Synthesis and anticonvulsant activity of novel 2,6-diketopiperazine derivatives. Part 1: Perhydropyrrole[1,2-a]pyrazines

Article abstract of DOI:10.1016/j.ejmech.2011.07.027

A number of novel pyrrole[1,2-a]pyrazine derivatives were synthesized and evaluated in in vivo animal models of epilepsy. Among them, several compounds displayed promising seizure protection in the maximal electroshock seizure (MES), subcutaneous metrazol seizure (scMET), 6 Hz and pilocarpine-induced status prevention (PISP) tests, with ED₅₀ values comparable to the reference anticonvulsant drugs (AEDs). A critical influence of the stereochemistry and conformational preferences of the pyrrole[1,2-a]pyrazine core on in vivo pharmacological activity was observed. The mechanism of the anticonvulsant action of the agents synthesized is most probably not via inhibition of the voltage-dependent sodium (Na⁺) currents.

Full text of DOI:10.1016/j.ejmech.2011.07.027

European Journal of Medicinal Chemistry 46 (2011) 4859e4869
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anticonvulsant activity of novel 2,6-diketopiperazine derivatives.
Part 1: Perhydropyrrole[1,2-a]pyrazines
,
a
a
a
Maciej Dawidowski a , Franciszek Herold , Andrzej Chodkowski , Jerzy Kleps ,
*
Pawe1 Szulczyk b, Marcin Wilczek c
a Department of Drug Technology and Pharmaceutical Biotechnology, Faculty of Pharmacy, Medical University of Warsaw, Banacha 1 Str., 02-097 Warszawa, Poland
b
ꢀ
Department of Physiology and Pathophysiology, The Medical University of Warsaw, Pawinskiego 3C Str., 02-106 Warszawa, Poland
c Laboratory of NMR Spectroscopy, Faculty of Chemistry, University of Warsaw, Pasteura 1 Str., 02-093 Warszawa, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of novel pyrrole[1,2-a]pyrazine derivatives were synthesized and evaluated in in vivo animal
models of epilepsy. Among them, several compounds displayed promising seizure protection in the
maximal electroshock seizure (MES), subcutaneous metrazol seizure (scMET), 6 Hz and pilocarpine-
induced status prevention (PISP) tests, with ED50 values comparable to the reference anticonvulsant
drugs (AEDs). A critical influence of the stereochemistry and conformational preferences of the pyrrole
[1,2-a]pyrazine core on in vivo pharmacological activity was observed. The mechanism of the anticon-
vulsant action of the agents synthesized is most probably not via inhibition of the voltage-dependent
sodium (Naþ) currents.
Received 13 April 2011
Received in revised form
27 June 2011
Accepted 16 July 2011
Available online 22 July 2011
Keywords:
2,6-Diketopiperazine
2,6-Piperazinedione
Pyrrole[1,2-a]pyrazine
Anticonvulsant activity
Chirality
Ó 2011 Elsevier Masson SAS. All rights reserved.
Conformational analysis
1. Introduction
capable of identifying classes of compounds with previously
unknown mechanisms of action, which are hoped to be ‘truly
According to the International League Against Epilepsy (ILAE),
epilepsy is defined as a brain disorder characterized by an enduring
predisposition to generate epileptic seizures and by the neurobio-
logical, cognitive, psychological and social consequences of this
condition [1]. Currently, the main treatment for epileptic disorder is
the long-term and consistent administration of anticonvulsant
drugs (AEDs). Although over 30 AEDs are available, 25e30% of
patients fail to achieve adequate seizure control, while others
experience disturbing adverse effects of the treatment. As a result,
intensive research efforts aim to find new, more effective and safer
therapeutics [2].
Despite the growing knowledge of the pathomechanisms of
epilepsy, the main routes for the discovery of new AEDs are:
random screening of novel compounds in animal models of
epilepsy and modifications of available therapeutics with already
known mechanism(s) of action (see Fig. 1). The first approach is
innovative’. The second approach primarily leads to the discovery
of ‘evolutionary AEDs’ with expected improved safety or efficacy
(e.g. fosphenytoin, fluorofelbamate, selectracetam and brivar-
acetam). However, it should be noted that even slight modifications
of a lead structure have often generated substantial changes in the
pharmacological properties of a novel drug candidate. Another
approach involves synthesizing compounds that act via a specific,
preferably novel, mechanism. There has been much serendipity in
the history of AED discovery and development and, from a histor-
ical point of view, both ‘mechanistic’ and ‘non-mechanistic’
approaches have resulted in new agents being found with diverse
mechanisms of action and clinical efficacy [3e6].
Many of the clinically useful therapeutics act via several possible
molecular targets and only some of them have been directly linked
to a specific receptor within the brain. Thus, the early-stage iden-
tification of novel antiseizure agents mainly employs in vivo
screening tests in animal models of epilepsy. The most widely
recognized of these models are the maximal electroshock seizure
(MES) and the subcutaneous pentylenetetrazole seizure (scMet)
tests. It should be emphasized that, to date, nearly all of the
* Corresponding author. Tel.: þ48 22 5720646; fax: þ48 22 5720643.
E-mail address: maciej.dawidowski@wum.edu.pl (M. Dawidowski).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.027

4860
M. Dawidowski et al. / European Journal of Medicinal Chemistry 46 (2011) 4859e4869
A
b)
O
a)
NH2
O
HN
S
O
NH
O
NH2
O
O
O
O
O
H
O
NH2
O
Phenytoin
O
H
H
O
O
Topiramate
Felbamate
O
O
O
NH
N
O
O
Ethosuximide
Trimethadione
B
O
O
NH2
NH2
O
HN
NH
O
O
N
O
O
CONH2
Felbamate
Phenytoin
Levetiracetam
F
F
O
NH2
O
O
HN
O
F
P
HO
N
OH
O
O
O
NH2
N
N
O
O
CONH2
CONH2
Selectracetam
Fosphenytoin
Brivaracetam
Fluorofelbamate
Fig. 1. (A) ‘Non-mechanistic’ approaches to novel AEDs. (a) Examples of AEDs generated through random screening. (b) Examples of AEDs generated through modification of a lead
structure, but with different modes of action to the parent compound. (B) Examples of ‘evolutionary AEDs’. The derivatization points are marked in red (for interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article).
clinically useful AEDs are efficient in at least one of these tests.
Moreover, as long as the molecular targets associated with drug
resistant epilepsy remain unclear, in vivo screening remains an
important tool in the discovery of new AEDs [3,7].
2. Chemistry
Synthesis of the target 2,6-DKP derivatives was accomplished as
depicted in Scheme 1.
The present study demonstrates an attempt to identify novel
lead structures for AEDs in the chemical group perhydropyrrole
[1,2-a]pyrazine-1,3-dione, derivatives of 2,6-diketopiperazine (2,6-
DKP) with fused five-membered aliphatic ring. The ‘non-mecha-
nistic’ approach was adapted to design novel AED candidates. It was
based on the structural similarities with known antiseizure agents:
currently marketed AEDs and compounds in clinical or pre-clinical
stages of development. The putative novel structures incorporate
acetamide, phenylacetamide, glycineamide, phenylglycineamide
and imide motifs in a rigid, heterobicyclic system. These afore-
mentioned structural patterns are common pharmacophores of
many anticonvulsant agents, such as those depicted in Fig. 2. The
derivatives synthesized were evaluated in in vivo animal models of
epilepsy and their antiseizure potency was compared with those of
the reference AEDs. The most active compounds were further
subjected to derivatization, for example N-alkylation of the imide
nitrogen and changes in the substituents at carbon C-4 (equivalent
L
-Proline was subjected to esterification, followed by ammo-
nolysis of the resulting methyl ester. The -prolineamide (2S)-1
obtained was used as the starting material for subsequent
reactions.
L
Pure diastereomers of (4R,8aS)-3 and (4S,8aS)-3 were obtained
in two steps from (2S)-1 and (S)- or (R)-2-(4-toluenesulphonyloxy)-
phenylacetic acid methyl esters, respectively, by the methods
described elsewhere [8]. N-Alkylation of the imide nitrogen of
(4R,8aS)-3 or (4S,8aS)-3 with appropriate alkyl halides was accom-
plished under solid-liquid phase transfer catalysis (S-L PTC) condi-
tions [9], yielding the desired (4R,8aS)-4a,b and (4S,8aS)-4a,b,c,d.
The reactions in the presence of the PTC catalyst were found to
proceed more rapidly than in the case of the non-catalysed
processes. Furthermore, only a slight degree of accompanying
base-induced epimerization at the stereogenic centre C-4 of the
products was observed by TLC analysis. The resulting small amounts
of unwanted opposite diastereoisomers were efficiently removed by
column chromatography to furnish stereochemically pure (4R,8aS)-
4a,b and (4S,8aS)-4a,b,c,d, as judged by TLC and 1H NMR. Basing on
previous findings for similar heterocyclic systems [10], racemization
at the stereogenic centre C-8a of the perhydropyrrole[1,2-a]pyrazine
core was not likely to occur under the reaction conditions employed.
Thus, no enantiomeric mixtures were formed.
of the
cations were aimed at affecting such parameters as lipophillicity of
the molecule and the number of -interaction sites. As most of the
a-position of the glycine moiety). These structural modifi-
p
pyrrole[1,2-a]pyrazine derivatives investigated possess two ster-
eogenic centres, there was a particular interest in studying the
relationship between their pharmacological activity and stereo-
chemistry. An initial attempt towards determination of the mech-
anism of action of this novel class of antiseizure agents is also
presented.
