Anticonvulsant evaluation and mechanism of action of benzylamino enaminones

Article abstract of DOI:10.1016/j.bmc.2006.03.049

The mechanism of anticonvulsant action was evaluated for the benzylamino enaminones. The most potent enaminone in this series was the unsubstituted benzylamine analog (30; methyl 4-benzylamino-6-methyl-2-oxocyclohex-3-en-1-oate) which had an oral effective dose (ED₅₀) in rats of 27 mg/kg against maximal electroshock seizures, and a concentration 10-fold less than this dose depressed excitatory synaptic transmission, and action potential firing in the rat brain in vitro.

Full text of DOI:10.1016/j.bmc.2006.03.049

Bioorganic & Medicinal Chemistry 14 (2006) 5266–5272
Anticonvulsant evaluation and mechanism of action of
benzylamino enaminones
Ivan O. Edafiogho,* Kethireddy V. V. Ananthalakshmi and Samuel B. Kombian
Faculty of Pharmacy, Kuwait University, PO Box 24923, Safat 13110, Kuwait
Received 2 November 2005; revised 25 March 2006; accepted 29 March 2006
Available online 18 April 2006
Abstract—The mechanism of anticonvulsant action was evaluated for the benzylamino enaminones. The most potent enaminone in
this series was the unsubstituted benzylamine analog (30; methyl 4-benzylamino-6-methyl-2-oxocyclohex-3-en-1-oate) which had an
oral effective dose (ED50) in rats of 27 mg/kg against maximal electroshock seizures, and a concentration 10-fold less than this dose
depressed excitatory synaptic transmission, and action potential firing in the rat brain in vitro.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
complete anticonvulsant evaluation of benzylamino
enaminones, and the neurophysiological effects of a
highly active analog (compound 30), a moderately active
analog (compound 31), and an inactive analog (com-
pound 21) on glutamate-mediated synaptic responses
and neuronal excitability in neurons of the nucleus
accumbens (NAc).
Enaminones are known to possess a variety of medicinal
properties including anticonvulsant, antimalarial, anti-
inflammatory, and cardiovascular effects.1 Aniline
enaminones possess anticonvulsant properties which dif-
fer with para-substitutions on the phenyl moiety. Thus,
the unique enaminone pharmacophore presents an
excellent opportunity to develop anticonvulsant com-
pounds that are devoid of neurotoxicity and yet possess
wider margin of safety than conventional antiepileptic
drugs such as valproate, carbamazepine, and
phenytoin.1
2. Chemistry
Generally, the reaction of b-hydroxyketo compounds
with benzylamine derivatives yielded the benzylamino
enaminones 1–37 (Scheme 1 and Table 1). In this report,
the benzylamino enaminones are displayed completely
with their physicochemical, C log P, and in vivo anti-
convulsant data on one table for ease of identifying
the quantitative structure activity relationship (QSAR)
of the analogs, as compiled from our previous
reports.2,7–9 This approach presents the benzylamino
enaminones as a unique class of anticonvulsant enami-
nones in their own rights. Typical synthesis of the ben-
zylamino enaminones involved the removal of water
by Dean–Stark trap, and the use of excess amino com-
pounds in the reactions to obtain the corresponding
benzylamino enaminones with the amide group on the
cyclohexenone ring.
In a continuing evaluation of the enaminones for use in
seizure disorders, a comparison of enaminones from
various unsubstituted and para-substituted benzylamino
enaminones was undertaken with the aim of elucidating
the essential structural parameters necessary for anti-
convulsant activity. In addition, we tested the hypothe-
sis that benzylamino enaminones interacted with
glutamate and/or its receptors to produce anticonvul-
sant effects in rats and mice.2,3 This hypothesis was
based on the possible role of glutamate in the genesis
of seizures4,5 as well as computer modeling of enaminon-
es and crystallographic studies, which suggest that anti-
convulsant enaminones would fit a putative glutamate
receptor site.3,6 In this study, we hereby report a
The benzylamino enaminones consist of substituents in
which R1 = H, CH3; R2 = H, CH3; R3 = H,
CO2C(CH3)3, CO2C2H5, CO2CH3, CONHCH2C6H5,
CONHCH2C6H4(p-F), and R4 = H, F, Cl, CN, CH3,
OCH3, CO2H, NO2. The calculated partition coefficient
Keywords: Anticonvulsants; Enaminones; Mechanism; QSAR.
