Design, synthesis and anticonvulsant properties of new N-Mannich bases derived from 3-phenylpyrrolidine-2,5-diones

Article abstract of DOI:10.1016/j.bmc.2013.07.029

The synthesis and anticonvulsant properties of new N-Mannich bases of 3-phenyl- (9a-d), 3-(2-chlorophenyl)- (10a-d), 3-(3-chlorophenyl)- (11a-d) and 3-(4-chlorophenyl)-pyrrolidine-2,5-diones (12a-d) were described. The key synthetic strategies involve the

Full text of DOI:10.1016/j.bmc.2013.07.029

Bioorganic & Medicinal Chemistry 21 (2013) 6821–6830
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and anticonvulsant properties of new N-Mannich
bases derived from 3-phenylpyrrolidine-2,5-diones
⇑
Krzysztof Kami n´ ski , Jolanta Obniska, Iwona Chlebek, Beata Wiklik, Sabina Rzepka
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:
The synthesis and anticonvulsant properties of new N-Mannich bases of 3-phenyl- (9a–d), 3-(2-chloro-
phenyl)- (10a–d), 3-(3-chlorophenyl)- (11a–d) and 3-(4-chlorophenyl)-pyrrolidine-2,5-diones (12a–d)
were described. The key synthetic strategies involve the formation of 3-substituted pyrrolidine-2,5-
diones (5–8), and then aminoalkylation reaction (Mannich-type) with formaldehyde and corresponding
secondary amines, which let to obtain the final compounds 9a–d, 10a–d, 11a–d and 12a–d in good yields.
Initial anticonvulsant screening was performed in mice (ip) using the maximal electroshock (MES) and
subcutaneous pentylenetetrazole (scPTZ) seizures tests. The most effective compounds in mice were
tested after oral administration in rats. The acute neurological toxicity was determined applying the min-
imal motor impairment rotarod test. The in vivo results revealed that numerous compounds were effec-
tive especially in the MES test (model of human tonic-clonic seizures). The most active in the MES
seizures in rats was 1-[(4-benzyl-1-piperidyl)methyl]-3-(2-chlorophenyl)pyrrolidine-2,5-dione (10c)
which showed ED50 value of 37.64 mg/kg. It should be stressed that this molecule along with 9a, 9d
and 10d showed protection in the psychomotor seizure test (6-Hz), which is known as an animal model
of therapy-resistant epilepsy. Furthermore compounds 9a, 9d and 10d were also tested in the pilocar-
pine-induced status prevention (PISP) test to assess their potential effectiveness in status epilepticus.
For the most promising molecule 9d an influence on human CYP3A4 isoform of P-450 cytochrome was
studied in vitro.
Received 23 April 2013
Revised 11 July 2013
Accepted 12 July 2013
Available online 23 July 2013
Keywords:
N-Mannich bases
3
-Phenylpyrrolidine-2,5-diones
Succinimides
Anticonvulsant activity
In vivo studies
In vitro studies
Epilepsy
Ó 2013 Elsevier Ltd. All rights reserved.
1
. Introduction
ity of life. Additionally such substance should possess antiepilepto-
genic properties. The currently used antiepileptic drugs (AEDs) can
Epilepsy is the most prevalent neurological disorder, affecting
be classified into four categories on the basis of the main molecular
mechanisms of action, as follow: modulation of voltage-dependent
approximately 50 million people worldwide.1 It is characterized
by recurrent seizures of cerebral origin with episodes of sensory,
motor or autonomic phenomena, which proceed with or without
loss of consciousness. Even though significant advances have been
made in epilepsy research, convulsions in almost one third of pa-
,2
+
2+
Na and/or Ca channels, enhancement of GABA-mediated inhibi-
tion or other effect on the GABA system, inhibition of synaptic exci-
tation mediated by ionotropic glutamate receptors and modulation
5
of synaptic release.
3
tients are inadequately controlled by standard drug therapy. Fur-
The incomplete information on the pathogenesis of human epi-
lepsy and the complex mechanism of action of majority AEDs make
it difficult to use rational methodologies of drug discovery, which
are based on three-dimensional structure of biological target. Thus
the most useful for the design of new anticonvulsants is ligand-
based approach that relies on the use of different pharmacophores
established through the analysis of structural characteristics of
clinically effective AEDs as well as other anticonvulsant active mol-
thermore, compliance is often limited by adverse side effects most
notably related to central nervous system exposure like dimin-
ished attention, executive function, intelligence, language skills,
4
memory and processing speed. In recent times several new drugs,
for example, levetiracetam, felbamate, lamotrigine, gabapentin,
and topiramate, have been approved to treat epilepsy. Although
these drugs have been shown to be effective in epileptic syn-
dromes in a number of patients, their efficacy does not appear to
be superior to that of the established antiepileptic drugs. Therefore
the ideal antiepileptic should prevent different types of seizures
without producing side effects that affect adversely patients’ qual-
6
ecules. The two past decades have demonstrated many attempts
to identify the structural features of compounds crucial for anti-
convulsant activity. As a result it was proved that important core
fragment is defined by nitrogen heteroatomic system, usually
imide or lactam, at least of one carbonyl group and phenyl or alkyl
7
,8
⇑
Corresponding author. Tel.: +48 12 620 54 59; fax: +48 12 620 54 58.
E-mail addresses: kaminskik@cm-uj.krakow.pl, k.kaminski@uj.edu.pl (K. Kami n´ ski).
substituents attached to the heterocyclic system. This template
is noticeable in the structures of the first generation AEDs such
0
968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.07.029

6
822
K. Kami n´ ski et al. / Bioorg. Med. Chem. 21 (2013) 6821–6830
as ethosuximide and phenytoin which act as T-type calcium chan-
nel or sodium channel blockers, respectively, as well as between
the newest drugs, for example, levetiracetam, brivaracetam or sel-
etracetam with novel mechanism of action that relies on the bind-
ing to a synaptic vesicle glycoprotein SV2A, and also inhibition of
presynaptic calcium channels (Fig. 1).
2.2. Biological studies
The initial anticonvulsant evaluation was performed within the
Antiepileptic Drug Development (ADD) Program in the Epilepsy
Branch, National Institutes of Health, National Institute of Neuro-
logical Disorders and Stroke (NIH/NINDS), Rockville, MD, USA.
The development of new chemical agents for the treatment of epi-
lepsy is based mainly on the use of predictable animal models.
Such models fall into two main categories, namely models of acute
seizures (non-epileptic 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 electroshock sei-
zure (MES), the subcutaneous pentylenetetrazole (scPTZ) and the
kindling model. From these tests, the MES and scPTZ screens are
still recognized as the ‘gold standard’ in the early stages of testing
of new anticonvulsants. Furthermore, the maximal electroshock
(MES) and subcutaneous pentylenetetrazole (scPTZ) tests are
claimed to detect compounds affording protection against human
generalized tonic–clonic seizures and generalized absence sei-
The previous studies from our laboratory demonstrated diversi-
fied anticonvulsant activities among variously substituted pyrroli-
9
–12
dine-2,5-diones.
Nevertheless, the most promising were
N-Mannich bases with an aromatic area at position-3 and the
phenylpiperazines, especially those with electron-withdrawing chlo-
rine and fluorine atoms or the trifluoromethyl group at position-1 of
pyrrolidine-2,5-dione (Fig. 2).1
3–16
Following these findings, in aim to search for new, effective
anticonvulsants as well as to continue systematic structure–activ-
ity relationship studies among differently substituted succini-
mides, in the current work we have synthesized the focused
library of new N-Mannich bases derived from 3-phenyl-, 3-(2-chlo-
rophenyl)-, 3-(3-chlorophenyl)- and 3-(4-chlorophenyl)-pyrroli-
1
3–16
dine-2,5-diones. In relation to compounds described earlier,
the main modification relied on the different N-containing hetero-
cyclic ring functions, namely phenylpiperazines moieties have
been changed into 1-(2-pyrimidyl)piperazine, 1-cyclohexylpiper-
azine, 1-benzylpiperidine or morpholine.
1
9
zures, respectively.
Bearing in mind the above, the anticonvulsant activity of com-
pounds 9a–d, 10a–d, 11a–d and 12a–d was studied in the MES
and scPTZ tests after intraperitoneal injection in mice at doses of
30, 100 or 300 mg/kg. The observation was carried out at two dif-
ferent 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.
