Design, synthesis, and anticonvulsant effects evaluation of nonimidazole histamine H3 receptor antagonists/inverse agonists containing triazole moiety

Article abstract of DOI:10.1080/14756366.2020.1774573

Histamine H₃ receptors (H₃R) antagonists/inverse agonists are becoming a promising therapeutic approach for epilepsy. In this article, novel nonimidazole H₃R antagonists/inverse agonists have been designed and synthesised via hybriding the H₃R pharmacophore (aliphatic amine with propyloxy chain) with the 1,2,4-triazole moiety as anticonvulsant drugs. The majority of antagonists/inverse agonists prepared here exerted moderate to robust activities in cAMP-response element (CRE) luciferase screening assay. 1-(3-(4-(3-Phenyl-4H-1,2,4-triazol-4-yl)phenoxy)propyl)piperidine (3l) and 1-(3-(4-(3-(4-chlorophenyl)-4H-1,2,4-triazol-4-yl)phenoxy)propyl)piperidine (3m) displayed the highest H₃R antagonistic activities, with IC₅₀ values of 7.81 and 5.92 nM, respectively. Meanwhile, the compounds with higher H₃R antagonistic activities exhibited protection for mice in maximal electroshock seizure (MES)-induced convulsant model. Moreover, the protection of 3m against the MES induced seizures was fully abrogated when mice were co-treated with RAMH, a CNS-penetrant H₃R agonist, which suggested that the potential therapeutic effect of 3m was through H₃R. These results indicate that the attempt to find new anticonvulsant among H₃R antagonists/inverse agonists is practicable.

Full text of DOI:10.1080/14756366.2020.1774573

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Design, synthesis, and anticonvulsant effects
evaluation of nonimidazole histamine H3 receptor
antagonists/inverse agonists containing triazole
moiety
Mingxia Song, Rui Yan, Yanhui Zhang, Dongfu Guo, Naiming Zhou &
XianQing Deng
To cite this article: Mingxia Song, Rui Yan, Yanhui Zhang, Dongfu Guo, Naiming Zhou & XianQing
Deng (2020) Design, synthesis, and anticonvulsant effects evaluation of nonimidazole histamine H3
receptor antagonists/inverse agonists containing triazole moiety, Journal of Enzyme Inhibition and
Medicinal Chemistry, 35:1, 1310-1321, DOI: 10.1080/14756366.2020.1774573
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1310–1321
https://doi.org/10.1080/14756366.2020.1774573
RESEARCH PAPER
Design, synthesis, and anticonvulsant effects evaluation of nonimidazole
histamine H3 receptor antagonists/inverse agonists containing triazole moiety
a
a
a
a
b
a
ꢀ
ꢀ
Mingxia Song , Rui Yan , Yanhui Zhang , Dongfu Guo , Naiming Zhou and XianQing Deng
aMedical College, Jinggangshan University, Ji’an, China; College of Life Sciences, Zhejiang University, Hangzhou, China
b
ABSTRACT
ARTICLE HISTORY
Received 7 February 2020
Revised 8 May 2020
Accepted 19 May 2020
Histamine H3 receptors (H3R) antagonists/inverse agonists are becoming a promising therapeutic approach
for epilepsy. In this article, novel nonimidazole H3R antagonists/inverse agonists have been designed and
synthesised via hybriding the H3R pharmacophore (aliphatic amine with propyloxy chain) with the 1,2,4-
triazole moiety as anticonvulsant drugs. The majority of antagonists/inverse agonists prepared here
exerted moderate to robust activities in cAMP-response element (CRE) luciferase screening assay. 1-(3-(4-
(3-Phenyl-4H-1,2,4-triazol-4-yl)phenoxy)propyl)piperidine (3l) and 1-(3-(4-(3-(4-chlorophenyl)-4H-1,2,4-triazol-
4-yl)phenoxy)propyl)piperidine (3m) displayed the highest H3R antagonistic activities, with IC50 values of
7.81 and 5.92 nM, respectively. Meanwhile, the compounds with higher H3R antagonistic activities exhib-
ited protection for mice in maximal electroshock seizure (MES)-induced convulsant model. Moreover, the
protection of 3m against the MES induced seizures was fully abrogated when mice were co-treated with
RAMH, a CNS-penetrant H3R agonist, which suggested that the potential therapeutic effect of 3m was
through H3R. These results indicate that the attempt to find new anticonvulsant among H3R antagonists/
inverse agonists is practicable.
KEYWORDS
Epilepsy; anticonvulsant; H3
receptor antagonists/inverse
agonists; hybrid; 1,2,4-
triazole
1. Introduction
related to epilepsy inextricably12,13. Therefore, more attention has
been focussed on H3R as an attractive therapeutic target for epi-
lepsy treatment14.
Epilepsy,
a very common neurologic disorder, affects about
around 1% of world population1,2. Presently, antiepileptic drugs
(AEDs) are the main strategy of therapy. However, the AEDs avail-
able in the clinic such as phenytoin, carbamazepine, sodium val-
proate, topiramate, and oxcarbamazepine are only effective in
approximately 70% of the patients with epilepsy. Moreover, their
use is long-term and often accompanied with severely side effects,
including naupathia, headache, and ataxia, even threaten the life
of patients3–5. Investigations for more effective and safer AEDs are
still a formidable and urgent task for medicinal chemists.
A large number of experimental studies involved in acute and
chronic models of epilepsy confirmed the anticonvulsive potential
of H3R antagonists/inverse agonists. They showed the protection
against experimental seizures by feedback increase of histamine
release and binding with H1 receptors15,16. Besides, other mecha-
nisms might be involved in their anticonvulsive action, such as
facilitating of GABA release17–19, increasing histidine decarboxylase
(HDC) activity20,21 and synergism with AEDs17,18,22
.
Early, anticonvulsant activity of some imidazole H3R antagonists
such as thioperamide and clobenpropit was confirmed in models
of epilepsy (Figure 1)16,19,20,23. Recently, a large number of non-
imidazole H3R antagonists such as DL77 (Figure 1) prepared by a
group/team of Kiec-Kononowicz exhibited excellent anticonvulsant
activity in the electrically-induced seizures model and subcutane-
ously pentylenetetrazole (PTZ)-induced seizure model at dose-
dependent, and the therapeutic action was proved through
H3R24–28. Sadek et al. synthesised some histamine H3R ligands
(Figure 1, I) incorporating different antiepileptic structural motifs
to investigate if the H3R pharmacophore could be combined to
some antiepileptic molecules, and give some new anticonvulsants
by the multiple-target approaches. The results were encouraging,
which indicated that the H3R pharmacophore successfully com-
bined to the antiepileptic molecules, maintaining the H3R affinity
and anticonvulsant activity, although the anticonvulsant activity
decreased compared to the prototypal antiepileptic molecules
The role of central histaminergic system being concerned in
epilepsy have been demonstrated in many experimental and
epidemiological studies, in which histamine regulated seizure sus-
ceptibility as an anticonvulsant neurotransmitter6–8. For example,
H1-antagonists such as pyrilamine, ketotifen that decrease brain
histamine levels increased the duration of convulsive phase in
electrically-induced convulsions model9. Histidine, as the precursor
of histamine, showed protection against chemically-induced con-
vulsions in rats, via activating the histamine H1 receptors10.
