Synthesis and biological evaluation of phenylpropanoid derivatives

Article abstract of DOI:10.1007/s00044-016-1554-6

In this work, a series of oxime ether phenylpropanoid derivatives were synthesized. Their anti-hepatitis B virus (HBV) activity in HepG 2.2.15 cells was determined, and anti-cancer potential against three human cancer cell lines was evaluated. All the synthesized derivatives showed great efficiency against HBV. Compound 4d demonstrated the most effective anti-HBV activity, performing strong potent inhibitory not only on the secretion of HBsAg (IC₅₀?=?50.45?μM, SI?=?9.18) and HBeAg (IC₅₀?=?50.11?μM, SI?=?9.24), but also on the HBV DNA replication (IC₅₀?=?51.80?μM, SI?=?8.94). Besides, the synthetic compounds also displayed obvious anti-cancer activity. Moreover, the docking study of all synthesized compounds inside the related protein active site was conducted to explore the molecular interactions and a molecular target for activity using a MOE-docking technique. This study identified a new class of potent anti-HBV and anti-cancer agents.

Full text of DOI:10.1007/s00044-016-1554-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1554-6
ORIGINAL RESEARCH
Synthesis and biological evaluation of phenylpropanoid
derivatives
Sheng Liu1,2 Yubin Li1 Wanxing Wei2 Jingchen Wei3
•
•
•
Received: 24 August 2015 / Accepted: 3 March 2016
Ó Springer Science+Business Media New York 2016
Abstract In this work, a series of oxime ether phenyl-
propanoid derivatives were synthesized. Their anti-hepati-
tis B virus (HBV) activity in HepG 2.2.15 cells was
determined, and anti-cancer potential against three human
cancer cell lines was evaluated. All the synthesized
derivatives showed great efficiency against HBV. Com-
pound 4d demonstrated the most effective anti-HBV
activity, performing strong potent inhibitory not only on
the secretion of HBsAg (IC50 = 50.45 lM, SI = 9.18) and
HBeAg (IC50 = 50.11 lM, SI = 9.24), but also on the
HBV DNA replication (IC50 = 51.80 lM, SI = 8.94).
Besides, the synthetic compounds also displayed obvious
anti-cancer activity. Moreover, the docking study of all
synthesized compounds inside the related protein active
site was conducted to explore the molecular interactions
and a molecular target for activity using a MOE-docking
technique. This study identified a new class of potent anti-
HBV and anti-cancer agents.
Introduction
Natural products and their derivatives possessing various
skeletons could provide a great opportunity for novel drugs
development (Gao et al., 2011; Du et al., 2011; Wang
et al., 2012). Lignans, one of the natural products, have
been associated with several bioactivities ranging from
anti-HBV, anti-HIV, anti-cancer, antioxidant and anti-in-
flammatory activities (Manat et al., 2007; Saleem et al.,
2005; Yamashita et al., 1992). Our previous study deter-
mined that lignans niranthin, nirtetralin, nirtetralin A and
nirtetralin B isolated from Phyllanthus niruri L. exhibited
significant anti-HBV activity (Wei et al., 2012; Liu et al.,
2014a, b). Lignans hypophyllanthin, phyllanthin and a
series of lignans isolated from Cornus kousa Burg. were
found to possess anti-cancer and anti-inflammatory activity
(Parvathaneni et al., 2014). Globoidnan A from Eucalyptus
globoidea and a series of lignans from Kadsura interior
were reported as potent anti-HIV agents (Chen et al., 1997;
Ovenden et al., 2004). These lignans are the most
promising alternative to current novel drugs. However,
only few of these complex products lignans exist in the
nature. Hence, a method to synthesize these derivatives is
necessary to approach their clinical function.
Keywords Synthesis Á Phenylpropanoid derivatives Á
Anti-HBV activity Á Anti-cancer activity Á
Molecular docking
Hepatitis and cancer are two of the main life-threatening
problems in the world, leading to millions of deaths every
year (Lavanchy, 2004). Although various of drugs have
been developed in HBV and cancer treatments, there is still
a growing need for more effective agent with highly
selectivity.
& Sheng Liu
liusheng87@126.com
& Wanxing Wei
wxwei@gxu.edu.cn
1
College of Science, Guangdong Ocean University,
Zhanjiang 524088, People’s Republic of China
Lignans are composed of two molecular phenyl-
propanoid with the common C6-C3 structure. Lignans with
anti-HBV and anti-cancer function are observed to possess
some similar fragments in their molecular structure,
including 3,4-dimethoxyphenyl, 3,4,5-trimethoxyphenyl,
2
Department of Chemistry, Guangxi University,
Nanning 530004, People’s Republic of China
3
Department of Pharmacology, Guilin Medical University,
Guilin 541004, People’s Republic of China
123

