Excavating precursors from the traditional Chinese herb Polygala tenuifolia and Gastrodia elata: Synthesis, anticonvulsant activity evaluation of 3,4,5-trimethoxycinnamic acid (TMCA)ester derivatives

Article abstract of DOI:10.1016/j.bioorg.2019.03.006

Epilepsy is a group of neurological disorders characterized by recurrent seizures that disturbs about 60 million people worldwide. In this article, a novel series of 3,4,5-trimethoxycinnamic acid (TMCA)ester derivatives 1–35 were designed inspired from the traditional Chinese herb pair drugs Polygala tenuifolia and Gastrodia elata and synthesized followed by in vivo and in silico evaluation of their anticonvulsant potential. All the synthesized derivatives were biologically evaluated for their anticonvulsant potential using two acute model of seizures induced in mice, the maximal electroshock (MES)and sc-pentylenetetrazole (PTZ)models. Simultaneously, the motor impairment as a surrogate of acute neurotoxicity and in vitro screening of cytotoxicity against HepG-2 cells line were assessed through the rotarod performance test and CCK-8 assay, respectively. In addition, the physicochemical and pharmacokinetic parameters of the active compounds were determined. Our results showed that compounds 5, 7, 8, 13, 20, 25, 28, 30 and 32 exhibited preferable anticonvulsant activity in primary evaluation, with compounds 28 and 32 being the most promising anticonvulsant agents in according to results of subsequent pharmacology and toxicity evaluation. Additionally, the molecular modeling experiments predicted good binding interactions of part of the obtained active molecules with the gamma-aminobutyric acid (GABA)transferas. Therefore, it could be concluded that the synthesized derivatives 28 and 32 would represent useful lead compounds for further investigation in the development of anticonvulsant agents.

Full text of DOI:10.1016/j.bioorg.2019.03.006

Accepted Manuscript
Excavating precursors from the traditional Chinese herb Polygala tenuifolia and
Gastrodia elata: Synthesis, anticonvulsant activity evaluation of 3,4,5-trime-
thoxycinnamic acid (TMCA) ester derivatives
Zefeng Zhao, Yajun Bai, Jing Xie, Xufei Chen, Xirui He, Ying Sun, Yujun Bai,
Yangyang Zhang, Shaoping Wu, Xiaohui Zheng
PII:
S0045-2068(18)31365-8
DOI:
https://doi.org/10.1016/j.bioorg.2019.03.006
YBIOO 2832
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 November 2018
27 February 2019
2 March 2019
Please cite this article as: Z. Zhao, Y. Bai, J. Xie, X. Chen, X. He, Y. Sun, Y. Bai, Y. Zhang, S. Wu, X. Zheng,
Excavating precursors from the traditional Chinese herb Polygala tenuifolia and Gastrodia elata: Synthesis,
anticonvulsant activity evaluation of 3,4,5-trimethoxycinnamic acid (TMCA) ester derivatives, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.03.006
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Excavating precursors from the traditional Chinese herb Polygala tenuifolia and
Gastrodia
elata:
Synthesis,
anticonvulsant
activity
evaluation
of
3,4,5-trimethoxycinnamic acid (TMCA) ester derivatives
1
1
3,
1
Zefeng Zhao 1, a, Yajun Bai 1,2, a, Jing Xie , Xufei Chen , Xirui He *, Ying Sun ,
Yujun Bai 1, Yangyang Zhang 1, Shaoping Wu 1, * and Xiaohui Zheng 1*
1 School of Pharmacy, Biomedicine Key Laboratory of Shaanxi Province, Northwest
University, Xi’an 710069, China;
2 College of Chemistry and Materials Science, Northwest University, Xi’an 710069,
China
3 Honghui Hospital, Xi’an Jiaotong University, Xi’an 710054, China
*
Correspondence:
Hxrhist@163.com,
wushaoping@nwu.edu.cn
and
Zhengxh@nwu.edu.cn;
a These authors contributed equally to this paper and share co-first authorship.
Tel.: +86-29-8830-2686 (X.Z.)

Abstract: Epilepsy is a group of neurological disorders characterized by recurrent
seizures that disturbs about 60 million people worldwide. In this article, a novel series
of 3,4,5-trimethoxycinnamic acid (TMCA) ester derivatives 1-35 were designed
inspired from the traditional Chinese herb pair drugs Polygala tenuifolia and
Gastrodia elata and synthesized followed by in vivo and in silico evaluation of their
anticonvulsant potential. All the synthesized derivatives were biologically evaluated
for their anticonvulsant potential using two acute model of seizures induced in mice,
the maximal electroshock (MES) and sc-pentylenetetrazole (PTZ) models.
Simultaneously, the motor impairment as a surrogate of acute neurotoxicity and in
vitro screening of cytotoxicity against HepG-2 cells line were assessed through the
rotarod performance test and CCK-8 assay, respectively. In addition, the
physicochemical and pharmacokinetic parameters of the active compounds were
determined. Our results showed that compounds 5, 7, 8, 13, 20, 25, 28, 30 and 32
exhibited preferable anticonvulsant activity in primary evaluation, with compounds 28
and 32 being the most promising anticonvulsant agents in according to results of
subsequent pharmacology and toxicity evaluation. Additionally, the molecular
modeling experiments predicted good binding interactions of part of the obtained
active molecules with the gamma-aminobutyric acid (GABA) transferas. Therefore, it
could be concluded that the synthesized derivatives 28 and 32 would represent useful
lead compounds for further investigation in the development of anticonvulsant agents.
Keywords: synthesis; Polygala tenuifolia; Gastrodia elata; 3,4,5-trimethoxycinnamic

acid; ester derivatives; anticonvulsant; docking.

1.Introduction
Epilepsy is a grievous and chronic brain neurological disorder which disturbs
0.5-1.0% of population around the whole world during their lifetime [1, 2]. Up to now,
utilizing anti-epileptic drugs (AEDs) is still the primary therapy for the treatment of
epilepsy. In spite of the fact that AEDs are general applicated, side effects of using
AEDs on symptoms including headache and memory deterioration should not be
neglected, in addition, a significant number of epileptic patients are refractory to
current pharmacological treatment. Faced with the limitation above, investigators
make efforts to seek natural medicine to alleviate the side effects of epilepsy.
Traditional Chinese Medicine (TCM) is an important source of kind of natural
product which meets the requirement for safe and effective and alleviates the side
effects of the existing epilepsy treatment and to make it more effective [3]. TCM has
been used for the treatment of epilepsy for more than two thousand years in China.
Among the TCM treating for epilepsy, the pair herb drugs Polygala tenuifolia
(Yuanzhi in Chinese) and Gastrodia elata (Tianma in Chinese) is a classical
combination. The combining of Polygala tenuifolia - Gastrodia elata are listed in
classical TCM prescription and used for curing epilepsy such as “Qian Jin Bao Ming
Dan (recorded in Yifang Dacheng, Yuan dynasty)” and “Ding Xian Wan (recorded in
Yixue Xinwu, Qing dynasty)” [4]. Recent years, studies about the sedative activity of
Polygala tenuifolia [5] and the anticonvulsant effect of Gastrodia elata have been
reported [6]. 3,4,5-Trimethoxycinnamic acid (TMCA) isolated from Polygala
tenuifolia, gastrodin and vanillyl alcohol isolated form Gastrodia elata are considered

