400
300
200
100
0
CC50
HBsAgIC50
HBeAgIC50
Compounds
Figure 1. Anti-HBV activities and toxicity of compounds in vitro (concentration of compounds in
µM).
Figure 2. Three-dimensional (3D) representation showing the binding mode of compound 4A-3 into
the binding site of HLA-A2. The hydrogen bond formed is colored in green.

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Figure 3. Two-dimensional (2D) representation showing the docking pose interactions of compound
4A-3 in the HLA-A2 active site.
600
HBsAgIC50
HBeAgIC50
ΔG×20
400
200
0
4A-1 4A-2 4A-3 4B-1 4B-2 4B-3 4C-1 4C-2 4C-3 4D-1 4D-2 4D-3
-200
-400
-600
Compounds
Figure 4. Affinity energy and anti-HBV activities of compounds in vitro (concentration of compounds
in µM).
3. Materials and Methods
Melting points were measured on an uncorrected WRX-4 electrothermal melting point apparatus
(Shanghai, China). 1H NMR and 13C NMR spectra were recorded on Bruker AVANCE III HD600
(1H/13C, 600MHz/150MHz) spectrometer (Bruker, Bremerhaven, Germany) using TMS as an internal
standard. The mass spectra were recorded on a Finnigan LCQ Deca XP MAX mass spectrometer
(Thermo Fisher, San Jose, CA, USA) equipped with an ESI source and an ion trap analyzer in the
positive ion mode/in the negative ion. Silica gel GF-254 was used in thin-layer chromatography, and
silica gel H used in flash column chromatography (Qingdao Haiyang Chemical, Qingdao, China).

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All solvents and reagents were analytical grade. The purity of target compounds was assessed on the
basis of analytical HPLC (Thermo Fisher Ultimate 3000), and the results were > 95%.
3.1. Chemistry
3.1.1. Preparation of 4- oxo-4-(substituted phenyl) Butanoic Acids (2) and Their Esters (3)
Preparation of 4- oxo-4-(substituted phenyl) butanoic acids (
2). These Compounds were
synthesized according to reports [26 27]. Succinic anhydride (1 equiv, 25 mmol) in 30 mL CH2Cl2
,
reacted with substituted benzenes (1, 25 mmol) under stirring for 0.5 h in ice-water bath, then anhydrous
aluminum chloride (37.5 mmol) was added to the CH2Cl2 solution (Scheme 1). The reaction was then
kept on room temperature for 6 h. After the reaction was stopped, icy aqueous hydrochloric acid solution
(0.1 mol/L, 10 mL) was drop to resultant solution under stirring for 10 min. Organic phase solution was
evaporated to give crude product. The crude product was further purified by crystallization repeatedly
with 70% ethanol solution to afford 4-oxo-4-phenylbutanoic acid (2A) and4-oxo-4-(p-tolyl)butanoic
acid (2B) [26], 4-(4-Methoxyphenyl)-4-oxobutanoic acid (2C) and 4-(4-Chlorophenyl)-4-oxobutanoic
acid (2D) [27].
OR
N
O
O
O
c
O
OH
b
a
O
R1
O
O
R1
4
R1
R1
3
2
1
4A-1: R=H
4A-2: R=CH3
4A-3: R=benzyl
4B-1: R=H
4B-2: R=CH3
4B-3: R=benzyl
2A: R1 = H
R1=H
R1=CH3
R1=Cl
2B: R1= CH3
2C: R1= OCH3
2D: R1= Cl
4C-1: R=H
4D-1: R=H
4C-2: R=CH3
4C-3: R=benzyl
4D-2: R=CH3
4D-3: R=benzyl
R1=OCH3
Scheme 1. Synthesis of Oximes and their Ethers.
Reagents and conditions: (a) Succinic anhydride, AlCl3, CH2Cl2, 0.5h, 0 ◦C, then 6h rt;
(b) Cyclohexanol, TsOH, CH2Cl2, 4h, reflux; (c) Hydroxylamine hydrdochloride (methoxylamine
hydrochloride, benzylhydroamine hydrochloride), pyridine, CH2Cl2,12h, reflux.