*
Corresponding author. Tel.: +965 498 6065; fax: +965 498 6840;
e-mail: ivanoe@hsc.edu.kw
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.03.049

I. O. Edafiogho et al. / Bioorg. Med. Chem. 14 (2006) 5266–5272
5267
R4
NHCH2
OH
R 3
R1
R2
R4
H2NCH2
+
R1
R2
O
O
R3
β-Hydroxyketo compounds Benzylamine derivatives
Scheme 1. General synthesis of benzylamino enaminones.
Benzylamino enaminones
Table 1. Physicochemical, C log P, and anticonvulsant data for benzylamine enaminones7–9
NHCH2
R4
R1
R2
O
R3
Compound
R1
R2
R3
R4
Yield
Mp (°C)
C log P
ASPa
1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
25
25
15
48
44
47
39
32
59
35
68
82
57
34
43
37
38
44
38
74
77
58
76
48
47
69
64
55
77
81
62
63
79
66
8
125–127
137.5–139
124–127
170–172
186–187
159–162
153–155
146–148
139–140
159–160
160–162
159–160
156–159
168–171
215–217
172–175
184–186
204–208
174–175
154–157
168.5–172
123–126
134–135
160–163
182–185
169–172
173–174
152–155
134–135
154–155
174–176
231–235 dec
138–139
174–176
183–184
193–195
219–220
2.41
2.93
3.45
3.12
3.64
4.16
2.91
3.43
3.95
2.33
2.85
3.37
1.84
2.36
2.90
3.46
2.75
2.22
3.95
3.24
2.71
4.53
3.82
3.29
4.74
4.03
2.79
4.03
3.32
2.79
2.86
2.46
3.32
2.46
3.94
3.60
4.49
1
1
1
4
4
2
2
3
3
2
3
3
2
1
3
3
3
3
3
3
3
1
3
3
3
3
2
3
3
1
2
3
2
3
3
3
3
2
CH3
CH3
H
H
H
3
CH3
H
H
Cl
4
5
CH3
CH3
H
H
Cl
Cl
6
CH3
H
7
CH3
CH3
CH3
OCH3
OCH3
OCH3
CN
CN
CN
CN
CN
CN
OCH3
OCH3
OCH3
CH3
CH3
CH3
Cl
8
CH3
CH3
H
H
9
CH3
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
CH3
CH3
H
H
CH3
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C6H5
H
CH3
H
CO2C(CH3)3
CO2C2H5
CO2CH3
H
H
H
CO2C(CH3)3
CO2C2H5
CO2CH3
H
H
H
CO2C(CH3)3
CO2C2H5
CO2CH3
H
H
H
CO2C(CH3)3
CO2C2H5
CO2CH3
H
Cl
Cl
H
H
CO2C(CH3)3
CO2C2H5
CO2CH3
H
H
H
H
H
H
CO2CH3
CO2CH3
F
H
CO2H
H
CH3
H
CO2CH3
CO2CH3
NO2
F
H
CONHCH2C6H4(p-F)
CONHCH2C6H5
CONHCH2C6H5
H
H
81
22
H
H
a Anticonvulsant Screening Project (ASP) classifications in Phase 1 evaluation in mice against maximal electroshock seizures (MES) are as follows: (1)
anticonvulsant activity at 100 mg/kg or less; (2) anticonvulsant activity at doses greater than 100 mg/kg; (3) inactive at doses of 300 mg/kg; (4)
activity inconsistent.

5268
I. O. Edafiogho et al. / Bioorg. Med. Chem. 14 (2006) 5266–5272
(C log P) values were determined using the CS Chem-
Draw Ultra version 6.01.10
(C log P value) and anticonvulsant effect. The C log P
range for all 37 benzylamine enaminones was from
1.84 (for compound 13) to 4.74 (for compound 25).
