Synthesis and anticonvulsant activity of enaminones. 4. Investigations on isoxazole derivatives

Article abstract of DOI:10.1016/S0223-5234(02)01377-6

Due to the exceptional anticonvulsant activity displayed by substituted aniline enaminones, related pyridine derivatives and phenothiazines synthesised in our laboratories, the further investigation of various aromatic heterocycles was undertaken. Condensation of cyclic 1,3-diketo esters with 3-, and 5-aminoisoxazole derivatives led to a series of potent anti-maximal electroshock (MES) analogues, three of which occurred in the 3-amino series: ethyl ester (10), orally (po) active in rats [ED₅₀ 68.9 mg kg^-1, TD₅₀>500 mg kg^-1, protective index (PI=TD₅₀/ED₅₀)>49.6]; methyl ester (9), ED₅₀ 68.9 mg kg^-1 intraperitoneally (ip) in mice, TD₅₀>500 mg kg^-1, PI>7.3, and tert-butyl ester (8), ED₅₀ 28.1 mg kg^-1 po in rats, TD₅₀>500 mg kg^-1, PI>17.8. Sodium channel binding studies, as well as evaluations against pentylenetetrazol, bicuculline, and picrotoxin on isoxazole 10 were all negative, leading to an unknown mechanism of action. X-ray diffraction patterns of a representative of the 3-amino series (isoxazoles 6-11) unequivocally display the existence of intramolecular hydrogen bonding of the nitrogen to the vinylic proton in the cyclohexene ring, providing a pseudo three ring structure which was also shown previously with the vinylic benzamides. Physicochemical-permeability across the BBB suggested an efflux mechanism for the previously synthesised aniline enaminones, but not with isoxazole 10.

Full text of DOI:10.1016/S0223-5234(02)01377-6

Eur. J. Med. Chem. 37 (2002) 635–648
www.elsevier.com/locate/ejmech
Original article
Synthesis and anticonvulsant activity of enaminones.
4. Investigations on isoxazole derivatives
Natalie D. Eddington a, Donna S. Cox b, Ralph R. Roberts c, Raymond J. Butcher d,
Ivan O. Edafiogho e, James P. Stables f, Neville Cooke g, Angela M. Goodwin g,
Carlynn A. Smith g, K.R. Scott g,*
a Department of Pharmaceutical Sciences, School of Pharmacy, Uni6ersity of Maryland, Baltimore, MD 21201-4403, USA
b DuPont Pharmaceuticals, Drug Metabolism and Pharmacokinetics Section, S112/36, 1090 Elkton Road, P.O. Box 30, Newark,
DE 19713, USA
c Science Research Laboratory, 3M Corporate Research, 3M Center, Building 201-2N-21, St. Paul, MN 55144-1000, USA
d Department of Chemistry, Graduate School, Howard Uni6ersity, Washington, DC 20059, USA
e Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Uni6ersity of Kuwait, Jabriya, Kuwait
f Epilepsy Branch, Di6ision of Con6ulsi6e, De6elopmental and Neuromuscular Disorders, National Institute of Neurological Disorders and Stroke,
Bethesda, MD 20892, USA
g Department of Pharmaceutical Sciences, School of Pharmacy, Howard Uni6ersity, Washington, DC 20059, USA
Received 29 October 2001; received in revised form 21 March 2002; accepted 27 March 2002
Abstract
Due to the exceptional anticonvulsant activity displayed by substituted aniline enaminones, related pyridine derivatives and
phenothiazines synthesised in our laboratories, the further investigation of various aromatic heterocycles was undertaken.
Condensation of cyclic 1,3-diketo esters with 3-, and 5-aminoisoxazole derivatives led to a series of potent anti-maximal
electroshock (MES) analogues, three of which occurred in the 3-amino series: ethyl ester (10), orally (po) active in rats [ED50 68.9
mg kg−1, TD50\500 mg kg−1, protective index (PI=TD50/ED50)\49.6]; methyl ester (9), ED50 68.9 mg kg−1 intraperitoneally
(ip) in mice, TD50\500 mg kg−1, PI\7.3, and tert-butyl ester (8), ED50 28.1 mg kg−1 po in rats, TD50\500 mg kg−1
,
PI\17.8. Sodium channel binding studies, as well as evaluations against pentylenetetrazol, bicuculline, and picrotoxin on
isoxazole 10 were all negative, leading to an unknown mechanism of action. X-ray diffraction patterns of a representative of the
3-amino series (isoxazoles 6–11) unequivocally display the existence of intramolecular hydrogen bonding of the nitrogen to the
vinylic proton in the cyclohexene ring, providing a pseudo three ring structure which was also shown previously with the vinylic
benzamides. Physicochemical-permeability across the BBB suggested an efflux mechanism for the previously synthesised aniline
´
enaminones, but not with isoxazole 10. © 2002 Published by Editions scientifiques et me´dicales Elsevier SAS.
Keywords: Enaminones; Isoxazoles; Maximal electroshock seizure test; Anticonvulsant activity; X-ray crystallography; Structure–activity
relationship; Physicochemical permeability
Abbre6iations: ADD, Antiepileptic drug development; AP, apical; BBMEC, bovine brain microvessel endothelial cells; BL, basolateral; b.p.,
boiling point; Clog P, calculated log P; CSD, Cambridge Structural Database; ED50, effective dose for 50% of test animals; EtOAc, ethyl acetate;
[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; ip, intraperitoneal; MDR, multidrug resistance; MES, maximal electroshock seizure; m.p., melting point; NINDS, National
Institutes of Neurological Disorders and Stroke; Pgp, P glycoprotein; PI, protective index; scBic, subcutaneous bicuculline; scPic, subcutaneous
picrotoxin; scPTZ, subcutaneous pentylenetetrazol; TD50, toxic dose for 50% of test animals; TD50/ED50; THF, tetrahydrofuran; Tox, neurologic
toxicity; TTE, threshold tonic extension.
* Correspondence and reprints
E-mail address: kscott@fac.howard.edu (K.R. Scott).
´
0223-5234/02/$ - see front matter © 2002 Published by Editions scientifiques et me´dicales Elsevier SAS.
