Synthesis, neuronal activity and mechanisms of action of halogenated enaminones

Article abstract of DOI:10.1016/j.ejmech.2014.02.002

Due to the excellent anticonvulsant activity of previously synthesized halogenated enaminones, more disubstituted analogs were synthesized and evaluated in vitro. The new enaminones either had no effect, depressed, or enhanced population spike (PS) amplitude in the rat hippocampus in a concentration-dependent manner. Structure-activity relationship (SAR) analysis indicated that compounds 21 and 25 (with dibromo substituents) were equipotent, and more potent than compound 2 (with dichloro substituents), with compound 25 being the most efficacious of all tested compounds. Both diiodo derivatives 30 and 31 tested produced no significant effect on PS. For PS depression, phenyl substitution on the cyclohexenone ring produced the most efficacious compound 25. PS depressing analogues also depressed evoked excitatory postsynaptic current (EPSC) and action potential firing frequency. Removal of phenyl or methyl group from position 6 on the cyclohexenone ring of enaminone esters produced compound 28 which exhibited pro-convulsant effects. There was no direct correlation between C log P values and anticonvulsant activity of the halogenated enaminones. The mechanisms of anticonvulsant activity were the indirect suppression of excitatory synaptic transmission by enhancing extracellular GABA, and the direct suppression of action potential firing of the neurons.

Full text of DOI:10.1016/j.ejmech.2014.02.002

European Journal of Medicinal Chemistry 76 (2014) 20e30
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, neuronal activity and mechanisms of action of halogenated
enaminones
,
b
b
Ivan O. Edafiogho a , Mohamed G. Qaddoumi , Kethireddy V.V. Ananthalakshmi ,
*
Oludotun A. Phillips c, Samuel B. Kombian b
a Department of Pharmaceutical Sciences, School of Pharmacy, University of Saint Joseph, Hartford, CT 06103, United States
b Department of Pharmacology and Therapeutics, Faculty of Pharmacy, Kuwait University, Kuwait
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kuwait University, Kuwait
a r t i c l e i n f o
a b s t r a c t
Article history:
Due to the excellent anticonvulsant activity of previously synthesized halogenated enaminones, more
disubstituted analogs were synthesized and evaluated in vitro. The new enaminones either had no effect,
depressed, or enhanced population spike (PS) amplitude in the rat hippocampus in a concentration-
dependent manner. Structureeactivity relationship (SAR) analysis indicated that compounds 21 and
25 (with dibromo substituents) were equipotent, and more potent than compound 2 (with dichloro
substituents), with compound 25 being the most efficacious of all tested compounds. Both diiodo de-
rivatives 30 and 31 tested produced no significant effect on PS. For PS depression, phenyl substitution on
the cyclohexenone ring produced the most efficacious compound 25. PS depressing analogues also
depressed evoked excitatory postsynaptic current (EPSC) and action potential firing frequency. Removal
of phenyl or methyl group from position 6 on the cyclohexenone ring of enaminone esters produced
compound 28 which exhibited pro-convulsant effects. There was no direct correlation between C log P
values and anticonvulsant activity of the halogenated enaminones. The mechanisms of anticonvulsant
activity were the indirect suppression of excitatory synaptic transmission by enhancing extracellular
GABA, and the direct suppression of action potential firing of the neurons.
Received 12 August 2013
Received in revised form
22 January 2014
Accepted 3 February 2014
Available online 4 February 2014
Keywords:
Dihalogenated enaminones
Neuronal activity
Synthesis
Published by Elsevier Masson SAS.
1. Introduction
Epilepsy is a neurological disorder characterized by the onset
of spontaneous convulsant and non-convulsant seizures that
Enaminones are synthetic compounds that consist of an amino
group, joined through an alkene group to a ketone group [1e3].
Enaminones possess a range of pharmacological effects including
antimalarial and anticonvulsant activities [1]. However, enami-
nones are largely devoid of neurotoxicity [4]. Halogenated enami-
nones have halogens such as fluoro, chloro, bromo, and iodo
moieties incorporated in the molecules [5].
result from neuronal hyperexcitability and hypersynchronous
neuronal firing [6]. About 50 million people worldwide are
affected by epilepsy, and it is estimated that 30% of the patients
suffer from therapy-resistant epilepsy. Resistance to antiepileptic
drugs (AEDs) and the side effects associated with the current
AEDs are the most serious problems in the treatment of epilepsy
[7e9]. Therefore, there is an urgent need to design and synthesis
novel anticonvulsants for the development of more effective and
safer AEDs [6].
In light of the emerging importance of enaminones as potential
AEDs [5], the rationale of the current study was to synthesize and
evaluate disubstituted phenylamino enaminones. Thus, we
assessed the effect of modification of 2,4-dihalogenation of the
phenylamino moiety of the investigated enaminones. In our cur-
rent study, nine new disubstituted phenylamino enaminones were
synthesized, and their anticonvulsant activity and neurotoxicity
evaluated to establish if some of these enaminones might become
lead compounds for further development into new AEDs.
Abbreviations: aCSF, artificial cerebrospinal fluid; ADD, antiepileptic drug
development; AED, antiepileptic drug; DMSO-d6, dimethyl sulfoxide-deuterated;
EPSC, excitatory postsynaptic current; EPSP, excitatory postsynaptic potential;
GABA, gamma aminobutyric acid; GABA-T, GABA transaminase; GAT, GABA trans-
porters; GCeMS, gas chromatographyemass spectrometry; ND, not determined;
NMR, nuclear magnetic resonance; mp, melting point; PS, population spike; SEM,
standard error of the mean; TMS, tetramethylsilane; TTX, tetrodotoxin.
* Corresponding author.
E-mail addresses: iedafiogho@usj.edu (I.O. Edafiogho), mqaddoumi@hsc.edu.kw
(M.G. Qaddoumi), anu@hsc.edu.kw (K.V.V. Ananthalakshmi), dphillips@hsc.edu.kw
(O.A. Phillips), kombian@hsc.edu.kw (S.B. Kombian).
