Synthesis and anticonvulsant activity of new N-(alkyl/Sub-stituted aryl)-N'-[4-(5-cyclohexylamino)-1,3,4-thiadiazole-2-ylphenyl]thioureas

Article abstract of DOI:10.1002/ardp.200800118

A series of novel thiourea derivatives carrying the 5-cylohexylamino-1,3,4- thiadiazole moiety was synthesized and their anticonvulsant activity was evaluated. Structures of the synthesized compounds have been confirmed by IR, ¹H-NMR, and eleme

Full text of DOI:10.1002/ardp.200800118

48
Arch. Pharm. Chem. Life Sci. 2009, 342, 48–53
Full Paper
Synthesis and Anticonvulsant Activity of New N-(Alkyl/Sub-
stituted aryl)-N'-[4-(5-cyclohexylamino)-1,3,4-thiadiazole-2-
yl)phenyl]thioureas
1
1
2
2
ˇ
ˇ
Sevgi Karakus , Bedia Koꢀyıgıt-Kaymakcioglu , Hale Z. Toklu , Feyza Aricioglu , and
Sevim Rollas1
1 Department of Pharmaceutical Chemistry, Marmara University, Faculty of Pharmacy, Istanbul, Turkey
2 Department of Pharmacology, Faculty of Pharmacy, Marmara University, Istanbul, Turkey
A series of novel thiourea derivatives carrying the 5-cylohexylamino-1,3,4-thiadiazole moiety
was synthesized and their anticonvulsant activity was evaluated. Structures of the synthesized
1
compounds have been confirmed by IR, H-NMR, and elemental analysis. All of the compounds
were administered at a dose of 50 mg/kg. Some of the active compounds have different effects in
pentylenetetrazole (PTZ) and maximal electroshock (MES) tests, indicating the therapeutical
potential in petit mal seizures, but not in grand mal seizures. Compounds 10, 11, 13, and 14
carrying 2-methylphenyl, 4-chlorophenyl, allyl, and 4-methylphenyl on the thiourea pharmaco-
phore, increased the survival rate in the PTZ model. The ED50 values of the active compounds 10,
11, 13, and 14 were found 68.42, 43.75, 18.75 and 25 mg/kg, respectively.
Keywords: Anticonvulsant activity / Maximal electroshock seizure / Pentylenetetrazole / 1,3,4-Thiadiazoles / Thioureas
/
Received: June 17, 2008; accepted: September 12, 2008
DOI 10.1002/ardp.200800118
The anticonvulsant-drug design was based on the pre-
Introduction
sumption that for the activity in maximal electroshock
(MES) evaluation at least one phenyl or similar aromatic
group in close proximity to two electron-donor atoms
and for activity in the pentylenetetrazole (PTZ) evalua-
tion an alkyl group substituted close to two electron-
donor atoms was required [17]. It has been hypothesized
that ureas and thioureas displaying anticonvulsant activ-
ity interact at locations on the putative binding site des-
ignated as aryl binding site, a hydrogen-bonding domain,
and an auxiliary aryl or other hydrophobic binding site
[14]. N-Substituted-N9-(3,5-di/and 1,3,5-trimethylpyrazole)-
thiourea and urea derivatives were reported as anticon-
vulsants in which the most active compound was to
found N-ethyl-N9-(3,5-dimethylpyrazole-4-yl)thiourea in
our previous study [15]. In view of the above results, the
thiourea pharmacophore has been attached to a thiadia-
zole ring and to an aryl or alkyl group. Thus, the aim of
the present study was to prepare a series of thiourea
derivatives and evaluate their anticonvulsant properties
by using MES and PTZ models in mice.
N,N9-Disubstituted thioureas belong to biologically
highly active skeletons. Various thiourea derivatives
were extensively studied for their antitubercular, antivi-
ral, antifungal, anti-HIV, and antihypertensive activities
[1–13]. Recently, compounds containing thiourea and
urea group have emerged as structurally novel anticon-
vulsants [14, 15].
Despite the apparent success of the current discovery
process, a significant need persists for more efficacious
and less toxic anti-epileptic drugs (AEDs). Besides, new
AEDs are needed for the patients whose seizures remain
refractory to the currently available AEDs [16].