PII: S0223-5234(02)01377-6

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N.D. Eddington et al. / European Journal of Medicinal Chemistry 37 (2002) 635–648
1. Introduction
2. Chemistry
The search for antiepileptic compounds with more
selective activity and lower toxicity continues to be an
area of investigation in medicinal chemistry [1]. Many
patients with epilepsy fail to experience adequate con-
trol of their seizures, despite the optimal use of availa-
ble antiepileptic drugs. Other patients do so only at the
expense of significant toxic side effects [2]. In a continu-
ing study of potential anticonvulsants [3–7], we have
extended the initial evaluation of the enaminones bear-
ing the aniline and benzylamine moieties to aromatic
heterocycles, initially with the pyridine system [4], and
currently the isoxazole nucleus. A preliminary report of
this latter investigation has been published [8]. The
study of anticonvulsant isoxazoles has been reported in
a number of laboratories [1,9–11], and principally in-
volved condensation of an acid synthon with an
aminoisoxazole, effectively producing an amide. Our
work, however, produces a secondary amine which, in
our laboratory, has previously been shown to produce a
distinctly different spectrum of anticonvulsant activity.
The active benzylamines, in our hands, retained anti-
convulsant activity exclusively in the maximal elec-
troshock seizure (MES) evaluation, while the active
benzamides possessed dual activity in both the elec-
troshock and the subcutaneous pentylenetetrazol
(scPTZ) models [7].
2.1. Synthesis
The synthesis of the isoxazole enaminones which
appear in Table 1 is indicated in the following scheme
(Scheme 1). The b-hydroxy keto esters, 1–3, were pre-
pared as previously reported [3–7] and were refluxed
with the requisite 3-amino isoxazoles, 4, and 5, or the
5-amino isoxazoles (commercially available 3-methy-5-
aminoisoxazole) or previously unreported 3-ethyl-5-
aminoisoxazole (13), either as previously reported, or
more recently, in a mixture of benzene, or toluene, and
ethyl acetate (70:30) in a Dean-Stark trap, the reaction
monitored by the water formation. The unreported
3-ethyl-5-aminoisoxazole (13), was synthesised by mod-
ification of the method of Nishiwaki and Saito [12],
condensing ethyl propionate with acetonitrile in the
presence of sodium hydride to form cyanoketone, 12,
which was subsequently reacted with hydroxylamine
hydrochloride and sodium acetate to form 3-ethyl-5-
aminoisoxazole (13), in reasonable yields. Condensation
of 3-ethyl-5-aminoisoxazole (13) with the b-keto esters
(1–3) under previously indicated conditions provided
the requisite 3-ethyl isoxazoles (14–19). The stereo-
chemistry of the products (6–11, and 14–19) indicates
that they exist as trans racemates, which was previously
reported by Friary et al. [13] and by us earlier [14].
Table 1
Anticonvulsant screening project (ASP): anticonvulsant results
a
Compound Clog P
3-Aminoisoxazoles
Anticonvulsant results b
6
0.89
phase I (mice): Class 2 (1 death at 30 mg kg−1 in scPTZ test). Active in the rat at po 30 mg kg−1 (3/4) at 2 h (0/4
toxic at all time periods at 30 mg kg−1
)
7
8
9
1.42
2.13
1.39
phase I (mice): Class 1 (MES) (1 tonic extension at 100 mg kg−1 is scPTZ test)
phase I (mice): Class 2 (1 death at 100 mg kg−1 in scPTZ test)
phase I (mice): Class 1 (MES) Mice: ED50 149.4 mg kg−1; TD50\500 mg kg−1 (at 1 h) Rats: ED50 28.1 mg kg−1
TD50\500 mg kg−1 (at 1 h)
;
10
11
1.91
2.62
phase I (mice): Class 1 (MES) Mice: ED50 68.9 mg kg−1; TD50\500 mg kg−1 (at 0.25 h)
phase I (mice): Class 1 (MES) Mice: ED50 119.9 mg kg−1; TD50\500 mg kg−1 (at 0.25 h) (repeat ED50 149.9 mg
kg−1) Rats: ED50 10.1 mg kg−1; TD50\500 mg kg−1 (at 4 h)
5-Aminoisoxazoles
14
1.39
phase I (mice): Class 1 (MES). Active in the rat at po 30 mg kg−1 (2/4) at 0.25, 0.5 and 1 h (0/4 toxic at all time
periods at 30 mg kg−1
)
15
16
1.91
2.62
phase I (mice): Class 3 (MES)
phase I (mice): Class 1 (MES). Active in the rat at po 30 mg kg−1 (3/4) at 4 h (0/4 toxic at all time periods at 30
mg kg−1
)
17
18
19
1.91
2.44
3.15
phase I (mice): Class 3 (MES)
phase I (mice): Class 3 (MES)
phase I (mice): Class 3 (MES)
a Calculated from reference [28].
b Phase I in mice activity-class 1=activity at 100 mg kg−1 or B; class 2=activity between 100 and 300 mg kg−1; class 3=no activity at 300
mg kg−1
.

N.D. Eddington et al. / European Journal of Medicinal Chemistry 37 (2002) 635–648
637
Scheme 1.
(20a), (l 5.48 ppm) when compared with the pyrro-
lidino enaminone (22), (l 5.08 ppm), the cyclohexy-
lamino enaminone (23) (l 5.17 ppm), and the
moropholino enaminone (24) (l 5.30 ppm), all of which
are devoid of aromatic protons [14]. In addition to the
X-ray studies which follow, Edafiogho performed an
X-ray crystallographic study on methyl 4-[(4-bro-
mophenyl)amino]-6-methyl-2-oxocyclohex-3-ene-1-car-
boxylate (20b) (Fig. 1, R3=4-Br) and determined that
the phenyl ring is planar with the cyclohexenone ring
and, as with the 4-chloro enaminone (20a), is not likely
to be in a conformation where the vinylic proton would
be above the aromatic ring, to cause a high-field shift
due to anisotropy of the aromatic ring [15].