Histamine H3 receptors (H3R) as a G-protein coupled receptor
(GPCR) binding to histamine like other histamine receptors, is
expressed mainly in the central nervous system, where it acts as
an auto-receptor in histaminergic neurons, and negatively regu-
lates the synthesis and release of histamine11. What is more, as a
inhibitory heteroreceptor, H3R also regulates the release of other
neurotransmitters including dopamine, acetylcholine, serotonin,
norepinephrine, c-aminobutyric acid, and glutamate. These neuro-
transmitters, especially c-aminobutyric acid and glutamate, are (Figure 1)29,30
.
CONTACT XianQing Deng
dengxianqing1121@126.com
Medical College, Jinggangshan University, Ji’an, China; Naiming Zhou
zhounaiming@zju.edu.cn
College of Life Sciences, Zhejiang University, Hangzhou, China
ꢀ
Research Center of Chinese Medicinal Resources and Functional Molecules of Jinggangshan University, Ji’an, 343009, China.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1311
Figure 1. Structures of histamine H3 receptor ligands with anticonvulsant activity, triazole derivatives with anticonvulsant activity and target compounds 3a-3q
designed.
Pitolisant (PIT), a H3R antagonist/inverse agonist, has been sub- anticonvulsive effects of 4-alkyl-5-aryl-1,2,4-triazole-3-thione deriv-
jected into clinical Phase III for the treatment of epilepsy31. When
used alone or in combination with other AEDs in the human
photosensitivity model at dose ranges of 30–60 mg, PIT showed a
favourable EEG profile in a dose-dependent manner32.
atives and suggested that the influence on the voltage-gated Naþ
channels was involved in them at least41,42. Therefore, in this
work, our strategy was to design molecules combining pharmaco-
phores of H3R antagonists and another anticonvulsant active
pharmacophore (e.g. 1,2,4-triazole moiety) into one skeleton, and
then produced a synergism for anticonvulsant active.
Supported by the above results, in this work, we designed and
synthesised some novel H3R antagonists/inverse agonists by
hybriding the H3R pharmacophore (aliphatic amine with propyloxy
chain) with the 1,2,4-triazole, the latter have been identified as an
important and effective anticonvulsive fragment in recent years
(Figure 1, II and III)33–37. According to Quan’s reports, the 1,2,4-tri-
azole derivatives were likely to have several mechanisms of action
such as inhibiting voltage-gated sodium ions channel and modu-
2. Results and discussion
2.1. Chemistry
According to Schemes 1 and 2, the target compounds (3a-3q)
lating GABAergic activity38–40. And a group of Plech illustrated the were synthesised smoothly. In brief, formyl hydrazine reacted with

1312
M. SONG ET AL.
Scheme 1. The synthesis route and conditions for the preparation of compounds 3a-3j.
Scheme 2. The synthesis route and conditions for the preparation of compounds 3k-3q.
4-aminophenol in dimethoxyl-N,N-dimethyl formamide (DMF-DMA) Their structures were characterised and confirmed by1H-NMR,
13 C-NMR, and HR-MS.
to give the 4-(4H-1,2,4-triazol-4-yl)phenol (1a). Compound 1a
underwent a nucleophilic substitution with 1-bromo-3-chloropro-
pane to get 4-(4-(3-chloropropoxy)phenyl)-4H-1,2,4-triazole (2a).
The reaction was conducted in the presence of potassium hydrox-
ide in dimethyl sulfoxide (DMSO) at room temperature to ensure
the formation of single-substituted derivatives. Finally, proper
amines reacted with compound 2a in the presence of K2CO3 and
KI in the solvent of CH3CN to give the desired compounds 3a-3j.
To enrich the structure–activity relationship, we also prepared the
derivatives of 3h via introducing the substituents at the triazole
ring and adjusting the length of the link. The reaction conditions
used to prepare these compounds (3k-3q) were the same as
above. Compounds (3a, 3b, 3c, 3d, 3e, 3 g, 3k, 3o, and 3p)
2.2. Biological activities
2.2.1. Evaluation of H3R antagonistic activity
cAMP-response element (CRE) reporter gene assay has been
extensively used to evaluate the efficacy of GPCR antagonists or
agonists. In this work, the H3R antagonistic activities of the pre-
pared 3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)-propylamine derivatives
have been screened by CRE-driven luciferase assay, in which the
HEK-293 cells expressing the human H3R and a reporter gene con-
sisting of the firefly luciferase coding region were used43,44
.
obtained as the form of oil were transformed to hydrochlorate. Ciproxifan (CXP) and Pitolisant (PIT) were employed as the positive

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1313
Table 1. H3R antagonistic activity of compounds 3a-3q.
% Antagonisma
Compounds
R
n
100 nM
1 lM
H3R antagonistic activity (IC50, lM)
3a
3b
49.85 9.73
13.24 4.83
223.76 7.56
15.02 4.03
2.99
NTb
3c
32.23 5.69
11.33 5.86
143.98 16.16
79.4 4.50
0.553
NT
3d
3e
3f
9.86 0.87
9.96 10.9
21.78 3.04
24.41 6.30
NT
NT
3g
3h
3i
ꢁ0.76 4.87
10.56 7.94
5.19 2.06
20.24 7.56
239.79 3.17
16.45 1.90
NT
0.127
NT
3j
ꢁ4.2 3.11
6.12 2.05
NT
3k
3l
3m
3n
3o
Me
C6H5
C6H4(p-Cl)
Biphenyl
H
H
H
1
1
1
1
0
2
3
133.91 8.91
148.62 4.43
201.12 12.63
1.03 7.79
45.04 3.61
25.00 3.95
1.88 12.38
33.96 8.59
54.16 7.33
183.54 14.41
169.04 15.04
203.92 8.44
3.36 1.27
94.20 2.60
90.52 5.71
2.39 13.85
63.05 9.33
246.29 21.48
0.021
0.00781
0.00592
NT
0.25
3.44
NT
0.082
0.51
3p
3q
CPXc
PITd
a% Antagonism, value represented as mean standard deviation of three independent experiments.
bNT, IC50 was not tested.
cCPX, an antagonist of H3R ciproxifan.
dPIT, an antagonist/inverse agonist of H3R pitolisant.
controls. Initially, compounds and positive controls were tested at prominent compounds IC50 values were determined at additional
two concentrations (100 nM and 1 mM) to obtain the preliminary assays.
investigation of their H3R antagonistic activities. In the assays, the
As seen in Table 1, majority of the synthesised compounds dis-
antagonistic activities was positively correlated with the rise of the played gentle to robust H3R antagonistic activities and eight of
fluorescence value and indicated by the % antagonism. For the them exhibited micromolar inhibitory activity. The antagonistic

1314
M. SONG ET AL.
activities of all compounds depended on the concentration concentrations. Cytotoxicity appeared at 100 mM. As shown in
treated. It is worth mentioning that compounds 3l (IC50
Figure 3, PIT and compound 3m showed effective H3R inverse
¼
7.81 nM) and 3m (IC50 ¼ 5.92 nM) displayed the most potent H3R agonistic activity with an EC50 value of 403 and 129 nM,
antagonistic activities, with the much stronger potency than that respectively.
of CXP (IC50 ¼ 0.082 mM) and PIT (IC50 ¼ 0.5 mM) in the CRE
Simple structure–activity relationships (SARs) could be obtained
from Table 1. In the series of 3a-3j, the different tertiary amines
significantly influenced the H3R antagonistic activities. The N-ethyl
derivative 3a showed an IC50 of 2.9 mM, while the activity declined
sharply for the N-propyl derivative 3b. Interestingly, compounds
containing piperazine or morpholine (3d-3g) exhibited weaker
activities than those with piperidine or pyrrolidine (3c and 3h).