Preparation of Cyclohexyl 4-(4-substituted phenyl) -4-oxobutanoates (3) Compound 2 (2A, 2B, 2C,
2D, 15 mmol) reacted with cyclohexanol (30 mmol) in CH2Cl2 (20 mL) with p-methylbenzenesulfonic
acid (TsOH) (2g, 11.6 mmol) under refluxing for 4 h (Scheme 1). After stopping, the resultant solution
was washed with icy aqueous sodium bicarbonate solution (3%, 15 mL) for three times, then the organic
solution was dried with anhydrous sodium sulfate evaporated to give crude product. This crude
product was purified by flash column chromatography eluting with an eluent of ethyl acetate/petroleum
ether (1:7 v/v) to afford 3A, 3B, 3C, and 3D, respectively.
1
Cyclohexyl 4-oxo-4-phenylbutanoate (3A) Colorless liquid, yield 91.6%. H NMR (CDCl3)
δ 7.98
(d, J = 7.3 Hz, 2H, H-2, 6), 7.55 (t, J = 7.4 Hz, 1H, H-4), 7.45 (t, J = 7.8 Hz, 2H, H-3, 5), 4.81–4.75 (m, 1H,
H-14), 3.29 (t, J = 6.7 Hz, 2H, H-8), 2.74 (t, J = 6.6 Hz, 2H, H-9), 1.84 (dd, J = 12.6, 4.2 Hz, 2H, H-15), 1.70
(dd, J = 8.9, 4.3 Hz, 2H, H-19), 1.52 (dd, J = 8.9, 3.9 Hz, 1H, H-17), 1.45–1.32 (m, 4H, H-16,18), 1.24 (dd,
J = 20.5, 7.5 Hz, 1H, H-17). 13C NMR (CDCl3)
δ 198.23 (C-7), 172.29 (C-10), 136.66 (C-1), 133.14 (C-4),
128.59 (C-2, 6), 128.01 (C-3, 5), 72.90 (C-14), 33.44 (C-8), 31.57 (C-15, 19), 28.69 (C-9), 25.37 (C-17), 23.71
(C-16, 18) [28].
Cyclohexyl 4-oxo-4-(p-tolyl) butanoate (3B). Colorless crystal, yield 90.8%. m.p. 40.8–41.6 ◦C.
1H NMR (MeOD)
δ 7.90 (d, J = 8.2 Hz, 2H, H-2, 6), 7.31 (d, J = 8.0 Hz, 2H, H-3, 5), 4.76–4.72 (m, 1H,
H-15), 3.32–3.28 (m, 2H, H-8), 2.70–2.68 (m, 2H, H-9), 2.41 (s, 3H, H-14, -phCH3), 1.82 (dd, J = 11.8,
5.7 Hz, 2H, H-16), 1.71 (dd, J = 9.7, 3.3 Hz, 2H, H-20), 1.53 (dd, J = 11.0, 8.0 Hz, 1H, H-18), 1.45–1.33
(m, 4H, H-17,19), 1.29 (dd, J = 12.8, 9.8 Hz, 1H, H-18). 13C NMR (CDCl3)
δ 197.88 (C-7), 172.41 (C-10),

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143.92 (C-1), 134.22 (C-4), 129.27 (C-2, 6), 128.15 (C-3,5), 72.89 (C-15), 33.34 (C-8), 31.59 (C-16, 20), 28.76
(C-9), 25.39 (C-18), 23.73 (C-17, 19), 21.64 (C-14). ESIMS: m/z 275.1646 [M + H]+, 297.1461 [M + Na]+,
calculated for C17H22O3 (274.36).
Cyclohexyl 4-(4-methoxyphenyl)-4-oxobutanoate (3C). Colorless crystal, yield 90.2%. m.p.
