Synthesis and anticonvulsant activity of trans- and cis-2-(2,6- dimethylphenoxy)-N-(2- or 4-hydroxycyclohexyl)acetamides and their amine analogs

Article abstract of DOI:10.1016/j.bmc.2011.09.014

A group of trans- and cis-2-(2,6-dimethylphenoxy)-N-(2-hydroxycyclohexyl) acetamides (1-7) and -ethylamines (8-9) have been synthesized and investigated for their anticonvulsant activity. One of them, racemic trans-2-(2,6- dimethylphenoxy)-N-(2-hydroxycyclohexyl)acetamide proved to be the most effective in MES (mice, ip), exhibiting ED₅₀ = 42.97 mg/kg b.w. and TD₅₀ = 105.67 mg/kg b.w. It also proved protection in focal seizures (electric kindling, rats, ip) and it raises seizure threshold. The mechanism of action is inhibition of voltage-gated sodium currents and enhancement of GABA effect. Safety pharmacology assay on threshold tonic extension revealed no lowering of the seizure threshold.

Full text of DOI:10.1016/j.bmc.2011.09.014

Bioorganic & Medicinal Chemistry 19 (2011) 6927–6934
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and anticonvulsant activity of trans- and cis-2-(2,6-dimethylphen-
oxy)-N-(2- or 4-hydroxycyclohexyl)acetamides and their amine analogs
a
b,
⇑
Elzbieta Pe˛kala , Anna M. Waszkielewicz , Edward Szneler c, Maria Walczak d, Henryk Marona b
_
a Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian University-Medical College, 30-688 Krakow, Poland
b Department of Bioorganic Chemistry, Chair of Organic Chemistry, Faculty of Pharmacy, Jagiellonian University-Medical College, 30-688 Krakow, Poland
c Faculty of Chemistry, Jagiellonian University, 30-060 Krakow, Poland
d Department of Pharmacokinetics and Physical Pharmacy, Faculty of Pharmacy, Jagiellonian University-Medical College, 30-688 Krakow, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 May 2011
Revised 25 August 2011
Accepted 8 September 2011
Available online 14 September 2011
A group of trans- and cis-2-(2,6-dimethylphenoxy)-N-(2-hydroxycyclohexyl)acetamides (1–7) and -eth-
ylamines (8–9) have been synthesized and investigated for their anticonvulsant activity. One of them,
racemic trans-2-(2,6-dimethylphenoxy)-N-(2-hydroxycyclohexyl)acetamide proved to be the most effec-
tive in MES (mice, ip), exhibiting ED50 = 42.97 mg/kg b.w. and TD50 = 105.67 mg/kg b.w. It also proved
protection in focal seizures (electric kindling, rats, ip) and it raises seizure threshold. The mechanism
of action is inhibition of voltage-gated sodium currents and enhancement of GABA effect. Safety pharma-
cology assay on threshold tonic extension revealed no lowering of the seizure threshold.
Ó 2011 Elsevier Ltd. All rights reserved.
Dedicated to the memory of Professor
Marian Eckstein
Keywords:
Aminocyclohexanol
New amides
Epilepsy
Seizures
Anticonvulsant
NIH
MES
Preclinical
1. Introduction
anticonvulsant agents are discovered through conventional screen-
ing and/or structure modification rather than a mechanism–driven
Epilepsy is one of the major neurological disorders. Antiepilep-
tic drugs (AEDs) exert their action by various mechanisms, for
example, they can influence the inhibitory or excitatory neuro-
transmitter systems (GABA or glutamic and aspartic acid, respec-
tively), or the ion transport across cell membranes. Conventional
antiepileptic drugs: valproate, phenytoin, carbamazepine, or ben-
zodiazepines, are widely used but exhibit an unfavorable side
effect profile and fail to control seizures adequately. In the recent
years, several new drugs, such as: lamotrigine,1 pregabaline,2 briv-
aracetam,3 or lacosamide4 have been added to the list of therapeu-
tic agents against epilepsy. However, there is a significant group of
patients (up to 30%) who are resistant to the available antiepileptic
drugs.5 Hence, there is an unmet need to develop new AEDs exhib-
iting more selective activity and lower toxicity. Chemical diversity
and various mechanisms of action of anticonvulsants make it
difficult to find a common way of identifying new drugs. Novel
design. Drug identification is conducted via in vivo screening tests,
on the basis of seizure type.
Numerous new compounds are synthesized and screened for
their anticonvulsant activities each year. In order to make the dis-
covery of new AEDs more rational, several investigators identify
structural fragments that may enhance anticonvulsant properties
and allow orientating the synthesis of novel compounds, in which
some of these active fragments can appear.6 One of the structural
features that play a significant role in relation to antiepileptic
activity is an amide group.7–9
Lately, new AEDs have come up, exhibiting the above men-
tioned amide group. Among many examples there are valpromide
with its new generation valrocemide and valnoctamide,9–12 leveti-
racetam with brivaracetam and seletracetam,13–16 as well as
retigabine,17,18 ameltolide,19,20 and rufinamide21 (Fig. 1).
Structure–activity studies performed by our team have shown
that anticonvulsant activity could be derived from N-substituted
derivatives of appropriate chiral 1,2-aminoalkanols. As an
example, appropriate derivatives of (S)-(+)-amino-1-butanol22
and (R)-(ꢀ)-2-amino-1-propanol23 displayed anti-MES activity
⇑
Corresponding author. Tel./fax: +48 12 6205594.
E-mail address: awaszkie@cm-uj.krakow.pl (A.M. Waszkielewicz).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.014

6928
E. Pe˛kala et al. / Bioorg. Med. Chem. 19 (2011) 6927–6934
is performed on the asymmetric center and leads to an oxazolidine
H3C
H3C
derivative, due to presence of the rigid cyclohexane moiety. Cycli-
zation reactions in this case do not cause racemization nor reten-
tion, however, they cause isomerization. Closing the ring is
performed through cis-oxazoline, which after hydrolysis opens to
an appropriate ester. Then the acyl group is rearranged from the
oxygen atom (in the carboxyl group) to the amine group and as a
result appropriate cis-amides (4–6) are formed. The above reaction
proceeds as the effect of anchimeric support of the adjacent
group.25 Compounds 8 and 9 were achieved in two ways: by reduc-
tion of the amide 1 and 7, respectively, using LiAlH4 in diethyl
ether solution under nitrogen atmosphere at room temperature
(yield 50%) and by N-alkylation of trans-2-amino-1- or trans-2-
amino-4-cyclohexanol with 2,6-dimethylphenoxyethyl bromide
(yield 65%). Physico-chemical data of 1–9 are presented in Table 1.