This probably attributed to the increase of the molecular polarity.
The introduction of phenyl or amide group on the piperidine ring
of compound 3h, gave the compounds 3i and 3j, which also
decreased the H3R antagonistic activities when compared to com-
pound 3h. Based on the facts above, it could be concluded that
the N,N-diethyl group, pyrrolidine and piperidine were more of
benefit to the H3R antagonistic activities of the 3-(4-(4H-1,2,4-tria-
zol-4-yl)phenoxy)-propylamine skeleton, and piperidine derivative
(3h) was the best one with the IC50 of 0.127 mM.
reporter gene assay.
Surprisingly, antagonism percent of some compounds as well
PIT were above 100%. It is well known that H3R is a GPCR coupled
with Gai. When the ligand (histamine) binds to H3R, the dissoci-
ated Gai inhibits the activity of adenylate cyclase (AC) and down-
regulates the level of intracellular cAMP. In this CREs driven luci-
ferase assay, when cells were pre-treated by H3R antagonists, the
downregulation of cAMP would be inhibited, and the level of
intracellular cAMP would regain to the initial level. However, in
some cases, the cAMP levels raised above the initial level, giving
the % antagonism greater than 100%. Our first speculation was
that these compounds might stimulate the AC directly and up-
regulate the level of cAMP. However, an additional assay indicated
that these compounds didn’t have effects on the level of cAMP
when pre-treated alone. As shown in Figure 2, forskolin (2 mM)
treated group gave more than 200 times rise for the cAMP level
when compared to the control group. While Histamine, ciproxifan,
and compounds 3a, 3c, 3h, 3k, 3l, and 3m have no significant
effects on the level of cAMP when carried out comparisons by
ANOVA followed by Dunnett’s test.
Another explanation is that these compounds may be inverse
agonists when binding to H3R, which not only antagonise the
function of histamine, but also give the inverse agonistic perform-
ance. Actually, PIT is a well-known H3R antagonist and reverse
agonist. So, the above results make sense. Based on the above,
we further assessed the H3R inverse agonist activity of compound
3m and PIT by using CRE-luciferase assay. In this experiment,
transfected HEK-293 cells were stimulated with 10 mM forskolin or
10 mM forskolin plus different concentrations of compound 3m.
The raise of luciferase activity after adding compound represented
the inverse agonistic activity. The EC50 was calculated by seven
To enrich the structure–activity relationships, we prepared the
derivatives of 3h via introducing the substituents at the triazole
ring and adjusting the length of the link.
Compounds 3k, 3l, 3m, and 3n were substituted on 3-position
of 1,2,4-triazole ring with methyl, phenyl, para-chlorophenyl, and
biphenyl, respectively. Encouragingly, the introduction of methyl,
phenyl, and para-chlorophenyl groups significantly increased the
H3R antagonistic activities, giving the two prominent compounds
3l and 3m with nanomolar IC50 values. While the biphenyl substi-
tuted compound 3n showed weaker activity when compared to
3h. Replacing the three-carbon link in the compound 3h with
two-carbon, four-carbon and five-carbon links, gave the com-
pounds 3o, 3p, and 3q, respectively. It could be seen that the
length of the link had a direct impact on H3 receptor antagonistic
activities of the 3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)-propylamine
derivatives. The activity order of the link length of carbon
was 3 > 2 > 4 ꢂ 5.
To investigate the molecular determinants that manage the
antagonistic activities of the tested compounds, molecular dock-
ing studies of PIT, 3h, and 3m with the H3R homology model
were carried out. The homology model was constructed from the
crystal structure of the H1 receptor (PDB ID: 3RZE)45. The docking
results are shown in Figure 4.
300
200
100
3
2
As shown in Figure 4(A), PIT bound to H3R through two critical
H-bond interactions with Tyr115 and Glu206, and other interac-
tions with amino acid residues Arg381, Phe193, Met378 and so
on. Figure 4(B) revealed that compound 3h had a similar binding
pattern to PIT, interacting with the same amino acid residues
Glu206, Tyr115, Arg381, and Met378. Surprisingly, the compound
1
0
Figure 2. Effects of compounds 3a, 3c, 3h, 3k, 3l, and 3m on the level of intra-
cellular cAMP administrated alone.
3m
Pitolisant
4.5×106
5.0×106
EC50 = 0.129 mM
EC50 = 0.403 mM
4.0×106
3.5×106
3.0×106
2.5×106
4.5×106
4.0×106
3.5×106
3.0×106
-12
-10
-8
-6
-4
-12
-10
-8
-6
-4
Log[M]
Log[M]
Figure 3. The H3R inverse agonistic activity (EC50, lM) of Pitolisant and compound 3m.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1315
Figure 4. The predicted configurations for PIT (A), 3h (B) and 3m (C) binding with H3R, and their overlying pattern (D).
3m with the highest H3R antagonistic activity showed a different intraperitoneally (i.p.) to mice at dosage of 10 mg/kg in the both
binding pattern to PIT (as seen in Figure 4(C)). The overlying pat-
tern of PIT, 3h, and 3m was shown in Figure 4(D). The piperidine
group of 3m was not involved in the formation of the salt bridge
or hydrogen-bond interactions with Glu206, which was generally
considered as the critical residue of H3R46,47. The unexpected
binding pattern of 3m might be due to the phenyl group on the
triazole ring, which did not fit into the hydrophobic cavity in TMs
3-5-6 region of H3R, even though the compound 3m showed a
forceful binding with H3R via another mode. The triazole nitrogen
established an ionic bond with Glu206, and a hydrogen bond was
observed between piperidine nitrogen and Tyr115. p-p shaped,
and alkyl interactions with Trp371, Tyr343, Arg381, His187,
Leu199, and ALA202 were observed to support the forceful bind-
ing with H3R.
models. PIT and valproic sodium (VPA) were used as positive con-
trols in the tests.
