Development of the 1,2,4-triazole-based anticonvulsant drug candidates acting on the voltage-gated sodium channels. Insights from in-vivo, in-vitro, and in-silico studies

Article abstract of DOI:10.1016/j.ejps.2018.12.018

The treatment of epilepsy remains difficult mostly since almost 30% of patients suffer from pharmacoresistant forms of the disease. Therefore, there is an urgent need to search for new antiepileptic drug candidates. Previously, it has been shown that 4-al

Full text of DOI:10.1016/j.ejps.2018.12.018

EuropeanJournalofPharmaceuticalSciences129(2019)42–57
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Development of the 1,2,4-triazole-based anticonvulsant drug candidates
acting on the voltage-gated sodium channels. Insights from in-vivo, in-vitro,
and in-silico studies
Barbara Kaprońa, Jarogniew J. Łuszczkib, Anita Płazińskac, Agata Siwekd, Tadeusz Karcze,
Anna Grybośd, Gabriel Nowakd, Anna Makuch-Kockaf, Katarzyna Walczakf, Ewa Langnerf,
Karolina Szalastf, Sebastian Marciniakf, Magdalena Paczkowskag, Judyta Cielecka-Piontekg,
Lukasz M. Cieslah, Tomasz Plechf,⁎
a Department of Clinical Genetics, Medical University of Lublin, Lublin, Poland
b Department of Pathophysiology, Medical University of Lublin, Lublin, Poland
c Department of Biopharmacy, Medical University of Lublin, Lublin, Poland
d Department of Pharmacobiology, Jagiellonian University Medical College, Cracow, Poland
e Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Cracow, Poland
f Department of Pharmacology, Medical University of Lublin, Lublin, Poland
g Department of Pharmacognosy, Poznan University of Medical Sciences, Poznań, Poland
h Department of Biological Sciences, The University of Alabama, Tuscaloosa, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Epilepsy
Cytotoxicity
Permeability
Genotoxicity
Molecular modeling
The treatment of epilepsy remains difficult mostly since almost 30% of patients suffer from pharmacoresistant
forms of the disease. Therefore, there is an urgent need to search for new antiepileptic drug candidates.
Previously, it has been shown that 4-alkyl-5-substituted-1,2,4-triazole-3-thione derivativatives possessed strong
anticonvulsant activity in a maximal electroshock-induced seizure model of epilepsy. In this work, we examined
the effect of the chemical structure of the 1,2,4-triazole-3-thione-based molecules on the anticonvulsant activity
and the binding to voltage-gated sodium channels (VGSCs) and GABAA receptors. Docking simulations allowed
us to determine the mode of interactions between the investigated compounds and binding cavity of the human
VGSC. Selected compounds were also investigated in a panel of ADME-Tox assays, including parallel artificial
membrane permeability assay (PAMPA), single cell gel electrophoresis (SCGE) and cytotoxicity evaluation in
HepG2 cells. The obtained results indicated that unbranched alkyl chains, from butyl to hexyl, attached to 1,2,4-
triazole core are essential both for good anticonvulsant activity and strong interactions with VGSCs. The com-
bined in-vivo, in-vitro and in-silico studies emphasize 4-alkyl-5-substituted-1,2,4-triazole-3-thiones as promising
agents in the development of new anticonvulsants.
1. Introduction
molecular mechanism(s) of action. Unfortunately, this becomes difficult
because many AEDs have multiple molecular targets (Czapiński et al.,
Lack of effectiveness of currently available antiepileptic drugs
(AEDs) in nearly 30% of patients justifies the efforts undertaken to
discover and develop better pharmaceuticals to treat epilepsy (Tang
et al., 2017; Alexopoulos, 2013). One of the most sucessfully employed
strategies in the process of novel drug development is the so called
„receptor-based drug design” approach. However, this method requires
the knowledge of the chemical structure of the drug target protein
(Mavromoustakos et al., 2011; Katsila et al., 2016). Thus, in the case of
new potential antiepileptics it is crucial to precisely define their
2005).
In our recent studies, we have demonstrated that alkyl derivatives of
1,2,4-triazole-3-thione represent a group of promising antiepileptic
drug candidates (Plech et al., 2013; Plech et al., 2014). Apart from their
anticonvulsant activity, exhibited in mouse maximal electroshock-in-
duced seizure (MES) model, these compounds were also characterized
by low neuro- and cellular toxicity. Both electrophysiological and
radioligand binding experiments have shown that 4-alkyl-1,2,4-tria-
zole-3-thione derivatives did not enhance gabaergic neurotransmission.
⁎ Corresponding author at: Department of Pharmacology, Faculty of Health Sciences, Medical University of Lublin, Chodźki 4a, 20-093 Lublin, Poland.
E-mail address: tomasz.plech@umlub.pl (T. Plech).
https://doi.org/10.1016/j.ejps.2018.12.018
Received 24 October 2018; Received in revised form 26 November 2018; Accepted 24 December 2018
Availableonline28December2018
0928-0987/©2018ElsevierB.V.Allrightsreserved.

B. Kaproń et al.
EuropeanJournalofPharmaceuticalSciences129(2019)42–57
Simultaneously, it was shown that 5-(3-chlorophenyl)-4-hexyl-2,4-di-
hydro-3H-1,2,4-triazole-3-thione (TP-315) possesses a strong affinity
towards batrachotoxin binding site at voltage gated sodium channels
(VGSCs) (Kaproń et al., 2017). To the extent of our knowledge, this was
the first reported confirmation of molecular mechanism of action of the
aforementioned group of compounds. However, more data is still
needed to reliably connect the anticonvulsant activity of 4-alkyl-1,2,4-
triazole-3-thiones, with their influence on the VGSCs. Additionally, it
should be emphasized that all previously performed studies concerned
1,2,4-triazole derivatives with linear (non-branched) alkyl substituents.
In those studies, the effect of alkyl chain length on pharmacological
activity was the main consideration. The influence of the branching of
the alkyl groups linked to 1,2,4-triazole-3-thione core has not been
considered thus far. Branching of the aliphatic substituent has been
previously shown to be crucial for potent anticonvulsant effect (Hen
et al., 2012). In addition, some authors have argued that compounds
containing α- or β-branched alkyl groups seem to undergo slower me-
tabolism and therefore cause longer pharmacological effect (Perlman
and Goldstein, 1984). Valproic acid is a good example of how the
structure of alkyl substituent influences its pharmacological activity.
The type and degree of alkyl fragment branching of valproic acid not
only influences its antiepileptic activity but also influences character-
istic side-effects of the drug (i.e., hepatotoxicity and teratogenicity)
(Bojic et al., 1996). Taking all the above into consideration, we decided
to examine how further modifications of the structure of the alkyl
substituent (including chain branching and incorporation of heteroa-
toms) influence the anticonvulsant activity and toxicity of 4-alkyl-1,2,4-
triazole-3-thione derivatives. Moreover, we aimed to investigate more
thoroughly the character of interactions between the discussed deri-
vatives and the VGSCs. To shed more light on this issue, we also added
our previously synthesized 4-alkyl-5-substituted-1,2,4-triazole-3-thione
derivatives to the current analyses. We hope that such comprehensive
studies may facilitate the design of improved antiepileptic agents.
1535, 1238, 739.
1-(3-Chlorobenzoyl)-4-isobutylthiosemicarbazide (TPR-3)
Compound firstly described in (Shah et al., 1969); M = 285.79;
Yield: 82%; m.p. 186–188 °C; 1H NMR (300 MHz, DMSO‑d6):0.83 (d,
6H, 2 × CH3, J = 6.6 Hz), 1.87–2.07 (m, 1H, CH), 3.25 (t, 2H, CH2,
J = 6.3 Hz), 7.54 (t, 1H, ArH, J = 7.8 Hz), 7.62–7.69 (m, 1H, ArH),
7.82–7.97 (m, 2H, ArH), 8.17 (t, 1H, NH, J = 5.1 Hz), 9.27 (s, 1H, NH),
10.44 (s, 1H, NH). 13C NMR (75 MHz, DMSO‑d6): 20.50, 28.04, 51.52,
127.01, 128.13, 130.78, 132.07, 133.52, 135.10, 165.14, 178.96. IR
(KBr, cm−1): 3363, 3251, 3145, 2961, 2870, 1680, 1574, 1543, 1231,
749.
4-(tert-Butyl)-1-(3-chlorobenzoyl)thiosemicarbazide (TPR-5)
M = 285.79; Yield: 77%; m.p. 140–141 °C; 1H NMR (300 MHz,
DMSO‑d6): 1.46 (s, 9H, 3 × CH3), 7.54 (t, 1H, ArH, J = 7.8 Hz),
7.62–7.69 (m, 1H, ArH), 7.82–7.88 (m, 1H, ArH), 7.94 (s, 1H, NH), 9.13
(s, 1H, NH), 10.41 (s, 1H, NH). 13C NMR (75 MHz, DMSO‑d6): 28.39,
52.82, 126.24, 127.34, 130.52, 131.72, 133.28, 134.40, 164.65,
181.69. IR (KBr, cm−1): 3366, 3294, 3157, 2974, 2927, 1680, 1640,
1526, 1249, 742. GC-EI-MS, m/z (Abundance %): 287 (3.25), 286
(2.07), 285 (M+, 8.42),170 (41.34), 139 (100), 111 (28.27), 75
(14.64), 57 (60.29), 41 (29.58).
1-(3-Chlorobenzoyl)-4-(2-methylbutyl)thiosemicarbazide (TPR-
7)
M = 299.82; Yield: 85%; m.p. 142–144 °C; 1H NMR (300 MHz,
DMSO‑d6): 0.73–0.89 (m, 6H, 2CH3), 0.93–1.42 (m, 2H, CH2),
1.64–1.81 (m, 1H, CH), 3.13–3.44 (m, 2H, CH2), 7.47–7.98 (m, 4H,
ArH), 8.11 (t, 1H, NH, J = 5.6 Hz), 9.24 (s, 1H, NH), 10.41 (s, 1H, NH);
13C NMR (75 MHz, DMSO‑d6): 11.18, 16.88, 26.45, 33.94, 49.47,
126.52, 127.62, 130.27, 131.54, 133.02, 134.60, 164.65, 181.69. IR
(KBr, cm−1): 3365, 3245, 3164, 2963, 2929, 2874, 1681, 1571, 1545,
1222, 748. GC-EI-MS, m/z (Abundance %): 301 (7.41), 300 (4.65), 299
(M+, 20.73), 170 (26.06), 156 (15.64), 139 (100), 111 (30.98), 86
(30.49), 71 (18.04), 57 (15.88), 43 (49.03), 41 (22.09).
2. Materials and methods
2.1. Chemistry
Chemicals and reagents used for the synthesis were purchased from
Sigma-Aldrich (St. Louis, MO, USA) and Alfa-Aesar (Ward Hill, USA).
Melting temperatures of the synthesized compounds were measured
using Fisher-Johns apparatus (Fisher Scientific, Schwerte, Germany).
NMR spectra were recorded on a Bruker Avance spectrometer (Bruker
BioSpin GmbH, Germany) using DMSO‑d6 as solvent and TMS as in-
ternal standard. The FT-IR spectra were recorded using KBr pellets on a
Perkin-Elmer 1725X FTIR spectrophotometer. GC-EI-MS spectra of the
selected compounds were obtained on Finnigan MAT95XP (Thermo-
Fisher Scientific, Waltham, MA, USA).
