Synthesis and anticonvulsant properties of new n-mannich bases derived from 3,3-diphenyl- and 3-ethyl-3-methyl-pyrrolidine-2,5-diones. Part III

Article abstract of DOI:10.1002/ardp.201200265

Twenty-four new N-[(4-phenylpiperazin-1-yl)-methyl] derivatives of 3,3-diphenyl- (7-18) and 3-ethyl-3-methyl-pyrrolidine-2,5-dione (19-30) were synthesized and evaluated for their anticonvulsant activity in the maximum electroshock (MES) and subcutaneous

Full text of DOI:10.1002/ardp.201200265

Arch. Pharm. Chem. Life Sci. 2013, 346, 71–82
71
Full Paper
Synthesis and Anticonvulsant Properties of New
N-Mannich Bases Derived from 3,3-Diphenyl- and
3-Ethyl-3-methyl-pyrrolidine-2,5-diones. Part III
1
Jolanta Obniska , Iwona Chlebek , Krzysztof Kaminski , and Janina Karolak-Wojciechowska
1
1
2
´
1
´
Department of Medicinal Chemistry, Jagiellonian University, Medical College, Krakow, Poland
´
Institute of General and Ecological Chemistry, Technical University, Łodz, Poland
2
Twenty-four new N-[(4-phenylpiperazin-1-yl)-methyl] derivatives of 3,3-diphenyl- (7–18) and 3-ethyl-3-
methyl-pyrrolidine-2,5-dione (19–30) were synthesized and evaluated for their anticonvulsant activity
in the maximum electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizure tests.
The acute neurological toxicity was determined using the rotorod screen. Eleven compounds were
active and revealed protection only in electrically induced seizures (MES). In the whole series the
most effective compound was N-[{4-(3-trifluoromethylphenyl)-piperazin-1-yl}-methyl]-3,3-diphenyl-
pyrrolidine-2,5-dione (14) with an ED50 value of 30.3 mg/kg (p.o. rats) in the MES test. To explain
the possible mechanism of action, for chosen active derivatives 7, 8, 9, 11, 14, 23, and 26, their
influence on NaV1.2 sodium channel currents was evaluated in vitro. The crystallographic structures
for several molecules (8, 10, and 11) were solved.
Keywords: 3,3-Disubstituted-pyrrolidine-2,5-diones / Anticonvulsant activity / In vitro studies / In vivo studies / Mannich
bases / Sodium channels
Received: July 1, 2012; Revised: October 7, 2012; Accepted: October 9, 2012
DOI 10.1002/ardp.201200265
Introduction
rational methodologies of discovery that are based on the
three-dimensional structure of a biological target. Thus the
most useful for the design of new anticonvulsants are ligand-
based approaches that rely on the use of different pharma-
cophores established through the analysis of structural
characteristics of clinically effective antiepileptic drugs
(AEDs) as well as other anticonvulsant active compounds
[5]. The two past decades have demonstrated many attempts
to identify the structural features of compounds crucial for
anticonvulsant activity. As a result it was proved that one of
the important core fragments is defined by a nitrogen het-
eroatomic system, usually imide or lactam, with at least one
carbonyl group and phenyl or alkyl groups attached to the
heterocyclic system [6, 7]. This common template is present
in the structures of both first generation AEDs such as etho-
suximide and phenytoin as well as among the newest drugs,
e.g., levetiracetam, brivaracetam, or seletracetam (Fig. 1).
The currently used AEDs can be classified into four
categories on the basis of the main molecular mechanisms
of action, as follow: (i) modulation of voltage-dependent Naþ
and/or Ca2þ channels, (ii) enhancement of GABA-mediated
inhibition or other effect on the GABA system, (iii) inhibition
Epilepsy is the most prevalent neurological disorder, affect-
ing approximately 50 million people worldwide [1]. Even
though significant advances have been made in epilepsy
research, convulsions in about 30% of epileptics are still
inadequately controlled by standard drug therapy [2, 3].
Furthermore, compliance is often limited by adverse side
effects most notably related to CNS exposure like diminished
attention, executive function, intelligence, language skills,
memory, and processing speed [4]. For these reasons, constant
attempts are made to investigate new chemical agents and
mechanisms through which epilepsy can be effectively con-
trolled. The incomplete information on the pathogenesis of
human epilepsy and the complex mechanism of action of
the majority the antiepileptic drugs makes it difficult to use
Correspondence: Jolanta Obniska, Department of Medicinal Chemistry,
´
Jagiellonian University Medical College, 9 Medyczna Street, Krakow 30-
688, Poland.
E-mail: mfobnisk@cyf-kr.edu.pl
Fax: þ48126570262
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J. Obniska et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 71–82
F
F
O
O
O
H
N
N
N
N
O
NH2
NH2
NH2
O
N
H
O
N
H
H
O
O
H
H
O
O
Ethosuximide
Seletracetam
Phenytoin
Levetiracetam
Brivaracetam
Figure 1. Structures of known AEDs bearing the imide or lactam scaffold.
of synaptic excitation mediated by ionotropic glutamate
receptors, and (iv) modulation of synaptic release [8].
Regardless of the complex modes of action, the discovery
of new anticonvulsants is still particularly focused on target-
ing voltage-gated sodium channels (VGSCs), since they play a
fundamental role in establishing and regulating the excit-
ability of neurons within the central nervous system (CNS).
Furthermore the changes in the expression pattern and
behavior of different sodium channel isoforms, especially
NaV1.2, are observed in epilepsy and could play a role in
seizure generation and spread [9, 10]. Thus they are of great
importance for the control and suppression of seizures [11].
Since many years our studies have been focused on the
search for new anticonvulsant agents among ethosuximide
analogues. The previous research from our laboratory has
demonstrated diversified anticonvulsant activities among
the differently substituted pyrrolidine-2,5-diones [12–16].
The most promising activity was observed among N-Mannich
bases with an aromatic ring at position 3 and phenylpiperazine
moieties (especially with electron-withdrawing chloro atom
or trifluoromethyl group) at position 1 of pyrrolidine-2,5-
dione (Fig. 2) [17–19].
As a continuation of systematic SAR studies among differ-
ently substituted succinimides as potential anticonvulsant
agents, in the present work we synthesized a new series
of N-Mannich bases of 3,3-diphenyl- (7–18) and 3-ethyl-3-
methyl-pyrrolidine-2,5-diones (19–30). These compounds
were designed as analogues of molecules described pre-
viously in which a second aromatic ring was introduced
at position 3 of pyrrolidine-2,5-dione. Furthermore, to
Cl
CF3
O
N
O
N
Cl
N
N
N
N
O
O
1
2
ED50 = 20.78 mg/kg
ED50 = 21.40 mg/kg
(MES test, p.o. rats)
(MES test, p.o. rats)
O
N
O
N
Cl
N
N
N
N
O
O
3
4
ED50 = 9.0 mg/kg
ED50 = 19.57 mg/kg
(MES test, p.o. rats)
(MES test, p.o. rats)
Figure 2. The structures of the model compounds 1–4.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 71–82
New Pyrrolidine-2,5-diones as Potential Anticonvulsants
73
investigate the influence of the above phenyl rings on anti-
convulsant activity in the next step of studies, their 3-ethyl-3-
methyl-analogues were obtained. Based on the previous
results differently substituted 4-phenylpiperazines (both
with positive and negative input into anticonvulsant
activity), were introduced at the imide nitrogen atom by a
methylene spacer. Taking into consideration the structures
of compounds 7–18 that correspond to molecules active
in the MES test (e.g., phenytoin – 5,5-diphenylhydantoin),
the assessment of their influence on NaV1.2 sodium channel
currents was planned as the most plausible mechanism of
anticonvulsant action.
reaction from the 3,3-disubstituted-pyrrolidine-2,5-diones
(5, 6), formaldehyde and corresponding 4-phenylpiperazines.
The reaction was carried out in ethanol at room temperature
for ca. 6–12 h. The crude products were crystallized from
96% ethanol. The final compounds were obtained in yields
ranging from 65 to 75%. Their purity was assessed by TLC.
The structures of the synthesized compounds were con-
firmed by both spectral and elemental analysis. The detailed
physical and analytical data are listed in the Experimental
section.
The synthesis and physicochemical properties of com-
pounds 19 and 24 were described previously [21]. These
molecules were not tested for anticonvulsant activity.