2.2.2.1. Protective effects of H3R antagonists/inverse agonists 3a-
3q on MES-induced convulsions. Protection for the mice was
defined as the reduction or abolition of the tonic hind limb exten-
sion (THLE) in the MES model in mice. As seen in Figure 5, com-
pounds 3a, 3 g, 3h, 3l, 3m, and 3o showed moderate protection
for the electro-stimulated mice with significant difference from
that of the control group (p < 0.05, p < 0.01, or p < 0.001). Mice
pre-treated with PIT (10 mg/kg, i.p.) and VPA (300 mg/kg, i.p.) were
moderately or potently protected, respectively. Generally, the anti-
convulsant activities of these compounds in MES model correlated
directly to their H3R antagonistic activities. For example, the antie-
pileptic activity obtained of compound 3m was the highest, and
in vitro H3R antagonistic activity measured for 3m with IC50 of
5.92 nM was also the highest. Compounds 3a, 3h, 3l, and 3o,
showing anticonvulsant activity in the MES model, also showed
2.2.2. Anticonvulsant activity evaluation
To investigate the anticonvulsive effects, all the target compounds
(3a-3q) were screened in the MES-induced and PTZ-induced con-
vulsion models in mice. Compounds were administered good H3R antagonistic activities. Compound 3c, 3k, and 3p

1316
M. SONG ET AL.
30
20
10
0
25
20
15
10
5
*#
*
*
*
*
**
**
***
0
****
Figure 8. Protective effects of compound 3m (10mg/kg, i.p.) against MES-
induced convulsions when pre-treatment of RAMH (10mg/kg, i.p.). Protection in
the test was defined as the reduction or abolition of the THLE in mice. Results
are showed as mean SEM with seven animals in each group. Values are consid-
ered significant at p < 0.01 as compared to saline-treated group, and #p < 0.01
ꢀ
Figure 5. Effects of H3R antagonists/inverse agonists 3a-3q (10mg/kg, i.p.), PIT
(10mg/kg, i.p.) and anticonvulsant drug VPA (300 mg/kg, i.p.) against MES-
induced convulsions. Protection for mice was defined as the reduction or aboli-
tion of the tonic hind limb extension (THLE) in MES model. Results are showed
as mean SEM with seven animals in each group. Values are considered signifi-
cant at p < 0.05,
saline-treated group.
as compared to 3m þ RAMH treated group.
reduced the average duration of THLE, although they did not
achieve a significant difference from the control group.
ꢀ
ꢀꢀ
p < 0.01,
ꢀꢀꢀ ꢀꢀꢀꢀ
p < 0.001, p < 0.0001 as compared to
2.2.2.2. Protective effects of H3R antagonists/inverse agonists 3a-
3q on PTZ-induced convulsions. Some experiments indicated that
H3R antagonists/inverse agonists could protect animals in PTZ-
induced convulsions model26,29. So the compounds 3a-3q, PIT,
and VPA were also screened in the PTZ model in mice.
Unfortunately, all compounds tested at the dose of 10 mg/kg (i.p.)
did not show any protection against the seizures induced by PTZ.
PIT also failed to protect the PTZ-treated mice as well at the same
conditions. By contrast, anticonvulsant agent VPA showed full pro-
tection against the PTZ-induced convulsions (Figure 6).
5
4
3
2
1
0
&
2.2.2.3. Effects of compound 3m on MES-induced convulsions in
dose dependent manner. In a further experiment, compound 3m,
as the most active one in the MES-induced seizure model, was
chosen to verify its protective effect in different doses.
Encouragingly, the 3m-provided protections were observed and
were dose dependent. The standard antagonist PIT also displayed
anticonvulsive activity dose-dependently at the same condition.
Notably, when pre-treated with 20 mg/kg dose, PIT could fully
abrogate the tonic hind limb extension induced by electro-stimu-
lation, showing its potential anticonvulsant activity (Figure 7). To
exclude the possibility that the anticonvulsant activity of 3m was
connected with sedative effect, we carried out a rotarod test for
3m. The result showed that compound 3m had no neurotoxicity
at the maximum dose of 10 and 20 mg/kg (the details could be
seen in Support Table 1).
PTZ (85 mg/kg, i.p.)
Figure 6. Effects of compounds 3a-3q (10mg/kg, i.p.), and reference drug PIT
(10mg/kg, i.p.) and VPA (300 mg/kg, i.p.) against PTZ-induced convulsions.
Results are showed as mean SEM of seven mice in each group. & represent full
protection.
30
20
*
*
**
***
2.2.2.4. Effects of RAMH pre-treatment on the compound 3m-pro-
vided protection in MES-induced seizure model. To investigate the
correlation between the anticonvulsant activity and H3R antagon-
istic activity of compound 3m, the protection provided by com-
pound 3m against MES-induced seizure was reassessed after the
administration of RAMH (10 mg/kg, i.p.), a CNS penetrant hista-
mine H3R agonist. The results indicated that when co-administra-
tion with RAMH, compound 3m lost its original protective effect
(Figure 8). Administration of RAMH alone also did not affect the
duration of THLE of mice with p > 0.05 for saline versus RAMH.
The above findings suggested that H3R antagonism was the main
contributor for the anticonvulsant activity of compound 3m in
MES model. When the H3R was blocked by H3R antagonist 3m,
10
&
0
Saline 3 mg
10 mg 20 mg
3m
3 mg
10 mg 20 mg
PIT
Figure 7. Protective effects of compound 3m and reference drug PIT against
MES-induced convulsions in different doses. Protection in the test was defined as
the reduction or abolition of the THLE in mice. Results were showed as
mean SEM with seven animals in each group. Values are considered significant
ꢀ
ꢀꢀ
ꢀꢀꢀ
p < 0.001 when compared to saline-treated group.
at p < 0.05, p < 0.01,
&PIT, at 20 mg/kg dose, fully abrogate the THLE for all the tested mice.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1317
histamine or other neurotransmitter such as GABA in the CNS indicated by the TLC (developing agent ratio: CH2Cl2/CH3OH ¼
15/1), the mixture was poured into 30 mL of water. The solution
was extracted with dichloromethane three times. The organic
layers were combined, washed with saturated salt water, dried
over MgSO4, filtered, and concentrated. Purification by column
chromatography (silica gel, 0-5% methanol in CH2Cl2) gave the
compound 2a. Chemical formula: C11H12ClN3O (MW ¼ 237.69).
increased, finally leading to anticonvulsive effects.
3. Conclusion
To identify novel H3R antagonists/inverse agonists with potential
anticonvulsant activities, a series of 3-(4-(4H-1,2,4-triazol-4-yl)phe-
noxy)-propylamine derivatives were designed through combining
pharmacophore of H3R antagonists and another anticonvulsant
active pharmacophore (1,2,4-triazole moiety) into one molecule.
The majority of those prepared compounds displayed moderate to
robust H3R antagonistic activities. The SAR analysis revealed that
piperidine and triazolephenol linked by three-carbon chain was
benefit for the H3R antagonistic activity, and substitution by aro-
matic nucleus on the 3-position of 1,2,4-triazole further increased
the H3R antagonistic activities. The most potent H3R antagonists/
inverse agonists 3l and 3m exhibited nanomolar H3R antagonistic
activities with IC50 of 7.81 nM and 5.92 nM, respectively. Molecular
docking analysis demonstrated that 3m strongly bound to H3R via
interactions with Tyr115, Glu206, Trp371, Tyr343, and so on,
although its binding mode was not similar to PIT. The anticonvul-
sive screens in vivo indicated that compounds with higher H3R
antagonistic activities showed more protection in the MES-induced
convulsant model in mice, while no one was observed protective
effect in PTZ-induced convulsant model. Moreover, the protection
of 3m in the seizure model was fully abrogated when mice were
co-treated with a H3R agonist RAMH, which suggested that its
potential therapeutic effect was through H3R.
m.p. 102–104 ꢃC, yield 81%. 1H-NMR (300 MHz, DMSO-d6):
d
2.15–2.23 (m, 2H, OCH2CH2), 3.80 (t, 2H, J ¼ 6.5 Hz, ClCH2), 4.16 (t,
2H, J ¼ 6.1 Hz, OCH2), 7.13 (d, 2H, J ¼ 9.0 Hz, Ph-H), 7.60 (d, 2H,
J ¼ 9.0 Hz, Ph-H), 9.00 (s, 2H, N¼CH). 13 C-NMR (75 MHz, DMSO-d6):
d 158.40, 142.03, 127.75, 123.40, 116.03, 65.22, 42.35, 32.05. As
mentioned above, replaced the reactant 1a by the alternative 4-
(3-substituted-4H-1,2,4-triazol-4-yl)phenols (1b-1e) to give the
compounds 2b-2e. Compounds 2f, 2g, 2h were obtained by the
same method as above just replacing 1-bromo-3-chloropropane
by 1-bromo-2-chloroethane, 1-bromo-4-chlorobutane, 1-bromo-2-
chloropentane, respectively.
