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

Article abstract of DOI:10.1002/ardp.201200092

A library of 21 new N-Mannich bases of 3,3-diphenyl- (5a-g), 3-methyl-3-phenyl- (6a-g), and 3-ethyl-3-methylpyrrolidine-2,5-diones (7a-g) were synthesized and evaluated for their anticonvulsant activity in the maximum electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizure tests after intraperitoneal injection into mice. The acute neurological toxicity was determined applying the rotarod screen. The results in mice showed that 13 compounds were effective in the MES or/and scPTZ screen. From these, seven molecules were tested in the MES seizures after oral administration in rats. The quantitative studies showed that N-[{4-(2-hydroxyethyl)-piperazin-1-yl}-methyl] -3-methyl-3-phenylpyrrolidine-2,5-dione (6c) and N-[(4-benzylpiperidin-1-yl)- methyl]-3-methyl-3-phenylpyrrolidine-2,5-dione (6f) revealed higher protection in the MES and scPTZ tests than valproic acid or ethosuximide which were used as reference antiepileptic drugs. Four compounds (5c, 6c, 6e, 6f) showed high effectiveness in the 6-Hz psychomotor seizure model of partial and therapy resistant epilepsy.

Full text of DOI:10.1002/ardp.201200092

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
1
Full Paper
Synthesis and Anticonvulsant Properties of New Mannich
Bases Derived from 3,3-Disubstituted Pyrrolidine-2,5-diones.
Part IV
´
Jolanta Obniska, Iwona Chlebek, and Krzysztof Kaminski
´
Department of Medicinal Chemistry, Jagiellonian University Medical College, Krakow, Poland
A library of 21 new N-Mannich bases of 3,3-diphenyl- (5a–g), 3-methyl-3-phenyl- (6a–g), and 3-ethyl-3-
methylpyrrolidine-2,5-diones (7a–g) were synthesized and evaluated for their anticonvulsant activity
in the maximum electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizure tests after
intraperitoneal injection into mice. The acute neurological toxicity was determined applying the rotarod
screen. The results in mice showed that 13 compounds were effective in the MES or/and scPTZ screen. From
these, seven molecules were tested in the MES seizures after oral administration in rats. The quantitative
studies showed that N-[{4-(2-hydroxyethyl)-piperazin-1-yl}-methyl]-3-methyl-3-phenylpyrrolidine-2,5-dione
(6c) and N-[(4-benzylpiperidin-1-yl)-methyl]-3-methyl-3-phenylpyrrolidine-2,5-dione (6f) revealed higher
protection in the MES and scPTZ tests than valproic acid or ethosuximide which were used as
reference antiepileptic drugs. Four compounds (5c, 6c, 6e, 6f) showed high effectiveness in the 6-Hz
psychomotor seizure model of partial and therapy resistant epilepsy.
Keywords: Anticonvulsant activity / In vivo studies / N-Mannich bases / Pyrrolidine-2,5-diones / Succinimides
Received: March 8, 2012; Revised: April 18, 2012; Accepted: April 25, 2012
DOI 10.1002/ardp.201200092
Introduction
the majority antiepileptic drugs makes it difficult to use
rational methodologies of discovery. Therefore, conceptually,
there are two different methods of obtaining new anticon-
vulsants namely ligand-based and screening approaches [4].
The ligand-based approach relies on the use of different
pharmacophores that were established through the analysis
of structural characteristics of clinically effective AEDs as
well as other anticonvulsant active compounds. The seren-
dipitous approach involves a comprehensive screening proc-
ess that utilizes rodent models. Traditionally, most screening
programs employ mice and rats to assess efficacy against
either electrically (e.g., maximal electroshock, MES) or
chemically (e.g., pentylenetetrazol, bicuculline, or picro-
toxin) induced seizures [5, 6]. The number of new AEDs
currently available, 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 [7]. One example supporting this hypothesis is
provided by levetiracetam, which has demonstrated efficacy
in refractory human partial epilepsy [5, 8]. It was found to be
inactive against MES and PTZ seizures even at high doses,
whereas it showed high effectiveness in the 6-Hz psychomo-
Epilepsy is a chronic disorder characterized by recurrent
unprovoked seizures which are defined as a discrete clinical
event arising from transient, hypersynchronous, and abnor-
mal neuronal behavior. Epilepsy then is not a disease but
rather a syndrome arising from a group of non-specific,
dysfunctional events in the brain. The treatment of patients
with epilepsy has been a long-term and consistent adminis-
tration of anticonvulsant drugs. Unfortunately current drugs
are ineffective for approximately 30% of the patients. Many
continue to have seizure, while others experience disturbing
side effects as drowsiness, dizziness, nausea, and liver
damage [1–3]. Thus, there is a need for more efficacious
and less toxic drugs.
The incomplete information on the pathogenesis of
human epilepsy and the complex mechanism of action of
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: þ48 12 620-54-58
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
tor seizure model of partial epilepsy [9]. Thus it is suggested
that the 6-Hz model might be capable of identifying anti-
seizure agents with a novel spectrum of activity and
unknown mechanism of anticonvulsant action.
The past decades have demonstrated many attempts to
identify the structural features of compounds crucial for
anticonvulsant activity. It is well documented that one of
the important core fragments of anticonvulsants is defined
by a nitrogen heterocyclic system, usually imide or lactam,
and phenyl or alkyl groups attached to the heterocyclic
system [10–12]. This common template is present in the
structures of old, however well-established anticonvulsants
(AEDs), such as ethosuximide and phenytoin, as well as
among the newest drugs, e.g., levetiracetam, brivaracetam,
or seletracetam (Fig. 1) [13, 14].
