Synthesis and anticonvulsant activity of new N-Mannich bases derived from 5-cyclopropyl-5-phenyl-hydantoins

Article abstract of DOI:10.1002/ardp.201000241

Synthesis, physicochemical and anticonvulsant properties of new N-Mannich bases 3-24 derived from 5-cyclopropyl-5-phenyl- and 5-cyclopropyl-5-(4- chlorophenyl)-hydantoins were described here. Initial anticonvulsant screening was performed using intraperitoneal (i.p.) maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizures tests. Selected derivatives were also screened in the 6-Hz test. The neurotoxicity was determined applying the rotorod test. The pharmacological results revealed that the majority of compounds were effective in MES and/or scPTZ tests. The quantitative studies after oral administration into rats showed that several molecules were more potent than phenytoin and ethosuximide which were used as reference antiepileptic drugs. From the whole series the most active was 3-[(4-phenylpiperazin-1-yl)-methyl]-5-cyclopropyl-5-phenyl-imidazolidine-2, 4-dione (3) with the ED₅₀ value of 5.29 mg/kg in the MES test.

Full text of DOI:10.1002/ardp.201000241

Arch. Pharm. Chem. Life Sci. 2011, 11, 231–241
231
Full Paper
Synthesis and Anticonvulsant Activity of New N-Mannich
Bases Derived from 5-Cyclopropyl-5-phenyl-hydantoins
´
Hanna Byrtus, Jolanta Obniska, Anna Czopek, and Krzysztof Kaminski
Department of Medicinal Chemistry, Jagiellonian University, Medical College, Krakow, Poland
Synthesis, physicochemical and anticonvulsant properties of new N-Mannich bases 3–24 derived from
5-cyclopropyl-5-phenyl- and 5-cyclopropyl-5-(4-chlorophenyl)-hydantoins were described here. Initial
anticonvulsant screening was performed using intraperitoneal (i.p.) maximal electroshock (MES) and
subcutaneous pentylenetetrazole (scPTZ) seizures tests. Selected derivatives were also screened in the
6-Hz test. The neurotoxicity was determined applying the rotorod test. The pharmacological results
revealed that the majority of compounds were effective in MES and/or scPTZ tests. The quantitative
studies after oral administration into rats showed that several molecules were more potent than
phenytoin and ethosuximide which were used as reference antiepileptic drugs. From the whole series
the most active was 3-[(4-phenylpiperazin-1-yl)-methyl]-5-cyclopropyl-5-phenyl-imidazolidine-2,4-dione
(3) with the ED50 value of 5.29 mg/kg in the MES test.
Keywords: Anticonvulsant activity / 5-Cyclopropyl-5-phenyl-hydantoins / Imidazolidine-2,4-diones / N-Mannich bases
Received: August 24, 2010; Revised: October 13, 2010; Accepted: October 15, 2010
DOI 10.1002/ardp.201000241
Introduction
The rational design of new anticonvulsants is based on the
use of different pharmacophores that were established
through the analysis of structural characteristics of clinically
effective AEDs as well as other anticonvulsant active com-
pounds. Their appearance in the structure may determine or
enhance anticonvulsant properties. Thus one of the impor-
tant core fragments is defined by a nitrogen heteroatomic
system, usually a cyclic imide, at least of one carbonyl group
and phenyl or alkyl groups attached to the heterocyclic
system [4–6]. This common template is present in the struc-
tures of old generation of AEDs such as phenobarbital (I),
ethosuximide (II) or phenytoin (III) as well as in the newest
drugs e.g. levetiracetam (IV) and its analogues, being cur-
rently under the clinical trials, namely brivaracetam (V) or
seletracetam (VI) [7–9] (Fig. 1).
Epilepsy is a common neurological disorder with chronic and
progressive course that affects about 1% of the world’s popu-
lation. It has numerous causes, may occur at any age, typi-
cally require lifelong treatment and may significantly limit
patient autonomy and quality of life. Despite the increasing
understanding of the pathogenesis of seizures up to 30% of
epilepsies are poorly treated with the available antiepileptic
drugs (AEDs) [1]. Moreover, a large number of new AEDs
marked during recent years, did not change the proportion
of patients responding to the treatment and many of those
medications cause serious side effects, which include ataxia,
nausea, mental dulling and hepatotoxicity [2, 3]. The ideal
antiepileptic would prevent seizures without producing side
effects that adversely affect the patient’s quality of life.
Therefore with all of above findings in mind the continued
search for safer and more effective AEDs is urgently necessary.
Previous researches in our group have identified the differ-
ently substituted pyrrolidine-2,5-diones or imidazolidine-2,4-
diones as targets for new antiepileptic drugs. Many of these
compounds were effective in the maximal electroshock (MES)
or/and subcutaneous pentylenetetrazole (scPTZ) screens that
are the most popular seizure models in the early stages of
testing of new anticonvulsants. The SAR studies have dem-
onstrated the potent anticonvulsant activity among the N-
Mannich bases containing the differently substituted arylpi-
perazines as amine function [10–12] (Fig. 2). These amines
Correspondence: Jolanta Obniska, Department of Medicinal Chemistry,
Jagiellonian University Medical College, Medyczna 9, 30-688 Krakow,
Poland.
E-mail: mfobnisk@cyf-kr.edu.pl
Fax: þ48 12 657-02-62
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

232
H. Byrtus et al.
Arch. Pharm. Chem. Life Sci. 2011, 11, 231–241
and 5-cyclopropyl-5-(4-chlorophenyl)-hydantoins (2) were
O
obtained from the cyclopropyl-phenyl- or cyclopropyl-
H
N
´´
4-chlorophenyl-ketones by means of the Bucherer–Berg
NH
O
O
reaction with modification described by Goodson et al.
[16]. In the next step twenty four new Mannich bases with
the 5-cyclopropyl-5-phenyl- (3–10, 19–21) and 5-cyclopropyl-
5-(4-chlorophenyl)-hydantoin (11–18, 22–24) as a core fragment
were prepared. The aminoalkylation of the acidic proton
(N3H) in the imidazolidine-2,4-dione ring was carried out
in the presence of formaldehyde (40% solution) and variously
substituted 4-aryl- (3–6, 11–14) or 4-benzyl-piperazines (7–10,
15–18). The 4-arylpiperazine fragment was also replaced by
4-methyl- (19, 22), 4-ethyl- (20, 23), and 4-(2-hydroxy)-ethyl-
pipreazines (21, 24). The reaction was carried out in ethanol
at a room temperature for ca. 6–12 h. The crude products,
as racemic mixtures, were crystallized from ethanol giving
the final compound in yield about 70%. Their purities were
assessed by TLC chromatography and the structures were
confirmed by both spectral and elemental analysis.
The detailed physical and analytical data are listed in the
experimental section.
N
H
O
N
O
N
H
O
O
H
Phenytoin (III)
Phenobarbital (I)
Ethosuximide (II)
F
F
O
N
O
N
O
N
NH2
NH2
NH2
H
H
H
O
O
O
Levetiracetam (IV)
Brivaracetam (V)
Seletracetam (VI)
Figure 1. Structures of known anticonvulsants.
were presented in structures of many other anticonvulsant
active agents [13–15].
Furthermore the comparison of pharmacological results
obtained for pyrrolidine-2,5-diones and respective imidazoli-
dine-2,4-dione analogues revealed higher activity of com-
pounds bearing in their structures the latter core
fragment [13].
