Design, synthesis, and anticonvulsant activity of new N-Mannich bases derived from spirosuccinimides and spirohydantoins

Article abstract of DOI:10.1016/j.bmc.2010.06.064

The synthesis and anticonvulsant properties of new N-Mannich bases of [7,8-f]benzo-2-aza-spiro[4.5]decane-1,3-diones (5a-h) and [7,8-f]benzo-1,3- diaza-spiro[4.5]decane-2,4-diones (7a-h) were described. Initial anticonvulsant screening was performed using

Full text of DOI:10.1016/j.bmc.2010.06.064

Bioorganic & Medicinal Chemistry 18 (2010) 6134–6142
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and anticonvulsant activity of new N-Mannich bases
derived from spirosuccinimides and spirohydantoins
a
a
a
b
Jolanta Obniska a, , Hanna Byrtus , Krzysztof Kaminski , Maciej Pawłowski , Małgorzata Szczesio ,
*
´
Janina Karolak-Wojciechowska b
a Department of Medicinal Chemistry, Jagiellonian University, Medical College, 9 Medyczna Street, 30-688 Kraków, Poland
Institute of General and Ecological Chemistry, Technical University, 116 Zeromskiego Street, 90-924 Łódz, Poland
b
_
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 January 2010
Revised 8 June 2010
Accepted 16 June 2010
Available online 30 June 2010
The synthesis and anticonvulsant properties of new N-Mannich bases of [7,8-f]benzo-2-aza-
spiro[4.5]decane-1,3-diones (5a–h) and [7,8-f]benzo-1,3-diaza-spiro[4.5]decane-2,4-diones (7a–h) were
described. Initial anticonvulsant screening was performed using intraperitoneal (ip) maximal electro-
shock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizures tests. The neurotoxicity was deter-
mined applying the rotarod test. The majority of compounds were effective in the MES or/and scPTZ
screen. The quantitative studies showed that several molecules were more potent than phenytoin, used
as reference drug. Selected derivatives were screened in the 6-Hz test and also assessed for potential
activity against nerve agents using the Pilocarpine Induced Status Prevention model. To explain the pos-
sible mechanism of anticonvulsant action, for chosen active derivatives, their influence on voltage-depen-
dent Na+ channel were tested in vitro.
Keywords:
Anticonvulsant activity
Pyrrolidine-2,5-diones
Imidazolidine-2,4-diones
Spirosuccinimides
Spirohydantoins
Ó 2010 Elsevier Ltd. All rights reserved.
In vivo studies
In vitro studies
1. Introduction
of synaptic release.7 In general, the discovery of new anticonvulsants
is still particularly focused on targeting voltage-dependent Na+
The past decades have demonstrated many attempts to identify
the structural features of compounds crucial for anticonvulsant
activity. It’s well documented that one of the important core
fragments of anticonvulsants is defined by nitrogen heteroatomic
system, usually a cyclic imide, at least of one carbonyl group and
phenyl or alkyl groups attached to the heterocyclic system.1–3 This
common template is present in the structures of two old, however
well-established anticonvulsants (AEDs) such as ethosuximide and
phenytoin as well as among the newest drugs, for example, leveti-
racetam, brivaracetam, or seletracetam4–6 (Fig. 1).
The incomplete information about the cellular mechanism of hu-
man epilepsy as well as the complex mechanism of action of most
AEDs makes it difficult to use of rational methodologies of discovery.
Therefore an alternative rational design of new AEDs is based on
mentioned pharmacophoric patterns and enables to overcome the
above difficulties. The currently used AEDs can be classified into four
categories on the basis of the main molecular mechanisms of action,
as follow: (i) modulation of voltage-dependent Na+ and/or Ca2+
channels, (ii) enhancement of GABA-mediated inhibition or other
effect on the GABA system, (iii) inhibition of synaptic excitation
mediated by ionotropic glutamate receptors, and (iv) modulation
channels since they play a fundamental role in establishing and reg-
ulation the excitability of neurons within the central nervous system
(CNS). Furthermore the changes in the expression pattern and
behavior of different Na+ channels isoforms are observed in epilepsy
and could play a role in seizure generation and spread.8,9
Previous research in our group has identified the pyrrolidine-
2,5-dione system differently substituted at position-1 and -3 as
targets for new antiepileptic drugs. Many of these compounds
were effective in the maximal electroshock (MES) or/and subcuta-
neous pentylenetetrazole (scPTZ) screens that are known as the
most popular seizure models in the early stages of the search of
new anticonvulsants.10–12
Recent studies from our laboratory have demonstrated the po-
tent anticonvulsant activity among the N-(4-arylpiperazin-1-yl)-
alkyl-azaspiranes.13,14 In this group of derivatives the most active
was compound 1 with the ED50 value of 23 mg/kg and its trifluoro-
methyl analogue 2 that showed the ED50 of 205 mg/kg in the MES
test (Fig. 1). Taking into consideration the above, as part of our ef-
forts to design new anticonvulsant agents, in the present study we
have synthesized a new series of compounds with the [7,8-f]ben-
zo-2-aza-spiro[4.5]decane-1,3-dione (5a–h) system as
a core
fragment. Based on results obtained previously,13,14 at the nitrogen
atom of succinimide we have introduced, by methylene spacer
amines favorable for anticonvulsant activity: 4-phenyl-piperazines
* Corresponding author. Tel.: +48 12 620 54 53; fax: +48 12 657 02 62.
E-mail address: mfobnisk@cyf-kr.edu.pl (J. Obniska).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.064

J. Obniska et al. / Bioorg. Med. Chem. 18 (2010) 6134–6142
6135
H
N
O
O
N
O
N
H
H
O
Phenytoin
Ethosuximide
F
F
O
O
O
N
N
N
NH2
NH2
NH2
H
H
H
O
O
O
Levetiracetam
Brivaracetam
Seletracetam
Cl
CF3
O
O
N
N
O
O
(CH2)2
(CH2)2
N
N
N
N
1
2
ED50 = 23 mg/kg (MES test)
PI = 2.60
ED50 = 205 mg/kg (MES test)
PI = 1.95
Figure 1. Lead structures for target compounds 5a–h and 7a–h.
(unsubstituted or with electron-withdrawing Cl, F atoms or CF3
group), benzyl-piperazine, 2-hydroxyethyl-piperazine or morpho-
line. On the other hand, taking into account well documented
anticonvulsant properties many of hydantoins15–17 and spirohyd-
antoins,18–20 as a second step of our investigation, we have synthe-
sized a series of analogues 5a–h in which the succinimide core
fragment has been changed into the hydantoin ring (7a–h). Such
modification enabled the evaluation of the role of mentioned het-
erocyclic rings for the anticonvulsant activity.
by both spectral and elemental analysis. The detailed physical
and analytical data are listed in the experimental section.