2
2
. Results and discussion
.1. Chemistry
The series of N-Mannich bases of 3-phenyl- (9a–d), 3-(2-chloro-
phenyl)- (10a–d), 3-(3-chlorophenyl)- (11a–d) and 3-(4-chloro-
Except for compounds 9a, 9b and 12a, which were devoid of
activity, all other compounds showed protection in the MES screen
(9c, 10a, 10b, 11a–d, 12b–d) or were effective in both tests ap-
plied—9d, 10c and 10d. Compounds that revealed protection in
the MES test, indicating the ability of a substance to prevent sei-
zure spread, at a dose of 100 mg/kg included 9c, 9d, 10a–d, 11a,
11c, 11d and 12b–d, whereas 11b provided anti-MES protection
at a dose of 300 mg/kg. In this series, 9d was active only at 0.5 h
after ip administration whereas 10a, 10b, 11a, 12b and 12c showed
protection only at time point 4 h. Nevertheless, majority of the
compounds, namely 9c, 10c, 10d, 11b–d and 12d showed activity
in both time intervals, indicating quick onset and long duration
of the anticonvulsant activity.
Apart from the anti-MES protection, compounds 9d, 10c and
10d were found to inhibit seizures in the scPTZ test, which identi-
fies substances elevating the seizure threshold. Among these mol-
ecules, 10c and 10d showed activity at the dose of 100 mg/kg at
time points 4 and 0.5 h, respectively. It should be stressed here that
compound 10d revealed equivalent protection to ethosuximide,
the model antiepileptic drug effective in the scPTZ test, and fur-
thermore was active in the MES test at dose of 100 mg/kg in both
time intervals. Thus 10d joins the biological properties of phenyt-
oin and ethosuximide. Compound 9d was less active and showed
protection at a dose of 300 mg/kg.
phenyl)-pyrrolidine-2,5-diones
according to Scheme 1.
(12a–d)
was
synthesized
The starting 2-phenyl- (1), 2-(2-chlorophenyl)- (2), 2-(3-chloro-
phenyl)- (3) and 2-(4-chlorophenyl)- (4) succinic acids were pre-
1
7
pared according to the method described by Miller and Long.
The cyclocondensation reaction of dicarboxylic acids 1–4 with
2
3
2
5% ammonia yielded in 3-phenyl- (5), 3-(2-chlorophenyl)- (6),
-(3-chlorophenyl)- (7) and 3-(4-chlorophenyl) (8) pyrrolidine-
,5-diones. The physicochemical and spectral data of intermedi-
18
ates 5–8 have been described in a separate paper. Final com-
pounds 9a–d, 10a–d, 11a–d and 12a–d were obtained in the
Mannich-type reaction from equimolar amounts of the imides 5–
8
, formaldehyde and corresponding secondary amines. The reac-
tion was carried out in ethanol at room temperature for ca. 12 h.
The crude products were crystallized from 96% ethanol. The final
compounds were obtained in good yields (58–82%). All compounds
were prepared as racemic mixtures. Their purity and homogeneity
were assessed by TLC and gradient HPLC chromatography. The
structures of compounds synthesized were confirmed by both
1
13
spectral ( H NMR, C NMR, LC–MS) and elemental (C, H, N) anal-
yses. The detailed physical and analytical data are listed in the
Section 4.
F
F
O
H
O
O
O
O
HN
O
O
O
O
N
N
N
N
H
N
H
O
NH2
NH2
NH2
Ethosuximide
Phenytoin
Levetiracetam
Brivaracetam
Seletracetam
Figure 1. Structures of known AEDs containing nitrogen heterocyclic ring.

K. Kami n´ ski et al. / Bioorg. Med. Chem. 21 (2013) 6821–6830
6823
O
O
N
N
N
N
O
N
O
N
CF3
Cl
I
II
ED50 = 9.0 mg/kg (MES test, p.o. rats)
TD50 > 250 mg/kg (rotarod test, p.o. rats)
PI (TD50 / ED50) > 27.78
ED50 = 19.57 mg/kg (MES test, p.o. rats)
TD50 > 500 mg/kg (rotarod test, p.o. rats)
PI (TD50 / ED50) > 25.55
Cl
Cl
O
O
N
N
Cl
N
N
O
N
O
N
Cl
III (14)*
IV (20)*
ED50 = 14.18 mg/kg (MES test, p.o. rats)
TD50 < 125 mg/kg (rotarod test, p.o. rats)
PI (TD50 / ED50) < 8.8
ED50 = 21.40 mg/kg (MES test, p.o. rats)
TD50 > 500 mg/kg (rotarod test, p.o. rats)
PI (TD50 / ED50) > 23.37
Figure 2. Model compounds I–IV obtained in the previous studies.14–16 ( The numbers of compounds III and IV in Table 1).
⁄
R
R
O
COOH
COOH
a
N
O
H
1
-4
5-8
b
N
N
R
N
N
N
N
N
N
9a, 10a, 11a, 12a
9b, 10b, 11b, 12b
9c, 10c, 11c, 12c
R:
O
H - 9a-d
2
3
4
-Cl 10a-d
-Cl 11a-d
-Cl 12a-d
N
O
CH2
O
9d, 10d, 11d, 12d
Scheme 1. Synthesis of intermediates 5–8 (Ref. 18), and final compounds 9a–d, 10a–d, 11a–d and 12a–d. Reagents and conditions: (a) 25% NH
4
OH, 180 °C, 1 h (b) 1-(2-
pyrimidyl)piperazine, 1-cyclohexylpiperazine, 1-benzylpiperidine or morpholine, formaldehyde, 96% ethyl alcohol, room temperature ca. 12 h.
In the rotorod test (NT) for acute neurological toxicity,
compounds 9d, 10a, 10c, 10d, 11d and 12a showed motor
impairment in the maximum dose administered (300 mg/kg).
The rest of molecules were devoid of neurotoxic properties. It
should be emphasized that eight molecules, namely 9c, 10b,
As shown in Table 2 the most active was compound 10c,
which protected 75% of animals at 0.5 and 1 h. This compound
was also active in twenty-five percent of animals at time points
0.25 and 2 h. The other molecules were less active and protected
50% of animals at 0.25 and 2 h (11c) or at 0.5, 1, 2 and 4 h (11d).
25% protection was observed for compounds 11c at 1 h and 10d
at 0.5 and 1 h. All derivatives tested did not cause motor impair-
ment when they were given orally. The in vivo data in rats
confirmed that compounds tested are absorbed from gastrointes-
tinal tract and are also capable to penetrate to central nervous
system.
On the basis of the preliminary data in rats, compound 10c was
chosen for quantification of the pharmacological parameters (ED50
and TD50) after p.o. application. The quantitative evaluation of the
MES median effective dose (ED50) and toxic dose (TD50) were
performed at previously estimated TPE. Results of the quantitative
1
1a–c and 12b–d emerged as anticonvulsants without neurotoxic
activity.
A valuable property of the candidate anticonvulsant is its ability
to inhibit convulsions when it is given by the oral route. This
screen discloses the time of onset, the approximate time of peak ef-
fect (TPE) and the duration of anticonvulsant activity or neurotox-
icity. On the basis of the ip screening data in mice, and according to
Anticonvulsant Screening Project (ASP) disposition, eight com-
pounds 9a, 9d, 10a, 10c, 10d, 11a, 11c and 11d were selected
and examined in the MES test as well as rotorod screen after p.o.
administration in rats at a dose of 30 mg/kg (Table 2).

6
824
K. Kami n´ ski et al. / Bioorg. Med. Chem. 21 (2013) 6821–6830
Table 1
Anticonvulsant and neurotoxicity screening after ip administration in mice
O
O
R1
X
N
N
R
N
N
R1
R
N
O
O
9
a-d, 10a-d, 11a-d, 12a-d
13-27
No.