Anticonvulsant activity
The preclinical discovery and development of new chemical
agents for the treatment of epilepsy are based mainly on the
use of predictable animal models. Such models fall into two
main categories, namely models of acute seizures (non-epi-
leptic animals induced to have a seizure by an electrical or
chemical stimulus) and models of chronic epilepsy (animals
induced to have enhanced seizure susceptibility or spon-
taneous seizures). At the present time there are three in-vivo
screens used routinely that include the maximal electro-
shock seizure (MES), the subcutaneous pentylenetetrazole
(scPTZ) and the kindling model. From these tests the MES
and scPTZ screens are recognized as the ‘‘gold standards’’ in
the early stages of testing. Furthermore, the MES and scPTZ
tests are claimed to detect compounds affording protection
Taking into consideration the above findings in the present
study a library of twenty four new N-Mannich bases derived
from 5-cyclopropyl-5-phenyl- or 5-cyclopropyl-5-(4-chloro-
phenyl)-hydantoins containing the 4-substituted piperazines
as amine function has been synthesized. The results of anti-
convulsant screening enabled the evaluation of structure-
activity relationships in this series of derivatives.
Results and discussion
Chemistry
The synthesis of compounds 1–24 was accomplished as
shown in Scheme 1. The starting 5-cyclopropyl-5-phenyl- (1)
H
N
CF3
O
O
N
N
N
O
O
N
N
N
VII
VIII
ED50 = 9.00 mg/kg (MES test)
PI > 27
ED50 = 1,88 mg/kg (MES test)
PI > 265
Figure 2. Structures of active compounds VII and VIII obtained in previous studies.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2011, 11, 231–241
Anticonvulsant Activity of Certain Hydantoins
233
R = H, 19-21
R = Cl, 22-24
R
H
N
O
N
O
N
R
N
R2
R
b
19-24
R2
cmpd
H
N
a
-CH3 19, 22
-C2H5 20, 23
O
O
N
O
H
-(CH2)2-OH 21, 24
c
1, R = H 2, R = Cl
R
H
N
O
N
O
R = H, 3-10
R = Cl, 11-18
R1
N
N
X
3-18
R1
cmpd
X
H
3, 11
4, 12
5, 13
-
-
-
-
2-F
4-F
6, 14
7, 15
8, 16
9, 17
10, 18
3-CF3
H
-CH2-
-CH2- 2,6-Cl
4-Cl
-CH2-
-CH2-
3-CH3
Reagents and reaction conditions: (a) KCN, (NH4)2CO3, 50% ethyl alcohol; (b, c) 4-substituted piperazines,
formaldehyde, 96% ethyl alcohol, 6–12 h room temperature.
Scheme 1. Synthetic procedures of compounds 3–24.
against generalized tonic-clonic seizures and generalized
absence seizures, respectively [17, 18].
tests used. The activity in the MES test indicates the ability
of substance to prevent seizure spread. The anti-MES pro-
tection was observed at a dose of 30 mg/kg: 5 and 7; 100 mg/kg:
3, 4, 6, 8–11, 13–15, and 19–21 or 300 mg/kg: 17, 18, 23, and
24. Compounds 3, 5–8, 10, 15, 18, 20, 21, 23, and
24 were found to be active in the scPTZ test which identifies
substances elevating seizure threshold. Among these 3, 5–
8, 10, 15, 18, 20, 21, 23, and 24 were active at a dose of
100 mg/kg or 300 mg/kg comparable to ethosuximide
used as reference anticonvulsant. The activity in MES or
scPTZ screens was observed mainly at 4 h or in both time
intervals that indicates long duration of anticonvulsant
action.
The profile of anticonvulsant activity of all examined com-
pounds (3–24) was established in the MES and scPTZ tests,
after intraperitoneal (i.p.) injection into mice at doses of 30,
100, and 300 mg/kg. An observation was carried out at two
different time intervals, namely 0.5 h and 4 h. The acute
neurological toxicity (NT) was determined in the minimal
motor impairment–rotorod screen. The results are shown in
Tables 1 and 2.
The anticonvulsant evaluation showed that majority of
compounds were effective in MES or/and scPTZ screens.
In the whole series only 16 and 22 were inactive in both
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

234
H. Byrtus et al.
Arch. Pharm. Chem. Life Sci. 2011, 11, 231–241
Table 1. The results after intraperitoneal administration to mice (3–18).
H
N
O
R1
N
O
N
N X
R
3-18
Comp.
R
R1
X
MESa
scPTZb
NTc
0.5 h
4 h
0.5 h
4 h
0.5 h
4 h
3
4
5
6
7
8
9
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
2-F
4-F
3-CF3
H
2,6-Cl
4-Cl
3–CH3
H
2-F
4-F
3-CF3
H
2,6-Cl
4-Cl
3–CH3
–
–
–
–
–
–
300
–
100
100
–
300
–
100
100
30
100
30
100
100
100
100
300
100
100
100
–
300
–
–
–
100
–
–
100
–
–
–
–
–
–
–
–
–
100
–
–
–
–
100
100
300
–
3001,14
–
300
300
–
–
–
10025
30025
100
30025
–
100
300
30014
30014
30014,33
–
–CH2
–CH2–
–CH2–
–CH2–
–
–
–
–
–CH2
–CH2
–CH2–
–CH2–
10
11
12
13
14
15
16
17
18
PHTd
ETXd
300
–
–
–
–
30014
–
–
–
–
300
–
300
30014
–
30014,33
100
100
–
–
–
–
–
30
–
300
–
300
300
30
–
–
30025
–
30
100
–
–
300
Doses of 30, 100, and 300 mg/kg were administrated intraperitoneally in mice. The figures indicate the minimum dose whereby
anticonvulsant activity or neurotoxicity was demonstrated in 100% of the animals. A dash indicates the absence of anticonvulsant
a
activity and neurotoxicity at the maximum dose administered (300 mg/kg). Maximal electroshock test. Subcutaneous penty-
b
c
lenetetrazole test. Neurotoxicity screening using rotorod test. PHT – phenytoin, ETX – ethosuximide reference drugs, data from
d
1
[23]. Response comments: death, unable to grasp rotorod, myoclonic jerks, tremors.
14
25
33
In the neurotoxicity screen (NT), active compounds 3, 4, 10,
13, and two inactive derivatives 16, 22 did not show neuro-
toxicity at the highest dose administered (300 mg/kg).
Compounds 5–9, 11, 12, 14, 15, 17, 19–21, and 23 exhibited
motor impairment at a dose of 100 mg/kg and/or 300 mg/kg,
whereas 18 and 24 at 30 mg/kg.
Compounds 3, 5, 7, 8, 10, and 14 protected 100% of rats at
2 h or/and 4 h and from 25% to 75% of animals in other time
intervals. These molecules showed comparable activity to
phenytoin used as reference drug. Satisfactory protection
was also observed for 11 and 20. Except of compound 5 all
other were non-neurotoxic when given orally.
A valuable property of a candidate anticonvulsant is its
ability to inhibit convulsions when given by the oral route.
This screen discloses the time of onset, the approximate time
of peak effect (TPE) and the duration of anticonvulsant
activity or neurotoxicity. Therefore on the basis of the data
obtained in i.p. screen in mice and according to the anticon-
vulsant screening project (ASP) disposition, twelve com-
pounds 3, 5, 7–11, 13–15, 20, and 24 were examined for
their activity (MES test) and neurotoxicity after p.o. adminis-
tration into rats at a dose of 30 mg/kg. The results obtained
are presented in Table 3.
