Synthesis and anticonvulsant activity of enaminones - Part 7. Synthesis and anticonvulsant evaluation of ethyl 4-[(substituted phenyl)amino]-6-methyl-2-oxocyclohex-3-ene-1-carboxylates and their corresponding 5-methylcyclohex-2-enone derivatives

Article abstract of DOI:10.1016/S0223-5234(02)00006-5

Further investigation of the potential anticonvulsant activity of the enaminones was attempted to discern the possible role of metabolites as the active/co-active entities of the esters of the enaminones. A series of 5-methyl-2-cyclohexene enaminones, the

Full text of DOI:10.1016/S0223-5234(02)00006-5

z
European Journal of Medicinal Chemistry 38 (2003) 49ꢂ64
/
www.elsevier.com/locate/ejmech
Original paper
Synthesis and anticonvulsant activity of enaminones
Part 7. Synthesis and anticonvulsant evaluation of ethyl 4-
[(substituted phenyl)amino]-6-methyl-2-oxocyclohex-3-ene-1-
carboxylates and their corresponding 5-methylcyclohex-2-enone
derivatives
Natalie D. Eddington a, Donna S. Cox a,1, Manoj Khurana a, Noha N. Salama a,
James P. Stables b, Sylvia J. Harrison c, Abraham Negussie c, Robert S. Taylor c,
Uy Q. Tran c, Jacqueline A. Moore c, Judith C. Barrow d, K.R. Scott c,*
a Department of Pharmaceutical Sciences, Pharmacokinetics Biopharmaceutics Laboratory, School of Pharmacy, University of Maryland at Baltimore,
Baltimore, MD 21201-4403, USA
b Epilepsy Branch, Division of Convulsive, Developmental and Neuromuscular Disorders, National Institute of Neurological Disorders and Stroke,
Bethesda, MD 20892, USA
c Department of Pharmaceutical Sciences, School of Pharmacy, Howard University, Washington, DC 20059, USA
d Department of Chemical Engineering, College of Engineering, Architecture and Computer Science, Howard University, Washington, DC 20059, USA
Received 23 September 2002; received in revised form 4 November 2002; accepted 4 November 2002
Abstract
Further investigation of the potential anticonvulsant activity of the enaminones was attempted to discern the possible role of
metabolites as the active/co-active entities of the esters of the enaminones. A series of 5-methyl-2-cyclohexene enaminones, the
hypothesised metabolites corresponding to a sequence of active and inactive esters were synthesised and evaluated for
anticonvulsant activity. With two exceptions, ethyl 4-[(4-cyanophenyl)amino]-6-methyl-2-oxocyclohex-3-ene-1-carboxylate (1k),
and 3-[N-(4-cyanophenyl)amino]-5-methyl-2-cyclohexenone (3g), and ethyl 4-(phenylamino)-6-methyl-2-cyclohexenone (1n), and 3-
N-(phenylamino)-5-methyl-2-cyclohexenone (3j), anticonvulsant screening data were parallel, with the ester and their putative
decarboxylated analogue displaying similar activity. The most active analogue evaluated in this series, ethyl 4-[(4-chloropheny-
l)amino]-6-methyl-2-oxocyclohex-3-ene-1-carboxylate (1e), which displayed an ED50 of 16.7 mg kgꢀ1 and a TD50 of 110.7 mg kgꢀ1
(protective index, PIꢁ
/
TD50/ED50
250 mg kgꢀ1 (PI ꢀ
our laboratories. Pharmacokinetic evaluation of compound 1e in rats using LC/MS analysis unequivocally provides evidence that
ꢁ
6.6) in the maximal electroshock seizure (MES) test in mice and an ED50 of 3.0 mg kgꢀ1 and a
83.3) in rats in the same evaluation, making this compound the most potent enaminone emanating from
/
TD50
ꢀ
/
/
this compound is converted into the decarboxylated analogue 3a in the brain and the urine.
´
# 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Enaminones; Maximal electroshock seizure test; Anticonvulsant activity; Structureꢂactivity relationship; LC/MS analysis
/
Abbreviations: MES, maximal electroshock seizure; TD50, toxic dose for 50% of test animals; ED50, effective dose for 50% of test animals; ip,
intraperitoneal; PI, protective index, TD50/ED50; scPTZ, subcutaneous pentylenetetrazol; Tox, neurologic toxicity; ADD, Antiepileptic Drug
Development; NINDS, National Institutes of Neurological Disorders and Stroke; TTE, threshold tonic extension; AUC, area under the curve;
Clog P, calculated log P; [3H]BTX-B, [3H]batrachotoxinin A 20 a-benzoate; IC50, 50% in vitro inhibition of binding of [3H]BTX-B to sodium
channels in rat brain synaptoneurosomes; m.p., melting point; b.p., boiling point; EtOAc, ethyl acetate; 2-PrOH, 2-propanol.
* Corresponding author. Present address: Department of Pharmaceutical Sciences, College of Pharmacy, Howard University, Washington, DC
20059, USA.
E-mail address: kscott@fac.howard.edu (K.R. Scott).
1
Present address: DuPont Pharmaceuticals, Drug Metabolism and Pharmacokinetics Section, S112/36, 1090 Elkton Road, P.O. Box 30, Newark,
DE 19713, USA.
´
0223-5234/02/$ - see front matter # 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S 0 2 2 3 - 5 2 3 4 ( 0 2 ) 0 0 0 0 6 - 5

50
N.D. Eddington et al. / European Journal of Medicinal Chemistry 38 (2003) 49ꢂ64
/
1. Introduction
pared as previously reported [2,4,5,8ꢂ
refluxed with appropriate aniline derivatives under
standard conditions. Enaminones (3aꢂ3t, Table 1)
were prepared from 5-methylcyclohexane-1,3-dione (5),
formed by the decarboxylation of 4-carbo-tert-butoxy-
/
11,16] and were
Despite optimal use of available antiepileptic drugs,
many patients with epilepsy fail to experience seizure
control and other do so only at the expense of significant
toxic side effects. Estimates suggest that available
medication controls the seizures in only 50% of patients
or decreases the incidence in only 75% of patients [1]. In
a continuing study of potential enaminone anticonvul-
/
5-methylcyclohexane-1,3-dione (4b, RꢁC(CH3)3) [18].
/
These reactions are shown in Fig. 2. As previously
reported by Friary [18] and by us [6], the stereochemistry
of the esters (1eꢂ1x, Table 1) indicated that they exist as
/
sants [2ꢂ
/
17], we have postulated a biotransformation
trans racemates. The NMR analyses of the enaminones
at 300 MHz were consistent with their assigned struc-
tures.
pathway in which the carboalkoxy substituent 1 (Fig. 1)
was a bioactive intermediate, and that a two step
reaction involving deesterification 2 and decarboxyla-
tion would occur, producing a second active enaminone
metabolite 3 [8]. Specifically, compound 1, being more
lipophilic than enaminone 3, would more readily
3. Pharmacology
penetrate the bloodꢂbrain barrier to exert an initial
/
3.1. Preliminary pharmacological testing
anticonvulsant effect. Subsequently, the ester 1, would
react with non-specific esterases to form the unstable b-
keto acid intermediate 2, which would subsequently
decarboxylate to form the corresponding enaminone 3.
Although this hypothesis was previously demonstrated
in vitro with methyl 4-[(4-chlorophenyl)amino]-6-meth-
The Antiepileptic Drug Development (ADD) Pro-
gram, Epilepsy Branch, Neurological Disorders Pro-
gram, National Institute of Neurological Disorders and
Stroke (NINDS) have provided preliminary pharmaco-
logical testing of the reported compounds. These testing
yl-2-oxocyclohex-3-ene-1-carboxylate (1a, Rꢁ
R1ꢁ
4-Cl) [8], it was not apparent in vivo [9]. In the
initial study, only the corresponding R1ꢁ
4-Cl ketone
/CH3,
procedures have been described [19ꢂ21]. Phase I studies
/
/
of the reported enaminones involved three tests: max-
imal electroshock seizure (MES), subcutaneous penty-
lenetetrazol (scPTZ), and neurologic toxicity (Tox). The
Anticonvulsant Screening Project (ASP) classifications
/
(3a, Table 1), was synthesised [8]. However, this current
study includes an extensive synthesis and anticonvulsant
evaluation of the potential 5-methyl metabolites (3aꢂ
/3t,
are as follows: class 1ꢁ
class 2ꢁactivity ꢀ
100 mg kgꢀ1, but less than 300 mg
kgꢀ1; class 3ꢁ
no activity at doses up to and including
300 mg kgꢀ1. As previously reported [2,3,7ꢂ
9], class 1
/
activity at 100 mg kgꢀ1 or less;
Table 1) emanating for the esters (1eꢂ1x, Table 1). In
/
/
/
addition, we herein report the results of the LC/MS
analysis that verified the biotransformation of ethyl 4-
[(4-chlorophenyl)amino]-6-methyl-2-oxocyclohex-3-ene-
1-carboxylate (1e) to the 5-methyl ketone (3a), in the
brain and urine of rats.
/
/
or 2 active compounds in the Phase I evaluation were
advanced to Phase II mice quantitation for ED50 and
TD50 evaluation. Further, to differentiate the results
between distinct rodent species, the active analogues
were subsequently evaluated for oral (po) activity (Phase
VIA) in the rat. Data for these compounds is shown in
Table 2. Quantitation of the Phase I intraperitoneal (ip)
and/or po activity (Phase VIB) is provided, where
2. Chemistry
2.1. Synthesis
appropriate, as well as the protective index (PIꢁ
/
The synthesis of the enaminones that appear in Table
1 is indicated in Fig. 2. The requisite b-hydroxy keto
TD50/ED50). In one case, ethyl 4-[(3-iodophenyl)a-
mino]-6-methyl-2-oxocyclohex-3-ene-1-carboxylate
(1q), an initially inactive compound in Phase I was
esters (4a, Rꢁ
/
C2H5, or 4b, RꢁC(CH3)3), were pre-
/
Fig. 1.

