Synthesis, characterization and preliminary anticonvulsant evaluation of some 4-alkyl-1,2,4-triazoles

Article abstract of DOI:10.1016/j.ejmech.2012.11.026

Designed and synthesized 4-alkyl-1,2,4-triazole-3-thione derivatives showed significant anticonvulsant activity, determined in the maximal electroshock-induced seizure (MES) test. The chemical structure of all new compounds was confirmed by spectral methods (¹H NMR, ¹³C NMR, IR, MS). A sensitive and selective method was elaborated for the determination of the anticonvulsant compounds levels in mice brain tissue, based on HPLC with diode array detector (DAD). Chromatographic tests showed that lack of anticonvulsant effect of two derivatives (15, 16) with long alkyl chains at N-4 position of the 1,2,4-triazole ring was due to the inability to cross the blood-brain barrier (BBB).

Full text of DOI:10.1016/j.ejmech.2012.11.026

European Journal of Medicinal Chemistry 60 (2013) 208e215
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Synthesis, characterization and preliminary anticonvulsant evaluation
of some 4-alkyl-1,2,4-triazoles
,
b
,
c
a
d
ꢀ d
Tomasz Plech a , Jarogniew J. Luszczki , Monika Wujec , Jolanta Flieger , Magdalena Pizon
*
a
ꢀ
Department of Organic Chemistry, Faculty of Pharmacy, Medical University of Lublin, Chodzki 4a, 20-093 Lublin, Poland
b Department of Pathophysiology, Medical University of Lublin, Jaczewskiego 8, 20-090 Lublin, Poland
c Isobolographic Analysis Laboratory, Institute of Rural Health, Jaczewskiego 2, 20-950 Lublin, Poland
d
ꢀ
Department of Analytical Chemistry, Medical University of Lublin, Chodzki 4a, 20-093 Lublin, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Designed and synthesized 4-alkyl-1,2,4-triazole-3-thione derivatives showed significant anticonvulsant
activity, determined in the maximal electroshock-induced seizure (MES) test. The chemical structure of
all new compounds was confirmed by spectral methods (1H NMR, 13C NMR, IR, MS). A sensitive and
selective method was elaborated for the determination of the anticonvulsant compounds levels in mice
brain tissue, based on HPLC with diode array detector (DAD). Chromatographic tests showed that lack of
anticonvulsant effect of two derivatives (15, 16) with long alkyl chains at N-4 position of the 1,2,4-triazole
ring was due to the inability to cross the bloodebrain barrier (BBB).
Received 28 June 2012
Received in revised form
18 October 2012
Accepted 18 November 2012
Available online 23 November 2012
Keywords:
s-Triazole
Ó 2012 Elsevier Masson SAS. All rights reserved.
Bloodebrain barrier
MES test
Chimney test
HPLC/DAD
1. Introduction
It is also known that the teratogenic effect of valproic acid is
caused by the presence of: (i) a carboxyl group; (ii) a branched
It is estimated that approximately 50 million people worldwide
suffer from epilepsy. Most of them (90%) are residents of the
so-called developing countries [1,2]. Despite the introduction of
a dozen of novel antiepileptic drugs (AEDs) in the recent years [3],
still in almost 30% of those patients the disease remains resistant to
standard therapy (using 1st and 2nd generation AEDs) [4,5]. Side
effects related to the use of those drugs also seem to be a problem.
Therefore, there still is a need for ongoing research of novel anti-
epileptic medications.
One of the most commonly used classic AED is valproic acid
and its salts. The drug is known to increase endogenic GABA
synthesis and inhibits its decomposition. Yet it also induces
adverse effects, e.g. hepatotoxicity and teratogenicity [6,7]. The
relatively simple structure of valproic acid suggests that three
fragments of the molecule play an important role in its antiepi-
leptic activity. These are the alkyl, the hydrogen bond donor
(HBD) fragment and the hydrogen bond acceptor (HBA) fragment.
aliphatic chain at carbon C-2; and (iii) a hydrogen atom at carbon
C-2 [8,9].
Using the above data, a group of compounds was synthesized in
which an aliphatic fragment (varying in length) was coupled with
the HBA and HBD fragments, while there would be no elements
whose presence might induce the teratogenic effect. To this end,
the aliphatic (not branched) chain was joined with the 1,2,4-
triazole-3-thione ring. The C]S and NH fragments played the
role of hydrogen bond acceptor and donor, respectively. An addi-
tional reason to use the 1,2,4-triazole ring in designing our
compounds was the fact that the ring may bind the GABA-A
receptor through nitrogens N-1 and N-2 [10]. Moreover, our
previous studies demonstrated that the 3-chlorophenyl fragment
bound to the 1,2,4-triazole-3-thione ring improves the antiepileptic
activity of the obtained derivatives [11,12].
Anticonvulsant 1,2,4-triazole derivatives designed to date
usually have two aryl components in their structure, since it was
believed that the second aryl component increases biological
activity [13e16]. However, little is known about the anticonvulsant
effect of alkyl 1,2,4-triazole derivatives. Therefore, the aim of our
study was to elucidate how the presence and the size of the alkyl
fragment affect anticonvulsant activity.
* Corresponding author. Tel.: þ48 081 532 05 19; fax: þ48 081 532 45 46.
E-mail address: tomasz.plech@umlub.pl (T. Plech).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.11.026

T. Plech et al. / European Journal of Medicinal Chemistry 60 (2013) 208e215
209
2. Results and discussion
of partial convulsions with or without secondary generalization in
humans. Compounds 9e17 were administered to mice at the dose
of 300 mg/kg at four pretreatment times (i.e., 15, 30, 60, 120 min)
prior to the test. This method allowed to determine the percentage
of animals (in a group consisting of 8 mice), which were protected
against seizures induced by electric current (Fig. 1).
5-(3-Chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione
(17) having no alkyl substituent, lacked the protective anticonvul-
sant effect. Whereas, introduction of the alkyl fragment in position
4 of the 1,2,4-triazole nucleus resulted in achievement of a group of
derivatives showing strong anticonvulsant activity. The character-
istic feature of all active compounds (9e14) was rapid onset and
long lasting effects (up to 2 h). Fifteen minutes after i.p. adminis-
tration of compounds 9e14, 100% of mice were protected against
MES-induced convulsions. Slight weakening of the activity, after
30 min of the MES test, was observed for 5-(3-chlorophenyl)-4-
ethyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (9). Whereas, the
decyl (15) and dodecyl (16) derivatives were completely inactive in
this test.
In the second stage of the pharmacological research median
effective doses (ED50), median toxic doses (TD50) and PI (protective
index) values for compounds 9e14 were determined (Table 1).