Med Chem Res
benzo[d][1,3]dioxol-5-yl and 4-methoxybenzo[d][1,3]dioxole-
5-yl. Therefore, in this work, (E)-3-(3,4-dimethoxyphenyl)-
acrylic acid (a), (E)-3-(3,4,5-trimethoxyphenyl)acrylic acid (b),
(E)-3-(benzo[d][1,3]dioxol-5-yl)acrylic acid (c) and (E)-3-(7-
methoxybenzo[d][1,3]dioxol-5-yl)acrylic acid (d) were chosen
as the main scaffold for the design and synthesis of our anti-
HBV and anti-cancer agents. Adamantyl substituent com-
pounds were reported to possess special effects in clinical
and used in flu virus, antitumor, artificial blood, treatment of
Parkinson’s disease and so on (Balzarini et al., 2009). Thus,
the acrylicacid and adamantane were considered to construct
the scaffold in order to find stronger activity drugs.
According to the molecular hybridization principle, esteri-
fication of natural compounds is a promising approach for
derivatives synthesis, by which two active parts can be easily
hybridized to enhance activity (Viegas-Junior, et al., 2007;
Chen et al., 2014). Moreover, using oxime ester compounds
obtained from the four acrylic acids and adamantine for anti-
HBV and anti-cancer treatment were not reported before
(Wu et al., 2012; Kim et al., 2000; Panda et al., 2012a, b).
Hence, in this work, a series of phenyl acryloyl type oxime
esters based on the four acrylic acids were synthesized. The
anti-HBV and anti-cancer activities of the compound were
also evaluated in this work.
silica gel plates. Yields referred to isolated pure products
and were not maximized. On the basis of NMR and HPLC
(Thermo Fisher UItiMate 3000, USA) data, all final com-
pounds reported in the manuscript were [95 % pure.
Chemistry (Scheme 1)
Preparation of 2-adamantanone oxime
Hydroxylamine hydrochloride (1.2 equiv, 12 mmol) and
sodium acetate (1.2 equiv, 12 mmol) were added to a
solution of the aldehyde (1 equiv, 10 mmol) in EtOH
(50 mL). The reaction was stirred at 60 °C for 2 h. After
the EtOH was removed by evaporating the solution in
vacuo, the residue was suspended in DCM (50 mL) and
washed with 1 M HCl solution (3 9 30 mL), H2O
(3 9 30 mL) and brine solution. The organic phase was
dried (Na2SO4) and then concentrated at reduced pressure.
The oximes 1–3 were purified by recrystallization.
2-adamantanone oxime Yield 100 %. m.p. 163–164 °C.
ESIMS: m/z 166.4 [M ? H]?, calc. for C10H15NO
(165.23) (Terent’ev et al., 2006).
Preparation for compounds 2a–2d
The study was further extended to prediction by a QSAR
study to help know about the molecular properties (Santos
et al., 2009; Babu et al., 2012). In addition, docking study
was carried out to explore the molecular interactions and
molecular targets for the activities of phenylpropanoid
derivatives with the protein using molecular operating
environment (MOE).
A mixture of compound 1 (1 equiv, 10 mmol), malonic
acid (1.2 equiv, 12 mmol) and two drops of piperidine in
pyridine (25 mL) was refluxed for 4 h and evaporated to
remove pyridine. The residue was suspended in H2O
(30 mL) and extracted with EtOAc (2 9 50 mL) which
was further purified by recrystallization to afford 2a–d.
(E)-3-(3,4-dimethoxyphenyl)acrylic acid (2a) Yield
82 %. m.p. 181–183 °C. ESIMS: m/z 208.0600 [M]?,
calc. for C11H12O4 (208.21) (Brittelli et al., 1981).
(E)-3-(3,4,5-trimethoxyphenyl)acrylic acid (2b) Yield
90 %. m.p. 126–127 °C. ESIMS: m/z 238.6 [M]?, 237.6
[M–H]?, calc. for C12H14O5 (238.24) (Peterson et al.,
1988).
Materials and methods
General
Melting points were determined using electrothermal
melting point apparatus WRX-4 (Shanghai, China) and
were uncorrected. Mass spectroscopy was performed on a
Finnigan LCQ Deca XP MAX mass spectrometer (Thermo
Fisher, San Jose, CA, USA) which is equipped with an ESI
source and an ion trap analyzer in the positive ion mode/in
the negative ion. NMR spectra were recorded on Bruker
AM 400 MHz (1H/13C, 400 MHz/100 MHz) or Bruker
DRX 500 MHz (1H/13C, 500 MHz/125 MHz) spectrome-
ter (Bruker, Bremerhaven, Germany) and chemical shifts
were quoted in d as parts per million (ppm) downfield with
tetramethylsilane (TMS) as internal reference. Column
chromatography (CC): silica gel (200–300 mesh; Qingdao
Makall Group Co., Ltd., Qingdao, China). All reactions
were monitored using thin layer chromatography (TLC) on
(E)-3-(benzo[d][1,3]dioxol-5-yl)acrylic acid (2c) Yield
85 %. m.p. 242–244 °C. ESIMS: m/z 192.2 [M]?, 408.4
[2 M ? Na]?, calc. for C10H8O4 (192.17) (Salum et al.,
2010).
(E)-3-(7-methoxybenzo[d][1,3]dioxol-5-yl)acrylic acid
(2d) Yield 80 %. m.p. 228–229 °C. ESIMS: m/z 221 [M–
H]?, calc. for C11H10O5 (222.19) (Salway, 1909).
Preparation for cinnamoyl chlorides 3a–3d
Substituted cinnamoyl chlorides were obtained by refluxing
the appropriate acid 2a–d (10 mmol) with thionyl chloride
(10 mL) for 5 h. After evaporation at reduced pressure, the
123

