Synthesis and anticonvulsant activity of new N-Mannich bases derived from 5-cyclopropyl-5-phenyl- and 5-cyclopropyl-5-(4-chlorophenyl)-imidazolidine-2,4- diones

Article abstract of DOI:10.1016/j.bmc.2011.08.017

Synthesis, physicochemical and anticonvulsant properties of new N-Mannich bases derived from 5-cyclopropyl-5-phenyl- and 5-cyclopropyl-5-(4-chlorophenyl)- imidazolidine-2,4-diones have been described. Initial anticonvulsant screening was performed using intraperitoneal (ip.) maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizure tests. The neurotoxicity was determined applying the rotarod test. The in vivo results in mice showed that all compounds were effective especially in the MES screen. The quantitative evaluation after oral administration in rats showed that the most active was 5-cyclopropyl-5-phenyl-imidazolidine-2,4-dione (1) with ED₅₀ values of 5.76 mg/kg (MES) and 57.31 mg/kg (scPTZ). This molecule was more potent than phenytoin and ethosuximide which were used as reference antiepileptic drugs. Additionally compound 1 with ED₅₀ of 26.06 mg/kg in psychomotor seizure test (6-Hz) in mice showed comparable activity to new generation anticonvulsant - levetiracetam.

Full text of DOI:10.1016/j.bmc.2011.08.017

Bioorganic & Medicinal Chemistry 19 (2011) 6149–6156
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and anticonvulsant activity of new N-Mannich bases derived
from 5-cyclopropyl-5-phenyl- and 5-cyclopropyl-5-(4-chlorophenyl)-
imidazolidine-2,4-diones
⇑
´
Hanna Byrtus, Jolanta Obniska , Anna Czopek, Krzysztof Kaminski, Maciej Pawłowski
Department of Medicinal Chemistry, Jagiellonian University, Medical College, 9 Medyczna Street, 30-688 Kraków, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis, physicochemical and anticonvulsant properties of new N-Mannich bases derived from 5-cyclo-
propyl-5-phenyl- and 5-cyclopropyl-5-(4-chlorophenyl)-imidazolidine-2,4-diones have been described.
Initial anticonvulsant screening was performed using intraperitoneal (ip.) maximal electroshock (MES)
and subcutaneous pentylenetetrazole (scPTZ) seizure tests. The neurotoxicity was determined applying
the rotarod test. The in vivo results in mice showed that all compounds were effective especially in
the MES screen. The quantitative evaluation after oral administration in rats showed that the most active
was 5-cyclopropyl-5-phenyl-imidazolidine-2,4-dione (1) with ED50 values of 5.76 mg/kg (MES) and
57.31 mg/kg (scPTZ). This molecule was more potent than phenytoin and ethosuximide which were used
as reference antiepileptic drugs. Additionally compound 1 with ED50 of 26.06 mg/kg in psychomotor sei-
zure test (6-Hz) in mice showed comparable activity to new generation anticonvulsant – levetiracetam.
Ó 2011 Published by Elsevier Ltd.
Received 26 May 2011
Revised 29 July 2011
Accepted 9 August 2011
Available online 16 August 2011
Keywords:
N-Mannich bases
5-Cyclopropyl-5-phenyl- and 5-
cyclopropyl-5-(4-chlorophenyl)-
imidazolidine-2,4-dione
Hydantoin
Anticonvulsant activity
1. Introduction
groups attached to the heterocyclic system.6–8 This common tem-
plate is present in the structures of two old, however, well-
During the last two decades several new antiepileptic drugs
(AEDs) have been marked but still about one third of patients with
epilepsy do not respond for the applied treatment. Furthermore
many of those medications cause serious side effects from which
more common are ataxia, nausea, mental dulling, and hepatotoxic-
ity.1–3 The currently used AEDs can be classified into four catego-
ries on the basis of the main molecular mechanisms of action, as
follow: (i) modulation of voltage-dependent Na+ and/or Ca2+ chan-
nels, (ii) enhancement of GABA-mediated inhibition or other effect
on the GABA system, (iii) inhibition of synaptic excitation medi-
ated by ionotropic glutamate receptors.4
Conceptually, there are two different methods of obtaining new
anticonvulsants namely knowledge-based approaches and screen-
ing approaches. Knowledge-based approaches rely on the use of dif-
ferent pharmacophores that were established through the analysis
of structural characteristics of clinically effective AEDs as well as
other anticonvulsant active compounds. Serendipitous approaches
involve screening of either diverse or focus compound libraries.5
It is well documented that the important core fragment of
anticonvulsants is defined by nitrogen heteroatomic system, usually
a cyclic imide, at least of one carbonyl group and phenyl or alkyl
established AEDs such as ethosuximide and phenytoin as well as
among the newest drugs, for example, levetiracetam, brivaracetam,
or seletracetam.9–11 Taking into consideration the above our re-
searches are focused on systematic structural modifications in a
group of pyrrolidine-2,5-dione and imidazolidine-2,4-dione deriva-
tives.12–17 The structure–activity relationships (SARs) studies per-
formed among these molecules showed higher activity for
differently substituted imidazolidine-2,4-diones in comparison to
respective pyrrolidine-2,5-dione analogs.18 Therefore in our recent
studies we have demonstrated the potent anticonvulsant activity
among the N-Mannich bases derived from 5-cyclopropyl-5-phe-
nyl-imidazolidine-2,4-diones.19 Several compounds showed activ-
ity in the MES test that was superior to phenytoin, used as a model
substance (Fig. 1).
Following these findings, as part of our efforts to design new
anticonvulsant agents in the present studies we have synthesized
a new series of N-Mannich bases derived from 5-cyclopropyl-
5-phenyl- (1) and 5-cyclopropyl-5-(4-chlorophenyl)-imidazoli-
dine-2,4-diones (2). These molecules have been designed as ana-
logs of compounds A and B (Fig. 1) in which an amine function
have been modified. The main goal was to obtain compounds
active both in maximal electroshock seizure (MES) and the subcu-
taneous pentylenetetrazole (scPTZ) screens. As basic fragments we
have introduced different piperazines with alkyl, alkylene, ketone,
⇑
Corresponding author. Tel. +48 12 620 54 53; fax: +48 12 657 02 62.
