Design, synthesis, and anticonvulsant activity of N-phenylamino derivatives of 3,3-dialkyl-pyrrolidine-2,5-diones and hexahydro-isoindole-1,3-diones

Article abstract of DOI:10.1016/j.bmc.2008.03.037

In the present study on the development of new anticonvulsants, the library of differently substituted N-phenylamino pyrrolidine-2,5-dione and hexahydro-isoindole-1,3-dione derivatives was synthesized. The anticonvulsant activity of all the compounds was evaluated using the maximal electroshock (MES) and pentylenetetrazole (scPTZ) screens, which are the most widely employed seizure models for early identification of candidate anticonvulsants. Their neurotoxicity was determined applying the rotorod test. The pharmacological results revealed that the majority of compounds were effective in electrical (MES) and/or pentylenetetrazole induced seizure (scPTZ) models. The quantitative in vivo anticonvulsant evaluation of N-phenylamino-3,3-dimethyl-pyrrolidine-2,5-dione (15), conducted at the time of peak pharmacodynamic activity (TPE), showed the MES ED₅₀ value of 69.89 mg/kg in rats. The median toxic dose (TD₅₀) was 500 mg/kg, providing compound 15 with a protective index (TD₅₀/ED₅₀) of 7.15 in the MES test.

Full text of DOI:10.1016/j.bmc.2008.03.037

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4921–4931
Design, synthesis, and anticonvulsant activity
of N-phenylamino derivatives of 3,3-dialkyl-pyrrolidine-2,5-diones
and hexahydro-isoindole-1,3-diones
*
´
Krzysztof Kaminski and Jolanta Obniska
Department of Medicinal Chemistry, Jagiellonian University, Medical College, 9 Medyczna Street, 30-688 Krako´w, Poland
Received 18 January 2008; revised 10 March 2008; accepted 14 March 2008
Available online 17 March 2008
Abstract—In the present study on the development of new anticonvulsants, the library of differently substituted N-phenylamino pyr-
rolidine-2,5-dione and hexahydro-isoindole-1,3-dione derivatives was synthesized. The anticonvulsant activity of all the compounds
was evaluated using the maximal electroshock (MES) and pentylenetetrazole (scPTZ) screens, which are the most widely employed
seizure models for early identification of candidate anticonvulsants. Their neurotoxicity was determined applying the rotorod test.
The pharmacological results revealed that the majority of compounds were effective in electrical (MES) and/or pentylenetetrazole
induced seizure (scPTZ) models. The quantitative in vivo anticonvulsant evaluation of N-phenylamino-3,3-dimethyl-pyrrolidine-
2,5-dione (15), conducted at the time of peak pharmacodynamic activity (TPE), showed the MES ED50 value of 69.89 mg/kg in rats.
The median toxic dose (TD50) was 500 mg/kg, providing compound 15 with a protective index (TD50/ED50) of 7.15 in the MES test.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
or alkyl substituents attached to the heterocyclic
system.4–6 This common template is present in the struc-
tures of older generation of AEDs such as phenobarbital
(1), primidone (2) or phenytoin (3) (Fig. 1), which are
known to be active against maximal electroshock seizure
test (MES), the experimental animal model for general-
ized tonic–clonic epilepsy. The extensive SAR studies of
AEDs revealed, however, that the phenyl substituents
attached at the heteroatomic imide or cyclic amide ring
are not necessary to retain the anticonvulsant activity.
An example of such molecules is ethosuximide (4), re-
cently marked AED–levetiracetam (5) or compounds
being currently under the clinical trials, namely briva-
racetam (ucb 34714) (6) and seletracetam (ucb 44212)
(7)7–10 (Fig. 1). In contrast to the drugs described above
these molecules were inactive in the MES screen. They
revealed protection in the subcutaneous pentylenetetra-
zole seizure test (scPTZ)-4, the animal model of absence
epilepsy, or in secondarily generalized motor seizures
screen in corneally kindled mice, 5–7, which is claimed
to detect compounds that can act to prevent the
development of seizures. Such substances could be
termed ‘antiepileptogenetic’.11
Epilepsy, a common neurological disorder characterized
by recurrent spontaneous seizures arising from excessive
electrical activity in some portion of the brain, is a
major, worldwide, public health problem, which affects
approximately 1% of the population.1 Despite the
increasing understanding of the pathogenesis of seizures
up to 30% of epilepsies are poorly treated with the avail-
able antiepileptic drugs (AEDs). Moreover, a large num-
ber of new AEDs marked during recent years did not
change the proportion of patients responding to the
treatment and many of those medications cause serious
side effects, which include ataxia, nausea, mental
dulling, and hepatotoxicity.2,3 Therefore, with all of
the above findings in mind the continued search for safer
and more effective AEDs is urgently necessary.
One of the important core fragments of anticonvulsants
is defined by a nitrogen heteroatomic system, usually
a cyclic imide, at least of one carbonyl group and phenyl
Keywords: Anticonvulsant activity; Pyrrolidine-2,5-diones; Succini-
mides; In vivo studies.
Previous studies from our laboratory have
demonstrated the potent anticonvulsant activity
among the N-phenylamino-azaspiranes, from which
*
Corresponding author. Tel.: +48 12 658 82 16; fax: +48 12 657 02
62; e-mail: kaminskik@cm-uj.krakow.pl
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.03.037

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K. Kaminski, J. Obniska / Bioorg. Med. Chem. 16 (2008) 4921–4931
4922
O
O
H
N
NH
NH
O
O
N
N
N
H
O
O
O
H
H
Primidone (2)
Phenytoin (3)
Phenobarbital (1)
F
F
O
O
O
N
N
N
O
NH2
NH2
NH2
N
O
H
H
H
H
O
O
O
Ethosuximide (4)
Levetiracetam (5)
Brivaracetam (6)
Seletracetam (7)
O
N
O
N
H
8
ED50=76.27 mg/kg (MES test)
PI=3.44
Figure 1. Structures of known anticonvulsants and model compound 8.
the N-phenylamino-2-azaspiro[4.4]nonane-1,3-dione (8)
showed ED50 = 76.27 mg/kg and a protective index PI
(TD50/ED50) of 3.44 in the MES test in rats.12,13 Fol-
lowing these results, as part of our efforts to design
new anticonvulsant agents which would be active both
in MES and in scPTZ tests and according to the fore-
going effective in different types of seizures, in the
present study we have synthesized a library of com-
pounds with N-phenylamino pyrrolidine-2,5-dione sys-
tem as a core fragment. At the position-3 of the
succinimide ring we have introduced different alkyl
substituents, which approximate the designed mole-
cules to the structure of ethosuximide, one of the
not numerous drugs effective in absence epilepsy.14
On the other hand, we have changed additionally
the manner of connection between the cyclohexane
ring and the imide system from the described spiro
carbon atom for two common carbon atoms observed
for bicyclic hexahydro-isoindole-1,3-diones.12
Germany) (14), as reaction substrate. Titled imides
15–51 were obtained in a simple one-pot cyclization
reaction of the prepared 2,2-dialkyl-succinic acids
(9–13) or 1,2-cyclohexanedicarboxylic anhydride (14)
with appropriately substituted phenyl-hydrazines
(15–50) or methyl-hydrazine (51) by heating them at
ca. 180 ꢁC for 1 h. The crude products were crystallized
from isopropanol and purified by column chromatogra-
phy, giving the final compounds in yields ranging from
61% to 81%. Their purity was assessed by TLC and gra-
dient HPLC chromatography. The structures of com-
pound synthesized were confirmed by both spectral
and elemental analysis. The detailed physical and ana-
lytical data are listed in Section 4.
2.2. Anticonvulsant activity
The preclinical discovery and development of new chem-
ical 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 rou-
tinely that include the maximal electroshock seizure
(MES), the subcutaneous pentylenetetrazole (scPTZ)
and the kindling model. From these tests the MES and
scPTZ screens are recognized as the ‘gold standards’ in
the early stages of testing.17,18
2. Results and discussion
2.1. Chemistry
The synthesis of compounds 15–51 was accomplished as
presented in Scheme 1. The starting materials for 15–44,
2,2-dimethyl- (9), 2-ethyl-2-methyl- (10), 2-methyl-2-
propyl- (11), 2-butyl-2-methyl- (12) or 2,2-diethyl- (13)
succinic acid derivatives were prepared by the using
methods reported elsewhere.15,16 Compounds 45–50
were synthesized by use of commercially available 1,2-
cyclohexanedicarboxylic anhydride (Merck, Darmstadt,
The profile of anticonvulsant activity of all the com-
pounds was established in the maximal electroshock
(MES) and subcutaneous pentylenetetrazole (scPTZ)
tests, after intraperitoneal 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.