◦
1
43.7–44.6 C. H NMR (MeOD)
δ 7.99 (d, J = 9.0 Hz, 2H, H-2, 6), 7.01 (d, J = 9.0 Hz, 2H, H-3,
5), 4.77–4.70 (m, 1H, H-16), 3.88 (s, 3H, H-15, OCH3), 3.30–3.26 (m, 2H, H-8), 2.70–2.67 (m, 2H, H-9),
1.82 (dd, J = 12.0, 5.7 Hz, 2H, H-17), 1.72 (dd, J = 8.0, 4.9 Hz, 2H, H-21), 1.56–1.50 (m, 1H, H-19), 1.44
(dd, J = 14.4, 8.0 Hz, 2H, H-18), 1.38 (dd, J = 9.9, 6.7 Hz, 2H, H-20), 1.32–1.26 (m, 1H, H-19). 13C NMR
(151 MHz, MeOD) δ 197.60 (C-7), 172.77 (C-10), 163.91 (C-4), 130.04 (C-2, 6), 129.55 (C-1), 113.49 (C-3, 5),
72.64 (C-16), 54.68 (C-15), 32.61 (C-8), 31.14 (C-17, 21), 28.29 (C-9), 25.09 (C-19), 23.25 (C-18, 20). ESIMS:
m/z 291.1593 [M + H]+, 313.1413 [M + Na]+, calculated for C17H22O4 (290.36).
Cyclohexyl 4-(4-chlorophenyl)-4-oxobutanoate (3D). Colorless crystal, yield 89.5%. m.p.
◦
1
67.5–68.7 C. H NMR (CDCl3)
δ 7.92 (d, J = 8.7 Hz, 2H, H-2, 6), 7.44 (d, J = 8.6 Hz, 2H, H-3,
5), 4.80-4.75 (m, 1H, H-14), 3.26 (t, J = 6.6 Hz, 2H, H-8), 2.74 (t, J = 6.6 Hz, 2H, H-9), 1.84 (dd, J = 11.4,
5.4 Hz, 2H, H-19), 1.71 (dd, J = 8.8, 4.4 Hz, 2H, H-15), 1.55-1.51 (m, 1H, H-17), 1.44–1.32 (m, 4H, H-16,18),
1.26 (t, J = 11.6 Hz, 1H, H-17). 13C NMR (CDCl3)
δ 197.04 (C-7), 172.16 (C-10), 139.58 (C-4), 134.99 (C-1),
129.45 (C-2, 6), 128.91 (C-3, 5), 73.01 (C-14), 33.40 (C-8), 31.57 (C-15, 19), 28.61 (C-9), 25.36 (C-17), 23.72
(C-16, c18). ESIMS: m/z 295.1102 [M + H]+, 317.0922 [M + Na]+, calculated for C16H19ClO3 (294.78).
3.1.2. Preparation of Oximes and Their Ethers
Compound 3 (3A, 3B, 3C, and 3D, 3.84mmol) and substituted hydroxylamine hydrochloride
(7.7 mmol) in CH2Cl2 (20 mL) with pyridine (2 mL) was refluxed for 12 h (Scheme 1). After stopping,
the resultant solution was evaporated to give a residue under vacuum. This residue was dissolved in
ethyl acetate (20 mL), then washed with saturated sodium chloride solution (20 mL
×
3). The organic
phase was dried with anhydrous sodium sulfate, and evaporated to give a crude product. This crude
product was purified by flash column chromatography with an eluent of ethyl acetate/petroleum ether
(1:5 to 1:13 v/v) to afford ethers.
Cyclohexyl (E)-4-hydroxyimino-4-phenylbutanoate (4A-1). Colorless crystal, yield 88.1%. m.p.
100.8–102.0 ◦C. 1H NMR (CDCl3)
δ
9.23 (s, 1H, H-21, COOH), 7.63 (d, J = 3.7 Hz, 2H, H-2, 6), 7.44–7.39
(m, 3H, H-3, 4, 5), 4.81–4.74 (m, 1H, H-14), 3.18-3.13 (m, 2H, H-8), 2.65-2.60 (m, 2H, H-9), 1.83 (dd,
J = 8.7, 3.8 Hz, 2H, H-15), 1.72 (dd, J = 9.8, 4.0 Hz, 2H, H-19), 1.57–1.52 (m, 1H, H-17), 1.38 (dd, J = 22.6
12.5 Hz, 4H, H-16, 18), 1.26 (dd, J = 16.3, 6.4 Hz, 1H, H-17). 13C NMR (CDCl3)
172.15 (C-10), 158.24
,
δ
(C-7), 135.21 (C-1), 129.42 (C-4), 128.67 (C-2, 6), 126.36 (C-3, 5), 73.04 (C-14), 31.56 (C-15, 19), 31.00
(C-9), 25.36 (C-8), 23.73 (C-17), 22.03 (C-16, 18). ESIMS: m/z 276.1601 [M + H]+, 298.1420 [M + Na]+,
calculated for C16H21NO3 (275.35) [29].