1-(3-Chlorobenzoyl)-4-(3-methoxypropyl)thiosemicarbazide
(TPR-13)
CAS: 891097–90-8; M = 301.79; Yield: 82%; m.p. 137–139 °C; 1H
NMR (300 MHz, DMSO‑d6): 1.74 (quint, 2H, CH2, J = 6.8 Hz), 3.18 (s,
3H, CH3), 3.32 (t, 2H, CH2, J = 6.2 Hz), 3.48 (q, 2H, CH2, J = 6.4 Hz),
7.54 (t, 1H, ArH, J = 7.8 Hz), 7.63–7.69 (m, 1H, ArH), 7.83–7.87 (m,
1H, ArH), 7.96 (t, 1H, ArH, J = 1.7 Hz), 8.13 (t, 1H, NH, J = 5.2 Hz),
9.34 (s, 1H, NH), 10.47 (s, 1H, NH). 13C NMR (75 MHz, DMSO‑d6):
29.26, 41.81, 58.34, 70.33, 127.97, 128.11, 130.82, 132.13, 133.53,
134.98, 165.09, 179.13. IR (KBr, cm−1): 3264, 3141, 2923, 2890,
2828, 1662, 1544, 1235, 747. GC-EI-MS, m/z (Abundance %): 303
(6.35), 302 (3.90), 301 (M+, 16.63), 268 (13.86), 170 (29.28), 156
(10.34), 139 (100), 111 (36.01), 88 (18.34), 72 (19.63), 56 (17.68), 45
(55.84), 41 (30.28).
2.1.1. General procedure for the synthesis of 4-alkyl-1-substituted-
thiosemicarbazide derivatives
Equimolar amounts of the respective alkyl isothiocyanates and 3-
chlorobenzoic acid hydrazide or 3-chlorophenylacetic acid hydrazide
were mixed together and heated without solvent for 5 min at 110 °C.
The obtained solid was washed with diethyl ether, filtered and crys-
tallized from anhydrous ethanol.
1-(3-Chlorobenzoyl)-4-[(3-methylthio)propyl]thiosemicarba-
zide (TPR-15)
1-(3-Chlorobenzoyl)-4-isopropylthiosemicarbazide (TPR-1)
CAS: 891050-70-7; M = 271.77; Yield: 84%; m.p. 182–183 °C; 1H
NMR (300 MHz, DMSO‑d6): 1.14 (d, 6H, 2 × CH3, J = 6.6 Hz),
4.37–4.56 (m, 1H, CH), 7.54 (t, 1H, ArH, J = 7.8 Hz), 7.62–7.68 (m,
1H, ArH), 7.78–7.90 (m, 2H, ArH), 7.97 (t, 1H, NH, J = 1.8 Hz), 9.23 (s,
1H, NH), 10.38 (s, 1H, NH). 13C NMR (75 MHz, DMSO‑d6): 22.41,
46.42, 127.06, 128.15, 130.70, 132.03, 133.52, 135.10, 165.07,
179.57. IR (KBr, cm−1): 3345, 3270, 3173, 2972, 2930, 1675, 1567,
M = 317.86; Yield: 89%; m.p. 128–130 °C; 1H NMR (300 MHz,
DMSO‑d6): 1.77 (quint, 2H, CH2, J = 7.1 Hz), 2.03 (s, 3H, CH3), 2.44 (t,
2H, CH2, J = 7.3 Hz), 3.51 (q, 2H, CH2, J = 6.7 Hz), 7.54 (t, 1H, ArH,
J = 7.8 Hz), 7.63–7.69 (m, 1H, ArH), 7.82–7.90(m, 1H, ArH), 7.96 (t,
1H, ArH, J = 1.7 Hz), 8.13 (t, 1H, NH, J = 5.2 Hz), 9.34 (s, 1H, NH),
10.47 (s, 1H, NH); 13C NMR (75 MHz, DMSO‑d6): 15.08, 28.68, 30.99,
43.21, 127.03, 128.13, 130.81, 132.11, 133.53, 135.00, 165.09,
43

B. Kaproń et al.
EuropeanJournalofPharmaceuticalSciences129(2019)42–57
180.13. IR (KBr, cm−1): 3345, 3254, 3131, 2950, 2912, 1663, 1564,
1239, 750. GC-EI-MS, m/z (Abundance %): 319 (4.53), 318 (2.58), 317
(M+, 10.03), 270 (31.94), 171 (41.62), 139 (100), 111 (50.89), 101
(44.54), 75 (28.91), 61 (40.52), 41 (56.16).
13C NMR (75 MHz, DMSO‑d6): 28.75, 41.18, 57.88, 69.77, 126.46,
128.08, 129.08, 129.92, 132.72, 137.89, 169.36, 181.69. IR (KBr,
cm−1): 3250, 3044, 2971, 2929, 2833, 1684, 1545, 1214. GC-EI-MS,
m/z (Abundance %): 317 (10.21), 316 (6.47), 315 (M+, 29.89), 282
(14.73), 264 (33.58), 184 (31.45), 132 (73.66), 125 (98.05), 89
(42.30), 71 (34.46), 56 (30.34), 45 (100), 41 (53.71).
1-[(3-Chlorophenyl)acetyl]-4-isopropylthiosemicarbazide
(TPR-19)
1-[(3-Chlorophenyl)acetyl]-4-[(3-methylthio)propyl]thiosemi-
carbazide (TPR-29)
M = 285.79; Yield: 88%; m.p. 168–170 °C; 1H NMR (300 MHz,
DMSO‑d6): 1.09 (d, 6H, 2CH3, J = 6.6 Hz), 4.27–4.50 (m, 1H, CH),
7.20–7.39 (m, 4H, ArH), 7.50 (d, 1H, NH, J = 7.4 Hz), 9.12 (s, 1H, NH),
9.91 (s, 1H, NH); 13C NMR (75 MHz, DMSO‑d6): 21.89, 45.65, 126.49,
128.00, 128.99, 130.01, 132.74, 138.07, 169.28, 181.02. IR (KBr,
cm−1): 3308, 3193, 2970, 2933, 1690, 1544, 1222, 773. GC-EI-MS, m/z
(Abundance %): 287 (5.56), 286 (3.44), 285 (M+, 16.17), 184 (8.17),
170 (20.67), 125 (54.10), 117 (40.65), 89 (21.00), 58 (100), 43
(58.68), 41 (24.97).
M = 331.88; Yield: 84%; m.p. 106–108 °C; 1H NMR (300 MHz,
DMSO‑d6): 1.75 (quint, 2H, CH2, J = 7.4 Hz), 2.02 (s, 3H, OCH3), 2.42
(t, 2H, CH2, J = 7.4 Hz), 3.43–3.56 (m, 4H, 2CH2), 7.20–7.40 (m, 4H,
ArH), 8.02 (t, 1H, NH, J = 5.2 Hz), 9.20 (s, 1H, NH), 9.95 (s, 1H, NH);
13C NMR (75 MHz, DMSO‑d6): 14.62, 28.24, 30.52, 42.65, 126.46,
128.09, 129.08, 129.93, 132.72, 137.87, 169.36, 181.69. IR (KBr,
cm−1): 3293, 3172, 2916, 2873, 2840, 1688, 1544, 1217. GC-EI-MS,
m/z (Abundance %): 333 (6.26), 332 (3.83), 331 (M+, 14.55), 313
(23.20), 284 (37.87), 266 (77.81), 185 (43.69), 125 (100), 101 (51.94),
89 (60.23), 73 (30.67), 61 (51.17), 45 (26.41), 41 (79.71).
1-[(3-Chlorophenyl)acetyl]-4-isobutylthiosemicarbazide (TPR-
21)
M = 299.82; Yield: 86%; m.p. 162–164 °C; 1H NMR (300 MHz,
DMSO‑d6): 0.81 (d, 6H, 2CH3, J = 6.6 Hz), 1.78–1.98 (m, 1H, CH), 3.23
(t, 2H, CH2, J = 6,1 Hz), 3.49 (s, 2H, CH2), 7.20–7.39 (m, 4H, ArH),
7.90 (t, 1H, NH, J = 5.5 Hz), 9.15 (s, 1H, NH), 9,94 (s, 1H, NH); 13C
NMR (75 MHz, DMSO‑d6): 19.87, 27.49, 50.80, 126.45, 128.07, 129.06,
129.93, 132.74, 137.96, 169.36, 181.69. IR (KBr, cm−1): 3286, 3162,
2959, 2928, 2873, 1681, 1550, 1215. GC-EI-MS, m/z (Abundance %):
301 (17.19), 300 (9.89), 299 (M+, 43.88), 184 (31.98), 170 (20.22),
152 (15.93), 131 (42.55), 125 (100), 89 (31.80), 72 (81.51), 57
(81.79), 41 (52.18).
2.1.2. General procedure for the synthesis of 4-alkyl-5-substituted-2,4-
dihydro-3H-1,2,4-triazole-3-thiones
The thiosemicarbazide derivatives obtained in the previous step
were heated under reflux in 2% NaOH for 2 h. After cooling, the solvent
was neutralized with 3 M HCl to form a solid, which was filtered, dried
and crystallized from anhydrous ethanol.
5-(3-Chlorophenyl)-4-isopropyl-2,4-dihydro-3H-1,2,4-triazole-
3-thione (TPR-2)
The compound was firstly described in (Shah et al., 1962).
M = 253.75; Yield: 75%; m.p. 187–188 °C; 1H NMR (300 MHz,
DMSO‑d6): 1.38 (d, 6H, 2CH3, J = 7.0 Hz), 4.54–4.68 (m, 1H, CH),
7.51–7.71 (m, 4H, ArH), 13.91 (s, 1H, NH); 13C NMR (75 MHz,
DMSO‑d6): 17.35, 46.71, 126.33, 126.52, 127.30, 128.32, 128.42,
131.03, 147.83, 163.84. IR (KBr, cm−1): 3160, 3094, 2974, 2937,
1502, 1460, 1299, 1264, 774.
4-(tert-Butyl)-1-[(3-chlorophenyl)acetyl]thiosemicarbazide
(TPR-23)
M = 299.82; Yield: 72%; m.p. 120–122 °C; 1H NMR (300 MHz,
DMSO‑d6): 1.37 (s, 9H, 3CH3), 3.49 (s, 2H, CH2), 7.22–7.34 (m, 4H,
ArH), 7.39 (s, 1H, NH), 9.07 (s, 1H, NH), 10.00 (s, 1H, NH); 13C NMR
(75 MHz, DMSO‑d6): 28.48, 52.62, 126.57, 127.86, 128.85, 130.10,
132.86, 138.01, 169.14, 181.24. IR (KBr, cm−1): 3360, 3264, 3153,
2966, 1678, 1534, 1225, 766. GC-EI-MS, m/z (Abundance %): 301
(4.57), 300 (2.68), 299 (M+, 12.53), 184 (45.84), 125 (55.66), 89
(20.12), 57 (100), 41 (49.81).
5-(3-Chlorophenyl)-4-isobutyl-2,4-dihydro-3H-1,2,4-triazole-3-
thione (TPR-4)
The compound was firstly described in (Shah et al., 1969).