In Table 2, the agreement between the 3-aminoisoxa-
zoles (9–11; R1=CH3) and the comparable 5-
aminoisoxazoles (14–16; R1=CH3) is quite good and
clearly shows the deshielding effect noted with the
pyridine analogue (21). This deshielding effect was most
marked with the unsubstituted 3-aminoisoxazoles (9–
11; R1=H) and was significantly diminished with the
ethyl analogues (17–19; R1=C2H5). This latter effect
2.2. NMR analysis
The NMR results at 300 MHz of the isoxazolyl
enaminones were consistent with the assigned struc-
tures. We had previously reported [4] that in comparing
methyl 4-[(4-chlorophenyl)amino]-6-methyl-2-oxocyclo-
hex-3-ene-1-carboxylate, 20a (Fig. 1, R3=4-Cl) with
the analogous 2-pyridine analogue (21), there was a
difference in the vinyl proton assignment. The vinyl
proton for the 4-chlorophenyl derivative (20a), ap-
peared at l 5.48 ppm, while the vinyl proton in the
pyridine analogue (21) was deshielded and appeared at
l 6.82 ppm, indicating involvement of this proton in
intramolecular hydrogen bonding. Table 2 summarises
the vinyl proton assignments for the reported isoxa-
zoles. In our previous NMR studies on the enaminones
[14], we have shown that hydrogen-bonding with nitro-
gen or oxygen atoms attached to enaminone system
display deshielding of the vinylic proton from l values
B6 to \6 ppm. This is also noted in Table 2. An-
isotropy of the aromatic ring has little influence on
shielding the vinylic proton of the 4-chloro enaminone

638
N.D. Eddington et al. / European Journal of Medicinal Chemistry 37 (2002) 635–648
between the NMR analysis and the X-ray crystal
structure.
2.3. X-ray crystallography
To further define the role of three dimensional struc-
ture to anticonvulsant activity, an X-ray diffraction
study of a representative 3-aminoisoxazole (10) was
performed as well as that of the 2-amino-5-chloropy-
ridine analogue (21). As noted in Fig. 2 of the isoxazole
compound (10) and in Fig. 3 of the 5-chloropyridine
analogue (21), strong hydrogen-bonding occurred be-
tween the vinyl protons and the nitrogen atoms. This
has been seen previously with respect to earlier struc-
tures [6,7]. In either case, the pseudo three ring configu-
ration was unequivocally shown. Tables 3 and 4
provide the experimental data detailing the crystal data,
data collection, and refinement data for isoxazole 10
and the pyridine analogue 21, respectively. Addition-
ally, Table 5A and Table 6A provide selected torsion
angles which illustrate the pseudo three-ring configura-
tion in isoxazole 10 and the pyridine compound 21,
respectively. Molecule A and Molecule B are the enan-
tiomeric forms of each compound, and Table 5B and
Table 6B provide the most important hydrogen bonds
supporting the hypothesis for isoxazole 10 and pyridine
compound 21, respectively. While X-ray crystal data on
our enaminones are modest, the hydrogen bonding
ensures the rings are planar. It is apparent that plana-
rity of the molecules is an essential factor in the deter-
mination of biological activity, because as shown in the
NMR studies for that non-planar pyrrolidino analogue
22, the cyclohexylamino analogue 23 and the mor-
pholino compound 24 have been shown to be inactive
in our hands.
Fig. 1. NMR structural correlation for vinylic coupling in compounds
20a (R3=4-Cl), 21 [4,14], 22–24 [14], and 23 [7]. X-ray analysis of 10
is shown in Fig. 2 and of 21 in Fig. 3.
could be correlated to the uniform inactivity of this
latter series. Recently [7], we showed that the vinyl
deshielding effect also occurred when the benzylamines
(25), were compared with the analogous benzamides,
(26). In the latter series, the hydrogen bonding occurred
between the carbonyl oxygen and the vinylic proton,
whereas, intramolecular hydrogen bonding did not oc-
cur with the benzylamines (25). This pseudo three ring
phenomenon was further verified by X-ray diffraction
analysis of two benzamides [7]. Thus, in this latter
study, we were able to provide a direct correlation
As further justification of the pseudo three ring hy-
pothesis, a search of the Cambridge Structural Data-
base (CSD) [16] for potential intermolecular or
intramolecular hydrogen bonding interactions between
Table 2
Vinyl proton assignments for the isoxazole enaminones
Compound
6
7
8
9
R
CH3
R1
H
H
H
CH3
CH3
CH3
l, ppm
6.39
6.38
6.31
6.02
Compound
R
CH3
R2
l, ppm
6.03
6.14
6.10
5.54
14
15
16
17
18
19
CH3
CH3
CH3
C2H5
C2H5
C2H5
C2H5
C(CH3)3
CH3
C2H5
C(CH3)3
C2H5
C(CH3)3
CH3
C2H5
C(CH3)3
10
11
6.02
6.01
5.54
5.54

N.D. Eddington et al. / European Journal of Medicinal Chemistry 37 (2002) 635–648
639
Fig. 2. X-ray crystal structure of ethyl 4-[(5-methyl-3-isooxazoly)amino]-6-methyl-2-oxo-3-cyclohexane-1-carboxyalte (10). Note dotted lines
indicating the N···H bonding interaction.
Fig. 3. X-ray crystal structure of methyl 4-[(5-chloro-2-amino)pyridyl]-6-methyl-2-oxo-3-cyclohexene-1-carboxylate (21). Note dotted lines
indicating the N···H bonding interaction.
vinyl protons as donors and oxygen atoms (selecting a
111.413 and 154.125° for the intra- and intermolecular
interactions, respectively). In all of the other parame-
ters, the average values agree very well between the two
different situations. While the CSD search was global,
it is acknowledged that the protons of the enamines
represent a small percentage of the total search. Never-
theless, it is feasible to assume that the vinylic protons
of the enamines play a role in hydrogen-bonding. Our
argument would have been conclusive if the X-ray
structure of the inactive isomeric isoxazole (Fig. 1, 15)
could be determined, however, single crystals of the
compound could not be grown in sufficient size to
afford a definitive analysis. X-ray structural data for
both molecules, 10 and 21, have been sent to the
Cambridge Crystallographic Data Centre and are desig-
nated CCDC 182110 and CCDC 182109 respectively.
,
screening H···O distance between 1.8 and 2.5 A) as
acceptors gave a total of 11 241 hits. While some of
these interactions could be discarded as not being hy-
drogen-bonding in character based on their small
CꢀH···O angle, nevertheless the mean H···O distance
,
was 2.448 A. The distribution of CꢀH···O angles were
bimodal with a minor cluster centred between 95 and
100° and a main cluster centred between 155 and 160°.
