Synthesis, and anticonvulsant activity of new amides derived from 3-methyl- or 3-ethyl-3-methyl-2,5-dioxo-pyrrolidin-1-yl-acetic acids

Article abstract of DOI:10.1016/j.bmc.2016.02.026

This paper describes the synthesis of the library of 22 new 3-methyl- and 3-ethyl-3-methyl-2,5-dioxo-pyrrolidin-1-yl-acetamides as potential anticonvulsant agents. The maximal electroshock (MES) and the subcutaneous pentylenetetrazole (scPTZ) seizure models were used for screening all the compounds. The 6 Hz model of pharmacoresistant limbic seizures was applied for studying selected derivatives. Six amides were chosen for pharmacological characterization of their antinociceptive activity in the formalin model of tonic pain as well as local anesthetic activity was assessed in mice. The pharmacological data indicate on the broad spectra of activity across the preclinical seizure models. Compounds 10 (ED₅₀ = 32.08 mg/kg, MES test) and 9 (ED₅₀ = 40.34 mg/kg, scPTZ test) demonstrated the highest potency. These compounds displayed considerably better safety profiles than clinically relevant antiepileptic drugs phenytoin, ethosuximide, or valproic acid. Several molecules showed antinociceptive and local anesthetic properties. The in vitro radioligand binding studies demonstrated that the influence on the sodium and calcium channels may be one of the essential mechanisms of action.

Full text of DOI:10.1016/j.bmc.2016.02.026

Accepted Manuscript
Synthesis, and anticonvulsant activity of new amides derived from 3-methyl- or
3-ethyl-3-methyl-2,5-dioxo-pyrrolidin-1-yl-acetic acids
Jolanta Obniska, Anna Rapacz, Sabina Rybka, Małgorzata Góra, Krzysztof
Kamiński, Kinga Sałat, Paweł Żmudzki
PII:
S0968-0896(16)30116-X
http://dx.doi.org/10.1016/j.bmc.2016.02.026
BMC 12828
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 December 2015
16 February 2016
20 February 2016
Please cite this article as: Obniska, J., Rapacz, A., Rybka, S., Góra, M., Kamiński, K., Sałat, K., Żmudzki, P.,
Synthesis, and anticonvulsant activity of new amides derived from 3-methyl- or 3-ethyl-3-methyl-2,5-dioxo-
pyrrolidin-1-yl-acetic acids, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.
2016.02.026
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Synthesis, and anticonvulsant activity of new amides derived from 3-
methyl- or 3-ethyl-3-methyl-2,5-dioxo-pyrrolidin-1-yl-acetic acids
Jolanta Obniskaa*, Anna Rapaczb, Sabina Rybkaa, Małgorzata Góraa, Krzysztof Kamińskia,
Kinga Sałatb, Paweł Żmudzkia
a Department of Medicinal Chemistry, Faculty of Pharmacy, Jagiellonian University, Medical
College, 9 Medyczna Street, 30-688 Kraków, Poland
b Department of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University Medical
College, 9 Medyczna Street, Kraków 30-688, Poland
*Corresponding author:
Tel.: + 48 12 620 54 53; fax: +48 12 657 02 62; E–mail: mfobnisk@cyf-kr.edu.pl (J. Obniska)
1

Abstract
This paper describes the synthesis of the library of 22 new 3-methyl- and 3-ethyl-3-methyl-
2,5-dioxo-pyrrolidin-1-yl-acetamides as potential anticonvulsant agents. The maximal
electroshock (MES) and the subcutaneous pentylenetetrazole (scPTZ) seizure models were
used for screening all the compounds. The 6 Hz model of pharmacoresistant limbic seizures
was applied for studying selected derivatives. Six amides were chosen for pharmacological
characterization of their antinociceptive activity in the formalin model of tonic pain as well as
local anesthetic activity was assessed in mice. The pharmacological data indicate on the broad
spectra of activity across the preclinical seizure models. Compounds 10 (ED50 = 32.08 mg/kg,
MES test) and 9 (ED50 = 40.34 mg/kg scPTZ test) demonstrated the highest potency. These
compounds displayed considerably better safety profiles than clinically relevant antiepileptic
drugs phenytoin, ethosuximide, or valproic acid. Several molecules showed antinociceptive
and local anesthetic properties. The in vitro radioligand binding studies demonstrated that the
influence on the sodium and calcium channels may be one of the essential mechanisms of
action.
Keywords: Succinimides; Anticonvulsant activity; Antinociceptive properties; Anesthetic
activity; Ion channels
2

1. Introduction
The past decades have demonstrated many attempts to identify the structural features
of compounds crucial for anticonvulsant activity. It is well documented that important core
fragment of anticonvulsants is defined by nitrogen heteroatomic system, usually an imide,
with at least one carbonyl group and phenyl or alkyl groups attached to the heterocyclic
system.1–3 This common template is present in the structures of old-generation, however, well-
established antiepileptic drugs (AEDs) such as ethosuximide and phenytoin as well as
levetiracetam, brivaracetam, or seletracetam, e.g., among the newest drugs (Figure 1). In the
previous studies, we have found the pyrrolidine-2,5-diones differently substituted at position-
1 and -3 as targets for new AEDs. Many of these compounds were effective in the MES and
scPTZ tests that are still recognized as the “gold standard” in the early stages of testing new
drug candidates.4–8
F
F
H
N
O
O
O
N
N
N
O
O
NH2
NH2
NH2
N
N
H
O
O
H
H
H
H
O
O
O
Phenytoin
Ethosuximide
Levetiracetam
Brivaracetam
Seletracetam
Figure 1. Structures of ADEs with five-member heterocyclic ring.
Recent studies on the structure-activity relationship (SAR) demonstrated the potent
anticonvulsant activity of the N-(4-arylpiperazin-1-yl)-methyl-3,3-disubstituted-pyrrolidine-
2,5-dio-nes among which the most active were 1-[{4-(3-chlorophenyl)-piperazin-1-
yl}methyl]-3-methyl-3-phenyl-pyrrolidine-2,5-dione (I) and its 3,4-dichloro analog (II),
showing protection exclusively in the MES test.9 Further SAR analysis revealed that exchange
3

of the methylene linker between imide and amine function into acetamide moiety yielded
compounds active in both MES and PTZ seizure models or predominantly in the PTZ test –
compound III (Figure 2).10
Figure 2. Anticonvulsants obtained in the previous studies.
Considering the aforementioned results, as part of our efforts to design new
anticonvulsant agents, in the present study, we have synthesized a new series of analogs in
which we introduced one methyl or ethyl and methyl substituents at position-3 of imide ring.
The proposed structural modifications enable to assess the influence of alkyl groups on
anticonvulsant properties in this series of derivatives. With the aim of ensuring the reliable
SAR discussion as an amine function, variously substituted 4-arylpiperazines have been
introduced.
Note that several AEDs such as pregabalin, gabapentin, phenytoin, lacosamide, and
topiramate are clinically effective for the treatment of certain kinds of pain (neuropathic pain,
migraine prophylaxis).11 Using different models of pain, studies performed by various groups
found that AEDs can inhibit sensitized signaling associated with allodynia and
hyperalgesia.12–14 Moreover, local anesthetics, which act by blocking nerve conduction by
altering the function of voltage-gated sodium channels, are used as adjuvant analgesics for the
treatment of neuropathic pain.15 Therefore, selected compounds were examined toward their
antinociceptive as well as local anesthetic activity.
4

2. Results and discussion
2.1. Chemistry
The final compounds 5–26 were synthesized according to Scheme 1. The starting
material 3-methylsuccinic acid (1) was commercially purchased from Merck (Darmstadt,
Germany) whereas 3-ethyl-3-methyl-succinic acid (2) was prepared as previously reported.16
In the next step, the condensation reaction of 1 or 2 with aminoacetic acid yielded in 3-
methyl- or 3-ethyl-3-methyl-2,5-dioxo-pyrrolidin-1-yl-acetic acids (3, 4). These intermediates
were converted to final compounds 5–26 in the coupling reaction with equimolar amounts of
appropriate 4-phenylpiperazines in the presence of carbonyldiimidazole (CDI). The reaction
was carried out at room temperature in dry DMF for 24 h. The crude products were
crystalized from 2-propanol. Their purity and homogeneity were assessed by TLC and
gradient HPLC chromatography. The structures of compounds synthesized were confirmed by
both spectral (1H NMR, LC-MS) and elemental analyses. The detailed physical and analytical
data are listed in the experimental section.
5

