Synthesis and anticonvulsant properties of new mannich bases derived from 3-aryl-pyrrolidine-2,5-diones. Part 1

Article abstract of DOI:10.1002/ardp.200900250

A series of new Mannich bases of N-[(4-arylpiperazin-1-yl)-methyl]-3- (chlorophenyl)-pyrrolidine-2,5-diones 10-23 have been synthesized and evaluated for their anticonvulsant activity in maximum electroshock (MES) and subcutaneous pentylenetetrazole (scPT

Full text of DOI:10.1002/ardp.200900250

Arch. Pharm. Chem. Life Sci. 2010, 343, 333–341
J. Obniska et al.
333
Full Paper
Synthesis and Anticonvulsant Properties of New Mannich
Bases Derived from 3-Aryl-pyrrolidine-2,5-diones. Part 1
´
Jolanta Obniska, Maciej Kopytko, Agnieszka Zagꢀrska, Iwona Chlebek, and Krzysztof Kaminski
Department of Medicinal Chemistry, Jagiellonian University, Medical College, Krakꢀw, Poland
A series of new Mannich bases of N-[(4-arylpiperazin-1-yl)-methyl]-3-(chlorophenyl)-pyrrolidine-
2,5-diones 10–23 have been synthesized and evaluated for their anticonvulsant activity in max-
imum electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizure threshold tests.
Their neurotoxicity was determined using a rotorod screen. Several molecules showed a promis-
ing anticonvulsant profile especially in the MES-test. In this model of seizures, the most active
were N-[{4-(4-chlorophenyl)-piperazin-1-yl}-methyl]-3-(3-chlorophenyl)-pyrrolidine-2,5-dione 16
and
N-[{4-(3-trifluoromethylphenyl)-piperazin-1-yl}-methyl]-3-(3-chlorophenyl)-pyrrolidine-2,5-
dione 17 with ED50 values of 21.4 mg/kg and 28.83 mg/kg, respectively. Selected derivatives 10,
14, and 16 were tested in the psychomotor seizure 6-Hz test from which N-[{4-(2-chlorophenyl)-
piperazin-1-yl}-methyl]-3-(2-chlorophenyl)-pyrrolidine-2,5-dione 10 revealed the highest protec-
tion with an ED50 of 78 mg/kg. Compounds 10, 12, and 17 were also tested in the pilocarpine-
induced status PIPS test. Furthermore, 17 was examined in the hippocampal kindling screen
after i.p. administration to rats.
Keywords: Anticonvulsant activity / N-(4-Arylpiperazin-1-yl)-methyl-pyrrolidine-2,5-dione / Mannich bases / 3-Phenyl-
pyrrolidine-2,5-dione /
Received: October 20, 2009; Accepted: January 15, 2010
DOI 10.1002/ardp.200900250
mentioned disadvantages in mind, the continued search
for safer and more effective AEDs is urgently needed.
Continued efforts are being made in the development
of antiepileptic drugs employing a range of strategies,
including modification of the structure of existing drugs,
targeting novel molecular substrates, as well as nonme-
chanism-based drug screening of compounds in animal
models [5]. Among the experimental models developed
to evaluate the anticonvulsant activity, conventional
acute seizure models are the most widely used, among
which the maximal electroshock (MES) and the subcuta-
neous pentylenetetrazole (scPTZ) tests are the most popu-
lar. Additionally, the use of alternative antiepileptic
models e.g., 6-Hz, genetic or kindled models enabled the
discovery of new, very efficient and safe drugs like levetir-
acetam or brivaracetam, which were inactive in the tradi-
tional animals seizure models (MES and scPTZ) [6, 7].
One of the important core fragments of anticonvul-
sants is defined by a nitrogen heteroatomic system usu-
ally a cyclic imide, consisting of at least one or two car-
bonyl groups and phenyl or alkyl substituents attached
Introduction
Epilepsy is not a disease but a syndrome of different cere-
bral disorders of the central nervous system (CNS), which
is characterized by recurrent spontaneous seizures aris-
ing from excessive electrical activity in some portion of
the brain [1]. Despite of the introduction of many new
antiepileptic drugs (AEDs) and remarkable strides in this
research field, more than 30% of the patients with epi-
lepsy remain refractory to available treatments [2]. More-
over, a large number of old and new AEDs cause serious
side effects, which include ataxia, nausea, mental dull-
ing, and hepatotoxicity [3, 4]. Therefore, with all of above-
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: +48 12 657-02-62
Abbreviations: antiepileptic drugs (AEDs); anticonvulsant screening
project (ASP); pilocarpine-induced status prevention (PISP); time-of-
peak-effect (TPE)
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J. Obniska et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 333–341
Figure 1. The structures of compounds 1, 2, and 3.
to the heterocyclic system [8–10]. This common template
is present in the structures of many older AEDs e.g., phe-
nobarbital, phenytoin, etosuximide, as well as in the
newest generation of anticonvulsants e.g., levetiracetam,
brivaracetam, or seletracetam.
The previous studies from our laboratory have demon-
strated the potent anticonvulsant activity in a group of
N-[4-(phenyl-piperazin-1-yl)-methyl]-3-aryl-pyrrolidine-2,5-
diones. These molecules were effective especially in elec-
trically induced seizures (MES). The most active among
these compounds were N-[{4-(3-chlorophenyl)-piperazin-
Scheme 1. General method for the synthesis of compounds 10–
23.
were prepared as previously reported [14]. The synthetic
procedures of the corresponding imides (7–9) have been
described in a separate report [15]. The target compounds
10–23 were prepared by a Mannich-type reaction from
the appropriately substituted imides 7–9, formaldehyde,
and the corresponding 4-arylpiperazines. The reaction
was carried out in ethanol at room temperature for
approx. 6–12 h. The crude products were crystallized
from 96% ethanol. Because of the oily form of com-
pounds 13 and 17, they were isolated as hydrochloride
salts and were recrystallized from anhydrous ethanol.