Med Chem Res
Scheme 1 Synthetic route to the series of compounds. Reagents and conditions: a CH2(COOH)2, piperidine, C5H5N, reflux, 4 h, 80–90 %;
b SOCl2, CH2Cl2, reflux, 5 h, 95 %; c H2NOH–HCl, AcONa, EtOH, 60 °C, 1 h, 98 %; d Et3N, CH2Cl2, rt, 12 h, 50–70 %
crude liquid residue was used for subsequent reactions
without purification.
(125 MHz, CDCl3): d 174.96 (C-9), 165.23 (C-10), 153.41
(C-3, 5), 145.42 (C-7), 140.21 (C-4), 129.88 (C-1), 115.41
(C-8), 105.30 (C-2, 6), 60.95 (C-21), 56.15 (C-20, 22),
38.87, 37.80, 36.18 (C-12, 14, 16, 17, 19), 36.16, 31.24,
27.49 (C-11, 13, 15, 18). DEPT135: d 174.96, 165.23,
153.41, 140.21, 129.88 (C), 145.42, 115.41, 105.30, 36.16,
31.24, 27.49 (CH), 38.87, 37.80, 36.18 (CH2), 60.95, 56.15
(CH3). ESIMS: m/z 386.3 [M ? H]?, 408.2 [M ? Na]?,
calc. for C22H27NO5 (385.45).
Preparation for compounds 4a–4d
Oxime (1 equiv, 10 mmol) was resolved in dry DCM
(20 mL), then triethylamine (TEA) (1.2 equiv, 12 mmol)
was added drop wise to the solution at 0 °C, and then
reaction mixture was stirred for 30 min at 0 °C. Appro-
priate acid chloride (1 equiv, 10 mmol) was added to the
mixture, and then reaction mixture was stirred for
10–20 min at 0 °C, for 12 h at room temperature. DCM
was evaporated to dryness. The residue was washed with
cold ether (5 mL) and hot water and then purified by col-
umn chromatography on silica gel eluting with ethyl
acetate/petroleum ether 1:1–3:1.
(E)-2-adamantanone-2-carbaldehyde O-3-(benzo[d][1,3]-
dioxol-5-yl)acryloyl oxime (4c) White crystal, yield 66 %.
C. 1H NMR d 1.82-1.99 (14H, m,
Æ
m.p. 119.6-120.0
H-adamantane), 6.05 (2H, s, H-20), 6.44 (1H, d, J = 15.90,
H-8), 6.85 (1H, d, J = 7.95, H-5), 6.95 (1H, s, H-2), 6.98
(1H, d, J = 7.95, H-6), 7.77 (1H, d, J = 15.90, H-7). 13C
NMR (100 MHz, CDCl3): d 174.76 (C-9), 166.80 (C-10),
150.80 (C-3), 148.84 (C-4), 145.40 (C-7), 127.31 (C-1),
121.67 (C-6), 113.84 (C-8), 109.93 (C-5), 106.95 (C-2),
101.85 (C-20), 38.88, 37.48, 36.51 (C-12, 14, 16, 17, 19),
36.23, 28.78, 27.86 (C-11, 13, 15, 18). DEPT135: d 174.76,
166.80, 150.80, 148.84, 127.31 (C), 145.40, 121.67, 113.84,
109.93, 106.95, 36.23, 28.78, 27.86 (CH), 101.85, 38.88,
37.48, 36.51 (CH2). ESIMS: m/z 678.3 [2 M?], 679.0
[2 M ? H]?, 701.1 [2 M ? Na]?, calc. for C20H21NO4
(339).
(E)-2-adamantanone-2-carbaldehyde O-3-(3,4-dimethox-
yphenyl)acryloyl oxime (4a) White crystal, yield 67 %. m.p.
1
127.6–127.8 °C. H NMR (400 MHz, CDCl3): d 1.86–2.00
(14H, m, H-adamantane), 3,89 (6H, each 3H, s, H-20, 21),
6.36 (1H, d, J = 15.91, H-8), 6.85 (1H, d, J = 8.28, H-5),
7.05 (1H, d, J = 1.79, H-2), 7.11 (1H, dd, J = 1.79, 8.28,
H-6), 7.71 (1H, d, J = 15.91, H-7). 13C NMR (100 MHz,
CDCl3): d 174.80 (C-9), 165.52 (C-10), 151.19 (C-3),
149.14 (C-4), 145.38 (C-7), 127.35 (C-1), 122.81 (C-6),
113.76 (C-8), 110.97 (C-5), 109.53 (C-2), 55.96, 55.86 (C-
20, 21), 38.85, 37.77, 36.17 (C-12, 14, 16, 17, 19), 36.14,
31.21, 27.48 (C-11, 13, 15, 18). ESIMS: m/z 356.188
[M ? H]?, 378.344 [M ? Na]?, 735.488 [2 M ? H?
Na]?, calc. for C21H25NO4 (355.43).
(E)-2-adamantanone-2-carbaldehyde O-3-(7-methoxybenzo
[d][1,3] dioxol-5-yl) acryloyl oxime (4d) White crystal, yield
1
65 %. m.p. 159.1–159.5 °C. H NMR (500 MHz, CDCl3): d
1.89–2.06 (14H, m, H-adamantane), 3.93 (3H, s, H-21), 6.02
(2H, s, H-20), 6.35 (1H, d, J = 15.84, H-8), 6.73 (1H, d,
J = 1.36, H-6), 6.78 (1H, d, J = 1.36, H-2), 7.67 (1H, d,
J = 15.84, H-7). 13C NMR (125 MHz, CDCl3): d 174.94 (C-
9), 165.38 (C-10), 149.38 (C-5), 145.23 (C-7), 143.71 (C-3),
137.49 (C-4), 129.26 (C-1), 114.68 (C-8), 109.35 (C-6),
102.03 (C-20), 101.33 (C-2), 56.63 (C-21), 38.90, 37.81, 36.22
(C-12, 14, 16, 17, 19), 36.18, 31.26, 27.53 (C-11, 13, 15, 18).
(E)-2-adamantanone-2-carbaldehyde O-3-(3,4,5-trimet-
hoxyphenyl)acryloyl oxime (4b) White crystal, yield 69 %.
m.p. 158.6–158.9 °C. 1H NMR (500 MHz, CDCl3): d
1.87–2.07 (14H, m, H-adamantane), 3.89 (9H, each 3H, s,
H-20, 21, 22), 6.42 (1H, d, J = 15.92, H-8), 6.79 (2H, s,
H-2, 6), 7.71 (1H, d, J = 15.92, H-7). 13C NMR
123