Correspondence: Sevim Rollas, Department of Pharmaceutical Chemis-
try, Marmara University, Faculty of Pharmacy, 34668 Istanbul, Turkey.
E-mail: sevim@sevimrollas.com
Fax: +90 216 345-2952
Abbreviations: anti-epileptic drugs (AEDs); maximal electroshock
(MES); pentylenetetrazole (PTZ)
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Arch. Pharm. Chem. Life Sci. 2009, 342, 48–53
Anticonvulsant Activity of 1,3,4-Thiadiazole Thioureas
49
Reactions and conditions: (a) C2H5-O-C2H5; (b) NH2NH2 N H2O, C2H5OH; (c) C6H11NCS, C2H5OH; (d) 50% H2SO4; D; (e) Ar(R)-NCS, CH3-CO-CH3.
Scheme 1. Synthetic protocol of the titled compounds.
migration of hydrogen atom on nitrogen to the other
nitrogen atom [15].
Results and discussion
Structure determination
Intermediate product 2-(4-aminophenyl)-4-cylohexyl- Anticonvulsant activity
All synthesized compounds were administered at the
amino-1,3,4-thiadiazole I was prepared following the
reported procedure [7]. The amine compounds were dose of 50 mg/kg. Besides, in case of anticonvulsant effect
reacted with substituted isothiocyanates in dry acetone the same compounds were also used in doses of 25 and
100 mg/kg and ED50 values were calculated for these com-
pounds.
The anticonvulsant activity results were shown in
medium and gave some novel N-(alkyl/substitutedaryl)-N9-
[4-(5-cyclohexylamino)-1,3,4-thiadiazole-2-yl)phenyl]th-
ioureas (Scheme 1). The structures of the obtained com-
pounds were elucidated by spectral analyses. In the IR Figs. 1 and 2. Compounds 2, 8, 10, 11, 13, and 14
decreased the mortality rate in the MES test. On the other
spectra, some significant stretching bands due to NH and
C=S were found at about 3231-3198 and 1346–1300 cm–1, hand, none of the compounds has exerted protective
respectively. In the 1H-NMR spectra, the thiourea NH pro- effect against MES-induced seizures. Moreover, com-
tons were observed at 7.91–10.17 and 9.54–10.20 ppm. pound 7, 9, 10 containing 4-nitrophenyl, 4-methoxy-
phenyl, and 2-methylphenyl, repectively, on the thiourea
The secondary amine NH proton was determined at
7.33–8.72 ppm as singlet. All the other aromatic and ali- pharmacophore, seemed to be proconvulsant in the MES
phatic protons were observed at expected regions. APCI- test (Figs. 1a and 1b). Therefore, none of the tested com-
MS spectra of the 3–5, 8–13 showed correct molecular pounds has a promissing activity on MES-induced convul-
sions.
Compounds 10, 11, 13, and 14 carrying 2-methyl-
ion peaks [M+1] which confirmed their molecular
weights. The major fragmentation pathway appeared by
the cleavage of NH-CS bond of thiourea moiety and the phenyl, 4-chlorophenyl, allyl, and 4-methylphenyl,
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S. Karakus et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 48–53
Figure 1. (a) The survival rate and (b) seizure prevention percent of the mice in the maximal electroshock seizures (MES) test
(60 Hz, 25 mA, 450 mS).
Figure 2. (a) The survival rate and (b) number of grade-5 seizures in the pentylenetetrazole induced seizure (PTZ) test (60 mg/kg).
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Arch. Pharm. Chem. Life Sci. 2009, 342, 48–53
Anticonvulsant Activity of 1,3,4-Thiadiazole Thioureas
51
were determined with a Bꢀchi 530 capillary apparatus (Bꢀchi
Labortechnik, Flawil, Switzerland). 1H-NMR spectra were
recorded in DMSO on a Bruker Avance-DPX-400 spectrometer
(Bruker Bioscience, Billerica, MA, USA) in DMSO-d6 and chemical
shifts were given in d ppm with tetramethylsilane (TMS). The
splitting patterns of 1H-NMR were designed as follows: s: singlet,
d: doublet, t: triplet, q: quartet, m: multiplet. The mass spectrom-
etry was performed using Agilent 1100 MSD spectrometer (Agi-
lent, Palo Alto, CA, USA) in the electrospray mode. All new com-
pounds were analyzed for C, H, N and the results were in an
acceptable range (1H-NMR, mass and elemental analysis were
provided by the Scientific and Technical Research Council of
Turkey, Tꢀbitak).
respectively, increased the survival rate in the PTZ test.