When the length of the alkyl chain was increased by two methylene
groups, from eC2H5 to eC4H9, a distinct increase in the anticon-
vulsant activity was observed. In such case the strength of anti-
convulsant effect increased approximately 4-fold (from 152 mg/kg
to 38.5 mg/kg). Further elongation of the alkyl chain did not cause
any increase at the maximum activity (in 15 min of the test), but
caused the decrease of ED50 value in subsequent time intervals
tested. This tendency is very beneficial in potentially new drugs, as
it means that the drug maintains relatively stable concentration in
the body. It was only after the increase of the length of the
substituent to eC9H19 that produced the pharmacological activity
after 15 min and 30 min following i.p. administration to decreased
by approximately two-fold. However, there were no significant
differences in ED50 values among the respective intervals (15, 30,
60, and 120 min). It is also worth mentioning that compounds 9e14
displayed much stronger anticonvulsant effect than valproate.
Elongation of the alkyl chain results in the increase of lipophilicity
of the subsequent 1,2,4-triazole derivatives. These changes may
justify the changes of activity in the respective time intervals. For
instance, 4-butyl-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-
3-thione (10), despite the fact that it showed the strongest anti-
convulsant activity after 15 min via systemic administration
(ED50 ¼ 38.5 ꢁ 3.9 mg/kg), its ED50 value increased to 311.2 mg/kg
2.1. Chemistry
4-Alkyl-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-
thiones were synthesized in a two step reaction (Scheme 1). In
the first step, a reaction between aliphatic isothiocyanates (with
alkyl chains varying in length) and 3-chlorobenzoic acid hydra-
zide was conducted to obtain 4-alkyl-1-(3-chlorobenzoyl)thio-
semicarbazide derivatives (1e8).
Our earlier studies demonstrated that 1,4-disubstituted thio-
semicarbazides may be quickly and efficiently obtained if anhy-
drous ethanol is used as reaction solvent [17]. Eliminating the need
for any kind of solvent would perhaps be a significant simplifica-
tion of the synthesis procedure. Moreover, such modification would
be in accordance with the idea of “green chemistry”, constantly
growing in popularity. Thus we decided to check whether it is
possible to use a solid-state synthesis method to obtain compounds
1e8. Synthesis optimization was performed by selecting appro-
priate temperature and time of substrate heating. The completion
of the reaction was monitored using thin-layer chromatography.
The best results were obtained with 3-chlorobenzhydrazide heated
with aliphatic isothiocyanates at 120 ꢀC. Under these conditions the
reaction was deemed complete in less than 1 min. Reaction effi-
ciency ranged between 80% and 94%. The results are comparable to
those usually obtained using microwave generators and much
better compared to syntheses conducted in various solvents, even
those employing ultrasound [18]. Undeniable advantages of our
method are its low cost, rapidity and simplicity. The synthesized 4-
alkyl-1-(3-chlorobenzoyl)thiosemicarbazide derivatives (1e8) were
subsequently subjected to alkaline dehydrocyclization which gave
rise to appropriate 4-alkyl-5-(3-chlorophenyl)-2,4-dihydro-3H-
1,2,4-triazole-3-thiones (9e16). 4-Unsubstituted analog e 5-(3-
chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (17) e was
obtained by prolonged heating of 4-benzoyl-1-(3-chlorobenzoyl)
thiosemicarbazide in 5% solution of sodium hydroxide. The ob-
tained compounds were fully characterized by elemental analyses,
1H NMR, 13C NMR, IR and MS spectra (details are shown in the
Section Experimental; see also Supplementary section).
2.2. Anticonvulsant activity
The anticonvulsant activity of compounds 9e17 was determined
using maximal electroshock-induced seizure (MES) test. This test is
thought to be an experimental model of toniceclonic seizures and
H
N
N
O
O
S
NH
NH
2% NaOH, 2h
120 °C, 1 min.
NH
N
NH
Alkyl
Alkyl-NCS
+
NH2
Alkyl
S
(1-8)
(9-16)
Cl
Cl
Cl
H
N
H
N
O
N
N
NH
NH
O
S
5% NaOH, reflux
S
NH
5% NaOH, reflux
N
N
H
S
O
(17)
Cl
Cl
Cl
Scheme 1. Synthetic route to compounds 9e17. Alkyl substituents: eC2H5 (1, 9), eC4H9 (2, 10), eC5H11 (3, 11), eC6H13 (4, 12), eC7H15 (5, 13), eC9H19 (6, 14), eC10H21 (7, 15), eC12H25
(8, 16).

210
T. Plech et al. / European Journal of Medicinal Chemistry 60 (2013) 208e215
of animals tested in the chimney test. Dividing the TD50 values of
the compounds 9e14 by those of valproate, one can calculate the
potency ratio for these derivatives. The lowest acute adverse-effect
(neurotoxic) potential, as compared to valproate, was observed in
the cases of ethyl (potency ratio ranged from 1.73 to 2.02) and nonyl
(potency ratio from 1.29 to 1.91) derivatives. In contrast, compound
11 (pentyl) was characterized by the highest neurotoxic potential
(potency ratio from 0.72 to 0.85) as compared to valproate.
The protective index (PI ¼ TD50/ED50) for the alkyl derivatives of
1,2,4-triazole-3-thione administered i.p. at various pretreatment
times (15, 30, 60, 120 min) ranged considerably, from 1.4 to 10.3.
The highest PI values were observed for butyl (10), hexyl (12) and
heptyl (13) derivatives administered at 15 min before the MES and
chimney tests (PI ¼ 10.3, 9.7, 7.5, respectively). Worth mentioning is
also the fact that, in case of compound 14, the least differences in PI
values at different pretreatment times were observed (PI ranged
from 4.6 to 6.1).
Fig. 1. Time-course of anticonvulsant effects of compounds 9e17 against maximal
electroshock (MES)-induced seizures in mice. Data are presented as percentage of
animals protected against MES-induced seizures out of 8 animals per group. The MES
test was performed at various pretreatment times (15, 30, 60, 120 min) after systemic
administration of the investigated compounds in a fixed dose of 300 mg/kg.
Taking into account the results of pharmacological studies of 4-
alkyl-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thiones
(9e14), one can suppose that these derivatives may be considered
as potential anticonvulsant agents. Obviously, more specific studies
are required to determine pharmacological and toxicological
profiles of the mentioned compounds.
(120 min). This suggests that the activity decreased approximately
eight fold after the concentration of this compound in the central
nervous decreased relatively quickly. The increased lipophilicity of
subsequent derivatives increases their affinity to brain structures,
and hence the elimination of these compounds is slower. Therefore,
in the cases of pentyl (11) and hexyl (12) derivatives no such
significant differences in activities were observed at various
pretreatment times (15, 30, 60, 120 min).
2.3. Chromatographic investigations
We have found itinteresting that 5-(3-chlorophenyl)-4-decyl-2,4-
dihydro-3H-1,2,4-triazole-3-thione (15) and 5-(3-chlorophenyl)-4-
dodecyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (16) were totally
deprived of any activity, despite the fact that compound 14 still
showed twice as high activity as valproate. We have considered two
possible reasons for this phenomenon: (1) the compounds cross the
bloodebrain barrier, but do not show affinity to respective receptors;
(2) the compounds are not capable of penetrating the brain, and
therefore they do not affect the receptors in the central nervous
system. To check this, we have developed and validated a chromato-
graphic method determining the concentration of compounds 15 and
16 in the mice brains. Moreover, for comparison purposes, the
concentration of the pharmacologically active compound e 5-(3-
chlorophenyl)-4-hexyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (12),
has been determined.