O
N
O
NH2
H2N
Valpromide
Carbamazepine
H3C
O
N
H
O
O
CH3
CH3
H2N
NH
Ameltolide
N
H
NH2
2.2. Pharmacology
F
Retigabine
O
Molecular chirality is a phenomenon that plays fundamental
important role in biological processes. A wide range of biological
and physical functions are generated through precise molecular
recognition because enzymes, receptors, and other natural binding
sites within biological systems interact with different enantiomers
in decisively diverse ways. As a result of such chiral recognition,
drug enantiomers may differ in their pharmacokinetic handling
and their pharmacological and/or toxicological profiles. For a drug
with a single stereogenic center, both enantiomers may be phar-
macologically active. However, if the main pharmacological effect
is observed only for one enantiomer, several possibilities exist for
the other one: inactivity, a qualitatively different effect, an antag-
onistic effect, or severe toxicity. For this reason we designed and
studied the racemic form (trans-1), their enantiomers (trans-2,3)
and corresponding geometric isomers (cis-4, 5, 6).
NH2
F
H
H3C
O
N
N
N
NH2
N
F
O
Rufinamide
Levetiracetam
Figure 1. Structures of known anticonvulsants with an amide group.
with protective index (TD50/ED50) of 4.55 and 6.23, respectively.
The results correspond to those for phenytoin, carbamazepine,
and valproate.24 With respect to the anticonvulsant activity of
1,2-aminoalkanol derivatives the aim of the study was to test
whether binding of this moiety into a cyclic ring would enhance
or diminish anticonvulsant activity. Such modification resulted in
stereochemical variability, an important aspect in optimizing bio-
logical activity of drug candidates. Moreover, elongation of the al-
kyl moiety between the amine and hydroxyl groups from 2 to 4
carbons could provide an interesting discussion on structure–
activity relationship.
We herein report synthesis and anticonvulsant activity of new
compounds which are amide derivatives of chiral (racemic and
enantiomeric) trans- or cis-2-amino-1-cyclohexanol (1–6) as well
as achiral 2-amino-4-cyclohexanol (7), which contain the common
2,6-dimethylphenoxyacetyl core. In order to compare the potential
anticonvulsant activities, their amine analogs have been
synthesized, too (8–9).
The new compounds 1–9 were tested in vivo by using three
screens (mice, rats): the MES, ScMet (anticonvulsant tests) and
TOX (neurotoxicity, rotarod) (Tables 2 and 3). For the selected com-
pounds (1, 2, 4, and 8) advanced quantitative tests (ED50 and TD50
)
in mice and/or rats were performed (Table 2).
Compounds 1, 2, 4, 7, and 8 have been evaluated in the time-to-
peak-effect (TPE) assay in rats (Table 4). Among them, 1, 2, 4, and 8
are the most active 15 min after administration (TPE = 0.25 h)
which can serve as a premise that the compound itself and not a
metabolite is biologically active. As seen in the TPE assay, 1 has
not revealed any neurotoxicity in rats given the doses up to
500 mg/kg b.w. po, observed for 24 h. This can be interpreted as
no neurotoxicity of the compound nor its potential metabolites.
The most interesting anticonvulsant results were observed for
trans-racemic-2-(2,6-dimethylphenoxy)-N-((1R,2R)-2-hydrox-
ycyclohexyl)acetamide (1) and its amine derivative (8). Compound
1 displayed anti-MES activity with protective index (TD50/ED50 of
2.47 (mice, ip) and 21.21 (rats, po). 8 exhibited TD50/ED50 of 3.90
(mice, ip) and 19.06 (rats, po) corresponding with PI for valproate
(1.70 and 2.2, respectively).3
2. Results and discussion
2.1. Chemistry
Since compounds 2 and 3 are enantiomers of 1, it could be ex-
pected that one of the enantiomers should be chosen for further
development. The screening results in MES, mice, ip (Table 2) re-
veal that compound 2 (1S,2S) is the one active and less neurotoxic
than 3 (1R,2R) in the dose of 100 mg/kg b.w., 0.5 h after adminis-
tration, giving premises for choice of 2 for development. However,
further in quantitative analysis of ED50, TD50 and PI (Table 2) it
turned out that although 2 has more favorable ED50 than 1
(35.47 and 42.97 mg/kg b.w., respectively), the PI of 1 is more
favorable than 2, proving that the racemate (1) would be slightly
safer to use. Moreover, considering the above results, costs of pro-
viding of the enantiomer would not be justified compared to costs
of synthesizing the racemate (1).
The title compounds 1–3 (Scheme 1) were prepared in our lab-
oratory as a result of acylation of trans racemic, (1R,2R)- or (1S,2S)-
2-amino-1-cyclohexanol, using the appropriate acid chloride in bi-
phasic medium (water–toluene). K2CO3 was used as a proton
acceptor. Racemic trans-2-amino-1-cyclohexanol was obtained
from cyclohexene oxide in the reaction with 25% ammonia.25 The
racemic trans-2-amino-1-cyclohexanol was separated into its opti-
cally active S-(+) and R-(ꢀ) enantiomers, using diastereoisomeric
pairs of salts (hydrogen (+)-tartrates). The process was possible
due to the difference in solubility of the both isomers in ethanol
and methanol.26 Compounds 4–6 were obtained from appropriate
1–3 with use of SOCl2. The reaction of trans-2-aminocyclohexanol

E. Pe˛kala et al. / Bioorg. Med. Chem. 19 (2011) 6927–6934
6929
OH
CH3
O
CH3
O
CH3
O
O
O
O
OH
Cl
ACH
N
H
toluene / water
K2CO3
SOCl2
CH3
CH3
CH3
trans-1-3,7
CH3
O
SOCl2
isomerization
HO
NH
O
LiAlH4 /
diethyl ether
CH3
cis-4-6
CH3
CH3
O
O
OH
Br
ACH
toluene / K2CO3
NH
CH3
CH3
trans-8,9
1,2-ACH or 1,4-ACH racemate as hydrochloride
1R,2R-ACH enantiomer as hydrogen (+)-tartrate
1S,2S-ACH enantiomer as hydrogen (+)-tartrate
Scheme 1. Synthesis of alkanoloamides 1–7 and alkanoloamines 8–9.