O
R
R
H2N
O
O
OH
H3C
H3C
N
OH
190 - 200oC / 1,5 h
O
OH
O
COOH
R = H (3)
R = H (1)
R = C2H5 (4)
R = C2H5 (2)
CDI
DMF
room temp.
R1
HN
N
24 h
R1
O
O
R = H, C2H5
N
N
R1 = H, 2-F, 4-F, 2-Cl, 3-Cl, 4-Cl,
N
R
2,3-Cl, 3,4-Cl, 3-CF3, 2-CH3, 3-CH3
H3C
O
5-26
Scheme 1. Synthetic protocol of the target compounds 5–26.
2.2. Anticonvulsant activity
The preclinical development of new chemical agents for the treatment of epilepsy is
based on the use of predictable animal seizure models, which correspond to different types of
human epilepsies. Such models can be categorized into two main categories: models of acute
seizures (non-epileptic animals induced to have a seizure by an electrical or chemical
stimulus) and models of chronic epilepsy (animals induced to have enhanced seizure
susceptibility or spontaneous seizures). For practical reasons, screening is often carried out
using acute seizure models, although various types of kindling models, a class of chronic
model, are commonly included in the battery of tests to which early stage compounds are
subjected. Despite the diversity of models that could potentially be used to screen for
anticonvulsant activity, the MES model and the scPTZ model are the most frequently used in
the early stages of testing. The MES test is the mechanism-independent animal seizure model
that enables the identification of compounds preventing seizure spread. This test uses an
electrical stimulus to produce generalized tonic-clonic seizures, and thus is thought to be an
6

experimental model of tonic-clonic epilepsy and of partial convulsions with or without
secondary generalization in humans. The scPTZ test uses chemically induced clonic seizures
and is proposed to identify the agents raising the seizure threshold. This test is related to
human generalized absence seizures.17–19 Therefore, all the final substances were screened in
the MES and scPTZ tests. Furthermore, in addition to the primary anticonvulsant screening,
the acute neurological toxicity (NT) was determined in mice by the rotarod test (Table 1).
Table 1. Anticonvulsant activity MES, scPTZ, and rotarod test in mice i.p.
MESa
scPTZb
NTc
Cmpd
R
R1
4 h
-
0.5 h
4 h
0.5 h
4 h
0.5 h
300
300
300
300
100
100
-
H
H
H
300
-
300
300
-
5*
6*
7*
8*
9
2-F
300
100
-
-
300
-
H
-
-
-
-
4-F
H
300
100
-
-
300
-
2-Cl
3-Cl
4-Cl
2,3-Cl
3,4-Cl
3-CF3
2-CH3
3-CH3
H
H
300
30
-
-
-
-
H
-
-
-
300
10*
11
12
13*
14
15
16
17
18
19
20
21
H
100
100
300
100
100
-
100
-
H
300
100
300
300
-
100
-
-
100
H
100
-
-
-
-
-
300
300
300
-
300
H
-
-
-
-
-
-
-
-
H
C2H5
C2H5
-
-
2-F
-
100
100
-
C2H5
C2H5
C2H5
C2H5
4-F
-
-
-
-
100
-
-
-
-
-
300
300
-
-
-
-
-
2-Cl
3-Cl
4-Cl
-
-
100
300
7

C2H5
C2H5
C2H5
C2H5
C2H5
-
2,3-Cl
3,4-Cl
3-CF3
2-CH3
3-CH3
-
-
-
-
-
-
-
-
-
-
22
-
100
-
-
-
23
100
100
-
-
-
300
300
-
-
24
100
-
-
-
25
-
-
300
-
-
-
26
ETXe
PHTe
VPAe
-
-
100
-
-
-
-
30
300
30
-
100
-
100
-
-
-
300
-
Doses of 100, and 300 mg/kg were administered intraperitoneally (i.p.). The data indicate the minimum dose
whereby anticonvulsant activity was demonstrated. A dash indicates the absence of anticonvulsant activity and
neurotoxicity at the maximum dose administered (300 mg/kg).
a Maximal electroshock test.
b Subcutaneous pentylenetetrazole test.
c Neurotoxicity screening - rotorod test.
d Reference drugs, data for ethosuximide (ETX), phenytoin (PHT), and valproic acid (VPA) from ref.20
The preliminary pharmacological data revealed that sixteen compounds (5–15, 18, 19,
21, 24, and 25) showed protection in half or more of the animals tested in the MES or/and
scPTZ tests. As Table 1 shows, nine molecules displayed a broad pharmacological activity in
both MES and PTZ seizures. In the MES test, compound 10 was most effective, displaying
anticonvulsant activity at a dose of 30 mg/kg at 4 h and 100 mg/kg at 0.5 h. The other
derivatives provided anti-MES protection at a dose of 100 mg/kg (7, 9, 12, 13, 18, 19, 24, and
25) or 300 mg/kg (5, 6, 8, 14, and 15). Compounds 5, 8, 9, 11–15, 18, 21, and 25 were found
to be active in the scPTZ test, which identify substances elevating seizure threshold. Among
these molecules, 9, 11, 12, 14, 15, 18, 21, and 25 showed protection at a dose of 100 mg/kg at
time point of 0.5 h (9, 14, 15, 18, 21, and 25) or in both time intervals (11 and 12). It should
be stressed that 11 and 12 were more potent than ethosuximide, which is known to be a model
AED in PTZ test. The other derivatives (5, 8, 13) exhibited anti-PTZ protection at a dose of
300 mg/kg. In the rotarod test for acute neurological toxicity, 7, 9, 11, 12, 16, 17, 20, 22, 23,
and 26 did not show neurotoxicity in the maximum dose administered (300 mg/kg). Among
these molecules, 16, 17, 20, 22, 23, and 26 were also inactive in the seizure tests. The rest of
8

the compounds revealed neurotoxicity at the highest dose of 300 mg/kg in different
pretreatment times. As a result, compounds 7, 9, 11, and 12 emerged as anticonvulsants
without neurotoxic properties.
Looking for new anticonvulsants with a wide spectrum of activity in the preclinical
studies, six chemically and biologically diversified compounds 7, 8, 18, 19, 21, and 24 were
studied in the psychomotor 6 Hz model of human pharmacoresistant limbic seizures. The
results are shown in Table 2.
Table 2. Anticonvulsant evaluation of compounds in the 6-Hz test (current 32 mA) following
intraperitoneal (i.p.) administration in mice.
Pretreatment timea
Cmpd
0.25 h
2/4
0.5 h
3/4
1 h
3/4
2/4
1/4
2/4
1/4
2/4
2 h
1/4
0/4
0/4
1/4
1/4
1/4
7
4/4
2/4
8
3/4
3/4
2/4
2/4
3/4
4/4
2/4
3/4
18
19
21
24
Dose of 100 mg/kg was administrated i.p. in mice.
a The data indicate: number of mice protected /number of mice tested.
As is shown in Table 2, the most potent anticonvulsant properties were observed for 8
and 19, which displayed two points of 100% protection at 0.25 and 0.5 h, respectively. High
efficacy with two peaks of 75% protection revealed 7 and 18.
In the next step of the pharmacological investigations, for the most potent compounds
in the screening studies (7, 9–12, 18, 19, and 24), the median effective (ED50 MES or/and
PTZ test) and toxic (TD50 rotarod test) doses were determined after intraperitoneal (i.p.)
injection in mice. The quantitative evaluation was performed at previously estimated time of
9