The acute adverse effects (neurotoxicity) of compounds 9e14
and valproate were evaluated and expressed as their median
toxic doses (TD50), producing deficits in motor coordination in 50%
Table 1
Time-course and doseeresponse effects of the active compounds in the MES and
chimney tests in mice.
Compound
R
Pretreatment ED50
TD50
PI
time (min)
(mg/kg)
(mg/kg)
(TD50/ED50)
9
eC2H5
15
30
60
120
15
30
152.6 ꢁ 17.3 730.6 ꢁ 48.1 4.8
203.6 ꢁ 27.0 753.0 ꢁ 45.9 3.7
251.5 ꢁ 27.7 741.6 ꢁ 47.1 2.9
320.1 ꢁ 23.6 887.1 ꢁ 42.8 2.8
38.5 ꢁ 3.9 397.9 ꢁ 26.9 10.3
77.8 ꢁ 5.5 405.4 ꢁ 27.2 5.2
156.1 ꢁ 10.2 413.8 ꢁ 27.6 2.7
311.2 ꢁ 11.7 429.2 ꢁ 34.0 1.4
46.5 ꢁ 3.7 309.8 ꢁ 55.9 6.7
65.2 ꢁ 4.1 309.8 ꢁ 55.9 4.8
69.5 ꢁ 4.3 302.8 ꢁ 38.1 4.4
147.4 ꢁ 15.7 391.5 ꢁ 43.0 2.7
47.6 ꢁ 3.8 462.9 ꢁ 20.0 9.7
68.3 ꢁ 10.3 462.9 ꢁ 20.0 6.8
98.1 ꢁ 16.4 456.9 ꢁ 19.7 4.7
159.7 ꢁ 21.7 448.1 ꢁ 21.7 2.8
55.1 ꢁ 5.2 415.8 ꢁ 24.1 7.5
65.2 ꢁ 4.6 422.7 ꢁ 22.8 6.5
101.6 ꢁ 16.8 429.5 ꢁ 19.0 4.2
134.6 ꢁ 11.8 439.5 ꢁ 19.1 3.3
144.2 ꢁ 21.9 666.8 ꢁ 37.1 4.6
130.4 ꢁ 17.6 712.7 ꢁ 39.5 5.5
104.7 ꢁ 16.5 640.2 ꢁ 47.1 6.1
142.8 ꢁ 14.6 659.1 ꢁ 59.9 4.6
189.0 ꢁ 17.3 363.3 ꢁ 14.2 1.9
216.9 ꢁ 9.4 372.9 ꢁ 16.9 1.7
10
eC4H9
The analyses were carried out on a reversed phase column
(C18, 150 mm ꢂ 4.6 mm, 5
mm), using a mobile phase composed
60
120
of methanol, acetonitrile, 2-propanol. Investigated compounds
administered to mice in the same doses: 300 mg kgꢃ1 were
extracted from tissue homogenate with solid phase extraction
(SPE) on octadecyl cartridges. There is no evidence that conditions
used in sample preparation such as HClO4 or heat lead to major
degradation of investigated compounds. No impurities were
observed during stress studies. The method provided a high
extraction yield of the examined compounds from brain tissue
homogenate with absolute recovery values greater than 90.0%
(Table 2). The detector response was linear over a concentration
11
eC5H11 15
30
60
120
12
eC6H13 15
30
60
120
13
eC7H15 15
30
60
120
range of 0.2e100.0
m
g mLꢃ1. Precision results, expressed by the
14
eC9H19 15
intra-day and the inter-day relative standard deviation values, were
satisfactory (less than 5%). Accuracy was satisfactory as well. This
method proved to be suitable for the analysis of investigated
compounds in mice brain tissues and for the study of their distri-
bution and pharmacokinetics in mice brain. All details of the
validation procedure will be published in a separate paper [19].
On the grounds of the findings (Table 3) it has been determined
that permeability of the bloodebrain barrier was the main factor
determining the lack of activity of two (C10 and C12) alkyl deriva-
tives (15, 16). The C10 derivative (15) concentration in the brain, was
found to be approx. 10 times less than the pharmacologically active
30
60
120
15
30
60
Valproate
218.4 ꢁ 18.9 417.3 ꢁ 9.5
1.9
120
246.6 ꢁ 21.4 512.3 ꢁ 20.2 2.1
Results are presented as median effective doses (ED50 ꢁ S.E.) and median toxic doses
(TD50 ꢁ S.E.) of the examined compounds. All compounds were administered i.p., at
four various pretreatment times (15, 30, 60, 120 min) prior to the appropriate
pharmacological test (MES or chimney test). In case of valproate (magnesium salt),
the results from the both tests have been presented in our earlier study [25].

T. Plech et al. / European Journal of Medicinal Chemistry 60 (2013) 208e215
211
Table 2
Validation parameters.
LOD
g/mL)
LOQ
g/mL)
Concentration
range ( g/mL)
r2 of linearity
equation
Mean of
extraction yield (%)
Repeatability
(RSD%)
Intermediate
precision (RSD%)
(
m
(
m
m
12 (eC6H13
15 (eC10H21
16 (eC12H25
)
0.01039
0.01305
0.1020
0.03467
0.04351
0.3400
0.2e100.0
0.2e100.0
0.2e100.0
0.9958
0.9992
0.9999
97.99
93.67
93.37
2.24
2.33
1.50
3.60
3.50
3.40
)
)
LOD e limit of detection; LOQ e limit of quantification.
C6 (hexyl) derivative (12). Also, the dodecyl (C12) analog (16) did not
permeate to the brain at all. The results obtained confirm the
known rule, namely molecules in which the number of rotatable
bonds is higher or equal to 10, have little chance to permeate
through biological membranes [20]. Data obtained from chro-
matographic investigations will be helpful during the tests inten-
ded to determine the mechanism of action of alkyl derivatives of
1,2,4-triazoles. Further, the suggested methods of determination of
1,2,4-triazole derivatives (12, 15, 16) may be used to test their
pharmacokinetic features.
heated in an oil bath at 120 ꢀC for 1 min. After cooling, the product
precipitated and was washed with diethyl ether and recrystallized
from ethanol.
4.2.1. 1-(3-Chlorobenzoyl)-4-ethylthiosemicarbazide (1)
Yield: 84%. CAS Registry Number: 26036-05-05.
4.2.2. 4-Butyl-1-(3-chlorobenzoyl)thiosemicarbazide (2)
Yield: 80%, CAS Registry Number: 891073-07-7.