Med Chem Res
Table 1 Anti-HBV activity and cytotoxicity of the phenylpropanoid derivatives in vitroa
Compound
CCb50 (lM)
HBsAgc
HBeAgd
HBV DNA replication
ICe50 (lM)
SIf
–
ICe50 (lM)
SIf
–
ICe50 (lM)
SIf
2-adamantanone
635.25
475.21
[1500
[1500
1289.31
1075.75
534.66
834.13
503.46
667.25
385.46
506.99
358.48
463.07
568.25
–
–
519.47
–
–
–
–
–
–
–
–
–
–
–
–
–
Oxime
1a
479.80
g
–
1.01
0.91
–
–
–
–
1b
–
–
–
–
–
–
1c
–
–
–
–
1d
–
–
–
–
–
2a
[600
[600
433.15
410.96
120.46
107.19
67.17
50.45
152.00
\0.89
\1.39
1.16
1.62
3.20
4.73
5.34
9.18
3.74
[600
[600
526.25
476.75
125.50
74.80
71.97
50.11
181.72
\0.89
\1.39
0.96
1.40
3.07
6.78
4.98
9.24
3.13
–
–
2b
2c
–
2d
–
4a
–
4b
141.46
97.36
51.80
6.86
3.58
4c
3.68
8.94
4d
3TCh
82.84
a
Values are means determined from at least two experiments
b
c
d
e
f
CC50 is 50 % cytotoxicity concentration in HepG2 2.2.15 cells
HBsAg: hepatitis B surface antigen
HBeAg, hepatitis B e antigen
IC50 is 50 % inhibitory concentration
SI (selectivity index) = CC50/IC50
No SI can be obtained
g
h
Lamivudine (3TC) as the positive control
DEPT135: d 174.94, 165.38, 149.38, 143.71, 137.49, 129.26
(C), 145.23, 114.68, 109.35, 101.33, 36.18, 31.26, 27.53 (CH),
102.03, 38.90, 37.81, 36.22 (CH2), 56.63 (CH3). ESIMS: m/z
370.1662 [M ? H]?, 392.1489 [M ? Na]?, calc. for
C21H23NO5 (369.41).
dependent experiment. Medium was taken at third day of
treatment (T3), the sixth day of treatment (T6) and the ninth
day of treatment (T9), and stored at -20 °C before analy-
sis. The IC50 and the selected index (SI) of each compound
were calculated, respectively. After drug treatment, the
cytotoxicity of the drugs was measured using the MTT
assay (Ferrari et al., 1990; Han et al., 2008).
Pharmacology
The levels of HBV surface antigen (HBsAg) and HBV e
antigen (HBeAg) were simultaneously detected using
ELISA kits (Rongsheng Biotechnology Co. Ltd, Shanghai,
China) according to the manufacturer’s instructions.
Inhibitory activity against HBV was determined using a
real-time fluorescence quantitative PCR (FQ-PCR). The
determination process was described in our previous work
(Liu et al., 2014a, b). Briefly, 2.0 lL of HBV DNA was
amplified in a 25 mL of mixture which contained 12.5 lL
2 9 SYBR Green Master (ROX) and 2 primers. These two
primers are specifically designed for HBV, including a
forward primer (50-AAC CAT TGA AGC AAT CAC TAG
AC-30) and a reverse primer (50-ATC TAT GGT GGC
TGC TCG AAC TA-30). The thermal program was com-
prised with an initial denaturation at 95 °C for 10 min
Anti-HBV potential determination
The anti-HBV activity of the compound was tested as
previously described (Liu et al., 2015). Briefly, the
HepG2.2.15 cells, provided by the Chinese Academy of
Medical Sciences (P.R. China), were selected as the tumor
cell model. 200 lL of the HepG2.2.15 suspension (Density:
1 9 105 cells/mL) was placed in each aperture of a 96-well
microtiter plates under 5 % CO2 at 37 °C. The HepG2.215
was incubated for 24 h before the addition of drug sample
at various concentrations. Lamivudine (3TC) was served as
the positive control. Cells were referred with drug-con-
taining fresh medium every 3 days for up to 9 days in time-
123

Med Chem Res
Fig. 1 Inhibitory effect of the phenylpropanoid derivatives on secretion of HBsAg (a) and HBeAg (b) in the HepG2.2.15 cell line. Data were
a
b
expressed as mean SE (n = 3). P \ 0.05, P \ 0.01 compared to control
followed by 40 amplification cycles with each of the two
final concentrations ranging from 0.0945 to 1.5 mM. After
the incubation, the culture media was removed, 180 lL
fresh media and 20 lL of MTT solution (5 mg/mL) were
added into each well. The suspension was then incubated for
another 4 h. After the incubation, the culture media was
removed again. After the addition of 100 lL of DMSO, the
OD values were measured using a microplate reader at
490 nm and the percent of cell death was calculated.
following steps: 95 °C for 15 s and 60 °C for 1 min.
Anti-cancer potential determination
Anti-cancer activities of the compounds were identified
using the MTT assay on lung cancer cell line A549, hepatic
cancer cell line SMMC-7721 and gastric cancer cell line
SMMG-803. Logarithmically growing cells were seeded in
96-well culture plates at a density of 1 9 105 cells/mL in
5 % CO2 at 37 °C. After 24-h incubation, various concen-
trations of drugs at different concentrations were added into
the cell sample. The mixture suspension was then incubated
for another 48 h. The synthesized compounds, with cis-
platinum as positive control, were evaluated in duplicate at
Molecular docking simulation
Ligand study was performed with HyperChem software.
The 3D structures of the synthesized compounds were
constructed and optimized, and then QSAR descriptors
were studied, which is a powerful lead optimization tool
123