E-mail address: mfobnisk@cyf-kr.edu.pl (J. Obniska).
0968-0896/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2011.08.017

6150
H. Byrtus et al. / Bioorg. Med. Chem. 19 (2011) 6149–6156
H
N
H
N
O
O
N
O
N
O
N
N
N
N
B
A
ED50 = 13.11 mg/kg (MES, rats, p.o.)
ED50 = 17.58 mg/kg (scPTZ, rats, p.o.)
ED50 = 5.29 mg/kg (MES, rats, p.o.)
H
N
O
N
H
O
Phenytoin
ED50 = 28.10 mg/kg (MES, rats, p.o.)
Figure 1. Structures of model compounds.
or ester moieties at position-4. Finally, the piperazine fragment has
been changed into tetrahydroisoquinoline or morpholine.
neous pentylenetetrazole (scPTZ) tests, after intraperitoneal injec-
tion into mice at doses of 30, 100, and 300 mg/kg. An observation
was carried out at two different time intervals, namely 0.5 and
4 h. The acute neurological toxicity (NT) was determined in the
minimal motor impairment – rotorod screen (NT). The results are
shown in Table 1.
2. Results and discussion
2.1. Chemistry
The most active in the mice ip. MES screen were 1, 3, and 14
that showed protection at dose of 30 mg/kg. It was comparable
with phenytoin used as reference drug preventing electrically
induced seizures. The lower anti-MES activity was observed for
4–9, 12, 16 (100 mg), and 2, 10, 11, 13, and 15 (300 mg/kg).
Second, routinely used convulsive screen is scPTZ test which
identifies substances elevating seizure threshold. The strongest
anti-scPTZ activity showed unsubstituted 5-cyclopropyl-5-
phenyl- and 5-cyclopropyl-5-(4-chlorophenyl)-imidazolidine-2,4-
diones (1, 2) that protected mice at a dose of 30 mg/kg. The other
molecules were less active, namely 100 mg/kg (4, 6, 8–10, 14,
and 16) or 300 mg/kg (3, 5, 7, and 13). Many of these compounds
revealed activity equivalent to ethosuximide used as a model
anticonvulsant for this type of seizures. Furthermore they were
active in both time intervals (0.5 and 4 h) that indicates quick
onset and long duration of anticonvulsant action. In the neuro-
toxicity screen (NT), compounds 5–7 and 15 did not show neuro-
The synthesis of compounds 1–16 was accomplished as shown
in Scheme 1. The starting 5-cyclopropyl-5-phenyl- (1) and 5-cyclo-
propyl-5-(4-chlorophenyl)-imidazolidine-2,4-dione (2) were ob-
tained from the appropriately substituted ketones by means of
the Bücherer–Berg reaction with modifications described by Good-
son et al.20 In the next step the aminoalkylation of the acidic pro-
ton (N3H) of 1 and 2 carried out in the present of formaldehyde and
appropriate amines enabled to obtain final compounds 3–16.
The following secondary amines were used: 1-(2-phenethyl)-
piperazine (3, 10), 1-(3-phenylprop-2-en-1-yl)-piperazine (4),
1-(1-phenethyl)-piperazine (5, 11), phenyl-(piperazin-1-yl)-meth-
anone (6), benzyl piperazine-1-carboxylate (7, 12), ethyl pipera-
zine-1-carboxylate (8), 1-(2-methoxyethyl)-piperazine (9),
tetrahydro-isoquinoline (13, 15) and morpholine (14, 16). The
reaction was carried out in ethanol at a room temperature for ca.
12 h. The crude products were crystallized from ethanol giving
the final compound in yields ranging from 46% to 85%. The struc-
tures of compounds synthesized were confirmed by both spectral
and elemental analysis. The detailed physical and analytical data
are listed in the experimental section.
toxicity in the maximum dose administered
– 300 mg/kg.
Compounds 1–4, 8, 11, 12, and 16 exhibited motor impairment
at the dose of 300 mg/kg. The other derivatives revealed neuro-
toxicity at doses of 30 mg/kg (9, 10) or 100 mg/kg (13, 14). Mol-
ecules neurotoxic at dose of 30 mg/kg have been excluded from
further testing.
2.2. Anticonvulsant activity
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. Therefore on
the basis of the data obtained in ip. screen in mice and according
to the Antiepileptic Drug Development (ADD) Program disposition,
seven compounds were selected and examined for their anticon-
vulsant activity in the MES and NT screens after p.o. administra-
tion into rats at a dose of 30 mg/kg (Table 2). The highest
activity was observed for 1 that protected 100% of animals in all
time intervals that means quick onset an long duration of anticon-
vulsant action. These molecule was more potent than phenytoin,
At the present time there are three in vivo screens used rou-
tinely that include the maximal electroshock seizure (MES), the
subcutaneous pentylenetetrazole (scPTZ) and the 6-Hz model.
