Preclinical evaluation of 1,2,4-triazole-based compounds targeting voltage-gated sodium channels (VGSCs) as promising anticonvulsant drug candidates

Article abstract of DOI:10.1016/j.bioorg.2019.103355

Epilepsy is a chronic neurological disorder affecting nearly 65–70 million people worldwide. Despite the observed advances in the development of new antiepileptic drugs (AEDs), still about 30–40% of patients cannot achieve a satisfactory seizure control. In our current research, we aimed at using the combined results of radioligand binding experiments, PAMPA-BBB assay and animal experimentations in order to design a group of compounds that exhibit broad spectrum of anticonvulsant activity. The synthesized 4-alkyl-5-substituted-1,2,4-triazole-3-thione derivatives were primarily screened in the maximal electroshock-induced seizure (MES) test in mice. Next, the most promising compounds (17, 22) were investigated in 6 Hz (32 mA) psychomotor seizure model. Protective effect of compound 22 was almost similar to that of levetiracetam. Moreover, these compounds did not induce genotoxic and hemolytic changes in human cells as well as they were characterized by low cellular toxicity. Taking into account the structural requirements for good anticonvulsant activity of 4-alkyl-5-aryl-1,2,4-triazole-3-thiones, it is visible that small electron-withdrawing substituents attached to phenyl ring have beneficial effects both on affinity towards VGSCs and protective activity in the animal models of epilepsy.

Full text of DOI:10.1016/j.bioorg.2019.103355

Journal Pre-proofs
Preclinical evaluation of 1,2,4-triazole-based compounds targeting voltage-ga-
ted sodium channels (VGSCs) as promising anticonvulsant drug candidates
Barbara Kaproń, Jarogniew J. Łuszczki, Agata Siwek, Tadeusz Karcz, Gabriel
Nowak, Mirosław Zagaja, Marta Andres-Mach, Anna Stasiłowicz, Judyta
Cielecka-Piontek, Janusz Kocki, Tomasz Plech
PII:
S0045-2068(19)31078-8
DOI:
https://doi.org/10.1016/j.bioorg.2019.103355
YBIOO 103355
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
15 July 2019
24 August 2019
9 October 2019
Please cite this article as: B. Kaproń, J.J. Łuszczki, A. Siwek, T. Karcz, G. Nowak, M. Zagaja, M. Andres-Mach,
A. Stasiłowicz, J. Cielecka-Piontek, J. Kocki, T. Plech, Preclinical evaluation of 1,2,4-triazole-based compounds
targeting voltage-gated sodium channels (VGSCs) as promising anticonvulsant drug candidates, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103355
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Preclinical evaluation of 1,2,4-triazole-based compounds targeting voltage-gated
sodium channels (VGSCs) as promising anticonvulsant drug candidates
Barbara Kaproń,1 Jarogniew J. Łuszczki,2 Agata Siwek,3 Tadeusz Karcz,4 Gabriel Nowak,3
Mirosław Zagaja,5 Marta Andres-Mach,5 Anna Stasiłowicz,6 Judyta Cielecka-Piontek,6 Janusz
Kocki,1 Tomasz Plech7,*
1Department of Clinical Genetics, Medical University of Lublin, Lublin, Poland
2Department of Pathophysiology, Medical University of Lublin, Lublin, Poland
3Department of Pharmacobiology, Jagiellonian University Medical College, Cracow, Poland
4Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical
College, Cracow, Poland
5Isobolographic Analysis Laboratory, Institute of Rural Health, Lublin, Poland
6Department of Pharmacognosy, Poznan University of Medical Sciences, Poznań, Poland
7Department of Pharmacology, Medical University of Lublin, Lublin, Poland
*Corresponding author:
Tomasz Plech, Department of Pharmacology, Faculty of Health Sciences, Medical University of
Lublin, Chodźki 4a, 20-093 Lublin, Poland. Phone/Fax.: +48 (81) 448 67 72.

Abstract: Epilepsy is a chronic neurological disorder affecting nearly 65-70 million people
worldwide. Despite the observed advances in the development of new antiepileptic drugs
(AEDs), still about 30-40% of patients cannot achieve a satisfactory seizure control. In our
current research, we aimed at using the combined results of radioligand binding experiments,
PAMPA-BBB assay and animal experimentations in order to design a group of compounds that
exhibit broad spectrum of anticonvulsant activity. The synthesized 4-alkyl-5-substituted-1,2,4-
triazole-3-thione derivatives were primarily screened in the maximal electroshock-induced
seizure (MES) test in mice. Next, the most promising compounds (17, 22) were investigated in
6 Hz (32 mA) psychomotor seizure model. Protective effect of compound 22 was almost similar
to that of levetiracetam. Moreover, these compounds did not induce genotoxic and hemolytic
changes in human cells as well as they were characterized by low cellular toxicity. Taking into
account the structural requirements for good anticonvulsant activity of 4-alkyl-5-aryl-1,2,4-
triazole-3-thiones, it is visible that small electron-withdrawing substituents attached to phenyl
ring have beneficial effects both on affinity towards VGSCs and protective activity in the
animal models of epilepsy.
Keywords: 6 Hz test; MES test, PAMPA-BBB assay; sodium channels; ADME-Tox
1. Introduction
Epilepsy is a chronic neurological disorder affecting nearly 65-70 million people worldwide [1].
Epileptics not only suffer from the disease’s symptoms, but they are also at an increased risk
of death [2]. Treatment of epilepsy is based mainly on the properly selected pharmacotherapy.
However, despite the observed advances in the development of new antiepileptic drugs
(AEDs), still about 30-40% of patients cannot achieve a satisfactory seizure control [3]. New
AEDs usually have milder side-effects, better pharmacokinetic profile and slightly improved
efficiency. Unfortunately, these drugs have not significantly improved the outcome of
treatment in patients with therapy-resistant epilepsy. Hence, there is an immense need for
new AEDs exhibiting efficiency against a broad spectrum of seizures.