Although the compounds did not effect the number of
PTZ-induced grade-3 and -4 seizures, compounds 3, 5, 6, 7,
8, 9, 10, 12, and 14 reduced the number of grade-5 seiz-
ures (Figs 2a and 2b). The ED50 values of the active com-
pounds 10, 11, 13, and 14 were 68.42, 43.75, 18.75, and
25 mg/kg, respectively. In terms of the effect of these
compounds on seizure severity, compounds 3, 6, 12, 13,
14 have a higher potential than the rest of the group.
None of the doses used had an effect on locomotor activ-
ity.
In order to investigate the possible effect of these com-
pounds in different seizure types, two different animal
models (MES and PTZ) were used in the present study.
Both tests provide some insight into the ability of a given
drug to penetrate the blood-brain barrier and exert a CNS
effect. Moreover, both models well suited for screening
anticonvulsant activity and both model systems display
clear and definable seizure endpoints [16]. These proper-
ties make them ideally suited to screen large numbers of
chemically diverse entities. However, these models may
differ in activity against different seizure types i. e., the
positive results obtained in the PTZ seizure test model
were historically considered suggestive of potential clini-
cal utility against generalized absence epilepsy, based
largely on the finding that drugs active in the clinical use
against spike-wave seizures (e. g., ethosuximide, trime-
thadione, valproic acid, benzodiazepines) were effective
at blocking clonic seizures induced by PTZ. In contrast,
sodium channel antagonists such as phenytoin and car-
bamazepine are ineffective.
Synthesis
Intermediate product 2-(4-aminophenyl)-4-cylohexylamino-
1,3,4-thiadiazole I was prepared according to literature [7]. Com-
pounds 1–14 were prepared according to a procedure, which
was described previously [15]. A solution of appropriate isothio-
cyanate (0.0085 mol) in anhydrous acetone (100 mL) was added
dropwise to a solution of the appropriate amine (0.0085 mol) in
anhydrous acetone (100 mL). The resulting mixture was refluxed
for 4-5 h and the solvent was evaporated in vacuo to give the
crude thioureas 1–14 which were purified by recrystallization
from ethanol.
N-{4-[5-Cyclohexylamino)-1,3,4-thiadiazole-2-yl)phenyl}-
N9-methylthiourea 1
Yield: 60%; m.p.: 211–2138C; IR (KBr) [cm– 1]: 3206 (NH), 1310
(C=S); 1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.15–2.14 (m, 10H,
CH2 protons), 2.91 (d, J = 4.3 Hz, 3H, CH3), 3.51 (s, 1H, CH proton),
7.52 (d, J = 8.5 Hz, 2H, aromatic protons), 7.70 (d, J = 8.6 Hz, 2H,
aromatic protons), 7.84 (s, 1H, NH), 7.86 (s, 1H, thiourea NH),
9.77 (s, 1H, thiourea NH). Anal. Calc.for C16H21N5S2: C, 55.30; H,
6.09; N, 20.15; S, 18.46. Found: C, 55.41; H, 6.01; N, 19.65; S,
17.06.
In conclusion, the results of the present study demon-
strate that thiourea derivatives 1–14 may have potential
anticonvulsant effects. Some of the active compounds
have different effects in PTZ and MES tests, indicating the
therapeutical potential in petit mal seizures, but not in
grand mal seizures. Further studies should be carried out
with these active compounds to evaluate the mecha-
nisms contributing the anticonvulsant effect.
N-{4-[5-Cyclohexylamino)-1,3,4-thiadiazole-2-yl)phenyl}-
N9-ethylthiourea 2
Yield: 65%; m.p.: 2198C; IR (KBr) [cm– 1]: 3207 (NH), 1310 (C=S); 1H-
NMR (400 MHz, DMSO-d6) d [ppm]: 1.13 (t, 3H, CH3), 1.18–2.06
(m, 10H, CH2 protons), 3.43–3.61 (m, 3H, CH2 and CH protons),
7.53 (d, J = 8.7 Hz, 2H, aromatic protons), 7.67 (d, J = 8.7 Hz, 2H,
aromatic protons), 7.85 (s, 1H, NH), 7.91 (s, 1H, thiourea NH),
9.65 (s, 1H, thiourea NH). Anal. Calc.for C17H23N5S2: C, 56.48; N,
19.37; S, 17.74. Found: C, 56.47; N, 19.11; S, 17.15.