0223-5234/$ e see front matter Published by Elsevier Masson SAS.
http://dx.doi.org/10.1016/j.ejmech.2014.02.002

I.O. Edafiogho et al. / European Journal of Medicinal Chemistry 76 (2014) 20e30
21
We had reported that anticonvulsant enaminones are very sta-
ble compounds at room temperature [10,11], and previous in-
vestigations indicated that cyclized enaminones were more potent
as anticonvulsants than the acyclic analogs. The NH proton was
mandatory for anticonvulsant activity, and that enaminones were
very promising as potential AEDs [12e20]. Initial evaluations of
halogenated enaminones revealed some important analogs of
which the most potent analog 20 was investigated for mechanisms
of anticonvulsant activity [21e23]. We have investigated further in
the current work the halogenated enaminones with more emphasis
on disubstituted phenyl enaminones. We hereby report our find-
ings that the 2,4-dibromophenyl enaminones are potent and effi-
cacious anticonvulsant agents.
stereoselective methods and evaluated pharmacologically as race-
mates. The synthesized compounds were stable solids and purified
by recrystallization from suitable solvents. 1H nuclear magnetic
resonance (NMR) spectra of the synthesized enaminones were in
CDCl3 or DMSO-d6 using TMS as an internal standard. The 13C NMR
spectra of representative compounds 2, 21, 25, 26 and 31, were also
performed. Elemental analyses were performed for all new enam-
inones (21e22, 25e31). Calculated log of partition coefficient (C log
P) values for the new compounds were performed using Chem-
DrawÒ Ultra version 8.0, 2003, Cambridge Soft Corporation.
3. Pharmacology
All experiments in this study were carried out on male Spra-
gueeDawley rats (100e150 g). Coronal slices (350 mm thick) con-
2. Chemistry
taining hippocampi were sliced in ice-cold artificial cerebrospinal
fluid (aCSF). Slices were perfused fully submerged at 2e3 mL/min
with aCSF (29e31 ꢀC) that was bubbled with 95% O2/5% CO2).
Tungsten bipolar stimulating electrodes were placed in the den-
dritic layer of area CA1 while recording glass electrodes were
placed in the cell body layer to record field population spikes (PS)
(Fig.1). All drugs were applied by bath perfusion. PS magnitude was
measured as the absolute amplitude from peak to trough and used
as a measure of neuronal excitation. All values are presented as
mean ꢁ SEM and P < 0.05 was taken as being statistically
significant.
The general synthetic routes for the halogenated enaminones is
shown in Scheme 1. Three independent routes of synthesis were
employed in unequivocal synthesis of the cyclized 4-
hydroxycyclohex-3-en-2-oxo-1-oates which were important in-
termediates for obtaining the desired halogenated enaminone es-
ters. The alkyl acrylate was reacted with alkyl acetoacetate in the
presence of freshly prepared sodium alkoxide to obtain the 4-
hydroxycyclohex-3-en-2-oxo-1-oate which existed as tautomers.
The second synthetic route involved the Michael addition reaction
of the alkylidene ketone and dialkyl malonate in sodium alkoxide to
give the 4-hydroxycyclohex-3-en-2-oxo-1-oate. The third route
involved the reaction of the alkylidene ketone with dialkyl malo-
nate under mild conditions to give the uncyclized Michael adduct
which was cyclized in the presence of sodium alkoxide to 4-
hydroxycyclohex-3-en-2-oxo-1-oate. The condensation of 4-
hydroxycyclohex-3-en-2-oxo-1-oates with appropriate amino
compounds yielded the halogenated enaminones as in Scheme 1,
and in Table 1. The halogenated enaminone derivatives (22, 23, 26
and 27) lacking the ester functionality were prepared from the
4. Results and discussion
The halogenated enaminones 1e31 were synthesized from b-
hydroxyketo starting materials, according to methods reported
previously [1,3,5]. The general synthesis is shown in Scheme 1. The
condensation of alkyl acrylate with alkyl acetoacetate, or alkyl vinyl
ketone with dialkyl malonate gave the
b-hydroxyketo in-
termediates, which were reacted with appropriate halogen
substituted-phenylamines to obtain the halogenated enaminones.
Since compound 2 was completely characterized in this study (mp,
UV, IR, 1H NMR, 13C NMR, and MS), its synthesis was markedly
different from reported literature [5].
commercially available
dimethylcyclohexane-1,3-dione,
b
-diketo compounds, namely, 4,4-
5,5-dimethylcyclohexane-1,3-
dione, cyclohexane-1,3-dione and 5-methylcyclohexane-1,3-
dione. The chiral enaminones were synthesized by non-
Scheme 1. Synthesis of halogenated enaminones. Reagents and conditions: (i) Na/MeOH for methyl esters; and Na/EtOH for ethyl esters, reflux, 2e4 h, (ii) Potassium carbonate, (iii)
appropriate amino compound (NH2R4).

22
I.O. Edafiogho et al. / European Journal of Medicinal Chemistry 76 (2014) 20e30
Table 1
Structure, and anticonvulsant activity of halogenated enaminones.
Cpd. R1
R2
R3
R4
MP (ꢀC)
ADDa
1
eCOOCH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2,4-dichlorophenyl
2,4-dichlorophenyl
2,5-dichlorophenyl
2,5-dichlorophenyl
3,4-dichlorophenyl
3,4-dichlorophenyl
2,6-dichlorophenyl
2,6-dichlorophenyl
3,5-dichlorophenyl
3,5-dichlorophenyl
153e155 1b
153e155 1b
190e192 1b
160e162 3b
160e161 1b
171e173 2b
205e210 3b
186e189 3b
212e215 3b
191e193 3b
2d
3
eCOOC2H5
eCOOCH3
eCOOC2H5
eCOOCH3
eCOOC2H5
eCOOCH3
eCOOC2H5
eCOOCH3
eCOOC2H5
eCOOCH3
eCOOC2H5
eCOOCH3
eCOOC2H5
eCOOCH3
eCOOC2H5
eCOOCH3
eCOOC2H5
eCOOC2H5
eCOOCH3
eCOOC2H5
Geminal CH3
H
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20d
21d
22d
23
24
25d
26d
27d
28
29
30
31
2,3,4-trichlorophenyl 205e207 3b
2,3,4-trichlorophenyl 165e167 2b
2,4,5-trichlorophenyl 200e201 1b
2,4,5-trichlorophenyl 183e184 3b
3,4,5-trichlorophenyl 181e183 1b
3,4,5-trichlorophenyl 168e170 2b
2,4,6-trichlorophenyl 204e213 3b
2,4,6-trichlorophenyl 190e197 3b
4-chlorophenyl
4-bromophenyl
2,4-dibromophenyl
2,4-dibromophenyl
Fig. 1. Experimental setup of hippocampal slice preparation (A) and sample PS trace
recorded in the cell body layer of the rat hippocampus (B).