Table 1
Physico-chemical data of N-acyl (1–7) and N-alkyl (8–9) derivatives of 2-amino-1- or 2-amino-4-cyclohexanol
D
Compds.
Stereoisomer
Racemic
(1S,2S)
Configuration
Mp [°C] crystal. solvent
Yield [%]
TLC Rf
0.68a
0.68a
0.68a
[
a
]
c [%]
Elemental analysis
H calc./found
C calc./found
N calc./found
1
2
3
trans
106–108
n-hexane/toluene
(1:1)
110–111
n-hexane/toluene
(1:1)
108–110
n-hexane/toluene
(1:1)
62
55
57
-
69.24
69.01
8.36
8.44
5.05
4.94
trans
3.176
5, CHCl3
69.24
69.07
8.35
8.34
5.05
5.19
(1R,2R)
trans
-3.259
5, CHCl3
69.24
69.26
8.35
8.65
5.05
4.94
4
Racemic
(1R,2S)
cis
88–90
60
48
52
57
65
87
64
0.79a
0.80a
0.80a
0.4b
-
69.24
69.42
69.24
69.19
69.24
69.38
69.42
69.50
64.09
63.87
72.94
72.83
64.09
63.88
8.35
8.52
8.35
8.41
8.35
8.57
8.35
8.36
8.74
8.47
9.57
9.39
8.74
8.75
5.05
5.17
5.05
5.23
5.05
5.93
5.05
5.06
4.69
4.86
5.34
5.41
4.69
4.49
n-hexane
125–127
n-hexane
126–128
n-hexane
160–162
n-hexane
90–91
5
cis
-31.186
5, CHCl3
31.06
5, CHCl3
-
6
(1S,2R)
cis
7
Racemic
Racemic
Racemic
Racemic
trans
trans
trans
trans
822
8a
9
0.52c
0.61c
0.62c
-
n-heptane
174–176
84–86
n-heptane
-
Molecular weight for compounds: 1–7: 277.34 g ꢁ molꢀ1 (C16H23NO3); 8–9: 263.37 g ꢁ molꢀ1 (C16H25NO2); 8a 299.83 g ꢁ molꢀ1 (C16H25NO2 xHCl).
a
CHCl3/acetone (7:3).
Toluene/acetone (5:1).
Toluene/methanol (5:1).
b
c
Therefore, among 1–3, compound 1 was chosen for quantitative
assays (ED50, TD50, PI) in rats, po (Table 2). Additionally, quantita-
tive studies in rats, po have been performed for compound 8 – the
amide analog of 1 (Table 2). Comparing the ED50s between 1 and 8,
it can be noticed that there is no real difference between them
(23.56 and 26.23 mg/kg/b.w., respectively), TD50s are >500 mg/kg
b.w. in both cases. Therefore, both compounds were advanced to
further testing.
Due to the structure similarity of 1 and 8 (common dimethyl-
phenol moiety) to mexiletine – a known antiarrythmic drug which
lowers seizure threshold – it is necessary to perform safety phar-
macology assays in order to exclude such effect from antiepileptic
drug candidates.27 Therefore, threshold tonic extension (TTE) test
was performed for 1 and 8, in order to assess whether the com-
pounds would enhance susceptibility to seizures. The results are
shown in Figures 2 and 3, respectively.

6930
E. Pe˛kala et al. / Bioorg. Med. Chem. 19 (2011) 6927–6934
Table 2
Anticonvulsant activity of N-acyl (1–7) and N-alkyl (8–9) derivatives of 2-amino-1- or 2-amino-4-cyclohexanol
B
C
D
Compds.
Dose mg/kg mice, ip
MES 0.5 h
MES 4 h
ScMET 0.5 h
ScMET 4 h
TOX 0.5 h
TOX 4 h
ASP classA
1
TD50
ED50
PI(MES)
131
30
0/1
2/3
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
3/5
1/1
0/1
3/5
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/1
0/1
0/1
1/1
0/1
0/1
0/4
2/8
4/4
0/4
0/8
0/4
0/4
2/8
2/4
0/4
0/8
0/4
0/4
0/8
0/4
0/4
0/8
2/4
0/4
7/8
4/4
0/4
0/4
4/4
8/8
4/4
0/4
7/8
4/4
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
105.61a
>500b
42.97a
23.56b
2.475a
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
30
3
>21.218b
231
331
4
0/1
1/3
1/1
0/1
0/3
1/1
0/1
0/3
1/1
0/1
1/3
1/1
0/1
2/3
1/1
0/1
3/3
1/1
0/4
3/4
1/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1
2
1
1
1
1
1
68.70a
35.47a
1.937a
nd
nd
nd
117.73a
nd
78.83a
nd
1.493a
nd
5
6
nd
nd
nd
7
nd
nd
nd
8
28.36a
>500b
7.73a
3.90a
10
30
100
26.23b
>19.06b
0/1
0/1
0/1
0/1
0/2
9
30
100
300
0/1
3/3
0/1
0/3
0/1
0/1
0/1
0/1
0/2
0/4
1
nd
nd
nd
PHTE
CBZE
VPAE
34.45a
>500b
47.8a
361b
6.48a
32.2b
9.85a
3.57b
287a
395b
6.60a
>22b
4.90a
101b
1.70a
2.2b
483a
859b
MES – maximal electroshock seizure: number of animals protected/number of animals tested; ScMET – subcutaneous pentylenetetrazole seizure: number of animals
protected/number of animals tested; TOX – neurotoxicity screen: number of animals exhibiting toxicity/number of animals tested in the rotorod test.
nd, not determined; PHT, phenytoin; CBZ, carbamazepine; VPA, valproate.
A
The ASP classification (Anticonvulsant Screening Project) is as follows: 1 – anticonvulsant activity at 100 mg/kg or less; 2 – anticonvulsant activity at doses greater than
100 mg/kg; 3 – compound inactive at 300 mg/kg; 4 – toxicity at doses 30 mg/kg.
B
TD50 – dose (mg/kg) eliciting evidence of minimal neurological toxicity in 50% of animals; 95% confidence interval is shown in parentheses; the slope regression line is
shown in brackets.
C
ED50 – dose (mg/kg) eliciting the MES protection in 50% of animals.