R
X
R1
Intraperitoneal administration in micea
MESb
scPTZc
NTd
0.5 h
4 h
0.5 h
4 h
0.5 h
4 h
9
9
9
9
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
a
b
c
d
H
H
H
H
N
N
C
O
N
N
C
O
N
N
C
O
N
N
C
O
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
2-Pyrimidinyl
Cyclohexyl
Benzyl
—
—
100
100
—
—
—
300
—
—
—
—
300
—
—
—
100
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
100
—
—
—
300
—
—
—
—
300
—
—
—
—
—
—
0a
0b
0c
0d
1a
1b
1c
1d
2a
2b
2c
2d
3
4
5
6
7
8
9
0
2-Cl
2-Cl
2-Cl
2-Cl
3-Cl
3-Cl
3-Cl
3-Cl
4-Cl
4-Cl
4-Cl
4-Cl
2-Cl
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-Pyrimidinyl
Cyclohexyl
Benzyl
100
100
100
100
100
300
100
100
—
100
100
300
—
30041
—
—
—
—
300
100
—
300
300
100
—
—
—
100
—
—
10025
300
—
300
30019
—
—
—
300
300
—
—
—
—
100
—
—
300
—
—
300
—
—
—
—
—
—
—
—
—
300
—
—
—
—
—
—
300
-
300
300
300
—
—
100
—
—
2-Pyrimidinyl
Cyclohexyl
Benzyl
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
300
-
2-Pyrimidinyl
Cyclohexyl
Benzyl
—
2-Cl
g
h,i
3-Cl
4-Cl
100
300
100
100
—
g
—
—
—
—
g
3-CF
2-F
3
g
g
2-Cl
3-Cl
4-Cl
3-CF
g
—
—
—
—
gi
300
100
—
—
—
—
—
—
30
—
100
100
30
—
—
100
—
100
30
g
1
2
3
4
5
6
7
3
300
100
300
300
300
—
300
100
—
g
3-CH
3
g
2-OCH
2-Cl
3
g
g
3-Cl
4-Cl
g
g
3-CF
3
e
Phenytoin
Ethosuximidef
—
Response comments: 19sedated, 25myoclonic jerks, 41diarrhea.
a
Doses of 30, 100, and 300 mg/kg were administered. The data indicate the minimum dose whereby bioactivity was demonstrated in half or more animals. The animals
were examined at 0.5 and 4.0 h. A dash indicates the absence of anticonvulsant activity or neurotoxicity at the maximum dose administered (300 mg/kg).
b
Maximal electroshock test.
Subcutaneous pentylenetetrazole test.
Neurotoxicity screening using rotorod test.
Phenytoin, reference antiepileptic drug tested by use of ADD Program procedures in NIH/NINDS, data from Ref. 30.
Ethosuximide, reference antiepileptic drug tested by use of ADD Program procedures in NIH/NINDS, data from Ref. 30.
Compounds 13, 15–27 data from Ref. 15.
Compound 14, data from Ref. 16.
Compounds 14 and 20 are shown in Figure 2 as III and IV, respectively.
c
d
e
f
g
h
i
tests along with the data for the standard AEDs—valproic acid,
ethosuximide and phenytoin, are shown in Table 3.
toxin) induced seizures. The number of new AEDs currently avail-
able, or in development, for the management of epilepsy certainly
attests to the success of this approach. However, this method may
overlook novel compounds that would be uniquely effective in the
therapy-resistant population. One example supporting this hypoth-
esis is provided by levetiracetam, which has demonstrated efficacy
in refractory human partial epilepsies. It was found to be inactive
against MES and scPTZ seizures even at high doses, whereas showed
high effectiveness in the 6-Hz psychomotor seizure model of partial
epilepsy. Thus itis suggestedthat the6-Hz modelmight becapable
for identifying anti-seizure agents with a novel spectrum of activity
and unknown mechanism of anticonvulsant action.
Taking into consideration the above, and in accordance with
ASP dispositions, six compounds 9a, 9c, 9d, 10c, 10d and 12c were
The quantitative data showed that 10c had comparable ED50
with phenytoin (MES test) and ethosuximide (scPTZ test), and fur-
thermore was distinctly more potent than valproic acid in both
screens. These resulted also in more favorable protection indexes
in comparison with model anticonvulsants.
The previous studies among differently substituted pyrrolidine-
2
,5-diones revealed that the most plausible mechanism of action
2
0
for these type of molecules is connected with the inhibition of
sodium channel current.14
Traditionally, most screening programs employ mice and rats to
assessefficacyagainsteitherelectrically(e.g., maximalelectroshock,
MES) or chemically (e.g., pentylenetetrazol, bicuculline, or picro-

K. Kami n´ ski et al. / Bioorg. Med. Chem. 21 (2013) 6821–6830
6825
Table 2
Test results in rats after oral administration at a dose of 30 mg/kg
Table 4
Psychomotor seizure test in mice (6-Hz, current 32 mA)
No.
MESa
1 h
No.
Dose (mg/kg)
Intraperitoneal injectiona
0.25 h
0.5 h
2 h
4 h
0.25 h
0.5 h
1 h
2 h
4 h
9
9
1
1
1
1
1
1
a
d
0
0
0
1
0
0
2
0
1
0
0
0
3
1
0
0
2
4
0
0
0
3
1
0
1
2
3
0
0
0
1
0
0
2
2
3
0
0
0
0
0
0
0
2
3
9a
9c
9d
10c
10d
10d
12c
100
100
100
75
50
100
100
3
0
3
0
4
4
0
2
0
0
3
3
4
0
2
1
1
0
3
4
0
2
1
0
1
3
4
3
0
0
0
0
0
4
1
0a
0c
0d
1a
1c
1d
a
b
The data indicate the number of mice out of four that were protected.
Phenytoin
The data indicate the number of rats out of four that were protected.
dose of 200 mg/kg (ip route of administration). Compound 9d
showed also protection in a sustained seizure model (time 0.5 h)
at a dose of 400 mg/kg. For 9d which revealed activity in more than
a
Maximal electroshock test.
b
Phenytoin, reference antiepileptic drug, tested by use of ADD Program proce-
dures in NIH/NINDS, data from Ref. 31.
5
0% of animals the ED50 value was determined at time point 0.5 h
and the data are shown in Table 7.
Many drug–drug interactions are metabolism based and medi-
ated primarily via the microsomal cytochrome P450 (CYP) family
of enzymes. Ten CYP isoforms are expressed in a typical human li-
ver, and six of them (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6
and CYP3A4) appear to be the most commonly responsible for the
metabolism of most drugs and the associated drug–drug interac-
tions. The inhibition of these enzymes may decrease the metabolic
clearance of a co-administered drug resulting in elevated blood
concentration, which may cause adverse drug effects or toxicity.
As detailed in the FDA’s Draft Guidance document for Drug–Drug
Interactions, the FDA has placed emphasis on evaluating the inhi-
bition potential of a new chemical entity (NCE) at an earlier stage
in drug development in order to avoid developing compounds with
the potential to yield adverse drug interactions.
Taking into consideration the above, according to the ASP dispo-
sition, the most promising 1-(morpholinomethyl)-3-phenyl-pyr-
rolidine-2,5-dione (9d) (active in the MES, scPTZ, 6-Hz and PISP
test), was tested in the human cytochrome P450 inhibition studies.
The studies were carried out for CYP3A4 subtype of the cyto-
chrome P450, and the outcome was the inhibition of metabolite
formation, namely 6-b-OH-testosterone from the probe sub-
strate—testosterone. The results are shown in Table 8.
selected for the evaluation of anticonvulsant activity in the 6-Hz
test in mice (ip). The results obtained are presented in Table 4.
In the 6-Hz model the most potent was compound 10d which
protected 100% of mice in all time intervals at dose of 100 mg/kg.
This molecule showed also 100% protection (0.25 h) and 75% (0.5,
1
, 2 h) at dose of 50 mg/kg. The other molecules 9a, 9d, 10c and
1
2c showed one peak of 75% protection in different time points.
It is worthy of note that 9a was inactive in mice MES and scPTZ
tests, however showed activity in the psychomotor seizures. Final-
ly, the weakest protection—25% (at 1 and 2 h) was observed for
compound 9c.
On the basis of preliminary screening four molecules 9a, 9d, 10c
and 10d were selected for quantification of ip ED50 values in the
psychomotor seizure test (6-Hz) in mice. At the same time the
TD50 values were determined in the rotorod test (Table 5).
As it is shown in Table 5 compounds 9a, 9d, 10c and 10d
showed on the one hand lower activity and on the other hand high-
er neurotoxicity in comparison with the results observed for leveti-
racetam (a model anticonvulsant active in the 6-Hz seizures).
In continuation of the biological studies, compounds 9a, 9d and
2
1
1
0d were additionally assessed for potential activity against nerve
agents using the pilocarpine-induced status prevention (PISP) test.
The results are presented in Table 6.
The data revealed that compound 9d showed only slight inhibi-
tion of CYP3A4 activity in vitro and is unlikely to show inhibitory
interactions with other CYP3A4 metabolized drugs in vivo.
The PISP model shares many characteristics with nerve agent
induced seizures since both initiation and early expression of nerve
agent induced seizures are cholinergic followed by the recruitment
of other neurotransmitter systems that serve to reinforce recurring
seizure activity progressing to status epilepticus (SE). The pilocar-
pine models are one of the most recognized animal models of sta-
tus epilepticus (SE).
2
.3. Structure activity relationships
The results of the preliminary screening in mice (see Table 1),
enabled to draw some general conclusions about the relationships
between structure and anticonvulsant properties. Taking into
consideration the substitution of the 3-phenyl ring, the highest
In the PISP test two compounds 9a and 9d were found to be ac-
tive and protected, respectively 5 and 4 rats at time-zero using the
Table 3
Quantification data in rats after p.o. administration
No.