161e163 1b
188e190 1b
155e157
1
156e161 NDc
144e147 NDc
196e198 NDc
4.1. C log P values and anticonvulsant activity
CH3 CH3 2,4-dibromophenyl
eCOOC2H5
eCOOCH3
H
eCOOCH3
eCOOC2H5
H
Ph
Ph
H
CH3
H
CH3
Ph
Ph
H
H
H
H
H
H
H
H
2,4-dibromophenyl
2,4-dibromophenyl
2,4-dibromophenyl
2,4-dibromophenyl
2,4-dibromophenyl
2,4-dibromophenyl
2,4-diiodophenyl
2,4-diiodophenyl
To investigate whether a correlation between lipophilicity and
anticonvulsant activity of the new enaminones existed, C log P
calculations for the new enaminones were performed (Table 2). The
C log P values would provide us with a prediction of the effec-
tiveness of our new enaminones to penetrate the central nervous
system (CNS) to produce anticonvulsant activity [24,25]. However,
we found that there was no direct correlation between the C log P
values of the halogenated enaminones and anticonvulsant activity
(see Table 2). The highest C log P value was 6.83 for compound 31,
while the lowest value was 4.51 for compound 26. The C log P data
for the six active enaminones 2, 21e22, 25e27 on Table 2 ranged
from 4.51 for compound 26 to 5.79 for compound 25, which also
covered the range of 4.91 for compound 28 and 5.03 for compound
29 both of which had pro-convulsant activity. The inactive enam-
inones had C log P values of 6.31 and 6.83 for compounds 30 and 31,
respectively. The high values of C log P for compounds 30 and 31
probably make these enaminones so lipophilic that they were not
available for action in the rat hippocampus. However, the most
potent anticonvulsant enaminone 25 possessed a C log P value of
5.79. Therefore, it appears that C log P data or lipophilicity of the
compounds could not be used to predict anticonvulsant activity of
the halogenated enaminones since no direct correlation was
observed. This supports our previous finding for other types of
enaminones [20]. Instead, steric effects of the substituents on the
cyclohexenone ring appeared to be more important in predicting
the anticonvulsant activity of the compounds.
220e222
1
150e153 NDc
128e130
2
128e130 NDc
191e192 NDc
234e238 NDc
190e192 NDc
eCOOCH3
eCOOC2H5
a
Anticonvulsant Drug Development classification:
1
¼
active <100 mg/kg;
2 ¼ active between 100 and 300 mg/kg and 3 ¼ inactive at 300 mg/kg in vivo.
b
Reference [5].
c
ND ¼ not determined in the ADD evaluation.
d
Compounds 2, 20, 21, 22, 25, 26, and 27 are active in suppressing rat hippo-
campal population spikes in vitro (see Fig. 2).
The synthesis of compound 2 was markedly different from the
literature [5] hence it was completely characterized in this study,
and then evaluated for anticonvulsant activity in vivo, and in vitro.
The 1H NMR and 13C NMR of compounds 25 and 31 were repre-
sentative of the 2,4-dihalogenated enaminones. Based on the
observed coupling constants, the cyclohexenone substituents
appear to occupy a trans-relationship to one another. Out of the
twenty compounds (1e20) initially reported, 8 of them, com-
pounds 1e3, 5, 13, 15, 19e20 were potent (class 1) anticonvulsant
enaminones. The 4-bromo analogue compound 20 was selected
and investigated extensively [1,17,19,21e23]. Due to the previously
reported excellent anticonvulsant profile of compound 20 that
possessed one bromo group in its chemical structure, it was
decided to synthesize and evaluate the anticonvulsant activity of
halogenated enaminones with 2,4-disubstitution of halogen moi-
ety on the phenylamino backbone. It was expected that the anti-
convulsant activity of the 2,4-dibromo enaminones would be more
potent. Accordingly, the new halogenated enaminones compounds
21e22, 25e31 were synthesized, their calculated log of partition
coefficients (C log P, values) were estimated, and then screened for
their neuronal activity.
4.2. Structureeactivity relationship (SAR) of halogenated
enaminones
The synthesis and anticonvulsant evaluation of enaminones
possessing mono-, di- or tri-halogen substitutions on the phenyl-
amino moiety as in compounds 1e20 in Table 1 were previously

I.O. Edafiogho et al. / European Journal of Medicinal Chemistry 76 (2014) 20e30
23
Table 2
Structures of halogenated enaminones, C log P values, and percent inhibition of population spikes in rat hippocampus at 10
m
M in vitro.
Cpd.
Structure
C log P values
Percent (%) inhibition at 10 mM
2
5.13
ꢂ24.6
21
5.43
ꢂ34.7
22
25
5.55
5.79
ꢂ18.2
ꢂ72.2
26
4.51
ꢂ23.7
ꢂ25.0
27
4.99
28
4.91
þ35
29
5.03
þ8.0
(continued on next page)

24
I.O. Edafiogho et al. / European Journal of Medicinal Chemistry 76 (2014) 20e30
Table 2 (continued )
Cpd.
Structure
C log P values
Percent (%) inhibition at 10 mM
30
31
6.31
6.83
þ8.5
þ7.1
reported to result in several lead compounds 1e3, 5, 13e14, 19e20
[1,3,5].
Following structural elucidation and characterization, the syn-
thesized enaminones in Table 2 were screened in vitro for potential
anticonvulsant activity by examining their effects on population
spikes (combination of excitatory synaptic responses and com-
pound action potentials) and whole cell synaptic and neuronal
responses recorded [19,22] in the rat hippocampal slices (Fig. 1).
The synthesized series of phenylamino enaminones possess
different halogen substitutions on the phenylamino moiety and
phenyl and methyl substituents on the cyclohexenone ring (com-
pounds 2, 21e22, 25e31 in Table 2). The effects of these compounds
on neuronal excitation ranged from suppression of population
spikes (PS) through no effect at all to enhancement in PS. Struc-
tureeactivity relationship (SAR) analysis revealed certain trends.
Fig. 1A shows the set-up of hippocampal slice preparation for
recording from the CA1 layer of the rat hippocampus, while Fig. 1B
shows an example of PS recorded from the rat hippocampus.
Population spike amplitudes were reduced following 5e6 min bath
application of inhibitory halogenated enaminones (25, 21, 27, 26, 2,
22) as illustrated by compound 25 in Fig. 2A while they were
increased by excitatory enaminones (28, 29, 30). These effects
showed partial recovery upon washing out of the compounds.