Med Chem Res
Table 2 Anti-cancer activity of the phenylpropanoid derivatives in vitro
Compound
Concentration/mM
A549
SMMC-7721
Inhibition/%
SMMG-803
Inhibition/%
Inhibition/%
IC50/mM
–
IC50/mM
–
IC50/mM
–
4a
0.094
0.188
0.375
0.75
86.27
87.00
87.92
88.91
88.93
22.54
32.96
51.20
58.57
71.34
31.98
37.13
60.11
69.76
77.79
5.92
82.47
84.27
85.24
85.47
85.98
83.72
85.06
85.15
85.61
86.16
83.16
84.59
84.59
85.10
86.07
16.74
18.93
84.46
86.39
85.82
83.76
83.90
84.29
85.26
85.29
84.88
84.97
85.53
85.72
85.77
35.88
44.24
55.30
74.71
84.37
59.45
76.53
83.25
83.62
85.39
1.40
1.5
4b
0.094
0.188
0.375
0.75
0.443
0.275
0.513
–
–
0.225
1.5
4c
0.094
0.188
0.375
0.75
–
–
1.5
4d
0.094
0.188
0.375
0.75
0.276
0.407
9.97
12.13
84.27
86.33
85.72
67.90
83.11
84.97
85.16
85.49
29.83
72.52
88.08
65.71
70.04
70.53
72.56
74.83
1.5
Cis-platinum
0.094
0.188
0.375
0.75
–
–
1.5
Table 3 Molecular descriptors of derivatives from QSAR study
Ligand Molecular weight
(Da)
LogP Molar refractivity
Surface area
(A )
Volume
(A )
Hydration energy
(Kcal/mol)
Polarizability
(A )
3
˚
2
˚
3
˚
3
˚
(A )
3TC
2a
229.25
208.21
238.24
192.17
222.19
355.43
385.45
339.39
369.41
-0.55 55.14
525.55
306.89
348.16
303.06
348.41
555.34
609.17
566.06
612.68
606.87
324.12
346.69
302.58
330.85
1036.38
1098.41
961.22
1030.68
48.20
55.67
56.46
53.96
54.82
60.65
60.41
58.30
57.18
21.71
21.41
23.89
18.80
21.28
38.38
40.85
35.77
38.24
-0.6
59.95
2b
2c
-1.59 66.32
-0.14 52.79
-1.40 59.16
2.32 103.17
1.32 109.54
2.51 96.01
1.51 102.38
2d
4a
4b
4c
4d
that can quantitatively relate variations in biological
activity to changes in molecular properties.
The target compounds were built using the builder interface
of the MOE program and subjected to energy minimization.
The crystal structure of protein was retrieved from Protein
Data Bank (http://www.rcsb.org/pdb/home/home.do) (Liu
Molecular docking study was performed using MOE
2008.10 to illuminate the ligand–protein interactions in detail.
123

Med Chem Res
Table 4 Docking score and binding into 3OX8
˚
Distance (A)
Ligand
3TC
S-score (kcal/mol)
No. of H-bonds
2
Amino acid involved
Molecular structure
-10.7074
2.03
2.29
2.94
3.03
1.94
3.01
2.92
2.05
2.80
3.09
1.98
2.87
3.01
2.07
TYR 27
TYR 27
SER 57
ASP 53
TYR 27
SER 57
ASP 53
TYR 27
SER 57
ASP 53
TYR 27
SER 57
ASP 53
TYR 27
O of –OH
O of –COC–
O of 3 –OCH3
O of –C=O
O of –ON
4a
4b
4c
4d
-13.2226
-15.6072
-15.6424
-16.9302
3
3
3
3
O of 3 –OCH3
O of –C=O
O of –ON
O of 3 –OCH3
O of –C=O
O of –ON
O of 3 –OCH3
O of –C=O
O of –ON
Fig. 2 Binding mode of 4d into the binding site of HLA-A. The hydrogen bond formed colored in green (Color figure online)
et al., 2011). HBV-related protein Human leukocyte antigen
(HLA-A) (PDB ID: 3OX8) was used to study the interactions
between ligand and HBV protein in the active site. Lung
cancer-related protein 3WDZ, hepatic cancer-related protein
3QBY and gastric cancer-related protein 4OUM were used
for anti-cancer study. The edited crystal structure after
removing water molecules was imported into MOE. Chain A
was considered for docking process as the protein is a dimer
consisting of A and B chains. The structure was protonated,
and polar hydrogens were added. And energy minimization
123

Med Chem Res
was performed till the gradient convergence 0.05 kcal/mol
that reached to get the stabilized conformation. The active site
was correlated with ‘Site Finder’ module of MOE to define
the docking site for the ligands. Docking procedure was fol-
lowed the standard protocol implemented in MOE 2008.10,
and the geometry of resulting complexes was studied using
the MOE’s Pose Viewer.
substituted phenylacrylic acid 2a–d and thionyl chloride
in DCM (Karakurt et al., 2012).
The structures of the newly synthesized compounds 4a–
4d were characterized by 1H NMR, 13CNMR and MS.
Corresponding data are presented in the experimental
1
Sect. H NMR spectra of the derivatives showed two dou-
blets at 6.35–6.44 and 7.67–7.77 ppm with J = 15.84–
15.92 Hz corresponding to trans hydrogens of CH=CH,
respectively. The singlet at 3.89–3.93 ppm attributed to O–
CH3 protons, and at 6.02–6.05 corresponded to OCH2O
protons. The chemical shifts of aromatic hydrogens of the
phenyl ring appeared as multiplets in the region d 6.73–7.11.
13C NMR chemical shifts for title compounds were observed
in their expected regions. 13C NMR spectrum for the
derivatives showed signals at 55.86–60.95, 101.85–102.03,
165.23–166.80 and 174.76–174.96 corresponding to CH3,
CH2, C=N and C=O, respectively.
Results and discussion
Chemistry
General synthesis for the intermediate and target com-
pounds is depicted in Scheme 1. 2-Adamantanone was
reacted with hydroxylamine hydrochloride in EtOH in the
presence of sodium acetate to yield 2-adamantanone
oxime in a good yield (Quan et al., 2013). Intermediates
2a–d were prepared by Knoevenagel condensation of
malonic acid and the aldehyde group of four benzalde-
hydes with yields of 80–90 % (Zou et al., 2010). The
final oxime ester derivatives 4a–4d were obtained by
reaction of oxime with cinnamoyl chloride 3a–d in the
presence of TEA, which was obtained by reaction of
Biological evaluation
Anti-HBV activity
All the newly synthesized derivatives were tested for their
anti-HBV activity, namely inhibiting the secretion of
Fig. 3 Binding mode of 4c into the binding site of HLA-A. The hydrogen bond formed colored in green (Color figure online)
123