These results clearly show that a hydrogen-bonding
interaction between a vinylic proton and oxygen is a
relatively common occurrence in the solid state. An
additional search of CSD found the following informa-
tion regarding ꢀCꢁCꢀH···N interactions: (a) intramolec-
,
ular bonding-195 hits; average ꢀCꢁCꢀ bond, 1.348 A;
,
average ꢀCꢀH bond, 0.993 A; average H···N distance,
,
2.410 A; average ꢀCꢁCꢀH bond angle, 119.916°; avera-
ge ꢀCꢀH···N bond angle, 111.413°; while (b) inter-
molecular bonding, 262 hits; average ꢀCꢁCꢀ bond,
1.348 A; average ꢀCꢀH bond, 1.0212 A; average H···N
distance, 2.420 A; average ꢀCꢁCꢀH bond angle,
3. Pharmacology
,
,
,
3.1. Preliminary pharmacological testing
123.030°; average ꢀCꢀH···N bond angle, 154.125°. In
the case of intramolecular interactions, the CꢀH···N
angle would be expected to be constrained to a much
smaller degree than would be the case where there no
such constraint existed (i.e. the CꢀH···N angles of
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-

640
N.D. Eddington et al. / European Journal of Medicinal Chemistry 37 (2002) 635–648
Table 4
logical testing of the reported compounds. These testing
procedures have been described [2,17,18]. Phase I stu-
dies of the isoxazoles involved three tests: MES, scPTZ,
and neurologic toxicity (Tox). Intraperitoneal (ip) ad-
ministration of the test compounds was as a suspension
in 0.5% methylcellulose. As previously reported [4–7],
active compounds in the Phase I evaluation were subse-
quently tested either for an ED50 quantitation in mice
(Phase II), or qualitatively in rats (Phase VIA). If
successful in Phase VI, rat quantitation (Phase VIB)
was undertaken. These data are provided in Table 1.
An initial screen of starting 3-amino-5-methyisoxazole
(3) indicated that it was inactive in the standard Phase
I evaluations as well as in the threshold tonic extension
(TTE) test [19].
Crystal data and structure refinement for methyl 4-[(5-chloro-2-
amino)pyridyl]-6-methyl-2-oxo-3-cyclohexene-1-carboxylate (21)
Parameter
Identification code
Empirical formula
Formula weight
Temperature (K)
Wavelength
Crystal system
Space group
Unit cell dimensions
21
C
14H15ClN2O3
294.73
293 (92)
0.71073
triclinic
P1
,
a (A)
6.6451 (10)
11.9653 (16)
18.984 (2)
107.585 (11)
91.289 (11)
96.323 (10)
1427.6 (3)
4
,
b (A)
,
c (A)
h (°)
i (°)
k (°)
3.2. Sodium channel binding studies
3
,
Volume (A )
Z
Density (calculated) (mg m−3
)
)
1.371
0.276
616
We had previously evaluated methyl 4-[(4-
chlorophenyl)amino]-6-methyl-2-oxocyclohex-3-ene-1-
Absorption coefficient (mm−1
F(000)
Crystal size (mm)
q Range for data collection (°)
Reflections collected
0.10×0.80×0.64
2.25–27.50
6548
Table 3
Crystal data and structure refinement for ethyl 4-[(5-methyl-3-isoxa-
zolyl)-amino)]-6-methyl-2-oxo-3-cyclohexene-1-carboxylate (10)
Independent reflections
Completeness to q=27.50°
Absorption correction
Maximum and minimum
transmission
6007 (Rint=0.0179)
91.3%
SHELXA
Parameter
0.8932 and 0.6365
Identification code
Empirical formula
Formula weight
Temperature (K)
Wavelength
10
Refinement method
full-matrix least-squares
on F2
6007/8/435
C14H18N2O4
278.30
293 (92)
0.71073
triclinic
P1
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I\2|(I)]
R indices (all data)
1.016
R1=0.0456, wR2=0.1111
R1=0.0683, wR2=0.1243
0.0036 (10)
Crystal system
Space group
Extinction coefficient
Unit cell dimensions
Largest diffraction peak and hole (e 0.293 and −0.266
,
a (A)
6.839 (3)
−3
,
A
)
,
b (A)
11.619 (5)
19.513 (7)
106.70 (11)
91.64 (11)
98.80 (10)
1463.2 (10)
4
1.259
0.093
592
,
c (A)
h (°)
i (°)
k (°)
carboxylate (20a) (R3=4-Cl) and methyl 4-[(4-tri-
fluoromethoxyphenyl)amino]-6-methyl-2-oxocyclohex-
3-ene-1-carboxylate (20c) (R3=4-OCF3) as inhibitors
of the specific binding of [3H]batrachotoxinin A 20
a-benzoate to neurotoxin site 2 of the voltage-depen-
dent sodium channel [5]. The IC50 data indicated that
both were sodium channel inhibitors (the 4-chloro
enaminone (20a), at 489 mM; the 4-trifluoromethoxy
enaminone (20c), at 170 mM). In view of the similarity
in their initial pharmacological results, the ethyl ester of
the 3-amino series (10), was evaluated in this assay. It
was, however, found to be inactive under the test
conditions [20,21].
3
,
Volume, Z (A )
Z
Density (calculated) (mg m−3
)
)
Absorption coefficient (mm−1
F(000)
Crystal size (mm)
q Range for data collection (°)
Reflections collected
0.07×0.79×0.23
3.02–24.91
3873
Independent reflections
Completeness to q=24.91°
Absorption correction
Refinement method
3502 (Rint=0.0225)
68.7%
None
Full-matrix least-squares
on F2
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I\2|(I)]
R indices (all data)
3502/10/449
1.031
R1=0.0617, wR2=0.1336
R1=0.1080, wR2=0.1579
0.0034 (13)
3.3. Pharmacokinetics
Extinction coefficient
Eddington et al. [22,23] investigated the physiochemi-
cal permeability properties and cellular transport mecha-
nisms of the enaminones and, of relevance to this
Largest diffraction peak and hole (e 0.421 and −0.213
−3
,
A
)

N.D. Eddington et al. / European Journal of Medicinal Chemistry 37 (2002) 635–648
641
Table 5
microvessel endothelial cells (BBMEC) [24–27]. This
data is summarised in Table 7. As noted, Clog P [28],
chromatographic retention data (expressed as
log KIAM), apical (AP) to basolateral (BL) transport as
well as BL to AP were significantly different for these
active anticonvulsant compounds. These data suggests
that for the 4-chloro enaminone (20a), and other
anilino enaminones [22], their transport was active and
not passive and that there was an efflux mechanism
(e.g. Pgp or MRP) responsible for the high BL to AP
transport of these analogues. This efflux mechanism,
however, did not effect the isoxazole (10). While these
data explain the extended (ca. 4 h) protection of the
isoxazole (10), but does not explain how it does, in fact,
exerts its anticonvulsant effect.