From these tests the MES and scPTZ screens are recognized as
the ‘gold standards’ in the early stages of testing.21 Furthermore,
the maximal electroshock (MES) and subcutaneous pentylenetet-
razole (scPTZ) tests are claimed to detect compounds affording
protection against generalized tonic-clonic seizures and general-
ized absence seizures, respectively.22
The profile of anticonvulsant activity of all tested compounds
was established in the maximal electroshock (MES) and subcuta-

H. Byrtus et al. / Bioorg. Med. Chem. 19 (2011) 6149–6156
6151
R
X
R
cmpd
H
H
H
-CH2-CH2-
-CH2-CH=CH-
3
4
5
H
N
-CH(CH3)-
6
7
-CO-
-COOCH2-
H
H
Cl
Cl
O
N
O
-CH2-CH2-
-CH(CH3)-
-COOCH2-
10
11
N
N
X
12
Cl
3-7, 10-12
R
b
R1
R
cmpd
R
H
H
-COOC2 H5
-(CH2)2-OCH3
8
9
H
N
R
O
c
H
N
a
O
N
O
N
O
N
R1
N
8, 9
O
H
1, 2
cmpd
R
d
H
Cl
1
2
R
e
R
H
N
O
N
O
H
N
N
O
13, 15
N
O
N
cmpd
R
O
14, 16
H
H
Cl
Cl
13
14
15
16
Scheme 1. Synthetic pathways of compounds 1–16. Reagents and reaction conditions: (a) KCN, (NH4)2CO3, 50% ethyl alcohol; (b and c) 4-substituted piperazine derivatives,
formaldehyde, 96% ethyl alcohol, 12 h room temperature; (d) tetrahydroisoquinoline, formaldehyde, 96% ethyl alcohol, 12 h room temperature; (e) morpholine,
formaldehyde, 96% ethyl alcohol, 12 h room temperature.
used as a model substance effective in electrically induced sei-
zures. The other molecules 3, 5, 6, 7, 13, and 14 protected from
25% to 100% of rats. As a continuation of pharmacological studies
four molecules 1, 2, 14, and 16 active in pentylenetetrazole sei-
zures in mice were tested in scPTZ screen after p.o. administration
into rats. Initially a dose of 50 mg/kg was employed and the anti-
convulsant activity was assessed at five time points (Table 3). In
this test, the most effective were compounds 1, 2, and 16 that
showed equable profile of activity and protected up to 75% of
tested animals. These molecules were more potent in comparison
with ethosuximide which is known as a model anticonvulsant for
pentylenetetrazole seizures.
The analysis of quantitative p.o. data revealed that all compounds
tested showed higher activity than phenytoin in the electrically
induced seizures. Moreover 1 and 2 were more potent than ethosux-
imide in the scPTZ screen. These data confirmed potential effective-
ness of 1 in treating both generalized tonic–clonic epilepsy and
absence seizures. Unfortunately compound 1 displayed higher
neurotoxicity as well as lower protection index in comparison to
both phenytoin and ethosuximide. The inhibition of electrically
induced seizures that is characteristic for phenytoin and phenyt-
oin-like drugs may indicate the influence of compounds on volt-
age-depended Na+ channels as the most plausible mechanism of
anticonvulsant action.
From the whole series six compounds have been chosen for
quantification of the pharmacological parameters (ED50 and
TD50). The quantitative evaluation of the median effective dose
(ED50) in the MES and scPTZ tests as well as toxic dose (TD50) were
performed at previously estimated time of peak effect (TPE) after
oral administration into rats. Results of the quantitative tests along
with the data for the standard drugs phenytoin and ethosuximide
are shown in Table 4.
According to the Antiepileptic Drug Development (ADD)
Program disposition compounds 1, 2, and 16 were chosen for the
evaluation of anticonvulsant activity in the 6-Hz test. The 6-Hz
screen has been validated recently as a model of therapy-resistant
epilepsy. It was not used widely because of its lack clinical validity
since the hydantoins such as phenytoin failed to show protective
activity. Nevertheless, the clinically effective antiepileptic drug
levetiracetam, which is not active in the conventional MES and

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H. Byrtus et al. / Bioorg. Med. Chem. 19 (2011) 6149–6156
Table 1
Anticonvulsant activity after intraperitoneal administration to mice (1–16)
Cmpd
R
X
R1
logPa
MESb
scPTZc
NTd
0.5 h
4 h
0.5 h
4 h
0.5 h
4 h
1
2
3
4
5
6
7
8
H
Cl
H
H
H
H
H
H
H
Cl
Cl
Cl
H
H
Cl
Cl
—
—
—
—
—
—
—
—
—
1.66
2.63
2.98
3.30
3.19
2.17
2.92
1.76
1.46
3.97
3.90
3.65
3.02
1.33
3.71
2.00
30
—
—
30
300
30
30
300
—
100
—
100
30
30014
—
30014
300
300
30013
—
–CH2–CH2–
–CH2–CH@CH–
–CH(CH3)–
CO
–COOCH2–
—
—
–CH2–CH2–
–CH(CH3)–
–COOCH2–
—
—
—
—
30025
300
30025
30025
30025
300
10025
10025
—
—
100
300
100
300
100
100
300
300
—
300
100
—
100
—
100
100
100
100
100
100
300
300
100
300
30
300
100
100
100
100
300
30
30014
—
10025
30025
100
10025
30025
—
—
—
—
—
–COOC2H5
–(CH2)2OCH3
30033,14
30
30014
30
9
10
11
12
13
14
15
16
Ie
—
—
—
—
—
—
—
30
30
—
30014
300
100
10014
—
—
—
—
300
—
30025
300
—
100
—
100
300
—
—
—
—
100
30013
—
300
—
—
—
—
30014
—
—
—
—
30013
—
IIe
IIIe
IVe
PHTf
ETXg
—
—
—
30
—
30014
—
—
300
100
—
100
—
—
—
Doses of 30, 100, and 300 mg/kg were administrated intraperitoneally in mice. The figures in the table indicate the minimum dose whereby anticonvulsant activity or
neurotoxicity was demonstrated in 100% of the animals. A dash indicates the absence of anticonvulsant activity and neurotoxicity at the maximum dose administered
(300 mg/kg).
Response comments: 13loss of righting reflex, 14unable to grasp rotorod, 25myoclonic jerks, 33tremors.
a
Log P calculated by use of Pallas 3.2.1.4 program.
Maximal electroshock test.
Subcutaneous pentylenetetrazole test.