Results and discussion
Anticonvulsant activity in vivo studies
Chemistry
All the compounds synthesized have been evaluated for
anticonvulsant and neurotoxic properties within the
Antiepileptic Drug Development (ADD) Program in
Epilepsy Branch, National Institutes of Health, National
Institute of Neurological Disorders and Stroke (NIH/
NINDS), Rockville, MD, USA [22]. The profile of anticonvulsant
activity was established in the maximal electroshock (MES)
and subcutaneous pentylenetetrazole (scPTZ) seizure tests,
The synthesis of compounds 7–30 was accomplished as
shown in Scheme 1. The starting material 3,3-diphenyl-
phenyl-pyrrolidine-2,5-dione (5) was prepared as previously
reported [20]. Compounds 19–30 were synthesized by use
of commercially available 3-ethyl-3-methylsuccinimide (6)
(Sigma–Aldrich, St. Louis, USA) as reaction substrate. The
final compounds 7–30 were obtained in the Mannich-type
R1
R1
R
O
N
HN
O
R2
R2
N
R
N
HCHO, EtOH, room temp
O
N
N
O
H
_
7 30
5, 6
R
cmpd
_
R1 = R2 =
compounds 5, 7 18
H
7, 19
2-F
8, 20
_
R1 = -CH3, R2 = -C2H5 compounds 6, 19 30
4-F
2-Cl
9, 21
10, 22
11, 23
12, 24
13, 25
14, 26
15, 27
3-Cl
4-Cl
3,4-diCl
3-CF3
2-CH3
3-CH3
16, 28
17, 29
18, 30
2-OCH3
3-OCH3
Scheme 1. General method for the synthesis of compounds 7–30.
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74
J. Obniska et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 71–82
after intraperitoneal (i.p.) injection into mice at doses of 30,
100, and 300 mg/kg. An observation was carried out at two
different time intervals, namely 0.5 and 4 h. It should be
noticed that despite significant advances that have been
made in epilepsy research during the last years, the MES
and scPTZ screens are still recognized as the ‘‘gold standards’’
in the early stages of testing of new anticonvulsants. These
tests are capable to detect compounds affording protection
against generalized tonic–clonic seizures and generalized
absence seizures, respectively [23, 24]. The acute neurological
toxicity (NT) was determined in the minimal motor impair-
ment – rotorod screen (NT). The results are shown in Table 1.
Except for compounds 20–22 and 29 tested as hydrochlo-
ride salts, all other molecules were evaluated as free bases.
The compounds tested showed protection against electrically
induced seizures (7–9, 11, 12, 14–16, 23, 24, and 26) or were
inactive (10, 13, 17–22, 25, 27–30). All substances were devoid
of activity in the pentylenetetrazole (scPTZ) seizures. It was
especially surprising in relation to 3-ethyl-3-methyl-pyrroli-
dine-2,5-diones (19–30) that are close analogues of ethosux-
imide which is recognized as model anticonvulsant active in
the scPTZ test. The strongest anti-MES activity, indicative of
the ability of a substance to prevent seizure spread, was
observed for 14 that revealed protection at a dose of
30 mg/kg 4 h after i.p. administration. This result was com-
parable to phenytoin which is known as model AED effective
in the MES test. The other molecules were active at dose of
100 mg/kg at the same time point. The activity 4 h after
intraperitoneal administration indicates the slow onset
and long duration of anticonvulsant action. In the rotorod
test for acute NT all of the 3,3-diphenyl-pyrrolidine-2,5-diones
(7–18) and 25 representing series of 3-ethyl-3-methyl-
analogues did not show neurotoxicity at the maximum dose
administered (300 mg/kg). Among these compounds 7–9, 11,
12, 14–16 emerged as anticonvulsants without neurotoxic
properties. The other molecules exhibited motor impairment
at a dose of 100 mg/kg (19, 20, 22, 28) or 300 mg/kg (21, 23, 24,
26, 27, 29, 30). It should be stressed here that structure–
neurotoxic properties analysis showed remarkable growth
of neurotoxicity caused by introduction of ethyl and methyl
groups in place of phenyl rings at position 3 of pyrrolidine-
2,5-dione.
Table 1. Anticonvulsant and neurotoxicity screening after i.p.
administration in mice (7–30).
Compound
MESa)
NTb)
0.5 h
4 h
0.5 h
4 h
7
8
9
10
11
12
13
14
15
16
17
18
–
–
–
–
–
–
–
100
–
–
100
100
100
–
100
100
–
30
100
100
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
On the basis of mice i.p. data seven compounds (7, 8, 11, 12,
14, 23, and 26) active in the electrically induced seizures were
examined for anticonvulsant activity (MES screen) and neuro-
toxicity after p.o. administration in rats at a dose of 30 mg/kg.
This screen discloses the time of onset, the approximate time
of peak effect (TPE) and the duration of anticonvulsant
activity or neurotoxicity. The results are shown in Table 2.
–
–
–
–
–
–
19
20
21
22
23
24
25
26
27
28
29
30
–
–
–
–
–
–
–
–
–
–
–
–
30
–
–
–
–
–
100
100
–
100
–
–
–
–
30
–
100
100
300
100
300
300
–
300
300
100
300
300
100
–
–
–
–
–
–
300
–
300
–
–
Table 2. Anticonvulsant activity of selected compounds
administrated orally to rats at a dose of 30 mg/kg.
Compound
MESa)
1 h
0.25 h
0.5 h
2 h
4 h
300
–
–
Phenytoinc)
Ethosuximidec)
7
8
11
12
14
23
26
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
4
0
0
0
0
2
1
2
3
0
0
2
0
3
1
3
3
2
0
3
0
4
1
3
3
Doses of 30, 100, and 300 mg/kg were administered. The data
indicate the minimum dose whereby anticonvulsant activity
or neurotoxicity was demonstrated in half or more animals.
The animals were examined at 0.5 and 4 h. A dash indicates
the absence of anticonvulsant activity and neurotoxicity at the
maximum dose administered (300 mg/kg).
Phenytoinb)
a)
Maximal electroshock test.
Neurotoxicity screening using the rotorod test.
The data indicate the number of rats of four that were protected.
a)
b)
Maximal electroshock test.
Phenytoin, reference drug, tested by use of ADD Program
procedures in NIH/NINDS, data from ref. [44].
c)
b)
Phenytoin, ethosuximide – reference drugs, tested by use of
ADD Program procedures in NIH/NINDS, data from ref. [43].
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Arch. Pharm. Chem. Life Sci. 2013, 346, 71–82
New Pyrrolidine-2,5-diones as Potential Anticonvulsants
75
Table 4. Electrophysiological evaluation of selected compounds
for efficacy against Nav1.2 current.
Among these the most active was compound 14 that pro-
tected 100% of animals at 4 h, 75% at 2 h, and 50% at 1 h. This
molecule revealed however lower activity than phenytoin –
model AED. The others 7, 11, 23, 26 were less active and
inhibited convulsions in 75% of rats at 2 h (26) or/and 4 h
(11, 26); 50% of rats at 1 h (26), 2 h (11) or 4 h (7), and 25% of
rats at 0.5 h (26), 1 h, 2 h, and 4 h (23). All derivatives were
non-neurotoxic when given orally (data not indicated in
Table 2).
Compound
Percentage of blocking Nav1.2 sodium
channel currents (n ¼ 2)
0.1 mM
1.0 mM
10.0 mM
100 mM
7
8
9
11
14
23
26
4.17
11.45
11.20
15.60
11.00
1.76
9.25
14.38
4.58
28.39
4.63
2.58
25.87
40.78
34.09
58.47
42.48
33.47
53.90
43.13
38.85
80.69
91.19
86.04
79.19
87.91
Compound 14 as the most active molecule in previous tests
was chosen for phase II evaluation for quantification of the
pharmacological parameters (ED50 and TD50). The quantitat-
ive evaluation of the median effective dose (ED50) in the MES
seizures and neurotoxic dose (TD50) in the rotorod screen
were performed after p.o. administration to rats. The studies
were carried out at previously estimated TPE – 4 h. Results of
the quantitative tests along with the data for the standard
drug – phenytoin are shown in Table 3.
13.12
16.43
The data indicate the percentage of blocking Nav1.2 current. The
results are divided as follows:
<20% – inactive compound,
20–50% – compound with marginal activity,
>50% – active compound.
As shown in Table 3 compound 14 revealed equivalent
activity in comparison to the reference drug – phenytoin.
Moreover, it possessed relatively low rotorod toxicity com-
pared with the standard drug that was expressed in 3.6-fold
more favorable protection index. These results confirmed
also satisfactory absorption from the gastrointestinal tract
as well as penetration of the substance into the CNS.