Cyclohexyl (E)-4-methoxyimino-4-phenylbutanoate (4A-2). Colorless liquid, yield 81.5%. 1H
NMR (CDCl3) δ 7.64 (d, J = 1.9 Hz, 2H, H-2, 6), 7.40–7.34 (m, 3H, H-3, 4, 5), 4.78–4.74 (m, 1H, H-14),
4.00 (s, 3H, H-21), 3.08–3.04 (m, 2H, H-8), 2.57–2.53 (m, 2H, H-9), 1.83 (dd, J = 8.9, 3.7 Hz, 2H, H-15),
1.72 (dd, J = 9.7, 3.9 Hz, 2H, H-19), 1.54 (dd, J = 10.1, 2.5 Hz, 1H, H-17), 1.38 (dd, J = 22.0, 12.3 Hz, 4H,
H-16,18), 1.27 (dd, J = 13.6, 5.8 Hz, 1H, H-17). 13C NMR (CDCl3)
δ 172.03 (C-10), 156.98 (C-7), 135.27
(C-4), 129.19 (C-1), 128.53 (C-2, 6), 126.33 (C-3, 5), 72.90 (C-14), 62.01 (C-21), 31.57 (C-15, 19), 31.20
(C-9), 25.38 (C-8), 23.72 (C-17), 22.38 (C-16, 18). ESIMS: m/z 290.1748 [M + H]+, 312.1567 [M + Na]+
calculated for C17H23NO3 (289.38).
,
Cyclohexyl (E)-4-benzyloxyimino-4-phenylbutanoate (4A-3). Pale yellow liquid, yield 79.5%.
1H NMR CDCl3)
δ 7.69 (d, J = 3.3 Hz, 2H, H-2, 6), 7.46 (d, J = 7.1 Hz, 2H, H-3, 5), 7.43–7.38 (m, 5H,
H-23,24,25,26,27), 7.35 (t, J = 7.3 Hz, 1H, H-4), 5.29 (s, 2H, H-21), 4.78 (dt, J = 8.9, 4.6 Hz, 1H, H-14),
3.16–3.12 (m, 2H, H-8), 2.62–2.58 (m, 2H, H-9), 1.84 (dd, J = 9.7, 3.6 Hz, 2H, H-15), 1.75–1.72 (m, 2H,
H-19), 1.57 (dd, J = 7.9, 4.9 Hz, 1H, H-17), 1.44–1.35 (m, 4H, H-16, 18), 1.30 (dd, J = 16.6, 6.5 Hz, 1H,
H-17). 13C NMR (CDCl3)
δ
172.09 (C-10), 157.36 (C-7), 138.01 (C-22), 135.29 (C-1), 129.27 (C-4), 128.55
(C-24, 26), 128.41 (C-2, 6), 128.11 (C-3, 5), 127.81 (C-25), 126.42 (C-23, 27), 76.35 (C-21), 72.94 (C-14),

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31.59 (C-19), 31.25 (C-9), 25.41 (C-8), 23.75 (C-17), 22.57 (C-16, 18). ESIMS: m/z 366.2070 [M + H]+,
388.1886 [M + Na]+, calculated for C23H27NO3 (365.47).