Figure 2. Structures of active compounds obtained in previous
Taking into consideration the above our researches are
focused on a group of 1,3-substituted pyrrolidine-2,5-diones
as targets for new antiepileptic drugs [15–19]. Many of these
compounds were effective in the MES or/and subcutaneous
pentylenetetrazole (scPTZ) screens that are still recognized as
studies (data in rats after oral administration) [17, 21, 22].
the most popular seizure models in the early stages of the Results and discussion
search for new anticonvulsants [20–22]. The structure–
activity relationship (SAR) analysis showed that the most
active ones were N-[(4-phenylpiperazin-1-yl)-methyl]-3-phenyl-
pyrrolidine-2,5-diones with different electron withdrawing
substituents at both phenyl rings. The structures of chosen,
active compounds obtained in previous studies are shown in
Fig. 2.
Chemistry
The synthesis of compounds 5a–g, 6a–g, and 7a–g was accom-
plished as shown in Scheme 1.
The starting materials 2-methyl-2-phenyl- succinic acid (4)
and 3,3-diphenylpyrrolidine-2,5-dione (5) were prepared
using methods reported previously [23, 24]. The 3-methyl-3-
phenylpyrrolidine-2,5-dione (6) was obtained in the cycliza-
tion reaction of 4 with the 25% ammonia. The final
compounds 5a–g, 6a–g, and 7a–g were synthesized in amino-
alkylation reaction of the imide acidic proton, in the pres-
ence of formaldehyde and appropriate secondary amines
such as 4-substituted piperazines, morpholine, or 4-benzyl-
piperidine. The reaction was carried out in ethanol at
room temperature for ca. 12 h. Compounds 5a–g, 6a–g,
and 7a–g were obtained in yields ranging from 62 to
88%. Their purities were assessed by TLC chromatography.
Compounds 6a–g and 7a–g were synthesized as racemic
mixtures. The chemical structures were confirmed by both
As a continuation of systematic SAR discussion, in the
present work we synthesized a series of 21 new N-Mannich
bases derived from 3,3-diphenyl- (5a–g), 3-methyl-3-phenyl-
(6a–g), and 3-ethyl-3-methyl-(7a–g) pyrrolidine-2,5-diones.
The diverse substitution modes at position-3 of the imide
ring enabled to assess the influence of aromatic or alkyl
substituents on the anticonvulsant activity among the com-
pounds obtained. To continue comprehensive SAR analysis,
at the imide nitrogen atom we introduced through the
methylene spacer various secondary amines such as 4-
benzyl-, 4-pyrimid-2-yl-, 4-(2-hydroxyethyl)-, 4-cyclohexyl-, 4-
methyl-piperazines, 4-benzylpiperidine, or morpholine.
Figure 1. Structures of model AEDs.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
New Pyrrolidine-2,5-diones as Potential Anticonvulsants
3
Scheme 1. Synthetic procedures for target compounds 5a–g, 6a–g, and 7a–g.
spectral and elemental analysis. The detailed physical and
analytical data are listed in the experimental section.
impairment-rotarod screen (NT). The results are shown in
Table 1.
The data obtained revealed that anticonvulsant activity
was closely connected with the presence of at least one
phenyl ring at position-3 of pyrrolidine-2,5-dione (5a–g,
6a–g), whereas all 3-ethyl-3-methyl- analogs (7a–g) were inac-
tive. Except for N-[(4-benzyl-piperazin-1-yl)-methyl]-3-methyl-3-
phenylpyrrolidine-2,5-dione (6a), all other 3-phenyl-3-methyl-
and 3,3-diphenylpyrrolidine-2,5-diones (5a–g, 6b–g) were
effective in the MES test and several of them, namely 6b, 6c,
6e, and 6g, showed protection in the scPTZ screen, additionally.
Compounds that revealed protection in the MES test, indicative
of the ability of the substance to prevent seizure spread, at a
dose of 100 mg/kg included 5a–e, 5g, and 6b–g, whereas
molecule 5f provided anti-MES protection at a dose of
300 mg/kg. Except for compounds 6e and 6g, which were active
only at 0.5 h after i.p. administration, the other derivatives
showed activity at 4 h or in both time intervals (5a, 5c–e, 5g, 6b,
6c, and 6f), indicating quick onset and long duration of the
anticonvulsant activity. Apart from the anti-MES protection,
Anticonvulsant activity
The pre-clinical discovery and development of new chemical
agents for the treatment of epilepsy are based mainly on
the use of predictable animal models. At the present time
there are three in vivo screens used routinely that include the
maximal electroshock seizure (MES), the scPTZ and the kin-
dling model. From these tests the MES and scPTZ screens are
still recognized as the ‘‘gold standards’’ in the early stages of
testing and enabled to detect compounds affording protec-
tion against generalized tonic–clonic seizures and general-
ized absence seizures, respectively [6].
The profile of anticonvulsant activity of compounds 5a–g,
6a–g, and 7a–g was established in the MES and scPTZ tests,
after intraperitoneal (i.p.) injection in mice at doses of 30,
100, and 300 mg/kg. An observation was carried out at two
different time intervals – 0.5 and 4 h. The acute neurological
toxicity (NT) was determined by use of the minimal motor
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J. Obniska et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
Table 1. The results after i.p. administration into mice.
On the basis of data in mice and according to the
Anticonvulsant Screening Project disposition, seven com-
pounds 5a, 5d, 5e, 6b–d, and 6f were selected and examined
for their anticonvulsant activity (MES screen) and neurotox-
icity after p.o. administration into rats at a dose of 30 mg/kg.
The results obtained are presented in Table 2.
Compd
MESa)
scPTZb)
NTc)
0.5 h
4 h
0.5 h
4 h
0.5 h
4 h
5a
5b
5c
5d
5e
5f
5g
6a
6b
6c
6d
6e
6f
6g
300
–
100
100
100
–
100
–
300
100
–
100
100
100
–
–
–
100
100
100
100
100
300
100
–
100
300
100
–
300
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
30014
–
30014
30014
Among the derivatives tested, the most active ones were 6c
and 6f which showed a peak of 75% protection at 1 h (6c) and
at 0.5 h (6f), respectively. These compounds were also active
in 50% of animals at time points 1 h (6f), 2 h, and 4 h (6c).
The latter revealed also 25% protection at 0.5 h. The other
molecules were less active and protected up to 25% (5a, 5d,
6b) or 50% (6d) of rats in different time intervals. All com-
pounds given orally did not cause motor impairment in the
rotarod screen (data not indicated in Table 2).