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K. Kaminski, J. Obniska / Bioorg. Med. Chem. 16 (2008) 4921–4931
4923
H
O
N
H2N
N
O
180oC, 1 h
N
H
51
R2
H
N
R2
R1
H2N
O
R
N
O
R1
HOOC
COOH
N
180oC, 1 h
R
H
9-13
9: R1=R2=CH3, cmpd 15-21, 51
15-44
cmpd
R
10: R1=CH3, R2=C2H5, cmpd 22-29
11: R1=CH3, R2=C3H7, cmpd 30-34
12: R1=CH3, R2=C4H9, cmpd 35-38
13: R1=C2H5, R2=C2H5, cmpd 39-44
H
15, 22, 39
23
2-CH3
4-CH3
2-Cl
16, 24, 40
17, 25, 30, 35, 41
18, 26, 31
3-Cl
4-Cl
19, 27, 32, 36, 42
20, 28, 33, 37, 43
21, 29, 34, 38, 44
2,4-Cl
4-Br
H
N
H2N
O
R
N
O
180oC, 1.5 h
N
H
O
R
O
O
14
45-50
cmpd
R
H
45
46
47
48
49
50
4-CH3
2-Cl
3-Cl
4-Cl
2,4-Cl
Scheme 1. General method for the synthesis of succinimides 15–51.
Several molecules were also screened at 1 h (21, 28, and
43) and 2 h (42). The acute neurological toxicity (NT)
was determined in the minimal motor impairment-roto-
rod screen. The results are shown in Table 1.
22–26, 29–31, 35, 40, and 45 provided anti-MES protec-
tion at a dose of 300 mg/kg. The majority of these com-
pounds showed activity only at 0.5 h, indicating that
they have rapid onset and short duration of anticonvul-
sant action. From these series 19, 21, 28, 32, 34, and 43
were active in the MES model both at 0.5 and at 4 h,
whereas 20 and 50 showed activity only at 4 h. Deriva-
tives 21, 28, 42, and 43 revealed the anti-MES activity
at a dose of 100 mg/kg in different time periods, namely
1 h (21, 28, and 43) and 2 h (42), additionally.
The initial anticonvulsant evaluation showed that
excluding molecules 36, 39, 44, 46, 47, and 51, devoid
of anticonvulsant activity, all other derivatives were
effective in ip MES and/or scPTZ screens.
Compounds that revealed protection in the MES test,
indicative of the ability of substance to prevent seizure
spread, at a dose of 100 mg/kg include 17–21, 27, 28,
32, 34, 37, 42, 43, and 50, whereas molecules 15, 16,
Compounds 15–17, 19, 20, 22, 24, 27–35, 37, 38, 41–43,
45, 48, and 49 were found to be active in the scPTZ test,
which identify substances elevating seizure threshold.

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K. Kaminski, J. Obniska / Bioorg. Med. Chem. 16 (2008) 4921–4931
4924
Table 1. The results after intraperitoneal administration to mice
22, 24, 27, 30–32, 34, 35, 38, 41, 45, and 48 showed
activity against scPTZ screen at a dose of 300 mg/kg
at 0.5 h.
Compound
Intraperitoneal injection in micea
MESb scPTZc NTd
0.5 h 4 h 0.5 h 4 h 0.5 h 4 h
logkw
As shown in Table 1, the majority of compounds were
effective both in MES and in scPTZ tests; therefore
these molecules may be useful in treating not only gen-
eralized tonic–clonic epilepsy but also absence seizures.
It is also noteworthy that in each series of derivatives
the most lipophilic compounds were the most potent
and possessed the longest duration of anticonvulsant
activity. The lipophilic properties for all the com-
pounds were measured experimentally by the use of
the RP-HPLC method and expressed as log kw param-
eters (Table 1).
15
16
300
300
100
100
100
—
—
—
—
—
300
300
300
—
—
—
—
—
100
300
30
—
—
—
—
1.471
1.642
1.876
2.071
17
18
300
19
20
21f
300 100
100 100
300 300
30014 2.080
—
—
—
—
—
—
—
—
100
30014
100
100
300
300
300
100
—
—
2.179
1.864
1.695
1.864
1.928
2.029
2.183
2.201
300
300
300
300
300
300
100
300
300
300
300
100
—
300
—
—
—
—
—
—
—
22
23
300
—
—
100
—
24
25
300
—
—
—
26
27
28f
—
300
300
30014 2.334
In the neurotoxicity screen (NT), 33, 44, 45, 47, 49, and
51 did not show neurotoxicity in the maximum dose
administered (300 mg/kg). Among these molecules 44,
47, and 51 were inactive additionally. Compounds 16,
18, 19, 21, 24–26, 29, 30, 34–36, 38, 39, 42, 46, 48, and
50 were less neurotoxic than phenytoin and exhibited
motor impairment at the dose of 300 mg/kg at 0.5 h
and also 4 h (19, 26, 32, 34, 42, and 50). The other deriv-
atives revealed neurotoxicity at a dose of 100 mg/kg (15,
20, 22, 23, 27, 31, 32, 37, 40, 41, and 43) or 30 mg/kg,
namely 17 and 28. Compounds 33, 45, and 49 emerged
as anticonvulsants without neurotoxic properties.
300 100
300 30
100 30014
29
30
—
—
—
300
300
300
—
—
—
2.024
2.364
2.401
—
—
—
—
—
—
—
—
—
—
—
—
300
100
100
—
31
32
300 300
100
300 300
30014 2.445
33
34
—
—
300
—
—
—
—
—
—
—
300
—
—
—
—
—
—
—
300
—
100
—
2.647
2.403
2.566
2.638
2.896
2.539
1.826
1.999
2.244
2.314
2.552
2.221
1.937
2.028
2.162
2.173
2.224
2.425
ND
100
300
—
300
300
300
10014
300
300
10014
100
35
36
—
—
—
—
—
—
—
300
—
37
38
100
—
100
300
—
39
40
—
300
—
—
300
41
42f
43f
44
On the basis of the data obtained in ip screen in mice
and according to Anticonvulsant Screening Project
(ASP) disposition, six compounds 15, 21, 27, 28, 29,
and 45 were selected and examined for their anticonvul-
sant activity in the MES screen as well as neurotoxicity
after po administration into rats at a dose of 30 mg/kg.
The results obtained are presented in Table 2.
—
300 100
300 100
300 300
300
—
—
—
—
—
—
—
—
—
—
—
300
100
—
—
—
—
—
—
—
100
—
30
—
—
45
46
300
—
300
—
—
300
—
47
48
—
—
—
300
100
—
300
—
49
50
—
300
—
As can be seen from these data, compounds 27, 28, and
45 were moderately effective in rat MES oral screen and
protected only 25% of tested animals at the time of 0.5 h
(28 and 45), 1 h (27), 2 h (28) or 4 h (27). Compound 15
provided 25% at 0.25 h and one peak of 50% protection
at time point of 1 h. The other derivatives were more
effective and showed the total duration of anticonvul-
sant activity within 0.5–4 h (21), whereas 29 was active
in all time periods. Compound 21 protected 50% of rats
at the time of 2 and 4 h as well as 25% of animals tested
51
Phenytoine
Ethosuximidee
—
30
—
—
—
100
—
100
ND, not determined.
Response comments:14 unable to grasp rotarod.
a Doses of 30, 100, and 300 mg/kg were administered. The data indicate
the minimum dose whereby bioactivity was demonstrated. The ani-
mals were examined at 0.5 and 4.0 h. A dash indicates the absence of
anticonvulsant activity and neurotoxicity at the maximum dose
administered (300 mg/kg).
b Maximal electroshock test.
c Subcutaneous pentylenetetrazole test.
Table 2. Anticonvulsant activity (MES test) of selected compounds
administrated orally to rats
d Neurotoxicity screening (rotorod test).
e Reference drugs, data for phenytoin and ethosuximide Ref. 19.
f Molecules 21, 28, 43 showed anti-MES activity at a dose of 100 mg/kg
at 1 h, 42 at 2 h.
Compound
Oral administration to ratsa
0.25 h
0.5 h
1 h
2 h
4 h
15
21
1
0
0
0
3
0
1
0
1
0
1
2
1
4
2
1
1
0
1
0
3
0
2
0
1
1
0
3
0
2
1
0
1
0
3
Among these molecules, 19, 28, and 42 showed anti-
scPTZ activity at the dose of 100 and 300 mg/kg at
time periods 0.5 and 4 h that was equivalent to etho-
suximide used as reference anticonvulsant drug. Com-
parable results were obtained for 20, 33, 37, 43, and 49
providing protection at a dose of 100 mg/kg at 0.5 h as
well as 29 that was active at doses of 100 mg/kg (4 h)
and 300 mg/kg at 0.5 h. The other derivatives 15–17,
27
28
29
45
Phenytoinb
a The data in the oral MES screen indicate the number of rats of four
that were protected at a dose of 30 mg/kg.
b Reference drug, data from Ref. 32.

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K. Kaminski, J. Obniska / Bioorg. Med. Chem. 16 (2008) 4921–4931
4925
Table 4. Anticonvulsant evaluation, psychomotor seizure test (6 Hz)
after intraperitoneal injection into mice
at 0.5 and 1 h. Derivative 29 showed the peak of 75%
protection at 0.25 h and was also effective in 50% and
25% of animals at 0.5 or 1 h, 2 and 4 h, respectively.