In our recent papers, we have identified 4-alkyl-5-aryl-1,2,4-triazole-3-thiones as a promising
group of antiepileptic drug candidates [4-7]. It has been proved that anticonvulsant activity of
these compounds is due to their ability to interact with the voltage-gated sodium channels
(VGSCs) [6, 7]. Moreover, 4-alkyl-5-aryl-1,2,4-triazole-3-thione derivatives possessed
favorable pharmacological and toxicological profile, including low neurotoxicity and toxicity
against human cells, lack of genotoxic properties, fast onset of action, sustainable activity over
time, and ability to potentiate the anticonvulsant activity of valproate [7, 8]. The effect of the
alkyl chain structure on the pharmacological activity, toxicity, ability to penetrate the blood-
brain barrier, and the affinity towards VGSCs has been well-investigated and discussed in our
previous articles [4-7]. 1,2,4-Triazole derivatives with unbranched alkyl chains, containing
from 4 to 7 carbon atoms, turned out to possess the most beneficial activity during preclinical
studies. However, still little is known about the effects of structural modifications of the aryl
moiety attached to 1,2,4-triazole-3-thione core on the anticonvulsant activity since only
compounds with 3-chlorophenyl substituent (with or without a methylene linker) have been
tested thus far (Fig. 1).
Figure 1 (insert here)
Additionally, these derivatives were tested only in the mouse maximal electroshock-induced
seizure (MES) test, which is recognized model of generalized tonic-clonic seizures in humans.
The ability of these compounds to inhibit seizures in other types of epilepsy would significantly
improve the chances of 1,2,4-triazole derivatives to be investigated in further stages of drug
development process. Therefore, in our current studies, we aimed to comprehensively
analyze the role of aryl moiety attached to 1,2,4-triazole-3-thione in binding to molecular
target and anticonvulsant effect of the synthesized compounds. The most promising 1,2,4-
triazole derivatives were also evaluated in the mouse 6 Hz (32 mA) model of psychomotor
seizures. We believe that the results presented in this manuscript will provide valuable insights
on the design and development of new AED candidates.
2. Results and discussion
2.1. Chemistry

The investigated compounds were synthesized in moderate to high yields (i.e., 48 – 90%),
starting from the respective carboxylic acids (Scheme 1). These acids were converted into the
corresponding esters, which subsequently were subjected to hydrazinolysis in order to obtain
carboxylic acid hydrazides. Reaction between respective hydrazides and alkyl isothiocyanates
resulted in 1,4-disubstituted thiosemicarbazide derivatives. Dehydrocyclization of the
respective thiosemicarbazides in an alkaline environment led to the formation of 4-alkyl-5-
substituted-1,2,4-triazole-3-thiones (1-22). The purity of the title compounds was checked
1
using elemental analysis (C, H, N), while their structures were elucidated on the basis of H-
and 13C-NMR spectra. The obtained results of the elemental analyses were within ± 0.4% of
the theoretical values. The results of spectral analyses of the intermediates (i.e., 1,4-
disubstituted thiosemicarbazides) are given as Supplementary Material.
Scheme 1 (insert here)
2.2. Anticonvulsant activity
From the hitherto synthesized 4-alkyl-5-substituted-1,2,4-triazole-3-thiones, which had
protective effect in mouse MES model of epilepsy, all possessed electron-withdrawing groups
(EWGs) at the phenyl ring attached to 1,2,4-triazole core [4-7]. Therefore, in order to examine
the effect of structural modifications of aryl substituent on the anticonvulsant activity of 1,2,4-
triazole-based derivatives, a series of compounds (1-8) with electron-donating groups (EDGs)
were obtained in the first stage of the study. EDGs differed in their electron-donating
properties (-OH, -OCH3) and position in the phenyl ring.
Table 1 (insert here)
Among 1,2,4-triazoles (1-8), only a few compounds exhibited weak protective activity against
MES-induced seizures (Tab. 1). This indicates that EDGs (regardless of their location)
negatively affect their anticonvulsant activity measured in the MES test in mice. Therefore,
during further studies the EWGs were selected for structure modifications. Only bromine and
fluorine derivatives were synthesized (Tab. 2) since the role of chlorophenyl moiety, with or
without a methylene linker, was extensively investigated in our previously published studies
[4, 5, 7]. To better understand the mechanism of the anticonvulsant effect (or its lack) of the

compounds 1-22, an initial evaluation of their affinity towards the batrachotoxin-binding site
of the VGSCs and their ability to permeate through the blood-brain barrier (BBB) has been
performed (Table 3). These results confirmed that both compounds containing EDGs and
EWGs can be classified as BBB+ (or CNS+) since good permeation through BBB is expected for
compounds with Pe > 5.19 cm/s [9]. Therefore weak activity of compounds 1-8 in mouse MES
test was associated rather with low or no affinity towards VGSCs (see Table 3).
Table 2 (insert here)
Table 3 (insert here)
The anticonvulsant effect of the compounds containing EWGs (9-22) was clearly dependent
on the type of halogen atom. Smaller substituents (-F, Cl) appear to have beneficial effect on
the protective activity of 1,2,4-triazole-3-thiones in MES test in mice. This can be associated
with their higher affinity towards VGSCs (lower IC50 values for the chlorine [6, 7] and fluorine
derivatives). Compounds with bromophenyl substituents (9-14) mostly possessed weak or no
anticonvulsant activity (Tab. 2). Notably, fluorophenyl derivatives (15-17) were characterized
by much stronger activity than valproate. The elongation of the alkyl substituent in
compounds 15-17 had significant impact on their affinity towards batrachotoxin-binding site
in VGSCs. Interestingly, both butyl and heptyl derivatives exhibited the same anticonvulsant
potency at their peak activity (ED50 = 67.8 mg/kg), although these compounds showed
apparently different affinity for VGSCs (i.e., 190 vs 18.2 µM). This may suggest the existence
of an additional molecular mechanism responsible for anticonvulsant effect. Such
phenomenon is very common in the case of AEDs. For instance, the antiepileptic activity of
valproic acid and its salts is associated with blockade of Na+ and T-type Ca2+ channels,
inhibition of NMDA receptors-mediated excitation, enhancement of GABA-ergic
neurotransmission and stimulation of K+ currents [10].
In our recent studies, we have observed that introduction of a methylene linker into the 4-
alkyl-5-aryl-1,2,4-triazole-3-thione derivatives significantly improved their pharmacological
properties and decreased neurotoxic effects [5, 7]. With this in mind, we analyzed the
influence of two types of linkers, i.e., methylene and ethylene ones, on the anticonvulsant
activity of 4-alkyl-5-(3-fluorophenyl)-1,2,4-triazole-3-thiones. Contrary to our expectations,
the replacement of 3-fluorophenyl moiety by 3-fluorobenzyl group did not result in an