Cyclohexyl (E)-4-hydroxyimino-4-(4-methylphenyl)butanoate (4B-1). Colorless crystal, yield
88.2%. m.p. 80.1–80.7 ◦C. 1H NMR (MeOD)
δ 7.51 (d, J = 8.2 Hz, 2H, H-2, 6), 7.20 (d, J = 8.0 Hz, 2H,
H-3, 5), 4.70 (dt, J = 8.7, 4.5 Hz, 1H, H-15), 3.08-3.05 (m, 2H, H-8), 2.54 (t, J = 7.9 Hz, 2H, H-9), 2.36 (s,
3H, H-14), 1.80 (d, J = 9.3 Hz, 2H, H-16), 1.75–1.71 (m, 2H, H-20), 1.57–1.54 (m, 1H, H-18), 1.39 (dd,
J = 17.8, 8.7 Hz, 4H, H-17, 19), 1.32 (d, J = 11.5 Hz, 1H, H-18). 13C NMR (CDCl3)
δ 172.15 (C-10), 158.23
(C-7), 139.48 (C-1), 132.32 (C-4), 129.35 (C-2, 6), 126.24 (C-3, 5), 72.96 (C-15), 31.57 (C-16, 20), 31.06
(C-9), 25.37 (C-8), 23.73 (C-18), 21.89 (C-17, 19), 21.27 (C-14). ESIMS: m/z 290.1754 [M + H]+, 312.1573
[M + Na]+, calculated for C17H23NO3 (289.38).
Cyclohexyl (E)-4-methoxyimino-4-(4-methoxyphenyl)butanoate (4B-2). Colorless liquid, yield
1
81.7%. H NMR (CDCl3)
δ 7.55 (d, J = 8.2 Hz, 2H, H-2, 6), 7.19 (d, J = 8.0 Hz, 2H, H-3, 5), 4.80–4.73 (m,
1H, H-15), 3.99 (s, 3H, H-22), 3.06-3.03 (m, 2H, H-8), 2.56–2.53 (m, 2H, H-9), 2.38 (s, 3H, H-14), 1.83 (dd,
J = 8.8, 3.8 Hz, 2H, H-20), 1.73 (dd, J = 9.0, 4.7 Hz, 2H, H-16), 1.55 (dd, J = 9.6, 6.5 Hz, 1H, H-18), 1.39
(dd, J = 22.4, 12.4 Hz, 4H, H-17, 19), 1.29–1.24 (m, 1H, H-18). 13C NMR (CDCl3)
δ 172.11 (C-10), 156.95
(C-7), 139.22 (C-4), 132.40 (C-1), 129.24 (C-2, 6), 126.22 (C-3, 5), 72.87 (C-15), 61.93 (C-22), 31.59 (C-16,
20), 31.27 (C-9), 25.39 (C-8), 23.73 (C-18), 22.34 (C-17, 19), 21.25 (C-14). ESIMS: m/z 326.1726 [M+Na]+,
calculated for C18H25NO3 (303.40).
Cyclohexyl (E)-4-benzyloxyimino-4-(4-methylphenyl)butanoate (4B-3). Pale yellow liquid, yield
1
79.3%. H NMR (CDCl3)
δ 7.37–7.29 (m, 7H, H-2, 6, 24, 25, 26, 27, 28), 7.22 (d, J = 7.9 Hz, 2H, H-3,
5), 5.09 (s, 2H, H-22), 4.74 (dt, J = 8.9, 4.6 Hz, 1H, H-15), 2.87–2.83 (m, 2H, H-8), 2.57–2.54 (m, 2H,
H-9), 2.38 (s, 3H, H-14), 1.84-1.78 (m, 2H, H-16), 1.72 (dd, J = 9.4, 3.6 Hz, 2H, H-20), 1.55 (dd, J = 6.9,
3.7 Hz, 1H, H-18), 1.41–1.33 (m, 4H, H-17, 19), 1.27 (t, J = 7.0 Hz, 1H, H-18). 13C NMR (CDCl3)
δ 172.16
(C-10), 155.68 (C-7), 138.81 (C-4), 138.41 (C-23), 130.80 (C-1), 128.86 (C-3, 5), 128.22 (C-25, 27), 127.89
(C-26), 127.82 (C-24, 28), 127.47 (C-2, 6), 75.89 (C-22), 72.64 (C-15), 31.61 (C-16, 20), 31.45 (C-9), 30.75
(C-8), 25.40 (C-18), 23.76 (C-17, 19), 21.37 (C-14). ESIMS: m/z 380.2223 [M + H]+, 402.2037 [M + Na]+,
calculated for C24H29NO3 (379.50).
