Synthesis and in vitro anti-epileptic activities of novel [1,2,4]-triazolo[1,5-a]pyrimidin-7(4H)-one derivatives

Article abstract of DOI:10.1080/10286020.2018.1529030

In this investigation, eight novel 2,5-disubstituted [1,2,4]-triazolo[1,5-a]pyrimidin-7(4H)-one and eight novel 2,5-disubstituted [1,2,4]-triazolo[1,5-a]pyrimidine amine derivatives were synthesized based on the novel marine natural product Essramycin. Their anti-epileptic activities were evaluated by 4-aminopyridine (4-AP)-induced hyper excitability model in primary cultured neocortical neurons. Five compounds with [1,2,4]-triazolo[1,5-a]pyrimidin-7(4H)-one skeleton showed remarkable anti-epileptic activities. The preliminary structure–activity relationship (SAR) showed that the pyrimidine-7(4H)-one motif is the necessary “active core” of anti-epileptic activity. To understand the action mechanism of anti-epileptic activity of [1,2,4]-triazolo[1,5-a]pyrimidin-7(4H)-one compounds, docking studies using the model of GABA_A as docking scaffolds were performed and the docking results were in concordance with the experiment observations. (Figure presented.).

Full text of DOI:10.1080/10286020.2018.1529030

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Synthesis and in vitro anti-epileptic activities of
novel [1,2,4]-triazolo[1,5-a]pyrimidin-7(4H)-one
derivatives
Jing Ding, Feng-De Cao, Yan-Ru Geng, Yuan Tian, Peng Li, Xiu-Fen Li & Long-
Jiang Huang
To cite this article: Jing Ding, Feng-De Cao, Yan-Ru Geng, Yuan Tian, Peng Li, Xiu-Fen Li
& Long-Jiang Huang (2018): Synthesis and inꢀvitro anti-epileptic activities of novel [1,2,4]-
triazolo[1,5-a]pyrimidin-7(4H)-one derivatives, Journal of Asian Natural Products Research, DOI:
10.1080/10286020.2018.1529030
To link to this article: https://doi.org/10.1080/10286020.2018.1529030
Published online: 28 Dec 2018.
Submit your article to this journal
Article views: 5
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ganp20

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2018.1529030
Synthesis and in vitro anti-epileptic activities of novel
[1,2,4]-triazolo[1,5-a]pyrimidin-7(4H)-one derivatives
Jing Dinga, Feng-De Caoa, Yan-Ru Genga, Yuan Tiana, Peng Lia, Xiu-Fen Lia and
Long-Jiang Huanga,b
aCollege of Chemical Engineering, Qingdao University of Science and Technology, Qingdao, 266061,
b
China; State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing,
100050, China
ABSTRACT
ARTICLE HISTORY
Received 14 August 2018
Accepted 24 September 2018
In this investigation, eight novel 2,5-disubstituted [1,2,4]-triazolo
[1,5-a]pyrimidin-7(4H)-one and eight novel 2,5-disubstituted
[1,2,4]-triazolo[1,5-a]pyrimidine amine derivatives were synthesized
based on the novel marine natural product Essramycin. Their
anti-epileptic activities were evaluated by 4-aminopyridine (4-AP)-
induced hyper excitability model in primary cultured neocortical
neurons. Five compounds with [1,2,4]-triazolo[1,5-a]pyrimidin-
7(4H)-one skeleton showed remarkable anti-epileptic activities.
The preliminary structure–activity relationship (SAR) showed that
the pyrimidine-7(4H)-one motif is the necessary “active core” of
anti-epileptic activity. To understand the action mechanism of
anti-epileptic activity of [1,2,4]-triazolo[1,5-a]pyrimidin-7(4H)-one
compounds, docking studies using the model of GABAA as
docking scaffolds were performed and the docking results were
in concordance with the experiment observations.
KEYWORDS
1,2,4-triazolo[1,5-a]
pyrimidine-7(4H)-one;
synthesis; anti-epileptic;
molecular docking
1. Introduction
Epilepsy, a common neurological disorder characterized by recurrent spontaneous
seizures, is considered to be a major health problem that affects approximately
1–2% of the population worldwide [1]. Despite the considerable progress in our
CONTACT Long-Jiang Huang
huanglj@qust.edu.cn
College of Chemical Engineering, Qingdao University of
Science and Technology, Qingdao 266061, China
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
J. DING ET AL.
Figure 1. Examples of triazole derivatives with anti-epileptic activity.
Figure 2. The modification strategy based on Essramycin.
understanding of pathophysiology and pharmacotherapy of seizures and epilepsy, the
cellular basis of human epilepsy remains an enigma [2]. Over years, success has been
made in the development of anti-epileptic drugs (AEDs). However, current AEDs
either exhibit unfavorable side effect profiles or fail to adequately control seizures [3].
It is necessary and urgent to develop new generation of effective AEDs [4].
1,2,4-triazole is a key pharmacophore in pesticide and pharmaceutical synthesis,
and several AEDs with 1,2,4-triazole moiety have successfully entered the pharmaceut-
ical market such as Alprazolam, Estazolam, and Etizolam [5–9]. Due to their import-
ance in medicinal chemistry, especially in the field of anti-epilepsy, the heterocyclic
compounds bearing the 1,2,4-triazole moiety have received considerable attentions
[10–13]. For example, 1,2,4-triazole derivatives I, II, and III (Figure 1) showed
remarkable anti-epileptic activities in cell or animal models [10,11]. Interestingly,
most of these compounds showed anti-epileptic activity through activating GABAA
receptors or GABA-mediated mechanism (direct or indirect) [10,11,14–17].