4.2.3. 1-(3-Chlorobenzoyl)-4-pentylthiosemicarbazide (3)
Yield: 91%, m.p. 134e135 ꢀC, 1H NMR (DMSO-d6): 0.84 (t, 3H,
CH3, J ¼ 7.10 Hz), 1.13e1.42 (m, 4H, 2CH2), 1.40e1.54 (m, 2H, CH2),
3.36e3.47 (m, 2H, CH2), 7.46e8.12 (m, 4H, AreH), 9.24, 9.68, 10.40
(3s, 3H, 3NH). 13C NMR (DMSO-d6): 14.04, 21.99, 28.52, 33.12, 43.60,
126.65, 127.76, 130.38, 131.68, 133.18, 134.62, 164.85, 184.10. IR (KBr,
cmꢃ1): 3411 (NH), 3100, 3003 (CHarom.), 2958 (CHaliph.), 1697 (C]
O), 1319 (C]S). MS (m/z): 299 [M]þ, 301 [M þ 2]þ. Anal. Calc. for
C13H18ClN3OS: C 52.08, H 6.05, N 14.02. Found: C 52.10, H 6.16, N
14.21.
3. Conclusions
4-Alkyl-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-
thiones (9e16) were obtained in a two step synthesis. The first
stage was optimized in such manner that the time of reaction was
shorter than 1 min. It has been proven that introduction of an
alkyl substituent in N-4 position leads to pharmacologically
active compounds. The anticonvulsant effect of the compounds
against maximal electroshock-induced seizures in mice was
greater than that of valproate. Chromatographic tests allow one to
state that the lack of permeability through the bloodebrain
barrier was the reason for the lack of activity of some
compounds in this series.
4.2.4. 1-(3-Chlorobenzoyl)-4-hexylthiosemicarbazide (4)
Yield: 88%, m.p. 141e142 ꢀC, 1H NMR (DMSO-d6): 0.83 (t, 3H,
CH3, J ¼ 7.11 Hz), 1.13e1.31 (m, 6H, 3CH2), 1.37e1.52 (m, 2H, CH2),
3.35e3.47 (m, 2H, CH2), 7.36e8.11 (m, 4H, AreH), 9.24 (s, 2H, 2NH),
10.42 (s, 1H, NH). 13C NMR (DMSO-d6): 13.99, 22.15, 25.98, 28.74,
31.12, 43.68, 126.63, 127.75, 130.41, 131.73, 133.16, 134.58, 164.53,
181.29. IR (KBr, cmꢃ1): 3356 (NH), 3076 (CHarom.), 2989, 2881
(CHaliph.), 1676 (C]O), 1331 (C]S). MS (m/z): 313 [M]þ, 315
[M þ 2]þ. Anal. Calc. for C14H20ClN3OS: C 53.58, H 6.42, N 13.39.
Found: C 53.73, H 6.53, N 13.24.
4. Experimental
4.1. General
All reagents were purchased from Lancaster (Ward Hill, USA)
and Merck Co. (Darmstadt, Germany). Melting points (uncorrected)
were determined by using Fischer-Johns apparatus (Sanyo, Japan).
The 1H NMR (250 MHz) and 13C NMR (90 MHz) spectra were
recorded on a Bruker Avance instrument using DMSO-d6 as
a solvent and TMS as an internal standard. The IR spectra were
recorded in KBr discs using a PerkineElmer 1725X FTIR spec-
trometer. The mass spectra were obtained on a Finnigan Trace DSQ
spectrometer operating at 70 eV. Elemental analyses were per-
4.2.5. 1-(3-Chlorobenzoyl)-4-heptylthiosemicarbazide (5)
Yield: 94%, m.p. 138e139 ꢀC, 1H NMR (DMSO-d6): 0.83 (t, 3H,
CH3, J ¼ 7.03 Hz), 1.13e1.30 (m, 8H, 4CH2), 1.39e1.51 (m, 2H, CH2),
3.36e3.46 (m, 2H, CH2), 7.46e8.14 (m, 4H, AreH), 9.24 (s, 2H, 2NH),
10.40 (s, 1H, NH). 13C NMR (DMSO-d6): 16.26, 24.33, 28.47, 30.76,
30.98, 33.54, 45.88, 128.83, 129.96, 132.62, 133.93, 135.38, 136.80,
167.02, 183.76. IR (KBr, cmꢃ1): 3420, 3348 (NH), 3121, 3039
(CHarom.), 2955, 2809 (CHaliph.), 1675 (C]O), 1327 (C]S). MS (m/z):
327 [M]þ, 329 [M þ 2]þ. Anal. Calc. for C15H22ClN3OS: C 54.95, H
6.76, N 12.82. Found: C 54.89, H 6.78, N 12.95.
ꢀ
formed on an AMZ 851 CHX analyser (PG, Gdansk, Poland) and the
results were within ꢁ0.4% of the theoretical value.
4.2. General procedure for the synthesis of 4-alkyl-1-(3-
chlorobenzoyl)thiosemicarbazides (1e8)
4.2.6. 1-(3-Chlorobenzoyl)-4-nonylthiosemicarbazide (6)
Yield: 84%, m.p. 126e127 ꢀC, 1H NMR (DMSO-d6): 0.82 (t, 3H,
CH3, J ¼ 7.00 Hz), 1.12e1.32 (m, 12H, 6CH2), 1.38e1.54 (m, 2H, CH2),
A mixture of 0.01 mol (1.70 g) of 3-chlorobenzhydrazide and
equimolar amount of the respective alkyl isothiocyanate was
Table 3
Concentration of investigated compounds (12, 15, 16) in brain of mice treated with a fixed dose of 300 mg/kg body weight.
Compound
m
g of compound
Average value of analyte in 1 mg of the
brain tissue homogenate of a mouse
treated with the dose of 300 mg/kg
Standard deviation
value (ꢁ)
found (range)
12 (eC6H13
15 (eC10H21
16 (eC12H25
)
1.8074e13.3031
0.6939e0.9704
Not found
7.2017
0.7029
Not found
0.1000
0.0900
e
)
)

212
T. Plech et al. / European Journal of Medicinal Chemistry 60 (2013) 208e215
3.39 (t, 2H, CH2, J ¼ 7.35 Hz), 7.44e8.12 (m, 4H, AreH), 9.23 (s, 2H,
2NH), 10.41 (s, 1H, NH). 13C NMR (DMSO-d6): 14.06, 22.16, 26.31,
27.34, 28.73, 28.91, 29.06, 31.38, 43.66,126.61, 127.77, 130.38, 131.71,
133.18, 134.58, 164.80, 181.51. IR (KBr, cmꢃ1): 3370 (NH), 3023
(CHarom.), 2834, 2792 (CHaliph.), 1680 (C]O), 1340 (C]S). MS (m/z):
355 [M]þ, 357 [M þ 2]þ. Anal. Calc. for C17H26ClN3OS: C 57.37, H
7.36, N 11.81. Found: C 57.38, H 7.46, N 11.95.
3461 (NH), 3000 (CHarom.), 2925 (CHaliph.), 1586 (C]N),1326 (C]S).
MS (m/z): 295 [M]þ, 297 [M þ 2]þ. Anal. Calc. for C14H18ClN3S: C
56.84, H 6.13, N 14.20. Found: C 56.95, H 6.01, N 14.32.