peak effect (TPE). Results of the quantitative tests along with the data for the standard drugs
such as phenytoin, ethosuximide, and valproic acid are shown in Table 3.
Table 3. Quantitative data following i.p. administration in mice.
ED50 MES
(mg/kg)b
ED50 scPTZ
(mg/kg)b
TD50 Rotarod
(mg/kg)b
Cmpd
7*
TPE (h)a
1.0
PIc
87.36
-
>300
>3.43 (MES)
(46.82–197.10)
82.74
40.34
323.34
3.91(MES)
8.0 (scPTZ)
>15.59 (MES)
>2.0 (scPTZ)
1.0
9
(54.53–125.52)
32.08
(29.33–55.51)
(237.66–439.93)
4.0
>250
>500
10*
11
(26.7–36.1)
74.65
(42.28–131.80)
54.13
343.28
(311.62–378.16)
263.21
1.0
–
4.60 (scPTZ)
75.60
(53.84–106.16)
69.05
3.48 (MES)
4.86 (scPTZ)
2.76 (MES)
1.47 (scPTZ)
1.0
12
(37.41–78.30)
129.50
(188.52–367.79)
190.62
0.5
18
(63.54–75.02)
122.04
(114.21–146.84)
(138.42–262.50)
131.66
0.5
–
–
-
1.08 (MES)
4.06 (MES)
8.56 (MES)
2.26 (scPTZ)
19
(100.26–148.56)
70.08
(94.26–183.89)
284.56
0.5
24
(64.77–75.82)
6.65
(242.67– 333.68)
56.9
PHTd
ETXd
VPAd
1.0
(5.42–8.16)
(48.5–66.7)
318.01
140.40
0.25
0.5
-
(115.81–170.21)
239.45
(295.80–341.89)
430.77
252.74
1.70 (MES)
(220.1–290.22)
(209.18–274.10)
(407.92–454.90)
1.80 (scPTZ)
a Time to peak effect.
b Results are represented as mean ± SEM at 95% confidence limit.
c Protection index (TD50/ED50).
d Model AEDs: ethosuximide (ETX), Phenytoin (PHT), Valproic acid (VPA) tested in the same condition.
The analysis of quantitative data revealed that excluding compound 10, all other derivatives
(9, 11, 12, and 18) were more potent than ethosuximide in the scPTZ test. In the MES model,
10

none of the compounds tested was more active than phenytoin used as the reference
antiepileptic drug for MES seizures; however, all the substances were more effective
compared to valproic acid. In the MES test, the most potent was 10 with ED50 value of 32.1
mg/kg, TD50 > 500 mg/kg that resulted in favorable protection index (TD50/ED50) > 15.6. In
the scPTZ test, the best ED50 values revealed 9, namely, 40.3 mg/kg, TD50 = 323.3 mg/ kg,
and PI = 8.0.
Given high activity in the 6-Hz test, compounds 8, 19, 21, and 24 were selected for the
quantification of pharmacological parameters in the aforementioned seizure model. As it is
shown in Table 4, two compounds 19 and 24 were more effective than valproic acid,
nevertheless less active compared to levetiracetam.
Table 4. Quantitative anticonvulsant data test 6–Hz in mice dosed i.p.
ED50 6-Hz
(mg/kg)b
TD50 Rotarod
(mg/kg)b
Cmpd
8
TPE (h)a
0.25
0.5
PIc
>3.29
1.80
1.53
3.15
3.30
>31
151.82
>500
(114.07–235.25)
73.26
131.66
(94.26–183.89)
195.74
19
(57.41–93.48)
128.22
0.5
21
(94.76–173.49)
90.26
(164.56–232.84)
284.56
0.5
24
(66.48–122.54)
130.64
(242.67–333.68)
430.77
VPAd
LEVd
0.5
(117.61–145.19)
15.73
(407.92–454.90)
1
>500
(10.45–23.69)
a Time to peak effect.
b Results are represented as mean ± SEM at 95% confidence limit.
c Protection index (TD50/ED50).
d Model AEDs: Valproic acid (VPA), Levetiracetam (LEV) tested in the same condition.
11

Based on the aforementioned pharmacological data, several SAR conclusions can be
deduced. In general, visibly higher activity was observed for 3-methyl-pyrrolidine-2,5-
diones in comparison to their 3-ethyl-3-methyl- analogs. The most active were compounds
with electronegative fluorine and chlorine atoms or trifluoromethyl group at the 4-
arylpiperazine fragment. It was in agreement with our previous studies that have demonstrated
that the presence of electron-attracting substituents increases anticonvulsant activity. The
replacement of the above substituents by the electron-donating group decreased the
anticonvulsant activity.
2.3. Antinociceptive activity in the formalin hind paw test
Because of anticonvulsant activity of compounds 7, 9, 10, 12, 18, and 24 in
electrically evoked seizures, their analgesic properties were investigated at doses equal to the
respective ED50 values obtained in MES test. A significant antinociceptive activity was
observed for each of the substance tested (Figure 3A–F).
A
B
80
60
40
20
0
80
60
40
20
0
vehicle
vehicle
9
7
***
*
*
**
*
*
Time after formalin (min)
Time after formalin (min)
12

C
D
80
60
40
20
0
80
60
40
20
0
vehicle
vehicle
12
10
**
****
*
*
*
*
**
Time after formalin (min)
Time after formalin (min)
E
F
80
60
40
20
0
80
60
40
20
0
vehicle
vehicle
18
24
*
***
**
*
**
**
Time after formalin (min)
Time after formalin (min)
Figure 3. A–F. Time course of the antinociceptive activity of compounds 7, 9, 10, 12, 18, and
24 in the formalin test. Statistical analysis of the result was conducted using two-way repeated
measures ANOVA, followed by Bonferroni’s multiple comparison. Results compared to
vehicle-treated mice at the same time points: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
In the first (neurogenic) phase, four compounds reduced the duration of the licking response
up to 76%. In this phase, derivative 10, which was tested at the dose of 32.08 mg/kg, had the
highest antinociceptive efficacy (76%, p < 0.0001). Compounds 9, 12, and 24, tested at the
doses of 82.74 mg/kg, 75.60 mg/kg, and 70.08 mg/kg, respectively, reduced the duration of
licking response by 67% (p < 0.001), 61% (p < 0.01), and 40% (p < 0.05), respectively.
Compounds 7 and 18 did not attenuate the nocifensive response in statistically significant
manner. In the second (late) phase of the formalin test, a strong antinociceptive effect was
observed for all compounds because they significantly reduced the duration of the licking
response. The evaluation of the time course of the antinociceptive activity at selected time
13

intervals showed that between 15 and 20 min of the observation, all compounds diminished
the pain responses 7 (95%, p < 0.05), 9 (98%, p < 0.01), 10 (91%, p < 0.05), 12 (100%, p <
0.01), 18 (93%, p < 0.01), and 24 (100%, p < 0.001). Next, between 20 and 25 min of the
assay, a statistically significant reduction of the licking response was observed for derivatives
9 and 12. Their analgesic activity ranged from 95% (12) to 100% (9). Between 25 and 30 min
of the test, all the molecules decreased the pain reaction significantly: 7 (98%, p < 0.05), 9
(96%, p < 0.05), 10 (86%, p < 0.05), 12 (95%, p < 0.05), 18 (92%, p < 0.05), and 24 (99%, p
< 0.01) (Figure 1A–F). In the previous study carried out in the same experimental condition
morphine used as a reference drug significantly reduced the duration of the licking response
in both phases of the formalin test.21,22 Moreover, new anticonvulsant drug – lacosamide
tested previously in our laboratory displayed antinociceptive activity in this test of tonic
pain.23
2.4. Local anesthetic activity
The effect of selected derivatives 7, 9, 10, 12, 18, and 24 on local anesthesia was
examined using the tail immersion test. In this test, compounds 10 and 18 at a concentration
of 2% significantly prolonged the animals’ reaction time to a heat stimulus by 124% and 61%,
respectively. Phenytoin, a well-known sodium channel blocker, in the same concentration
prolonged the time reaction by 84%. Furthermore, mepivacaine, used as reference drug was
highly active in this test. The results obtained are presented in Table 5.
Table 5. Local anesthetic activity in the tail immersion test.
Reaction time (s) to
painful stimulus ( ± SEM)
4.83 ± 0.60
Prolongation
Cmpd
Concentration (%)
of the time reaction (%)
vehicle
0.5 % MC
2
-
5.41 ± 0.53
12
7
14