Encouraged by anti-epileptic activities of 1,2,4-triazole compounds, we hypothesized
that the novel marine natural product essramycin bearing [1,2,4]-triazolo[1,5-a]
pyrimdin-7(4H)-one motif (Figure 2) possesses anti-epileptic activity. Just as we

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
expected, in our preliminary experiments, essramycin showed better anti-epileptic
activity (IC50 17 lM) than positive control carbamazepine (CBZ, IC50 35 lM) in 4-AP-
induced hyperexcitability model in primary cultured neocortical neurons. The result
prompted us to synthesize a series of compounds bearing [1,2,4]-triazolo[1,5-a]
pyrimidin-7(4H)-one motif as potential anti-epileptic agents.
In this work, a series of [1,2,4]-triazolo[1,5-a]pyrimidin-7(4H)-one derivatives
(Figure 2 structure A) based on essramycin scaffold were synthesized and their
anti-epileptic activities were evaluated. The preliminary SAR was studied by modify-
ing active structure A to structures B and C (Figure 2). In order to test whether the
anti-epileptic activity of our [1,2,4]-triazolo[1,5-a]pyrimidin-7(4H)-one derivatives
act through A-type GABA receptors (GABAA) like many other AEDs with the 1,2,4-
triazolo moiety, we performed docking studies on a native GABAA model.
2. Results and discussion
2.1. Chemistry
Essramycin was prepared according to the procedure reported by Huang L. H. [18].
The synthetic routes of [1,2,4]-triazolo[1,5-a]pyrimidin-7(4H)-one motif 6a-h were
showed in Scheme 1. Acids 1a-d were reacted with (COCl)2/DMF to afford acyl
chlorides 2a-d. The acyl chlorides 2a-d reacted with aminoguanidine bicarbonate
under base condition to afford the products 3a-d. Compounds 3a-d were cyclized
under acid condition to afford 2-amino-1,2,4-triazole derivatives 4a-d. 2-amino-1,2,4-
triazole derivatives 4a-d reacted with b-ketone esters 5a-b to afford 6a-h with [1,2,4]-
triazolo[1,5-a]pyrimidin-7(4H)-one motif.
The synthetic route of [1,2,4]-triazolo[1,5-a]pyrimidin-7-amine derivatives 8a-h was
showed in Scheme 2. The [1,2,4]-triazolo[1,5-a]pyrimidin-7-one derivatives 6b and 6e
were reacted with POCl3 in reflux under catalytic amount of DMF to afford
compounds 7a-b. Compounds 7a-b reacted with different amines to afford the targets
8a-h.
Scheme 1. The synthetic route of [1,2,4]-triazolo[1,5-a]pyrimidin-7(4H)-one motifs 6a–h. 1a–4a:
R1¼H; 1b–4b:R1¼4-CH3; 1c–4c:R1¼4-OCH3; 1d–4d: R1¼2-F; 5a:R2¼H; 5b:R2¼4-Cl; 6a:R1¼H, R2¼H;
6b:R1¼4-CH3, R2¼H; 6c:R1¼4-OCH3, R2¼H; 6d:R1¼2-F, R2¼H; 6e:R1¼H, R2¼4-Cl; 6f:R1¼4-CH3,
R2¼4-Cl; 6g:R1¼2-F, R2¼4-Cl; 6h: R1¼4-OCH3, R2¼4-Cl. Reagents and conditions: i: oxaloyl chloride,
DCM, rt, 2.5h, yield 75%–85%; ii: aminoguanidine carbonate, dry DCM, rt, 12 h, yield 50–90%; iii:
water, 100ꢀC, 12 h, yield 80–90%; iiii: n-BuOH, TsOH, 125ꢀC, 24 h, yield of 70–98%.

4
J. DING ET AL.
Scheme 2. The synthetic route of [1,2,4]-triazolo[1,5-a]pyrimidin-7-amine derivatives 8a–h.
7a:R1¼4-CH3, R2¼H; 7b: R1¼ H, R2¼4-Cl; 8a:R1¼4-CH3, R2¼H, R3¼-NHCH2CH3; 8b:R1¼4-CH3, R2¼H,
R3¼-NC5H10; 8c:R1¼4-CH3, R2¼H, R3¼-NC6H5; 8d:R1¼4-CH3, R2¼H, R3¼-NHCH2C6H5; 8e:R1¼H,
R2¼4-Cl, R3¼-NHCH2CH3; 8f: R1¼H, R2¼4-Cl, R3¼-NC5H10; 8g:R1¼H, R2¼4-Cl, R3¼-NHC6H5; 8h:
R1¼H, R2¼4-Cl, R3¼-NHCH2C6H5. Reagents and conditions: i: POCl3, DMF, 90 ꢀC, 5 h, yield 80–90%;
ii: Et3N, amine, EtOH, rt, yield 50–80%.
2.2. Anti-epileptic activity
Primary cultured neocortex neurons can form neuronal networks and display
spontaneous Ca2þ oscillations (SCOs). These SCOs occur simultaneously with action
potential generation. SCO activity is dependent on the balanced excitatory/inhibitory
neuronal inputs. 4-AP can induces hyper-excitability in neurons, therefore has been
considered to be an in vitro seizurogenic model widely used for in vitro anti-epileptic
activity test [19]. The activities toward 4-AP-induced SCO changes of 17 compounds
were tested in primary cultured neocortical neurons. Compounds 6a-h containing
[1,2,4]-triazolo[1,5-a]pyrimidin-7(4H)-one motif inhibited the intracellular calcium
ion concussion of mouse neuronal cells. Compounds 6a, 6b, 6c, 6d, and 6e showed
inhibitory effect in 4-AP induced hyper excitability model in primary cultured
neocortical neurons (Figure 3). In order to determine the “active core” of these
compounds, we selected the best active compound 6b as the model and modified
the structure of compound 6b to 7a and 8a-d (Scheme 2). Interestingly, all of the
compounds 7a and 8a-d showed no any inhibitory effects on 4-AP induced SCO
changes (Figure 4). The result indicated that the pyrimidine-7(4H)-one motif in the
structure of 6a-e is the probably “active core”. In order to confirm this, the active
compound 6e was modified to compounds 7b and 8e-h by using the same strategy
with compound 6b (Scheme 2). Compounds 7b and 8e-h had no effect on 4-AP-
induced SCO changes (Figure 4). All of the results indicated that the pyrimidin-
7(4H)-one motif in the structure of 6a-e is necessary to their activity.