These compounds were less active and showed shorter
duration of satisfactory action than phenytoin. They
were non-toxic when given orally.
Compound
Intraperitoneal injection into micea
0.25 h
0.5 h
1 h
2 h
4 h
35
38
0
0
2
1
0
0
0
0
0
0
a Dose of 100 mg/kg was administrated. The data indicate the number
of mice of four that were protected.
Three molecules 33, 38, and 45, which were active only
in the ip scPTZ (33 and 38) or in both MES and scPTZ
screens (45) in mice, were evaluated in the subcutaneous
pentylenetetrazole test after oral administration into
rats. Initially a dose of 50 mg/kg was employed and
the anticonvulsant activity was assessed in several time
points (Table 3). The results obtained were compared
with standard drug ethosuximide, which is the model
substance effective in this screen. As can be seen, 33 pro-
tected 50% of tested animals at 0.5 h and 25% at 0.25
and 4 h, whereas 38 and 45 showed 25% protection at
time periods 0.5 and 0.25 h, respectively. These mole-
cules were less active than ethosuximide and did not ex-
hibit also neurotoxicity at the tested dose of 50 mg/kg.
previously estimated time of peak effect (TPE) after oral
administration into rats. Results of the quantitative tests
for compound 15, along with the data for the standard
drug-phenytoin, are shown in Table 5.
In the rat oral MES screen compound 15 revealed an
ED50 value of 69.89 mg/kg and a TD50 of 500 mg/kg,
resulting in protection index (TD50/ED50) of 7.15. The
time to peak effect for this molecule was 0.5 h. Com-
pound 15 was less active than reference drug.
According to the ASP dispositions compounds 18 and
46 were assessed for potential activity against nerve
agents using the Pilocarpine Induced Status Prevention
(PISP) screen, which is one of the animal models of sta-
tus epilepticus (SE). The outcome measures are determi-
nation of ‘protection’ or ‘no-protection’. The results are
shown in Table 6.
Selected compounds 35 and 38 were chosen for the eval-
uation of anticonvulsant activity in the 6-Hz test. The
selection was made randomly as a part of the search of
molecules providing anti 6-Hz protection among chemi-
cally diversified compounds pursued in the NIH/NINDS.
The 6-Hz screen has been validated as a model of therapy-
resistant epilepsy, recently. It was not used widely
because of its lack of clinical validity since the hydantoins
such as phenytoin failed to show protective activity.
Nevertheless, the clinically effective antiepileptic drug
levetiracetam, which is not active in the conventional
MES and scPTZ tests, does exhibit protective activity in
the 6-Hz model. This suggested that the 6-Hz model
might be capable for identifying antiseizure agents with
a novel spectrum of activity and unknown mechanism
of anticonvulsant action.20 The results obtained for
compounds 35 and 38 are shown in Table 4.
In the Pilocarpine Induced Status Prevention (PISP)
screen compound 18 was found to be protective in 7/8
animals at time-zero using the dose of 65 mg/kg,
whereas 46 protected 6/7 rats at a dose of 450 mg/kg.
It is noteworthy that molecule 46 was inactive in ip.
MES and scPTZ in mice but displayed the anticonvul-
sant activity in the PISP model. To determine the acute
motor impairment doses of 30, 100, and 300 mg/kg were
administrated via the ip. route. Two rats per each dose
were employed. The animals were observed in the fol-
lowing time periods: 0.25, 0.5, 1, 2, and 4 h. The result
of acute toxicity screens revealed that both compounds
18 and 46 were negligibly neurotoxic and caused the mo-
tor impairment at a dose of 300 mg/kg within the time
range 0.25–2 h (18) or 0.25–1.0 h (46).
As can be seen from the above data, 35 and 38 adminis-
trated intraperitoneally at a dose of 100 mg/kg into mice
protected 50% (35) or 25% (38) of animals tested at
0.25 h.
Compound 15 was chosen for quantification of the
pharmacological parameters (ED50 and TD50). The
quantitative evaluation of the MES median effective
dose (ED50) and toxic dose (TD50) was performed at
2.3. Structure–activity relationships
The results of the preliminary anticonvulsant screening
revealed that the most active were compounds with
Table 3. Anticonvulsant activity (scPTZ test) of selected compounds
administrated orally to rats
Table 5. Quantification studies of compound 15 in the MES and
neurotoxicity test (NT) in rats after oral administration
Compound
Oral administration to ratsa
Compound Route TPEa MES ED50
NT TD50
(mg/kg)
PIc
b
b
0.25 h
0.5 h
1 h
2 h
4 h
(h)
(mg/kg)
33
38
1
0
1
0
225
1
0
0
0
1
0
0
0
1
1
0
0
0
15
Oral
0.5
2.0
69.89 1.64 500
23.2 4.28 >500
7.15
>21.6
Phenytoind Oral
45
Ethosuximideb
0
a Time to peak effect.
2
b Compound 15 was examined at 0.5 h after administration and results
are represented as means SEM at 95% confidence limit (MES,
maximal electroshock test; NT, neurotoxicity).
c Protection index (TD50/ED50).
Response comments:25 Myoclonic jerks.
a Dose of 50 mg/kg was administrated. The data in the oral scPTZ
screen indicate the number of rats of four that were protected.
b Reference drug, data from Ref. 19.
d Reference drug, data from Ref. 32.

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K. Kaminski, J. Obniska / Bioorg. Med. Chem. 16 (2008) 4921–4931
4926
Table 6. Pilocarpine Induced Status Prevention (PISP) model
Compound
Dose (mg/kg)
Time (h)a
Protected rats
Non-protected rats
Average weight change SEM (g)b
Protected rats
Non-protected rats
18
46
65
450
0
0
7
6
8
7
15 0.0
10.2 1.2
12.9 2.2
12.0 0.0
The results for compounds 18 and 46 after ip administration (Test 71).
a Post-first stage III seizure.
b Weight change 24 h post-first stage III seizure.
two methyl; ethyl-methyl or methyl-propyl substituents
at the position-3 of pyrrolidine-2,5-dione ring. The
replacement of the methyl group into the ethyl substitu-
ent among the N-phenylamino derivatives of 3,3-diethyl-
pyrrolidine-2,5-diones (39–44) or the introduction of the
longer butyl chain into position-3 of succinimide moiety
(35–38) decreased the activity. The similar effect was ob-
served in the case of removal of one 3-alkyl group,
namely for N-phenylamino-3-methylpyrrolidine-2,5-
dione, that may be recognized as close analogue of the
most active compound 15.21 The anticonvulsant proper-
ties depended also on the kind and location of the sub-
stituents at the phenyl ring. In general, the most effective
were molecules with highly electronegative chloro atoms
at position-2,4 (20, 28, 33, 37, 43, and 50) or -4 (19, 27,
32, and 42). The replacement of the chloro substituents
into isosteric bromo atoms caused similar pharmacolog-
ical properties. The respective ortho- and meta-chloro
derivatives as well as compounds with methyl substitu-
ents and unsubstituted molecules were less active. Fur-
thermore, comparison of results obtained previously
for the N-phenylamino-2-azaspiro[4.4]nonane- and
[4.5]decane-1,3-diones12 and compounds described here-
in, which may be recognized as analogues or respective
spirosuccinimides with degraded cycloalkyl ring showed
that such modification substantially increased the anti-
convulsant activity. This structural feature seems to be
especially important for anti-scPTZ protection. The
change of connection of cyclohexane ring with pyrroli-
dine-2,5-dione moiety from spiro carbon atom to two
common carbon atoms among the hexahydro-isoin-
dole-1,3-diones (45–50) did not influence the anticonvul-
sant activity. The lack of anticonvulsant activity for
molecule 51, which may be recognized as an analogue
of active compound 15, proved that the presence of
the aromatic moiety at the imide nitrogen is crucial for
the anticonvulsant properties of such type of
compounds.
moiety. Several molecules were effective in the 6-Hz
model, a model of therapy-resistant epilepsy as well as
the Pilocarpine Induced Status Prevention (PISP) screen
that is recognized as the animal model of status
epilepticus.
4. Experimental
4.1. Chemistry
All the chemicals and solvents were purchased from
Merck (Darmstadt, Germany) and were used without
further purification. Melting points (mp) were deter-
mined in open capillaries on a Buchi 353 melting point
¨
apparatus (Buchi Labortechnik, Flawil, Switzerland)
¨
and are uncorrected. The purity and homogeneity of
the compounds were assessed by TLC and gradient
HPLC chromatography. The thin-layer chromatogra-
phy (TLC) was performed on Merck silica gel 60 F254
aluminum sheets (Merck, Darmstadt, Germany), using
developing system consisting of chloroform/acetone
(9:1, v/v). Spots were detected by their absorption under
UV light (k = 254 nm) and by visualization with
0.05 mol I2 in 10% HCl. Analytical HPLC were run on
a Waters Alliance HPLC instrument, equipped with a
Chromolith SpeedROD column (4.6 · 50 mm). Stan-
dard conditions were eluent system A (water), system
B (acetonitrile). A flow rate of 5 ml/min and a gradient
of (0–100%) B over 5 min were used, detection 214 nm.