4.3.5. 5-(3-Chlorophenyl)-4-heptyl-2,4-dihydro-3H-1,2,4-triazole-
3-thione (13)
Yield: 87%, m.p. 80e81 ꢀC, 1H NMR (DMSO-d6): 0.77 (t, 3H, CH3,
J ¼ 7.00 Hz), 0.99e1.19 (m, 8H, 4CH2),1.42e1.55 (m, 2H, CH2), 4.00 (t,
4.2.7. 1-(3-Chlorobenzoyl)-4-decylthiosemicarbazide (7)
2H, CH2, J ¼ 7.58 Hz), 7.55e7.78 (m, 4H, AreH), 14.00 (s, 1H, NH). 13
C
Yield: 87%, m.p. 122e123 ꢀC, 1H NMR (DMSO-d6): 0.83 (t, 3H,
CH3, J ¼ 6.95 Hz), 1.10e1.31 (m, 14H, 7CH2), 1.38e1.54 (m, 2H, CH2),
3.34e3.47 (m, 2H, CH2), 7.46e8.15 (m, 4H, AreH), 9.26 (s, 2H, 2NH),
10.40 (s, 1H, NH). 13C NMR (DMSO-d6): 14.11, 21.46, 25.47, 26.80,
28.15, 28.51, 29.10, 31.62, 44.38, 127.04, 128.20, 129.16, 130.87,
131.38,134.03,164.70,180.23. IR (KBr, cmꢃ1): 3420, 3367 (NH), 3107
(CHarom.), 2961, 2821 (CHaliph.), 1675 (C]O), 1329 (C]S). MS (m/z):
369 [M]þ, 371 [M þ 2]þ. Anal. Calc. for C18H28ClN3OS: C 58.44, H
7.63, N 11.36. Found: C 58.34, H 7.70, N 11.47.
NMR (DMSO-d6): 16.16, 24.21, 27.77, 29.49, 30.05, 33.17, 45.78,
129.72,130.47,130.69,133.06,133.33,136.04,152.23,169.39. IR (KBr,
cmꢃ1): 3453 (NH), 3105, 3023 (CHarom.), 2968 (CHaliph.),1581 (C]N),
1333 (C]S). MS (m/z): 309 [M]þ, 311 [M þ 2]þ. Anal. Calc. for
C15H20ClN3S: C 58.14, H 6.51, N 13.56. Found: C 58.00, H 6.50, N 13.48.
4.3.6. 5-(3-Chlorophenyl)-4-nonyl-2,4-dihydro-3H-1,2,4-triazole-
3-thione (14)
Yield: 86%, 88e90 ꢀC, 1H NMR (DMSO-d6): 0.81 (t, 3H, CH3,
J ¼ 7.04 Hz),1.00e1.27 (m,12H, 6CH2),1.39e1.54 (m, 2H, CH2), 4.00 (t,
4.2.8. 1-(3-Chlorobenzoyl)-4-dodecylthiosemicarbazide (8)
Yield: 88%, m.p. 108e109 ꢀC, 1H NMR (DMSO-d6): 0.83 (t, 3H,
CH3, J ¼ 6.86 Hz), 1.14e1.29 (m, 18H, 9CH2), 1.37e1.52 (m, 2H, CH2),
3.34e3.45 (m, 2H, CH2), 7.48e8.12 (m, 4H, AreH), 9.23 (s, 2H, 2NH),
10.42 (s, 1H, NH). 13C NMR (DMSO-d6): 12.28, 20.17, 24.86, 25.77,
26.10, 27.19, 27.36, 27.56, 27.78, 30.84, 42.05, 127.14, 127.87, 128.30,
129.08, 129.98, 132.26, 165.87, 181.06. IR (KBr, cmꢃ1): 3390 (NH),
3111, 3027 (CHarom.), 2939, 2788 (CHaliph.), 1670 (C]O), 1337 (C]S).
MS (m/z): 398 [M]þ. Anal. Calc. for C20H32ClN3OS: C 60.35, H 8.10, N
10.56. Found: C 60.46, H 8.18, N 10.53.
2H, CH2, J ¼ 7.66 Hz), 7.51e7.89 (m, 4H, AreH), 13.99 (s, 1H, NH). 13
C
NMR (DMSO-d6): 14.01, 22.14, 25.56, 27.25, 28.15, 28.57, 28.65, 31.27,
43.56, 127.49, 128.25,128.45,130.84, 131.11, 133.82,150.02,167.20. IR
(KBr, cmꢃ1): 3415 (NH), 3054 (CHarom.), 2896 (CHaliph.), 1582 (C]N),
1321 (C]S). MS (m/z): 337 [M]þ, 339 [M þ 2]þ. Anal. Calc. for
C17H24ClN3S: C 60.42, H 7.16, N 12.44. Found: C 60.51, H 7.01, N 12.30.
4.3.7. 5-(3-Chlorophenyl)-4-decyl-2,4-dihydro-3H-1,2,4-triazole-
3-thione (15)
Yield: 80%, m.p. 91e92 ꢀC, 1H NMR (DMSO-d6): 0.84 (t, 3H, CH3,
J ¼ 7.01 Hz), 1.08e1.27 (m, 14H, 7CH2), 1.47e1.52 (m, 2H, CH2), 4.02
(t, 2H, CH2, J ¼ 7.85 Hz), 7.57e7.88 (m, 4H, AreH), 14.01 (s, 1H, NH).
13C NMR (DMSO-d6): 14.02, 22.15, 25.55, 27.25, 28.14, 28.71, 28.88,
31.33, 43.58, 127.48, 128.25, 128.46, 130.84, 131.10, 133.82, 150.03,
167.20. IR (KBr, cmꢃ1): 3397 (NH), 3015 (CHarom.), 2868, 2804
(CHaliph.), 1586 (C]N), 1322 (C]S). MS (m/z): 351 [M]þ, 353
[M þ 2]þ. Anal. Calc. for C18H26ClN3S: C 61.43, H 7.45, N 11.94.
Found: C 61.55, H 7.52, N 12.05.
4.3. Generalprocedure for thesynthesis of4-alkyl-5-(3-chlorophenyl)-
2,4-dihydro-3H-1,2,4-triazole-3-thione derivatives (9e16)
4-Alkyl-1-(3-chlorobenzoyl)thiosemicarbazides (1e8) were
dissolved in 2% NaOH and the resulting solution was refluxed for
2 h. After cooling, the mixture was neutralized with 3 M HCl. The
precipitate that formed was filtered and washed with distilled
water. The compounds were recrystallized from EtOH.
4.3.8. 5-(3-Chlorophenyl)-4-dodecyl-2,4-dihydro-3H-1,2,4-
triazole-3-thione (16)
4.3.1. 5-(3-Chlorophenyl)-4-ethyl-2,4-dihydro-3H-1,2,4-triazole-3-
thione (9)
Yield: 81%, m.p. 68e70 ꢀC, 1H NMR (DMSO-d6): 0.82 (t, 3H, CH3,
J ¼ 6.97 Hz), 1.00e1.29 (m, 18H, 9CH2), 1.39e1.55 (m, 2H, CH2), 4.00
(t, 2H, CH2, J ¼ 7.91 Hz), 7.52e7.81 (m, 4H, AreH), 14.01 (s, 1H, NH).