As usually in our practices,23–26 the molecular structure of se-
lected compounds, 5a and 5h, has been crystallographically con-
firmed. In both molecules the mutual positions of [7,8-f]benzo-2-
aza-spiro[4.5]decane-1,3-dione moiety with respect to 4-aryl-
piperazine (5a) or morpholine (5h) are almost the same (Fig. 2).
2.2. Anticonvulsant activity
The pre-clinical discovery and development of new chemical
agents for the treatment of epilepsy are based mainly on the use
of predictable animal models. At the present time there are three
in vivo screens used routinely that include the maximal electro-
shock 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.27
The profile of anticonvulsant activity of compounds 5a–h and
7a–h was established in the maximal electroshock (MES) and sub-
cutaneous pentylenetetrazole (scPTZ) tests, after intraperitoneal
injection into mice at doses of 30, 100, and 300 mg/kg. An observa-
tion was carried out at two different time intervals—0.5 and 4 h.
The acute neurological toxicity (NT) was determined by use of
the minimal motor impairment-rotarod screen (NT). The results
are shown in Table 1.
2. Results and discussion
2.1. Chemistry
The synthesis of compounds 5a–h and 7a–h was accomplished
as shown in Scheme 1. The starting materials 2-carboxy-1,2,3,4-
tetrahydronaphtalene-2-carboxylic acid (3) and [7,8-f]benzo-2-
azaspiro[4.5]decane-1,3-dione (4) were prepared using methods
reported by Faust et al.21 The b-tetralonehydantoin ([7,8-f]benzo-
1,3-diaza-spiro-decane-2,4-dione) (6) was prepared from the 3,4-
dihydronaphtalen-2-on (b-tetralone) by the Bücherer–Berg reac-
tion with modifications described by Goodson et al.22 In the next
step, we prepared two series of new N-Mannich bases derived from
spirosuccinimide (5a–h) or spirohydantoin (7a–h). For aminoalky-
lation of the acidic proton, in the presence of formaldehyde, we
used various 4-substituted piperazines or morpholine. The reaction
was carried out in ethanol at a room temperature for ca. 12 h. The
final 5a–h and 7a–h compounds were obtained in yields ranging
from 62% to 88%. Their purity was assessed by TLC chromatogra-
phy. The structures of compounds synthesized were confirmed
The initial anticonvulsant evaluation of spirosuccinimides (5a–
h) showed, that excluding 5a and 5b, devoid of anticonvulsant
activity, all other derivatives were effective in MES test (5c–h)
and several of them, namely 5c, 5g, and 5h were also active in
the scPTZ screen. Compounds that revealed protection in the MES

6136
J. Obniska et al. / Bioorg. Med. Chem. 18 (2010) 6134–6142
b
a
O
O
COOH
N
N
O
O
HOOC
H
N
N
3
4
R
5a-g
H
N
H
N
O
cmpd
b
R
O
N
O
N
H
-C6H5
2-F-C6H4
4-F-C6H4
5a, 7a
5b, 7b
5c, 7c
5d, 7d
5e, 7e
5f, 7f
N
O
N
R
6
7a-g
3-Cl-C6H4
3-CF3-C6H4
-CH2-C6H5
-(CH2)2-OH
5g, 7g
X
c
X
O
O
N
N
O
O
H
N
O
X = CH2 (5h)
X = NH (7h)
X = CH2 (4)
X = NH (6)
Scheme 1. Synthetic protocol of the target compounds 5a–h and 7a–h. Reagents and conditions: (a) 25% NH4OH, 190 °C, 2 h, (b) 4-substituted piperazine derivatives,
formaldehyde, 96% ethyl alcohol, 12 h room temperature, and (c) morpholine, formaldehyde, 96% ethyl alcohol, 12 h room temperature.
Figure 2. ORTHEP drawing of the 5a and 5h molecules.
test, indicative the ability of substance to prevent seizure spread, at
a dose of 100 mg/kg included 5c–e, 5g, and 5h, whereas molecule
5f provided anti-MES protection at a dose of 300 mg/kg. Except of
compound 5c, which was active only at 0.5 h after ip administra-
tion, the other derivatives 5d–h showed activity at 4 h, indicating
long duration of anticonvulsant activity. Apart from the anti-MES
protection, compounds 5c, 5g, and 5h were found to be active in
the scPTZ test, which identify substances elevating seizure thresh-
old. Among these molecules, 5c and 5h showed anti-scPTZ activity
at the dose 100 mg/kg that was equivalent to ethosuximide used as
reference anticonvulsant. In the neurotoxicity screen (NT), only 5f
did not show neurotoxicity in the maximum dose administered
(300 mg/kg). The other derivatives 5c–e, 5g, and 5h showed neuro-
toxicity at a dose of 300 mg/kg. At the same dose mice were unable
to grasp rotarod after administration of 5g and 5h.
The change of core fragment from [7,8-f]benzo-2-aza-spiro-
[4.5]decane-1,3-dione (spirosuccinimide) to [7,8-f]benzo-1,3-dia-
za-spiro[4.5]decane-2,4-dione (spirohydantoin) yielded in more
potent, however at the same time more neurotoxic compounds
7a–h. In this series only 7c was inactive that was in contrary to
its succinimide analogue 5c, active both in the MES and scPTZ test.
The other derivatives inhibited seizures only in the MES—7a, 7b,
7d, 7e, and 7f or in both MES and scPTZ screens—7g and 7h. In this
series, the most active was 7h which showed anti-MES protection

J. Obniska et al. / Bioorg. Med. Chem. 18 (2010) 6134–6142
6137
Table 1
Table 2
Anticonvulsant screening project (ASP): phase I results for compounds 5a–h and 7a–h
Anticonvulsant activity (MES test) of selected compounds administrated orally to rats
(30 mg/kg)
Compd
Intraperitoneal injection in micea
ASP classe
Compd
Oral administration to ratsa
MESb
scPTZc
NTd
0.25 h
0.5 h
1 h
2 h
4 h
0.5 h 4 h
0.5 h
4 h
0.5 h
4 h
5c
5d
5e
5g
5h
7a
7f
7g
0
0
0
1
2
1
1
0
0
1
0
0
0
1
1
2
2
1
1
4
0
1
3
0
1
2
3
2
2
3
1
2
3
1
1
4
2
4
3
3
1
2
4
2
2
4
2
4
4
3
5a
5b
5c
5d
5e
5f
5g
5h
7a
7b
7c
7d
7e
7f
7g
7h
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
100
300
300
300
—
300
—
300
—
300
—
—
3
3
1
1
1
2
1
1
1
1
4
4
4
1
1
1
100
300
—
300
100
100
—
—
—
—
300
100
100
30
30
—
10025
—
100
100
300
100 300
300 100
30
100
—
—
—
—
30014
30014
300
—
300
7h
—
—
—
—
—
—
Phenytoinb
30033 30012,34
a
30
30
30
30
30
—
—
—
The data in the oral MES screen indicate the number of rats of four that were
protected at a dose of 30 mg/kg.