Through a continuous assay procedure, different concentrations of compounds
6a-e were tested in 4-AP-induced hyper excitability model in primary cultured

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Figure 3. Effects of compounds 6a–h on 4-AP-induced SCO changes in neocortical neurons.
Figure 4. Effects of compounds 8a–h and 7a on 4-AP-induced SCO changes in neocortical
neurons.
neocortical neurons. As shown in Figure 5, it was found that with the increase of the
drug concentration, the compounds (6a, 6b, 6c, 6d, 6e) can inhibit the spontaneous
calcium oscillation of primary cortical neurons (Figure 5(A)) and 4-AP induced
hyper excitability model in primary cultured neocortical neurons (Figure 5(B)) in a
dose-dependent manner. Compounds 6a, 6b, 6c, 6d, and 6e showed inhibitory effect
on the intracellular calcium ion concussion of mouse neuronal cells with IC50 values
between 1.68 lM and 7.24 lM (Table 1). In the 4-AP induced hyper excitability
model in primary cultured neocortical neurons, IC50 values of compounds 6a, 6b, 6c,
6d, and 6e were ranged between 2.35 lM and 15.40 lM (Table 1). Compounds 6b
and 6e (IC50 2.35 lM and 3.21 lM, respectively) significantly outperformed essramy-
cin (IC50 17 lM) and postive control-CBZ (IC50 35 lM).
2.3. Molecular docking studies
CBZ can inhibit seizures by enhancing the transmission or activity of GABAergic
neurons and activating GABAA-mediated mechanisms [20], most of 1,2,4-triazole

6
J. DING ET AL.
Figure 5. A: Concentration response curve for compounds 6a–6e inhibiting the frequency of
SCO in primary cortical neuron; B: Concentration response curve for compounds 6a–6e inhibiting
4-AP-induced SCOs in neocortical neurons.
Table 1. The IC50 values of the active compounds against 4-AP-induced neuron excitability.
4-AP primary cerebral cortex
Primary cortical cells
95%CI (lM)
hyperstimulation model
Compd
IC50 (lM)
IC50 (lM)
95%CI (lM)
6a
6b
6c
6d
6e
CBZ
2.04
2.27
7.24
3.23
1.68
–
1.44–2.88
1.67–3.08
4.87–10.77
2.49–4.20
0.58–4.87
–
11.03
2.35
12.35
15.40
3.21
6.39–19.04
1.77–3.12
8.46–18.03
8.79–26.98
2.70–3.83
35.0
32.21–37.79
95% CI: 95% confidence intervals.
derivatives with anti-epileptic activity also have a similar mechanism [21–23]. In
order to study the action mechanism of compounds 6a-e, the docking mode of
GABAA protein with compounds 6a-e was studied. Compounds 6a-e were docked to
subunits of extracellular domain of GABAA interacting with CBZ. [20].
As shown in Figure 6, from top view of GABAA protein model, all of compounds
6a-e can bind with active region of GABAA and the binding sites of compounds 6a,
6c, 6d with GABAA were different. Compounds 6b and 6e shared same binding sites
with GABAA. The detailed binding sites of compounds 6b and 6e with the amino
acid residues of GABAA were shown in Figures 7 and 8.
In 2017, Meeta Sahu et al. proposed binding interactions of standard drug CBZ
with GABAA receptor (PDB ID: 4COF) and the result showed that CBZ made crucial
H-bonds with Arg216 and Ile218 of GABAA active site [20]. In our experiment, the
most effective molecules 6b (as showed in Figure 7) and 6e (as showed in Figure 8)
made the same crucial H-bonds with Arg216 and Ile218 of GABAA active site. In
addition, the methyl group on the benzene ring attached on the trizole ring of
compound 6b had a hydrophobic effect with residues Pro-276, the pyrimidone ring
and two benzene rings have pi-Cation, pi-Sigma, and pi-Alkyl interactions with the
amino acid residues Lys-215, Pro-276 and Leu-145, respectively. Same with the
compound 6b, the pyrimidone ring and two benzene rings of compound 6e had
pi-Cation, pi-Sigma, and pi-Alkyl interactions with the amino acid residues Lys-215,
Pro-276 and Leu-145, respectively.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
Figure 6. Top view of GABAA protein model (PDB Code: 4COF): active region of GABAA and 6a-e
docking conformation with GABAA.
Figure 7. Docking conformation of compound 6b in the active pocket of GABAA.
The docking results indicated that compounds 6b and 6e had same action
mechanism with the receptor protein GABAA. Compounds 6a, 6c, and 6d bound the
different active sites of GABAA receptor as compared with 6b and 6e. The H-bonds
binding energy of compounds 6a-e to the amino acid residues of GABAA receptor
was calculated and shown in Table 2. These docking results were in concordance
with the observations on the 4-AP induced hyper excitability model in primary
cultured neocortical neurons.

8
J. DING ET AL.
Figure 8. Docking conformation of compound 6e in the active pocket of GABAA.
Table 2. The H-bonds binding energy of compounds 6a–e to the amino acid residues of
GABAA receptor.