0c
TPEa (h)
1
ED50 MESb (mg/kg)
37.64
ED50 scPTZb (mg/kg)
TD50 NTb (mg/kg)
>500
PIc (TD50/ED50
)
1
106.83
(71.14–164.65)
646
(466–869)
167
>13.28 (MES)
>4.68 (scPTZ)
1.6 (MES)
1.2 (scPTZ)
>2.99 (scPTZ)
(
22.86–58.51)
485
324–677)
Valproic acidd
Ethosuximided
Phenytoind
1
1
1
784
(503–1176)
>500
(
>500
(
116–237)
28.10
>500
>100
>3.6 (MES)
(
20.7–35.2)
a
Time to peak effect.
b
Results are presented as mean ± SEM at 95% confidence limit (MES-maximal electroshock test; scPTZ—subcutaneous pentylenetetrazole test; NT-neurotoxicity, rotarod
test).
c
Protection index (TD50/ED50).
Reference antiepileptic drugs tested by use of ADD Program procedures in NIH/NINDS, data from Ref. 19.
d

6
826
K. Kami n´ ski et al. / Bioorg. Med. Chem. 21 (2013) 6821–6830
Table 5
Quantification data—psychomotor seizure tests (6-Hz, current 32 mA) after ip injection into mice
No.
TPEa (h)
1
ED50 6 Hzb (mg/kg)
TD50 NTb (mg/kg)
nd
PIc (TD50/ED50
—
)
9
9
1
1
1
a
103.05
(
79.0–147.25)
77.20
66.3–81.6)
84.59
54.96–148.65)
31.06
27.46–36.48)
55.63
51.33–66.74)
19.40
9.90–36.0)
d
0.25
0.5
0.25
0.5
1
292.90
(273.3–336)
275.33
(226.98–325.02)
nd
3.79
3.26
—
(
0c
(
0d
(
0d (Current 44 mA)
nd
—
(
Levetiracetamd
>500
>25.77
(
Nd—no data.
a
Time to peak effect.
b
Results are presented as mean ± SEM at 95% confidence limit (6 Hz- psychomotor seizure tests; NT-neurotoxicity, rotarod test).
Protection index (TD50/ED50).
Levetiracetam, reference antiepileptic drug tested by use of ADD Program procedures in NIH/NINDS, data from Ref. 19.
c
d
Table 6
Pilocarpine induced status prevention (PISP) model results
No.
Dose (mg/kg)
Timea (h)
Protected rats
Non-protected rats
Average weight change ± SEMb (g)
Protected rats
Non-protected rats
9
9
9
1
a
d
d
0d
200
200
400
100
0
0
0.5
0.5
5
4
5
0
8
6
7
8
11.7 ± 4.1
10.0 ± 2.5
23.0 ± 1.6
—
—
10.0 ± 0.0
25.0 ± 2.5
20.6 ± 1.5
a
Post first stage III seizure.
Weight change 24 h Post first stage III seizure.
b
Table 7
Pilocarpine induced status prevention (PISP) model, quantification data for compound
at the most in two in vivo screens, namely MES, 6-Hz or PISP. It
should be noticed, as described previously, the 4-phenylpiper-
azine derivatives of 3-(2-chlorophenyl)-, 3-(3-chlorophenyl)- and
1
5
9
d
No.
Time (h)
0.5
ED50 PISPa (mg/kg)
342.2
3
-(4-chlorophenyl)-pyrrolidine-2,5-diones were active exclusively
9
d
in the maximal electroshock test (the data for selected compounds
(
265.4–436.6)
1
3–27 from Refs. 15,16 are shown in Table 1).
a
Result is presented as mean ± SEM at 95% confidence limit.
3
. Conclusion
Table 8
In the current studies the focused library of sixteen new N-Man-
Human cytochrome P450 inhibition studies for compound 9d
nich bases of 3-phenyl-pyrrolidine-2,5-diones were synthesized
and assessed for their anticonvulsant activity in several animal
models of epilepsy. The results revealed that majority of com-
pounds showed protection in the maximal electroshock tests
Compound 9d concentration (
l
M)
[S] = 200
4,225.1a
l
M
[S] = 50 lM
0
1,839.7
(0)
1,532.6
(17)
(
0)
3,417.8
19)
5
00
(
MES). Several compounds were also active in the pentylenetetraz-
ole (scPTZ) and psychomotor (6-Hz) seizures. The most active 1-
morpholinomethyl)-3-phenyl-pyrrolidine-2,5-dione (9d) was
(
a
Activities are reported in protein/min. In parentheses below each activity is (%)
inhibition relative to the activity determined at the same substrate concentration
S] in the absence of 9d (0 M).
(
effective in the MES, scPTZ, 6-Hz tests as well as in the pilocar-
pine-induced status prevention (PISP) screen which may indicate
its potential usefulness in the tonic-clonic, absence and refractory
epilepsy, and also status epilepticus. This molecule as the most
promising from the whole series showed only slight inhibition of
CYP3A4 activity in vitro and is unlikely to show inhibitory interac-
tions with other CYP3A4 metabolized drugs in vivo.
[
l
activity in the maximal electroshock (MES) test was observed for
-chlorophenyl- (10a–d) and 3-chlorophenyl (11a–d) derivatives
whereas weaker protection revealed 4-chlorophenyl- analogs
12a–d) and unsubstituted molecules (9a–d). Moreover, the
2
(
3
-chlorophenyl-
(11a–d)
and
4-chlorophenyl-pyrrolidine-
2
,5-diones (12a–d) were active only in the MES screen, whereas
4. Experimental
4.1. General
several compounds representing morpholine (9d, 10d) or 4-ben-
zyl-1-piperidine (10c) derivatives from the remaining series were
also effective in the pentylenetetrazole seizures (scPTZ). These
molecules showed additionally potent activity in the psychomotor
All chemicals and solvents were purchased from Sigma–Al-
drich (St. Louis, USA) and were used without further purification.
Melting points (mp) were determined in open capillaries on a
Büchi 353 melting point apparatus (Büchi Labortechnik, Flawil,
(
6-Hz) seizures.
The weaker protection was observed for 2-pyrimidinyl (9a–
1
2a) and cyclohexyl (9b–12b) derivatives that showed protection

K. Kami n´ ski et al. / Bioorg. Med. Chem. 21 (2013) 6821–6830
6827
Switzerland) and are uncorrected. The purity of the compounds
4.2.2.1.
3-Phenyl-1-{[4-(pyrimidin-2-yl)piperazin-1-
was confirmed by the thin-layer chromatography (TLC) per-
formed on Merck silica gel 60 F254 aluminum sheets (Merck;
Darmstadt, Germany), using subsequent developing systems:
yl]methyl}pyrrolidine-2,5-dione (9a).
70%; mp 118–120 °C; = 0.46 (S ),
0.769 min); H NMR (300 MHz, CDCl ) d: 2.70 (br s, 4H, pipera-
zine), 2.86 (dd, 1H, H , imide, J = 5.00 Hz), 3.22 (dd, 1H , imide,
J = 9.62 Hz), 3.82 (br s, 4H, piperazine), 4.04 (q, 1H , imide,
J = 4.88 Hz), 4.60 (s, 2H, CH ), 6.50 (t, 1H, ArH, J = 4.74 Hz), 7.19–
7.39 (m, 5H, ArH), 8.30 (d, 2H, ArH, J = 4.87 Hz);
(75 MHz, CDCl ) d: 36.98, 43.63, 46.00, 20.59, 60.41, 109.97,
127.30, 127.99, 129.24, 137.03, 157.69, 176.96. ESI-MS: 352.2
White solid. Yield:
R
f
1
R
f
= 0.91 (S ); HPLC (t
2
R
1
3
S
1
—chloroform:methanol (9:0.2, v/v),
S
2
—chloroform:2-propa-
b
a
nol:25% ammonia (9:11:2, v/v/v). Spots were detected by their
absorption under UV light (k = 254 nm) and by visualization with
c
2
13
0
.05 mol I
2
in 10% HCl. HPLC analyses were run on a HPLC
C NMR
Ò
Waters 2695 Separation Module equipped with photodiode ar-
ray detector (Waters 2998). A Chromolith RP-18 SpeedROD col-
3
Ò
+
umn (4.6 Â 50 mm) was used. Conditions applied were as follow:
eluent A (water/0.1% TFA), eluent B (acetonitrile/0.1% TFA); flow
rate of 5 ml/min, gradient of 0–100% B over 3 min were used,
21 5 2 21 5 2
(C19H N O [M+H] ). Anal. Calcd for C19H N O (351.41): C,
64.93; H, 6.02; N, 19.93. Found: C, 64.59; H, 6.08; N, 19.48.
injection volume was 10
l
L. Standard solutions (1 mg/ml) of each
4.2.2.2. 1-[(4-Cyclohexylpiperazin-1-yl)methyl]-3-phenylpyrr-
compound were prepared in analytical grade acetonitrile and
analysed at wavelengths 214 nm and 254 nm. Retention times
olidine-2,5-dione (9b).