The dichloro 2, and dibromo 21 and 25, derivatives having
substituents on the cyclohexenone ring, suppressed PS amplitude
in a concentration-dependent manner. Compounds 21 (6-methyl,
1-ethylcarboxylate) and 25 (6-phenyl, 1-methylcarboxylate) ana-
logs had similar potencies, with EC50 values of 1.9 and 2.1
respectively while 2 (6-methyl, 1-ethylcarboxylate) was less potent
with EC50 value of 10 M (Fig. 2). Compound 25 with a phenyl
substituent on the cyclohexenone ring was more efficacious with a
depression of 72.2 ꢁ 12.3% at 10 M compared to 34.7 ꢁ 3.9% for 21
with methyl substituent (Fig. 2). The rank order of efficacy (at
10 M) for halogen substituents on the phenylamino group was
mM,
m
m
m
dibromo-(25: ꢂ72%) > dichloro-(2: ꢂ25%) > diiodo-(31: þ4% e
inactive) > diiodo-(30: þ8% e inactive). Removal of the phenyl or
methyl substituent from position 6 on the cyclohexenone ring of
enaminone esters (28: þ35%) resulted in a pro-convulsant com-
pound. Therefore, six halogenated enaminones 2, 21e22, 25e27
were anticonvulsant, two analogs 30e31 were inactive, and two
other compounds 28e29 were pro-convulsant. For all the com-
pounds that depressed the PS amplitude, concentrations beyond
Fig. 2. Effects of selected enaminones on population spike amplitude. (A) shows
sample PS in control, 6 min after application of Cpd 25 and 10 min after washing out
the compound. Below the traces is the concentrationeresponse curve for Cpd 25. Note
10
m
M i.e. ꢃ100
mM produced either a decrease in the amount of
depression (see doseeresponse curve in Fig. 2A for compound 25)
or even caused an enhancement in PS amplitude i.e. pro-convulsant
that at 100
curves for Cpds 2 and 21. (C) Summary bar graph showing the effects of several
enaminones at 10 M on the PS amplitude.
mM, the PS depressant effect is almost reversed. (B) Concentration-response
effects. As such, the concentration of 10
mM was selected to further
characterize the effects of the anticonvulsant enaminones.
m

I.O. Edafiogho et al. / European Journal of Medicinal Chemistry 76 (2014) 20e30
25
We further investigated the effects of compounds 21 and 25 (the
most potent and efficacious analogs) on excitatory postsynaptic
currents (EPSCs), action potential firing and postsynaptic neuronal
excitability by recording whole-cell responses in the CA1 pyramidal
cells using voltage and current clamp recording modes as appro-
priate. In voltage clamp mode, cells were held around their resting
membrane potential (ꢂ60 mV) and excitatory postsynaptic cur-
rents (EPSCs) were evoked by afferent stimulation and recorded
those reported earlier [21], we examined if the effects of com-
pounds 21 and 25 were dependent on GABAergic mechanisms. Bath
application of a saturating concentration of vigabatrin (500 mM)
depressed the evoked EPSC amplitude by 39.3 ꢁ 15.2% (n ¼ 4,
P < 0.05). In the presence of vigabatrin, compound 21 was no longer
able to depress EPSC amplitude (3.9 ꢁ 18.6%, P > 0.05, Fig. 5A
and B). In another set of experiments, vigabatrin (500 mM) again
depressed the evoked EPSC by 31.4 ꢁ 7.2% (n ¼ 7, P < 0.05). Here
again, vigabatrin (500 M) was chosen to saturate the response. In
its presence, compound 25 (10 M) was still able to depress the
(Fig. 3A and B inserts). At 10
mM, compound 25 depressed the EPSC
m
amplitude by 31.2 ꢁ 5.7% (n ¼ 6, P < 0.05, Fig. 3A and C) while
compound 21 depressed it by 47.5 ꢁ 14.2% (n ¼ 5, P < 0.05; Fig. 3B
and C) effects that, unlike those on PS, did not show appreciable
recovery following 15e20 min washout of the compounds (Fig. 3A
and B). At this same concentration, compound 21 depressed action
potential firing frequency by 76.3 ꢁ 11.9% (n ¼ 4, P < 0.05) while
compound 25 depressed it by 39.2 ꢁ 4.5% (n ¼ 4; P < 0.05, Fig. 4). To
further investigate if their mechanisms of action were similar to
m
evoked EPSC amplitude by 40.0 ꢁ 7.1% (n ¼ 7, P < 0.05 compared to
effect in presence of vigabatrin alone, Fig. 5C). To confirm a role for
GABA, we blocked GABAB receptors with a selective antagonist,
CGP55845 (1 mM) and asked if these compounds still depressed
EPSC amplitude. CGP55845 predictably enhanced the EPSC ampli-
tude by 41.6 ꢁ 4.8% (n ¼ 5, P < 0.05). At the peak of this CGP55845
effect, compound 25 was no longer able to depress EPSC amplitude
(16.6 ꢁ 10.1%, P > 0.05, Fig. 6A and B). In a similar experiment, the
ability of compound 21 to depress EPSC amplitude was also blocked
by CGP55845 (16.6 ꢁ 13.7%, n ¼ 4, P > 0.05, Fig. 6C and D).
4.3. Mechanisms of action of halogenated enaminones
By comparison to other enaminones and even a clinically
available anticonvulsant agent (vigabatrin), compounds 21 and 25
Fig. 3. Effects of Cpds 21 and 25 on evoked whole cell excitatory postsynaptic currents
recorded in pyramidal cells of the hippocampus. (A) Timeeeffect plot of 10
On top are sample EPSCs taken in control, in the presence of Cpd 25 and following
washout. (B) Timeeeffect plot for 10 M Cpd 21 as in (A). (C) Summary bar graph
showing the relatively long acting effects of Cpds 21 and 25.
m
M Cpd 25.
Fig. 4. Effects of Cpds 21 and 25 on action potential firing frequency. (A) Sample
voltage traces with action potential firing following 75 ms step current injection in
pyramidal neurons in control, presence of Cpd 21 and after washing out. (B) Summary
m
bar graph showing the effects of 10 mM each of Cpds 21 and 25.

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I.O. Edafiogho et al. / European Journal of Medicinal Chemistry 76 (2014) 20e30
Fig. 5. Blocking GABA transaminase enzyme with vigabatrin completely occludes Cpd
21 effects while partly occluding those of Cpd 25. (A) Sample traces taken from B. (B)
Timeeeffect plot of the effects of vigabatrin and Cpd 21 on EPSC amplitude. To the left
is a summary bar graph of this effect. (C) Simple plot as in B but with Cpd 25. Note that
Cpd 25 still had a significant effect, albeit smaller, in the presence of vigabatrin.
were the most potent analogs of the series synthesized and com-
pound 25 was the most efficacious. Both compounds were class 1
anticonvulsants in the in vivo model, and depressed PS amplitude in
the rat hippocampus in vitro. Accordingly, they were chosen as lead
compounds that can be developed into promising AEDs. It is
planned to carry out detailed pharmacological screening with
compounds 21 and 25 to determine how the two compounds affect
the spontaneous burst that usually accompanies multiple spikes.
The two compounds caused significant depression of PS amplitude
in the rat hippocampus. In addition, both compounds significantly
depressed EPSC amplitude recorded in the CA1 pyramidal cells and
significantly depressed the action potential firing frequency of the
pyramidal neurons. These enaminones depress synaptic excitation
by a gamma-aminobutyric acid (GABA)-mediated mechanism
involving GABAB receptors on glutamate terminals leading to
decrease in EPSC amplitude [1,21,26]. Therefore, one mechanism of
anticonvulsant action of the halogenated enaminones is to
reversibly suppress the glutamate-mediated EPSCs, most likely by
enhancing extracellular GABA levels rather than direct activation of
GABAB receptors [23]. We earlier reported that enaminone 20
interacted with adrenergic receptors, ultimately leading to GABAB
receptor-mediated synaptic depression. Here, we show that com-
pounds 21 and 25 depress PS and EPSC by a GABAergic mechanism.