13C NMR (DMSO-d6): 12.57, 20.72, 24.11, 25.80, 26.69, 27.24, 27.31,
27.45, 27.59, 29.91, 42.11, 126.03, 126.80, 126.99, 129.36, 129.64,
132.35, 148.53, 165.72. IR (KBr, cmꢃ1): 3438 (NH), 3105 (CHarom.),
2962 (CHaliph.), 1569 (C]N), 1341 (C]S). MS (m/z): 379 [M]þ, 381
[M þ 2]þ. Anal. Calc. for C20H30ClN3S: C 63.22, H 7.96, N 11.06.
Found: C 63.20, H 8.08, N 11.20.
Yield: 87%. CAS Registry Number: 26028-63-7.
4.3.2. 4-Butyl-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-
thione (10)
Yield: 80%. CAS Registry Number: 92696-66-7.
4.3.3. 5-(3-Chlorophenyl)-4-pentyl-2,4-dihydro-3H-1,2,4-triazole-
3-thione (11)
Yield: 90%, m.p. 88e89 ꢀC, 1H NMR (DMSO-d6): 0.72 (t, 3H, CH3,
J ¼ 6.96 Hz), 0.98e1.17 (m, 4H, 2CH2),1.41e1.56 (m, 2H, CH2), 4.00 (t,
4.4. Synthesis of 5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-
3-thione (17)
2H, CH2, J ¼ 7.62 Hz), 7.54e7.82 (m, 4H, AreH), 14.00 (s, 1H, NH). 13
C
NMR (DMSO-d6): 14.14, 21.24, 29.20, 34.14, 43.62, 125.76, 127.50,
130.00, 131.48, 134.19, 134.89, 152.21, 169.76. IR (KBr, cmꢃ1): 3423
(NH), 3058 (CHarom.), 2961, 2866 (CHaliph.),1569 (C]N),1328 (C]S).
MS (m/z): 281 [M]þ, 283 [M þ 2]þ. Anal. Calc. for C13H16ClN3S: C
55.41, H 5.72, N 14.91. Found: C 55.50, H 5.70, N 15.06.
4-Benzoyl-1-(3-chlorobenzoyl)thiosemicarbazide (CAS Registry
Number: 648860-52-0) was dissolved in 5% NaOH and the resulting
solution was refluxed for 10 h. After cooling, the mixture was
neutralized with 3 M HCl. The precipitate formed was filtered off
and washed with distilled water. Yield: 74%, CAS Registry Number:
117320-61-3.
4.3.4. 5-(3-Chlorophenyl)-4-hexyl-2,4-dihydro-3H-1,2,4-triazole-
3-thione (12)
Yield: 83%, m.p. 96e98 ꢀC, 1H NMR (DMSO-d6): 0.75 (t, 3H, CH3,
J ¼ 6.87 Hz), 0.99e1.18 (m, 6H, 3CH2), 1.40e1.55 (m, 2H, CH2), 4.02
(t, 2H, CH2, J ¼ 7.75 Hz), 7.55e7.78 (m, 4H, AreH), 13.88 (s, 1H, NH).
13C NMR (DMSO-d6): 14.23, 23.40, 26.18, 29.87, 34.45, 44.29, 127.56,
129.06, 131.34, 132.90, 133.66, 135.36, 152.77, 170.20. IR (KBr, cmꢃ1):
4.5. Pharmacology
4.5.1. General
Adult male Swiss mice (weighing 22e26 g), that were kept in
colony cages with free access to food and tap water, housed

T. Plech et al. / European Journal of Medicinal Chemistry 60 (2013) 208e215
213
under standardized housing conditions, were used. After seven
days of adaptation to laboratory conditions, the animals were
randomly assigned to experimental groups each comprised of 8
mice. Each mouse was used only once. Procedures involving
animals and their care were conducted in accordance with
current European Community and Polish legislation on animal
experimentation. The experimental protocols and procedures
described in this manuscript were approved by the First Local
Ethics Committee at the Medical University in Lublin and the
Second Local Ethics Committee at the University of Life Sciences
in Lublin and complied with the European Communities Council
Directive of 24 November 1986 (86/609/EEC).
test, by the respective ED50 value, as determined in the MES test.
The protective index is considered as an index of the margin of
safety and tolerability between anticonvulsant doses and doses
of the compounds exerting acute adverse effects e.g., sedation,
motor coordination impairment, ataxia or other neurotoxic mani-
festations [24].
4.6. Determination of the concentration of compounds 12, 15, 16 in
the mice brain tissue
4.6.1. Preparation of the mice brain homogenates
Respective 1,2,4-triazole-3-thione derivatives: 12 (pharmaco-
logically active compound) and 15, 16 (inactive ones) were sus-
pended in a 1% aqueous solution of Tween 80 (Sigma, St. Louis, MO,
USA) and administered intraperitoneally in a dose of 300 mg/kg in
a volume of 5 mL/kg body weight. The control animals received
adequate amounts of vehicle. Mice pretreated with appropriate
compounds (or with the vehicle only) were decapitated at time
reflecting the peak of maximum anticonvulsant effect for the active
derivative (12). The whole brains of mice were removed from skulls,
weighed, harvested and homogenized using Abbott buffer (1:2
weight/volume; Abbott Laboratories, North Chicago, IL, USA) in an
Ultra-Turrax T8 homogenizer.
4.5.2. Maximal electroshock seizure test
The investigated compounds (9e17) were suspended in a 1%
solution of Tween 80 in distilled water and administered intra-
peritoneally (i.p.) as a single injection, in a volume of 5 mL/kg body
weight. Valproate (magnesium salt), used as a reference drug, was
directly dissolved in distilled water. Fresh drug solutions were
prepared on each day of experimentation and administered at 15,
30, 60 and 120 min before the initiation of maximal electro-
convulsions. The pretreatment times before testing of the investi-
gated compounds and the route of i.p. administration were based
upon information from the Anticonvulsant Screening Program [21].
Electroconvulsions were produced by a current (0.2 s stimulus
duration; 500 V, 50 Hz, fixed current intensity of 25 mA) delivered
via ear-clip electrodes by a Rodent Shocker generator (constant-
current stimulator Type 221, Hugo Sachs Elektronik, Freiburg,
Germany). The criterion for the occurrence of seizure activity was
the tonic hind limb extension. The animals were administered with
a constant dose of 300 mg/kg of each of the examined compounds
and were subjected to MES-induced seizures. The anticonvulsant
activity of the active compounds administered i.p. at various
pretreatment times was determined as their median effective doses
(ED50 values in mg/kg) in the MES-induced seizure test in mice. The
animals were administered with different doses of the tested
compounds so as to obtain a variable percentage of protection
against maximal electroconvulsions, allowing the construction of
doseeresponse relationship lines for each examined compound
administered i.p. at various pretreatment times, according to
Litchfield and Wilcoxon [22]. Each ED50 value represents the dose
of the studied compounds that is required to protect 50% of the
animals tested against MES-induced seizures.