166 °C; R = 0.59 (S ), R
(300 MHz, CDCl ) d: 1.18 (t, 5H, cyclohexane, J = 9.75 Hz), 1.62 (d,
White solid. Yield: 75%; mp 164–
0.721 min); 1H NMR
f
1
f
= 0.90 (S ); HPLC (t
2
R
(
t
R
) are given in minutes. Elemental analysis for C, H, and N were
3
carried out by a micro method using the elemental Vario EI III Ele-
mental analyser (Hanau, Germany). The results of elemental anal-
1H, cyclohexane, J = 11.28 Hz), 1.57–1.69 (m, 4H, cyclohexane),
2.19 (t, 1H, cyclohexane, J = 6.62 Hz), 2.57 (br s, 4H, piperazine),
1
13
yses were within ±0.4% of the theoretical values. H NMR and
NMR spectra were obtained in a Varian Mercury spectrometer
Varian Inc., Palo Alto, CA, USA), in CDCl or DMSO, operating at
00 MHz and 75 MHz, respectively. Chemical shifts are reported
C
2.67 (br s, 4H, piperazine), 2.84 (dd, 1H, H
3.20 (dd, 1H, H , imide, J = 9.74 Hz), 4.02 (q, 1H, H
J = 5.02 Hz), 4.55 (s, 2H, CH
(75 MHz, CDCl ) d: 25.83, 26.26, 28.83, 28.94, 36.60, 45.48, 48.74,
51.09, 60.40, 63.45, 125.55, 127.88, 128.21, 130.45, 134.99,
a
imide, J = 5.39 Hz),
b
c
, imide,
1
3
(
3
2
), 7.22–7.40 (m, 5H, ArH); C NMR
3
3
1
in d values (ppm) relative to TMS d = 0 ( H), 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 (doublet of doublets), t (triplet), q (quar-
tet), m (multiplet). The mass spectra for compounds 9a–d, 10a–d,
+
138.65, 176.52, 177.97. ESI-MS: 356.2 (C21
29 3 2
H N O [M+H] ); Anal.
Calcd for C21 (355.47): C, 70.95; H, 8.22; N, 11.82. Found:
29 3 2
H N O
C, 70.94; H, 8.31; N, 11.73.
1
1a–d and 12a–d were obtained on Waters ACQUITY™ TQD sys-
4.2.2.3. 1-[(4-Benzyl-1-piperidyl)methyl]-3-phenyl-pyrrolidine-
tem with the TQ Detector (Waters, Milford, USA). The ACQUITY
2,5-dione (9c).
= 0.58 (S ),
(300 MHz, CDCl
piperidine), 2.50 (d, 2H, CH
J = 4.87 Hz), 2.98 (br s, 2H, piperidine), 3.21 (dd, 1H, H
J = 9.62 Hz), 4.02 (q, 1H, H , imide, J = 4.79 Hz), 4.52 (s, 2H, CH
White solid. Yield: 68%; mp 132–134 °C;
1
UPLC BEH C18, 1.7
Milford, USA).
l
m, 2.1 Â 50 mm column was used (Waters,
R
f
1
R
f
= 0.86 (S
) d: 1.27 (br s, 2H, piperidine), 1.59–1.64 (m, 5H,
, J = 6.92 Hz), 2.85 (dd, 1H, H , imide,
, imide,
),
) d: 32.05,
32.12, 36.99, 37.31, 43.01, 46.01, 51.40, 51.45, 60.86, 125.81,
27.32, 127.95, 128.15, 129.07, 129.22, 137.29, 140.48, 177.06,
2
); HPLC (t
R
0.769 min);
H NMR
3
The starting 2-phenyl- (1), 2-(2-chlorophenyl)- (2), 2-(3-chloro-
phenyl)- (3) and 2-(4-chlorophenyl)- (4) succinic acids were pre-
pared by use of method described by Miller and Long.17
2
b
a
c
2
1
3
7
.10–7.40 (m, 10H, ArH); C NMR (75 MHz, CDCl
3
4
4
.2. Chemical synthesis
1
+
.2.1. General procedure for the synthesis of 3-phenyl-, 3-(2-
178.64. ESI-MS: 363.2 (C23
26 2 2
H N O [M+H] ); Anal. Calcd for
chlorophenyl)-, 3-(3-chlorophenyl)- and 3-(4-chlorophenyl)-
pyrrolidine-2,5-diones (5–8)
C H N O (362.47): C, 76.20; H, 7.23; N, 7.73. Found: C, 76.35;
H, 7.38; N, 7.70.
23 26 2 2
A total of 0.05 mol of the 2-phenyl- (1), 2-(2-chlorophenyl)- (2),
2
-(3-chlorophenyl)- (3) or 2-(4-chlorophenyl)- (4) succinic acids
4.2.2.4. 1-(Morpholinomethyl)-3-phenyl-pyrrolidine-2,5-dione
were suspended in 20 ml of water and 0.05 mol of the 25% ammo-
nia was gradually added. The mixture was heated in a term-regu-
lated sand bath (ST 72 Roth, Karlsruhe, Germany) with
simultaneous distillation of water. After complete removal of
water, the temperature of the reaction mixture raised up to
(9d).
= 0.92 (S
2.63 (q, 4H, morpholine, J = 5.26 Hz), 2.89 (dd, 1H, H
J = 4.87 Hz), 3.24 (dd, 1H, H imide, J = 9.62 Hz), 3.67 (t, 4H, mor-
pholine, J = 4.62 Hz), 4.06 (q, 1H, H , imide, J = 4.87 Hz), 4.62 (s,
2H, CH ), 7.22–731 (m, 2H, ArH), 7.33–7.42 (m, 3H, ArH);
NMR (75 MHz, CDCl ) d: 36.24, 44.97, 51.08, 60.60, 66.89, 127.58,
129.53, 130.17, 130.39, 133.62, 134.82, 176.59, 178.07. ESI-MS:
White solid. Yield: 65%; mp 106–107 °C; R
f
= 0.40 (S
0.698 min); H NMR (300 MHz, CDCl ) d:
, imide,
1
),
1
R
f
2
); HPLC (t
R
3
b
a
c
13
1
80 °C and was maintained for 1 h. The crude products were
2
C
recrystallized from methanol. The physicochemical and spectral
3
data of intermediates 5–8 have been described in a separate
1
8
+
paper.
18 2 3 18 2 3
274.1 (C15H N O [M+H] ); Anal. Calcd for C15H N O (274.32):
C, 65.68; H, 6.61; N, 10.21. Found: C, 65.43; H, 6.56; N, 10.11.
4
9
.2.2. General procedure for the synthesis of final compounds
a–d, 10a–d, 11a–d and 12a–d
4.2.2.5.
yl)methyl]pyrrolidine-2,5-dione
(10a). White solid. Yield: 80%; mp 239–241 °C; R
= 0.72 (S
2.72 (t, 4H, piperazine, J = 5.64 Hz), 2.82 (dd, 1H, H
J = 6.15 Hz),3.23 (dd, 1H, H , imide, J = 9.87 Hz), 3.82 (t, 4H, piper-
azine, J = 5.13 Hz), 4.32 (q, 1H, H , imide, J = 5.95 Hz), 4.62 (s, 2H,
CH ), 7.16–7.19 (m, 1H, ArH), 7.20–7.37 (m, 3H, ArH), 7.38–7.42
(m, 1H, pyrimidine), 8.29 (d, 2H, pyrimidine, J = 4.62 Hz), 11.09
3-(2-Chlorophenyl)-1-[(4-pyrimidin-2-ylpiperazin-1-
monohydrochloride
= 0.67 (S ),
1.068 min); H NMR (300 MHz, CDCl ) d:
, imide,
The mixture of appropriately substituted 3-phenyl-succini-
mide (5–8) (0.01 mol), 40% formaldehyde solution (0.01 mol)
and 1-(2-pyrimidyl)piperazine, 1-cyclohexylpiperazine, 1-ben-
zylpiperidine or morpholine in 96% ethanol (20 ml) was stirred
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
f
1
1
R
f
2
); HPLC (t
R
3
b
a
c
2
9
6% ethanol. Compound 10a was obtained as light oil which was
1
3
converted into solid hydrochloride salt in anhydrous ethanol sat-
urated with HCl gas. The obtained precipitate was crystallized
from anhydrous ethanol.