as effective ingredients that play a role in anti-seizure according to the previous
literatures published. TMCA exhibited anticonvulsant effect in MES and PTZ model
in vivo, with a potential mechanism of interacting with GABAA/benzodiazepine (BZD)
receptor complex [7]. Gastrodin exerted anticonvulsant activity in seizure-prone
gerbils via decreasing GABA transaminase, succinic semialdehyde dehydrogenase
(SSADH) and succinic semialdehyde reductase (SSAR) immunoreactivity in
hippocampus, suggesting that the antiepileptic effect of gastrodin may be related to
increased GABA turnover [8]. Vanillol prevented injure from ferric chloride-induced
seizure, meanwhile, the activity of scavenging free radical in brain was revealed [9].
Adopting the strategy of Combination of Traditional Chinese Medicine Chemistry
(CMTCC), we introduce the mentioned natural product as the precursor for further
investigation. Previously, we have developed promising compounds including
α-asaronol from Acorus gramineu [10], tanshinol borneol ester inspired by the pair
herb drugs Salvia miltiorrhiza and borneol [11, 12].
In previous research [13], we have reported the anticonvulsant potential of
TMCA amide derivatives, which provide to us the evidence of the favourable
anticonvulsant potential for TMCA derivatives. In this study, we design a series of
TMCA fatty esters and benzyl esters (Scheme 1, 1-35). Among the alcohols used for
the synthesis of TMCA ester, hydroxy benzyl alcohol (19) and vanillol (20) are the
constituents isolated from Gastrodia elata, which is correspond to the idea that
creating novel anticonvulsant agents via the combination of effective ingredients
obtaining from traditional Chinese medicines. Acegastrodine (28), which is a kind of

marked sedative drug in China approved by CFDA (H20013040), is also introduced
into the research to investigate the anticonvulsant effect. Besides, geraniol (8),
menthol (12) and borneol (13) are derivatived from natural products as well.
Furthermore, we designed compounds (29-32) to explore influence of the
etherification to the anticonvulsant effect of vanillin and 4-hydroxy benzaldehy. In
China, vanillin is also used to treat epileptic petit mal approved by CFDA
(H32025802). Propyl methyl group and 3-methyl-butenyl group are the usual
pharmacophore, thus we introduce them into our derivatives as well [14, 15]. To
investigate the influence of substituent group of cinnamic acid to the potential
anticonvulsant effect, we designed and synthesized compounds (33-35).

Scheme 1. Synthesis of compounds 1-35
2. Results and discussion
2.1. Chemistry
All titled compounds 1-35 were successfully synthesized using the synthetic
protocols presented in Scheme 1. Identification of compounds 1-35 is listed as follow:
(E)-methyl 3-(3,4,5-trimethoxyphenyl)acrylate (1) White solid. mp: 96-97 °C. Yield:
1
85 %; Rf = 0.5 (Hexane/EtOAc = 4:1, v/v); H NMR (600 MHz, CDCl3) δ ppm:
7.63-7.59 (m, 1H), 6.74 (d, J = 2.8 Hz, 2H), 6.34 (dd, J = 15.8, 2.3 Hz, 1H), 3.90-3.86
(m, 9H), 3.80 (d, J = 2.4 Hz, 3H); 13C NMR (151 MHz, CDCl3) δ ppm: 167.56,
153.64, 145.03, 140.38, 130.07, 117.24, 105.47, 61.16, 56.36, 51.89; HRMS(ESI) m/z
calcd for C13H16O5 ([M+Na]+): 275.0890; found: 275.0893.
(E)-ethyl 3-(3,4,5-trimethoxyphenyl)acrylate (2) White solid. mp: 59.7-60.1 °C. Yield:
84 %; Rf = 0.5 (Hexane/EtOAc = 4:1, v/v); 1H NMR (600 MHz, CDCl3) δ ppm: 7.60
(d, J = 15.8 Hz, 1H), 6.75 (s, 1H), 6.34 (d, J = 15.7 Hz, 1H), 4.26 (d, J = 7.2 Hz, 2H),
3.88 (d, J = 4.1 Hz, 9H), 1.34 (t, J = 7.0 Hz, 3H);13C NMR (151 MHz, CDCl3) δ ppm:
167.20, 153.64, 144.77, 140.42, 130.16, 117.73, 105.54, 105.42, 61.24, 60.77, 56.49,
56.39, 14.69; HRMS(ESI) m/z calcd for C14H18O5 ([M+H]+): 267.1227;
found:267.1206; ([M+Na] +): 289.1046; found: 289.1023.

(E)-ethyl 3-(3,4,5-trimethoxyphenyl)acrylate (3) White solid. mp: 87-89 °C. Yield:
71 %; Rf = 0.5 (Hexane/EtOAc = 4:1, v/v); 1H NMR (600 MHz, CDCl3) δ ppm: 7.65
(d, J = 15.9 Hz, 1H), 6.76 (s, 2H), 6.38 (d, J = 15.9 Hz, 1H), 4.47 (t, J = 5.7 Hz, 2H),
3.89 (d, J = 4.1 Hz, 9H), 3.76 (t, J = 5.7 Hz, 2H);13C NMR (151 MHz, CDCl3) δ ppm:
167.20, 153.64, 144.76, 140.42, 130.31, 117.86, 105.49, 105.42, 61.32, 60.77, 56.39,
56.36, 29.94; HRMS(ESI) m/z calcd for C14H17ClO5 ([M+H]+): 301.0837; found:
301.0835; ([M+Na] +): 323.0657; found: 323.0664.
(E)-propyl 3-(3,4,5-trimethoxyphenyl)acrylate (4) Yellow solid. mp: 72-73 °C. Yield:
78 %; Rf = 0.6 (Hexane/EtOAc = 4:1, v/v); 1H NMR (600 MHz, CDCl3) δ ppm: 7.60
(d, J = 15.9 Hz, 1H), 6.75 (s, 2H), 6.35 (dd, J = 15.9, 2.1 Hz, 1H), 4.17 (t, J = 6.7 Hz,
2H), 3.89 (s, 5H), 3.88 (s, 4H), 1.73 (h, J = 7.2 Hz, 2H), 1.00 (t, J = 7.4 Hz, 3H);13C
NMR (151 MHz, CDCl3) δ ppm: 167.25, 153.64, 144.74, 140.29, 130.19, 117.74,
105.42, 66.37, 61.18, 56.37, 22.33, 10.68; HRMS(ESI) m/z calcd for C15H20O5
([M+H]+): 281.1384; found: 281.1382; ([M+Na]+): 303.1203; found: 303.1203.
isopropyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (5) White solid. mp: 68-69 °C.
Yield: 70 %; Rf = 0.6 (Hexane/EtOAc = 4:1, v/v); 1H NMR (600 MHz, CDCl3) δ ppm:
7.58 (d, J = 15.9 Hz, 1H), 6.75 (s, 2H), 6.33 (d, J = 15.9 Hz, 1H), 5.14 (p, J = 6.3 Hz,
1H), 3.88 (d, J = 3.2 Hz, 9H), 1.31 (d, J = 6.3 Hz, 6H).;13C NMR (151 MHz, CDCl3)
δ ppm: 166.67, 153.64, 144.51, 140.23, 130.26, 118.28, 105.39, 68.03, 61.19, 56.37,
22.20; HRMS(ESI) m/z calcd for C15H20O5 ([M+H]+): 281.1384; found: 281.1280.