According to the ASP dispositions four compounds 11, 14,
23, 24 were selected randomly and assessed for potential
activity against nerve agents using the pilocarpine model
of epilepsy as an introductory screen. This 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
pilocarpine models are one of the most recognized animal
models of SE. The results revealed however that all molecules
were inactive in this screen.
might be controlled. The fundamental role in establishing
and regulating excitability of CNS neurons as well as sup-
pression of seizures is ascribed to VGSCs [9]. Thus the brain
ion channels are the molecular targets of a number of
chemically diverse AEDs from which the most important
are phenytoin, lamotrigine, carbamazepine, and oxcarbaze-
pine. These AEDs are sodium channel blockers and have been
shown to inhibit NaV1.2 Na channel currents [25–27]. It
should be stressed here that such mode of action is charac-
teristic for compounds active in the maximal electroshock
seizure (MES) test. Taking into consideration the activity of
compounds synthesized in electrically induced seizures,
for seven, active compounds 7, 8, 9, 11, 14, 23, and 26 the
influence on NaV1.2 sodium channel currents was studied
in vitro. The results are shown in Table 4.
According to the method applied [28, 29], the percentage of
blocking the NaV1.2 channel currents for active compound
should be higher than 50%. Taking into consideration the
above the most effective blockers were 11 and 26 that showed
activity in concentration of 10.0 mM (blocking percentage
>50%). These molecules revealed also the highest inhibition
in concentration of 100 mM – 91.19% (11) and 87.91% (26).
The other active molecules include 9, 14, and 23 (100 mM),
In vitro sodium channel electrophysiological studies
Intensive studies into the physiological and biochemical
events taking place during epileptic seizures have provided
insight into the molecular mechanisms by which these
Table 3. Quantification studies of 14 in the MES and neurotoxicity tests (NT) in rats after p.o. administration.
c)
Compound
TPE (h)a)
ED50 MES (mg/kg)b)
TD50 (mg/kg)b)
PI (TD50/ED50)
14
Phenytoind)
4
2
30.3 (19.40–54.50)
28.1 (20.7–35.2)
<400
>100
<13.20
>3.60
a)
Time to peak effect.
Results are represented as mean ꢀ SEM at 95% confidence limit (MES, maximal electroshock test; NT, neurotoxicity, rotarod test).
b)
c)
PI, protective index (TD50/ED50).
Phenytoin, reference drug, tested by use of ADD Program procedures in NIH/NINDS, data from ref. [45].
d)
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J. Obniska et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 71–82
Table 5. H-bonds geometry description.
whereas 7 and 8 showed only moderate activity in concen-
tration of 10.0 and 100 mM. Despite of activity the compari-
son of electrophysiological and in vivo data revealed no
direct correlation between NaV1.2 channel inhibition and
results obtained in animals. It was especially visible for 14
that showed the most potent anti-MES protection in mice
and rats however was a weaker NaV1.2 channel blocker than
the less active molecules 11 and 26. Furthermore 7 and 8
which were moderately active on sodium channels possessed
equal in vivo activity with other compounds tested. These
findings suggest that the mechanism of anticonvulsant
action may be connected with the influence on other
molecular targets beyond sodium channels or interaction
˚
Compound
8
X–H. . . Y
HꢁꢁꢁY (A)
X–HꢁꢁꢁY (8)
C4–H4B. . .O2
C12–H12. . .O2
C15–H15. . .O5
C4–H4B. . .O2
C12–H12. . .O2
15–H15. . .O5
C23–H23A. . .Cl36
C4–H4B. . .O2
3.186 (2)
3.253 (2)
3.365 (2)
3.255 (2)
3.363 (3)
3.432 (3)
3.795 (2)
3.086 (4)
3.659(4)
157
154
143
161
154
146
149
135
174
10
11
C
C20–H20A. . .O5
with specific sodium channel gating modes such as the slow Structure–activity relationships
inactivation mode which is known to be important for AED
activity [30].
The preliminary pharmacological screening revealed that
anticonvulsant activity depended strongly on the substitu-
ents at position 3 of the imide ring. The most active were
compounds with two phenyl rings at position 3 of the pyr-
rolidine-2,5-dione moiety (7–18). The exchange of the men-
tioned phenyl substituents into alkyl – ethyl and methyl
groups made the compounds less active or inactive (19–30).
The second factor influencing anticonvulsant activity is the
presence of electron-withdrawing atoms especially at position
3 or 4 of the 4-phenylpiperazine moiety. In general, in
both series of compounds the most active were the 3-CF3
derivatives (14, 26) and compounds with a chloro atom
located at the meta- (11, 23) or para-position (12, 24).
The respective ortho-chloro isomers (10, 22) were devoid of
activity. The weaker activity was observed for unsubstituted
(7, 19) and fluoro compounds 8, 9 20, and 21, among
which only 3,3-diphenyl-pyrrolidine-2,5-diones (7, 8, 9)
showed anticonvulsant protection. The replacement of
electron-withdrawing fragments into electron-donating
CH3 or OCH3 substituents (17, 18, 27, 28, 29, 30) yielded
inactive molecules, in general (an exception was observed
X-ray structure analysis
As a continuation of discerning structural studies [17, 31–34],
the molecular structures of selected compounds 8, 11, and 10
were crystallographically confirmed. The presence of the 4-
phenylpiperazine moiety influences different properties of
the compounds including the ability of intermolecular inter-
actions. In aim to confirm the above assumption the struc-
tures of molecules with ortho-fluoro- (8), ortho-chloro- (10), and
meta-chloro- (11) substituents were examined. In three struc-
tures obtained, similarly to pyrrolidine-2,5-diones described
previously [17, 31–34], the main motive for crystal construc-
tion is based on C–H. . .O hydrogen bonds (Fig. 3).
As shown in Table 5, the bond of C4–H. . .O2 (anchored
oxygen O2, imide ring C4 atom), is the strongest in each
structure. Moreover this intermolecular interaction becomes
weaker in the following order: meta-chloro- (11) < ortho-fluoro-
(8) < ortho-chloro- (10) substituted molecules.
Figure 3. ORTEP drawing of the 8, 10, and 11 molecules.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 71–82
New Pyrrolidine-2,5-diones as Potential Anticonvulsants
77
Hertz (Hz). Signal multiplicities are represented by the following
abbreviations: s (singlet), brs (broad singlet), d (doublet), dd
(double doublets), t (triplet), m (multiplet). The mass spectra
for compounds 7–30 were obtained on a Waters ACQUITYTM
TQD system with the TQ detector (Waters, Milford, USA). The
ACQUITY UPLC BEH C18, 1.7 mm, 2.1 ꢂ 50 mm column was used
(Waters, Milford, USA).
for active 2-CH3 (15) and 3-CH3 (16) derivatives in series of
3,3-diphenyl-pyrrolidine-2,5-diones).
Summing up, the modifications applied that relied on
introduction of two phenyl rings or ethyl and methyl sub-
stituents at position 3 of the pyrrolidine-2,5-dione ring did
not improve anticonvulsant activity in comparison with model
compounds 1–4 described previously [17–19]. Therefore it
seems that the most favorable for anticonvulsant activity
is the presence of one aromatic system in the vicinity of
the C-3 imide carbon atom.
General procedure for preparation of compounds 7–30
To a mixture of 3,3-diphenyl- (5) or 3-ethyl-3-methyl-pyrrolidine-
2,5-dione (6) (0.01 mol) and 40% solution of formaldehyde
(0.01 mol) in 96% ethanol (20 ml), corresponding 4-phenylpiper-
azines (0.01 mol) dissolved in 96% ethanol were added. The
mixture was left for ca. 6–12 h at room temperature and then
refrigerated at ca. ꢃ108C for 24 h. The precipitated crude prod-
ucts were washed with cold ethanol, separated by filtration, and
recrystallized from 96% ethanol. Compounds 20–22 and 29 due
to their oily character were converted into hydrochloride salts
in anhydrous ethanol saturated with HCl gas. The obtained
precipitate salts were crystallized from anhydrous ethanol.
Conclusion
Twenty four new N-[(4-phenylpiperazin-1-yl)-methyl]- deriva-
tives of 3,3-diphenyl- (7–18) and 3-ethyl-3-methyl-pyrrolidine-
2,5-dione (19–30) were synthesized and evaluated for their
anticonvulsant and neurotoxic properties. The in vivo results
revealed that 11 compounds were active and revealed pro-
tection only in electrically induced seizures. Moreover all
effective 3,3-diphenyl-pyrrolidine-2,5-diones did not show
acute NT in the rotorod screen. The most active were N-[{4-
(3-trifluoromethylphenyl)-piperazin-1-yl}-methyl]-3,3-diphenyl-
pyrrolidine-2,5-dione (14) with ED50 of 30.30 mg/kg in
rats (p.o.). The SAR studies revealed that anticonvulsant
activity was closely connected with the presence of phenyl
substituents at position 3 of the imide ring as well as
electron-withdrawing atoms especially at position 3 or 4
of the phenylpiperazine moiety. The in vitro electrophysio-
logical studies demonstrated that compounds active in the
MES test had activity against the NaV1.2 sodium channel
isoform.
