Synthesis, antiepileptic effects, and structure-activity relationships of α-asarone derivatives: In vitro and in vivo neuroprotective effect of selected derivatives

Article abstract of DOI:10.1016/j.bioorg.2021.105179

In the present study, we compared the antiepileptic effects of α-asarone derivatives to explore their structure-activity relationships using the PTZ-induced seizure model. Our research revealed that electron-donating methoxy groups in the 3,4,5-position on phenyl ring increased antiepileptic potency but the placement of other groups at different positions decreased activity. Besides, in allyl moiety, the optimal activity was reached with either an allyl or a 1-butenyl group in conjugation with the benzene ring. The compounds 5 and 19 exerted better neuroprotective effects against epilepsy in vitro (cell) and in vivo (mouse) models. This study provides valuable data for further exploration and application of these compounds as potential anti-seizure medicines.

Full text of DOI:10.1016/j.bioorg.2021.105179

Bioorganic Chemistry 115 (2021) 105179
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, antiepileptic effects, and structure-activity relationships of
α
-asarone derivatives: In vitro and in vivo neuroprotective effect of
selected derivatives
Jian Zhang, Keman Mu, Peng Yang, Xinqian Feng, Di Zhang, Xiangyu Fan, Qiantao Wang*,
Shengjun Mao*
Key Laboratory of Drug Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and
Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
In the present study, we compared the antiepileptic effects of α-asarone derivatives to explore their structure-
Structure-activity relationships
activity relationships using the PTZ-induced seizure model. Our research revealed that electron-donating
methoxy groups in the 3,4,5-position on phenyl ring increased antiepileptic potency but the placement of
other groups at different positions decreased activity. Besides, in allyl moiety, the optimal activity was reached
with either an allyl or a 1-butenyl group in conjugation with the benzene ring. The compounds 5 and 19 exerted
better neuroprotective effects against epilepsy in vitro (cell) and in vivo (mouse) models. This study provides
valuable data for further exploration and application of these compounds as potential anti-seizure medicines.
α
-asarone
Epilepsy
PTZ
1. Introduction
propenyl-2,4,5-trimethoxy-benzene, see Fig. 1 A) is one of the active
components extracted from Acorus Tatarinowii, showing distinct anti-
Epilepsy is a chronic neurological disorder characterized by spon-
taneous recurrent seizures with abnormal electrical discharges in the
brain [1]. It is one of the most common neurological diseases affecting
about 68 million people worldwide [2].
convulsant and anti-epilepsy effects [12,13]. Also, α-asaronol ((E)-3-
(2,4,5-trimethoxyphenyl)prop-2-en-1-ol, see Fig. 1 B), which is derived
from ASA and possess an anticonvulsant activity [14]. So, we expect to
modify ASA’s chemical structure to get new antiepileptic candidates
with more powerful effects and lower toxicity.
Antiepileptic drugs (AEDs) therapy is still the primary medical treat-
ment for patients with epilepsy, of whom nearly 70% have benefit from
AEDs therapy [3,4]. However, uncontrolled seizures occur in nearly one-
third of epileptic patients suffering from various adverse effects or drug
resistance [5,6]. Indeed, the side effects of currently available AEDs [7],
including headache, depression, cognitive impairment, sedation, memory
deterioration, and loss of appetite [8,9], cannot be ignored. In addition,
epileptic patients, their caretakers, and society endure huge burdens ow-
ing to uncontrolled seizures and severe medication hangover [10].
Therefore, it is still urgently needed and quite challenging to develop
promising AEDs with high efficacy and minimal side effects.
Given this, we designed and synthesized a series of ASA derivatives
with promising biological activities in the present study (Scheme 1-5, 1-
30). Since more of the methoxybenzaldehyde derivatives could poten-
tially be made from commercially available starting materials, it was
decided to begin with these compounds. 2–22 were synthesized from
methoxybenzaldehyde derivative, of which small part (2a, 6a-8a, 10a-
13a) were prepared in our laboratory because they were not available
commercially, and the corresponding alkyl triphenylphosphonium bro-
mide via Wittig reaction. Besides, synthesis of 23–28 was achieved from
commercially available starting materials methoxybenzaldehyde deriv-
ative (6c). The preparation of 29–30 was performed from compounds 5
and 19 via Pd-catalyzed hydrogenation. These structures were charac-
terized using 1H NMR and 13C NMR techniques. We evaluated these
derivatives’ antiepileptic activity by establishing the pentylenetetrazol
(PTZ)-induced seizure models, elucidating the structure-activity
Natural products have attracted much attention in searching for
bioactive compounds to develop new drugs in the last decades. The
rhizome of Acorus Tatarinowii that belongs to Araceae Juss’s family is
widely used in the clinic treatment for neurologic disorders such as
epilepsy, amnesia, senile dementia, etc. [11]. Alpha asarone (ASA, (E)-1-
* Corresponding authors.
E-mail addresses: qwang@scu.edu.cn (Q. Wang), xinba789@163.com (S. Mao).
https://doi.org/10.1016/j.bioorg.2021.105179
Received 17 March 2021; Received in revised form 11 July 2021; Accepted 12 July 2021
Available online 15 July 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

J. Zhang et al.
Bioorganic Chemistry 115 (2021) 105179
carried out via methyl esterification of (E)-3-(3,4,5-trimethoxyphenyl)
acrylic acid (26b) and reduction reaction of methyl (E)-3-(3,4,5-trime-
thoxyphenyl)acrylate (26a) formed. Trifluoromethylation of 23 26b
afforded compounds 27 and 28 in the presence of copper sulfate pen-
tahydrate
(CuSO4⋅5H2O),
sodium
trifluoromethanesulfinate
(NaSO2CF3), and tert-butyl hydroperoxid (TBHP) at reflux [22],
respectively. Finally, the preparation of compounds 29 and 30 was
performed via reduction of 5 and 19 in the presence of 10% Pd/C and a
pure hydrogen atmosphere, outlined in Scheme 5.
Fig. 1. Structure of α-asarone and α-asaronol.
2.2. Structure-activity relationship
relationships. We aimed to obtain active compounds through these
procedures.
The ASA structure consists of three moieties of A, B, and C (Fig. 2).
Each part structure optimization and modification was carried out,
respectively. We evaluated the types, the position, and the number of the
substituted groups on the phenyl ring in A and then the types of sub-
stituent R2 in C. Next, we explored the effect of double bonds in B on the
biological activity. All the synthesized compounds were screened in a
pentylenetetrazole (PTZ) -induced seizure test [23]. The results were
shown in Table 1. It was clear that compounds 5 and 19 had superior
antiepileptic activity.
2. Results and discussion
2.1. Chemistry
The compounds 1 were synthesized according to Scheme 1. 1-(2,3,6-
trimethoxyphenyl)propan-1-ol (1a) was synthesized by reaction of
1,2,4-trimethoxybenzene (1b) with propionaldehyde in the presence of
n-butyllithium (n-BuLi), and N,N,N’,N’-Tetramethylethylenediamine
(TMEDA). Then, 1 was prepared by dehydration of 1a by anhydrous
CuSO4 [15].
2.2.1. Effect of changing the position of trimethoxy substitution on phenyl
ring in A
We first evaluated the importance of the three methoxy groups’
substitution position on the phenyl ring to optimize antiepileptic activity
further. Therefore, five isomers 1–5 with different substituted positions
of methoxy groups on the phenyl ring were synthesized. From Table 1, it
can be seen that compound 5 significantly prolonged the latent time of
seizures when compared with the negative control (NC) group (P <
0.05), and this compound could also remarkably reduced Racine’s sore
(P < 0.01) and tonic seizure rate (TSR) (P < 0.01). Further, compared
with ASA, the latent time, Racine’s sore, and TSR of compound 5 per-
formed slightly better than ASA’s (Fig. 3). The structural activity anal-
ysis showed that 3,4,5-trimethoxy-substituted compound 5 exhibited
great antiepileptic potency compared to the other four isomers. The fact
that the change of the trimethoxy group’s position on the phenyl ring
significantly affected the activity suggested 3,4,5-trimethoxy substitu-
tion makes a special contribution that might be due to its ability to orient
and bind with the target proteins.
The synthetic routes of intermediates 2a, 6a-8a, 10a-13a, and final
compounds 2–17 were outlined in Scheme 2. 2,3,5-trimethoxybenzalde-
hyde (2a) was prepared by Dakin reaction[16], Duff reaction [17], and
alkylation reaction of 2,4-dimethoxybenzaldehyde (2d). The interme-
diate 2-ethoxy-4,5-dimethoxybenzaldehyde (6a) was synthesized by
demethylation of 2,4,5-trimethoxybenzaldehyde (6c) with boron tri-
bromide (BBr3) [18], and ethylation of 2-hydroxy-4,5-dimethoxybenzal-
dehyde (6b) with iodoethane. Reaction of 1-(2,4-dimethoxyphenyl)
ethan-1-one (7c) with triethylsilane (Et3SiH) in trifluoroacetic acid
(CF3COOH) yielded 1-ethyl-2,4-dimethoxybenzene (7b), followed by
treatment with phosphorus oxychloride (POCl3) in N,N-dime-
thylformamide (DMF) to obtain 7a [19]. The preparation of 8a was
achieved via reaction of 2d with ethyltriphenylphosphonium bromide in
the presence of both potassium tert-butoxide (t-BuOK) and tetrabuty-
lammonium bromide (TBAB), reduction reaction by catalyst (10% Pd/C)
and formylation of 8b formed. 10a, 11a, and 13a were synthesized by
alkylation of the corresponding starting material. 12b was prepared by
Duff reaction of 2,6-diisopropylphenol (12c), of which methylation
afforded 12a with iodomethane. 2a-17a were converted to final com-
pounds 2–17 in the reaction with ethyltriphenylphosphonium bromide
in the presence of both t-BuOK and TBAB at 90–95 ◦C in DMF. The final
compounds 18–22 were also obtained by the reaction of 18a-22a with
5a followed the same conditions described for the compounds 2–17
synthesis, which was illustrated in Scheme 3.
2.2.2. Effect of different substituents on phenyl ring in A
After assessment of the trimethoxy substitution position on phenyl
ring, the replacement of the methoxy group on the phenyl ring by other
substitutions, such as ethoxy (6, 10), ethyl (7), n-propyl (8), hydroxyl
(9), n-propoxy (11), disubstituted isopropyl (12), disubstituted methyl
(13) and bromo (14), was performed to explore the structure-activity
relationships of these compounds. However, Table 1 shows that the
difference was not statistically significant in the latent time, Racine’s
sore (except for compound 6), CSR, and TSR of these compounds versus
the negative control group. Although there was a statistical difference in
the Racine’s sore of compound 6, no significant difference for the latent
time, CSR, and TSR could be seen. These data indicated that they were
inactive. Structural activity analysis showed that the enlarged molecular
volume of ethoxy (6,10) and propoxyl (11) substituted compounds
would result in steric hindrance to the target proteins binding. Alkyl
The total synthesis of the compounds 23–28 was given in Scheme 4.
23 was synthesized by the reaction of 6c and propanedioic acid [20].
The compound 24 was prepared by the reaction of 6c and 3-bromopro-
pene in the presence of palladium acetate (Pd(OAc)2), cuprous oxide
(Cu2O), and silver carbonate (Ag2CO3) at 110 ◦C in dimethyl sulfoxide
(DMSO) and toluene, which was based on prior literature [21]. Synthesis
of 25 was achieved by methyl esterification of 23. Preparation of 26 was
Scheme 1. Synthesis of compound 1. Reagents and conditions: (a) propionaldehyde, n-BuLi, TMEDA, THF, 5–10 ◦C; (b) CuSO₄, toluene, reflux.