2
5.66 ± 0.60
17
9
1
6.07 ± 1.13
26
10
10
12
18
24
PHT
2
10.80 ± 1.74**
5.53 ± 1.06
124
14
2
2
7.79 ± 0.95*
7.06 ± 0.76
61
2
46
1
5.72 ± 0.63
18
2
8.90 ± 0.80***
7.26 ± 2.17
84
vehicle
0.5 % MC
-
1
2
14.13 ± 2.67
15.73 ± 2.09**
94.63
116.67
Mepivacainea
Each value represents the mean ± SEM obtained from 8 mice. Significant difference compared to the vehicle-
treated group: t-test for cmpds. 7, 9, 12, 18, and 24 or one-way analysis of variance ANOVA followed by
Dunnett’s post hoc test (cmpd 10 and phenytoin - PHT): *p < 0.05, **p < 0.01, ***p < 0.001.
a Data from the previous study.23
2.5. In vitro radioligand binding studies
Intensive studies into the physiological and biochemical events occurring during
epileptic seizures have provided insight into the molecular mechanisms by which these might
be controlled. The fundamental role in establishing and regulating excitability of CNS
neurons as well as suppression of seizures is ascribed to voltage-gated sodium channels
(VGSCs) and voltage-dependent calcium channels (VDCC).24 Thus, the brain ion channels are
the molecular targets of a number of chemically diverse antiepileptic drugs from which the
most important are phenytoin, lamotrigine, carbamazepine, and oxcarbazepine that are known
to be a sodium channel blockers.25–27 It should be stressed that such mode of action is
characteristic for compounds active in the MES test. Considering the activity of compounds
synthesized in the electrically induced convulsions as well as in pentylenetetrazole test for six
molecules 7, 9, 10, 12, 18, and 24 with various activity in the preclinical studies, their binding
to Na+ and Ca2+ (L-type) channels was assessed in vitro.
15

Table 6. In vitro binding assays (concentration 100 μM).
% Inhibition of control specific bindinga
Voltage-sensitive Na+
Voltage-sensitive L-type Ca2+
Cmpd
channel (site 2)b
channel (antagonist site) c
36.6
28.5
20.9
33.4
32.5
63.5
53.9
13.5
17.2
4.2
7
9
10
12
71.0
30.8
82.4
57.8
18
24
Phenytoind
a
Results showing an inhibition higher than 50% are considered to represent significant effects of the test
compounds; results showing an inhibition between 25% and 50% are indicative of moderate effect; results
showing an inhibition lower than 25% are not considered significant and mostly attributable to variability of the
signal around the control level.
b
Compounds were evaluated in synaptoneurosomal preparations from rat cerebral cortex as inhibitors of the
specific binding of [3H]BTX to the voltage-sensitive sodium channel.
c
Compounds were evaluated in synaptoneurosomal preparations from rat cerebral cortex as inhibitors of the
specific binding of [3H]nitrendipine to the voltage-sensitive Ca2+ channel.
d Reference drug tested in the same conditions.
As indicated in Table 6, the most effective binder to Na+ and Ca2+ (L-type) channels was 24,
which showed activity only in MES test. High affinity to calcium channel demonstrated also
12, which was active in both MES and scPTZ tests. It should be highlighted that 12 and 24
were stronger sodium and calcium channel blockers than phenytoin.
Other compounds revealed only a moderate effect on both ion channels. The lack of the
correlation between in vivo results and binding studies may suggest an additional mechanism
of action responsible for anticonvulsant activity beyond the interaction with Na+ and Ca2+ (L-
type) channels.
3. Conclusion
In the current study, the series of 26 new compounds was synthesized and tested for
anticonvulsant and neurotoxic properties. The obtained results revealed that the majority of
16

compounds exhibited anticonvulsant activity in the MES test and/or in the PTZ test. Several
substances were also active in 6-Hz screen. The most potent compounds were more effective
than ethosuximide and valproic acid used as the reference AEDs. Six molecules showed a
strong antinociceptive activity and two of them also revealed local anesthetic properties. The
in vitro binding studies revealed that the mechanism of anticonvulsant action may be partially
connected with the influence on voltage-sensitive sodium and L-type calcium channels. The
results obtained in the current study proved that compounds based on the pyrrolidine-2,5-
dione template may be recognized as candidates for new effective AEDs.
4. Experimental Part
4.1. General
All the chemicals and solvents were purchased from Merck (Darmstadt, Germany) and
were used without further purification. Melting points (mp:) were determined in open
capillaries on a Büchi 353 melting point apparatus (Büchi Labortechnik, Flawil, Switzerland)
and are uncorrected. The purity and homogeneity of the compounds were confirmed by use of
the thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC).
The TLC was performed on Merck silica gel 60 F254 aluminium sheets (Merck; Darmstadt,
Germany). Spots were detected by their absorption under UV light (λ = 254 nm) and by
visualization with 0.05 mol I2 in 10% HCl. The mass spectra were obtained on Waters
ACQUITY™ TQD system with the TQ Detector (Waters, Milford, USA). The ACQUITY
UPLC BEH C18, 1.7 μm, 2.1  100 mm column was used (Waters, Milford, USA).
Preparative column chromatography was performed using silica gel 60 (particle size 0,063-
0,200; 70-230 Mesh ATM) purchased from Merck (Darmstadt, Germany). 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%
17

1
of the theoretical values. H NMR spectra were obtained in a Varian Mercury spectrometer
(Varian Inc., Palo Alto, CA, USA), in CDCl3 operating at 300 MHz. Chemical shifts are
reported in δ values (ppm) relative to TMS δ = 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), m (multiplet).
4.2. Chemical synthesis
4.2.1. General procedure for the preparation of the 3-methyl- (3) and 3-ethyl-3-methyl-
2,5-dioxo-pyrrolidin-1-yl-acetic acids (4)
2-Methyl- or 2-ethyl-2-methyl-succinic acids (0.04 mol) were dissolved in 20 ml of
water and 2-aminoacetic acid (0.04 mol) was gradually added. The mixtures were heated in a
term-regulated sand bath with simultaneous distillation of water. After complete removal of
water, the temperature of the reaction mixture raised up to 180°C and was maintained for ca.
1.5 h. The crude products, 3-methyl-2,5-dioxo-pyrrolidin-1-yl-acetic acid (3) was
recrystallized from methanol, while 3-ethyl-3-methyl-2,5-dioxo-pyrrolidin-1-yl-acetic acid (4)
was purified by column chromatography (S1: dichloromethane: methanol, 9 : 1, v/v) to afford
intermediates 4 as yellow oils.
4.2.1.1. 3-Methyl-2,5-dioxo-pyrrolidin-1-yl-acetic acid (3). White solid; yield: 82%;
1
Rf=0.58 (S1); mp: 86–88ºC; HPLC: tR=3.2 min.; H NMR (300 MHz, CDCl3): δ 1.37 (d, 3H
CH3, J=6.9 Hz), 2.41 (d, 1H, Hb imide, J=13.5 Hz), 2.95–3.08 (m, 2H, imide), 4.28 (s, 2H,
CH2), 9.32 (brs, 1H, COOH); MS: m/z [M+H]+ calcd for C7H9N1O4:172.15, found: 172.12.
4.2.1.2. 3-Ethyl-3-methyl-2,5-dioxo-pyrrolidin-1-yl-acetic acid (4). Yellow oil, yield: 70%;
Rf=0.46 (S1); HPLC: tR=3.08 min., 1H NMR (300 MHz, CDCl3): δ 0.87 (t, 3H, CH3, J=7.5
18