D
PI – neurotoxic dose/median effective dose (TD50/ED50) for an anticonvulsant test. In each case 32 mice or 32 rats were used for determination of ED50 and toxicity (TOX).
E
Data from Ref.3
a
Values for mice after ip administration.
Values for rats after po administration.
b
Table 3
Nevertheless, it can be stated that compound 1 does not have the
side effect of lowering seizure threshold. The doses tested were
Anticonvulsant Screening Project: phase VIa. The results in rats (dose 30 mg/kg, po)
equivalent to ED50 in MES (mice, ip) = 43 mg/kg and TD50 in MES
(mice, ip) = 106 mg/kg. It is interesting that compound 8 exhibits
the opposite effect – it lowers seizure threshold (Fig. 3), and the
difference between the structures is in the presence of the amide
group. Lowering seizure threshold may cause increasing suscepti-
bility of patients to seizures, therefore such effect eliminated com-
pound 8 from further development.
Compound 1 was advanced for further assays as the most prom-
ising one. It was investigated for prevention of expression and
acquisition of focal seizures in the hippocampal kindling test in
rats (Fig. 4). The greatest activity was observed 15 and 45 min after
administration (the Racine score of 5 decreased below 3 which is
considered protection).28 Therefore, compound 1 can be termed
‘antiepileptogenic’.29 The activity 15 min after administration is
consistent with TPE in the MES test, in spite of different routes of
administration.
Compds.
Test
Time [h]
1.0
0.25
0.5
2.0
4.0
1
2
3
4
7
8
MESa
TOXa
MES
TOX
MES
TOX
MES
TOX
MES
TOX
MES
TOX
3/4
0/4
1/4
0/4
0/4
0/4
2/4
0/4
4/4
0/4
1/4
0/4
3/4
0/4
3/4
0/4
1/4
0/4
0/4
0/4
4/4
0/4
3/4
0/4
2/4
0/4
2/4
0/4
1/4
0/4
0/4
0/4
3/4
0/4
1/4
0/4
2/4
0/4
2/4
0/4
1/4
0/4
0/4
0/4
3/4
0/4
1/4
0/4
2/4
0/4
1/4
0/4
1/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
a
see Table 1.
It can be observed in Figure 2 that the more dose of compound 1
is administered, the more pentylenetetrazole is needed to cause a
twitch or clonus, although the dependence is not linear.
In order to have the first insight into molecular mechanisms of
action, compound 1 was tested in electrophysiological studies. The
a

E. Pe˛kala et al. / Bioorg. Med. Chem. 19 (2011) 6927–6934
6931
Table 4
Anticonvulsant Screening Project: time to peak effect for 1, 2, 4, 7 and 8
Compds.
Test
Dose (mg/kg) Animals
(route of administration)
Time [h]
2.0
0.25
3/4
0.50
1/4
1.0
4.0
6.0
8.0
24
MESa
MES
TOXa
TOX
TOX
TOX
MES
MES
TOX
MES
MES
TOX
MES
TOX
TOX
TOX
MES
TOX
15
rats (po)
30
rats (po)
62.50
rats (po)
125
rats (po)
250
rats (po)
500
rats (po)
90
mice (ip)
130
mice (ip)
130
mice (ip)
60
mice (ip)
90
mice (ip)
90
mice (ip)
70
mice (ip)
110
mice (ip)
300
mice (ip)
100
rats (ip)
30
rats (ip)
30
4/4
0/2
0/2
0/2
0/2
8/8
4/4
8/8
8/8
4/4
7/8
3/4
3/8
8/8
7/8
4/4
3/4
4/4
0/2
0/2
0/2
0/2
7/8
4/4
0/8
1/8
4/4
4/8
0/4
3/8
8/8
8/8
4/4
2/4
1/4
0/2
0/2
0/2
0/2
1/4
0/2
0/2
0/2
0/2
1/4
0/2
0/2
0/2
0/2
1
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
2/4
0/8
0/4
0/8
0/4
2
4
0/4
3/8
0/4
1/4
2/8
0/4
0/4
2/8
0/8
5/8
7/8
4/4
0/4
0/8
5/8
0/4
0/4
0/8
7
8
0/4
0/4
rats (ip)
a
see Table 1.
purpose for the assays was to correlate with the data from the ani-
mal seizure models. The assays evaluate compound’s interactions
with sodium, glutamate or GABA ion channels.30 The methodology
uses whole cell patch-clamp electrophysiological measurement on
single neurons.
The assays performed on neuroblastoma N1E-115 cells revealed
voltage-gated sodium channel blocking effect at 100 lmol concen-
tration (Table 5), when voltage of ꢀ60 or ꢀ90 mV was used as
holding potential. The results show that activity measured as per-
cent of control stimulation (samples without the tested com-
pound) fall in the range for known antiepileptic sodium channel
blockers such as carbamazepine, lamotrigine, and phenytoin, with
statistical significance p <0.05. The shown activity is consistent
with in vivo activity in MES of compound 1.
Studies on murine cortical neurons examined neuronal re-
sponse to stimuli of inhibitory (GABA), as well as excitatory (NMDA
and kainate) ion channels in the presence of the tested compound.
The results presented in Table 6 proved enhancement of GABA ef-
fect as another mechanism of action of 1 (100 lmol concentration).
It is shown in the results that structural modification of com-
pounds containing 2,6-dimethylphenoxy group (present also in
antiarrythmic and anticonvulsant mexiletine) may reduce mexile-
tine’s adverse event of lowering seizure threshold. Moreover, sig-
nificant anticonvulsant activity can be observed among these
compounds. Therefore, further investigation within this group of
derivatives and further structural modifications of compound 1
are justified.
Figure 2. Results of threshold tonic extension test for 1. After compound 1 (ED50 in
MES (mice, ip) = 43 mg/kg and TD50 in MES (mice, ip) = 106 mg/kg) is administered
intraperitoneally to mice, infusion of pentylenetetrazol is started and lasts until the
first twich and the first clonus. The doses of administered PTZ were calculated from
the speed of infusion. As it can be seen, the higher dose of 1, the more PTZ is needed
to cause seizures. Therefore, 1 raises seizure threshold. Results are presented as
average of eight animals S.E.M.