4.6.2. Chromatographic conditions
Experiments were performed using a LaChrom HPLC Merck
Hitachi (E. Merck, Darmstadt, Germany) model equipped with
diode array detector, L-7350 column oven and L-7612 solvent
degasser. The column (150 mm ꢂ 4.6 mm I.D.) was packed with 5-
2
ꢁ
m
m Zorbax Extend-C18 (pore size: 80 A, surface area: 180 m /g)
Agilent Technologies (Santa Clara, CA, USA); its void volume was
determined to be 1.31 mL, by the injection of thiourea in
acetonitrileewater (50:50) eluent system. The column was ther-
mostated at 20 ꢀC ꢁ 0.1. Mobile phase was a mixture of acetonitrile,
methanol or 2-propanol in water. The phase was filtered through
a Nylon 66 membrane filter (0.45
mm) Whatman (Maidstone,
England). Retention data were recorded at
a
flow-rate of
1 mL minꢃ1 for 80% methanol (retention time of the first
compound, 12 was 4.7 min) and 85% acetonitrile (retention time for
the second compound, 15 was 6.01 min) and 0.5 mL minꢃ1 for 80%
2-propanol (retention time for the third compound, 16 was
5.17 min) used as a mobile phase. The detection of the compounds
was set at appropriate wavelength (lmax ¼ 254 for the first and
second compound and 260 nm for the third compound) chosen in
accord with the recorded spectra. Typical injection volumes were
4.5.3. Chimney test
The chimney test of Boissier et al. [23] was used to quantify the
acute adverse-effect potential of compounds 9e14 and valproate on
motor performance in mice. In this test, the animals had to climb
backwards up a plastic tube (3 cm inner diameter, 30 cm length), and
motor performance impairment was indicated by the inability of the
mice to climb backward up the transparent tube within 60 s. The
acute adverse (neurotoxic) effects of compounds 9e14 and valproate
administered alone were expressed as their median toxic doses
(TD50), representing the doses, at which the investigated compounds
impaired motor coordination in 50% of the animals tested in the
chimney test. To evaluate each TD50 value, at least four groups of
animals (each group consisted of 8 mice) injected with various doses
of the appropriate compound were challenged with the chimney
test. A doseeresponse relationship line was calculated on the basis of
the percentage of mice showing motor deficits by means of the log-
probit method according to Litchfield and Wilcoxon [22].
20 mL.
4.6.3. Solutions
Stock solutions of the analytes at concentration of 300 m
g mLꢃ1
were prepared in methanol, acetonitrile or 2-propanol for
compounds 12, 15, 16, respectively. The stock solutions were stored
at refrigerator in glass vials. Standard solutions were prepared daily
by diluting the stock solutions by the use of appropriate organic
solvent.
4.6.4. Sample preparation
Analyte standard solutions of the first compound (12) were
prepared as follows: 200
was mixed with 50 L of standard solution. Obtained mixture was
incubated in 37.1 ꢀC for 60 min. After that 1 mL of methanol and
250
L of 3% HClO4 were added and the sample was heated to 60 ꢀC
mL of the blank brain tissue homogenate
m
m
for 30 min. The mixture was centrifuged at 9000ꢂ g for 15 min.
Aliquot of the supernatant was further analyzed by SPE procedure.
The brain homogenate of the mouse treated by the dose of 300 mg/
kg was weighted and mixed with 4 mL of methanol and 1 mL of 3%
4.5.4. Protective index (PI)
The protective index for the investigated compounds was
calculated by dividing a TD50 value, as determined in the chimney

214
T. Plech et al. / European Journal of Medicinal Chemistry 60 (2013) 208e215
HClO4 (the ratio of methanol to perchloric acid was 4:1 to receive
the best extraction efficiency). An additional procedure was carried
out for a blank sample. The preparation of samples of the second
compound (15) required application of acetonitrile instead of
methanol, the ratio of organic solvent to 3% perchloric acid was 2:1
and temperature was established as 70 ꢀC. In the case of the last
compound (16), the following changes were made in the procedure
of sample preparation: 2-propanol was used as organic solvent, the
ratio of organic solvent to 3% perchloric acid was 1:1 and incubation
temperature was 80 ꢀC.
precision results were satisfactory. RSD% for repeatability was
always lower than 3% and for intermediate precision e lower than
5%. All collected precision values are presented in Table 2.
Acknowledgments
The project was partially supported by grant from the Institute
of Rural Health (Lublin, Poland). Professor J.J. Luszczki is a Member
of the Academy of Young Scientists (Polish Academy of Sciences,
Warszawa, Poland).
4.6.5. SPE procedure
Appendix A. Supplementary data
Solid-phase extraction (SPE) was carried out using
BAKERBONDÔ spe Octadecyl (C18) J.T. Baker cartridges (200 mg,
3 mL and 500 mg, 6 mL) on a Baker spe-12G apparatus. The C18
cartridges were activated and conditioned with 2 ꢂ 1 mL of
methanol and 2 ꢂ 1 mL of water respectively and dried applying full
vacuum for 1 min (ꢃ30 kPa). Aliquots of the supernatant were
loaded onto the conditioned cartridge. The whole was then washed
with 2 ꢂ 1 mL of water in the case of compound (12) and addi-
tionally by the use of 1 mL of 50% methanol in the case of
compound (15) or 1 mL of 50% acetonitrile in the case of (16).
Analyte elution was carried out with 2 mL of methanol, acetonitrile
or 2-propanol in the case of the first, the second and the third
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.11.026.
References
[1] P. Kwan, M.J. Brodie, Epilepsy after the first drug fails: substitution or add-on?
Seizure 9 (2000) 464e468.
[2] http://www.who.int/mediacentre/factsheets/fs999/en/.
[3] E. Perucca, J. French, M. Bialer, Development of new antiepileptic drugs:
challenges, incentives, and recent advances, Lancet Neurol. 6 (2007) 793e
804.
[4] D. Schmidt, W. Löscher, Drug resistance in epilepsy: putative neurobiologic
and clinical mechanisms, Epilepsia 46 (2005) 858e877.
[5] C.A. Hovinga, Novel anticonvulsant medications in development, Expert Opin.
Investig. Drugs 11 (2002) 1387e1406.
compound respectively, applying full vacuum for 1 min. 20
eluate was injected directly into the HPLC column.
mL of the
[6] H. Nau, R.-S. Hauck, K. Ehlers, Valproic acid induced neural tube defects in
mouse and human: aspects of chirality, alternative drug development,
pharmacokinetics and possible mechanisms, Pharmacol. Toxicol. 69 (1991)
310e321.
[7] H. Nau, H. Siemens, Differentiation between valproate-induced anticonvul-
sant effect, teratogenicity and hepatotoxicity, Pharm. Weekbl. Sci. 14 (1992)
101e105.