The final compounds were obtained in yields ranging
from 65% to 75%. Their purity was assessed by TLC chro-
matography. The structures of the compounds synthe-
sized were confirmed by spectral and elemental analyses.
The detailed physical and analytical data are listed in the
experimental section.
1-yl}-methyl]-3-(2-chlorophenyl)-pyrrolidine-2,5-dione
1
with an ED50 = 14.18 mg/kg, protective index PI (TD50/
ED50) of 8.8 and N-[4-(phenyl-piperazin-1-yl)-methyl]-3-(2-
trifluoromethyl-phenyl)-pyrrolidine-2,5-dione
2
that
showed an ED50 value of 20.78 mg/kg and PI of 24.1 (Fig. 1;
[11, 12]). In continuation of our systematic SAR studies of
these series of derivatives, recently, we have obtained sev-
eral analogues of the active compounds 1 and 2, which
were devoid of electron-withdrawing substituents at the
3-phenyl ring. Surprisingly enough, all of these mole-
cules were inactive (compound 3, Fig. 1; [13]). Taking into
consideration the above, it is justified to claim that the
presence of electron-withdrawing substituents at the 3-
phenyl ring of pyrrolidine-2,5-dione seems to be crucial
for the anticonvulsant activity of such type compounds.
Following these results and as part of our efforts to
design new anticonvulsant agents, in the present study, a
small library of N-Mannich bases with a 3-phenyl-pyrroli-
dine-2,5-dione system as a core fragment and a chloro
atom at positions 2, 3, or 4 of the phenyl ring has been
synthesized. At the imide nitrogen atom through the
methylene bridge, we have introduced 4-arylpiperazines
with different, electron-withdrawing (F, Cl, CF3) or elec-
tron-donating (CH3, OCH3) groups.
Anticonvulsant activity
The initial anticonvulsant evaluation was performed
within the Antiepileptic Drug Development (ADD) Pro-
gram in Epilepsy Branch, National Institutes of Health,
National Institute of Neurological Disorders and Stroke
(NIH/NINDS), Rockville, MD, USA. The pre-clinical discov-
ery and development of new chemical agents for the
treatment of epilepsy are based mainly on the use of pre-
dictable animal models. At the present time, there are
three in-vivo screens used routinely that include the max-
imal electroshock seizure (MES), the subcutaneous penty-
lenetetrazole (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 [16, 17].
Results and discussion
Chemistry
The synthesis of compounds 10–23 was accomplished as
shown in Scheme 1. The starting 3-(2-chlorophenyl)- 4, 3-
(3-chlorophenyl)- 5, 3-(4-chlorophenyl)- 6 succinic acids
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Arch. Pharm. Chem. Life Sci. 2010, 343, 333–341
Anticonvulsant Activity of Certain Succinimides
335
Table 1. Anticonvulsant screening project (ASP) phase I in mice
(compounds tested 10–23).
at a dose of 100 mg/kg include 12, 13, 16, 17, 21, and 23,
whereas 11 provided anti-MES protection at a dose of 300
mg/kg. In this series, 16 and 17 were active in both time
intervals – 0.5 and 4 h – however, the majority 11–13, 18,
21, and 23 showed activity at 4 h. This indicates the slow
onset and long duration of the anticonvulsant action. It
is worth to note that only 18 revealed the anti-MES activ-
ity at a dose of 30 mg/kg, on the other hand, however, it
was neurotoxic at a dose of 100 mg/kg. In the neurotoxic-
ity screen (NT) compounds 10–12, 14–16, and 22 did not
show neurotoxicity in the maximum dose administered
(300 mg/kg). Among these, 10, 14, 15, and 22 were inac-
tive. Compounds 13, 17, 19–21, and 23 exhibited motor
impairment at the dose of 300 mg/kg at 0.5 and 4 h. The
results of the neurotoxicity screening showed that 12
and 16 emerged as anticonvulsants without neurotoxic
properties.
A valuable property of a candidate anticonvulsant is its
ability to inhibit convulsions when given orally. This
screen discloses the time of onset, the approximate time-
of-peak-effect (TPE) and the duration of anticonvulsant
activity or neurotoxicity. On the basis of the i.p.-screen-
ing data in mice, and according to the Anticonvulsant
Screening Project (ASP) disposition, six compounds 13,
16–18, 21, and 23 were selected and examined in the MES
test as well as neurotoxicity screen after p.o. administra-
tion into rats at a dose of 30 mg/kg. The results obtained
are presented in Table 2. The most active were com-
pounds 16, 17, and 18; they protected 100% of the ani-
mals at 0.5 h (17) or 4 h (16 and 18). The following mole-
cules provided 75% protection at 2 h: 16, 50% protection
at 1 h and 2 h: 17 and 18 or at 4 h: 17. 25% protection was
given at 0.25 h by: 17, at 0.5 h 18, and 1 h 16. The other
compounds were less active, namely 21 showed a peak of
75% protection at 1 and 4 h; compound 23 was effective
in 25, 75, and 50% of the animals at 1 h, 2 h, and 4 h,
respectively. Finally, compound 13 protected 25% of the
tested animals at the time of 0.5 h and 1 h as well as 50%
of rats at 2 and 4 h. Except for 18, all other derivatives
were non neurotoxic when given orally.