M = 267.78; Yield: 80%; m.p. 131–132 °C; 1H NMR (300 MHz,
DMSO‑d6): 0.63 (d, 6H, 2CH3, J = 6.8 Hz), 1.74–1.92 (m, 1H, CH), 3.94
(d, 2H, CH2, J = 7.6 Hz), 7.53–7.81 (m, 4H, ArH), 13.96 (s, 1H, NH);
13C NMR (75 MHz, DMSO‑d6): 17.13, 24.53, 48.06, 125.17, 126.13,
128.38, 128.66, 131.35, 147.72, 165.44. GC-EI-MS, m/z (Abundance
%): 269 (12.77), 268 (8.58), 267 (M+, 35.81), 211 (100.00), 152
(21.76), 125 (5.81), 111 (6.87), 55 (9.33), 41 (13.73).
1-[(3-Chlorophenyl)acetyl]-4-(2-methylbutyl)thiosemicarba-
zide (TPR-25)
M = 313.85; Yield: 80%; m.p. 142–144 °C; 1H NMR (300 MHz,
DMSO‑d6): 0.77 (d, 3H, CH3, J = 6.6 Hz), 0.83 (t, 3H, CH3, J = 7.2 Hz),
0.92–1.42 (m, 2H, CH2), 1.58–1.80 (m, 1H, CH), 3.11–3.42 (m, 2H,
CH2),3.49 (s, 2H, CH2) 7.16–7.39 (m, 4H, ArH), 7.86 (t, 1H, NH,
J = 5.4 Hz), 9.14 (s, 1H, NH), 9.93 (s, 1H, NH); 13C NMR (75 MHz,
DMSO‑d6): 11.13, 16.74, 26.37, 33.99, 49.46, 126.47, 128.08, 129.08,
129.93, 132.75, 137.97, 169.32, 181.86. IR (KBr, cm−1): 3291, 3163,
2960, 2927, 2876, 1681, 1552, 1241. GC-EI-MS, m/z (Abundance %):
315 (14.19), 314 (8.21), 313 (M+, 37.88), 184 (29.02), 170 (20.83),
145 (44.17), 125 (95.33), 86 (59.07), 71 (36.01), 57 (30.42), 43 (100),
41 (45.79).
4-(tert-Butyl)-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-tria-
zole-3-thione (TPR-6)
M = 267.78; Yield: 83%; m.p. 102–104 °C; 1H NMR (300 MHz,
DMSO‑d6): 1.77 (s, 9H, 3CH3), 7.21–7.42 (m, 4H ArH), 13.69 (s, 1H,
NH); 13C NMR (75 MHz, DMSO‑d6): 24.09, 40.49, 125.30, 125.74,
126.27, 128.44, 128.65, 131.36, 147.67, 164.92. IR (KBr, cm−1): 3112,
2938, 2913, 1539, 1481, 1415, 1263, 803, 736. GC-EI-MS, m/z
(Abundance %): 269 (10.17), 268 (7.04), 267 (M+, 32.02), 252
(100.00), 212 (13.67), 137 (12.41), 111 (8.80), 73 (24.28), 61 (13.27),
41 (53.00).
1-[(3-Chlorophenyl)acetyl]-4-(3-methoxypropyl)thiosemi-
carbazide (TPR-27)
M = 315.82; Yield: 87%; m.p. 116–118 °C; 1H NMR (300 MHz,
DMSO‑d6): 1.72 (quint, 2H, CH2, J = 6.6 Hz), 3.20 (s, 3H, OCH3), 3.31
(t, 2H, CH2, J = 6.2 Hz), 3.40–3.51 (m, 4H, 2CH2), 7.20–7.39 (m, 4H,
ArH), 7.95 (t, 1H, NH, J = 5.4 Hz), 9.19 (s, 1H, NH), 9.95 (s, 1H, NH);
5-(3-Chlorophenyl)-4-(2-methylbutyl)-2,4-dihydro-3H-1,2,4-
triazole-3-thione (TPR-8)
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3-thione (TPR-24)
M = 281.80; Yield: 73%; m.p. 106–108 °C; 1H NMR (300 MHz,
DMSO‑d6): 0.72 (t, 3H, CH3, J = 7,1 Hz), 0.89 (d, 3H, CH3, J = 6,7 Hz),
1.11–1.32 (m, 2H, CH2), 1.63–1.80 (m, 1H, CH), 3.11–3.34 (m, 2H,
CH2), 6.97–7.26 (m, 4H, ArH), 13.65 (s, 1H, NH); 13C NMR (75 MHz,
DMSO‑d6): 11.28, 15.46, 26.42, 32.82, 47.00, 123.39, 127.60, 130.25,
131.62, 133.85, 134.65, 151.24, 167.88. IR (KBr, cm−1): 3182, 3081,
2940, 2913, 1553, 1461, 1261, 800, 745. GC-EI-MS, m/z (Abundance
%): 283 (8.49), 282 (5.22), 281 (M+, 20.38), 248 (16.50), 211
(100.00), 152 (16.16), 138 (10.80), 125 (4.68), 111 (5.70), 75 (5.98),
55 (13.24), 41 (19.84).
M = 281.80; Yield: 82%; m.p. 198–200 °C; 1H NMR (700 MHz,
DMSO‑d6): 1.78 (s, 9H, 3CH3), 4.31 (s, 2H, CH2), 7.07 (d, 1H ArH,
J = 7.5 Hz), 7.22 (s, 1H, ArH), 7.33 (d, 1H, ArH, J = 7.8 Hz), 7.38 (t,
1H, ArH, J = 7.8 Hz), 13.56 (s, 1H, NH); 13C NMR (175 MHz,
DMSO‑d6): 28.64, 35.34, 61.84, 127.15, 127.64, 128.84, 130.80,
133.57, 140.05, 150.95, 167.99. IR (KBr, cm−1): 3108, 3077, 2924,
1377, 1283, 1209, 772, 684. GC-EI-MS, m/z (Abundance %): 283
(15.10), 282 (3.52), 281 (M+, 5.82), 225 (100.00), 190 (10.61), 166
(4.03), 131 (6.91), 125 (12.40), 89 (7.48), 74 (6.69), 57 (76.56), 41
(36.77).
5-(3-Chlorophenyl)-4-(3-methoxypropyl)-2,4-dihydro-3H-1,2,4-
triazole-3-thione (TPR-14)
5-(3-Chlorobenzyl)-4-(2-methylbutyl)-2,4-dihydro-3H-1,2,4-
triazole-3-thione (TPR-26)
M = 283.78; Yield: 80%; m.p. 112–114 °C; 1H NMR (300 MHz,
DMSO‑d6): 1.83 (quint, 2H, CH2, J = 6.2 Hz), 3.06 (s, 3H, OCH3), 3.20
(t, 2H, CH2, J = 5.9 Hz), 4.1 (t, 2H, CH2, J = 1.3 Hz), 7.59–7.80 (m, 4H,
ArH), 13.99 (s, 1H, NH); 13C NMR (75 MHz, DMSO‑d6): 27.99, 42.04,
58.19, 69.09, 128.00, 128.62, 128.96, 131.14, 131.38, 134.11, 150.56,
167.74. IR (KBr, cm−1): 3183, 2882, 1490, 1446, 1424, 1115, 1073,
493, 795, 775, 693, 593, 543. GC-EI-MS, m/z (Abundance %): 285
(4.93), 284 (2.97), 283 (M+, 12.94), 250 (100.00), 224 (18.50), 212
(23.08), 152 (9.31), 138 (11.69), 125 (4.48), 111 (7.31), 71 (10.89), 45
(26.47), 41 (18.93).
M = 295.83; Yield: 70%; m.p. 132–134 °C; 1H NMR (300 MHz,
DMSO‑d6): 0.77 (d, 3H, CH3, J = 6.8 Hz), 0.83 (t, 3H, CH3, J = 7.4 Hz),
1.09–1.32 (m, 2H, CH2), 1.92–1.98 (m, 1H, CH), 3.71–3.82 (m, 2H,
CH2), 4.13 (s, 2H, CH2), 7.19–7.43 (m, 4H, ArH), 13.62 (s, 1H, NH); 13
C
NMR (75 MHz, DMSO‑d6): 11.57, 16.78, 26.71, 30.81, 33.71, 49.06,
127.51, 128.12, 129.32, 130.85, 133.60, 138.01, 151.53, 167.80. IR
(KBr, cm−1): 3092, 3037, 2958, 2930, 1567, 1493, 1453, 1433, 1274,
1080, 766, 676. GC-EI-MS, m/z (Abundance %): 297 (16.30), 296
(10.46), 295 (M+, 43.05), 262 (41.65), 225 (100.00), 190 (25.82), 166
(16.19), 131 (16.26), 125 (29.24), 89 (11.97), 55 (25.13), 41 (29.88).
5-(3-Chlorophenyl)-4-[(3-methylthio)propyl]-2,4-dihydro-3H-
1,2,4-triazole-3-thione (TPR-16)
5-(3-Chlorobenzyl)-4-(3-methoxypropyl)-2,4-dihydro-3H-1,2,4-
triazole-3-thione (TPR-28)
M = 299.84; Yield: 78%; m.p. 105–106 °C; 1H NMR (300 MHz,
DMSO‑d6): 1.84 (quint, 2H, CH2, J = 7.3 Hz), 1.92 (s, 3H, SCH3), 2.37
(t, 2H, CH2, J = 6.8 Hz), 4.12 (t, 2H, CH2, J = 7.5 Hz), 7.59–7.82 (m,
4H, ArH), 14.01 (s, 1H, NH); 13C NMR (75 MHz, DMSO‑d6): 14.74,
26.91, 30.47, 43.31, 128.09, 128.54, 129.08, 131.24, 131.44, 134.17,
150.47, 167.76. IR (KBr, cm−1): 3106, 3060, 2912, 1538, 1480, 1414,
1339, 1261, 801, 737, 684. GC-EI-MS, m/z (Abundance %): 301
(13.44), 300 (7.04), 299 (M+, 32.86), 284 (11.80), 266 (30.90), 252
(100.00), 224 (11.71), 212 (14.77), 137 (13.66), 111 (9.79), 88
(10.86), 73 (25.02), 61 (14.10), 45 (14.53), 41 (54.06).
M = 297.80; Yield: 86%; m.p. 100–102 °C; 1H NMR (700 MHz,
DMSO‑d6): 1.76 (quint, 2H, CH2, J = 7.2 Hz), 3.22 (s, 3H, OCH3), 3.31
(t, 2H, CH2, J = 6.0 Hz), 3.92 (t, 2H, CH2, J = 7.4 Hz), 4.13 (s, 2H,
CH2), 7.21–7.40 (m, 4H, ArH), 13.62 (s, 1H, NH); 13C NMR (75 MHz,
DMSO‑d6): 27.72, 30.69, 41.32, 58.28, 69.42, 127.56, 128.09, 129.36,
130.90, 133.66, 137.89, 151.33, 167.23. IR (KBr, cm−1): 3094, 3046,
2920, 2879, 1572, 1503, 1109, 1077, 777, 699, 675. GC-EI-MS, m/z
(Abundance %): 299 (9.28), 298 (6.27), 297 (M+, 25.23), 264 (100),
238 (14.90), 226 (32.64), 125 (40.91), 89 (14.35), 71 (16.56), 45
(35.96), 41 (24.42).