Neurotoxicity screening – rotorod test.
b
c
d
e
Data from Ref. 19: I-3-[(4-Phenylpiperazin-1-yl)-methyl]-5-cyclopropyl-5-phenyl-imidazolidine-2,4-dione, II-3-[{4-(2-Fluorophenyl)-piperazin-1-yl}-methyl]-5-cyclo-
propyl-5-phenyl-imidazolidine-2,4-dione, III-3-[(4-Phenyl-piperazin-1-yl)-methyl]-5-cyclopropyl-5-(4-chlorophenyl)-imidazolidine-2,4-dione, IV-3-[{4-(2-Fluorophenyl)-
piperazin-1-yl}-methyl]-5-cyclopropyl-5-(4-chlorophenyl)-imidazolidine-2,4-dione.
f
PHT-phenytoin, reference drug, data from Ref. 29.
ETX-ethosuximide, reference drug, data from Ref. 29.
g
Table 2
Table 3
Anticonvulsant activity (MES test) of selected compounds administrated orally to rats
Anticonvulsant activity (scPTZ test) of selected compounds administrated orally to
rats
Cmpd
MESa
NTb
Cmpd
scPTZa
NTb
0.25 h 0.5 h 1 h 2 h 4 h 0.25 h 0.5 h 1 h 2 h 4 h
0.25 h 0.5 h 1 h 2 h 4 h 0.25 h 0.5 h 1 h 2 h 4 h
1
3
5
6
4
0
2
0
0
0
2
1
4
0
1
0
0
0
2
4
4
3
2
3
1
2
2
3
4
3
3
3
1
3
3
3
4
4
3
3
4
4
2
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
14
16
ETXc
0
0
0
0
0
2
2
0
2
2
2
1
0
3
1
3
2
4
2
1
2
3
1
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
7
13
14
PHTc
a
The data indicate the number of rats of four that were protected at a dose of
50 mg/kg.
Neurotoxicity screening – rotorod test. The data in indicate the number of rats
of four in which neurotoxicity was observed at a dose of 50 mg/kg.
a
b
The data indicate the number of rats of four that were protected at a dose of
30 mg/kg.
Neurotoxicity screening – rotorod test. The data in indicate the number of rats
of four in which neurotoxicity was observed at a dose of 30 mg/kg.
b
c
ETX-ethosuximide, reference drug, data from Ref. 29.
c
PHT-phenytoin, reference drug, data from Ref. 30.
2.3. Structure–activity relationships
scPTZ tests, does exhibit protective 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.23 The results obtained are
shown in Table 5. As can be seen from mice ip. data, the most
active was compounds 1 that showed maximal 100% protection
at time points 0.5, 1, and 2 h. The other molecules were less active
and protected up to 75% of mice. The quantitative studies (Table 6)
performed for 5-cyclopropyl-5-phenyl-imidazolidine-2,4-dione (1)
revealed comparable ED50 value with result observed for leveti-
racetam that was used as model anticonvulsant. It should be
noticed that levetiracetam is one of the newest and the most
effective antiepileptic drugs marked during last years.
The results of preliminary anticonvulsant screening in mice
enable to draw some general conclusions about the relations
between structure and activity. From the whole series the most
active was unsubstituted 5-cyclopropyl-5-phenyl-imidazolidine-
2,4-dione (1), therefore the presence of the acidic proton (N3H)
seems to be important, however, not crucial for anticonvulsant
activity. In general, introduction of chloro atom at para position of
the 5-phenyl ring (2, 10–12, 15, and 16) decreased activity in com-
parison to unsubstituted compounds (1, 3–9, 13, and 14). Further
analysis showed that compounds with dihydro-1H-isoquinoline
moiety (13, 15) revealed lower protection in comparison to pipera-
zine (3–12) or morpholine (14, 16) derivatives. Except of 11 and 12

H. Byrtus et al. / Bioorg. Med. Chem. 19 (2011) 6149–6156
6153
Table 4
Quantification studies of selected compounds in rats after oral administration
b
b
c
Cmpd
TPE (h)a
2
MES ED50 (mg/kg)
5.76 (3.81–7.50)e
scPTZ ED50 (mg/kg)
NT TD50 (mg/kg)
PId
1
57.31 (38.61–82.88)
77.57 (53.36–110.57)
13.47 (MES)
1.35 (scPTZ)
>1.60 (MES)
>5.86 (MES)
>18.30 (MES)
>5.98 (MES)
<10.79 (MES)
>35.58 (MES)
>3.0 (scPTZ)
2
3
5
7
14
PTHf
ETXf
2
4
2
6
4
1
2
ND
84.98 (44.87–336.42)
ND
<100
ND
ND
>500
>135
>100
>200
>60
<150
>1000
>500
17.06 (10.13–28.38)
10.92 (5.91–19.16)
10.03 (3.88–20.25)
13.90 (8.72–24.90)
28.1 (27.7–35.20)
>500
167.00 (116.00–237.00)
ND – not determined.
a
Time to peak effect.
b
c
d
e
f
ED50 – median effective dose required to assure anticonvulsant protection in 50% animals.
TD50 – median toxic dose eliciting minimal neurological toxicity in 50% animals.
PI – protective index (TD50/ED50).
95% Confidence limits given in parentheses.
Reference drugs, data for phenytoin (PHT) and ethosuximide (ETX), Ref. 31.
Table 5
additionally superior or comparable activity to other model anti-
convulsants, namely ethosuximide and levetiracetam.