Hz), 1.29 (s, 3H, CH3), 1.59 (m, 1H, CH2), 1.73 (m, 1H, CH2), 2.57 (dd, 2H, imide, J=7.4
Hz), 4.22 (s, 2H, CH2), 7.44 (brs, 1H, COOH); MS: m/z [M+H]+ calcd for C9H13N1O4: 200.08,
found: 200.10.
4.2.2. General procedure for the synthesis of compounds 5–26
The obtained 3-methyl- or 3-ethyl-3-methyl-2,5-dioxo-pyrrolidin-1-yl-acetic acids (3,
4) (0.01 mol) were dissolved in 20 ml of DMF and N,N-carbonyl-diimidazole (0.01 mol) was
added. The mixtures were stirred for 0.5 h at a room temperature. Afterwards, the appropriate
4-arylpiperazine (0.01 mol) was added. After 24 h of stirring the reaction mixtures were left in
an ice-cold bath. The products were precipitated out with cold water and were purified by
recrystallization from isopropyl alcohol giving the final compounds in yields ranging from
50% to 80%.
4.2.2.1. 3-Methyl-1-[2-oxo-2-(4-phenyl-piperazin-1-yl)-ethyl]-pyrrolidine-2,5-dione (5).
1
White powdery crystals; yield: 58%; Rf=0.71 (S1); HPLC: tR=4.56 min.; mp: 131–132ºC; H
NMR (300 MHz, CDCl3): δ 1.41 (d, 3H, CH3, J=7.2 Hz), 2.43 (d, 1H, imide, J=13.6 Hz),
2.97–3.07 (m, 2H, imide), 3.17 (t, 2H, piperazine, J=5.0 Hz), 3.25 (t, 2H, piperazine, J=5.1
Hz), 3.64 (t, 2H, piperazine, J=5.0 Hz), 3.76 (t, 2H, piperazine, J=5.0 Hz), 4.34 (s, 2H, CH2),
6.90–6.95 (m, 3H, ArH), 7.26–7.32 (m, 2H, ArH); MS: m/z [M+H]+ calcd for C17H21N3O3:
316.16, found: 316.18.
4.2.2.2. 1-{2-[4-(2-Fluorophenyl)-piperazin-1-yl]-2-oxo-ethyl}-3-methyl-pyrrolidine-2,5-
dione (6). White powdery crystals; yield: 65%; Rf =0.70 (S1); HPLC: tR=4.99 min.; mp: 118–
1
120ºC; H NMR (300 MHz, CDCl3): δ 1.41 (d, 3H, CH3, J=7.2 Hz), 2.43 (d, 1H, imide,
J=13.6 Hz), 2.97–3.04 (m, 2H, imide), 3.07 (t, 2H, piperazine, J=5.0 Hz), 3.15 (t, 2H,
19

piperazine, J=5.0 Hz), 3.65 (t, 2H, piperazine, J=5.0 Hz), 3.77 (t, 2H, piperazine, J=5.0 Hz),
13
4.34 (s, 2H, CH2), 6.90–7.10 (m, 4H, ArH); C NMR (75 MHz, CDCl3) δ: 16.77, 34.91,
36.48, 39.48, 42.35, 44.90, 50.13, 116.12, 119.26, 123.27, 124.55, 139.30, 154.09, 157.35,
163.30, 176.13, 180.24; MS: m/z [M+H]+ calcd for C17H20N3O3F1: 334.37, found: 334.35.
4.2.2.3. 1-{2-[4-(4-Fluorophenyl)-piperazin-1-yl]-2-oxo-ethyl}-3-methyl-pyrrolidine-2,5-
dione (7). White powdery crystals; yield: 62%; Rf =0.75 (S1); HPLC: tR=4.79 min.; mp: 166–
1
168ºC; H NMR (300 MHz, CDCl3): δ 1.41 (d, 3H, CH3, J=7.2 Hz), 2.43 (d, 1H, imide,
J=13.6 Hz), 2.97–3.04 (m, 2H, imide), 3.07 (t, 2H, piperazine, J=5.0 Hz), 3.15 (t, 2H,
piperazine, J=5.0 Hz), 3.65 (t, 2H, piperazine, J=5.0 Hz), 3.77 (t, 2H, piperazine, J=5.0 Hz),
4.34 (s, 2H, CH2), 6.86–6.99 (m, 2H, ArH), 7.01–7.27 (m, 2H, ArH); MS: m/z [M+H]+ calcd
for C17H20N3O3F1: 334.37, found: 334.35.
4.2.2.4. 1-{2-[4-(2-Chlorophenyl)-piperazin-1-yl]-2-oxo-ethyl}-3-methyl-pyrrolidine-2,5-
dione (8). White powdery crystals; yield: 68%; Rf =0.73 (S1); HPLC: tR=5.62 min.; mp: 133–
1
134ºC; H NMR (300 MHz, CDCl3): δ 1.41 (d, 3H, CH3, J=7.2 Hz), 2.43 (d, 1H, imide,
J=13.3 Hz), 2.95–3.07 (m, 4H, 2H imide, 2H piperazine), 3.11 (t, 2H, piperazine, J=5.1 Hz),
3.65 (t, 2H, piperazine, J=5.0 Hz) 3.78 (t, 2H, piperazine, J=4.8 Hz), 4.34 (s, 2H, CH2), 6.99–
7.05 (m, 2H, ArH), 7.21–7.27 (m, 1H, ArH), 7.37–7.40 (m, 1H, ArH); 13C NMR (75 MHz,
CDCl3) δ: 16.78, 34.92, 36.49, 39.53, 42.54, 45.04, 50.79, 51.20, 120.60, 124.43, 127.73,
128.96, 130.71, 148.45, 163.37, 176.13, 180.25; MS: m/z [M+H]+ calcd for C17H20N3O3Cl1:
350.12, found: 350.37.
4.2.2.5. 1-{2-[4-(3-Chlorophenyl)-piperazin-1-yl]-2-oxo-ethyl}-3-methyl-pyrrolidine-2,5-
dione (9). White powdery crystals; yield 70%; Rf =0.71 (S1); HPLC: tR=5.68 min.; mp: 139–
20

1
141ºC; H NMR (300 MHz, CDCl3): δ 1.39 (d, 3H, CH3, J=7.2 Hz), 2.42 (d, 1H, imide,
J=13.6 Hz), 2.91–3.06 (m, 2H, imide), 3.18 (t, 2H, piperazine, J=4.7 Hz), 3.26 (d, 2H,
piperazine, J=4.6 Hz), 3.62 (d, 2H, piperazine, J=5.1 Hz), 3.73 (d, 2H, piperazine, J=4.6 Hz),
4.33 (s, 2H, CH2), 6.76–6.88 (m, 3H, ArH), 7.18 (t, 1H, ArH, J=8.33 Hz); MS: m/z [M+H]+
calcd for C17H20N3O3Cl1:350.12, found: 350.37.
4.2.2.6. 1-{2-[4-(4-Chlorophenyl)-piperazin-1-yl]-2-oxo-ethyl}-3-methyl-pyrrolidine-2,5-
dione (10). White powdery crystals; yield: 72%; Rf =0.73 (S1); HPLC: tR=5.61 min.; mp: 140–
1
142ºC; H NMR (300 MHz, CDCl3): δ 1.40 (d, 3H, CH3, J=7.2 Hz), 2.43 (d, 1H, imide,
J=13.6 Hz), 2.97–3.06 (m, 2H, imide), 3.13 (t, 2H, piperazine, J=5.1 Hz), 3.21 (t, 2H,
piperazine, J=5.1 Hz), 3.63 (t, 2H, piperazine J=5.0 Hz), 3.75 (t, 2H, piperazine, J=5.1 Hz),
4.33 (s, 2H, CH2), 6.82–6.87 (m, 2H, ArH), 7.21–7.25 (m, 2H, ArH); MS: m/z [M+H]+ calcd
for C17H20N3O3Cl1: 350.12, found: 350.37.
4.2.2.7. 1-{2-[4-(2,3-Dichlorophenyl)-piperazin-1-yl]-2-oxo-ethyl}-3-methyl-pyrrolidine-
2,5-dione (11). White powdery crystals; yield: 69%; Rf =0.73 (S1); HPLC: tR=6.29 min.; mp:
148–150ºC; 1H NMR (300 MHz, CDCl3): δ 1.40 (d, 3H, CH3, J=6.9 Hz), 2.43 (d, 1H, imide,
J=13.6 Hz), 2.99–3.09 (m, 6H, 2H, imide, 4H piperazine), 3.66 (brs, 2H piperazine), 3.77
(brs, 2H, piperazine), 4.33 (s, 2H CH2), 6.92 (d, 1H, ArH, J=7.2 Hz), 7.14–7.19 (m, 2H,
ArH); MS: m/z [M+H]+ calcd for C17H19N3O3Cl2: 384.27, found: 384.34.
4.2.2.8. 1-{2-[4-(3,4-Dichlorophenyl)-piperazin-1-yl]-2-oxo-ethyl}-3-methyl-pyrrolidine-
2,5-dione (12). White powdery crystals; yield: 77%; Rf =0.70 (S1); HPLC: tR=6.31 min; mp:
136–138°C; 1H NMR (300 MHz, CDCl3): δ 1.20 (d, 3H, CH3, J=6.6 Hz), 2.36 (d, 1H, imide,
J=13.3 Hz), 2.87–2.99 (m, 2H, imide), 3.17 (d, 2H, piperazine, J=4.8 Hz), 3.25 (brs, 2H,
21