100
30
100
b
100
Reference drug, data from Ref. 40.
3001
300 100
300 10014
100 100
30
30
—
100
—
100
Phenytoinf
Ethosuximidef
—
300
100
—
100
—
100% of animals at 4 h, 75% at 2 h, 50% at 1 h, and 25% at 0.5 h. Fi-
nally 7f showed one peak of 75% protection at 1 h and was also
effective in 50% of animals at 0.5, 2, and 4 h. As can be seen from
the data above, the compounds tested were characterized by de-
layed onset and long time of anticonvulsant action. Furthermore,
several molecules showed activity comparable to observed for phe-
nytoin. When given orally they were non-neurotoxic (data not
indicated in Table 2).
Sprosuccinimides 5c and 5h, active in both MES and scPTZ
screens in mice, were evaluated in the subcutaneous pentylenetet-
razole test after oral administration into rats. Initially a dose of
50 mg/kg was employed and the anticonvulsant activity was as-
sessed in five time points (Table 3).
As can be seen, 5c protected 25% of animals at 2 and 4 h,
whereas 5h showed 25% protection time intervals from 0.5 to
4 h. These molecules were less active than ethosuximide, which
is the model substance effective in the pentylenetetrazole seizures.
These molecules did not exhibit neurotoxicity at the dose applied.
Compounds 5d, 5e, 5h, 7a, 7g, and 7h were chosen for quantifi-
cation of the pharmacological parameters (ED50 and TD50) after
oral administration into rats. The quantitative evaluation of the
MES and scPTZ median effective dose (ED50) and toxic dose
(TD50) were performed at previously estimated time of peak effect
(TPE). Results of the quantitative tests along with the data for the
standard drugs phenytoin and ethosuximide are shown in Table 4.
The analysis of quantitative data revealed that except of 5h the
other compounds were more potent than phenytoin in the MES
test. In the applied range of doses none of them was enough effec-
tive to determine the ED50 in the pentylenetetrazole seizures. In
this group the most active was 7a with ED50 value of 1.88 mg/kg
and TD50 of >500 mg/kg, that resulted in very promising protection
index (TD50/ED50) of 265.78, especially when comparing to 35.58 of
phenytoin. It should be pointed out that calculated PI values have
only an approximate character because in general low neurotox-
Response comments: 1death, 12ataxia, 14unable to grasp rotarod, 25myoclonic jerks,
33tremors, 34muscle spasms.
a
Doses of 30, 100, and 300 mg/kg were administered. The data indicate the
minimum dose whereby anticonvulsant activity or neurotoxicity was demonstrated
in half or more animals. The animals were examined at 0.5 and 4 h. A dash indicates
the absence of anticonvulsant activity and neurotoxicity at the maximum dose
administered (300 mg/kg).
b
Maximal electroshock test.
Subcutaneous pentylenetetrazole test.
Neurotoxicity screening—rotarod test.
The ASP classification is as follows: 1—anticonvulsant activity at doses of
c
d
e
100 mg/kg or less; 2—anticonvulsant activity at doses of 300 mg/kg; 3—compound
inactive at doses of 300 mg/kg; 4—compound active but toxic at a dose of 30 mg/kg
or only toxic at the same dose.
f
Reference drugs, data for phenytoin and ethosuximide Ref. 39.
at a dose of 30 mg/kg in both time intervals that was equal to activ-
ity of phenytoin used as reference drug. Additionally, it was also
effective in the scPTZ screen at a dose of 100 mg/kg at 0.5 and
4 h. Furthermore, the anti-MES activity at doses of 30 mg/kg was
observed for 7a and 7e, however the letter was neurotoxic at the
same dose unfortunately. The 2-fluoro derivative 7b was active
at a dose of 100 mg/kg but at a dose of 300 mg/kg provoked muscle
spasms, tremors, and ataxia. As presented in Table 1, compounds
7d and 7f inhibited seizure at a dose of 100 mg/kg (MES). The
wider activity was observed for 7g, which showed anti-MES and
anti-scPTZ protection at doses of 100 mg/kg (0.5 h) and 300 mg/
kg (4 h), respectively. This derivative exhibited neurotoxicity at a
dose of 100 mg/kg—mice were unable to grasp rotarod. It is worthy
of note that compounds active both in MES and scPTZ tests, may be
useful in treating not only generalized tonic-clonic epilepsy but
also absence seizures.
On the basis of data obtained in mice and according to Anticon-
vulsant Screening Project (ASP) disposition, nine compounds 5c–e,
5g, 5h, 7a, 7f, 7g, and 7h were selected and examined for their anti-
convulsant activity (MES screen) and neurotoxicity after po admin-
istration into rats at a dose of 30 mg/kg. The results obtained are
presented in Table 2.
Table 3
Anticonvulsant activity (scPTZ test) of selected compounds administrated orally to
rats (50 mg/kg)
As can be seen from these data, in the series of spirosuccini-
mides the most active was 5e that showed one peak of 100% pro-
tection at 4 h and 75% protection at time intervals 1 and 2 h. The
other derivatives (5d, 5g, and 5h) were less active and protected
at most of 50% animals. Much higher activity was observed for spi-
rohydantoin derivatives. In this series the most active were 7a and
7g that protected 100% of animals at 2 and 4 h. Compound 7a pro-
tected also 50% of rats (0.5 h, 1 h) and 25% (0.25 h), whereas 7g—
50% (1 h) and 25% (0.5 h). A little less activity revealed 7h, namely
Compd
Oral administration to ratsa
0.25 h
0.5 h
1 h
2 h
4 h
5c
5h
0
0
0
0
1
2
0
1
1
1
1
1
1
1
0
Ethosuximideb
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. 39.

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J. Obniska et al. / Bioorg. Med. Chem. 18 (2010) 6134–6142
Table 4
Quantitative anticonvulsant data in rats dosed orally for compounds 5d, 5e, 5h, 7a, 7g, and 7h
b
b
b
Compd
TPE (h)a
ED50 MES (mg/kg)
ED50 scPTZ (mg/kg)
TD50 (mg/kg)
PIc MES (TD50/ED50
)
5d
5e
5h
7a
7g
7h
6.0
2.0
0.5
6.0
4.0
6.0
1.0
19.57 (9.23–34.80)
9.00 (4.81–14.55)
>160
1.88 (0.93–3.43)
14.93 (10.73–18.77)
10.65 (6.86–16.09)
28.1 (27.7–35.20)
>500
ND
ND
>150
ND
>250
>200
>500
167 (116–237)
>500
>250
>500
>500
>300
400
25.55
>27.78
—
>265.78
>20.09
37.55
>3.6
Phenytoind
Etosuximided
>100
>500
>2.99
ND—no data.
a
Time to peak effect.
b
Results are represented as mean SEM at 95% confidence limit (MES-maximal electroshock test; scPTZ—subcutaneous pentylenetetrazole test; neurotoxicity).