3
(br s, 1H, 1H, +NH); C NMR (75 MHz, CDCl ) d: 36.99, 43.62,
46.01, 20.60, 60.43, 109.99, 127.32, 128.01, 129.26, 137.05,
+
5 2
157.70, 176.98. ESI-MS: 386.1 (C19H20ClN O [M+H] ); Anal. Calcd

6
828
K. Kami n´ ski et al. / Bioorg. Med. Chem. 21 (2013) 6821–6830
for C19
H
21Cl
2
N
5
O
2
(422.30): C, 54.04; H, 5.01; N, 16.58. Found: C,
cyclohexane), 2.21 (br s, 1H, cyclohexane), 2.57 (br s, 4H, pipera-
zine), 2.66 (br s, 4H, piperazine), 2.81 (dd, 1H, H , imide,
J = 5.51 Hz), 3.20 (dd, 1H, H , imide, J = 9.74 Hz), 4.01 (q, 1H, H
imide, J = 5.37 Hz), 4.55 (s, 2H, CH ), 7.11–7.16 (m, 1H, ArH),
C NMR (75 MHz, CDCl ) d: 25.83,
5
4.14; H, 5.19; N, 16.49.
b
a
c
,
4
.2.2.6.
3-(2-Chlorophenyl)-1-[(4-cyclohexylpiperazin-1-yl)
White solid. Yield:
= 0.72 (S ); HPLC (t
) d: 1.06 (m, 5H, cyclohexane),
.62 (d, 1H, cyclohexane, J = 7.18 Hz), 1.57–1.68 (m, 4H, cyclohex-
2
1
3
methyl]pyrrolidine-2,5-dione (10b).
7.24–7.34 (m, 3H, ArH);
3
8
0
1
2%; mp 159–160 °C;
.82 min); H NMR (300 MHz, CDCl
R
f
= 0.55 (S
1
),
R
f
2
R
26.27, 28.87, 28.97, 36.62, 45.48, 48.77, 51.13, 60.40, 63.46,
1
3
125.58, 127.89, 128.23, 130.47, 135.00, 138.69, 176.56, 177.99.
+
ESI-MS: 390.2 (C21
3
H28ClN O
2
[M+H] ); Anal. Calcd for C21
H28ClN3-
ane), 2.21 (d, 1H, cyclohexane, J = 7.18 Hz), 2.57–2.69 (m, 4H,
piperazine), 2.71 (br s, 4H, piperazine), 2.76 (dd, 1H, H , imide,
J = 6.03 Hz), 3.23 (dd, 1H, H , imide, J = 9.87 Hz), 4.35 (q, 1H, H
imide, J = 6.03 Hz), 4.57 (s, 2H, CH ), 7.18–7.28 (m, 3H, ArH),
C NMR (75 MHz, CDCl ) d: 25.82,
O
10.83.
2
(389.92): C, 64.68; H, 7.24; N, 10.77. Found: C, 64.50; H, 7.30; N,
b
a
c
,
2
4.2.2.11. 1-[(4-Benzyl-1-piperidyl)methyl]-3-(3-chlorophenyl)
pyrrolidine-2,5-dione (11c). White solid. Yield: 58%; mp
= 0.68 (S ); HPLC (t
1.056 min); 1
) d: 1.21–1.30 (m, 2H, piperidine), 1.31–
13
7
2
1
.38–7.44 (m, 1H, ArH);
3
6.27, 28.85, 28.95, 36.61, 45.49, 48.76, 51.10, 60.42, 63.47,
81–83 °C; R
f
= 0.53 (S
1
), R
f
2
R
H
25.57, 127.89, 128.24, 130.46, 135.01, 138.68, 176.55, 177.98.
NMR (300 MHz, CDCl
3
+
ESI-MS: 390.2 (C21
3
H28ClN O
2
[M+H] ); Anal. Calcd for C21
H28ClN3-
1.48 (m, 1H, piperidine), 1.59–1.64 (m, 2H, piperidine), 2.06–2.20
(m, 2H, piperidine), 2.51 (d, 2H, CH , J = 6.92 Hz), 2.82 (dd, 1H,
, imide, J = 4.87 Hz), 2.95–3.00 (m, 2H, piperidine), 3.20 (dd,
H, H , imide, J = 9.74 Hz), 4.00 (q, 1H, H , imide, J = 4.87 Hz),
4.51 (s, 2H, CH ), 7.10–7.32 (m, 9H, ArH); C NMR (75 MHz, CDCl
O
2
(389.92): C, 64.68; H, 7.24; N, 10.77. Found: C, 64.45; H, 7.32; N,
2
1
0.79.
H
b
1
a
c
1
3
4
.2.2.7.
1-[(4-Benzyl-1-piperidyl)methyl]-3-(2-chlorophenyl)
White solid. Yield: 70%; mp
2
3
)
pyrrolidine-2,5-dione (10c).
1–73 °C; R = 0.49 (S ), R = 0.86 (S
NMR (300 MHz, CDCl ) d: 1.23–1.32 (m, 2H, piperidine), 1.45–
.65 (m, 5H, piperidine), 2.52 (d, 2H, CH , J = 7.18 Hz), 2.81 (dd,
H, H , imide, J = 5.90 Hz), 3.02 (d, 2H, piperidine, J = 12.05 Hz)
.22 (dd, 1H, H , imide, J = 9.87 Hz), 4.31 (q, 1H, H , imide,
), 7.11–7.29 (m, 4H, ArH), 7.30–7.39
d: 32.05, 32.12, 36.99, 37.33, 43.00, 46.01, 51.42, 51.47, 60.85,
125.81, 127.35, 127.96, 128.15, 129.07, 129.24, 137.29, 140.48,
177.06, 178.65. ESI-MS: 397.2 (C23
for C23 25ClN
69.41; H, 6.44; N, 7.20.
1
7
f
1
f
2
); HPLC (t
R
1.055 min);
H
+
3
2
H25ClN O
2
[M+H] ); Anal. Calcd
1
1
3
2
H
2
O
2
(396.92): C, 69.59; H, 6.35; N, 7.06. Found: C,
b
a
c
J = 6.03 Hz), 4.53 (s, 2H, CH
m, 4H, ArH), 7.41–7.43 (m, 1H, ArH); C NMR (75 MHz, CDCl
d: 32.06, 32.12, 36.98, 37.32, 43.01, 46.02, 51.41, 51.46, 60.86,
2
4.2.2.12. 3-(3-Chlorophenyl)-1-(morpholinomethyl)pyrrolidine-
13
(
3
)
2,5-dione (11d).
= 0.46 (S ),
(300 MHz, CDCl
, imide, J = 5.13 Hz) 3.24 (dd, 1H, H
4H, morpholine, J = 4.62 Hz), 4.04 (q, 1H, H
4.51 (s, 2H, CH ), 7.11 (t, 1H, ArH, J = 2.31 Hz), 7.13–7.16 (m, 1H,
ArH), 7.26–7.36 (m, 2H, ArH); C NMR (75 MHz, CDCl
44.97, 51.08, 60.60, 66.91, 127.58, 129.55, 130.18, 130.41, 133.62,
White solid. Yield: 70%; mp 98–100 °C;
1
R
f
1
R
f
= 0.77 (S
) d: 2.55–2.66 (m, 4H, morpholine), 2.85 (dd, 1H,
, imide, J = 9.40 Hz), 3.66 (t,
, imide, J = 5.13 Hz),
2
); HPLC (t
R
0.998 min);
H NMR
1
1
25.82, 127.33, 127.96, 128.14, 129.08, 129.23, 137.28, 140.49,
3
+
77.07, 178.64. ESI-MS: 397.1 (C23
25ClN (396.92): C, 69.59; H, 6.35; N, 7.06. Found: C,
9.38; H, 6.54; N, 7.23.
2
H25ClN O
2
[M+H] ); Anal. Calcd
H
b
a
for C23
6
H
2
O
2
c
2
1
3
3
) d: 36.24,
4
2
R
.2.2.8. 3-(2-Chlorophenyl)-1-(morpholinomethyl)pyrrolidine-
,5-dione (10d). White solid. Yield: 78%; mp 99–101 °C;
= 0.42 (S ),
+
134.83, 176.61, 178.07. ESI-MS: 309.2 (C15
Anal. Calcd for C15 17ClN (308.76): C, 58.35; H, 5.55; N, 9.07.
Found: C, 58.49; H, 5.42; N, 9.16.