N-[(4-Phenyl-piperazin-1-yl)-methyl]-3,3-diphenyl-
pyrrolidine-2,5-dione (7)
White solid. Yield: 79%; mp 160–1618C; TLC: Rf ¼ 0.81 (S1);
1H NMR (300 MHz, CDCl3) d: 2.75 (t, 4H, piperazine,
J ¼ 5.0 Hz), 3.14 (t, 4H, piperazine, J ¼ 5.0 Hz), 3.47 (s, 2H,
imide), 4.61 (s, 2H, –CH2–), 6.83–6.89 (m, 3H, ArH), 7.22–7.38
(m, 12H, ArH); 13C NMR (75 MHz, CDCl3) d: 44.96, 49.19, 50.56,
57.30, 60.42, 116.19, 119.85, 127.41, 127.76, 128.92, 129.13,
141.64, 151.15, 176.11, 179.49. ESI-MS: 426.18. (C27H27N3O2
[MþH]þ). Anal. calcd. for C27H27N3O2 (425.54): C: 76.21,
H: 4.57, N: 9.87. Found C: 76.48, H: 6.38, N: 9.85.
N-[{4-(2-Fluorophenyl)-piperazin-1-yl}-methyl]-3,3-
diphenyl-pyrrolidine-2,5-dione (8)
White solid. Yield: 70%; mp 160–1628C; TLC: Rf ¼ 0.68 (S1);
1H NMR (300 MHz, CDCl3) d: 2.77 (t, 4H, piperazine,
J ¼ 4.74 Hz), 3.05 (t, 4H, piperazine, J ¼ 4.87 Hz), 3.50 (s, 2H,
imide), 4.60 (s, 2H, –CH2–), 6.88–6.99 (m, 4H, ArH), 7.01–7.39
(m, 10H, ArH). 13C NMR (75 MHz, CDCl3) d: 44.93, 50.20, 50.56,
57.28, 60.37, 115.36, 115.66, 117.87, 117.95, 127.35, 127.75,
128.92, 141.61, 147.77, 155.63, 158.75, 176.05, 179.46.
19F NMR (282 MHz, CDCl3) d: ꢃ124.40 (s, 1F). ESI-MS: 444.10.
(C27H26N3O2F [MþH]þ). Anal. calcd. for C27H26N3O2F (443.53):
C: 73.12, H: 5.91, N: 9.47. Found C: 72.85, H: 5.89, N: 9.46.
Experimental
Chemistry
All the chemicals and solvents were purchased from Sigma–
Aldrich (St. Louis, USA) and were used without further purifi-
cation. Melting points (mp) were determined in open capillaries
on a Bu¨chi 353 melting point apparatus (Bu¨chi Labortechnik,
Flawil, Switzerland) and are uncorrected. The purity of the com-
pounds was confirmed by thin-layer chromatography (TLC) per-
formed on Merck silica gel 60 F254 aluminum sheets (Merck;
Darmstadt, Germany), using a developing system consisting of
chloroform/acetone (9:1 v/v) (S1) and chloroform/isopropanol/
ammonia (9:11:2 by volume) (S2). Spots were detected by their
absorption under UV light (l ¼ 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 micromethod using the elemental Vario EI
III elemental analyzer (Hanau, Germany). The results of the
elemental analyses were within ꢀ0.4% of the theoretical values.
1H NMR, 13C NMR, and 19F NMR spectra were obtained in a Varian
Mercury spectrometer (Varian Inc., Palo Alto, CA, USA), in CDCl3
or DMSO, operating at 300, 282, and 75 MHz, respectively.
Chemical shifts are reported in d values (ppm) relative to TMS
d ¼ 0 (1H), as internal standard. The J values are expressed in
N-[{4-(4-Fluorophenyl)-piperazin-1-yl}-methyl]-3,3-
diphenyl-pyrrolidine-2,5-dione (9)
White solid. Yield: 68%; mp 106–1078C; TLC: Rf ¼ 0.75 (S1);
1H NMR (300 MHz, CDCl3) d: 2.75 (t, 4H, piperazine,
J ¼ 4.87 Hz), 3.55 (t, 4H, piperazine, J ¼ 4.74 Hz), 3.49 (s, 2H,
imide), 4.59 (s, 2H, –CH2–), 6.80–6.85 (m, 2H, ArH), 6.91–6.97
(m, 2H, ArH), 7.26–7.37 (m, 10H, ArH). 13C NMR (75 MHz,
CDCl3) d: 44.94, 50.19, 50.55, 57.29, 60.38, 115.38, 115.67,
117.89, 117.99, 127.39, 127.76, 128.91, 141.63, 147.78, 155.62,
158.79, 176.09, 179.48. 19F NMR (282 MHz, CDCl3) d: ꢃ124.38
(s, 1F). ESI-MS: 444.10. (C27H26N3O2F [MþH]þ). Anal. calcd. for
C27H26N3O2F (443.53): C: 73.12, H: 5.91, N: 9.47. Found C: 73.22,
H: 5.92, N: 9.46.
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78
J. Obniska et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 71–82
(75 MHz, CDCl3) d: 44.95, 48.68, 50.33, 57.29, 60.30, 112.23,
112.28, 115.89, 115.94, 118.80, 127.38, 127.77, 128.92, 129.56,
131.62, 141.58, 151.18, 176.04, 179.48. 19F NMR (282 MHz, CDCl3)
d: ꢃ62.71 (s, 3F, CF3). ESI-MS: 494.11 (C28H26N3O2F3 [MþH]þ).
Anal. calcd. for C28H26N3O2F3 (493.54): C: 68.14, H: 5.31,
N: 8.51. Found C: 68.10, H: 5.30, N: 8.48.
N-[{4-(2-Chlorophenyl)-piperazin-1-yl}-methyl]-3,3-
diphenyl-pyrrolidine-2,5-dione (10)
White solid. Yield: 78%; mp 147–1498C; TLC: Rf ¼ 0.73 (S1);
1H NMR (300 MHz, CDCl3) d: 2.76 (t, 4H, piperazine,
J ¼ 4.61 Hz), 2.99 (t, 4H, piperazine, J ¼ 4.49 Hz), 3.51 (s, 2H,
imide), 4.61 (s, 2H, –CH2–), 6.93–7.01 (m, 2H, ArH), 7.17–7.39
(m, 12H, ArH). 13C NMR (75 MHz, CDCl3) d: 44.91, 48.71,
50.30, 57.31, 60.38, 113.92, 115.80, 119.31, 127.33, 127.76,
128.93, 130.03, 134.92, 141.55, 152.10, 176.02, 179.45.
ESI-MS: 460.11. (C27H26N3O2Cl [MþH]þ). Anal. calcd. for
C27H26N3O2Cl (459.98): C: 70.50, H: 5.70, N: 9.14. Found C:
70.25, H: 5.71, N: 9.18.
N-[{4-(2-Methylphenyl)-piperazin-1-yl}-methyl]-3,3-
diphenyl-pyrrolidine-2,5-dione (15)
White solid. Yield: 68%; mp 139–1418C; TLC: Rf ¼ 0.71 (S1);
1H NMR (300 MHz, CDCl3) d: 2.21 (s, 3H, 3-CH3), 2.70–2.71
(m, 4H, piperazine), 2.84 (t, 4H, piperazine, J ¼ 4.74 Hz), 3.51
(s, 2H, imide), 4.61 (s, 2H, –CH2–), 6.94–6.99 (m, 2H, ArH), 7.15
(t, 2H, ArH, J ¼ 7.05), 7.26–7.40 (m, 10H, ArH). 13C NMR (75 MHz,
CDCl3): d 21.76, 44.95, 49.26, 50.59, 57.26, 60.42, 113.30, 117.01,
120.76, 127.40, 127.73, 128.92, 128.98, 138.80, 141.61, 151.19,
176.05, 179.45. ESI-MS: 440.08 (C28H29N3O2 [MþH]þ). Anal. calcd.
for C28H29N3O2 (439.56): C: 76.51, H: 6.65, N: 9.56. Found C: 76.65,
H: 6.67, N: 9.54.