Selected torsion angles for ethyl 4-[(5-methyl-3-isoxazolyl)-amino)]-6-
methyl-2-oxo-3-cyclohexene-1-carboxylate, (10) and methyl 4-[(5-
chloro-2-amino)pyridyl]-6-methyl-2-oxo-3-cyclohexene-1-carboxylate
(21)
(A)
Molecule A
H(2AA) C(2A) C(1A) N(1A)
C(1A) N(1A) C(7A) N(2A)
3.0
−2.8
Molecule B
H(2BA) C(2B) C(1B) N(1B)
C(1B) N(1B) C(7B) N(2B)
3.1
−4.1
(B)
Molecule A
H(7AA) C(7A) C(6A) N(2A)
C(6A) N(2A) C(5A) N(1A)
2.5
−2.1
Molecule B
H(7BA) C(7B) C(6B) N(2B)
C(6B) N(2B) C(5B) N(1B)
2.8
−8.4
4. Results and discussion
Pharmacological comparison of the isomeric 5-
methyl-substituted isoxazoles (9–11) with the 3-methyl-
substituted isoxazoles (14–16) in Table 1 reveal both
similarities and differences. Both methyl esters, (9) and
(14), were MES-active in both rodent species, while
only the ethyl ester of the 3-amino series (10), was
active. The tert-butyl ester in the 3-amino series (11)
gave irreproducible results in mice, but excellent results
in rats, with the best PI in that series of all analogues
tested, while the comparable 5-amino analogue (16),
also produced an active compound in both species. The
presence or absence of the alkyl substitution, R1, on the
isoxazole ring, can further differentiate the 3-amino
series. Clearly, the anti-MES activity for unsubstituted
isoxazoles (6–8) was comparable to the 5-methyl-sub-
stituted isoxazoles (14–16), however, the unsubstituted
series resulted in highly toxic compounds in the scPTZ
evaluation. As noted in Table 1, deaths occurred with
isoxazoles (6) and (8) under standard assay conditions.
In the 5-amino series, extension of the alkyl substitu-
tion, producing 3-ethyl analogues (17–19), resulted in
inactive compounds.
Table 6
Selected torsion angles for ethyl 4-[(5-methyl-3-isoxazolyl)-amino)]-6-
methyl-2-oxo-3-cyclohexene-1-carboxylate (10) and methyl 4-[(5-
chloro-2-amino)pyridyl]-6-methyl-2-oxo-3-cyclohexene-1-carboxylate
(21)
,
,
,
CꢀH (A)
H···N (A)
CꢀH···N (°)
C···N (A)
(A)
Molecule A
0.930
2.277
2.317
125.1
124.9
2.914
2.949
Molecule B
0.930
(B)
Molecule A
0.930
2.326
2.295
123.1
123.3
2.939
2.912
Molecule B
0.930
study, compared the 4-chloro prototype (20a) with the
ethyl ester of the 3-amino series (10), in the in vitro
primary cell culture model consisting of bovine brain
The mechanism of action of the isoxazoles in this
study remains to be determined. As expected from
Table 7
Chemical, pharmacological and permeability (Papp) properties of isoxazole 10 and enaminone 20a (R3=4-Cl) a
b
Compound
ED50 (rat, po, mg kg−1
)
Clog P
log KIAM
Papp
AP“BL c
BL“AP
BL“AP/AP“BL
10
20a
10.1
5.9
1.91
2.98
−1.60
0.11
23.47 (90.72) d
8.33 (90.43)
18.41 (90.32)
19.63 (90.54)
0.78
2.36
a Calculated from reference [23].
b Calculated from reference [28].
c Papp coefficients listed as 10−5 cm s−1
.
d Data listed as the mean at n=3 (9S.D.).

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N.D. Eddington et al. / European Journal of Medicinal Chemistry 37 (2002) 635–648
Table 8
Prelimiary hippocampal kindling screen in rats, ip, of isoxazole 11 a
Seizure score, pre-drug
Seizure score, drug After discharge duration (s), pre-drug
After discharge duration (s), drug
Rat
c1
Rat
c2
5
5
3
4
58–71
51–79
36
83
a Dose=300 mg kg−1; time of maximum effect=45 min.
TD50\500 mg kg−1 for benzamide (29), and similarly
to our series, displayed greater sensitivity in the rat
than the mouse. As noted in Table 1, Clog P [28]
measurements of the reported isoxazoles varied from
0.89 to 3.68, while the most active compound was the
tert-butyl ester of the 3-aminoisxazole series (11), being
2.62, while the other 3-methylisoxazoles, the methyl
ester (9) and the ethyl ester (10), were 1.39 and 1.91,
respectively. These data suggest that these compounds
may be taken into the brain via the same mechanism.
The Clog P [28] measurements for the active amides in
Lepage’s study are 1.38 for compound 28 and 1.88 for
compound 29, respectively. These latter values agree
well with our active compounds. Hansch has indicated
that the maximum potency of drugs acting on the
central nervous system was obtained with congeners
having an optimum lipophilicity (log P0) near 2.0 [32].
This factor was clearly evident in the time of peak effect
of our reported analogues (Table 1), as the most
lipophilic compound, the tert-butyl isoxazole (11), was
active at 4 h.
previous studies with the enaminones [3–7], Phase I test
against pentylenetetrazol was consistently negative. The
compounds were subsequently evaluated against bicu-
culline and picrotoxin, compounds which delineate
whether the test substance acts through GABA in-
hibitory function, or chloride channel inhibition, re-
spectively. The 3-aminoisoxazole compounds (9, 10 and
11) were all uniformly inactive in the subcutaneous
bicuculline (scBic) and the subcutaneous picrotoxin
(scPic) evaluation. Compound 9 produced death fol-
lowing continuous seizures in the animals at 250 and
500 mg kg−1 of test dose in the scBic test; isoxazole 10
produced death following continuous seizure in the
animals at 125 mg kg−1 in the same test; while the
tert-butyl isoxazole (11), did not produce any fatalities,
but was inactive in both tests in doses up to 500 mg
kg−1
. Compound 11 was evaluated in isolated
hippocampal slices [29,30]. This method has the advan-
tage of recording important electrical activity under a
variety of experimental conditions and tissue penetra-
tion by the drug. However, this evaluation has the
disadvantages of cell viability and the lack of interac-
tions with the neural circuits found in the intact animal
[31]. The hippocampal results on tert-butyl isoxazole
(11) are shown in Table 8 and suggested only a weak
ability to block the expression of fully kindled seizures.