Other derivatives of 2,6-DKP, (8aS)-6, (8aS)-7, (4R,8aS)-9 and
(4S,8aS)-9, were synthesized in several steps from
(2S)-1 and ethyl bromoacetate or methyl (ꢀ)- -bromopropionate.
L-prolineamide
a

M. Dawidowski et al. / European Journal of Medicinal Chemistry 46 (2011) 4859e4869
4861
pre-clinical
'first generation'
of AEDs
candidates
F
F
O
F
H
N
O
O
N
NH
NH
N
O
O
O
O
N
Primidone
O
Ethosuximide
O
N
O
O
HN
N
NH
H
O
N 8a
O
4
NH
R
O
H
O
Phenytoin
H2N
H
N
O
O
O
H
N
O
N
N
H
Remacemide
NH2
O
NH
NH2
Lacosamide
Levetiracetam
O
Milacemide
clinical
'second' or 'third generation'
of AEDs
candidates
Fig. 2. Examples of structural similarities (blue) between known AEDs, compounds in clinical or pre-clinical [11,12] development and the pyrrole[1,2-a]pyrazine heterocyclic system
(for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
The reaction of (2S)-1 with ethyl bromoacetate in the presence
of Hunig’s base resulted in the amidoester (2S)-5. The subsequent
intramolecular cyclocondensation furnished the 2,6-DKP derivative
(8aS)-6. N-Alkylation of the imide nitrogen with 2-
bromoethylbenzene under the S-L PTC conditions gave (8aS)-7.
by raising the seizure threshold. Acute neurological impairment
(TOX) was evaluated in the rotorod test. In order to further differ-
entiate the activity between rodent species the most active
compounds were tested in rats. Moreover, this form of screening
determines the ability of a compound to inhibit convulsions when
given via the oral route, which is regarded a valuable feature of
a candidate anticonvulsant. The compounds that exhibited signif-
icant activity and low neurological toxicity levels were subse-
quently subjected to a quantitative determination of the median
effective dose (ED50) and toxic dose (TD50) at previously estimated
times of peak effect (TPE).
Some of the compounds were subjected to an evaluation of
anticonvulsant activity in the minimal clonic seizure (6 Hz) test. It
was recently found that Levetiracetam, a novel AED used in patients
with therapy-resistant epilepsy, is ineffective in both of the classic
MES and scMET tests and that it exhibits significant activity in the
6 Hz model at the same time. Therefore, an expanded approach,
currently employed by the ASP, was to utilize this model as
a secondary screening procedure to examine the compounds that
are inactive in conventional tests, but which can possess novel
activity spectra. The results of the aforementioned tests are
summarized in Tables 1e6.
Apart from negligible activity in the 6 Hz model (1/4 at 100 mg/
kg, at 4 h), (4R,8aS)-3 did not exhibit an anticonvulsant effect or
neurotoxicity in any of the tests (see Tables 1 and 2). An increase in
lipophilicity by imide nitrogen substitution with either the benzyl
((4R,8aS)-4a) or phenylethyl ((4R,8aS)-4b) group did not enhance
the activity of this particular diastereomer.
In contrast to (4R,8aS)-3, its diastereomer (4S,8aS)-3 revealed
significant anticonvulsant activity in mice (see Tables 1e4). The
ED50 values in the MES and scMET models were 69.5 mg/kg and
87.9 mg/kg, respectively, at 0.25 h, suggesting a possible broad-
N-Alkylation of (2S)-1 with racemic methyl
a-bromopropionate
gave a mixture of (2S, R)-8 and (2S, S)-8 in a 1:1 ratio, as judged by
a
a
1H NMR analysis. Unfortunately, attempts to isolate the pure
isomers by column chromatography or fractional recrystallization
failed. Therefore, the diastereomeric mixture was used for the
subsequent intramolecular cyclocondensation, with the expecta-
tion that the resulting constrained cyclic (4R,8aS)-9 and (4S,8aS)-9
would be separable. Indeed, the equimolar (1H NMR) mixture of
(4R,8aS)-9 and (4S,8aS)-9 was efficiently resolved by column
chromatography (see Section 5).
3. Results and discussion
3.1. Biological evaluation
3.1.1. In vivo pharmacological evaluation
In vivo pharmacological testing of the synthesized compounds
in animal models of epilepsy was performed by the Anticonvulsant
Screening Program (ASP) of Epilepsy Branch, National Institutes of
Health, National Institute of Neurological Disorders and Stroke
(NIH/NINDS). Initially, anticonvulsant activity was assessed by the
maximal electroshock seizure (MES) and the subcutaneous
metrazole (scMET) tests in mice. It is generally accepted that the
former model employs an electrical stimulus to induce generalized
toniceclonic seizures and identifies which compounds prevent the
spread of seizures. The latter model utilizes chemically induced
myoclonic seizures and is capable of recognizing the agents that act

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M. Dawidowski et al. / European Journal of Medicinal Chemistry 46 (2011) 4859e4869
Scheme 1. Synthesis of compounds 1e9. Reagents and conditions: i. SOCl2, MeOH, reflux, 2 h, then AcOEt, Et3N, rt; ii. NH3, MeOH, rt, 72 h; iii. (R)-2-(4-toluenesulphonyloxy)-
phenylacetic acid methyl ester for (2S, S)-2 or (S)-2-(4-toluenesulphonyloxy)-phenylacetic acid methyl ester for (2S, R)-2, Py, MeCN, reflux, 2 h; iv. NaOH, EtOH, rt, 10 min; v. RX,
a
a
K2CO3, TBAB, acetone, reflux, 30e45 min.; vi. BrCH2COOEt, DIPEA, MeCN, rt, 17 h; vii. NaOH, EtOH, rt, 10 min.; viii. PhCH2CH2Br, K2CO3, TBAB, acetone, reflux, 1 h, then Et2O, HCl; ix.
(ꢀ)-CH3CH(Br)COOMe, Py, MeCN, reflux, 2 h; x. NaOH, EtOH, rt, 10 min, chromatographic resolution.
spectrum efficacy of anticonvulsant action. The TD50 of (4S,8aS)-3
was 121.5 mg/kg at 0.25 h, giving a fair protective index (PI > 1.7)
for the MES model. The compound proved to be very potent in the
6 Hz test, with an ED50 value of 9.93 mg/kg at 0.25 h (PI ¼ 12.2). By
oral administration to rats, (4S,8aS)-3 produced significant seizure
protection (MES ED50 ¼ 39.7 mg/kg, scMET ED50 ¼ 114.0 mg/kg, at
0.25 h) with no evidence of concomitant neurotoxicity at doses up
to 500 mg/kg (PI > 12.6 for the MES model, see Table 5). Due to its
high efficacy in the MES, scMET and 6 Hz models, the (4S,8aS)-3
diastereoisomer was subjected to further structural modifications.
The increase in lipophilicity achieved by the substitution of the
imide nitrogen of the active (4S,8aS)-3 isomer with either the
benzyl ((4S,8aS)-4a) or phenylethyl ((4S,8aS)-4b) group only
produced slight changes in the pharmacological effect of the parent
compound (see Table 1). Compared to (4S,8aS)-3, (4S,8aS)-4a did
not produce any neurotoxicity in mice. At the same time, a signifi-
cant loss of activity in the MES model and a lack of scMET-induced
seizure protection was observed. The compound proved to be
inactive when orally administered to rats. The slightly more lipo-
philic (4S,8aS)-4b was almost equally efficient in the MES test in
observed at the same dose. The N-ethoxycarbonylmethyl derivative
(4S,8aS)-4d was potent in the MES test in mice (5/7 and 4/5 at
100 mg/kg and 300 mg/kg, respectively, at 0.5 h) and produced
some neurotoxicity (2/4 at 300 mg/kg, at 0.5 h, see Table 1). It is
worth mentioning, that the compound was very active in the 6 Hz
test (ED50 ¼ 55.6 mg/kg, at 0.25 h), with a good protective index
(PI ¼ 3.2, see Tables 2 and 4).
Replacement of the phenyl substituent at carbon C-4 in inactive
(4R,8aS)-3 or active (4S,8aS)-3 with the methyl group afforded
(4R,8aS)-9 and (4S,8aS)-9, respectively. Despite the stereochemical
differences between the resulting compounds, both of them were
devoid of the desired antiseizure effects (see Table 1). This was
most probably as a result of their lower lipophilicity or the lack of
a
p-donor group adjacent to carbon C-4 compared to the parent
compounds. Similarly, replacement of the phenyl substituent at
carbon C-4 of (4S,8aS)-3 with a hydrogen atom afforded inactive
(8aS)-6. Interestingly, it was observed that its N-phenylethyl
analogue ((8aS)-7) displayed a MES activity comparable with the
most active derivatives, such as (4S,8aS)-4b (see Table 1). Therefore,
it seems probable that in the pyrrole[1,2-a]pyrazines investigated
mice (ED50 ¼ 80.6 mg/kg), with
a
similar neurotoxic effect
the p-donor group adjacent to the carbon C-4 atom was not critical
(TD50 ¼ 178.1 mg/kg) as (4S,8aS)-3 (see Table 3). However, unlike
the parent compound, (4S,8aS)-4b did not suppress scMET-induced
seizures and it did not reveal any activity in the MES test when
orally administered to rats. The N-methyl analogue of (4S,8aS)-3
((4S,8aS)-4c) revealed some anticonvulsant effects in the MES and
scMET models in mice (in either case 1/1 at 300 mg/kg, at 0.5 h, see
Table 1). However, significant neurotoxicity (4/4 at 0.5 h) was
for the efficacy in in vivo animal models of epilepsy and that the
lack of the desired antiseizure effects in (4R,8aS)-9, (4S,8aS)-9 and
(8aS)-6 was a result of the dramatic decrease in lipophilicity.
