Studies on the anticonvulsant activity and influence on GABA-ergic neurotransmission of 1,2,4-triazole-3-thione-based compounds

Article abstract of DOI:10.3390/molecules190811279

The anticonvulsant activity of several 1,2,4-triazole-3-thione derivatives on mouse maximal electroshock-induced seizures was tested in this study. Characteristic features of all active compounds were rapid onset of action and long lasting effect. Structure-activity observations showed that the probability of obtaining compounds exerting anticonvulsant activity was much higher when at least one of the phenyl rings attached to 1,2,4-triazole nucleus had a substituent at the para position. The obtained results, moreover, permit us to conclude that despite the structural similarity of loreclezole (second-generation anticonvulsant drug) and the titled compounds, their anticonvulsant activity is achieved via completely different molecular mechanisms.

Full text of DOI:10.3390/molecules190811279

Molecules 2014, 19, 11279-11299; doi:10.3390/molecules190811279
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Studies on the Anticonvulsant Activity and Influence on
GABA-ergic Neurotransmission of 1,2,4-Triazole-3-thione-
Based Compounds
Tomasz Plech 1,*, Barbara Kaproń 1, Jarogniew J. Łuszczki 2,3, Monika Wujec 1, Agata Paneth 1,
Agata Siwek 4, Marcin Kołaczkowski 5, Maria Żołnierek 4 and Gabriel Nowak 4
1
Department of Organic Chemistry, Medical University of Lublin, Chodźki 4a, Lublin 20-093,
Poland; E-Mails: baska_k@o2.pl (B.K.); monika.wujec@umlub.pl (M.W.);
agata.siwek@umlub.pl (A.P.)
2
Department of Pathophysiology, Medical University of Lublin, Jaczewskiego 8, Lublin 20-090;
Poland; E-Mail: jarogniew.luszczki@gmail.com
3
Isobolographic Analysis Laboratory, Institute of Rural Health, Jaczewskiego 2,
Lublin 20-950, Poland
4
Department of Pharmacobiology, Jagiellonian University Medical College, Medyczna 9,
Kraków 30-688, Poland; E-Mails: asiwku@wp.pl (A.S.); mfzolnie@cyf-kr.edu.pl (M.Ż.);
nowak@if-pan.krakow.pl (G.N.)
5
Department of Pharmaceutical Chemistry, Jagiellonian University Medical College, Medyczna 9,
Kraków 30-688, Poland; E-Mail: mkolacz@tlen.pl
* Author to whom correspondence should be addressed; E-Mail: tomasz.plech@umlub.pl;
Tel./Fax: +48-081-535-7351.
Received: 3 July 2014; in revised form: 22 July 2014 / Accepted: 23 July 2014 /
Published: 31 July 2014
Abstract: The anticonvulsant activity of several 1,2,4-triazole-3-thione derivatives on
mouse maximal electroshock-induced seizures was tested in this study. Characteristic
features of all active compounds were rapid onset of action and long lasting effect.
Structure-activity observations showed that the probability of obtaining compounds
exerting anticonvulsant activity was much higher when at least one of the phenyl rings
attached to 1,2,4-triazole nucleus had a substituent at the para position. The obtained
results, moreover, permit us to conclude that despite the structural similarity of loreclezole
(second-generation anticonvulsant drug) and the titled compounds, their anticonvulsant
activity is achieved via completely different molecular mechanisms.

Molecules 2014, 19
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Keywords: 30-688 s-triazoles; Mannich bases; maximal electroshock-induced seizure
(MES) test; blood-brain barrier (BBB); radioligand binding assay
1. Introduction
Epilepsy is a chronic disease of the central nervous system caused by paroxysmal, recurrent
disturbances of the bioelectrical activity of the brain. It is estimated that approximately 50 million
people worldwide have epilepsy [1]. An important point is the fact that epilepsy affects young children
and adolescents more than any other age group [2]. As epileptic seizures are the result of an imbalance
between neurotransmitters in the brain, the mechanism of action of some antiepileptic drugs consists in
modulating the activity of GABA-ergic and glutamatergic systems [3]. Moreover, a large group of
antiepileptic drugs is constituted by substances influencing the activity of voltage-dependent sodium
channels [4–7]. The same drug is often capable of influencing different molecular targets (receptors,
enzymes, etc.), which makes it difficult to define these elements of the drug’s structure, which
determine its pharmacological activity to the largest extent.
However, extensive theoretical studies aimed at defining the relationship between molecular
structure and bioactivity of the antiepileptic drugs available on the market have shown that (i) spatially
remote hydrophobic domains (usually phenyl rings); (ii) the hydrogen bonding domain; and (iii) the
electron-donating fragment constitute the elements that are necessary for good anticonvulsant
activity [8–10]. On the other hand, Wingrove and co-workers put forward a hypothesis that the activity
of loreclezole (second-generation antiepileptic drug) is dependent on the interaction between the
triazole moiety and the amide group of asparagine (Asn-289), which is located on the β2 subunit of the
GABAA receptor [11]. Our pilot studies moreover showed that 1,2,4-triazole derivatives administered
systemically in completely non-toxic doses, significantly elevated the threshold for electroconvulsions
and intensified the anticonvulsant activity of classic antiepileptic drugs [12,13]. Many other
compounds bearing a 1,2,4-triazole moiety were also found to possess anticonvulsant properties in
various animal models of epilepsy [14–17]. These observations inspired us to: (i) search for triazole-based
compounds endowed with anticonvulsant activity; and (ii) to check whether the obtained derivatives
may act via a mechanism analogous to that of loreclezole, based on the allosteric modulation of
GABAA receptors. The results thus obtained also served as the grounds of theoretical considerations on
structural requirements conditioning anticonvulsant activity of 1,2,4-triazole-3-thione derivatives.
2. Results and Discussion
2.1. Chemistry
The synthetic pathway of 1,2,4-triazole-3-thione based compounds is presented in Schemes 1 and 2.
The substrates for the synthesis were the commercially available hydrazides, 3-chlorobenzhydrazide
and 4-chlorobenzhydrazide. Reaction between the above-mentioned hydrazides and appropriate aryl
isothiocyanates yielded 1,4-disubstituted thiosemicarbazide derivatives (1–18). The use of a highly
efficient and quick method of synthesis [18] in anhydrous ethanol allowed us to obtain thiosemicarbazides