Compounds 7 and 10 both active in pentylenetetrazole
seizures in mice were tested in the scPTZ screen after p.o.
administration in rats (dose of 50 mg/kg). As shown in Table 4
these molecules revealed comparable, however, longer pro-
tection to the reference anticonvulsant ethosuximide. These
compounds did not exhibit neurotoxicity at the tested dose
of 50 mg/kg.
Compounds 3, 7, and 10 have been chosen for quantifi-
cation of the pharmacological parameters (ED50 and TD50) in
rats after oral application (Table 5). The quantitative evalu-
ation of the median effective dose (ED50) in the MES and scPTZ
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2011, 11, 231–241
Anticonvulsant Activity of Certain Hydantoins
235
Table 2. The results after intraperitoneal administration to mice (19–24).
H
N
O
N
O
N
N
R1
R
19-24
Comp.
R
R1
MESa
scPTZb
NTc
0.5 h
4 h
0.5 h
4 h
0.5 h
4 h
19
20
21
22
23
24
H
H
H
Cl
Cl
Cl
–CH3
–C2H5
–(CH2)2-OH
–CH3
–C2H5
100
100
100
–
100
100
100
–
300
300
30
–
–
30
300
–
300
–
100
30014
100
–
30014
–
30014
–
300
300
–
300
300
–
–
30014
30
–
–(CH2)2-OH
300
30
–
10014
100
–
PHTd
ETXd
–
300
100
–
–
100
a
c
14
For , b, , d, see Table 1. Response comments: unable to grasp rotorod.
tests as well as toxic dose (TD50) were performed at previously
estimated time of peak effect (TPE) after p.o. administration.
The quantitative p.o. data revealed that all compounds tested
showed higher activity than phenytoin in the electrically
induced seizures. Moreover 7 and 10 were more active than
ethosuximide in the scPTZ screen. These data suggest poten-
tial effectiveness of 7 and 10 in treating both generalized
tonic-clonic epilepsy and absence seizures. The range of doses
applied enabled the determination of TD50 values only for
compounds 3 and 7. These molecules displayed higher pro-
tection index in comparison to reference antiepileptic drugs.
According NIH/NINDS disposition compounds 4 and 10
were screened in the 6-Hz test. The selection was made ran-
domly as a part of the search of molecules providing anti-6-Hz
protection among chemically diversified compounds. The 6-
Hz model was not used widely because of its lack clinical
Table 3. Anticonvulsant activity (MES test) of selected compounds administrated orally to rats.
Comp.
MESa
1 h
NTb
1 h
0.25 h
0.5 h
2 h
4 h
0.25 h
0.5 h
2 h
4 h
3
5
7
8
1
2
0
0
1
0
2
0
0
0
0
0
1
2
2
3
3
1
3
0
0
0
1
4
2
4
3
2
4
3
3
2
1
1
2
0
1
0
3
4
4
4
3
2
4
1
1
4
1
4
0
3
4
4
4
4
3
4
4
3
4
0
2
3
3
0
1
0
0
0
0
0
0
0
0
0
0
ND
0
3
0
0
0
0
0
0
0
0
0
0
ND
0
2
0
0
0
0
0
0
0
0
0
0
ND
0
1
0
0
0
0
0
0
0
0
0
0
ND
0
1
0
0
0
0
0
0
0
0
0
0
ND
9
10
11
13
14
15
20
24
PHTc
a
The data indicate the number of rats of four that were protected at a dose of 30 mg/kg. b Neurotoxicity screening using rotorod test.
c
The data in indicate the number of rats of four in which neurotoxicity was observed at a dose of 30 mg/kg. PHT – phenytoin,
reference drug, data from [23]. ND – No data
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

236
H. Byrtus et al.
Arch. Pharm. Chem. Life Sci. 2011, 11, 231–241
Table 4. Anticonvulsant activity (scPTZ test) of selected
compounds administrated orally to rats.
Table 6. Psychomotor seizure 6-Hz test (dose of 100 mg/kg,
current ꢀ32 mA).
Comp.
Oral administration to ratsa
0.5 h 1 h 2 h
Comp.
Intraperitoneal injection into micea
0.25 h
4 h
0.25 h
0.5 h
1 h
2 h
4 h
7
10
ETXb
2
0
0
1
0
2
3
0
1
1
2
1
2
2
0
4
10
0
0
0
1
2
0
1
0
4
1
a
Dose of 100 mg/kg was administrated. The data indicate the
number of mice of four that were protected.
a
Dose of 50 mg/kg was administrated. The data indicate the
number of rats out of four that were protected. b ETX – ethosux-
imide, reference drug, data from [23].
validity since the hydantoins such as phenytoin failed to
show protective activity. However, it is worthy of note that
the newest anticonvulsants such as levetiracetam, which is
not active in the conventional MES test, does exhibit protec-
tive activity in the 6-Hz model. This suggested that the 6-Hz
model might be capable for identifying anti-seizure agents
with a novel spectrum of activity and unknown mechanism
of anticonvulsant action. The results are shown in Table 6.
As can be seen from the data above, compound 4 admin-
istrated intraperitoneally at a dose of 100 mg/kg into mice
protected 100% of animals tested at 4 h. This molecule was
also effective in 50% of mice at 1 h and 25% at 2 h. Compound
10 were less active and protected only 25% of animals at 0.5 h
and 4 h.
para-position of the 5-phenyl ring at the imidazolidine-2,4-
dione. The SAR studies have shown higher activity of the 5-
cyclopropyl-5-phenyl-hydantoins (3–10 and 19–21) in com-
parison to respective 5-(4-chlorophenyl) analogues 11–18
and 22–24. In the series of 5-cyclopropyl-5-phenyl-hydantoins
two compounds 4 and 9 were active only in the maximal
electroshock test (MES) whereas all other derivatives revealed
protection in both MES and scPTZ screens in mice. The
majority of these molecules were active at 0.5 h and 4 h after
i.p. administration that means rapid onset and long duration
of anticonvulsant action. An exception was observed for 4, 6,
and 9 that were active only at time point 4 h. The structure–
activity relationships were more pronounced for 5-cyclo-
propyl-5-(4-chlorophenyl)-hydantoins. In this series, all 4-phe-
nyl-piperazine derivatives (11–14) were effective only in the
MES test. The introduction of methylene spacer between
piperazine and phenyl ring among 4-benzylpiperazine
derivatives (15–18) caused activity in both screens (15, 18)
made compound less active (17) or inactive (16). The 4-phenyl-
or 4-benzylpiperazines showed activity only at 4 h after i.p.
administration that means delayed onset however long anti-
convulsant action. In this series different pharmacological
Structure activity relationships
The results of preliminary anticonvulsant screening enable
to draw general conclusions about the relationships between
structure and anticonvulsant activity. The spectrum and
duration of activity depended on kind of the substituents
at position-4 of piperazine moiety e.g., phenyl, benzyl, alkyl or
hydroxyalkyl as well as the presence of a chloro atom at the
Table 5. Quantification studies of selected compounds in the MES, scPTZ, and neurotoxicity tests after oral administration in rats.