N.D. Eddington et al. / European Journal of Medicinal Chemistry 38 (2003) 49ꢂ
/
64
51
Table 1
Enaminone esters, 1, and enaminones, 3
a
Compound 1 (Rꢁ
/
C2H5)
Compound 3
b
b
Compound
R1
Clog P
M.p. (8C)
Compound
R1
Clog P
M.p. (8C)
c
c
c
1e
1f
4-Cl
4-Br
3.46
3.61
3.87
2.89
3.86
3.66
2.58
161ꢂ
/
163
3a
3b
3c
3d
3e
3f
4-Cl
4-Br
3.07
3.22
3.48
2.50
3.47
3.27
2.19
198ꢂ
213ꢂ
225ꢂ
185ꢂ
205ꢂ
174ꢂ
234ꢂ
208ꢂ
194ꢂ
/
199.5
d
151ꢂ
/
154
/
215
227
e
d
1g
1h
1i
4-I
4-F
160.5ꢂ
150ꢂ151.5
184.5ꢂ
/
162.5
4-I
4-F
/
c
f
/
/
187
c
d
4-CF3
4-OCF3
4-CN
4-NO2
4-CH3
H
/
186
4-CF3
4-OCF3
4-CN
4-NO2
4-CH3
H
/
206
175
c
f
1j
162ꢂ
190ꢂ
173ꢂ
/
164
193
175
/
e
c
f
1k
1l
/
3g
3h
3i
/
236
209
f
ꢀ
/
0.84
2.95
2.45
3.46
3.61
3.87
2.89
3.86
3.66
2.58
0.84
2.95
2.55
/
ꢀ
/
1.24
2.55
2.06
3.07
3.22
3.48
2.50
3.47
3.27
2.19
1.24
2.55
2.16
/
c
d
1m
1n
1o
1p
1q
1r
1s
1t
134.5ꢂ
155ꢂ
137ꢂ
135ꢂ
120ꢂ
138ꢂ
164ꢂ
151ꢂ
165ꢂ
155ꢂ
125ꢂ
110ꢂ
/
135.5
c
/
195
f
/
158
138
137
124
140
166
152
167
157
127
113
3j
155 (soften); 158ꢂ
/
160
e
e
e
e
e
e
e
e
e
e
f
f
f
f
3-Cl
3-Br
/
3k
3l
3-Cl
3-Br
177ꢂ
172ꢂ
185ꢂ
163ꢂ
179ꢂ
156ꢂ
/
179
174
187
165
/
/
3-I
3-F
/
3m
3n
3o
3p
3q
3r
3s
3t
3-I
3-F
/
/
/
f
f
f
3-CF3
3-OCF3
3-CN
3-NO2
3-CH3
3-OCH3
/
3-CF3
3-OCF3
3-CN
3-NO2
3-CH3
3-OCH3
/
180.5
f
160
/
/
1u
1v
1w
1x
/
175.5ꢂ177
/
f
ꢀ/
/
ꢀ
/
187ꢂ
147ꢂ
140ꢂ
/
189
148.5
/
/
f
/
/
142
The infrared and 1H-NMR spectra (300 MHz) were consistent with assigned structures. All compounds gave satisfactory C, H, N, and halogen
a
(when required) analyses (9
/
0.4%). Recrystallisation solvents as indicated.
b
c
d
e
f
Calculated from Ref. [24].
Ref. [8].
2-Propanol.
Ref. [9].
Ethyl acetate.
subsequently shown to be active in the threshold tonic
extension (TTE) test [22]. The TTE evaluation was also
employed in the evaluation of ethyl 4-[(3-cyanopheny-
l)amino]-6-methyl-2-oxocyclohex-3-ene-1-carboxylate
(1u); however, this compound remained inactive.
CH3, R1ꢁ4-Cl; ED50 26.2 mg kgꢀ1 ip in mice, ED50
/
5.9 mg kgꢀ1 po in rats) [2,3]. Based on the promising
pharmacological results obtained with compound 1e,
the present studies were conducted to understand its
distribution in the target organ, the brain, and its
metabolism and pharmacokinetics in rats. Based on
the pharmacological activity data obtained with ip
administration, and to verify the hypothesis delineated
in Fig. 1, a disposition study was carried out via the
same route of administration in rats. Therefore, this
data will provide relevant information on the uptake of
the target compound 1e, by the target organ, its
metabolism and pharmacokinetic profile and aid in
further development of the target compound as a
candidate drug. Preliminary in vitro studies on target
compound 1e were performed. Treatment of compound
1e with porcine liver esterase (EC 3.1.1.1) at pH 7.4 at
3.2. Metabolism and pharmacokinetics
Ethyl 4-[(4-chlorophenyl)amino]-6-methyl-2-oxo-3-
cyclohexene-1-carboxylate (1e) was found to be the
most promising enaminone analogue in the aniline series
in both the rat and mouse models during preliminary
pharmacological evaluation providing an ED50 16.7 mg
kgꢀ1 ip in mice, ED50 3.0 mg kgꢀ1 po in rats, almost
twice as effective than previously for the reported
prototype analogue, methyl 4-[(4-chlorophenyl)amino]-
6-methyl-2-oxo-3-cyclohexene-1-carboxylate (1a, Rꢁ
/

52
N.D. Eddington et al. / European Journal of Medicinal Chemistry 38 (2003) 49ꢂ64
/
extent of 20% over 24 h [8]. As noted by us earlier, the
enaminones are quite stable at pH 7.0 and 7.4 in the
absence of esterase [8]. Edafiogho et al., in a stability-
indicating assay of methyl 4-[(4-bromophenyl)amino]-6-
methyl-2-oxocyclohex-3-ene-1-carboxylate (1b, Rꢁ
/
CH3, R1ꢁ
/
4-Br) noted that the compound was stable
in both 0.1 M NaOH as well as in neutral media, but
decomposed rapidly in acidic (0.1 M HCl) media [23].
Data on the pharmacokinetic parameters of ethyl 4-
[(4-chlorophenyl)amino]-6-methyl-2-oxocyclohex-3-ene-
1-carboxylate (1e) following a single ip dose (10 mg
kgꢀ1) in rats is shown in Table 3, data on mass spectra
and daughter ion spectra of compound 1e is shown in
Table 4, the brain and plasma concentrations of
compound 1e following a single ip dose (10 mg kgꢀ1
)
is shown in Fig. 3, a representative mass spectra of
compound 1e and its metabolites is shown in Figs. 4 and
5 provides the major metabolic pathways of compound
1e in rats.
Fig. 2. Conditions: (a) D, amine; (b) RꢁC(CH3)3, H2SO4, D.
/
3.3. Sodium channel binding
37 8C and monitoring by ultraviolet analysis resulted in
a 30% conversion of compound 1e to enaminone 3a, in 8
h. This was consistent with previous studies in our
laboratories with methyl 4-[(4-chlorophenyl)amino]-6-
Ethyl 4-[(4-chlorophenyl)amino]-6-methyl-2-oxocy-
clohex-3-ene-1-carboxylate (1e) was evaluated in a
synaptoneurosomal preparation in N1E-115 neuroblas-
methyl-2-oxocyclohex-3-ene-1-carboxylate (1a, Rꢁ
/
toma cells [25ꢂ29]. This assay was similar to literature
procedure [29] and measured the ability of the test
compound to inhibit the specific binding of [3H]batra-
/
CH3, R1ꢁ
identical enaminone 3a and was metabolised to the
/
4-Cl) which was also converted into the
Table 2
Anticonvulsant evaluation of enaminone esters, 1, enaminones, 3 in mice and rats
a
b
c
Compound
Quadrant
ASP class
1
Anticonvulsant results
Esters
1e
ꢃ
/
s, ꢃ
/
p
Phase II: ED50 16.7, TD50 110.7, PI 6.6, TPE 0.25
Phase VIB: ED50 3.0, TD50 250, PI ꢀ83.3, TPE 1.0
Phase II: ED50 7.9, TD50 86.9, PI 10.9, TPE 0.25
Phase VIB: ED50 6.6, TD50 220, PI ꢀ33.3, TPE 0.5
400, PI ꢀ10.8, TPE 1.0
500, PI ꢀ75.8, TPE 2.0
ꢀ
/
/
1f
1g
1j
ꢃ
/
s, ꢃ
s, ꢃ
s, ꢃ
/
p
1
1
1
ꢃ
/
/
p
ꢀ
/
/
ꢃ/
/
p
Phase II: ED50 37.1, TD50
ꢀ
/
/
Phase VIB: ED50 6.6, TD50
ꢀ/
/
1k
1o
1u
ꢃ
/
s, ꢀ
s, ꢃ
s, ꢀ
/
p
1
1
Phase II: ED50 63.1, TD50 159.5, PI 2.1, TPE 0.25
ꢃ
/
/
p
Phase VIB: ED50 11.5, TD50 424, PI ꢀ36.9, TPE 0.25
ꢀ/
/
d
ꢃ/
/
p
1
ED50 (rats) 7.7, TD50 not determined, TPE 0.25
5-Methyl ketones
3a
ꢃ
/
s, ꢃ
/
p
1
Phase II: ED50 40.4, TD50 93.6, PI 2.3, TPE 0.25
Phase VIB: ED50 14.7, TD50
Phase VIB: ED50 7.6, TD50
Phase II: ED50 77.0, TD50 343.6, PI 4.5, TPE 0.25
ꢀ/
186, PI ꢀ
/
12.7, TPE 1.0
3b
3c
ꢃ
/
s, ꢃ
/
p
1
1
ꢀ/
500, PI ꢀ65.8, TPE 2.0
/
ꢃs, ꢃ
/
/
p
Phase VIB: ED50 17.6, TD50
Phase VIB: ED50 10.3, TD50
ꢀ
/
500, PI ꢀ
500, PI ꢀ
400, PI ꢀ
500, PI ꢀ
500, PI ꢀ
/
28.3, TPE 2.0
48.4, TPE 1.0
3e
3f
3h
3t
ꢃ
/
s, ꢃ
s, ꢃ
s, ꢃ
s, ꢃ
/
p
1
1
1
2
ꢀ
/
/
ꢃ
/
/
p
Phase VIB: ED50 6.5, TD50
Phase VIB: ED50 18.4, TD50
Phase VIB: ED50 39.4, TD50
ꢀ
/
/
62.0, TPE 0.5
ꢃ
/
/
p
ꢀ
/
/
27.4, TPE 4.0
12.7, TPE 0.5
ꢃ/
/
p
ꢀ/
/
a
Refs. [34,35].
b
Phase I activity in mice-class 1ꢁ
/
activity at 100 mg kgꢀ1 or B
/
; class 2ꢁ
/
activity between 100 and 300 mg kgꢀ1; class 3ꢁ
no activity up to and
/
including 300 mg kgꢀ1
.
c
All results vs. MES, Phase II in mice, Phase VIB in rats; ED50, median effective dose in mg kgꢀ1; TD50, median toxic dose in mg kgꢀ1; TPE,
time of peak effect in hours.
d
Result of TTE evaluation (Ref. [33]).