Since enaminones have been reported not to interact with GABAB
receptors [27], the dihalogenated enaminones in this series most
likely also produced their synaptic depressant effect by enhancing
Fig. 6. Effects of Cpds 21 and 25 are blocked by
a GABAB receptor antagonist,
CGP55845. (A) Sample traces taken from B. (B) Timeeeffect plot of the effects of CGP
and Cpd 25 on EPSC amplitude. (C) Simple plot as in B but with Cpd 21. (D) Summary
bar graph showing the blockade of Cpds 21 and 25 effects by CGP.
extracellular GABA levels rather than by direct interaction with
GABAB receptors. The increase in extracellular GABA levels may be
through one or several mechanisms including inhibition of GABA
transporters (GAT); inhibition of the GABA transaminase (GABA-T)
enzyme or through interaction with a-2 adrenoceptors [1]. Com-
pound 25, being more efficacious, may produce this effect by
several mechanisms since profound inhibition of GABA trans-
aminase with 500 mM vigabatrin only partially occluded its effect.
The second mechanism of action is a direct effect on neuronal
excitability independent of synaptic transmission. These dihalo-
genated enaminones suppress compound action potential ampli-
tudes (in PS) and action potential firing frequency of single
pyramidal neurons [22]. These non-synaptic effects of halogenated
enaminones were through the inhibition of tetrodotoxin (TTX)-
sensitive sodium channel currents involved in action potential
firing as previously reported [22].

I.O. Edafiogho et al. / European Journal of Medicinal Chemistry 76 (2014) 20e30
27
5. Conclusion
parts per million (ppm) downfield from tetramethylsilane (TMS) as
an internal reference or DMSO-d6
¼ 2.5; 39.7) as solvent.
(
d
Enaminones are a class of compounds that have been reported
to possess anticonvulsant activity, and the enaminone pharmaco-
phore appears to provide the potential for anticonvulsant activity in
in vivo and in vitro models [28]. Enaminones, when evaluated in the
rat provided broad protection in both test models, i.e. MES and
scPTZ. However, it was also noted that the protection was greater by
the intraperitoneal (ip) route than orally, indicating differences in
absorption and distribution by different routes of administration
[26]. The mechanism of resistance to AEDs is currently the subject
of intense investigations with suggestions for a radical rethink of
antiepileptic therapy to include antiepileptogenic agents [29].
Furthermore, pharmacokinetic and enzyme induction studies are
required to fully characterize these new and promising anticon-
vulsant compounds and to determine if they possess potential for
further development into clinical trials [30].
In this paper, we report the synthesis and neuronal evaluation of
halogenated enaminones in vitro. Not all enaminones in this series
are anticonvulsant even at low concentrations. There was no direct
correlation between C log P values and anticonvulsant activity of
the halogenated enaminones. Phenyl and methyl substitution on
position 6 of the cyclohexenone ring of enaminone esters yielded
analogs with actions consistent with anticonvulsant potential.
Dibromo substitution on the phenylamino ring yielded the most
potent and most efficacious analogs with anticonvulsant activity.
Enaminones with dibromo substitution on the phenylamino ring
and phenyl substitution on position 6 of the cyclohexenone ring
hold the greatest potential for development as anticonvulsant
agents. The mechanisms of anticonvulsant action of the haloge-
nated enaminones include the indirect suppression of excitatory
synaptic transmission by enhancing extracellular GABA; and the
direct suppression of action potential firing of pyramidal neurons in
the hippocampus, a seizure relevant brain region of the CNS.
Chemical structures of newly synthesized compounds were
assessed by 1H NMR and elemental analyses. The melting point was
determined on a Stuart Scientific melting point apparatus (SMP1,
UK). Elemental analyses were performed on an Elementar Vario
Micro Cube CHN Analyzer (Elementar, Germany). Elemental anal-
ysis (C, H, N) was used to confirm the purity of all newly synthe-
sized compounds (>95%). Analyses indicated by the symbols of the
elements or functions were within ꢁ0.4 of theoretical values.
6.3. General procedure for the synthesis of halogenated enaminones
The following procedure is typical for the synthesis of the
halogenated enaminones, starting with the preparation of the in-
termediate
b-hydroxyketo compound that is condensed with
appropriate amino compounds carrying halogen substituents to
give the products.
6.3.1. Ethyl 4-hydroxy-6-methyl-2-oxocyclohex-3-en-1-oate
To a freshly prepared solution of sodium (17.8 g, 0.77 g-atom) in
absolute ethanol (350 mL) was added ethyl acetoacetate (100.2 g,
0.77 mol) over 30 min and the mixture stirred on an ice bath for an
additional 30 min. Ethyl crotonate (96%,100 mL, 0.77 mol) was added
dropwise and the mixture stirred at room temperature for an addi-
tional 30 min. After refluxing for 2 h, the reaction mixture was cooled
and the white precipitate which formed from the reaction mixture
wascollected, dissolvedina minimumamountof coldwater, acidified
with 2 M sulfuric acid (480 mL), and extracted with dichloromethane
(2 ꢄ 900 mL). The dichloromethane extract was dried over anhydrous
sodium sulfate and filtered. The filtrate was evaporated using rotary
evaporator, and the residue recrystallized from toluene to yield 56 g
(40%): white crystals; mp 95e98 ꢀC (mixed mp with authentic sam-
ple); 1H NMR (CDCl3)
d
1.03 (3H, d, J ¼ 6 Hz, CH3CH), 2.50 (2H, m, CH2),
3.20 (2H, m, 2 ꢄ CH), 3.70 (3H, t, J ¼ 7 Hz, OCH2CH3), 4.08 (2H, q,
6. Experimental section
J ¼ 7 Hz, OCH2CH3), 5.03 (1H, s, ]CH); IR (CHCl3 solution) 3150 (br),
3020 (CH), 1730, 1660, 1610 cmꢂ1
.
6.1. Chemicals
6.3.2. Ethyl 4-(20,40-dibromophenylamino)cyclohex-3-en-6-methyl-
2-oxo-1-oate (21)
The starting materials 1,3-cyclohexanedione, 5-methyl-1,3-
cyclohex-anedione, 5,5-dimethyl-1,3-cyclohexanedione, and 4,4-
dimethyl-1,3-cyclohexanedione; and common reagents including
2,4-dibromoaniline, 2,4-diiodoaniline, dichloromethane and so-
dium metal; and solvents including ethyl acetate, petroleum ether,
methanol, absolute ethanol and diethyl ether were obtained from
SigmaeAldrich (United States).