Cyclohexyl (E)-4-hydroxyimino-4-(4-methoxyphenyl)butanoate (4C-1). Colorless crystal, yield
89.0%. m.p. 67.7–68.5 ◦C. 1H NMR (CDCl3)
δ 8.69 (s, 1H, H-23), 7.59 (d, J = 8.9 Hz, 2H, H-2, 6), 6.93 (d,
J = 8.9 Hz, 2H, H-3, 5), 4.79-4.75 (m, 1H, H-16), 3.85 (s, 3H, H-15), 3.13- 3.10 (m, 2H, H-8), 2.62-2.59
(m, 2H, H-9), 1.83 (dd, J = 8.8, 4.0 Hz, 2H, H-17), 1.72 (dd, J = 8.7, 3.7 Hz, 2H, H-21), 1.54 (dd, J = 5.7,
3.2 Hz, 1H, H-19), 1.38 (dd, J = 23.1, 11.8 Hz, 4H, H-18, 20), 1.26 (dd, J = 13.0, 3.1 Hz, 1H, H-19). 13C
NMR (CDCl3) δ 172.27 (C-10), 160.61 (C-4), 157.68 (C-7), 127.71 (C-2, 6), 127.63 (C-1), 114.05 (C-3, 5),
73.00 (C-16), 55.33 (C-15), 31.57 (C-17, 21), 31.08 (C-9), 25.37 (C-8), 23.73 (C-19), 21.95 (C-18, 20). ESIMS:
m/z 306.1701 [M + H]+, 328.1521 [M + Na]+, calculated for C17H23NO4 (305.37).
Cyclohexyl (E)-4-methoxyimino-4-(4-methoxyphenyl)butanoate (4C-2). Colorless liquid, yield
1
80.7%. H NMR (MeOD)
δ 7.59 (d, J = 8.9 Hz, 2H, H-2, 6), 6.94 (d, J = 8.9 Hz, 2H, H-3, 5), 4.72–4.64
(m, 1H, H-16), 3.95 (s, 3H, H-23), 3.83 (s, 3H, H-15), 3.03–2.97 (m, 2H, H-8), 2.52-2.46 (m, 2H, H-9),
1.82-1.76 (m, 2H, H-17), 1.74-1.67 (m, 2H, H-21), 1.55 (dd, J = 10.7, 4.6 Hz, 1H, H-19), 1.42-1.33 (m, 4H,
H-18,20), 1.29 (dd, J = 9.6, 3.0 Hz, 1H, H-19). 13C NMR (MeOD)
δ 172.28 (C-10), 160.78 (C-4), 156.67
(C-7), 127.48 (C-2, 6), 127.35 (C-1), 113.53 (C-3, 5), 72.89 (C-16), 60.84 (C-23), 54.42 (C-15), 31.16 (C-17,
21), 30.99 (C-9), 25.07 (C-8), 23.34 (C-19), 21.81 (C-18, 20). ESIMS: m/z 320.1845 [M + H]+, 342.1661
[M + Na]+, calculated for C18H25NO4 (319.40).
Cyclohexyl (E)-4-benzyloxyimino-4-(4-methoxyphenyl)butanoate (4C-3). Pale yellow liquid, yield
1
76.9%. H NMR (MeOD)
δ 7.58 (d, J = 8.9 Hz, 2H, H-2, 6), 7.41 (d, J = 7.1 Hz, 2H, H-25, 29), 7.36 (t,
J = 7.5 Hz, 2H, H-26, 28), 7.30 (t, J = 7.3 Hz, 1H, H-27), 6.92 (d, J = 8.9 Hz, 2H, H-3, 5), 5.19 (s, 2H, H-23),
4.66 (dt, J = 8.7, 4.5 Hz, 1H, H-16), 3.81 (s, 3H, H-15), 3.06-3.03 (m, 2H, H-8), 2.52-2.49 (m, 2H, H-9), 1.75
(dd, J = 13.6, 6.0 Hz, 2H, H-17), 1.68 (dd, J = 9.2, 4.9 Hz, 2H, H-21), 1.55–1.50 (m, 1H, H-19), 1.39–1.32
(m, 4H, H-18,20), 1.30–1.24 (m, 1H, H-19). 13C NMR (MeOD)
δ
172.35 (C-10), 160.82 (C-4), 157.11 (C-7),
138.08 (C-24), 127.99 (C-26, 28), 127.79 (C-2, 6), 127.52 (C-27), 127.43 (C-25, 29), 127.38 (C-1), 113.50 (C-3,

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5), 75.75 (C-23), 72.87 (C-16), 54.39 (C-15), 31.09 (C-17, 21), 30.99 (C-9), 25.04 (C-8), 23.28 (C-19), 22.02
(C-18, 20). ESIMS: m/z 396.2179 [M + H]+, 418.1994 [M +Na]+, calculated for C24H29NO4 (395.50).