Anticonvulsant activity – psychomotor seizure test (6-Hz, current 32 mA)
Cmpd
Intraperitoneal injection into micea
0.25 h
0.5 h
1 h
2 h
4 h
4. Experimental section
4.1. Chemistry
1
2
16
1
0
0
4
0
2
4
2
3
4
2
2
2
1
2
a
Dose of 100 mg/kg was administrated. The data indicate the number of mice of
All chemicals and solvents were obtained from Merck (Darms-
tadt, Germany) and were used without purification. Melting points
(mp) were determined in open capillaries on a Büchi 353 melting
point apparatus (Büchi Labortechnik, Flawil, Switzerland) and are
uncorrected. The purity and homogeneity of the compounds were
assessed by the thin-layer chromatography (TLC) performed on
Merck silica gel 60 F254 aluminum sheets (Merck, Darmstadt,
Germany), using the developing system consisted of S1: methanol:
25% ammoniac (10:1.5, v/v). Spots were detected by their absorp-
tion under UV light (k = 254 nm) and by visualization with
0.05 mol I2 in 10% HCl. The structures were confirmed by both
spectral (1H NMR, LC/MS) and elemental analysis. Elemental anal-
ysis for C, H, and N were carried out by a micro method using the
elemental Vario EI III elemental analyzer (Hanau, Germany). The
results of elemental analyses were within 0.4% of the theoretical
values. 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) relative to TMS d = 0. The J values are expressed
in Hertz (Hz). Signal multiplicities are represented by the following
abbreviations: s (singlet), br s (broad singlet), d (doublet), t (trip-
let), m (multiplet). The liquid chromatography/mass spectrometry
(LC/MS) spectra for chosen compounds were obtained on Applied
Biosystem/MDS-SCIEX API 2000 with Agilent HPLCs.
four that were protected.
Table 6
Quantification data
injection into mice
– psychomotor seizure test (6 Hz, current 32 mA) after ip.
PId
(TD50/
ED50
b
c
Cmpd
TPE
(h)a
ED50
TD50
(mg/kg)
(mg/
kg)
)
1
2
1
26.06 (18.80–
26.16)
19.40 (9.90–36.0)
ND
ND
Levetiracetame
>500
>26
ND – not determined.
a
Time to peak effect.
b
ED50 – median effective dose required to assure anticonvulsant protection in
50% animals.
c
TD50 median toxic dose eliciting minimal neurological toxicity in 50% animals.
PI – protective index (TD50/ED50).
d
the introduction of alkyl, alkylene, ketone or ester linkers between
piperazine and phenyl ring extended anticonvulsant protection to
pentylenetetrazole seizures as well as made compounds active in
both time intervals (0.5 and 4 h in mice). It should be noticed as
described previously,19 the 4-phenylpiperazine derivatives were
active exclusively in maximal electroshock (MES) test and showed
activity only at 4 h after ip. administration in mice (the data for
selected compounds I–IV from Ref. 19 are shown in Table 1).
The synthesis, physicochemical and spectral data of 5-cyclopro-
pyl-5-phenyl-imidazolidine-2,4-dione (1) and 5-cyclopropyl-5-
(4-chlorophenyl)-imidazolidine-2,4-dione (2) were described
elsewhere.19
3. Conclusion
4.1.1. General procedure for preparation of compounds 3–16
To a mixture of 5-cyclopropyl-5-phenyl- (0.01 mol; 1) or 5-
cyclopropyl-5-(4-chlorophenyl)-imidazolidine-2,4-dione
(0.01 mol; 2), 40% solution of formaldehyde (0.01 mol) in 96%
ethanol (20 mL), corresponding 4-substituted piperazines
(0.01 mol), tetrahydroisoquinoline (THIQ) or morpholine dissolved
in 96% ethanol (10 mL) were added. The mixture was left for ca.
12 h at room temperature and then refrigerated at ca. À10 °C for
24 h. The precipitated crude products were washed with cold
ethanol, separated by filtration and recrystallized from 96% ethanol.
The library of sixteen new N-Mannich bases derived from
5-cyclopropyl-5-phenyl- and 5-cyclopropyl-5-(4-chlorophenyl)-
imidazolidine-2,4-dione derivatives has been synthesized and
tested for anticonvulsant activity. The results of anticonvulsant
screening revealed that some of derivatives were effective in the
MES test. The quantitative studies in rats after oral administration
showed that five compounds were more potent than phenytoin in
the maximal electroshock test. The highest activity was observed
for 5-cyclopropyl-5-phenyl-imidazolidine-2,4-dione that showed

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H. Byrtus et al. / Bioorg. Med. Chem. 19 (2011) 6149–6156
4.1.1.1. 3-[(4-Phenethyl-piperazin-1-yl)-methyl]-5-cyclopropyl-
5-phenyl-imidazolidine-2,4-dione (3). White powdery crystals.
TLC: Rf = 0.59 (S1), yield: 66%, mp 86–88 °C. 1H NMR (300 MHz)
CDCl3: d 0.32–0.38 (m, 1H, cyclopropane), 0.53–0.59 (m, 1H,
cyclopropane), 0.64–0.75 (m, 2H, cyclopropane), 1.58–1.68 (m,
1H, cyclopropane), 2.40–2.82 (m, 10H, 8H, piperazine, 2H, CH2),
3.65–3.74 (m, 2H, CH2), 4.46 (s, 2H, CH2), 6.00 (s, 1H, N1H),
7.15–7.26 (m, 5H, ArH), 7.36–7.40 (m, 3H, ArH), 7.49–7.52 (m,
2H, ArH). Anal. Calcd for C25H30N4O2: C, 71.74; H, 7.22; N, 13.39.
Found: C, 71.00; H, 7.33; N, 12.78.
(m, 3H, ArH), 7.54–7.58 (m, 2H, ArH). Anal. Calcd for C20H26N4O4:
C, 62.15; H, 6.78; N, 14.49. Found: C, 61.83; H, 6.68; N, 14.34.
4.1.1.7. 3-[4-(2-Methoxyethyl-piperazin-1-yl)-methyl]-5-cyclo-
propyl-5-phenyl-imidazolidine-2,4-dione (9). White powdery
crystals. TLC: Rf = 0.68 (S1), yield: 83%, mp 97–103 °C. 1H NMR
(300 MHz) CDCl3: d 0.31–0.38 (m, 1H, cyclopropane), 0.54–0.59
(m, 1H, cyclopropane), 0.63–0.72 (m, 2H, cyclopropane),
1.64–1.73 (m, 1H, cyclopropane), 2.42–2.57 (m, 6H, 4H, piperazine,
2H, CH2), 2.69 (t, 4H, piperazine, J = 4.90 Hz), 3.31 (s, 3H, CH3), 3.50
(s, 2H, CH2), 4.49 (s, 2H, CH2), 5.80 (s, 1H, N1H), 7.32–7.58 (m, 5H,
ArH). Anal. Calcd for C20H28N4O3: C, 64.49; H, 7.58; N, 15.04.