Compound
Binding energy
Amino acid residues
6a
6b
6c
6d
6e
8.69
7.85
7.38
7.38
7.39
ASN-217, GLU-182
ARG-216, ILE-218
ILE-218
ARG-142, ASP-146
ARG-216, ILE-218
3. Experimental
3.1. Chemistry
All of the solvents and materials were analytical grade and used without further
1
purification. General H NMR and 13C NMR spectra were recorded on a 500 MHz,
125 MHz (Bruker Avance III) instrument, respectively, and chemical shifts were
given in ppm relative to tetramethylsilane. Mass spectra were measured on an
UPLC-Q/TOF Xevo G2-XS (Waters, MA, USA). All chemical shifts are reported as d
(ppm) values. Splitting patterns are designated as follows: s: singlet; d: doublet; t:
triplet; q: quartet; and m: multiplet.
3.2. Synthesis of the compounds
3.2.1. 3-Amino-1,2,4-triazole derivatives 4a-d
To a solution of benzoic acid derivatives (1a-d) (132 mmol) in dry dichloromethane
(200 ml), was added oxalyl chloride (158.4 mmol) and a catalytic amount of DMF
(two drops) at 0 ꢀC, and the mixture was allowed to warm to room temperature and
stirred at room temperature for 2.5 h. The resulting mixture was concentrated under
vacuum to afford benzoyl chloride derivatives (2a-d) as light yellow oil, which was
used directly without further purification.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
To a solution of aminoguanidine carbonate (20.0 mmol) in pyridine (50 ml) was
added benzoyl chloride derivatives 2a-d (10.0 mmol), and the reaction mixture was
stirred for 12 h at room temperature. The resulting mixture was concentrated under
vacuum to give a residue, which was dissolved in water (30 ml) and adjusted to pH
12 with sodium hydroxide. The product was precipitated and collected by filtration
and dried in an oven at 40 ꢀC under reduced pressure to afford 3a-d.
A solution of compounds 3a-d (10.75 mmol) in water (100 ml) was stirred
overnight at 100 ꢀC. The reaction was cooled down to room temperature; the product
was precipitated and collected by filtration and dried in an oven at 40 ꢀC under
reduced pressure to afford 4a-d.
3.2.2. Methyl benzoylacetate derivatives 5a–b
To a solution of NaH (166.7 mmol) in toluene (40 ml) was added dimethyl carbonate
(166.7 mmol). After the mixture was heated to 100 ꢀC, a solution of acetophenone
derivatives (83.3 mmol) in toluene (20 ml) was added dropwise over 0.5 h, and the
reaction mixture was stirred for 2 h at 100 ꢀC. The reaction was cooled down to room
temperature. Glacial acetic acid (30 ml) was added dropwise and a heavy pasty solid
separated. Ice-cold water was slowly added until the solid was dissolved completely.
Then, the reaction system was diluted with 200 ml of EtOAc. The organic layer was
separated, washed with water (20 ml) and brine (20 ml) and dried over Na2SO4. After
the solvent was evaporated, the residue was purified by flash chromatography on
silica gel with petroleum ether/ethyl acetate (20:1) as eluent to give the desired aryl
b-ketoesters 5a-b.
3.2.3. [1,2,4]-Triazolo[1,5-a]pyrimidin-7(4H)-one motif 6a–h
To a solution of 4a-d (5.3 mmol) in n-BuOH (20 ml) was added 5a-b (10.6 mmol)
and TsOH (0.53 mmol), and the reaction mixture was stirred at 125 ꢀC for 24 h.
The solids were collected by filtration and washed with methanol (10 ml) to afford
[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one 6a-h.
3.2.3.1. 2,5-Diphenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one (6a). White solid,
1
yield 98%; H-NMR (DMSO, 500MHz) d (ppm): 8.14 (t, J ¼ 6.5Hz, 2H), 7.89 (d,
J ¼ 6.5Hz, 2H), 7.57–7.53 (m, 6H), 6.34 (s, 1H). 13C-NMR (DMSO, 125MHz) d
(ppm): 156.3, 152.2, 131.7, 130.9, 129.4 (6C), 127.9 (3C), 127.1 (3C), 98.1. HRESIMS:
m/z 289.1094 [M þ H]þ (calcd for C17H13N4O, 289.1089).
3.2.3.2. 5-Phenyl-2-(p-tolyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one (6b). White
solid, yield 95%; 1H-NMR (DMSO, 500MHz) d (ppm): 13.55 (s, 1H), 8.03 (d,
J ¼ 7.5HZ, 2H), 7.90 (d, J ¼ 6.0Hz, 2H), 7.57 (t, J ¼ 7.0Hz, 3H), 7.36 (d, J ¼ 7.5Hz,
2H), 6.34 (s, 1H), 2.39 (s, 3H); 13C-NMR (DMSO, 125MHz) d (ppm): 156.3, 152.1,
140.7, 131.6, 130.0 (3C), 129.4 (3C), 127.9 (3C), 127.0 (3C), 98.1, 21.5. HRESIMS:
m/z 303.1247 [M þ H]þ (calcd for C18H15N4O, 303.1246).
3.2.3.3. 2-(4-Methoxyphenyl)-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one (6c).
1
White solid, yield 97%; H-NMR (DMSO, 500MHz) d (ppm): 8.08 (d, J ¼ 8.5Hz, 2H),

10
J. DING ET AL.
7.91 (d, J ¼ 6.0Hz, 2H), 7.56 (d, J ¼ 6.5Hz, 3H), 7.11 (d, J ¼ 8.5Hz, 2H), 6.33 (s, 1H),
3.84 (s, 3H). 13C-NMR (DMSO, 125MHz) d (ppm): 161.6, 156.2, 152.2, 131.5, 129.4
(3C), 128.7 (3C), 127.9 (3C), 114.8 (3C), 97.7, 55.8. HRESIMS: m/z 319.1198
[M þ H]þ (calcd for C18H15N4O2, 319.1195).