Molecules 2014, 19
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(1–18) with an efficiency exceeding 85% in most cases. Subsequently, compounds (1–18)
were dissolved in 2% NaOH solution and heated under reflux for 2 h, which resulted in obtaining
4,5-diaryl-1,2,4-triazole-3-thiones (1a–18a). Finally, the compounds (19a–30a) were synthesized
using the so-called Mannich reaction. Its mechanism has been extensively described by Almajan and
co-workers [19]. The structures of the obtained compounds were confirmed on the basis of
spectroscopic data and the results of elemental analyses.
Scheme 1. Synthetic pathway for the synthesis of 1,2,4-triazole-3-thione derivatives.
O
O
NCS
NH NH
S
EtOH (anhydrous)
reflux, 5 minutes
NH
NH
NH
+
(1-18)
2
R
R
1
R
R
2
1
2
2% NaOH, reflux, 2 h
R1
R2
R1
R2
4-CH3
4-COCH3
2-F
1/1a
2/2a
3/3a
4/4a
5/5a
6/6a
7/7a
8/8a
9/9a
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
2-F
3-F
4-F
4-Cl
2-Br
4-Br
4-I
10/10a
11/11a
12/12a
13/13a
14/14a
15/15a
16/16a
17/17a
18/18a
3-Cl
3-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
NH
N
S
N
(1a-18a)
2,4-diF
2,4-diCl
2-Br
R
1
4-Br
R
2
2-CH3
3-CH3
4-SCH3
4-N(C2H5)2
Scheme 2. Synthetic route to 1,2,4-triazole based Mannich bases (19a–30a). Reagents and
conditions: (a) morpholine, HCHO, EtOH, 1 h, r.t.; (b) 1-phenylpiperazine, HCHO, EtOH,
1 h, r.t.; (c) 1-(4-fluorophenyl)piperazine, HCHO, EtOH, 1 h, r.t.
O
N
N
N
1 9 a . R 1
2 2 a . R 1
2 5 a . R 1
2 8 a . R 1
=
=
=
=
3 -C l, R 2
3 -C l, R 2
4 -C l, R 2
4 -C l, R 2
=
=
=
=
4 -B r
4 -C H 3
2 -B r
S
N
4 -B r
R
1
R
2
(a)
N
N
S
N
N
N
N H
N
2 0 a . R1
=
=
=
=
3 - Cl, R 2
3 - Cl, R 2
4 - Cl, R 2
4 - Cl, R 2
=
=
=
=
4- Br
4- CH
2- Br
4- Br
S
(b)
2 3 a . R1
2 6 a . R1
2 9 a . R1
3
N
R
1
R
1
R
2
R
2
(c)
F
N
N
S
N
N
2 1 a . R 1
2 4 a . R 1
2 7 a . R 1
3 0 a . R 1
=
=
=
=
3 -C l, R2
3 -C l, R2
4 -C l, R2
4 -C l, R2
=
=
=
=
4 - Br
4 - CH 3
2 - Br
N
4 - Br
R
1
R
2

Molecules 2014, 19
11282
2.2. Pharmacology
2.2.1. Anticonvulsant Activity
Compounds (1a–30a) were evaluated in vivo using the mouse maximal electroshock-induced
seizure model-an experimental model of human generalized tonic-clonic seizures. Procedures
involving animals and their care were conducted in accordance with current European Community and
Polish legislation on animal experimentation. Additionally, all efforts were made to minimize animal
suffering and to use only the number of animals necessary to produce reliable scientific data.
In the initial stage of the study, groups of eight mice were supplied with the compounds
(administered intraperitoneally) in a dose of 300 mg/kg and then exposed to electrical impulse at fixed
intervals (15, 30, 60, 120 min). The anticonvulsant effects of the compounds were considered
important if at least 50% of the animals tested were protected against electroshock-induced seizures
(Table 1). In the cases of compounds 3a and 14a, administered in a dose of 300 mg/kg, it was
impossible to establish their protective effect in the maximal electroshock-induced seizure (MES) test,
as the dose resulted in the occurrence of acute adverse effects. Once the doses of both compounds were
decreased to lower concentrations, which did not induce neurotoxic effects, only Compound 3a
retained its anticonvulsant activity. Nine of the 30 tested compounds (1a–30a) exhibited various levels
of anticonvulsant activity. All active derivatives (3a, 4a, 6a, 10a, 12a, 13a, 15a, 16a, 22a) were
characterized by a quick onset of action. A significant level of protection against maximal-electroshock
induced seizures was observed as early as 15 min after their parenteral administration. From a
preclinical point of view, the long-lasting anticonvulsant activity is also noteworthy. Apparently, the
highest ability to remain in the central nervous system was presented by 13a and 16a, whose time to
peak of the anticonvulsant action was observed at 120 min after their systemic administration.
In the second stage of the pharmacological research median effective doses (ED50), median toxic
doses (TD50), and protective index (PI) values for compounds 3a, 4a, 6a, 10a, 12a, 13a, 15a, 16a, and
22a were determined (Table 2). The most potent activity (approximately five and a half times higher
than that of valproate) was exhibited by 5-(3-chlorophenyl)-4-(4-fluorophenyl)-2,4-dihydro-3H-1,2,4-
triazole-3-thione (3a) whose ED50 equaled 35.2 mg/kg. Time-course and dose-response relationship of
action of 3a revealed that the time to peak of anticonvulsant action was observed at
15 min after its i.p. administration. The level of toxicity (TD50) of the compound, ranging from
136.7 to 201.0 mg/kg makes long-term use of this derivative impossible. However, due to the fast
onset of action, low ED50 value, as well as the favorable protection index value (PI = 3.9),
Compound 3a should undergo further investigation for its potential use in interrupting status
epilepticus. Among the remaining derivatives, only compounds 6a and 22a exhibited activity slightly
weaker than that of valproate. The former compound, despite its less potent anticonvulsant activity,
significantly (i.e., by 70%) elevated the threshold for electroconvulsions and intensified the
anticonvulsant action of carbamazepine, phenobarbital, and valproate [12]. A very beneficial activity
profile, expressed by low variability in the ED50 values at different pretreatment times (15, 30, 60,
120 min), was observed in the cases of 2-fluorophenyl (12a) and 2,4-difluorophenyl (13a) derivatives.
The ED50 values for these derivatives were 186.4–217.8 mg/kg and 80–108.9 mg/kg, respectively. It is
also noteworthy that 5-(4-chlorophenyl)-4-(2-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (12a)

Molecules 2014, 19
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was the best tolerated compound among all tested 1,2,4-triazole derivatives. Its median toxic doses
(TD50) ranged from 418.5 to 534.3 mg/kg. In most cases, the toxicological profile of the discussed
compounds was similar to that of valproate (PI = 1.7–2.1). A more beneficial value of the protection index
(PI = 4.5–5.9) was observed for 5-(3-chlorophenyl)-4-(4-methylphenyl)-2,4-dihydro-3H-1,2,4-triazole-
3-thione (10a). Additionally, our previous studies demonstrated pharmacologically beneficial
interactions of 10a with valproate that were pharmacokinetic in nature [13]. Sub-protective doses
(i.e., the doses that per se did not affect the seizure threshold) of compound 10a intensified the
anticonvulsant action of valproate as the total brain concentration of this drug was elevated by 52%.
Table 1. Time-course of anticonvulsant effects of compounds (1a–30a) against maximal
electroshock-induced seizure (MES)-induced seizures in mice. Data is presented as the
number of animals protected against maximal electroshock (MES)-induced seizures out of
eight animals per group. The MES test was performed at various pretreatment times
(15, 30, 60, 120 min) after systemic administration of the investigated compounds in a
fixed dose of 300 mg/kg. p-number of animals protected against MES-induced seizures;
t-number of animals tested. “*” - Substance administered at a dose of 300 mg/kg produced
acute neurotoxic effects in mice and it was impossible to determine its anticonvulsant
action in the mouse MES model.
Pretreatment Time (min)
Compounds
15
30
60
120
p/t (%)
p/t (%)
p/t (%)
p/t (%)
1a
2a
0/8 (0)
0/8 (0)
*
0/8 (0)
0/8 (0)
*
1/8 (12.5)
0/8 (0)
1/8 (12.5)
0/8 (0)
3a
*
*
4a
8/8 (100)
0/8 (0)
4/8 (50)
0/8 (0)
0/8 (0)
0/8 (0)
8/8 (100)
1/8 (12.5)
6/8 (75)
8/8 (100)
*
8/8 (100)
0/8 (0)
6/8 (75)
0/8 (0)
0/8 (0)
0/8 (0)
8/8 (100)
1/8 (12.5)
6/8 (75)
8/8 (100)
*
7/8 (87.5)
1/8 (12.5)
6/8 (75)
0/8 (0)
7/8 (87.5)
1/8 (12.5)
3/8 (37.5)
0/8 (0)
5a
6a
7a
8a
0/8 (0)
0/8 (0)
9a
0/8 (0)
1/8 (12.5)
6/8 (75)
0/8 (0)
10a
11a
12a
13a
14a
15a
16a
17a
18a
19a
20a
21a
22a
23a
8/8 (100)
1/8 (12.5)
7/8 (87.5)
8/8 (100)
*
7/8 (87.5)
8/8 (100)
*
6/8 (75)
5/8 (62.5)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
4/8 (50)
0/8 (0)
8/8 (100)
7/8 (87.5)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
5/8 (62.5)
0/8 (0)
8/8 (100)
8/8 (100)
1/8 (12.5)
1/8 (12.5)
0/8 (0)
7/8 (87.5)
8/8 (100)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
3/8 (37.5)
0/8 (0)
2/8 (25)
0/8 (0)