Com-
pound
R
R1
Intraperitoneal injection
into mice
MESa)
4 h
TOXb)
4 h
0.5 h
0.5 h
10
11
12
13
14
15
16
17
18
19
20
21
22
23
2-Cl
2-Cl
2-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
4-Cl
4-Cl
4-Cl
4-Cl
–
2-Cl
4-Cl
3-CF3
2-F
–
–
–
–
–
–
300
100
–
–
–
–
–
–
30
–
–
–
–
–
–
–
–
–
–
–
300
–
300
300
300
–
–
100
300
100
100
–
3001
–
2-Cl
3-Cl
4-Cl
3-CF3
3-CH3
2-OCH3
2-Cl
3-Cl
4-Cl
3-CF3
–
–
–
–
100
100
30
–
–
100
–
300
100
300
300
300
–
100
30
300
100
Pheny-
toinc)
The figures in the table indicate the minimum dose whereby
bioactivity or neurotoxicity was demonstrated in half or more
of the animals. A dash indicates the absence of activity or neu-
rotoxicity at the maximum dose administrated (300 mg/kg).
a) Maximal electroshock test; b) rotorod toxicity; c) reference
drug, data for phenytoin, Ref. [27]; Response comments: 1death.
The anticonvulsant activity profile of all the com-
pounds was established with maximal electroshock
(MES) and subcutaneous pentylenetetrazole (scPTZ) tests,
after intraperitoneal injection of the test compounds
into mice at doses of 30, 100, and 300 mg/kg. An observa-
tion 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. The results are shown in Table 1.
The compounds studied revealed diversified anticon-
vulsant properties, nevertheless, they were effective
mainly in the MES test. The initial evaluation in mice
(phase I) showed that excluding molecules 10, 14, 15, 19,
20, and 22 which were devoid of activity, all other deriv-
atives were effective in the i. p. MES screen.
On the basis of oral data from rats, compound 16 and
17 were chosen for phase-II evaluation for the quantifica-
tion of the pharmacological parameters (ED50 and TD50).
The quantitative evaluations of the MES median effective
dose (ED50) and toxic dose (TD50) were performed after
oral administration to rats at the previously estimated
time-of-peak-effect (TPE) – 4 h for 16 and 0.5 h for 17.
Results of the quantitative tests along with the data for
the standard drug phenytoin are shown in Table 3. Also
shown there, compound 16 revealed an ED50 of 21.40 mg/
kg and TD50 > 500 mg/kg, resulting in a protection index
(TD50/ED50) of 23.37, whereas 17 was less active and more
The MES test is claimed to detect compounds affording
protection against generalized tonic-clonic seizures.
Compounds that revealed protection in the MES test indi-
cating the ability of a substance to prevent seizure spread
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J. Obniska et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 333–341
Table 2. Anticonvulsant activity of selected compounds administrated p.o. to rats at a dose of 30 mg/kg.
Compound
MES)
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
13
16
17
18
21
23
1
0
1
0
0
0
1
1
0
4
1
0
0
4
0
1
2
2
3
1
3
2
3
2
2
0
3
3
2
4
2
4
3
2
3
0
0
0
0
0
0
ND
0
0
0
3
0
0
ND
0
0
0
0
0
0
ND
0
0
0
0
0
0
ND
0
0
0
0
0
0
ND
Phenytoinc)
a) Maximal electroshock test, number of animals protected/number of animals tested; b) rotorod test for neurological toxicity,
number of animals exhibiting toxicity/number of animals tested; c) reference drug, data for phenytoin, Ref. [28]; ND: no data.
Table 3. Quantification studies of compounds 16 and 17 in the MES and neurotoxicity test (NT) in rats after p.o. administration.
TPE (h)a)
ED50b) (MES)
(mg/kg)
TD50
PId)(MES)
(TD50/ED50)
c)
Compound
(mg/kg)
16
4
21.4
>500
23.37
5.30
(12.93–40.05)e)
28.83
17
0.5
2
152.8
(121.45–193.98)
>500
(16.28–46.75)
23.20
Phenytoinf)
>21.60
(21.4–25.4)
(36.9–46.1)
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; d) PI – protective index (TD50/ED50); e) 95% confidence limits
given in parentheses; f) reference drug, data for phenytoin, Ref. [29].
Table 4. Quantification studies of compound 16 in the MES, scPTZ, and neurotoxicity test (NT) in mice after i. p. administration.
c)
Compound
TPE
(h)a)
ED50b) (MES)
(mg/kg)
ED50b) (scPTZ)
(mg/kg)
TD50
PId) (MES)
(TD50/ED50)
(mg/kg)
16
6
1
71.84
376.32
(260.62–539.15)
>500
519.16
(477.40–593.75)
41.2
7.23 (MES)
1.38 (scPTZ)
6.52
(53.14–96.89)e)
5.32
Phenytoinf)
(5.44–7.23)
(36.9–46.1)
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; d) PI – protective index (TD50/ED50); e) 95% confidence limits
given in parentheses; f) reference drug, data for phenytoin, Ref. [29].
toxic and revealed an ED50 value of 28.83 mg/kg, a TD50 mg/kg. It is worth to note that despite of the lower activ-
value of 152.8 mg/kg, and a protective index (PI) of 5.30. ity of 16 in the MES screen comparing to phenytoin, this
The quantitative data showed that compound 16 was molecule was also active in the pentylenetetrazole seiz-
more potent and safer (higher PI index) than phenytoin ures, which may suggest its potential effectiveness in
which was used as reference drug for anticonvulsants different types of human epilepsies. Compound 17
active in the MES test.
underwent further studies in the hippocampal-kindled
Continuing the pharmacological studies of compound rats test after i.p. administration. Kindling is a phenom-
16, the quantitative evaluation of the MES and scPTZ, enon whereby repeated, low level, initially subconvul-
ED50 and TD50 values were determined after intraperito- sive, electrical brain stimulation results in a progressive
neal administration into mice (TPE = 6 h) (Table 4). Com- intensification of seizure activity, eventually leading to
pound 16 showed ED50 values of 71.84 mg/kg (MES, PI = fully generalized electrographic and motor seizures [18].