piperazine), 3.53 (brs, 2H, piperazine), 3.62 (d, 2H, piperazine, J=4.8 Hz), 4.29 (s, 2H, CH2),
6.92–6.96 (m, 1H, ArH), 7.15 (d, 1H, ArH, J=2.5 Hz), 7.40 (d, 1H, ArH, J=8.9 Hz); MS: m/z
[M+H]+ calcd for C17H19N3O3Cl2: 384.27, found: 384.34.
4.2.2.9. 1-{2-[4-(3-Trifluoromethylphenyl)-piperazin-1-yl]-2-oxo-ethyl}-3-methyl-
pyrrolidine-2,5-dione (13). White powdery crystals; yield: 80%; Rf =0.77 (S1); HPLC:
1
tR=6.05 min.; mp: 123–124ºC; H NMR (300 MHz, CDCl3): δ 1.41 (d, 3H, CH3, J=7.2 Hz),
2.44 (d, 1H, imide, J=13.6 Hz), 2.95–3.07 (m, 2H, imide), 3.23 (t, 2H, piperazine, J=5.1 Hz),
3.31 (t, 2H, piperazine, J=5.0 Hz), 3.66 (t, 2H, piperazine J=5.0 Hz), 3.77 (t, 2H, piperazine,
J=5.1 Hz), 4.34 (s, 2H, CH2), 7.06–7.16 (m, 3H, ArH), 7.38 (t, 1H, ArH, J=7.9 Hz); 13C NMR
(75 MHz, CDCl3) δ: 16.75, 34.92, 34.94, 36.47, 39.45, 41.96, 44.45, 48.84, 112.91, 116.87,
119.42, 129.76, 139.30, 150.86, 163.43, 176.11, 180.23; MS: m/z [M+H]+ calcd for
C18H20N3O3F3: 383.36, found: 384.40.
4.2.2.10. 1-{2-[4-(2-Methylphenyl)-piperazin-1-yl]-2-oxo-ethyl}-3-methyl-pyrrolidine-2,5-
dione (14). White powdery crystals; yield: 79%; Rf =0.78 (S1); HPLC: tR=5.67 min; mp: 115–
1
117°C; H NMR (300 MHz, CDCl3): δ 1.41 (d, 3H, CH3, J=7.7 Hz), 2.32 (s, 3H, CH3), 2.43
(d, 1H, imide, J=13.6 Hz), 2.89 (t, 2H, imide, J=5.0 Hz), 2.95–3.05 (m, 4H, piperazine), 3.62
(t, 2H, piperazine, J=4.8 Hz), 3.74 (t, 2H, piperazine, J=4.5 Hz), 4.34 (s, 2H, CH2), 6.98–7.05
(m, 2H, ArH), 7.15–7.21 (m, 2H, ArH); 13C NMR (75 MHz, CDCl3) δ: 16.79, 17.78, 34.92,
36.50, 39.56, 42.81, 45.28, 51.64, 119.29, 123.92, 126.74, 131.18, 132.72, 150.65, 163.37,
176.16, 180.28; MS: m/z [M+H]+ calcd for C18H23N3O3: 330.39, found: 330.42.
4.2.2.11. 1-{2-[4-(3-Methylphenyl)-piperazin-1-yl]-2-oxo-ethyl}-3-methyl-pyrrolidine-2,5-
dione (15). White powdery crystals; yield: 75%; Rf =0.71 (S1); HPLC: tR=5.10 min.; mp: 126–
22

1
128°C; H NMR (300 MHz, CDCl3): δ 1.40 (d, 3H, CH3, J=7.2 Hz), 2.32 (s, 3H, CH3), 2.43
(d, 1H, imide, J=13.3 Hz), 2.94–3.06 (m, 2H, imide), 3.16 (t, 2H, piperazine, J=5.1 Hz), 3.23
(t, 2H, piperazine, J=5.1 Hz), 3.63 (t, 2H, piperazine, J=5.1 Hz), 3.74 (t, 2H, piperazine, J=5.0
Hz), 4.33 (s, 2H, CH2), 6.72–6.75 (m, 3H, ArH), 7.15–7.20 (m, 1H, ArH); 13C NMR (75 MHz,
CDCl3) δ: 16.78, 21.72, 34.91, 36.48, 39.50, 42.21, 44.69, 49.34, 49.52, 113.89, 117.67,
121.64, 129.09, 138.99, 150.83, 163.33, 176.13, 180.25; MS: m/z [M+H]+ calcd for
C18H23N3O3: 330.39, found: 330.42.
4.2.2.12. 3-Ethyl-3-methyl-1-[2-oxo-2-(4-phenyl-piperazin-1-yl)-ethyl]-pyrrolidine-2,5-
dione (16). White powdery crystals; yield: 65%; Rf =0.84 (S1); HPLC: tR=5.65 min.; mp: 134–
135°C; 1H NMR (300 MHz, CDCl3): δ 0.95 (t, 3H, CH3, J=7.5 Hz), 1.36 (s, 3H, CH3), 1.52–
1.60 (m, 2H, CH2), 2.50 (d, 1H imide, J=18.2 Hz), 2.73 (d, 1H, imide, J=18.8 Hz), 3.17 (t,
2H, piperazine, J=5.1 Hz), 3.24 (t, 2H, piperazine, J=4.6 Hz), 3.64 (t, 2H piperazine, J=5.2
Hz), 3.74 (t, 2H, piperazine, J=5.3 Hz), 4.33 (s, 2H, CH2), 6.88–6.97 (m, 2H, ArH), 7.26–7.32
(m, 3H, ArH); MS: m/z [M+H]+ calcd for C19H25N3O3:344.19, found: 344.12.
4.2.2.13. 1-{2-[4-(2-Fluorophenyl)-piperazin-1-yl]-2-oxo-ethyl}-3-ethyl-3-methyl-
pyrrolidine-2,5-dione (17). White powdery crystals; yield: 55%; Rf =0.83 (S1); HPLC:
1
tR=5.90 min.; mp: 118–119°C; H NMR (300 MHz, CDCl3): δ 0.95 (t, 3H, CH3, J=7.4 Hz),
1.36 (s, 3H, CH3), 1.60–1.84 (m, 2H, CH2), 2.50 (d, 1H imide, J=18.2 Hz), 2.73 (d, 1H,
imide, J=18.3 Hz), 3.06 (t, 2H, piperazine, J=5.1 Hz), 3.14 (t, 2H, piperazine, J=4.8 Hz), 3.64
(t, 2H piperazine, J=5.4 Hz), 3.75 (t, 2H, piperazine, J=5.1 Hz), 4.33 (s, 2H, CH2), 6.90–7.02
(m, 2H, ArH), 7.03–7.12 (m, 2H, ArH); 13C NMR (75 MHz, CDCl3) δ: 8.71, 23.93, 30.80,
39.39, 40.43, 42.33, 44.54, 50.14, 116.11, 119.24, 123.24, 124.55, 139.31, 154.08, 157.34,
163.33, 175.85, 182.52; MS: m/z [M+H]+ calcd for C19H24N3O3F1: 362.18, found: 362.13.
23