Found: C, 64.15; H, 7.63; N, 14.93. LC/MS: m/z calcd for
4.1.1.2. 3-[{4-(3-Phenylallyl)-piperazin-1-yl}-methyl]-5-cyclo-
propyl-5-phenyl-imidazolidine-2,4-dione (4). White powdery
crystals. TLC: Rf = 0.84 (S1), yield: 46%, mp 138–140 °C. 1H NMR
(300 MHz) CDCl3: d 0.30-0.38 (m, 1H, cyclopropane), 0.52–0.73
(m, 3H, cyclopropane), 1.64–1.73 (m, 1H, cyclopropane), 2.48 (t,
4H, piperazine, J = 4.75 Hz), 2.68 (t, 4H, piperazine, J = 4.80 Hz),
3.12 (d, 2H, CH2, J = 7.00 Hz), 4.45 (s, 2H, CH2), 5.06 (s, 1H, N1H),
6.17–6.27 (m, 1H, CH), 6.52 (d, 1H, CH, J = 16.00 Hz), 7.18–7.42
(m, 8H, ArH), 7.55–7.57 (m, 2H, ArH). Anal. Calcd for C26H30N4O2:
C, 72.35; H, 7.00; N, 12.98. Found: C, 71.35; H, 6.58; N, 12.92. LC/
MS: m/z calcd for C26H30N4O2 [M+H]+: 431.24, found: 431.30.
C
20H28N4O3 [M+H]+: 473.22, found: 473.50.
4.1.1.8. 3-[(4-Phenethyl-piperazin-1-yl)-methyl]-5-cyclopropyl-
5-(4-chlorophenyl)-imidazolidine-2,4-dione (10). White pow-
dery crystals. TLC: Rf = 0.59 (S1), yield: 83%, mp 84–87 °C. 1H
NMR (300 MHz) CDCl3:
d
0.31–0.38 (m, 1H, cyclopropane),
0.54–0.62 (m, 1H, cyclopropane), 0.65–0.76 (m, 2H, cyclopropane),
1.60–1.69 (m, 1H, cyclopropane), 2.17–2.76 (m, 10H, 8H pipera-
zine, 2H, CH2), 3.67–3.76 (m, 2H, CH2), 4.50 (s, 2H, CH2), 6.00 (s,
1H, N1H), 7.17–7.30 (m, 5H, ArH), 7.39 (d, 2H, ArH, J = 4.60 Hz)
7.55 (d, 2H, ArH, J = 4.60 Hz). Anal. Calcd for C25H29ClN4O2: C,
66.28; H, 6.45; N, 12.37. Found: C, 66.54; H, 6.70; N, 12.46. LC/
MS: m/z calcd for C25H29ClN4O2 [M+H]+: 453.20, found: 453.50.
4.1.1.3. 3-[4-(1-Phenyl-ethyl-piperazin-1-yl)-methyl]-5-cyclo-
propyl-5-phenyl-imidazolidine-2,4-dione (5). White powdery
crystals. TLC: Rf = 0.58 (S1), yield: 80%, mp 73–75 °C. 1H NMR
(300 MHz) CDCl3: d 0.32-0.38 (m, 1H, cyclopropane), 0.48–0.72
(m, 2H, cyclopropane), 1.32 (s, 3H, –CH3), 1.60–1.75 (m, 2H, cyclo-
propane), 2.40–2.59 (m, 8H, piperazine), 3.66–3.72 (m, 1H, CH),
4.46 (s, 2H, CH2), 6.05 (s, 1H, N1H), 7.12–7.26 (m, 5H, ArH),
7.34–7.42 (m, 3H, ArH), 7.53–7.57 (m, 2H, ArH). Anal. Calcd for
4.1.1.9. 3-[4-(1-Phenyl-ethyl-piperazin-1-yl)-methyl]-5-cyclo-
propyl-5-(4-chlorophenyl)-imidazolidine-2,4-dione
(11). White powdery crystals. TLC: Rf = 0.73 (S1), yield: 67%, mp
73–78 °C. 1H NMR (300 MHz) CDCl3: d 0.32–1.20 (m, 5H, cyclopro-
pane), 1.32 (d, 3H, CH3), 2.38–2.60 (m, 8H, piperazine), 3.67–3.73
(m, 1H, CH), 4.41 (s, 2H, CH2), 6.01 (s, 1H, N1H), 7.24–7.39 (m,
5H, ArH), 7.40 (d, 2H, ArH, J = 2.20 Hz), 7.52 (d, 2H, ArH,
J = 2.20 Hz). Anal. Calcd for C25H29ClN4O2: C, 66.28; H, 6.45; N,
12.37. Found: C, 66.07; H, 6.71; N, 11.96.
C25H30N4O2: C, 71.74; H, 7.22; N, 13.39. Found: C, 71.54; H, 7.20;
N, 12.68.
4.1.1.4. 3-[(4-Benzoyl-piperazin-1-yl)-methyl]-5-cyclopropyl-5-
phenyl-imidazolidine-2,4-dione (6). White powdery crystals.