7.23), 376.32 mg/kg (scPTZ, PI = 1.38), and TD50 of 519.16 The hippocampal-kindled rat test is an excellent animal
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Arch. Pharm. Chem. Life Sci. 2010, 343, 333–341
Anticonvulsant Activity of Certain Succinimides
337
Table 5. Anticonvulsant evaluation – psychomotor seizure tests (6-Hz) after i.p. injection into mice.
Compound
Intraperitoneal injection into micea)
0.25 h
0.5 h
1 h
2 h
4 h
10
14
16
3
1
0
3
3
0
4
1
0
2
0
2
0
0
2
a) Dose of 100 mg/kg was administrated. The data indicate the number of mice out of four that were protected.
Table 6. Quantification data – psychomotor seizure tests (6-Hz) after i.p. injection into mice.
TPE (h)a)
ED50b) (6-Hz)
(mg/kg)
TD50
PId)
(TD50/ED50)
c)
Compound
(mg/kg)
10
1
1
78.00
>600
(477.40–593.75)
>500
>7
(54.20–109.30)e)
19.40
Levetiracetamf)
>26
(9.90–36.00)
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; d) PI – protective index (TD50/ED50); e) 95%-confidence limits
given in parentheses; f) data for levetiracetam from ref. [17].
model for the study of seizure activity and is an accept- mice at 0.25 h and 1 h as well as 75% at 0.5 h. It is worth
able model for the study of human partial epilepsy, noticing that 10 and 14 were inactive in mice MES and
which is still one of the most important therapeutic chal- scPTZ i. p. screens. Finally, 16 protected 50% of the ani-
lenges. Compound 17 has demonstrated high activity – mals in two time intervals – 2 h and 4 h. Compound 10,
ED50 of 28.5 mg/kg (95% confidence interval: 14.9–44.1 as the most active, was selected for quantification of i.p.
mg/kg), whereas motor impairment was observed at a ED50 and TD50 values at TPE = 1 h in mice. This molecule
dose of 300 mg/kg.
showed an ED50 = 78 mg/kg, TD50 > 600 mg/kg and a pro-
According to the ASP dispositions, compounds 10 and tective index (PI) > 7 (Table 6).
14 inactive both in MES and scPTZ test as well as 16 (active
According to the ASP dispositions, compounds 10, 12,
in the MES screen), were chosen for the evaluation of 16, and 17 were assessed for potential activity against
anticonvulsant properties in the 6-Hz test. The selection nerve agents using the pilocarpine-induced status pre-
was made randomly as part of the search for molecules vention (PISP) screen, which is one of the animal models
providing anti 6-Hz protection among chemically diversi- of status epilepticus (SE). The outcome measures are the
fied compounds pursued in the NIH/NINDS. The 6-Hz determination of “protection” or “no-protection”. The
screen has been validated as a model of therapy-resistant results are shown in Table 7. In the PISP screen, only 12
epilepsy recently. It was not used widely because of its was found to be active and protected seven animals (rats)
lack of clinical validity, since the hydantoins such as phe- at the time-zero-dose of 200 mg/kg (i.p.) . The other com-
nytoin failed to show protective activity. Nevertheless, pounds 10, 16, and 17 were inactive. To determine the
the clinically effective antiepileptic drug levetiracetam, acute motor impairment doses of 100 mg/kg, 300 mg/kg,
which is not active in the conventional MES test, does and 600 mg/kg the compounds were administrated via
exhibit protective activity in the 6-Hz model. This sug- the i.p. route. Two rats per each dose were used. The ani-
gested that the 6-Hz model might be capable for identify- mals were observed during the following time periods:
ing antiseizure agents with a novel spectrum of activity 0.25, 0.5, 1, 2, and 4 h. The result of the neurotoxicity
and unknown mechanism of anticonvulsant action [19]. screens revealed that compound 12 caused motor impair-
The results obtained for compounds 10, 14, and 16 are ment at a dose of 100 and 300 mg/kg at the time of 2 h,
shown in Table 5. The results obtained revealed that 10 whereas 17 caused the effect at a dose of 600 mg/kg at a
was the most active compound, which, administrated time ranging from 1 to 4 h. Compounds 10 and 16 were
intraperitoneally into mice at a dose of 100 mg/kg, pro- non-neurotoxic at all doses applied.
tected 75% of animals at 0.25 h and 0.5 h, 100% at 1 h,
In conclusion, the results of the preliminary screening
and 50% at 2 h. Its 3-chloro analogue 14 protected 25% of revealed that anticonvulsant activity depended on the
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338
J. Obniska et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 333–341
Table 7. Pilocarpine-induced status prevention (PISP) model. The results for compound 12 after i.p. administration into mice.