4.1.3. Synthesis of compounds 3a-3q
Taking compound 3a as an example: in a 100 mL round-bottom
flask with 15 mL of acetonitrile, compound 2a (0.40 g, 1.68 mmol),
diethylamine (0.245 g, 3.36 mmol), K2CO3 (0.46 g, 3.36 mmol) and
potassium iodide (0.56 g, 3.36 mmol) were added one by one. The
mixture was heated up to 110 ꢃC for 12–16 h. After cooing the
mixture to 40 ꢃC, it was filtered and dried by vacuum to obtain a
residue. Purification by column chromatography (silica gel, 0–20%
methanol in CH2Cl2) gave the compound 3a. The same conditions
were used to prepare the compounds 3b-3q. Compounds 3a, 3c,
3d, 3e, 3 g, 3k, 3o, and 3p obtained as oils were transformed into
the corresponding hydrochloride by hydrogen chloride in CH2Cl2.
4. Experimental section
4.1. Synthesis
All the chemical solvents and reagents were purchased from sup-
plier and used as received. Unless otherwise specified, reactions
were monitored by thin-layer chromatography (TLC). All NMR
spectrum was carried out on an AV-300 spectrometer with
300 MHz. High resolution mass spectra were measured on an
MALDI-TOF/TOF mass spectrometer.
4.1.4. Characterisation for the target compounds
4.1.4.1. 3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)-N,N-diethylpropan-1-
amine hydrochloride (3a). Chemical formula: C15H22N4O ꢄ HCl
(MW ¼ 310.83). m.p. 105–106 ꢃC, yield 82%. 1H-NMR (300 MHz,
DMSO-d6): d 1.25 (t, 6H, J ¼ 7.2 Hz, CH3), 2.18 (t, 2H, J ¼ 9.0 Hz,
OCH2CH2), 3.08–3.16 (m, 6H, N(CH2)3), 4.17 (t, 2H, J ¼ 6.0 Hz,
OCH2), 7.19 (d, 2H, J ¼ 8.8 Hz, Ph-H), 7.75 (d, 2H, J ¼ 8.8 Hz, Ph-H),
9.84 (s, 2H, N¼CH), 10.96 (s, 1H, HCl). 13 C-NMR (75 MHz, DMSO-
d6): d 159.34, 142.39, 126.44, 124.35, 116.08, 66.00, 48.04, 46.59,
23.40, 8.87. ESI-HRMS calculated for C15H23N4Oþ ([M-Cl]þ):
275.1866; found: 275.1860.
4.1.1. Synthesis of compounds 1a-1e
Taking compound 1a as an example: dimethoxyl-N, N-dimethyl
formamide (DMF-DMA, 1.31 g, 11 mmol) and formyl hydrazine
(0.65 g, 11 mmol) were added into a flask containing 30 ml of
acetonitrile. The mixture was heated up to 60 ꢃC for 30 min, then
4-aminophenol (0.60 g, 5.5 mmol) and acetic acid (3 mL) were
added and heated up to 120 ꢃC for 9 h. The mixture was cooled,
filtered and washed by acetonitrile to give the product 1a.
Chemical formula: C8H7N3O (MW ¼ 161.16). m.p. 270–272 ꢃC, yield
4.1.4.2. 3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)-N,N-dipropylpropan-1-
amine hydrochloride (3b). Chemical formula: C17H26N4O ꢄ HCl
(MW ¼ 338.88). m.p. 180–183 ꢃC, yield 62%. 1H-NMR (300 MHz,
CDCl3,):
d
1.05 (t, 6H, J ¼ 7.3 Hz, CH3), 1.77–1.90 (m, 4H,
1
N(CH2CH2)2), 2.27–2.37 (m, 2H, OCH2CH2), 3.15 (t, 4H, J ¼ 8.0 Hz,
N(CH2)2), 3.40 (t, 2H, J ¼ 8.3 Hz, NCH2), 4.18 (t, 2H, J ¼ 5.6 Hz,
OCH2), 7.05 (d, 2H, J ¼ 8.9 Hz, Ph-H), 7.43 (d, 2H, J ¼ 8.9 Hz, Ph-H),
8.62 (s, 2H, CH¼N)., 9.51 (s, 1H, HCl). 13 C-NMR (75 MHz, CDCl3): d
163.08, 146.48, 132.18, 128.41, 120.58, 69.93, 59.42, 55.13, 28.54,
21.98, 15.92. ESI-HRMS calculated for C17H27N4Oþ ([M-Cl]þ):
303.2179; found: 303.2178.
73%. H-NMR (300 MHz, DMSO-d6): d 6.90 (d, 2H, J ¼ 8.9 Hz, Ph-H),
7.46 (d, 2H, J ¼ 8.9 Hz, Ph-H), 8.94 (s, 2H, N ¼ CH), 9.88 (s, 1H, OH).
13 C-NMR (DMSO-d6, 75 MHz): 157.75, 142.14, 126.18, 123.65,
116.60. The compounds 1 b-1e were obtained according to the
above method using the other hydrazides.
4.1.2. Synthesis of compounds 2a-2h
4.1.4.3.
4-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl)-4H-1,2,4-triazole
Taking compound 2a as an example: compound 1a (0.50 g,
3.1 mmol) and KOH (0.35 g, 6.2 mmol) were put into a flask with
5 mL of DMSO. The mixture was stirred for 5 min at 20 ꢃC. Then
added 1-bromo-3-chloropropane (0.98 g, 6.2 mmol) into the mix-
hydrochloride (3c). Chemical formula: C15H20N4O ꢄ HCl (MW ¼
308.81). m.p. 120–122 ꢃC, yield 56%. 1H-NMR (300 MHz, DMSO-d6):
d 1.92–2.00 (m, 4H, NCH2CH2), 2.19–2.22 (m, 2H, OCH2CH2),
ture and continued the reaction. After completion of the reaction 3.00–3.53 (m, 6H, N(CH2)3), 4.17 (t, 2H, J ¼ 5.7 Hz, OCH2), 7.17 (d,

1318
M. SONG ET AL.
2H, J ¼ 8.7 Hz, Ph-H), 7.72 (d, 2H, J ¼ 8.7 Hz, Ph-H), 9.75 (s, 2H, 124–126 ꢃC, yield 50%. 1H-NMR (300 MHz, DMSO-d6): d 1.88–2.10
N¼CH), 11.45 (s, 1H, HCl). 13 C-NMR (75 MHz, DMSO-d6): d 159.25,
142.36, 126.57, 124.25, 116.07, 66.03, 53.21, 51.46, 25.53, 23.24.
ESI-HRMS calculated for C15H21ClN4Oþ ([M-Cl]þ): 273.1710; found:
273.1711.