N-[{4-(3-Chlorophenyl)-piperazin-1-yl}-methyl]-3,3-
diphenyl-pyrrolidine-2,5-dione (11)
White solid. Yield: 74%; mp 105–1078C; TLC: Rf ¼ 0.76 (S1);
1H NMR (300 MHz, CDCl3) d: 2.71 (t, 4H, piperazine,
J ¼ 5.0 Hz), 3.13 (t, 4H, piperazine, J ¼ 5.0 Hz), 3.49 (s, 2H,
imide), 4.59 (s, 2H, –CH2–), 6.71–6.81 (m, 3H, ArH), 7.14 (t, 1H,
ArH, J ¼ 7.47 Hz), 7.25–7.37 (m, 10H, ArH). 13C NMR (75 MHz,
CDCl3) d: 44.95, 48.69, 50.31, 57.29, 60.31, 113.99, 115.84, 119.36,
127.39, 127.78, 128.92, 130.04, 134.94, 141.59, 152.12, 176.05,
179.49. ESI-MS: 460.11. (C27H26N3O2Cl [MþH]þ). Anal. calcd. for
C27H26N3O2Cl (459.98): C: 70.50, H: 5.70, N: 9.14. Found C: 70.58,
H: 5.68, N: 9.13.
N-[{4-(3-Methylphenyl)-piperazin-1-yl}-methyl]-3,3-
diphenyl-pyrrolidine-2,5-dione (16)
White solid. Yield: 72%; mp 139–1418C; TLC: Rf ¼ 0.82 (S1);
1H NMR (300 MHz, CDCl3) d: 2.31 (s, 3H, 3-CH3), 2.74 (t, 4H,
piperazine, J ¼ 4.88 Hz), 3.13 (t, 4H, piperazine, J ¼ 5.0 Hz),
3.49 (s, 2H, imide), 4.61 (s, 2H, –CH2–), 6.67–6.71 (m, 3H, ArH),
7.11–7.17 (m, 1H, ArH), 7.26–7.38 (m, 10H, ArH). 13C NMR
(75 MHz, CDCl3): d 21.78, 44.96, 49.27, 50.61, 57.29, 60.44,
113.33, 117.02, 120.77, 127.41, 127.75, 128.91, 128.97, 138.82,
141.64, 151.22, 176.11, 179.49. ESI-MS: 440.10 (C28H29N3O2
[MþH]þ). Anal. calcd. for C28H29N3O2 (439.56): C: 76.51, H: 6.65,
N: 9.56. Found C: 76.55, H: 6.63, N: 9.54.
N-[{4-(4-Chlorophenyl)-piperazin-1-yl}-methyl]-3,3-
diphenyl-pyrrolidine-2,5-dione (12)
White solid. Yield: 77%; mp 154–1558C; TLC: Rf ¼ 0.73 (S1);
1H NMR (300 MHz, CDCl3) d: 2.73 (t, 4H, piperazine,
J ¼ 5.0 Hz), 3.01 (t, 4H, piperazine, J ¼ 5.0 Hz), 3.51 (s, 2H,
imide), 4.59 (s, 2H, –CH2–), 6.78 (d, 2H, ArH, J ¼ 8.97), 7.18
(d, 2H, ArH, J ¼ 9.23), 7.21–7.37 (m, 10H, ArH). 13C NMR
(75 MHz, CDCl3) d: 44.96, 48.70, 50.34, 57.28, 60.34, 113.90,
115.85, 119.36, 127.40, 127.77, 128.91, 130.14, 134.90, 141.61,
152.14, 176.08, 179.51. ESI-MS: 460.11. (C27H26N3O2Cl [MþH]þ).
Anal. calcd. for C27H26N3O2Cl (459.98): C: 70.50, H: 5.70, N: 9.14.
Found C: 70.28, H: 5.71, N: 9.15.
N-[{4-(2-Methoxyphenyl)-piperazin-1-yl}-methyl]-3,3-
diphenyl-pyrrolidine-2,5-dione (17)
White solid. Yield: 66%; mp 123–1258C; TLC: Rf ¼ 0.70 (S1);
1H NMR (300 MHz, CDCl3) d: 2.82 (t, 4H, piperazine,
J ¼ 4.75 Hz), 3.03–3.04 (m, 4H, piperazine), 3.49 (s, 2H, imide),
3.82 (s, 3H, –OCH3), 4.61 (s, 2H, –CH2–), 6.83–7.02 (m, 4H, ArH),
7.26–7.38 (m, 10H, ArH). 13C NMR (75 MHz, CDCl3) d: 24.23,
44.97, 49.29, 50.63, 57.30, 60.46, 113.35, 117.04, 120.79,
127.43, 127.74, 128.92, 128.98, 138.83, 141.65, 151.24, 176.12,
179.50. ESI-MS: 456.18 (C28H29N3O3 [MþH]þ). Anal. calcd. for
C28H29N3O3 (455.56): C: 73.82, H: 6.42, N: 9.22. Found C: 73.68,
H: 6.39, N: 9.25.
N-[{4-(3,4-Dichlorophenyl)-piperazin-1-yl}-methyl]-3,3-
diphenyl-pyrrolidine-2,5-dione (13)
White solid. Yield: 65%; mp 143–1458C; TLC: Rf ¼ 0.68 (S1);
1H NMR (CDCl3) d: 2.70 (t, 4H, piperazine, J ¼ 5.0 Hz), 3.11
(t, 4H, piperazine, J ¼ 5.0 Hz), 3.49 (s, 2H, imide), 4.59 (s, 2H,
–CH2–), 6.66–6.70 (dd, 1H, ArH, J ¼ 2.82, Hz, J ¼ 8.97 Hz),
6.89 (d, 1H, ArH, J ¼ 2.82 Hz), 7.23 (s, 1H, ArH), 7.25–7.37
(m, 10H, ArH). 13C NMR (75 MHz, CDCl3) d: 44.98, 48.72,
50.36, 57.30, 60.36, 113.92, 115.87, 119.38, 127.42, 127.80,
128.92, 130.16, 134.91, 141.63, 152.15, 176.12, 179.54. ESI-MS:
494.05 (C27H25N3O2Cl2 [MþH]þ). Anal. calcd. for C27H25N3O2Cl2
(494.43): C: 65.59, H: 5.10, N: 8.50. Found C: 65.95, H: 5.08,
N: 8.48.
N-[{4-(3-Methoxyphenyl)-piperazin-1-yl}-methyl]-3,3-
diphenyl-pyrrolidine-2,5-dione (18)
White solid. Yield: 60%; mp 178–1808C; TLC: Rf ¼ 0.78 (S1);
1H NMR (300 MHz, CDCl3) d: 2.74 (t, 4H, piperazine,
J ¼ 4.87 Hz), 3.13 (t, 4H, piperazine, J ¼ 4.87 Hz), 3.49 (s, 2H,
imide), 3.78 (s, 3H, –OCH3), 4.59 (s, 2H, –CH2–), 6.40–6.43
(m, 2H, ArH), 6.48–6.51 (m, 1H, ArH), 7.13–7.18 (m, 1H, ArH),
7.26–7.38 (m, 10H, ArH). 13C NMR (75 MHz, CDCl3) d: 24.26,
44.98, 49.28, 50.62, 57.30, 60.40, 113.33, 117.03, 120.78,
127.41, 127.75, 128.91, 128.99, 138.84, 141.67, 151.24, 176.13,
179.49. ESI-MS: 456.12 (C28H29N3O3 [MþH]þ). Anal. calcd.
for C28H29N3O3 (455.56): C: 73.82, H: 6.42, N: 9.22. Found C:
73.75, H: 6.39, N: 9.19.
N-[{4-(3-Trifluoromethylphenyl)-piperazin-1-yl}-methyl]-
3,3-diphenyl-pyrrolidine-2,5-dione (14)
White solid. Yield: 60%; mp 78–808C; TLC: Rf ¼ 0.76 (S1); 1H NMR
(300 MHz, CDCl3) d: 2.73 (t, 4H, piperazine, J ¼ 4.87 Hz), 3.17
(t, 4H, piperazine, J ¼ 4.87 Hz), 3.49 (s, 2H, imide), 4.60 (s, 2H,
–CH2–), 6.98–7.08 (m, 4H, ArH), 7.24–7.37 (m, 10H, ArH). 13C NMR
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2013, 346, 71–82
New Pyrrolidine-2,5-diones as Potential Anticonvulsants
79
1.32 (s, 3H, CH3), 1.54–1.81 (m, 2H, –CH2–), 2.46 (d, 1H, imide,
J ¼ 18.21 Hz), 2.70 (d, 1H, imide, J ¼ 18.21 Hz), 2.75 (brs, 4H,
piperazine), 3.17 (brs, 4H, piperazine), 4.52 (s, 2H, –CH2–), 6.74–
6.86 (m, 3H, ArH), 7.12–7.26 (m, 1H, ArH). 13C NMR (75 MHz,
CDCl3) d: 8.93, 24.29, 31.03, 40.34, 44.38, 48.73, 50.41,
59.69, 114.04, 115.89, 119.45, 130.0, 134.91, 152.22, 177.10,
193.89. ESI-MS: 350.12 (C18H24ClN3O2 [MþH]þ). Anal. calcd. for
C18H24N3O2Cl (349.87): C: 61.79, H: 6.91, N: 12.01. Found C: 62.08,
H: 6.92, N: 11.99.