3.2.3.4. 2-(2-Fluorophenyl)-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one (6d).
1
Light yellow solid, yield 71%; H-NMR (DMSO, 500MHz) d (ppm): 13.64 (s, 1H),
8.13 (t, J ¼ 7.5Hz, 1H), 7.90 (d, J ¼ 7.0Hz, 2H), 7.57 (t, J ¼ 7.5Hz, 3H), 7.43–7.38
(m, 2H), 6.36 (s, 1H). 13C-NMR:(DMSO,125MHz) d (ppm) 161.5, 159.3, 158.1, 156.2,
151.9, 132.7, 132.2, 131.7, 130.8, 129.4 (2C), 128.0 (2C), 125.2, 118.4, 117.2, 98.1.
HRESIMS: m/z 307.0998 [M þ H]þ (calcd for C17H12N4OF, 307.0995).
3.2.3.5. 5-(4-Chlorophenyl)-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one (6e).
1
White solid, yield 98%; H-NMR (DMSO, 500MHz) d (ppm): 8.14 (d, J ¼ 6.0Hz, 2H),
7.92 (d, J ¼ 8.0Hz, 2H), 7.63 (d, J ¼ 8.0Hz, 2H), 7.55 (s, 3H), 6.38 (s, 1H). 13C-NMR
(DMSO, 125MHz) d (ppm): 156.2, 152.1, 136.4, 131.0, 129.79 (3C), 129.41 (6C),
127.05 (3C), 98.47. HRESIMS: m/z 323.0702 [M þ H]þ (calcd for C17H12N4
OCl, 323.0700).
3.2.3.6. 5-(4-Chlorophenyl)-2-(p-tolyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one (6f).
1
White solid, yield 94%; H-NMR (DMSO, 500MHz) d (ppm): 8.03 (d, J ¼ 8.0Hz, 2H),
7.93 (d, J ¼ 8.5Hz, 2H), 7.63 (d, J ¼ 8.5Hz, 2H), 7.37 (d, J ¼ 8.0Hz, 2H), 6.39 (s, 1H),
2.39 (s, 3H). 13C-NMR (DMSO, 125MHz) d (ppm): 156.2, 152.1, 140.9, 136.3, 130.0
(3C), 129.8 (3C), 129.4 (3C), 127.0 (3C), 98.5, 21.5. HRESIMS: m/z 337.0858
[M þ H]þ (calcd for C18H14N4OCl, 337.0856).
3.2.3.7. 5-(4-Chlorophenyl)-2-(2-fluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-
1
one (6g). Light yellow solid, yield 71%; H-NMR (DMSO, 500MHz) d (ppm): 8.11
(t, J ¼ 7.0Hz, 1H), 7.91 (d, J ¼ 8.5Hz, 2H), 7.64–7.57 (m, 3H), 7.43-7.37 (m, 2H), 6.39
(s, 1H).13C-NMR (DMSO, 125MHz) d (ppm): 161.4, 159.4, 157.8, 156.1, 151.9, 136.5,
132.8, 131.4, 130.8, 129.8 (2C), 129.4 (2C), 125.2, 118.2, 117.4, 98.4. HRESIMS: m/z
341.0607 [M þ H]þ (calcd for C17H11N4OFCl, 341.0605).
3.2.3.8.
5-(4-Chlorophenyl)-2-(4-methoxyphenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-
1
7(4H)-one (6h). White solid, yield 98%; H-NMR (DMSO, 500MHz) d (ppm): 8.08
(d, J ¼ 8.5Hz, 2H), 7.93 (d, J ¼ 8.0Hz, 2H), 7.63 (d, J ¼ 8.0Hz, 2H), 7.12 (d, J ¼ 8.5Hz,
2H), 6.4 (s, 1H), 3.84 (s, 3H), 13C-NMR (DMSO, 125MHz) d (ppm): 161.7, 156.2,
152.0, 136.3, 129.7 (3C), 129.4 (3C), 128.7 (3C), 114.9 (3C), 98.5, 55.8. HRESIMS:
m/z 353.0808 [M þ H]þ (calcd for C18H14N4O2Cl, 353.0805).
3.2.4. [1,2,4]-Triazolo[1,5-a]pyrimidine amine 8a–h
To a solution of 6b or 6e (2.7 mmol) in POCl3 (6 ml) was added 0.1 ml DMF, the
resulting mixture was stirred at 90 ꢀC for 5 h. After cooling to room temperature,
ice was added to quench the reaction, the water phase was extracted with ethyl
acetate 2–3 times. The organic phase was combined and dried with anhydrous

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
11
sodium sulfate, and the organic layer was evaporated to afford the yellow solid 7a-b.
To a solution of 7a-b (0.44 mmol) in EtOH, was added Et3N and amine (0.88 mmol),
the resulting mixture was stirred at room temperature overnight. The resulting
mixture was concentrated under vacuum and water was added, the solid was filtered,
and the filter cake was washed with water and dried under vacuum to afford yellow
solid 8a-h.
3.2.4.1. N-Ethyl-5-phenyl-2-(p-tolyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (8a).
1
White solid, yield 80%; H-NMR (DMSO, 500MHz) d (ppm): 8.21 (d, J ¼ 5.0Hz, 2H),
8.11 (d, J ¼ 8.0Hz, 2H), 7.52 (s, 3H), 7.36 (d, J ¼ 7.5Hz, 2H), 6.97 (s, 1H), 3.62–3.58
(m, 2H), 2.37 (s, 3H), 1.27 (t, J ¼ 7.0Hz, 3H). 13C-NMR (DMSO, 125MHz) d (ppm):
163.5, 160.6, 156.4, 148.1, 140.4, 137.9, 130.8, 129.8 (2C), 129.1 (2C), 128.4, 127.8
(2C), 127.2 (2C), 85.4, 37.0, 21.5, 14.7. HRESIMS: m/z 330.1722 [M þ H]þ (calcd for
C20H20N5, 330.1719).