(2E)-(E)-but-2-enyl 3-(3,4,5-trimethoxyphenyl)acrylate (6) White solid. mp: 82-84 °C.
Yield: 82 %; Rf = 0.6 (Hexane/EtOAc = 4:1, v/v); 1H NMR (600 MHz, CDCl3) δ ppm:
7.60 (d, J = 15.9 Hz, 1H), 6.74 (s, 2H), 6.35 (d, J = 16.0 Hz, 1H), 5.93-5.63 (m, 1H),
4.67-4.62 (m, 2H), 3.87 (d, J = 1.2 Hz, 10H), 1.75 (dt, J = 6.6, 1.3 Hz, 3H); 13C NMR
(151 MHz, CDCl3) δ ppm: 166.88, 153.63, 144.94, 140.32, 131.76, 130.13, 125.35,
117.56, 105.42, 65.47, 61.16, 56.34, 18.01; HRMS(ESI) m/z calcd for C16H20O5
([M+H]+): 293.1384; found: 293.1386.
(E)-cyclopropylmethyl 3-(3,4,5-trimethoxyphenyl)acrylate (7) White solid. mp:
1
87-89 °C. Yield: 86 %; Rf = 0.7 (Hexane/EtOAc = 1:1,v/v); H NMR (600 MHz,
CDCl3) δ ppm: 7.61 (d, J = 15.4 Hz, 1H), 6.75 (d, J = 9.2 Hz, 2H), 6.38 (d, J = 15.8
Hz, 1H), 3.92-3.82 (m, 10H), 1.25 (s, 2H), 0.61 (d, J = 7.7 Hz, 2H), 0.33 (d, J = 4.9
Hz, 2H);13C NMR (151 MHz, CDCl3) δ ppm: 167.26, 153.64, 144.86, 130.19, 128.52,
117.71, 105.45, 69.58, 61.17, 56.36, 10.10, 3.50; HRMS(ESI) m/z calcd for C16H20O5
([M+H]+): 293.1384; found: 293.1381.
(E)-3,7-dimethylocta-2,6-dien-1-yl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (8) Yellow
liquid. Yield: 60 %; Rf = 0.7 (Hexane/EtOAc = 4:1, v/v); 1H NMR (600 MHz, CDCl3)
δ ppm: 7.58 (d, J = 15.8 Hz, 1H), 6.73 (s, 2H), 6.34 (d, J = 15.9 Hz, 1H), 5.40 (s, 1H),
5.08-5.06 (m, 1H), 4.70 (d, J = 7.1 Hz, 2H), 3.85 (s, 9H), 1.72 (s, 3H), 1.66 (s, 5H),
1.58 (s, 4H); 13C NMR (151 MHz, CDCl3) δ ppm: 167.08, 153.53, 144.73, 142.58,

131.93, 130.10, 124.04, 123.87, 118.42, 117.60, 105.34, 59.46, 56.23, 39.67, 26.44,
25.79, 17.81, 16.64; HRMS(ESI) m/z calcd for C22H30O5 ([M+Na]+): 397.1998; found:
397.1998.
(tetrahydrofuran-2-yl)methyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (9) Pale-yellow
solid. mp: 77-79 °C. Yield: 68 %; Rf = 0.6 (Hexane/EtOAc = 4:1, v/v); 1H NMR (600
MHz, CDCl3) δ ppm: 7.63 (d, J = 15.9 Hz, 1H), 6.75 (s, 2H), 6.41 (d, J = 15.9 Hz,
1H), 4.32 (dd, J = 11.4, 3.3 Hz, 1H), 4.23-4.06 (m, 2H), 3.88 (d, J = 1.5 Hz, 11H),
2.09-1.61 (m, 4H); 13C NMR (151 MHz, CDCl3) δ ppm: 167.08, 153.64, 145.24,
130.11, 117.36, 105.47, 68.72, 66.83, 61.19, 56.37, 29.92, 28.24, 25.90; HRMS(ESI)
m/z calcd for C17H23O6 ([M+H]+): 323.1495; found: 323.1468.
pyridin-2-ylmethyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (10) Pale-yellow solid. mp:
1
70-71 °C. Yield: 65 %; Rf = 0.6 (Hexane/EtOAc = 1:1, v/v); H NMR (600 MHz,
CDCl3) δ ppm: 8.69 (d, J = 2.2 Hz, 1H), 8.59 (d, J = 4.8 Hz, 1H), 7.74 (dt, J = 7.7, 2.0
Hz, 1H), 7.64 (d, J = 15.9 Hz, 1H), 7.31 (dd, J = 7.9, 4.9 Hz, 1H), 6.74 (s, 2H), 6.37
(d, J = 15.9 Hz, 1H), 5.26 (s, 2H), 3.87 (s, 9H); 13C NMR (151 MHz, cdcl3) δ ppm:
166.71, 153.65, 149.96, 149.89, 145.82, 140.54, 136.21, 131.89, 129.84, 123.65,
116.77, 105.53, 63.98, 61.17, 56.36; HRMS(ESI) m/z calcd for C18H19NO5 ([M+H]+):
330.1341; found: 330.1343.

pyridin-3-ylmethyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (11) White solid. mp:
70-72 °C. Yield: 86 %; Rf = 0.6 (Hexane/EtOAc = 1:1, v/v); 1H NMR (600 MHz,
CDCl3) δ ppm: 8.69 (d, J = 2.2 Hz, 1H), 8.59 (d, J = 4.8 Hz, 1H), 7.74 (dt, J = 7.7, 2.0
Hz, 1H), 7.64 (d, J = 15.9 Hz, 1H), 7.31 (dd, J = 7.9, 4.9 Hz, 1H), 6.74 (s, 2H), 6.37
(d, J = 15.9 Hz, 1H), 5.26 (s, 2H), 3.87 (s, 9H); 13C NMR (151 MHz, cdcl3) δ 166.71,
153.65, 149.96, 149.89, 145.82, 140.54, 136.21, 131.89, 129.84, 123.65, 116.77,
105.53, 63.98, 61.17, 56.36; HRMS(ESI) m/z calcd for C18H19NO5 ([M+H]+):
330.1341; found: 330.1350.
2-isopropyl-5-methylcyclohexyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (12) White
solid. mp: 70-72 °C. Yield: 64 %; Rf = 0.7 (Hexane/EtOAc = 4:1, v/v); 1H NMR (600
MHz, CDCl3) δ ppm: 7.58 (d, J = 15.9 Hz, 1H), 6.75 (s, 2H), 6.34 (d, J = 15.9 Hz,
1H), 4.83 (td, J = 10.9, 4.4 Hz, 1H), 3.89 (s, 6H), 3.88 (s, 3H), 2.10-1.00 (m, 9H),
0.94-0.77 (m, 9H); 13C NMR (151 MHz, CDCl3) δ ppm: 166.73, 153.62, 144.55,
140.22, 130.26, 118.20, 105.40, 74.48, 61.18, 56.37, 47.50, 41.30, 34.54, 31.66, 26.56,
23.74, 22.27, 21.02, 16.63; HRMS(ESI) m/z calcd for C22H32O5 ([M+H]+): 377.2328;
found: 377.2340; ([M+Na]+): 399.2147; found: 399.2164.
1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (13)
1
White solid. mp: 69-71 °C. Yield: 55 %; Rf = 0.7 (Hexane/EtOAc = 4:1, v/v); H
NMR (600 MHz, CDCl3) δ ppm: 7.58 (d, J = 15.9 Hz, 1H), 6.76 (s, 2H), 6.37 (d, J =
15.8 Hz, 1H), 5.02 (dt, J = 9.9, 2.8 Hz, 1H), 4.00 (dt, J = 10.0, 2.7 Hz, 1H), 3.90 (s,