Protection index (TD50/ED50).
Reference drug, data from Ref. 41.
c
d
icity of all substances, including standard drugs, made impossible
systems that serve to reinforce recurring seizure activity progress-
ing to status epilepticus. To determine if the tested substances 5c–e
can prevent acute pilocarpine induced status in the Pilocarpine In-
duced Status Prevention (PISP) model an initial qualitative efficacy
screens were performed. These molecules were given to rats via
the ip route of administration. Then a challenge dose of pilocarpine
was administrated observing for treatment-effects of the sub-
stance. The outcome measures are determination of ‘protection’
or ‘no-protection’. The seizure severity is determined using the
well known Racine scale,29 as follow: (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 accomplished by bilateral forelimb
clonus; and (V) all of the above plus loss of balance and falling,
accomplished by generalized clonic seizures. The anticonvulsant
activity of compounds 5c–e was assessed at time zero, namely
the time from the first stage III seizures. The results are shown in
Table 6.
the precise estimation of TD50 parameters. However it is justified
to claim that 5d, 5e, 7a, 7g, and 7h were equal or more potent than
reference phenytoin.
Selected compounds 5d, 5e, and 5f were chosen for the evalua-
tion 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 chemically diversified compounds pursued
in the NIH/NINDS. The 6-Hz screen has been validated recently as a
model of therapy-resistant epilepsy. It was not used widely be-
cause of its lack clinical validity since the hydantoins such as phe-
nytoin 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 pro-
tective activity in the 6-Hz model. This suggested that the 6-Hz
model might be capable for identifying anti-seizure agents with a
novel spectrum of activity and unknown mechanism of anticonvul-
sant action.28 The results obtained are shown in Table 5.
As can be seen compounds 5d, 5e, and 5f administrated intra-
peritoneally at a dose of 100 mg/kg revealed marginal activity
and protected 50% mice (5d at 4 h, 5e at 2 h) and 25% at 1 h (5e, 5f).
The ASP has employed recently a series of models designed to
arrest benzodiazepine resistant status epilepticus including: pilo-
carpine induced status, neuroprotection against excitotoxins such
In the PISP screen, all molecules were found to be inactive.
During further studies compounds, 5d and 5h were examined in
the in vitro hippocampal slice culture neuroprotection assay, how-
ever did not exhibit neuroprotection against KA and NMDA.
2.3. In vitro sodium channels binding assays
as kainic acid (KA) or N-methyl-D-aspartate (NMDA) in in vitro hip-
pocampal slice culture model, non-convulsive electrophysiology
monitoring and isobolographic studies. These models were devel-
oped and incorporated into the ASP screening capabilities with
the intent to find compounds useful against both convulsive and
non-convulsive status.
According to the ASP dispositions, compounds 5c–e were as-
sessed for potential activity against nerve agents using the Pilocar-
pine Induced Status Prevention (PISP) model. This model shares
many characteristics with nerve agent induced seizures since both
initiation and early expression of nerve agent induced seizures are
cholinergic followed by the recruitment of other neurotransmitter
Intensive studies into the physiological and biochemical events
taking place during epileptic seizures have provided insight into
the molecular mechanisms by which these might be controlled.
Inhibition of neuronal conductance via block of voltage-gated so-
dium channels is proven, however not only mechanism, by which
anticonvulsants such phenytoin, carbamazepine or lamotrigine act
to control seizures. To search for possible mechanism of action of
chosen, active derivatives 5d, 5e, 7a, and 7h, their affinities for so-
dium channels were assessed in vitro. The sodium channels were
Table 6
Table 5
Pilocarpine Induced Status Prevention (PISP) model. Results for compounds 5c–e after
Results of anticonvulsant activity by psychomotor seizure test (6-Hz, dose of 100 mg/
kg)
ip administration (Test 71)
Compd Dose
(mg/
Time Protected Non-
(h)a rats
protected
Average weight
Compd
Intraperitoneal injection into micea
change SEMb (g)
kg)
rats
0.25 h
0.5 h
1 h
2 h
4 h
Protected Non-protected
rats
rats
5d
5e
5f
0
0
0
0
0
0
0
1
1
0
215
0
2
0
0
5c
5d
5e
600
600
600
0
0
0
0
0
0
6
8
7
0
0
0
20 1.1
20.6 1.1
15.0 0.0
Response comments: 15minimal motor impairment.
Dose of 100 mg/kg was administrated. The data indicate the number of mice of
four that were protected.
a
a
Post first stage III seizure.
Weight change 24 h Post first stage III seizure.
b

J. Obniska et al. / Bioorg. Med. Chem. 18 (2010) 6134–6142
6139
chosen from many molecular targets for anticonvulsants on the ba-
4. Experimental part
4.1. Chemistry
sis of in vivo data that showed high activity of compounds tested in
the maximal electroshock test. These studies were based on the
ability of compound to displace [3H]batrachotoxin from neurotoxin
site 2 of voltage-gated sodium channels in rat forebrain tissue. The
IC50 values of 5d, 5e, 7a, and 7h together with phenytoin used as
standard drug effective in the electrically induced seizures are
listed in Table 7.
As shown in Table 7 compounds 5d, 5e, and 7a revealed higher
affinities than phenytoin whereas 7h did not bind to the voltage-
gated sodium channels. The radioligand binding experiments sug-
gest that anticonvulsant activity of compounds tested may be par-
tially mediated by influence on the sodium channels. It should be
emphasized that it is one but not only mechanism of action.
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 assessed with TLC. The thin-layer chromatog-
raphy (TLC) was performed on Merck silica gel 60 F254 aluminum
sheets (Merck; Darmstadt, Germany), using developing systems
consisted of S1 chloroform/acetone (9:1, v/v), S2—benzene/ethyl
acetate/acetone (10:5:1, v/v/v), S3—methanol/25% ammonia
(100:1.5, v/v).
2.4. Structure–activity relationships
Spots were detected by their absorption under UV light
(k = 254 nm) and by visualization with 0.05 mol I2 in 10% HCl. Ele-
mental analysis for C, H, and N were carried out by a micro method
using the elemental Vario EI III Elemental analyzer (Hanau, Ger-
many). The results of elemental analyses were within 0.4% of
the theoretical values.