Compound
Dose (mg/kg)
Time (h)a)
Protected
rats
Nonprotected
rats
Average Weight Change
€ SEM (g)b)
Protected
Nonprotected
rats
rats
12
200
0
7
8
15.7 € 2.1
20.0 € 0.0
a) Post first stage III seizure; b) weight change 24 h post first stage III seizure.
Switzerland) and are uncorrected. The purity of the compounds
was confirmed by thin-layer chromatography (TLC) performed
on Merck silica gel 60 F254 aluminum sheets (Merck), using
chloroform / acetone (9:1, v/v) as developing system. Spots were
detected by their absorption under UV light (k = 254 nm) and by
visualization with 0.05 mol I2 in 10% HCl. Elemental analyses for
C, H, and N were carried out by a micro method using the ele-
mental Vario EI III Elemental analyser (Heraeus, Hanau, Ger-
many). The results of elemental analyses were within € 0.4% of
the theoretical values. 1H-NMR spectra were obtained in a Varian
Mercury spectrometer (Varian Inc., Palo Alto, CA, USA), in CDCl3,
operating at 300 MHz. Chemical shifts are reported in d values
(ppm) relative to TMS d = 0 (1H), as internal standard. The J values
are expressed in Hertz (Hz). Signal multiplicities are represented
by the following abbreviations: s (singlet), brs (broad singlet), d
(doublet), dd (double doublets), t (triplet), q (quartet), m (multip-
let).
position of the chloro atom at the succinimide 3-phenyl
ring and also on the kind and position of the substituents
at the 4-phenyl-piperazine moiety. In general, the most
active compounds were those with a chloro atom located
at the meta-position of the 3-phenyl ring. The respective
ortho- and para-analogs were less active. Moreover, taking
into consideration the results of our previous studies
which showed the lack of activity among unsubstituted
3-phenyl-pyrrolidine-2,5-diones [13] or low activity for
the respective 3-(methylphenyl)-pyrrolidine-2,5-diones, it
seems that the presence of an electron-withdrawing
chloro atom in this fragment is decisive for anticonvul-
sant activity. Considering the 4-phenyl-piperazine moi-
ety, the most potent compounds were those with a para-
chloro or meta-trifluoromethyl substitution. The intro-
duction of a chloro atom or a methoxy group at the ortho-
position yielded inactive molecules (one exception was
observed for the 2-fluoro derivative 13 that was active in
the MES test).
General procedure for preparation of compounds 10–23
To a mixture of 3-(2-chlorophenyl)- (0.01 mol; 7), 3-(3-chloro-
phenyl)- (0.01 mol; 8), or 3-(4-chlorophenyl)-pyrrolidine-2,5-dione
(0.01 mol; 9) in 96% ethanol (20 mL) and 40% solution of formal-
dehyde (0.01 mol), corresponding 4-arylpiperazines (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 approx-
imately –108C for 24 h. The precipitated crude products were
washed with cold ethanol, separated by filtration, and recrystal-
lized from 96% ethanol (Scheme 1). Compounds 13 and 17 were
obtained as light oils. These molecules were converted to hydro-
chloride salts in anhydrous ethanol saturated with HCl gas.
They were crystallized from anhydrous ethanol.
Conclusion
The results obtained revealed that number of N-[(4-arylpi-
perazin-1-yl)-methyl]-3-(chlorophenyl)-pyrrolidine-2,5-dio-
nes were effective in the MES screen. The most active
were N-[{4-(4-chlorophenyl)-piperazin-1-yl}-methyl]-3-(3-
chlorophenyl)-pyrrolidine-2,5-dione with ED50 of 21.4
mg/kg, TD50 > 500 mg/kg and a protection index (TD50/
ED50) of 23.37 mg/kg. This molecule was more potent and
safer than phenytoin, which was used as reference drug.
Several compounds revealed high activity in the 6-Hz
seizure test and pilocarpine-induced status prevention
(PISP) screen, which may indicate their potential efficacy
in the refractory epilepsy as well as status epilepticus.
N-[{4-(2-Chlorophenyl)-piperazin-1-yl}-methyl]-3-(2-
chlorophenyl)-pyrrolidine-2,5-dione 10
1
White powdery crystals. Yield: 70%; m.p.: 107–1098C; H-NMR
(300 MHz, CDCl3) d: 2.85 (t, 4H, piperazine, J = 5.38 Hz), 2.92 (dd,
1H, imide, J = 4.36 Hz, J = 18.21 Hz), 3.06 (t, 4H, piperazine, J =
4.49 Hz), 3.24 (dd, 1H, imide, J = 5.39 Hz, J = 10.00 Hz), 4.38 (q, 1H,
imide, J = 5.90 Hz), 4.64 (t, 2H, -CH2-, J = 13.08 Hz), 6.94–7.05 (m,
2H, ArH), 7.21–7.25 (m, 6H, ArH). C21H21N3O2Cl2 (418.33).
N-[{4-(4-Chlorophenyl)-piperazin-1-yl}-methyl]-3-(2-
Experimental
chlorophenyl)-pyrrolidine-2,5-dione 11
1
White powdery crystals. Yield: 75%; m.p.: 106–1088C; H-NMR
Chemistry
(300 MHz, CDCl3) d: 2.82–2.85 (m, 4H, piperazine), 2.95 (dd, 1H,
imide, J = 5.90 Hz, J = 17.65 Hz), 3.16 (brs, 4H, piperazine), 3.26
(dd, 1H, imide, J = 9.75 Hz), 4.35 (q, 1H, imide, J = 6.16 Hz), 4.61 (t,
2H, -CH2-, J = 13.33 Hz), 6.83 (d, 2H, ArH, J = 8.98 Hz), 7.17–7.39
(m, 5H, ArH), 7.40–7.44 (m, 1H, ArH). C21H21N3O2Cl2 (418.33).