6H), 3.88 (s, 3H), 2.46-2.39 (m, 1H), 2.27 (ddt, J = 15.0, 8.1, 2.4 Hz, 1H), 2.06 (ddd,
J = 13.3, 9.4, 4.4 Hz, 1H), 1.89 (dd, J = 15.2, 10.1, 4.5 Hz, 1H), 1.71 (t, J = 4.7 Hz,
2H), 1.62 (s, 1H), 0.87-0.83 (m, 9H); 13C NMR (151 MHz, CDCl3) δ ppm: 167.50,
153.63, 144.39, 140.22, 130.26, 118.25, 105.42, 80.22, 61.18, 56.39, 49.18, 48.10,
45.31, 39.25, 28.49, 26.12, 18.89, 13.53; HRMS(ESI) m/z calcd for C22H32O5
([M+H]+): 375.2171; found: 375.2181; ([M+Na]+): 397.1991; found: 397.1997.
4-methoxybenzyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (14) White solid. mp:
1
65-67 °C. Yield: 80 %; Rf = 0.5 (Hexane/EtOAc = 4:1, v/v); H NMR (600 MHz,
CDCl3) δ ppm: 7.62 (d, J = 15.9 Hz, 1H), 7.38-7.33 (m, 2H), 6.94-6.70 (m, 4H), 6.37
(d, J = 15.8 Hz, 1H), 5.18 (s, 2H), 3.87 (d, J = 2.4 Hz, 9H), 3.81 (d, J = 6.7 Hz, 3H);
13C NMR (151 MHz, cdcl3) δ ppm: 166.99, 159.90, 153.61, 145.14, 140.33, 130.37,
130.08, 128.33, 117.48, 114.20, 105.42, 66.43, 61.15, 56.33, 55.50; HRMS(ESI) m/z
calcd for C20H22O6 ([M+H]+): 359.1495; found: 359.1495; ([M+Na]+): 381.1309;
found: 381.1324.
benzo[d][1,3]dioxol-5-ylmethyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (15) White
solid. mp: 88-90 °C. Yield: 76 %; Rf = 0.5 (Hexane/EtOAc = 4:1, v/v); 1H NMR (600
MHz, CDCl3) δ ppm: 7.62 (d, J = 15.9 Hz, 1H), 6.94 – 6.71 (m, 5H), 6.37 (d, J = 15.9
Hz, 1H), 5.96 (d, J = 11.4 Hz, 2H), 5.14 (s, 2H), 3.88 (s, 9H).; 13C NMR (151 MHz,
CDCl3) δ ppm: 166.91, 153.64, 148.06, 147.90, 145.30, 140.39, 130.04, 129.98,

122.55, 117.35, 109.31, 108.50, 105.46, 101.39, 77.44, 66.58, 61.18, 56.36.;
HRMS(ESI+) m/z calcd for C20H20O7 ([M+H]+): 373.1287; found: 373.1295.
4-chlorobenzyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (16) White solid. mp:
1
88-91 °C. Yield: 82 %; Rf = 0.5 (Hexane/EtOAc = 4:1, v/v); H NMR (600 MHz,
CDCl3) δ ppm: 7.63 (d, J = 15.9 Hz, 1H), 7.35 (d, J = 0.8 Hz, 3H), 6.75 (s, 2H), 6.38
(d, J = 15.9 Hz, 1H), 5.21 (s, 2H), 3.88 (s, 9H); 13C NMR (151 MHz, CDCl3) δ ppm:
166.81, 153.67, 145.60, 140.52, 134.78, 134.41, 129.88, 129.01, 128.46, 117.04,
105.53, 65.74, 61.19, 56.38; HRMS(ESI+) m/z calcd for C19H19ClO5 ([M+H]+):
363.0999; found: 363.0993.
4-fluorobenzyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (17) White solid. mp: 70-72 °C.
Yield: 80 %; Rf = 0.5 (Hexane/EtOAc = 4:1, v/v); 1H NMR (600 MHz, CDCl3) δ ppm:
7.59 (d, J = 15.9 Hz, 1H), 7.34-7.22 (m, 5H), 6.74 (s, 2H), 6.33 (d, J = 15.9 Hz, 1H),
4.44 (t, J = 7.1 Hz, 2H), 3.89 (s, 6H), 3.88 (s, 3H), 3.03 (t, J = 7.1 Hz, 2H);13C NMR
(151 MHz, CDCl3) δ ppm: 167.07, 153.66, 145.06, 138.08, 129.12, 128.75, 126.80,
117.49, 105.49, 65.20, 61.20, 56.40; HRMS(ESI) m/z calcd for C19H19FO5 ([M+H]+):
347.1295; found: 347.1289; ([M+Na]+): 369.1114; found: 369.1119.
4-bromobenzyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (18) White solid. mp:
1
86-88 °C. Yield: 82 %; Rf = 0.5 (Hexane/EtOAc = 4:1, v/v); H NMR (600 MHz,
CDCl3) δ ppm: 7.63 (d, J = 15.9 Hz, 1H), 7.54 – 7.48 (m, 2H), 7.29 (d, J = 8.1 Hz,

2H), 6.75 (s, 2H), 6.38 (d, J = 15.9 Hz, 1H), 5.19 (s, 2H), 3.88 (d, J = 0.9 Hz, 9H);13C
NMR (151 MHz, CDCl3) δ ppm: 166.78, 153.66, 145.61, 140.52, 135.29, 131.96,
130.16, 129.93, 122.52, 117.01, 105.54, 65.75, 61.18, 56.38; HRMS(ESI) m/z calcd
for C19H19BrO5 ([M+H]+): 407.0489; found: 407.0493.
4-hydroxybenzyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (19) White solid. mp:
1
90-92 °C. Yield: 60 %; Rf = 0.4 (Hexane/EtOAc = 1:1, v/v); H NMR (600 MHz,
CDCl3) δ ppm: 7.78 (d, J = 15.9 Hz, 1H), 7.40 (d, J = 8.1 Hz, 2H), 7.15 (d, J = 8.1 Hz,
2H), 6.81 (s, 2H), 6.62-6.44 (m, 1H), 4.78-4.66 (m, 2H), 3.90 (s, 9H); 13C NMR (151
MHz, CDCl3) δ ppm: 165.64, 153.73, 150.43, 146.86, 138.68, 129.82, 128.33, 121.95,
116.59, 105.73, 65.03, 61.22, 56.42.; HRMS(ESI) m/z calcd for C19H20O6 ([M+H]+):
345.1338; found: 345.1340.
4-hydroxy-3-methoxybenzyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (20) White solid.
1
mp: 198-200 °C. Yield: 63 %; Rf = 0.4 (Hexane/EtOAc = 1:1, v/v); H NMR (600
MHz, CDCl3) δ ppm: 7.79 (d, J = 15.9 Hz, 1H), 7.12-7.05 (m, 2H), 6.95 (dd, J = 8.0,
1.9 Hz, 1H), 6.82 (s, 2H), 6.58 (d, J = 15.9 Hz, 1H), 4.71-4.69 (m, 2H), 3.93-3.84 (m,
12H); 13C NMR (151 MHz, CDCl3) δ ppm: 165.25, 153.69, 151.55, 146.82, 140.66,
139.32, 129.94, 123.09, 119.24, 116.36, 111.31, 105.71, 65.33, 61.22, 56.40;
HRMS(ESI) m/z calcd for C20H22O7 ([M+H]+): 375.1438; found: 375.1441.