Comp. TPE (h)a
MES ED50b (mg/kg)
scPTZ ED50b (mg/kg)
>250
TD50b (mg/kg)
PIc
3
4
5.29
(3.48–7.74)
13.11
(8.21–21.78)
12.06
(6.47–23.32)
28.1
(27.7–35.20)
>500
114.4
(81.75–138.57)
150
21.62 (MES)
7
4 (MES)
1 (scPTZ)
2 (MES)
1 (scPTZ)
1
17.58
(8.88–28.46)
30.78
(12.0–108.36)
>500
11.44 (MES)
8.53 (scPTZ)
>8.29 (MES)
>3.25 (scPTZ)
>3.60 (MES)
10
>100
>100
>500
PHTd
ETXd
2
167.00
(116.00–237.00)
>3.0 (scPTZ)
a
b
Time to peak effect. Results are represented as mean ꢁ SEM at 95% confidence limit (MES – maximal electroshock test; scPTZ –
subcutaneous pentylenetetrazole test; TD50 – neurotoxicity). c Protection index (TD50/ED50). d PHT – phenytoin, ETX – ethosuximide
reference drugs, data from [24].
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2011, 11, 231–241
Anticonvulsant Activity of Certain Hydantoins
237
properties were observed for compounds with the alkyl or
hydroxyalkyl substituents at the piperazine ring (22–24).
Excluding inactive methyl derivative 22 the other showed
activity in MES and additionally in scPTZ screens in both time
intervals 0.5 h and 4 h. Similar results were observed for 5-
cyclopropyl-5-phenyl analogues (19–21). It proves that the
introduction of alkyl or hydroxyalkyl groups at the pipera-
zine moiety is especially important for protection against
pentylenetetrazole seizures.
General procedure for the synthesis of 5-cyclopropyl-5-
phenyl- (1) and 5-cyclopropyl-5-(4-chlorophenyl)-
hydantoins (2) [16]
A solution of cyclopropyl-phenyl or cyclopropyl-(4-chloro-
phenyl)-ketone (0.33 mol) and ammonium carbonate
(1 mol) in 50% ethanol was warmed to 508C, at which time
potassium cyanide (0.35 mol) dissolved in 50 mL of water was
dropped in over a period of 15 min. The mixture was heated
under reflux at 56–608C for more than 20 h. The reflux
condenser was then replaced by an air condenser and the
temperature rose to 808C for 1 h to remove the excess
ammonium carbonate. Then the reaction solution was
cooled and acidified. The precipitated solids were filtered
off, washed with water, and recrystallized from a mixture
of ethanol and water.
Conclusion
The library of twenty two new N-Mannich bases derived from
5-cyclopropyl-5-phenyl-imidazolidine-2,4-dione has been syn-
thesized and tested for anticonvulsant activity. The results of
anticonvulsant screening revealed that majority of deriva-
tives were effective in the MES or/and scPTZ screens, the most
widely employed seizure models for early identification of
candidate anticonvulsants. The quantitative studies in rats
after oral administration showed that three compounds were
more potent than phenytoin in the maximal electroshock
test. The highest activity was observed for 3 with ED50 of
5-Cyclopropyl-5-phenyl-imidazolidine-2,4-dione (1)
White powdery crystals. Yield 70%; mp 216–2178C; TLC:
Rf ¼ 0.53 (S1); 1H-NMR (300 MHz, DMSO) d 0.28–0.56 (m,
4H, cyclopropane), 1.54–1.63 (m, 1H, cyclopropane), 7.32–
7.55 (m, 5H, ArH), 8.33 (m, 1H, N1H), 10.76 (s,
1H, N3H). C12H12N2O2 (216.42).
5.29 mg/kg (MES),
7 with ED50 of 13.11 mg/kg (MES),
17.58 mg/kg (scPTZ) and 10 with ED50 of 12.06 mg/kg
(MES), 30.78 mg/kg (scPTZ). These molecules were more active
in comparison with standard anticonvulsants - phenytoin or/
and ethosuximide.
5-Cyclopropyl-5-(4-chlorophenyl)-imidazolidine-2,4-dione
(2)
White powdery crystals. Yield 64%; mp 255–2568C; TLC:
Rf ¼ 0.59 (S1); 1H-NMR (300 MHz, DMSO) d 0.29–0.52 (m,
4H, cyclopropane), 1.52–1.61 (m, 1H, cyclopropane), 7.45–
7.48 (m, 2H, ArH), 7.52–7.57 (m, 2H, ArH), 8.39 (s, 1H, N1H)
10.93 (s, 1H, N3H). C12H11N2O2Cl (250.68).
Experimental protocol
Chemistry
All chemicals and solvents were obtained from Merck
(Darmstadt, Germany) and were used without purification.
The purity of the compounds was confirmed by the thin-layer
chromatography (TLC) performed on Merck silica gel 60 F254
aluminum sheets (Merck; Darmstadt, Germany), using the
developing system: S1 benzene/ethylacetate/acetone in the
ratio of 10:5:1 (v/v/v) and S2 methanol/25% ammonia in the
ratio of 10:1.5 (v/v). The structures were confirmed by both
spectral (1H-NMR, LC/MS) and elemental analysis and the data
were within ꢁ0.4% of the theoretical values. Elemental
analyses for C, H, N were carried out with an Elementar
Vario EL III (Hanau, Germany). 1H-NMR spectra were obtained
in a Varian Mercury 300 MHz spectrometer (Varian Inc., Palo
Alto, CA, USA), in CDCl3, with TMS as an internal standard.
Chemical shifts are reported in d values (ppm) and J values in
Hertz (Hz). Signal multiplicities are represented by the fol-
lowing abbreviations: s (singlet), brs (broad singlet), d
(doublet), t (triplet), m (multiplet). Additionally, the liquid
chromatography/mass spectrometry (LC/MS) spectra for
chosen compounds were obtained on Applied Biosystem/
MDS-SCIEX API 2000 with Agilent HPLCs.
General procedure for the preparation of compounds 3–24
To a mixture of 5-cyclopropyl-5-phenyl- (1) or 5-cyclopropyl-5-
(4-chlorophenyl)-hydantoin (2) (0.01 mol), 40% solution of
formaldehyde (0.01 mol) and corresponding 4-substituted
piperazines (0.01 mol) dissolved in 96% ethanol, were added.
The mixture was left for ca. 6–12 h at room temperature and
then refrigerated at ca. ꢀ108C for 24 h. The precipitated
crude products were washed with cold ethanol and separated
by filtration. The final compounds were purified by and
crystallization from 96% ethanol.
[R,S]3-[(4-Phenylpiperazin-1-yl)-methyl]-5-cyclopropyl-5-
phenyl-imidazolidine-2,4-dione (3)
White powdery crystals. Yield: 70%; mp 188–1908C; TLC:
Rf ¼ 0.45 (S1); 1H-NMR (300 MHz, CDCl3) d 0.32–0.41 (m,
1H, cyclopropane), 0.52–0.78 (m, 3H, cyclopropane), 1.68–
1.77 (m, 1H, cyclopropane), 2.76–2.80 (t, 4H, piperazine,
J ¼ 4.99 Hz), 3.15–3.18 (t, 4H, piperazine, J ¼ 5.00 Hz), 4.57
(s, 2H, –CH2–), 6.00 (s, 1H, N1H), 6.82–6.91 (m, 3H, ArH), 7.21–
7.42 (m, 5H, ArH), 7.55–7.59 (m, 2H, ArH). C23H26N4O2
(390.49), [M þ H]þ ¼ 391.8.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

238
H. Byrtus et al.
Arch. Pharm. Chem. Life Sci. 2011, 11, 231–241
[R,S]3-[{4-(2-Fluorophenyl)-piperazin-1-yl}-methyl]-
5-cyclopropyl-5-phenyl-imidazolidine-2,4-dione (4)
White powdery crystals. Yield: 81%; mp 188–1908C; TLC:
Rf ¼ 0.61 (S1); 1H-NMR (300 MHz, CDCl3) d 0.34–0.42 (m,
1H, cyclopropane), 0.54–0.78 (m, 3H, cyclopropane), 1.68–
1.78 (m, 1H, cyclopropane), 2.79–2.82 (t, 4H, piperazine,
J ¼ 5.10 Hz), 3.05–3.08 (t, 4H, piperazine, J ¼ 5.10 Hz),
4.55 (s, 2H, –CH2–), 6.19 (s, 1H, N1H), 6.88–7.07 (m, 5H,
ArH), 7.32–7.44 (m, 2H, ArH), 7.56–7.61 (m, 2H,
ArH). C23H25N4O2F (408.48).