5-(3-Chlorobenzyl)-4-isopropyl-2,4-dihydro-3H-1,2,4-triazole-
3-thione (TPR-20)
5-(3-Chlorobenzyl)-4-[(3-methylthio)propyl]-2,4-dihydro-3H-
1,2,4-triazole-3-thione (TPR-30)
M = 267.78; Yield: 78%; m.p. 186–188 °C; 1H NMR (700 MHz,
DMSO‑d6): 1.41 (d, 6H, 2CH3, J = 7.1 Hz), 4.20 (s, 2H, CH2), 4.70–4.81
(m, 1H, CH), 7.20–7.39 (m, 4H, ArH), 13.56 (s, 1H, NH); 13C NMR
(175 MHz, DMSO‑d6): 19.81, 31.33, 48.57, 127.48, 128.05, 129.15,
130.83, 133.60, 138.42, 151.08, 166.71. IR (KBr, cm−1): 3102, 3043,
2940, 1493, 1289, 1263, 768, 682, 553. GC-EI-MS, m/z (Abundance %):
269 (29.92), 268 (17.88), 267 (M+, 88.35), 225 (100.00), 190 (27.52),
166 (14.11), 152 (19.72), 131 (20.74), 125 (32.35), 89 (18.25), 74
(13.96), 41 (31.06).
M = 313.87; Yield: 78%; m.p. 94–96 °C; 1H NMR (700 MHz,
DMSO‑d6): 1.73 (quint, 2H, CH2, J = 7.4 Hz), 2.00 (s, 3H, SCH3), 2.44
(t, 2H, CH2, J = 7.2 Hz), 3.95 (t, 2H, CH2, J = 7.6 Hz), 4.16 (s, 2H,
CH2), 7.24–7.42 (m, 4H, ArH), 13.63 (s, 1H, NH); 13C NMR (175 MHz,
DMSO‑d6): 14.90, 27.08, 30.49, 30.72, 42.79, 127.59, 128.09, 129.29,
130.98, 133.69, 137.95, 151.24, 167.24. IR (KBr, cm−1): 3098, 3040,
2913, 1568, 1476, 1452, 1425,1265, 753, 680. GC-EI-MS, m/z
(Abundance %): 315 (11.94), 314 (8.30), 313 (M+, 30.06), 298
(13.84), 280 (19.56), 266 (100.00), 226 (21.87), 125 (25.03), 89
(12.90), 73 (19.15), 61 (12.06), 45 (11.96), 41 (33.01).
5-(3-Chlorobenzyl)-4-isobutyl-2,4-dihydro-3H-1,2,4-triazole-3-
thione (TPR-22)
M = 281.80; Yield: 72%; m.p. 108–110 °C; 1H NMR (300 MHz,
DMSO‑d6): 0.84 (d, 6H, CH3, J = 6.6 Hz), 2.17–2.26 (m, 1H, CH), 3.73
(d, 2H, CH2, J = 7.40 Hz), 4.13 (s, 2H, CH2), 7.16–7.47 (m, 4H, ArH),
13.62 (s, 1H, NH); 13C NMR (75 MHz, DMSO‑d6): 19.98, 27.40, 30.78,
50.27, 127.52, 128.17, 129.42, 130.80, 133.57, 138.00, 151.49,
167.72. IR (KBr, cm−1): 3091, 3036, 2957, 2927, 1570, 1494, 1455,
1432, 1273, 1256, 762, 677. GC-EI-MS, m/z (Abundance %): 283
(20.02), 282 (13.06), 281 (M+, 52.32),225 (100.00), 190 (26.80), 166
(16.55), 131 (17.61), 125 (26.29), 89 (13.12), 55 (15.45), 41 (23.13).
2.2. Anticonvulsant activity evaluation in mice maximal electroshock-
induced seizure (MES) model
Anticonvulsant activity of the investigated compounds was de-
termined according to the same procedure as described in (Plech et al.,
2013; Plech et al., 2014). 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). In brief, the
experiments were conducted on adult Swiss albino mice (males)
weighing 20–25 g, which were kept in colony cages with free access to
4-(tert-Butyl)-5-(3-chlorobenzyl)-2,4-dihydro-3H-1,2,4-triazole-
45

B. Kaproń et al.
EuropeanJournalofPharmaceuticalSciences129(2019)42–57
Scheme 1. Synthetic route to the investigated 4-alkyl-1,2,4-triazole derivatives.
food and tap water. After one week of acclimatization to laboratory
conditions, the animals were randomly divided into groups consisting
of 8 mice. The investigated compounds were suspended in a 1% solu-
tion of Tween 80 in distilled water and administered intraperitoneally in
a volume of 5 ml/kg. During preliminary evaluation of the antic-
onvulsant activity, 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 alter-
nating current (0.2 s stimulus duration; 500 V, 50 Hz, fixed current in-
tensity of 25 mA) delivered via ear-clip electrodes by a Type 221 Ro-
dent Shocker generator (Hugo Sachs Elektronik, Freiburg, Germany).
The abolition of a hind-limb tonic extension (HLTE) 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.
reoptimized at the PM6 (Stewart, 2007) level of theory using Gaussian
09 (Frisch et al., 2009). All tested compounds (N = 14) were docked
into the crystal structure of the mammalian Nav1.7 (PDB ID: 5EK0) by
assuming the flexibility of their molecules, i.e. allowing the rotation
around the selected dihedral angles. The docking simulations were
performed in the Molegro Virtual Docker software (MVD v. 2010.4.0.0).
The procedure of docking was performed using the binding cavity
within the sphere of a radius 11 Å, covering the inhibitor molecule
originally co-crystallized with Nav1.7 (GX-936). The GX-936 molecule
which was co-crystalized with Nav1.7 was used as a template for
docking tested inhibitors and, additionally, for the initial validation of
the docking procedure. The obtained root mean square deviation
(RMSD) parameter calculated for position of all atoms of GX-936 co-
crystalized with Nav1.7 (PDB ID: 5EK0) and that docked into Nav1.7
was equal to 0.84 Å, which proved that the docking procedure is correct
(Fig. S2). Docking simulations were performed according to the meth-
odology described in our previous studies (Plazinska et al., 2013). The
MolDock SE search algorithm was applied during docking procedure.
The estimation of the ligand-protein interactions was described by the
MVD-implemented scoring function (MolDock Score [kJ/mol]). The
predicted favorable positions of the ligands docked into Nav1.7 are
characterized by the lowest MolDock Score values that corresponds to
the high values of the ligand-protein binding energy.
2.3. Motor coordination impairment determination in a rotarod test in mice
The acute neurotoxic effects of the investigated compounds were
measured by the rotarod test in mice according to (Luszczki et al.,
2005). 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).
2.7. Permeability studies
Gastrointestinal permeability of TP-315, TP-427, TPR-4 and TPR-22
compounds was investigated using a PAMPA method (parallel artificial
membrane permeability assay) in regards to GIT (gastrointestinal tract)
model. As reference values of permeability were used these which were
determined for phenytoin and valproic acid. The PAMPA system con-
sisted of a 96-well microfilter plate and a 96-well filter plate and 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 a 20%
(w/v) dodecane solution of a lecithin mixture (Pion, Inc.). Solutions of
all compounds (0.10 mmol/L) in water were prepared in a different 96-
well filter plate and added to the donor compartments. The donor so-
lution was adjusted to pH 1.2 and 6.8 (NaOH-treated universal buffer).
The plates were put together and incubated at 37 °C for 60 min in a
humidity-saturated atmosphere. The concentrations of TP-315, TP-427,
TPR-4 and TPR-22 compounds as well as phenytoin and valproic acid
were determined using HPLC-DAD method in the donor and acceptor
compartments after 15 min and 60 min of incubation.
2.4. Radioligand binding assay for Na+ channel - site 2 using [3H]
batrachotoxin
Radioligand binding assay was performed according to the method
described in details in our recent paper (Kaproń et al., 2017).
2.5. Patch-clamp recordings of GABAA-mediated chloride currents
Patch clamp recordings were performed on QPatch16X automatic
patch clamp platform (Sophion Biosciences) using HEK-293 cells with
stable expression of α1β2γ2 subunits of the human GABAA receptors.
The experimental procedure was described in details in our previous
paper (Kaproń et al., 2017).
2.6. Docking methodology
The molecular models of tested inhibitors were prepared by using
Avogadro 1.1.1 (Hanwell et al., 2012) and optimized using the UFF
force field (Rappe et al., 1992). Subsequently, their geometries were
The Ultimate 3000 LC system (DionexThermoline Fisher Scientific,
Germany) equipped with a high pressure pump (UltiMate 3000), an
autosampler (UltiMate 3000) and DAD detector (UltiMate 3000) was
46

B. Kaproń et al.
EuropeanJournalofPharmaceuticalSciences129(2019)42–57
Table 1
Anticonvulsant activity (MES test), neurotoxicity (rotarod test) and affinity towards batrachotoxin-binding site on sodium channels of the investigated 4-alkyl-5-
substituted-1,2,4-triazole-3-thione derivatives.
Compound
Anticonvulsant activity
Neurotoxicity
Na+ channel (site 2) affinity
Pretreatment time
(min)
ED50 (mg/kg)
SEM TD50 (mg/kg)
SEM PI (TD50
/
% inhibition
(n = 2)
SEM at 100 μM IC50 (μM)
SEM
ED50
)
TPR-2
15
30
60
120
320.0
310.3
> 500
> 500
38.9
20.2
422.7
406.7
389.3
448.9
22.8
19.7
19.1
22.3
1.3
1.3
< 1
< 1
61
7
~ 100
TPR-4
TPR-6
15
30
60
120
286.9
221.7
279.0
395.1
26.6
18.4
24.3
25.2
120.0
135.6
187.1
297.5
20.3
15.2
18.8
21.8
< 1
< 1
< 1
< 1
71
55
6
6
> 50
~100
15
30
60
120
391.5
295.1
440.4
> 500
43.0
27.3
66.5
462.9
347.6
288.6
246.7
20.0
21.4
19.0
21.6
1.2
1.2
< 1
< 1
TPR-14
TPR-16
15
30
60
120
286.9
208.9
274.8
328.2
26.6
27.3
20.7
33.2
181.7
232.5
272.1
309.3
18.0
18.3
22.1
22.3
< 1
1.1
< 1
< 1
44
68
2
8
~100
> 50
15
30
60
120
252.3
201.0
297.2
380.6
24.5
20.3
21.4
24.1
135.6
221.7
306.0
320.1
15.2
21.8
19.8
23.6
< 1
1.1
1.0
< 1
TPR-22
TPR-28
15
30
60
120
130.4
130.4
159.9
195.7
17.6
17.6
21.9
21.5
306.0
314.5
325.9
329.9
19.8
22.0
23.1
24.2
2.3
2.4
2.0
1.7
89
93
7
6
18.9
25.9
1.2
3.9
15
30
60
120
132.4
144.2
149.0
210.7
27.5
15.5
16.3
31.7
320.1
314.5
312.1
336.7
23.6
22.0
20.4
23.5
2.4
2.2
2.1
1.6
TPR-30
TP-315⁎
15
30
60
120
320.0
322.9
347.6
397.9
9.6
7.9
21.4
21.4
471.7
462.9
446.1
477.4
23.5
20.0
22.1
22.8
1.5
1.4
1.3
1.2
63
92
7
4
> 50
15
30
60
120
47.6
68.3
98.1
159.7
3.8
10.3
16.4
462.9
462.9
456.9
448.1
20.0
20.0
19.7
21.7
9.7
6.8
4.7
2.8
6.21
0.80
21.7
TP-423⁎⁎
15
30
60
120
44.5
43.2
61.5
102.3
8.0
10.3
3.2
325.9
310.2
297.5
324.9
26.6
26.2
21.8
26.0
7.3
7.2
4.8
3.2
99
4
15.8
0.1
10.1
(continued on next page)
47