Retention times (tr) are given in minutes. Elemental
analysis for C, H, and N were carried out by a micro-
method using the elemental Vario EI III Elemental ana-
lyser (Hanau, Germany). The results of elemental
analyses were within 0.4% of the theoretical values.
1H NMR spectra were obtained in a Varian Mercury
spectrometer (Varian Inc., Palo Alto, CA, USA), in
CDCl3, operating at 300 MHz. Chemical shifts are re-
ported 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 follow-
ing abbreviations: s (singlet), br s (broad singlet), d
(doublet), t (triplet), dd (double doublet), td (triple dou-
blet), ddd (double double doublet), and m (multiplet).
For chosen compounds 15, 17, 20, 21, 23, 25, 28, 36,
42, 43, and 49 the mass spectra (MS) were recorded on
AMD—604 Mass Spectrometer operating at 70 eV.
3. Conclusion
The results obtained revealed that number of N-phe-
nylamino-3,3-dialkyl-pyrrolidine-2,5-diones and hexa-
hydro-isoindole-1,3-diones were effective in the MES
and he scPTZ screens. The most active was N-phenyla-
mino-3,3-dimethyl-pyrrolidine-2,5-dione that showed
the ED50 value of 69.89 mg/kg and a protective index
(TD50/ED50) of 7.15 in the MES test in rats. The anti-
convulsant activity depended on the kind and position
of substituents at the aryl moiety as well as the length
of alkyl substituents at the position-3 of succinimide
4.1.1. General procedure for the synthesis of compounds
15–51. To a suspension of 0.01 mol of 2,2-dimethyl-
succinic acid (9), 2-ethyl-2-methyl-succinic acid (10),
2-methyl-2-propyl-succinic acid (11), 2-butyl-2-methyl-

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K. Kaminski, J. Obniska / Bioorg. Med. Chem. 16 (2008) 4921–4931
4927
succinic acid (12), 2,2-diethyl-succinic acid (13) or 1,2-
cyclohexanedicarboxylic anhydride (Merck, Darmstadt,
Germany) (14) in 10 ml of water, the appropriately
substituted phenyl-hydrazines (compounds 15–50) or
methyl-hydrazine (compound 51) was gradually added.
The mixture was heated in an oil bath with simultaneous
distillation of water. After complete removal of the
water the temperature of the reaction mixture rose up
to 180 ꢁC and was maintained for 1 h. The crude prod-
ucts were crystallized from isopropanol. The obtained
solid residues were purified by column chromatography
on Silica gel 60 (Merck, Darmstadt, Germany) using
chloroform/acetone mixture (9:1, v/v), as a solvent.
After evaporation of the solvents the oil products were
recrystallized from isopropanol.
C2,6-ArH), 7.18–7.22 (m, 2H, C3,5-ArH); C12H13ClN2O2
(252.70).
4.1.1.6. N-(2,4-Dichlorophenyl)-amino-3,3-dimethyl-
pyrrolidine-2,5-dione (20). White powdery crystals.
1
Yield: 76%; HPLC (tR 2.77 min); mp 158–160 ꢁC; H
NMR (300 MHz, CDCl3): d 1.42 (s, 6H, –CH3, –CH3),
2.70 (s, 2H, imide), 6.46 (d, 1H, C6-ArH, J = 8.72 Hz),
6.52 (br s, 1H, NH), 7.12 (dd, 1H, C5-ArH,
J = 2.28 Hz, J = 8.70 Hz), 7.34 (d, 1H, C3-ArH,
J = 2.28 Hz); MS m/z 290 [M+4]+, 288 [M+2]+, 286
[M]+; C12H12Cl2N2O2 (287.15).
4.1.1.7. N-(4-Bromophenyl)-amino-3,3-dimethylpyrrol-
idine-2,5-dione (21). White powdery crystals. Yield: 78%;
HPLC (tR 2.52 min); mp 157–159 ꢁC; d 1.41 (s, 6H,
–CH3, –CH3), 2.68 (s, 2H, imide), 6.10 (br s, 1H, NH),
4.1.1.1. N-Phenylamino-3,3-dimethylpyrrolidine-2,5-
dione (15). White powdery crystals. Yield: 76%; HPLC
(tR 1.99 min); mp 127–129 ꢁC; 1H NMR (300 MHz,
CDCl3): d 1.41 (s, 6H, –CH3, –CH3), 2.68 (s, 2H, imide),
6.11 (br s, 1H, NH), 6.75–6.78 (m, 2H, C2,6-ArH), 6.96–
7.01 (m, 1H, C4-ArH), 7.22–7.27 (m, 2H, C3,5-ArH); MS
m/z 219 [M+1]+, 218 [M]+; C12H14N2O2 (218.26).
6.63–6.68 (m, 2H, C2,6-ArH), 7.32–7.36 (m, 2H, C3,5
-
ArH); MS m/z 299 [M+2]+, 297 [M]+; C12H13BrN2O2
(297.15).
4.1.1.8. N-Phenylamino-3-ethyl-3-methyl-pyrrolidine-
2,5-dione (22). White powdery crystals. Yield: 73%;
HPLC (tR 2.24 min); mp 68–70 ꢁC; 1H NMR
(300 MHz, CDCl3): d 0.95 (t, 3H, –CH3, J = 7.46 Hz),
1.38 (s, 3H, –CH3), 1.62–1.88 (m, 2H, –CH2–), 2.53 (d,
1H, imide, J = 18.37 Hz), 2.75 (d, 1H, imide,
J = 18.38 Hz), 6.17 (br s, 1H, NH), 6.75–6.79 (m, 2H,
C2,6-ArH), 6.95–7.01 (m, 1H, C4-ArH), 7.21–7.27 (m,
2H, C3,5-ArH); C13H16N2O2 (232.28).
4.1.1.2. N-(4-Methylphenyl)-amino-3,3-dimethylpyr-
rolidine-2,5-dione (16). White powdery crystals. Yield:
1
70%; HPLC (tR 2.27 min); mp 116–118 ꢁC; H NMR
(300 MHz, CDCl3): d 1.40 (s, 6H, –CH3, –CH3), 2.26
(s, 3H, Ar-4CH3), 2.65 (s, 2H, imide), 6.05 (br s, 1H,
NH), 6.69–6.72 (m, 2H, C3,5-ArH), 7.03–7.06 (m, 2H,
C2,6-ArH); C13H16N2O2 (232.28).
4.1.1.9. N-(2-Methylphenyl)-amino-3-ethyl-3-methyl-
pyrrolidine-2,5-dione (23). White powdery crystals.
Yield: 67%; HPLC (tR 2.49 min); mp 96–98 ꢁC; 1H
NMR (300 MHz, CDCl3): d 0.97 (t, 3H, –CH3,
J = 7.46 Hz), 1.39 (s, 3H, –CH3), 1.66–1.89 (m, 2H,
–CH2–), 2.35 (s, 3H, Ar-2CH3), 2.55 (d, 1H, imide,
J = 18.36 Hz), 2.77 (d, 1H, imide, J = 18.37 Hz), 6.03
(br s, 1H, NH), 6.50 (d, 1H, C6-ArH, J = 8.21 Hz),
6.89 (td, 1H, C4-ArH, J = 1.11 Hz, J = 7.44 Hz), 7.06–
7.13 (m, 2H, C3,5-ArH); MS m/z 247 [M+1]+, 246
[M]+; C14H18N2O2 (246.31).
4.1.1.3. N-(2-Chlorophenyl)-amino-3,3-dimethylpyr-
rolidine-2,5-dione (17). White powdery crystals. Yield:
79%; HPLC (tR 2.36 min); mp 138–140 ꢁC; H NMR
(300 MHz, CDCl3): d 1.43 (s, 6H, –CH3, –CH3), 2.70
(s, 2H, imide), 6.53 (dd, 1H, C6-ArH, J = 1.54 Hz,
J = 8.21 Hz), 6.58 (br s, 1H, NH), 6.91 (td, 1H, C4-
ArH, J = 1.45 Hz, J = 8.08 Hz), 7.11–7.17 (m, 1H, C5-
ArH), 7.33 (dd, 1H, C3-ArH, J = 1.38 Hz,
J = 7.97 Hz); MS m/z 254 [M+2]+, 252 [M]+;
C12H13ClN2O2 (252.70).
1
4.1.1.4. N-(3-Chlorophenyl)-amino-3,3-dimethylpyr-
rolidine-2,5-dione (18). White powdery crystals. Yield:
70%; HPLC (tR 2.47 min); mp 70–72 ꢁC; 1H NMR
(300 MHz, CDCl3): d 1.42 (s, 6H, –CH3, –CH3), 2.69
(s, 2H, imide), 6.13 (br s, 1H, NH), 6.64 (ddd, 1H, C6-
ArH, J = 0.72 Hz, J = 2.26 Hz, J = 8.13 Hz), 6.72 (t,
1H, C2-ArH, J = 2.08 Hz), 6.95 (ddd, 1H, C4-ArH,
J = 0.78 Hz, J = 1.82 Hz, J = 7.97 Hz), 7.16 (t, 1H, C5-
ArH, J = 8.05 Hz); C12H13ClN2O2 (252.70).