4-(trifluoromethyl)benzyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (21) White solid. mp:
1
120-122 °C. Yield: 73 %; Rf = 0.5 (Hexane/EtOAc = 4:1, v/v); H NMR (600 MHz,
CDCl3) δ ppm: 7.84-7.63 (m, 3H), 7.53 (d, J = 7.9 Hz, 2H), 6.76 (s, 2H), 6.40 (d, J =
15.9 Hz, 1H), 5.30 (s, 2H), 3.89 (d, J = 1.4 Hz, 9H); 13C NMR (151 MHz, CDCl3) δ
ppm: 166.74, 153.69, 145.88, 143.86, 142.32, 140.60, 132.12, 129.88, 128.43, 125.78,
116.81, 105.57, 65.56, 61.22, 56.40; HRMS(ESI+) m/z calcd for C20H19F3O5
([M+H]+): 397.1263; found: 397.1260.
2,4-dimethoxybenzyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (22) White solid. mp:
1
74-76 °C. Yield: 67 %; Rf = 0.5 (Hexane/EtOAc = 4:1, v/v); H NMR (600 MHz,
CDCl3) δ ppm: 7.61 (dd, J = 15.9, 3.3 Hz, 1H), 7.30 (d, J = 8.9 Hz, 1H), 6.74 (d, J =
10.3 Hz, 2H), 6.49 (dt, J = 3.9, 2.1 Hz, 1H), 6.37 (dd, J = 23.1, 15.9 Hz, 1H), 5.23 (s,
2H), 3.91-3.79 (m, 16H);13C NMR (151 MHz, CDCl3) δ ppm: 167.21, 161.56, 159.26,
153.60, 144.79, 140.22, 131.73, 130.23, 117.82, 116.96, 105.38, 104.31, 98.83, 61.99,
61.16, 56.33, 55.63; HRMS(ESI) m/z calcd for C21H14O7 ([M+Na]+): 411.1414; found:
411.0951.
2,4,5-trimethoxybenzyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (23) White solid. mp:
1
68-70 °C. Yield: 60 %; Rf = 0.4 (Hexane/EtOAc = 4:1, v/v); H NMR (600 MHz,
CDCl3) δ ppm: 7.61 (d, J = 15.8 Hz, 1H), 6.94 (s, 1H), 6.73 (s, 2H), 6.55 (s, 1H), 6.39
(d, J = 16.0 Hz, 1H), 3.90 (s, 4H), 3.85 (d, J = 8.4 Hz, 16H); 13C NMR (151 MHz,
cdcl3) δ ppm: 167.18, 153.58, 152.75, 150.32, 144.90, 143.12, 140.24, 130.13, 117.65,

115.69, 114.90, 105.38, 97.74, 61.84, 61.13, 56.30; HRMS(ESI) m/z calcd for
C22H26O8 ([M+Na]+): 441.1528; found: 441.1521.
2,3,4-trimethoxybenzyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (24) White solid. mp:
1
91-93 °C. Yield: 67 %; Rf = 0.4 (Hexane/EtOAc = 4:1, v/v); H NMR (600 MHz,
Chloroform-d) δ 7.61 (d, J = 15.7 Hz, 1H), 7.12 – 7.07 (m, 1H), 6.74 (d, J = 11.1 Hz,
2H), 6.68 (d, J = 8.5 Hz, 1H), 6.37 (d, J = 16.1 Hz, 1H), 5.22 (d, J = 2.3 Hz, 2H), 3.94
(d, J = 2.2 Hz, 3H), 3.87 (dd, J = 11.5, 2.0 Hz, 16H); 13C NMR (151 MHz, cdcl3) δ
167.02, 154.50, 153.61, 152.86, 145.00, 142.46, 140.33, 130.11, 125.38, 122.28,
117.60, 107.36, 105.43, 62.12, 61.65, 61.14, 56.34; HRMS(ESI) m/z calcd for
C22H26O8 ([M+H]+): 419.1706; found: 419.1769.
3,4,5-trimethoxybenzyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (25) White solid. mp:
1
72-74 °C. Yield: 67 %; Rf = 0.4 (Hexane/EtOAc = 4:1, v/v); H NMR (600 MHz,
CDCl3) δ ppm: 7.64 (d, J = 15.9 Hz, 1H), 6.75 (s, 2H), 6.64 (s, 2H), 6.40 (d, J = 15.8
Hz, 1H), 5.16 (s, 2H), 3.88-3.86 (m, 16H), 3.84 (d, J = 1.9 Hz, 3H).;13C NMR (151
MHz, CDCl3) δ ppm: 166.90, 153.63, 145.45, 140.44, 138.26, 131.70, 129.96, 117.17,
105.91, 105.48, 66.90, 61.15, 56.36; HRMS(ESI) m/z calcd for C22H26O8 ([M+Na]+):
441.1525; found: 441.1426.
phenethyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (26) White solid. mp: 65-68 °C.
Yield: 86 %; Rf = 0.5 (Hexane/EtOAc = 2:1,v/v); 1H NMR (600 MHz, CDCl3) δ ppm:

7.59 (d, J = 15.9 Hz, 1H), 7.34-7.22 (m, 5H), 6.74 (s, 2H), 6.33 (d, J = 15.9 Hz, 1H),
4.44 (t, J = 7.1 Hz, 2H), 3.89 (s, 6H), 3.88 (s, 3H), 3.03 (t, J = 7.1 Hz, 2H);13C NMR
(151 MHz, CDCl3) δ ppm: 167.07, 153.66, 145.06, 138.08, 129.12, 128.75, 126.80,
117.49, 105.49, 65.20, 61.20, 56.40; HRMS(ESI) m/z calcd for C20H22O5 ([M+H]+):
343.1545; found: 343.1567.
3-phenylpropyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (27) White solid. mp:
1
69-72 °C. Yield: 86 %; Rf = 0.6 (Hexane/EtOAc = 2:1,v/v); H NMR (600 MHz,
CDCl3) δ ppm: 7.59 (d, J = 15.9 Hz, 1H), 7.29-7.20 (m, 5H), 6.76 (s, 2H), 6.35 (d, J =
15.9 Hz, 1H), 4.24 (t, J = 6.5 Hz, 2H), 3.90 (s, 6H), 3.89 (s, 3H), 2.76 (t, J = 7.7 Hz,
2H), 2.09-2.01 (m, 2H);13C NMR (151 MHz, CDCl3) δ ppm: 167.18, 153.66, 144.91,
141.45, 140.35, 130.14, 128.64, 128.62, 126.24, 117.58, 105.47, 77.44, 64.15, 61.20,
56.40, 32.49, 30.53; HRMS(ESI) m/z calcd for C21H24O5 ([M+H]+): 357.1702; found:
357.1698.
(2R,3R,4S,5R,6S)-2-(acetoxymethyl)-6-(4-((((E)-3-(3,4,5-trimethoxyphenyl)acryloyl)o
xy)methyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (28) Yellow solid. mp:
1
143-146 °C. Yield:63 %; Rf = 0.5 (Hexane/EtOAc = 1:2, v/v); H NMR (600 MHz,
CDCl3) δ ppm: 7.62 (d, J = 15.9 Hz, 1H), 7.36 (d, J = 8.3 Hz, 2H), 7.00 (d, J = 8.5 Hz,
2H), 6.73 (s, 2H), 6.36 (d, J = 15.8 Hz, 1H), 5.28 (t, J = 8.2 Hz, 2H), 5.19 (s, 2H),
5.09 (d, J = 7.2 Hz, 1H), 4.23 (dd, J = 56.0, 3.8 Hz, 1H), 3.87 (s, 13H), 2.18-1.98 (m,
13
12H); C NMR (151 MHz, CDCl3) δ ppm: 170.74, 170.43, 169.58, 169.46, 166.90,

157.04, 153.65, 145.39, 140.44, 131.29, 130.27, 129.98, 117.27, 117.23, 105.47,
99.24, 77.44, 72.89, 72.30, 71.37, 68.48, 66.06, 62.14, 61.18, 56.37; MS(ESI) m/z
calcd for C33H38O15 ([M+ Na]+): 697.2103; found: 697.2099.
4-(cyclopropylmethoxy)benzyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (29) Yellow
liquid. Yield: 88 %; Rf = 0.6 (Hexane/EtOAc = 4:1, v/v); 1H NMR (600 MHz, CDCl3)
δ ppm: 7.59 (dd, J = 15.9, 3.8 Hz, 1H), 7.38-7.27 (m, 2H), 6.89 (dd, J = 8.2, 5.2 Hz,
2H), 6.72 (d, J = 3.7 Hz, 2H), 6.35 (dd, J = 15.9, 2.9 Hz, 1H), 5.15 (d, J = 4.4 Hz, 2H),
3.88-3.77 (m, 11H), 0.97-0.79 (m, 1H), 0.62 (dd, J = 7.9, 5.2 Hz, 2H), 0.33 (t, J = 5.1
Hz, 2H); 13C NMR (151 MHz, CDCl3) δ ppm: 166.90, 159.29, 153.56, 145.04, 140.29,
130.28, 130.03, 128.20, 117.45, 114.79, 105.39, 72.94, 66.37, 56.26, 10.40, 3.33;
HRMS(ESI) m/z calcd for C23H26O6 ([M+Na]+): 421.1622; found: 421.1622.
4-(cyclopropylmethoxy)-3-methoxybenzyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (30)
1
White solid. mp: 79-82 °C. Yield: 88 %; Rf = 0.6 (Hexane/EtOAc = 4:1, v/v); H
NMR (600 MHz, CDCl3) δ ppm: 8.00 (d, J = 6.3 Hz, 1H), 7.61 (dd, J = 16.0, 6.2 Hz,
1H), 6.94 (d, J = 6.6 Hz, 2H), 6.85 (d, J = 8.2 Hz, 1H), 6.73 (d, J = 6.6 Hz, 2H),
6.41-6.34 (m, 1H), 5.15 (s, 1H), 3.87 (d, J = 15.5 Hz, 10H), 2.94 (s, 2H), 1.24 (s, 3H),
0.62 (q, J = 5.9 Hz, 2H), 0.34 (t, J = 5.2 Hz, 2H); 13C NMR (151 MHz, CDCl3) δ ppm:
162.68, 153.59, 149.73, 148.89, 145.18, 140.32, 130.02, 128.79, 121.50, 117.39,
113.43, 112.56, 105.41, 74.22, 66.72, 61.12, 56.31, 10.46, 3.59; HRMS(ESI) m/z
calcd for C24H28O7 ([M+H]+): 429.1913; found: 429.1882.

4-((3-methylbut-2-en-1-yl)oxy)benzyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (31)
Yellow liquid. Yield: 70 %; Rf = 0.5 (Hexane/EtOAc = 5:1, v/v); 1H NMR (600 MHz,
CDCl3) δ ppm: 7.62 (d, J = 15.9 Hz, 1H), 7.39-7.31 (m, 2H), 6.95-6.91 (m, 2H), 6.74
(s, 2H), 6.37 (d, J = 15.9 Hz, 1H), 5.53-5.43 (m, 1H), 5.18 (s, 2H), 4.52 (d, J = 6.7 Hz,
2H), 3.87 (d, J = 1.7 Hz, 10H), 1.80 (s, 3H), 1.74 (s, 3H); 13C NMR (151 MHz,
CDCl3) δ ppm: 167.01, 159.22, 153.63, 145.12, 138.53, 130.35, 130.10, 128.23,
119.75, 117.53, 114.95, 105.44, 66.48, 65.03, 61.17, 56.35, 26.03, 18.41; HRMS(ESI)
m/z calcd for C24H28O6 ([M+Na]+): 435.1778; found: 435.1721.
3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)benzyl
(E)-3-(3,4,5-trimethoxyphenyl)acrylate (32) White solid. mp: 47-51 °C. Yield: 77 %;
Rf = 0.6 (Hexane/EtOAc = 4:1, v/v); 1H NMR (600 MHz, CDCl3) δ ppm: 7.59 (d, J =
15.9 Hz, 1H), 6.92 (d, J = 11.0 Hz, 2H), 6.84 (d, J = 8.0 Hz, 1H), 6.71 (s, 2H), 6.36 (d,
J = 15.9 Hz, 1H), 5.47 (t, J = 6.7 Hz, 1H), 5.14 (s, 2H), 4.55 (d, J = 6.6 Hz, 2H), 3.85
(s, 3H), 3.83 (d, J =2.1 Hz, 9H), 1.71 (d, J = 22.3 Hz, 6H); 13C NMR (151 MHz,
CDCl3) δ ppm: 166.93, 153.44, 149.51, 148.48, 145.12, 137.78, 129.91, 128.49,
121.35, 119.87, 117.23, 112.95, 112.22, 105.31, 77.23, 66.64, 65.82, 60.96, 56.16,
25.83, 18.24; HRMS(ESI) m/z calcd for C25H30O7 ([M+Na]+): 465.1884; found:
465.1889.