This study was supported by Marmara University Scientific
Research Projects Commission (BAPKO, Project number SAG-
YY-270206-0027-2006).
The authors have declared no conflict of interest.
N-{4-[5-Cyclohexylamino)-1,3,4-thiadiazole-2-yl)phenyl}-
N9-propylthiourea 3
Yield: 57%; m.p.: 1878C; IR (KBr) [cm– 1]: 3213 (NH), 1301 (C=S); 1H-
NMR (400 MHz, DMSO-d6) d [ppm]: 0.90 (t, 3H, CH3 ), 1.27–3.55
(m, 15H, CH and CH2 protons), 7.33–7.80 (m, 5H, aromatic pro-
tons and NH), 7.94 (s, 1H, thiourea NH), 9.68 (s, 1H, thiourea NH);
MS (APCI): m/z 376 [M + 1], 343, 342, 317, 292, 277, 276, 275, 261,
260, 236, 235, 194, 193, 151, 134, 119, 118, 83, 75.
Experimental
Chemistry
All chemicals and solvents were purchased from Merck, Aldrich
(both Germany), and Fluka (Flawil, Switzerland). Melting points
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S. Karakus et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 48–53
N-{4-[5-Cyclohexylamino)-1,3,4-thiadiazole-2-yl)phenyl}-
N-{4-[5-Cyclohexylamino)-1,3,4-thiadiazole-2-yl)phenyl}-
N9-(4-fluorophenyl)thiourea 4
N9-(2-methylphenyl)thiourea 10
Yield: 53%; m.p.: 1948C; IR (KBr) [cm– 1]: 3209 (NH), 1310 (C=S); 1H-
NMR (400 MHz, DMSO-d6) d [ppm]: 1.18-2.11 (m, 10H, CH2), 3.56
(m, 1H, CH proton), 7.07–7.78 (m, 8H, aromatic protons), 7.85 (s,
1H, NH), 8.15 (s, 1H, thiourea NH), 10.17 (s, 1H, thiourea NH),
9.92 and 10.02 (two s, 1H, thiourea NH); MS (APCI): m/z 428 [M +
1], 413, 412, 325, 317, 277, 276, 275, 243, 235, 195, 194, 193, 151,
134, 119, 118, 83, 75.
Yield: 54%; m.p.: 170–1738C; IR (KBr) [cm– 1]: 3208 (NH), 1310
(C=S); 1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.10–2.10 (m, 10H,
CH2 protons), 2.25 (s, 3H, CH3), 3.50 (s, 1H, CH proton), 6.70–7.80
(m, 8H, aromatic protons), 8.15 (bs, 1H, NH), 9.51 (s, 1H, thiourea
NH), 9.90 (s, 1H, thiourea NH); MS (APCI): m/z 424 [M + 1], 379,
317, 275, 193.
N-{4-[5-Cyclohexylamino)-1,3,4-thiadiazole-2-yl)phenyl}-
N-{4-[5-Cyclohexylamino)-1,3,4-thiadiazole-2-yl)phenyl}-
N9-(4-chlorophenyl)thiourea 11
N9-(4-bromophenyl)thiourea 5
Yield: 63%; m.p.: 2028C; IR (KBr) [cm– 1]: 3198 (NH),1308 (C=S); 1H-
NMR (400 MHz, DMSO-d6) d [ppm]: 1.19–2.11 (m, 10H, CH2 pro-
tons), 3.55 (s, 1H, CH proton), 7.40 (d, J = 8.8 Hz, 2H, aromatic pro-
tons), 7.53 (d, J = 8.8 Hz, 2H, aromatic protons), 7.64 (d, J = 8.7 Hz,
2H, aromatic protons), 7.74 (d, J = 8.7 Hz, 2H, aromatic protons),
8.41 (s, 1H, NH), 10.04 (s, 1H, thiourea NH), 10.12 (s, 1H, thiourea
NH); MS (APCI): m/z 376 [M + 1], 317, 275, 193, 156.