Molecules 2014, 19
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Table 1. Cont.
Pretreatment Time (min)
Compounds
15
30
60
120
p/t (%)
p/t (%)
p/t (%)
p/t (%)
24a
25a
26a
27a
28a
29a
30a
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
0/8 (0)
Table 2. Time-course and dose-response effects of the active compounds in the MES and
chimney tests in mice. Results are presented as median effective doses (ED50 ± S.E.) and
median toxic doses (TD50 ± S.E.) of the examined compounds. All compounds were
administered i.p., at four various pretreatment times (15, 30, 60, 120 min.) prior to the
appropriate pharmacological test (MES or chimney test). n - the total number of animals
used at those doses whose anticonvulsant/toxic effects ranged between 4 and 6 probits.
Pretreatment
ED50 ± S.E.
(mg/kg)
TD50 ± S.E.
(mg/kg)
Compounds
n
n
PI (TD50/ED50)
Time (min)
15
35.2 ± 5.3
8
136.7 ± 19.9
148.8 ± 19.9
171.3 ± 21.4
201.0 ± 20.3
354.9 ± 21.5
365.3 ± 24.0
361.0 ± 20.2
375.8 ± 26.1
372.5 ± 22.6
383.7 ± 18.7
389.3 ± 19.1
424.7 ± 21.4
338.1 ± 12.0
338.1 ± 14.7
333.4 ± 18.6
395.1 ± 25.2
534.3 ± 25.9
496.8 ± 31.7
418.5 ± 34.3
483.8 ± 25.9
195.7 ± 21.8
191.2 ± 21.1
176.1 ± 17.2
217.8 ± 21.7
24
32
24
32
24
16
32
32
16
16
16
32
24
16
16
24
40
32
32
40
8
3.9
1.6
-
30
93.6 ± 9.4
16
-
3a
60
>200
120
15
>200
-
-
90.1 ± 15.7
135.0 ± 19.8
171.3 ± 21.4
183.8 ± 28.0
297.5 ± 21.1
217.8 ± 21.7
208.9 ± 27.3
335.7 ± 14.0
57.0 ± 9.4
16
8
3.9
2.7
2.1
2.0
1.2
1.8
1.9
1.3
5.9
4.5
1.8
1.4
2.6
2.7
1.9
2.3
1.9
1.9
1.6
2.7
30
4a
6a
60
24
24
24
16
32
32
16
16
16
32
32
32
16
32
8
120
15
30
60
120
15
30
74.5 ± 8.1
10a
12a
13a
60
187.1 ± 18.8
281.4 ± 13.6
207.5 ± 25.3
186.4 ± 24.6
217.8 ± 21.7
208.9 ± 27.3
104.0 ± 15.8
100.0 ± 30.1
108.9 ± 11.9
80.0 ± 10.9
120
15
30
60
120
15
30
8
8
60
16
24
16
16
120

Molecules 2014, 19
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Table 2. Cont.
Pretreatment ED50 ± S.E.
TD50 ± S.E.
(mg/kg)
Compounds
15a
n
n
PI (TD50/ED50)
Time (min)
(mg/kg)
15
30
182.7 ± 25.1
104.7 ± 16.5
136.7 ± 19.9
175.5 ± 25.0
272.5 ± 21.4
181.7 ± 44.3
152.6 ± 23.5
140.9 ± 25.1
223.5 ± 20.9
228.3 ± 14.7
274.2 ± 10.9
448.1 ± 21.4
189.0 ± 17.3
216.9 ± 9.4
218.4 ± 18.9
246.6 ± 21.6
24
24
24
24
32
8
278.9 ± 34.8
250.0 ± 26.9
247.9 ± 39.5
240.9 ± 27.3
430.9 ± 27.9
471.7 ± 26.7
455.8 ± 21.7
404.4 ± 24.0
341.0 ± 24.8
310.2 ± 26.2
433.2 ± 26.5
455.5 ± 29.7
363.3 ± 14.2
372.9 ± 16.9
417.3 ± 9.5
512.3 ± 20.2
32
24
32
24
32
32
24
24
32
32
32
32
24
16
16
32
1.5
2.4
1.8
1.4
1.6
2.6
3.0
2.9
1.5
1.4
1.6
1.0
1.9
1.7
1.9
2.1
60
120
15
30
16a
22a
60
24
16
24
16
8
120
15
30
60
120
15
24
24
16
24
24
30
Valproate
60
120
Based on the data presented in Tables 1 and 2, it may be assumed that the anticonvulsant activity of
1,2,4-triazole derivatives depends on the structure of substituents at positions N2, N4 and C5 of the
triazole ring. In the group of monosubstituted derivatives of 4-aryl-5-(3-chlorophenyl)-2,4-dihydro-3H-
1,2,4-triazole-3-thione (1a–11a), the most advantageous position for the substituent seemed to be the
para position. Simultaneously, the electronic nature of the substituents was much less important as
regards the effect on the anticonvulsant potency (since derivatives with both electron-withdrawing and
electron-donating substituents were active). Moreover, the ED50 values for para substituted derivatives
(3a, 4a, 6a, 7a) allow the observation of the negative correlation between the anticonvulsant activity
and the size of halogen substituent. Following this correlation, the 4-fluorophenyl derivative (3a)
exhibited the most potent activity, whereas the 4-iodophenyl derivative (7a) was inactive. This may
suggest that a large iodine atom constitutes a steric hindrance, which prevents the 4-halogenophenyl
fragment from binding to appropriate molecular targets. Comparison of the activity of 2-fluorophenyl
(1a vs. 12a) and 2-bromophenyl (5a vs. 15a) derivatives is more evidence confirming that the
probability of obtaining compounds exerting anticonvulsant activity is much higher when at least one
of the phenyl rings attached to the 1,2,4-triazole nucleus has a substituent at para position. Exchanging
3-chlorophenyl moiety (1a, 5a) for a 4-chlorophenyl group (12a, 15a) resulted in the emergence of
anticonvulsant activity in the cases of 2-substituted derivatives.
In order to study the effect of the presence of a substituent at position N2, four active 1,2,4-triazoles
(6a, 10a, 15a, 16a) were transformed into their appropriate N2-aminomethyl derivatives (19a–30a).
Among the compounds obtained this way, only compound 22a exhibited a weak anticonvulsant
activity, much less potent than that of its predecessor (10a). The remaining N2-aminomethyl
derivatives were inactive. According to Waterbeemd and co-workers, the essential factors affecting the
drug delivery across the blood-brain barrier (BBB) are molecular weight (MW) and polar surface area
(PSA) [20]. Based on a study of marketed central nervous system (CNS)- and non-CNS drugs, it was