C log P measurements were for the protonated benzyl-
amines, which predominated at physiological pH. A cor-
relation could not be established between lipophilicity of
the benzylamine enaminones and anticonvulsant activi-
ty. The C log P values of classes 1 and 2 anticonvulsants
varied between 1.84 and 4.53, while the C log P values of
the inactive (class 3) enaminones varied from 2.22 to
4.74. The main factor affecting the anticonvulsant effect
of the benzylamino enaminones was the steric effect.2,7
Generally however, the highly lipophilic compounds
were inactive. In addition to steric effects, the lack of
strong anticonvulsant activity in the para-substituted
benzylamine series may be due to the lack of inductive
effect with the para-substituents arising from the pres-
ence of the methylene bridge which effectively blocks
this electronic contribution.
3. Anticonvulsant evaluations
The anticonvulsant evaluation of all 37 benzylamino
enaminones was determined by in vivo methods,2,7–9
and analogs 21, 30, and 31 were selected in the present
work, and evaluated directly on neurons by in vitro
methods.
3.1. In vivo anticonvulsant evaluation
Anticonvulsant evaluations were performed by the Anti-
convulsant Screening Program at the Epilepsy Branch of
the National Institutes of Neurological, Communicative
Disorders, and Stroke (NINCDS) in Bethesda, USA, by
methods that have been previously reported.2,7 Phase 1
evaluation of the benzylamino enaminones in mice is
shown in Table 1. To differentiate the results between
different rodent species, the most active enaminone
(30) was evaluated for oral (po) activity (Phase VIA)
in the rat.
4.3. In vivo anticonvulsant evaluation
According to the anticonvulsant screening program
(ASP),7 six benzylamino enaminones 1–3, 14, 22, and
30 were class 1 anticonvulsants, and they possessed sig-
nificant protective effect against maximal electroshock
seizures (MES) induced in mice. Seven benzylamino
enaminones including 6, 7, 10, 13, 27, 31, and 33 were
class 2 anticonvulsants and were moderately active.
Twenty-one of these compounds (8, 9, 11, 12, 15–21,
23–26, 28, 29, and 34–37) were class 3 enaminones and
were inactive. Two compounds 4 and 5 gave inconsistent
results, and were grouped in Class 4. Although we
reported certain aspects of the benzylamino enaminones
in our previous work,2,7–9 we hereby provide a more
complete QSAR for the in vivo anticonvulsant activity
for the compounds, and the possible mechanisms of ac-
tion of benzylamino enaminones that may underlie their
anticonvulsant activity.
3.2. In vitro anticonvulsant evaluation
To examine the effects of these compounds on excitatory
synaptic transmission and neuronal excitability, gluta-
mate-mediated excitatory postsynaptic currents (EPSC)
and action potentials (APs) were recorded in nucleus
accumbens (NAc) cells in vitro for the most active ana-
log (30), the moderately active analog (31), and the inac-
tive analog (21) according to the methods of Kombian
et al.11
4. Results and discussion
4.1. Chemistry
4.3.1. Quantitative structure activity relationships
(QSAR). The para-substitution pattern for this benzyla-
mino enaminones included chloro (+r, +p), methyl
(Àr, +p), cyano (+r, Àp), and methoxy (Àr, Àp)
groups which were compared to the unsubstituted ana-
log (0r, 0p).12 Class 4 benzylamines in Table 1 which
included compounds 4 and 5 were found to be sparingly
soluble and presented problems establishing a uniform
dosage, thus accounting for inconsistent results. The
enaminone ester with para-methyl substituent (22) pos-
sessed class 1 anticonvulsant activity, while one enami-
none ester with para-chloro substituent (27) showed
moderate, class 2 activity.7
The physicochemical, C log P, and anticonvulsant data
for benzylamine enaminones (1–37) are summarized in
Table 1. These compounds were completely character-
ized, and the UV, IR, and NMR data confirmed the
structures.2,7–9 The detailed syntheses of typical unsub-
stituted benzylamino enaminone, substituted benzyl-
amino ester, and benzylamino amide are described
under Experimental section. The benzylamino enami-
nones contain one or two chiral centers; and the com-
pounds with one chiral center would have racemic
mixture of two diastereomers, while the compounds
with two chiral centers would have racemic mixtures
of four possible diastereomers. However, we were un-
able to separate the compounds into individual stereo-
isomers, hence the in vivo and in vitro studies were
carried out on the racemic enaminones.