2 3
H17ClN O [M+H] );
1
f
1
R
f
= 0.91 (S
) d: 2.60–2.72 (m, 4H, morpholine), 2.86 (dd, 1H,
, imide, J = 6.03 Hz), 3.25 (dd, 1H, H , imide, J = 10.00 Hz) 3.68
t, 4H, morpholine, J = 4.74 Hz), 4.34 (q, 1H, imide,
J = 5.90 Hz), 4.53 (s, 2H, CH ), 7.19–7.24 (m, 1H, ArH), 7.28–7.32
m, 2H, ArH), 7.40–7.44 (m, 1H, ArH); C NMR (75 MHz, CDCl
d: 36.25, 44.98, 51.08, 60.61, 66.90, 127.59, 129.55, 130.18,
30.40, 133.63, 134.83, 176.60, 178.08. ESI-MS: 309.1 (C15
2
); HPLC (t
R
0.951 min);
H
NMR
H
2 3
O
(
300 MHz, CDCl
3
H
(
b
a
H
c
,
4.2.2.13. 3-(4-Chlorophenyl)-1-[(4-pyrimidin-2-ylpiperazin-1-
2
yl)methyl]pyrrolidine-2,5-dione (12a).
65%; mp 154–155 °C; R = 0.63 (S ),
1.061 min); H NMR (300 MHz, CDCl ) d: 2.60–2.70 (m, 4H, piper-
azine), 2.81 (dd, 1H, H , imide, J = 5.13 Hz), 3.21 (dd, 1H, H , imide,
J = 9.74 Hz), 3.79 (t, 4H, piperazine, J = 5.13 Hz), 4.01 (q, 1H, H
imide, J = 5.13 Hz), 4.58 (s, 2H, CH ), 6.49 (t, 1H, pyrimidine,
J = 4.74 Hz), 7.13–7.17 (m, 2H, ArH), 7.29–7.33 (m, 2H, ArH), 8.29
White solid. Yield:
13
(
3
)
f
1
R
f
= 0.72 (S ); HPLC (t
2
R
1
3
1
O
H
17ClN2-
b
a
+
3
[M+H] ); Anal. Calcd for C15
H
17ClN
2
O
3
(308.76): C, 58.35; H,
c
,
5
.55; N, 9.07. Found: C, 58.52; H, 5.45; N, 9.15.
2
1
3
4
.2.2.9. 3-(3-Chlorophenyl)-1-[(4-pyrimidin-2-ylpiperazin-1-yl)
(d, 2H, pyrimidine, J = 4.62 Hz);
3
C NMR (75 MHz, CDCl ) d:
methyl]pyrrolidine-2,5-dione (11a).
mp 118–120 °C; R = 0.40 (S ), R = 0.80 (S
H NMR (300 MHz, CDCl ) d: 2.49–2.58 (m, 4H, piperazine) 2.86
dd, 1H, imide, J = 6.10 Hz), 3.18 (dd, 1H, imide,
J = 9.86 Hz), 3.70 (t, 4H, piperazine, J = 5.12 Hz), 4.27 (q, 1H, H
imide, J = 5.89 Hz), 4.38 (s, 2H, CH ), 6.60 (t, 1H, pyrimidine,
J = 4.62 Hz), 7.24–7.42 (m, 3H, ArH), 8.34 (d, 2H, pyrimidine,
J = 4.78 Hz), 7.59–7.63 (m, 1H, ArH); 13C NMR (75 MHz, CDCl
) d:
6.98, 43.63, 46.02, 20.60, 60.44, 109.99, 127.33, 128.02, 129.26,
White solid. Yield: 82%;
25.82, 26.27, 28.88, 28.97, 36.61, 45.47, 48.78, 51.14, 60.41,
f
1
f
2
); HPLC (t 1.1044 min);
R
63.45, 125.57, 127.88, 128.24, 130.46, 135.02, 138.68, 176.58,
1
+
3
177.98. ESI-MS: 386.2 (C19
20ClN
5.20; N, 18.10.
H
20ClN
5
O
2
[M+H] ); Anal. Calcd for C19-
(
H ,
b
H
a
,
H
5 2
O (385.85): C, 59.12; H, 5.22; N, 18.14. Found: C, 59.25; H,
c
,
2
4.2.2.14.
3-(4-Chlorophenyl)-1-[(4-cyclohexylpiperazin-1-yl)
3
methyl]pyrrolidine-2,5-dione (12b).
White solid. Yield:
= 0.83 (S = 0.90 (S ); HPLC (t
3
1.054 min); H NMR (300 MHz, CDCl ) d: 1.15–1.25 (m, 5H, cyclo-
3
1
68%; mp 134–136 °C;
R
f
1
),
R
f
2
R
+
1
37.06, 157.71, 176.99. ESI-MS: 386.1 (C19
20ClN (385.85): C, 59.12; H, 5.22; N,
8.14. Found: C, 59.24; H, 5.18; N, 18.00.
5
H20ClN O
2
[M+H] );
Anal. Calcd for C19
H
5
O
2
hexane), 1.60–1.64 (m, 1H, cyclohexane), 1.77–1.87 (m, 4H, cyclo-
hexane), 2.24 (br s, 1H, cyclohexane), 2.58 (br s, 4H, piperazine),
1
2
.67 (br s, 4H piperazine), 2.80 (dd, 1H, H
3.20 (dd, 1H, H , imide, J = 9.74 Hz), 4.00 (q, 1H, H
J = 5.26 Hz), 4.54 (s, 2H, CH ), 7.19 (d, 2H, ArH, J = 8.46 Hz), 7.35
b
, imide, J = 5.51 Hz),
4
.2.2.10.
3-(3-Chlorophenyl)-1-[(4-cyclohexylpiperazin-1-
White solid. Yield:
= 0.77 (S ); HPLC (t
) d: 1.09–1.24 (m, 5H, cyclo-
a
c
, imide,
yl)methyl]pyrrolidine-2,5-dione (11b).
2
1
3
6
9%; mp 130–131 °C;
R
f
= 0.36 (S
1
),
R
f
2
R
3
(d, 2H, ArH, J = 8.46 Hz); C NMR (75 MHz, CDCl ) d: 25.84,
1
1
.033 min); H NMR (300 MHz, CDCl
3
26.28, 28.88, 28.97, 36.63, 45.49, 48.78, 51.14, 60.41, 63.46,
125.59, 127.90, 128.24, 130.48, 135.01, 138.68, 176.57, 177.97.
hexane), 1.62 (d, 1H, cyclohexane,J = 11.80 Hz), 1.76–1.86 (m, 4H,

K. Kami n´ ski et al. / Bioorg. Med. Chem. 21 (2013) 6821–6830
6829
+
ESI-MS: 390.1 (C21
(389.92): C, 64.68; H, 7.24; N, 10.77. Found: C, 64.57; H, 7.31; N,
0.82.
H
28ClN
3
O
2
[M+H] ); Anal. Calcd for C21
H
28ClN3-
4.3.1.1. Maximal electroshock seizure test (MES).
In the
O
1
2
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.
4
.2.2.15. 1-[(4-Benzyl-1-piperidyl)methyl]-3-(4-chlorophenyl)
White solid. Yield: 78%; mp
pyrrolidine-2,5-dione (12c).
18–120 °C; R = 0.47 (S ), R = 0.73 (S
NMR (300 MHz, CDCl ) d: 1.18–1.29 (m, 2H, piperidine), 1.38–
.43 (m, 1H, piperidine), 1.61 (d, 2H, piperidine, J = 12.57 Hz),
.04–2.18 (m, 2H, piperidine), 2.50 (d, 2H, CH , J = 6.92 Hz), 2.80
, imide, J = 5.13 Hz), 2.96 (br s, 2H, piperidine), 3.20
, imide, J = 9.75 Hz), 4.00 (q, 1H, H , imide, J = 5.13 Hz),
.50 (s, 2H, CH ), 7.10–7.20 (m, 4H, ArH), 7.24–7.29 (m, 3H, ArH),
.33–7.37 (m, 2H, ArH);
4.3.1.2.
Subcutaneous
pentylenetetrazole
seizure
test
1
1
f
1
f
2
); HPLC (t
R
1.049 min);
H
(scPTZ).
This screen utilizes a dose of pentylenetetrazole
3
(85 mg/kg in mice and 70 mg/kg in rats) that produces clonic sei-
zures lasting for a period of at least five seconds in 97% (CD97) of
animals tested. At the anticipated time of testing the convulsant
is administered subcutaneously.
1
2
2
(
(
dd, 1H, H
dd, 1H, H
b
a
c
4
7
3
1
1
2
4.3.1.3. Neurological toxicity (NT).
The neurological toxicity
13
3
C NMR (75 MHz, CDCl ) d: 32.06,
induced by a compound was detected in mice or rats using the
2
5
2.13, 36.98, 37.34, 43.01, 46.02, 51.42, 51.46, 60.87, 125.82,
27.35, 127.97, 128.16, 129.08, 129.24, 137.28, 140.48, 177.07,
78.66. ESI-MS: 397.1 (C23
standarized rotorod test. 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.