Med Chem Res
Fig. 4 Binding mode of 4b into the binding site of HLA-A. The hydrogen bond formed colored in green (Color figure online)
HBsAg, and HBeAg in HepG 2.2.15 cells. Positive control
was conducted using lamivudine (3TC, a clinically popular
anti-HBV agent) against the same HBV samples. The anti-
HBV activity of each compound was evaluated using their
IC50 concentration against the secretion of HBsAg and
HBeAg. And the cytotoxicity of each compound was
determined by their concentration at which they could
inhibit 50 % (CC50) of the HepG 2.2.15 cells. Ratios
between CC50 and IC50 of each compound was calculated
to obtain their selectivity index (SI). The results of their
anti-HBV activity and cytotoxicity are summarized in
Table 1. The start reactants substituted benzaldehyde 1a–d,
2-adamantanone, intermediates 2a–d and oximes 1–3
showed low suppressant properties on the HBV while most
of the derivatives showed high potency activity against of
the secretion of HBsAg and HBeAg as shown in Table 1.
The treatment of HBV-transfected HepG2.2.15 cells
with various concentrations of drugs for 9 d exhibited a
time- and dose-dependent inhibitory effect on the secretion
of HBsAg and HBeAg (Fig. 1). All the derivatives were
observed to show higher inhibitory activity against the
secretion of HBsAg and HBeAg than lamivudine. Com-
pound 4d showed the most potent anti-HBV activity,
demonstrating potent inhibitory effect on the secretion of
HBsAg (IC50 = 50.45 lM, SI = 9.18) and HBeAg
(IC50 = 50.11 lM, SI = 9.24).
Importantly, the inhibition of these compounds on
HBV DNA replication was also investigated using lami-
vudine as the reference drug. Compounds 4b, 4c, and 4d
exhibited anti-HBV activity with their IC50 values against
HBV DNA replication of 141.46, 97.36, 51.80 lM,
respectively. Compounds 4b, 4c, and 4d displayed
inhibiting not only HBsAg and HBeAg secretion but also
HBV DNA replication. 3TC showed more significant
activity against HBV DNA replication (IC50 = 6.86),
while it showed little inhibitory on HBsAg and HBeAg
secretion.
Anti-cancer activity
The in vitro anti-cancer activities of the synthesized com-
pounds against three human cancer cell lines (lung cancer
cell line A549, hepatic cancer cell line SMMC-7721 and
gastric cancer cell line SMMG-803) were evaluated using
an MTT assay with cis-platinum as positive control. The
results are shown in Table 2.
Compounds 4a displayed the most potent anti-cancer
activity against cell lines A549, SMMC-7721, SMMG-803
123

Med Chem Res
Fig. 5 Binding mode of 4a into the binding site of HLA-A. The hydrogen bond formed colored in green (Color figure online)
Table 5 Docking score and binding into 3WDZ, 3QBY and 4OUM
Ligand
S-score (kcal/mol)
3WDZ
3QBY
4OUM
4a
4b
4c
4d
-15.3414
-15.5431
-16.1699
-16.9997
-15.8125
-15.9114
-14.3159
-15.6494
-14.6212
-15.1279
-14.4864
-15.0533
with a mean growth inhibitions of 88.93, 85.98 and
85.77 %, respectively. The inhibition was higher than that
of the positive control. The half maximal (50 %) inhibitory
concentration (IC50) of these compounds was measured as
well. Compounds 4d exhibited anti-cancer activity with
their IC50 values against A549, SMMC-7721 and SMMG-
803 of 0.513, 0.276, 0.407 mM, respectively.
optimized. Molecular descriptors were determined by
QSAR study, including logP, molar refractivity, surface
area, volume, hydration energy and polarizability, and the
results showing that all molecules have drug like properties
(Table 3). All the compounds have the molecular weight in
the range from 190 to 390 Da. The log P values of these
compounds laid in the region between 1.59 and 2.51,
indicating their superior to act as drug. Their molar
refractivity is in the range of 50–110.
QSAR study
The 3D structures of all the compounds were generated
using Built Optimum option of Hyperchem (version 8.0).
Subsequently energy was minimized using MM ? force
field. The structures of all the compounds were fully
Molecular docking
Molecular docking studies of phenylpropanoid derivatives
were carried out using MOE 2008.10 as the docking
123

Med Chem Res
Fig. 6 Compounds Binding
into the active pockets of 3WDZ
(a), 3QBY (b) and 4OUM (c)
software in order to rationalize the biological activity
results and understand the various interactions between
ligand and protein in the active site in detail. The crystal
structure of HLA-A protein (PDB ID: 3OX8), which is
associated with severe liver inflammation in Chinese
patients with chronic HBV infection, was used for anti-
HBV study. Lung cancer-related protein 3WDZ, hepatic
cancer-related protein 3QBY and gastric cancer-related
protein 4OUM were used to identify the anti-cancer
activity of our compounds. The ‘Site Finder’ tool of the
program was used to search for its active site. We per-
formed three docking procedures for each ligand and the
best configuration of each of the ligand-receptor complexes
was selected based on energetic grounds. The affinity
scoring function dG was used to assess and rank the
receptor–ligand complexes.
The synthesized series derivatives had dock score with
3OX8 ranging from -13.2226 to -16.9302 (Table 4).
Compound 4d was showing the best least docking score of
-16.9302, and the next best least docking score was found
with 4c followed by 4b. Compound 4d was found to be
forming three hydrogen bonds of lengths 2.87, 3.01 and
˚
2.07 A each with 3-OCH3 in benzene ring of Ser57, car-
bonyl (C=O) of Asp53 and O–N in oxime ester group of
Tyr27, respectively (Fig. 2). Compound 4c also formed
˚
three hydrogen bonds of lengths 2.80, 3.09 and 1.98 A with
3-OCH3 in benzene ring of Ser57, carbonyl (C=O) of
Asp53 and O–N in oxime ester group of Tyr27 (Fig. 3).
123