–
30014
–
30014
30014
–
–
–
300
–
–
–
–
–
–
–
3001
300
300
30014,1
–
10025
10025
–
300
–
100
–
–
–
–
300
–
3001
300
–
Compounds 6c and 6f were chosen for quantification of the
pharmacological parameters (ED50 and TD50) after i.p. admin-
istration into mice. Quantitative evaluation of the MES and
7a
7b
7c
7d
–
–
–
–
–
–
–
scPTZ median effective dose (ED50) and neurotoxic dose (TD50
)
7e
7f
7g
–
–
–
30
–
–
–
–
30
–
–
–
–
–
300
100
–
–
–
–
–
300
–
–
–
was performed at the previously estimated time of peak effect
(TPE). Results of the quantitative tests along with the data for
the standard drugs ethosuximide and valproic acid are
shown in Table 3.
30014
–
Phenytoind)
Valproic acidd)
Ethosuximidee)
100
–
–
100
–
–
–
–
The quantitative data revealed that 6c and 6f were more
potent than the reference drugs in the MES test. Furthermore
6c showed also higher activity in the PTZ seizures in com-
parison with standard substances. Such in vivo profile may
indicate potential effectiveness of 6c in treating different
types of seizures including human generalized tonic–clonic,
myoclonic, and absence seizures. Both compounds revealed
lower rotarod toxicity than valproic acid and ethosuximide
that resulted in favorable protection index.
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 of the animals.
A dash indicates the absence of anticonvulsant activity and
neurotoxicity at the maximum dose administered (300 mg/kg).
Response comments: 1death, 14unable to grasp rotarod, 25myo-
clonic jerks.
a)
Maximal electroshock test.
Subcutaneous pentylenetetrazole test.
Neurotoxicity screening – rotarod test.
Reference drugs, data from [29].
Reference drugs, data from [30].
b)
c)
In the next step of pharmacological investigations, com-
pound 6f was tested in the MES, scPTZ, and rotarod screens
after p.o. administration to rats. The quantitative data are
shown in Table 4.
d)
e)
compounds 6b, 6c, 6e, and 6g were found to be active in the
scPTZ test, which identifies substances elevating the seizure
threshold. Among these molecules 6b, 6c, and 6g showed anti-
scPTZ activity at the dose 100 mg/kg that was equivalent to
ethosuximide used as reference anticonvulsant. Unfortunately,
despite the protection in PTZ seizures, 6b and 6c provoked
myoclonic jerks at the same dose. Compound 6d was less active
and inhibited seizures at a dose of 300 mg/kg.
Table 2. Anticonvulsant activity (MES test) of selected compounds
administrated orally to rats (30 mg/kg).
Compd
0.25 h
0.5 h
1 h
2 h
4 h
5a
5d
5e
6b
6c
6d
6f
0
1
0
0
0
0
0
1
0
1
2
0
1
0
3
4
1
0
1
1
3
0
2
3
0
0
0
0
2
2
0
3
0
0
0
1
2
0
0
3
In the rotarod test (NT) for acute neurological toxicity 5b,
5f, 5g, 6e, 6g as well as 7c–e and 7g did not show neuro-
toxicity at the maximum dose administered (300 mg/kg). The
other derivatives 5a, 5c–e, 5g, 6a–d, 6f, 7a, 7b, and 7f showed
neurotoxicity at a dose of 300 mg/kg. At the same dose mice
were unable to grasp the rotarod after administration of 5a,
5c–e, 6d, and 7f.
Phenytoina)
The data in the oral MES screen indicate the number of rats of
four that were protected at a dose of 30 mg/kg.
a)
Reference drug, data from [31].
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
New Pyrrolidine-2,5-diones as Potential Anticonvulsants
5
Table 3. Quantitative anticonvulsant data in mice after i.p. administration (6c and 6f).
Compd
TPE
(h)a)
ED50b) MES (mg/kg)
ED50b) scPTZ (mg/kg)
TD50b) (mg/kg)
PIc) (TD50/ED50
)
6c
6f
0.5
0.5
1.0
1.0
76.15 (60.56–97.80)
95.61 (85.12–111.03)
263 (237–282)
>500
132.90 (106.01–153.76)
>195.0
289.28 (251.16–316.50)
212.24 (184.98–259.11)
398 (356–445)
3.8 (MES), 2.17 (scPTZ)
2.22 (MES), >1.08 (scPTZ)
1.5 (MES), 1.8 (scPTZ)
2.50 (scPTZ)
Valproic acidd)
Etosuximided)
220 (177–268)
136 (101–184)
341 (290–384)
a)
Time to peak effect.
Results are represented as mean ꢀ SEM at 95% confidence limit (MES, maximal electroshock test; scPTZ, subcutaneous pentylene-
b)
tetrazole test; neurotoxicity-rotarod screen).
c)
Protection index (TD50/ED50).
Reference drug, data from [32].
d)
Table 4. Quantitative anticonvulsant data in rats after p.o. administration (6f).
Compd
TPE
(h)a)
ED50a) MES (mg/kg)
ED50b) scPTZ (mg/kg)
TD50b) (mg/kg)
PIc) (TD50/ED50
)
6f
0.5
1.0
1.0
52.56 (30.21–99.99)
485 (324–677)
>500
96.43 (80.06–112.4)
646 (466–869)
167 (116–237)
>500
784 (503–1176)
>500
>9.5 (MES), >5.18 (scPTZ)
1.6 (MES), 1.2 (scPTZ)
>2.99
Valproic acidd)
Etosuximided)
a)
Time to peak effect.
Results are represented as mean ꢀ SEM at 95% confidence limit (MES, maximal electroshock test; scPTZ, subcutaneous pentylene-
b)
tetrazole test; neurotoxicity-rotarod screen).
c)
Protection index (TD50/ED50).
Reference drug, data from [32].
d)
Compound 6f given orally to rats showed superior activity
and lower neurotoxicity in comparison with model anticon-
vulsants – valproic acid and ethosuximide. These results
confirmed also satisfactory absorption from the gastrointes-
tinal tract as well as penetration of the substance into the
central nervous system.