2

J. Zhang et al.
Bioorganic Chemistry 115 (2021) 105179
substituted compounds (7, 8, 12, and 13) decreased the electron density
of the benzene ring compared with trimethoxy substituted compounds,
underscoring the importance of electron-rich substituents on the ben-
zene ring. Interestingly, in this inference, the electron-rich hydroxyl
substituted compound (9) should have better activity, but things go
contrary to one’s wishes. Since it had a low CLogP value, it showed a
decreased distribution in the brain. It further confirmed our inference
that the electron-deficient bromo-substituted compound (14) did not
have any antiepileptic activity due to the further decrease of the benzene
ring’s electron cloud density.
decreased methoxy substituents on the phenyl ring, indicating that the
antiepileptic activity was also reduced. It was suggested that decreasing
the number of methoxy substituents reduced the electron cloud density
of benzene rings, ultimately leading to lower activity. The fact was
consistent with the above conclusion.
2.2.4. Effect of the length of the carbon chain in C
We next evaluated the effect of the length of the R2 carbon chain in C
based on the fact that compound 5 was found optimal by studying the
types, the position, and the number of the substituted groups on the
phenyl ring in A. Accordingly, we synthesized a series of compounds
(18–21) with different carbon chain lengths. Among these compounds,
only compound 19 could prolong the latency of epileptic seizures (P <
0.01) and decrease Racine’s sore (P < 0.01) and TSR (P < 0.05) when
compared with the negative control group. Further, compared with ASA,
compound 19 showed a very significant prolongation of the latent time
(P < 0.01), and its Racine’s sore also performed slightly lower than
ASA’s (Fig. 3). However, the other compounds’ antiepileptic activities
2.2.3. Effect of the number of methoxy substituents on phenyl ring in A
To evaluate the effect of the number of methoxy substituents on the
phenyl ring, we had synthesized dimethoxy-substituted compounds (15,
16) and mono-methoxy substituted compounds (17). Although these
compounds could prolong the latent time of seizures and reduce
Racine’s sore, these differences did not reach statistical significance
(Table 1). The Racine’s sore and TSR’s upward trend was revealed with
Scheme 2. Synthesis of compound
2–17. Reagents and conditions: (a) 30%
H2O2, 98% H2SO₄, MeOH, RT; (b)
HMTA, AcOH, 90–95 ◦C; (c) K2CO3,
CH3I, DMF, 70–80 ◦C; (d) BBr3, DCM,
ꢀ 10 ◦C; (e) K2CO3, CH3CH2I, DMF,
70–80 ◦C; (f) triethylsilane, TFA,
60–65 ◦C; (g) POCl3, DMF, 60–65 ◦C; (h)
ethyltriphenylphosphonium bromide, t-
BuOK, TBAB, DMF, 90–95 ◦C; (i) 10%
Pd/C, H2, MeOH, RT; (j) POCl3, DMF,
60–65 ◦C; (k) K2CO3, CH3CH2I, DMF,
70–80 ◦C; (l) K2CO3, CH3CH2CH2I,
DMF, 70–80 ◦C; (m) HMTA, AcOH,
90–95 ◦C; (n) K2CO3, CH3I, DMF,
70–80 ◦C; (o) K2CO3, CH3I, DMF,
70–80
triphenylphosphonium
BuOK, TBAB, DMF, 90–95 ◦C.
◦C;
(p)
ethyl-
t-
bromide,
3

J. Zhang et al.
Bioorganic Chemistry 115 (2021) 105179
were reduced. Therefore, the optimum R2 was methyl or ethyl group in
this part of the study.
neurogenesis abnormalities [25–27]. Further, a growing body of evi-
dence showed that a substantial number of neurons within hippocampal
regions are most prone to neuronal apoptosis and loss following recur-
rent epileptic seizures. It is also well recognized that apoptotic cell death
was a common consequence of brain injury and contributed to func-
tional deficits following experimental seizures [28,29]. In this light, we
evaluated the effects of the active compounds on PTZ-induced neuronal
damage in the CA1 region of the hippocampus by using Nissl staining. As
demonstrated in Fig. 4A, NC group had the fewest number of Nissl
bodies in the CA1, which was also significantly reduced compared to BC
group (P < 0.01). Both compound 5 and 19 remarkably increased the
Nissl bodies’ number compared with NC group (5 p < 0.05, 19 p <
0.05). Further, the difference versus the BC group was not statistically
significant. These results substantiated the idea that both compounds 5
and 19 might be capable of preventing PTZ-induced hippocampal
neuronal damage.
2.2.5. Effect of different substituents of R2 in C
We further evaluated the effect of different substituents of R2 in C.
Acetyl substituted compound (22), carboxyl substituted compound (23),
methoxycarbonyl substituted compound (25), hydroxymethyl com-
pound (26) and trifluoromethyl substituted compounds (27–28) were
synthesized accordingly. As shown in Table 1, compounds 26 and 28
significantly prolonged the latent time of seizures (26 P < 0.05, 28 P <
0.05), whereas only compound 28 remarkably reduced Racine’s sore (P
< 0.01). However, compound 28 did not have a statistically significant
difference in TSR compared with NC group. Except for Racine’s sore (P
< 0.01), none of the other indexes are statistically different for com-
pound 27. On the other hand, compounds 22, 23, which were the me-
tabolites of ASA [24], and 25 did not show antiepileptic activities in the
tests. The result indicated that all the substituents of these compounds,
except 26, were the electron-withdrawing group, which reduced the
conjugated system’s electron cloud density, resulting in a decreased
antiepileptic activity. Although the hydroxyl group of compound 26 was
an electron-donating group, the anticonvulsant activity decreased
compared to ASA. One possible reason would be that it had a low CLogP
value, which caused a decreased distribution in the brain. Interestingly,
2.4. Immunohistochemical analysis of caspase-3 expression
Caspase-3, a member of cysteine proteases, played key effector roles
in apoptosis in mammalian cells [30,31]. The activation and expression
of caspase-3 preceded neuronal damage and led to cell apoptosis [32].
Therefore, these active compounds’ effect on caspase-3 expression was
also explored to test if the apoptotic responses could be interrupted after
seizure-induced by PTZ. The results of the expression of caspase-3 in the
hippocampus CA1 region are shown in Fig. 4B. The more expression of
the caspase-3 protein, the severer neuronal damage in the CA1 region of
the hippocampus. Compared with the BC, NC was significantly increased
(P < 0.001). After administration, the expression of the caspase-3 pro-
tein in compounds 5 and 19 significantly decreased (5P < 0.001, 19P <
0.01) when compared to NC group. Further, there was no significant
difference between the administration groups and BC group. The
immunohistochemical findings further confirmed that compounds 5 and
19 protected neurons from injury.
the only difference of the chemical structure of 26 and
B) is the position of the methoxyl group on the phenyl ring. However,
there was significant difference in antiepileptic activity. We s-
α-asaronol (Fig. 1
a
peculate that the latter would be more prone to form electrostatic in-
teractions and hydrophobic interactions with the target proteins.
Instead, the R2 groups of compounds 5 and 19 were methyl and ethyl
groups, respectively, which increased the conjugated system’s electron
cloud density and thereby increased antiepileptic activities.
2.2.6. Effect of reduction and position of unsaturated double bonds unit in
B
After studying a series of ASA derivatives (1–23 and 25–28), we
planned to reduce the active derivatives’ carbon–carbon double bonds
(5 and 19). Compound 29, which was obtained via a reduction of 5,
significantly prolonged the latent time of seizures (P < 0.01) and
decreased Racine’s sore (P < 0.01), but its TSR dramatically increased
from 0% to 50%, meaning that its antiepileptic activity is lower than the
latter’s. Similarly, the reduced product (30) of compound 19 also proved
futile. Finally, compound 24, which was synthesized by a shift in the
double bond position of the ASA side chain, had no benefit in any of the
parameters. The results suggested that the carbon–carbon double bonds
and their position played a critical role in these derivatives for antiep-
ileptic activities.
2.5. The active compound protected primary hippocampal neurons from
Mg-deficient induced neurotoxicity
After confirming the effects of compounds 5 and 19 in vivo, an
appropriate cell experiment was warranted to evaluate their cell-
protective effect for epilepsy. It has been reported that brief exposure
of hippocampal neurons in culture to Mg2+-free media elicited perma-
nently spontaneous recurrent seizures in these populations of neurons
[33]. This seizure state caused the death of neurons due to excitotox-
icity. Hence, the study of the neuroprotective effect of compounds 5 and
19 in the Mg2+-free induced neuronal damage model was illustrative.
We firstly ensured that both compounds 5 and 19 at their effective
concentrations had no significant cytotoxicity on normal neuron sur-
vival (Fig. 5A). The neuronal viability was significantly declined
following treatment with Mg2+-free media for 1 h compared to the
control group (P < 0.001). In contrast, both doses of compound 5
significantly attenuated the neuron cell damage versus the model group
2.3. The active compounds block hippocampal neuronal damage in the
PTZ-induced seizure model
The hippocampus had long been a focus of epilepsy since seizures
impacted neural network structure and caused hippocampal
Scheme 3. Synthesis of compound 18–22. Reagents and conditions: (a) t-BuOK, TBAB, DMF, 90–95 ◦C.
4

J. Zhang et al.
Bioorganic Chemistry 115 (2021) 105179
(Fig. 5B, low-dose group, P < 0.001; high-dose group, P < 0.001). Also,
both doses of compound 19 show very similar effects (Fig. 5B low-dose
group, P < 0.001; high-dose group, P < 0.01). We observed reduced
numbers of shrinking cells with synaptic reticular structure loss by
compound 5 or 19 treatment (Fig. 5C). The result indicated that both 5
and 19 had a neuroprotective effect that alleviated permanently spon-
taneous recurrent seizures in vitro, preventing the neuron from
overexcitation.
Scheme 5. Synthesis of compound 29–30. Reagents and conditions: (a) 10%
Pd/C, H2, MeOH, RT. 5, 29: R1 = CH3;19, 30: R1 = CH2CH3.
2.6. Toxicity risks using in silico analysis
Toxicity risks were an essential factor in determining whether
compounds could eventually become a drug. Osiris Property Explorer
[34] was a knowledge-based algorithm that predicted mutagenic,
tumorigenic, irritant, and reproductive effect of compounds, regarding
chemical fragment data of existing drugs and non-drugs as published. As
shown in Table 2, compounds 5 and 19 exhibited lower toxicity, which
is in good agreement with in vivo and in vitro results as previously
observed.
Fig. 2. Three moieties of α-asarone.
2.7. Neurotoxicity test
effect in vitro. Due to these positive results, these compounds will be
further evaluated as potential anti-seizure drug candidates.
The results (Table 2) indicated that 5 and 19 showed lower acute
neurotoxicity in the rotarod test, because their antiepileptic doses (100
4. Experimental section
mg/kg) were far less than TD50
. According to pharmacology and
neurotoxicity, 5 and 19 were considered the most promising anti-seizure
4.1. Chemistry
drug candidates.
The chemicals and reagents were used as purchased from commer-
cial suppliers such as J&K Scientific, Macklin and Energy Chemical. The
reaction’s progress was monitored by analytical thin-layer chromatog-
raphy (TLC, petroleum ether: ethyl acetate-20:1 to 1:1), which was
performed using silica gel G plates, and visualization of spots was
accomplished with UV light (256 nm). The mixtures were purified by
flash column chromatography on silica gel (Qingdao Haiyang Chemical
Co., Ltd. 200–300 mesh) chromatography. 1H NMR and 13C NMR
spectra were obtained by Varian UNITY INOVA-400 using CDCl3 as a
solvent and TMS as an internal standard. High-resolution mass spectra
(HRMS) were executed on Bruker TOF Premier by the ESI method.
3. Conclusion
In this study, a series of ASA derivatives were designed, synthesized,
and assessed for antiepileptic ability. Subsequently, structure-activity
relationship studies were conducted, which showed that methoxylated
compounds in the 3,4,5-position on the phenyl ring had better antiepi-
leptic activities than those replaced by other groups on phenyl ring.
Further, its activities were optimal when R2 was methyl or ethyl group.