N-[(4-Phenyl-piperazin-1-yl)-methyl]-3-ethyl-3-methyl-
pyrrolidine-2,5-dione (19)
White solid. Yield: 70%; mp 65–678C; TLC: Rf ¼ 0.60 (S1); 1H NMR
(300 MHz, CDCl3) d: 0.90 (t, 3H, CH3, J ¼ 7.44 Hz), 1.33 (s, 3H,
CH3), 1.54–1.82 (m, 2H, –CH2–), 2.46 (d, 1H, imide, J ¼ 18.47 Hz),
2.67 (d, 1H, imide, J ¼ 18.46 Hz), 2.79 (brs, 4H, piperazine), 3.18
(brs, 4H, piperazine), 4.53 (s, 2H, –CH2–), 6.91–6.94 (m, 3H, ArH),
7.24–7.29 (m, 2H, ArH). 13C NMR (75 MHz, CDCl3) d: 8.91, 24.25,
31.04, 40.36, 44.36, 49.10, 50.57, 55.15, 59.74, 102.58, 104.59,
108.93, 129.76, 152.58, 160.54, 177.10, 183.89. ESI-MS: 316.09
(C18H25N3O2 [MþH]þ). Anal. calcd. for C18H25N3O2 (315.42):
C: 68.54, H: 7.99, N: 13.32. Found C: 68.29, H: 8.02, N: 13.38.
N-[{4-(4-Chlorophenyl)-piperazin-1-yl}-methyl]-3-ethyl-3-
methyl-pyrrolidine-2,5-dione (24)
White solid. Yield: 68%; mp 154–1558C; TLC: Rf ¼ 0.58 (S1);
1H NMR (300 MHz, CDCl3) d: 0.90 (t, 3H, CH3, J ¼ 7.56 Hz),
1.33 (s, 3H, CH3), 1.54–1.81 (m, 2H, –CH2–), 2.46 (d, 1H, imide,
J ¼ 18.21 Hz), 2.74 (d, 1H, imide, J ¼ 18.46 Hz), 2.78 (brs, 4H,
piperazine), 3.13 (brs, 4H, piperazine), 4.52 (s, 2H, –CH2–), 6.83–
6.85 (m, 2H, ArH), 7.12–7.26 (m, 2H, ArH), C18H24N3O2Cl (349.87).
13C NMR (75 MHz, CDCl3) d: 8.94, 24.28, 31.01, 40.36, 44.40, 48.71,
50.40, 59.67, 114.02, 115.87, 119.44, 130.02, 134.89, 152.20,
177.11, 193.88. ESI-MS: 350.11 (C18H24ClN3O2 [MþH]þ). Anal.
calcd. for C18H24N3O2Cl (349.87): C: 61.79, H: 6.91, N: 12.01.
Found C: 61.81, H: 6.92, N: 12.03.
N-[{4-(2-Fluorophenyl)-piperazin-1-yl}-methyl]-3-ethyl-3-
methyl-pyrrolidine-2,5-dione monohydrochloride (20)
White solid. Yield: 78%; mp 165–1688C; TLC: Rf ¼ 0.80 (S2); 1H NMR
(300 MHz, DMSO): d 0.78 (t, 3H, CH3, J ¼ 7.55 Hz), 1.24 (s, 3H, CH3),
1.41–1.89 (m, 2H, –CH2–), 2.39 (d, 1H, imide, J ¼ 18.21 Hz), 2.47–2.61
(m, 2H, piperazine), 2.74 (d, 1H, imide, J ¼ 18.47 Hz), 3.46 (brs, 2H,
piperazine), 3.69 (brs, 4H, piperazine), 4.63 (s, 2H, –CH2–), 6.96–7.20
(m, 4H, ArH), 10.66 (brs, 1H, þNH). 13C NMR (75 MHz, DMSO) d: 8.94,
24.30, 31.10, 40.38, 44.35, 48.69, 50.40, 59.68, 114.05, 115.85,
119.43, 130.05, 134.89, 152.26, 177.12, 193.88. ESI-MS: 334.15
(C18H24N3O2F [MþH]þ). Anal. calcd. for C18H24N3O2F ꢂ HCl
(369.87): C: 58.45, H: 6.54, N: 11.36. Found C: 58.25, H: 6.57, N: 11.38.
N-[{4-(3,4-Dichlorophenyl)-piperazin-1-yl}-methyl]-3-ethyl-
3-methyl-pyrrolidine-2,5-dione (25)
White solid. Yield: 75%; mp 82–848C; TLC: Rf ¼ 0.64 (S1); 1H NMR
(300 MHz, CDCl3) d: 0.90 (t, 3H, CH3, J ¼ 7.44 Hz), 1.32 (s, 3H,
CH3), 1.53–1.79 (m, 2H, –CH2–), 2.45 (d, 1H, imide, J ¼ 18.21 Hz),
2.67 (d, 1H, imide, J ¼ 18.21 Hz), 2.72–2.74 (m, 4H, piperazine),
3.10–3.19 (m, 4H, piperazine), 4.51 (s, 2H, –CH2–), 6.68–6.77 (m,
1H, ArH), 6.91–6.97 (m, 1H, ArH), 7.24–7.28 (m, 1H, ArH). 13C NMR
(75 MHz, CDCl3) d: 8.95, 24.29, 31.02, 40.38, 44.43, 48.72, 50.43,
59.69, 114.05, 115.89, 119.47, 130.07, 134.91, 152.22, 177.12,
193.89. ESI-MS: 384.10 (C18H23N3O2Cl2 [MþH]þ). Anal. calcd.
for C18H23N3O2Cl2 (384.31): C: 56.26, H: 6.03, N: 10.94. Found
C: 56.19, H: 6.04, N: 10.99.
N-[{4-(4-Fluorophenyl)-piperazin-1-yl}-methyl]-3-ethyl-3-
methyl-pyrrolidine-2,5-dione monohydrochloride (21)
White solid. Yield: 58%; mp 152–1558C; TLC: Rf ¼ 0.72 (S2);
1H NMR (300 MHz, DMSO): d 0.94 (t, 3H, CH3, J ¼ 7.44 Hz),
1.41 (s, 3H, CH3), 1.62–1.86 (m, 2H, –CH2–), 2.59 (d, 1H, imide,
J ¼ 18.47 Hz), 2.74 (d, 1H, imide, J ¼ 18.47 Hz), 3.37 (brs, 4H,
piperazine), 3.53 (brs, 4H, piperazine), 4.63 (s, 2H, –CH2–), 7.12 (t,
2H, ArH, J ¼ 8.47Hz), 7.58 (brs, 2H, ArH), 10.68 (brs, 1H, þNH).
13C NMR (75 MHz, DMSO) d: 8.93, 24.27, 31.05, 40.38, 44.39,
48.69, 50.41, 59.66, 114.03, 115.88, 119.43, 130.03, 134.88,
152.21, 177.10, 193.81. 19F NMR (282 MHz, CDCl3) d: ꢃ124.18
(s, 1F). ESI-MS: 334.13 (C18H24N3O2F [MþH]þ). Anal. calcd.
for C18H24N3O2F ꢂ HCl (369.87): C: 58.45, H: 6.54, N: 11.36.
Found C: 58.55, H: 6.54, N: 11.35.