increased efficiency of the obtained compounds (18-20) in the MES test in mice. Moreover, in
the case of heptyl derivative (20) significant decrease in anticonvulsant activity has been
observed. However, the introduction of methylene linker led to a beneficial time-course
profile of anticonvulsant potency, similarly as in the case of previously investigated 4-alkyl-5-
(3-chlorobenzyl)-1,2,4-triazole-3-thiones [5]. Respective hexyl and heptyl derivatives (19, 20)
lacked significant fluctuations of anticonvulsant activity at consecutive time points (i.e., 15,
30, 60, 120 min). Since in-vitro studies have shown that introduction of the ethylene linker
leads to a further increase in affinity towards VGSCs, respective (3-fluorophenyl)ethyl
derivatives (21, 22) were also evaluated against MES-induced seizures. These compounds
were also characterized by the highest ability to cross the blood-brain barrier as shown in
PAMPA-BBB model (Tab. 3). Both compounds (21, 22) exhibited stronger anticonvulsant
activity than valproate. Additionally, 5-[(3-fluorophenyl)ethyl]-4-hexyl-2,4-dihydro-3H-1,2,4-
triazole-3-thione (22) produced stable anticonvulsant effect, devoid of fluctuations after its
administration to mice. This may confirm the hypothesis formulated in our earlier paper [5]
that the presence of linker between aryl (i.e., halogenphenyl) moiety and 1,2,4-triazole core
results in a slower elimination of the compounds from the central nervous system.
The most promising compounds (i.e., 17 and 22), which activity was above 2-fold higher than
that of valproate, were subsequently investigated in the mouse 6 Hz (32 mA) psychomotor
seizure model of partial epilepsy. The mentioned test is one of a preclinical screening tool for
evaluation of novel AEDs with diverse mechanisms of action. It has been used for several years
and by numerous groups as a model of refractory epilepsy to evaluate preclinical AEDs
pharmacology [11-13]. However, according to Metcalf et al. [14] the 6 Hz (44 mA) seizure test
is much more discriminatory pharmacoresistant screening model when compared to 32 mA.
The latest cohort studies have shown that despite the introduction of over a dozen of new
AEDs, the overall seizure control in patients with epilepsy has not essentially changed [15].
Vast majority of these new AEDs are „me-too drugs”, i.e. they are structurally similar to
already known ones. Although „me-too drugs” show a better profile of side effects and usually
offer beneficial pharmacokinetics, it is highly unlikely that they may constitute an effective
alternative in the treatment of refractory epilepsy. Therefore, 4-alkyl-5-substituted-1,2,4-
triazole-3-thione derivatives, that are structurally unrelated to currently marketed AEDs have
a chance to become promising drug candidates for further clinical testing. As presented in

Table 4, both 17 and 22 turned out to be highly effective in 6 Hz psychomotor seizure model.
It is worth mentioning that the anticonvulsant activities observed were quite stable over the
investigated periods of time (i.e., 15, 30, 60, 120 min) (Figure 2).
Table 4 (insert here)
Figure 2 (insert here)
Importantly, the protective effect of compound 22 in the above-mentioned model of
psychomotor seizures was much stronger when compared to valproate and carbamazepine
and was almost similar to that of levetiracetam, which is considered a model drug in 6 Hz (32
mA) test [11, 16, 17] (Table 5). Compound 22 had also substantially better benefit-to-risk ratio
than valproate and carbamazepine as indicated by the respective PIs values. Out of the AEDs
presented in Table 5, only levetiracetam was found to be much safer than 22 in terms of the
impairment of motor coordination evaluated in the rotarod test. According to Barton et al.
[11] TD50 of levetiracetam was higher than 500 mg/kg, while the experiments performed in
our laboratory by Luszczki et al. [25] gave TD50 as high as 1601 mg/kg.
Table 5 (insert here)
Considering the structure-activity (and toxicity) relationships, it is visible that the exchange of
3-fluorophenyl substituent by (3-fluorophenyl)ethyl moiety resulted in a more potent activity
against 6 Hz-induced seizures. Introduction of ethylene linker resulted also in an increase of
PI values to 16.6, which indicates that such a structural modification has beneficial effect not
only on the anticonvulsant activity but also on the safety of compound 22.
2.3. ADME-Tox properties of compounds 17 and 22
Since toxicity studies constitute an important part of preclinical testing of new drug
candidates, the most promising drug candidates (17, 22) were analyzed for their possible
cytotoxic, genotoxic and hemolytic effects. Cytotoxicity was monitored after 24 h incubation
of liver cells (NMu3Li, HepG2) and human embryonic kidney cells (HEK-293) with the increased
concentrations (0 – 125 µg/ml) of the investigated compounds. Although HepG2 cells have
cancerous characteristics, they retain many liver-specific functions and are widely used for

studying hepatotoxic properties of drugs and drug-candidates [18]. According to the
literature, toxicity threshold was defined as the concentration of the compound at which the
viability of cells decreased by at least 30% [19]. Exceeding of this threshold for all of the tested
cell lines was observed above 16 µg/ml (Fig. 3). Pharmacokinetic (PK) studies for compounds
17 and 22 were not performed yet, thus their in-vivo concentrations are unknown. However,
the maximal concentration of the previously examined 5-(3-chlorobenzyl)-4-hexyl-2,4-
dihydro-3H-1,2,4-triazole-3-thione, structurally related to 17 and 22, in mouse plasma was
4.232 µg/ml [7]. Therefore, it is likely that the above-mentioned 1,2,4-triazole derivatives
would be non-toxic for living cells in concentrations necessary to generate the anticonvulsant
effect in-vivo.
Figure 3 (insert here)
Evaluation of genotoxic potential of 17 and 22 was performed using a single cell gel
electrophoresis (SCGE) technique, widely known as comet assay. This method allows
detection of DNA damages (e.g., single- and double-strand breaks, AP sites, DNA-DNA and
DNA-protein cross-links) in living cells exposed to the investigated factors [20]. The main
principle of the comet assay is based on different migration speeds of damaged and
undamaged DNA under electrophoretic conditions. The presence and intensity of the comet
tail reflect the number of DNA breaks. As can be seen in Figure 4, there have not been
observed any genotoxic changes in HEK-293 cells exposed to compounds 17 and 22 (10 µg/ml)
for 24 h.
Figure 4 (insert here)
Finally, the assessment of hemolytic potential of these compounds was performed by
spectrophotometric measurement of hemoglobin released from red blood cells (RBCs). Free
hemoglobin may cause serious damage to kidneys, liver, heart and spleen [21]. It also exerts
neurotoxic activity which is mediated by heme [22]. Drug-induced lysis of RBCs not only
prevents i.v. administration of the drug but also intensifies its toxicity when administered by
other routes [23]. Although toxic hemolysis caused by the antiepileptic drugs is rather rare
[24], it is desirable to exclude the risk of such side-effects in the case of each drug candidate.
Compounds 17 and 22 in concentrations non-toxic for the cells evaluated in MTT assay (i.e. 5,
10, 15 µg/ml), were tested during in-vitro hemolysis study. The investigated derivatives did