Antiepileptic activities also depended strongly on carbon–carbon double
bonds and their position where the carbon–carbon double bonds formed
conjugation with the benzene ring. More importantly, the active com-
pounds 5 and 19 displayed the ability to prevent PTZ-induced hippo-
campal neuronal damage by Nissl staining and immunohistochemical
analysis of caspase-3 expression. The MTT cell proliferation assay
further demonstrated that both 5 and 19 exerted a neuroprotective
4.1.1. General procedure A
To a stirred mixture of phenol derivative (6.6 mmol) and potassium
carbonate (K2CO3) (1.8 g, 13.2 mmol) in DMF (40 mL) was added the
corresponding iodoalkyl derivative (9.9 mmol) under the protection of
Scheme 4. Synthesis of compound 23–28. Reagents and conditions: (a) propanedioic acid, pyridine, piperidine, reflux; (b) 98% H2SO₄, MeOH, reflux;(c) DIBAL-H,
THF, ꢀ 40 ◦C; (d) 3-bromopropene, Pd(OAc)2, Cu2O, Ag2CO3, Tol, DMSO, 110 ◦C; (e) CuSO4⋅5H2O, NaSO2CF3, TBHP, DCM, H2O, reflux.
5

J. Zhang et al.
Bioorganic Chemistry 115 (2021) 105179
DMF (10 mL) was added the corresponding methoxybenzaldehyde de-
rivative (6.0 mmol) under the protection of nitrogen at 0–5 ◦C and
stirring was continued at room temperature (RT) for 0.5 h. The reaction
mixture was heated at 90–95 ◦C for 4 h. After completing the reaction
(TLC monitoring), the reaction mixture was quenched with 70 mL H2O.
Then the mixture was extracted three times with 70 mL EtOAc. The
organic phase was collected, dried over anhydrous MgSO4, filtered, and
concentrated in reduced pressure. The residue was purified using silica
gel chromatography (Pet. Ether/EtOAc, v/v = 40–20:1).
Table 1
Anti-seizure activity in the PTZ-induced seizure model (100 mg/kg) and ClogP
values of compounds administered i.p. to mice.
Compound
Latent time
Racine’s score
CSR%a
TSR%b
CLogPc
BCd
NCe
1
0
0
0
0
—
57.2 ± 17.0
107.3 ± 42.1
84.7 ± 27.0
119.8 ± 25.8
95.7 ± 22.2
290.8 ± 65.5*
164.7 ± 72.3
65.2 ± 14.3
78.8 ± 18.6
62.7 ± 12.7
71.7 ± 19.9
65.7 ± 27.3
53.0 ± 7.2
5.0 ± 0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
83.3
100.0
83.3
—
4.7 ± 0.8
4.5 ± 1.2
4.8 ± 0.4
3.7 ± 1.2*
1.8 ± 0.4**
3.2 ± 1.3**
4.8 ± 0.4
4.5 ± 1.2
4.8 ± 0.4
4.3 ± 1.2
5.0 ± 0.0
5.0 ± 0.0
4.8 ± 0.4
5.0 ± 0.0
3.5 ± 1.6
3.5 ± 1.8
4.2 ± 2.0
4.7 ± 0.8
1.5 ± 1.2**
2.5 ± 1.4**
4.7 ± 0.5
5.0 ± 0.0
4.8 ± 0.4
4.8 ± 0.4
4.8 ± 0.4
4.8 ± 0.4
2.3 ± 1.4**
3.2 ± 0.8**
2.7 ± 1.5**
4.3 ± 1.2
2.0 ± 1.1**
3.05
3.05
2.70
3.40
2.70
3.57
4.43
4.96
2.36
3.22
3.75
5.37
4.31
3.76
3.05
3.40
3.31
2.17
3.22
3.75
4.28
1.45
1.89
2.87
2.12
0.91
3.01
2.66
3.00
3.53
3.05
2
83.3
3
100.0
50.0
4
5
0**
4.1.3. General procedure C
6
50.0
7
100.0
83.3
POCl3 (1.7 g, 11.2 mmol) and 10 mL dry DMF were placed in a 50 mL
three-mouth flask with a magnetic stirrer under the protection of ni-
trogen, and stirring was continued at room temperature for 30 min. To
the reaction mixture was added dropwise (over 10 min) alkyldime-
thoxybenzene (10.2 mmol) in dry DMF (5 mL) under the protection of
nitrogen at 0–5 ◦C. The reaction mixture was heated at 60–65 ◦C for 8 h.
After completing the reaction (TLC monitoring), the reaction mixture
was quenched with 30 mL H2O. Then the mixture was extracted three
times with 30 mL EtOAc. The organic phase was collected, dried over
anhydrous MgSO4, filtered, and concentrated in reduced pressure. The
residue was purified using silica gel chromatography (Pet. Ether/EtOAc,
v/v = 10:1).
8
9
100.0
66.7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
ASA
100.0
100.0
100.0
100.0
50.0
57.8 ± 12.3
63.2 ± 10.2
250.0 ± 226.6
247.0 ± 230.4
258.7 ± 237.3
130.2 ± 36.2
427.8 ± 63.0**
141.8 ± 35.1
83.8 ± 15.8
58.7 ± 11.8
67.5 ± 9.7
50.0
83.3
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
83.3
16.7*
33.3
100.0
100.0
100.0
100.0
100.0
100.0
33.3
4.1.4. General procedure D
113.0 ± 22.0
95.3 ± 39.7
285.7 ± 82.6*
227.3 ± 82.9
112.8 ± 14.0*
181.3 ± 27.6**
80.0 ± 13.1
176.8 ± 30.5**
(E)-methoxy-(propenyl)benzene analogue (16.8 mmol), 0.4 g of 10%
of palladium-on-carbon (Pd/C), and methanol (MeOH) (15 mL) were
placed in a 50 mL three-mouth flask with a magnetic stirrer under a
hydrogen atmosphere, and stirring was continued at RT for 5 h. After
completing the reaction (TLC monitoring), the reaction mixture was
filtered and concentrated in reduced pressure. The residue was purified
using silica gel chromatography (Pet. Ether/EtOAc, v/v = 10:1).
33.3
50.0
83.3
16.7*
a
CSR: clonic seizures rate.
TSR: tonic seizure rate.
b
c
4.1.5. General procedure E
ClogP was calculated by the ChemDraw Professional (v19.0) (PerkinElmer
To a stirred mixture of phenolic compound (41.5 mmol) in acetic
acid (AcOH) (130 mL) was added hexamethylenetetramine (HMTA)
(11.6 g, 83.0 mmol) under the protection of nitrogen. The reaction
mixture was heated at 90–95 ◦C for 6 h. After completing the reaction
(TLC monitoring), the reaction mixture was quenched with 500 mL H2O,
added 50 mL 1 N HCl, and stirred for an additional 1 h. Then the mixture
was extracted three times with 300 mL EtOAc. The organic phase was
collected, dried over anhydrous MgSO4, filtered, and concentrated in
reduced pressure. The residue was purified using silica gel chromatog-
raphy (Pet. Ether/EtOAc, v/v = 10:1).
Informatics. Inc, USA).
d
e
Blank control group.
Negative control group.
*
P < 0.05.
**
P < 0.01.
nitrogen, and stirring was continued at 70–80 ◦C for 4 h. After the
completion of the reaction (TLC monitoring), the reaction was treated
with 50 mL H2O. Then the mixture was extracted three times with 50 mL
ethyl acetate (EtOAc). The organic phase was collected, dried over
anhydrous MgSO4, filtered, and concentrated in reduced pressure. The
residue was purified using silica gel chromatography (Pet. Ether/EtOAc,
v/v = 5:1).
4.1.6. General procedure F
To a stirred mixture of alkyl triphenylphosphonium bromide (32.9
mmol) in dry tetrahydrofuran (THF) (60 mL) was added dropwise (over
10 min) 2.5 M n-BuLi in hexanes (13.1 mL, 32.9 mmol) under the pro-
tection of nitrogen at 0–5 ◦C and stirring was continued. After 30 min,
the corresponding benzaldehyde derivative (27.4 mmol) in dry THF (30
mL) was then added dropwise (over 10 min) to the reaction mixture at
4.1.2. General procedure B
To a stirred mixture of alkyl triphenylphosphonium bromide (9.0
mmol), t-BuOK (2.7 g, 24.0 mmol), and TBAB (0.1 g, 0.3 mmol) in dry
Fig. 3. Antiepileptic effects of compounds 5
and 19 in PTZ-induced seizure test. (A) The
latency of onset time, (B) Racine’s sore, and
(C) tonic seizure number of seizures were
shown in the PTZ test. Graphical represen-
tations of the data (A and B) were made with
box-and-whisker plots, where the whiskers
show the minimum and maximum, respec-
tively (n = 6 mice in each group). Graphical
representations of the data (C) were made
with bar plots (n = 6 mice in each group). NC
represents negative control group and N.S.
means not significant, * P < 0.05, ** P <
0.01.
6

J. Zhang et al.
Bioorganic Chemistry 115 (2021) 105179
Fig. 4. Effects of compounds 5 and 19
on hippocampal neuronal damage eli-
cited by PTZ. (A) Representative images
of histological examination by Nissl
staining showing the survival of neurons
in the hippocampus CA1 region. (B)
Representative images of immunohisto-
chemistry showing caspase-3 protein
distribution in the hippocampus CA1
region. Graphical representations of the
data were made with box-and-whisker
plots, where the whiskers indicate the
minimum and maximum, respectively
(n = 4 mice, in each group). BC, blank
control group; NC, negative control
group. N.S. means not significant, * P <
0.05, ** P < 0.01, *** P < 0.001.
0–5 ◦C. The reaction mixture was heated to RT for 4 h. After completing
the reaction (TLC monitoring), the reaction mixture was quenched with
100 mL H2O. Then the mixture was extracted three times with 100 mL
EtOAc. The organic phase was collected, dried over anhydrous MgSO4,
filtered, and concentrated in reduced pressure. The residue was purified
using silica gel chromatography (Pet. Ether/EtOAc, v/v = 10:1).
4.1.9. (E)-1,2,4-trimethoxy-3-(prop-1-en-1-yl)benzene (1)
1,2,4-trimethoxybenzene (1b) (3.0 g, 18 mmol) and 60 mL dry THF
were placed in a 100 mL three-mouth flask with a magnetic stirrer under
the nitrogen protection. After the mixture was cooled down to 5 ◦C, the
2.5 M solution of n-BuLi in hexane (19.7 mL, 49.2 mmol) was added
dropwise (over 15 min), and stirring was continued at 5–10 ◦C. An hour
later, TMEDA (2.5 mL, 16.3 mmol) and propionaldehyde (1.9 mL, 26.7
mmol) were then added dropwise (over 20 min) to the reaction mixture
at 5–10 ◦C. After the completion of the reaction (TLC monitoring), the
reaction was quenched with 3 mL H2O. The solvent was removed in a
vacuum under pressure, and the residue obtained was treated with 60
mL EtOAc and 20 mL 1 N HCl. The organic phase was collected, and the
aqueous phase was extracted with 60 mL EA. Organic phases were
combined, dried over anhydrous MgSO4, filtered, concentrated in
reduced pressure, and purified on silica gel column chromatography
(Pet. Ether/EtOAc, v/v = 20:1) to obtain 2.3 g (10.2 mmol; yield:
56.7%) 1-(2,3,6-trimethoxyphenyl)propan-1-ol (1a).
4.1.7. General procedure G
To a stirred mixture of trimethoxyphenylacrylic acid (4.2 mmol) in
MeOH (20 mL) was added dropwise (over 10 min) concentrated sulfuric
acid (0.6 g, 12.0 mmol) in MeOH (10 mL) at 0–5 ◦C. The reaction
mixture was heated at reflux for 2 h. After completing the reaction (TLC
monitoring), the reaction mixture was quenched with 100 mL H2O. Then
the mixture was extracted three times with 100 mL EtOAc. The organic
phase was collected, dried over anhydrous MgSO4, filtered, and
concentrated in reduced pressure to obtain the target product.