Nevertheless, (8aS)-7 displayed a notable neurotoxic effect and it
was not investigated further.
The open-chain analogues of (4R,8aS)-3 and (4S,8aS)-3, (2S,
aR)-
2 and (2S, S)-2 did not suppress seizures in either the MES or
a

M. Dawidowski et al. / European Journal of Medicinal Chemistry 46 (2011) 4859e4869
4863
Table 1
scMET models. Although (2S,
a
R)-2 displayed a negligible activity in
Anticonvulsant activity and neurotoxicity of compounds in the MES and scMET
models following intraperitoneal (i.p.) administration in mice.
the 6 Hz model (1/4 at 100 mg/kg, at 0.5 h), at higher doses this
compound produced notable neurotoxicity (3/4 at 300 mg/kg) and
proconvulsant effects.
Compound
Dose (mg/kg)
MESa
scMETb,c
0.5 h
TOXc,d
0.5 h
ClogPe
Compounds (4S,8aS)-3 and (4S,8aS)-4b were further assessed
for their potential efficacy against nerve agents using the
pilocarpine-induced status prevention (PISP) screening proce-
dure, which is one of the animal models of pharmacoresistant
status epilepticus. In order to determine whether or not the
substance prevented acute pilocarpine-induced status, it was
administered to male SpragueeDawley rats via the intraperitoneal
route, followed by the application of a challenge dose of pilocar-
pine immediately (0 min) and 30 min after treatment with a drug
candidate. The outcome measures were ‘protection’ or ‘no
protection’ from epileptic seizures. The results are summarized in
Table 6: (4S,8aS)-3 was found to provide full protection (8/8 rats)
at time zero when a dose of 65 mg/kg was used. Two of the eight
animals were protected at 30 min after a post-first stage III seizure,
at a dose of 130 mg/kg, whereas (4S,8aS)-4b was inactive in this
test (1/8 rats were protected at time zero when a dose of 600 mg/
kg was used).
0.5 h
ef
e
4 h
(4R,8aS)-3
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
e
e
e
e
e
e
e
e
e
e
e
e
e
eh
e
e
e
e
e
e
e
e
e
e
e
e
1/1
e
e
e
e
e
1/1
e
e
1/1
e
e
1.03
e
e
e
e
e
(4S,8aS)-3
(8aS)-6
e
e
e
1.03
3/3
1/1
e
2/5
1/1
e
1/8
4/4g
e
ꢁ0.94
ꢁ0.40
ꢁ0.40
3.06
e
e
e
e
e
e
(4R,8aS)-9
(4S,8aS)-9
(4R,8aS)-4a
(4S,8aS)-4ai
(4R,8aS)-4b
(4S,8aS)-4bi
(4S,8aS)-4ci
(4S,8aS)-4d
(8aS)-7i
e
e
e
e
e
e
e
eh
e
1/4
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
3.06
1/3
1/1
e
e
e
e
e
3.1.2. In vitro investigations of voltage-dependent Naþ current
modulation
e
e
3.31
e
e
e
Voltage-gated Naþ channels (VGNCs) are among the most
important targets for a number of clinically important anticonvul-
sant drugs. It is suggested that therapeutically useful inhibitors of
VGNCs should selectively suppress only the overactive tissue
without affecting the activity of VGNCs in healthy cells. This
property is referred to as ‘use-dependent blocking’, which means
that only frequently used Naþ channels are inhibited by up to 80%
by an effective compound [14]. Based on the structural similarities
between the pyrrole[1,2-a]pyrazine derivatives obtained and some
antiseizure agents known to act via voltage-dependent Naþ current
inhibition, this was the first mechanism proposed. Compound
(4S,8aS)-3 was chosen for the preliminary in vitro investigations as
the most active compound from the series.
e
e
e
e
e
e
3.31
3/3
1/1
e
e
e
e
e
e
e
1.28
e
e
e
1/1
e
1/1
e
4/4
e
1.20
5/7
4/5
e
e
e
e
2/4
e
e
1.34
2/3
1/1
e
1/1j
e
4/4h
a
Maximal electroshock test (number of animals protected/number of animals
tested) at 0.5 h and 4 h post administration.
b
Subcutaneous pentylenetetrazole test (number of animals protected/number of
In the first set of experiments, low frequency voltage-dependent
animals tested) at 0.5 h post administration.
Naþ currents were evoked (0.1 Hz). After 5 min of incubation with
c
No effect at 4 h post administration.
Neurotoxicity test (number of animals exhibiting neurological toxicity/number
d
10 mM (Fig. 3Aa) or 100 mM (Fig. 3Ab) (4S,8aS)-3, the amplitude of
the Naþ currents hardly changed (an increase of 3.0% and 8.4%,
respectively, n ¼ 3). Furthermore, the Naþ currents were evoked by
100 pulses with a frequency of 10 Hz. Under control conditions and
of animals tested) at 0.5 h post administration.
e
Theoretical log P value calculated by a logarithm included in the HyperChem 7.5
package [13].
f
Not active.
Sedated.
during application of the test compound (10
the Naþ current decreased by 4.2% and 5.3%, respectively (Fig. 3Ba,
M (4S,8aS)-3 was investigated, the Naþ currents
mM), the amplitude of
g
h
Death following tonic extension in at least one of the animals tested.
Tested as a hydrochloride salt.
Myoclonic jerks.
i
n ¼ 3). When 100
m
j
were reduced by 14.1 ꢀ3% compared to 8 ꢀ 2.3% under control
conditions after the application of 100 pulses at a frequency of
10 Hz (Fig. 3Bb, n ¼ 3).
Compound (4S,8aS)-3 did not significantly affect the voltage-
dependent Naþ channel current activated with either a low
frequency (0.1 Hz) or with a frequency at which the ‘use-dependent
blocking’ should be clearly revealed (10 Hz). Thus, it was concluded
that the mechanism of anticonvulsant action of this compound is
not via inhibition of the voltage-dependent Naþ channel currents in
overactive cells.
Table 2
Anticonvulsant activity and neurotoxicity of compounds in the 6Hz model following
intraperitoneal (i.p.) administration in mice.
Compound
Testa
0.25 h
0.5 h
1.0 h
2.0 h
4.0 h
(4R,8aS)-3
6 Hzb
TOXc
6 Hz
6 Hz
TOX
6 Hz
TOX
e
e
e
e
1/4
e
1/4
4/4
e
e
e
e
(4S,8aS)-3
(4R,8aS)-4b
4/4
1/4
e
e
e
e
e
e
e
3.2. Conformational analysis
e
e
e
e
(4S,8aS)-4d
4/4
e
4/4
2/4
2/4
1/4
1/4
e
e
The structures of the diastereoisomers (4S,8aS)-3 and (4R,8aS)-
3 have previously been investigated, both in the solid state and in
solution [8]. Nevertheless, the very pronounced dependence of
anticonvulsant activity on the stereochemistry of the pyrrole[1,2-a]
pyrazine system prompted a careful search for additional structural
features that could at least partially explain this phenomenon.
1/4
a
At a dose of 100 mg/kg, except compound (4S,8aS)-3 for which a dose of 75 mg/
kg was applied.
b
c
6 Hz test, 32 mA (number of animals protected/number of animals tested).
Neurotoxicity test (number of animals exhibiting neurological toxicity/number
of animals tested).

4864
M. Dawidowski et al. / European Journal of Medicinal Chemistry 46 (2011) 4859e4869
Table 3
Quantification studies of (4S,8aS)-3 and (4S,8aS)-4b in the MES, scMET and neurotoxicity tests following intraperitoneal (i.p.) administration in mice.
Compound
ED50 MESa (mg/kg)
ED50 scMETb (mg/kg)
TD50 (mg/kg)c,d
PIe MES
TPEf MES (h)
(4S,8aS)-3
69.5 (60.9e77.9)
80.6 (71.1e92.8)
5.64 (4.74e6.45)
21.8 (15.0e25.5)
>500
87.9 (72.2e100.4)
>200
>50
13.2 (5.87e15.9)
136 (100e184)
>25
121.5 (104.9e142.8)
178.1 (121.0e220.3)
41.0 (39.4e43.0)
69.0 (62.8e72.9)
341 (290e384)
1.7
2.2
7.3
3.2
2.5i
6.0
0.25
0.25
1.0
(4S,8aS)-4bg
Phenytoinh
Phenobarbitalh
Ethosuximideh
Lacosamidek
1.0
0.25j
0.5
4.46 (3.72e5.46)
26.8 (25.5e28.0)
Values in parentheses are 95% confidence intervals determined by probit analysis.
a
Maximal electroshock test.
Subcutaneous pentylenetetrazole test.
ED50 for the neurotoxicity test.
Response comments: sedated, unable to grasp rotorod, loss of righting reflex.
Protective index (TD50/ED50 MES).
Time to peak effect.
Tested as a hydrochloride salt.
Data from Ref. [15].
b
c
d
e
f
g
h
i
PI calculated for the scMET test.
TPE for the scMET test.
Data from Ref. [16].
j
k
1
The temperature H NMR spectra (ꢁ60 to 50 ꢂC, CDCl3) of
On the basis of the above findings, it seemed probable that the
lack of anticonvulsant potency of (4R,8aS)-3 vs. (4S,8aS)-3 diaste-
reoisomers could be associated with the different spatial arrange-
ments of pharmacophores, such as the aromatic ring, the lone pair of
bridgehead nitrogen atoms and the imide moiety, as well as with the
equiplanar orientation of the coupled pyrrole and pyrazine rings.