4.1.1.10. N-(4-Methylphenyl)-amino-3-ethyl-3-methyl-
pyrrolidine-2,5-dione (24). White powdery crystals.
Yield: 69%; HPLC (tR 2.51 min); mp 99–101 ꢁC; 1H
NMR (300 MHz, CDCl3): d 0.93 (t, 3H, –CH3,
J = 7.46 Hz), 1.37 (s, 3H, –CH3), 1.63–1.87 (m, 2H,
–CH2–), 2.26 (s, 3H, Ar-4CH3), 2.51 (d, 1H, imide,
J = 18.35 Hz), 2.73 (d, 1H, imide, J = 18.36 Hz), 6.06
(br s, 1H, NH), 6.70–6.73 (m, 2H, C2,6-ArH), 7.03–
7.06 (m, 2H, C3,5-ArH); C14H18N2O2 (246.31).
4.1.1.5. N-(4-Chlorophenyl)-amino-3,3-dimethylpyr-
rolidine-2,5-dione (19). White powdery crystals. Yield:
81%; HPLC (tR 2.41 min); mp 148–150 ꢁC; H NMR
(300 MHz, CDCl3): d 1.41 (s, 6H, –CH3, –CH3), 2.68
(s, 2H, imide), 6.10 (br s, 1H, NH), 6.68–6.73 (m, 2H,
4.1.1.11. N-(2-Chlorophenyl)-amino-3-ethyl-3-methyl-
pyrrolidine-2,5-dione (25). White powdery crystals.
Yield: 71%; HPLC (tR 2.62 min); mp 116–118 ꢁC; H
NMR (300 MHz, CDCl3): d 0.97 (t, 3H, –CH3,
J = 7.46 Hz), 1.40 (s, 3H, –CH3), 1.64–1.90 (m, 2H,
–CH2–), 2.56 (d, 1H, imide, J = 18.43 Hz), 2.78 (d, 1H,
imide, J = 18.42 Hz), 6.54 (dd, 1H, C6-ArH,
J = 1.37 Hz, J = 8.11 Hz), 6.59 (br s, 1H, NH), 6.91
(td, 1H, C4-ArH, J = 1.43 Hz, J = 7.77 Hz), 7.11–7.16
(m, 1H, C5-ArH), 7.33 (dd, 1H, C3-ArH, J = 1.35 Hz,
1
1
Compound 15 was described by Levy,22 mp 131–132 ꢁC, no spectral
data available.

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K. Kaminski, J. Obniska / Bioorg. Med. Chem. 16 (2008) 4921–4931
4928
J = 7.96 Hz); MS m/z 268 [M+2]+, 266 [M]+;
C13H15ClN2O2 (266.73).
J = 7.21 Hz), 1.20–1.80 (m, 7H, –C2H4–, –CH3), 2.56
(d, 1H, imide, J = 18.42 Hz), 2.78 (d, 1H, imide,
J = 18.41 Hz), 6.10 (br s, 1H, NH), 6.65 (ddd, 1H, C6-
ArH, J = 0.70 Hz, J = 2.26 Hz, J = 8.15 Hz), 6.73 (t,
1H, C2-ArH, J = 2.09 Hz), 6.95 (ddd, 1H, C4-ArH,
J = 0.74 Hz, J = 1.80 Hz, J = 7.96 Hz), 7.16 (t, 1H, C5-
ArH, J = 8.05 Hz); C14H17ClN2O2 (280.76).
4.1.1.12. N-(3-Chlorophenyl)-amino-3-ethyl-3-methyl-
pyrrolidine-2,5-dione (26). White powdery crystals.
1
Yield: 71%; HPLC (tR 2.65 min); mp 103–105 ꢁC; H
NMR (300 MHz, CDCl3): d 0.96 (t, 3H, –CH3,
J = 7.46 Hz), 1.39 (s, 3H, –CH3), 1.63–1.89 (m, 2H,
–CH2–), 2.55 (d, 1H, imide, J = 18.43 Hz), 2.77 (d, 1H,
imide, J = 18.43 Hz), 6.12 (br s, 1H, NH), 6.65 (ddd,
1H, C6-ArH, J = 0.89 Hz, J = 2.30 Hz, J = 8.13 Hz),
6.73 (t, 1H, C2-ArH, J = 2.08 Hz), 6.95 (ddd, 1H, C4-
ArH, J = 0.88 Hz, J = 1.90 Hz, J = 7.98 Hz), 7.16 (t,
1H, C5-ArH, J = 8.05 Hz); C13H15ClN2O2 (266.73).
4.1.1.18. N-(4-Chlorophenyl)-amino-3-methyl-3-pro-
pyl-pyrrolidine-2,5-dione (32). White powdery crystals.
Yield: 66%; HPLC (tR 2.91 min); mp 90–92 ꢁC; 1H
NMR (300 MHz, CDCl3): d 0.95 (t, 3H, –CH3,
J = 7.20 Hz), 1.18–1.78 (m, 7H, –C2H4–, –CH3), 2.54
(d, 1H, imide, J = 18.43 Hz), 2.77 (d, 1H, imide,
J = 18.42 Hz), 6.10 (br s, 1H, NH), 6.70–6.73 (m, 2H,
C2,6-ArH), 7.19–7.22 (m, 2H, C3,5-ArH); C14H17ClN2O2
(280.76).
4.1.1.13. N-(4-Chlorophenyl)-amino-3-ethyl-3-methyl-
pyrrolidine-2,5-dione (27). White powdery crystals.
Yield: 66%; HPLC (tR 2.65 min); mp 80–82 ꢁC; 1H
NMR (300 MHz, CDCl3): 1H NMR (300 MHz,
CDCl3): d 0.94 (t, 3H, –CH3, J = 7.45 Hz), 1.38 (s,
3H, –CH3), 1.67–1.88 (m, 2H, –CH2–), 2.53 (d, 1H,
imide, J = 18.43 Hz), 2.75 (d, 1H, imide, J = 18.42 Hz),
6.11 (br s, 1H, NH), 6.70–6.74 (m, 2H, C2,6-ArH),
7.19–7.21 (m, 2H, C3,5-ArH); C13H15ClN2O2 (266.73).
4.1.1.19. N-(2,4-Dichlorophenyl)-amino-3-methyl-3-pro-
pyl-pyrrolidine-2,5-dione (33). White powdery crystals.
1
Yield: 73%; HPLC (tR 2.91 min); mp 108–110 ꢁC; H
NMR (300 MHz, CDCl3): d 0.96 (t, 3H, –CH3,
J = 7.20 Hz), 1.20–1.79 (m, 7H, –C2H4–, –CH3), 2.57
(d, 1H, imide, J = 18.46 Hz), 2.79 (d, 1H, imide,
J = 18.46 Hz), 6.47 (d, 1H, C6-ArH, J = 8.70 Hz), 6.52
(br s, 1H, NH), 7.12 (dd, 1H, C5-ArH, J = 2.09 Hz,
J = 2.49 Hz), 7.34 (d, 1H, C3-ArH, J = 2.28 Hz);
C14H16Cl2N2O2 (315.2).
4.1.1.14. N-(2,4-Dichlorophenyl)-amino-3-ethyl-3-
methyl-pyrrolidine-2,5-dione (28). White powdery crys-
tals. Yield: 77%; HPLC (tR 2.99 min); mp 138–140 ꢁC;
1H NMR (300 MHz, CDCl3): d 0.96 (t, 3H, –CH3,
J = 7.45 Hz), 1.39 (s, 3H, –CH3), 1.63–1.89 (m, 2H,
–CH2–), 2.56 (d, 1H, imide, J = 18.46 Hz), 2.78 (d,
1H, imide, J = 18.47 Hz), 6.48 (d, 1H, C6-ArH,
J = 8.70 Hz), 6.53 (br s, 1H, NH), 7.11 (dd, 1H, C5-
ArH, J = 2.28 Hz, J = 8.69 Hz), 7.35 (d, 1H, C3-ArH,
J = 2.29 Hz); MS m/z 304 [M+4]+, 302 [M+2]+, 300
[M]+; C13H14Cl2N2O2 (301.17).
4.1.1.20. N-(4-Bromophenyl)-amino-3-methyl-3-propyl-
pyrrolidine-2,5-dione (34). White powdery crystals. Yield:
1
66%; HPLC (tR 2.98 min); mp 115–117 ꢁC; H NMR
(300 MHz, CDCl3): d 0.95 (t, 3H, –CH3, J = 7.20 Hz),
1.18–1.78 (m, 7H, –C2H4–, –CH3), 2.55 (d, 1H, imide,
J = 18.43 Hz), 2.77 (d, 1H, imide, J = 18.42 Hz), 6.07
(br s, 1H, NH), 6.65–6.67 (m, 2H, C2,6-ArH), 7.34–7.37
(m, 2H, C3,5-ArH); C14H17BrN2O2 (325.21).
4.1.1.15. N-(4-Bromophenyl)-amino-3-ethyl-3-methyl-
pyrrolidine-2,5-dione (29). White powdery crystals.