TLC: Rf = 0.80 (S1), yield: 56%, mp 142–144 °C. 1H NMR
(300 MHz) CDCl3: d 0.32–0.42 (m, 2H, cyclopropane), 0.48–0.57
(m, 1H, cyclopropane), 0.63–0.68 (m, 1H, cyclopropane),
1.23–1.32 (m, 1H, cyclopropane), 2.63–2.72 (m, 4H, piperazine),
3.46–3.74 (m, 2H, piperazine), 3.75–3.84 (m, 2H, piperazine),
4.53 (s, 2H, CH2), 6.00 (s, 1H, N1H), 7.21–7.36 (m, 5H, ArH),
7.40–7.49 (m, 3H, ArH), 7.54–7.57 (m, 2H, ArH). Anal. Calcd for
4.1.1.10. 3-[4-(Benzyloxycarbonyl-piperazin-1-yl)-methyl]-
5-cyclopropyl-5-(4-chlorophenyl)-imidazolidine-2,4-dione
(12). White powdery crystals. TLC: Rf = 0.55 (S1), yield: 75%, mp
121–123 °C. 1H NMR (300 MHz) CDCl3: d 0.35–0.37 (m, 1H, cyclo-
propane), 0.50–0.62 (m, 1H, cyclopropane), 0.65–0.69 (m, 2H,
cyclopropane), 1.61–1.64 (m, 1H, cyclopropane), 2.58 (t, 4H, piper-
azine, J = 4.90 Hz), 3.48 (t, 4H, piperazine, J = 5.05 Hz), 4.50 (s, 2H,
CH2), 5.10 (s, 2H, COOCH2), 6.48–6.59 (br s, 1H, N1H), 7.32–7.37
(m, 7H, ArH), 7.49–7.52 (d, 2H, ArH). Anal. Calcd for C25H27ClN4O4:
C, 62.17; H, 5.63; N, 11.60. Found: C, 62.45; H, 5.80; N, 11.37.
C
24H26N4O2: C, 68.88; H, 6.26; N, 13.39. Found: C, 68.83; H, 6.15;
N, 12.97. LC/MS: m/z calcd for C24H26N4O2 [M+H]+: 419.20, found:
419.50.
4.1.1.5.
3-[(4-Benzyloxycarbonyl-piperazin-1-yl)-methyl]-5-
cyclopropyl-5-phenyl-imidazolidine-2,4-dione (7). White pow-
dery crystals. TLC: Rf = 0.72 (S1), yield: 65%, mp 67–69 °C. 1H NMR
(300 MHz) CDCl3: d 0.34–0.38 (m, 1H, cyclopropane), 0.42–0.56
(m, 1H, cyclopropane), 0.60–0.76 (m, 2H, cyclopropane), 1.61–
1.70 (m, 1H, cyclopropane), 2.58 (t, 4H, piperazine, J = 4.74 Hz),
3.48 (t, 4H, piperazine, J = 4.80 Hz), 4.49 (s, 2H, CH2), 5.10 (s, 2H,
COOCH2) 6.48 (br s, 1H, N1H), 7.29–7.34 (m, 5H, ArH), 7.40–7.44
(m, 3H, ArH) 7.48–7.52 (m, 2H, Ph). Anal. Calcd for C25H28N4O4:
C, 66.95; H, 6.29; N, 12.49. Found: C, 66.38; H, 6.54; N, 12.13.
4.1.1.11. 3-[(3,4-Dihydro-1H-isoquinolin-2-yl)-methyl]-5-cyclo-
propyl-5-phenyl-imidazolidine-2,4-dione (13). White powdery
crystals. TLC: Rf = 0. 64 (S1), yield: 85%, mp 171–174 °C. 1H NMR
(300 MHz) CDCl3: d 0.34–0.38 (m, 1H, cyclopropane), 0.57–0.83
(m, 3H, cyclopropane), 1.77–1.78 (m, 1H, cyclopropane),
2.95–3.00 (t, 4H, isoquinoline J = 4.13 Hz), 3.87 (s, 2H, isoquino-
line), 4.78 (s, 2H, CH2), 5.59 (s, 1H, N1H), 7.06–7.19 (m, 4H, ArH),
7.39–7.48 (m, 3H, ArH), 7.60–7.64 (m, 2H, ArH). Anal. Calcd for
C22H23N3O2: C, 71.10; H, 6.25; N, 11.73. Found: C, 71.98; H, 6.13;
N, 11.72.
4.1.1.6. 3-[(4-Ethoxycarbonyl-piperazin-1-yl)-methyl]-5-cyclo-
propyl-5-phenyl-imidazolidine-2,4-dione (8). White powdery
crystals. TLC: Rf = 0.77 (S1), yield: 53%, mp 79–81 °C. 1H NMR
(300 MHz) CDCl3: d 0.32–0.39 (m, 1H, cyclopropane), 0.43–0.59
(m, 1H, cyclopropane), 0.61–0.74 (m, 2H, cyclopropane), 1.13
(s, 3H, CH3), 1.65–1.75 (m, 1H, cyclopropane), 2.58 (t, 4H, pipera-
zine, J = 5.00 Hz), 3.42 (t, 4H, piperazine, J = 5.00 Hz), 4.09 (q, 2H,
CH2, J = 7.00 Hz), 4.48 (s, 2H, CH2), 6.54 (s, 1H, N1H), 7.32–7.43
4.1.1.12. 3-[(Morpholin-4-yl)-methyl]-5-cyclopropyl-5-phenyl-
imidazolidine-2,4-dione (14). White powdery crystals. TLC:
Rf = 0.38 (S1), yield: 70%, mp 110–112 °C. 1H NMR (300 MHz)
CDCl3: d 0.35–0.43 (m, 1H, cyclopropane), 0.50–0.76 (m, 3H, cyclo-
propane), 1.68–1.77 (m, 1H, cyclopropane), 2.59–2.61 (t, 4H, mor-
pholine, J = 3.80 Hz), 3.63–3.66 (t, 4H, morpholine, J = 3.80 Hz),
4.46 (s, 2H, CH2), 6.73 (s, 1H, N1H), 7.26–7.59 (m, 5H, ArH). Anal.