(4S,8aS)-3 and (4R,8aS)-3 did not reveal any peak broadening,
which suggested that nitrogen-inversion was not likely to occur for
either of the respective diastereoisomers and thus that they
seemed to exist in single conformations. Most probably, similar to
the solid state [17], the pyrrolidine and piperazine rings of (4R,8aS)-
3 were equiplanar, with a trans-diaxial arrangement of the lone N-5
nitrogen pair and the hydrogen at annelation (H-8a). This
assumption was also in agreement with the torsion angles of
H8aeC8aeC8eH8 and H8aeC8aeC8eH08 of w30ꢂ and w140ꢂ,
respectively, estimated according to the Karplus curve [18,19].
Unfortunately, other than in the case of the (4R,8aS)-3 diaste-
reomer, (4S,8aS)-3 did not form crystals suitable for X-ray diffrac-
tion (XRD) analysis. Thus, further structural elucidations were
limited to the 1H NMR experiments. From the two hypothetical
conformations of (4S,8aS)-3 (see Fig. 4), the cis-invertomer was
proposed as the more feasible one. Most of all, the signal of proton
H-8a appeared as a doublet of doublets with the two vicinal
4. Conclusions
A series of novel, chiral derivatives of pyrrole[1,2-a]pyrazine
were designed, synthesized and evaluated in in vivo animal models
of epilepsy. A very pronounced impact of the absolute configuration
of the stereogenic centres on the biological activity of this novel,
promising class of potential anticonvulsant agents was observed.
The (4R,8aS)-3 diastereoisomers investigated proved to be inactive,
whereas several compounds with the (4S,8aS) stereochemistry
possessed an antiseizure potency comparable to the reference
AEDs. The conformational analysis of the respective isomers
revealed differences in the mutual relationships of fused pyrroli-
dine and pyrazine rings, which could be associated with the
aforementioned structure-activity dependence.
Compound (4S,8aS)-3 seemed to be most potent in the different
models of epilepsy, suggesting a potential broad-spectrum anti-
convulsant activity. Its mechanism of action is most probably not
via the inhibition of voltage-dependent Naþ channel currents.
Compound (4S,8aS)-3 and its ethoxycarbonylmethyl ((4S,8aS)-4d)
and 2-phenylethyl ((4S,8aS)-4b) derivatives will be subjected to
structural optimization in order to increase their antiseizure
potencies and ameliorate their protective indices. Furthermore, in
the future, structural investigations of adequately designed close
analogues could help to better understand the basis of the
stereochemistry-activity dependence observed.
coupling constants of 3JH8aeH8 ¼ 8.5 and 3JH8aeH 8 ¼ 3.2 (in Hz). The
0
consequential values of the torsion angles H8aeC8aeC8eH8 and
H8aeC8aeC8eH08 of w30ꢂ and w80ꢂ, respectively, suggested the
position of the H8a-C8a bond inside the H8-C8-H08 plane, which
was only possible for the cis-(4S,8aS)-3 invertomer. Furthermore,
the difference-nuclear Overhauser effect (difference-nOe) experi-
ment showed no enhancement between the H-8a and H-6 protons.
Other than in the case of the hypothetical trans-(4S,8aS)-3, the
interatomic distance in cis-(4S,8aS)-3 could prove to be too large for
the spinespin interaction to occur through space.
Table 4
Quantification studies of (4S,8aS)-4d and (4S,8aS)-4d in the 6 Hz and neurotoxicity
tests following intraperitoneal (i.p.) administration in mice.
Compound
ED50 6 Hz (mg/kg) TD50 (mg/kg)a
PIb 6 Hz TPEc (h)
5. Experimental section
(4S,8aS)-3
(4S,8aS)-4d
9.93 (5.97e14.55) 121.5 (104.9e142.8)
12.2
3.2
2.0
0.25
0.25
0.25
0.5
55.6 (42.7e74.9)
178.0 (163.5e199.1)d
341 (290e384)
69.0 (62.8e72.9)
>500
5.1. Chemistry
Ethosuximidee 167 (114e223)
Phenobarbitale 14.8 (8.9e23.9)
Levetiracetame 19.4 (9.90e36.0)
4.7
>25.8
2.7
1.0
0.5
Melting points were determined on an Electrothermal 9100
apparatus with open capillary tubes and were uncorrected.
Elemental analyses were performed on a PerkineElmer 2400
analyzer and the results were within ꢀ0.4% of the theoretical
values. The IR spectra (KBr, thin film) were obtained on a Shimadzu
FTIR-8300 spectrometer. The NMR spectra were recorded on
Lacosamidef
9.99 (7.73e12.78)
26.8 (25.5e28.0)
Values in parentheses are 95% confidence intervals determined by probit analysis.
a
ED50 for the neurotoxicity test.
Protective index (TD50/ED50
Time to peak effect for the 6 Hz test.
0.5 h Post administration.
Data from Ref. [20].
b
6 Hz).
c
d
e
f
a Varian Unity Plus 500 MHz spectrometer. Chemical shifts (
expressed in parts per million relative to tetramethylsilane, which
d) were
Data from Ref. [16].

M. Dawidowski et al. / European Journal of Medicinal Chemistry 46 (2011) 4859e4869
4865
Table 5
Quantification studies of (4S,8aS)-3 in the MES, scMET and neurotoxicity tests following oral administration (p.o.) in rats.
Compound
ED50 MESa (mg/kg)
ED50 scMETb (mg/kg)
TD50c (mg/kg)
PId MES
TPEe MES (h)
(4S,8aS)-3
39.7 (19.3e70)
28.1 (20.7e35.2)
9.14 (7.58e11.9)
>500
114.0
>500
11.6 (7.74e15.0)
167 (116e237)
>250
>500
>1000
61.1 (43.7e95.9)
>500
>500
>12.6
>36.6
6.7
0.25
4.0
Phenytoinf
Phenobarbitalf
Ethosuximidef
Lacosamidei
5.0
>3.0g
>128.0
0.25h
0.5
3.90 (2.58e6.20)
Values in parentheses are 95% confidence intervals determined by probit analysis.
a
Maximal electroshock test.
Subcutaneous pentylenetetrazole test.
ED50 for the neurotoxicity test.
Protective index (TD50/ED50 MES).
Time to peak effect.
Data from Ref. [15].
PI calculated for the scMET test.
TPE for the scMET test.
b
c
d
e
f
g
h
i
Data from Ref. [16].
2
was used as the internal reference. The following abbreviations
were used to describe the peak patterns when appropriate: s
(singlet), d (doublet), t (triplet), q (quartet), qt (quintet), m
(multiplet), p (pseudo-), and b (broad-). Coupling constants (J) were
in hertz (Hz). The electrospray ionization high resolution mass
spectra (ESI-HRMS) analyses were recorded on an LCT TOF
(Micromass) instrument. Optical rotations were measured using
a PerkineElmer 241 polarimeter at 20 ꢂC. Thin-layer chromatog-
raphy (TLC) was performed on Merck Silica gel-60 F254 plates. The
spots were visualized by UV light (254 nm) or iodine vapour. Flash
column chromatography (FC) was performed on Merck Silica gel 60
(230e400 mesh ASTM). All reagents were purchased from
commercial sources and used as received. Solvents were dried and
purified by standard methods. Petroleum ether referred to the
fraction boiling at 40e60 ꢂC. Unless otherwise stated, the chemical
yields referred to pure (dr ꢃ 99/1) stereoisomers.
2J ¼ 17.0, 1H, H-
a
), 3.54 (d, J ¼ 17.0, 1H, H0-
a
), 4.18 (q, 3J ¼ 7.0, 2H,
OCH2CH3), 5.48 (bs, 1H, CONH), 7.52 (bs, 1H, CONH0); 13C NMR (125
MHz, CDCl3):
d
14.4 (OCH2CH3), 25.1 (C-4), 31.2 (C-3), 54.4 (C-5),
), 171.4 (CONH2), 178.2
55.7 (C-2), 61.0 (OCH2CH3), 67.1 (C-
a
(COOCH3); HRMS (ESI) calculated for C9H16N2O3Na: 223.1059
(M þ Na)þ found 223.1032.