All chemicals and solvents were purchased from Merck (Darm-
stadt, Germany) and were used without further purification.
Melting points (m.p.) were determined in open capillaries on a
Bꢀchi 353 melting point apparatus (Bꢀchi Labortechnik, Flawil,
i 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2010, 343, 333–341
Anticonvulsant Activity of Certain Succinimides
339
N-[{4-(3-Trifluoromethylphenyl)-piperazin-1-yl}-methyl]-3-
N-[{4-(3-Methylphenyl)-piperazin-1-yl}-methyl]-3-(3-
(2-chlorophenyl)-pyrrolidine-2,5-dione 12
chlorophenyl)-pyrrolidine-2,5-dione 18
White powdery crystals. Yield: 65%; m.p. 118–1208C; 1H-NMR
(300 MHz, CDCl3) d: 2.85–2.93 (m, 5H, 4H piperazine; 1H imide,),
3.23–3.32 (m, 5H, 4H piperazine; 1H imide,), 4.36 (q, 1H, imide, J
= 5.90 Hz), 4.65 (t, 2H, -CH2-, J = 13.33 Hz), 7.05–7.11 (m, 3H, ArH),
7.21–7.43 (m, 5H, ArH). C22H21N3O2Cl1F3 (451.88).
White powdery crystals. Yield: 76%; m.p.: 81–838C; 1H-NMR (300
MHz, CDCl3) d: 2.31 (s, 3H, -CH3), 2.78 (brs, 4H, piperazine), 2.84
(dd, 1H, imide, J = 5.13 Hz, J = 18.72 Hz), 3.16 (t, 4H, piperazine, J
= 4.99 Hz), 3.23 (dd, 1H, imide, J = 9.49 Hz, J = 18,46 Hz), 4.03 (q,
1H, imide, J = 5.13 Hz), 4.60 (t, 2H, -CH2-, J = 13.59 Hz), 6.68–6.73
(m, 3H, ArH), 7.09–7.18 (m, 2H, ArH), 7.29–7.34 (m, 3H, ArH).
C22H24N3O2Cl1 (397.90).
Monohydrochloride N-[{4-(2-fluorophenyl)-piperazin-1-
yl}-methyl]-3-(3-chlorophenyl)-pyrrolidine-2,5-dione 13
N-[{4-(2-Methoxyphenyl)-piperazin-1-yl}-methyl]-3-(3-
1
White powdery crystals. Yield: 72%; m.p.: 157–1598C; H-NMR
chlorophenyl)-pyrrolidine-2,5-dione 19
(300 MHz, CDCl3) d: 2.92 (dd, 1H, imide, J = 6.41 Hz, J = 17.95 Hz),
3.06 (brs, 4H, piperazine), 3.21–3.48 (m, 5H, 4H piperazine; 1H
imide), 4.37 (q, 1H, imide, J = 6.15 Hz), 4.70 (s, 2H, -CH2-), 6.97–
7.19 (m, 3H, ArH), 7.34–7.40 (m, 4H, ArH), 7.56 (s, 1H, ArH), 11.36
(s, 1H, HCl). C21H22N3O2Cl2F1 (438.34).
White powdery crystals. Yield: 67%; m.p.: 99–1018C; 1H-NMR
(300 MHz, CDCl3) d: 2.85–2.89 (m, 5H, 4H piperazine; 1H imide),
3.09 (brs, 4H, piperazine), 3.25 (dd, 1H, imide, J = 9.49 Hz, J =
18.46 Hz), 3.85 (s, 3H, -OCH3), 4.07 (q, 1H, imide, J = 5.39 Hz), 4.62
(t, 2H, -CH2-, J = 13.20 Hz), 6.85–7.03 (m, 4H, ArH), 7.16 (d, 1H,
ArH, J = 6.15 Hz), 7.29–7.36 (m, 3H, ArH). C22H24N3O3Cl1 (413.90).
N-[{4-(2-Chlorophenyl)-piperazin-1-yl}-methyl]-3-(3-
chlorophenyl)-pyrrolidine-2,5-dione 14
N-[{4-(2-Chlorophenyl)-piperazin-1-yl}-methyl]-3-(4-
White powdery crystals. Yield: 78%; m.p. 95–978C; 1H-NMR (300
MHz, CDCl3) d: 2.85 (brs, 4H, piperazine), 2.91 (dd, 1H, imide, J =
5.13 Hz, J = 18.35 Hz), 3.06 (brs, 4H, piperazine), 3.28 (dd, 1H,
imide, J = 9.75 Hz, J = 18.46 Hz), 4.08 (q, 1H, imide, J = 5.13 Hz),
4.62 (t, 2H, -CH2-, J = 13.34 Hz), 6.94–7.04 (m, 2H, ArH), 7.14–7.36
(m, 6H, ArH). C21H21N3O2Cl2 (418.33).
chlorophenyl)-pyrrolidine-2,5-dione 20
1
White powdery crystals. Yield: 72%; m.p.: 114–1168C; H-NMR
(300 MHz, CDCl3) d: 2.82 (brs, 4H, piperazine), 2.89 (dd, 1H,
imide, J = 5.13 Hz, J = 18.46 Hz), 3.05 (brs, 4H, piperazine), 3.27
(dd, 1H, imide, J = 9.74 Hz, J =18.46 Hz), 4.07 (q, 1H, imide, J = 5.13
Hz), 4.61 (t, 2H, -CH2-, J = 13.34 Hz), 6.94–7.04 (m, 2H, ArH), 7.18–
7.24 (m, 3H, ArH), 7.33–7.39 (m, 3H, ArH). C21H21N3O2Cl2 (418.33).