N.D. Eddington et al. / European Journal of Medicinal Chemistry 38 (2003) 49ꢂ
/
64
53
Table 3
concentrations to a site (or sites) on the sodium channel
that is allosterically linked to neurotoxin site 2, resulting
Pharmacokinetic parameters of ethyl 4-[(4-chlorophenyl)amino]-6-
methyl-2-oxocyclohex-3-ene-1-carboxylate (1e) in plasma and brain
values and Cmax observed after single ip dose (10 mg kgꢀ1) in male
in the inhibition of binding of [3H]BTX-B [25ꢂ
29]. It
was previously shown that methyl 4-[(4-chlorophenyl)a-
/
SpragueꢂDawley rats
/
mino]-6-methyl-2-oxocyclohex-3-ene-1-carboxylate (1a,
CH3, R1ꢁ
/4-Cl) and methyl 4-[(4-trifluoromethox-
Parameter
Plasma
Brain
Rꢁ
yphenyl)amino]-6-methyl-2-oxocyclohex-3-ene-1-car-
4-OCF3) were inhibitors of
/
Cmax (mg Lꢀ1
Tmax (h)
)
11 0429
0.25
/
2973
10 3949
0.25
/
1387
boxylate (1c, Rꢁ
/
CH3, R1ꢁ
/
AUC0 ꢂ ꢁ (mg h Lꢀ1
)
17 487
0.57
0.62
15 660
0.63
0.80
the specific binding of [3H]BTX-B; the IC50 for the 4-
chloro analogue 1a at 489 mM; the 4-trifluoromethoxy
analogue 1c, at 170 mM [9]. The results of the sodium
channel study on compound 1e are shown in Table 5.
Cl Fꢀ1 (L hꢀ1 kgꢀ1
)
Vd Fꢀ1 (L kgꢀ1
t1/2 (h)
MRT (h)
)
0.75
1.9
0.84
1.9
Cmax, mean maximum concentration; Tmax, time to reach maximum
concentration; AUC, area under the curve; Cl Fꢀ1, plasma clearance;
t1/2, time required to reach 0.5 maximum concentration; MRT, mean
residence time.
3.4. Hippocampal slice
Several reported 5-methyl ketones [4-chloro (3a), 4-
bromo (3b), 4-iodo (3c), 4-trifluoromethyl (3e), 4-
trifluoromethoxy (3f), and 4-nitro (3h)] were evaluated
Table 4
ESI mass spectra and daughter ion spectra of ethyl 4-[(4-chlorophe-
nyl)amino]-6-methyl-2-oxocyclohex-3-ene-1-carboxylate (1e) and its
metabolites excreted in rat urine
in isolated hippocampal slices [19,30ꢂ33]. This method
/
has the advantage of recording important electrical
activity under a variety of experimental conditions and
tissue penetration by the drug. However, this evaluation
has the disadvantages of cell viability and the lack of
interactions with other neural circuits found in the intact
animal [33]. The hippocampal results for these analo-
gues are shown in Table 6. Seizures were scored
Retention time
(min)
m/z of parent ion
[Mꢃ1]
m/z of daughter ions
[Mꢃ1]
/
/
16.5
19.2
27.7
45.8
280
280
236
308
262, 234
262, 234
193, 178
262, 234
according to the following criteria: stage 1ꢁ
and facial clonus; stage 2ꢁstage 1 plus head nodding;
stage 3ꢁstage 2 plus forelimb clonus; stage 4ꢁstage 3
plus rearing; stage 5ꢁstage 4 plus repeated rearing and
falling [32].
/
mouth
chotoxinin A 20 a-benzoate ([3H]BTX-B) to neurotoxin
site 2 of the voltage-dependent sodium channel [9].
Local anesthetics, class I antiarrhythmics and class 1
anticonvulsants bind at pharmacologically relevant
/
/
/
/
Fig. 3. Brain and plasma concentrations of ethyl 4-[(4-chlorophenyl)amino]-6-methyl-2-oxocyclohex-3-ene-1-carboxylate (1e) after administration
(10 mg kgꢀ1) to rats (mean9
SD, nꢁ3).
/
/

54
N.D. Eddington et al. / European Journal of Medicinal Chemistry 38 (2003) 49ꢂ64
/
Fig. 4. Representative mass spectra of ethyl 4-[(4-chlorophenyl)amino]-6-methyl-2-oxocyclohex-3-ene-1-carboxylate (1e) and its metabolites in rat
urine analysed via LC-MS/MS.
4. Results and discussion
(1w)]; and [(0s, 0p) (1n)]. The activity or inactivity for
compounds 3aꢂ3t should therefore parallel these data.
As noted in Table 1, Clog P [24] data indicates that
analogues 3aꢂ3t are more hydrophilic than the corre-
sponding enaminones 1eꢂ1x. Thus, the activity or
inactivity of the former enaminones 3aꢂ3t may also be
/
4.1. Structure activity relationship
/
/
The ester analogues chosen for this study provided a
range of anticonvulsant activity from the highly active
/
attributed to this physiochemical phenomena as well as
to an efflux mechanism previously reported [12,14,16].
The lipophilic nature of the enaminone ester 1 (Fig. 1),
(class 1) [(1eꢂ
/
1j), (1l), (1oꢂ1q), and (1v)], to moderately
/
active (class 2) [(1k), (1m), (1s) and (1t)], to inactive
[(1n), (1r), (1v) and (1w)]. In addition, these analogues
represent the following positions in the Craig plot
permits passive diffusion across the bloodꢂbrain bar-
/
rier; however, the active efflux mechanism militates
[38,39]: [(ꢃ
/
s, ꢃ
/
p) (1eꢂ
/
1j), (1l), (1oꢂ
/
1t), (1v), and
p) (1m), and
(1x)]; [(ꢃs, ꢀ
/
/
p) (1k), and (1u)], [(ꢀ
/
s, ꢃ
/
against a sustained anticonvulsant activity. Biotransfor-

N.D. Eddington et al. / European Journal of Medicinal Chemistry 38 (2003) 49ꢂ
/
64
55
Fig. 5. Major metabolic pathways of ethyl 4-[(4-chlorophenyl)amino]-6-methyl-2-oxocyclohex-3-ene-1-carboxylate (1e) in rats.
mation within the brain (Fig. 1), however, could
produce the corresponding enaminone 3, the more
hydrophilic moiety that opposes the efflux mechanism
and provides a sustained response. Experimental data
with the isomeric chloro analogues illustrates this point.
As noted in Fig. 6, comparing the time course of
anticonvulsant effect in rats for the 4-chloro analogues:
ester 1e; and the putative metabolite 3a. As was
expected, 75% of the rats were protected by the ester
1e at 15 min and remained the same at 30 min,
increasing to a 100% maximum at 1 h, thereafter the
activity declined rapidly. The more hydrophilic enam-
inone 3a, however, developed its protective effect slowly,
but sustained its activity for 2 h, declining slower than
the more lipophilic ester 1e. In Fig. 7, the results were
more dramatic, the 3-chloro ester 1o being immediately
active at 15 min, but declined rapidly thereafter, while
the corresponding enaminone 3k, maintained significant
anticonvulsant activity up to 2 h.
the ip toxicity evaluation at 100 mg kgꢀ1 over a 24 h
period [8]. In comparing the other 4-substituted halo
ketones, the 4-bromo analogue 3b, appeared to be more
toxic than the 4-iodo compound 3c in Phase I analysis,
however, Phase VIB ED50 data (Table 2) disclosed that
the 4-bromo compound 3b was more potent than the
iodo compound 3c. The 4-fluoro compound 3d, while
moderately active, did not possess much separation
between the protective and toxic effects in the MES
screen to be considered further. The trifluoromethyl
analogue 3e was quite active in both mice and rats and
was more active than the isomeric 3-substituted analo-
gue 3o. However, as noted in Table 2, the rat po ED50
for this compound 3e was less active than the 4-
trifluoromethoxy derivative 3f, one of the most active
compounds in the series. This latter compound 3f
possessed potency paralleled the ED50 of the putative
parent ester 1j [(ED50 6.5 mg kgꢀ1, PI ꢀ
/
62.0 for 3f vs.
75.8 for 1j] in rats. The 3-
ED50 6.6 mg kgꢀ1, PI ꢀ
/
5-Methyl ketone analysis*
/
an ip study in mice of the
methoxy keto analogue 3t displayed moderate potency
(class 2) comparable to the parent ester (1x, class 2) and
provided a reasonable ED50 in rats (Table 2). As noted
previously, the 4-trifluoromethoxy compound 3f was
more active than its 3-substituted isomer 3p. This
consistent pattern was also noted within other unrelated
4-chloro analogue 3a, revealed notable toxicity at 60 mg
kgꢀ1 with 4/16 animals displaying neurologic deficit, at
90 (3/8), 120 (12/16) and 200 mg kgꢀ1 (7/8). This factor
may explain the toxic effects previously reported with
the carbomethoxy analogue (1a, Rꢁ
/
CH3, R1ꢁ
4-Cl) in
/
Table 5
Effect of ethyl 4-[(4-chlorophenyl)amino]-6-methyl-2-oxocyclohex-3-ene-1-carboxylate (1e) on voltage-dependent sodium channels in N1E-115
neuroblastoma cells
Drug concentration (mM)
Holding potential (mV)
Number of cells
% Control (9S.E.M.)
/
a
100
100
ꢀ
/
60
5
5
859
/
5
ꢀ90
/
989
/
4
a
Significantly different from control, P B0.05.
/