4.1.8. General procedure H
To a stirred mixture of anhydrous copper sulfate (0.7 g, 4.4 mmol) in
toluene (30 mL) was added 1a (1.0 g, 4.4 mmol). The reaction mixture
was heated at reflux for 5 h. After completing the reaction (TLC moni-
toring), the mixture was filtered, concentrated in reduced pressure and
purified on silica gel column chromatography (Pet. Ether/EtOAc, v/v =
30:1) to obtain 0.6 g (2.9 mmol; total yield: 36.9%) (E)-1,2,4-trime-
thoxy-3-(prop-1-en-1-yl)benzene (1). 1H NMR (400 MHz, Chloroform-d)
δ = 6.72–6.63 (m, 2H), 6.61–6.54 (m, 2H), 3.81 (s, 3H), 3.78 (d, J = 2.0,
6H), 1.93 (dd, J = 6.2, 1.4, 3H); 13C NMR (100 MHz, Chloroform-d) δ =
152.10, 147.69, 147.34, 131.33, 121.40, 121.23, 110.36, 105.95, 60.22,
56.30, 55.91, 20.10; HRMS (ESI) m/z calcd for C12H16O3 ([M + Na]+):
231.0992; found: 231.0992.
To a stirred mixture of trimethoxyphenylacrylic acid (8.4 mmol),
CuSO4⋅5H2O (0.2 g, 0.8 mmol), NaSO2CF3 (3.9 g, 25.2 mmol) in
dichloromethane (DCM) (25 mL) and H2O (10 mL) was added TBHP
(5.1 mL, 42.0 mmol) at RT under the protection of nitrogen. The reac-
tion mixture was heated at reflux for 6 h. After the completion of the
reaction (TLC monitoring), the reaction mixture was quenched with
sodium thiosulfate (5.3 g, 33.5 mmol) in H2O (30 mL). Then the mixture
was extracted three times with 40 mL DCM. The organic phase was
collected, dried over anhydrous MgSO4, filtered, and concentrated in
reduced pressure. The residue was purified using silica gel chromatog-
raphy (Pet. Ether/EtOAc, v/v = 30:1).
7

J. Zhang et al.
Bioorganic Chemistry 115 (2021) 105179
Fig. 5. The protective effects of 5 and 19 on hippocampal neuronal injury induced by Mg-deficient media. (A) Cell viability was assessed by MTT assay in primary
hippocampal neurons. Both 5 and 19 exhibited no cytotoxicity on the normal primary hippocampal neurons (n = 6). (B) Both 5 and 19 treatments improved neuronal
viability impaired by Mg-deficient media (n = 6). (C) Representative hippocampal neuron morphology was observed by an inverted optical microscope. 5 or 19
treatments rescued axonal and dendritic arbors injured by Mg-deficient media. The control group (CG), the model group (MG), the low-dose of 5 (LDG of 5), high-
dose of 5 (HDG of 5), LDG of 19, and HDG of 19 were treated with normal media, Mg2+-free media, Mg2+-free media containing 2.5
μ
g/mL of 5, Mg2+-free media
containing 5.0
μ
g/mL of 5, Mg2+-free media containing 2.5
μ
g/mL of 19 and Mg2+-free media containing 5.0
μg/mL of 19, respectively. Graphical representations of
the data were made with scatter plots. N.S. means not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.
dropwise (over 20 min) to the reaction mixture at 0–5 ◦C and stirring
was continued at RT for 20 h. After the completion of the reaction (TLC
monitoring), the reaction was quenched with sodium sulfite (2.5 g, 19.8
mmol). The reaction mixture was filtered, concentrated in reduced
pressure and purified on silica gel column chromatography (Pet. Ether/
EtOAc, v/v = 10:1) to obtain 6.9 g (44.8 mmol; yield: 74.4%) 2,4-dime-
thoxyphenol (2c). Finally, the synthesis of 2 was carried out via a three
steps of the general procedure E, the general procedure A and the gen-
eral procedure B from 2c to obtain 0.9 g (4.3 mmol; total yield: 19.8%)
of the target product. 1H NMR (400 MHz, Chloroform-d) δ = 6.70 (dq, J
= 15.9, 1.8, 1H), 6.54 (d, J = 2.8, 1H), 6.38 (d, J = 2.8, 1H), 6.24 (dq, J
= 15.9, 6.7, 1H), 3.83 (s, 3H), 3.78 (s, 3H), 3.74 (s, 3H), 1.91 (dd, J =
6.7, 1.8, 3H); 13C NMR (100 MHz, Chloroform-d) δ = 156.06, 153.61,
140.40, 131.82, 127.12, 125.22, 100.29, 98.93, 61.01, 55.75, 55.47,
18.81; HRMS (ESI) m/z calcd for C12H16O3 ([M + Na]+): 231.0992;
found: 231.0992.
Table 2
Drug likeliness properties and toxicity risks of compounds 5 and 19 according to
Osiris Property Explorer tool and their acute neurotoxicity.
Cpds
Drug-
Drug-
score
Toxicity risksa
TOX-ED50 (mg/kg)
likeness
Mb
Tc
Id
Re
5
1.04
0.19
0.79
0.68
+
+
+
+
352.0
(253.9–489.0)
368.9
19
+
+
+
+
(285.2–477.1)
a
(+) represent no bad effect, (–) represent bad effect.
M: mutagenic effect.
b
c
T: tumorigenic effect.
d
e
I: irritant effect.
R: reproductive effect.
4.1.10. (E)-1,2,5-trimethoxy-3-(prop-1-en-1-yl)benzene (2)
2,4-dimethoxybenzaldehyde (2d) (10.0 g, 60.2 mmol) and 80 mL
MeOH were placed in a 100 mL three-mouth flask with magnetic stirrer.
After the mixture was cooled down to 0 ◦C, 30% H2O2 (8.9 g, 78.5 mmol)
in MeOH (20 mL) was added dropwise (over 15 min) at 0–5 ◦C. Then 1.2
mL concentrated sulfuric acid (98%) in MeOH (20 mL) was added
4.1.11. (E)-1,2,3-trimethoxy-4-(prop-1-en-1-yl)benzene (3)
3 was synthesized according to the general procedure B from 3a to
obtain 1.5 g (7.2 mmol; yield: 62.1%) of the target product. 1H NMR
(400 MHz, Chloroform-d) δ = 7.10 (d, J = 8.7, 1H), 6.63 (d, J = 8.7, 1H),
6.58 (dd, J = 15.8, 1.9, 1H), 6.13 (dq, J = 15.9, 6.6, 1H), 3.87 (s, 3H),
8

J. Zhang et al.
Bioorganic Chemistry 115 (2021) 105179
3.85 (s, 3H), 3.84 (s, 3H), 1.88 (dd, J = 6.6, 1.8, 3H); 13C NMR (100
MHz, Chloroform-d) δ = 152.52, 150.89, 142.34, 125.16, 125.13,
124.99, 120.46, 107.72, 61.06, 60.88, 56.02, 18.84; HRMS (ESI) m/z
calcd for C12H16O3 ([M + Na]+): 231.0992; found: 231.0991.
4.1.16. (E)-1,5-dimethoxy-2-(prop-1-en-1-yl)-4-propylbenzene (8)
The synthesis of 8 was carried out via a five process of the general
procedure B, the general procedure D, the general procedure C and the
general procedure B from 2d to obtain 0.7 g (3.2 mmol; total yield:
21.8%) of the target product. 1H NMR (400 MHz, Chloroform-d) δ =
7.17 (s, 1H), 6.65 (dq, J = 15.8, 1.7, 1H), 6.43 (s, 1H), 6.12 (dq, J = 15.8,
6.6, 1H), 3.85 (s, 3H), 3.84 (s, 3H), 2.56–2.50 (m, 2H), 1.90 (dd, J = 6.6,
1.8, 3H), 1.67–1.53 (m, 2H), 0.96 (t, J = 7.4, 3H); 13C NMR (100 MHz,
Chloroform-d) δ = 157.29, 155.27, 127.64, 125.33, 123.92, 123.19,
119.02, 95.46, 55.86, 55.53, 31.68, 23.33, 18.86, 14.06; HRMS (ESI) m/
z calcd for C14H20O2 ([M + H]+): 221.1537; found:221.1539.
4.1.12. (E)-1,3,5-trimethoxy-2-(prop-1-en-1-yl)benzene (4)
4 was synthesized according to the general procedure B from 4a to
obtain 1.3 g (6.2 mmol; yield: 60.7%) of the target product. 1H NMR
(400 MHz, Chloroform-d) δ = 6.55 (dd, J = 16.0, 1.5, 1H), 6.51–6.41 (m,
1H), 6.13 (s, 2H), 3.82 (s, 6H), 3.81 (s, 3H), 1.89 (dd, J = 6.2, 1.3, 3H);
13C NMR (100 MHz, Chloroform-d) δ = 159.37, 158.76, 128.02, 120.94,
108.43, 90.76, 55.70, 55.29, 20.06; HRMS (ESI) m/z calcd for C12H16O3
([M + H]+): 209.1173; found: 209.1173.
4.1.17. (E)-2,6-dimethoxy-4-(prop-1-en-1-yl)phenol (9)
9 was synthesized according to the general procedure F from 9a to
obtain 0.4 g (2.1 mmol; yield: 55.6%) of the target product. 1H NMR
(400 MHz, Chloroform-d) δ = 6.57 (s, 2H), 6.31 (dq, J = 15.7, 1.8, 1H),
6.09 (dq, J = 15.5, 6.5, 1H), 5.46 (s, 1H), 3.89 (s, 6H), 1.86 (dd, J = 6.6,
1.7, 3H); 13C NMR (100 MHz, Chloroform-d) δ = 147.08, 133.95,
130.94, 129.60, 123.86, 102.60, 56.22, 18.32; HRMS (ESI) m/z calcd for
4.1.13. (E)-1,2,3-trimethoxy-5-(prop-1-en-1-yl)benzene (5)
5 was synthesized according to the general procedure B from 5a to
obtain 1.1 g (5.3 mmol; yield: 69.8%) of the target product. 1H NMR
(400 MHz, Chloroform-d) δ = 6.55 (s, 2H), 6.32 (dq, J = 15.6, 1.7, 1H),
6.15 (dq, J = 15.7, 6.5, 1H), 3.86 (s, 6H), 3.83 (s, 3H), 1.87 (dd, J = 6.5,
1.6, 3H); 13C NMR (100 MHz, Chloroform-d) δ = 153.27, 137.16,
133.78, 130.91, 125.31, 102.84, 60.91, 56.02, 18.37. HRMS (ESI) m/z
calcd for C12H16O3 ([M + Na]+): 231.0992; found: 231.0992.
C
11H14O3 ([M + Na]+): 217.0836; found: 217.0833.
4.1.18. (E)-2-ethoxy-1,3-dimethoxy-5-(prop-1-en-1-yl)benzene (10)
The synthesis of 10 was carried out via a two process of the general
procedure A and the general procedure B from 9a to obtain 0.8 g (3.6
mmol; total yield: 53.1%) of the target product. 1H NMR (400 MHz,
Chloroform-d) δ = 6.55 (s, 2H), 6.33 (dt, J = 15.6, 1.8, 1H), 6.15 (dq, J
= 15.7, 6.5, 1H), 4.04 (q, J = 7.1, 2H), 3.85 (s, 6H), 1.87 (dd, J = 6.6,
1.7, 3H), 1.35 (t, J = 7.1, 3H); 13C NMR (100 MHz, Chloroform-d) δ =
153.54, 136.12, 133.53, 130.93, 125.10, 102.91, 68.88, 56.02, 18.31,
15.47; HRMS (ESI) m/z calcd for C13H18O3 ([M + Na]+): 245.1149;
found: 245.1148.