(m, 6H, NCH2CH2), 2.17–2.22 (m, 2H, NCH2), 2.56 (t, 1H, J ¼ 6.0 Hz,
NCH2CH2CH), 2.64 (t, 2H, J ¼ 7.0 Hz, NCH2), 3.14 (t, 2H, J ¼ 6.4 Hz,
NCH2), 4.11 (t, 2H, J ¼ 6.0 Hz, OCH2), 7.03-7.32 (m, 9H, Ph-H), 8.47
(s, 2H, CH¼N). 13 C-NMR (75 MHz, DMSO-d6): d 159.49, 146.04,
141.92, 128.46, 126.83, 126.71, 126.24, 123.98, 115.85, 66.88, 55.36,
54.42, 42.51, 33.23, 26.68. ESI-HRMS calculated for C22H27N4Oþ
([M þ H]þ): 363.2179; found: 363.2178.
4.1.4.4.
1-(3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)propyl)piperazine
dihydrochloride (3d). Chemical formula: C15H21N5O ꢄ 2HCl (MW ¼
323.83). m.p. 146–148 ꢃC, yield 52%. 1H-NMR (300 MHz, CDCl3): d
2.20–2.30 (m, 2H, J ¼ 7.0 Hz, OCH2CH2), 3.32 (t, 2H, J ¼ 7.8 Hz,
NCH2), 3.37–3.57 (m, 8H, Piperazine-H), 4.17 (t, 2H, J ¼ 5.8 Hz,
OCH2), 7.16 (d, 2H, J ¼ 8.9 Hz, Ph-H), 7.68 (d, 2H, J ¼ 8.9 Hz, Ph-H),
9.44 (s, 2H, CH¼N), 10.04 (s, 2H, HCl). 13 C-NMR (75 MHz, DMSO-
d6): d 158.91, 142.29, 127.01, 123.96, 116.10, 65.89, 53.41, 48.17,
45.78, 23.50. ESI-HRMS calculated for C15H22N5Oþ ([M-2HCl þ H]þ):
288.1819; found: 288.1820.
4.1.4.10. 1-(3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)propyl)piperidine-4-
carboxamide (3j). Chemical formula: C17H23N5O2 (MW ¼ 329.40).
1
m.p. 196–198 ꢃC, yield 68%. H-NMR (300 MHz, CDCl3): d 1.74–2.06
(m, 7H, NCH2(CH2)3CH), 2.14–3.00 (m, 6H, N(CH2)3), 4.07 (d, 2H,
J ¼ 6.5 Hz, OCH2), 5.70 (s, 2H, NH2), 7.02 (d, 2H, J ¼ 8.9 Hz, Ph-H),
7.28 (d, 2H, J ¼ 8.9 Hz, Ph-H), 8.39 (s, 2H, CH¼N). 13 C-NMR
(75 MHz, CDCl3): d 177.53, 159.53, 141.87,126.30, 123.98, 115.81,
66.76, 54.96, 53.23, 42.71, 28.94, 26.71. ESI-HRMS calculated for
C17H24N5O2 ([M þ H]þ): 330.1925; found: 330.1926.
þ
4.1.4.5. 1-(3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)propyl)-4-methylpi-
perazine hydrochloride (3e). Chemical formula: C16H23N5O ꢄ HCl
(MW ¼ 337.85). m.p. 220–223 ꢃC, yield 65%. 1H-NMR (300 MHz,
CDCl3): d 2.26 (s, 2H, OCH2CH2), 2.84 (s, 3H, CH3), 3.35-3.66 (m,
10H, N(CH2)5), 4.17 (t, 2H, J ¼ 5.2 Hz, OCH2), 7.16 (d, 2H, J ¼ 8.7 Hz,
Ph-H), 7.68 (d, 2H, J ¼ 8.7 Hz, Ph-H), 9.46 (s, 2H, CH¼N), 10.57 (s,
1H, HCl). 13 C-NMR (75 MHz, DMSO-d6): d 158.93, 142.30, 126.96,
123.99, 116.10, 65.90, 54.53, 48.75, 45.75, 23.60, 15.53. ESI-HRMS
calculated for C16H24N5Oþ ([M-Cl]þ): 302.1975; found: 302.1976.
4.1.4.11. 1-(3-(4–(3-methyl-4H-1,2,4-triazol-4-yl)phenoxy)propyl)pi-
peridine hydrochloride (3k). Chemical formula: C17H24ClN4O ꢄ HCl
(MW ¼ 336.86). m.p. 240–242 ꢃC, yield 61%. 1H-NMR (300 MHz,
CDCl3 þ DMSO-d6): d 1.86–2.43 (m, 8H, NCH2CH2CH2), 2.64 (s, 3H,
N¼CCH3), 2.94–3.55 (m, 6H, N(CH2)3), 4.22 (s, 2H, OCH2), 7.13 (d,
2H, J ¼ 5.8 Hz, Ph-H), 7.55 (d, 2H, J ¼ 5.8 Hz, Ph-H), 9.33 (s, 1H,
CH¼N), 11.31 (s, 1H, HCl). 13 C- NMR (75 MHz, CDCl3 þ DMSO-d6):
d 164.95, 156.81, 149.03, 132.21, 129.15, 120.75, 70.66, 59.08,
57.74, 28.43, 27.48, 26.65, 15.04. ESI-HRMS calculated for
C17H25N4Oþ ([M-Cl]þ): 301.2023; found: 301.2025.
4.1.4.6.
1-(3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)propyl)-4-phenylpi-
perazine (3f). Chemical formula: C21H25N5O (MW ¼ 363.47). m.p.
172–174 ꢃC, yield 64%. 1H-NMR (300 MHz, DMSO-d6): d 1.91–1.97
(m, 2H, OCH2CH2), 2.47–2.55 (m, 6H, N(CH2)3), 3.12–3.15 (m, 4H,
N(CH2)2), 4.09 (t, 2H, J ¼ 6.2 Hz, OCH2), 6.76 (t, 1H, J ¼ 7.2 Hz, Ph-H),
6.92 (d, 2H, J ¼ 8.0 Hz, Ph-H), 7.10 (d, 2H, J ¼ 8.9 Hz, Ph-H), 7.2 (t,
2H, J ¼ 7.9 Hz, Ph-H), 7.58 (d, 2H, J ¼ 8.9 Hz, Ph-H), 9.00 (s, 2H,
CH¼N). 13 C-NMR (75 MHz, DMSO-d6): d 158.70, 151.50, 142.03,
129.36, 127.52, 123.37, 119.21, 116.00, 115.77, 66.81, 54.82, 53.26,
48.68, 26.60. ESI-HRMS calculated for C21H26N5Oþ ([M þ H]þ):
364.2132; found: 364.2133.
4.1.4.12. 1-(3-(4-(3-phenyl-4H-1,2,4-triazol-4-yl)phenoxy)propyl)pi-
peridine (3l). Chemical formula: C22H26N4O (MW ¼ 362.48). m.p.