3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)benzyl
3-(3,4,5-trimethoxyphenyl)propanoate (33) Yellow liquid. Yield: 73 %; Rf = 0.6
1
(Hexane/EtOAc = 4:1, v/v); H NMR (600 MHz, CDCl3) δ ppm: 6.81 (ddd, J = 8.8,
5.9, 2.4 Hz, 3H), 6.36 (d, J = 5.2 Hz, 2H), 5.47 (t, J = 6.6 Hz, 1H), 5.01 (s, 2H), 4.53
(d, J = 6.9 Hz, 2H), 3.83-3.74 (m, 12H), 2.87 (t, J = 7.6 Hz, 2H), 2.63 (t, J = 7.8 Hz,
2H), 1.72 (d, J = 5.8 Hz, 3H), 1.69 (d, J = 5.9 Hz, 3H);13C NMR (151 MHz, CDCl3) δ
ppm: 172.65, 153.12, 149.37, 148.35, 137.65, 136.35, 136.20, 128.30, 121.09, 119.81,
112.78, 111.98, 105.17, 66.43, 65.68, 60.72, 55.83, 35.97, 31.25, 25.76, 18.16;
HRMS(ESI) m/z calcd for C25H32O7 ([M+Na]+): 467.204； found: 467.206.
3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)benzyl
(E)-3-(benzo[d][1,3]dioxol-5-yl)acrylate (34) Yellow liquid. Yield: 73 %; Rf = 0.7
(Hexane/EtOAc = 5:1, v/v); 1H NMR (600 MHz, CDCl3) δ ppm: 7.62 (d, J = 15.9 Hz,
1H), 7.03-6.98 (m, 2H), 6.96-6.92 (m, 2H), 6.87 (d, J = 8.0 Hz, 1H), 6.80 (d, J = 8.0
Hz, 1H), 6.30 (d, J = 15.9 Hz, 1H), 6.00 (s, 2H), 5.56-5.49 (m, 0H), 5.16 (s, 2H), 4.59
(d, J = 6.7 Hz, 2H), 3.89 (s, 3H), 1.77 (d, J = 1.6 Hz, 3H), 1.73 (d, J = 1.4 Hz, 3H);
13C NMR (151 MHz, cdcl3) δ ppm: 155.41, 153.41, 150.43, 145.96, 142.40, 130.06,
128.71, 127.20, 121.38, 120.02, 117.72, 112.98, 112.22, 110.62, 105.41, 66.77, 65.98,
56.26, 29.93, 26.06; HRMS(ESI) m/z calcd for C25H32O7 ([M+Na]+): 419.1465; found:
419.1476.

3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)benzyl cinnamate (35) White solid. mp:
1
51-52 °C. Yield: 63 %; Rf = 0.7 (Hexane/EtOAc = 5:1, v/v); H NMR (600 MHz,
CDCl3) δ ppm: 7.49 (d, J = 16.0 Hz, 1H), 7.26 (dp, J = 7.6, 2.9, 2.4 Hz, 2H), 7.12 (h,
J = 3.7, 2.9 Hz, 3H), 6.74 (d, J = 7.6 Hz, 2H), 6.65 (d, J = 7.8 Hz, 1H), 6.25 (d, J =
16.0 Hz, 1H), 5.30 (tt, J = 5.7, 2.3 Hz, 1H), 4.96 (s, 2H), 4.35 (d, J = 6.5 Hz, 2H),
3.65 (s, 3H), 1.54 (s, 3H), 1.50 (s, 3H);13C NMR (151 MHz, CDCl3) δ ppm: 166.74,
149.45, 148.39, 144.98, 137.58, 134.32, 130.27, 128.84, 128.04, 121.21, 119.91,
117.96, 112.83, 112.07, 66.46, 65.70, 55.84, 25.78, 18.18; HRMS(ESI) m/z calcd for
C22H24O4 ([M+Na]+): 375.1567; found: 375.1571.
2.2. Pharmacology
2.2.1. Maximal electroshock test (MES)
In initial MES test, all the compounds exhibited protection of the tested mice
after 0.5 h except compounds 2 and 4 (Table 1), indicative of their ability to prevent
seizure spread. Compounds which were significantly active at 100 mg/kg after 0.5 h
in MES test including 5, 7, 8, 13, 20, 25, 28, 30 and 32, indicative of their good ability
to protect from seizure. Among these compounds, only compounds 7 and 28 were
effective at the same dose after 4 h. This result revealed that compounds 7 and 28
have quick onset and long duration of action at relatively higher dose. Furthermore,
we evaluated the protective effect of the derivatives at lower dosage. The ED50 values
of potent active compounds 5, 7, 8, 13, 20, 25, 28, 30 and 32 were 62.5, 12.2, 53.0,
32.9, 46.1, 40.4, 19.5, 26.0 and 21.9 mg/kg, respectively (Table 2). The therapeutic

effect of the most active compounds 7 and 28 were close to CBZ (ED50 = 8.8 mg/kg,
Table 2) used as reference drug. As for the protective index (PI), except compound 5,
all the active compounds exhibited higher value than CBZ. Compounds 13, 28 and 32
exerted highest value of protective index among the test compounds (>30), suggesting
a combination of good anticonvulsant effect and high safety.
Table 1. Anticonvulsant activity in the MES model (100 mg/kg) and ClogP values
of compounds administered i.p. to mice
Comp 0.5h
ound
1h
2h
3h
4h
ClogPa
0b/4c
0/4
6/6
1/4
0/4
3/4
0/4
2/4
1/4
3/4
3/4
1/4
1/4
1/4
0/4
4/6
0/4
0/4
3/4
0/4
1/4
0/4
1/4
3/4
1/4
1/4
0/4
0/4
4/6
0/4
0/4
1/4
0/4
0/4
0/4
1/4
1/4
0/4
0/4
0/4
0/4
2/6
0/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
-
NS
5/6
1/4
0/4
3/4
0/4
4/4
3/4
4/4
4/4
3/4
1/4
2/4
2.38
1.77
2.29
1.88
2.82
2.60
3.07
2.74
5.50
2.21
2.03
2.03
CBZ
1
2
3
4
5
6
7
8
9
10
11

Comp 0.5h
ound
1h
2h
3h
4h
ClogPa
2/4
4/4
1/4
3/4
1/4
2/4
1/4
2/4
4/4
3/4
2/4
3/4
2/4
4/4
3/4
2/4
4/4
2/4
4/4
3/4
1/4
2/4
0/4
1/4
0/4
1/4
0/4
1/4
2/4
0/4
2/4
3/4
2/4
2/4
3/4
1/4
3/4
2/4
3/4
2/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
2/4
2/4
1/4
2/4
1/4
0/4
2/4
0/4
1/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
1/4
1/4
1/4
1/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
5.76
5.63
3.45
3.40
4.25
3.68
4.40
2.87
2.72
4.42
3.54
2.83
2.83
2.83
3.86
4.24
0.50
4.42
4.16
5.15
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31