Molecules 2014, 19
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established that the transport of a drug to the brain is possible when (T)PSA < 90Å, while MW < 450.
Among the Mannich bases (19a–30a), only compound 22a met these requirements (Table 3).
Therefore we speculate that the most probable cause of the loss of anticonvulsant activity, once the
hydrogen atom is substituted with an aminomethyl moiety, is the loss of the ability of such compounds
to penetrate across the blood brain barrier (BBB). Apart from the BBB, another important biological
barrier the permeability of which (or lack thereof) affects drug efficiency, is the intestinal epithelium,
which modulates drug absorption after oral administration. Most antiepileptic drugs are administered
orally, with the exception of drugs interrupting status epilepticus. Therefore, newly synthesized
compounds with antiepileptic potential should be efficiently absorbed from the digestive system. It has
been demonstrated experimentally that intestinal absorption of drugs is significantly correlated with
the (T)PSA parameter. Palm et al., have proven, based on Caco-2 cell studies, that the drugs with a
(T)PSA below 60 are completely absorbed in the intestine [21]. In the cases of active compounds (3a,
4a, 6a, 10a, 12a, 13a, 15a, 16a, and 22a), the PSA values ranged from 33.62 to 35.23 Å2 and the level
of intestinal absorption according to the algorithm described by Zhao et al., [22] was in the range of
96.8%–97.4% (Table 3). Using these calculations as a theoretical model it is highly likely that
1,2,4-triazole-based compounds could be considered as promising drug candidates, although further
experiments should confirm their applicability in clinical settings.
Table 3. Physicochemical parameters of the studied 1,2,4-triazole-3-thiones.
Compounds
1a
MW
logP TPSA ABS (%)
305.765 3.817 33.617
305.765 3.841 33.617
305.765 3.654 33.617
322.22 4.168 33.617
366.671 4.462 33.617
366.671 4.299 33.617
413.671 4.573 33.617
301.802 4.102 33.617
301.802 4.126 33.617
301.802 3.938 33.617
329.812 3.388 50.688
305.765 3.841 33.617
323.755 3.981 33.617
356.665 5.009 33.617
366.671 4.486 33.617
366.671 4.32 33.617
333.869 3.947 33.617
358.898 4.368 36.855
465.804 4.23 35.232
540.918 5.973 29.236
558.908 6.136 29.236
400.935 3.869 35.232
476.049 5.612 29.236
97.40
97.40
97.40
97.40
97.40
97.40
97.40
97.40
97.40
97.40
91.51
97.40
97.40
97.40
97.40
97.40
97.40
96.28
96.84
98.91
98.91
96.84
98.91
2a
3a
4a
5a
6a
7a
8a
9a
10a
11a
12a
13a
14a
15a
16a
17a
18a
19a
20a
21a
22a
23a

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Table 3. Cont.
Compounds
24a
MW
logP TPSA ABS (%)
494.039 5.776 29.236
465.804 4.417 35.232
540.918 6.16 29.236
558.908 6.324 29.236
465.804 4.254 35.232
540.918 5.997 29.236
558.908 6.16 29.236
98.91
96.84
98.91
98.91
96.84
98.91
98.91
25a
26a
27a
28a
29a
30a
ABS (%)—absorption after oral administration.
2.2.2. Radioligand Binding Assay
Epileptic seizures are caused by disruption of the balance between excitatory and inhibitory systems
in the central nervous system. The most important neurotransmitter inhibiting neuronal processes is
γ-aminobutyric acid (GABA). Among three types of GABA receptors (i.e., GABAA, GABAB,
GABAC), the main role in preventing seizures is played by the GABAA receptors [23,24]. Antiepileptic
drugs acting directly on GABAA receptors, enhancing the synthesis or inhibiting the metabolism of
GABA, as well as blocking the reuptake of GABA from the synaptic cleft, cause an increase in the
GABA-ergic neurotransmission [25]. Our studies conducted to date [12,13,26,27], involving the
design and synthesis of 1,2,4-triazole derivatives endowed with anticonvulsant activity, have led to
promising results, which in turn has encouraged us to attempt the elucidation of their mechanism of
action. A representative group of the synthesized 1,2,4-triazole-based compounds (including active and
inactive ones) was tested with the aim to establish whether the anticonvulsant activity of these
derivatives was a result of their interactions with GABAA receptor complex and/or their affinity to
benzodiazepine (BDZ) binding sites. The degree of specific binding of the compounds to GABAA
receptors and BDZ-binding sites was low and ranged from 2% to 24% (Table 4). This forced us to
exclude the possibility of a direct involvement of GABAA receptors in the occurrence of the
anticonvulsant effect. Similarly, the possibility of allosteric modulation of the function of this receptor via
the interactions of the investigated 1,2,4-triazole derivatives with BDZ-binding sites should be excluded.
Activation of the GABAA receptor may be due to binding of various chemical substances to the
allosteric sites on the receptor surface [28]. Barbiturates and benzodiazepines are the best-known
allosteric modulators of GABAA receptors. However, a similar mechanism of action has also been
described for loreclezole – a second-generation antiepileptic drug. Yet, the binding sites for this drug
are distinct from those of the barbiturates and benzodiazepines. Loreclezole enhances GABAA-mediated
chlorine currents via specific modulatory sites present on the β2 and β3 subunits of the GABAA
receptor [29]. Moreover, Wingrove and co-workers put forward a hypothesis that the activity of
loreclezole is dependent on the interaction between its 1,2,4-triazole moiety and the amide group of
asparagine (Asn-289), which is located on the β2 subunit of the GABAA receptor [11]. This fact
prompted us to check, whether the obtained 1,2,4-triazole derivatives may act via a mechanism
analogous to that of loreclezole. For this study, two derivatives (6a and 10a) were selected, as they are
known to significantly elevate the seizure threshold and enhance the protective activity of classic
antiepileptic drugs [12,13]. In the radioligand binding assay, the affinity of GABA administered alone

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or in combination with 1,2,4-triazole derivatives (6a, 10a), was compared. The IC50 and Ki values for
GABA administered alone were 406.5 ± 42.0 nM and 96.6 ± 9.9 nM, respectively (Table 5). The
addition of 6a and 10a did not lead to an increase in the affinity of GABA to GABAA receptors. The
obtained results permit us to conclude that despite the structural similarity of loreclezole and compounds
(1a–30a), their anticonvulsant activities are achieved via a completely different molecular mechanism.
Table 4. Affinity of the selected 1,2,4-triazole derivatives to GABAA receptors and
benzodiazepine (BDZ)-binding sites.
Percent of Specific Binding (%)
Compounds
(Concentration 1 × 10−6 M)
GABAA
BDZ
14
12
20
9
1a
5a
8
2
6a
17
2
10a
12a
13
8
18
21
24
6
15a
16a
17
21
6
17a
22a
12
7
23a
23
21
82
-
24a
13
-
GABA
Diazepam
Zolpidem
83
73
-
Table 5. Effect of 6a and 10a on affinity of γ-aminobutyric acid (GABA) to
GABAA receptor.
Treatment
Ki ± S.E. [nM]
96.6 ± 9.9
IC50 ± S.E. [nM]
406.5 ± 42.0
370.3 ± 42.7
469.0 ± 55.9
496.0 ± 57.0
474.0 ± 36.2
358.5 ± 6.5
GABA
GABA + 6a (1 × 10−5 M)
GABA + 6a (1 × 10−6 M)
GABA + 6a (1 × 10−7 M)
GABA + 10a (1 × 10−5 M)
GABA + 10a (1 × 10−6 M)
GABA + 10a (1 × 10−7 M)
99.7 ± 11.6
108.0 ± 11.2
155.0 ± 16.5
112.3 ± 8.5
85.5 ± 1.5
107.0 ± 5.0
452.0 ± 22.0
3. Experimental Section
3.1. Chemistry
All reagents were purchased from Alfa-Aesar and Sigma-Aldrich and used without further
purification. Melting points were determined by using Fischer-Johns apparatus (Sanyo, Japan) and are
1
uncorrected. The H-NMR spectra were recorded on a Bruker Avance instrument using DMSO-d6 or
CDCl3 as solvents and tetramethylsilane (TMS) as an internal standard. Chemical shifts are expressed
as δ (ppm). The infrared (IR) spectra were recorded in KBr using a Perkin-Elmer 1725X Fourier