7.57 (m, 2H, ArH). [M þ H]þ: 473.8. C24H26N4O2Cl2 (473.41),
[M þ H]þ ¼ 473.8.
[R,S]3-[{4-(4-Chlorobenzyl)-piperazin-1-yl}-methyl]-
5-cyclopropyl-5-phenyl-imidazolidine-2,4-dione (9)
White powdery crystals. Yield 67%; m.p. 169–1718C; TLC:
Rf ¼ 0.80 (S2); 1H-NMR (300 MHz, CDCl3) d 0.36–0.48 (m,
1H, cyclopropane), 0.58–0.79 (m, 3H, cyclopropane), 1.69–
1.79 (m, 1H, cyclopropane), 2.42 (brs, 4H, piperazine), 2.68
(m, 4H, piperazine), 3.42 (s, 2H, –CH2–), 4.52 (s, 2H, –CH2–),
6.02 (s, 1H, N1H), 7.23–7.32 (m, 3H, ArH), 7.36–7.46 (m, 4H,
ArH), 7.59–7.62 (m, 2H, ArH). C24H27N4O2Cl (438.96).
[R,S]3-[{4-(4-Fluorophenyl)-piperazin-1-yl}-methyl]-
5-cyclopropyl-5-phenyl-imidazolidine-2,4-dione (5)
White powdery crystals. Yield: 69%; mp 157–1598C; TLC:
Rf ¼ 0.54 (S1); 1H-NMR (300 MHz, CDCl3) d 0.33–0.41 (m,
1H, cyclopropane), 0.52–0.77 (m, 3H, cyclopropane), 1.63–
1.76 (m, 1H, cyclopropane), 2.78–2.92 (t, 4H, piperazine,
J ¼ 5.02 Hz), 3.05–3.08 (t, 4H, piperazine, J ¼ 4.90 Hz), 4.58
(s, 2H, –CH2–), 5.90–5.98 (brs, 1H, N1H), 6.78–6.88 (m, 2H,
ArH), 6.90–6.98 (m, 2H, ArH), 7.33–7.43 (m, 3H, ArH), 7.52–
7.54 (m, 2H, ArH). C23H25N4O2F (408.48).
[R,S]3-[{4-(3-Methylbenzyl)-piperazin-1-yl}-methyl]-
5-cyclopropyl-5-phenyl-imidazolidine-2,4-dione (10)
White powdery crystals. Yield 79%; mp 165–1678C; TLC:
Rf ¼ 0.47 (S1); 1H-NMR (300 MHz, CDCl3) d 0.36–0.43 (m,
1H, cyclopropane), 0.61–0.79 (m, 3H, cyclopropane), 1.71–
1.77 (m, 1H, cyclopropane), 2.36 (s, 3H, –CH3), 2.47 (t, 4H,
piperazine, J ¼ 5.00 Hz), 2.69 (t, 4H, piperazine, J ¼ 4.85 Hz),
3.49 (s, 2H, –CH2–), 4.53 (s, 2H, –CH2–), 6.62 (s, 1H, N1H), 7.07–
7.22 (m, 4H, ArH), 7.38–7.44 (m, 3H, ArH), 7.59–7.60 (m, 2H,
ArH). C25H30N4O2 (418.54).
[R,S]3-[{4-(3-Trifluoromethylphenyl)-piperazin-1-yl}-
methyl]-5-cyclopropyl-5-phenyl-imidazolidine-2,4-dione
(6)
[R,S]3-[(4-Phenyl-piperazin-1-yl)-methyl]-5-cyclopropyl-
5-(4-chlorophenyl)-imidazolidine-2,4-dione (11)
White powdery crystals. Yield: 67%; mp 143–1478C; TLC:
Rf ¼ 0.59 (S1); 1H-NMR (300 MHz, CDCl3) d 0.33–0.41 (m,
1H, cyclopropane), 0.51–0.72 (m, 3H, cyclopropane), 1.65–
1.74 (m, 1H, cyclopropane), 2.68–2.72 (t, 4H, piperazine,
J ¼ 4.97 Hz), 3.18–3.28 (m, 4H, piperazine), 4.58 (s, 2H,
–CH2–), 6.55 (s, 1H, N1H), 6.98–7.15 (m, 3H, ArH), 7.25–7.39
White powdery crystals. Yield 54%; mp 185–1878C; TLC:
Rf ¼ 0.59 (S1); 1H-NMR (300 MHz, CDCl3) d 0.34–0.38 (m,
1H, cyclopropane), 0.63–0.73 (m, 3H, cyclopropane), 1.63–
1.72 (m, 1H, cyclopropane), 2.75 (t, 4H, piperazine,
J ¼ 4.70 Hz), 3.16 (t, 4H, piperazine, J ¼ 4.78 Hz), 4.56 (s,
2H, –CH2–), 6.64 (s, 1H, N1H), 6.88–6.91 (m, 3H, ArH), 7.25–
7.26 (m, 2H, ArH) 7.35–7.38 (m, 2H, ArH), 7.51–7.54 (m, 2H,
ArH). C23H25N4O2Cl (424.93), [M þ H]þ ¼ 425.7.
(m, 4H, ArH),7.45–7.54 (m, 2H, ArH).
(458.49).
C24H25N4O2F3
[R,S]3-[(4-Benzylpiperazin-1-yl)-methyl]-5-cyclopropyl-
5-phenyl-imidazolidine-2,4-dione (7)
[R,S]3-[{4-(2-Fluorophenyl)-piperazin-1-yl}-methyl]-
5-cyclopropyl-5-(4-chlorophenyl)-imidazolidine-2,4-dione
(12)
White powdery crystals. Yield 55%; mp 161–1638C; TLC:
Rf ¼ 0.27 (S1); 1H-NMR (300 MHz, CDCl3) d 0.33–0.41 (m,
1H, cyclopropane), 0.54–0.75 (m, 3H, cyclopropane), 1.66–
1.75 (m, 1H, cyclopropane), 2.42 (brs, 4H, piperazine), 2.65
(brs, 4H, piperazine), 3.48 (s, 2H, –CH2–), 4.49 (s, 2H, –CH2–),
6.32 (s, 1H, N1H), 7.21–7.42 (m, 8H, ArH), 7.55–7.59 (m, 2H,
ArH). C24H28N4O2 (405.50), [M þ H]þ ¼ 405.6.