N-[{4-(3-Trifluoromethylphenyl)-piperazin-1-yl}-methyl]-3-
ethyl-3-methyl-pyrrolidine-2,5-dione (26)
White solid. Yield: 61%; mp 78–808C; TLC: Rf ¼ 0.63 (S1); 1H NMR
(300 MHz, CDCl3) d: 0.89 (t, 3H, CH3, J ¼ 7.44 Hz), 1.33 (s, 3H,
CH3), 1.54–1.81 (m, 2H, –CH2–), 2.46 (d, 1H, imide, J ¼ 18.47 Hz),
2.68 (d, 1H, imide, J ¼ 18.47 Hz), 2.74 (brs, 4H, piperazine), 3.25
(brs, 4H, piperazine), 4.53 (s, 2H, –CH2–), 7.02–7.08 (m, 3H, ArH),
7.26–7.36 (m, 1H, ArH). 13C NMR (75 MHz, CDCl3) d: 8.91, 24.29,
31.02, 40.33, 44.38, 48.73, 50.41, 59.68, 112.29, 112.35, 115.96,
116.01, 118.85, 129.53, 151.26, 177.10, 183.88. ESI-MS: 384.10
(C19H24N3O2F3 [MþH]þ). Anal. calcd. for C19H24N3O2F3 (383.43):
C: 59.52, H: 6.31, N: 10.96. Found C: 59.63, H: 6.32, N: 11.00.
N-[{4-(2-Chlorophenyl)-piperazin-1-yl}-methyl]-3-ethyl-3-
methyl-pyrrolidine-2,5-dione monohydrochloride (22)
White solid. Yield: 58%; mp 147–1498C; TLC: Rf ¼ 0.77 (S2);
1H NMR (300 MHz, DMSO) d: 0.84 (t, 3H, CH3, J ¼ 7.18 Hz),
1.24 (s, 3H, CH3), 1.41–1.89 (m, 2H, –CH2–), 2.39 (d, 1H, imide,
J ¼ 18.21 Hz), 2.75 (d, 1H, imide, J ¼ 18.46 Hz), 3.16 (brs, 4H,
piperazine), 3.44 (brs, 4H, piperazine), 4.62 (s, 2H, –CH2–), 7.03–
7.21 (m, 2H, ArH), 7.27–7.35 (m, 1H, ArH), 7.39–7.46 (m, 1H, ArH),
10.54 (brs, 1H, þNH). 13C NMR (75 MHz, DMSO) d: 8.93,
24.29, 31.0, 40.35, 44.38, 48.70, 50.41, 59.66, 114.03, 115.86,
119.42, 130.01, 134.88, 152.21, 177.10, 193.87. ESI-MS: 350.10
(C18H24ClN3O2 [MþH]þ). Anal. calcd. for C18H24ClN3O2 ꢂ HCl
(386.32): C: 51.14, H: 5.72, N: 9.94. Found C: 51.18, H: 5.70, N: 9.92.
N-[{4-(2-Methylphenyl)-piperazin-1-yl}-methyl]-3-ethyl-3-
methyl-pyrrolidine-2,5-dione (27)
White solid. Yield: 64%; mp 73–758C; TLC: Rf ¼ 0.67 (S1); 1H NMR
(300 MHz, CDCl3): d 0.90 (t, 3H, CH3, J ¼ 7.44 Hz), 1.23 (s, 3H,
CH3), 1.53–1.79 (m, 2H, –CH2–), 2.18 (s, 3H, CH3), 2.45 (d, 1H,
imide, J ¼ 18.21 Hz), 2.67 (d, 1H, imide, J ¼ 18.21 Hz), 2.72–2.74
(m, 4H, piperazine), 3.10–3.19 (m, 4H, piperazine), 4.51 (s, 2H,
–CH2–), 6.91–6.97 (m, 2H, ArH), 7.24–7.28 (m, 2H, ArH). 13C NMR
(75 MHz, CDCl3) d: 8.90, 21.25, 24.24, 31.06, 40.37, 44.37, 49.28,
N-[{4-(3-Chlorophenyl)-piperazin-1-yl}-methyl]-3-ethyl-3-
methyl-pyrrolidine-2,5-dione (23)
White solid. Yield: 78%; mp 105–1078C; TLC: Rf ¼ 0.65 (S1);
1H NMR (300 MHz, CDCl3) d: 0.90 (t, 3H, CH3, J ¼ 7.44 Hz),
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80
J. Obniska et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 71–82
19.29, 50.65, 51.57, 59.75, 113.33, 117.05, 120.82, 128.93, 138.77,
151.24, 177.14, 183.89. ESI-MS: 330.17 (C19H27N3O2 [MþH]þ).
Anal. calcd. for C19H27N3O2 (329.45): C: 69.27, H: 8.26, N: 12.75.
Found C: 69.51, H: 8.25, N: 12.71.
anticonvulsant activity and neurotoxicity assessment at 0.5
and 4 h after administration. The results are presented in
Table 1.
Selected derivatives (7, 8, 11, 12, 14, 23, and 26) were admin-
istrated orally into rats using four animals at a fixed dose of
30 mg/kg (MES test). The motor impairment was studied in
parallel. Rats were tested at five time periods ranging from
15 min to 4 h post substance administration. The results are
shown in Table 2.
N-[{4-(3-Methylphenyl)-piperazin-1-yl}-methyl]-3-ethyl-3-
methyl-pyrrolidine-2,5-dione (28)
White solid. Yield: 72%; mp 64–668C; TLC: Rf ¼ 0.64 (S1); 1H NMR
(CDCl3): d 0.85 (t, 3H, CH3, J ¼ 10.0 Hz), 1.23 (s, 3H, CH3), 1.47–
1.67 (m, 2H, –CH2–), 2.18 (s, 3H, CH3), 2.57 (brs, 1H, imide), 2.61–
2.63 (m, 4H, piperazine), 2.69 (brs, 1H, imide), 2.75–2.80 (m, 4H,
piperazine), 4.33 (s, 2H, –CH2–), 6.90–6.99 (m, 2H, ArH), 7.09–7.14
(m, 2H, ArH). 13C NMR (75 MHz, CDCl3) d: 8.91, 21.24, 24.26,
31.05, 40.38, 44.36, 49.29, 19.28, 50.66, 51.59, 59.77, 113.32,
117.02, 120.80, 128.94, 138.78, 151.25, 177.12, 183.90. ESI-MS:
330.16 (C19H27N3O2 [MþH]þ). Anal. calcd. for C19H27N3O2
(329.45): C: 69.27, H: 8.26, N: 12.75. Found C: 69.18, H: 8.23,
N: 12.77.
Maximal electroshock test (MES)
The MES is a model for generalized tonic–clonic seizures and
provides an indication of a compound’s ability to prevent seizure
spread when all neuronal circuits in the brain are maximally
active. These seizures are highly reproducible and are electro-
physiologically consistent with human seizures. For all tests
based on MES convulsions, 60 Hz of alternating current
(50 mA in mice and 150 mA in rats) is delivered for 2 s by corneal
electrodes which have been primed with an electrolyte solution
containing an anesthetic agent (0.5% tetracaine HCl). An animal
is considered ‘‘protected’’ from MES-induced seizures upon
abolition of the hindlimb tonic extensor component of the
seizure [37, 38].
N-[{4-(2-Methoxyphenyl)-piperazin-1-yl}-methyl]-3-ethyl-
3-methyl-pyrrolidine-2,5-dione monohydrochloride (29)
White solid. Yield: 66%; mp 160–1638C; TLC: Rf ¼ 0.76 (S2);
1H NMR (300 MHz, DMSO): d 0.76–0.86 (m, 3H, CH3), 1.26
(s, 3H, CH3), 1.41–1.70 (m, 2H, –CH2–), 2.36–3.17 (m, 6H, 2H,
imide, 4H piperazine), 3.28–3.53 (m, 4H, piperazine), 3.79
(s, 3H, OCH3), 4.67 (s, 2H, –CH2–), 6.86–7.09 (m, 4H, ArH),
11.09 (s, 1H, 1H, þNH). 13C NMR (75 MHz, DMSO) d: 8.94,
25.22, 28.26, 32.14, 40.65, 44.55, 49.42, 50.30, 50.65, 51.58,
59.78, 113.31, 117.12, 121.10, 128.96, 138.85, 151.34, 177.29,
184.07. ESI-MS: 346.08 (C19H27N3O2 [MþH]þ). Anal. calcd.
for C19H27N3O2 ꢂ HCl (381.90): C: 59.76, H: 7.13, N: 11.00.
Found C: 59.59, H: 7.14, N: 10.98.
Subcutaneous pentylenetetrazole seizure test (scPTZ)
Subcutaneous injection of the convulsant pentylenetetrazole
(a GABAA receptor antagonist) produces clonic seizures in
laboratory animals. The scPTZ test detects the ability of a test
compound to raise the seizure threshold of an animal and thus
protect it from exhibiting a clonic seizure in response to a
normally convulsant dose of pentylenetetrazole. Animals are
pretreated with various doses of the test compound given by
i.p. injection or through oral dosing. At various times after
dosing with the test compound, the dose of pentylenetetrazole
which will induce convulsions in 97% of animals (CD97:
85 mg/kg in mice or 56.4 mg/kg in rats) is injected into a
loose fold of skin in the midline of the neck. The animals are
placed in isolation cages to minimize stress and observed
for the next 30 min for the presence or absence of a seizure.