N-[{4-(3-Chlorophenyl)-piperazin-1-yl}-methyl]-3-(3-
chlorophenyl)-pyrrolidine-2,5-dione 15
N-[{4-(3-Chlorophenyl)-piperazin-1-yl}-methyl]-3-(4-
White powdery crystals Yield: 67%; m.p.: 81–838C; 1H-NMR (300
MHz, CDCl3) d: 2.78 (brs, 4H, piperazine), 2.86 (dd, 1H, imide, J =
5.38 Hz, J = 18.72 Hz), 3.18 (t, 4H, piperazine, J = 4.87 Hz), 3.24
(dd, 1H, imide, J = 9.74 Hz, J =18.72 Hz), 4.05 (q, 1H, imide, J = 5.13
Hz), 4.60 (t, 2H, -CH2-, J = 13.59 Hz), 6.76–6.85 (m, 4H, ArH), 7.10–
7.18 (m, 2H, ArH), 7.30–7.35 (m, 2H, ArH). C21H21N3O2Cl2 (418.33).
chlorophenyl)-pyrrolidine-2,5-dione 21
1
White powdery crystals. Yield: 70%; m.p.: 120–1228C; H-NMR
(300 MHz, CDCl3) d: 2.79 (brs, 4H, piperazine), 2.85 (dd, 1H,
imide, J = 5.13 Hz, J = 18.46 Hz), 3.19 (brs, 4H, piperazine), 3.25
(dd, 1H, imide, J = 9.49 Hz, J = 18.46 Hz), 4.05 (q, 1H, imide, J =
5.13 Hz), 4.60 (s, 2H, -CH2-), 6.76–6.97 (m, 3H, ArH), 7.14–7.23 (m,
3H, ArH), 7.33–7.37 (m, 2H, ArH). C21H21N3O2Cl2 (418.33).
N-[{4-(4-Chlorophenyl)-piperazin-1-yl}-methyl]-3-(3-
chlorophenyl)-pyrrolidine-2,5-dione 16
N-[{4-(4-Chlorophenyl)-piperazin-1-yl}-methyl]-3-(4-
1
White powdery crystals. Yield: 70%; m.p.: 113–1158C; H-NMR
chlorophenyl)-pyrrolidine-2,5-dione 22
(300 MHz, CDCl3) d: 2.74–2.78 (m, 4H, piperazine), 2.85 (dd, 1H,
imide, J = 5.38 Hz, J = 18.72 Hz), 3.13 (t, 4H piperazine, J = 4.87
Hz), 3.24 (dd, 1H, imide, J = 9.49 Hz, J = 18.46 Hz), 4.03 (q, 1H,
imide, J = 5.13 Hz), 4.59 (t, 2H, -CH2-, J = 13.59 Hz), 6.78–6.86 (m,
2H, ArH), 7.10–7.13 (m, 1H, ArH), 7.16–7.21 (m, 2H, ArH), 7.30–
7.34 (m, 3H, ArH). C21H21N3O2Cl2 (418.33).
1
White powdery crystals. Yield: 75%; m.p.: 115–1178C; H-NMR
(300 MHz, CDCl3) d: 2.78 (brs, 4H, piperazine), 2.84 (dd, 1H,
imide, J = 5.13 Hz, J = 18.46 Hz), 3.14 (t, 4H, piperazine, J = 5.00
Hz), 3.24 (dd, 1H, imide, J = 9.74 Hz, J = 18.72 Hz), 4.04 (q, 1H,
imide, J = 5.13 Hz), 4.59 (t, 2H, -CH2-, J = 13.37 Hz), 6.83 (d, 2H,
ArH, J = 8.72 Hz), 7.16–7.22 (m, 4H, ArH), 7.33–7.37 (m, 2H, ArH).
C21H21N3O2Cl2 (418.33).
Monohydrochloride N-[{4-(3-trifluoromethylphenyl)-
piperazin-1-yl}-methyl]-3-(3-chlorophenyl)-pyrrolidine-
2,5-dione 17
N-[{4-(3-Trifluoromethylphenyl)-piperazin-1-yl}-methyl]-3-
(4-chlorophenyl)-pyrrolidine-2,5-dione 23
1
White powdery crystals. Yield: 63%; m.p.: 168–1708C; H-NMR
White powdery crystals. Yield: 68%; m.p.: 87–898C; 1H-NMR (300
MHz, CDCl3) d: 2.80 (brs, 4H, piperazine), 2.85 (dd, 1H, imide, J =
5.13 Hz, J = 18.46 Hz), 3.20 (brs, 4H, piperazine), 3.25 (dd, 1H,
imide, J = 9.74 Hz, J =18.46 Hz), 4.05 (q, 1H, imide, J = 5.13 Hz),
4.60 (t, 2H, -CH2-, J = 13.33 Hz), 7.03–7.19 (m, 3H, ArH), 7.20–7.24
(m, 2H, ArH), 7.31–7.37 (m, 3H, ArH). C22H21N3O2Cl1F3 (451.88).