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transform infrared (FTIR) spectrometer. The purity of the compounds was checked by thin-layer
chromatography (TLC) on plates precoated with silica gel Si 60 F254, produced by Merck Co.
(Darmstadt, Germany). The spots were detected by exposure to UV-lamp at λ = 254 nm. Elemental
analyses were performed on AMZ 851 CHX analyzer and the results were within ±0.4% of the theoretical
value. Molecular properties (i.e., MW, TPSA, and logP) of the compounds were calculated using
Molinspiration online tool [30].
3.1.1. General Procedure for the Synthesis of Thiosemicarbazide Derivatives (1–18)
A solution of 0.01 mol of 3-chlorobenzhydrazide or 4-chlorobenzhydrazide and equimolar amount
of appropriate aryl isothiocyanate in 25 mL of anhydrous ethanol (EtOH) was heated under reflux for
5 min. Next, the solution was cooled and the solid formed was filtered off, washed with diethyl ether
and crystallized from EtOH. Information on already known compounds may be retrieved in the
Chemical Abstract Service database (CAS numbers are given below).
1-(3-Chlorobenzoyl)-4-(2-fluorophenyl)thiosemicarbazide (1): Yield: 88%. CAS Registry Number:
894226-73-4.
1-(3-Chlorobenzoyl)-4-(3-fluorophenyl)thiosemicarbazide (2): Yield: 90%. CAS Registry Number:
894226-67-6.
1-(3-Chlorobenzoyl)-4-(4-fluorophenyl)thiosemicarbazide (3): Yield: 85%. CAS Registry Number:
316151-86-7.
1-(3-Chlorobenzoyl)-4-(4-chlorophenyl)thiosemicarbazide (4): Yield: 86%. CAS Registry Number:
351877-21-9.
4-(2-Bromophenyl)-1-(3-chlorobenzoyl)thiosemicarbazide (5): Yield: 86%, CAS Registry Number:
1263379-27-6.
4-(4-Bromophenyl)-1-(3-chlorobenzoyl)thiosemicarbazide (6): Yield: 85%. CAS Registry Number:
351877-22-0.
1-(3-Chlorobenzoyl)-4-(4-iodophenyl)thiosemicarbazide (7): Yield: 96%, CAS Registry Number:
1263379-28-7.
1-(3-Chlorobenzoyl)-4-(2-methylphenyl)thiosemicarbazide (8): Yield: 87%. CAS Registry Number:
891083-31-1.
1-(3-Chlorobenzoyl)-4-(3-methylphenyl)thiosemicarbazide (9): Yield: 86%. CAS Registry Number:
905231-64-3.
1-(3-Chlorobenzoyl)-4-(4-methylphenyl)thiosemicarbazide (10): Yield: 94%. CAS Registry Number:
891082-71-6

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4-(4-Acetylphenyl)-1-(3-chlorobenzoyl)thiosemicarbazide (11): Yield: 86%. CAS Registry Number:
891560-05-7
1-(4-Chlorobenzoyl)-4-(2-fluorophenyl)thiosemicarbazide (12): Yield: 93%. CAS Registry Number:
894226-79-0.
1-(4-Chlorobenzoyl)-4-(2,4-difluorophenyl)thiosemicarbazide (13): Yield: 85%. CAS Registry
Number: 891552-00-4.
1-(4-Chlorobenzoyl)-4-(2,4-dichlorophenyl)thiosemicarbazide (14): Yield: 90%. CAS Registry
Number: 891643-33-7.
4-(2-Bromophenyl)-1-(4-chlorobenzoyl)thiosemicarbazide (15): Yield: 94%, m.p. 174–176 °C, 1H-NMR
(300 MHz, DMSO-d6): 7.16–7.23 (m, 2H, Ar-H), 7.40–7.57 (m, 3H, Ar-H), 7.68–7.81 (m, 3H, Ar-H),
9.66 (s, 1H, 1NH, D2O exchangeable), 9.80 (s, 1H, 1NH, D2O exchangeable), 10.65 (s, 1H, NH, D2O
exchangeable). IR (KBr, cm−1): 3367 (NH), 3110, 2991 (CHarom.), 1652 (C=O), 1296 (C=S). EI-MS
(m/z): 383 [M+]. Anal. Calc. for C14H11BrClN3OS (%): C 43.71, H 2.88, N 10.92. Found: C 43.65, H
2.72, N 11.05.
4-(4-Bromophenyl)-1-(4-chlorobenzoyl)thiosemicarbazide (16): Yield: 88%. CAS Registry Number:
356576-31-3.
1-(4-Chlorobenzoyl)-4-[4-(methylsulfanyl)phenyl]thiosemicarbazide (17): Yield: 96%, m.p.
1
180–182 °C, H-NMR (300 MHz, DMSO-d6): 2.26 (s, 3H, SCH3), 7.19 (d, 2H, Ar-H, J = 8.56 Hz),
7.30 (d, 2H, Ar-H, J = 8.55 Hz), 7.38–7.68 (m, 4H, Ar-H), 9.86 (s, 2H, 2NH, D2O exchangeable),
10.74 (s, 1H, NH, D2O exchangeable). IR (KBr, cm−1): 3410 (NH), 3076 (CHarom), 2930 (CHaliph.),
1661 (C=O), 1311 (C=S). EI-MS (m/z): 351 [M+]. Anal. Calc. for C15H14ClN3OS2 (%): C 51.20, H
4.01, N 11.94. Found: C 51.03, H 4.11, N 11.98.
1-(4-Chlorobenzoyl)-4-[4-(diethylamino)phenyl]thiosemicarbazide (18): Yield: 87%, m.p. 158–159 °C,
1H-NMR (300 MHz, DMSO-d6): 1.08 (t, 6H, 2CH3, J = 6.80 Hz), 3.10 (q, 4H, 2CH2, J = 6.80 Hz),
6.94 (d, 2H, Ar-H, J = 8.65 Hz), 7.10 (d, 2H, Ar-H, J = 8.65 Hz), 7.45–7.81 (m, 4H, Ar-H), 9.56, 9.68,
10.54 (3s, 3H, 3NH, D2O exchangeable). IR (KBr, cm−1): 3386, 3350 (NH), 3064 (CHarom), 2951, 2863
(CHaliph), 1658 (C=O), 1304 (C=S). Anal. Calc. for C18H21ClN4OS (%): C 57.36, H 5.62, N 14.87.
Found: C 57.42, H 5.79, N 14.76.
3.1.2. General Procedure for the Synthesis of 1,2,4-triazole-3-thione Derivatives (1a–18a)
Suitable 1,4-disubstituted thiosemicarbazides (1–18) (0.01 mol) were dissolved in 2% solution of
sodium hydroxide and the resulting solution was refluxed for 2 h. After cooling, the mixture was
neutralized with 3M hydrochloric acid. The precipitate formed was filtered and washed with distilled
water. The compounds were crystallized from EtOH.
5-(3-Chlorophenyl)-4-(2-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (1a): Yield: 81%, m.p.
1
170–171 °C, H-NMR (300 MHz, DMSO-d6): 7.18–7.77 (m, 8H, Ar-H), 14.17 (s, 1H, NH, D2O