56
N.D. Eddington et al. / European Journal of Medicinal Chemistry 38 (2003) 49ꢂ64
/
Table 6
Preliminary hippocampal kindling screen in rats (ip)
a
Compound
Dose (mg kgꢀ1
)
Time (min)
Seizure score
Pre-drug
ADD
Pre-drug
Rat #1: 57ꢂ
Drug
Drug
b
b
3a
100
100
100
30
165
165
Rat #1: 5
Rat #2: 5
Rat #1: 4
Rat #2: 3
/
67
Rat #1: 103
Rat #2: 68
Rat #2: 50ꢂ
/
84
3b
Rat #1: 4ꢂ
/5
Rat #1: 4
Rat #2: 3
Rat #1: 76ꢂ
Rat #2: 52ꢂ
/
87
Rat #1: 66
Rat #2: 66
Rat #2: 4
/
81
3c
15ꢂ/135
Rat #1: 5
Rat #1: 4ꢂ
Rat #2: 4ꢂ
/5
Rat #1: 30ꢂ
Rat #2: 19ꢂ
/
80
Rat #1: 50ꢂ
/
61
99
Rat #2: 4ꢂ
/5
/5
/
68
Rat #2: 47ꢂ
/
3e
15ꢂ/135
Rat #1: 4ꢂ
/5
Rat #1: 4
Rat #1: 42ꢂ
/
62
Rat #1: 54ꢂ
/
74
87
Rat #2: 4ꢂ
/5
Rat #2: 4ꢂ
/5
Rat #2: 60ꢂ
/
73
Rat #2: 62ꢂ
/
3f
30
165
105
Rat #1: 5
Rat #1: 1
Rat #2: 4
Rat #1: 45ꢂ
/
52
Rat #1: 26
Rat #2: 57
Rat #2: 4ꢂ
/
5
Rat #2: 42ꢂ
/
64
3h
100
Rat #1: 5
Rat #1: 3
Rat #2: 4
Rat #1: 68ꢂ
/
76
Rat #1: 71
Rat #2: 58
Rat #2: 4ꢂ
/5
Rat #2: 74ꢂ
/
80
c
e
e
d
Carbamazepine
c
Phenytoin
30
30
5
0.8
3
54ꢂ
/
59
23ꢂ
/
28
54
d
5
59ꢂ
/
65
46ꢂ
/
a
After-discharge duration (s).
Toxic.
Data from Ref. [32].
b
c
d
e
Nꢁ/5 animals.
Not stated.
compounds in the ADD database [36]. By far the most
active, in terms of time of peak effect (TPE), was the 4-
nitro analogue 3h, which provided activity up to 4 h and
displayed an ED50 of 18.4 mg kgꢀ1 with no evidence of
toxicity up to 500 mg kgꢀ1. As was expected, the
isomeric 3-nitro analogue 3r, was inactive in mice, but
Fig. 6. Time vs. MES protection graph. Nꢁ
ip); , 3-[N-(4-chlorophenyl)amino]-5-methylcyclohex-2-enone (3a, 40 mg/kg, ip); , points of coincidence.
/
4 rats. D, Ethyl 4-[(4-chlorophenyl)amino]-6-methyl-2-oxocyclohex-3-en-1-carboxylate (1e, 10 mg/kg,

N.D. Eddington et al. / European Journal of Medicinal Chemistry 38 (2003) 49ꢂ
/
64
57
Fig. 7. Time vs. MES protection graph. Nꢁ
ip); , 3-[N-(3-chlorophenyl)amino]-5-methylcyclohex-2-enone (3k, 50 mg/kg, ip).
/
4 rats. D, Ethyl 4-[(3-chlorophenyl)amino]-6-methyl-2-oxocyclohex-3-en-1-carboxylate (1o, 50 mg/kg,
was somewhat active in rats. Nonetheless, an ED50
could not be obtained for compound 3r because of poor
suspension properties.
The 5-methyl compound 3j was the most toxic of all
decarboxylated ketone derivatives in the study; one
animal died in the scPTZ evaluation at 300 mg kgꢀ1
without seizure and one died at 300 mg kgꢀ1 in the
toxicity evaluation.
Anomalous results were obtained in two instances: (1)
the 4-cyano analogues: ester 1k, and ketone 3g; and (2)
the unsubstituted analogues: ester 1n, and ketone 3j. In
the 4-cyano series, the enaminone ester 1k was active,
while the decarboxylated entity 3g was inactive. It
should also be noted that the ester 1k displayed dual
activity, being active in the scPTZ test at 30 (1/5), 100 (3/
5) and at 300 mg kgꢀ1 (5/5) and in the MES evaluation
at 100 (3/3) and 300 mg kgꢀ1 (1/1). It is suspected that
due to the strong electron withdrawing property of the
nitrile group, the decarboxylation reaction was inhib-
ited. This fact is substantiated by the work of Dougherty
4.2. Pharmacokinetics
The results of the pharmacokinetic evaluation of ethyl
4-[(4-chlorophenyl)amino]-6-methyl-2-oxocyclohex-3-
ene-1-carboxylate (1e) in rats revealed after ip adminis-
tration a rapid absorption into the systemic circulation
with Cmax appearing at 15 min post dose. The mean
plasma and brain concentration versus time profiles for
compound 1e after ip dose is presented in Fig. 3 and the
pharmacokinetic parameters are summarised in Table 3.
After an ip administration, compound 1e was rapidly
absorbed into the systemic circulation with Cmax
appearing at 15 min post dose. Compound 1e was
eliminated from plasma with a half-life of ca. 0.75 h and
a plasma clearance (Cl Fꢀ1) was around 0.57 L hꢀ1
kgꢀ1. Mean maximum plasma and brain concentrations
et al. [37ꢂ39] who has shown that in the simple
/
molecule, benzonitrile, the electron withdrawing char-
acter of the cyano group was deactivating, more so than
fluorine. In the second case, i.e. the unsubstituted pair
ester 1n and ketone 3j, the reverse trend was noted, with
the parent ester 1n being inactive, while the decarboxy-
lated analogue 3j being highly active. It should also be
of 11 042ꢂ
/
2973 mg Lꢀ1 and 10 394ꢂ
/
1387 mg kgꢀ1
,
noted that the carbomethoxy analogue (1d, Rꢁ
/
CH3,
R1ꢁ
in Phase I analysis in mice) [2], but was subsequently
/H) was initially determined to be inactive (class 3
respectively were observed at 15 min post dose indicat-
ing a rapid uptake of the drug in the target organ. The
Cl Fꢀ1 and t1/2 were found to be 0.63 L hꢀ1 kgꢀ1 and
0.84 h, respectively in the brain. A scatter plot (not
shown) [17] of brain concentration versus plasma
shown to have a po rat ED50 of 7.8 mg kgꢀ1 and a TD50
ꢀ
/
500 mg kgꢀ1 (PI ꢀ
the oral route apparently activated the compound 1d.
/
64.2) [12]. It was concluded that

58
N.D. Eddington et al. / European Journal of Medicinal Chemistry 38 (2003) 49ꢂ64
/
concentration observed in individual rats at various time
points revealed a good correlation (r2ꢁ
0.998) between
the two variables. The overall brain to plasma ratio
conclusion regarding efficacy against focal seizures
could be drawn. As indicated by Lothman et al. [32],
the time period for selection of the hippocampal test is
also critical. Clearly, more extensive testing with this
procedure is warranted. We had initially proposed [2]
that the enaminones possessed an anticonvulsant profile
similar to phenytoin. The data presented in Table 6
further confirms this hypothesis.
/
compound 1e obtained from brain to plasma concentra-
tion ratios of individual rats was found to be 1.0ꢂ0.3,
/
which was similar to the ratio (0.9) obtained by
comparison of AUC0ꢂꢁ in brain versus plasma. The
metabolite ratio calculated as AUCcompound 1e/AUCCom-
in plasma was found to be 0.008.
pound 3a
The results of mass spectral analysis are shown in Fig.
4 and Table 4. Fig. 2 shows the daughter ion spectra of
metabolite(s) M1 (m/z 280), M2 (m/z 280), the dia-
steromeric acids, 2, noted in the HPLC analysis (not
shown), M3 (m/z 236), the 5-methyl ketone 3a, the
putative metabolite, and target compound 1e (m/z 308).
In ESI-MS and MS/MS spectra of target compound 1e
and its metabolites, the presence of Cl resulted in m/z
5. Conclusions
The pharmacokinetic evaluation of ethyl 4-[(4-chloro-
phenyl)amino]-5-methyl-2-oxocyclohex-3-ene-1-carbox-
ylate (1e) conclusively indicates its conversion into the 5-
methyl analogue 3a. Analysis of the anticonvulsant
activity of the various enaminone esters (1eꢂ
1) and their comparable 5-methyl analogue metabolites
(3aꢂ3t, Table 1) is pervasive and differed in only two
/
1x, Table
values with typical isotopic pattern for the [MꢃH] ion.
/
Inversely, the presence of this pattern was used to
identify the possible metabolites during the LC-MS/
MS study. The typical fragmentation of target com-
pound 1e in positive ion mode resulted in a major
/
cases, one of which can be theoretically explained, while
the second may have resulted from an error in animal
testing. It is clear that although the 4-chloro enaminone
ester 1e, was active in the sodium channel assay, in a
fragment with m/z 234 (loss of 28 amu; Ã
/
CO) forming
carboxylic acid 2. The fragmentation of metabolites M1
and M2 [17] also resulted in an ion with m/z of 234 (loss
similar manner as to ester homologues 1a (Rꢁ
/
CH3,
R1ꢁ CH3, R1ꢁ
/
4-Cl) and 1c (Rꢁ 4-OCF3), this action
/
/
of 28; Ã
/
CO), which was similar to the pattern observed
alone cannot fully explain its principal mechanism of
action.
with the parent compound 1e. This pattern is shown in
Fig. 5. Of theoretical concern was the absence of the
isomeric 37Cl (mꢃ
2) fragment for compound 1e. As
/
noted, however, carboxy metabolite 2 and keto meta-
bolite 3a did produce the isomeric fragments. In a
separate in vitro study, the LC-MS analysis of com-
pounds 1a, 1b and 3a was performed [40]. In all cases we
did find the two tops for the halogens. We can only
attribute the absence to the differences in the scanning
modes.
6. Experimental
6.1. Chemistry
Melting points were determined on a ThomasꢂHo-
/
over capillary melting point apparatus and were un-
corrected. Observed b.p.s were also uncorrected. IR
spectra were recorded in Nujol, as diluted CHCl3
4.3. Sodium channel binding
solutions in matched NaCl cells, or neat on a Perkinꢂ
/
1
Elmer 1660 series FTIR spectrophotometer. H-NMR
As noted in Table 5, ethyl 4-[(4-chlorophenyl)amino]-
6-methyl-2-oxocyclohex-3-ene-1-carboxylate (1e) was
evaluated for sodium channel binding activity. While
target compound 1e provided a statistically significant
spectra were recorded on a General Electric QE 300-
MHz spectrometer in deuterated solvents using TMS as
an internal reference. Coupling patterns are described as
follows: s, singlet; bs, broad singlet; d, doublet; dd,
doubled doublet; t, triplet; q, quartet; m, multiplet; and
1H, 2H, 3H, etc. as the number of hydrogens integrated
within a given coupling pattern. The chemical shifts
were measured to two decimal points, while the coupling
constants were rounded off to one decimal place. TLC
block (P B
/
0.05) at ꢀ60 mV, the blockade was not
/
strong enough to conclude that this was its principal
mechanism of action, however.
4.4. Hippocampal slice
analysis employed EtOAcꢂ
/
cyclohexane (3:1) elution
20 cm2 fluorescent
The hippocampal results for the test analogues 3aꢂ
/3c,
solvent mixture and 5ꢄ10 and 5ꢄ
/
/
3e, 3f and 3h are shown in Table 6 with carbamazepine
and phenytoin included for comparative purpose [32]. In
all cases, including that provided for compound 3c,
these data suggest only a weak ability to block the
expression of fully kindled seizures for these analogues.
Further testing would be required before any definitive
plates (Whatman silica gel 60A). Elemental analyses (C,
H, N, and halogen, where appropriate) were performed
by Schwarzkopf Microanalytical Laboratory, Wood-
side, NY 11377. The analytical results for the elements
were within 9
/
0.4% of the theoretical values. Enzymatic
hydrolysis studies were conducted on a Milton Roy 1201