(300 MHz, CDCl3) d: 2.92 (dd, 1H, imide, J = 6.41 Hz, J = 17.95 Hz),
3.22–3.45 (m, 5H, 1H imide; 4H piperazine), 3.57 (brs, 4H, pipera-
zine), 4.37 (q, 1H, imide, J = 6.41 Hz), 4.75 (s, 2H, -CH2-), 7.09–7.15
(m, 1H, ArH), 7.21–7.32 (m, 2H, ArH), 7.34–7.45 (m, 5H, ArH),
11.35 (brs, 1H, HCl). C22H22N3O2Cl2F3 (488.35).
i 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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340
J. Obniska et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 333–341
Pharmacology
Hippocampal-kindled rats test
Compounds 10–23 were pharmacologically pre-evaluated
within the Antiepileptic Drug Development (ADD) Program, Epi-
lepsy Branch, Neurological Disorders Program, National Insti-
tutes of the Neurological and Communicative Disorders and
Stroke (NINCDS), Rockville, MD, USA using procedures described
elsewhere [20, 21].
The hippocampal kindling paradigm as described by Lothman et
al. [23] offers a distinct advantage over other kindling models.
This procedure requires the surgical placement of bipolar elec-
trodes in the ventral hippocampus of adult male Sprague-Daw-
ley rats (Charles River, USA). Stage-five behavioral seizures
(Racine scale [24]) are produced by using a stimulus consisting of
a 50 Hz, 10 s train of 1 ms biphasic 200 lA pulses delivered every
30 min for 6 h (12 stimuli per day) on alternating days for a total
of 60 stimulations (five stimulus days). Prior to evaluating a can-
didate's anticonvulsant activity, a drug-free control period con-
sisting of supramaximal stimulations is recorded to verify the
stability of a stage-five generalized seizure. A single dose of the
candidate compound is then administered intraperitoneally
(i. p.), 15 min following the last control stimulation. The anticon-
vulsant activity of the drug is assessed every 30 min for three to
four hours starting 15 min after administering the test material.
After each stimulation, individual Racine-seizure scores and
after-discharge durations are recorded. Rats are used again in
drug trials after four to five drug-and stimulus-free days.
Male albino mice (CF-1 strain) and male albino rats (Sprague-
Dawley) were used as experimental animals; they were obtained
from Charles River, Portage, MI, USA. The animals were housed
in metabolic cages and allowed free access to food and water.
The compounds were suspended in 0.5% methylcellulose/water
mixture. All the compounds were injected intraperitoneally
into mice at the dose levels of 30, 100, and 300 mg/kg with anti-
convulsant activity and neurotoxicity assessment at 0.5 and 4 h
after administration. The results are presented in Table 1.
Selected derivatives (13, 16–18, 21, and 23) were administrated
orally to the rats using four animals at a fixed dose of 30 mg/kg
(MES test; Phase VIa). The motor impairment was studied in par-
allel. Rats were tested at five time periods ranging from 15 min
to 4 h post-substance administration. The results are shown in
Table 2.
The 6-Hz model
This screen was carried out according to the protocol originally
described by Brown et al. [25] and, more recently, by Barton et al.
[19] and Kaminski et al. [26]. It is an alternative electroshock para-
digm that uses low-frequency (6 Hz), long-duration (3 s) electrical
stimulation. 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 move-
ments, 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 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 stimulation [26].
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 at 60 Hz) is delivered via corneal
electrodes primed with an electrolyte solution containing an
anesthetic agent.
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 five seconds in 97% (CD97) of the animals
tested. At the anticipated time of testing, the convulsant is
administered subcutaneously.
Neurological toxicity (NT)
Neurological toxicity induced by the compounds was detected
in mice or rats using the standardized rotorod test [22].
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 animal to maintain equilibrium for a given time.
The pilocarpine-induced status prevention (PISP) model
Male albino rats (Sprague-Dawley, 150–180 g; Charles River,
USA) were used as experimental animals. The compounds were
administrated via the i. p. route of administration. Then, a chal-
lenge dose of pilocarpine is given observing for treatment-effects
of the substance tested. The seizure severity is determined using
the well known Racine scale [24] as follows: (I) immobility, eye
closure, twitching of vibrissae, sniffing, and facial clonus; (II)
head nodding associated with more severe facial clonus; (III) clo-
nus of one of the fore limbs; (IV) rearing often accomplished by
bilateral forelimb clonus, and (V) all of the above plus loss of bal-
ance and falling, accomplished by generalized clonic seizures.
The anticonvulsant activity of compounds 10, 12, 16, and 17 was
assessed at time zero, namely the time from the first-stage III
seizures (Test 71). The outcome measures are the determination
of “protection” or “no protection” (Table 7).
Quantification studies
The quantitative determination of ED50 and TD50 values for 16
and 17 was performed at the previously estimated time-of-peak-
effect after oral administration to rats. Groups of eight rats
received various doses of the compound to be tested until at
least three points were established in the range of 10–90% seiz-
ure protection or minimal neurotoxicity. From the plot of the
data obtained, the respective ED50 and TD50 values, 95% confi-
dence intervals, slope of the regression line, and standard error
of the slope were calculated by means of a computer program
written at NINDS/NIH. For compound 16, the ED50 and TD50 were
also determined after intraperitoneal administration into mice.
The results are shown in Tables 3 and 4.
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 Disor-
ders and Stroke, National Institutes of Health, Rockville, MD, USA).
i 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2010, 343, 333–341
Anticonvulsant Activity of Certain Succinimides
341
The project is financed from the Jagiellonian University Medical Col-
lege grand No K/ZDS/000711.
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1998, 55, 223–231.
The authors have declared no conflict of interest.
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www.archpharm.com