3.2.4.2. 5-Phenyl-7-(piperidin-1-yl)-2-(p-tolyl)-[1,2,4]triazolo[1,5-a]pyrimidine (8b).
Light yellow solid, yield 75%; 1H-NMR (CDCl3, 500MHz) d (ppm): 8.26 (d,
J ¼ 8.0Hz, 2H), 8.17–8.15 (m, 2H), 7.49–7.48 (m, 3H), 7.29 (d, J ¼ 8.0Hz, 2H), 6.7 (s,
1H), 3.92 (t, J ¼ 5.4Hz, 4H), 2.42 (s, 3H), 1.89-1.87 (m, 4H), 1.82–1.81 (m, 2H).
13C-NMR (DMSO, 125MHz) d (ppm): 163.3, 160.4, 158.4, 150.6, 140.5, 137.8, 130.9,
129.9 (2C), 129.2 (2C), 128.4, 127.9 (2C), 127.1 (2C), 91.8, 55.3 (2C), 25.6 (2C), 24.3,
21.5. HRESIMS: m/z 370.2032 [M þ H]þ (calcd for C23H24N5, 370.2030).
3.2.4.3. N,5-Diphenyl-2-(p-tolyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (8c). Light
1
yellow solid, yield 70%; H-NMR (DMSO, 500MHz) d (ppm): 8.18 (d, J ¼ 8.0Hz, 2H),
8.0–7.9 (m, 2H), 7.57–7.48 (m, 7H), 7.38 (d, J ¼ 8.0Hz, 2H), 7.33 (t, J ¼ 7.0Hz, 1H),
6.86 (s, 1H), 2.38 (s, 3H). 13C-NMR (DMSO, 125MHz) d (ppm): 164.2, 160.8, 156.9,
146.9, 140.6, 137.8, 137.3, 130.9, 130.1 (2C), 129.9 (2C), 129.3 (2C), 128.3, 127.6 (2C),
127.4 (2C), 126.7, 124.9 (2C), 86.8, 21.3. HRESIMS: m/z 378.1718 [M þ H]þ (calcd
for C24H20N5, 378.1719).
3.2.4.4. N-Benzyl-5-phenyl-2-(p-tolyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (8d).
1
White solid, yield 78%; H-NMR (CDCl3, 500MHz) d (ppm): 8.23 (d, J ¼ 8.0Hz, 2H),
8.11–8.09 (m, 2H), 7.47–7.38 (m, 8H), 7.29 (d, J ¼ 8.0Hz, 2H), 6.6 (s, 1H), 6.57 (s,
1H), 4.72 (d, J ¼ 6.0Hz, 2H), 2.42 (s, 3H). 13C-NMR (DMSO, 125MHz) d (ppm):
164.1, 160.4, 156.7, 148.2, 140.4, 138.4, 137.9, 130.8, 129.9 (2C), 129.3, 129.1 (2C),
129.0 (3C), 128.5, 127.7 (3C), 127.2 (2C), 85.7, 45.1, 21.5. HRESIMS: m/z 392.1874
[M þ H]þ (calcd for C25H22N5, 392.1875).
3.2.4.5. 5-(4-Chlorophenyl)-N-ethyl-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine
1
(8e). Light yellow solid, yield 59%; H-NMR (CDCl3, 500MHz) d (ppm): 8.36–8.34
(m, 2H), 8.12 (d, J ¼ 8.5Hz, 2H), 7.50–7.46 (m, 5H), 6.51 (s, 1H), 3.62–3.58 (m, 2H),
1.51 (t, J ¼ 7.5Hz, 3H). 13C-NMR (DMSO, 125MHz) d (ppm): 163.9, 159.2, 156.7,
148.1, 136.9, 135.6, 131.3, 130.7, 129.6 (2C), 129.2 (2C), 129.1 (2C), 127.2 (2C), 85.2,
37.0, 14.7. HRESIMS: m/z 350.1173 [M þ H]þ (calcd for C19H17N5Cl, 350.1172).

12
J. DING ET AL.
3.2.4.6.
5-(4-Chlorophenyl)-2-phenyl-7-(piperidin-1-yl)-[1,2,4]triazolo
[1,5-a]
1
pyrimidine (8f). Light yellow solid, yield 60%; H-NMR (CDCl3, 500MHz) d (ppm):
8.37 (d, J ¼ 8.0Hz, 2H), 8.12 (d, J ¼ 8.0Hz, 2H), 7.48 (t, J ¼ 8.0Hz, 5H), 6.60 (s, 1H),
3.94 (t, J ¼ 4.5Hz, 4H), 1.89–1.57 (m, 6H). 13C-NMR (DMSO, 125MHz) d (ppm):
163.2, 159.1, 158.3, 150.6, 136.5, 135.8, 131.0, 130.8, 129.7 (2C), 129.3 (2C), 129.2
(2C), 127.2 (2C), 91.7, 49.6 (2C), 25.7 (2C), 24.3. HRESIMS: m/z 390.1486 [M þ H]þ
(calcd for C22H21N5Cl, 390.1485).
3.2.4.7.