Compound 30 (R1, R4 = H, R2 = CH3, R3 = CO2CH3)
was the most potent anticonvulsant benzylamino com-
pound in this series, displaying an intraperitoneal (ip)
ED50 in mice of 64.7 mg/kg, and a TD50 > 500 mg/kg
and an oral (po) ED50 in rats of 26.8 mg/kg with no tox-
icity noted at dosages up to 500 mg/kg.7 Given that bio-
availability is often higher via a parenteral route as
compared to the oral route, the current finding indicates
that compound 30 is more potent in rats than in mice.
4.2. C log P data and anticonvulsant activity
When the C log P data of the benzylamino enaminones
(1–37) were compared to in vivo anticonvulsant activity,
there was no distinct correlation between lipophilicity

I. O. Edafiogho et al. / Bioorg. Med. Chem. 14 (2006) 5266–5272
5269
Furthermore, in corneal kindling model, compound 30
provided an ED50 of 78.3 mg/kg compared to phenyto-
in, 48.3 mg/kg, under the same conditions.7 Kubicki
et al.13 in the X-ray analysis of compound 30 observed
intermolecular N–H. . .O bonding, resulting in an
extended ‘sofa’ conformation without intramolecular
vinyl proton involvement. In determining the QSAR,
it was found that in the same carbomethoxy series,
para-substitutions led to less active or inactive com-
pounds. However, the p-fluoro compound 31
(R1 = CH3, R2 = H, R3 = CO2CH3, R4 = F) was active,
providing an ip ED50 of 159 mg/kg in mice, and a po
ED50 of 49.3 mg/kg and a TD50 > 230 mg/kg in rats.
The activity of this analog is consistent with Topliss’s
findings that p-fluoro substitution produces a minimal
change in r and p effects compared to the unsubstituted
compound.12 The p-chloro (27, R1 = CH3, R2 = H,
R3 = CO2CH3, R4 = Cl; +r, +p) provided spurious re-
sults, probably due to solubility problems, while the p-
tolyl
(24,
R1 = CH3,
R2 = H,
R3 = CO2CH3,
R4 = CH3; Àr, +p) and p-carboxy (32, R1 = CH3,
R2 = H, R3 = CO2CH3, R4 = CO2H; +r, Àp) analogs
were inactive. Further, in the dimedone series, the
unsubstituted
benzylamine
compound
(3,
R1 = R2 = CH3, R3 = H, R4 = H) was active with an
ED50 of 53 mg/kg and a TD50 of 148 mg/kg in mice,
and the comparable cyclohexenone derivative (1,
R1 = R2 = R3 = H, R4 = H) also showed class 1 activity.
Within the 6-methyl series of the enaminones, the
importance of the ester functionality in contributing to
anticonvulsant activity was shown in compounds 28,
29, and 30. Compound 30 (C log P 2.79) with a methyl
ester group was highly active,2,7 while changing the ester
functionality to the ethyl ester 29 (C log P 3.32) or the
tert-butyl ester 28 (C log P 4.03) completely abolished
activity. None of the enaminone amides (35–37) was
anticonvulsant probably due to their high lipophilicity
and steric hindrance.