B. Kaproń et al.
EuropeanJournalofPharmaceuticalSciences129(2019)42–57
Table 1 (continued)
Compound
Anticonvulsant activity
Neurotoxicity
Na+ channel (site 2) affinity
Pretreatment time
(min)
ED50 (mg/kg)
SEM TD50 (mg/kg)
SEM PI (TD50
/
% inhibition
(n = 2)
SEM at 100 μM IC50 (μM)
SEM
ED50
)
TP-425**
TP-427⁎⁎
15
30
60
120
48.5
62.5
68.4
84.2
5.9
8.2
10.0
9.6
370.8
322.3
329.0
347.6
21.5
22.4
18.9
21.7
7.6
5.2
4.8
4.1
100
3
7.49
0.53
15
30
60
120
72.1
74.5
83.6
97.9
7.0
8.1
3.8
10.9
> 1000
> 1000
540.7
> 13.9
> 13.4
6.5
89
4
6.17
1.43
0.35
20.9
21.4
548.5
5.6
TP-429⁎⁎
15
30
60
120
49.7
76.3
70.6
89.6
4.8
11.7
8.1
506.7
640.2
518.8
442.3
42.5
47.1
40.3
43.0
10.2
8.4
7.3
103
2
7.67
4.3
4.9
Valproate⁎⁎
15
30
60
120
189.0
216.9
218.4
246.6
17.3
9.4
18.9
21.4
363.3
372.9
417.3
512.3
14.2
16.9
9.5
1.9
1.7
1.9
2.1
nd.
nd.
30.2
Veratridine
Lidocaine
100
26
2
2
16.7
390
1.8
126
Carbamazepine
17.4 (Kamiński et al., 2015)
131 (Willow and
Catterall, 1982)
Results of anticonvulsant activity are presented as median effective doses (ED50 in mg/kg
SEM) of the examined compounds, protecting 50% of animals tested in
the MES test. Neurotoxicity of the compounds is presented as median toxic doses (TD50) that impair motor coordination in 50% of the mice challenged with the
⁎
rotarod test. Both ED50 and TD50 were calculated using Litchfield and Wilcoxon method (Litchfield and Wilcoxon, 1949); nd. – not determined; - values of ED50
,
TD50, and PI (i.e., protective index) for TP-315 were obtained from our previously published paper (Plech et al., 2013), IC50 for TP-315 was obtained from (Kaproń
et al., 2017); ⁎⁎ - values of ED50, TD50, and PI (i.e., protective index) for TP-423, TP-425, TP-427, TP-429, and valproate were obtained from our previously published
paper (Plech et al., 2014).
used. For data processing and acquisition, Chromeleon software version
7.0 from Dionex Thermoline Fisher Scientific (US) was used. The se-
paration was achieved on a Kinetex (4.6 × 150 mm, 5 μm) column
using a mobile phase composed of acetonitrile – 0.1% formic acid
(60:40 V/V) at a flow rate of 1.0 ml/min−. The injection volume and
temperature of column were 5.0 μL and 30 °C, respectively. The wave-
length of detection was controlled at 220 nm for phenytoin and valproic
acid while the wavelength of detection of TP-315, TP-427, TPR-4 and
TPR-22 compounds was 254 nm. The HPLC-DAD method was validated
in regards to all compounds determination.
Experiments were performed in accordance with the international
standard ISO-10993-5:2009. HepG2 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 strep-
tomycin (PenStrep, Sigma Aldrich). Cells were incubated at 37 °C in a
humidified atmosphere of 5% CO2. Stock solutions of the tested com-
pounds were prepared by dissolving in a sterile dimethyl sulfoxide
(DMSO) to the concentration of 100 mg/ml. The cells were seeded into
96-well sterile plates (Nunc) at a cell density of 1 × 105 cells/ml. After
24 h of incubation the medium was removed from each well and then
cells were incubated for the next 24 h with different concentrations of
the tested compounds in a respective medium containing 2% FBS.
Control cells were cultured only with medium containing 2% addition
of FBS. Toxicity of the compounds was evaluated using MTT assay,
which is based on the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) into dark-blue formazan crystals.
Briefly, after 24 h incubation of cells with varying concentrations of
tested compounds all culture media were removed from the plates. Cells
were washed with PBS, and then 100 μL of medium containing 10%
MTT solution (5 mg/ml) was added to each well. Cells were incubated
for the next 4 h at 37 °C in an atmosphere of 5% CO2. Next, 100 μL (per
well) of 10% SDS buffer solution was added to dissolve formazan
crystals and after an overnight incubation the absorbance was mea-
sured at 570 nm using a microplate reader (Epoch, BioTek Instruments).
Experiments were repeated twice, and the measurements in each ex-
periment were run in triplicate.
The apparent permeability coefficient (Papp) was calculated using
the following equation:
C
A
−ln 1 −
(
)
C
equilibrium
P
app
=
1
V
D
1
A
S ×
+
× t
(
)
V
where VD – donor volume, VA – acceptor volume, Cequilibrium – equili-
brium concentration Cequilibrium
tion, CA – acceptor concentration, S – membrane area, t – incubation
time (in seconds). Compounds with Papp < 1 × 10−6 cm/s are classi-
fied as ones with low permeability and those with
Papp > 1 × 10−6 cm/s as ones with high permeability (Yee, 1997).
C
D
× V + C × V
D
V
D
A
=
A , CD – donor concentra-
+ V
A
2.8. Cytotoxicity evaluation
Cellular toxicity of the compounds was analyzed using MTT assay
on murine (NMu3Li cell line) and human (HepG2 cell line) liver cells.
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manufacturer's protocol. In brief, HepG2 cells (1 × 105 cells/ml in ice-
cold PBS) previously exposed (for 24 h) to the investigated compounds
were combined with liquefied low melting agarose at 37 °C and trans-
fered onto the Oxiselect comet slides. After storing for 15 min at 4 °C,
the slides were immersed in a pre-chilled Lysis Buffer (for 45 min at
4 °C, in the dark) and Alkaline Solution (for 30 min at 4 °C, in the dark).
In the next step, the Alkaline Solution was replaced with pre-chilled
TBE electrophoresis solution. After immersing for 5 min, the slides were
transferred into the horizontal electrophoresis chamber and covered
with TBE electrophoresis buffer. The electrophoresis was run for 15 min
at 1 V/cm. Finally, DNA was stained with Vista Green DNA Dye for
15 min at room temparature and observed under fluorescence micro-
scope (Olympus BX63). The images of cells were captured using XM10
digital camera (Olympus). The DNA damage was measured quantita-
tively using OpenComet v1.3.1 software (CometBio).
3. Results and discussion
3.1. Chemistry
The investigated compounds were synthesized through a simple,
two-step reaction of aromatic acid hydrazides with alkyl iso-
thiocyanates, followed by subsequent alkaline cyclization of the ob-
tained thiosemicarbazide derivatives into respective 4-alkyl-5-(3-
chlorophenyl/3-chlorobenzyl)-1,2,4-triazole-3-thiones (Scheme 1).
This method allowed for relatively fast and effective synthesis of alkyl
derivatives of 1,2,4-triazole-3-thiones (Plech et al., 2013; Plech et al.,
2014). The structures of the final compounds were confirmed based on
1H NMR, 13C NMR, IR and MS spectroscopic data. Other compounds
used in the experiments described in this article were synthesized and
described in our earlier papers (Plech et al., 2013; Plech et al., 2014).
3.2. Pharmacology
The designed compounds contained 3-chlorophenyl or 3-chlor-
obenzyl fragments at position 5 of the 1,2,4-triazole-3-thione core and
branched alkyl substituents or non-branched chains enriched with
heteroatoms at position 4. Our earlier studies showed that 3-chlor-
ophenyl/3-chlorobenzyl substituents increase the anticonvulsant ac-
tivity of 4-alkyl-5-substituted-1,2,4-triazole-3-thiones (Plech et al.,
2013; Plech et al., 2014). The introduction of a methylene linker (as a
part of 3-chlorobenzyl group) between the 1,2,4-triazole ring and 3-
chlorophenyl moiety resulted in the highest increase of potency and
improvement of the time-course profile of the anticonvulsant activity
(Plech et al., 2014). All of the synthesized compounds were evaluated
for their protective activity in a mouse maximal electroshock-induced
seizure (MES) model of epilepsy. During preliminary examinations, the
compounds were administered intraperitoneally (i.p.) in a fixed dose of
300 mg/kg at four pretreatment times (i.e., 15, 30, 60, 120 min). 1,2,4-
Triazole-3-thione derivatives that turned out to protect mice from MES-
Fig. 1. Effect of introduction of heteroatoms (O, S) into the alkyl chain of 5-(3-
chlorophenyl)-4-pentyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (upper graph)
or 5-(3-chlorobenzyl)-4-pentyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (lower
graph). ED50 values for all the investigated compounds were determined in the
maximal electroshock-induced seizure (MES) test. Results of anticonvulsant
activity of the respective pentyl derivatives were taken from our earlier articles
(Plech et al., 2013; Plech et al., 2014).
2.9. Genotoxicity evaluation
Measurement of DNA damage in HepG2 cells was done using
OxiSelect Comet Assay Kit (Cell Biolabs, Inc.) according to the
Fig. 2. Representative whole-cell patch-clamp recordings for agonist-mode experiments at GABAA receptor.