not produce statistically significant changes in the level of hemoglobin released to the medium
(Tab. 6). Therefore, it can be concluded that the most promising anticonvulsant drug
candidates have no tendency to disrupt membranes of RBCs.
Table 6 (insert here)
3. Conclusions
Based on the results of MES and 6 Hz (32 mA) tests, a group of 4-alkyl-5-substituted-1,2,4-
triazole-3-thione derivatives with broad anticonvulsant activity have been selected for further
mechanistic and ADME-Tox studies. The most promising compounds (17, 22) exhibited strong
anticonvulsant activity both in generalized tonic-clonic and psychomotor seizures. These
compounds did not induce genotoxic and hemolytic changes in human cells as well as they
were non-toxic for living cells in concentrations less than 16 µg/ml. Taking into account the
structural requirements for good anticonvulsant activity of 4-alkyl-5-aryl-1,2,4-triazole-3-
thiones, it is visible that small EWGs attached to phenyl ring have beneficial effects both on
affinity towards VGSCs and protective activity in the animal models of epilepsy.
4. Materials and methods
4.1. Chemistry
All reagents and solvents were purchased from Sigma-Aldrich and were used without further
purification. Melting points of the compounds were measured using Fisher-Johns apparatus.
NMR spectra of the compounds dissolved in DMSO-d6 were recorded on a 700 MHz Bruker
Avance II Plus spectrometer (Bruker BioSpin, Germany). Chemical shifts were reported in parts
per million (δ) in relation to tetramethylsilane (TMS) as an internal standard. Elemental
analyses were performed on AMZ 851 CHX analyzer (PG, Gdańsk, Poland) and the results were
within ± 0.4% of the theoretical values. The results of spectral characterization of the
intermediates (i.e., 1,4-disubstituted thiosemicarbazides) are given as Supplementary
Material.

Synthesis of the compounds 1-22
Equimolar amounts of various alkyl isothiocyanates and carboxylic acid hydrazides (obtained
from the respective carboxylic acids) were mixed together and heated to 110ºC until
a product was formed (i.e., for 5 min). The obtained thiosemicarbazide derivatives were
washed with diethyl ether, filtered and crystallized from anhydrous ethanol. These
compounds were subsequently dissolved in 2% NaOH and heated under reflux for 2 hours.
After cooling to room temperature, the solvent was neutralized with 3M HCl to form a solid
of appropriate 4-alkyl-5-substituted-1,2,4-triazole-3-thione derivative, which was filtered,
dried and crystallized from anhydrous ethanol.
4-Hexyl-5-(2-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (1)
1
Yield: 59%, m.p. 86-88ºC, H-NMR (700 MHz): 0.74 (t, 3H, CH3, J = 7.2 Hz), 1.00-1.26 (m, 6H,
3CH2), 1.44-1.49 (m, 2H, CH2), 3.85 (t, 2H, CH2, J = 7.5 Hz), 6.95 (td, 1H, ArH, J = 7.4 Hz, J = 1.1
Hz), 7.02 (dd, 1H, ArH, J = 8.2 Hz, J = 0.9 Hz), 7.30 (dd, 1H, ArH, J = 7.6 Hz, J = 1.7 Hz), 7.40-7.43
(m, 1H, ArH), 10.30 (s, 1H, OH), 13.78 (s, 1H, NH). 13C-NMR (175 MHz): 14.20, 22.16, 25.76,
27.64, 30.81, 43.92, 113.90, 116.49, 119.79, 131.81, 132.83, 150.36, 156.28, 166.88. Anal.
C14H19N3OS (C, H, N).
4-Heptyl-5-(2-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (2)
1
Yield: 48%, m.p. 72-74ºC, H-NMR (700 MHz): 0.87 (t, 3H, CH3, J = 7.1 Hz), 1.08-1.38 (m, 8H,
4CH2), 1.45-1.49 (m, 2H, CH2), 3.79 (t, 2H, CH2, J = 7.1 Hz), 7.04-7.54 (m, 4H, ArH), 10.26 (s, 1H,
OH), 13.78 (s, 1H, NH). 13C-NMR (175 MHz): 14.06, 22.53, 25.22, 27.63, 27.92, 31.65, 47.81,
115.11, 117.60, 119.79, 122.90, 131.01, 145.44, 156.28, 170.27. Anal. C15H21N3OS (C, H, N).
4-Hexyl-5-(3-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (3)
1
Yield: 64%, m.p. 90-92ºC, H-NMR (700MHz): 0.78 (t, 3H, CH3, J = 7.2 Hz), 1.10-1.19 (m, 6H,
3CH2), 1.46-1.51 (m, 2H, CH2), 3.95 (t, 2H, CH2, J = 7.3 Hz), 6.72-6.82 (m, 2H, ArH), 7.29-7.33
(m, 2H, ArH), 9.90 (s, 1H, OH), 14.00 (s, 1H, NH). 13C-NMR (175MHz): 12.11, 19.37, 21.65,
24.05, 27.79, 43.47, 111.78, 113.21, 118.30, 125.07, 127.97, 150.03, 156.87, 165.91. Anal.
C14H19N3OS (C, H, N).