Cyclohexyl (E)-4-hydroxyimino-4-(4-chlorophenyl)butanimidate (4D-1). Colorless crystal, yield
86.3%. m.p. 92.9-93.6 ◦C. 1H NMR (CDCl3)
δ 9.12 (s, 1H, H-21), 7.57 (d, J = 8.6 Hz, 2H, H-2, 6), 7.37 (d,
J = 8.6 Hz, 2H, H-3, 5), 4.76 (dt, J = 8.9, 4.6 Hz, 1H, H-14), 3.12-3.09 (m, 2H, H-8), 2.62–2.59 (m, 2H,
H-9), 1.84–1.80 (m, 2H, H-15), 1.71 (dd, J = 9.5, 3.7 Hz, 2H, H-19), 1.54 (dd, J = 9.7, 3.2 Hz, 1H, H-17),
1.37 (dd, J = 20.0, 11.6 Hz, 4H, H-16, 18), 1.28–1.23 (m, 1H, H-17). 13C NMR (CDCl3)
δ 172.06 (C-10),
157.39 (C-7), 135.48 (C-4), 133.65 (C-1), 128.87 (C-3, 5), 127.68 (C-2, 6), 73.19 (C-14), 31.56 (C-15,19),
30.91 (C-9), 25.34 (C-8), 23.73 (C-17), 21.89 (C-16, 18). ESIMS: m/z 332.1024 [M + Na]+, calculated for
C16H20ClNO3 (309.79).
Cyclohexyl (E)-4 -methoxyimino-4-(4-chlorophenyl)butanoate (4D-2). Colorless liquid, yield
1
75.5%. H NMR (CDCl3)
δ 7.59 (d, J = 8.6 Hz, 2H, H-2, 6), 7.34 (d, J = 8.6 Hz, 2H, H-3, 5), 4.75 (dt,
J = 9.0, 4.6 Hz, 1H, H-14), 3.99 (s, 3H, H-21), 3.03-3.00 (m, 2H, H-8), 2.53 (dd, J = 8.6, 7.5 Hz, 2H, H-9),
1.83-1.79 (m, 2H, H-19), 1.71 (dd, J = 9.3, 3.5 Hz, 2H, H-15), 1.56-1.51 (m, 1H, H-17), 1.37 (dd, J = 18.8,
10.5 Hz, 4H, H-16,18), 1.28-1.22 (m, 1H, H-17). 13C NMR (CDCl3)
δ 171.90 (C-10), 155.87 (C-7), 135.19
(C-4), 133.71 (C-1), 128.70 (C-2, 6), 127.61 (C-3, 5), 72.99 (C-14), 62.13 (C-21), 31.57 (C-15, 19), 31.10
(C-9), 25.35 (C-8), 23.72 (C-17), 22.16 (C-16, 18). ESIMS: m/z 324.1370 [M + H]+, 346.1187 [M + Na]+,
calculated for C17H22ClNO3 (323.82).