6932
E. Pe˛kala et al. / Bioorg. Med. Chem. 19 (2011) 6927–6934
Table 6
Anticonvulsant Screening Project: electrophysiology studies for compound 1. Effect
on whole cell currents of mouse cortical neurons (14–21 days, in vitro). Part II
Effect evoked by:
1 mmol GABA
No. of cells
% of control ( SEM)
179 15*
5
6
6
10
l
mol NMDA +1
mol kainate
lmol glycine
104
98
6
3
100
l
Holding potential ꢀ70 mV; compound concentration:100
l
mol.
*
Significantly different from control, p <0.05; potent GABA enhancement.
2.3. Conclusions
The seven new compounds with an amide group and their two
amine analogs derived from chiral or achiral aminocyclohexanol
have been synthesized and tested for anticonvulsant activity. The
results of anticonvulsant screening revealed that all derivatives
were effective in the MES screen. The quantitative studies in mice
after ip administration showed that two compounds (1 and its
amine derivative 8) were more potent than valproate in the max-
imal electroshock test (MES). The highest activity was observed
for 1 with ED50 of 42.97 mg/kg (MES) and 8 with ED50 of
7.73 mg/kg (MES). Compound 1 can also be termed ‘antiepilepto-
genic’ and it raises seizure threshold. Its observed mechanisms of
action are block of sodium currents and enhancement of GABA
effect.
Figure 3. Results of threshold tonic extension test for 8. After compound 8 (ED50 in
MES (mice, ip) = 8 mg/kg and TD50 in MES (mice, ip) = 28 mg/kg) is administered
intraperitoneally to mice, infusion of pentylenetetrazol is started and lasts until the
first twitch and the first clonus. The doses of administered PTZ were calculated from
the speed of infusion. As it can be seen, the higher dose of 8, the less PTZ is needed
to cause seizures. Therefore, 8 lowers seizure threshold. Results are presented as
average of 8 animals S.E.M.; p value <0.05 at 28 mg/kg b.w.
3. Experimental
3.1. Chemistry
3.1.1. General
1H and 13C NMR spectra were recorded on a Bruker spectrome-
ter 500 or 125 MHz, respectively using the signal from DMSO in
DMSO-d6 and TMS in CDCl3 as internal standards. The IR spectra
were recorded on a Perkin Elmer or Jasco FT/IR 410 spectrometer
(KBr pellets). Melting points were determined using a Büchi
SMP-20 apparatus and are uncorrected. Analyses of C, H, N were
within 0.4% of the theoretical values. Analytical TLC was carried
out on precoated plates (silica gel, 60 F-254 Merck) using the sol-
vent systems: aCHCl3/acetone (7:3), btoluene/acetone (5:1) and
ctoluene /methanol (5:1). Spots were visualized with UV light.
Measurements of optical rotation ([a]D) were carried out on Jasco
DIP-1000 (k = D (546 nm)).
The reagents and solvents – except for the described above R-
(ꢀ) and S-(+)-trans-2-amino-1-cyclohexanol – were commercially
available materials of reagent grade.
Figure 4. Compound 1 was administered ip to fully kindled rats (score 5 in the
Racine’s scale) at the dose 100 mg/kg b.w. Seizures were induced 15, 45, 75, 105,
Isomeric trans-2-amino-1-cyclohexanols ½a 20
ꢂ
546: R-(ꢀ) = ꢀ40.2°;
and 135 min after administration. Scores below
3 are considered protection.
Therefore, the compound is effective 15–45 min after administration.
S-(+) = +40.2° and isomeric hydrogen (+)-tartrates of trans-2-ami-
no-1-cyclohexanol ½a 20
ꢂ546: R-(ꢀ) = ꢀ4.3°; S-(+) = +38.96° were ob-
tained according to literature.26 The initial 2,6-dimethylpheno
xyacetic acid (mp 128–130 °C) was obtained by a classical method
using the appropriate sodium phenolate and chloroacetic acid. 2,
6-dimethylphenoxyethyl bromide (bp 121–123 °C) was synthesized
from 2,6-dimethylphenoxyethanol with tribromophosphine.
Table 5
Anticonvulsant Screening Project: electrophysiology studies for 1. Anticonvulsant
drugs on voltage dependent sodium currents in N1E-115 neuroblastoma cells. Part I
Compds.
Holding potential [mV]
No. of cells
% of control ( SEM)
39 2*
85 5*
78 2**
67 5**
78 2**
3.1.2. General procedure for trans-amides (1–3, 7)
1
1
CBZ
LMG
PHT
ꢀ60
ꢀ90
ꢀ90
ꢀ90
ꢀ90
8
7
5
8
6
The appropriate salt of trans-2-amino-1- or trans-2-amino-4-
cyclohexanol (20 mmol) and 5.6 g (40 mmol) of powdered anhy-
drous K2CO3 was suspended in 50 mL of cooled water. Next, a
solution of 25 mmol of 2,6-dimethylphenoxyacetic chloride in
50 mL of dry toluene was added with vigorous stirring at
10–12 °C. The reaction mixture was stirred for 2 h at room temper-
ature, next it was heated to the boiling point and then left to cool
down. The precipitated amide deposit was filtered off, then it was
Drugs concentration: 100 lmol.
The results from several individual cells were averaged, the SEM was calculated and
statistical significance was determined using the Student’s t-test.
*
Significantly different from control, p <0.05.
Significantly different from control, p <0.001.
**

E. Pe˛kala et al. / Bioorg. Med. Chem. 19 (2011) 6927–6934
6933
stirred with a 10% solution of NaHCO3, and after drying it was
recrystallized from n-hexane or -hexane/toluene.
to warm to room temperature and left for 3 h. Then the mixture
was carefully poured into ca. 100 mL of distilled water. The result-
ing aqueous solution was filtered and the filtrate was refluxed for
10 min. Then the solution was cooled in an ice-salt-bath, filtered,
and potassium hydroxide (6 mol/L) was added to the filtrate in or-
der to yield the final cis-amide, which afterward was recrystallized
from n-hexane.