[8] U. Bojic, M.M.A. Elmazar, R.-S. Hauck, H. Nau, Further branching of valproate-
related carboxylic acids reduces the teratogenic activity, but not the anti-
convulsant effect, Chem. Res. Toxicol. 9 (1996) 866e870.
[9] M. Bialer, New antiepileptic drugs that are second generation to existing
antiepileptic drugs, Expert Opin. Investig. Drugs 15 (2006) 637e647.
[10] P.B. Wingrove, K.A. Wafford, C. Bain, P.J. Whiting, The modulatory action of
4.6.6. Method validation
4.6.6.1. Linearity. Analyte standard solutions at six different
concentrations were added to 200 L of blank sample, and sub-
m
jected to the SPE and HPLC procedure. The analyte peak area was
plotted against the corresponding concentrations and the calibra-
tion curves determined by means of the least-squares method.
Good linearity values with r2 > 0.995 were found for all examined
compounds. Complete results of linearity, limit of detection, limit of
quantification are presented in Table 2. Limit of quantification
(LOQ) and limit of detection (LOD) values were also determined as
the analyte concentrations which gave rise to peaks whose height is
10 and 3 times that of the baseline and noise, respectively.
loreclezole at the
g-aminobutyric acid type A receptor is determined by
a single amino acid in the b2 subunit, Proc. Natl. Acad. Sci. U. S. A. 91 (1994)
4569e4573.
[11] J.J. Luszczki, T. Plech, M. Wujec, Influence of 5-(3-chlorophenyl)-4-(4-
methylphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione on the anticonvulsant
action of four classical antiepileptic drugs in the mouse maximal
electroshock-induced seizure model, Pharmacol. Rep. 64 (2012) 970e978.
[12] J.J. Luszczki, T. Plech, M. Wujec, Effect of 4-(4-bromophenyl)-5-(3-
chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione on the anticonvulsant
action of different classical antiepileptic drugs in the mouse maximal
electroshock-induced seizure model, Eur. J. Pharmacol. 690 (2012) 99e106.
[13] M. Shalini, P. Yogeeswari, D. Sriram, J.P. Stables, Cyclization of the semi-
carbazone template of aryl semicarbazones: synthesis and anticonvulsant
activity of 4,5-diphenyl-2H-1,2,4-triazol-3(4H)-one, Biomed. Pharmacother.
63 (2009) 187e193.
4.6.6.2. Recovery. Spiked blank samples were prepared as follows:
50
to 200
m
L of standard solution of different concentrations were added
L of the blank brain tissue homogenate and incubated at
m
37.1 ꢀC for 30 min. At least three concentrations were prepared and
analyzed for each compound, corresponding to the lower, middle
and upper limit of the corresponding linearity curve. These samples
were analyzed as described above. The determined analyte peak
areas were substituted into calibration curve equation and the
percentage extraction yield was calculated.
[14] A. Almasirad, S.A. Tabatabai, M. Faizi, A. Kebriaeezadeh, N. Mehrabi,
A. Dalvandi, A. Shafiee, Synthesis and anticonvulsant activity of new 2-
substituted-5-[2-(2-fluorophenoxy)phenyl]-1,3,4-oxadiazoles
triazoles, Bioorg. Med. Chem. Lett. 14 (2004) 6057e6059.
and
1,2,4-
4.6.6.3. Accuracy. Analyte standard solutions at three different
concentrations were added to 200 mL of the brain tissue homoge-
[15] N. Siddiqui, M.S. Alam, W. Ahsan, Synthesis, anticonvulsant and toxicity
evaluation of 2-(1H-indol-3-yl)acetyl-N-(substituted phenyl)hydrazine car-
bothioamides and their related heterocyclic derivatives, Acta Pharm. 58
(2008) 445e454.
[16] N. Siddiqui, W. Ahsan, Triazole incorporated thiazoles as a new class of
anticonvulsants: design, synthesis and in vivo screening, Eur. J. Med. Chem. 45
(2010) 1536e1543.
[17] T. Plech, M. Wujec, A. Siwek, U. Kosikowska, A. Malm, Synthesis and anti-
microbial activity of thiosemicarbazides, s-triazoles and their Mannich
bases bearing 3-chlorophenyl moiety, Eur. J. Med. Chem. 46 (2011) 241e
248.
nate and the obtained mixture was subjected to further SPE and
HPLC procedure. Recovery values were calculated according to the
following formula: 100 ꢂ [(after spiking ꢃ before spiking)/added].
The procedure was repeated three times at each concentration level
to obtain standard deviation values. The mean recovery values
were almost always higher than 90% and standard deviation
values ꢄ 3%. All data are summarized in Table 2.
[18] Y.X. Li, W.G. Zhao, Z.-M. Li, S.H. Wang, W.L. Dong, Microwave and ultrasound
irradiation-assisted synthesis of novel disaccharide-derived arylsulfonyl thi-
osemicarbazides, Synth. Commun. 36 (2006) 1471e1477.
4.6.6.4. Precision. Spiked blank samples were prepared and
analyzed according to procedure described above. The analysis was
repeated six times giving intraday precision values and six times in
another day giving intermediate precision values both expressed as
percentage relative standard deviation value-RSD%. Obtained
ꢀ
[19] J. Flieger, M. Pizon, T. Plech, J.J. quszczki, Analysis of new potential anticon-
vulsant compounds in mice brain tissue by SPE/HPLC/DAD, J. Chrom. B 909
(2012) 26e33.
[20] H. Pajouhesh, G.R. Lenz, Medicinal chemical properties of successful central
nervous system drugs, NeuroRx 2 (2005) 541e553.

T. Plech et al. / European Journal of Medicinal Chemistry 60 (2013) 208e215
215
[21] J.P. Stables, H.J. Kupferberg, Chapter 16
e
the NIH anticonvulsant drug
[24] W. Löscher, B. Nolting, The role of technical, biological and pharmacological
factors in the laboratory evaluation of anticonvulsant drugs IV. Protective
indices, Epilepsy Res. 9 (1991) 1e10.
[25] J.J. Luszczki, E. Wojda, M. Andres-Mach, W. Cisowski, M. Glensk,
K. Glowniak, S.J. Czuczwar, Anticonvulsant and acute neurotoxic effects of
imperatorin, osthole and valproate in the maximal electroshock seizure
and chimney tests in mice: a comparative study, Epilepsy Res. 85 (2009)
293e299.
development (ADD) program: preclinical anticonvulsant screening project, in:
G. Avanzini, G. Regesta, P. Tanganelli, M. Avoli (Eds.), Molecular and Cellular
Targets for Anti-epileptic Drugs, John Libbey, London, 1997.
[22] J.T. Litchfield, F. Wilcoxon, A simplified method of evaluating dose-effect
experiments, J. Pharmacol. Exp. Ther. 96 (1949) 99e113.
[23] J.R. Boissier, J. Tardy, J.C. Diverres, Une nouvelle méthode simple pour explorer
l’action tranquilisante: le test de la cheminée, Med. Exp. 3 (1960) 81e84.