Yield: 66%; HPLC (tR 2.71 min); mp 122–124 ꢁC; H
NMR (300 MHz, CDCl3): d 0.95 (t, 3H, –CH3,
J = 7.44 Hz), 1.38 (s, 3H, –CH3), 1.64–1.88 (m, 2H,
–CH2–), 2.54 (d, 1H, imide, J = 18.46 Hz), 2.75 (d, 1H,
imide, J = 18.46 Hz), 6.07 (br s, 1H, NH), 6.65–6.68
(m, 2H, C2,6-ArH.), 7.34–7.38 (m, 2H, C3,5-ArH);
C13H15BrN2O2 (311.18).
4.1.1.21. N-(2-Chlorophenyl)-amino-3-butyl-3-methyl-
pyrrolidine-2,5-dione (35). White powdery crystals.
Yield: 64%; HPLC (tR 3.17 min); mp 78–80 ꢁC; 1H
NMR (300 MHz, CDCl3): d 0.91 (t, 3H, –CH3,
J = 7.05 Hz), 1.17–1.82 (m, 9H, –C3H6–, –CH3), 2.56
(d, 1H, imide, J = 18.42 Hz), 2.78 (d, 1H, imide,
J = 18.42 Hz), 6.55 (dd, 1H, C6-ArH, J = 1.41 Hz,
J = 8.12 Hz), 6.59 (br s, 1H, NH), 6.91 (td, 1H, C4-
ArH, J = 1.42 Hz, J = 7.76 Hz), 7.11–7.17 (m, 1H, C5-
ArH), 7.33 (dd, 1H, C3-ArH, J = 1.38 Hz,
J = 7.96 Hz); C15H19ClN2O2 (294.78).
1
4.1.1.16. N-(2-Chlorophenyl)-amino-3-methyl-3-pro-
pyl-pyrrolidine-2,5-dione (30). White powdery crystals.
Yield: 67%; HPLC (tR 2.90 min); mp 96–98 ꢁC; 1H
NMR (300 MHz, CDCl3): d 0.96 (t, 3H, –CH3,
J = 7.21 Hz), 1.19–1.80 (m, 7H, –C2H4–, –CH3), 2.57
(d, 1H, imide, J = 18.42 Hz), 2.80 (d, 1H, imide,
J = 18.42 Hz), 6.53 (dd, 1H, C6-ArH, J = 1.42 Hz,
J = 8.11 Hz), 6.58 (br s, 1H, NH), 6.91 (td, 1H, C4-
ArH, J = 1.42 Hz, J = 7.76 Hz), 7.11–7.17 (m, 1H, C5-
ArH), 7.32 (dd, 1H, C3-ArH, J = 1.40 Hz,
J = 7.97 Hz). C14H17ClN2O2 (280.76).
4.1.1.22. N-(4-Chlorophenyl)-amino-3-butyl-3-methyl-
pyrrolidine-2,5-dione (36). White powdery crystals.
Yield: 66%; HPLC (tR 3.15 min); mp 94–96 ꢁC; 1H
NMR (300 MHz, CDCl3): d 0.90 (t, 3H, –CH3,
J = 7.06 Hz), 1.14–1.80 (m, 9H, –C3H6–, –CH3), 2.55
(d, 1H, imide, J = 18.43 Hz), 2.76 (d, 1H, imide,
J = 18.43 Hz), 6.08 (br s, 1H, NH), 6.71–6.74 (m, 2H,
C2,6-ArH), 7.19–7.22 (m, 2H, C3,5-ArH); MS m/z 297
[M+2]+, 295 [M]+; C15H19ClN2O2 (294.78).
4.1.1.17. N-(3-Chlorophenyl)-amino-3-methyl-3-pro-
pyl-pyrrolidine-2,5-dione (31). White powdery crystals.
Yield: 67%; HPLC (tR 2.95 min); mp 58–60 ꢁC; 1H
NMR (300 MHz, CDCl3): d 0.96 (t, 3H, –CH3,
4.1.1.23. N-(2,4-Dichlorophenyl)-amino-3-butyl-3-
methyl-pyrrolidine-2,5-dione (37). White powdery crys-
tals. Yield: 69%; HPLC (tR 3.45 min); mp 68–70 ꢁC;

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K. Kaminski, J. Obniska / Bioorg. Med. Chem. 16 (2008) 4921–4931
4929
1H NMR (300 MHz, CDCl3): d 0.91 (t, 3H, –CH3,
J = 7.01 Hz), 1.15–1.81 (m, 9H, –C3H6–, –CH3), 2.56
(d, 1H, imide, J = 18.46 Hz), 2.78 (d, 1H, imide,
J = 18.47 Hz), 6.48 (d, 1H, C6-ArH, J = 8.70 Hz), 6.52
(br s, 1H, NH), 7.11 (dd, 1H, C5-ArH, J = 2.26 Hz,
J = 8.69 Hz), 7.34 (d, 1H, C3-ArH, J = 2.28 Hz);
C15H18Cl2N2O2 (329.23).
J = 8.71 Hz), 7.34 (d, 1H, C3-ArH, J = 2.28 Hz); MS
m/z 318 [M+4]+, 316 [M+2]+, 314 [M]+; C14H16Cl2N2O2
(315.20).
4.1.1.30. N-(4-Bromophenyl)-amino-3,3-diethyl-pyr-
rolidine-2,5-dione (44). White powdery crystals. Yield:
1
72%; HPLC (tR 2.92 min); mp 136–138 ꢁC; H NMR
(300 MHz, CDCl3): d 0.92 (t, 6H, –CH3, –CH3,
J = 7.46 Hz), 1.64–1.88 (m, 4H, –CH2–, –CH2–), 2.63
(s, 2H, imide), 6.12 (br s, 1H, NH), 6.65–6.69 (m, 2H,
C2,6-ArH), 7.33–7.36 (m, 2H, C3,5-ArH); C14H17BrN2O2
(325.21).
4.1.1.24. N-(4-Bromophenyl)-amino-3-butyl-3-methyl-
pyrrolidine-2,5-dione (38). White powdery crystals.
Yield: 67%; HPLC (tR 3.22 min); mp 104–106 ꢁC; H
NMR (300 MHz, CDCl3): d 0.90 (t, 3H, –CH3,
J = 7.05 Hz), 1.14–1.79 (m, 9H, –C3H6–, –CH3), 2.54
(d, 1H, imide, J = 18.43 Hz), 2.76 (d, 1H, imide,
J = 18.43 Hz), 6.09 (br s, 1H, NH), 6.65–6.68 (m, 2H,
C2,6-ArH), 7.33–7.37 (m, 2H, C3,5-ArH); C15H19BrN2O2
(339.23).
1
4.1.1.31. N-Phenylamino-hexahydro-isoindole-1,3-dione
(45).à White powdery crystals. Yield: 74%; HPLC (tR
2.32 min); mp 156–158 ꢁC; 1H NMR (300 MHz,
CDCl3): d 1.47–1.53 (m, 4H, cyclohexane), 1.81–1.97
(m, 4H, cyclohexane), 2.95–3.03 (m, 2H, imide), 6.09
(br s, 1H, NH), 6.76–6.80 (m, 2H, C2,6-ArH), 6.94–
7.00 (m, 1H, C4-ArH), 7.21–7.27 (m, 2H, C3,5-ArH);
C14H16N2O2 (244.30).
4.1.1.25. N-Phenylamino-3,3-diethyl-pyrrolidine-2,5-
dione (39). White powdery crystals. Yield: 73%; HPLC
(tR 2.49 min); mp 68–70 ꢁC; 1H NMR (300 MHz,
CDCl3): d 0.93 (t, 6H, –CH3, –CH3, J = 7.46 Hz),
1.61–1.88 (m, 4H, –CH2–, –CH2–), 2.63 (s, 2H, imide),
6.19 (br s, 1H, NH), 6.76–6.79 (m, 2H, C2,6-ArH),
4.1.1.32. N-(4-Methylphenyl)-amino-hexahydro-isoin-
dole-1,3-dione (46). White powdery crystals. Yield: 70%;
HPLC (tR 2.55 min); mp 136–138 ꢁC; 1H NMR
(300 MHz, CDCl3): d 1.48–1.51 (m, 4H, cyclohexane),
1.84–1.91 (m, 4H, cyclohexane), 2.26 (s, 3H, –CH3),
2.96–2.99 (m, 2H, imide), 6.00 (br s, 1H, NH); 6.70–
6.74 (m, 2H, C2,6-ArH), 7.03–7.06 (m, 2H, C3,5-ArH);
C15H18N2O2 (258.32).
6.94–7.00 (m, 1H, C4-ArH), 7.21–7.25 (m, 2H, C3,5
ArH); C14H18N2O2 (246.31).