The results of the preliminary anticonvulsant screening of spi-
rosuccinimides revealed that the most active were compounds
with 3-chloro atom (5d) and 3-CF3 (5e) group at the 4-arylpiper-
azine fragment that showed ED50 values of 19.23 and 9.00 mg/kg,
respectively. It was in agreement with our previous studies in
which we have demonstrated that presence of electron-attracting
substituents, especially CF3 groups, increases anticonvulsant activ-
ity.10,12–14 The removal or replacement of the above substituents
decreased anticonvulsant activity (5c) or made compounds inac-
tive (5a, 5b). The similar effect was observed in respect to the ben-
zyl-piperazine analogue 5f. The change of phenyl ring at position-4
of piperazine into hydroxyethyl chain (5g) has extended the profile
of anticonvulsant activity and made compound active in both MES
and scPTZ seizures. Further data revealed that introduction of the
spirohydantoin moiety as core fragment yielded in more potent,
however at the same time more neurotoxic, derivatives. In this ser-
ies the most active were unsubstituted compound 7a, and deriva-
tives with 2-hydroxyethyl-piperazine- (7g) and morpholine (7h)
with an ED50 values of 1.88 mg/kg (7a), 14.93 mg/kg (7g) and
10.65 mg/kg (7h) in the MES test. It should be pointed out that
7d and 7e, obtained as analogues of the most effective spirosuccin-
imides (5d and 5e), showed high activity, however due to the
strong neurotoxicity were excluded from further testing.
1H NMR spectra were obtained in a Varian Mercury spectrome-
ter (Varian Inc., Palo Alto, CA, USA), in CDCl3, operating at 300 MHz.
Chemical shifts are reported in d values (ppm) relative to TMS d = 0
(1H), as internal standard. The J values are expressed in Hertz (Hz).
Signal multiplicities are represented by the following abbrevia-
tions: s (singlet), br s (broad singlet), d (doublet), t (triplet), dd
(double doublet), q (quartet), m (multiplet).
4.1.1. 2-Carboxy-1,2,3,4-tetrahydronaphtalene-2-carboxylic
acid (3), [7,8-f]benzo-2-azaspiro[4.5]decane-1,3-dione (4), [7,8-
f]benzo-1,3-diaza-spiro-decane-2,4-dione (6)
The starting materials 2-carboxy-1,2,3,4-tetrahydronaphtalene-
2-carboxylic acid (3) or [7,8-f]benzo-2-azaspiro[4.5]decane-1,3-
dione (4) were prepared using methods reported by Faust et al.21
The b-tetralonehydantoin ([7,8-f]benzo-1,3-diaza-spiro-decane-
2,4-dione) (6) was prepared from the 3,4-dihydronaphtalen-2-on
(b-tetralone) by the Bücherer–Berg reaction with modifications de-
scribed by Goodson et al.22
4.1.2. General procedure for the synthesis of compounds 5a–h
and 7a–h
3. Conclusion
The mixture of [7,8-f]benzo-2-aza-spiro[4.5]decane-1,3-dione
(0.01 mol) (4), or [7,8-f]benzo-1,3-diaza-spiro[4.5]decane-2,4-
dione (0.01 mol) (6), 40% formaldehyde solution (0.01 mol) and
corresponding 4-substituted piperazine (0.01 mol) or morpholine
(0.01 mol) in 96% ethanol (40 ml) was left for ca. 12 h and then
refrigerated ca. ꢀ10 °C for 24 h. The products were washed with
cold ethanol and the solid products (5a–h and 7a–h) were sepa-
rated by filtration and recrystallized from 96% ethanol.
The results obtained revealed that majority of compounds syn-
thesized exhibited high anticonvulsant activity, especially in the
maximal electroshock test, which is the animal model of human
generalized tonic-clonic seizures. The ED50 values for the most ac-
tive compounds were in range of 1.88–19.57 mg/kg. Several mole-
cules were more potent and safer than phenytoin used as reference
antiepileptic drug. In summary, it is justified to claim that the spi-
rosuccinimide and spirohydantoin moieties enable the design of
new prototypic molecules, their consequent structural modifica-
tions and for that reason they play an essential role in the search
for novel anticonvulsants.
4.1.2.1. N-[(4-Phenylpiperazin-1-yl)-methyl]-[7,8-f]benzo-2-aza-
spiro[4,5]decane-1,3-dione (5a). White powdery crystals. Yield:
70%; mp 145–147 °C; TLC: Rf = 0.66 (S1); 1H NMR (300 MHz,
CDCl3): d 1.91 (br s, 1H cyclohexane), 2.18–2.26 (m, 1H cyclohex-
ane), 2.52–2.99 (m, 7H, 1H cyclohexane, 2H imide, 4H piperazine),
3.00–3.06 (m, 2H cyclohexane), 3.22 (br s, 4H piperazine), 3.30 (d,
1H cyclohexane, J = 16.40 Hz), 4.60 (s, 2H, –CH2–), 6.91–7.10 (m,
2H, ArH), 7.13–7.20 (m, 4H, ArH), 7.27–7.34 (m, 3H, ArH);
Table 7
Results of in vitro sodium channels binding
assays
Compd
IC50 (lM)
C24H27N3O2 (389.50).
5d
5e
7a
2.45
9.28
9.35
4.1.2.2. N-[{4-(2-Fluorophenyl)-piperazin-1-yl}-methyl]-[7,8-f]-
benzo-2-aza-spiro[4.5]decane-1,3-dione (5b). White powdery
crystals. Yield: 69%; mp 115–117 °C; TLC: Rf = 0.86 (S1); 1H NMR
(300 MHz, CDCl3): d 1.88 (t, 1H cyclohexane, J = 7.43 Hz), 2.19–
7h
No affinity
40.0
Phenytoina
a
Reference drug, data from Ref. 42.

6140
J. Obniska et al. / Bioorg. Med. Chem. 18 (2010) 6134–6142
2.30 (m, 1H cyclohexane), 2.52 (d, 1H cyclohexane, J = 18.20 Hz)
2.68 (q, 2H imide, J = 8.46 Hz), 2.84–99 (m, 5H, 1H cyclohexane,
4H piperazine), 3.00–3.10 (m, 1H cyclohexane), 3.22 (br s, 4H
piperazine), 3.30 (d, 1H cyclohexane, J = 16.40 Hz), 4.60 (s, 2H,
–CH2–), 6.94–6.99 (m, 2H, ArH), 7.02–7.16 (m, 3H, ArH), 7.20–
7.24 (m, 3H, ArH); C24H26FN3O2 (407.49).
morpholine, J = 4.49 Hz), 4.50 (s, 2H –CH2–), 7.06–7.20 (m, 4H,
ArH); C18H22N2O3 (314.39).
4.1.2.9. N-[(4-Phenylpiperazin-1-yl)-methyl]-[7,8-f]benzo-1,3-
diaza-spiro[4.5]decane-2,4-dione (7a). White powdery crystals.