88–90 ꢃC, yield 59%. 1H-NMR (300 MHz, DMSO-d6): d 1.68 (s, 2H,
NCH2CH2CH2), 1.84–1.92 (m, 4H, NCH2(CH2)2, 2.18–2.30 (m, 2H,
OCH2CH2), 3.18-3.25 (m, 6H, N(CH2)3), 4.13 (t, 2H, J ¼ 6.0 Hz, OCH2),
7.00 (d, 2H, J ¼ 8.9 Hz, Ph-H), 7.23 (d, 2H, J ¼ 8.9 Hz, Ph-H), 7.32-
7.46 (m, 5H, Ph-H), 8.45 (s, 1H, CH¼N). 13 C-NMR (75 MHz, CDCl3): d
163.64, 157.72, 150.07, 134.62, 133.40, 133.26, 132.35, 132.05,
131.39, 120.31, 70.19, 59.33, 58.11, 28.99, 28.09, 26.66. ESI-HRMS
calculated for C22H27N4Oþ ([M þ H]þ): 363.2179; found: 363.2178.
4.1.4.7.
4-(3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)propyl)morpholine
hydrochloride (3g). Chemical formula: C15H20N4O2 ꢄ HCl (MW ¼
324.81). m.p. 237–239 ꢃC, yield 51%. 1H-NMR (300 MHz, CDCl3): d
2.27 (q, 2H, J ¼ 6.0 Hz, OCH2CH2), 3.12-3.48 (m, 6H, N(CH2)3), 3.92
(t, 4H, J ¼ 7.7 Hz, OCH2), 4.16 (t, 2H, J ¼ 6.0 Hz, OCH2), 7.14 (d, 2H,
J ¼ 8.9 Hz, Ph-H), 7.67 (d, 2H, J ¼ 8.9 Hz, Ph-H), 9.52 (s, 2H, N¼CH),
11.44 (s, 1H, HCl). 13 C-NMR (75 MHz, DMSO-d6): d 159.02, 142.22,
126.81, 123.93, 116.01, 65.94, 63.63, 53.95, 51.55, 23.37. ESI-HRMS
4.1.4.13. 1-(3-(4-(3-(4-chlorophenyl)-4H-1,2,4-triazol-4-yl)phenoxy)-
propyl)piperidine (3m). Chemical formula: C22H25ClN4O (MW ¼
396.92). m.p. 165–167 ꢃC, yield 75%. 1H-NMR (300 MHz, CDCl3): d
1.54 (s, 2H, NCH2CH2CH2), 1.72 (s, 4H, NCH2(CH2)2), 2.09–2.14 (m,
2H, OCH2CH2), 3.08–3.33 (m, 6H, N(CH2)3), 4.10 (t, 2H, J ¼ 6.0 Hz,
OCH2), 7.06 (d, 2H, J ¼ 8.8 Hz, Ph-H), 7.34 (d, 2H, J ¼ 8.8 Hz, Ph-H),
7.41 (d, 2H, J ¼ 8.6 Hz, Ph-H), 7.48 (d, 2H, J ¼ 8.6 Hz, Ph-H), 8.79 (s,
1H, CH¼N). 13 C-NMR (75 MHz, DMSO-d6): d 159.14, 151.96, 146.43,
135.01, 130.54, 129.21, 128.04, 127.56, 126.12, 115.85, 65.98, 54.08,
52.99, 29.45, 24.31, 23.56. ESI-HRMS calculated for C22H26ClN4Oþ
([M þ H]þ): 397.1790; found: 397.1791.
calculated for C15H21N4O2 ([M - Cl]þ): 289.1659; found: 289.1658.
þ
4.1.4.8. 1–(3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)propyl)piperidine (3h).
Chemical formula: C16H22N4O (MW ¼ 286.38). m.p. 249–252 ꢃC,
yield 67%. 1H-NMR (300 MHz, DMSO-d6): d 1.38–1.91 (m, 6H,
NCH2CH2(CH2)2), 2.20–2.30 (m, 2H, OCH2CH2), 2.83–3.44 (m, 6H,
N(CH2)3), 4.15 (t, 2H, J ¼ 6.0 Hz, OCH2), 7.17 (d, 2H, J ¼ 8.9 Hz, Ph-
H), 7.70 (d, 2H, J ¼ 8.9 Hz, Ph-H), 9.62 (s, 2H, CH¼N). 13 C-NMR
(75 MHz, DMSO-d6): d 159.09, 142.30, 126.78, 124.09, 116.07, 66.20,
53.67, 52.40, 23.59, 22.71, 21.90. ESI-HRMS calculated for
C16H23N4Oþ ([M þ H]þ): 287.1866; found: 287.1867.
4.1.4.14. 1-(3-(4-(3-([1,1’-biphenyl]-4-yl)-4H-1,2,4-triazol-4-yl)phe-
noxy)propyl)piperidine (3n). Chemical formula: C28H30N4O (MW ¼
438.58). m.p. 66–68 ꢃC, yield 58%. 1H-NMR (300 MHz, DMSO-d6): d
1.73 (s, 2H, NCH2CH2CH2), 2.01–2.09 (m, 4H, NCH2CH2), 2.39–2.48
(m, 2H, OCH2CH2), 3.12–3.20 (m, 6H, N(CH2)3), 4.14 (t, 2H,
J ¼ 6.0 Hz, OCH2), 6.96 (d, 2H, J ¼ 8.8 Hz, Ph-H), 7.20 (d, 2H,
J ¼ 8.8 Hz, Ph-H), 7.35-7.58 (m, 9H, Ph-H), 8.34 (s, 1H, CH¼N). 13 C-
NMR (75 MHz, DMSO-d6): d 158.97, 153.04, 145.16, 142.55, 139.81,
4.1.4.9.
1-(3-(4-(4H-1,2,4-triazol-4-yl)phenoxy)propyl)-4-phenylpi-
peridine (3i). Chemical formula: C22H26N4O (MW ¼ 362.48). m.p. 128.89, 128.86, 127.91, 127.63, 127.63, 127.22, 127.01, 125.05,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1319
115.61, 65.70, 55.17, 53.69, 24.32, 23.08, 22.30. ESI-HRMS calculated mice were purchased from Changsha tianqin Biotechnology Co.,
for C28H31N4Oþ ([M þ H]þ): 439.2492; found: 439.2494.
Ltd, China and used with the body weight 20–25 g. Procedures
involving animals were performed according to the Guide for the
Care and Use of Laboratory Animals (8th Edition, National
Academies Press, Washington, DC), and was approved by the local
animal ethics committee (Institutional Animal Ethics Committee of
Jinggangshan University, approval number: 201906018).
4.1.4.15.
1-(2-(4–(4H-1,2,4-triazol-4-yl)phenoxy)ethyl)piperidine
hydrochloride (3o). Chemical formula: C15H20N4O ꢄ HCl (MW ¼
308.81). m.p. 85–87 ꢃC, yield 72%. 1H-NMR (300 MHz, CDCl3): d
1.63–1.96 (m, 6H, NCH2(CH2)2CH2), 2.95–3.07 (m, 2H, OCH2CH2),
3.48 (t, 4H, J ¼ 5.5 Hz, N(CH2)2), 4.55 (t, 2H, J ¼ 5.1 Hz, OCH2), 7.23
(d, 2H, J ¼ 8.8 Hz, Ph-H), 7.75 (d, 2H, J ¼ 8.8 Hz, Ph-H), 9.69 (s, 2H,
CH¼N), 11.21 (s, 1H, HCl). 13 C-NMR (75 MHz, CDCl3): d 158.38,
142.33, 127.14, 124.20, 116.33, 63.37, 54.89, 53.01, 22.70, 21.68.