Comp 0.5h
ound
1h
2h
3h
4h
ClogPa
4/4
3/4
3/4
1/4
3/4
2/4
2/4
1/4
2/4
1/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
4.90
4.72
5.56
5.59
32
33
34
35
a
Notes: ClogP was calculated by utilizing the ChemBioDraw Ultra (v13.0)
(Cambridgesoft, Billerica, MA, USA);
b protect mice;
c total mice;
2.2.2 Pentylenetetrazole (PTZ)-induced seizure test
The anticonvulsant activity of active compounds 5, 7, 8, 13, 20, 25, 28, 30 and 32
were further evaluated in scPTZ-induced seizure in mice (Table 2). The result
exhibited that except compound 5, all the compounds could effectively inhibit the
scPTZ-induced seizure in latent time at the dose of 100 mg/kg (P<0.01). The ED50
values of potent active compounds 5, 7, 8, 13, 20, 25, 28, 30 and 32 were 280.7, 135.8,
143.3, 278.6, 208.4, 223.2, 178.0, 209.9 and 201.3 mg/kg, respectively. Compounds 7
and 8 displayed the strongest inhibitory effect in this model.
2.2.3. Acute neurotoxicity screening
In the neurotoxicity screen, compounds 13, 20, 28 and 32 showed weak
neurotoxicity (>500 mg/kg). Compounds 13 and 28 even possessed no obvious
neurotoxicity in the maximum dose administered (1000 mg/kg) (Table 2). To our

dismay, the most active compound 7 exerted marked toxicity, indicating that this
compound needed further modification for more safety. Generally, substituted groups
derivatived from natural product tend to exert lower neurotoxicity. According to the
results of in vivo pharmacology and neurotoxicity, compounds 28 and 32 were
regarded as the most promising compounds.
Table 2. Further screening for some active compounds.
Com. MES ED50
Sc-PTZ
TOX ED50
PIa
(MES)
Latent time ED50
(100mg/kg, s)
---
150.8±16.3
---
---
---
NS
CZBb
5
8.8 (5.5-14.1) ---
>100
71.6 (45.9-135) 8.1
62.5
176.3±16.3
280.7
417.6
6.7
(5.4-124.7)
12.2
(124.9-374.0)
(306.8-471.3)
291.7
275.8±12.4** 135.8
(86.4-276.5)
289.3±18.4** 143.3
(64.9-232.9)
237.8±11.5** 278.6
(123.5-365.1)
263.3±16.8** 208.4
(88.6-397.8)
251.0±19.8** 223.2
24.0
8.0
7
(7.5-18.7)
53.0
(253.1-349.5)
422.2
8
(26.6-113.2)
32.9
(345.3-514.4)
>1000
30.4
14.8
11.9
13
20
25
(17.9-61.5)
46.1
684.3
(23.5-79.7)
40.4
(598.9-810.1)
479.5

Com. MES ED50
Sc-PTZ
TOX ED50
PIa
(MES)
Latent time ED50
(100mg/kg, s)
(19.4-72.9)
(97.1-346.8)
274.0±15.6** 178.0
(67.6-315.9)
246.8±20.5** 209.9
(148.0-394.7)
253.2±22.3** 201.3
(149.4-275.9)
(396.6-587.3)
>1000
19.5
51.2
18.3
31.9
28
30
32
(9.8-38.9)
26.0
476.4
(16.6-41.3)
21.9
(397.3-609.4)
697.6
(16.7-37.2)
(558.1-786.8)
Notes: a PI: protective index (TD50/ED50);
b Reference drug, data from [16];
**: P<0.01, 95% confidence intervals given in parentheses.
2.2.4. Cytotoxicity
As we can see in Table 3, except for compounds 7 and 25, most of the active
compounds exerted weak cytotoxicity (< 10%) against HepG-2 cells at the dosage of
20 μM, suggesting the safety and potential for test compounds to develop as novel
anticonvulsant agents.
Table 3. The measured results cytotoxicity for HepG-2 cells (μM).
Com.
Inhibition rate Compound Inhibition
rate
（%）
（%）
37.5
14.5
5-Fu
25

3.7
18.4
9.8
8.4
5.7
6.7
9.2
7.1
5
28
30
32
7
8
13
20
Notes: 5-Fu: 5-Fluorouracil is used as positive controlled drug.
2.2.6. Molecular docking analysis
Based on the pharmacology evaluation results, the active compounds 5, 7, 8, 13,
20, 25, 28, 30 and 32 were selected as ligand examples. As shown in Table 4, total
scores of compounds were evaluated, and vitamin B6 phosphate was used as
reference standard. When the score was more than 5 indicated that the drug could
interact with the combination of domains for the protein. The docking result showed
that except for compounds 13 and 28, other the tested compounds could bond to the
protein with good scores. While the compound 13 and 28 did not interact with the
combination of domains for the protein, suggesting that compounds 13 and 28 could
possess anticonvulsant effect mediating by other targets. Based on the results of score
and H-boding, compound 32 displayed the optimal interaction with the protein. The
intermolecular hydrogen-bonding interactions of 32 were mainly seen with the
GLN301, TRP354, ASN140 (dotted yellow line in Figure 1), which were moderately
similar to the original ligand of 1OHW. Compound 32 could be observed in the cavity
of 1OHW (Figure 1), which demonstrated the rationality of the docking study.
Table 4. Molecular docking score for active compounds.

Compound
Score
11.53
5.74
9.43
7.51
<0
H-boding Residues
4
AGR192, THR353, TRP354, GLN301,
1OHW-ligand
2
TRP354, ASN140
5
3
SER137, GLY136, GLN301
LYS329, SER269, GLN301
SER137, GLY136, GLN301
LYS329, GLY136, SER137
GLN301, ASN352
7
3
8
6
13
20
25
28
7.87
6.51
<0
4
2
11
GLU270, SER269, GLN301, TRP354,
AGR100, ASN140
5.50
9.24
4
5
AGR192, SER137, LYS329
GLN301, TRP354, ASN140
30
32
(A)
(B)
Figure 1. Docking mode of compound 32 at GABA transferas. The (A) shows the 2D
interaction patterns. The legend inset represents the type of interaction between the
ligand atoms and the amino acid residues of the protein. The (B) shows the 3D
docking of the compound in the binding pocket.
2.2.6. Predicting pharmacokinetic properties of synthesized compounds in silico

In the discovery of new CNS-active drugs including anticonvulsant agents,
favorable oral bioavailability and acceptable BBB permeability of lead compounds
are important factors for transduction of them into the successful drug candidates [17].
The drug-like properties of designed compounds based on Lipinski’s “Rule of five”
[18]. As the results presented in Table 5, except for compound 28, all active
compounds follow the Lipinski's rule without causing any violation, which supposes
important information about the potential druglikeness of these novel TMCA
derivatives. As it was detailed in Table 5, all compounds were predicted for a certain
extent transmittance for BBB, which is essential for anticonvulsant activity in brain.
Results of CYP2C19 and P-gp inhibition prediction revealed that most of tested
compounds could inhibit the activity of CYP2C19 and P-gp.
Table 5. Calculated physicochemical and pharmacokineti parameters of the
active compounds.
Comp LogP MW
ound
TPSA nO
N
nOHN BBB Caco CYP P-gp
H
0
0
0
0
1
0
0
2
2C19
3.96
3.03
5.68
4.82
3.09
3.38
3.87
278.35 36.94
4
5
5
5
7
8
0.12
0.05
0.33
0.10
0.10
0.36
56.59
56.10
57.05
53.67
46.80
56.56
+
+
+
-
-
5
292.33 54.01
374.48 54.01
374.48 54.01
374.39 83.47
418.44 81.71
-
7
+
+
+
+
+
8
13
20
25
28
+
+
+
674.65 177.69 15
0.074 39.67