4-Heptyl-5-(3-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (4)
1
Yield: 52%, m.p. 86-88ºC, H-NMR (700MHz): 0.85 (t, 3H, CH3, J = 7.2 Hz), 1.06-1.24 (m, 6H,
3CH2), 1.58-1.64 (m, 4H, 2CH2), 3.92 (t, 2H, CH2, J = 7.4 Hz), 6.94-6.97 (m, 1H, ArH), 7.25-7.35
(m, 3H, ArH), 10.05 (s, 1H, OH), 14.02 (s, 1H, NH). 13C-NMR (175MHz): 11.79, 19.64, 23.85,
25.05, 27.28, 32.54, 113.77, 115.40, 120.21, 124.81, 127.70, 150.06, 156.65, 166.58. Anal.
C15H21N3OS (C, H, N).
4-Hexyl-5-(4-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (5)
1
Yield: 55%, m.p. 86-88ºC, H-NMR (700 MHz): 0.73 (t, 3H, CH3, J = 7.2 Hz), 1.08-1.18 (m, 6H,
3CH2), 1.49-1.54 (m, 2H, CH2), 4.00 (t, 2H, CH2, J = 7.4Hz), 6.92 (dd, 2H, ArH, J = 6.3 Hz, J = 1.9
Hz), 7.47 (dd, 2H, ArH, J = 6.3 Hz, J = 1.9 Hz), 10.07 (s, 1H, OH), 13.75 (s, 1H, NH). 13C-NMR (175
MHz): 14.21, 22.26, 25.78, 27.80, 30.85, 43.91, 116.23, 117.33, 130.61, 152.00, 159.95,
167.29. Anal. C14H19N3OS (C, H, N).
4-Heptyl-5-(4-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (6)
1
Yield: 61%, m.p. 76-78ºC, H-NMR (700 MHz): 0.90 (t, 3H, CH3, J = 7.2 Hz), 1.07-1.27 (m, 6H,
3CH2), 1.64-1.72 (m, 4H, 2CH2), 4.10 (bs, 2H, CH2), 6.92 (dd, 2H, ArH, J = 6.3 Hz, J = 2.1 Hz), 7.42
(d, 2H, ArH, J = 6.2 Hz, J = 2.2 Hz), 10.21 (s, 1H, OH), 13.75 (s, 1H, NH). 13C-NMR (175 MHz):
19.23, 25.19, 25.62, 25.94, 29.41, 32.36, 57.57, 114.47, 116.51, 117.73, 119.57, 132.42,
149.96, 157.09, 166.07. Anal. C15H21N3OS (C, H, N).
4-Butyl-5-(3-methoxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (7)
Yield: 80%, m.p. 94-96°C, 1H-NMR (700 MHZ): 0.71 (t, 3H, CH3, J = 7.2 Hz), 1.10 (sext, 2H, CH2,
J = 7.3 Hz), 1.40 (quint, 2H, CH2, J = 7.1 Hz), 3.71 (s, 3H, OCH3), 3.96 (t, 2H, CH2, J = 7.3 Hz),
7.04-7.43 (m, 4H, ArH), 13.92 (s, 1H, NH). 13C-NMR (175 MHz): 11.91, 19.58, 29.07, 43.46,
55.21, 112.11, 114.12, 118.44, 125.38, 127.30, 147.52, 159.84, 166.18. Anal. C13H17N3OS (C, H,
N).
4-Hexyl-5-(3-methoxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (8)
Yield: 89%, m.p. 118-120°C, 1H-NMR (700 MHz): 0.75 (t, 3H, CH3, J = 6.8 Hz), 1.01-1.20 (m, 6H,
3CH2), 1.40-1.58 (m, 2H, CH2), 3.79 (s, 3H, OCH3), 4.01 (t, 2H, CH2, J = 7.5 Hz), 7.10-7.25 (m, 3H,

ArH), 7.42-7.52 (m, 1H, ArH), 13.90 (s, 1H, NH). 13C-NMR (175 MHz): 11.39, 19.44, 22.97, 24.95,
28.05, 41.24, 53.07, 111.49, 114.18, 118.45, 125.12, 127.96, 148.71, 157.10, 164.67. Anal.
C15H21N3OS (C, H, N).
5-(2-Bromophenyl)-4-butyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (9)
1
Yield: 81%, m.p. 144-146°C, H-NMR (700 MHz): 0.67 (t, 3H, CH3, J = 7.3 Hz), 1.08 (sext, 2H,
CH2, J = 7.3 Hz), 1.43 (quint, 2H, CH2, J = 7.3 Hz), 3.76 (t, 2H, CH2, J = 7.4 Hz), 7.54 (m, 2H, ArH),
7.66-7.70 (m, 1H, ArH), 7.83-7.88 (m, 1H, ArH), 14.02 (s, 1H, NH). 13C-NMR (175 MHz): 13.66,
19.38, 29.96, 43.69, 123.81, 127.79, 128.69, 133.13, 133.50, 133.55, 150.33, 167.13. Anal.
C12H14BrN3S (C, H, N).
5-(2-Bromophenyl)-4-pentyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (10)
Yield: 88%, m.p. 154-156°C, 1H-NMR (700 MHz): 0.70 (t, 3H, CH3, J = 6.9 Hz), 1.01-1.10 (m, 4H,
2CH2), 1.39-1.50 (m, 2H, CH2), 3.76 (t, 2H, CH2, J = 7.5 Hz), 7.53-7.63 (m, 2H, ArH), 7.66-7.70
(m, 1H, ArH), 7.83-7.88 (m, 1H, ArH), 14.02 (s, 1H, NH). 13C-NMR (175 MHz): 13.99, 21.76,
27.43, 28.14, 43.89, 123.80, 127.80, 128.68, 133.12, 133.51, 133.55, 150.31, 167.11. Anal.
C13H16BrN3S (C, H, N).
5-(3-Bromophenyl)-4-butyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (11)
Yield: 81%, m.p. 106-108°C, 1H-NMR (700 MHz): 0.74 (t, 3H, CH3, J = 7.3 Hz), 1.05-1.19 (m, 2H,
CH2), 1.43-1.55 (m, 2H, CH2), 4.02 (t, 2H, CH2, J = 7.6 Hz), 7.53 (t, 1H, ArH, J = 7.8 Hz), 7.68-7.73
(m, 1H, ArH), 7.79-7.84 (m, 1H, ArH), 7.91 (t, 1H, ArH, J = 1.7 Hz), 14.00 (s, 1H, NH). 13C-NMR
(175 MHz): 13.69, 19.45, 29.95, 43.82, 122.56, 128.21, 128.88, 131.65, 131.69, 134.12, 150.29,
167.70. Anal. C12H14BrN3S (C, H, N).
5-(3-Bromophenyl)-4-pentyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (12)
Yield: 88%, m.p. 108-110°C, 1H-NMR (700 MHz): 0.74 (t, 3H, CH3, J = 6.9 Hz), 1.03-1.19 (m, 4H,
2CH2), 1.53 (t, 2H, CH2, J = 7.3 Hz), 4.01 (t, 2H, CH2, J = 7.6 Hz), 7.53 (t, 1H, ArH, J = 7.9 Hz),
7.68-7.73 (m, 1H, ArH), 7.79-7.84 (m, 1H, ArH), 7.90 (t, 1H, ArH, J = 1.6 Hz), 14.00 (s, 1H, NH).
13C-NMR (175 MHz): 14.06, 21.76, 27.45, 28.23, 44.00, 122.66, 128.21, 128.89, 131.67, 134.10,
134.11, 150.28, 167.69. Anal. C13H16BrN3S (C, H, N).