However, further testing would be required before any
definitive conclusion regarding efficacy against focal
seizures could be drawn. In addition, isoxazole (11) was
evaluated for ip toxicity in rats and was completely
non-toxic at dosages up to 300 mg kg−1 for periods up
to 4 h.
5. Conclusions
The anticonvulsant analysis of the series of ena-
minone-derived isoxazoles provide a potent, orally ac-
tive class of compounds with a hitherto distinct mecha-
nism of activity which, as yet, is undefined. Their
effectiveness in the MES evaluation and extended dura-
tion of action warrants further study.
The activity–inactivity of the 3-amino and 5-amino
isoxazoles may be due to orientation of the pseudo
three ring system.
Our synthetic approach was focused on alkyl substi-
tution on the isoxazole ring and its effect on anticon-
vulsant activity. Lepage and co-workers [1] reported on
the anticonvulsant activity of the isoxazole amides,
from which emerged two promising candidates, N-(2,6-
dimethylphenyl)-5-methyl-3-isoxazolecarboxamide (28),
and N-(3-heremethyl-5-isoxazolyl)-2,6-dimethylbenza-
mide (29). These isosteres provided MES protection at
po rat ED50 5.5 mg kg−1 and TD50\500 mg kg−1 for
carboxamide (28) and an ED50 of 8.9 mg kg−1 and a
6. Experimental
6.1. Chemistry
Melting points (m.p.) were determined on
a
Thomas–Hoover capillary m.p. apparatus and are

N.D. Eddington et al. / European Journal of Medicinal Chemistry 37 (2002) 635–648
643
uncorrected. Observed boiling points (b.p.) were also
uncorrected. IR spectra were recorded in Nujol, as
diluted chloroform solutions in matched sodium chlo-
ride cells, or neat with a Perkin–Elmer 1330 spec-
duced pressure yielded a yellow–brown oil which dis-
tilled at 109–110 °C (100 mm) and proved to be the
crude cyanomethylethyl ketone (12), 16.6 g (25%); H-
1
NMR (CDCl3): l 1.00 (3H, t, J=7.0 Hz, CH3), 2.60
(2H, t, CH2ꢀCꢁO), 3.35 (2H, s, CH2ꢀCN). To a solu-
tion of sodium acetate (29.4 g, 0.36 mol) and hydroxy-
lamine hydrochloride (22.1 g, 0.32 mol) in 200 mL of
water was added the above ketone (7.49 g, 0.07 mol),
dissolved in 200 mL of ethanol, added over 30 min and
the mixture stirred and refluxed for 1.5 h. The alcohol
was removed under reduced pressure and the sodium
chloride removed and concentration repeated until a
crude residue, m.p.B100 °C resulted. Recrystallisation
from methanol–petroleum ether (b.p. 60–80 °C) pro-
duced a product, 13; m.p. 92–96 °C, 4.0 g (50%).
1H-NMR (CDCl3): l 1.23 (3H, t, J=7.0 Hz, CH3),
2.59 (2H, q, J=7.0 Hz, CH2), 6.85 (1H, s, CH), 7.30
(2H, s, NH2). Calc. for (C5H8N2O) C, H, N.
1
trophotometer. H-NMR spectra were recorded on a
General Electric QE 300-MHz spectrometer in deuter-
ated solvents using tetramethylsilane as an internal
reference. Coupling patterns are described as follows: s,
singlet; bs, broad singlet; d, doublet; dd, doubled dou-
blet; 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 mea-
sured to two decimal points, while the coupling con-
stants were rounded off to one decimal place. TLC
analysis employed ethyl acetate:cyclohexane (3:1) elu-
tion solvent mixture and 5×10-cm fluorescent plates
(Whatman silica-gel 60A). Column chromatography
employed silica gel (100–200 mesh, Fisher Scientific)
and eluted with ethyl acetate:cyclohexane (3:1). Ele-
mental analyses (C, H, and N) were performed by
Schwarzkopf Microanalytical Laboratory, Woodside,
NY 11377 USA. The analytical results for the elements
were within 90.4% of the theoretical values. X-ray
crystal analysis was performed on a Nicolet P4 diffrac-
tometer. Methyl 6-methyl-2,4-dioxocyclohexane-1-car-
boxylate, (1) (R=CH3) [3], ethyl 6-methyl-2,4-
dioxocyclohexane-1-carboxylate (2) (R=C2H5) [3], and
tert-butyl 6-methyl-2,4-dioxocyclohexane-1-carboxylate
(3) (R=C(CH3)3) [13], were prepared by literature
methods. 3-Aminoisoxazole (4) (R1=H), and 5-amino-
3-methylisoxazole were obtained from Aldrich Chemi-
cal Company and 3-amino-5-methylisoxazole (5)
(R1=CH3), was obtained from Fluka Chemika AG
(Germany) and used without further purification.
6.3. General procedure for the preparation of
3-isoxazolylamino enaminones
6.3.1. Method A
6.3.1.1. Methyl 4-[(5-methyl-3-isoxazolyl)amino]-6-
methyl-2-oxo-3-cyclohexene-1-carboxylate, (9). A mix-
ture of methyl 6-methyl-2,4-dioxocyclohexane-1-
carboxylate (1) (5.0 g, 27 mmol), and 3-amino-5-methy-
isoxazole (5) (3.24 g, 33 mmol), was added to a mixture
of absolute ethanol (100 mL) and ethyl acetate (100
mL) and the solution refluxed and stirred for 6 h.
Evaporation under reduced pressure yielded a yellow
solid which was recrystallised three times from EtOAc
to yield isoxazole 9, 4.31 g (58%) of white crystals; m.p.
220–222 °C. 1H-NMR (DMSO d6): l 0.98 (3H, d,
J=5.9 Hz, CH3 on C6), 2.36 (3H, s, CH3 on isoxazole
ring), 2.38–2.48 (3H, m, CH2+CH of cyclohexene
ring), 3.14 (1H, d, J=11.6 Hz, CH on C1), 3.64 (3H, s,
OCH3), 6.02 (1H, s, ꢁCH), 6.24 (1H, s, CH on isoxazole
ring), 9.64 (1H, bs, NH). Anal. (C13H16N2O4) C, H, N.
6.2. General procedure for the preparation of
3-substituted 5-aminoisoxazoles
6.2.1. 3-Ethyl-5-aminoisoxazole (13)
The procedure of Nishiwaki and Saito [12] was
modified as follows. Into a 500 mL single neck flask
equipped with a Y tube, a condenser, a 125 mL pres-
sure equalising dropping funnel and a magnetic stirring
bar was introduced 100 mL dry THF under nitrogen.