4.2.2.14. 1-{2-[4-(4-Fluorophenyl)-piperazin-1-yl]-2-oxo-ethyl}-3-ethyl-3-methyl-
pyrrolidine-2,5-dione (18). White powdery crystals; yield: 50%; Rf =0.73 (S1); HPLC:
1
tR=5.78 min.; mp: 122–124°C; H NMR (300 MHz, CDCl3): δ 0.95 (t, 3H, CH3, J=7.4 Hz),
1.36 (s, 3H, CH3), 1.56–1.84 (m, 2H, CH2), 2.50 (d, 1H imide, J=18.2 Hz), 2.73 (d, 1H,
imide, J=18.3 Hz), 3.08 (t, 2H, piperazine, J=5.2 Hz), 3.15 (t, 2H, piperazine, J=5.0 Hz), 3.63
(t, 2H piperazine, J=4.9 Hz), 3.75 (t, 2H, piperazine, J=5.3 Hz), 4.32 (s, 2H, CH2), 6.86–6.91
(m, 2H, ArH), 6.96–7.02 (m, 2H, ArH); MS: m/z [M+H]+ calcd for C19H24N3O3F1: 362.18,
found: 362.13.
4.2.2.15. 1-{2-[4-(2-Chlorophenyl)-piperazin-1-yl]-2-oxo-ethyl}-3-ethyl-3-methyl-
pyrrolidine-2,5-dione (19). White powdery crystals; yield: 55%; Rf =0.76 (S1); HPLC:
1
tR=6.57 min.; mp: 124–126°C; H NMR (300 MHz, CDCl3): δ 0.95 (t, 3H, CH3, J=7.4 Hz),
1.37 (s, 3H, CH3), 1.54–1.80 (m, 2H, CH2), 2.51 (d, 1H imide, J=18.5 Hz), 2.74 (d, 1H,
imide, J=18.2 Hz), 3.03 (t, 2H, piperazine, J=4.9 Hz), 3.11 (t, 2H, piperazine, J=4.9 Hz), 3.66
(t, 2H piperazine, J=5.4 Hz), 3.77 (t, 2H, piperazine, J=4.8 Hz), 4.33 (s, 2H, CH2), 7.00–7.03
(m, 2H, ArH), 7.21–7.40 (m, 2H, ArH); MS: m/z [M+H]+ calcd for C19H24N3O3Cl1: 378.15,
found: 378.08.
4.2.2.16. 1-{2-[4-(3-Chlorophenyl)-piperazin-1-yl]-2-oxo-ethyl}-3-ethyl-3-methyl-
pyrrolidine-2,5-dione (20). White powdery crystals; yield: 65%; Rf =0.83(S1); HPLC:
1
tR=6.59 min.; mp: 107–109°C; H NMR (300 MHz, CDCl3): δ 0.95 (t, 3H, CH3, J=7.4 Hz),
1.36 (s, 3H, CH3), 1.62–1.84 (m, 2H, CH2), 2.50 (d, 1H imide, J=18.2 Hz), 2.73 (d, 1H,
imide, J=18.5 Hz), 3.18 (t, 2H, piperazine, J=5.1 Hz), 3.25 (t, 2H, piperazine, J=5.0 Hz), 3.63
(t, 2H, piperazine, J=5.1 Hz), 3.75 (t, 2H, piperazine, J=4.6 Hz), 4.32 (s, 2H, CH2), 6.77–6.88
24

(m, 3H, ArH), 7.16–7.19 (m, 1H, ArH); 13C NMR (75 MHz, CDCl3) δ: 8.70, 23.92, 30.80,
39.36, 40.43, 41.93, 44.43, 44.54, 48.70, 48.81, 114.50, 116.47, 130.20, 135.02, 151.76,
163.44, 175.81, 182.49; MS: m/z [M+H]+ calcd for C19H24N3O3Cl1: 378.15: found: 378.08.
4.2.2.17. 1-{2-[4-(4-Chlorophenyl)-piperazin-1-yl]-2-oxo-ethyl}-3-ethyl-3-methyl-
pyrrolidine-2,5-dione (21). White powdery crystals; yield: 57%; Rf =0.62 (S1); HPLC:
1
tR=6.51 min.; mp: 141–143°C; H NMR (300 MHz, CDCl3): δ 0.95 (t, 3H, CH3, J=7.4 Hz),
1.36 (s, 3H, CH3), 1.67–1.84 (m, 2H, CH2), 2.50 (d, 1H imide, J=18.2 Hz), 2.73 (d, 1H,
imide, J=18.2 Hz), 3.13 (t, 2H, piperazine, J=5.4 Hz), 3.21 (t, 2H, piperazine, J=5.1 Hz), 3.63
(t, 2H piperazine, J=5.3 Hz), 3.75 (t, 2H, piperazine, J=5.1 Hz), 4.32 (s, 2H, CH2), 6.82–6.87
(m, 2H, ArH), 7.21–7.26 (m, 1H, ArH); MS: m/z [M+H]+ calcd for C19H24N3O3Cl1: 378.15,
found: 378.08.
4.2.2.18. 1-{2-[4-(2,3-Dichlorophenyl)-piperazin-1-yl]-2-oxo-ethyl}-3-ethyl-3-methyl-
pyrrolidine-2,5-dione (22). White powdery crystals; yield: 58%; Rf =0.76 (S1); HPLC:
1
tR=7.15 min.; mp: 148–150°C; H NMR (300 MHz, CDCl3): δ 0.95 (t, 3H, CH3, J=7.5 Hz),
1.37 (s, 3H, CH3), 1.67–1.85 (m, 2H, CH2), 2.51 (d, 1H imide, J=18.2 Hz), 2.73 (d, 1H,
imide, J=18.2 Hz), 3.02 (t, 2H, piperazine, J=5.1 Hz), 3.10 (t, 2H, piperazine, J=5.2 Hz), 3.66
(t, 2H piperazine, J=5.1 Hz), 3.78 (t, 2H, piperazine, J=4.7 Hz), 4.33 (s, 2H, CH2), 6.92–6.95
(m, 1H, ArH), 7.17–7.21 (m, 2H, ArH); 13C NMR (75 MHz, CDCl3) δ: 8.70, 23.92, 30.81,
39.41, 39.41, 40.45, 42.48, 44.55, 44.99, 50.89, 51.29, 118.83, 125.35, 127.60, 134.18,
150.38, 163.46, 175.85, 182.52; MS: m/z [M+H]+ calcd for C19H23N3O3Cl2: 412.11, found:
412.04.
25