5-(4-Bromophenyl)-4-butyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (13)
CAS: 701946-13-6, yield: 68%, m.p. 128-130°C, 1H-NMR (700 MHz): 0.74 (t, 3H, CH3, J = 7.3 Hz),
1.12 (sext, 2H, CH2, J = 7.3 Hz), 1.49 (quint, 2H, CH2, J = 7.2 Hz), 4.02 (t, 2H, CH2, J = 7.5 Hz),
7.64 (dd, 2H, ArH, J = 2.0 Hz, J = 6.6 Hz), 7.77 (dd, 2H, ArH, J = 1.9 Hz, J = 6.6 Hz), 13.99 (s, 1H,
NH). 13C-NMR (175 MHz): 13.75, 19.48, 30.03, 43.87, 124.93, 125.92, 131.10, 132.61, 150.78,
167.73. Anal. C12H14BrN3S (C, H, N).
5-(4-Bromophenyl)-4-pentyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (14)
Yield: 81%, m.p. 124-126°C, 1H-NMR (700 MHz): 0.74 (t, 3H, CH3, J = 7.1 Hz), 1.02-1.19 (m, 4H,
2CH2), 1.50 (quint, 2H, CH2, J = 7.3 Hz), 4.01 (t, 2H, CH2, J = 7.6 Hz), 7.64 (dd, 2H, ArH, J = 2.1
Hz, J = 6.6 Hz), 7.78 (dd, 2H, ArH, J = 1.9 Hz, J = 6.6 Hz), 13.98 (s, 1H, NH). 13C-NMR (175 MHz):
14.10, 21.85, 27.52, 28.27, 44.03, 124.92, 125.93, 131.11, 132.60, 150.77, 167.71. Anal.
C13H16BrN3S (C, H, N).
4-Butyl-5-(3-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (15)
Yield: 78%, m.p. 70-72°C, 1H-NMR (700 MHz): 0.74 (t, 3H, CH3, J = 7.4 Hz), 1.13 (sext, 2H, CH2,
J = 7.3 Hz), 1.49 (quint, 2H, CH2, J = 7.4 Hz), 4.02 (t, 2H, CH2, J = 7.5 Hz), 7.44-7.66 (m, 4H, ArH),
14.00 (s, 1H, ArH). 13C-NMR (175 MHz): 13.69, 19.42, 29.98, 43.82, 116.12 (d, J = 23.52 Hz),
118.24 (d, J = 21.09 Hz), 125.40 (d, J = 2.9 Hz), 128.69 (d, J = 8.5 Hz), 131.84 (d, J = 8.6 Hz),
150.47, 161.80 (d, J = 245.4 Hz), 167.80. Anal. C12H14FN3S (C, H, N).
5-(3-Fluorophenyl)-4-hexyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (16)
1
CAS: 54543-33-8, yield: 76%, m.p. 76-78°C, H-NMR (700 MHz): 0.77 (t, 3H, CH3, J = 7.1 Hz),
1.07-1.17 (m, 6H, 3CH2), 1.47-1.54 (m, 2H, CH2), 3.88 (t, 2H, CH2, J = 7.3 Hz), 7.45-7.67 (m, 4H,
ArH), 13.99 (s, 1H, NH). 13C-NMR (175 MHz): 14.19, 22.20, 25.70, 27.68, 30.80, 43.98, 116.17
(d, J = 23.1 Hz), 118.24 (d, J = 21.0 Hz), 125.44 (d, J = 2.9 Hz), 128.80 (d, J = 8.6 Hz), 131.83 (d,
J = 8.5 Hz), 150.44, 162.48 (d, J = 243.8 Hz), 167.75. Anal. C14H18FN3S (C, H, N).
5-(3-Fluorophenyl)-4-heptyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (17)
1
Yield: 79%, m.p. 64-66°C, H-NMR (700 MHz): 0.80 (t, 3H, CH3, J = 7.3 Hz), 1.06-1.20 (m, 8H,
4CH2), 1.50 (quint, 2H, CH2, J = 7.2 Hz), 4.05 (t, 2H, CH2, J = 7.5 Hz), 7.44-7.66 (m, 4H, ArH),

13.99 (s, 1H, NH). 13C-NMR (175 MHz): 14.31, 22.38, 25.97, 27.71, 28.26, 31.34, 43.98, 116.19
(d, J = 24.0 Hz), 118.23 (d, J = 22.4 Hz), 125.44 (d, J = 2.9 Hz), 128.80 (d, J = 8.6 Hz), 131.81 (d,
J = 8.5 Hz), 150.48, 162.49 (d, J = 246.7 Hz), 167.77. Anal. C15H20FN3S (C, H, N).
4-Butyl-5-[(3-fluorophenyl)methyl]-2,4-dihydro-3H-1,2,4-triazole-3-thione (18)
Yield: 76%; m.p. 76-78°C, 1H-NMR (700MHz): 0.81 (t, 3H, CH3, J = 7.3 Hz), 1.22 (sext, 2H, CH2,
J = 7.5 Hz), 1.31-1.37 (m, 2H, CH2), 3.84 (t, 2H, CH2, J = 7.8 Hz), 4.16 (s, 2H, CH2), 7.10-7.41 (m,
4H, ArH), 13.60 (s, 1H, NH). 13C-NMR (175MHz): 13.91, 19.71, 29.87, 30.86, 43.46, 114.41 (d, J
= 20.6 Hz), 116.24 (d, J = 22.3 Hz), 125.46 (d, J = 2.8 Hz), 131.08 (d, J = 8.5 Hz), 138.38 (d, J =
2.8 Hz), 151.26, 162.67 (d, J = 243.2 Hz), 167.22. Anal. C13H16FN3S (C, H, N).
5-[(3-Fluorophenyl)methyl]-4-hexyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (19)
Yield: 82%, m.p. 122-124°C, 1H-NMR (700 MHz): 0.83 (t, 3H, CH3, J=7.2 Hz), 1.12-1.25 (m, 6H,
3CH2), 1.34 (quint, 2H, CH2, J=7.7 Hz), 3.83 (t, 2H, CH2, J=7.8 Hz), 4.15 (s, 2H, CH2), 7.09-7.42
(m, 4H, ArH), 13.60 (s, 1H, NH). 13C-NMR (175 MHz): 14.28, 22.31, 26.00, 27.67, 30.83, 31.18,
43.69, 114.43 (d, J = 20.9 Hz), 116.22 (d, J = 21.7 Hz), 125.42, 131.08 (d, J = 8.4 Hz), 138.42 (d,
J = 7.8 Hz), 151.46, 162.74 (d, J = 245.2 Hz), 167.22. Anal. C15H20FN3S (C, H, N).
5-[(3-Fluorophenyl)methyl]-4-heptyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (20)
1
Yield: 84%, m.p. 82-84°C, H-NMR (700 MHz): 0.85 (t, 3H, CH3, J = 7.3 Hz), 1.13-1.19 (m, 6H,
3CH2), 1.23 (sext, 2H, CH2, J = 7.1 Hz), 1.26-1.35 (m, 2H, CH2), 3.83 (t, 2H, CH2, J = 7.9 Hz), 4.16
(s, 2H, CH2), 7.05-7.19 (m, 3H, ArH), 7.37-7.42 (m, 1H, ArH), 13.60 (s, 1H, NH). 13C-NMR (175
MHz): 14.39, 22.43, 26.29, 27.71, 28.61, 30.82, 31.48, 43.69, 114.42 (d, J = 21.1 Hz), 116.19 (d,
J = 21.7 Hz), 125.40 (d, J = 2.5 Hz), 131.08 (d, J = 8.3 Hz), 138.42 (d, J = 7.8 Hz), 151.17, 162.75
(d, J = 244.8 Hz), 167.23. Anal. C16H22FN3S (C, H, N).
4-Butyl-5-[2-(3-fluorophenyl)ethyl]-2,4-dihydro-3H-1,2,4-triazole-3-thione (21)
Yield: 88%, m.p. 86-88°C, 1H-NMR (700MHz): 0.88 (t, 3H, CH3, J = 7.4 Hz), 1.28 (sext, 2H, CH2,
J = 7.5 Hz), 1.54-1.59 (m, 2H, CH2), 2.98-3.04 (m, 4H, 2CH2), 3.88 (t, 2H, CH2, J = 7.7 Hz), 7.00-
7.04 (m, 1H, ArH), 7.11-7.17 (m, 2H, ArH), 7.31-7.34 (m, 1H, ArH), 13.50 (s, 1H, NH). 13C-NMR
(175MHz): 14.00, 19.80, 26.37, 30.23, 31.23, 43.04, 113.44 (d, J = 21.2 Hz), 115.70 (d, J = 21.2