Sodium hydride (9 g of a 50% suspension in mineral oil,
0.19 mol), and acetonitrile (7.0 g, 0.17 mol) in 20 mL of
dry THF were slowly added maintaining reflux condi-
tions. The mixture was refluxed for a total of 4 h after
addition and was allowed to stir at room temperature
(r.t.) for 8 h. The reaction was quenched with 200 mL
of ether and the solid obtained was filtered and air
dried. The residue from four reactions were combined
and dissolved in a minimum volume of water. Acidifica-
tion by the dropwise addition of HCl yielded an oil that
was extracted with ether (4×100 mL) and dried over
anhydrous sodium sulphate. Evaporation under re-
6.3.1.2.
Ethyl
4-[(5-methyl-3-isoxazolyl)amino]-6-
methyl-2-oxo-3-cyclohexene-1-carboxylate, (10). In a
similar procedure, ethyl 6-methyl-2,4dioxocyclohexane-
1-carboxylate (2), and 3-amino-5-methyiisoxazole (5),
produced isoxazole 10. Three recrystallisations from
ethanol provided an analytical sample: yield 5.68 g
(76%) of white crystals; m.p. 195–197 °C. 1H-NMR
(DMSO d6): l 1.00 (3H, d, J=5.7 Hz, CH3 on C6),
1.20 (3H, t, J=7.1 Hz, CH3 of ethyl group), 2.35 (3H,
s, CH3 on isoxazole ring), 2.38–2.55 (3H, m, CH2+
CH of cyclohexene ring), 3.09 (1H, d, J=11.0 Hz, CH
on C1), 4.12 (2H, q, J=7.1 Hz, CH2 of ethyl group),
6.02 (1H, s, ꢁCH), 6.25 (1H, s, CH on isoxazole ring),
9.63 (1H, bs, NH). Anal. (C14H18N2O4) C, H, N.

644
N.D. Eddington et al. / European Journal of Medicinal Chemistry 37 (2002) 635–648
6.3.1.3. Tert-butyl 4-[(5-methyl-3-isoxazolyl)amino]-6-
methyl-2-oxo-3-cyclohexene-1-carboxylate, (11). Simi-
ring), 2.51 (1H, m, CH of cyclohexene ring), 2.93 (1H,
d, J=11.0 Hz, CH on C1), 6.31 (1H, s, ꢁCH), 6.27 (1H,
d, J=1.6 Hz, CH on isoxazole ring at C4), 8.41 (1H, d,
J=1.6 Hz, CH on isoxazole ring at C3), 9.60 (1H, bs,
NH). Anal. (C15H20N2O4) C, H, N.
larly,
tert-butyl
6-methyl-2,4-dioxocyclohexane
carboxylate (3), and 3-amino-5-methylisoxazole (5),
produced isoxazole 11, as white crystals after three
recrystallisations from EtOAc; m.p. 197–201 °C (with
1
effervescence); yield, 3.57 g (43%). H-NMR (DMSO
6.4. General procedure for the preparation of
5-isoxazolylamino enaminones
d6): l 1.00 (3H, d, J=5.7 Hz, CH3 on C6), 1.42 (9H, s,
3×CH3), 2.35 (3H, s, CH3 on isoxazole ring), 2.44 (2H,
m, CH2 of cyclohexene ring), 2.51 (1H, m, CH of
cyclohexene ring), 2.93 (1H, d, J=11.0 Hz, CH on C1),
6.01 (1H, s, ꢁCH), 6.23 (1H, s, CH on isoxazole ring),
9.58 (1H, bs, NH). Anal. (C16H22N2O4) C, H, N.
6.4.1. Method B
6.4.1.1. Methyl 4-[(3-methyl-5-isoxazolyl)amino]-6-
methyl-2-oxo-3-cyclohexene-1-carboxylate, (14).
A
mixture of ketone 1 (5.0 g, 27 mmol), and 5-amino-3-
methylisoxazole (3.24 g, 33 mmol), was added to a
mixture of benzene (50 mL) and ethyl acetate (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 r.t. Crystals formed which resisted recrys-
tallisation with EtOAc and remained as a gel. Removal
of the solvent under reduced pressure yielded an amor-
phous product that was dissolved in chloroform and
eluted through a silica-gel G column (100–200 mesh)
with ethyl acetate:cyclohexane (3:1). Recrystallisation
of the individual 50 mL aliquots with EtOAc resulted in
an analytical sample of isoxazole 14, m.p. 189–190 °C;
yield, 3.95 g (55%) as light yellow plates. 1H-NMR
(DMSO d6): l 1.08 (3H, d, J=6.0 Hz, CH3 on C6),
2.36 (3H, s, CH3 on isoxazole ring), 2.74 (3H, complex
m, CH2+CH of cyclohexene ring), 3.25 (1H, d, J=
11.0 Hz, CH on C1), 3.81 (3H, s, OCH3), 6.03 (1H, s,
ꢁCH), 7.40 (1H, s, CH on isoxazole ring), 8.11 (1H, bs,
NH). Anal. (C13H16N2O4) C, H, N.
6.3.1.4. Methyl 4-[(3-isoxazolyl)amino]-6-methyl-2-oxo-
3-cyclohexene-1-carboxylate, (6). In a similar proce-
dure, ester 1 and 3-aminoisoxazole (4), produced
isoxazole 6. Three recrystallisations from EtOAc pro-
vided an analytical sample: yield 3.44 g (51%) of white
1
crystals; m.p. 203–205.5 °C. NMR indicated the com-
pound existed as a diastereomeric mixture in a ratio of
87:13 mol%. The major isomer gave the following
1H-NMR (DMSO d6): l 1.08 (3H, d, J=6.8 Hz, CH3
on C6), 2.38 (1H, dd, one of CHCH2), 2.50–2.70 (2H,
complex m, other CH2+CH of cyclohexene ring), 3.05
(1H, d, J=11.6 Hz, CH on C1), 3.74 (3H, s, OCH3),
6.28 (1H, d, J=1.6 Hz, CH on isoxazole ring at C4),
6.39 (1H, s, ꢁCH), 8.36 (1H, d, J=1.6 Hz, CH on
isoxazole ring at C3), 9.52 (1H, bs, NH). Anal.
(C12H14N2O4) C, H, N.