4.1.14. (E)-1-ethoxy-4,5-dimethoxy-2-(prop-1-en-1-yl)benzene (6)
To a stirred mixture of 2,4,5-trimethoxybenzaldehyde (6c) (10.0 g,
51.0 mmol) in dry DCM (150 mL) was added dropwise (over 30 min)
BBr3 (12.8 g, 51.0 mmol) in dry DCM (50 mL) under the protection of
nitrogen at ꢀ 10 ◦C and stirring was continued at RT for 4 h. After the
completion of the reaction (TLC monitoring), the reaction mixture was
quenched with 80 mL H2O. Then the mixture was extracted three times
with 50 mL DCM. The organic phase was collected, dried over anhy-
drous MgSO4, filtered, concentrated in reduced pressure and purified on
silica gel column chromatography (Pet. Ether/EtOAc, v/v = 5:1) to
obtain 4.5 g (24.7 mmol; yield: 48.5%) 2-hydroxy-4,5-dimethoxybenzal-
dehyde (6b). Finally, the synthesis of 6 was carried out via a two steps
process of the general procedure A and the general procedure B from 6b
to obtain 0.8 g (3.6 mmol; total yield: 27.8%) of the target product. 1H
NMR (400 MHz, Chloroform-d) δ = 6.94 (s, 1H), 6.66 (dq, J = 15.9, 1.8,
1H), 6.49 (s, 1H), 6.11 (dq, J = 15.9, 6.7, 1H), 4.01 (q, J = 7.0, 2H), 3.86
4.1.19. (E)-1,3-dimethoxy-5-(prop-1-en-1-yl)-2-propoxybenzene (11)
The synthesis of 11 was carried out via a two process of the general
procedure A and the general procedure B from 9a to obtain 0.7 g (3.0
mmol; total yield: 56.7%) of the target product. 1H NMR (400 MHz,
Chloroform-d) δ = 6.55 (s, 2H), 6.32 (dq, J = 15.6, 1.7, 1H), 6.14 (dq, J
= 15.7, 6.5, 1H), 3.91 (t, J = 6.9, 2H), 3.84 (s, 6H), 1.87 (dd, J = 6.6, 1.6,
3H), 1.77 (h, J = 7.3, 2H), 1.00 (t, J = 7.4, 3H); 13C NMR (100 MHz,
Chloroform-d) δ = 153.52, 136.55, 133.47, 130.98, 125.05, 103.06,
75.18, 56.08, 23.32, 18.36, 10.36; HRMS (ESI) m/z calcd for C14H20O3
([M + Na]+): 259.1305; found: 259.1307.
(d, J = 3.3, 6H), 1.89 (dd, J = 6.6, 1.8, 3H), 1.42 (t, J = 7.0, 3H); 13
C
NMR (100 MHz, Chloroform-d) δ = 149.95, 148.63, 143.50, 125.16,
124.08, 119.49, 109.45, 99.52, 65.60, 56.41, 56.06, 18.82, 15.08;
HRMS (ESI) m/z calcd for C13H18O3 ([M + Na]+): 245.1149; found:
245.1147.
4.1.20. (E)-1,3-diisopropyl-2-methoxy-5-(prop-1-en-1-yl)benzene (12)
The synthesis of 12 was carried out via a three process of the general
procedure E, the general procedure A and the general procedure B from
12c to obtain 0.4 g (1.7 mmol; total yield: 29.4%) of the target product.
1H NMR (400 MHz, Chloroform-d) δ = 7.07 (s, 2H), 6.38 (dq, J = 15.8,
1.7, 1H), 6.16 (dq, J = 15.7, 6.6, 1H), 3.73 (s, 3H), 3.32 (p, J = 6.9, 2H),
1.88 (dd, J = 6.6, 1.7, 3H), 1.25 (d, J = 7.0, 12H); 13C NMR (100 MHz,
Chloroform-d) δ = 153.68, 141.62, 134.10, 131.08, 124.48, 121.60,
62.25, 26.51, 24.06, 18.44; HRMS (ESI) m/z calcd for C16H24O ([M +
H]+): 233.1900; found: 233.1900.
4.1.15. (E)-1-ethyl-2,4-dimethoxy-5-(prop-1-en-1-yl)benzene (7)
To a stirred mixture of 1-(2,4-dimethoxyphenyl)ethan-1-one (7c)
(3.6 g, 20.0 mmol) in CF3COOH (20 mL) was added Et3SiH (6.4 mL,
20.0 mmol) at RT. The reaction mixture was heated at 60–65 ◦C for 8 h.
After the completion of the reaction (TLC monitoring), the reaction
mixture was quenched with 80 mL H2O. Then the mixture was extracted
three times with 50 mL EtOAc. The organic phase was collected, dried
over anhydrous MgSO4, filtered, concentrated in reduced pressure and
purified on silica gel column chromatography (Pet. Ether/EtOAc, v/v =
30:1) to obtain 2.0 g (12.0 mmol; yield: 60.2%) 1-ethyl-2,4-dimethoxy-
benzene (7b). Finally, the synthesis of 7 was carried out via a two steps
process of the general procedure C and the general procedure B from 7b
to obtain 0.9 g (4.4 mmol; total yield: 22.2%) of the target product. 1H
NMR (400 MHz, Chloroform-d) δ = 7.18 (s, 1H), 6.64 (dq, J = 15.9, 1.8,
1H), 6.42 (s, 1H), 6.11 (dq, J = 15.9, 6.6, 1H), 3.85 (s, 3H), 3.84 (s, 3H),
2.57 (q, J = 7.5, 2H), 1.88 (dd, J = 6.6, 1.8, 3H), 1.17 (t, J = 7.5, 3H);
13C NMR (100 MHz, Chloroform-d) δ = 157.12, 155.21, 126.74, 125.25,
124.71, 123.97, 119.08, 95.41, 55.91, 55.53, 22.67, 18.89, 14.57;
HRMS (ESI) m/z calcd for C13H18O2 ([M + H]+): 207.1380; found:
207.1382.
4.1.21. (E)-2-methoxy-1,3-dimethyl-5-(prop-1-en-1-yl)benzene (13)
The synthesis of 13 was carried out via a two process of the general
procedure A and the general procedure B from 13b to obtain 0.9 g (5.1
mmol; total yield: 55.6%) of the target product. 1H NMR (400 MHz,
Chloroform-d) δ = 7.00 (s, 2H), 6.31 (dd, J = 15.8, 2.0, 1H), 6.13 (dq, J
= 15.7, 6.5, 1H), 3.72 (s, 3H), 2.28 (s, 6H), 1.87 (dd, J = 6.6, 1.7, 3H);
13C NMR (100 MHz, Chloroform-d) δ = 156.02, 133.58, 130.73, 130.50,
126.25, 124.53, 59.73, 18.47, 16.12; HRMS (ESI) m/z calcd for C12H16
([M + H]+): 177.1274; found: 177.1277.
O
4.1.22. (E)-1-bromo-2,3-dimethoxy-5-(prop-1-en-1-yl)benzene (14)
14 was synthesized according to the general procedure B from 14a to
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J. Zhang et al.
Bioorganic Chemistry 115 (2021) 105179
obtain 0.6 g (2.3 mmol; yield: 54.8%) of the target product. 1H NMR
(400 MHz, Chloroform-d) δ = 7.09 (s, 1H), 6.80 (s, 1H), 6.26 (dt, J =
15.6, 1.8, 1H), 6.21–6.07 (m, 1H), 3.85 (s, 3H), 3.83 (s, 3H), 1.86 (dt, J
= 6.4, 1.5, 3H); 13C NMR (100 MHz, Chloroform-d) δ = 153.57, 145.28,
135.31, 129.49, 126.55, 122.16, 117.73, 109.01, 60.63, 56.00, 18.40;
HRMS (ESI) m/z calcd for C11H13BrO2 ([M + H]+): 257.0172; found:
257.0183.
4.1.29. (E)-5-(hex-1-en-1-yl)-1,2,3-trimethoxybenzene (21)
21 was synthesized according to the general procedure B from 21a to
obtain 2.2 g (8.8 mmol; yield: 59.1%) of the target product. 1H NMR
(400 MHz, Chloroform-d) δ = 6.57 (s, 2H), 6.30 (d, J = 15.9, 1H), 6.14
(dt, J = 15.6, 6.8, 1H), 3.87 (s, 6H), 3.83 (s, 3H), 2.26–2.15 (m, 2H),
1.52–1.31 (m, 4H), 0.93 (t, J = 7.1, 3H); 13C NMR (100 MHz,
Chloroform-d) δ = 153.27, 137.18, 133.77, 130.78, 129.60, 102.90,
60.92, 56.04, 32.63, 31.54, 22.28, 13.97; HRMS (ESI) m/z calcd for
4.1.23. (E)-1,2-dimethoxy-4-(prop-1-en-1-yl)benzene (15)
C
15H22O3 ([M + Na]+): 273.1462; found: 273.1464.
15 was synthesized according to the general procedure B from 15a to
obtain 0.7 g (3.9 mmol; yield: 73.5%) of the target product. 1H NMR
(400 MHz, Chloroform-d) δ = 6.91–6.81 (m, 2H), 6.78 (d, J = 8.2, 1H),
6.33 (dq, J = 15.6, 1.7, 1H), 6.10 (dq, J = 15.7, 6.6, 1H), 3.88 (s, 3H),
3.85 (s, 3H), 1.86 (dd, J = 6.6, 1.7, 3H); 13C NMR (100 MHz,
Chloroform-d) δ = 148.97, 148.16, 131.14, 130.64, 123.73, 118.67,
111.18, 108.46, 55.87, 55.74, 18.38; HRMS (ESI) m/z calcd for
4.1.30. (E)-4-(3,4,5-trimethoxyphenyl)but-3-en-2-one (22)
22 was synthesized according to the general procedure B from 22a to
obtain 3.1 g (13.1 mmol; yield: 50.4%) of the target product. 1H NMR
(400 MHz, Chloroform-d) δ = 7.43 (d, J = 16.2, 1H), 6.77 (s, 2H), 6.63
(d, J = 16.2, 1H), 3.89 (s, 6H), 3.88 (s, 3H), 2.38 (s, 3H); 13C NMR (100
MHz, Chloroform-d) δ = 198.27, 153.49, 143.51, 140.31, 129.88,
126.55, 105.39, 60.99, 56.17, 27.44; HRMS (ESI) m/z calcd for
C
11H14O2 ([M + Na]+): 201.0886; found: 201.0887.
C
13H16O4 ([M + Na]+): 259.0941; found: 259.0942.
4.1.24. (E)-1,3-dimethoxy-5-(prop-1-en-1-yl)benzene (16)
16 was synthesized according to the general procedure B from 16a to
obtain 0.9 g (5.0 mmol; yield: 72.1%) of the target product. 1H NMR
(400 MHz, Chloroform-d) δ = 6.50 (d, J = 2.3, 2H), 6.39–6.30 (m, 2H),
6.23 (dq, J = 15.7, 6.4, 1H), 3.79 (s, 6H), 1.88 (dd, J = 6.5, 1.5, 3H); 13C
NMR (100 MHz, Chloroform-d) δ = 160.88, 140.02, 131.01, 126.34,
103.95, 99.08, 55.29, 18.43; HRMS (ESI) m/z calcd for C11H14O2 ([M +
H]+):179.1067; found:179.1073.
4.1.31. (E)-3-(2,4,5-trimethoxyphenyl)acrylic acid (23)
To a stirred mixture of 6c (1.2 g, 5.5 mmol) and propanedioic acid
(1.1 g, 10.9 mmol) in pyridine (20 mL) was added piperidine (0.2 mL,
2.2 mmol) at RT. The reaction mixture was heated at reflux for 5 h. After
the completion of the reaction (TLC monitoring), the reaction mixture
pH was adjusted to 3.0 using 1 N HCI. Then the mixture was extracted
three times with 100 mL EtOAc. The organic phase was collected, dried
over anhydrous MgSO4, filtered and concentrated in reduced pressure to
obtain 1.0 g (4.2 mmol; yield: 68.6%) 23. 1H NMR (400 MHz,
Chloroform-d) δ = 8.07 (d, J = 16.0, 1H), 7.03 (s, 1H), 6.50 (s, 1H), 6.38
(d, J = 16.0, 1H), 3.93 (s, 3H), 3.88 (s, 3H), 3.87 (s, 3H); 13C NMR (100
MHz, Chloroform-d) δ = 173.16, 154.26, 152.57, 143.28, 141.88,
114.67, 114.59, 110.98, 96.72, 56.43, 56.33, 56.08; HRMS (ESI) m/z
calcd for C12H14O5 ([M + Na]+): 261.0734; found: 261.0735.