-
4.1.1.26. N-(4-Methylphenyl)-amino-3,3-diethyl-pyr-
rolidine-2,5-dione (40). White powdery crystals. Yield:
75%; HPLC (tR 2.71 min); mp 76–78 ꢁC; 1H NMR
(300 MHz, CDCl3): d 0.92 (t, 6H, –CH3, –CH3,
J = 7.45 Hz), 1.63–1.85 (m, 4H, –CH2–, –CH2–), 2.26
(s, 3H, Ar-4CH3), 2.61 (s, 2H, imide), 6.06 (br s, 1H,
NH), 6.70–6.74 (m, 2H, C2,6-ArH), 7.03–7.05 (m, 2H,
C3,5-ArH); C15H20N2O2 (260.34).
4.1.1.33. N-(2-Chlorophenyl)-amino-hexahydro-isoin-
dole-1,3-dione (47). White powdery crystals. Yield:
1
74%; HPLC (tR 2.64 min); mp 156–158 ꢁC; H NMR
(300 MHz, CDCl3): d 1.49–1.55 (m, 4H, cyclohexane),
1.82–1.97 (m, 4H, cyclohexane), 2.98–3.06 (m, 2H,
imide), 6.55–6.58 (m, 2H, 1H, NH, 1H, C6-ArH), 6.90
(td, 1H, C4-ArH, J = 1.42 Hz, J = 7.72 Hz), 7.10–7.16
(m, 1H, C5-ArH), 7.32 (dd, 1H, C3-ArH, J = 1.37 Hz,
J = 7.96 Hz); C14H15ClN2O2 (278.74).
4.1.1.27. N-(2-Chlorophenyl)-amino-3,3-diethyl-pyr-
rolidine-2,5-dione (41). White powdery crystals. Yield:
1
67%; HPLC (tR 2.82 min); mp 106–108 ꢁC; H NMR
(300 MHz, CDCl3): d 0.96 (t, 6H, –CH3, –CH3,
J = 7.47 Hz), 1.64–1.90 (m, 4H, –CH2–, –CH2–), 2.66
(s, 2H, imide), 6.56 (dd, 1H, C6-ArH, J = 1.45 Hz,
J = 8.12 Hz), 6.60 (br s, 1H, NH), 6.90 (td, 1H, C4-
ArH, J = 1.46 Hz, J = 7.69 Hz), 7.10–7.15 (m, 1H, C5-
ArH), 7.32 (dd, 1H, C3-ArH, J = 1.38 Hz,
J = 7.96 Hz); C14H17ClN2O2 (280.76).
4.1.1.34. N-(3-Chlorophenyl)-amino-hexahydro-isoin-
dole-1,3-dione (48). White powdery crystals. Yield:
1
70%; HPLC (tR 2.69 min); mp 168–170 ꢁC; H NMR
(300 MHz, CDCl3): d 1.50–1.57 (m, 4H, cyclohexane),
1.84–1.96 (m, 4H, cyclohexane), 3.00–3.04 (m, 2H,
imide), 6.07 (br s, 1H, NH), 6.65 (ddd, 1H, C6-ArH,
J = 0.88 Hz, J = 2.29 Hz, J = 8.12 Hz), 6.73 (t, 1H, C2-
ArH, J = 2.07 Hz), 6.95 (ddd, 1H, C4-ArH,
J = 0.88 Hz, J = 1.90 Hz, J = 7.98 Hz), 7.17 (t, 1H, C5-
ArH, J = 8.04 Hz); C14H15ClN2O2 (278.74).
4.1.1.28. N-(4-Chlorophenyl)-amino-3,3-diethyl-pyr-
rolidine-2,5-dione (42). White powdery crystals. Yield:
1
72%; HPLC (tR 2.85 min); mp 113–115 ꢁC; H NMR
(300 MHz, CDCl3): d 0.92 (t, 6H, –CH3, –CH3,
J = 7.46 Hz), 1.64–1.88 (m, 4H, –CH2–, –CH2–), 2.63
(s, 2H, imide), 6.12 (br s, 1H, NH), 6.70–6.75 (m, 2H,
C2,6-ArH), 7.19–7.22 (m, 2H, C3,5-ArH); MS m/z 283
[M+2]+; 281 [M]+; C14H17ClN2O2 (280.76).
4.1.1.35. N-(4-Chlorophenyl)-amino-hexahydro-isoin-
dole-1,3-dione (49). White powdery crystals. Yield:
1
73%; HPLC (tR 2.63 min); mp 188–190 ꢁC; H NMR
(300 MHz, CDCl3): d 1.48–1.53 (m, 4H, cyclohexane),
1.85–1.93 (m, 4H, cyclohexane), 2.98–3.01 (m, 2H,
imide), 6.04 (br s, 1H, NH), 6.72–6.75 (m, 2H, C2,6-
4.1.1.29.
N-(2,4-Dichlorophenyl)-amino-3,3-diethyl-
pyrrolidine-2,5-dione (43). White powdery crystals.
Yield: 71%; HPLC (tR 3.18 min); mp 98–100 ꢁC; 1H
NMR (300 MHz, CDCl3): d 0.95 (t, 6H, –CH3, –CH3,
J = 7.46 Hz), 1.66–1.88 (m, 4H, –CH2–, –CH2–), 2.65
(s, 2H, imide), 6.49 (d, 1H, C6-ArH, J = 8.71 Hz), 6.53
(br s, 1H, NH), 7.11 (dd, 1H, C5-ArH, J = 2.27 Hz,
à
Compound 45 was described by Baddar et al.,23 mp 173–174 ꢁC, no
1HNMR data available.

´
K. Kaminski, J. Obniska / Bioorg. Med. Chem. 16 (2008) 4921–4931
4930
ArH), 7.19–7.22 (m, 2H, C3,5-ArH); MS m/z 281
(NIH/NINDS), Bethesda, MD, USA, by using proce-
dures described elsewhere.26,27
[M+2]+, 279 [M]+; C14H15ClN2O2 (278.74).
4.1.1.36. N-(2,4-Dichlorophenyl)-amino-hexahydro-
isoindole-1,3-dione (50). White powdery crystals. Yield:
69%; HPLC (tR 2.99 min); mp 165–167 ꢁC; H NMR
(300 MHz, CDCl3): d 1.51–1.58 (m, 4H, cyclohexane),
1.81–1.98 (m, 4H, cyclohexane), 3.00–3.04 (m, 2H,
imide), 6.49–6.52 (m, 2H, 1H, NH, 1H, C6-ArH), 7.11
(dd, 1H, C5-ArH, J = 2.25 Hz, J = 8.90 Hz), 7.34 (d,
1H, C3-ArH, J = 2.29 Hz); C14H14Cl2N2O2 (313.19).
Male albino mice (CF-1 strain) and male albino rats
(Sprague–Dawley) were used as experimental animals.
The animals were housed in metabolic cages and al-
lowed free access to food and water. The compounds
were suspended in 0.5% methylcellulose/water mixture.
1
Phase I studies in mice involved two convulsant tests:
maximal electroshock seizure test (MES), subcutaneous
pentylenetetrazole seizure test (scPTZ) and rotorod test
for neurological toxicity (NT).
4.1.1.37. N-Methylamino-3,3-dimethyl-pyrrolidine-
2,5-dione (51). White powdery crystals. Yield: 61%; mp
1
4.3.1. The maximal electroshock test (MES). In the MES
screen, an electrical stimulus of 0.2 s in duration (50 mA
in mice and 150 mA in rats at 60 Hz) is delivered via cor-
neal electrodes primed with an electrolyte solution con-
taining an anesthetic agent.
61–63 ꢁC; H NMR (300 MHz, CDCl3): d 1.34 (s, 6H,
–CH3, –CH3), 2.54 (s, 2H, imide), 2.72 (s, 3H, –CH3),
3.02 (br s, 1H, NH); C7H12N2O2 (156.19).
4.2. Lipophilicity determination
4.3.2. The subcutaneous pentylenetetrazole seizure test
(scPTZ). This screen utilizes a dose of pentylenetetraz-
ole (85 mg/kg in mice and 70 mg/kg in rats) that pro-
duces clonic seizures lasting for a period of at least
five seconds in 97% (CD97) of animals tested. At the
anticipated time of testing the convulsant is adminis-
tered subcutaneously.
The analytical system consisted of a Waters (Milford,
USA) instrument, equipped with 515 HPLC pumps, a
600S flow controller, 486 Tunable Absorbance Detector
and injector: Rheodyne 7161 valve (Cotati, CA, USA).
The measurements were performed on a LiChrospher
C18 end-capped column, 250 · 4.6 mm (Merck, Darms-
tadt, Germany). Acetonitrile for measurements was of
gradient grade and was purchased from Merck (Darms-
tadt, Germany). The water was filtrated under vacuum
a 0.45 lm HA Millipore filter (Millipore, Milford, MA,
USA). For chromatographic analysis stock solutions of
1.0 mg/ml of the samples in acetonitrile/water (1:1, v/v)
were prepared and filtrated through 0.2 lm Millipore fil-
ter (Millipore, Milford, MA, USA). These solutions were
kept in Eppendorf tubes at room temperature. Isocratic
runs were carried out with mixtures containing increasing
acetonitrile volume fractions from 40% to 95% v/v in 5%
increment. Flow rate 1 ml/min. The injection volume was
5ll in all cases. Uracil (Merck, Darmstadt, Germany)
was used as the unretained compound to determine the
dead volume at the different acetonitrile concentrations
in the mobile phase. The retention times were measured
at room temperature by the UV detector at the kmax of
the analytes (214 nm). Three sets of measurements were
conducted for each compound and the mean value was
used for further calculations. The capacity factors log k
were calculated by us of equation: k = (tr À t0)/t0, where
tr and t0 are the retention times for solute and unretained
compound (uracil), respectively. The log k values were
extrapolated to 100% water concentration that enabled
the estimation of relative retention parameters log kw.