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Fig. 3. Allosteric potentiation of GABA-mediated chloride currents by the increasing concentrations (0.001–100 μM) of the selected 4-alkyl-1,2,4-triazole derivatives
in HEK-293 cells with stable expression of α1β2γ2 subunits of the human GABAA receptors.
induced seizures (i.e., TPR-2, 4, 6, 14, 16, 22, 28, 30) were subjected to
the dose-response analyses to determine their median effective doses
(ED50). These compounds were also assessed for motor coordination
impairment in rotarod test (Table 1).
properties, both in terms of anticonvulsant potency and neurotoxicity
level, were observed in case of respective 3-chlorobenzyl derivatives
(TPR-22, 28, 30). Such results are in line with our previous results in
other 4-alkyl-5-substituted-1,2,4-triazole-3-thione derivatives con-
taining methylene linker (Plech et al., 2014). Respective isobutyl (TPR-
22) and 3-methoxypropyl (TPR-28) derivatives protected mice from
electrically-induced convulsions greater than the referenced antic-
It is evident from the obtained data that the introduction of bran-
ched alkyl chains into the triazole-based compounds highly decreases
their anticonvulsant activity in the MES test. Additionally, these com-
pounds were characterized by higher neurotoxicity than respective
unbranched derivatives, which were described in our earlier papers
(Plech et al., 2013; Plech et al., 2014). All active 4-alkyl-5-(3-chlor-
ophenyl)-1,2,4-triazole-3-thiones (i.e., TPR-2, 4, 6, 14, 16) impaired
motor coordination in mice at doses lower than their ED50 values,
suggesting that such compounds are of no interest as possible antic-
onvulsant drug candidates. Improvement of pharmacological
onvulsant agent
– magnesium valproate. Additionally, the antic-
onvulsant activity of these compounds was relatively stable in all in-
vestigated pretreatment times. The introduction of heteroatoms into the
alkyl chains in the position 4 of the 1,2,4-triazole scaffold was intended
to favorably affect the interaction of the molecules with the possible
molecular target, e.g. through the formation of hydrogen bonds invol-
ving free-electron pairs of oxygen and sulfur atoms. The presence of
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Fig. 4. (Right) The crystal structure of the Nav1.7 VSD4-NavAb channel in complex either with the antagonist molecule, GX-936 (ball-and-stick, colored by element)
co-crystalized with the protein (PDB ID: 5EK0) or with TP-315 (ball, colored in magenta). (Left) Detailed view of the Nav1.7 VSD4 binding cavity with GX-936 (stick,
colored by element) and TP-315 (ball-and-stick, colored by element). (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
Fig. 5. The extracellular side view highlighting key interactions with TP-315 (A) and TP-427 (B). The Nav1.7 VSD4 receptor binding site is shown with selected
sidechains rendered as sticks (colored by element). The green arrows represent HBs created between the ligands and the protein. The two different positions of the
docked ligand, characterized by the lowest energy values, are shown. The sulfur atom of the ligand (colored in yellow) can create HBs with Arg1608 (S4) and Ser1578
(backbone) (A) or Arg1608 (S4) and Arg1602 (backbone) (B). Arg1608 (S4) and Ser1578 (S3) (A) of TP-315 exhibit the π-π interactions with the phenyl moiety of
Tyr1537 (S2) (A). The chlorine atom of TP-427 can create HB with the carboxyl moiety of Asp1586 (S3) (B). (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
Fig. 6. The TPR-16-Nav (A), TPR-30-Nav (B) and TPR-24-Nav (C) complexes. The ligand molecules are presented as sticks. The carbon atoms are colored in orange,
the sulfur in yellow, the chlorine in green. The residues of Nav that create HBs with the docked ligands, are shown as sticks (colored by element). (For interpretation
of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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B. Kaproń et al.
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Table 2
substituents. Additionally, we extended the panel of the investigated
compounds to 5-(3-chlorobenzyl)-4-hexyl-1,2,4-triazole-3-thione (TP-
427), which was found the most promising compound from the group of
1,2,4-triazole-3-thione alkyl derivatives (Plech et al., 2014). As it is
shown in Fig. 2, none of the three tested compounds evoked GABAA-
mediated chloride currents. Their anticonvulsant activity was also not
associated with allosteric potentiation of GABAA receptors response to
endogenous γ-aminobutyric acid (GABA) (Fig. 3). Only compound TPR-
22 in a concentration of 100 μM, used in combination with GABA
(10 μM), caused an increase of current peak maximum when compared
to recordings obtained for GABA administered alone. Therefore, it
should be concluded that TPR-22 works as a very weak positive allos-
teric modulator of GABAA receptors. Since such effect was observed
only for the highest dose of 1,2,4-triazole-based compound, it is un-
likely that it would be responsible for its anticonvulsant activity.
The lowest MolDock Score values obtained during docking of
the tested compounds to the Nav1.7 VSD4 binding site (PDB:
5EK0).
Compound
MolDock score [kJ/mol]
TP-315
TP-423
TP-425
TP-427
TPR-2
−132.05
−118.66
−126.7
−137.16
−101.54
−97.39
TPR-4
TPR-6
−83.93
TPR-14
TPR-16
TPR-20
TPR-22
TPR-24
TPR-28
TPR-30
GX-936
veratridine
−102.55
−110.32
−98.41
−117.38
−101.29
−108.92
−114.06
−136.73
−97.98
3.3.2. Affinity of 4-alkyl-5-substituted-1,2,4-triazole-3-thiones towards
batrachotoxin-binding site on voltage-gated sodium channels (VGSCs)
The anticonvulsant activity of compounds protecting animals from
MES-induced seizures is often connected with their influence on vol-
tage-gated sodium channels. These channels are molecular targets for
many antiepileptic drugs, local anesthetics, antiarrhythmics, and nat-
ural neurotoxins (Stevens et al., 2011). The affinity of the synthesized
compounds towards VGSCs was determined by measuring the dis-
placement of [3H]-batrachotoxin from its binding site (Fig. S1; Table 1).
The strength of the binding of the investigated 1,2,4-triazole-3-thione
derivatives to VGSCs was correlated with their anticonvulsant activity
in the MES-induced seizure test (Table 1). In order for a comprehensive
assessment of the role of the interactions of 1,2,4-triazole-based com-
pounds with VGSCs in their anticonvulsant effect to be possible, we also
performed similar radioligand binding experiments with our previously
obtained 4-alkyl-1,2,4-triazole-3-thiones, characterized by improved
anticonvulsant potency. The determined IC50 values (Table 1) show
that unbranched alkyl groups containing from 4 to 7 carbon atoms
constitute the most preferred alkyl substituents at position 4 of the
1,2,4-triazole-3-thione core. Their strong anticonvulsant potency was in
accordance with low IC50 values. The ability to displace [3H]-ba-
trachotoxin from its binding site on VGSCs increased when the carbon
chain length increased from butyl to hexyl. Further extension of the
alkyl chain length, however, did not result in more favorable affinity of
heteroatoms should also influence the hydrophilic-lipophilic properties
of 4-alkyl-1,2,4-triazole derivatives due to the facilitated solvation of
heteroalkyl moiety. Contrary to our expectations, none of the com-
pounds containing heteroatoms in alkyl substituents showed better
anticonvulsant activity than respective derivatives with hydrocarbon
moiety (Fig. 1). Considering all of the observations outlined above, it
may be concluded that there are no benefits resulting from the in-
troduction of branched or heteroatom-enriched alkyl substituents into
the 1,2,4-triazole-3-thione core. Respective compounds containing un-
branched alkyl chains, described previously (Plech et al., 2013; Plech
et al., 2014), were characterized by much stronger anticonvulsant ac-
tivity and better tolerability profile.
3.3. Mechanistic studies
3.3.1. Effect of 4-alkyl-5-substituted-1,2,4-triazole-3-thiones on GABAA-
mediated chloride currents
Increase of the gabaergic neurotransmission is one of the most
common mechanism of action of antiepileptic drugs. In our recent
publication, we showed that one of the previously synthesized alkyl
derivatives of 1,2,4-triazole-3-thione (namely TP-315) did not evoke
GABAA-mediated chloride current during electrophysiological experi-
ments (Kaproń et al., 2017). Similar studies have also been performed
for currently obtained compounds TPR-22 and TPR-28, which had the
strongest anticonvulsant activity among the 1,2,4-triazole-3-thione
derivatives containing branched or heteroatom-enriched alkyl
the respective heptyl derivative (IC50 = 7.67
0.35 μM). Thus, it
must be stated that unbranched hexyl group interacts with the VGSCs in
the most optimal manner. The affinity of ligands towards the molecular
target does not have to be strictly correlated with the strength of re-
spective biological effect since the observed activity is also determined
by other parameters, e.g. the ability of the compound to permeate
through the biological barriers, deactivation by the enzymes, etc. We
Fig. 7. The veratridine-Nav complex (A), veratridine-TPR-30-Nav complex (B). The veratridine is colored in dark blue, TPR-30 is presented as balls (the carbon atoms
are colored in orange, nitrogen in dark blue and sulfur in yellow). (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
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Fig. 8. Cytotoxic effect of the investigated 1,2,4-triazole derivatives against HepG2 cells measured in MTT test after 24 h incubation. Viability of liver cells are
represented as mean standard error of the mean (SEM).
have observed it when comparing the activity of TP-423 (n-butyl de-
rivative) and TPR-22 (isobutyl derivative) compounds. Although these
derivatives had similar IC50 values (i.e., 15.8 μM vs. 18.9 μM), they
differed significantly in the anticonvulsant effects in MES-induced sei-
zure model of epilepsy.
residues 1608 and 1602, belonging to the S4 transmembrane segment
of the protein. Thus, the Nav inhibition may involve the interactions
between the negatively charged sulfur atom located at the 1,2,4-triazole
moiety of the tested compounds and positively charged S4 arginines
(Arg1608, Arg1602). Moreover, the nitrogen atoms of the 1,2,4-triazole
moiety can create hydrogen bonds (HB) with Ser1578 located at S3,
Tyr1537 (S2) (Fig. 5) as well as with Arg1602 (S4) (Fig. 6). The 1,2,4-
triazole-3-thione moiety of the ligands can exhibit two different or-
ientations, i.e. the sulfur atom can interact (HB) either with: (i) both
Arg1608 (S4) and Ser1578 (S3) (Fig. 5A) or (ii) both Arg1602 and
Arg1608 (S4) (Fig. 5B). These two different positions of the triazole
moiety result in the two different orientations of both the alkyl chain
and phenyl group of the ligands, where: (i) the phenyl moiety exhibits
the π-π interactions with the phenyl moiety of Tyr1537 (S2) (Fig. 6A),
(ii) the chlorine atom creates HB with Glu1589 (S3), Tyr1537 (S2) or
Asp1586 (S3) (Figs. 5B, 6B). The incorporation of the methylene linker
between the 1,2,4-triazole-3-thione ring and phenyl groups of the tested
compounds results in: (i) lower MolDock Score values (Table 2) and (ii)
larger number of contacts with Nav in comparison to the compounds
without the mentioned linker (Fig. 5).
Additionally, we observed that the reduction of the n-alkyl chain
length causes the increase of the obtained MolDock Score values which
corresponds to the reduced inhibition towards Nav. This is in ac-
cordance with the experimental data for compounds TP-423, TP-425
and TP-427, which suggests the decrease in the IC50 values correlated
with the substitution of the ligand n-alkyl chain by the methyl group.
According to the docking simulations, the ligand with the longest n-
alkyl chain (TP-427), exhibits the simultaneous interactions with the
residues located at S2 (Tyr1537), S3 (Asp1586) and S4 (Arg1602,
Arg1608) (Fig. 5B). The compound with the shortest n-alkyl chain does
not interact with Tyr1537 (S2). This suggests that the simultaneous
interactions with the residues located at S2, S3 and S4 are crucial for
3.3.3. Molecular modeling of 1,2,4-triazole-based ligands
interactions
– VGSC
Mammalian NaV channels consist of four homologous domains (I-
IV). Each of them contains six transmembrane segments (S1-S6) and a
membrane-reentrant pore loop between the S5 and S6 segments.