4.1.25. (E)-1-methoxy-4-(prop-1-en-1-yl)benzene (17)
17 was synthesized according to the general procedure B from 17a to
obtain 1.0 g (6.7 mmol; yield: 67.2%) of the target product. 1H NMR
(400 MHz, Chloroform-d) δ = 7.27–7.19 (m, 2H), 6.85–6.76 (m, 2H),
6.32 (dq, J = 15.7, 1.8, 1H), 6.06 (dq, J = 15.7, 6.6, 1H), 3.75 (s, 3H),
1.83 (dd, J = 6.6, 1.7, 3H); 13C NMR (100 MHz, Chloroform-d) δ =
158.66, 130.86, 130.44, 126.95, 123.47, 113.95, 55.26, 18.48; HRMS
(ESI) m/z calcd for C10H12O ([M + H]+): 149.0961; found: 149.0963.
4.1.32. 1-allyl-2,4,5-trimethoxybenzene (24)
To a stirred mixture of 3-bromopropene (4.6 g, 37.7 mmol), Pd
(OAc)2 (0.8 g, 3.7 mmol), Cu2O (0.03 g, 0.2 mmol) and Ag2CO3 (16.0 g,
56.6 mmol) in toluene (360 mL) and DMSO (18 mL) was added 6c (4.0 g,
18.9 mmol) at RT. The reaction mixture was heated at 110 ◦C for 6 h.
After the completion of the reaction (TLC monitoring), the reaction
mixture was quenched with 100 mL H2O. Then the mixture was
extracted three times with 100 mL EtOAc. The organic phase was
collected, dried over anhydrous MgSO4, filtered, concentrated in
reduced pressure and purified on silica gel column chromatography
(Pet. Ether/EtOAc, v/v = 20:1) to obtain 0.2 g (1.0 mmol; yield: 4.7%)
24. 1H NMR (400 MHz, Chloroform-d) δ = 6.69 (s, 1H), 6.53 (s, 1H),
5.96 (ddt, J = 15.7, 10.8, 6.5, 1H), 5.09–4.99 (m, 2H), 3.88 (s, 3H), 3.83
(s, 3H), 3.80 (s, 3H), 3.33 (dt, J = 6.5, 1.6, 2H); 13C NMR (100 MHz,
Chloroform-d) δ = 151.32, 147.90, 143.01, 137.33, 120.02, 115.20,
113.96, 98.01, 56.62, 56.60, 56.25, 33.67; HRMS (ESI) m/z calcd for
4.1.26. 1,2,3-trimethoxy-5-vinylbenzene (18)
18 was synthesized according to the general procedure F from 18a to
obtain 0.5 g (2.6 mmol; yield: 53.2%) of the target product. 1H NMR
(400 MHz, Chloroform-d) δ = 6.69–6.58 (m, 3H), 5.66 (dd, J = 17.5, 0.8,
1H), 5.21 (dd, J = 10.8, 0.8, 1H), 3.88 (s, 6H), 3.85 (s, 3H); 13C NMR
(100 MHz, Chloroform-d) δ = 153.27, 137.99, 136.73, 133.28, 113.22,
103.26, 60.88, 56.04; HRMS (ESI) m/z calcd for C11H14O3 ([M + Na]+):
217.0836; found: 217.0835.
4.1.27. (E)-5-(but-1-en-1-yl)-1,2,3-trimethoxybenzene (19)
19 was synthesized according to the general procedure B from 19a to
obtain 1.1 g (4.9 mmol; yield: 75.9%) of the target product. 1H NMR
(400 MHz, Chloroform-d) δ = 6.57 (s, 2H), 6.31 (dt, J = 15.7, 1.5, 1H),
6.18 (dt, J = 15.7, 6.4, 1H), 3.87 (s, 6H), 3.83 (s, 3H), 2.29–2.17 (m,
2H), 1.09 (t, J = 7.4, 3H); 13C NMR (100 MHz, Chloroform-d) δ =
153.28, 137.19, 133.76, 132.26, 128.70, 102.91, 60.93, 56.04, 25.98,
13.69; HRMS (ESI) m/z calcd for C13H18O3 ([M + Na]+): 245.1149;
found: 245.1149.
C
12H16O3 ([M + H]+): 209.1173; found: 209.1177.
4.1.33. Methyl (E)-3-(2,4,5-trimethoxyphenyl)acrylate (25)
25 was synthesized according to the general procedure G from 23 to
obtain 0.7 g (2.8 mmol; yield: 91.1%) of the target product. 1H NMR
(400 MHz, Chloroform-d) δ = 7.96 (d, J = 16.1, 1H), 7.00 (s, 1H), 6.49
(s, 1H), 6.36 (d, J = 16.1, 1H), 3.92 (s, 3H), 3.87 (s, 3H), 3.85 (s, 3H),
3.79 (s, 3H); 13C NMR (100 MHz, Chloroform-d) δ = 168.21, 153.90,
152.12, 143.24, 139.74, 115.37, 114.89, 110.88, 96.86, 56.43, 56.35,
56.06, 51.52; HRMS (ESI) m/z calcd for C13H16O5 ([M + Na]+):
275.0890; found: 275.0886.
4.1.28. (E)-1,2,3-trimethoxy-5-(pent-1-en-1-yl)benzene (20)
20 was synthesized according to the general procedure B from 20a to
obtain 2.1 g (8.9 mmol; yield: 55.6%) of the target product. 1H NMR
(400 MHz, Chloroform-d) δ = 6.57 (s, 2H), 6.31 (dt, J = 15.8, 1.5, 1H),
6.14 (dt, J = 15.7, 6.9, 1H), 3.87 (s, 6H), 3.83 (s, 3H), 2.18 (qd, J = 7.1,
1.4, 2H), 1.50 (h, J = 7.4, 2H), 0.96 (t, J = 7.4, 3H); 13C NMR (100 MHz,
Chloroform-d) δ = 153.28, 137.21, 133.75, 130.56, 129.80, 102.92,
60.93, 56.05, 35.05, 22.56, 13.77; HRMS (ESI) m/z calcd for C14H20O3
([M + Na]+): 259.1305; found: 259.1306.
4.1.34. (E)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-ol (26)
Methyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (26a) was synthe-
sized according to the general procedure G from (E)-3-(3,4,5-trime-
thoxyphenyl)acrylic acid (26b) to obtain 3.0 g (11.9 mmol; yield:
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J. Zhang et al.
Bioorganic Chemistry 115 (2021) 105179
90.8%) of the target product.
Institutes of Health Guide for the Care and Use of Laboratory Animals.
All male KunMing mice (weight 25–30 g) and newborn SD rats (weight
5–8 g) were purchased from Chengdu Dossy Experimental Animals Co.,
Ltd. (Sichuan, China). In the PTZ-induced seizure test, a dose of PTZ 75
mg/kg was used to induce tonic seizures (Racine’s score ≥ 4) lasting for
at least 5 s in all mice tested. PTZ was dissolved in saline, and all the
tested compounds were prepared with 5–10% Solutol HS-15 and
90–95% saline, which was intraperitoneally (i.p.) injected in a standard
volume of 0.1 mL/10 g body weight [35,36]. In the PTZ test, all the
compounds were injected i.p. into mice at the dose of 100 mg/kg body
weight, and then PTZ was injected i.p. into mice at 3 min after the
treatment of compounds. The antiepileptic activities were evaluated
using the average Racine’s score at the endpoint of 0.5 h after injection
of PTZ [37]. The Racine grading criteria was stage 0 (normal non-
epileptic activity), stage 1 (facial movements, wet dog shaking, or
scratching), stage 2 (head nodding or tail flicking), stage 3 (one forelimb
extension or one limb clonus), stage 4 (multiple limb clonus or tonic)
and stage 5 (falling, tumbling and generalized tonic-clonic seizures). The
latency of onset time of seizure (Racine’s score ≥ 1) were also recorded.
The clonic seizures rate (CSR) and the tonic seizure rate (TSR) were
calculated according to the seizure severity that Racine’s score (1–3)
were classified as the clonic seizures and Racine’s score (4–5) as the
tonic seizure. Besides synthesized compounds, the ASA group (dissolved
in 10% Solutol HS-15 and 90% saline) and the solvent group were
included as a clinical drug control and the negative control group (NC).
The solvent was injected i.p. into mice in a volume of 0.1 mL/10 g body
weight, and then saline was injected i.p. into mice at 3 min after the
treatment of solvent, which was used as a blank control group (BC).
To a stirred mixture of 26a (2.3 g, 9.1 mmol) in dry THF (30 mL) was
added dropwise (over 20 min) 1.5 M solution of diisobutyl aluminium
hydride (DIBAL-H) in hexanes (14.0 mL, 21.0 mmol) kept at ꢀ 40 ◦C
under the protection of nitrogen and stirring was continued for 10 min.
After the completion of the reaction (TLC monitoring), the reaction
mixture was quenched with 200 mL H2O. The pH of the mixture was
adjusted to 3.0 by dilute hydrochloric acid. Then the mixture was
extracted three times with 200 mL EtOAc. The organic phase was
collected, dried over anhydrous MgSO4, filtered, concentrated in
reduced pressure and purified on silica gel column chromatography
(Pet. Ether/EtOAc, v/v = 20:1) to obtain 1.3 g (5.8 mmol; total yield:
57.7%) 26. 1H NMR (400 MHz, Chloroform-d) δ = 6.59 (s, 2H), 6.51 (dt,
J = 15.9, 1.6, 1H), 6.26 (dt, J = 15.7, 5.7, 1H), 4.30 (dd, J = 5.7, 1.5,
2H), 3.84 (s, 6H), 3.82 (s, 3H), 1.86 (s, 1H); 13C NMR (100 MHz,
Chloroform-d) δ = 153.32, 137.88, 132.46, 131.08, 128.09, 103.53,
63.61, 60.95, 56.08; HRMS (ESI) m/z calcd for C12H16O4 ([M + Na]+):
247.0941; found: 247.0942.
4.1.35. (E)-1,2,4-trimethoxy-5-(3,3,3-trifluoroprop-1-en-1-yl)benzene
(27)
27 was synthesized according to the general procedure H from 23 to
obtain 1.0 g (3.8 mmol; yield: 46.9%) of the target product. 1H NMR
(400 MHz, Chloroform-d) δ = 7.37 (dq, J = 16.1, 2.2, 1H), 6.92 (s, 1H),
6.51 (s, 1H), 6.17 (dq, J = 16.2, 6.7, 1H), 3.92 (s, 3H), 3.87 (s, 6H); 13
C
NMR (100 MHz, Chloroform-d) δ = 153.21, 151.58, 143.23, 132.53,
132.46, 132.39, 132.32, 128.25, 125.58, 122.91, 120.24, 114.25,
113.92, 113.78, 113.59, 113.26, 110.99, 96.98, 56.53, 56.32, 56.06;
HRMS (ESI) m/z calcd for C12H13F3O3 ([M + Na]+): 285.0709; found:
285.0712.
4.3. Brain tissue preparation
4.1.36. (E)-1,2,3-trimethoxy-5-(3,3,3-trifluoroprop-1-en-1-yl)benzene
(28)
Five days after conducting the behavioral tests, each group randomly
selected four mice in BC, NC, compounds 5, and 19 groups. The selected
mouse was deeply anesthetised by injecting 10% chloral hydrate (4 mL/
kg) i.p. and then perfused through the left ventricle with 20 mL saline,
followed by 20 mL 4% paraformaldehyde (PFA). The brain tissues were
carefully removed and then were fixed with 4% PFA for 72 h. The fixed
tissues were dehydrated sequentially in 70%, 80%, 90% alcohol for 90
min. The tissue blocks were paraffin-embedded, and serial coronal sec-
tions were cut with a thickness of 5 um for later use [38].