Yield: 58%; m.p.: 1848C; IR (KBr) [cm– 1]: 3220 (NH),1308 (C=S); 1H-
NMR (400 MHz, DMSO-d6) d [ppm]: 1.04–2.11 (m, 10H, CH2 pro-
tons), 3.57 (m, 1H, CH proton), 7.31–7.89 (m, 8H, aromatic pro-
tons), 8.42 (s, 1H, NH), 10.04 (s, 1H, thiourea NH), 10.13 and 10.20
(two s, 1H, thiourea NH); MS (APCI): m/z 488 [M + 1], 428, 413,
412, 363, 317, 275, 193, 79, 64.
N-{4-[5-Cyclohexylamino)-1,3,4-thiadiazole-2-yl)phenyl}-
N-{4-[5-Cyclohexylamino)-1,3,4-thiadiazole-2-yl)phenyl}-
N9-cyclohexylthiourea 6
N9-benzylthiourea 12
Yield: 40%; m.p.: 2248C; IR (KBr) [cm– 1]: 3217 (NH), 1301 (C=S);1H-
NMR (400 MHz, DMSO-d6) d [ppm]: 1.09–2.08 (m, 20H, CH2 pro-
tons), 3.54 (s, 1H, CH proton), 4.10 (1H, CH proton), 7.58 (d, J =
8.7 Hz, 2H, aromatic protons), 7.67 (d, J = 8.7 Hz, 2H, aromatic
protons), 7.74–7.90 (m, 2H, NH and thiourea NH), 9.54 (s, 1H, thi-
ourea NH). Anal. Calc. for C21H29N5S2: C, 60.69; H, 7.03; N, 16.85;
S, 15.43. Found: C, 60.01; H, 6.26; N, 16.57; S, 16.30.
Yield: 69%; m.p.: 2198C; IR (KBr) [cm– 1]: 3231 (NH), 1346 (C=S);1H-
NMR (400 MHz, DMSO-d6) d [ppm]: 1.18–2.00 (m, 10H, CH2 pro-
tons), 3.55 (s, 1H, CH proton ), 4.75 (d, J = 6.0 Hz, 2H, -CH2-), 7.29–
7.86 (m, 10H, aromatic protons and NH), 8.34 (s, 1H, thiourea
NH), 9.83 (s, 1H, thiourea NH); MS (APCI): m/z 424 [M + 1], 390,
318, 317, 277, 275, 269, 267, 193.
N-{4-[5-Cyclohexylamino)-1,3,4-thiadiazole-2-yl)phenyl}-
N-{4-[5-Cyclohexylamino)-1,3,4-thiadiazole-2-yl)phenyl}-
N9-allylthiourea 13
N9-(2-phenylethyl)thiourea 7
Yield: 53%; m.p.: 209–2118C; IR (KBr) [cm– 1]: 3218 (NH), 1300
(C=S); 1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.12–2.15 (m, 10H,
CH2 protons), 3.56 (s, 1H, CH proton), 4.15 (s, 2H, CH2), 5.11 (d, J
=11.0 Hz, 1H, =CH2), 5.14 (d, J =17.0 Hz, 1H, =CH2), 5.73–6.03 (m,
1H, =CH-), 7.62–7.78 (m, 4H, aromatic protons) 7.85 (d, J = 7.3 Hz,
1H, NH), 8.03 (s, 1H, thiourea NH), 9.75 (s, 1H, thiourea NH); MS
(APCI): m/z 375 [M + 1], 374, 317, 275, 193.
Yield: 52%; m.p.: 2288C; IR (KBr) [cm– 1]: 3210 (NH),1300 (C=S); 1H-
NMR (400 MHz, DMSO-d6) d [ppm]: 1.15–2.15 (m, 10H, CH2 pro-
tons ), 2.89 (t, 2H, CH2), 3.43–3.62 (bs, 1H, CH proton ), 3.63–3.80
(bs, 2H, CH2 ), 7.10–7.37 (m, 5H, aromatic protons), 7.47 (d, J =
8.7 Hz, 2H, aromatic protons), 7.65 (d, J = 8.7 Hz, 2H, aromatic
protons), 7.80–8.05 (m, 2H, thiourea NH and secondary amine
NH), 9.80 (s, 1H, thiourea NH). Anal. Calc.for C23H27N5S2: C, 63.12;
H, 6.22; N, 16.00; S, 14.65. Found: C, 62.60; H, 6.20; N, 15.76; S,
14.42.