5-(4-Chlorophenyl)-N,2-diphenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine
1
(8g). White solid, yield 60%; H-NMR (CDCl3, 500MHz) d (ppm): 8.40 (d, J ¼ 6.0Hz,
2H), 8.05 (d, J ¼ 8.5Hz, 2H), 7.97 (s, 1H), 7.58–7.39 (m, 10H), 6.91 (s, 1H). 13C-NMR
(DMSO, 125MHz) d (ppm): 164.3, 159.6, 157.0, 147.0, 137.2, 136.6, 135.7, 131.1,
130.9, 130.1 (2C), 129.4 (2C), 129.3 (4C), 127.4 (2C), 126.7, 124.9 (2C), 86.80.
HRESIMS: m/z 398.1175 [M þ H]þ (calcd for C23H17N5Cl, 398.1172).
3.2.4.8. N-Benzyl-5-(4-chlorophenyl)-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine
(8h). Light yellow solid, yield 75%; 1H-NMR (CDCl3, 500MHz) d (ppm): 8.34
(t, J ¼ 3.7Hz, 2H), 8.04 (d, J ¼ 8.5Hz, 2H), 7.49–7.38 (m, 10H), 6.65 (s, 1H), 6.52 (s,
1H), 4.73 (d, J ¼ 5.5Hz, 2H). 13C-NMR (DMSO, 125MHz) d (ppm): 163.8, 159.2,
156.4, 148.4, 138.3, 136.6, 135.7, 131.0, 130.8, 129.5 (2C), 129.2 (2C), 129.1 (2C),
129.0 (2C), 127.7 (3C), 127.3 (2C), 85.9, 45.1. HRESIMS: m/z 412.1330 [M þ H]þ
(calcd for C24H19N5Cl, 412.1329).
3.3. Pharmacology
3.3.1. Primary neocortical neuron culture
All animal experiment protocols were carried out in accordance with the National
Institutes of Health guide for the care and use of Laboratory animals (NIH
Publications No. 8023, revised 1978) approved by the China Pharmaceutical
University Institutional Animal Care and Use Committee. Animals were treated
humanly and with regard for alleviation of suffering. Neocortical neuron cultures
were obtained from embryonic day 16 C57 BL/6 mice. Briefly, neocortices were
collected, stripped off meninges, minced by trituration with a Pasteur pipette, and
treated with trypsin for 25 min at 37 ꢀC. The cells were then dissociated by two
successive trituration and sedimentation steps in soybean trypsin inhibitor and DNase
I-containing isolation buffer, centrifuged, and resuspended in neurobasal complete
medium (neurobasal medium supplemented with 2% NS21 [24], 1 mM L-glutamine,
1% HEPES with 5% fetal bovine serum), Cells were planted onto poly-L-lysine-coated
clear-bottomed, black-wall, 96-well plates (Corning Life Sciences, Acton, MA) at a
density of 5.6 ꢁ 105 cells/cm2 and incubated at 37 ꢀC in a 5% CO2 and 95% humidity
atmosphere. A final concentration of ARA-C (10 lM) was added to the culture
medium after planting for 24–36 h to prevent the astrocytes proliferation. The
culture medium was half-replaced on days 5 and 7 with serum-free growth medium
containing neurobasal medium supplemented with 2% NS21, 1% HEPES, and 0.2 mM
L-glutamine.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
13
3.3.2. Intracellular Ca2þ concentration determination
Neocortical neurons at 8–9 d in vitro (DIV) were used to investigate the alteration of
compounds on intracellular Ca2þ dynamics (Huang QL, PhytoChemistry, 2018) [25].
Briefly, the growth medium was removed and replaced with dye loading buffer
(100 ll/well) containing 4 lM Fluo-8/AM and 0.5% BSA in Locke’s buffer (in mM:
HEPES 8.6, KCl 5.6, NaCl 154, D-glucose 5.6, MgCl2 1.0, CaCl2 2.3, and glycine 0.1,
adjust pH to 7.4). After 45 min incubation in dye loading buffer, the neurons were
washed four times with fresh Locke’s buffer (200 ll/well) and placed in a Fluorescent
Imaging Plate Reader (FLIPRTetra; Molecular Devices, Sunnyvale, CA) incubation
chamber. Basal fluorescence levels were acquired in Locke’s buffer for 5 min at a
sampling rate of 1 Hz followed by an addition of vehicle or compounds using a
programmable 96-channel pipetting robotic system, and the fluorescent signals were
recorded for an additional 8 min. To investigate influence of compounds on 4-AP-
induced Ca2þ response, a second addition of 4-AP or was made and the fluorescent
signals were recorded for an additional 8 min.
The background fluorescence of the plate was determined from a sister well
without Fluo-8/AM loading, and all the fluorescence signals were corrected by
subtracting the background fluorescence. Data were presented as F/F0, where F is the
fluorescent signal at different time point minus background fluorescence, whereas
F0 is the basal fluorescence minus the background fluorescence. To determine the
compounds response on SCOs, the SCO frequency from an epoch of 300 s after
addition of vehicle (0.1% DMSO) or compounds for 60 s was manually counted. All
the data were presented as % vehicle. To determine the compounds response on
4-AP-induced Ca2þ oscillation, the Ca2þ oscillation frequency was manually counted
from an epoch of 300 s in the presence or absence of compounds after addition of
4-AP for 120 s. The data were presented as % of 4-AP. Events having F/F0 >0.05
units were included in the analyses of SCO frequency.
3.3.3. Molecular docking studies
The crystal protein of GABA receptor protein was from Protein Data Bank database
(PDB), PDB code: 4COF. Autodock 4.2 (http://autodock.Scripps.edu/) was used as
molecular docking software and the software for PDB crystal protein processing was
Autodock tools 1.5.6 (http://autodock.scripps.edu/) during docking process. The
render software package was Discovery Studio 4.5 Client (http://accelrys.com/) and
algorithm was Genetic Algorithm in autodock 4.2.