ESI-HRMS calculated for C15H21N4Oþ ([M-Cl]þ): 273.1710; 273.1711.
4.2.2.2. MES-induced seizure. Ear stimulation with alternating cur-
rent (0.2 s, 60 Hz, 50 mA) was used to induce the seizures in mice.
The reduction or abolition of the hind limb tonic extension (THLE)
of mice was considered protective against the MES-induced seiz-
ures29,48. Test compounds and positive drugs VPA (300 mg/kg)
and PIT (10 mg/kg) were i.p. administrated half an hour prior to
the electric stimulation. To investigate the mechanism of action,
the most promising one 3m was chose for a further test. In one
group of animals of seven mice, compound 3m (10 mg/kg) co-
injected with RAMH 10 mg/kg with 5 min interval. The animals in
other three groups were single treated with RAMH 10 mg/kg,
compound 3m 10 mg/kg, and vehicle, respectively.
4.1.4.16.
1-(4–(4-(4H-1,2,4-triazol-4-yl)phenoxy)butyl)piperidine
hydrochloride (3p). Chemical formula: C17H24N4O ꢄ HCl (MW ¼
336.86). m.p. 80–82 ꢃC, yield 69%. 1H-NMR (300 MHz, CDCl3): d
1.88–2.21 (m, 8H, NCH2(CH2)3CH2), 2.22–2.36 (m, 2H, OCH2CH2),
2.82–3.73 (m, 6H, N(CH2)3), 4.08 (t, 2H, J ¼ 5.8 Hz, OCH2), 7.00 (d,
2H, J ¼ 8.9 Hz, Ph-H), 7.30 (d, 2H, J ¼ 8.9 Hz, Ph-H), 8.49 (s, 2H,
CH¼N). 10.22 (s, 1H, HCl). 13 C-NMR (75 MHz, CDCl3): d 158.93,
141.86, 126.87, 123.86, 115.89, 67.27, 56.93, 53.26, 26.37, 22.50,
21.88, 20.76. ESI-HRMS calculated for C17H25N4Oþ ([M-Cl]þ):
301.2023; found: 301.2021.
4.2.2.3. PTZ-induced seizures. PTZ (85 mg/kg) was injected sub-
cutaneously to induce seizures. Firstly, vehicle, tested compounds
3a-3q (10 mg/kg), and positive controls (VPA 300 mg/kg, PIT
4.1.4.17. 1-(5-(4-(4H-1,2,4-triazol-4-yl)phenoxy)pentyl)piperidine (3q). 10 mg/kg) were administered i.p. After 30 min, PTZ was injected to
Chemical formula: C18H26N4O ꢄ HCl (MW ¼ 314.43). m.p. 70–74 ꢃC,
all the animals. Animals were observed for 30 min (experiment
yield 77%. 1H-NMR (300 MHz, CDCl3):
d 1.56–2.09 (m, 14H,
period) for any convulsion signs, and graded scores were used to
assess the seizures severity. The following are the specific mean-
ing of graded scores: score 0 ¼ normal, score 1 ¼ eyelids or facial
twitches, score 2 ¼ agitation with body twisting, score 3 ¼ myo-
clonic jerks or rearing, score 4 ¼ turn over into one side position
or running violently, and score 5 ¼ turn over into back position,
limbs tonic extension, or die during the experiment period30.
NCH2(CH2)3(CH2)2CH2), 3.04–3.09 (m, 4H, N(CH2)2), 4.01 (t, 2H,
J ¼ 6.0 Hz, OCH2), 7.00 (d, 2H, J ¼ 8.8 Hz, Ph-H), 7.30 (d, 2H,
J ¼ 8.8 Hz, Ph-H), 8.44 (s, 2H, CH¼N). 13 C-NMR (75 MHz, CDCl3): d
159.25, 141.88, 126.69, 123.91, 115.86, 67.76, 57.25, 53.28, 28.32,
23.37, 22.49, 21.92, 21.88. ESI-HRMS calculated for C18H27N4Oþ
([M þ H]þ): 315.2179; found: 315.2180.
4.2. Pharmacology
4.2.3. Statistics
GraphPad Prism was used for statistical analysis. All data in vivo
were presented as the mean standard error of mean (SEM). One-
way analysis of variance (ANOVA), followed by the Dunnett’s post-
test were conducted for multiple comparisons. The statistical sig-
nificance was defined as p values <0.05.
4.2.1. In vitro screening
4.2.1.1. Cell culture and transfection. Human thalamus poly-A RNA
(Clontech, Palo Alto, CA, USA) was used to clone the hH3R gene
by RT-PCR. DNA PCR primers were designed in the light of the
reported human histamine receptor gene sequences (GenBank
accession no.AF140538). HEK-293 cells were cultured and trans-
fected for the luciferase assay. The detailed procedures were
described in the previous publication40.
4.2.4. Docking
4.2.4.1. Homology modelling. Crystal structure of the histamine H1
receptor (PDB ID: 3RZE) was used to construct the H3 receptor
homology model45. The hH3R primary sequence was downloaded
from the Universal Protein Resource (UniProt ID: Q9Y5N1). DS
MODELLER (Discovery Studio 2019) was used to construct a 3D
model of the H3R. Then the model was assessed in accordance
with the PDF Total Energy and the Profile-3D procedure. The 3D
model of H3R with the lowest PDF total energy was selected for
the next docking test.
4.2.1.2. CRE-driven reporter gene assay. Stable HEK-293 cells,
which had been co-transfected with H3R and pCRE-Luc, were
seeded in a 96-well plate overnight, and were grown to 90–95%
confluence43,44. Then the cells were treated with various concen-
trations of tested compounds in serum-free DMEM and incubated
for 20 min. Cells were then stimulated with 100 nM Histamine in
serum-free DMEM containing 2 mM Forskolin and incubated for 4 h
at 37 ꢃC. Firefly luciferase assay kits (Ken-real, Shanghai, China)
were used to determine the luciferase activity.
4.2.4.2. Molecular docking. To consider the conformation of the
protein and its ligands, Flexible Docking (Discovery Studio 2019)
was used for the docking procedure. The initial ligand and water
were removed, and hydrogen atoms were added. Three-dimen-
sional structures of compounds 3h, 3m as well as PIT were gener-
ated and then placed into the protein structure during the
molecular docking procedure. Interactions of the protein with 3h,
4.2.2. In vivo pharmacology
4.2.2.1. Drugs and animals. Valproic acid (VPA) was obtained from
melongpharma, Dalian, China. Ciproxifan maleate was purchased
from Shanghai Hanxiang Biotechnology Co., Ltd, China. R-
(a)-methyl-histamine
(RAMH),
Pitolisant
(PIT),
and
Pentylenetetrazol (PTZ) were bought from Macklin Co. KunMing 3m, and PIT were analysed.

1320
M. SONG ET AL.
16. Vohora D, Pal SN, Pillai KK. Thioperamide, a selective hista-
mine H3 receptor antagonist, protects against PTZ-induced
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18. Vohora D, Pal SN, Pillai KK. Histamine and selective H3-
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Acknowledgements
The School of Life Sciences, Jinggangshan University was appreci-
ated for providing the BIOVIA Discovery Studio software in this
study.
Disclosure statement
The authors declare that there is no conflict of interest.
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Funding
Thanks for the financial support provided by the National Natural
Science Foundation of China [No. 21562028].
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