Med Chem Res
Brittelli David R (1981) Phosphite-mediated in situ carboxyvinyla-
tion: a new general acrylic acid synthesis. J Org Chem
46:2514–2520
Chen DF, Zhang SX, Xie L et al (1997) Anti-AIDS agents—XXVI.
Structure-activity correlations of gomisin-G-related anti-HIV
lignans from Kadsura interior and of related synthetic analogues.
Bioorgan Med Chem 5:1715–1723
Chen H, Ma YB, Huang XY, Geng CA, Zhao Y, Wang LJ, Guo RH,
Liang WJ, Zhang XM, Chen JJ (2014) Synthesis, structure–
activity relationships and biological evaluation of dehydroan-
drographolide and andrographolide derivatives as novel anti-
hepatitis B virus agents. Bioorg Med Chem Lett 24:2353–2359
Du NN, Li X, Wang YP, Liu F, Liu YX, Li CX, Peng ZG, Gao LM,
Jiang JD, Song DQ (2011) Synthesis, structure-activity relation-
ship and biological evaluation of novel N-substituted matrinic
acid derivatives as host heat-stress cognate 70 (Hsc70) down-
regulators. Bioorg Med Chem Lett 21:4732–4735
Docking study of compound 4b and 4a with HLA-A
receptor is shown in Figs. 4 and 5, respectively. In the
docking study, we also found that the O of O–N in the
oxime ester group interacted with Tyr27 by hydrogen bond.
In order to predict the possible binding mode of the
compounds of the three kinds of cancers, compounds were
docked into the related protein 3WDZ, 3QBY and 4OUM
binding site of related cancer. The docking results are
shown in Table 5. As shown in Fig. 6, compounds were
completely binded into the active pockets of 3WDZ, 3QBY
and 4OUM. The compounds interacted with 3WDZ
through Van der waals interaction with residue ArgA380,
GlyA433, CysA434 and GlnB356. Then, they interacted
with 3QBY by interaction with residue MelA1, LysA6,
AspA9 and HisA3.
Ferrari M, Fornasiero MC, Isetta AM (1990) MTT colorimetric assay
for testing macrophage cytotoxic activity in vitro. J Immunol
Methods 131:165–172
Gao LM, Han YX, Wang YP, Li YH, Shan YQ, Li X, Peng ZG, Bi
CW, Zhang T, Du NN, Jiang JD, Song DQ (2011) Design and
synthesis of oxymatrine analogues overcoming drug resistance in
hepatitis B virus through targeting host heat stress cognate 70.
J Med Chem 54:869–876
Han YQ, Huang ZM, Yang XB, Liu HZ, Wu GX (2008) In vivo and
in vitro anti-hepatitis B virus activity of total phenolics from
Oenanthe javanica. J Ethnopharmacol 18:148–153
Conclusion
In summary, our design and synthesis have led to four
oxime ether phenylpropanoid derivatives by attaching of
2-adamantanone oxime to cinnamic acids. Then we
screened their anti-HBV activity in HepG 2.2.15 cells and
anti-cancer activities against three human cancer cell lines.
All the derivatives displayed potent anti-HBV activity with
the SIHBsAg values from 3.20 to 9.18 and SIHBeAg values
from 3.07 to 9.24, in which compound 4d showed the most
potent anti-HBV activity, demonstrating potent inhibitory
¨
Karakurt A, Mehmet AA, Burcu S, C¸ alıs U, Dalkara S (2012)
Synthesis of some novel 1-(2-naphthyl)-2-(imidazol-1-yl)etha-
none oxime ester derivatives and evaluation of their anticonvul-
sant activity. Eur J Med Chem 57:275–282
Kim SN, Lee JY, Kim HJ, Shin CG, Parka H, Lee YS (2000)
Synthesis and HIV-1 integrase inhibitory activities of caffeoyl-
glucosides. Bioorg Med Chem Lett 10:1879–1882
Lavanchy D (2004) Hepatitis B virus epidemiology, disease burden,
treatment, and current and emerging prevention and control
measures. J Viral Hepat 11:97–107
Liu JX, Kenneth Y, Chen ECR (2011) Structural insights into the
binding of hepatitis B virus core peptide to HLA-A2 alleles:
towards designing better vaccines. Eur J Immunol 41:2097–2106
Liu S, Wei WX, Shi KC, Cao X, Zhou M, Liu ZP (2014a) In vitro and
in vivo anti-hepatitis B virus activities of the lignan niranthin
effect not only on the secretion of HBsAg (IC50
=
50.45 lM, SI = 9.18) and HBeAg (IC50 = 50.11 lM,
SI = 9.24) but also on HBV DNA replication
(IC50 = 51.80 lM, SI = 8.94). Then the synthetic com-
pounds also exhibited potent anti-cancer activity. In addi-
tion, the docking study of all synthesized compounds inside
the related protein active site was carried out to explore the
molecular interactions and a molecular target for the
activity using a MOE-docking technique. This study
identified a new class of potent anti-HBV and anti-cancer
agents.
isolated from Phyllanthus niruri L.
155:1061–1067
J
Ethnopharmacol
Liu S, Wei WX, Li YB, Lin X, Shi KC, Cao X, Zhou M (2014b)
In vitro and in vivo anti-hepatitis B virus activities of the lignan
nirtetralin B isolated from Phyllanthus niruri L. J Ethnopharma-
col 157:62–68
Liu S, Wei WX, Li YB, Liu X, Cao XJ, Lei KC, Zhou M (2015)
Design, synthesis, biological evaluation and molecular docking
studies of phenylpropanoid derivatives as potent anti-hepatitis B
virus agents. Eur J Med Chem 95:473–482
Manat M, Patil B (2007) Evaluation of antiinflammatory activity of
methanol extract of Phyllanthus amarus in experimental animal
models. Indian J Pharm Sci 69:33
Acknowledgments This work was financially supported by the
national natural science foundation of China, natural science foun-
dation of Guangxi province, China, and science research and tech-
nology development foundation of Guangxi province, China.
Ovenden SP, Yu J, San WS et al (2004) Globoidnan A: a lignan from
Eucalyptus globoidea inhibits HIV integrase. Phytochemistry
65:3255–3259
References
Babu KR, Rao VK, Kumar YN, Polireddy K, Subbaiah KV, Bhaskar
M, Lokanatha V, Raju CN (2012) Identification of substituted [3,
2-a] pyrimidines as selective antiviral agents: molecular mod-
eling study. Antivir Res 95:118–127
Panda P, Appalashetti M, Natarajan M, Mary CP, Venkatraman SS,
Judeh ZMA (2012a) Synthesis and antiproliferative activity of
helonioside A,30,40,60-tri-O-feruloylsucrose, lapathoside C and
their analogs. Eur J Med Chem 58:418–430
Balzarini J, Orzeszko-Krzesinska B, Maurin JK, Orzeszko A (2009)
Synthesis and anti-HIV studies of 2- and 3-adamantyl-substitut-
edthiazolidin-4-ones. Eur J Med Chem 44:303–311
Panda P, Appalashetti M, Natarajan M, Chan-Park MB, Venkatraman
SS, Judeh ZMA (2012b) Synthesis and antitumor activity of
lapathoside D and its analogs. Eur J Med Chem 53:1–12
123