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exchangeable). IR (KBr, cm−1): 3453 (NH), 3012 (CHarom), 1485 (C=N), 1321 (C=S). EI-MS (m/z):
305 [M+]. Anal. Calc. for C14H9ClFN3S (%): C 54.99, H 2.97, N 13.74. Found: C 55.06, H 2.76, N 13.88.
5-(3-Chlorophenyl)-4-(3-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (2a): Yield: 83%, m.p.
1
221–222 °C, H-NMR (300 MHz, DMSO-d6): 7.17–7.62 (m, 8H, Ar-H), 14.27 (s, 1H, NH, D2O
exchangeable). IR (KBr, cm−1): 3461 (NH), 3002 (CHarom.), 1483 (C=N), 1320 (C=S). EI-MS (m/z):
305 [M+]. Anal. Calc. for C14H9ClFN3S (%): C 54.99, H 2.97, N 13.74. Found: C 55.08, H 2.81,
N 13.85.
5-(3-Chlorophenyl)-4-(4-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (3a): Yield: 90%. CAS
Registry Number: 720667-75-4.
4-(4-Chlorophenyl)-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (4a): Yield: 82%. CAS
Registry Number: 879074-80-3.
4-(2-Bromophenyl)-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (5a): Yield: 85%. CAS
Registry Number: 1263379-33-4.
4-(4-Bromophenyl)-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (6a): Yield: 95%. CAS
Registry Number: 1263379-32-3.
5-(3-Chlorophenyl)-4-(4-iodophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (7a): Yield: 87%. CAS
Registry Number: 1263379-34-5.
5-(3-Chlorophenyl)-4-(2-methylphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (8a): Yield: 87%, m.p.
1
186–188 °C, H-NMR (300 MHz, DMSO-d6): 2.13 (s, 3H, CH3), 7.12–7.86 (m, 8H, Ar-H), 14.17 (s,
1H, NH, D2O exchangeable). IR (KBr, cm−1): 3387 (NH), 3046 (CHarom.), 2931 (CHaliph), 1494 (C=N),
1332 (C=S). EI-MS (m/z): 301 [M+]. Anal. Calc. for C15H12ClN3S (%):C 59.70, H 4.01, N 13.92.
Found: C 59.79, H 3.87, N 14.03.
5-(3-Chlorophenyl)-4-(3-methylphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (9a): Yield: 83%, m.p.
1
200–201 °C, H-NMR (300 MHz, DMSO-d6): 2.32 (s, 3H, CH3), 7.10–7.53 (m, 8H, Ar-H), 14.09 (s,
1H, NH, D2O exchangeable). IR (KBr, cm−1): 3415 (NH), 3068 (CHarom.), 2864 (CHaliph.), 1476 (C=N),
1322 (C=S). EI-MS (m/z): 301 [M+]. Anal. Calc. for C15H12ClN3S (%):C 59.70, H 4.01, N 13.92.
Found: C 59.81, H 4.09, N 14.13.
5-(3-Chlorophenyl)-4-(4-methylphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (10a): Yield: 80%.
CAS Registry Number: 893725-08-1.
4-(4-Acetylphenyl)-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (11a): Yield: 84%, m.p.
1
218–220 °C, H-NMR (300 MHz, DMSO-d6): 2.64 (s, 3H, CH3), 7.21–8.14 (m, 8H, Ar-H), 14.27 (s,
1H, NH, D2O exchangeable). IR (KBr, cm−1): 3421 (NH), 3041 (CHarom.), 2924 (CHalif.), 1666 (C=O),
1490 (C=N), 1328 (C=S). Anal. Calc. for C16H12ClN3OS (%): C 58.27, H 3.67, N 12.74. Found: C
58.14, H 3.58, N 12.67.

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5-(4-Chlorophenyl)-4-(2-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (12a): Yield: 86%,
1
m.p. 240–242 °C, H-NMR (300 MHz, DMSO-d6): 7.40 (d, 2H, Ar-H, J = 8.70 Hz), 7.52 (d, 2H,
Ar-H, J = 8.69 Hz), 7.58–7.86 (m, 4H, Ar-H), 14.32 (s, 1H, NH, D2O exchangeable). IR (KBr, cm−1):
3387 (NH), 3034 (CHarom.), 1489 (C=N), 1328 (C=S). Anal. Calc. for C14H9ClFN3S (%): C 54.99, H
2.97, N 13.74. Found: C 55.10, H 3.00, N 13.61.
5-(4-Chlorophenyl)-4-(2,4-difluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (13a): Yield: 86%,
1
m.p. 196–198 °C, H-NMR (300 MHz, DMSO-d6): 7.24 (d, 2H, Ar-H, J = 8.63 Hz), 7.42 (d, 2H,
Ar-H, J = 8.61 Hz), 7.58–7.81 (m, 3H, Ar-H), 14.29 (s, 1H, NH, D2O exchangeable). IR (KBr, cm−1):
3420 (NH), 3100 (CHarom.), 1483 (C=N), 1336 (C=S). EI-MS (m/z): 323 [M+]. Anal. Calc. for
C14H8ClF2N3S (%): C 51.94, H 2.49, N 12.98. Found: C 52.13, H 2.50, N 12.81.
5-(4-Chlorophenyl)-4-(2,4-dichlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (14a): Yield: 88%,
1
m.p. 238–240 °C, H-NMR (300 MHz, DMSO-d6): 7.39 (d, 2H, Ar-H, J = 8.65 Hz), 7.55 (d, 2H,
Ar-H, J = 8.66 Hz), 7.70–7.95 (m, 3H, Ar-H), 14.39 (s, 1H, NH, D2O exchangeable). IR (KBr, cm−1):
3387 (NH), 3049 (CHarom.), 1490 (C=N), 1318 (C=S). Anal. Calc. for C14H8Cl3N3S (%): C 47.15, H
2.26, N 11.78. Found: C 47.13, H 2.30, N 11.66.
4-(2-Bromophenyl)-5-(4-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (15a): Yield: 90%,
1
m.p. 250–252 °C, H-NMR (300 MHz, DMSO-d6): 7.36 (d, 2H, Ar-H, J = 8.61 Hz), 7.50 (d, 2H,
Ar-H, J = 8.61 Hz), 7.67–7.91 (m, 4H, Ar-H), 14.30 (s, 1H, NH, D2O exchangeable). IR (KBr, cm−1):
3401 (NH), 3105 (CHarom), 1496 (C=N), 1342 (C=S). EI-MS (m/z): 365 [M+]. Anal. Calc. for
C14H9BrClN3S (%): C 45.86, H 2.47, N 11.46. Found: C 45.65, H 2.53, N 11.60.
4-(4-Bromophenyl)-5-(4-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (16a): Yield: 84%,
CAS Registry Number: 537017-82-6.
5-(4-Chlorophenyl)-4-[4-(methylsulfanyl)phenyl]-2,4-dihydro-3H-1,2,4-triazole-3-thione (17a): Yield:
88%, m.p. 214–216 °C, 1H-NMR (300 MHz, DMSO-d6): 2.42 (s, 3H, CH3), 7.23–7.85 (m, 8H, Ar-H),
14.29 (s, 1H, NH, D2O exchangeable). IR (KBr, cm−1): 3412 (NH), 3075 (CHarom), 2886 (CHaliph.),
1568 (C=N), 1320 (C=S). EI-MS (m/z): 333 [M+]. Anal. Calc. for C15H12ClN3S2 (%): C 53.96, H 3.62,
N 12.59. Found: C 54.10, H 3.54, N 13.02.
5-(4-Chlorophenyl)-4-[4-(diethylamino)phenyl]-2,4-dihydro-3H-1,2,4-triazole-3-thione (18a): Yield:
1
80%, m.p. 264–266 °C, H-NMR (300 MHz, DMSO-d6): 1.10 (t, 6H, 2 × CH3, J = 7.08 Hz), 3.34 (q,
4H, 2CH2, J = 7.10 Hz), 7.10 (d, 2H, Ar-H, J = 8.80 Hz), 7.21 (d, 2H, Ar-H, J = 8.80 Hz), 7.28–7.61
(m, 4H, Ar-H), 14.23 (s, 1H, NH, D2O exchangeable). IR (KBr, cm−1): 3422 (NH), 3041 (CHarom.),
2926, 2864 (CHaliph.), 1559 (C=N), 1330 (C=S). Anal. Calc. for C18H19ClN4S (%): C 60.24, H 5.34, N
15.61. Found: C 60.31, H 5.48, N 15.60.
3.1.3. General Procedure for the Synthesis of Mannich Bases (19a–30a)
Ten mmol of the 1,2,4-triazole derivative (6a, 10a, 15a, 16a) was dissolved (with heating) in 20 mL
of anhydrous ethanol and then equimolar amounts of appropriate secondary amine (morpholine,