N.D. Eddington et al. / European Journal of Medicinal Chemistry 38 (2003) 49ꢂ
/
64
59
UV spectrophotometer. HPLC analysis was performed
on a Waters-Millipore (Milford, MA) model 515 HPLC
system supplied with a Waters-Millipore model 717
autosampler. Solutes were detected with a Waters-
yellow plates, m.p. 225ꢂ
(DMSO-d6): d 1.05 (3H, d, Jꢁ
2.95 (5H, m, cyclohexene ring), 5.53 (1H, s Ä
/
227 8C (64%). 1H-NMR
6.15 Hz, CH3), 2.10ꢂ
CH), 6.27ꢂ
/
/
/
/
7.40 (4H, m, C6H4), 8.87 (1H, s, NH). Anal. Calc. for
Millipore model 486 UV detector (lꢁ307 nm). Chro-
/
(C13H14INO) C, H, I, N.
matograms were recorded on a Hewlett-Packard (Rock-
ville, MD) model 3390A integrator. All samples were
6.2.3. 3-[N-(4-Fluorophenyl)amino]-5-methyl-2-
cyclohexenone (3d)
run on a reverse phase ODS ‘Luna’ C18 column (250ꢄ
/
4.5 mm, 5 mm, 440 m2 gꢀ1, 19% carbon load,
Phenomenex, Torrance, CA) equipped with a guard
column (Phenomenex). The LC-MS comprised of a
Micromass Quattro triple quadrupole mass spectro-
meter coupled to a Waters 2695 separation module
equipped with a Waters dual wavelength tunable
absorbance detector in series. The separations were
achieved at ambient temperature on a reverse phase
In a similar procedure, ketone 5 and 4-fluoroaniline
produced the 4-fluoro enaminone 3d. After three
recrystallisations from EtOAc, the product occurred as
yellow crystals, m.p. 185ꢂ
(DMSO-d6): d 1.06 (3H, d, Jꢁ
2.92 (5H, m, cyclohexene ring), 5.54 (1H, s Ä
/
187 8C (44%). 1H-NMR
6.55 Hz, CH3), 1.90ꢂ
CH), 6.47ꢂ
/
/
/
/
6.74 (4H, m, C6H4), 8.24 (1H, s, NH). Anal. Calc. for
(C13H14FNO) C, H, F, N.
C
18 column (Waters XTERRA, 150ꢄ
4.6 mm2, i.d. 5 m)
/
preceded by a SupelguardTM guard cartridge column 20
6.2.4. 3-[N-(4-Trifluoromethylphenyl)amino]-5-methyl-
2-cyclohexenone (3e)
In a similar procedure, ketone 5 and 4-trifluoro-
methylaniline produced the 4-trifluoromethyl enami-
none 3e. After three recrystallisations from 2-PrOH,
mm, 5 m. 4-Carbo-tert-butoxy-5-methylcyclohexane-1,3-
dione (4, Fig. 2, Rꢁ
/
C(CH3)3) [18], 4-carbethoxy-5-
methylcyclohexane-1,3-dione (4, Rꢁ/C2H5) [2] and 5-
methyl-1,3-cyclohexandione (5) [18] were prepared by
literature methods. Aniline was distilled under reduced
pressure from NaOH pellets before use. Enaminone
the product occurred as a gray amorphous powder, m.p.
1
205ꢂ
d, Jꢁ
ring), 5.50 (1H, s Ä
/
206 8C (31%). H-NMR (DMSO-d6): d 1.06 (3H,
esters 1e, 1f, 1oꢂ
/
1q [8], 1g [2], and 1hꢂ
/
1n, 1rꢂ/x [9], were
/
6.15 Hz, CH3), 1.95ꢂ
/
2.90 (5H, m, cyclohexene
prepared by literature methods. 3-N-[(4-Chloro)pheny-
lamino]-5-methyl-2-oxocyclohexenone (3a), was pre-
pared by the literature method [8].
/
CH), 6.37ꢂ
/7.22 (4H, m, C6H4), 8.91
(1H, s, NH). Anal. Calc. for (C14H14F3NO) C, H, F, N.
6.2.5. 3-[N-(4-Trifluoromethoxyphenyl)amino]-5-
methyl-2-cyclohexenone (3f)
In a similar procedure, ketone 5 and 4-trifluoro-
methoxyphenylaniline produced the 4-trifluoromethox-
yphenyl enaminone 3f. After three recrystallisations
from EtOAc, the product occurred as a gray amorphous
6.2. General procedure for the preparation of 5-methyl-2-
cyclohexene enaminones
6.2.1. 3-[N-(4-Bromophenyl)amino]-5-methyl-2-
cyclohexenone (3b)
A mixture of 5-methyl-1,3-cycohexanedione (5) [18]
(3.41 g, 27 mmol) and 4-bromoaniline (5.70 g, 33 mmol),
were added to a mixture of C6H6 (50 mL) and EtOAc
(15 mL) in a 100 mL single neck flask connected to a
Dean-stark trap and the mixture stirred and refluxed
until the evolution of water ceased (ca. 3 h). The mixture
was concentrated to ca. 1/2 its volume and allowed to
stand overnight at room temperature (r.t.). Crystal-
lisation occurred spontaneously and after separation
and drying, three recrystallisations from 2-PrOH pro-
powder, m.p. 174ꢂ
d 1.00 (3H, d, Jꢁ
cyclohexene ring), 5.54 (1H, s Ä
/
175 8C (39%). 1H-NMR (DMSO-d6):
6.45 Hz, CH3), 1.95ꢂ2.92 (5H, m,
CH), 6.35ꢂ6.55 (4H, m,
/
/
/
/
C6H4), 8.71 (1H, s, NH). Anal. Calc. for (C14H14F3NO2)
C, H, F, N.
6.2.6. 3-[N-(4-Cyanophenyl)amino]-5-methyl-2-
cyclohexenone (3g)
In a similar procedure, ketone 5 and 4-aminobenzo-
nitrile produced the 4-cyanophenyl enaminone 3g. After
three recrystallisations from EtOAc, the product oc-
vided an analytical sample of 3b as amber crystals, m.p.
1
213ꢂ
(3H, d, Jꢁ
ring), 5.52 (1H, s Ä
/
215 8C (6.4 g, 84%). H-NMR (DMSO-d6): d 1.06
curred as an orangeꢂ
/
brown powder, m.p. 234ꢂ
(14%). H-NMR (DMSO-d6): d 0.98 (3H, d, Jꢁ
Hz, CH3), 1.92ꢂ2.90 (5H, m, cyclohexene ring), 5.60
(1H, s ÄCH), 6.64ꢂ7.25 (4H, m, C6H4), 8.98 (1H, s,
/
236 8C
1
/
6.7 Hz, CH3), 1.89ꢂ
CH), 6.68 (1H, s, NH), 6.35ꢂ
(4H, m, C6H4). Anal. Calc. for (C13H14BrNO) C, H, Br,
/
2.55 (5H, m, cyclohexene
7.04
/6.15
/
/
/
/
/
N.
NH). Anal. Calc. for (C14H14N2O) C, H, N.
6.2.2. 3-[N-(4-Iodophenyl)amino]-5-methyl-2-
cyclohexenone (3c)
6.2.7. 3-[N-(4-Nitrophenyl)amino]-5-methyl-2-
cyclohexenone (3h)
In a similar procedure, ketone 5 and 4-iodoaniline
produced the 4-iodo enaminone 3c. After three recrys-
tallisations from 2-PrOH, the product occurred as bright
In a similar procedure, ketone 5 and 4-nitroaniline
produced the 4-nitrophenyl enaminone 3h. After three
recrystallisations from EtOAc, the product occurred as a