To a solution of ethyl 4-hydroxy-6-methyl-2-oxocyclohex-3-en-
1-oate (1 g, 5 mmol) in 80 mL of absolute ethanol was added a
solution of 2,4-dibromoaniline (98%, 1.28 g, 5 mmol) in 50 mL of
absolute ethanol and the reaction mixture refluxed with stirring for
8 h. The reaction mixture was evaporated using rotary evaporator,
and the residue recrystallized from ethyl acetate/petroleum ether
bp 60e80 ꢀC to obtain 0.60 g of enaminone 21, 28% yield. 1H NMR
6.2. Materials and methods
(DMSO, 400 MHz):
d
(ppm) d, 3H, 1.00, J ¼ 1 Hz, 6-methyl group),
1.19 (3H, t, J ¼ 2 Hz, OCH2CH3), 2.33e2.35 (4H, m, cyclohexenone
ring), 4.10 (2H, q, J ¼ 2 Hz, OCH2CH3), 4.66 (1H, s, ]CH), 7.30e8.01
(3H, m, C6H3 phenyl ring); 8.94 (1H, bs, NH). 13C NMR (DMSO-d6):
The mass spectra were recorded on a Thermo Scientific DFS High
Resolution Gas Chromatography/Mass Spectrometer (DFS GCeMS).
One crystal of the sample is loaded to the direct inlet probe, heated
from room temperature to 350 ꢀC at the rate of 30 ꢀC/minute and
inserted to the ion source with electron impact ionization setup.
The Infrared spectra of solids (KBr) were recorded on JASCO FT-IR-
6300 (JASCO, Japan) spectrometer. The 1H NMR spectra in DMSO-d6
using tetramethylsilane (TMS) as the internal standard, were
recorded on a Bruker DPX 400 MHz NMR spectrometer. Chemical
d
191.31, 171.21, 163.38, 136.91, 135.79, 132.26, 131.04, 122.51, 120.43,
97.43, 60.52, 60.42, 34.87, 32.35, 19.68, 14.61. IR (KBr pellet) 1470,
1518, 1562, 1598 enaminone system, 3224 NH; UV lmax EtOH
95% ¼ 296 nm; MS 431 (Mþ). Anal. (C16H17NO3Br2) C, H, N, O.
6.3.3. Ethyl 4-(2,4-dichlorophenylamino)-6-methyl-2-oxocyclohex-
3-en-1-oate (2)
shifts (
d
scale) are reported in parts per million (ppm) relative to
(0.82 g, 82.8% yield), mp 150e155 ꢀC. 1H NMR (DMSO-d6,
residual TMS. Multiplicity is indicated as follows: s (singlet),
d (doublet), t (triplet), m (multiplet), dd (doublet of doublet), and br
m (broad multiplet). Coupling constants (J) are given in (Hz). In
addition, the 13C NMR spectrum of representative compounds 2, 21,
25, 26 and 31, were recorded on Bruker Avance II 600 NMR spec-
trometer. Chemical shifts of protons and carbons are reported in
600 MHz):
d
1.00 (d, 3H, J ¼ 6.1 Hz, CH3), 1.19 (t, 3H, J ¼ 7.1 Hz,
CO2CH2CH3), 2.36e2.58 (m, 3H, cyclohexene), 3.04 (d, 1H,
J ¼ 11.4 Hz, cyclohexene H), 4.10 (q, 2H, J ¼ 7.1 Hz, CO2CH2CH3), 4.71
(s, 1H, ]CH), 7.40 (d, 1H, J ¼ 2.3 Hz, phenyl H), 7.49 (dd, 1H,
J ¼ 2.4 Hz, 8.6 Hz, phenyl H), 7.77 (d, 1H, J ¼ 2.3 Hz, phenyl H), 8.96
(s, 1H, NH). 13C NMR (DMSO-d6):
d 191.41, 171.20, 163.33, 135.01,

28
I.O. Edafiogho et al. / European Journal of Medicinal Chemistry 76 (2014) 20e30
131.98, 131.47, 130.41, 130.23, 128.80, 97.53, 60.51, 60.42, 34.88,
protons); 8.68 (1H, bs, amino proton). MS 358.9 (Mþ). Anal.
(C13H12NOBr2) C, H, N, O.
32.35, 19.67, 14.61. IR (KBr pellet, cmꢂ1):
n 3452, 1735, 1600, 1567.
UV EtOH lmax ¼ 296 nm; MS 341.1 (Mþ). Anal. (C16H17Cl2NO3) C, H,
N, O.
6.3.10. Methyl 4-(2,4-diiodophenylamino)-2-oxo-6-
phenylcyclohex-3-en-1-oate (30)
(0.25 g, yield 31.5%), mp 234e238 ꢀC. 1H NMR (DMSO-d6,
6.3.4. 3-(2,4-dibromophenylamino)-6,6-dimethylcyclohex-2-enone
(22)
600 MHz):
d
2.60 (dd, 1H, J ¼ 4.0 Hz, 16.5 Hz, cyclohexene H), 2.94
(0.22 g, 31.5% yield), mp 156e161 ꢀC. 1H NMR (DMSO-d6,
(m, 1H, J ¼ 12.7 Hz, 16.2 Hz, cyclohexene H), 3.39 (s, 3H, CO2CH3),
3.51e3.59 (m, 1H, cyclohexene H), 3.80 (d, 1H, J ¼ 12.5 Hz, cyclo-
hexene H), 4.67 (s,1H, ]CH), 7.12 (d,1H, J ¼ 8.3 Hz, phenyl H), 7.21e
7.40 (m, 5 H, phenyl H), 7.81 (dd, 1H, J ¼ 1.9 Hz, 8.3 Hz, phenyl H),
8.31 (d, 1H, J ¼ 1.9 Hz, phenyl H), 8.99 (br. s, 1H, NH). IR (KBr pellet,
600 MHz):
d
1.00 (s, 6H, cyclohexene 2(CH3)), 1.76 (t, 2H, J ¼ 6.2 Hz,
cyclohexene CH2), 2.50e2.54 (m, 2H, cyclohexene CH2, overlaps
with the DMSO signal), 4.57 (s, 1H, ]CH), 7.31 (d, 1H, J ¼ 8.5 Hz,
phenyl H), 7.62 (dd, 1H, J ¼ 2.2 Hz, 8.5 Hz, phenyl H), 7.98 (d, 1H,
cmꢂ1):
n
3188, 1736, 1587, 1570, 1530. UV lmax ¼ 298 nm. MS 573.0
J ¼ 2.2 Hz, phenyl H), 8.60 (s, 1H, NH). IR (KBr pellet, cmꢂ1):
n 3444,
(Mþ). Anal. (C20H17I2NO3) C, H, N, O.