This procedure utilized an equation: log k = log kw À Su,
where S is the slope, u is the volume fraction of the organ-
ic modifier. The log kw parameters characterize the parti-
tion of the compound between a non-polar hydrocarbon
stationary phase and pure water.24,25
All the compounds were injected intraperitoneally into
mice at the dose levels of 30, 100, and 300 mg/kg with
anticonvulsant activity and neurotoxicity assessment at
0.5 and 4 h after administration. The results are pre-
sented in Table 1.
Selected derivatives were administrated orally into rats
using four animals at a fixed dose of 30 mg/kg (MES
test) and 50 mg/kg (scPTZ test) (Phase VIa). This screen
discloses the time of onset, the approximate time of peak
effect (TPE) and the duration of anticonvulsant activity.
For both doses the motor impairment was studied in
parallel. Rats were tested at five time periods ranging
from one quarter to 4 h post-substance administration.
The results are shown in Tables 2 and 3.
4.3.3. The neurological toxicity (NT). induced by com-
pound was detected in mice or rats using standardized
rotorod test.28 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 a given time.
4.3.4. Quantification studies. The quantitative determi-
nation of ED50 and TD50 values for 15 was performed
at previously estimated time of peak effect after oral
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 protec-
tion 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. The re-
sults are shown in Table 4.
4.3. Pharmacology
The initial anticonvulsant evaluation was performed
within the Antiepileptic Drug Development (ADD) Pro-
gram in Epilepsy Branch, National Institutes of Health,
National Institute of Neurological Disorders and Stroke

´
K. Kaminski, J. Obniska / Bioorg. Med. Chem. 16 (2008) 4921–4931
4931
2. Lo¨scher, W.; Schmidt, D. Epilepsy Res. 2002, 50, 3.
3. Greenwood, R. S. Epilepsia 2000, 41, S42.
4. Wong, M. G.; Defina, J. A.; Andrews, P. R. J. Med.
Chem. 1986, 29, 562.
5. Camerman, A.; Camerman, N. In Antiepileptic Drugs:
Mechanisms of Action; Glaser, G. H., Penry, J. K.,
Woodbury, D. M., Eds.; Raven Press: New York, 1980;
p 223.
6. Tasso, S. M.; Bruno-Blanch, L. E.; Moon, S. C.; Estiu´, G.
L. J. Mol. Struct. 2000, 504, 229.
7. Kenda, B. M.; Matagne, A. C.; Talaga, P. E.; Pasau, P.
M.; Differding, E.; Lallemand, B. I.; Frycia, A. M.;
Moureau, F. G.; Klitgaard, H. V.; Gillard, M. R.; Fuks,
B.; Michel, P. J. Med. Chem. 2004, 47, 530.
8. Bialer, M.; Johannessen, S. I.; Kupferberg, H. J.;
Levy, R. H.; Perucca, E.; Tomson, T. Epilepsy Res.
2007, 73, 1.
9. Malawska, B.; Kulig, K. Curr. Opin. Investig. Drugs 2005,
6, 740.
10. Bennett, B.; Matagne, A.; Michel, P.; Leonard, M.;
Cornet, M.; Meeus, M. A.; Toublanc, N. Neurotherapeu-
tics 2007, 4, 117.
4.3.5. The 6-Hz model. This screen was carried out
according to the protocol originally described by Brown
et al.29 and more recently by Barton et al.20 and Kaminski
et al.30 It is an alternative electroshock paradigm that uses
low-frequency (6 Hz), long-duration (3 s) electrical stimu-
lation. Corneal stimulation (0.2 ms-duration monopolar
rectangular pulses at 6-Hz for 3 s) was delivered by a con-
stant-current device. During the stimulation, mice were
manually restrained and released into the observation
cage immediately after the current application. The sei-
zures manifest in ‘stunned’ posture associated with rear-
ing, 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 behav-
ior within 10 s from the stimulation.30
11. Rogawski, M. A. Epilepsy Res. 2006, 68, 22.
12. Kaminski, K.; Obniska, J.; Dybala, M. Eur. J. Med.
Chem. 2008, 43, 53.
4.3.6. The Pilocarpine Induced Status Prevention (PISP)
model. The male albino rats (Sprague–Dawley, 150–
180 g) were used as experimental animals. The com-
pounds were administrated via the ip route of administra-
tion. Then a challenge dose of pilocarpine is given
observing for treatment-effects of the substance tested.
The seizure severity is determined using the well-known
Racine scale,31 as follows: (I) immobility, eye closure,
twitching of vibrissae, sniffing, and facial clonus; (II) head
nodding associated with more severe facial clonus; (III)
clonus of one of the fore limbs; (IV) rearing often accom-
plished by bilateral forelimb clonus and (V) all of the
above plus loss of balance and falling, accomplished by
generalized clonic seizures. The anticonvulsant activity
of compounds 18 and 46 was assessed at time zero, namely
the time from the first stage III seizures (Test 71). The out-
come measures are determination of ‘protection’ or ‘no
protection’.
´
13. Obniska, J.; Dzierzawska-Majewska, A.; Zagorska, A.;
Zajdel, P.; Karolak-Wojciechowska, J. Il Farmaco 2005,
60, 529.
14. Gibbs, J. W., III; Zhang, Y. F.; Ahmed, H. S.; Coulter, D.
A. Epilepsia 2002, 43, 342.
15. Smith, P. A. S.; Horowitz, J. P. J. Am. Chem. Soc. 1949,
71, 3418.
16. Le Moal, H. Bull. Soc. Chim. Fr. 1956, 1, 418.
17. White, H. S. Epilepsia 2003, 44, 2.
18. White, H. S.; Woodhead, J. H.; Wilcox, K. S.;
Stables, J. P.; Kupferberg, H. J.; Wolf, H. H. In
Antiepileptic Drugs; Levy, R. H., Mattson, R. H.,
Meldrum, B. S., Perucca, E., Eds.; Lippincott: Phila-
delphia, PA, 2002; pp 36–48.
19. Thirumurugan, R.; Sriram, D.; Saxena, A.; Stables,
J.; Yogeeswari, P. Bioorg. Med. Chem. 2006, 14,
3106.
20. Barton, M. E.; Klein, B. D.; Wolf, H. H.; White, H. S.
Epilepsy Res. 2001, 47, 217.
4.4. Statistical analysis
21. Kornet, M. J. J. Pharm. Sci. 1984, 73, 405.
22. Levy, E. Justus Liebigs Ann. Chem. 1887, 242, 201.
23. Baddar, F. G.; El–Newaihy, M. F.; Salem, M. R. J. Chem.
Soc. 1971, 716.
24. Liu, X.; Tanaka, H.; Yamauchi, A.; Testa, B.; Chuman,
H. J. Chromatogr. A 2005, 1091, 51.
Statistical analyses were performed using the computer
program Statistica version 5, 1997 Edition, Copyrightꢀ
Statsoft, Inc. 1984–1997.
25. Dross, K.; Rekker, R. F.; Vries, G.; Mannhold, R. QSAR
1999, 18, 549.
Acknowledgments
26. Krall, R. L.; Penry, J. K.; White, B. G.; Kupferberg, H. J.;
Swinyard, E. A. Epilepsia 1978, 19, 409.
27. Kupferberg, H. J. Epilepsia 1989, 30, 51.
28. Dunham, N. W.; Miya, T. A. J. Am. Pharm. Assoc. Sci.
1957, 46, 208.
29. Brown, W. C.; Schiffman, D. O.; Swinyard, E. A.;
Goodman, L. S. J. Pharmacol. Exp. Ther. 1953, 107,
273.
The authors thank Dr James Stables for providing them
with pharmacological data through the Antiepileptic
Drug Development Program (Epilepsy Branch, Na-
tional Institute of Neurological Disorders and Stroke,
National Institutes of Health, Bethesda, MD, USA).
The project is co-financed from the European Union
Funds and National Budget.
30. Kaminski, R. F.; Livingood, M. R.; Rogawski, M. A.
Epilepsia 2004, 45, 864.
References and notes
31. Racine, R. J. Electroencephalogr. Clin. Neurophysiol. 1972,
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1. McNamara, O. J. In The Pharmacological Basis of
Therapeutics; Hardman, J. G., Limbird, L. E., Gilman,
A. G., Eds.; McGraw-Hill: New York, 2001; p 521.
32. Yogeeswari, P.; Sriram, D.; Thirumurugan, R.; Ragha-
vendran, J. V.; Sudhan, K.; Pavana, P. K.; Stables, J. P.
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