3.1.2.1. ( )-Trans-2-(2,6-dimethylphenoxy)-N-(2-hydroxycycloh
exyl)acetamide (1). IR (cmꢀ1): 3300, 1650, 1260; 1H NMR
(500.13 MHz, DMSO-d6): d 7.71 (1H, d, J = 7.8 Hz, NH), 7.03 (2H,
AA0BX3X03, JAB = 7.5 Hz; JAX = 0.6 Hz, H-3, H-5), 6.94 (1H, AA0BX3X03,
JAB = 7.5 Hz, H-4), 4.85 (1H, d, J = 5.3 Hz, OH), 4.21 (1H, d,
J = 14.2 Hz, Ar–O–CHH–), 4.16 (1H, d, J = 14.2 Hz, Ar–O–CHH), 3.54–
3.46 (1H, m, H –C–NH), 3.42–3.32 (1H, m, H–C–OH), 2.22 (6H, dt,
J = 0.6 Hz; J = 0.2 Hz, Ar–CH3), 1.92–1.79 (2H, m, H-60 (eq), H-30
(eq)), 1.68–1.54 (2H, m, H-50 (eq), H-40 (eq)), 1.30–1.14 (4H, m, H-30
(ax), H-40 (ax), H-50 (ax), H-60 (ax)); 13C NMR (500.13 MHz, DMSO-
d6): d 167.40 (CO), 154.93 (C-1), 130.24 (C-2, C-6), 128.69 (C-3, C-5),
124.02 (C-4), 70.76 (Ar–O–CH2–), 70.76 (C-10 (C–OH cyclohexane)),
54.28 (C-20 (C–NH cyclohexane)), 34.27 (C-60), 30.87 (C-50), 24.16
(C-40),23.83 (C-30), 15.90 (2x CH3).
3.1.3.1. ( )-Cis-2-(2,6-dimethylphenoxy)-N-(2-hydroxycyclohex
yl)acetamide (4). IR (cmꢀ1): 3420, 1640, 1260; 1H NMR (500.
13 MHz, DMSO-d6): d 7.50 (1H, d, J = 8.2 Hz, NH), 7.02 (2H, A2B,
J = 7.4 Hz; J = 7.4 Hz, H-3, H-5), 6.95 (1H, A2B, J = 7.4 Hz; H-4),
4.89 (1H, d, J = 4.0 Hz, OH), 4.24 (2H, d, J = 14.3 Hz, Ar–O–CHH–),
4.20 (2H, d, J = 14.3 Hz, Ar–O–CHH –), 3.75–3.79 (1H, m, H –C–
NH), 3.71–3.75 (1H, m, H–C–OH), 2.22 (6H, s, 2x Ar–CH3), 1.22–
1.73 (8H, m, cyclohexane), 13C NMR (500.13 MHz, DMSO-d6): d
166.72 (CO), 154.75 (C-1), 130.12 (C-2, C-6), 128.76 (C-3, C-5),
124.13 (C-4), 70.58 (Ar–O–CH2–), 66.50 (C-10 (C–OH cyclohexane)),
50.21 (C-20 (C–NH cyclohexane)), 31.84 (C-60), 26.54 (C-50), 23.71
(C-40), 19.12 (C-30), 15.91 (2x CH3).
3.1.2.2.(1S,2S)-Trans-2-(2,6-dimethylphenoxy)-N-(2-hydroxycyc
lohexyl)acetamide (2). IR (cmꢀ1): 3420, 3380, 1650, 1265; 1H NMR
(500.13 MHz, DMSO-d6): d 7.71 (1H, d, J = 7.7 Hz, NH), 7.02 (2H,
A2BX3Y03, JAB = 7.5 Hz; JAX = 0.6 Hz, JAY = 0.7 Hz, H-3, H-5), 6.94 (1H,
3.1.3.2. (1R,2S)-Cis-2-(2,6-dimethylphenoxy)-N-(2-hydroxycyclo
hexyl)acetamide (5). IR (cmꢀ1): 3420, 1650,1260; 1H NMR
(500.13 MHz, DMSO-d6): d 7.50 (1H, d, J = 8.2 Hz, NH), 7.02 (2H,
A2B, J = 7.5 Hz; J = 7.4 Hz, H-3, H-5), 6.95 (1H, A2B, J = 7.5 Hz; H-
4), 4.88 (1H, d, J = 4.0 Hz, OH), 4.25 (2H, d, J = 14.3 Hz, Ar–O–CH
H–), 4.20 (2H, d, J = 14.3 Hz, Ar–O–CHH–), 3.75–3.80 (1H, m, H–
C–NH), 3.71–3.75 (1H, m, H –C–OH), 2.22 (6H, s, 2x Ar–CH3),
1.22–1.73 (8H, m, cyclohexane), 13C NMR (500.13 MHz, DMSO-
d6): d 166.72 (CO), 154.74 (C-1), 130.13 (C-2, C-6), 128.76 (C-3,
C-5), 124.14 (C-4), 70.56 (Ar–O–CH2–), 66.48 (C-10 (C-OH cyclohex-
ane)), 50.20 (C-20 (C–NH cyclohexane)), 31.84 (C-60), 26.54 (C-50),
23.72 (C-40), 19.11 (C-30), 15.92 (2x CH3).
0
A2 BX3Y03, JAB = 7.5 Hz; H-4), 4.57 (1H, d, J = 5.2 Hz, OH), 4.22 (2H,
d, J = 14.2 Hz, Ar–O–CHH–), 4.15 (2H, d, J = 14.2 Hz, Ar–O–CHH –),
3.56–3.43 (1H, m, H–C–NH), 3.43–3.33 (1H, m, H–C–OH), 2.22 (3H,
d, J = 0.6 Hz, Ar–CH3), 2.22 (3H, d, J = 0.7 Hz, Ar–CH3) 1.91–1.80
(2H, m, H-60 (eq), H-30 (eq)), 1.67–1.54 (2H, m, H-50 (eq), H-40 (eq)),
1.29–1.12 (4H, m, H-30 (ax), H-40 (ax), H-50 (ax), H-60 (ax)); 13C
NMR (500.13 MHz, DMSO-d6): d 167.40 (CO), 154.93 (C-1), 130.24
(C-2, C-6), 128.69 (C-3, C-5), 124.02 (C-4), 70.76 (Ar–O–CH2–),
70.76 (C-10 (C–OH cyclohexane)), 54.28 (C-20 (C–NH cyclohexane)),
34.28 (C-60), 30.87 (C-50), 24.17 (C-40), 23.84 (C-30), 15.91 (2x CH3).