Figure 1. Effect of compounds 21, 30, and 31 on evoked EPSC
amplitude. (A) A time-effect plot of the EPSC amplitude of a
representative cell that was first exposed to compound 21 and
subsequently compound 30 after recovery. Each compound was
applied for 6 min. Inserts are sample synaptic responses taken at the
times indicated by letters in the main graph. (B) An averaged time-
effect graph showing the effect of compound 30 on EPSC amplitude in
7 cells. (C) Average bar graph summarizing the effects of compounds
4.4. In vitro anticonvulsant evaluation
In our in vitro electrophysiology studies, 10 lM of com-
pounds 21, 30, and 31 was tested on isolated EPSCs and
on neuronal excitability. This concentration is 10 times
less than the effective therapeutic dose of the most active
compound 30. Bath application of 10 lM of compound
21, the methoxy-substituted benzylamine derivative,
either did not affect EPSC amplitude or increased them
in some cells resulting in an average change in EPSC
amplitude of 19.5 12.9% (n = 4; p > 0.05, paired
Student’s t test; Fig. 1). However, 10 lM of the unsub-
stituted benzylamine derivative compound 30, or the flu-
oro-substituted derivative (compound 31) which
demonstrated excellent and intermediate in vivo anti-
convulsant activity, respectively, reversibly depressed
the evoked EPSC amplitude by À23.6 5.9% (n = 7,
p < 0.05, paired Student’s t test, Fig. 1) and À30.8
4.6% (n = 5, p < 0.05, paired Student’s t test, Fig. 1C),
respectively. Furthermore, both compounds 30 and 31,
at this same concentration, also reversibly reduced AP
firing frequency. However, while the most active com-
pound 30 depressed the AP firing frequency by
21, 30, and 31 on EPSC amplitude. represents statistical significance
*
at p < 0.05.
À62.5 14.7% (n = 5, p < 0.05, paired Student’s t test,
Fig. 2), the intermediately potent compound 31 only
produced À33 11.0% (n = 5, p < 0.05, paired Student’s
t test, Fig. 2B) suppression of AP firing frequency.
Finally, compound 21, the inactive analog had no signif-
icant effect on AP firing (À5.3 7.5%, n = 4; p > 0.05;
paired Student’s t test). In both the EPSC and AP exper-
iments, the onset of action was about 2 min with maxi-
mum effect observed at 5–6 min (Figs. 1 and 2).
Recovery from the EPSC and action potential frequency
depression was complete with responses returning to
baseline levels in 8–15 min after the commencement of
washout (Figs. 1 and 2). The effects of compounds 30
and 31 on EPSC and AP firing frequency at the tested
concentration of 10 lM were without significant
changes in holding current or membrane potential.

5270
I. O. Edafiogho et al. / Bioorg. Med. Chem. 14 (2006) 5266–5272
similar EPSC depression but lesser AP firing suppres-
sion than the more potent compound 30 (see Figs. 1C
and 2B). Therefore, our in vitro data on synaptic
transmission and neuronal excitability are consistent
with the results of our in vivo studies which indicate
that the unsubstituted benzylamino enaminone com-
pound 30 is a potent (class 1) anticonvulsant agent,
while the less potent fluoro-substituted compound 31
displayed less suppression of neuronal excitability.
By comparison, the substituted analog compound 21
is inactive as an anticonvulsant, as it does not depress
evoked EPSCs, nor reduce the AP firing frequency in
the neurons of NAc.
Compound 21 is inactive, while compound 30 is anti-
convulsant in vivo. The structural difference between
these enaminones is that compound 21 has a para-sub-
stitution of an electron-donating substituent, while com-
pound 30 has no para-substitution (Scheme 2).
Accordingly, compound 30 is sterically less hindered to
interact at the molecular level and this may be the opti-
mum structural requirement for the benzylamino enami-
nones to depress excitatory synaptic transmission and
neuronal excitation in the rat brain. Furthermore, this
structure also enables this compound to suppress AP fir-
ing which could be via interaction with sodium channels.
Both these effects may then contribute to or underlie its
anticonvulsant property.
Figure 2. Compounds 30 and 31 decrease action potential firing
frequency in neurons. (A) APs generated by a depolarizing current
injection into a cell in control conditions (upper panel), 5 min after
bath application of compound 30 (10 lM; middle panel), and 15 min
after washing out compound 30 (lower panel). (B) Summary bar graph
from five (5) neurons each treated with either compound 30 or 31 as in
(A) showing the decrease and subsequent recovery in AP firing
frequency induced by compounds 30 and 31. Compound 21 did not
4.4.2. Electrophysiological data support mechanisms of
action. We have performed initial molecular dynamic
studies with a variety of common targets for the action
of anticonvulsant compounds.3,14 Out of these, the glu-
tamate receptor (GLUR) best recognized the enaminon-
es, and optimally distinguished between active and
inactive enaminones. These molecular dynamic studies
suggested that the observed anticonvulsant activity of
benzylamine enaminone 27 may be due to inhibition
of the glutamate machinery in the central nervous sys-
tem. The in vitro electrophysiological studies seem to
support the involvement of glutamate in the actions of
enaminones to produce their anticonvulsant effects.