White powdery crystals. Yield 76%; mp 190–1928C; TLC:
Rf ¼ 0.52 (S1); 1H-NMR (300 MHz, CDCl3) d 0.35–0.38 (m,
1H, cyclopropane), 0.57–0.73 (m, 3H, cyclopropane), 1,65–
1.71 (m, 1H, cyclopropane), 2.79 (t, 4H, piperazine,
J ¼ 4.61 Hz), 3.00 (t, 4H, piperazine, J ¼ 4.85 Hz), 4.55 (s,
2H, –CH2–), 6.13–6,18 (brs, 1H, N1H), 6.91–7.04 (m, 4H,
ArH), 7.38–7.39 (d, 2H ArH, J ¼ 2.05 Hz) 7.52–7.53 (d, 2H,
ArH, J ¼ 2.05 Hz). C23H24N4O2ClF (442.92).
[R,S]3-[{4-(2,6-Dichlorobenzyl)-piperazin-1-yl}-methyl]-
5-cyclopropyl-5-phenyl-imidazolidine-2,4-dione (8)
White powdery crystals. Yield 46%; mp 75–778C; TLC:
Rf ¼ 0.89 (S2); 1H-NMR (300 MHz, CDCl3) d 0.25–0.38 (m,
1H, cyclopropane), 0.50–0.78 (m, 3H, cyclopropane), 1.68–
1.74 (m, 1H, cyclopropane), 2.46–2.57 (m, 8H, piperazine),
3.74 (s, 2H, –CH2–), 4.46 (s, 2H, –CH2–), 5.95 (brs, 1H, N1H),
7.12 (t, 1H, ArH, J ¼ 7.80 Hz), 7.26–7.40 (m, 5H, ArH), 7.53–
[R,S]3-[{4-(4-Fluorophenyl)-piperazin-1-yl}-methyl]-
5-cyclopropyl-5-(4-chlorophenyl)-imidazolidine-2,4-dione
(13)
White powdery crystals. Yield 81%; mp 188–1908C; TLC:
Rf ¼ 0.49 (S1); 1H-NMR (300 MHz, CDCl3) d 0.33–0.38 (m,
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2011, 11, 231–241
Anticonvulsant Activity of Certain Hydantoins
239
1H, cyclopropane), 0.56–0.73 (m, 3H, cyclopropane), 1,64–
1.69 (m, 1H, cyclopropane), 2.76 (t, 4H piperazine,
J ¼ 4.80 Hz), 3.07(t, 4H piperazine, J ¼ 4.88 Hz), 4.55 (s, 2H,
–CH2–), 5.97 (s, 1H, N1H), 6.83–6.86 (m, 2H, ArH), 6.91–9.95
(m, 2H, ArH) 7.38 (d, 2H, ArH, J ¼ 2.31 Hz), 7.51 (d, 2H, ArH,
J ¼ 2.31 Hz). C23H24N4O2ClF (442.92).
[R,S]3-[{4-(3-Methylbenzyl)-piperazin-1-yl}-methyl]-
5-cyclopropyl-5-(4-chlorophenyl)-imidazolidine-2,4-dione
(18)
White powdery crystals. Yield 43%; mp 138–1408C; TLC:
Rf ¼ 0.42 (S1); 1H-NMR (300 MHz, CDCl3) d 0.32–0.38 (m,
1H, cyclopropane), 0.54–0.61 (m, 1H, cyclopropane), 0.62–
0.76 (m, 2H, cyclopropane), 1,60–1.69 (m, 1H, cyclopropane),
2.33 (s, 3H, –CH3), 2.43 (brs, 4H, piperazine), 2.64 (brs, 4H,
piperazine), 3.46 (s, 2H, –CH2–), 4.47 (s, 2H, –CH2–), 6.20
(s, 1H, N1H), 7.04–7.21 (m, 4H, ArH), 7.40 (d, 2H, ArH,
J ¼ 4.60 Hz) 7.55 (d, 2H, ArH, J ¼ 4.60 Hz). C25H29N4O2Cl
(452.99), [M þ H]þ ¼ 453.5.
[R,S]3-[{4-(3-Trifluoromethylphenyl)-piperazin-1-yl}-
methyl]-5-cyclopropyl-5-(4-chlorophenyl)-imidazolidine-
2,4-dione (14)
White powdery crystals. Yield 63%; mp 155–1568C; TLC:
Rf ¼ 0.31 (S1); 1H-NMR (300 MHz, CDCl3)
d 0.35–0.38
(m, 1H, cyclopropane), 0.65–0.72 (m, 3H, cyclopropane),
1,65–1.72 (m, 1H, cyclopropane), 2.75 (t, 4H piperazine,
J ¼ 4.70 Hz), 3.07 (t, 4H piperazine, J ¼ 4.70 Hz), 4.55
(s, 2H, –CH2–), 6.66 (s, 1H, N1H), 7.01–7.07 (m, 3H, ArH),
7.30–7.38 (m, 3H, ArH) 7.45–7.54 (m, 2H, ArH).
[R,S]3-[(4-Methyl-piperazin-1-yl)-methyl]-5-cyclopropyl-
5-phenyl-imidazolidine-2,4-dione (19)
White powdery crystals. Yield 53%; mp 62–648C; TLC:
Rf ¼ 0.59 (S2); 1H-NMR (300 MHz, CDCl3)
d 0.36–0.38
C
24H24N4O2ClF3 (492.93).
(m, 1H, cyclopropane), 0.52–0.59 (m, 1H, cyclopropane),
0.60–0.76 (m, 2H, cyclopropane), 1.64–1.74 (m, 1H, cyclopro-
pane), 2.26 (s, 3H, –CH3), 2.42 (brs, 4H, piperazine), 2.68 (brs,
4H, piperazine), 4.45 (s, 2H, –CH2–), 6.02 (s, 1H, N1H),
7.35–7.60 (m, 5H, ArH). C18H24N4O2 (328.42).
[R,S]3-[(4-Benzylpiperazin-1-yl)-methyl]-5-cyclopropyl-
5-(4-chlorophenyl)-imidazolidine-2,4-dione (15)
White powdery crystals. Yield 52%; mp 156–1588C; TLC:
Rf ¼ 0.43 (S1); 1H-NMR (300 MHz, CDCl3) d 0.34–0.40 (m,
1H, cyclopropane), 0.56–0.61 (m, 1H, cyclopropane), 0.65–
0.73 (m, 2H, cyclopropane), 1.63–1.69 (m, 1H, cyclopropane),
2.43 (brs, 4H, piperazine), 2.64 (brs, 4H, piperazine), 3.49
(s, 2H, –CH2–), 4.43 (s, 2H, –CH2–), 5.90 (s, 1H, N1H), 7.20–
7.36 (m, 4H, ArH), 7.39–7.42 (m, 2H, ArH) 7.50–7.55 (m, 3H,
ArH). C24H27N4O2Cl (438.95), [M þ H]þ ¼ 439.4.
[R,S]3-[(4-Ethyl-piperazin-1-yl)-methyl]-5-cyclopropyl-
5-phenyl-imidazolidine-2,4-dione (20)
White powdery crystals. Yield 77%; mp 78–808C; TLC:
Rf ¼ 0.72 (S2); 1H-NMR (300 MHz, CDCl3)
d 0.31–0.39
(m, 1H, cyclopropane), 0.51–0.59 (m, 1H, cyclopropane),
0.60–0.73 (m, 2H, cyclopropane), 1.04 (t, 3H, –CH3,
J ¼ 6.0 Hz), 1.63–1.72 (m, 1H, cyclopropane), 2.35–2.42
(m, 6H, 4H, piperazine, 2H –CH2–), 2.67 (t, 4H, piperazine,
J ¼ 5.00 Hz), 4.42 (s, 2H, –CH2–), 6.20 (s, 1H, N1H), 7.31–7.58
(m, 5H, ArH). C19H26N4O2 (342.45).