3.1.2.3. (1R,2R)-Trans-2-(2,6-dimethylphenoxy)-N-(2-hydroxycy
clohexyl)acetamide (3). IR (cmꢀ1): 3290, 1650, 1260; 1H NMR
(500.13 MHz, DMSO-d6): d 7.76 (1H, d, J = 7.9 Hz, NH), 7.03 (2H,
AA0BX3X03, JAB = 7.5 Hz; JAX = 0.8 Hz, H-3, H-5), 6.94 (1H, AA0BX3X03,
JAB = 7.5 Hz; H-4), 4.61 (1H, d, J = 5.4 Hz, OH), 4.22 (2H, d,
J = 14.2 Hz, Ar–O–CH H–), 4.16 (2H, d, J = 14.2 Hz, Ar–O–CHH –),
3.53–3.46 (1H, m, H –C–NH), 3.41–3.33 (1H, m, H –C–OH), 2.22
(6H, d, J = 0.8 Hz, Ar–CH3), 1.91–1.80 (2H, m, H-60 (eq)), H-30
(eq)), 1.68–1.54 (2H, m, H-50 (eq), H-40 (eq)), 1.30–1.13 (4H, m,
H-30 (ax), H-40 (ax), H-50 (ax), H-60 (ax)); 13C NMR (500.13 MHz,
DMSO-d6): d 167.40 (CO), 154.93 (C-1), 130.24 (C-2, C-6), 128.69
(C-3, C-5), 124.01 (C-4), 70.76 (Ar–O–CH2–), 70.76 (C-10 (C–OH
cyclohexane)), 54.28 (C-20 (C–NH cyclohexane)), 34.27 (C-60),
30.87 (C-50), 24.16 (C-40), 23.84 (C-30), 15.90 (2x CH3).
3.1.3.3. (1S,2R)-Cis-2-(2,6-dimethylphenoxy)-N-(2-hydroxycyclo
hexyl)acetamide (6). IR (cmꢀ1): 3420, 3360, 1650, 1260; 1H NMR
(500.13 MHz, DMSO-d6): d 7.48 (1H, d, J = 8.1 Hz, NH), 7.03 (2H,
A2BX6, JAB = 7.4 Hz; JAX = 0.6 Hz; JBX = 0.2, H-3, H-5), 6.94 (1H,
A2BX6, JAB = 7.4 Hz; JAX = 0.6 Hz; JBX = 0.2; H-4), 4.85 (1H, d,
J = 4.3 Hz, OH), 4.26 (2H, d, J = 14.3 Hz, Ar–O–CHH–), 4.18 (2H, d,
J = 14.3 Hz, Ar–O–CHH), 4.69–4.81 (2H, m, H–C–NH + H–C–OH),
2.22 (6H, td, J = 0.6 Hz; J = 0.2 Hz, 2x Ar–CH3), 1.20–1.75 (8H, m,
cyclohexane), 13C NMR (500.13 MHz, DMSO-d6): d 166.72 (CO),
154.74 (C-1), 130.12 (C-2, C-6), 128.76 (C-3, C-5), 124.13 (C-4),
70.58 (Ar–O–CH2–), 66.50 (C-10 (C–OH cyclohexane)), 50.21 (C-20
(C–NH cyclohexane)), 31.84 (C-60), 26.55 (C-50), 23.71 (C-40),
19.12 (C-30), 15.91 (2x CH3).
3.1.2.4. ( )-Trans-2-(2,6-dimethylphenoxy)-N-(4-hydroxycycloh
exyl)acetamide (7). IR (cmꢀ1): 3374, 3285, 1652, 1265, 1238; 1H
NMR (500.13 MHz, DMSO-d6): d 7.80 (1H, d, J = 8.2 Hz, NH), 6.97–
7.06 (2H, m, H-3, H-5), 6.90–6.96 (1H, m, H-4), 4.49 (1H, d,
J = 4.4 Hz, OH), 4.16 (2H, s, O–CH2–C@O), 3.57–3.70 (1H, m, C-40
(H–C–OH (cyclohexane))), 3.33–3.43 (1H, m, C-10 (H–C–OH (cyclo-
hexane))), 2.22 (6H, s, 2x Ar–CH3), 1.79–1.86 (2H, m, C-30 (eq), C-50
(eq)), 1.71–1.79 (2H, m, C-20 (eq), C-60 (eq)), 1.32–1.43 (2H, m, C-30
(ax), C-50 (ax)), 1.16–1.27 (2H, m, C-20 (ax), C-60 (ax)); 13C NMR
(500.13 MHz, DMSO-d6): d 167.35 (CO), 155.49 (C-1), 130.72 (C-
2, C-6), 129.20 (C-3, C-5), 124.52 (C-4), 71.28 (Ar–O–CH2–), 68.54
(C-40 (CH–OH cyclohexane)), 47.45 (C-10 (CH–NH cyclohexane)),
34.49 (C-30), 30.87 (C-50), 30.54 (C-20,C-60), 16.36 (2x CH3).
3.1.4. General procedure for trans-alkanoloamines (8–9)
2,6-Dimethylphenoxyethyl bromide (20 mmol) was dissolved
in 80 mL of toluene. Then 24 mmol of appropriate trans-2-amino-
1- or trans-2-amino-4-cyclohexanol was added and refluxed for
5–6 h in presence of 16 mmol of anhydrous K2CO3. The inorganic
salt precipitate was filtered from the hot mixture and washed with
hot toluene (2 ꢃ 10 mL). The precipitate was separated from the
cooled filtrate, filtered off and recrystallized from n-heptane. Com-
pound 8 was converted into salt (hydrochloride – 8a) in n-propanol
with excess of EtOH saturated with HCl.