60
N.D. Eddington et al. / European Journal of Medicinal Chemistry 38 (2003) 49ꢂ64
/
1
bright yellow plates, m.p. 208ꢂ
(DMSO-d6): d 1.00 (3H, d, Jꢁ
2.94 (5H, m, cyclohexene ring), 5.50 (1H, s Ä
/
209 8C (59%). H-NMR
6.29 Hz, CH3), 1.92ꢂ
CH), 6.28ꢂ
white powder, m.p. 185ꢂ
(DMSO-d6): d 1.00 (3H, d, Jꢁ
2.90 (5H, m, cyclohexene ring), 5.50 (1H, s Ä
6.47ꢂ7.00 (4H, m, C6H4), 8.79 (1H, s, NH). Anal. Calc.
/
187 8C (44%). 1H-NMR
6.3 Hz, CH3), 1.90ꢂ
CH),
/
/
/
/
/
/
/
6.68 (4H, m, C6H4), 8.22 (1H, s, NH). Anal. Calc. for
/
(C13H14N2O) C, H, N.
for (C13H14BrNO) C, H, I, N.
6.2.8. 3-N-[(4-Tolyl)amino]-5-methyl-2-cyclohexenone
(3i)
6.2.13. 3-[N-(3-Fluorophenyl)amino]-5-methyl-2-
cyclohexenone (3n)
In a similar procedure, ketone 5 and 4-toluidine
produced the 4-tolyl enaminone 3i. After three recrys-
tallisations from EtOAc, the product occurred as a
In a similar procedure, ketone 5 and 3-fluoroaniline
produced the 3-fluorophenyl enaminone 3n. After three
recrystallisations from EtOAc, the product occurred as a
brown crystals, m.p. 194ꢂ
(DMSO-d6): d 1.00 (3H, d, Jꢁ
2.85 (5H, m, cyclohexene ring), 2.35 (3H, s, CH3 on
CH), 6.20ꢂ6.85 (4H, m, C6H4), 8.38
/
195 8C (79%). 1H-NMR
6.35 Hz, CH3), 1.89ꢂ
white powder, m.p. 163ꢂ
(DMSO-d6): d 0.98 (3H, d, Jꢁ
2.90 (5H, m, cyclohexene ring), 5.54 (1H, s Ä
/
165 8C (77%). 1H-NMR
6.55 Hz, CH3), 1.90ꢂ
CH), 6.55ꢂ
/
/
/
/
/
/
ring), 5.50 (1H, s Ä
/
/
7.00 (4H, m, C6H4), 8.47 (1H, s, NH). Anal. Calc. for
(1H, s, NH). Anal. Calc. for (C14H17NO) C, H, N.
(C13H14FNO) C, H, F, N.
6.2.9. 3-N-(Phenyl)amino]-5-methyl-2-cyclohexenone
(3j)
6.2.14. 3-[N-(3-Trifluoromethylphenyl)amino]-5-
methyl-2-cyclohexenone (3o)
In a similar procedure, ketone 5 and aniline (freshly
distilled under reduced pressure) produced the unsub-
stituted phenyl enaminone 35. After three recrystallisa-
In a similar procedure, ketone 5 and 3-trifluoro-
methylaniline produced the 3-trifluorophenyl enami-
none 3o. After three recrystallisations from EtOAc, the
tions from EtOAc, the product occurred as a yellow
product occurred as a gray powder, m.p. 179ꢂ
/
180.5 8C
(69%). H-NMR (DMSO-d6): d 1.08 (3H, d, Jꢁ6.67
Hz, CH3), 1.92ꢂ2.90 (5H, m, cyclohexene ring), 5.54
(1H, s ÄCH), 6.46ꢂ6.94 (4H, m, C6H4), 8.79 (1H, s,
1
1
powder, m.p. 155 8C (soften), 158ꢂ
NMR (DMSO-d6): d 0.98 (3H, d, Jꢁ
1.94ꢂ2.55 (5H, m, cyclohexene ring), 5.35 (1H, s Ä
6.34 (1H, s, NH), 6.44ꢂ7.00 (5H, m, C6H4). Anal. Calc.
/
160 8C (32%). H-
/
/6.56 Hz, CH3),
/
/
/
CH),
/
/
/
NH). Anal. Calc. for (C14H14F3NO) C, H, F, N.
for (C13H15NO) C, H, N.
6.2.15. 3-[N-(3-Trifluoromethoxyphenyl)amino]-5-
methyl-2-cyclohexenone (3p)
In a similar procedure, ketone 5 and 3-trifluoro-
methoxyaniline produced the 3-trifluoromethoxyphenyl
enaminone 3p. After three recrystallisations from
6.2.10. 3-[N-(3-Chlorophenyl)amino]-5-methyl-2-
cyclohexenone (3k)
In a similar procedure, ketone 5 and 3-chloroaniline
produced the 3-chlorophenyl enaminone 3k. After three
recrystallisations from EtOAc, the product occurred as a
EtOAc, the product occurred as off-white crystals,
1
yellow crystalline mass, m.p. 177ꢂ
NMR (DMSO-d6): d 1.08 (3H, d, Jꢁ
1.90ꢂ2.95 (5H, m, cyclohexene ring), 5.35 (1H, s Ä
6.34 (1H, s, NH), 6.44ꢂ7.00 (4H, m, C6H4). Anal. Calc.
/
179 8C (44%). 1H-
6.35 Hz, CH3),
CH),
m.p. 156ꢂ
(3H, d, Jꢁ
ene ring), 5.55 (1H, s Ä
/
160 8C (38%). H-NMR (DMSO-d6): d 1.06
/
/
6.16 Hz, CH3), 1.92ꢂ
/2.92 (5H, m, cyclohex-
/
/
/
CH), 6.00ꢂ
/6.90 (4H, m, C6H4),
/
8.28 (1H, s, NH). Anal. Calc. for (C14H14F3NO2) C, H,
for (C13H14ClNO) C, H, Cl, N.
F, N.
6.2.11. 3-[N-(3-Bromophenyl)amino]-5-methyl-2-
cyclohexenone (3l)
6.2.16. 3-[N-(3-Cyanophenyl)amino]-5-methyl-2-
cyclohexenone (3q)
In a similar procedure, ketone 5 and 3-bromoaniline
produced the 3-bromophenyl enaminone 3l. After three
recrystallisations from EtOAc, the product occurred as a
In a similar procedure, ketone 5 and 3-aminobenzyni-
trile produced the 3-cyanophenyl enaminone 3q. After
three recrystallisations from EtOAc, the product oc-
yellow crystalline mass, m.p. 172ꢂ
NMR (DMSO-d6): d 1.06 (3H, d, Jꢁ
1.89ꢂ2.55 (5H, m, cyclohexene ring), 5.52 (1H, s Ä
6.68 (1H, s, NH), 6.35ꢂ7.04 (4H, m, C6H4). Anal. Calc.
/
174 8C (84%). 1H-
6.70 Hz, CH3),
CH),
curred as light yellow crystals, m.p. 175.5ꢂ
1H-NMR (DMSO-d6): d 1.00 (3H, d, Jꢁ
6.60 Hz, CH3),
1.79ꢂ2.88 (5H, m, cyclohexene ring), 5.50 (1H, s ÄCH),
6.85ꢂ7.55 (4H, m, C6H4), 8.49 (1H, s, NH). Anal. Calc.
/
177 8C (54%).
/
/
/
/
/
/
/
/
for (C13H14BrNO) C, H, Br, N.
for (C14H14N2O) C, H, N.
6.2.12. 3-[N-(3-Iodophenyl)amino]-5-methyl-2-
cyclohexenone (3m)
6.2.17. 3-[N-(3-Nitrophenyl)amino]-5-methyl-2-
cyclohexenone (3r)
In a similar procedure, ketone 5 and 3-iodoaniline
produced the 3-iodophenyl enaminone 3m. After three
recrystallisations from EtOAc, the product occurred as a
In a similar procedure, ketone 5 and 3-nitroaniline
produced the 3-nitrophenyl enaminone 3r. After three
recrystallisations from EtOAc, the product occurred as

N.D. Eddington et al. / European Journal of Medicinal Chemistry 38 (2003) 49ꢂ
/
64
61
1
yellow crystalline plates, m.p. 187ꢂ
NMR (DMSO-d6): d 0.98 (3H, d, Jꢁ
1.90ꢂ2.90 (5H, m, cyclohexene ring), 5.50 (1H, s Ä
6.47ꢂ7.00 (4H, m, C6H4), 8.79 (1H, s, NH). Anal. Calc.
/
189 8C (68%). H-
6.55 Hz, CH3),
CH),
was also noted for the determination of ED50 and TD50
values. All test data are listed in Table 2.
/
/
/
/
6.4. Sodium channel binding
for (C14H13N2O3) C, H, N.
The procedure for the sodium channel study is as
follows. The N1E-115 neuroblastoma cell line was
maintained at 35 8C in Dulbecco’s modified Eagles
Medium supplemented with 5% fetal calf serum, 20
mM HEPES, 80 mg mLꢀ1 gentamicin, and 4 mM
glutamine. Prior to electrophysiological studies, cells
6.2.18. 3-[N-(3-Tolyl)amino]-5-methyl-2-
cyclohexenone (3s)
In a similar procedure, ketone 5 and 3-toluidine
produced the 3-tolyl enaminone 3s. After three recrys-
tallisations from EtOAc, the product occurred as brown
1
were plated and incubated for 3ꢂ5 days in a differentia-
/
crystals, m.p. 147ꢂ
d6): d 0.98 (3H, d, Jꢁ
cyclohexene ring), 2.35 (3H, s, CH3 on ring), 5.54 (1H, s
CH), 6.27ꢂ6.90 (4H, m, C6H4), 8.24 (1H, s, NH).
/
148.5 8C (79%). H-NMR (DMSO-
tion medium similar to the above solution, however,
with reduced (2.5%) fetal calf serum and 2% DMSO.
Recordings were carried out at r.t. in a bath solution
containing 130 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1
mM MgCl2, 5 mM glucose, and 5 mM HEPES. The
solution also contained 0.1 mM CdCl2 and 25 mM
tetraethylammonium chloride to block voltage-gated
Ca2ꢃ and Kꢃ channels, respectively. Whole-cell record-
/
6.68 Hz, CH3), 1.90ꢂ2.90 (5H, m,
/
Ä
/
/
Anal. Calc. for (C14H17NO) C, H, N.
6.2.19. 3-[N-(3-Methoxyphenyl)amino]-5-methyl-2-
cyclohexenone (3t)
In a similar procedure, ketone 5 and 3-anisidine
produced the 3-methoxyphenyl enaminone 45. After
three recrystallisations from EtOAc, the product oc-
ings were obtained using patch electrodes (1ꢂ2 MV)
/
filled with the intracellular solution described above.
The currents were filtered at 5 kHz and analysed on a
computer using PClamp 6 (Axon Instruments, Union
City, CA, USA). Series resistance and capacitance
currents were compensated using the internal clamp
curred as an off-white powder, m.p. 140ꢂ
1H-NMR (DMSO-d6): d 0.98 (3H, d, Jꢁ
6.55 Hz, CH3),
1.90ꢂ2.90 (5H, m, cyclohexene ring), 3.74 (3H, s,
methoxy group on ring), 5.54 (1H, s ÄCH), 5.98ꢂ6.99
/
142 8C (44%).
/
/
/
/
circuitry. The series resistance was 3ꢂ
of the series resistance was compensated. Cells were
voltage clamped [41] at ꢀ70 mV and ethyl 4-[(4-
/
5 mV and 80ꢂ/90%
(4H, m, C6H4), 8.62 (1H, s, NH). Anal. Calc. for
(C14H17NO2) C, H, N.
/
chlorophenyl)amino]-6-methyl-2-oxocyclohex-3-ene-1-
carboxylate (1e) was applied using the perfusion system
described above. To activate voltage-gated Na channels,
6.3. Pharmacology
Initial evaluations for anticonvulsant activity were
performed by the ADD Program, Epilepsy Branch,
Neurological Disorders Program, NINDS performed
initial evaluations for anticonvulsant activity and in-
cluded Phases I, II, VIA and VIB test procedures which
cells were hyperpolarised to ꢀ
depolarised to potentials from ꢀ
mV increments in control and at 1 and 2 min following
incubation with target compound 1e. The process was
repeated, hyperpolarizing at ꢀ60 mV. The results from
/
90 mV for 90 ms and then
/
80 to ꢃ60 mV in 10
/
/
have been described [19ꢂ
either in male Carworth Farms no. 1 (CF1) mice
(weighing 18ꢂ25 g) or male SpragueꢂDawley rats
(weighing 100ꢂ150 g). Phase I, a qualitative antic-
/
21]. These tests were performed
the individual cells were averaged, the S.E.M. was
calculated, and statistical significance was determined
using the Student’s t-test. Significance was taken to be
/
/
/
in the range of P values B0.01. Data for target
/
onvulsant ip evaluation in mice included three tests:
MES, scPTZ and the rotorod test for Tox. Compounds
were suspended in 0.5% aq. methylcellulose and were
administered at three dosage levels (30, 100, and 300 mg
kgꢀ1) with anticonvulsant activity and motor impair-
ment noted 30 min and 4 h after administration. Phase
VIA was a similar qualitative evaluation to the Phase I
evaluation, however the test drug was administered
orally (po) in rats utilizing the three tests noted
previously. Phase II test quantitated the anticonvulsant
activity and motor impairment observed for the most
promising compounds in Phase I while Phase VIB
quantitated the activity obtained in Phase VIA, respec-
tively. An ip test in rats was performed at 30 mg kgꢀ1
for activity and toxicity. The TPE ip in mice and po rats
compound 1e is shown in Table 5.
6.5. Hippocampal slice
The procedure for hippocampal slices is as follows. A
bipolar stimulating electrode was stereotactically im-
planted in the ventral hippocampus (AP ꢀ
DV ꢀ5.0 from dura, incisor bar ꢃ5) of adult male
SpragueꢂDawley rats (250ꢂ300 g) under ketamineꢂ
/
3.6, ML 4.9,
/
/
/
/
/
xylazine anesthesia. Three anchor screws were attached
to the skull and the electrode assembly anchored to the
skull with dental acrylic cement. After the incision was
closed with sutures, the animal received a single dose of
bicillin (60 000 U, im) and returned to the home cage to
recover. Animals were kindled according to the proce-