Med Chem Res
Parvathaneni M, Battu GR, Gray AI et al (2014) Investigation of
anticancer potential of hypophyllanthin and phyllanthin against
breast cancer by in vitro and in vivo methods. Asian Pac J Trop
Dis 4:S71–S76
Peterson JR, Russell ME, Surjasasmita IB (1988) Synthesis and
experimental ionization energies of certain (E)-3-arylpropenoic
acids and their methyl esters. J Chem Eng Data 33:534–537
Quan VV, Trenerry C, Rochfort S, Wadeson J, Leyton C, Hughes AB
(2013) Synthesis and anti-inflammatory activity of aromatic
glucosinolates. Bioorgan Med Chem 21:5945–5954
chloronitroso- and gem-chloronitrocycloalkanes. Synthesis 22:
3819–3824
Viegas-Junior C, Danuello A, Bolzani VS, Barreir EJ, Fraga CAM
(2007) Molecular hybridization: a useful tool in the design of
new drug prototypes. Curr Med Chem 14:1829–1852
Wang LJ, Geng CA, Ma YB, Huang XY, Luo J, Chen H, Guo RH,
Zhang XM, Chen JJ (2012) Synthesis, structure–activity rela-
tionships and biological evaluation of caudatin derivatives as
novel anti-hepatitis B virus agents. Bioorg Med Chem Lett
20:2877–2888
Saleem M, Kim HJ, Ali MS et al (2005) An update on bioactive plant
lignans. Nat Prod Rep 22:696–716
Salum ML, Robles CJ, Erra-Balsells R (2010) Photoisomerization of
ionic liquid ammonium cinnamates: one-pot synthesis-isolation
of Z-cinnamic acids. Org Lett 12:4808–4811
Wei WX, Li XR, Wang KW, Zheng ZW, Zhou M (2012) Lignans
with anti-hepatitis B virus activities from phyllanthus niruri L.
Phytother Res 26:964–968
Wu ZR, Zheng LF, Li Y, Su F, Yue XX, Tang W, Ma XY, Nie JY, Li
HY (2012) Synthesis and structure–activity relationships and
effects of phenylpropanoid amides of octopamine and dopamine
on tyrosinase inhibition and antioxidation. Food Chem 134:
1128–1131
Yamashita K, Nohara Y, Katayama K et al (1992) Sesame seed
lignans and gamma-tocopherol act synergistically to produce
vitamin E activity in rats. J Nutr 122:2440–2446
Zou HB, Wu H, Zhang XN, Zhao Y, Joachim S, Lou YJ, Yu YP
(2010) Synthesis, biological evaluation, and structure-activity
relationship study of novel cytotoxic aza-caffeic acid derivatives.
Bioorgan Med Chem 18:6351–6359
Salway AH (1909) Synthesis of substances allied to cotarnine.
J Chem Soc Trans 95:1204–1220
Santos FD, Abreu P, Castro HC, Paixao ICPP, Cirne-Santos CC,
Giongo V, Barbosa JE, Simonetti BR, Garrido V, Bou-Habib
DC, Silva DD, Batalha PN, Temerozo JR, Souza TM, Nogueira
CM, Cunha AC, Rodrigues CR, Ferreira VF, Souza MCBV
(2009) Synthesis, antiviral activity and molecular modeling of
oxoquinoline derivatives. Bioorgan Med Chem 17:5476–5481
Terent’ev AO, Krylov IB, Ogibin YN, Nikishin GI (2006) Chlorina-
tion of oximes with aqueous H2O2/HCl system: facile synthe-
sis of gem-chloronitroso- and gem-chloronitroalkanes, gem-
123