[R,S]3-[{4-(2,6-Dichlorobenzyl)-piperazin-1-yl}-methyl]-
5-cyclopropyl-5-(4-chlorophenyl)-imidazolidine-2,4-dione
(16)
White powdery crystals. Yield 71%; mp 159–1618C; TLC:
Rf ¼ 0.59 (S1); 1H-NMR (300 MHz, CDCl3)
d 0.30–0.34
[R,S]3-[(4-Hydroxyethyl-piperazin-1-yl)-methyl]-
(m, 1H, cyclopropane), 0.56–0.71 (m, 3H, cyclopropane),
1,61–1.64 (m, 1H, cyclopropane), 2.54–2.56 (m, 8H, pipera-
zine), 3.72 (s, 2H, –CH2–), 4.47 (s, 2H, –CH2–), 5.93 (s, 1H, N1H),
7.26–7.34 (m, 3H, ArH), 7.36–7.378 (d, 2H, ArH, J ¼ 2.05 Hz)
7.49–7.50 (d, 2H, ArH, J ¼ 2.31 Hz). C24H25N4O2Cl3 (507.85).
5-cyclopropyl-5-phenyl-imidazolidine-2,4-dione (21)
White powdery crystals. Yield 48%; mp 168–1708C; TLC:
Rf ¼ 0.44 (S1); 1H-NMR (300 MHz, CDCl3) d (ppm) 0.32–0.40
(m, 1H, cyclopropane), 0.52–0.77 (m, 3H, cyclopropane), 1.67–
1.76 (m, 2H, 1H, cyclopropane, 1H, OH), 2.48–2.52 (m, 6H, 4H,
piperazine, 2H, –CH2–), 2.64 (t, 4H, piperazine, J ¼ 5.02 Hz),
3.55 (t, 2H, –CH2–, J ¼ 7.0 Hz), 4.50 (s, 2H, –CH2–), 6.09
(s, 1H, N1H), 7.32–7.43 (m, 3H, ArH), 7.54–7.58 (m, 2H,
ArH). C19H26N4O3 (358.44).
[R,S]3-[{4-(4-Chlorobenzyl)-piperazin-1-yl}-methyl]-
5-cyclopropyl-5-(4-chlorophenyl)-imidazolidine-2,4-dione
(17)
White powdery crystals. Yield 70%; mp 158–1608C; TLC:
Rf ¼ 0.84 (S2); 1H-NMR (300 MHz, CDCl3)
d 0.31–0.38
(m, 1H, cyclopropane), 0.58–0.61 (m, 1H, cyclopropane),
0.63–0.79 (m, 2H, cyclopropane), 1.61–1.70 (m, 1H, cyclopro-
pane), 2.41 (brs, 4H, piperazine), 2.63 (brs, 4H, piperazine),
3.46 (s, 2H, –CH2–), 4.52 (s, 2H, –CH2–), 5.93 (s, 1H, N1H), 7.24–
7.35 (m, 4H, ArH), 7.40 (d, 2H, ArH, J ¼ 4.50 Hz) 7.54 (d, 2H,
ArH, J ¼ 4.50 Hz). C24H25N4O2Cl2 (472.40).
[R,S]3-[(4-Methyl-piperazin-1-yl)-methyl]-5-cyclopropyl-
5-(4-chlorophenyl)-imidazolidine-2,4-dione (22)
White powdery crystals. Yield 92%; mp 68–718C; TLC:
Rf ¼ 0.84 (S2); 1H-NMR (300 MHz, CDCl3)
d 0.38–0.39
(m, 1H, cyclopropane), 0.53–0.58 (m, 1H, cyclopropane), 0.67–
0.69 (m, 2H, cyclopropane), 1.61–1.67 (m, 1H, cyclopropane),
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

240
H. Byrtus et al.
Arch. Pharm. Chem. Life Sci. 2011, 11, 231–241
2.35 (s, 3H, –CH3), 2.56 (brs, 4H, piperazine), 2.74 (brs, 4H,
piperazine), 4.49 (s, 2H, –CH2–), 6.42–6.52 (brs, 1H, N1H),
7.36 (d, 2H, ArH, J ¼ 8.72 Hz), 7.51–7.54 (m, 2H,
ArH). C18H23N4O2Cl (362.86), [M þ H]þ ¼ 363.4.
Maximal electroshock test (MES)
In the MES screen, an electrical stimulus of 0.2 s in duration
(50 mA in mice and 150 mA in rats) is delivered via corneal
electrodes primed with an electrolyte solution containing an
anesthetic agent.
[R,S]3-[(4-Ethyl-piperazin-1-yl)-methyl]-5-cyclopropyl-
5-(4-chlorophenyl)-imidazolidine-2,4-dione (23)
Subcutaneous pentylenetetrazole seizure test (scPTZ)
The scPTZ test utilizes a dose of pentylenetetrazole (85 mg/kg
in mice and 70 mg/kg in rats) that produces clonic seizures
lasting for a period of at least five seconds in 97% (CD97) of
animals tested. At the anticipated time of testing the penty-
lenetetrazole is administrated subcutaneously.
White powdery crystals. Yield 57%; mp 83–868C; TLC:
Rf ¼ 0.39 (S2); 1H-NMR (300 MHz, CDCl3)
d 0.37–0.38
(m, 1H, cyclopropane), 0.53–0.57 (m, 1H, cyclopropane),
0.66–0.69 (m, 2H, cyclopropane), 1.09–1.15 (m, 3H, –CH3),
1.61–1.66 (m, 1H, cyclopropane), 2.54–2.65 (m, 6H, 4H, piper-
azine, 2H, –CH2), 2.74 (brs, 4H, piperazine), 4.48 (s, 2H, –CH2–),
6.44 (s, 1H, N1H), 7.35–7.38 (d, 2H, ArH, J ¼ 8.72 Hz), 7.52–
7.55 (m, 2H, ArH). C19H25N4O2Cl (376.89).
Neurological toxicity (NT)
The neurological toxicity test (NT) induced by a compound
was detected in mice using standardized rotorod test [21].
Untreated control mice or rats, when placed on the rod, can
maintain their equilibrium for a prolonged time period.
The acute motor impairment can be demonstrated by the
inability of the animal to maintain equilibrium for 1 min in
each of three successive trials.
[R,S]3-[(4-Hydroxyethyl-piperazin-1-yl)-methyl]-
5-cyclopropyl-5-(4-chlorophenyl)-imidazolidine-2,4-dione
(24)
White powdery crystals. Yield: 63%; mp 189–1918C; TLC:
Rf ¼ 0.46 (S1); 1H-NMR (300 MHz, CDCl3)
d 0.31–0.38
(m, 1H, cyclopropane), 0.68–0.77 (m, 3H, cyclopropane),
1.62–1.71 (m, 2H, 1H, cyclopropane, 1H, OH), 2.49–2.51
(m, 6H, 4H, piperazine, 2H, –CH2–), 2.58 (t, 4H, piperazine,
J ¼ 4.90 Hz), 3.57 (t, 2H –CH2–, J ¼ 5.20 Hz), 4.49 (s, 2H,
–CH2–), 6.01 (s, 1H, N1H), 7.37–7.42 (m, 2H, ArH), 7.52–7.60
(m, 2H, ArH). C19H25N4O3Cl (392.89).