An episode of clonic spasms, approximately 3–5 s, of the
fore and/or hindlimbs, jaws, or vibrissae is taken as the
endpoint. Animals which do not meet this criterion are con-
sidered protected [39].
N-[{4-(3-Methoxyphenyl)-piperazin-1-yl}-methyl]-3-ethyl-
3-methyl-pyrrolidine-2,5-dione (30)
White solid. Yield: 65%; mp 103–1058C; TLC: Rf ¼ 0.54 (S1);
1H NMR (300 MHz, CDCl3): 1H NMR (CDCl3): d 0.90 (t, 3H, CH3,
J ¼ 7.44 Hz), 1.32 (s, 3H, CH3), 1.58–1.78 (m, 2H, –CH2–), 2.44
(d, 1H, imide, J ¼ 18.21 Hz), 2.66 (d, 1H, imide, J ¼ 18.21 Hz),
2.72–2.75 (m, 4H, piperazine), 3.13–3.16 (m, 4H, piperazine), 3.77
(s, 3H, OCH3), 4.51 (s, 2H, –CH2–), 6.39–6.53 (m, 3H, ArH), 7.13–
7.18 (m, 1H, ArH). 13C NMR (75 MHz, CDCl3) d: 8.95, 25.24, 28.28,
32.15, 40.68, 44.58, 49.45, 50.31, 50.66, 51.61, 59.80, 113.36,
117.13, 121.12, 128.98, 138.88, 151.35, 177.31, 184.11. ESI-MS:
346.12 (C19H27N3O2 [MþH]þ). Anal. calcd. for C19H27N3O2
(345.44): C: 66.06, H: 7.88, N: 12.16. Found C: 66.10, H: 7.78,
N: 12.21.
Neurological toxicity (NT)
Neurological toxicity induced by a compound was detected in
mice or rats using the standardized rotorod test [40]. When an
untreated control mouse is placed on a rod that rotates at a speed
of 6 rpm, the animal can maintain its equilibrium for long
periods of time. The animal is considered neurotoxic if it falls
off this rotating rod three times during a 1-min period. In rats,
minimal motor impairment is indicated by ataxia, which is
manifested by an abnormal, uncoordinated gait. Rats used for
evaluating toxicity are examined before the test drug is admin-
istered, since individual animals may have peculiarities in gait,
equilibrium, placing response, etc., which might be attributed
erroneously to the test substance. In addition to MMI, animals
may exhibit a circular or zigzag gait, abnormal body posture
and spread of the legs, tremors, hyperactivity, lack of exploratory
behavior, somnolence, stupor, catalepsy, loss of placing response,
and changes in muscle tone.
In vivo studies
Compounds 7–30 were pharmacologically pre-evaluated within
the Antiepileptic Drug Development (ADD) Program, realized
in NINDS/NIH, Rockville, USA, using procedures described else-
where [35, 36].
Male albino mice (CF-1 strain) and male albino rats (Sprague-
Dawley) were used as experimental animals. Groups of eight
mice or four rats were employed. 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
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Arch. Pharm. Chem. Life Sci. 2013, 346, 71–82
New Pyrrolidine-2,5-diones as Potential Anticonvulsants
81
˚
(l ¼ 0.71073 A) at room temperature. The structures were solved
by direct method and refined with SHELXTL [42]. E-maps pro-
vided positions for all non-H-atoms. The full-matrix least-squares
refinement was carried out on F2’s using anisotropic tempera-
ture factors for all non-H-atoms. All C-bound H atoms were placed
in idealized locations and refined using a riding model, with
Quantification studies
The quantitative determination of ED50 and TD50 values was
performed at previously estimated TPE after p.o. administration
into rats. Groups of 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 impair-
ment. From the plot of the data obtained, the respective ED50 and
TD50 values, 95% confidence intervals, slope of the regression
line, and standard error of the slope were calculated by means of
a computer program written at NINDS/NIH. The results are
shown in Table 3.
˚
C–H ¼ 0.93 A and Uiso(H) ¼ 1.2Ueq(C).
Crystal data for 8: C27H26N3O2F, M ¼ 443.51, monoclinic,
˚
˚
space
group
C2/c,
a ¼ 17.8890(5) A,
b ¼ 6.3931(2) A,
3
˚
˚
c ¼ 40.1879(11) A, b ¼ 94.293(1)8, V ¼ 4583.2(2) A , Z ¼ 8,
Dx ¼ 1.286 g/cm3, T ¼ 293 K, m ¼ 0.088 mmꢃ1, l ¼ 0.71073 A,
˚
data/parameters ¼ 3950/299; final R1 ¼ 0.0396.
Crystal data for 10: C27H26N3O2Cl, M ¼ 459.96, monoclinic,
The Pilocarpine Induced Status Prevention (PISP) model
˚
˚
space
group
C2/c,
a ¼ 18.0187(5) A,
b ¼ 6.5138(2) A,
3
˚
˚
c ¼ 39.9615(11) A, b ¼ 92.690(4)8, V ¼ 4685.1(2) A , Z ¼ 8,
˚
A – Acute toxicity
Dx ¼ 1.308 g/cm3, T ¼ 293 K, m ¼ 0.193 mmꢃ1, l ¼ 0.71073 A,
data/parameters ¼ 4163/299; final R1 ¼ 0.0501.
Acute motor impairment will be assessed following the intra-
peritoneal (i.p.) administration of doses starting at 100 and
300 mg/kg of the test substance. Individual Sprague Dawley rats
are evaluated for acute toxicity over several time points follow-
ing administration of test drugs (unless there is previously
obtained i.p. toxicity data available). The results obtained from
this initial study determine whether any dose adjustments may
be required. The behavior of the animals is observed closely and
recorded over a 4-h period. Routinely, a minimum number of
four (4) rats, two per dose, will be employed in this acute screen.
Crystal data for 11: C27H26N3O2Cl, M ¼ 459.96, monoclinic,
˚
˚
space
group
P21/n,
a ¼ 17.713(2) A,
b ¼ 6.2223(5) A,
3
˚
˚
c ¼ 22.0686(17) A, b ¼ 103.330(9)8, V ¼ 2366.8(4) A , Z ¼ 4,
Dx ¼ 1.291 g/cm3, T ¼ 293 K, m ¼ 0.191 mmꢃ1, l ¼ 0.71073 A,
˚
data/parameters ¼ 4199/299; final R1 ¼ 0.0671.
The authors wish to thank Dr. James Stables for providing them with
pharmacological data through the Antiepileptic Drug Development
Program (Epilepsy Branch, National Institute of Neurological Disorders
and Stroke, National Institute of Health, Rockville, MD, USA), and Prof.
Katarzyna Kiec´-Kononowicz (Department of Technology and Biotechnology
of Drugs, Jagiellonian University Medical College, Krako´w, Poland), the
coordinator of the ADD Project from the Polish site. We are pleased to
acknowledge the generous financial support of this work by the grant of the
Minister Committee for Scientific Research, Poland (grant no N405 298536).
B – Status intervention
To determine if the test substance can halt the acute pilocarpine-
induced status an initial qualitative efficacy screen is performed.
A challenge dose of pilocarpine (50 mg/kg) is administered i.p.
and animals are observed until the first convulsive (e.g., Stage 3,
4, or 5) seizure (time zero). The seizure severity is determined
using the well-established Racine scale [41], as follows: (1) immo-
bility, eye closure, twitching of vibrissae, sniffing, and facial
clonus; (2) head nodding associated with more severe facial
clonus; (3) clonus of one of the fore limbs; (4) rearing often
accomplished by bilateral forelimb clonus and (5) all of the above
plus loss of balance and falling, accomplished by generalized
clonic seizures. At this point a minimally toxic dose of the
candidate drug is administered to a group of 8 male albino
Sprague Dawley rats (150–180 mg) via the i.p. route of adminis-
tration. Efficacy is defined by the ability of an investigational
drug to halt the further expression of pilocarpine induced con-
vulsive seizures (e.g., Stage 3, 4, or 5). The anticonvulsant activity
of compounds 11, 14, 23, and 24 was assessed at time zero,
namely the time from the first stage III seizures (Test 71). The
outcome measures are determination of ‘‘protection’’ or ‘‘no
protection’’.
The authors have declared no conflict of interest.
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