Molecules 2014, 19
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1-phenylpiperazine, 1-[4-fluorophenyl]piperazine) and formaldehyde solution (37%) were added. The
obtained mixture was stirred at room temperature for 1 h. Next, 5 mL of distilled water was added, the
precipitate was filtered off, washed with distilled water, and crystallized from ethanol. Spectral and
physicochemical data for compounds 19a–24a, and 28a are available in our earlier articles [31,32].
4-(4-Bromophenyl)-5-(3-chlorophenyl)-2-(morpholin-4-ylmethyl)-2,4-dihydro-3H-1,2, 4-triazole-3-
thione (19a): Yield: 75%. CAS Registry Number: 1349172-90-2.
4-(4-Bromophenyl)-5-(3-chlorophenyl)-2-[(4-phenylpiperazin-1-yl)methyl]-2,4-dihydro-3H-1,2, 4-
triazole-3-thione (20a): Yield: 78%. CAS Registry Number: 1349172-92-4.
4-(4-Bromophenyl)-5-(3-chlorophenyl)-2-{[4-(4-fluorophenyl)piperazin-1-yl]methyl}-2, 4-dihydro-3H-
1,2,4-triazole-3-thione (21a): Yield: 82%. CAS Registry Number: 1349172-94-6.
5-(3-Chlorophenyl)-4-(4-methylphenyl)-2-(morpholin-4-ylmethyl)-2,4-dihydro-3H-1,2, 4-triazole-3-
thione (22a): Yield: 72%. CAS Registry Number: 1349172-89-9.
5-(3-Chlorophenyl)-4-(4-methylphenyl)-2-[(4-phenylpiperazin-1-yl)methyl]-2,4-dihydro-3H-1,2, 4-
triazole-3-thione (23a): Yield: 79%. CAS Registry Number: 1349172-91-3.
5-(3-Chlorophenyl)-2-{[4-(4-fluorophenyl)piperazin-1-yl]methyl}-4-(4-methylphenyl)-2,4-dihydro-3H-
1,2,4-triazole-3-thione (24a): Yield: 83%. CAS Registry Number: 1349172-93-5.
4-(2-Bromophenyl)-5-(4-chlorophenyl)-2-(morpholin-4-ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-
1
thione (25a): Yield: 80%, m.p. 180–181 °C, H-NMR (300 MHz, CDCl3): 2.93 (t, 4H, morpholine,
J = 4.70 Hz), 3.75 (t, 4H, morpholine, J = 4.70 Hz), 5.25 (s, 2H, CH2), 7.20 (d, 2H, Ar-H, J = 8.60 Hz),
7.26–7.34 (m, 4H, Ar-H), 7.65 (d, 2H, Ar-H, J = 8.60 Hz). 13C-NMR (75 MHz, CDCl3): 50.86, 66.90,
70.05, 123.49, 124.20, 129.23, 129.43, 129.53, 129.77, 129.89, 133.14, 133.85, 137.21, 148.21,
170.61. EI-MS (m/z): 464 [M+]. Anal. Calc. for C19H18BrClN4OS (%): C 48.99, H 3.90, N 12.03.
Found: C 49.12, H 3.78, N 12.00.
4-(2-Bromophenyl)-5-(4-chlorophenyl)-2-[(4-phenylpiperazin-1-yl)methyl]-2,4-dihydro-3H-1,2,
1
4-triazole-3-thione (26a): Yield: 73%, m.p. 140–142 °C, H-NMR (600 MHz, CDCl3): 3.09–3.20 (m,
4H, piperazine), 3.27 (t, 4H, piperazine, J = 4.9 Hz), 5.31 (d, 1H, CH2, J = 13.2 Hz), 5.48 (d, 1H, CH2,
J = 13.2 Hz), 6.89 (t, 1H, Ar-H, J = 7.2 Hz), 6.97 (d, 2H, Ar-H, J = 7.4 Hz), 7.27–7.32 (m, 5H, Ar-H),
7.34 (dd, 2H, Ar-H, J = 1.9 Hz, 5.9 Hz), 7.42–7.48 (m, 2H, Ar-H), 7.55 (td, 1H, Ar-H, J = 1.2 Hz,
13
7.6 Hz), 7.75 (dd, 1H, Ar-H, J = 1.2 Hz, 7.9 Hz). C NMR (150 MHz, CDCl3): 49.48, 50.40, 69.63,
116.41, 123.35, 123.87, 129.07, 129.16, 129.19, 130.92, 131.91, 134.20, 134.40, 137.12, 148.20,
170.43. IR (KBr, cm−1): 3084, 3006 (CHarom), 2892, 2784 (CHaliph), 1495 (C=N), 1314 (C=S). Anal.
Calc. for C25H23BrClN5S (%):C 55.51, H 4.29, N 12.95. Found: C 55.57, H 4.12, N 12.79.
4-(2-Bromophenyl)-5-(4-chlorophenyl)-2-{[4-(4-fluorophenyl)piperazin-1-yl]methyl}-2, 4-dihydro-3H-
1,2,4-triazole-3-thione (27a): Yield: 82%, m.p. 168–170 °C, 1H-NMR (600 MHz, CDCl3): 3.09–3.25 (m,
8H, piperazine), 5.30 (d, 1H, CH2, J = 13.4 Hz), 5.47 (d, 1H, CH2, J = 13.4 Hz), 6.89-6.96 (m, 2H, Ar-H),