Segments S5 and S6 and the membrane-reentrant pore loop form the
pore. They serve the principal role in the ion conduction, comprising
the gate and pore of the channel, while S1-S4 serve as the voltage-
sensing domain (VSD1-4) (Catterall, 2012; Marban et al., 1998;
Payandeh et al., 2011). The voltage-sensing S4 helix contains posi-
tively-charged arginines (Arg1599, Arg1602, Arg1605, Arg1608,
Arg1611) in repeated motifs which act as gating charges in the voltage-
sensing mechanism (Guy and Seetharamulu, 1986; Catterall, 1986a;
Catterall, 1986b). Recently resolved Nav crystal structure indicates the
significant role of S4 in binding of the small molecule inhibitors to Nav
(Ahuja et al., 2015). In order to clarify the character of interactions
between the investigated class of compounds and VGSCs,
a re-
presentative group of 4-alkyl-1,2,4-triazole-3-thiones (TP-315, TP-423,
TP-425, TP-427, TPR-2, TPR-4, TPR-6, TPR-14, TPR-16, TPR-20, TPR-
22, TPR-24, TPR-28, TPR-30) were subjected to computational studies.
Docking simulations revealed that all the tested compounds bind to the
activated conformer of voltage-sensor domain IV (VSD4) similarly to
the selective, aryl sulfonamide antagonist (GX-936), co-crystallized
with mammalian Nav1.7 (Fig. 4).
The 1,2,4-triazole core and sulfur atom located at the triazole
moiety of the ligands were always located in the vicinity of the arginine
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Fig. 9. DNA fragmentation in HepG2 cells exposed to 4-alkyl-5-substituted-1,2,4-triazole-3-thione derivatives detected by a single cell gel electrophoresis (comet
assay).
the Nav inhibition. Additionally, the presence of branched alkyl sub-
stituents (e.g., isopropyl, isobutyl, tert-butyl) leads to ligand displace-
ment in the VSD4 binding site and moving the triazole moiety closer to
S2; this is associated with the creation of HB with Tyr1537 (S2). The
additional HBs between the chlorine atom of the ligand phenyl moiety
and arginines of S4 (Arg1608, Arg1602) were observed (Fig. 6C). For
TPR-2, TPR-4, TPR-6, TPR-20 and TPR-24 HB with Ser1578 was no
longer observed (Fig. 6C). Moreover, the addition of the sulfur atom to
the alkyl chain in TPR-16 and TPR-30 caused the loss of interactions
with Ser1578 (S3) and Tyr1537 (S2) (Fig. 6A, B). HBs with arginines
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3.4. Preliminary ADME-Tox screening of selected 4-alkyl-5-substituted-
1,2,4-triazole-3-thiones
3.4.1. Cytotoxicity against human and murine hepatic cells
The most common route of delivery of antiepileptic drugs into the
human body is the oral route. After being absorbed from the gastro-
intestinal tract, the drug goes through the hepatic portal system into the
liver, where it reaches high concentrations. Therefore, liver cells are
highly exposed to cytotoxic substances, and therefore these cells are a
basic model for toxicological testing of new drug candidates. Taking
this into account, we evaluated cytotoxicity of selected 1,2,4-triazole
derivatives on murine (NMu3Li cell line) and human (HepG2 cell line)
liver cells. Experiments were performed in accordance with the inter-
national standard IS0-10993-5:2009. According to the practice con-
firmed in scientific literature (Adan et al., 2016; Dzitko et al., 2014), the
concentration of the investigated compound that reduced viability of
cells by 30% was assumed as a toxicity threshold. NMu3Li cells turned
out to be less sensitive to the compounds tested than human HepG2
cells. As can be seen from Fig. 8, compounds containing branched or
heteroatom-enriched alkyl chains were less toxic than respective deri-
vative with n-hexyl substituent (TP-427). Exceeding of the toxicity
threshold after 24 h incubation of NMu3Li cells with TP-427, TPR-22,
and TPR-28 was observed in concentrations higher than 32 μg/ml,
62.5 μg/ml, and 125 μg/ml, respectively. Pharmacokinetic analysis of
compound TP-427 showed that its maximal concentration in mice
Fig. 10. Quantitative representation of the DNA damage in HepG2 cells ex-
posed to the investigated compounds (10 μg/ml). Data are shown as
mean
SEM. Statistical significance was calculated using ANOVA analysis
followed by post-hoc Dunnett test (⁎⁎p < 0.05, ⁎⁎⁎⁎p < 0.0001 when compared
to the untreated cells).
Table 3
plasma (Cmax) was 4.232
0.213 μg/ml (unpublished results, data
Apparent coefficients of permeability of phenytoin sodium, valproic acid, TP-
315, TP-427, TPR-4 and TPR-22 determined using a parallel artificial mem-
brane permeability assay (PAMPA) in gastrointestinal tract model.
under preparation for publication). Since other examined compounds
(TPR-22, TPR-28) were less toxic than TP-427, it may be concluded that
these 1,2,4-triazole derivatives remain non-toxic for hepatic cells in
concentrations necessary to maintain anticonvulsant effect in-vivo.
(a)
Papp × 10−6 [cm/ s]
Acceptor fluids (pH 1.2)
15 min
60 min
3.4.2. Genotoxicity evaluation using a single cell gel electrophoresis (SCGE)
Single cell gel electrophoresis, usually identified as comet assay, is a
simple and sensitive technique that enables the detection of DNA da-
mage in individual cells. This method is often used to evaluate poten-
tially genotoxic drugs or drug candidates. To check the ability of 4-
alkyl-5-substituted-1,2,4-triazole-3-thiones to create the lesions in ge-
netic material of HepG2 cells, compounds TP-427, TPR-22, and TPR-28
were selected for SCGE assay. Additionally, etoposide (human DNA
topo II inhibitor/positive control) and sodium valproate were analyzed
as reference drugs. Degradation of DNA was assessed both qualitatively
(Fig. 9) and quantitatively (Fig. 10), as %DNA in the comet tail. As
presented in Fig. 10, none of the investigated 1,2,4-triazole derivatives
exhibited statistically significant genotoxic effect in HepG2 cells.
However, such effect was observed after a 24 h exposition of the cells to
antiepileptic drug – sodium valproate. The median %DNA in the comet
tail of HepG2 cells incubated with sodium valproate was 6.06% (vs.
3.74% for untreated control cells). This fact may relate to scientifically
proven mutagenic activity of valproic acid and its derivatives, which
are manifested in higher risk of developmental disorders in children of
women who have been exposed to valproate during pregnancy (Tomson
et al., 2015).
Mean values
SD
Mean values
SD
Phenytoin Na
Valproate
TP-315
TP-427
TPR-4
438.99
151.43
91.60
624.49
182.049
184.81
59.33
9.89
7.35
32.25
5.98
8.56
1210.78
765.92
125.50
711.60
1028.21
1025.93
69.52
54.10
2.51
96.60
48.72
88.06
TPR-22
(b)
P
app × 10−6 [cm/ s]
Acceptor fluids (pH 6.8)
15 min
60 min
Mean values
SD
Mean values
SD
Phenytoin Na
Valproate
TP-315
TP-427
TPR-4
562.73
255.10
41.01
229.11
337.30
1110.44
43.06
15.55
2.41
13.95
16.74
59.33
887.83
905.37
44.88
254.96
733.04
1416.04
72.38
23.75
1.74
2.39
23.43
54.51
TPR-22
1608 and 1602 appear to be essential for ligand binding to Nav.
However, in spite of preserving these HBs, the loss of the interactions
with Ser1578, Asp1586 and Tyr1537 leads to the increase of the IC50
values. In summary, all of the tested compounds are docked deeper in
the Nav1.7 cavity in comparison to the agonist, veratridine (Fig. 7). In
contrast to veratridine, the tested compounds interact with Arg1608
and Arg1602 similarly to the inhibitor co-crystalized with mammalian
Nav1.7 (PDB ID: 5EK0). Thus, we suppose that the interactions with
Arg1608 and Arg1602 are the most important in binding of inhibitors
and determine the Nav inhibition. Moreover, the activity of the tested
inhibitors can result from the simultaneously created interactions with
the residues of S2 (Tyr1537), S3 (Ser1578, Asp1586) and S4 (Arg1608
and Arg1602).
3.4.3. In-vitro gastrointestinal (GIT) permeability studies
The treatment of epilepsy is mainly based on oral medication, so it is
important to evaluate the ability of antiepileptic drug candidates to be
absorbed from the gastrointestinal tract at early stages of drug devel-
opment. The possibility of fast and reliable prediction of passive dif-
fusion across the membranes is given by a parallel artificial membrane
permeability assay (PAMPA). Permeation studies of the respective hexyl
(TP-315, TP-427) and isobutyl (TPR-4, TPR-22) derivatives were con-
ducted at pH 1.2 and 6.8, i.e. in the conditions reflecting those of
human gastrointestinal environment. Concentrations of the investigated
compounds on both sides of the artificial membrane were determined
using HPLC-DAD method after 15 and 60 min of incubation. The ap-
parent permeability coefficients (Papp) were also calculated for classical
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EuropeanJournalofPharmaceuticalSciences129(2019)42–57
Fig. 11. Apparent coefficients of permeability of phenytoin sodium, valproic acid, TP-315, TP-427, TPR-4 and TPR-22 determined using a parallel artificial
membrane permeability assay (PAMPA) in gastrointestinal tract model.
antiepileptics – sodium valproate and phenytoin sodium, which were
used as reference drugs. According to literature (Yee, 1997), com-
pounds with Papp < 1 × 10−6 cm/s are classified as ones with low
permeability and those with Papp > 1 × 10−6 cm/s – as ones with high
permeability. The obtained results (Table 3, Fig. 11) allow us to classify
the investigated 1,2,4-triazole-3-thione derivatives as compounds
characterized by a high ability to penetrate across the biological
membranes. Regardless of the pH level of acceptor fluid, the least
passive diffusion was observed for compound TP-315, while the in-
creased permeability through the artificial membrane was noticed for
the derivatives with branched alkyl substituents. Moreover, in the case
of these two isobutyl derivatives (TPR-4, TPR-22), a significant increase
in permeability was observed over longer incubation time (60 min). In
contrast, the elongation of incubation time for 1,2,4-triazole-3-thiones
containing unbranched substituents did not significantly affect the ki-
netic of passive diffusion process. These compounds were able to reach
maximal concentrations in an acceptor fluid at a faster rate, however,
their final concentrations were lower than in the case of respective
branched alkyl derivatives. Therefore, it can be concluded that for the
latter group of compounds, the passive diffusion process takes longer
but is more effective. The differences in permeability of the investigated
1,2,4-triazole-3-thione-based compounds observed in PAMPA assay are
also reflected in their anticonvulsant activity profile measured in the
MES model. While unbranched alkyl derivatives reached the peak of
anticonvulsant activity 15 min after i.p. administration, a prolonged
time to peak activity was observed in the case of compounds with
branched substituents at position 4 of 1,2,4-triazole-3-thione core.
Tyr1537 (S2), Ser1578 (S3), Asp1586 (S3), Arg1608 and Arg1602 (S4).