Yield: 82%; mp 220–222 °C; TLC: Rf = 0.21 (S1); Rf = 0.37 (S2); 1H
NMR (300 MHz, CDCl3): d 1.93–1.96 (m, 1H cyclohexane), 2.17–
2.34 (m, 1H, cyclohexane), 2.77–2.92 (m, 6H, 2H cyclohexane, 4H
piperazine), 2.96–3.26 (m, 5H, 1H cyclohexane, 4H piperazine),
3.46 (d, 1H cyclohexane, J = 16.37 Hz), 4.58 (s, 2H, –CH2–), 6.02
(br s, 1H, N1H), 6.90–6.94 (m, 3H, ArH), 7.05–7.31 (m, 6H, ArH);
4.1.2.3. N-[{4-(4-Fluorophenyl)-piperazin-1-yl}-methyl]-[7,8-f]-
benzo-2-aza-spiro[4.5]decane-1,3-dione (5c). White powdery
crystals. Yield: 77%; mp 173–175 °C; TLC: Rf = 0.45 (S1); 1H NMR
(300 MHz, CDCl3): d 1.88 (br s, 1H cyclohexane), 2.15–2.30 (m,
1H cyclohexane), 2.51 (d, 1H cyclohexane, J = 18.46), 2.66 (q, 2H,
imide, J = 8.63 Hz), 2.74–2.99 (m, 5H, 1H cyclohexane, 4H pipera-
zine), 3.00–3.11 (m, 5H, 1H cyclohexane, 4H piperazine), 3.33 (d,
1H cyclohexane, J = 16.15 Hz), 4.59 (t, 2H –CH2–, J = 13.59 Hz),
6.88–7.20 (m, 8H, ArH); C24H26FN3O2 (407.49).
C23H26O2N4 (389.48).
4.1.2.10. N-[{4-(2-Fluorophenyl)-piperazin-1-yl}-methyl]-[7,8-
f]benzo-1,3-diaza-spiro[4.5]decane-2,4-dione (7b). White pow-
dery crystals. Yield: 78%; mp 148–150 °C; TLC: Rf = 0.37 (S2),
Rf = 0.96 (S3); 1H NMR (300 MHz, CDCl3): d 1.86–1.92 (m, 1H, cyclo-
hexane), 2.18–2.29 (m, 1H, cyclohexane), 2.75 (t, 4H, piperazine,
J = 5.05 Hz), 2.81–2.86 (m, 2H cyclohexane), 2.91 (t, 4H piperazine,
J = 5.0 Hz), 3.03–3.16 (m, 1H cyclohexane), 3.42 (d, 1H cyclohex-
ane, J = 16.47 Hz), 4.56 (s, 2H, –CH2–), 6.58 (s, 1H, N1H), 6.82–
7.05 (m, 8H, ArH); C23H25O2N4F (408.48).
4.1.2.4. N-[{4-(3-Chlorophenyl)-piperazin-1-yl}-methyl]-[7,8-f]-
benzo-2-aza-spiro[4.5]decane-1,3-dione (5d). White powdery
crystals. Yield: 79%; mp 152–153 °C; TLC: Rf = 0.71 (S1); 1H NMR
(300 MHz, CDCl3): d 1.88 (br s, 1H cyclohexane), 2.16–2.30 (m,
1H cyclohexane), 2.47–2.55 (m, 1H cyclohexane), 2.62–2.99 (m,
7H, 1H cyclohexane, 2H imide, 4H piperazine), 3.00–3.06 (m, 1H
cyclohexane), 3.21–3.35 (m, 4H piperazine), 3.33 (d, 1H cyclohex-
ane, J = 16.16 Hz), 4.59 (s, 2H –CH2–), 6.79–6.99 (m, 3H, ArH), 7.06–
7.20 (m, 5H, ArH); C24H26ClN3O2 (423.94).
4.1.2.11. N-[{4-(4-Fluorophenyl)-piperazin-1-yl}-methyl]-[7,8-
f]benzo-1,3-diaza-spiro[4.5]decane-2,4-dione (7c). White pow-
dery crystals. Yield: 81%; mp 211–213 °C; TLC: Rf = 0.36 (S2),
Rf = 0.94 (S3); 1H NMR (300 MHz, CDCl3): d 1.85–1.91 (m, 1H cyclo-
hexane), 2.16–2.27 (m, 1H cyclohexane), 2.75 (t, 4H piperazine,
J = 4.95 Hz,), 2.80–2.85 (m, 2H cyclohexane), 2.91 (t, 4H piperazine,
J = 5.0 Hz), 3.02–3.15 (m, 1H cyclohexane), 3.42 (d, 1H cyclohex-
ane, J = 16.45 Hz), 4.55 (s, 2H, –CH2–), 6.58 (s, 1H, N1H), 6.81–
7.18 (m, 8H, ArH); C23H25O2N4F (408.48).
4.1.2.5.
N-[{4-(3-Trifluoromethylphenyl)-piperazin-1-yl}-
methyl]-[7,8-f]benzo-2-aza-spiro[4.5]decane-1,3-dione (5e).
White powdery crystals. Yield: 75%; mp 140–142 °C; TLC:
Rf = 0.67 (S1); 1H NMR (300 MHz, CDCl3): d 1.83–1.90 (m, 1H
cyclohexane), 2.19–2.30 (m, 1H cyclohexane), 2.52 (d, 1H cyclo-
hexane, J = 18.46 Hz), 2.68 (d, 2H imide, J = 18.72 Hz), 2.85–2.93
(m, 5H, 1H cyclohexane, 4H piperazine), 3.02 (dd, 1H cyclohex-
ane, J = 17.95 Hz) 3.16–3.36 (m, 5H, 1H cyclohexane, 4H pipera-
zine), 4.61 (s, 2H, –CH2–), 7.06–7.20 (m, 7H, ArH), 7.36 (t, 1H,
ArH, J = 8.08 Hz); C25H26F3N3O2 (457.51).
4.1.2.12. N-[{4-(3-Chlorophenyl)-piperazin-1-yl}-methyl]-[7,8-
f]benzo-1,3-diaza-spiro[4.5]decane-2,4-dione (7d). White pow-
dery crystals. Yield: 86%; mp 190–191 °C; TLC: Rf = 0.39 (S2),
Rf = 0.39 (S3); 1H NMR (300 MHz, CDCl3): d 1.84–1.94 (m, 1H cyclo-
hexane), 2.16–2.32 (m, 1H cyclohexane), 2.75 (t, 4H piperazine,
J = 4.95 Hz), 2.80–2.96 (m, 2H cyclohexane), 3.00–3.24 (m, 5H, 4H
piperazine, 1H cyclohexane), 3.42 (d, 1H cyclohexane,
J = 16.50 Hz) 4.58 (s, 2H, –CH2–), 6.02 (br s, 1H, N1H), 6.86–6.98
(m, 3H, ArH), 7.05–7.35 (m, 5H, ArH); C23H25ClO2N4 (424.93).