1611, 1597, 1561, 1527. UV EtOH lmax 296 nm. MS 373.0 (Mþ). Anal.
(C14H15Br2NO) C, H, N, O.
6.3.11. Ethyl 4-(2,4-diiodophenylamino)-2-oxo-6-phenylcyclohex-
3-en-1-oate (31)
6.3.5. Methyl 4-(2,4-dibromophenylamino)-2-oxo-6-
phenylcyclohex-3-en-1-oate (25)
(0.10 g, yield 6%), mp 190e192 ꢀC 1H NMR (DMSO-d6, 600 MHz):
d
0.90 (t, 3H, J ¼ 7.1 Hz, CO2CH2CH3), 2.59 (dd,1H, J ¼ 4.0 Hz,16.6 Hz,
(1.26 g, 25.6% yield), mp 220e228 ꢀC. 1H NMR (DMSO-d6,
cyclohexene H), 2.94 (dd, 1H, J ¼ 12.0 Hz, 16.4 Hz, cyclohexene H),
3.55 (tt, 1H, J ¼ 4.2 Hz, 12.3 Hz, cyclohexene H), 3.75 (d, 1H,
J ¼ 12.5 Hz, cyclohexene H), 3.85 (q,1H, J ¼ 7.1 Hz, CO2CH2CH3), 4.67
(s, 1H, ]CH), 7.12 (d, 1H, J ¼ 8.3 Hz, phenyl H), 7.23e7.40 (m, 5H,
phenyl H), 7.81 (dd, 1H, J ¼ 1.9 Hz, 8.2 Hz, phenyl H), 8.31 (d, 1H,
J ¼ 1.9 Hz, phenyl H), 8.98 (br. s, 1H, NH). 13C NMR (DMSO-d6):
600 MHz):
d
2.62 (dd, 1H, J ¼ 4.2 Hz, 16.3 Hz, cyclohexene H), 2.97
(dd, 1H, J ¼ 12.9 Hz, 16.4 Hz, cyclohexene H), 3.39 (s, 3H, CO2CH3),
3.56 (tt, 1H, J ¼ 4.2 Hz, 12.4 Hz, cyclohexene H), 3.82 (d, 1H,
J ¼ 12.5 Hz, cyclohexene H), 4.77 (s, 1H, ]CH), 7.23e7.41 (m, 6 H,
phenyl H), 7.67 (dd, 1H, J ¼ 2.2 Hz, 8.5 Hz, phenyl H), 8.04 (d, 1H,
J ¼ 2.2 Hz, phenyl H), 9.05 (s, 1H, NH). 13C NMR (DMSO-d6):
d
190.68, 170.37, 163.11,147.08,141.92, 140.53, 138.76,130.59, 128.91,
d
190.80, 191.77, 170.95, 163.11, 142.03, 136.84, 136.81, 135.35,
127.97, 127.56, 101.04, 97.10, 94.16, 60.08, 59.32, 35.38, 14.34. IR (KBr
132.33, 130.95, 129.00, 128.92, 127.85, 127.58, 122.40, 120.50, 97.26,
pellet, cm-1):
n 3256, 1736, 1607, 1582, 1547, 1508; UV EtOH.
59.24, 59.22, 51.70, 51.67, 43.37, 35.30, 21.23. IR (KBr pellet, cm-1):
n
lmax ¼ 299 nm; MS 587.0 (Mþ). Anal. (C21H19I2NO3) C, H, N, O.
3446, 1737, 1592, 1532.UV EtOH lmax 296 nm MS 479.0 (Mþ). Anal.
(C20H17Br2NO3) C, H, N, O.
6.4. Anticonvulsant evaluation in vivo
6.3.6. 3-(2,4-dibromophenylamino)cyclohex-2-enone (26)
The preliminary evaluations for the anticonvulsant activity of
the halogenated enaminones 1e20 were performed in vivo by the
Antiepileptic Drug Development (ADD) Program of the Anticon-
vulsant Screening Program of the National Institute of Neurological
Disorders and Stroke (NINDS). Phase 1 testing procedures were
performed in male Carworth Farms no 1 (CF1) mice with tests
consisting of maximal electroshock (MES), subcutaneous pentyle-
netetrazole (Metrazol) seizure threshold to assess anticonvulsant
activity, and the rotorod test to assess neurological toxicity. Com-
pounds were either dissolved or suspended in 30% polyethylene
glycol 400 and were administered by intraperitoneal injection at
three dosage levels (30, 100, and 300 mg/kg). Anticonvulsant ac-
tivity and neurotoxicity were noted 30 min and 4 h after admin-
istration. The results of the anticonvulsant activity for compounds
1e20 were reported previously [5] and included in this study for
comparison.
(1.55 g, 90% yield), mp 184e188 ꢀC. 1H NMR (DMSO-d6,
600 MHz):
d 1.86e1.92 (m, 2H, cyclohexene H), 2.14 (t, 2H,
J ¼ 6.4 Hz, cyclohexene CH2), 2.48e2.51 (m, 2H, cyclohexene CH2,
overlaps with the DMSO signal), 4.66 (s, 1H, ]CH), 7.30 (d, 1H,
J ¼ 8.5 Hz, phenyl H), 7.63 (dd, 1H, J ¼ 2.2 Hz, 8.5 Hz, phenyl H), 7.99
(d, 1H, J ¼ 2.2 Hz, phenyl H), 8.69 (s, 1H, NH). 13C NMR (DMSO-d6):
d
195.91, 163.50, 137.30, 135.72, 132.14, 130.90, 122.40, 119.95, 99.06,
36.84, 28.30, 21.99. IR (KBr pellet, cmꢂ1):
n 3230, 1607, 1589, 1556,
1508. UV EtOH lmax ¼ 296 nm MS 344.9 (Mþ). Anal. (C12H11Br2NO):
C, H, N, O.
6.3.7. Methyl 4-(20,40-dibromophenylamino)cyclohex-3-en-6-
methyl-2-oxo-1-oate (27)
1.02 g, 49% yield. 1H NMR (DMSO, 400 MHz):
d (ppm) 0.98e1.00,
(3H, d, J ¼ 1 Hz, 6-methyl group); 2.36e2.57 (4H, m, cyclohexenone
ring); 3.61 (3H, s, OCH3), 4.66 (1H, s, ¼CH proton); 7.31e8.01 (3H,
m, phenyl ring); 8.96 (1H, s, NH). MS 416.9 (Mþ). Anal.
(C15H15NO3Br2) C, H, N, O.
6.5. Anticonvulsant evaluation in vitro
6.5.1. General electrophysiology methodology
6.3.8. Ethyl 4-(20,40-dibromophenylamino)-2-oxocyclohex-3-en-1-
Extracellular electrophysiological experiments were performed
in coronal hippocampal slices generated from rats (100e150 g)
using previously published techniques and methods [19,21,22].