28 was synthesized according to the general procedure H from 26b
to obtain 1.1 g (4.2 mmol; yield: 43.1%) of the target product. 1H NMR
(400 MHz, Chloroform-d) δ = 7.06 (dq, J = 16.1, 2.2, 1H), 6.66 (s, 2H),
6.11 (dq, J = 15.9, 6.5, 1H), 3.88 (s, 6H), 3.87 (s, 3H); 13C NMR (100
MHz, Chloroform-d) δ = 153.50, 139.73, 137.76, 137.69, 137.63,
137.56, 128.95, 127.68, 125.01, 122.34, 119.66, 115.64, 115.30,
114.97, 114.63, 104.69, 60.93, 56.13; HRMS (ESI) m/z calcd for
C
12H13F3O3 ([M + Na]+): 285.0709; found: 285.0702.
4.4. Nissl staining
4.1.37. 1,2,3-trimethoxy-5-propylbenzene (29)
29 was synthesized according to the general procedure D from 5 to
obtain 1.9 g (9.0 mmol; yield: 81.2%) of the target product. 1H NMR
(400 MHz, Chloroform-d) δ = 6.39 (s, 2H), 3.84 (s, 6H), 3.81 (s, 3H),
2.56–2.48 (m, 2H), 1.70–1.56 (m, 2H), 0.94 (t, J = 7.3, 3H); 13C NMR
(100 MHz, Chloroform-d) δ = 152.99, 138.53, 135.92, 105.27, 60.81,
55.99, 38.50, 24.66, 13.88; HRMS (ESI) m/z calcd for C12H18O3 ([M +
Na]+): 233.1149; found: 233.1148.
Nissl staining was carried out according to the standard practice of
pathological examination. In brief, the coronal sections were immersed
in 3-aminopropyl-3-ethylylsilane (APES) to make the brain tissues
closely packed to the slides. After dewaxed and rehydrated, the sections
were stained with a toluidine blue solution at 50 ◦C for 20 min, washed
with distilled water, soaked in 70% alcohol, and differentiated in 95%
alcohol. After rapidly dehydrated with anhydrous alcohol, the sections
were cleaned with xylene until transparent and sealed tightly with
neutral gum. Nissl staining images were acquired by a Pannoramic 250
digital slice scanner (Danjier, China) and analyzed by Image-Pro Plus 6.0
(Media Cybernetics, USA). The mean density of neurons in the CA1 re-
gion of the hippocampus was calculated from up to three non-
overlapping and representative images obtained from each mouse’s
brain slice in different groups.
4.1.38. 5-butyl-1,2,3-trimethoxybenzene (30)
30 was synthesized according to the general procedure D from 19 to
obtain 1.8 g (8.0 mmol; yield: 85.6%) of the target product. 1H NMR
(400 MHz, Chloroform-d) δ = 6.39 (s, 2H), 3.84 (s, 6H), 3.82 (s, 3H),
2.59–2.50 (m, 2H), 1.59 (tt, J = 9.3, 6.9, 2H), 1.37 (h, J = 7.3, 2H), 0.93
(t, J = 7.4, 3H); 13C NMR (100 MHz, Chloroform-d) δ = 153.01, 138.76,
135.88, 105.22, 60.85, 56.02, 36.15, 33.77, 22.47, 13.98; HRMS (ESI)
m/z calcd for C13H20O3 ([M + Na]+): 247.1305; found: 247.1304.
4.5. Caspase-3 activity determination
4.2. Pentylenetetrazole (PTZ)-induced seizure test
The devoted coronal sections were dewaxed, treated with 3%
hydrogen peroxide in methanol for 10 min, and then washed with PBS
three times for 5 min each at RT. Subsequently, the sections were
immersed in 0.01 M citrate solution (pH 6.0), heated to boiling status in
a microwave oven, and then cooled at room temperature. After 5 min,
the heating–cooling operation was repeated once. When the sections
In this study, all experiment procedures for the use and care of the
animals were approved by the Sichuan University Ethics Committee for
the Use of Laboratory Animals with a license number as SYXK (Chuan)
2018–113 and were strictly conducted in compliance with the National
11

J. Zhang et al.
Bioorganic Chemistry 115 (2021) 105179
were cooled to room temperature, sections were washed with PBS twice
for 5 min each at RT. The sections were rinsed and added with normal
goat serum (ZSGB-BIO, Beijing, China) in drops for blocking at RT for 20
min, then incubated with primary antibody against caspase-3 (rabbit
polyclonal antibody, 1:50 dilution, abcam, UK) overnight at 4 ◦C. The
secondary antibody with biotin-labelled goat anti-rabbit (ZSGB-BIO,
Beijing, China) was added dropwise, incubated at 37 ◦C for 30 min, and
then washed with PBS three times for 5 min each time. The sections were
immediately developed with DAB chromogen kit (ZSGB-BIO, Beijing,
China) for 2 min at RT and then washed with distilled water. Finally, the
sections were slightly re-dyed with hematoxylin, dehydrated, trans-
parently treated, and sealed with neutral gum.
4.8. Neurotoxicity test
The rotarod test’s acute neurotoxicity of compounds 5 and 19 was
measured in mice [41]. The mice were trained to place on a diameter 4
cm rod rotating at a speed of 24 rpm. The trained mice were selected
and randomly grouped, one day later and administrated the tes-
ting compounds through the intraperitoneal route to obtain TD50 (the
median neurotoxic dose). 3, 30, and 60 min after drug treatment, each
mouse was placed on rotarod rotating at a speed of 24 rpm. Neurologic
toxicity is defined as the mouse not being kept on the rod for 3 min test
(Falling more than three times from the rotarod).
Images were collected by a Pannoramic 250 digital slice scanner
(Danjier, China) and analyzed by Image-Pro Plus 6.0 (Media Cyber-
netics, USA). Integrated optical density (IOD) and the positive area of all
images in the CA1 region of the hippocampus were measured from up to
three non-overlapping and representative images obtained from the
brain slice of each mouse in different groups. The mean optical density
(MOD) of each image was also calculated by dividing IOD by the positive
area.
4.9. Statistical analysis
All statistical analyses were performed using IBM SPSS Statistics 26.0
software, and the graphical representations were carried out in Graph-
Pad Prism 8.0. If the data were normally distributed and groups had
equal variances, multiple comparisons among groups were evaluated by
one-way analysis of variance (ANOVA) followed by LSD post hoc anal-
ysis. If the data were normally distributed and groups had unequal
variances, multiple comparisons among groups were performed using
one-way ANOVA with Dunnett’s T3 test. If data were not normally
distributed (Racine’s score), the test group and NC group comparisons
were carried out by the Mann-Whitney U test (nonparametric test). The
CSR and TSR were analyzed using fisher’s exact test between the test
group and NC group. The values of p < 0.05 were considered statistically
significant.
4.6. Culture of primary hippocampal neurons
Primary hippocampal neuron was isolated from the cerebral hippo-
campi of newborn SD rats according to the literature[39] with minor
modifications. Briefly, after dissection, the hippocampi were digested by
treatment with phosphate-buffered saline (PBS) containing 0.25% (m/v)
trypsin (Gibco, USA) for 1 h at 37 ◦C. After the digestion was terminated
by adding 1 mL fetal bovine serum (Gibco, USA), the cell suspension was
Declaration of Competing Interest
filtered through a 70 μm cell strainer (BD Biosciences, USA) to remove
chunks of undissociated tissue. The collected lysate containing hippo-
campal neurons was centrifugated at 1000 rpm/min for 10 min at 4 ◦C to
remove enzymes and cellular debris. The resulting cell pellet was
resuspended in DMEM/F12 (Gibco, USA) supplemented with 1% (v/v)
streptomycin-penicillin (Hyclone, USA) and 20% (v/v) fetal bovine
serum. The resuspended neurons (5.0 × 105 cells/well) were seeded into
a 24-well plastic culture dish pre-coated with 0.1 mg/mL poly-lysine
(Solarbio, Beijing, China), and incubated for 4 h at 37 ◦C. The culture
medium was then replaced by a serum-free Neurobasal™-A (Gibco,
USA) medium supplemented 2% (v/v) B27 (Gibco, USA) and 1% (v/v)
streptomycin-penicillin. Medium change was performed by replacing
one-half of the volume with a fresh medium every day. Within a few
days of incubation, the primary hippocampal cultures form a dense
network of neurites, and the neurons were usually used for the experi-
ments 8 days after seeding.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by the National Natural Science Foundation
of China (No. 81473168 and No. 81602954); Key Research and Devel-
opment Projects of Sichuan Department of Science and Technology,
China (No. 2019YFS0328); Sichuan Youth Science and Technology
Innovation Research Team Funding, China (No.2016TD0001); Sichuan
Science and Technology Program, China (No.2019YJ0036); and the
Fundamental Research Funds of the Central Universities, China (No.
YJ201561).
References
4.7. Cell viability assay
[1] S.D. Reddy, I. Younus, V. Sridhar, D.S. Reddy, Neuroimaging Biomarkers of
Experimental Epileptogenesis and Refractory Epilepsy, Int. J. Mol. Sci. 20 (2019),
https://doi.org/10.3390/ijms20010220.
The cells viability was measured to evaluate the neuroprotective
effect of compound 5 and 19 in Mg2+-free induced neuronal damage
model by quantitative colorimetric assay with MTT (3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma- Aldrich,
USA), which is an indicator of the mitochondrial activity of living cells
[40]. Morphological characteristics of primary neurons were observed
using an inverted microscope (Changfang optical, Shanghai, China). In
brief, primary hippocampal neurons were incubated with 5 or 19 (dis-
[2] C. Zhang, P. Kwan, The Concept of Drug-Resistant Epileptogenic Zone, Front.
Neurol. 10 (2019) 558, https://doi.org/10.3389/fneur.2019.00558.
[3] A. Golyala, P. Kwan, Drug development for refractory epilepsy: The past 25 years
and beyond, Seizure 44 (2017) 147–156, https://doi.org/10.1016/j.
seizure.2016.11.022.
[4] M. Bialer, S.I. Johannessen, R.H. Levy, E. Perucca, T. Tomson, H.S. White, Progress
report on new antiepileptic drugs: A summary of the Thirteenth Eilat Conference
on New Antiepileptic Drugs and Devices (EILAT XIII), Epilepsia 58 (2017)
181–221. http://doi.org/10.1111/epi.13634.
[5] 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-trimethoxy-
cinnamic acid (TMCA) ester derivatives, Bioorg. Chem. 88 (2019), 102832,
https://doi.org/10.1016/j.bioorg.2019.03.006.
solved in Mg2+-free media to the final concentration of 2.5
μg/mL or 5.0
μ
g/mL) for 1 h and then returned to Mg2+ containing media for 24 h at
37 ◦C. The control group (CG) and the model group (MG) were treated
with normal media and Mg2+-free media, respectively. Following neu-
[6] V. Franco, J.A. French, E. Perucca, Challenges in the clinical development of new
antiepileptic drugs, Pharmacol. Res. 103 (2016) 95–104, https://doi.org/10.1016/
j.phrs.2015.11.007.
rons exposure to 100 μL MTT (5 mg/mL) for 4 h, the supernatants were
carefully removed, and 500 µL dimethyl sulfoxide (Solarbio, Beijing,
China) was added to dissolve the purple formazan. Finally, cell viability
was measured by a microplate reader (Bio-Rad, Richmond, CA, USA) at
the wavelength of 570 nm.
[7] A. Verrotti, A. Scaparrotta, M. Cofini, F. Chiarelli, G.M. Tiboni, Developmental
neurotoxicity and anticonvulsant drugs: a possible link, Reprod. Toxicol.
(Elmsford, N.Y.) 48 (2014) 72–80, https://doi.org/10.1016/j.
reprotox.2014.04.005.