5.1.2. (2S,
propionic acid methyl ester (2S,
Methyl (ꢀ)-2-bromopropionate (1.5 mL, 13.45 mmol) was
added in one portion to a solution of -prolineamide (2S)-1 (1.86 g,
aR)- and (2S,
aS)-
a-(2-carbamoylpyrrolidinyl)-
a-
aR)-8 and (2S, S)-8
a
L
16.29 mmol) and pyridine (1.31 mL, 16.29 mmol) in acetonitrile
(100 mL). The mixture was stirred under reflux (3 h) and the
solvent was evaporated in vacuo. The residue was purified by FC
(gradient: petroleum ether:ethyl acetate 1:2 to ethyl acetate:me-
thanol 9.5:0.5) to give 1.50 g (46%) of a chromatographically
inseparable (
D
Rf ¼ 0) mixture of diastereomers: (2S,
aR)-8 and
1
5.1.1. (2S)-
Ethyl bromoacetate (1.1 mL, 9.95 mmol) was added drop-wise to
a stirred, cooled (0 ꢂC, ice-salt bath) mixture of
-prolineamide
a-(2-carbamoylpyrrolidinyl)-acetic acid ethyl ester (2S)-5
(2S,a
S)-8 (dr ¼ 1:1, H NMR). White powder; mp 105e107 ꢂC; IR
(KBr): 741, 1157, 1207, 1454, 1628, 1732, 2982, 3198, 3391; TLC (ethyl
L
acetate): Rf ¼ 0.17; 1H NMR (diastereomeric mixture, 500 MHz,
(1.40 g, 12.26 mmol), N,N-diisopropyl-N-ethylamine (1.58 mL,
12.26 mmol) and acetonitrile (100 mL) over 10 min. The mixture
was warmed to room temperature, stirred (17 h) and the solvent
was evaporated in vacuo. The residue was purified by FC (gradient:
petroleum ether:ethyl acetate 1:1 to ethyl acetate:methanol
9.5:0.5) to give 1.28 g (69%) of (2S)-5. White powder; mp 97e98 ꢂC;
CDCl3):
d
1.32 (d, 3J ¼ 7.0, 3H, H-b(2S, R)), 1.39 (d, 3J ¼ 7.0, 3H, H-
a
b(2S, S)), 1.79 (m, 4H, H-4(2S, R), H-4(2S, S), H0-4(2S, R), H0-4(2S, S)), 1.99
a
a
a
a
a
(m, 2H, H-3(2S, R), H-3(2S, S)), 2.19 (m, 2H, H0-3(2S, R), H0-3(2S, S)), 2.73
a
a
a
a
(m, 8 lines, 1H, H-5(2S, R)), 2.79 (m, 8 lines, 1H, H-5(2S, S)), 3.04 (m, 7
a
a
lines, 2J ¼ 9.0, 3J1 ¼6.5, J2 ¼ 2.0, 1H, H0-5(2S, R)), 3.13 (m, 7 lines,
3
a
2J ¼ 8.5, 3J1 ¼7.0, J2 ¼ 2.5, 1H, H0-5(2S, S)), 3.50 (dd, 3J1 ¼10.0,
3
a
[
a
]D ¼ ꢁ66.5 (c 1, CHCl3); IR (KBr): 752, 1207, 1416,1632, 1732, 2978,
3J2 ¼ 7.5, 1H, H-2(2S, S)), 3.54 (q, 3J ¼ 7.0, 1H, H-a(2S, S)), 3.59 (dd,
a
a
3194, 3387; TLC (ethyl acetate): Rf ¼ 0.37; 1H NMR (500 MHz,
3J1 ¼10.0, 3J2 ¼ 7.5, 1H, H-2(2S, S)), 3.62 (q, 3J1 ¼7.5, 1H, H-a(2S, R)),
a
a
CDCl3):
d
1.28 (t, 3J ¼ 7.0, 3H, OCH2CH3), 1.85 (m, 2H, H-4, H0-4), 2.00
3.70 (s, 3H, OCH3(2S, S)), 3.71 (s, 3H, OCH3(2S, R)), 5.57 (bs, 2H,
a a
(m, 1H, H-3), 2.25 (4t, 2J ¼ 13.0, 3J1 ¼8.0, J2 ¼ 10.5, 1H, H0-3), 2.62
(dt, 2J ¼ 3J1 ¼9.0, 3J2 ¼ 6.5, 1H, H-5), 3.25 (m, 2J ¼ 9.0, 3J1 ¼6.5,
3J2 ¼ 2.5, 1H, H0-5), 3.35 (dd, 3J1 ¼10.0, 3J2 ¼ 4.0, 1H, H-2), 3.39 (d,
CONH(2S, R), CONH(2S, S)), 7.29 (bs, 1H, CONH0(2S, S)), 7.38 (bs, 1H,
a a a
3
CONH0(2S, R)); 13C NMR (diastereomeric mixture, 125 MHz, CDCl3):
a
d
14.7 (C-b(2S, R)), 17.4 (C-b(2S, S)), 24.8 (C-4(2S, S)), 24.9 (C-4(2S, R)),
a a a a
31.4 (C-3(2S, S)), 31.5 (C-3(2S, R)), 49.6 (C-5(2S, S)), 50.7 (C-5(2S, R)),
a
a
a
a
51.7 (C-2(2S, S)), 51.9 (C-2(2S, R)), 58.8 (OCH3(2S, R)), 59.0 (OCH3(2S, S)),
a
a
a
a
63.2 (C-a(2S, R)), 64.7 (C-a(2S, S)), 174.0 (CONH2(2S, S)), 174.1 (CON-
a
a
a
Table 6
H2(2S, R)), 179.0 (COOCH3(2S, S)), 179.4 (COOCH3(2S, R)); HRMS (ESI)
a
a
a
Anticonvulsant activity of (4S,8aS)-3 in the pilocarpine-induced status prevention
model following intraperitoneal (i.p.) administration in rats.
calculated for C9H16N2O3Na: 223.1059 (M þ Na)þ; found 223.1055.
Compound Dose
Time Number of animals Average weight change
5.1.3. General procedure for the cyclocondensation of amidoesters
(2S)-5, (2S,aR)-8 and (2S,aS)-8
(mg/kg) (h)a protected/number (g) ꢀ S.E.Mb
of animals tested
Protected
rats
Non-protected
rats
A sodium hydroxide (1 equivalent) pellet was added to a stirred
solution of the appropriate amidoester (1 equivalent) in absolute
ethanol (7.5 mL per 1 mmol of amidoester) at room temperature.
After complete dissolution of the hydroxide, the mixture was stir-
red for an additional 5 min and quenched with a saturated aqueous
solution of ammonium chloride (50 mL). The resulting cloudy
solution was extracted with methylene chloride (2 ꢄ 25 mL) and
(4S,8aS)-3
(4S,8aS)-3
65
130
0
0.5
0
8/8
2/8
1/8
þ4.4 ꢀ 1.1
e
þ12.5 ꢀ 1.3 ꢁ22.5 ꢀ 1.0
ꢁ10.0 ꢀ 0.0 ꢁ18.8 ꢀ 1.7
(4S,8aS)-4bc 600
a
Post-first stage III seizure.
Weight change 24 h post-first stage III seizure.
Tested as a hydrochloride salt.
b
c

4866
M. Dawidowski et al. / European Journal of Medicinal Chemistry 46 (2011) 4859e4869
Fig. 3. (A) Effect of 10
mM (a) and 100
m
M (b) (4S,8aS)-3 on the amplitude of Naþ currents evoked with a frequency of 0.1 Hz. Vertical axis e normalized current amplitude;
horizontal axis e time in minutes. Application of the test compound was marked with a black horizontal line. Averaged results obtained from two neurons are presented. Inset to ‘a’:
voltage-dependent Naþ currents evoked by rectangular 30 ms voltage step to ꢁ20 mV preceded by 100 ms hyperpolarization to ꢁ100 mV. (B) Effect of 10
mM (a) and 100 mM (b)
(4S,8aS)-3 on the amplitude of Naþ currents evoked with a frequency of 10 Hz. Currents were recorded under control conditions (white points) or during application of the test
compound (black points). Vertical axis e normalized amplitude of the Naþ current. Horizontal axis e time in seconds (during 10 s 100-v steps were applied). The averaged results
were obtained from three neurons. Insets to ‘a’ and ‘b’e overlapping of the last two current traces recorded under control conditions and during the application of 10
100 M (b) (4S,8aS)-3.
mM (a) and
m
2
with
a
mixture of dichloromethane/methanol 70:30 (v/v)
1H, H-8a), 3.79 (d, J ¼ 17.5, 1H, H0-4), 7.99 (bs, 1H, NH); 13C NMR
(2 ꢄ 25 mL). The combined organic extracts were dried with
magnesium sulphate, filtered, and the solvent was evaporated in
vacuo. The resulting residue was purified by FC.
(125 MHz, CDCl3): d 22.4 (C-7), 26.3 (C-8), 52.5 (C-6), 53.7 (C-8a),
63.2 (C-4), 171.1 (C-3), 173.0 (C-1); elemental analysis: calculated
for C7H10N2O2: C 54.54%, H 6.54%, N 18.17%; found: C 54.19%, H
6.78%, N 18.00%.
5.1.3.1. (8aS)-perhydropyrrole[1,2-a]pyrazine-1,3-dione
(8aS)-6.
From (2S)-5 (0.65 g, 3.49 mmol); FC (petroleum ether:ethyl acetate
5.1.3.2. (4R,8aS)- and (4S,8aS)-4-methyl-perhydropyrrole[1,2-a]pyr-
1:3): yield 0.41 g (77%).
azine-1,3-dione (4R,8aS)-9 and (4S,8aS)-9. From an equimolar
White powder; mp 137e139 ꢂC; [
a]
¼ þ22.5 (c 1, CHCl3); IR
mixture of (2S,aR)-8 and (2S,aS)-8 (1.30 g, 6.50 mmol): yield 1.03 g
D
(KBr): 1258, 1331, 1693, 1713, 2835, 2966, 3560; TLC (ethyl acetate):
(94%), dr z 1:1 (1H NMR); FC (gradient: petroleum ether:ethyl
acetate 1:4 to ethyl acetate): yield 0.38 g (35%) of (S,S) isomer,
dr ꢃ 99:1 (1H NMR), 0.40 g (36%) of (R,S) isomer, dr ꢃ 99:1 (1H
NMR) and 0.25 g (23%) of diastereomeric mixture.