As can be seen, the most potent was 5c which protected
100% of mice in all time intervals at a dose of 100 mg after i.p.
administration. Compound 6e was less active and protected
100% of animals at 0.25 h, 0.5 h; 75% at 1 h and 50% at 2 h,
whereas 6c showed only one peak of 75% protection at 0.25 h.
It should be stressed here that compound 6f revealed 50%
effectiveness at 0.5 h, however in contrast with the other
molecules, it was tested at a dose of 50 mg/kg. Thus it was
selected for quantification of the median effective dose in
psychomotor seizure test (6-Hz) after i.p. administration in
mice (Table 6).
Selected compounds 5c, 6c, 6e, and 6f were chosen for the
evaluation of anticonvulsant activity in the 6-Hz test. The selec-
tion was made randomly as a part of the search for molecules
providing protection in the 6-Hz model among chemically diver-
sified compounds pursued in the NIH/NINDS. The 6-Hz screen
has been validated recently as a model of therapy-resistant
epilepsy [9]. The results obtained are shown in Table 5.
Compound 6f revealed higher activity in comparison with
valproic acid or ethosuximide however was less active than
levetiracetam which is known as model antiepileptic drug
effective in 6-Hz seizures.
Table 5. Anticonvulsant activity – i.p. psychomotor seizure test in
mice (6-Hz, current 32 mA).
Summing up, the potency and spectrum of anticonvulsant
activity depended mainly on the kind of substituents at
position-3 of pyrrolidine-2,5-dine. All the 3,3-diphenyl deriva-
tives (5a–g) provided protection only in electrically induced
seizures. The exchange of one phenyl ring into the methyl
group (compounds 6a–g) yielded molecules that showed
activity in both electrical and chemical (pentylenetetrazole)
seizures, in general. The removal of aromatic rings (7a–g)
caused lack of any anticonvulsant activity. These data enable
Compd
0.25 h
0.5 h
1 h
2 h
4 h
5c
6c
6e
6f
4
3
4
1
4
2
4
2
4
1
3
0
4
1
2
0
4
0
0
0
The data indicate the number of mice of four that were protected
at a dose of 100 mg/kg (5c, 6c, 6e) or 50 mg/kg (6f).
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J. Obniska et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
Table 6. Quantification data – psychomotor seizure test (6-Hz) after i.p. injection into mice.
Compd
TPE (h)a)
ED50b) 6 Hz (mg/kg)
TD50b) (mg/kg)
PIc) (TD50/ED50
)
6f
0.5
1
1
90.16 (40.32–167.15)
19.4 (9.90–36.0)
167 (114–223)
275.30 (256.10–300.53)
>500
3.05
>26.0
2.5
Levetiracetamd)
Ethosuximided)
Valproic acidd)
341 (290–384)
398 (356–445)
1
126.0 (94.5–152)
3.2
a)
Time to peak effect.
Results are represented as mean ꢀ SEM at 95% confidence limit (6-Hz – psychomotor seizure test; neurotoxicity-rotarod screen).
b)
c)
Protection index (TD50/ED50).
Reference drug, data from [32].
d)
to design new molecules, effective in different types of
(Merck; Darmstadt, Germany), using developing systems
consisting of S1 chloroform/acetone (9:1 v/v), S2 – chloroform/
isopropanol/ammonia (9:11:2 by volume).
seizures, in future studies. The in vivo results and the
structures of the active compounds that correspond to
known AEDs such as ethosuximide, phensuximide, or
phenytoin may suggest that the most possible mechanism
of anticonvulsant action is related with the influence on
voltage-depended sodium or/and calcium channels.
Spots were detected by their absorption under UV light
(l ¼ 254 nm). Elemental analysis for C, H, and N were
carried out by a micro-method using the elemental Vario
EI III Elemental analyzer (Hanau, Germany). The results of
elemental analyses were within ꢀ0.4% of the theoretical
values.
Conclusion
1H NMR spectra were obtained in a Varian Mercury
spectrometer (Varian, Inc., Palo Alto, CA, USA), in CDCl3
or DMSO, operating at 300 MHz. Chemical shifts are
reported in d values (ppm) relative to TMS d: 0 (1H), as
internal standard. The J values are expressed in Hertz (Hz).
Signal multiplicities are represented by the following
abbreviations: s (singlet), brs (broad singlet), d (doublet),
t (triplet), and m (multiplet).
In the present studies a library of twenty one new N-Mannich
bases of 3,3-diphenyl-, 3-methyl-3-phenyl-, and 3-ethyl-3-methyl-
pyrrolidine-2,5-diones were synthesized and evaluated for their
anticonvulsant activity in the maximum electroshock (MES)
and scPTZ seizure tests. The results of in vivo studies revealed
that the majority of compounds exhibited anticonvulsant
activity in the MES test, which is an animal model of human
generalized tonic–clonic seizures. Several molecules showed
activity in the scPTZ and 6-Hz screens that may indicate their
potential usefulness in absence seizures as well as partial and
therapy resistant epilepsy, respectively. From the whole
series the most promising compound was N-[(4-benzylpiper-
idin-1-yl)-methyl]-3-methyl-3-phenylpyrrolidine-2,5-dione (6f),
which revealed ED50 values of 95.61 mg/kg (MES, i.p. mice),
52.56 (MES, p.o. rats), 96.43 (scPTZ, p.o. rats), and 90.16 mg/kg
(6-Hz test, i.p. mice).
The starting materials 2-methyl-2-phenyl-succinic acid (4)
and 3,3-diphenylpyrrolidine-2,5-dione (5) were prepared
using methods described previously [23, 24].
Compounds 7a–g were obtained using as a starting
material commercially available 3-ethyl-3-methyl-pyrroli-
dine-2,5-dione (7) (Sigma–Aldrich, USA).