Cyclohexyl (E)-4-benzyloxyimino-4-(4-chlorophenyl)butanoate (4D-3). Pale yellow liquid, yield
1
70.8%. H NMR (CDCl3)
δ
7.60 (d, J = 8.4 Hz, 2H, H-2, 6), 7.42 (ddd, J = 19.1, 15.0, 6.3 Hz, 10H, H-25,
26, 27, 28, 29, 17, 18, 19, 20, 21), 7.22 (d, J = 7.8 Hz, 2H, H-3, 5), 5.29 (s, 2H, H-15), 5.14 (s, 2H, H-23),
3.19-3.16 (m, 2H, H-8), 2.71–2.65 (m, 2H, H-9), 2.41 (s, 3H, H-14). 13C NMR (CDCl3)
δ
172.52 (C-10),
157.05 (C-7), 139.37(C-4), 138.04 (C-24), 135.91 (C-16), 132.32 (C-4),129.30 (C-2, 6), 129.29 (C-26, 28),
128.60 (C-18, 20), 128.42 (C-27), 128.27 (C-19), 128.17 (C-25, 29), 127.81 (C-17, 21), 126.30 (C2, 6), 76.33
(C-23), 66.47 (C-15), 30.95(C-9), 22.45 (C-8), 21.32 (C-14). ESIMS: m/z 388.1918 [M + H]+, 410.1739
[M + Na]+, calculated for C25H25NO3 (387.48).
3.1.3. Evaluation of Compounds for Biological Activity in vitro
Cytotoxicity of Compounds In Vitro
These synthesized compounds were evaluated for anti-HBV activity with referring to reported
method [25,30]. The HepG2.2.15 cell, a human hepatoblastoma cell line stably transfected with cloned
HBV DNA, was obtained from Chinese Academy of Medical Sciences (Beijing, China), and was
cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum
and 1.5 g/L◦of sodium bicarbonate, 10 mL/L of streptomycin, 10 mL/L of penicillin, and 200 mg/L of
G418 at 37 C in a 5% CO2 atmosphere with 95–98% humidity. Then,◦HepG2.2.15 cell was plated
105 cells/mL in 96-well plates and incubated at 37 C for 24 h and then treated
at a density of 1
×
with variant concentration of compounds in 300, 150, 75, and 35.5 µM/mL; every three days, in a nine
day period, C (OD) values were recorded at 450 nm. After nine days, the percent of cell death was
measured using the method of MTT assay after drug treatment. Cytotoxicity of these compounds was
expressed as the concentration of compound required to kill 50% (CC50) of the HepG 2.2.15 cells.
Method for the 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 cells were determined by the
enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol (Shanghai
Kehua Biotech Co., Ltd., Shanghai, China). The synthesized compounds were expressed as the
concentration 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 a major pharmaceutical parameter of
estimates possible future clinical application.

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3.2. Statistical Analysis
SPSS 20.0 and MICROSOFT EXCEL 2010 were used for the statistical analysis. All data were
expressed as the mean standard error of mean (S.E.M.). The differences among the groups were
±
analyzed by one-way analysis of variance (ANOVA) with a Tukey post hoc test when comparing
multiple groups. P values lower than 0.05 were regarded as statistically significant.
4. Conclusions
A series of cyclohexyl (E)-4-(alkyl imino)-4-(4-substituted-phenyl) butanoates possessed obvious
and significant anti-HBV activities with SIHBsAg values from 0.89 to 13.41 and SIHBeAg values from 1.06
to 17.34 compared to control lamivudine in SIHBsAg values 2.20 and SIHBeAg values 2.07. Results also
revealed that oximes were weaker in inhibiting HBV, and more cytotoxic than their relative ethers;
meanwhile, methyl ethers were the most active in inhibiting HBV among compounds in every group.
Compound 4B-2 among these compounds showed the best inhibition of HBsAg and HBeAg secretion.
Author Contributions: Conceptualization, W.W. and X.-C.; Methodology, M.Z.; Software, C.L.; Validation,
X.C. and M.Z.; Formal Analysis, P.L.; Investigation, X.C. and Z.W.; Data Curation, J.T.; Writing-Original Draft
Preparation, X.C.; Writing-Review & Editing, M.Z.; Visualization, X.C. and M.Z.; Supervision, W.W.; Project
Administration, W.W.; Funding Acquisition, W.W.
Funding: This work was financially supported by the National natural science foundation of China (No. 81760635).
Acknowledgments: We are thankful to Jun Xu from the School of Chemical & Chemistry Engineering, Sun Yat
Sen University, for providing generously help in MOE docking.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to
publish the results.
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Sample Availability: Samples of the compounds are available from the authors.
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