4. Conclusion
In this work, eight [1,2,4]-triazolo[1,5-a]pyrimidin-7(4H)-one derivatives were synthe-
sized and their anti-epileptic activities were evaluated on the intracellular calcium ion
concussion of mouse neuronal cells and 4-AP induced hyper excitability model in
primary cultured neocortical neurons. Five of [1,2,4]-triazolo[1,5-a]pyrimidin-7(4H)-
one derivatives show remarkable inhibitory effects on these two models. The most
effective compounds 6b and 6e were obviously superior to marine natural product
essramycin and positive control CBZ. The preliminary SAR was studied by modifying

14
J. DING ET AL.
the compounds 6b and 6e to [1,2,4]-triazolo[1,5-a]pyrimidine amine 8a-h or
chloride [1,2,4]-triazolo[1,5-a]pyrimidine 7a-b and the results indicated that the
pyrimidin-7(4H)-one motif in the active compounds of 6a-6e is the necessary “active
core” of anti-epileptic activity. The molecular docking revealed that compounds 6b
and 6e have strong binding affinity with GABAA receptor, the result is agreement
with the experimental observations.
Disclosure statement
No potential conflict of interests was reported by the authors.
Funding
This work was financially supported by the Opening Foundation of State Key Laboratory of
Bioactive Substance and Function of Natural Medicine (No. GTZK201805) and the Shandong
Province Higher Educational Science and Technology Program (No. J16LC13).
References
[1] P. Kwan and M. J. Brodie, N. Engl. J. Med. 342, 314 (2000).
[2] E. Consortium, E. P. Project, A. S. Allen, S. F. Berkovic, and P. Cossette, Nature 501,
217 (2013).
[3] P. Kwan and M. J. Brodie, Seizure 9, 464 (2000).
[4] J. R. Pollard and J. French, Neurology 5, 1064 (2006).
[5] B. Chai, H. Q. Xu, S. Cao, H. D. Liu, and G. H. Song, Arkivoc. 36, 141 (2003).
[6] R. Chelamalla, A. Venkatesham, and M. Sarangapani, J. Pharm. Res. 5, 4739 (2012).
[7] H. B. Shivarama, R. B. Sooryanarayana, B. K. Sarojini, P. M. Akberali, and N. S.
Kumari, Eur. J. Med. Chem. 41, 657 (2006).
[8] A. Ayati, S. Emami, and A. Foroumadi, Eur. J. Med. Chem. 47, 380 (2016).
[9] V. K. Kamboj, P. K. Verma, A. Dhanda, and S. Ranjan, Cent. Nerv. Syst. Agents Med.
Chem. 15, 17 (2015).
[10] X. Q. Deng, C. X. Wei, F. N. Li, Z. G. Sun, and Z. S. Quan, Eur. J. Med. Chem. 45,
3080 (2010).
[11] T. Plech, B. Kapro, J. J. Luszczki, A. Paneth, A. Siwek, M. Kolaczkowski, M. Zolnierek,
and G. Nowak, Eur. J. Med. Chem. 86, 690 (2014).
[12] L. Zhang, L. P. Guan, X. Y. Sun, C. X. Wei, K. Y. Wei, and Z. S. Quan, Chem. Biol.
Drug. Des. 73, 313 (2009).
[13] A. Almasirad, S. A. Tabatabai, M. Faizi, A. Kebriaeezadeh, N. Mehrabi, A. Dalvandi,
and A. Shafiee, Bioorg. Med. Chem. Lett. 14, 6057 (2004).
[14] Y. Q. Fang, C. L. Sun, D. C. Liu, S. B. Wang, and Z. S. Quan, Iran. J. Pharm. Res. 14,
77 (2015).
[15] S. B. Wang, X. Q. Deng, Y. Zheng, H. J. Zhang, and Z. S. Quan, Arch. Pharm. Res. 36,
32 (2013).
[16] X. Q. Deng, L. N. Quan, M. X. Song, C. X. Wei, and Z. S. Quan, Eur. J. Med. Chem.
46, 2955 (2011).
[17] X. Q. Deng, Z. Q. Dong, M. X. Song, B. Shu, S. B. Wang, and Z. S. Quan, Arch.
Pharm. 345, 565 (2012).
[18] L. H. Huang, Y. F. Zheng, C. J. Song, Y. G. Wang, Z. Y. Xie, Y. W. Lai, Y. Z. Lu, and
H. M. Liu, Steroids 77, 367 (2012).
[19] Z. Y. Cao, X. H. Zou, Y. J. Cui, S. S. Hulsizer, P. J. Lein, H. K. Wulff, and I. N. Pessah,
Mol. Pharmacol. 87, 595 (2015).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
15
[20] M. Sahu, N. Siddiqui, M. J. Naim, O. Alam, M. S. Yar, V. Sharma, and S. Wakode,
Arch. Pharm. 350, 1 (2017).
[21] L. Battistin, M. Varotto, G. Berlese, and R. Giovanni, Neurochem. Res. 9, 225 (1984).
[22] R. L. Macdonal, J. H. Skerritt, and M. J. Mclean, Neuropharmacology 23, 843 (1984).
[23] P. Granger, B. Biton, C. Faure, X. Vige, H. Depoortere, D. Graham, S. Z. Langer, B.
Scatton, and P. Avenet, Mol. Pharmacol. 47, 1189 (1995).
[24] Y. C. Chen, B. Stevens, J. F. Chang, J. Milbrandt, B. A. Barres, and J. W. Hell, J.
Neurosci. Methods 171, 239 (2008).
[25] Q. L. Huang, J. Chen, W. J. Zhang, B. P Zhou, C. L. Zhang, W. H. Gerwick, and Z. Y.
Cao, Phytochemistry 150, 85 (2018).