Quantification studies
The quantitative determination of the median effective dose
(ED50) and the median neurotoxic dose (TD50) values were
performed at previously estimated time of peak effect after
oral administration into rats. Groups of eight rats received
various doses of the compound until at least three points
were established in the range of 10–90% seizure protection
and 0% protection or minimal neurotoxicity. From the plot of
the data obtained, the respective ED50 and TD50 values, 95%
confidence intervals, slope of the regression line and stan-
dard error of the slope were calculated by means of a com-
puter program written at NINDS/NIH.
Pharmacology
Compounds 3–24 were pharmacologically pre-evaluated
within the Antiepileptic Drug Development (ADD) Program
in Epilepsy Branch, Neurological Disorders Program,
National Institute of the Neurological and Communicative
Disorders and Stroke (NIH/NINDS), Rockville, MD, USA, by
using procedures described elsewhere [19, 20].
Phase I studies of the investigated compounds involved
three tests: Maximal electroshock (MES), subcutaneous
pentylenetetrazole (scPTZ), and rotorod test for neurologi-
cal toxicity (NT). Male albino mice (CF#1 strain), weighing
18–25 g) and male albino rats (Sprague-Dawley) were used
as experimental animals. The animals were housed in
metabolic cages and allowed free access to food and
water. The compounds were injected intraperitoneally
to mice as a suspension in 0.5% methylcellulose/water
mixture at a dose level of 30, 100, and 300 mg/kg with
anticonvulsant activity and neurotoxicity assessment at
0.5 and 4 h intervals after administration. Promising
derivatives from phase I underwent phase VIa in which
they were administrated orally into rats at a fixed dose of
30 mg/kg for both MES and the rotorod toxicity tests or
50 mg/kg in the scPTZ screen. Groups of eight mice or
four rats are employed.
The 6-Hz model
The 6-Hz model is an alternative electroshock paradigm that
uses low-frequency (6 Hz) long-duration (3 s) electrical stimu-
lation. Corneal stimulation (0.2 ms duration monopolar
rectangular pulses at 6-Hz for 3 s) was delivered by a con-
stant-current device. During the stimulation, mice were man-
ually restrained and released into the observation cage
immediately after the current application. The seizures mani-
fest in ‘‘stunned’’ posture associated with rearing, forelimb,
automatic movements and clonus, twitching the vibrissae
and Straub-tail. The duration of the seizure activity ranges
from 60 to 120 s in untreated animals. At the end of the
seizure, animals resume their normal exploratory behavior.
The experimental end point is protection against the seizure.
The animal is considered to be protected if it resumes its
normal exploratory behavior within 10 s from the stimu-
lation [22].
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2011, 11, 231–241
Anticonvulsant Activity of Certain Hydantoins
241
´
´
[11] J. Obniska, K. Kaminski, D. Skrzynska, J. Pichor, Eur. J. Med.
´
The authors wish to thank Dr James Stables for providing them with
pharmacological data through the Antiepileptic Drug Development
Program (Epilepsy Branch, National Institute of Neurological Disorders
and Stroke, National Institute of Health, Rockville, MD, USA).
Chem. 2009, 44, 2224–2233.
´
´
[12] J. Obniska, M. Kopytko, A. Zagorska, I. Chlebek, K. Kaminski,
Arch. Pharm. Chem. Life Sci. 2010, 343, 333–341.
´
[13] J. Obniska, H. Byrtus, K. Kaminski, M. Pawłowski, M.
We are pleased to acknowledge the generous financial support of this work
by the grant of the Minister Committee for Scientific Research, Poland
(Grant No N405 298536).
Szczesio, J. Karolak-Wojciechowska, Bioorg. Med. Chem.
2010, 18, 6134–6142.
´
[14] H. Marona, A. Gunia, K. Słoczynska, Anna Rapacz, Barbara
Filipek, Marek Cegła, W. Opoka, Acta Pol. Pharm. Drug Res.
2009, 66, 571–578.
The authors have declared no conflict of interest.
[15] T. Dylag, M. Zygmund, D. Macia˛g, J. Handzlik, M. Bendrski,
References
´
B. Filipek, K. Kiec-Kononowicz, Eur. J. Med. Chem. 2004, 39 (12),
1013–1027.
[1] O. J. McNamara, Drugs effective in the therapy of the
epilepsies, in The Pharmacological Basis of Therapeutics (Eds.:
J. G. Hardman, L. E. Limbird, A. G. Gilman), McGraw-Hill,
New York 2001, pp. 521–548.
[16] L. H. Goodson, J. L. Honigberg, J. L. Lehman, W. H. Burton,
J. Org. Chem. 1960, 25, 1920–1924.
[17] H. S. White, Epilepsia 2003, 44, 2–8.
[18] M. A. Rogawski, Epilepsy Res. 2006, 68, 23–26.
[2] W. Lo¨scher, D. Schmidt, Epilepsy Res. 2006, 69, 183–272.
[19] R. L. Krall, J. K. Penry, B. G. White, H. J. Kupferberg, E. A.
Swinyard, Epilepsia 1978, 19, 409–428.
[3] J. A. Cramer, S. Mintzer, J. Wheless, R. H. Mattson, Expert. Rev.
Neurother. 2010, 10 (6), 885–891.
[20] H. J. Kupferberg, Epilepsia 1989, 30 (Suppl.), 51–56.
[4] M. G. Wong, J. A. Defina, P. R. Andrews, J. Med. Chem. 1986, 29,
562–572.
[21] N. W. Dunham, T. A. Miya, J. Am. Pharm. Assoc. Sci. 1957, 46,
208–209.
[5] E. Estrada, A. Pena, Bioorg, Med. Chem. 2000, 8, 2755–2770.
[22] R. F. Kaminski, M. R. Livingood, M. A. Rogawski, Epilepsia
2004, 45, 864–867.
´
[6] L. Bruno-Blanch, J. Galvez, R. Garcia-Domenach, Bioorg. Med.
Chem. Lett. 2003, 13, 2749–2754.
[23] R. Thirumurugan, D. Sriram, A. Saxena, J. P. Stables, P.
Yogeeswari, Bioorg. Med. Chem. 2006, 14, 3106–3112.
[7] J. R. Pollard, J. French, Lancet Neurol. 2006, 5, 1064–1067.
[8] M. Bialer, S. I. Johannessen, H. J. Kupferberg, R. H. Levy,
E. Perucca, T. Tomson, Epilepsy Res. 2007, 73, 1–52.
[24] H. S. White, J. H. Woodhead, K. S. Wilcox, J. P. Stables, H. J.
Kupferberg, H. H. Wolf, Discovery and preclinical develop-
ment of antiepileptic drugs, in Antiepileptic Drugs (Eds.: R. H.
Levy, R. H. Mattson, B. S. Meldrum, E. Perucca), Lippincott,
Philadelphia 2002, pp. 36–48.
[9] B. Bennett, A. Matagne, P. Michel, M. Leonard, M. Cornet, M. A.
Meeus, N. Toublanc, Neurotherapeutics 2007, 4, 117–122.
´
[10] J. Obniska, A. Zagorska, Il Farmaco 2003, 58, 1227–1234.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com