Hz), 125.09 (d, J = 2.6 Hz), 130.60 (d, J = 8.5 Hz), 143.76 (d, J = 7.5 Hz), 151.90, 162.70 (d, J =
242.7 Hz), 166.80. Anal. C14H18FN3S (C, H, N).
5-[2-(3-Fluorophenyl)ethyl]-4-hexyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (22)
1
Yield: 90%, m.p. 64-66°C, H-NMR (700 MHz): 0.82 (t, 3H, CH3, J = 6.4 Hz), 1.15-1.33 (m, 6H,
3CH2), 1.43-1.65 (m, 2H, CH2), 2.90-3.06 (m, 4H, 2CH2), 3.85 (t, 2H, CH2, J = 7.6 Hz), 6.94-7.94
(m, 4H, ArH), 13.50 (s, 1H, NH). 13C-NMR (175 MHz): 11.54, 19.63, 23.32, 23.56, 25.25, 28.40,
40.40, 110.69 (d, J = 20.9 Hz), 112.89 (d, J = 20.9 Hz), 122.28 (d, J = 2.7 Hz), 127.82 (d, J = 8.4
Hz), 141.00 (d, J = 7.6 Hz), 149.11, 159.76 (d, J = 242.4 Hz), 163.97. Anal. C16H22FN3S (C, H, N).
4.2. Animal experimentations
All animal experiments were performed in accordance with EU Directive 2010/63/EU for
animal experiments and complied with the ARRIVE guidelines. All the procedures were also
approved by the Local Ethics Committee (Lublin, Poland, No. of approvals: 102/2017, 19/2018,
65/2018, 87/2018).
MES test
Anticonvulsant activity of the investigated compounds was determined according to the same
procedure as described in [4, 5, 7]. Experiments were conducted on adult Swiss albino mice
weighing 20-25 g. The animals were kept in colony cages with free access to food and tap
water. After one week of acclimatization, the animals were randomly divided into groups
consisting of 8 mice. The investigated compounds were suspended in a 1% solution of Tween
80 in distilled water and administered i.p. in a volume of 5 ml/kg. During experiment, the
compounds were administered i.p. in a fixed dose of 300 mg/kg at four pretreatment times
(15, 30, 60, 120 min) before the MES test. Electroconvulsions were produced by an alternating
current (0.2 s stimulus duration; 500 V, 50 Hz, fixed current intensity of 25 mA) delivered via
ear-clip electrodes by a Type 221 Rodent Shocker generator (Hugo Sachs Elektronik, Freiburg,
Germany). The abolition of a hind-limb tonic extension was taken as a criterion of the
anticonvulsant activity. Next, the active compounds (i.e., the compounds that exhibited

anticonvulsant effect in a dose of 300 mg/kg) were administered in increasing doses in order
to calculate ED50 values using the log-probit method by Litchfield and Wilcoxon.
6 Hz test
Psychomotor (limbic) seizures in mice were evoked by current (6 Hz, 0.2 ms rectangular pulse
width, 32 mA, 3 s duration) generated by an S48 Square Pulse Stimulator and CCU1 Constant
Current Unit (Grass Technologies, West Warwick, RI, USA). After application of ocular
anesthetic (0.5% solution of tetracaine hydrochloride) to the mouse corneas, the animals
treated with the increasing concentrations of 17 and 22 underwent corneal stimulation and
were placed separately in plexiglas cages (25 × 15 × 10 cm) for the observation of the presence
or absence of psychomotor seizures. Immediately following the 6 Hz corneal stimulation the
animals exhibited a “stunned” posture associated with rearing and automatic movements that
lasted from 60 to 120 s in untreated animals [11]. The mice were considered to be protected
from seizures if they resumed their normal exploratory behavior within 20 s after stimulation.
The anticonvulsant activity was evaluated as the ED50 value (median effective dose of the
compound, which protects 50% of mice against convulsions) calculated from the log-probit
method according to Litchfield and Wilcoxon.
Rotarod test
Motor coordination impairment (acute neurotoxicity) after i.p. administration of the
investigated compounds to mice was examined using the rotarod test as described in [25].
Neurotoxicity of the tested compounds is presented as median toxic doses (TD50) that impair
motor coordination in 50% of the mice challenged with the rotarod test.
4.3. PAMPA-BBB assay
Blood-brain barrier (BBB) permeability of the compounds was investigated using a PAMPA
method (parallel artificial membrane permeability assay). The PAMPA system consisted of a
96-well microfilter plate was divided into two chambers: a donor at the bottom and an
acceptor at the top, separated by a 120-μm-thick microfilter disc coated with BBB lipid
solution (Pion, Inc.). The solutions of each compound were prepared in dimethyl sulfoxide