4.2.2.19. 1-{2-[4-(3,4-Dichlorophenyl)-piperazin-1-yl]-2-oxo-ethyl}-3-ethyl-3-methyl-
pyrrolidine-2,5-dione (23). White powdery crystals; yield: 71%; Rf =0.84 (S1); HPLC:
1
tR=7.15 min.; mp: 140–142°C; H NMR (300 MHz, CDCl3): δ 0.95 (t, 3H, CH3, J=7.5 Hz),
1.36 (s, 3H, CH3), 1.59–1.84 (m, 2H, CH2), 2.50 (d, 1H imide, J=18.2 Hz), 2.73 (d, 1H,
imide, J=18.2 Hz), 3.16 (t, 2H, piperazine, J=4.9 Hz), 3.23 (t, 2H, piperazine, J=4.9 Hz), 3.63
(t, 2H piperazine, J=5.4 Hz), 3.74 (t, 2H, piperazine, J=4.9 Hz), 4.32 (s, 2H, CH2), 6.74 (dd,
1H, ArH, J=2.9 Hz), 6.96 (d, 1H, ArH, J=2.8 Hz), 7.30 (d, 1H, ArH, J=9.0 Hz); 13C NMR (75
MHz, CDCl3) δ: 8.71, 23.93, 30.81, 39.35, 40.43, 41.85, 44.35, 44.56, 48.71, 48.83, 115.94,
118.00, 123.33, 130.62, 132.95, 150.09, 163.44, 175.82, 182.50; MS: m/z [M+H]+ calcd for
C19H23N3O3Cl2: 412.11, found: 412.04.
4.2.2.20. 1-{2-[4-(3-Trifluoromethylphenyl)-piperazin-1-yl]-2-oxo-ethyl}-3-ethyl-3-
methyl-pyrrolidine-2,5-dione (24). White powdery crystals; yield: 65%; Rf =0.73 (S1);
HPLC: tR=6.88 min.; mp: 128–130°C; 1H NMR (300 MHz, CDCl3): δ 0.95 (t, 3H, CH3, J=7.5
Hz), 1.36 (s, 3H, CH3), 1.67–1.85 (m, 2H, CH2), 2.51 (d, 1H imide, J=18.2 Hz), 2.73 (d, 1H,
imide, J=18.2 Hz), 3.23 (t, 2H, piperazine, J=5.2 Hz), 3.30 (t, 2H, piperazine, J=5.1 Hz), 3.66
(t, 2H piperazine, J=5.0 Hz), 3.77 (t, 2H, piperazine, J=5.1 Hz), 4.33 (s, 2H, CH2), 7.05–7.16
(m, 3H, ArH), 7.38 (t, 1H, ArH, J=7.90 Hz); MS: m/z [M+H]+ calcd for C20H24N3O3F3:
412.18, found: 412.11.
4.2.2.21. 1-{2-[4-(2-Methylphenyl)-piperazin-1-yl]-2-oxo-ethyl}-3-ethyl-3-methyl-
pyrrolidine-2,5-dione (25). White powdery crystals; yield: 60%; Rf =0.53 (S1); HPLC:
1
tR=6.65 min.; mp: 137–139°C; H NMR (300 MHz, CDCl3): δ 0.95 (t, 3H, CH3, J=7.5 Hz),
1.37 (s, 3H, CH3), 1.67–1.85 (m, 2H, CH2), 2.32 (s, 3H, CH3), 2.51 (d, 1H, imide, J=18.2 Hz),
2.74 (d, 1H, imide, J=18.2 Hz), 2.89 (t, 2H, piperazine, J=5.0 Hz), 2.97 (t, 2H, piperazine,
26

J=5.0 Hz), 3.62 (t, 2H piperazine, J=5.0 Hz), 3.74 (brs, 2H, piperazine), 4.33 (s, 2H, CH2),
6.98–7.05 (m, 2H, ArH), 7.18–7.21 (m, 2H, ArH); 13C NMR (75 MHz, CDCl3) δ: 8.72, 17.78,
23.94, 30.81, 39.47, 40.45, 42.80, 44.55, 45.27, 51.48, 51.66, 119.29, 123.92, 126.73, 131.17,
132.72, 150.67, 163.42, 175.89, 182.56; MS: m/z [M+H]+ calcd for C20H27N3O3: 358.21,
found: 358.14.
4.2.2.22. 1-{2-[4-(3-Methylphenyl)-piperazin-1-yl]-2-oxo-ethyl}-3-ethyl-3-methyl-
pyrrolidine-2,5-dione (26). White powdery crystals; yield: 52%; Rf =0.38 (S1); HPLC:
1
tR=5.94 min.; mp: 110–112°C; H NMR (300 MHz, CDCl3): δ 0.95 (t, 3H, CH3, J=7.7 Hz),
1.36 (s, 3H, CH3), 1.62–1.84 (m, 2H, CH2), 2.32 (s, 3H, CH3), 2.50 (d, 1H imide, J=18.2 Hz),
2.73 (d, 1H, imide, J=18.2 Hz), 3.16 (t, 2H, piperazine, J=5.2 Hz), 3.23 (t, 2H, piperazine,
J=5.0 Hz), 3.63 (t, 2H piperazine, J=5.2 Hz), 3.75 (t, 2H, piperazine, J=5.0 Hz), 4.33 (s, 2H,
CH2), 6.72–6.75 (m, 3H, ArH), 7.17 (t, 1H, ArH, J=7.7 Hz); MS: m/z [M+H]+ calcd for
C20H27N3O3: 358.21, found: 358.14.
4.3. Pharmacology
Male albino mice (CD-1 strain) weighing 16–26 g were used as experimental animals.
The animals were housed in cages at room temperature of 22 ± 2°C, under a light/dark (12/12)
cycle and had free access to food and water. Ambient temperature of the room and humidity
were kept consistent throughout all tests. For the experiments, the animals were randomly
selected. Each group consisted of three-four animals (anticonvulsant screening) and each
mouse was used only once. 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 anticonvulsant activity and neurotoxicity assessment at 0.5 and 4
hours after administration. Compounds 5–8, 10, and 13 were pharmacologically pre-evaluated
27

within the Antiepileptic Drug Development (ADD) Program, realized in NINDS/NIH,
Rockville, USA, using procedures described elsewhere.28 Compounds 9, 11, 12, and 14–26
were tested at the Department of Pharmacodynamics, Jagiellonian University Medical
College. All the procedures were approved by the Local Ethical Committee on Animal
Testing at the Jagiellonian University in Krakow (no 16/2014 and 100/2014).
4.3.1. The maximal electroshock test (MES)
In the MES screen, electroconvulsions were produced by an electrical stimulus of 0.2 s
in duration at a frequency of 50 Hz delivered via standard auricular electrodes by an
electroshock generator (Hugo Sachs rodent shocker, Germany). The tonic hind-limb extension
was taken as the end-point. Mice not displaying hind-limb tonic extension were considered
protected from seizure.29
4.3.2. Subcutaneous pentylenetetrazole seizure test (scPTZ)
ScPTZ-induced seizure was performed by subcutaneously injection of
pentylenetetrazole (Sigma Aldrich, Poland) at a dose of 85 mg/kg. This produced clonic
convulsions lasting for at least five seconds in 97% of animal tested. The absence of clonic
convulsions in the observed time period of 30 min was interpreted as the compound’s ability
to protect against PTZ-induced seizure.30
4.3.3. The neurological toxicity (NT)
Neurological toxicity induced by compound was detected in mice using standardized
rotarod test. Untreated control mice when placed on the rod, can maintain their equilibrium
for a prolonged time period. The acute motor impairment can be demonstrated by the inability
of animal to maintain equilibrium on the rod revolting at 10 rpm for at least 1 minute.31
28

4.3.4. The 6–Hz model
This screen was carried out according to the protocol originally described by Brown et
al.32 and more recently by Barton et al.33 and Kaminski et al.34 It is an alternative electroshock
paradigm that uses low–frequency (6 Hz), long–duration (3 s) electrical stimulation. Corneal
stimulation (0.2 ms–duration monopolar rectangular pulses at 6–Hz for 3 s) was delivered by
a constant–current device. During the stimulation, mice were manually restrained and released
into the observation cage immediately after the current application. The seizures manifest in
“stunned” posture associated with rearing, forelimb, automatic movements and clonus,
twitching of the vibrissae and Straub–tail. The duration of the seizure activity ranges from 60
to 120 s in untreated animals. At the end of the 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.34
4.3.5. Quantification Studies
The ED50 is defined as the dose of a drug protecting 50% of animals against the MES,
scPTZ and 6-Hz seizures. Groups of six animals received various doses of the compound until
at least three points were established in the range of 10-90% seizure protection or minimal
motor impairment. Both ED50 and TD50 values with 95% confidence limits were calculated by
probit analysis.35 The PI (protective index) value was calculated as the ratio of TD50 to ED50
(PI = TD50/ED50).
4.3.6. Antinociceptive activity – the formalin test
Antinociceptive activity in the formalin hind paw test was examined according to the
method previously described.36 In mice intraplantarly injection of diluted formalin produces a
29