However, the interactions with Arg1608 and Arg1602 are the most
important for binding of the inhibitors. During preliminary ADME-Tox
studies, the investigated 1,2,4-triazole derivatives remained non-toxic
for hepatic cells in concentrations necessary to produce anticonvulsant
effect in-vivo. These compounds also did not cause genotoxic effects in
HepG2 cells, and were characterized by parameters indicating their
high absorption from the gastrointestinal tract. The presented results
collectively show that 4-alkyl-5-substituted-1,2,4-triazole-3-thiones
constitute a group of promising anticonvulsant drug candidates, whose
molecular mechanism of action depends on VGSCs inhibition. Based on
the totality of the data obtained, compounds TP-315, TP-423, TP-425,
TP-427, and TP-429 seem to be the most promising ones for further
development.
Acknowledgements
This work was financially supported by the National Science Centre,
Poland (DEC-2013/11/D/NZ7/01170).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ejps.2018.12.018.
References
Adan, A., Kiraz, Y., Baran, Y., 2016. Cell proliferation and cytotoxicity assays. Curr.
Pharm. Biotechnol. 17, 1216–1221.
Ahuja, S., Mukund, S., Deng, L., Khakh, K., Chang, E., Ho, H., Shriver, S., Young, C., Lin,
S., Johnson Jr., J.P., Wu, P., Li, J., Coons, M., Tam, C., Brillantes, B., Sampang, H.,
Mortara, K., Bowman, K.K., Clark, K.R., Estevez, A., Xie, Z., Verschoof, H., Grimwood,
M., Dehnhardt, C., Andrez, J.C., Focken, T., Sutherlin, D.P., Safina, B.S., Starovasnik,
M.A., Ortwine, D.F., Franke, Y., Cohen, C.J., Hackos, D.H., Koth, C.M., Payandeh, J.,
2015. Structural basis of Nav1.7 inhibition by an isoform-selective small-molecule
antagonist. Science 350, aac5464.
4. Conclusions
Based on obtained data, it can be observed that there are no benefits
from the replacement of unbranched alkyl moieties in 4-alkyl-5-sub-
stituted-1,2,4-triazole-3-thiones by branched or heteroatom-enriched
alkyl substituents. The compounds with unbranched aliphatic chains,
containing from 4 to 7 carbon atoms, are mostly preferred from the
point of anticonvulsant activity, neurotoxicity and affinity towards
VGSCs. Docking simulations collectively indicated that all the tested
compounds bind mostly to the VSD4 region, similarly to the inhibitor
co-crystalized with mammalian Nav1.7 (PDB ID: 5EK0). The docking
revealed that the activity of the tested inhibitors may result from the
simultaneously created interactions with the following residues:
Alexopoulos, A.V., 2013. Pharmacoresistant epilepsy: definition and explanation.
Epileptology 1, 38–42.
Bojic, U., Elmazar, M.M.A., Hauck, R.S., Nau, H., 1996. Further branching of valproate-
related carboxylic acids reduces the teratogenic activity, but not the anticonvulsant
effect. Chem. Res. Toxicol. 9, 866–870.
Catterall, W.A., 1986a. Voltage-dependent gating of sodium channels: correlating struc-
ture and function. Trends Neurosci. 9, 7–10.
Catterall, W.A., 1986b. Molecular properties of voltage-sensitive sodium channels. Annu.
Rev. Biochem. 55, 953–985.
Catterall, W.A., 2012. Voltage-gated sodium channels at 60: structure, function and
56

B. Kaproń et al.
EuropeanJournalofPharmaceuticalSciences129(2019)42–57
pathophysiology. J. Physiol. 590, 2577–2589.
test in mice. Eur. Neuropsychopharmacol. 15, 609–616.
Czapiński, P., Błaszczyk, B., Czuczwar, S.J., 2005. Mechanisms of action of antiepileptic
Marban, E., Yamagishi, T., Tomaselli, G.F., 1998. Structure and function of voltage-gated
drugs. Curr. Top. Med. Chem. 5, 3–14.
sodium channels. J. Physiol. 508, 647–657.
Dzitko, K., Paneth, A., Plech, T., Pawełczyk, J., Węglińska, L., Paneth, P., 2014. Triazole-
based compound as a candidate to develop novel medicines to treat toxoplasmosis.
Antimicrob. Agents Chemother. 58, 7583–7585.
Mavromoustakos, T., Durdagi, S., Koukoulitsa, C., Simcic, M., Papadopoulos, M.G.,
Hodoscek, M., Grdadolnik, S.G., 2011. Strategies in the rational drug design. Curr.
Med. Chem. 18, 2517–2530.
Gaussian 09, Revision E.01, Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E.,
Robb, M.A., Cheeseman, J.R., Scalmani, G., Barone, V., Mennucci, B., Petersson, G.A.,
Nakatsuji, H., Caricato, M., Li, X., Hratchian, H.P., Izmaylov, A.F., Bloino, J., Zheng,
G., Sonnenberg, J.L., Hada, M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J.,
Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H., Vreven, T., Montgomery Jr.,
J.A., Peralta, J.E., Ogliaro, F., Bearpark, M., Heyd, J.J., Brothers, E., Kudin, K.N.,
Staroverov, V.N., Kobayashi, R., Normand, J., Raghavachari, K., Rendell, A., Burant,
J.C., Iyengar, S.S., Tomasi, J., Cossi, M., Rega, N., Millam, J.M., Klene, M., Knox, J.E.,
Cross, J.B., Bakken, V., Adamo, C., Jaramillo, J., Gomperts, R., Stratmann, R.E.,
Yazyev, O., Austin, A.J., Cammi, R., Pomelli, C., Ochterski, J.W., Martin, R.L.,
Morokuma, K., Zakrzewski, V.G., Voth, G.A., Salvador, P., Dannenberg, J.J.,
Dapprich, S., Daniels, A.D., Farkas, Ö., Foresman, J.B., Ortiz, J.V., Cioslowski, J., Fox,
D.J., 2009. Gaussian, Inc., Wallingford CT.
Payandeh, J., Scheuer, T., Zheng, N., Catterall, W.A., 2011. The crystal structure of a
voltage-gated sodium channel. Nature 475, 353–358.
Perlman, B.J., Goldstein, D.B., 1984. Membrane disordering potency and anticonvulsant
action of valproic acid and other short-chain fatty acids. Mol. Pharmacol. 26, 83–89.
Plazinska, A., Kolinski, M., Wainer, I.W., Jozwiak, K., 2013. Molecular interactions be-
tween fenoterol stereoisomers and derivatives and the β₂-adrenergic receptor binding
site studied by docking and molecular dynamics simulations. J. Mol. Model. 19,
4919–4930.
Plech, T., Łuszczki, J.J., Wujec, M., Flieger, J., Pizoń, M., 2013. Synthesis, characteriza-
tion and preliminary anticonvulsant evaluation of some 4-alkyl-1,2,4-triazoles. Eur.
J. Med. Chem. 60, 208–215.
Plech, T., Kaproń, B., Łuszczki, J.J., Paneth, A., Siwek, A., Kołaczkowski, M., Żołnierek,
M., Nowak, G., 2014. Studies on the anticonvulsant activity of 4-alkyl-1,2,4-triazole-
3-thiones and their effect on gabaergic system. Eur. J. Med. Chem. 86, 690–699.
Rappe, A.K., Casewit, C.J., Colwell, K.S., Goddard III, W.A., Skiff, W.M., 1992. UFF, a full
periodic table force field for molecular mechanics and molecular dynamics simula-
tions. J. Am. Chem. Soc. 114, 10024–10035.
Guy, H.R., Seetharamulu, P., 1986. Molecular model of the action potential sodium
channel. Proc. Natl. Acad. Sci. U. S. A. 83, 508–512.
Hanwell, M.D., Curtis, D.E., Lonie, D.C., Vandermeersch, T., Zurek, E., Hutchison, G.R.,
2012. Avogadro: an advanced semantic chemical editor, visualization, and analysis
platform. Aust. J. Chem. 4 (1), 17.
Shah, M.H., Patki, V.M., Mhasalkar, M.Y., 1962. 1,2,4-Triazole derivatives as possible
Hen, N., Bialer, M., Yagen, B., 2012. Syntheses and evaluation of anticonvulsant activity
of novel branched alkyl carbamates. J. Med. Chem. 55, 2835–2845.
Kamiński, K., Zagaja, M., Łuszczki, J.J., Rapacz, A., Andres-Mach, M., Latacz, G., Kieć-
Kononowicz, K., 2015. Design, synthesis, and anticonvulsant activity of new hybrid
compounds derived from 2-(2,5-dioxopyrrolidin-1-yl)propanamides and 2-(2,5-di-
oxopyrrolidin-1-yl)butanamides. J. Med. Chem. 58, 5274–5286.
Kaproń, B., Łuszczki, J., Paneth, A., Wujec, M., Siwek, A., Karcz, T., Mordyl, B., Głuch-
Lutwin, M., Gryboś, A., Nowak, G., Pająk, K., Jóźwiak, K., Tomczykowski, A., Plech,
T., 2017. Molecular mechanism of action and safety of 5-(3-chlorophenyl)-4-hexyl-
2,4-dihydro-3H-1,2,4-triazole-3-thione – a novel anticonvulsant drug candidate. Int.
J. Med. Sci. 14, 741–749.
Katsila, T., Spyroulias, G.A., Patrinos, G.P., Matsoukas, M.T., 2016. Computational ap-
proaches in target identification and drug discovery. Comput. Struct. Biotechnol. J.
14, 177–184.
Litchfield, J.T., Wilcoxon, F., 1949. A simplified method of evaluating dose-effect ex-
periments. J. Pharmacol. Exp. Ther. 96, 99–113.
diuretic agents. J. Sci. Ind. Res. 21C, 76–78.
Shah, M.H., Mhasalkar, M.Y., Patki, V.M., Deliwala, C.V., Sheth, U.K., 1969. 1,2,4(H)-
Triazole derivatives as diuretic agents. J. Pharm. Sci. 58, 1398–1401.
Stevens, M., Peigneur, S., Tytgat, J., 2011. Neurotoxins and their binding areas on vol-
tage-gated sodium channels. Front. Pharmacol. 2, 71.
Stewart, J.J., 2007. Optimization of parameters for semiempirical methods V: modifica-
tion of NDDO approximations and application to 70 elements. J. Mol. Model. 13,
1173–1213.
Tang, F., Hartz, A.M.S., Bauer, B., 2017. Drug-resistant epilepsy: multiple hypotheses, few
answers. Front. Neurol. 8, 301.
Tomson, T., Marson, A., Boon, P., Canevini, M.P., Covanis, A., Gaily, E., Kälviäinen, R.,
Trinka, E., 2015. Valproate in the treatment of epilepsy in girls and women of
childbearing potential. Epilepsy 56, 1006–1019.
Willow, M., Catterall, W.A., 1982. Inhibition of binding of [3H]batrachotoxinin A 20-
alpha-benzoate to sodium channels by the anticonvulsant drugs diphenylhydantoin
and carbamazepine. Mol. Pharmacol. 22, 627–635.
Luszczki, J.J., Andres, M.M., Czuczwar, P., Cioczek-Czuczwar, A., Wojcik-Cwikla, J.,
Ratnaraj, N., Patsalos, P.N., Czuczwar, S.J., 2005. Levetiracetam selectively po-
tentiates the acute neurotoxic effects of topiramate and carbamazepine in the rotarod
Yee, S., 1997. In vitro permeability across Caco3 cells (colonic) can predict in vivo (small
intestinal) absorption in man - fact or myth. Pharm. Res. 14, 763–766.
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