The fact that compound 30, the most potent anticonvul-
sant in the MES test, was found to also depress gluta-
mate-mediated excitatory synaptic transmission
suggests that an interaction with glutamate may underlie
or contribute to its anticonvulsant activity. Recent neu-
ropharmacological evidence in our laboratory suggests
that this interaction may however be indirect through
the enhancement of gamma amino butyric acid (GABA)
affect the AP firing frequency in all four (4) cells tested. indicated
*
statistical significance at p < 0.05.
4.4.1. Mechanisms of anticonvulsant action of benzyla-
mino enaminones. In order to understand the mecha-
nism(s) underlying the anticonvulsant actions of the
benzylamine enaminones, their effects on neuronal
physiology were investigated. The results of our in vi-
tro study show that the most potent anticonvulsant
enaminone (compound 30) in the benzylamino series
caused a significant depression of both glutamate-med-
iated excitatory synaptic transmission and action po-
tential firing in cells of the central nervous system
(CNS). Furthermore, it was observed that the interme-
diately potent analog, compound 31, also produced a
CH2
O
F
NH
CH2
NH
CH2
O
CH
3
O
NH
H3C
H3C
O
H3C
COOCH3
COOCH3
COOCH3
Compound
31
Compound 30
Compound 21
Scheme 2. Complete structures of compounds 21, 30, and 31. The differences between the three compounds studied by electrophysiological recording
are the methoxy (OCH3) group in compound 21, hydrogen (H) atom in compound 30, and fluorine (F) atom in compound 31.

I. O. Edafiogho et al. / Bioorg. Med. Chem. 14 (2006) 5266–5272
5271
level in rat brains.11 Additional studies are required to
determine if indeed this series of enaminones employ
similar cellular mechanism(s) as those recently reported
for other enaminones.11 It is noted that the anilino
enaminones whose mechanisms of anticonvulsant action
were elucidated recently11 are structurally different from
the benzylamino enaminones which we are reporting in
this work. However, the enaminone system is common
to both types of moieties, and based on our current
and previous data, we postulate that the enaminone
pharmacophore existing in a sterically favored confor-
mation is very important for excitatory synaptic depres-
sion and anticonvulsant activity.
15 mmol) and the mixture refluxed for 3 h using a
Dean–Stark water separator. During the reaction,
0.25 mL of water was collected. Evaporation of the mix-
ture to dryness and two recrystallizations from ethyl
acetate provided 1.6 g (81%) of enaminone 30, mp
154–155 °C. Anal. (C, H, N).
6.1.3. Synthesis of benzylamino enaminone with amide
group; 4-benzylamino-6-methyl-2-oxocyclohex-3-ene(N-
benzyl) carboxamide (36). To a solution of methyl 4-hy-
droxyl-6-methyl-2-oxocyclohexen-3-en-1-oate
(4 g,
22 mmol) in 100 mL of toluene was added benzylamine
(6.4 g, 60 mmol) and the mixture refluxed for 6 h using a
Dean–Stark water separator. During the reaction,
0.4 mL of water was collected. Evaporation of the reac-
tion mixture to dryness and two recrystallizations from
methanol provided 6.2 g (81%) of enaminone 36, mp
193–195 °C. Anal. (C, H, and N).
5. Conclusions
Prior to our recent report,11 the mechanism of anticon-
vulsant action of the enaminones was unknown.20,21
Our evaluation of anticonvulsant benzylamino enami-
nones gave results which indicate that para-substitution
of the benzylamine moiety with either carboxyl, chloro,
cyano, fluoro, methoxy, methyl, or nitro groups decreas-
es or abolishes the anticonvulsant activity of the enami-
nones. The most potent benzylamino enaminone was the
unsubstituted benzylamine analog (30; R1, R4 = H,
R2 = CH3, R3 = CO2CH3). Furthermore, our in vitro
electrophysiological data showed that the active ben-
zylamino analogs (30 and 31) depressed glutamate-med-
iated excitation and action potential firing in neurons.