62
N.D. Eddington et al. / European Journal of Medicinal Chemistry 38 (2003) 49ꢂ64
/
dure of Lothman and Williamson [42]. Briefly, after 1
week, animals were stimulated with suprathreshold
trains of 200 mA for 10 s, 50 Hz, every 30 min for 6 h
on alternate days until the animals were fully kindled.
One week later the effect of a single dose of test
substance (50 mg kgꢀ1, ip) on the behavioral seizure
score (BSS) and after-discharge was assessed in a single
Dosing solution for ip administration in animals was
prepared by dissolving an appropriate amount of ethyl
4-[(4-chlorophenyl)amino]-6-methyl-2-oxocyclohex-3-
ene-1-carboxylate (1e) in a solution containing 19%
DMSO, 49% propylene glycol and 32% PBS.
6.7.2. Pharmacokinetics and brain distribution in rats
Pharmacokinetics and brain distribution of target
group of kindled rats (nꢁ
/
6ꢂ/8) at 15, 45, 75, 105, 135,
165, and 195 min after drug administration. Results
obtained at the various time points were compared with
the last control stimulus delivered 15 min prior to drug
administration. Thus, each animal served as its own
control. Seizures were scored according to the following
compound 1e was evaluated in male SpragueꢂDawley
/
rats. Overnight fasted rats were administered with a
single ip dose of target compound 1e (10 mg kgꢀ1).
Cohorts of three animals were sacrificed by CO2
asphyxiation at 15, 30, 60, 90, 120, 240, 480 and 1440
min post dose. Blood was collected using heparinised
syringes via cardiac puncture into heparinised polypro-
pylene centrifuge tubes (15 mL), which were later
centrifuged for 10 min to separate plasma. Brain tissue
samples were collected, blotted dry on a tissue paper and
weighed. The plasma and brain tissue samples were flash
criteria: Stage 1*
stage 1 plus head nodding; Stage 3*
forelimb clonus; Stage 4*stage 3 plus rearing; Stage
5*stage 4 plus repeated rearing and falling [32]. When
a drug treatment was observed to significantly lower
seizure score and decrease after-discharge, a doseꢂ
/
mouth and facial clonus; Stage 2*
/
/stage 2 plus
/
/
/
response study was initiated. The following 5-methyl
keto enaminones were evaluated: 4-Chloro (3a), 4-
bromo (3b), 4-iodo (3c), 4-trifluoromethyl (3e), 4-
trifluoromethoxy (3f) and 4-nitro (3h), and the data is
shown in Table 6.
frozen, stored at ꢀ70 8C and analysed using a validated
HPLC assay method described in Ref. [17].
/
6.7.3. Urinary excretion study in rats
In a separate study, a single ip dose of 10 mg kgꢀ1 of
target compound 1e was administered to five male
6.6. Metabolism
SpragueꢂDawley rats and the animals were housed in
/
individual metabolism cages designed to separately
collect feces and urine. Urine was collected for a period
A solution of target compound, ethyl 4-[(4-chloro-
phenyl)amino]-6-methyl-2-oxocyclohex-3-ene-1-carbox-
ylate (1e), was dissolved in 95% EtOH and diluted to 50
mL in the same solvent. Tris buffer (0.01 M, pH 8.0),
and esterase (EC 3.1.1.1, 200 units mgꢀ1, 19 mg,
dissolved in Tris buffer and diluted to 10 mL), was
employed. All experiments were performed in triplicate.
A volume of the stock EtOH solution of compound 1e
(0.2 mL), 1 mL buffer, and 0.1 mL of enzyme solution
were placed in a Teflon-capped reaction vial and
incubated at 37 8C for the following time periods:
0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 16.0 and 24.0 h. The
of 8 h post dose and stored at ꢀ70 8C until analysis.
/
6.7.4. Analytical procedures
Rat plasma and brain samples were assayed using a
validated HPLCꢂUV method for simultaneous analysis
/
of target compound 1e and the putative metabolite 3a
[17]. Carbamazepine was used as the internal standard.
In brief, 0.5 mL of plasma sample was extracted with
two mL of methyl tert-butyl ether. The organic phase
was separated by freezing the aqueous layer and
evaporated under a gentle stream of nitrogen. The dried
residue was reconstituted in 0.25 mL of mobile phase,
vortexed and centrifuged The 50 mL volume of super-
natant was injected into the HPLC. Brain tissue 0.1 g
(blank, spiked or test) was homogenised in 0.5 mL of
mobile phase using a tissue tearor (PowerGen 125
Homogeniser) for 30 s. The homogenate was centrifuged
at 14 000 rpm for 1 min using a tabletop microcentrifuge
(Eppendorf, Model 5415). The 50 mL volume of super-
natant was injected into the HPLC. The HPLC system
was a Model 515 Pump (Waters-Millipore, Milford,
MA, USA) coupled with a 717 Waters autosampler.
Analytes were separated at ambient temperature on a
blank (timeꢁ0 h) contained 0.2 mL of stock solution, 1
/
mL of buffer but no enzyme. After each time period, the
vials were removed from the incubator and quenched at
ꢀ80 8C until ready for analysis. As previously reported
/
[8], differential ultraviolet analysis of target compound
1e and the 5-methyl ketone metabolite 3a provided two
distinct lmax values that were used to calculate the %
conversion.
6.7. Pharmacokinetics
6.7.1. Animals
Male Spragueꢂ
/
Dawley rats (300ꢂ
/
350 g body weight)
reverse phase C18 column (Waters XTERRA, 150ꢄ4.6
/
was purchased from Harlan (Indianapolis, IN, USA)
and was provided free access to rodent chow and water
ad libitum. Care and use of the animals followed the
Guide for the Care and Use of Laboratory Animals [43].
mm2, i.d. 5 m) preceded by a SupelguardTM guard
cartridge column 20 mm, 5 m. Analytes were detected
using a Waters Model 486 Tunable absorbance detector
in the ultraviolet mode (lmax
ꢁ307 nm). Chromato-
/

N.D. Eddington et al. / European Journal of Medicinal Chemistry 38 (2003) 49ꢂ
/
64
63
graphic data acquisition and analysis was performed on
a Millenium Chromatography Manager (Waters Ver-
voltage of 2 V, RF lens voltage 0.5 V, cone gas flow 71 L
ꢀ1 and desolvation gas flow 513 L hꢀ1. Centroid mass
h
sion 3.2). The mobile phase consisted of MeCNꢂ
/
0.05 M
spectra were acquired between m/z 100 and 800 for 1 s.
In a pilot analysis, a full MS scan was obtained for all
the components eluting at different retention times to
determine their m/z values. Finally, the data was
acquired in MS and MS/MS mode and TIC scan and
daughter ion spectra were obtained for parent masses by
operating the first quadrupole in static mode for parent
ions with m/z 282, 236 and 308 as second function.
Fragmentation of the analyte ions during the full
acquisitions was achieved by using cone voltage and
collision energy ramps. Degradation of selected analyte
ion was performed by collision with Ar. System control
and data acquisition was performed using MassLynx
software Version 3.0.
NaH2PO4 buffer, pH 3.9 (32:68 v/v) and was degassed
under ultrasound for 30 min before use. Mobile phase
was pumped at a flow rate of 1 mL minꢀ1 with a run
time of 30 min per sample.
6.7.5. Urinary excretion study
Urine samples were analysed using the same HPLC
setup as described above except that mobile phase
consisted of 30% v/v MeCN in 0.05 M NaH2PO4 buffer,
pH 3.9. This assay method was validated for intra-day
recovery of target compound 1e and putative metabolite
3a with the primary objective to characterise the extent
of urinary excretion of target compound 1e. As a
secondary objective, the urine samples were subse-
quently analysed to obtain qualitative information
regarding the metabolites on LC-MS/MS discussed in
the later section. For chromatographic analysis utilizing
UV detection, the urine sample (blank, spiked or test; 50
mL) was diluted with 1 mL of mobile phase, vortex
mixed and centrifuged. The supernatant was transferred
into the microvial and 50 mL was injected into the
HPLC.
6.7.7. Data analysis
Plasma and brain concentrations of target compound
1e and the putative metabolite 3a were calculated by
internal standard method using the peak height ratios of
analyte to carbamazepine (IS). The unknown concen-
trations of compounds 1e and 3a were read from the
respective standard curves obtained by weighted linear
regression analysis (Wt. 1/x2) of anlayte/IS peak height
ratio versus concentration data on Microsoft Excel. The
weighing scheme was selected on the basis of residuals
and corresponding % error in the predicted concentra-
tions at each level. Since the study involved destructive
sampling, the trends in pharmacokinetic parameters
6.7.6. Identification of metabolites by LC-MS/MS
A pooled urine sample (2 mL) was applied onto a
Bakerbond octadecyl solid phase extraction cartridge
column (500 mg sorbent; 3 mL) previously conditioned
with 3 mL of MeOH and 3 mL of water. The cartridge
was washed twice with 1 mL of water and the retained
components were eluted with 2 mL of MeOH. The
MeOH eluent was evaporated to dryness under a gentle
stream of nitrogen at 34 8C and the dried residue was
reconstituted in 1 mL of mobile phase and analysed
using a LC-MS as described. The LC-MS comprised of
a Micromass Quattro triple quadrupole mass spectro-
meter coupled to a Waters 2695 separations module
equipped with a Waters dual wavelength tunable
absorbance detector in series. The separations were
achieved at ambient temperature on a reverse phase
were assessed from mean naıve-pooled plasma and
¨
brain concentration versus time data. Pharmacokinetic
parameters were determined using non-compartmental
analysis using WinNonlin professional software package
(Pharsight Corporation, NC, USA). The values of Cmax
and Tmax were calculated from the raw data as the
maximum observed concentration in the naıve pooled
¨
data and the time at which it was observed, respectively.
Brain uptake of target compound 1e and its metabolite
3a was determined both from the ratio of AUC0ꢂꢁ in
brain to that of plasma and also from average of brain
to plasma concentration ratio of each animal at different
time points. Data for the analysis is found in Tables 3
C
18 column (Waters XTERRA, 150ꢄ
4.6 mm2, i.d. 5 m)
/
preceded by a SupelguardTM guard cartridge column 20
mm, 5 m. The mobile phase consisted of 40% v/v MeCN
in CH3COONH4 buffer 10 mM, pH 3.9 and was
delivered at a flow rate of 0.2 mL minꢀ1 over a run
time of 60 min. The analytes were monitored simulta-
neously on an UV detector and the mass spectrometer.
The outlet flow from UV detector was directly intro-
duced into the ESI probe of mass spectrometer. Positive
ion electrospray mass spectra were obtained for target
compound 1e and its metabolites in urine. The mass
spectrometer was operated using source temperature of
80 8C, desolvation temperature of 150 8C, capillary
voltage of 3.2 kV, cone voltage of 40 V, extractor
and 4 and Figs. 3ꢂ5.
/
Acknowledgements
This research was supported by a grant from the
National Institutes of Health (1 R21 GM63494-01), for
which K.R.S. is the principal investigator. We also
thank the Graduate School for support of the high
resolution NMR.