4.1.2.6. N-[(4-Benzylpiperazin-1-yl)-methyl]-[7,8-f ]benzo-2-
aza-spiro[4.5]decane-1,3-dione (5f). White powdery crystals.
Yield: 78%; mp 132–134 °C; TLC: Rf = 0.42 (S1); 1H NMR
(300 MHz, CDCl3): d 1.86 (br s, 1H cyclohexane), 2.17–2.28 (m,
1H cyclohexane), 2.44–2.60 (m, 4H, piperazine), 2.63–2.66 (m,
6H, 2H imide, 4H piperazine), 2.80–2.90 (m, 2H cyclohexane),
2.92–3.05 (m, 1H cyclohexane), 3.31 (d, 1H cyclohexane,
J = 16.41 Hz), 3.51 (s, 2H, –CH2–), 4.51 (s, 2H, –CH2–), 7.09–7.19
(m, 4H, ArH), 7.31–7.54 (m, 5H, ArH); C25H29N3O2 (403.53).
4.1.2.13.
N-[{4-(3-Trifluoromethylphenyl)-piperazin-1-yl}-
methyl]-[7,8-f]benzo-1,3-diaza-spiro[4.5]decane-2,4-dione (7e).
White powdery crystals. Yield: 67%; mp 173–174 °C; TLC:
Rf = 0.33 (S2), Rf = 0.85 (S3); 1H NMR (300 MHz, CDCl3): d 1.88–
1.95 (m, 1H cyclohexane), 2.17–2.32 (m, 1H cyclohexane), 2.76
(t, 4H piperazine, J = 5.05 Hz), 2.82–3.06 (m, 3H cyclohexane),
3.12–3.24 (m, 4H piperazine), 3.40 (d, 1H cyclohexane,
J = 16.49 Hz), 4.60 (s, 2H, –CH2–), 6.26 (s, 1H, N1H), 7.04–7.27 (m,
7H, ArH), 7.35 (t, 1H, ArH, J = 7.95 Hz); C24H25O2N4F3 (458.49).
4.1.2.7.
N-[{4-(2-Hydroxyethyl)-piperazin-1-yl}-methyl]-[7,8-
f]benzo-2-aza-spiro[4.5]decane-1,3-dione (5g). White powdery
crystals. Yield: 80%; mp 46–48 °C; TLC: Rf = 0.66 (S1); 1H NMR
(300 MHz, CDCl3): d 1.85–1.91 (m, 1H cyclohexane), 2.08 (d, 1H
OH, J = 16.67 Hz), 2.18–2.27 (m, 1H cyclohexane), 2.51 (d, 1H
cyclohexane, J = 18.21 Hz), 2.63–2.99 (m, 12H, 8H piperazine, 2H
imide, 2H –CH2–), 3.00–3.15 (m, 1H cyclohexane), 3.32 (d, 1H
cyclohexane, J = 16.41 Hz), 3.49 (s, 1H cyclohexane), 3.71–3.78
(m, 2H, –CH2–), 4.52 (t, 2H, –CH2–, J = 13.59 Hz), 7.08–7.19 (m,
4H, ArH); C20H27N3O3 (357.46).
4.1.2.14. N-[(4-Benzylpiperazin-1-yl)-methyl]-[7,8-f]benzo-1,3-
diaza-spiro[4.5]decane-2,4-dione (7f). White powdery crystals.
Yield: 82%; mp 81–83 °C; TLC: = 0.21 (S1); Rf = 0.37 (S2); 1H NMR
(300 MHz, CDCl3): d 1.91–1.98 (m, 1H cyclohexane), 2.21–2.31
(m, 1H cyclohexane), 2.28–2.54 (m, 4H piperazine), 2.69–2.80 (m,
5H, 1H cyclohexane, 4H piperazine), 2.86–3.01 (m, 2H cyclohex-
ane), 3.38 (d, 1H cyclohexane, J = 16.41 Hz) 3.49 (s, 2H, –CH2–),
4.52 (s, 2H, –CH2–), 5.64 (br s, 1H, N1H), 7.01–7.19 (m, 5H, ArH),
7.26–7.33 (m, 4H, ArH); C24H28O2N4 (404.52).
4.1.2.8.
N-(Morpholin-1-yl)-methyl-[7,8-f]benzo-2-aza-spiro-
[4.5]decane-1,3-dione (5h). White powdery crystals. Yield: 70%;
mp 148–150 °C; TLC: Rf = 0.69 (S1); 1H NMR (300 MHz, CDCl3): d
1.80–1.88 (m, 1H cyclohexane), 2.18–2.29 (m, 1H cyclohexane),
2.50 (d, 1H cyclohexane, J = 18.21 Hz), 2.62–2.70 (m, 6H, 2H imide,
4H morpholine), 2.81–2.99 (m, 1H cyclohexane) 3.00–3.07 (m, 1H
cyclohexane), 3.33 (d, 1H cyclohexane, J = 16.16 Hz), 3.70 (t, 4H
4.1.2.15. N-[{4-(2-Hydroxyethyl)-piperazin-1-yl}-methyl]-[7,8-
f]benzo-1,3-diaza-spiro[4.5]decane-2,4-dione (7g). White pow-
dery crystals. Yield: 62%; mp 143–145 °C; TLC: Rf = 0.29 (S1);

J. Obniska et al. / Bioorg. Med. Chem. 18 (2010) 6134–6142
6141
Rf = 0.37 (S2); 1H NMR (300 MHz, CDCl3): d 1.89–1.96 (m, 1H cyclo-
hexane), 2.20–2.32 (m, 1H cyclohexane), 2.47–2.52 (m, 6H, 4H
piperazine, 2H –CH2–), 2.62–2.80 (m, 6H, 4H piperazine, 1H cyclo-
hexane, 1H OH), 2.95–3.10 (m, 2H cyclohexane), 3.40 (d, 1H cyclo-
hexane, J = 16.36 Hz), 3.60 (t, 2H, –CH2–, J = 5.30 Hz), 4.48 (s, 2H,
–CH2–), 6.32 (br s, 1H, N1H), 7.05–7.18 (m, 4H, ArH); C17H26O3N4
(358.44).
control mice or rats, when placed on the rod, can maintain their
equilibrium for a prolonged time period. The acute motor impair-
ment can be demonstrated by the inability of animal to maintain
equilibrium for given time.
4.2.4. Quantification studies
The quantitative determination of ED50 and TD50 values 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 protection or minimal neurotox-
icity. 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 com-
puter program written at NINDS/NIH. The results are shown in Ta-
ble 4.