(DMSO) at 4 mg/ml concentration and then diluted with Prisma buffer (pH = 7.4) to obtain the
donor drug solution with the final nominal concentration of 20 µg/ml. The donor solutions
were placed in donor plate. Acceptor plate contained Brain Sink Buffer (BSB). The plates were
put together and incubated at 37oC for 180 min in a humidity-saturated atmosphere. The
concentrations of the respective compounds were determined with a UV-reader (Multiskan
GO, Thermo Scientific) at 254 nm in the donor and acceptor compartments.
The permeability coefficient values (Pe) were calculated by using the following equation:
where VD– donor volume, VA – acceptor volume, Cequilibrium – equilibrium concentration,
, CD – donor concentration, CA – acceptor concentration, S –
membrane area, t – incubation time (in seconds).
4.4. Radioligand binding assay
Radioligand binding studies with [3H]batrachotoxin were performed using the same procedure
as described in our previous paper [6].
4.5. Cellular toxicity evaluation
Toxicity of the compounds was analyzed using MTT assay on murine (NMu3Li) and human
(HepG2, HEK-293) cells. Experiments were performed in accordance with the international
standard ISO-10993-5:2009. HEK-293 (human embryonic kidney) cells grew in Minimal
Essential Medium (MEM; Sigma-Aldrich, Saint Louis, MO, USA), HepG2 (hepatocellular
carcinoma) cells were cultured in Eagle’s Minimal Essential Medium containing L-glutamine
(Sigma-Aldrich), while NMu3Li cells were cultivated in DMEM-high glucose (Sigma Aldrich). All
media were supplemented with 10% fetal bovine serum (FBS, Sigma Aldrich), 100 U/mL of
penicillin and 100 mg/mL of streptomycin (PenStrep, Sigma Aldrich). Procedure of MTT test
was described in details in our previously published paper [7]. Experiments were repeated
twice, and the measurements in each experiment were run in quadruplicate.

4.6. Hemolytic activity evaluation
Human red blood cells (RBCs) concentrate was obtained from the Regional Blood Donation
and Transfusion Centre (Lublin, Poland). About 5 ml of RBCs concentrate was washed three
times with sterile PBS and each time centrifuged at 1500 rpm for 3 min. The obtained pellet
was resuspended using sterile PBS in order to obtain 2% suspension of RBCs. A volume of 1ml
of RBCs suspension was mixed with 1ml of different concentrations of the investigated
compounds. The mixtures were incubated at 37°C for 30 min and centrifuged at 2500 rpm for
10 min. The amount of free hemoglobin in supernatants was measured
spectrophotometrically at 405 nm. Negative and positive controls were performed by
incubating RBCs with sterile PBS and 0.1% Triton-X, respectively. Each experiment was run in
triplicate.
4.7. Genotoxicity evaluation
Assessment of DNA damages in HepG2 cells was performed using OxiSelect Comet Assay Kit
(Cell Biolabs, Inc.) according to the manufacturer’s protocol. HepG2 cells (1 x 105 cells/ml in
ice-cold phosphate buffer saline) previously exposed (for 24 h) to compounds 17 and 22 (10
µg/ml) were mixed with liquefied low melting agarose and transferred onto the Oxiselect
comet slide. After storing for 15 minutes at 4°C, the slides were immersed in a chilled Lysis
Buffer (for 45 min at 4°C, in the dark) and Alkaline Solution (for 30 min at 4°C, in the dark).
Next, the Alkaline Solution was replaced with pre-chilled TBE electrophoresis solution. After
immersing for 5 minutes, the slides were transferred into the horizontal electrophoresis
chamber and covered with TBE electrophoresis buffer. The electrophoresis was run for 15
minutes at 1 volt/cm. DNA released from the cells was stained with Vista Green DNA Dye for
15 minutes at room temparature and observed under fluorescence microscope (Olympus
BX63). The images of cells were captured using XM10 digital camera (Olympus).
Funding: This work was financially supported by the National Science Centre, Poland (DEC-
2013/11/D/NZ7/01170).

Conflicts of Interest: The authors declare no conflict of interest.
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Figure 1. Design of the target compounds based on the results of anticonvulsant activity
(ED50), neurotoxicity (TD50) and affinity towards batrachotoxin-binding sites of VGSCs (IC50) of
the most promising 4-alkyl-5-substituted-1,2,4-triazole-3-thione derivatives published so far.

Figure 2. Time-course effect of compounds 17 and 22 in the 6 Hz (32 mA) psychomotor seizure
test.

100
75
50
25
0
toxicity
threshold
NMu3Li
HepG2
HEK-293
0
1
2
4
8
6
2
5
.
5
2
1
3
2
1
6
Concentrations [g/ml]
100
75
50
25
0
toxicity
threshold
NMu3Li
HepG2
HEK-293
0
1
2
4
8
6
2
5
.
5
2
1
3
2
1
6
Concentrations [g/ml]
Figure 3. Toxicity of compounds 17 (upper graph) and 22 (lower graph) against NMu3Li, HepG2
and HEK-293 cells evaluated in MTT assay after 24 h incubation.

Control cells
Positive control
(etoposide)
Compound 17
Compound 22
Figure 4. Genotoxicity evaluation after 24 h treatment of HEK-293 cells with compounds 17
and 22 (10 µg/ml).

Design &
synthesis
H
N
N
linker
S
N
Alkyl
EWGs/EDGs
optimal length: 4-7 carbon atoms
decreases fluctuations
in anticonvulsant activity
In-vivo testing
• MES
test
Mechanistic studies ADME-Tox evaluation
• PAMPA-BBB
• VGSCs binding
• cytotoxicity
• genotoxicity
• 6 Hz
test
• hemolytic activity
• Rotarod
test

Highlights:




The obtained compounds were identified as sodium channel blockers
PAMPA-BBB assay confirmed their good permeability across the blood-brain barrier
Compounds 17 and 22 were highly active in MES and 6 Hz tests
Compounds 17 and 22 possessed favourable ADME-Tox properties

O
O
O
O
 min
EtOH/H+/

NH NH /EtOH/

2
2
R2 NCS
+
R1
R1
R1
NH
NH
R1
NH
R2
NH NH2
OC2H5
OH
S
1. 2% NaOH/

2. 3M HCl
NH
S
N
R1
N
(1- 22)
R2
Scheme 1. Synthetic route to compounds 1 – 22. Structures of R1 and R2 substituents are given
in Tables 1 & 2.

Table 1. Anticonvulsant activity of compounds 1 – 8 (containing EDGs) in the mouse maximal
electroshock-induced seizure (MES) test
R1
R2
Pretreatment time
MES test
[min]
ED50 [mg/kg] ± SEM
15
1.
2.
3.
hexyl
30
60
120
inactive
OH
15
30
60
> 500
heptyl
hexyl
430.6 ± 29.6
361.8 ± 27.8
330.2 ± 21.4
OH
120
15
30
inactive
60
120
OH
15
30
4.
heptyl
inactive
60
120
OH
15
30
60
120
15
30
60
120
15
374.4 ± 10.9
317.2 ± 9.5
356.4 ± 19.8
370.1 ± 9.6
5.
6.
7.
hexyl
heptyl
butyl
HO
HO
inactive
30
inactive
60
120
OCH3
15
30
60
462.9 ± 20.0
429.5 ± 19.0
454.8 ± 24.1
460.8 ± 22.6
8.
hexyl
OCH3
120
Valproate
15
30
60
189.0 ± 17.3
216.9 ± 9.4
218.4 ± 18.9
246.6 ± 21.4
120