General procedure for the synthesis of 3-methyl-3-
phenylpyrrolidine-2,5-dione (6)
A total of 0.05 mol of the 2-methyl-2-phenyl-succinic acid (4)
was dissolved in 50 mL of water and 0.05 mol of the 25%
ammonia was gradually added. The mixture was heated in a
sand bath with simultaneous distillation of water. After the
water was completely removed, the temperature of the reac-
tion rose up to 1908C and was maintained for 1.5 h. The solid
product was separated by crystallization from isopropanol
(Scheme 1).
Experimental protocol
Chemistry
All the chemicals and solvents were purchased from Merck
(Darmstadt, Germany) and were used without further puri-
fication. 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 and homogeneity of the compounds were assessed
with TLC. The thin-layer chromatography (TLC) was per-
formed on Merck silica gel 60 F254 aluminum sheets
3-Methyl-3-phenylpyrrolidine-2,5-dione (6)
White powdery crystals. Yield: 70%; mp 77–788C; TLC:
Rf ¼ 0.51 (S1); 1H NMR (CDCl3): d 1.76 (s, 3H, CH3), 2.90
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
New Pyrrolidine-2,5-diones as Potential Anticonvulsants
7
(d, 1H, imide, J ¼ 18.21 Hz), 3.17 (d, 1H, imide, J ¼ 18.46 Hz),
3.46 (s, 2H, imide), 4.54 (s, 2H, CH2), 7.24–7.37 (m, 10H,
ArH); C27H33O2 N3 (431.58).
7.26–7.39 (m, 5H, ArH), 8.00 (brs, 1H, NH); C11H11O2N (189.22).
General procedure for the synthesis of compounds 5a–f,
6a–f, and 7a–f
N-[(4-Methyl-piperazin-1-yl)-methyl]-3,3-
diphenylpyrrolidine-2,5-dione (5e)
The mixture of 3,3-disubstituted-succinimide (0.01 mol) (5, 6,
7), 40% formaldehyde solution (0.01 mol) and corresponding
4-substituted piperazine (0.01 mol), morpholine (0.01 mol),
or 4-benzyl-piperidine in 96% ethanol (40 mL) was left for ca.
12 h at room temperature and was then refrigerated at ca.
ꢁ108C for 24 h. The products were washed with cold ethanol
and the solid products (5a–g, 6a–g, and 7a–g) were
separated by filtration and recrystallized from 96% ethanol.
Compounds 5c, 6a, 6c, 6e, 7a, 7c, and 7e were obtained as
light oils. These molecules were converted to hydrochloride
salts in anhydrous ethanol saturated with HCl gas. They were
crystallized from anhydrous ethanol.
White powdery crystals. Yield: 75%; mp 103–1058C; TLC:
Rf ¼ 0.67 (S1); 1H NMR (300 MHz, CDCl3): d 2.25 (s, 3H,
CH3), 2.39 (brs, 4H, piperazine), 2.66 (brs, 4H, piperazine),
3.46 (s, 2H, imide), 4.55 (s, 2H, CH2), 7.25–7.37 (m, 10H,
ArH); C22H25O2N3 (363.46).
N-[(4-Benzylpiperidin-1-yl)-methyl]-3,3-
diphenylpyrrolidine-2,5-dione (5f)
White powdery crystals. Yield: 78%; mp 101–1038C; TLC:
Rf ¼ 0.69 (S1); 1H NMR (300 MHz, CDCl3): d 1.13–1.26
(m, 2H, piperidine), 1.28–1.37 (m, 1H, piperidine), 1.55–
1.58 (m, 2H, piperidine), 2.00–2.17 (m, 2H, piperidine),
2.47 (d, 2H, piperidine, J ¼ 6.67 Hz), 2.94 (d, 2H, CH2,
J ¼ 12.06 Hz), 3.45 (s, 2H, imide), 4.52 (s, 2H, CH2), 7.08–
7.38 (m, 10H, ArH); C29H30O2N2 (438.57).
N-[(4-Benzyl-piperazin-1-yl)-methyl]-3,3-
diphenylpyrrolidine-2,5-dione (5a)
White powdery crystals. Yield: 78%; mp 82–848C; TLC:
Rf ¼ 0.69 (S1); 1H NMR (300 MHz, CDCl3): d 2.41 (brs, 4H,
piperazine), 2.61 (brs, 4H, piperazine) 3.47 (s, 4H, 2H,
CH2, 2H, imide), 4.54 (2H, CH2), 7.21–7.39 (m, 15H,
ArH); C28H29O2N3 (439.56).
N-(Morpholin-1-yl)-methyl-3,3-diphenylpyrrolidine-2,5-
dione (5g)
White powdery crystals. Yield: 80%; mp 66 ꢁ 678C; TLC:
Rf ¼ 0.66 (S1); 1H NMR (300 MHz, CDCl3): d 2.56 (t, 4H,
morpholine, J ¼ 4.75 Hz), 3.49 (s, 2H, imide), 3.63 (t, 4H
morpholine, J ¼ 4.75 Hz), 4.52 (2H, CH2), 7.25–7.39
(m, 10H, ArH); C21H22O3N2 (350.41).
N-[(4-Pyrimid-2-yl-piperazin-1-yl)-methyl]-3,3-
diphenylpyrrolidine-2,5-dione (5b)
White powder-y crystals. Yield: 69%; mp 153–1548C; TLC:
Rf ¼ 0.68 (S1); 1H NMR (300 MHz, CDCl3): d 2.61 (t, 4H, piper-
azine J ¼ 5.13 Hz), 3.46 (s, 2H, imide), 3.76 (t, 4H piperazine
J ¼ 5.00 Hz), 4.59 (s, 2H, CH2), 6.48 (t, 1H, pyrimidine,
J ¼ 4.74 Hz), 7.22–7.34 (m, 10H, ArH), 8.28 (d, 2H, pyrimidine,
J ¼ 4.61 Hz); C25H25O2N5 (427.50).