Molecules 2014, 19
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6.99 (t, 2H, Ar-H, J = 8.1 Hz), 7.27–7.36 (m, 4H, Ar-H), 7.42–7.47 (m, 2H, Ar-H), 7.55 (td, 1H, Ar-H,
13
J = 1.3 Hz, 7.6 Hz), 7.75 (dd, 1H, Ar-H, J = 1.3 Hz, 8.0 Hz). C NMR (150 MHz, CDCl3): 50.32,
50.54, 69.51, 115.57, 115.72, 123.34, 123.83, 129.06, 129.16, 130.90, 131.92, 134.20, 134.38, 137.16,
148.23, 170.44. IR (KBr, cm−1): 3130, 3085 (CHarom.), 2956, 2879 (CHaliph.), 1486 (C=N), 1317 (C=S).
Anal. Calc. for C25H22BrClFN5S (%): C 53.73, H 3.97, N 12.53. Found: C 53.80, H 3.76, N 12.69.
4-(4-Bromophenyl)-5-(4-chlorophenyl)-2-(morpholin-4-ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-
thione (28a): Yield: 69%. CAS Registry Number: 1403137-49-4.
4-(4-Bromophenyl)-5-(4-chlorophenyl)-2-[(4-phenylpiperazin-1-yl)methyl]-2,4-dihydro-3H-1,2,4-
1
triazole-3-thione (29a): Yield: 78%, m.p. 178–180 °C, H-NMR (CDCl3): 3.12–3.15 (m, 4H,
piperazine), 3.25–3.28 (m, 4H, piperazine), 5.37 (s, 2H, CH2), 6.90 (t, 1H, Ar-H, J = 7.30 Hz), 6.96 (d,
2H, Ar-H, J = 8.20 Hz), 7.21 (d, 2H, Ar-H, J = 8.60 Hz), 7.25–7.39 (m, 6H, Ar-H), 7.69 (d, 2H, Ar-H,
J = 8.60 Hz). 13C NMR (CDCl3): 49.50, 50.60, 69.89, 116.51, 120.16, 123.56, 124.23, 129.23, 129.28,
129.61, 129.95, 133.19, 133.92, 137.20, 148.20, 151.31, 170.60. EI-MS (m/z): 539 [M+]. Anal. Calc.
for C25H23BrClN5S (%): C 55.51, H 4.29, N 12.95. Found: C 55.38, H 4.38, N 13.12.
4-(4-Bromophenyl)-5-(4-chlorophenyl)-2-{[4-(4-fluorophenyl)piperazin-1-yl]methyl}-2, 4-dihydro-3H-
1
1,2,4-triazole-3-thione (30a): Yield: 80%, m.p. 156–158 °C, H-NMR (CDCl3): 3.07–3.20 (m, 8H,
piperazine), 5.35 (s, 2H, CH2), 6.87–7.02 (m, 4H, Ar-H), 7.19 (d, 2H, Ar-H, J = 8.40 Hz), 7.24–7.32 (m,
13
4H, Ar-H), 7.66 (d, 2H, Ar-H, J = 8.40 Hz). C NMR (CDCl3): 50.50, 50.55, 69.78, 115.47, 115.77,
118.25, 118.35, 123.49, 124.24, 129.25, 129.54, 129.88, 133.18, 133.86, 137.23, 147.90, 148.20,
170.57. EI-MS (m/z): 557 [M+]. Anal. Calc. for C25H22BrClFN5S (%): C 53.73, H 3.97, N 12.53.
Found: C 53.87, H 4.11, N 12.45.
3.2. Pharmacology
3.2.1. General Information
Adult male Swiss mice (weighing 22–26 g) that were kept in colony cages with free access to food
and tap water, housed under standardized housing conditions, were used. After seven days of
adaptation to laboratory conditions, the animals were randomly assigned to experimental groups each
comprised of eight mice. Each mouse was used only once. Procedures involving animals and their care
were conducted in accordance with current European Community and Polish legislation on animal
experimentation. The experimental protocols and procedures described in this manuscript were
approved by the First Local Ethics Committee at the Medical University in Lublin and the Second
Local Ethics Committee at the University of Life Sciences in Lublin and complied with the European
Communities Council Directive of 24 November 1986 (86/609/EEC).
3.2.2. Maximal Electroshock Seizure Test
The investigated compounds (1a–30a) were suspended in a 1% solution of Tween 80 in distilled
water and administered intraperitoneally (i.p.) as a single injection, in a volume of 5 mL/kg body
weight. Magnesium valproate, used as a reference drug, was directly dissolved in distilled water. Fresh

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drug solutions were prepared on each day of experimentation and administered at 15, 30, 60 and
120 min. before the initiation of maximal electroconvulsions. Electroconvulsions were produced by a
current (0.2 s stimulus duration; 500 V, 50 Hz, fixed current intensity of 25 mA) delivered via ear-clip
electrodes by a Rodent Shocker generator (constant-current stimulator Type 221, Hugo Sachs
Elektronik, Freiburg, Germany). The criterion for the occurrence of seizure activity was the tonic hind
limb extension. The animals were administered with a constant dose of 300 mg/kg of each of the
examined compounds and were subjected to MES-induced seizures. The anticonvulsant activity of the
active compounds administered i.p. at various pretreatment times was determined as their median
effective doses (ED50 values in mg/kg) in the MES-induced seizure test in mice. The animals were
administered with different doses of the tested compounds so as to obtain a variable percentage of
protection against maximal electroconvulsions, allowing the construction of dose-response relationship
lines for each examined compound administered i.p. at various pretreatment times, according to
Litchfield and Wilcoxon [33]. Each ED50 value represents the dose of the studied compounds that is
required to protect 50% of the animals tested against MES-induced seizures.
3.2.3. Chimney Test
The chimney test of Boissier et al. [34] was used to quantify the acute adverse-effect potential of
compounds 3a, 4a, 6a, 10a, 12a, 13a, 15a, 16a, 22a, and valproate on motor performance in mice. In
this test, the animals had to climb backwards up a plastic tube (3 cm inner diameter, 30 cm length), and
motor performance impairment was indicated by the inability of the mice to climb backward up the
transparent tube within 60 s. The acute adverse effects of the 1,2,4-triazole derivatives and valproate
administered alone were expressed as their median toxic doses (TD50), representing the doses, at which
the investigated compounds impaired motor coordination in 50% of the animals tested in the chimney
test. To evaluate each TD50 value, at least four groups of animals (each group consisted of eight mice)
injected with various doses of the appropriate compound were challenged with the chimney test.
A dose-response relationship line was calculated on the basis of the percentage of mice showing motor
deficits by means of the log-probit method according to Litchfield and Wilcoxon [33].
3.2.4. Protective Index (PI)
The protective index for the investigated compounds was calculated by dividing a TD50 value, as
determined in the chimney test, by the respective ED50 value, as determined in the MES test. The
protective index is considered as an index of the margin of safety and tolerability between
anticonvulsant doses and doses of the compounds exerting acute adverse effects e.g., sedation, motor
coordination impairment, ataxia or other neurotoxic manifestations [35].
3.2.5. Radioligand Binding Assay
Binding experiments were conducted in 96-well microplates in a total volume of 200 μL of
appropriate buffer. The reaction mixture included 50 μL solution of test compound, 50 μL of
radioligand, and 150 μL the tissue suspension. Specific assay conditions for each receptor are shown in
the table below (Table 6). Rat cerebral cortex was used for each receptor. The radioactivity was

Molecules 2014, 19
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measured in MicroBeta TriLux 1450-liquid scintillation counter (PerkinElmer,Waltham, MA, USA).
Radioligand binding data were analyzed using iterative curve fitting routines (GraphPAD/Prism,
Version 3.0, San Diego, CA, USA). Ki values were calculated from the Cheng and Prusoff equation [36].
The concentrations of analyzed compound GABA (γ-aminobutyric acid) ranged from 1 × 10−11 to
1 × 10−3 M. The percentage of specific binding was calculated for analyzed compounds in the
screening test, which was determined in a concentration of 10−6 M.
Table 6. Radioligand binding assay conditions.
Blank
Incubation
Conditions
Receptor
GABAA
BDZ
Radioligand
Buffer
(nonspecific)
50 mM Tris-HCl
pH 7.4
[3H]muscimol
100 µM GABA
10 min, 0–4 °C
20 min, 0–4 °C
50 mM Tris-HCl
pH 7.4
[3H]flunitrazepam 10 µM diazepam
4. Conclusions
In conclusion, a series of 1,2,4-triazole-based compounds has been synthesized and their
anticonvulsant and neurotoxic effects have been evaluated. Nine out of 30 tested derivatives exhibited
various level of anticonvulsant activity. Median effective dose (ED50) for the most potent compound,
i.e., 5-(3-chlorophenyl)-4-(4-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione, equaled 35.2 mg/kg.
Based on the radioligand binding assay results, the hypothesis that the anticonvulsant activity of
the mentioned compounds results from the direct or allosteric modulation of GABAA receptors
should be excluded.
Acknowledgments
Part of the research was funded by the National Science Centre (decision number:
2013/11/D/NZ7/01170). J.J. Łuszczki is a Member of the Academy of Young Scholars of the Polish
Academy of Sciences (Warsaw, Poland). Tomasz Plech is a recipient of the Fellowship for Young
Researchers with Outstanding Scientific Achievements from the Medical University of Lublin
(Lublin, Poland).
Author Contributions
T.P. designed the research, synthesized 4,5-disubstituted 1,2,4-triazole-3-thiones, performed
theoretical calculations, and wrote the manuscript; B.K. synthesized Mannich bases and analyzed MS
spectra; J.J.Ł performed in vivo pharmacological experiments; M.W., A.P. analyzed IR and NMR
spectra; A.S, M.K., M.Ż, G.B. performed in vitro experiments.
Conflicts of Interest
The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are available from the authors.
© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).