+
H
25ClN
2
O
2
[M+H] ); Anal. Calcd for C23-
H
25ClN
2
O
2
(396.92): C, 69.59; H, 6.35; N, 7.06. Found: C, 69.45; H,
6
.41; N, 7.15.
4
2
.2.2.16. 3-(4-Chlorophenyl)-1-(morpholinomethyl)pyrrolidine-
4.3.1.4. The 6-Hz psychomotor seizure model.
This screen
,5-dione (12d).
= 0.38 (S ),
300 MHz, CDCl
, imide, J = 5.14 Hz), 3.24 (dd, 1H, H
t, 4H, morpholine, J = 4.62 Hz), 4.04 (q, 1H,
J = 5.13 Hz), 4.51 (s, 2H, CH ), 7.16–7.24 (m, 2H, ArH), 7.26–7.39
White solid. Yield: 68%; mp 94–96 °C;
was carried out according to the protocol originally described by
1
26
20
R
f
1
R
f
= 0.79 (S
) d: 2.54–2.66 (m, 4H, morpholine), 2.84 (dd, 1H,
, imide, J = 9.62 Hz), 3.66
imide,
2
); HPLC (t
R
0.997 min);
H
NMR
Brown et al. and more recently by Barton et al. and Kaminski
2
7
(
H
(
3
et al. The 6-Hz model is an alternative electroshock paradigm
that uses low-frequency (6-Hz) long-duration (3 s) electrical stim-
ulation. Corneal stimulation (0.2 ms duration monopolar rectangu-
lar pulses at 6 Hz for 3 s) was delivered by a constant-current
device. During the stimulation, mice were manually restrained
and released into observation cage immediately after the current
application. The seizures manifested in ‘stunned’ posture associ-
ated 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 stimulation (Table 4).
b
a
c
H ,
2
1
3
(
m, 2H, ArH); C NMR (75 MHz, CDCl
3
) d: 36.25, 44.98, 51.08,
6
1
0.61, 66.90, 127.58, 129.56, 130.19, 130.40, 133.63, 134.84,
+
76.63, 178.08. ESI-MS: 309.1 (C15
17ClN
8.42; H, 5.47; N, 9.15.
H
2 3
17ClN O [M+H] ); Anal. Calcd
for C15
H
2 3
O (308.76): C, 58.35; H, 5.55; N, 9.07. Found: C,
5
4
4
.3. Biological studies
.3.1. Animals and routes of administration
The initial anticonvulsant evaluation was performed within the
Antiepileptic Drug Development (ADD) Program in the Epilepsy
Branch, Neurological Disorders Program, National Institutes of
Health, National Institute of the Neurological Disorders and Stroke
4.3.1.5. The pilocarpine-induced status prevention (PISP) mod-
el.
Male albino rats (Sprague-Dawley, 150–180 g) were used
(
NIH/NINDS), Rockville, MD, USA, by using procedures described
as experimental animals. The compounds were administrated via
the ip route of administration. Then, a challenge dose of pilocar-
pine is given observing the treatment-effects of the substance
tested. The seizure severity is determined using the well-known
Racine scale as follows: (I) immobility, eye closure, twitching of
vibrissae, sniffing, and facial clonus; (II) head nodding associated
with more several facial clonus; (III) clonus of one of the forelimbs;
(IV) rearing often accomplished by bilateral forelimb clonus, and
(V) all of the above plus loss of balance and falling, accomplished
by generalized clonic seizures. The anticonvulsant activity of
compound 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 6).
2
2–24
elsewhere.
Male albino mice (CF-1 strain) and male albino rats (Spra-
gue-Dawley) were used as experimental animals. The animals
were housed in metabolic cages and allowed free access to food
and water. The compounds were suspended in 0.5% methylcel-
lulose/water mixture. The ASP (Anticonvulsant Screening
Project) 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 were employed. Phase I studies in mice
involved two convulsant tests: maximal electroshock (MES),
subcutaneous pentylenetetrazole (scPTZ) and rotorod test for
neurological toxicity (NT).
2
8
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. Selected derivatives were administrated orally into rats
using four animals at a fixed dose of 30 mg/kg (MES test). This
screen discloses the time of onset, the approximate time of peak ef-
fect (TPE) and the duration of anticonvulsant activity. For both
doses the motor impairment was studied in parallel. Rats were
tested at five time periods ranging from one quarter to 4 h post
substance administration. The results of preliminary anticonvul-
sant screening are shown in Tables 1 and 2.
4.3.1.6. Quantification studies.
Quantitative determination
of the median effective dose (ED50) and the median neurotoxic
dose (TD50) values was performed at previously estimated time
of peak effect (TPE) after p.o. administration into rats. Groups of
minimum eight animals received various doses of the compound
until at least three points were established in the range of 10–
90% seizure protection or minimal motor impairment. 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 Tables 3, 5 and 7.

6
830
K. Kami n´ ski et al. / Bioorg. Med. Chem. 21 (2013) 6821–6830
8. Siddiqui, N.; Ahsan, W.; Alam, M. S.; Ali, R.; Srivastava, K. Arch. Pharm. Chem.
4
.3.2. Cytochrome P450 inhibition studies
Life Sci. 2012, 345, 185.
Kami n´ ski, K.; Obniska, J.; Wiklik, B.; Atamanyuk, D. Eur. J. Med. Chem. 2012, 46,
The cytochrome P450 inhibition studies were carried out in Na-
tional Institutes of Health, National Institute of the Neurological
9
.
4
634.
10. Kami n´ ski, K.; Rzepka, S.; Obniska, J. Bioorg. Med. Chem. Lett. 2011, 21, 5800.
11.
Kami n´ ski, K.; Obniska, J. Bioorg. Med. Chem. 2008, 16, 4921.
Disorders and Stroke (NIH/NINDS), Rockville, MD, USA, by using
procedures described elsewhere.29 Compound tested was incu-
1
1
2. Kami n´ ski, K.; Obniska, J.; Dybała, M. Eur. J. Med. Chem. 2008, 43, 53.
3. Obniska, J.; Kami n´ ski, K.; Skrzy n´ ska, D.; Pichór, J. Eur. J. Med. Chem. 2009, 44,
2224.
bated together with probe substrate (testosterone) in the presence
of NADPH, and the inhibition of metabolite formation, namely 6-b-
OH-testosterone by CYP3A4 subtype of the cytochrome P450 was
determined. The results are shown in Table 8.
1
1
4. Obniska, J.; Byrtus, H.; Kami n´ ski, K.; Pawłowski, M.; Szczesio, M.; Karolak-
Wojciechowska, J. Bioorg. Med. Chem. 2010, 18, 6134.
5. Obniska, J.; Kopytko, M.; Zagórska, A.; Chlebek, I.; Kami n´ ski, K. Arch. Pharm.
Chem. Life Sci. 2010, 343, 333.
1
1
1
6. Obniska, J.; Zagórska, A. Il Farmaco 2003, 58, 1227.
7. Miller, C. A.; Long, L. M. J. Am. Chem. Soc. 1951, 73, 4895.
8. Obniska, J.; Kulig, K.; Zejc, A. Acta Pol. Pharm. Drug Res. 1998, 55, 223.
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), and Professor Katarzyna Kie c´ -Kononowicz (Department
of Technology and Biotechnology of Drugs, Jagiellonian University
Medical College, Kraków, Poland) the coordinator of the ADD Pro-
ject from Polish site.
19. White, S. H.; Woodhead, J. H.; Wilcox, K. S.; Stables, J. P.; Kupferberg, H. J.; Wolf,
H. H. Discovery and Preclinical Development of Antiepileptic Drugs. In
Antiepileptic Drugs; Levy, R. H., Mattson, R. H., Meldrum, B. S., Perucca, E.,
Eds., 5th ed.; Lippincott Williams and Wilkins: Philadelphia, 2002; pp 36–48.
20. Barton, M. E.; Klein, B. D.; Wolf, H. H.; White, H. S. Epilepsy Res. 2001, 47, 217.
21. USFDA Draft Guidance for Industry: Drug Interaction Studies-Study Design,
Data Analysis, and Implications for Dosing and Labeling, U.S. Food and Drug
Administration Publication, 2006.
2
2. White, H. S. Epilepsia 2003, 44, 2.
23. Krall, R. L.; Penry, J. K.; White, B. G.; Kupferberg, H. J.; Swinyard, E. A. Epilepsia
978, 19, 409.
1
2
2
2
4. Kupferberg, H. J. Epilepsia 1989, 30, 51.
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