N-[(4-Benzyl-piperazin-1-yl)-methyl]-3-methyl-3-
phenylpyrrolidine-2,5-dione hydrochloride (6a)
White powdery crystals. Yield: 70%; mp 208–2108C; TLC:
Rf ¼ 0.85 (S2); 1H NMR (300 MHz, DMSO): d 1.65 (s, 3H,
CH3), 2.77–3.05 (m, 8H, piperazine), 3.20–3.24 (m, 2H, imide),
4.26 (s, 2H, CH2), 4.39 (s, 2H, CH2), 7.24–7.29 (m, 1H, ArH),
7.35–7.45 (m, 7H, ArH), 7.60–7.63 (m, 2H, ArH), 11.38 (brs, 1H,
HCl); C23H28O2N3Cl (413.95).
N-{[4-(2-Hydroxyethyl)-piperazin-1-yl]-methyl}-3,3-
diphenylpyrrolidine-2,5-dione dihydrochloride (5c)
White powdery crystals. Yield: 77%; mp 121–1248C; TLC:
Rf ¼ 0.82 (S2); 1H NMR (300 MHz, DMSO): d 2.66–3.10
(m, 6H, 2H, CH2, 4H, piperazine), 3.24 (t, 1H, OH,
J ¼ 4.75 Hz), 3.39–3.80 (m, 8H, 2H, imide, 2H, CH2, 4H,
piperazine), 4.40 (s, 2H, CH2), 7.26–7.41 (m, 10H, ArH),
11.37 (brs, 1H, HCl), 11.56 (brs, 1H, HCl); C23H29O3N3Cl2
(466.42).
N-[(4-Pyrimid-2-yl-piperazin-1-yl)-methyl]-3-methyl-3-
phenylpyrrolidine-2,5-dione (6b)
White powdery crystals. Yield: 82%; mp 99–1018C; TLC:
Rf ¼ 0.39 (S1); 1H NMR (300 MHz, CDCl3): d 1.72 (s, 3H,
CH3), 2.66 (t, 4H, piperazine, J ¼ 4.36 Hz), 2.86 (d, 1H, imide,
J ¼ 18.21 Hz), 3.15 (d, 1H, imide, J ¼ 18.21 Hz), 3.78 (t, 4H,
piperazine, J ¼ 5.13 Hz), 4.59 (s, 2H, CH2), 6.48 (t, 1H,
pyrimidine, J ¼ 4.75 Hz), 7.24–7.36 (m, 5H, ArH), 8.29
(d, 2H, pyrimidine, J ¼ 4.62 Hz); C20H23O2N5 (365.44).
N-[(4-Cyclohexyl-piperazin-1-yl)-methyl]-3,3-
diphenylpyrrolidine-2,5-dione (5d)
White powdery crystals. Yield: 79%; mp 116–1188C; TLC:
Rf ¼ 0.67 (S1); 1H NMR (300 MHz, CDCl3): d 1.10–1.24
(m, 5H, cyclohexane), 1.58–1.63 (m, 1H, cyclohexane), 1.75–
1.85 (m, 4H, cyclohexane), 2.18–2.19 (m, 1H, cyclohexane),
2.54–2.55 (m, 4H, piperazine), 2.62–2.65 (m, 4H, piperazine),
N-{[4-(2-Hydroxyethyl)-piperazin-1-yl]-methyl}-3-methyl-3-
phenylpyrrolidine-2,5-dione hydrochloride (6c)
White powdery crystals. Yield: 78%; mp 159–1618C; TLC:
Rf ¼ 0.83 (S2); 1H NMR (300 MHz, DMSO): d 1.66 (s, 3H,
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8
J. Obniska et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
CH3), 2.77–3.12 (m, 8H, 2H, imide, 2H, CH2, 4H, piperazine),
3.25 (brs, 1H, OH), 3.42–3.80 (m, 6H, 2H, CH2, 4H, piperazine),
4.39 (s, 2H, CH2), 7.25–7.31 (m, 1H, ArH), 7.35–7.43 (m, 4H,
ArH), 11.38 (brs, 1H, HCl); C18H26O3N3Cl (367.88).
piperazine, 1H imide), 2.90 (d, 4H piperazine, J ¼ 12.05 Hz),
3.20 (d, 2H, CH2 J ¼ 11.03 Hz), 4.28 (s, 2H, CH2), 7.41–7.45
(m, 3H, ArH), 7.58–7.60 (m, 2H, ArH), 10.99 (brs, 1H,
HCl); C19H27O2N3 (329.45).
N-[(4-Cyclohexyl-piperazin-1-yl)-methyl]-3-methyl-3-
phenylpyrrolidine-2,5-dione (6d)
N-[(4-Pyrimid-2-yl-piperazin-1-yl)-methyl]-3-ethyl-3-
methylpyrrolidine-2,5-dione (7b)
White powdery crystals. Yield: 81%; mp 101–1038C; TLC:
Rf ¼ 0.53 (S1); 1H NMR (300 MHz, CDCl3): d 1.14–1.24
(m, 5H, cyclohexane), 1.54–1.63 (m, 1H, cyclohexane), 1.72
(s, 3H, CH3), 1.75–1.85 (m, 4H, cyclohexane), 2.20–2.21 (m, 1H,
cyclohexane), 2.55–2.56 (m, 4H, piperazine), 2.65–2.66 (m, 4H,
piperazine), 2.86 (d, 1H, imide, J ¼ 17.95 Hz), 3.13 (d, 1H
imide, J ¼ 18.21 Hz), 4.53 (s, 2H, CH2), 7.24–7.38 (m, 5H,
ArH); C22H31O2N3 (369.51).
White powdery crystals. Yield: 88%; mp 69–718C; TLC:
Rf ¼ 0.034 (S1); 1H NMR (300 MHz, CDCl3): d 0.87 (t, 2H,
CH2, J ¼ 7.44 Hz), 1.29 (s, 3H, CH3), 1.60–1.78 (m, 2H, CH2),
2.42 (d, 1H, imide, J ¼ 18.21 Hz), 2.60–2.68 (m, 5H, 4H, piper-
azine, 1H, imide), 3.79 (t, 4H, piperazine, J ¼ 4.87 Hz), 4.51
(s, 2H, CH2), 6.47 (t, 1H, pyrimidine, J ¼ 4.74 Hz), 8.28 (d, 2H,
pyrimidine, J ¼ 4.87 Hz); C16H23O2N5 (317.39).