3.1.4.1. ( )-Trans-2-(2-(2,6-dimethylphenoxy)ethylamino)cyclo-
hexanol (8). IR (cmꢀ1): 3293, 3176, 3021, 1288, 1265, 1234, 1207;
1H NMR (500.13 MHz, DMSO-d6): d 7.00 (2H, d, J = 7.5 Hz, H-3, H-5),
6.89 (1H, d, J = 7.5 H-4), 4.42 (1H, d, J = 5.1 Hz, OH), 3.75–3.84 (2H, m,
Ar–O–CH2), 3.08–3.17 (1H, m, H–C–OH (cyclohexane)), 2.88–2.95
(1H, m, H –C–NH), 2.78–2.86 (1H, m, CHH–N), 2.36 (1H, bbs, NH),
2.22–2.27 (1H, m, N–CH), 2.22 (6H, s, 2x Ar–CH3), 1.87–1.96 (1H,
3.1.3. General procedure for cis-amides (4–6)
The appropriate trans-2-(2,6-dimethylphenoxy)-N-(2-hydrox-
ycyclohexyl)acetamide (10 mmol) was added in small portions to
5 mL of thionyl chloride (cooled to 0 °C). The mixture was allowed

6934
E. Pe˛kala et al. / Bioorg. Med. Chem. 19 (2011) 6927–6934
m, cyclohexane), 1.75–1.86 (1H, m, (cyclohexane (ax))), 1.55–1.68
(1H, m, 0.97–1.22, (cyclohexane (ax))), 1.09–1.27 (2H, m, (cyclohex-
ane (ax))), 0.87–1.00 (3H, m, (cyclohexane (eq))), 0.88–0.89 (1H, m,
(cyclohexane (eq))); 13C NMR (500.13 MHz, DMSO-d6): d 155.50 (C-
1), 130.30 (C-2, C-6), 128.67 (C-3, C-5), 123.54 (C-4), 72.72 (Ar–O–
CH2–), 71.79 (C-10 (CH–OH cyclohexane)), 63.08 (C-20 (CH–NH
cyclohexane)), 46.73 (–CH2–N) 34.11 (C-60), 29.92 (C-50), 24.26 (C-
40), 24.14 (C-30), 15.99 (2x CH3).
was given another electrical stimulus of the mentioned parameters.
The behavior of the animal is again described by the scale of Racine.5
Electrophysiology studies were performed on single N1E-115 cells
and the murine cortical neurons were of patch-clamp type. The cells
were obtained from cultures, and the whole cell recordings were
performed on primary cultured neurons using borosilicate glass
electrodes as described by Hamill et al.30
The activity of studied compounds is presented in Tables 2–5
and Figures 2–4.
3.1.4.2. ( )-Trans-4-(2-(2,6-dimethylphenoxy)ethylamino)cyclo-
hexanol (9). IR (cmꢀ1): 3258, 3083, 3036, 1265, 1228, 1210; 1H
NMR (500.13 MHz, DMSO-d6): d 6.99 (2H, d, J = 7.4 Hz, H-3, H-5),
6.88 (1H, d, J = 7.4 H-4), 4.42 (1H, d, J = 3.4 Hz, OH), 3.75 (2H, t,
J = 5.7 Hz, Ar–O–CH2–), 3.32–3.44 (1H, m, H–C–NH (cyclohexane)),
2.21 (6H, s, 2x Ar–CH3), 1.74–1.89 (4H, m, (C-30 (eq), C-50 (eq), C-20
(eq), C-60 (eq)), 1.61 (1H, bs, NH), 1.22–1.11 (2H, m, C-30 (ax), C-50
(ax)), 1.09–0.97 (2H, m, C-20 (ax), C-60 (ax)); 13C NMR (500.13 MHz,
DMSO-d6): d 155.98 (C-1), 130.67 (C-2, C-6), 129.08 (C-3, C-5),
123.92 (C-4), 72.35 (Ar–O–CH2–), 69.24 (C-40 (CH–OH cyclohex-
ane)), 56.12 (C-40 (CH–NH cyclohexane)), 47.06 (–CH2–N) 34.27
(C-30, C-50), 31.39 (C-20, C-60), 16.35 (2x CH3).
Acknowledgments
The authors are grateful to J. Stables Ph.D for providing the
pharmacological data through the Antiepileptic Drug Development
program at the National Institutes of Neurological Disorders and
´
Stroke (Rockville, USA) and would like to thank Professor K. Kiec-
Kononowicz for coordination of the cooperation between the Na-
tional Institutes of Neurological Disorders and Stroke and the Fac-
ulty of Pharmacy, Jagiellonian University-Medical College.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.09.014.
3.2. Pharmacology
Antiepileptic activity and neurotoxicity assays were carried out
by the Antiepileptic Drug Development Program, Epilepsy Branch,
National Institute of Neurological and Communicative Disorders
and Stroke, National Institutes of Health in Rockville, USA. Com-
pounds were injected as suspensions in 0.5% methylcellulose at
three dosage levels (30, 100 and 300 mg/kg b.w.) intraperitoneally
(ip) to mice, and orally (po) to rats at dosage rates of 30 mg/kg
b.w. Phase I of the evaluation was a qualitative assay which used
small groups of animals (1–8) and included three tests: maximal
electroshock seizure (MES), subcutaneous pentylenetetrazol test
(ScMet), and neurotoxicity (rotarod), noted at 30 min and 4 h after
administration. The MES seizures were elicited by 60 Hz alternating
current at 50 mA (mice) or 150 mA (rats) delivered for 0.2 s via cor-
neal electrodes. A drop of 0.9% NaCl solution was placed into each
eye prior to applying the electrodes. Protection in the MES test
was defined as the abolition of the hindlimb tonic extension compo-
nent of the seizure. The ScMet was conducted by administration
85 mg/kg of pentylenetetrazole dissolved in 0.9% NaCl solution into
the posterior midline of mice. A minimal time of 30 min subsequent
to subcutaneous administration of pentylenetetrazole was used for
seizure detection. A failure to observe even a threshold seizure (a
single episode of clonic spasm of at least 5 s in duration) was re-
garded as protection. Neurological deficit was measured in mice
by the rotarod test. The mouse was placed on a 1 inch diameter
knurled plastic rod rotating at 6 rpm. Neurotoxicity was indicated
by the inability of the animal to maintain equilibrium on the rod
for at least 1 min in each of the three trials. In rats, neurological def-
icit was indicated by ataxia and loss of placing response and muscle
tone. Anticonvulsant quantification, that is, the doses of drug re-
quired to produce the biological responses in 50% of animals
(ED50), and the respective 95% confidence intervals, were deter-
mined for selected compounds displaying sufficient antiepileptic
activity and low neurotoxicity in the above primary evaluations,
by means of a computer program using probit analysis. Inhibition
of focal seizures was tested using hippocampal kindling in rats
(ip). Rat’s hippocampus was kindled with 50 Hz alternating current
at 200 mA per 1 s for 10 s every 0.5 h, for 6 h (12 cycles per day). This
procedure was repeated every other day so that within 10 days the
rat received 60 cycles, and as a result the animal developed 5th stage
of seizures described by Racine from 1 (corresponding with aura in
human) to 5 (corresponding with full tonic–clonicseizures). A tested
substance was administered ip and after the TPE time the animal
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