Briefly, male SpragueeDawley rats were deeply anesthetized with
halothane and killed by quick decapitation. The brains were quickly
removed and placed in ice cold (4 ꢀC) artificial cerebrospinal fluid
(aCSF) bubbled continuously with 95% O2 and 5% CO2 (Carbogen).
Using Leica VT 1000S (Leica Microsystems, Wetzlar, Germany) or
OTS-4000 (Electron Microscopy Sciences, Hatfield, PA, USA) tissue
oate (28)
0.75 g, 36% yield. 1H NMR (DMSO, 400 MHz):
d (ppm) 1.14e1.16
(3H, t, J ¼ 2 Hz, OCH2CH3); 1.85e2.58 (5H, m, cyclohexenone ring);
4.06 (2H, q, J ¼ 2 Hz OCH2CH3), 4.64 (1H, s, ]CH), 7.29e8.00 (3H, m,
phenyl protons); 8.89 (1H, bs, amino group). MS 416.9 (Mþ). Anal.
(C15H15NO3Br2) C, H, N, O.
6.3.9. 3-(20,40-dibromophenylamino)-5-methylcyclohex-2-en-1-
one (29)
slicers, 350 mm thick coronal slices of the forebrain containing the
1.04 g, 58% yield. 1H NMR (DMSO, 400 MHz):
d
(ppm) 1.02e1.04
hippocampus were cut from a block of brain tissue in ice cold aCSF.
Prior to recording, slices were incubated for 1 h in aCSF which was
continuously bubbled with carbogen at room temperature. Slices
(3H, d, J ¼ 2 Hz, 6-methyl protons); 1.89e2.54 (5H, m, cyclo-
hexenone protons); 4.65 (!H, s, ]CH); 7.29e8.00 (3H, m, phenyl

I.O. Edafiogho et al. / European Journal of Medicinal Chemistry 76 (2014) 20e30
29
methyl- 2-oxocyclohex-3-en-1-oate (ADD 906022), Chemical Society Re-
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were carefully trimmed of most cortical and midbrain tissue and
suspended on a nylon mesh in a 500 mL capacity recording
chamber. Bath temperature was tightly maintained at 29e31 ꢀC to
ensure that changes in responses were not due to variation in
temperature. Slices were perfused at a flow rate of 2e3 mL/min
with aCSF that was bubbled with carbogen. An extracellular field
recording glass electrode filled with 3M NaCl was placed in the
stratum pyramidale of area CA1 for recording and bipolar stimu-
lating electrodes were placed in the stratum radiatum near area CA1
to activate Schaffer collateral/commissural fibers afferents (see
setup in Fig. 2).
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The composition of the aCSF used for dissection, storage, PS and
whole cell recordings was (in mM) 120 NaCl, 3.3 KCl, 1.2 MgSO4, 1.3
CaCl2, 1.23 NaHPO4, 25 NaHCO3 and 10 D-glucose [19,21,22]. For PS,
each stored trace was an average of five successively triggered re-
sponses elicited at 10 s intervals. The amplitude of the PS was
measured from the peak of the positive going wave to the tip of the
negative going wave in both control and drug application. The data
were expressed as mean and standard error. For whole-cell
recording, blind patches were made by glass electrodes filled
with gluconate-based internal recording solution with the
following composition (in mM): K-gluconate (135), NaCl (8), EGTA
(0.2), HEPES (10), Mg-ATP (2) and GTP (0.2). pH and osmolarity
were adjusted to 7.3 (with KOH) and 270e280 mOsm respectively.
The patch electrodes had tip resistance between 3 and 10 MU. The
extracellular perfusing solution was identical to that used for the
extracellular recording. Recordings were performed on pyramidal
cells of the hippocampus which had resting membrane potentials
ranging from ꢂ55 to ꢂ63 mV. In voltage clamp experiments, they
were held around rest (ꢂ60 mV). Excitatory postsynaptic currents
(EPSCs) were recorded by activating the same afferents as in
extracellular recording above. Two successive responses (30 s
apart) were averaged and stored for off line analysis. For action
potential recording, these were done in current clamp mode by
applying increasing step depolarizing and hyperpolarizing currents
and recording the corresponding potentials. For these recordings,
no averaging was done.
Authors contribution to the manuscript
All authors have contributed equally to the manuscript.
[19] K.V.V. Ananthalakshmi, I.O. Edafiogho, S.B. Kombian, Anticonvulsant enami-
none E139 suppresses epileptiform activity in rat hippocampal slices, Epilepsy
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Conflict of interest
[20] I.O. Edafiogho, O.A. Phillips, E.E. Udo, S. Samuel, B. Rethish, Synthesis, anti-
bacterial, and anticonvulsant evaluations of some cyclic enaminones, Euro-
pean Journal of Medicinal Chemistry 44 (2009) 967e975.
All authors have no conflict of interest.
[21] S.B. Kombian, I.O. Edafiogho, V.V. Kethireddy, Anticonvulsant enaminones
depress excitatory synaptic transmission in the rat brain by enhancing
extracellular GABA levels, British Journal of Pharmacology 145 (2005) 945e
953.
[22] K.V.V. Ananthalakshmi, I.O. Edafiogho, S.B. Kombian, Concentration-depen-
dent effects of anticonvulsant enaminone methyl 4-(4’-bromophenyl)ami-
nocyclohex-3-en-6-methyl-2-oxo-1-oate on rat neuronal excitability in vitro,
Neuroscience 141 (2006) 345e356.
Acknowledgment
The research project was supported by Kuwait University Grant
number PR01/08, and the Instrument Grants GS01/01, GS01/03 and
GS01/05 awarded to the Science Analytical Facilities (SAF).
[23] S.B. Kombian, K.V.V. Ananthalakshmi, I.O. Edafiogho, Enaminones and
norepinephrine employ convergent mechanisms to depress excitatory syn-
aptic transmission in rat nucleus accumbens in vitro, European Journal of
Neuroscience 24 (2006) 2781e2788.
[24] J.P. Stables, H.J. Kupferberg, The NIH anticonvulsant drug development
(ADD) program: preclinical anticonvulsant screening project, in:
G. Avanzini, G. Regesta, O. Tanganelli, M. Avoli (Eds.), Molecular and
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[25] W. Loscher, H.H. Frey, Kinetics of penetration of common antiepileptic drugs
into cerebrospinal fluid, Epilepsia 25 (1984) 346e352.
[26] P.L. Jackson, C.D. Hansen, A.K. Farrel, R.J. Butcher, J.P. Stables, N.D. Eddington,
K.R. Scott, Enaminones 12. An explanation of anticonvulsant activity and
toxicity per Linus Pauling’s clathrate hypothesis, European Journal of Medic-
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.02.002.
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