64
N.D. Eddington et al. / European Journal of Medicinal Chemistry 38 (2003) 49ꢂ64
/
[20] R.L. Krall, J.K. Penry, B.G. White, H.J. Kupferberg, E.A.
Swinyard, Epilepsia 19 (1978) 409ꢂ428.
[21] R.J. Porter, J.J. Cereghino, G.D. Gladding, B.J. Hessie, H.J.
References
/
[1] J.J. Coatsworth, NINDS Monograph No. 12 HEW Publication
No. (NIH) 73-51, US Government Printing Office, 1971.
[2] I.O. Edafiogho, C.N. Hinko, H. Chang, J.A. Moore, D. Mulzac,
Kupferberg, B. Scoville, G.G. White, Cleveland Clin. Q. 51 (1984)
293ꢂ
[22] S.G. Piredda, J.H. Woodhead, E.A. Swinyard, J. Pharmacol. Exp.
Ther. 232 (1985) 741ꢂ745.
[23] I.O. Edafiogho, M.E. Abdel-Hamid, H. Hamza, K.R. Scott, J.
Liq. Chromatogr. Relat. Technol. 24 (2001) 565ꢂ577.
[24] MAC OGP Program, Version 4.0 BioByte Corp., Claremont, CA
91711, USA.
[25] M. Willow, E.A. Kuenzel, W.A. Catterall, Mol. Pharmacol. 25
(1984) 228ꢂ234.
[26] Y. Zhu, W.B. Im, R.A. Lewis, P.F. VonVoigtlander, Br. J.
Pharmacol. 106 (1992) 49ꢂ54.
[27] N. Natsuki, F.N. Quandt, R.E. Eick, J.Z. Yeh, J. Pharmacol.
Exp. Ther. 228 (1948) 523ꢂ530.
/305.
J.M. Nicholson, K.R. Scott, J. Med. Chem. 35 (1992) 2798ꢂ
[3] D. Mulzac, K.R. Scott, Epilepsia 34 (1993) 1141ꢂ1146.
[4] J.M. Nicholson, I.O. Edafiogho, J.A. Moore, V.A. Farrar, K.R.
Scott, J. Pharm. Sci. 83 (1994) 76ꢂ78.
[5] I.O. Edafiogho, J.A. Moore, V.A. Farrar, J.M. Nicholson, K.R.
Scott, J. Pharm. Sci. 83 (1994) 79ꢂ84.
[6] I.O. Edafiogho, J.A. Moore, M.S. Alexander, K.R. Scott, J.
Pharm. Sci. 83 (1994) 1155ꢂ1170.
[7] I.O. Edafiogho, M.S. Alexander, J.A. Moore, V.A. Farrar, K.R.
Scott, Curr. Med. Chem. 1 (1994) 159ꢂ175.
/2805.
/
/
/
/
L
/
/
/
/
/
[8] K.R. Scott, I.O. Edafiogho, E.L. Richardson, V.A. Farrar, J.A.
Moore, E.I. Tietz, C.N. Hinko, H. Chang, A. El-Assadi, J.M.
/
Nicholson, J. Med. Chem. 36 (1993) 1947ꢂ
/1955.
[28] J. Benes, A. Parada, A.A. Figueiredo, P.C. Alves, A.P. Freitas,
[9] K.R. Scott, G.O. Rankin, J.P. Stables, M.S. Alexander, I.O.
Edafiogho, V.A. Farrar, K.R. Kolen, J.A. Moore, L.D. Sims,
D.A. Learmonth, R.A. Cunha, J. Garrett, P. Soares-da-Silva, J.
Med. Chem. 42 (1999) 2582ꢂ
[29] J. Vamecq, P. Bac, C. Herrenknecht, P. Maurois, P. Delcourt, J.P.
Stables, J. Med. Chem. 43 (2000) 1311ꢂ1319.
[30] A.P. Oliver, B.J. Hoffer, R.J. Wyatt, Epilepsia 18 (1977) 543ꢂ
[31] S. Piredda, W.D. Yonekawa, T.S. Whittingham, H.J. Kupferberg,
Epilepsia 26 (1985) 167ꢂ174.
[32] E.W. Lothman, R.A. Salerno, J.B. Perlin, D.L. Kaiser, Epilepsy
Res. 2 (1988) 367ꢂ379.
/
2587.
A.D. Tonnu, J. Med. Chem. 38 (1995) 4033ꢂ4043.
/
[10] M.L. Laws, R.R. Roberts, J.M. Nicholson, R. Butcher, J.P.
Stables, A.M. Goodwin, C. Smith, K.R. Scott, Bioorg. Med.
/
/
548.
Chem. 6 (1998) 2289ꢂ2299.
/
[11] J.E. Foster, J.M. Nicholson, R. Butcher, J.P. Stables, I.O.
Edafiogho, A.M. Goodwin, M.C. Henson, C.A. Smith, K.R.
/
Scott, Bioorg. Med. Chem. 7 (1999) 2415ꢂ
[12] N.D. Eddington, D.S. Cox, R.R. Roberts, J.P. Stables, C.B.
Powell, K.R. Scott, Curr. Med. Chem. 7 (2000) 417ꢂ436.
[13] D.S. Cox, J. Du, K.R. Scott, H. Gao, N.E. Eddington, J.
Chromatogr. Sect. B 749 (2000) 191ꢂ196.
[14] D.S. Cox, K.R. Scott, H. Gao, S. Raje, N.D. Eddington, J.
Pharm. Sci. 90 (2001) 1540ꢂ1552.
[15] D.S. Cox, H. Gao, S. Raje, K.R. Scott, N.D. Eddington, Eur. J.
Pharm. Biopharm. 52 (2001) 145ꢂ150.
/
2425.
/
[33] H.J. Kupferberg, J.P. Stables, Mechanisms of action revisited:
/
drug discovery, testing and clinical prediction, in: H. Stefan, G.
Kramer, B. Mamoli (Eds.), Challenge in Epilepsy*
onvulsant Drugs, Blackwell Science Ltd, 1998, pp. 7ꢂ
/
New Antic-
27.
/
/
[34] P.N. Craig, J. Med. Chem. 14 (1971) 680ꢂ
[35] P.N. Craig, J. Med. Chem. 14 (1971) 144ꢂ
[36] J.P. Stables, personal communication.
[37] S. Mecozzi, A.P. West, Jr., D.A. Dougherty, Proc. Natl. Acad.
Sci. USA 93 (1996) 10566ꢂ10571.
[38] D.A. Dougherty, Science 271 (1996) 163ꢂ
[39] D.A. Dougherty, D.A. Stauffer, Science 250 (1990) 1558ꢂ
[40] M.E. Abdel-Hamid, I.O. Edafiogho, K.R. Scott, J. Pharm.
Biochem. Anal. 30 (2002) 1001ꢂ1011.
[41] O.P. Hamill, A. Marty, E. Neher, B. Sakmann, F.J. Sigworth,
Pfluger’s Arch. 391 (1981) 85ꢂ100.
[42] E.W. Lothman, J.M. Williamson, Brain Res. 649 (1994) 71ꢂ
/684.
/
/149.
/
[16] N.D. Eddington, D.S. Cox, R.R. Roberts, R.J. Butcher, I.O.
Edafiogho, J.P. Stables, N. Cooke, A.M. Goodwin, C.A. Smith,
/
/168.
K.R. Scott, Eur. J. Med. Chem. 37 (2002) 635ꢂ
[17] D.S. Cox, K.R. Scott, H. Gao, N.D. Eddington, J. Pharmacol.
Exp. Ther. 302 (2002) 1096ꢂ1104.
[18] R.J. Friary, J.M. Gilligan, R.P. Szajewski, K.J. Falci, R.W.
Franck, J. Org. Chem. 38 (1973) 3487ꢂ3491.
/
648.
/1560.
/
/
/
/
[19] J.P. Stables, H.J. Kupferberg, The NIH anticonvulsant drug
development (ADD) program: preclinical anticonvulsant screen-
ing project, in: G. Avanzini, P. Tanganelli, M. Avoli (Eds.),
Molecular and Cellular Targets for Anti-epileptic Drugs, John
/
84.
[43] Guide for the Care and Use of Laboratory Animals, Institute of
Laboratory Animal Resources, National Research Council, 1986,
NIH Publication No. 86-23.
Libbey and Co, 1997, pp. 191ꢂ198.
/