12

J. Zhang et al.
Bioorganic Chemistry 115 (2021) 105179
¨
´
[8] A. Easter, M.E. Bell, J.R. Damewood, W.S. Redfern, J.-P. Valentin, M.J. Winter,
C. Fonck, R.A. Bialecki, Approaches to seizure risk assessment in preclinical drug
discovery, Drug Discovery Today 14 (2009) 876–884, https://doi.org/10.1016/j.
drudis.2009.06.003.
[24] K. Bjornstad, A. Helander, P. Hulten, O. Beck, Bioanalytical investigation of
asarone in connection with Acorus calamus oil intoxications, J. Anal. Toxicol. 33
(2009) 604–609, https://doi.org/10.1093/jat/33.9.604.
[25] K.-O. Cho, Z.R. Lybrand, N. Ito, R. Brulet, F. Tafacory, L. Zhang, L. Good, K. Ure, S.
G. Kernie, S.G. Birnbaum, H.E. Scharfman, A.J. Eisch, J. Hsieh, Aberrant
hippocampal neurogenesis contributes to epilepsy and associated cognitive
decline, Nat. Commun. 6 (2015) 6606, https://doi.org/10.1038/ncomms7606.
[26] A.M. Dam, Epilepsy and neuron loss in the hippocampus, Epilepsia 21 (1980)
617–629, https://doi.org/10.1111/j.1528-1157.1980.tb04315.x.
¨
[9] W. Loscher, H. Klitgaard, R.E. Twyman, D. Schmidt, New avenues for anti-epileptic
drug discovery and development, Nat. Rev. Drug Discovery 12 (2013) 757–776,
https://doi.org/10.1038/nrd4126.
[10] A.K. Ngugi, C. Bottomley, I. Kleinschmidt, J.W. Sander, C.R. Newton, Estimation of
the burden of active and life-time epilepsy: a meta-analytic approach, Epilepsia 51
(2010) 883–890, https://doi.org/10.1111/j.1528-1167.2009.02481.x.
[11] Q.-X. Chen, J.-K. Miao, C. Li, X.-W. Li, X.-M. Wu, X.-P. Zhang, Anticonvulsant
activity of acute and chronic treatment with a-asarone from Acorus gramineus in
seizure models, Biol. Pharm. Bull. 36 (2013) 23–30, https://doi.org/10.1248/bpb.
b12-00376.
[27] K. Tsunashima, C. Schwarzer, E. Kirchmair, W. Sieghart, G. Sperk, GABAA receptor
subunits in the rat hippocampus III: altered messenger RNA expression in kainic
acid-induced epilepsy, Neuroscience 80 (1997) 1019–1032, https://doi.org/
10.1016/S0306-4522(97)00144-9.
[28] Y. Huang, X. Liu, Y. Liao, C. Luo, D. Zou, X. Wei, Q. Huang, Y. Wu, MiR-181a
influences the cognitive function of epileptic rats induced by pentylenetetrazol, Int.
J. Clin. Exp. Path. 8 (2015) 12861–12868.
[12] C. Huang, W.-G. Li, X.-B. Zhang, L. Wang, T.-L. Xu, D. Wu, Y. Li, α-asarone from
Acorus gramineus alleviates epilepsy by modulating A-type GABA receptors,
Neuropharmacology 65 (2013) 1–11, https://doi.org/10.1016/j.
neuropharm.2012.09.001.
[29] J. Yue, J. He, Y. Wei, K. Shen, K. Wu, X. Yang, S. Liu, C. Zhang, H. Yang, Decreased
expression of Rev-Erb
α
in the epileptic foci of temporal lobe epilepsy and
[13] Z.-J. Wang, S.R. Levinson, L. Sun, T. Heinbockel, Identification of both GABAA
receptors and voltage-activated Na(+) channels as molecular targets of
activation of Rev-Erb
α
have anti-inflammatory and neuroprotective effects in the
pilocarpine model, J. Neuroinflammat. 17 (2020) 43, https://doi.org/10.1186/
s12974-020-1718-7.
anticonvulsant α-asarone, Front. Pharmacol. 5 (2014) 40, https://doi.org/
10.3389/fphar.2014.00040.
[30] M. Krajewska, H.G. Wang, S. Krajewski, J.M. Zapata, A. Shabaik, R. Gascoyne, J.
C. Reed, Immunohistochemical analysis of in vivo patterns of expression of CPP32
(Caspase-3), a cell death protease, Cancer Res. 57 (1997) 1605–1613.
[31] D.W. Nicholson, A. Ali, N.A. Thornberry, J.P. Vaillancourt, C.K. Ding, M. Gallant,
Y. Gareau, P.R. Griffin, M. Labelle, Y.A. Lazebnik, Identification and inhibition of
the ICE/CED-3 protease necessary for mammalian apoptosis, Nature 376 (1995)
37–43, https://doi.org/10.1038/376037a0.
[14] X. He, Y. Bai, M. Zeng, Z. Zhao, Q. Zhang, N. Xu, F. Qin, X. Wei, M. Zhao, N. Wu,
Z. Li, Y. Zhang, T.-P. Fan, X. Zheng, Anticonvulsant activities of α-asaronol ((E)-3’-
hydroxyasarone), an active constituent derived from α-asarone, Pharmacol.
Reports PR 70 (2018) 69–74, https://doi.org/10.1016/j.pharep.2017.08.004.
[15] J. Poplawski, B. Lozowicka, A.T. Dubis, B. Lachowska, S. Witkowski, D. Siluk,
J. Petrusewicz, R. Kaliszan, J. Cybulski, M. Strzałkowska, Z. Chilmonczyk,
Synthesis and hypolipidemic and antiplatelet activities of alpha-asarone isomers in
humans (in vitro), mice (in vivo), and rats (in vivo), J. Med. Chem. 43 (2000)
3671–3676, https://doi.org/10.1021/jm000905n.
[32] M. Sakurai, T. Nagata, K. Abe, T. Horinouchi, Y. Itoyama, K. Tabayashi, Survival
and death-promoting events after transient spinal cord ischemia in rabbits:
induction of Akt and caspase3 in motor neurons, J. Thoracic Cardiovasc. Surg. 125
(2003) 370–377, https://doi.org/10.1067/mtc.2003.112.
[16] C.M.G. Azevedo, C.M.M. Afonso, J.X. Soares, S. Reis, D. Sousa, R.T. Lima, M.
H. Vasconcelos, M. Pedro, J. Barbosa, L. Gales, M.M.M. Pinto, Pyranoxanthones:
Synthesis, growth inhibitory activity on human tumor cell lines and determination
of their lipophilicity in two membrane models, Eur. J. Med. Chem. 69 (2013)
798–816, https://doi.org/10.1016/j.ejmech.2013.09.012.
[33] S. Sombati, R.J. Delorenzo, Recurrent spontaneous seizure activity in hippocampal
neuronal networks in culture, J. Neurophysiol. 73 (1995) 1706–1711, https://doi.
org/10.1152/jn.1995.73.4.1706.
[34] https://www.organic-chemistry.org/prog/peo/.
[17] S. Rahmani-Nezhad, M. Safavi, M. Pordeli, S.K. Ardestani, L. Khosravani,
Y. Pourshojaei, M. Mahdavi, S. Emami, A. Foroumadi, A. Shafiee, Synthesis, in
vitro cytotoxicity and apoptosis inducing study of 2-aryl-3-nitro-2H-chromene
derivatives as potent anti-breast cancer agents, Eur. J. Med. Chem. 86 (2014)
562–569, https://doi.org/10.1016/j.ejmech.2014.09.017.
[35] T. Hanada, Y. Hashizume, N. Tokuhara, O. Takenaka, N. Kohmura, A. Ogasawara,
S. Hatakeyama, M. Ohgoh, M. Ueno, Y. Nishizawa, Perampanel: a novel, orally
active, noncompetitive AMPA-receptor antagonist that reduces seizure activity in
rodent models of epilepsy, Epilepsia 52 (2011) 1331–1340, https://doi.org/
10.1111/j.1528-1167.2011.03109.x.
[18] C. Schultze, B. Schmidt, Prenylcoumarins in One or Two Steps by a Microwave-
Promoted Tandem Claisen Rearrangement/Wittig Olefination/Cyclization
Sequence, J. Organic Chem. 83 (2018) 5210–5224, https://doi.org/10.1021/acs.
joc.8b00667.
[36] J. Vamecq, D. Lambert, J.H. Poupaert, B. Masereel, J.P. Stables, Anticonvulsant
activity and interactions with neuronal voltage-dependent sodium channel of
analogues of ameltolide, J. Med. Chem. 41 (1998) 3307–3313, https://doi.org/
10.1021/jm9608772.
¨
¨
[19] T. de Paulis, Y. Kumar, L. Johansson, S. Ramsby, H. Hall, M. Sallemark, K. Angeby-
[37] R.J. Racine, Modification of seizure activity by electrical stimulation: Cortical
areas, Electroencephalogr. Clin. Neurophysiol. 38 (1975) 1–12, https://doi.org/
10.1016/0013-4694(75)90204-7.
¨
Moller, S.O. Ogren, Potential neuroleptic agents. 4. Chemistry, behavioral
pharmacology, and inhibition of 3Hspiperone binding of 3,5-disubstituted N-(1-
ethyl-2-pyrrolidinyl)methyl-6-methoxysalicylamides, J. Med. Chem. 29 (1986)
61–69, https://doi.org/10.1021/jm00151a010.
[38] S. Khamse, S.S. Sadr, M. Roghani, G. Hasanzadeh, M. Mohammadian, Rosmarinic
acid exerts a neuroprotective effect in the kainate rat model of temporal lobe
epilepsy: Underlying mechanisms, Pharm. Biol. 53 (2015) 1818–1825, https://doi.
org/10.3109/13880209.2015.1010738.
[20] K. Yang, Y. Li, Q. Tang, L. Zheng, D. He, Synthesis, mitochondrial localization of
fluorescent derivatives of cinnamamide as anticancer agents, Eur. J. Med. Chem.
170 (2019) 45–54, https://doi.org/10.1016/j.ejmech.2019.03.001.
[39] B. Ahlemeyer, E. Baumgart-Vogt, Optimized protocols for the simultaneous
preparation of primary neuronal cultures of the neocortex, hippocampus and
cerebellum from individual newborn (P0.5) C57Bl/6J mice, J. Neurosci. Methods
149 (2005) 110–120, https://doi.org/10.1016/j.jneumeth.2005.05.022.
[40] F. Denizot, R. Lang, Rapid colorimetric assay for cell growth and survival,
J. Immunol. Methods 89 (1986) 271–277, https://doi.org/10.1016/0022-1759
(86)90368-6.
[21] J. Wang, Z. Cui, Y. Zhang, H. Li, L.-M. Wu, Z. Liu, Pd(II)-catalyzed decarboxylative
allylation and Heck-coupling of arene carboxylates with allylic halides and esters,
Org. Biomol. Chem. 9 (2011) 663–666, https://doi.org/10.1039/c0ob00696c.
[22] Z. Li, Z. Cui, Z.-Q. Liu, Copper- and iron-catalyzed decarboxylative tri- and
difluoromethylation of α, β-unsaturated carboxylic acids with CF3SO2Na and
(CF2HSO2)2Zn via a radical process, Org. Lett. 15 (2013) 406–409, https://doi.
org/10.1021/ol3034059.
[41] Z. Zhao, Y. Bai, X. Chen, S. Wu, X. He, Y. Bai, Y. Sun, X. Zheng, Design, synthesis
and biological evaluation of (E)-3-(3,4,5-trimethoxyphenyl) acrylic acid (TMCA)
amide derivatives as anticonvulsant and sedative agents, Med. Chem. Res. 27
(2018) 2387–2396, https://doi.org/10.1007/s00044-018-2228-3.
¨
¨
[23] W. Loscher, D. Honack, C.P. Fassbender, B. Nolting, The role of technical,
biological and pharmacological factors in the laboratory evaluation of
anticonvulsant drugs III. Pentylenetetrazole seizure models, Epilepsy Res. 8 (1991)
171–189, https://doi.org/10.1016/0920-1211(91)90062-K.
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