Rf ¼ 0.46; 1H NMR (500 MHz, CDCl3):
d
1.89 (m, 2H, H-7, H0-7), 2.22
(m, 2H, H-8, H0-8), 2.71 (dt, 2J ¼ 9.0, 3J ¼ 7.0, 1H, H-6), 2.88 (dt, 1H,
2J ¼ 9.0, 3J ¼ 7.0,1H, H0-6), 3.51 (d, 2J ¼ 17.5,1H, H-4), 3.37 (t, 3J ¼ 6.0,
5.1.3.2.1. (4S,8aS)-4-methyl-perhydropyrrole[1,2-a]pyrazine-1,3-
dione (4S,8aS)-9. White powder; mp 133e134 ꢂC; [
a
]D ¼ ꢁ5.4 (c 1,
CHCl3); IR (KBr): 833, 1269, 1319, 1466, 1713, 2820, 2966, 3155; TLC
(ethyl acetate): Rf ¼ 0.41; 1H NMR (500 MHz, CDCl3):
d 1.47 (d,
3J ¼ 7.5, 3H, CH3), 1.85 (m, 2H, H-7, H0-7), 2.26 (m, 1H, H-8), 2.34 (m,
11 lines, 1H, H0-8), 2.60 (dd, 2J ¼ 8.5, 3J1 ¼17.5, 3J2 ¼ 0, 1H, H-6), 3.07
3
(m, 2J ¼ 9.0, 3J1 ¼8.0, J2 ¼ 3.5, 1H, H0-6), 3.72 (q, 3J ¼ 7.5, 1H, H-4),
3.88 (dd, 3J1 ¼8.5, 3J2 ¼ 2.5, 1H, H-8a), 7.94 (bs, 1H, NH); 13C NMR
(125 MHz, CDCl3):
d 17.0 (CH3), 22.4 (C-7), 27.4 (C-8), 52.0 (C-6),
56.6 (C-4), 56.9 (C-8a), 174.1 (C-3), 174.9 (C-1); elemental analysis:
calculated for C8H12N2O2: C 57.13%, H 7.19%, N 16.66%; found: C
57.12%, H 7.50%, N 16.51%.
5.1.3.2.2. (4R,8aS)-4-methyl-perhydropyrrole[1,2-a]pyrazine-1,3-
dione (4R,8aS)-9. White powder; mp 109e110 ꢂC; [
a]D ¼ ꢁ17.5 (c 1,
CHCl3); IR (KBr): 841, 1219, 1431, 1713, 2820, 2978, 3198; TLC (ethyl
acetate): Rf ¼ 0.52; 1H NMR (500 MHz, CDCl3):
d
1.47 (d, 3J ¼ 7.0, 3H,
Fig. 4. Computational geometries of the hypothetical cis- and trans-invertomers of
(4S,8aS)-3 obtained at the B3LYP level of theory with 6-311G(d,p) basis set. The
hydrogen atoms most relevant for conformational analysis are marked in red (for
interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article).
CH3), 1.90 (m, 2H, H-7, H0-7), 2.19 (m, 2H, H-8, H0-8), 2.51 (dd,
2J ¼ 8.0, 3J1 ¼16.5, 3J2 ¼ 0, 1H, H-6), 2.98 (m, 2J ¼ 8.5, 3J1 ¼8.5,
3J2 ¼ 4.5, 1H, H0-6), 3.27 (t, 3J ¼ 8.0, 1H, H-8a), 3.32 (q, 3J ¼ 7.0, 1H, H-

M. Dawidowski et al. / European Journal of Medicinal Chemistry 46 (2011) 4859e4869
4867
4), 7.93 (bs, 1H, NH); 13C NMR (125 MHz, CDCl3):
(C-7), 25.7 (C-8), 49.7 (C-6), 59.9 (C-4), 65.1 (C-8a),172.9 (C-3),173.8
(C-1); elemental analysis: calculated for C8H12N2O2: C 57.01%, H
7.22%, N 16.62%; found: C 57.12%, H 7.50%, N 16.51%.
d
14.9 (CH3), 21.6
18.26 mmol); FC (gradient: petroleum ether:ethyl acetate 9:1 to
petroleum ether:ethyl acetate 6:1): yield 0.61 g (63%).
White crystals; mp 172e174 ꢂC; [
a
]D ¼ ꢁ63.7 (c 1, CHCl3); IR
(KBr): 733, 1196, 1358, 1686, 1740, 2384, 2882; TLC (free base,
petroleum ether:ethyl acetate 6:1): Rf ¼ 0.56; 1H NMR (CDCl3,
5.1.4. General procedure for S-L PTC N-alkylation of 2,6-
diketopiperazine derivatives (8aS)-6, (4R,8aS)-3 and (4S,8aS)-3
A mixture of an appropriate 2,6-DKP (1 equivalent), potassium
carbonate (1 equivalent), tetrabutylammonium bromide (single
crystal), alkylating agent (7 equivalents) and acetone (1.5 mL per
1 mmol of 2,6-DKP) was vigorously stirred under reflux until TLC
showed no future reaction (30e60 min).1 The resulting suspension
was cooled, filtered and the solvent was evaporated in vacuo. The
residue was purified by FC.2
500 MHz): d
1.82 (m, 1H, H-7), 2.17 (m, 1H, H0-7), 2.40 (m, 1H, H-8),
2.62 (m, 1H, H0-8), 2.92 (bps, 1H, H-6), 3.06 (m, 2H, H-
b
, H0-
b
), 3.67
),
(bps,1H, H0-6), 4.22 (m, 8 lines, 2J ¼ 8.5, 3J1 ¼13.0, 3J2 ¼ 7.0,1H, H-
a
4.26 (pt, 1H, H-8a), 4.34 (m, 8 lines, 2J ¼ 8.5, 3J1 ¼13.0, 3J2 ¼ 7.0, 1H,
H0- ), 5.19 (s, 1H, H-4), 7.23e7.51 (m, 10H, HeAr); 13C NMR (free
a
base, 125 MHz, CDCl3):
d 22.7 (C-7), 29.0 (C-8), 34.2 (C-b), 40.4 (C-
a), 52.1 (C-6), 57.8 (C-8a), 64.5 (C-4), 126.8, 127.2, 128.4, 128.6, 129.0,
129.3, 135.0, 138.3 (CeAr), 171.6 (C-3), 173.8 (C-1); elemental
analysis: calculated for C21H22N2O2xHCl: C 68.01%, H 6.25%, N
7.55%; found: C 67.92%, H 6.19%, N 7.82%.
5.1.4.1. (4S,8aS)-2-benzyl-4-phenyl-perhydropyrrole[1,2-a]pyrazine-
1,3-dione hydrochloride (4S,8aS)-4a. From (4S,8aS)-3 (0.40 g,
1.74 mmol) and benzyl bromide (1.4 mL, 12.17 mmol); FC (gradient:
petroleum ether:ethyl acetate 9:1 to petroleum ether:ethyl acetate
6:1): yield 0.45 g (73%).
5.1.4.4. (4R,8aS)-2-(2-phenylethyl)-4-phenyl-perhydropyrrole[1,2-a]
pyrazine-1,3-dione (4R,8aS)-4b. From (4R,8aS)-3 (0.50 g, 2.17 mmol)
and 2-bromoethylbenzene (2.1 mL, 15.21 mmol); FC (gradient:
petroleum ether:ethyl acetate 9:1 to petroleum ether:ethyl acetate
6:1): yield 0.58 g (80%).
White powder; mp 176e178 ꢂC; [
a
]
¼ ꢁ42.5 (c 1, CHCl3); IR
D
(KBr): 733, 771, 1207, 1358, 1693, 1740, 2172, 2882, 2959; TLC (free
White powder; mp 105e107 ꢂC; [
a
]D ¼ ꢁ64.4 (c 1, CHCl3); IR
base, petroleum ether:ethyl acetate 6:1): Rf ¼ 0.54; 1H NMR (CDCl3,
(KBr): 744, 1165, 1327, 1686, 1736, 2957, 3028; TLC (petroleum
500 MHz):
d
1.93 (m, 1H, H-7), 2.22 (m, 1H, H0-7), 2.55 (m, 1H, H-8),
ether/ethyl acetate 6:1): Rf ¼ 0.48; 1H NMR (CDCl3, 500 MHz):
2.68 (m, 1H, H0-8), 3.12 (bps, 1H, H-6), 3.83 (bps, 1H, H0-6), 4.33 (pt,
1H, H-8a), 5.13 (s, 2H, H- , H-
0), 5.26 (s, 1H, H-4), 7.30e7.48 (m,
10H, HeAr); 13C NMR (free base, 125 MHz, CDCl3):
22.8 (C-7), 29.0
(C-8), 42.8 (C- ), 52.0 (C-6), 58.0 (C-8a), 64.5 (C-4), 127.3, 127.7,
d
1.79 (m, 2H, H-7, H0-7), 2.09 (pq, 2J ¼ 3J ¼ 9.0, 1H, H-6), 2.15 (m, 1H,
a
a
H-8), 2.23 (m, 1H, H0-8), 2.84 (m, 3H, H0-6, H- , H0-
b b), 3.07 (pt,
d
3J ¼ 8.0, 1H, H-8a), 3.93 (m, 8 lines, 2J ¼ 9.0, 3J1 ¼13.0, 3J2 ¼ 6.5, 1H,
a
H-
1H, H-a
a
), 4.00 (s, 1H, H-4), 4.07 (m, 8 lines, 2J ¼ 9.0, 3J1 ¼13.0, 3J2 ¼ 6.5,
128.4, 128.5, 128.6, 129.0, 129.1, 134.8, 137.1 (CeAr), 171.6 (C-3),
173.8 (C-1); elemental analysis: calculated for C20H20N2O2xHCl: C
67.32%, H 5.93%, N 7.85%; found: C 67.02%, H 6.12%, N 7.89%.