N-{4-[5-Cyclohexylamino)-1,3,4-thiadiazole-2-yl)phenyl}-
N9-(4-methylphenyl)thiourea 14
N-{4-[5-Cyclohexylamino)-1,3,4-thiadiazole-2-yl)phenyl}-
Yield: 91%; m.p.: 201–2028C; IR (KBr) [cm– 1]: 3203 (NH), 1316
N9-(4-nitrophenyl)thiourea 8
1
(C=S); H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.20–2.12 (m, 10H,
Yield: 75%; m.p.: 2208C; IR (KBr) [cm– 1]: 3225 (NH),1321 (C=S); 1H-
NMR (400 MHz, DMSO-d6) d [ppm]: 1.19–2.09 (m, 10H, CH2 pro-
tons), 3.53 (s, 1H, CH, proton), 7.69 (d, J = 7.9 Hz, 2H aromatic pro-
tons), 7.77 (d, J = 7.8 Hz, 2H, aromatic protons), 7.91 (d, J = 9.3 Hz,
2H, aromatic protons), 8.19 (d, J = 9.2 Hz, 2H, aromatic protons),
8.72 (s, 1H, NH), 10.55 (s, 1H, thiourea NH), 10.61 (s, 1H, thiourea
NH); MS (APCI): m/z 455 [M + 1], 317, 276, 275, 269, 81, 79.
CH2 protons), 2.29 (s, 3H, CH3 ), 3.51 (s, 1H, CH proton), 6.66–7.80
(m, 8H, aromatic protons), 8.11–8.38 (bs, 1H, NH), 9.90 (s, 1H, thi-
ourea NH), 9.95 (s, 1H, thiourea NH); MS (APCI): m/z 424 [M+1],
363, 317, 275, 193.
Pharmacology
Male and female adult Balb/C mice weighing 20–30 g were used.
The animals were housed in colongy cages, under standard labo-
ratory conditions, with free access to food and tap water. Room
temperature and relative humidity were maintained at 22 l 18C
and 60% respectively. A 12 hr/12 hr (8 a.m./8 p.m.) light-dark
cycle was used. All testing was conducted in the light phase.
After the adaption period of two days, experimental groups
were chosen randomly. Each mouse was used only once. The
experimental protocols were approved by the Animal Care and
Use Committee of Marmara University.
N-{4-[5-Cyclohexylamino)-1,3,4-thiadiazole-2-yl)phenyl}-
N9-(4-methoxylphenyl)thiourea 9
Yield: 85%; m.p.: 184–1868C; IR (KBr) [cm– 1]: 3203 (NH), 1321
1
(C=S); H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.06–2.10 (m, 10H,
CH2 protons), 3.60 (s, 1H, CH proton), 3.80 (s, 3H, OCH3), 6.90–
7.70 (m, 8H, aromatic protons), 8.30 (s, 1H, NH), 9.79 (s, 1H, thio-
urea NH), 9.87 (s, 1H, thiourea NH); MS (APCI): m/z 440 [M + 1],
317, 275, 193.
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Arch. Pharm. Chem. Life Sci. 2009, 342, 48–53
Anticonvulsant Activity of 1,3,4-Thiadiazole Thioureas
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Anticonvulsant activity
References
The anticonvulsant activity of the new compounds were deter-
mined by using PTZ and MES tests. These rodent models are
widely used as standard methods for predicting protection
against generalized tonic-clonic and generalized absence seiz-
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in 0.5% methyl cellulose and administered at the dose of 50 mg/
kg 30 minutes prior the tests. In case of anticonvulsant effect,
the same compounds were also used in doses of 25 and 100 mg/
kg intraperitoneally. Open field test for locomotor activity and
neurological examination for neurotoxicity in order to observe
possible changes in behavior have been done as previously
described [19, 20]. ED50 (effective dose 50) value for PTZ (60 mg/
kg) and CC50 (convulsive current 50) of animals and its 95% fidu-
cial limits were calculated by the method of Litchfield and Wil-
coxon [21].
˙
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shock, electrodes were attached to each animal's ears and the
animals lay on their backs, their tails being fixed. Thus observa-
tion of the tonic and clonic convulsions that appeared during
the seizure was ensured.
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