These actions may contribute to their anticonvulsant
activity.
6.2. In vivo anticonvulsant evaluations
Anticonvulsant evaluations of the benzylamino enami-
nones (1–37) were performed by the Antiepileptic Drug
Development (ADD) Program, Epilepsy Branch, Neu-
rological Disorders Program, National Institute of
Neurological Disorders and Stroke, and included Phase
I, II, VIA, and VIB test procedures which have been
described.16–18 These tests were performed in male Car-
worth Farms no. 1 (CF1) mice (Phases I and II) or male
Sprague–Dawley rats (Phases VIA and VIB). Phase I
and Phase VIA of the anticonvulsant evaluation com-
prised of maximal electroshock test, subcutaneous
pentylenetetrazole (ScMet) test, and the rotorod test
for neurological toxicity (Tox). Compounds were sus-
pended in 0.5% aqueous methylcellulose and were
administered at 30, 100, and 300 mg/kg dosage levels
with anticonvulsant activity and motor impairment
noted 30 min and 4 h after administration. Phase II
and Phase VIB quantitated the anticonvulsant activity
and motor impairment observed for the most promising
enaminones in Phase I. Phase II quantitated data in CF1
mice by intraperitoneal (ip) administration, while Phase
VIB quantitated oral rat data comparable to Phase II
intraperitoneal (ip) data in mice. Data for the anticon-
vulsant evaluations are shown in Table 1.
6. Experimental
6.1. Chemical synthesis
Melting points of the enaminone compounds were deter-
mined on a Thomas Hoover capillary melting point
apparatus and are uncorrected. Spectrometric (IR and
NMR) data and elemental analyses showed the appro-
priate peaks and chemical shifts, and confirmed the
molecular formulae for the benzylamino enaminones.
Typical experiments illustrating the general synthesis
of the enaminones are described below.
6.3. Electrophysiology
6.1.1. Synthesis of benzylamino enaminone with no ester
group; 3-benzylaminocyclohex-2-en-1-one (1). To a solu-
tion of 1,3-cyclohexanedione (7.5 g, 67 mmol) in
75 mL of ethanol and 100 mL of benzene was added
benzylamine (7.88 g, 70 mmol) and the mixture refluxed
for 4 h. Evaporation of the reaction mixture yielded a
dark brown oil which crystallized from diethyl ether.
Two recrystallizations of the crude product from 2-pro-
panol provided 3.4 g (25%) of greenish-yellow crystals of
enaminone 1, mp 125–127 °C (lit. 122–124 °C).15
Electrophysiological studies were performed with three
compounds of this series, compounds 21, 30, and 31
(see Scheme 2) using in vitro patch-clamp recording
techniques.11 In brief, forebrain slices were prepared
from male Sprague–Dawley rats using standard meth-
ods and pure glutamate-mediated excitatory postsynap-
tic currents (EPSCs) were recorded in voltage clamp
mode as previously reported.11 Pure glutamate-mediated
responses were isolated by bath application of picrotox-
in (50 lM) to eliminate the GABAA receptor-mediated
component of synaptic responses (see Kombian
et al.19). APs were recorded in current clamp mode
and a series of hyperpolarizing and depolarizing cur-
rents were applied to the cells at their resting potential
and the corresponding membrane responses were
6.1.2. Synthesis of benzylamino enaminone with ester
group; methyl 4-benzylamino-6-methyl-2-oxocyclohex-3-
en-1-oate (30). To a solution of methyl 4-hydroxyl-6-
methyl-2-oxocyclohexen-3-en-1-oate (2 g, 10.9 mmol)
in 100 mL of toluene was added benzylamine (1.61 g,

5272
I. O. Edafiogho et al. / Bioorg. Med. Chem. 14 (2006) 5266–5272
3. Abou-Zeid, L.; Edafiogho, I. O. Health Sciences Poster
Day 2002 and Ibn Sina Forum, Faculty of Medicine, Kuwait
University, Kuwait, 2002, p. 178.
4. Dingledine, R.; McBain, C. J.; McNamara, J. O. Trends
Pharmacol. Sci. 1990, 11, 334.