0), 7.18e7.39 (m, 10H, HeAr); 13C NMR (125 MHz, CDCl3):
d
20.9 (C-7), 25.7 (C-8), 34.2 (C-b), 41.3 (C-a), 52.7 (C-6), 65.2 (C-8a),
72.9 (C-4),126.6, 128.6,128.7,128.8, 129.0,129.2,137.1,138.5 (CeAr),
171.7 (C-3), 172.4 (C-1); elemental analysis: calculated for
C21H22N2O2: C 75.42%, H 6.63%, N 8.38%; found: C 75.72%, H 6.92%,
N 8.40%.
5.1.4.2. (4R,8aS)-2-benzyl-4-phenyl-perhydropyrrole[1,2-a]pyrazine-
1,3-dione (4R,8aS)-4a. From (4R,8aS)-3 (0.40 g, 1.74 mmol) and
benzyl bromide (1.4 mL, 12.17 mmol); FC (gradient: petroleum
ether:ethyl acetate 9:1 to petroleum ether:ethyl acetate 6:1): yield
0.47 g (84%).
5.1.4.5. (4S,8aS)-2-methyl-4-phenyl-perhydropyrrole[1,2-a]pyrazine-
1,3-dione hydrochloride (4S,8aS)-4c. From (4S,8aS)-3 (0.50 g,
2.17 mmol) and methyl iodide (1.5 mL, 15.22 mmol); FC (gradient:
petroleum ether:ethyl acetate 9:1 to petroleum ether:ethyl acetate
6:1): yield 0.43 g (71%).
White powder; mp 108e110 ꢂC; [
a
]D ¼ ꢁ129.1 (c 1, CHCl3); IR
(KBr): 753, 1177, 1315, 1678, 1732, 2827, 2978, 3012; TLC (petroleum
ether:ethyl acetate 6:1): Rf ¼ 0.46; 1H NMR (CDCl3, 500 MHz):
d
1.80 (m, 2H, H-7, H0-7), 2.10 (pq, 2J ¼ 3J ¼ 9.0, 1H, H-6), 2.21 (m, 2H,
White powder; mp 188e190 ꢂC; [
a
]D ¼ ꢁ27.9 (c 1, CHCl3); IR
H-8, H0-8), 2.84 (m,1H, H0-6), 3.12 (pt, 3J ¼ 8.5,1H, H-8a), 4.04 (s,1H,
(free base, KBr): 729, 1288, 1678, 1724, 2808, 2920, 2959; TLC (free
H-4), 4.88 (d, 2J ¼ 14.0, 1H, H-
a
), 5.01 (d, J ¼ 14.0, 1H, H0-
a
),
base, petroleum ether/ethyl acetate 5:1): Rf ¼ 0.53; 1H NMR (free
2
7.22e7.39 (m, 10H, HeAr); 13C NMR (125 MHz, CDCl3):
d
20.9 (C-7),
base, CDCl3, 500 MHz): d
1.88 (m, 2H, H-7, H0-7), 2.28 (m, 2H, H-8,
25.6 (C-8), 43.4 (C-
a), 52.8 (C-6), 65.3 (C-8a), 73.1 (C-4), 127.7, 128.6,
H0-8), 2.79 (dd, 2J ¼ 8.5, 3J1 ¼17.0, 3J2 ¼ 0, 1H, H-6), 3.16 (m, 2J ¼ 9.0,
128.7, 128.8, 129.0, 137.0, 137.1 (CeAr), 171.7 (C-3), 172.5 (C-1);
elemental analysis: calculated for C20H20N2O2: C 74.98%, H 6.29%, N
8.74%; found: C 75.53%, H 6.12%, N 8.89%.
3J1 ¼8.0, 3J2 ¼ 4.0, 1H, H0-6), 3.24 (s, 3H, 3ꢄH-
a
), 3.73 (dd, 3J1 ¼7.5,
3J2 ¼ 4.5, 1H, H-8a), 4.91 (s, 1H, H-4), 7.30e7.37 (m, 5H, HeAr); 13
C
NMR (free base,125 MHz, CDCl3): d 22.7 (C-7), 26.1 (C-a), 28.7 (C-8),
52.0 (C-6), 57.9 (C-8a), 64.6 (C-4), 127.3, 128.4, 129.0, 134.9 (CeAr),
171.9 (C-3), 174.1 (C-1); elemental analysis: calculated for
C14H16N2O2xHCl: C 68.83%, H 6.60%, N 11.47%; found: C 69.03%, H
6.82%, N 11.30%
5.1.4.3. (4S,8aS)-2-(2-phenylethyl)-4-phenyl-perhydropyrrole[1,2-a]
pyrazine-1,3-dione hydrochloride (4S,8aS)-4b. From (4S,8aS)-3
(0.60 g,
2.61 mmol)
and
2-bromoethylbenzene
(2.5 mL,
5.1.4.6. Ethyl (4S,8aS)-a-(1,3-dioxo-4-phenyl-perhydropyrrole[1,2-a]
pyrazin-2-yl)-acetate
(4S,8aS)-4d. From (4S,8aS)-3 (0.50 g,
1
It is essential to cool the reaction mixture immediately after no future reaction
is observed to avoid extensive epimerization on carbon C-4 upon prolonged heating
under basic conditions. However, the formation of small amounts (less than 10%,
TLC) of an opposite diastereomer is unavoidable and a carefully repeated FC is
required during purification.
2.17 mmol) and ethyl bromoacetate (1.7 mL, 15.22 mmol); FC
(gradient petroleum ether/ethyl acetate 9:1 to petroleum ether:ethyl
acetate 6:1): yield 0.49 g (71%)
White powder; mp 88e90 ꢂC; [
a]
¼ ꢁ120.6 (c 1, CHCl3); IR
D
2
(KBr): 750, 1203, 1690, 1728, 2831, 2963; TLC (petroleum ether:-
Free bases of compounds (8aS)-7, (4S,8aS)-4a, (4S,8aS)-4b and (4S,8aS)-4c were
ethyl acetate 5:1): Rf ¼ 0.42; 1H NMR (CDCl3, 500 MHz):
d 1.29 (t,
obtained as oils that tended to slightly decompose (TLC) upon prolonged storage.
They were converted to the corresponding stable hydrochlorides by the following
procedure: the compound was dissolved in diethyl ether (approximately 10 mL)
and cooled to 0 ꢂC. Then, a slight excess of a 1.5 M solution of hydrogen chloride in
diethyl ether was added while stirring. The resulting suspension was evaporated in
vacuo to afford a solid hydrochloride.
3J2 ¼ 7.0, 3H, OCH2CH3), 1.91 (m, 2H, H-7, H0-7), 2.28 (m, 2H, H-8, H0-
8), 2.97 (dd, 2J ¼ 8.0, 3J1 ¼16.5, 3J2 ¼ 0, 1H, H-6), 3.17 (dt, 2J ¼ 8.5,
3J1 ¼3J2 ¼ 6.0, 1H, H0-6), 3.75 (dd, 3J1 ¼8.0, 3J2 ¼ 3.5, 1H, H-8a), 4.22
(m, 12 lines, 3J1 ¼7.0, 2H, OCH2CH3), 4.57 (dd, 2J ¼ 17.0, 1H, H-
a),

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M. Dawidowski et al. / European Journal of Medicinal Chemistry 46 (2011) 4859e4869
2
4.63 (dd, J ¼ 17.0, 1H, H0-
a
), 4.96 (s, 1H, H-4), 7.31e7.46 (m, 5H,
14.3 (OCH2CH3), 22.6 (C-7),
a), 51.8 (C-6), 57.7 (C-8a), 61.8 (OCH2CH3), 64.5
5.2.1.3. The acute neurological impairment (TOX). Neurological
HeAr); 13C NMR (125 MHz, CDCl3):
28.6 (C-8), 40.3 (C-
(C-4), 127.5, 128.5, 129.0, 134.7 (CeAr), 168.0 (COOCH2CH3), 171.4
(C-3), 173.6 (C-1); elemental analysis: calculated for C17H20N2O4: C
64.54%, H 6.37%, N 8.86%; found: C 64.29%, H 6.55%, N 8.91%.
d
toxicity induced by a compound was detected in mice or rats using
the standardized rotorod test [21]. An untreated animal, when
placed on a 6 r.p.m. rotation rod, could maintain equilibrium for
a prolonged period of time. Mice were tested 30 min and 4 h after
doses of 30, 100 and 300 mg/kg of the test compound. Rats were
tested at time intervals between 0.25 and 4 h following a standard
oral dose of 30 mg/kg. Neurological impairment was demon-
strated by the inability of a mouse or rat to maintain equilibrium
for a given time.
5.1.4.7. (8aS)-2-(2-Phenylethyl)-perhydropyrrole[1,2-a]pyrazine-1,3-
dione hydrochloride (8aS)-7. From (8aS)-6 (0.28 g, 1.82 mmol) and
2-bromoethylbenzene (1.7 mL, 12.72 mmol); FC (gradient: petro-
leum ether:ethyl acetate 3:1 to petroleum ether:ethyl acetate 1:1):
yield 0.40 g (85%).
5.2.1.4. The minimal clonic seizure test (6 Hz). This screening
procedure was performed according to the protocol originally
described by Brown et al. [22] and more recently by Barton et al.
[20] and Kaminski et al. [23]. It is an alternative electroshock
paradigm that uses low-frequency (6 Hz), long-duration (3 s)
electrical stimulation. Mice were tested at time intervals between
0.25 and 4 h after doses of 100 mg/kg. Corneal stimulation (0.2-ms
duration monopolar rectangular pulses at 6 Hz for 3 s) was deliv-
ered by a constant-current device. During stimulation, the mice
were manually restrained and released into the observation cage
immediately after current application. The seizures manifested in
a ‘stunned’ posture associated with rearing, forelimb and automatic
movements and clonus, and twitching of the vibrissae and Straub-
tail. The duration of seizure activity ranged from 60 to 120 s in
untreated animals. At the end of the seizure, the animals resumed
their normal exploratory behaviour. The experimental end point
was protection against seizure. The animal was considered to be
protected if it resumed its normal exploratory behaviour within
10 s of stimulation [23].