4.1.2.16. N-(Morpholin-1-yl)-methyl-[7,8-f]benzo-1,3-diaza-spiro-
[4.5]decane-2,4-dione (7h). White powdery crystals. Yield: 88%;
mp 185–187 °C; TLC: Rf = 0.61 (S1), Rf = 0.28 (S2); 1H NMR
(300 MHz, CDCl3): d 1.87–1.94 (m, 1H, cyclohexane), 2.18–2.30
(m, 1H cyclohexane), 2.56 (t, 4H morfoline, J = 4.65 Hz), 2.77 (d,
1H cyclohexane, J = 18.20 Hz), 2.92–3.06 (m, 2H cyclohexane),
3.40 (d, 1H cyclohexane, J = 16.18 Hz), 3.62 (t, 4H morpholine,
J = 4.50 Hz), 4.41 (s, 2H, –CH2–), 6.54 (s, 1H, N1H), 7.04–7.14 (m,
4H, ArH); C15H21O3N3 (315.38).
4.2.5. The 6-Hz model
4.2. Pharmacology
This screen was carried out according to the protocol originally
described by Brown et al.33 and more recently by Barton et al.28
and Kaminski et al.34 It is an alternative electroshock paradigm
that uses low-frequency (6 Hz), long-duration (3 s) electrical stim-
ulation. Corneal stimulation (0.2 ms-duration monopolar rectan-
gular 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 cur-
rent application. The seizures manifest in ‘stunned’ posture associ-
ated with rearing, forelimb, automatic movements and clonus,
twitching of the vibrissae and Straub-tail. The duration of the sei-
zure 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 sei-
zure. The animal is considered to be protected if it resumes its nor-
mal exploratory behavior within 10 s from the stimulation34 (Table
5).
The initial anticonvulsant evaluation was performed within the
Antiepileptic Drug Development (ADD) Program in Epilepsy
Branch, National Institutes of Health, National Institute of Neuro-
logical Disorders and Stroke (NIH/NINDS), Rockville, MD, USA, by
using procedures described elsewhere.30,31
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 allowed free access to food and
water. The compounds were suspended in 0.5% methylcellulose/
water mixture.
The ASP initially evaluates anticonvulsant activity for newly
submitted compounds following intraperitoneal (ip) administra-
tion in mice and oral administration in rats. Groups of eight mice
or four rats are employed. Phase I studies in mice involved two
convulsant tests: maximal electroshock seizure test (MES), subcu-
taneous pentylenetetrazole seizure test (scPTZ) and rotarod test for
neurological toxicity (NT).
4.2.6. The Pilocarpine Induced Status Prevention (PISP) model
The male albino rats (Sprague–Dawley, 150–180 g) were used
as experimental animals. The compounds were administrated via
the ip rout of administration. 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,29 as follow: (I) immobility, eye closure, twitching of vibris-
sae, sniffing, and facial clonus; (II) head nodding associated with
more severe facial clonus; (III) clonus of one of the fore limbs;
(IV) rearing often accomplished by bilateral forelimb clonus; and
(V) all of the above plus loss of balance and falling, accomplished
by generalized clonic seizures. The anticonvulsant activity of tested
compounds was assessed at time zero, namely the time from the
first stage III seizures (Test 71). The outcome measures are deter-
mination of ‘protection’ or ‘no protection’. The results are given
in Table 6.
4.2.1. 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 rat at 60 Hz) is delivered via corneal
electrodes primed with an electrolyte solution containing an anes-
thetic agent.
4.2.2. The subcutaneous pentylenetetrazole seizure test (scPTZ)
This screen utilizes a dose of pentylenetetrazole (85 mg/kg in
mice and 70 mg/kg in rats) that produces clonic seizures lasting
for a period of at least five seconds in 97% (CD97) of animals tested.
At the anticipated time of testing the convulsant is administered
subcutaneously.
All the compounds were injected intraperitoneally into mice at
the dose levels of 30, 100, and 300 mg/kg with anticonvulsant
activity and neurotoxicity assessment at 0.5 and 4 h after adminis-
tration. The results are presented 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 anti-
convulsant 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.
4.2.7. In vitro hippocampal slice culture neuroprotection assay
Organotypic hippocampal brain slice cultures have been gaining
importance as an early stage drug screening tool in the neuropro-
tection arena.35 It is possible, with the use of propidium iodide (PI),
to quantitatively assess neuronal cell death in the slice cultures fol-
lowing treatment with the excitotoxic agents, kainic acid (KA) or
N-methyl-D-aspartate (NMDA). PI binds to the DNA of dead and dy-
ing cells and fluoresces. This assay enabled evaluation the ability of
novel compounds to prevent such excitotoxic cell death when
these compounds are added either in conjunction with, or follow-
ing, treatment with agents that induce excitotoxic cell death.
When organotypic slice are treated for 6 h with either KA
4.2.3. The neurological toxicity (NT)
The neurological toxicity (NT) induced by compound was de-
tected in mice or rats using standardized rotarod test.32 Untreated
(10 lm) or NMDA (10 lm), robust cell death occurs in the granule

6142
J. Obniska et al. / Bioorg. Med. Chem. 18 (2010) 6134–6142
cell layer of the dentategyrus and in the CA3 and CA1 pyramidal
cell layers of the organotypic hippocampal slice preparation and
this cell death can be quantified with PI. Treatment with agents
known to prevent excitotoxic cell death can significantly reduce
the percentage of total PI uptake in a dose-dependent manner.
Therefore, if new compounds can prevent cell death, the amount
of total PI will be significantly reduced. It is important to bear in
mind that these experiments are performed in an in vitro cell cul-
ture system. Thus, until confirmed in other experiments, it would
be premature to conclude that compounds effective in this system
would exhibit neuroprotection against excitotoxic agents in vivo.
The in vitro Hippocampal Slice Culture Neuroprotection Assay is
described in details in separate literature.35,36
include MOL files and InChiKeys of the most important compounds
described in this article.
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0
0
0
group P21/n, a = 17.284(3) ÅA, b = 6.0973(8) ÅA, c = 20.714(3) ÅA,
0
b = 111.157(4)°,
V = 2035.8(5) ÅA3,
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T = 293 K,
l
= 0.082 mmꢀ1, k = 0.71073 ÅA, data/parameters = 2813/
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Crystal data for 5h: C18H22N2O3, M = 314.38, monoclinic, space
0
0
0
group P21/c, a = 7.6388(6) ÅA, 0 b = 9.9055(9) ÅA, c = 21.960(2) ÅA,
b = 93.675(8)°, Z = 4,
V = 1658.2(3) ÅA3, Dx = 1.259 g cmꢀ3
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= 0.086 mmꢀ1, k = 0.71073 ÅA, data/parameters = 2877/
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_
_
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0
graphite-monochromated MoK
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The authors wish to thank Dr. James Stables for providing them
with pharmacological data through the Antiepileptic Drug Devel-
opment Program (Epilepsy Branch, National Institute of Neurolog-
ical Disorders and Stroke, National Institutes of Health, Rockville,
MD, USA).
The present work was supported by the Jagiellonian University
Medical College Grant No. K/ZDS/000711.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.06.064. These data