H. Byrtus et al. / Bioorg. Med. Chem. 19 (2011) 6149–6156
6155
Calcd for C17H21N3O3: C, 64.74; H, 6.71; N, 13.32. Found: C, 64.46;
H, 6.42; N, 13.30. LC/MS: m/z calcd for C17H21N3O3 [M+H]+: 316.16,
found: 316.40.
anticonvulsant activity. For both doses the motor impairment
was studied in parallel. Rats were tested at five time periods rang-
ing from one quarter to 4 h post substance administration. The re-
sults are shown in Tables 2 and 3.
4.1.1.13. 3-[(3,4-Dihydro-1H-isoquinolin-2-yl)-methyl]-5-
cyclopropyl-5-(4-chlorophenyl)-imidazolidine-2,4-dione
(15). White powdery crystals. TLC: Rf = 0. 67 (S1), yield: 74%, mp
166–168 °C. 1H NMR (300 MHz) CDCl3: d 0.32–0.36 (m, 1H, cyclo-
propane), 0.53–0.72 (m, 3H, cyclopropane), 1.65–1.68 (m, 1H,
cyclopropane), 2.90–2.93 (m, 4H, isoquinoline), 3.80 (s, 2H, iso-
quinoline), 4.69 (s, 2H, CH2), 6.08–6.12 (br s, 1H, N1H), 7.09–7.12
(m, 4H, ArH), 7.37–7.38 (d, 2H, ArH, J = 2.05), 7.50–7.51
(d, J = 2.05, 2H, ArH). Anal. Calcd for C22H22ClN3O2: C, 66.75; H,
5.60; N, 10.61. Found: C, 66.61; H, 5.76; N, 10.65. LC/MS: m/z calcd
for C22H22ClN3O2 [M+H]+: 369.14, found: 369.60.
4.2.3. Neurological toxicity (NT)
Neurological toxicity induced by a compound was detected in
mice using standardized rotorod test.26 Untreated control mice or
rats, when placed on the rod, can maintained their equilibrium
for a prolonged time period. The acute motor impairment can be
demonstrated by the inability of the animal to maintain equilib-
rium for a given time (1 min).
4.2.4. Quantification studies
The quantitative determination of ED50 and TD50 values was per-
formed at previously estimated time of peak effect after oral admin-
istration 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 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 standard
error of the slope were calculated by means of a computer program
written at NINDS/NIH. The results are shown in Table 4.
4.1.1.14. 3-[(Morpholin-4-yl)-methyl]-5-cyclopropyl-5-(4-chlo-
rophenyl)-imidazolidine-2,4-dione (16). White powdery crys-
tals. TLC: Rf = 0.39 (S1), yield: 67%, mp 75–79 °C. 1H NMR
(300 MHz) CDCl3: d 0.33–0.39 (m, 1H, cyclopropane), 0.62–0.73
(m, 2H, cyclopropane), 1.63–1.72 (m, 2H, cyclopropane),
2.54–2.64 (m, 4H, morpholine), 3.63–3.66 (t, 4H, morpholine,
J = 4.60 Hz), 4.46 (s, 2H, CH2), 6.57 (s, 1H, N1H), 7.36–7.39 (m, 2H,
ArH), 7.49–7.54 (m, 2H, ArH). Anal. Calcd for C17H20ClN3O3: C,
58.37; H, 5.76; N, 12.01. Found: C, 58.19; H, 5.92; N, 11.57.
4.2.5. The 6-Hz model
This screen was carried out according to the protocol originally
described by Brown et al.27 and more recently by Barton et al.23
and Kaminski et al.28 It is an alternative electroshock paradigm that
uses low-frequency (6 Hz), long-duration (3 s) electrical stimula-
tion. Corneal stimulation (0.2 ms-duration monopolar rectangular
pulses at 6 Hz for 3 s) was delivered by a constant-current device.
During the stimulation, mice were manually restrained and
released into the observation cage immediately after the current
application. The seizures manifest in ‘stunned’ posture associated
with rearing, forelimb, automatic movements and clonus, twitching
of 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 sei-
zure, animals resume their normal exploratory behavior. The exper-
imental 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 stimulation28 (Table 5).
4.2. Pharmacology
The initial anticonvulsant evaluation was performed within the
Antiepileptic Drug Development (ADD) Program in Epilepsy Branch,
Neurological Disorders Program, National Institute of the Neurolog-
ical and Communicative Disorders and Stroke (NIH/NINDS), Rock-
ville, MD, USA, by using procedures described elsewhere.24,25
Male albino mice (CF-1 strain) and male albino rats
(Sprague–Dawley) were used as experimental animals. The ani-
mals were housed in metabolic cages and allowed free access to
food and water. The compounds were suspended in 0.5% methyl-
cellulose/water mixture.
The ASP initially evaluates anticonvulsant activity for newly
submitted compounds following intraperitoneal (ip.) administra-
tion in mice and oral administration in rats. Groups of eight mice
or four rats are employed. Phase I studies in mice involved two
convulsant tests: maximal electroshock seizure test (MES), subcu-
taneous pentylenetetrazole seizure test (scPTZ) and rotarod test for
neurological toxicity (NT).
Acknowledgments
The authors wish to thank Dr. James Stables for providing them
with pharmacological data through the Antiepileptic Drug Devel-
opment Program (Epilepsy Branch, National Institute of Neurolog-
ical Disorders and Stroke, National Institute of Health, Rockville,
MD, USA).
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).
4.2.1. 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 rat at 60 Hz) is delivered via corneal
electrodes primed with an electrolyte solution containing an anes-
thetic agent.
4.2.2. Subcutaneous pentylenetetrazole seizure test (scPTZ)
This screen 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 5 s in 97% (CD97) of animals tested. At the
anticipated time of testing the convulsant is administered
subcutaneously.
All the compounds were injected intraperitoneally into mice at
the dose levels of 30, 100, and 300 mg/kg with anticonvulsant
activity and neurotoxicity assessment at 0.5 and 4 h after adminis-
tration. The results are presented in Table 1.
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