N-{[4-(2-Hydroxyethyl)-piperazin-1-yl]-methyl}-3-ethyl-3-
methylpyrrolidine-2,5-dione dihydrochloride (7c)
N-[(4-Methyl-piperazin-1-yl)-methyl]-3-methyl-3-
phenylpyrrolidine-2,5-dione dihydrochloride (6e)
White powdery crystals. Yield: 68%; mp 140–1428C; TLC:
1
White powdery crystals. Yield: 86%; mp 189–1918C; TLC:
Rf ¼ 0.84 (S2); 1H NMR (300 MHz, DMSO): d 1.65 (s, 3H
CH3), 2.67–2.79 (m, 5H, 3H, CH3, 2H, piperazine), 2.92–3.00
(m, 5H, 4H, piperazine, 1H, imide), 3.29–3.32 (m, 3H, 2H
piperazine, 1H, imide), 4.40 (s, 2H, CH2), 7.25–7.32 (m, 1H,
ArH), 7.35–7.41 (m, 4H, ArH), 11.04 (brs, 1H, HCl), 11.38
(brs, 1H, HCl); C17H24O2N3Cl (337.85).
Rf ¼ 0.93 (S2), H NMR (300 MHz, DMSO): d 0.79 (t, 3H, CH3,
J ¼ 7.44 Hz), 1.22 (s, 3H, CH3), 1.47–1.65 (m, 2H CH2), 2.50
(d, 1H imide, J ¼ 18.21 Hz), 2.68 (d, 1H, imide, J ¼ 18.21 Hz),
2.80 (d, 2H, piperazine, J ¼ 11.80 Hz), 2.93–3.03 (m, 4H,
piperazine), 3.11 (t, 2H, CH2, J ¼ 5.00 Hz), 3.23 (t, 1H, OH,
J ¼ 5.00 Hz), 3.44 (d, 2H, piperazine, J ¼ 11.80 Hz), 3.73
(t, 2H, CH2, J ¼ 5.13 Hz), 4.34 (s, 2H CH2), 10.58 (brs, 1H,
HCl); C14H26O3N3Cl (319.83).
N-[(4-Benzylpiperidin-1-yl)-methyl]-3-methyl-3-
phenylpyrrolidine-2,5-dione (6f)
N-[(4-Cyclohexyl-piperazin-1-yl)-methyl]-3-ethyl-3-
methylpyrrolidine-2,5-dione (7d)
White powdery crystals. Yield: 67%; mp 77–788C; TLC:
Rf ¼ 0.57 (S1); 1H NMR (300 MHz, CDCl3): d 1.17–1.29
(m, 2H, piperidine), 1.34–1.41 (m, 1H, piperidine), 1.58–
1.62 (m, 2H, piperidine), 1.72 (s, 3H, CH3), 2.04–2.14
(m, 2H, piperidine), 2.49 (d, 2H, piperidine, J ¼ 6.93 Hz),
2.85 (d, 1H, imide, J ¼ 18.21 Hz), 2.95–2.99 (m, 2H, CH2),
3.14 (d, 1H, imide, J ¼ 18.21 Hz), 4.51 (s, 2H, CH2), 7.09–
7.38 (m, 10H, ArH); C24H28O2N2 (376.50).
White powdery crystals. Yield: 81%; mp 77–798C; TLC:
Rf ¼ 0.52 (S1); 1H NMR (300 MHz, CDCl3): d 0.88 (t, 3H,
CH3, J ¼ 7.44 Hz) 1.04–1.23 (m, 5H, cyclohexane), 1.30
(s, 3H, CH3), 1.57–1.84 (m, 7H, 5H, cyclohexane, 2H,
CH2), 2.17–2.21 (m, 1H, cyclohexane), 2.41 (d, 1H, imide,
J ¼ 18.21 Hz), 2.54–2.55 (m, 4H, piperazine), 2.58–
2.64 (m, 5H, 4H, piperazine, 1H, imide), 4.45 (s, 2H,
CH2); C18H31O2N3 (321.46).
N-(Morpholin-1-yl)-methyl-3-methyl-3-phenylpyrrolidine-
2,5-dione (6g)
N-[(4-Methyl-piperazin-1-yl)-methyl]-3-ethyl-3-
methylpyrrolidine-2,5-dione dihydrochloride (7e)
White powdery crystals. Yield: 82%; mp 63–648C; TLC:
Rf ¼ 0.55 (S1); 1H NMR (300 MHz, CDCl3): d 1.75 (s, 3H,
CH3), 2.60 (t, 4H, morpholine, J ¼ 4.62 Hz), 2.89 (d, 1H, imide,
J ¼ 18.21 Hz), 3.17 (d, 1H, imide, J ¼ 18.21 Hz), 3.65 (t, 4H,
morpholine, J ¼ 4.62 Hz), 4.51 (2H, CH2), 7.25–7.40 (m, 10H,
ArH); C16H20O3N2 (288.34).
White powdery crystals. Yield: 86%; mp 178–1818C; TLC:
Rf ¼ 0.92 (S2); 1H NMR (300 MHz, DMSO): d 0.75–0.84
(m, 3H, CH3), 1.14 (s, 3H, CH3), 1.21 (s, 3H, CH3), 1.41–1.64
(m, 2H, CH2), 2.39 (d, 1H, imide, J ¼ 18.21 Hz), 2.54 (d, 1H,
imide, J ¼ 18.21 Hz), 2.65–2.92 (m, 8H, piperazine), 4.30
(s, 2H, CH2), 11.07 (brs, 2H, HCl); C13H25O2N3Cl2 (326.27).