Pale yellow powder; mp 139e141 ꢂC; [
a]
¼ ꢁ36.2 (c 1, CHCl3);
D
IR (KBr): 748, 1180, 1358, 1450, 1693, 1744, 2199, 2966, 3472; TLC
(free base, petroleum ether/ethyl acetate 1:1): Rf ¼ 0.29; 1H NMR
(500 MHz, CDCl3):
d
1.68 (m, 1H, H-7), 2.12 (m, 1H, H0-7), 2.45 (m,
1H, H-8), 2.53 (m, 2H, H-6, H0-8), 2.98 (t, 3J ¼ 7.5, 2H, H-
b b), 3.69
, H0-
(m, 1H, H0-6), 3.97 (d, 2J ¼ 17.5, 1H, H-4), 4.14 (dt, 2J ¼ 13.0, 3J ¼ 6.5,
1H, H-
a
), 4.26 (dt, 2J ¼ 13.0, 3J ¼ 7.5, 1H, H0-
a
), 4.50 (d, 2J ¼ 17.5, 1H,
H0-4), 4.62 (pd, 1H, H-8a), 7.18e7.30 (m, 5H, HeAr); 13C NMR (free
base, 125 MHz, CDCl3): d 22.2 (C-7), 27.1 (C-8), 34.2 (C-b), 40.5 (C-a),
52.7 (C-6), 54.8 (C-8a), 63.9 (C-4), 126.7, 128.6, 129.2, 138.5 (CeAr),
170.6 (C-3), 172.7 (C-1); elemental analysis: calculated for
C15H18N2O2xHCl: C 69.74%, H 7.02%, N, 10.84%, found C 69.33%, H
7.12%, N 11.01%.
5.2. Pharmacology
5.2.1. In vivo evaluation
The invivo screeningof the compounds for anticonvulsant activity
and neurotoxicity in the animal models of epilepsy was undertaken
by the National Institute of Neurological Disorders and Stroke,
Bethesda, USA, according to their well-established protocols [15].
Experiments were performed on male albino Carworth Farms
No. 1 mice (18e25 g) or albino Sprague-Dawley rats (100e150 g).
The animals had free access to food and water, except during the
actual testing period. Housing, handling and feeding were all in
accordance with recommendations contained in the ‘Guide for the
Care and Use of Laboratory Animals’.
5.2.1.5. Quantification studies. The quantitative determination of
the median effective (ED50) and toxic doses (TD50) were conducted
at previously calculated times of peak effect using the intraperito-
neal route in mice and the oral route in rats. Groups of at least eight
animals were tested using different doses of the test compound until
at least two points were determined between 100 and 0% protection
and minimal motor impairment. The dose of the candidate
substance required to produce the desired end point (abolition of
hindlimb tonic extensor component) in 50% of the animals in each
test, at a 95% confidence interval, were calculated by a computer
program based on the methods described by Finney [24].
The test compounds were suspended in 0.5% (v/v) of methyl-
cellulose in water.
5.2.1.1. The maximal electroshock seizure test (MES). In the MES test,
an electrical stimulus of 0.2 s duration (50 mA in mice and 150 mA
in rats, at 60 Hz) was delivered via corneal electrodes primed with
an electrolyte solution containing an anaesthetic agent (0.5%
butacaine hemisulphate in 0.9% sodium chloride). Mice were tested
at 30 min and 4 h after doses of 30, 100 and 300 mg/kg of the test
compound. Rats were tested at time intervals between 0.25 and 4 h
after a standard oral dose of 30 mg/kg. Abolition of the hindlimb
tonic extensor component indicated the test compound’s ability to
inhibit the spread of MES-induced seizures [15].
5.2.1.6. The pilocarpine-induced status prevention (PISP). Male
albino rats (SpragueeDawley, 150e180 g) were used as experi-
mental animals. The compounds were administrated via the
intraperitoneal route. Then, a challenge dose of pilocarpine was
given whilst watching for treatment effects of the substance being
tested. Seizure severity was determined using the well-known
Racine scale [25] 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 forelimbs;
(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 the
compounds was assessed at time zero, namely the time from the
first stage III seizures or at 30 min after a post-first stage III seizure.
The outcome measures were the determination of ‘protection’ or
‘no protection’.
5.2.1.2. The subcutaneous metrazol seizure test (scMET). This test
utilized a dose of metrazol (pentylenetetrazole, 85 mg/kg in mice
and 70 mg/kg in rats). This produced clonic seizures lasting for
a period of at least 5 s in 97% (CD97) of the animals tested. At the
anticipated time of testing, the convulsant was subcutaneously
administered. The test compound was intraperitoneally adminis-
tered in mice and orally in rats and the animals were observed
over a 30-min period. Mice were tested 30 min and 4 h after doses
of 30, 100 and 300 mg/kg of the test compound. The absence of
clonic spasms in the period of observation indicated a compound’s
ability to abolish the effect of pentylenetetrazol on the seizure
threshold [15].
5.2.2. In vitro voltage-gated Naþ channel inhibition assay
The experiments were performed on 3-week-old male Wistar
rats (WAG Cmd). The animals were decapitated and coronal slices
were prepared from cerebral prefrontal tissue using a vibrotome
(Vibrotome 1000, Pelco International, CA). Regions of the slices that

M. Dawidowski et al. / European Journal of Medicinal Chemistry 46 (2011) 4859e4869
4869
corresponded to the mPFC in rats were dissected (2.2e3.5 mm
anterior to Bregma, 3e5 mm below the upper cortical surface,
and 0.6e0.9 mm from the midline), mechanically dispersed and
placed in a recording chamber on the stage of a Nikon inverted
microscope (Nikon Instech Co., Ltd., Kawasaki, Kanagawa, Japan).
Cells were identified as pyramidal neurons under Hoffman optics
(magnification ꢄ400).
through the Antiepileptic Drug Development Program (Epilepsy
Branch, National Institute of Neurological Disorders and Stroke,
National Institutes of Health, Bethesda, MD, USA). Financial support
for the present study was provided by the Committee for Scientific
Research, Poland (Grant No. N N405 623138).
References
The recording of Naþ currents was performed using the whole-
cell voltage clamp method with an Axopatch 1D amplifier, a Dig-
idata 1322 acquisition system and PClamp 10.0 software. Recording
of Naþ currents was performed in a solution containing (in mM):
TEA-OH (90), NaCl (30), HEPES (10), MgCl2 (2), CaCl2 (2) and glucose
(15) (pH 7.4, osm 320). The pipette intracellular solution designed
to isolate the Naþ currents contained (mM): Cs methanesulphonate
(90), NaCl (15), EGTA (11), HEPES (10), CaCl2 (2), Na GTP (0.2),
MgATP (2), phosphocreatine (12) and leupeptin (0.1). The pipette
junction potential was nullified by immersing the pipette tip in the
bath. The path pipettes were sealed against the cell membranes. A
holding potential of ꢁ80 mV was applied and the membrane dis-
rupted spontaneously or by suction. An 80% series of resistance
compensation was applied.
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The effect of 5 min application of (4S,8aS)-3 at concentrations of
10 and 100 m
M on the Naþ current amplitude was tested. The Naþ
currents were evoked by a voltage step to ꢁ20 mV, lasting 30 ms,
preceded by a ꢁ100 mV prepulse lasting 1000 ms before and
during (4S,8aS)-3 delivery at a concentration of 10 mM (see inset to
Fig. 3A) [26]. The currents were evoked once every 10 s (0.1 Hz) and
continuously recorded. The first five peak Naþ current amplitude
measurements were averaged. The maximum peak Naþ current
measurements were expressed as a percentage of this average and
plotted as a function of time (Fig. 3Aa and Ab). The holding
potential was maintained at ꢁ80 mV.
Additionally, the effect of (4S,8aS)-3 was tested on the ampli-
tude of Naþ currents evoked by a series of 100 depolarizing pulses
from ꢁ100 mV to ꢁ20 mV, lasting 5 ms each, and applied at
a frequency 10 Hz. The Naþ current recordings were obtained under
control conditions (without application of the test compound) and
[23] R.F. Kaminski, M.R. Livingood, M.A. Rogawski, Epilepsia 45 (2004) 864e867.
[24] D.J. Finney, in: Probit Analysis, third ed. Cambridge University Press, London,
1971.
[25] R.J. Racine, Electroencephalogr. Clin. Neurophysiol. 32 (1972) 281e294.
[26] It was previously found that this voltage step evoked maximum Naþ current
in pyramidal neurons, as described in G. Witkowski, B. Szulczyk, R. Rola,
P. Szulczyk, Neuroscience 155 (2008) 53e63.
5 min after the application of 10 or 100 mM of (4S,8aS)-3 (Fig. 3Ba
and Bb, respectively). The normalized amplitude of Naþ currents
was plotted as a function of time.
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5.3. Molecular modelling
Density functional theory (DFT) optimization was performed
using Becke 3 LeeeYangeParr (B3LYP) functional and 6-311G(d,p)
basis set implemented in the Gaussian 03 package [27]. The
structures were visualized with HyperChem 7.5 [13].
Acknowledgments
The authors would like to thank Dr James Stables, Dr Tracy Chen
and Dr Jeff Jiang for providing them with pharmacological data