5. McNamara, J. O. Nature 1999, 399, A15.
captured. 10 mM stock solutions of compounds 21, 30,
and 31 were made by dissolving them in DMSO. These
were then aliquoted and stored frozen until application.
They were bath perfused at the final concentration of
10 lM by dissolving the 10 mM stock solution in artifi-
cial cerebrospinal fluid resulting in a 1000-fold dilution
of the DMSO (yielding <0.1% DMSO in the bath). This
final solution was then applied for 5–6 min. A concen-
tration of 10 lM of compounds 21, 30, and 31 was
selected for this study because we have previously
reported that other anticonvulsant enaminones of the
aniline series produced robust synaptic depression at
this concentration.11 Furthermore, this concentration
was calculated to be 10 times less than the equivalent
oral dose that demonstrated in vivo anticonvulsant
activity. Thus, if only 10% of administered compound
30 were absorbed following oral administration, and
this entire amount reached the brain, it would be expect-
ed to produce the anticonvulsant effect.
6. Edafiogho, I. O.; Denny, B. J.; Schwalbe, C. H.; Lowe, P.
R. Med. Principles Practice 2003, 12, 237.
7. Foster, J. E.; Nicholson, J. M.; Butcher, R.; Stables, J. P.;
Edafiogho, I. O.; Goodwin, A. M.; Henson, M. C.; Smith,
C. A.; Scott, K. R. Bioorg. Med. Chem. 1999, 7, 2415.
8. Scott, K. R.; Edafiogho, I. O.; Richardson, E. L.; Farrar,
V. A.; Moore, J. A.; Teitz, E. I.; Hinko, C. N.; Chang, H.;
El-Assadi, A.; Nicholson, J. M. J. Med. Chem. 1993, 36,
1947.
9. Edafiogho, I. O.; Moore, J. A.; Alexander, M. S.; Scott, K.
R. J. Pharm. Sci. 1994, 83, 1155.
10. CS ChemDraw Ultra version 6.01, C log P computer
software by CambridgeSoft.Com.
11. Kombian, S. B.; Edafiogho, I. O.; Ananthalakshmi, K. V.
V. Br. J. Pharmacol. 2005, 145, 945.
12. Topliss, J. G. J. Med. Chem. 1972, 15, 1006.
13. Kubicki, M.; Codding, P. W. Acta Cryst. C 1993, 49, 2045.
14. Armstrong, N.; Gouaux, E. Neuron 2000, 28, 165.
15. Kotera, K. Yakugaku Zasshi 1960, 80, 1275.
16. Anticonvulsant Screening Project, Antiepileptic Drug
Development program, National Institutes of Health,
DHEW publ (NIH) (U.S.), 1978, NIH 78-1093.
17. Porter, R. J.; Cereghino, J. J.; Gladding, G. D.; Hessie, B.
J.; Kupferberg, H. J.; Scoville, B.; White, B. G. Cleveland
Clin. Q 1984, 51, 293.
Acknowledgments
This work was supported by Kuwait University,
Research Grant Nos. PR 02/02 to IOE and PT 02/02
to KSB. We thank James P. Stables for the in vivo
anticonvulsant screening.
18. Krall, R. L.; Penry, J. K.; White, B. G.; Kupferberg, H. J.;
Swinyard, E. A. Epilepsia 1978, 19, 400.
References and notes
19. Kombian, S. B.; Ananthalakshmi, K. V. V.; Parvathy, S.
S.; Matowe, W. C. J. Neurophysiol 2003, 89, 728.
20. Malawska, B. Curr. Top. Med. Chem. 2005, 5, 69.
21. Anderson, A. J.; Nicholson, J. M.; Bakare, O.; Butcher, R.
J.; Wilson, T. L.; Scott, K. R. Bioorg. Med. Chem. 2006,
14, 997.
1. Eddington, N. D.; Cox, D. S.; Roberts, R. R.; Stables, J. P.;
Powell, C. B.; Scott, K. R. Curr. Med. Chem. 2000, 7, 417.
2. Edafiogho, I. O.; Hinko, C. N.; Chang, H.; Moore, J. A.;
Mulzac, D.; Nicholson, J. M.; Scott, K. R. J. Med. Chem.
1992, 35, 2798.