N-[(4-Benzyl-piperazin-1-yl)-methyl]-3-ethyl-3-
methylpyrrolidine-2,5-dione hydrochloride (7a)
White powdery crystals. Yield: 62%; mp 69–708C; TLC:
N-[(4-Benzylpiperidin-1-yl)-methyl]-3-ethyl-3-
methylpyrrolidine-2,5-dione (7f)
1
Rf ¼ 0.90 (S2); H NMR (300 MHz, CDCl3): d 0.80 (t, 3H, CH3,
White powdery crystals. Yield: 67%; mp 92–948C; TLC:
1
J ¼ 7.44 Hz), 1.20 (s, 3H, CH3), 1.46–1.63 (m, 2H, CH2), 2.42–
Rf ¼ 0.70 (S1); H NMR (300 MHz, CDCl3): d 0.88 (t, 3H, CH3,
2.49 (m, 3H, 2H, piperazine, 1H, imide), 2.49–2.73 (m, 3H, 2H
J ¼ 7.57 Hz), 1.29 (s, 1H, CH3), 1.32–1.37 (m, 2H, piperidine),
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
New Pyrrolidine-2,5-diones as Potential Anticonvulsants
9
1.39–1.43 (m, 1H, piperidine), 1.53–1.79 (m, 4H, 2H, CH2, 2H,
piperidine), 2.07–2.15 (m, 2H, piperidine), 2.42 (d, 1H, imide,
J ¼ 18.21 Hz), 2.50 (d, 2H, piperidine, J ¼ 6.67 Hz), 2.63
(d, 1H, imide, J ¼ 18.21 Hz), 2.99 (d, 2H, CH2, J ¼ 10.77 Hz),
4.44 (s, 2H, CH2), 7.09–7.38 (m, 5H, ArH); C20H28O2N2 (328.46).
Neurological toxicity (NT)
The neurological toxicity induced by a compound was
detected in mice using the standardized rotorod test [27].
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.
N-(Morpholin-1-yl)-methyl-3-ethyl-3-methylpyrrolidine-2,5-
dione (7g)
White powdery crystals. Yield: 82%; mp 48–508C; TLC:
1
Rf ¼ 0.53 (S1); H NMR (300 MHz, CDCl3): d 0.91 (t, 3H, CH3,
Quantification studies
J ¼ 7.44 Hz), 1.32 (s, 3H, CH3), 1.55–1.79 (m, 2H, CH2), 2.45
(d, 1H, imide, J ¼ 18.47 Hz), 2.57 (t, 4H, morpholine,
J ¼ 4.75 Hz), 2.66 (d, 1H, imide, J ¼ 18.47 Hz), 3.65 (t, 4H,
morpholine, J ¼ 4.75 Hz), 4.43 (2H, CH2); C12H20O3N2
(240.30).
Quantitative determination of the median effective dose
(ED50) and the median neurotoxic dose (TD50) values was
performed at previously estimated TPE after i.p. adminis-
tration into mice and p.o. administration into rats. Groups
of eight rats received various doses of the compound
until at least three points were established in the range of
10–90% seizure protection and 0% protection or minimal
neurotoxicity. From the plot of the data obtained, the
respective ED50 and TD50 values, 95% confidence intervals,
slope of the regression line and standard error of the slope
were calculated by means of a computer program written at
NINDS/NIH.
Pharmacology
Compounds 3–24 were pharmacologically pre-evaluated
within the Antiepileptic Drug Development (ADD) Program
in the Epilepsy Branch, Neurological Disorders Program,
National Institute of the Neurological and Communicative
Disorders and Stroke (NIH/NINDS), Rockville, MD, USA, by
using procedures described elsewhere [25, 26].
Phase I studies of the investigated compounds involved
three tests: MES, scPTZ and rotorod test for NT. Male albino
mice (CF#1 strain, weighing 18–25 g) and male albino rats
(Sprague–Dawley) were used as experimental animals. The
animals were housed in metabolic cages and allowed
free access to food and water. The compounds were
injected intraperitoneally into mice as a suspension in
0.5% methylcellulose/water mixture at dose levels of 30,
100, and 300 mg/kg with anticonvulsant activity and neuro-
toxicity assessment at 0.5 and 4 h intervals after adminis-
tration. Promising derivatives from phase I underwent phase
VIa in which they were administered orally to rats at a fixed
dose of 30 mg/kg for both MES and the rotorod toxicity tests
or 50 mg/kg in the scPTZ screen. Groups of eight mice or four
rats were employed.
The 6-Hz model
The 6-Hz model is an alternative electroshock paradigm
that uses low-frequency (6 Hz) long-duration (3 s) electrical
stimulation. Corneal stimulation (0.2 ms duration mono-
polar rectangular pulses at 6 Hz for 3 s) was delivered by a
constant-current device. During the stimulation, mice were
manually restrained and released into the observation cage
immediately after the current application. The seizures mani-
fest in ‘‘stunned’’ posture associated with rearing, forelimb,
automatic movements, and clonus, twitching the vibrissae
and Straub-tail. The duration of the seizure activity ranges
from 60 to 120 s in untreated animals. At the end of the
seizure, animals resume their normal exploratory behavior.
The experimental end point is protection against the seizure.
The animal is considered to be protected if it resumes
its normal exploratory behavior within 10 s from the
stimulation [28].
Maximal electroshock test (MES)
In the MES screen, an electrical stimulus of 0.2 s in duration
(50 mA in mice and 150 mA in rats) is delivered via corneal
electrodes primed with an electrolyte solution containing an
anesthetic agent.
The authors wish to thank the ADD Program (Epilepsy Branch, National
Institute of Neurological Disorders and Stroke, National Institute of
Health, Rockville, MD, USA) for providing pharmacological data,
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).
Subcutaneous pentylenetetrazole seizure test (scPTZ)
The scPTZ test utilizes a dose of pentylenetetrazole (85 mg/kg
in mice and 70 mg/kg in rats) that produces clonic seizures
lasting for a period of at least 5 s in 97% (CD97) of the animals
tested. At the anticipated time of testing the pentylene-
tetrazole is administrated subcutaneously.
The author has declared no conflict of interest.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
´
[18] K. Kaminski, J. Obniska, B. Wiklik, D. Atamanyuk, Eur. J. Med.
Chem. 2011, 46, 4634–4641.
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