6.3.1.5. Ethyl 4-[(3-isoxazolyl)amino]-6-methyl-2-oxo-
3-cyclohexene-1-carboxylate, (7). Compound 7, was
prepared from ester 2 and 3-aminoisoxazole (4), and
occurred as white crystals after three recrystallisations
from EtOAc; m.p. 180–182 °C; yield, 4.10 g (58%).
1H-NMR indicated the compound existed as
a
6.4.1.2.
Ethyl
4-[(3-methyl-5-isoxazolyl)amino]-6-
diastereomeric mixture in a ratio of 89:11 mol%. The
major isomer gave the following H-NMR (DMSO d6):
methyl-2-oxo-3-cyclohexene-1-carboxylate, (15). In like
manner, ester 2 and 5-amino-3-methyisoxazole pro-
vided isoxazole (15), m.p. 137 °C (dampens), 140–
142 °C; yield 42% (after chromatography and three
1
l 1.08 (3H, d, J=6.4 Hz, CH3 on C6), 1.26 (3H, t, CH3
of ethyl group), 2.42 (1H, dd, one of CHCH2), 2.60
(2H, complex m, other CH2+CH of cyclohexene ring),
3.00 (1H, d, J=11.6 Hz, CH on C1), 4.18 (2H, q, CH2
of ethyl group), 6.26 (1H, d, J=1.6 Hz, CH on isoxa-
zole ring at C4), 6.38 (1H, s, ꢁCH), 8.41 (1H, d, J=1.6
Hz, CH on isoxazole ring at C3), 9.54 (1H, bs, NH).
Anal. (C13H16N2O4) C, H, N.
1
recrystallisations from EtOAc) as yellow crystals. H-
NMR (DMSO d6): l 1.09 (3H, d, J=6.0 Hz, CH3 on
C6), 1.28 (3H, t, J=7.0 Hz, CH3 of ethyl group), 2.36
(3H, s, CH3 on isoxazole ring), 2.74 (3H, complex m,
CH2+CH of cyclohexene ring), 3.25 (1H, d, J=11.0
Hz, CH on C1), 4.20 (2H, q, J=7.0 Hz, CH2 of ethyl
group), 6.14 (1H, s, ꢁCH), 7.44 (1H, s, CH on isoxazole
ring), 8.20 (1H, bs, NH). Anal. (C14H18N2O4) C, H, N.
6.3.1.6. Tert-butyl 4-[(3-isoxazolyl)amino]-6-methyl-2-
oxo-3-cyclohexene-1-carboxylate, (8). Similarly, tert-
butyl 6-methyl-2,4-dioxocyclohexane carboxylate (3)
and 3-aminoisoxazole (4), produced isoxazole 8 as
white crystals after five recrystallisations from EtOAc;
m.p. 190–193 °C; yield, 4.67 g (58%). 1H-NMR
(DMSO d6): l 1.00 (3H, d, J=5.7 Hz, CH3 on C6),
1.44 (9H, s, 3×CH3), 2.44 (2H, m, CH2 of cyclohexene
6.4.1.3. Tert-butyl 4-[(3-methyl-5-isoxazolyl)amino]-6-
methyl-2-oxo-3-cyclohexene-1-carboxylate, (16). Simi-
larly, isoxazole 16 was prepared from ester 3 and
5-amino-3-methyisoxazole, m.p. 127–130 °C; yield
33% (after chromatography and three recrystallisations
from EtOAc) as a yellow amorphous powder. 1H-NMR

N.D. Eddington et al. / European Journal of Medicinal Chemistry 37 (2002) 635–648
645
(DMSO d6): l 1.08 (3H, d, J=5.7 Hz, CH3 on C6),
1.40 (9H, s, 3×CH3), 2.35 (3H, s, CH3 on isoxazole
ring), 2.42 (2H, m, CH2 of cyclohexene ring), 2.55 (1H,
m, CH of cyclohexene ring), 2.94 (1H, d, J=11.0 Hz,
CH on C1), 6.10 (1H, s, ꢁCH), 6.26 (1H, s, CH on
isoxazole ring), 9.80 (1H, bs, NH). Anal. (C16H22N2O4)
C, H, N.
chloro-2-amino)pyridyl]-6-methyl-2-oxo-3-cyclohexene-
1-carboxylate (21), were recrystallised from ethanol. All
experimental details related to the structural analysis
are provided Tables 3 and 4. The structures were solved
by direct methods of the SHELXTLPC program and
refined by the SHELXTL program [33]. There was no
intensity decay in the analysis.
6.4.1.4.
Methyl
4-[(3-ethyl-5-isoxazolyl)amino]-6-
6.6. Pharmacology
methyl-2-oxo-3-cyclohexene-1-carboxylate, (17).
A
mixture of 1 (5.0 g, 27 mmol) and 5-amino-3-ethylisox-
azole (13) (3.70 g, 33 mmol), provided isoxazole 17,
m.p. 112–113 °C; yield 15% (after chromatography
and two recrystallisations from 2-propanol) as white
Initial evaluations for anticonvulsant activity were
performed by the ADD program, Epilepsy Branch,
Neurological Disorders Program, NINDS and included
Phases I, II, VIA and VIB test procedures which have
been described [17,18]. These tests were performed ei-
ther in male Carworth Farms no. 1 (CF1) mice (weigh-
ing 18–25 g) or male Sprague–Dawley rats (weighing
100–150 g). Phase I, a qualitative anticonvulsant ip
evaluation in mice included three tests: MES, scPTZ,
and the rotorod test for neurological toxicity (Tox).
Compounds were suspended in 0.5% aqueous methyl-
cellulose and were administered at three dosage levels
(30, 100, and 300 mg kg−1) with anticonvulsant activity
and motor impairment noted 30 min and 4 h after
administration. Phase VIA evaluation was a similar
qualitative evaluation to the Phase I evaluation, how-
ever, the test drug was administered orally in rats
utilising the three tests noted previously. The Phase II
test quantitated the anticonvulsant activity and motor
impairment observed for the most promising com-
pounds in Phase I and VIA, respectively. An ip test in
rats was performed at 30 mg kg−1 for activity and
toxicity. The time of peak effect ip in mice and rats was
also noted for the determination of ED50 and TD50
values. All test data are listed in Table 1. Phase V of the
ADD testing protocol measured the ability of com-
pounds to provide protection against seizures induced
by subcutaneous injection of the CD97 of bicuculline
(2.7 mg kg−1) and picrotoxin (3.15 mg kg−1). A special
ip toxicity evaluation was performed on compound 11
in Sprague–Dawley rats. The TTE test [19] is described
as follows. Twenty mice were pretreated with 100 mg
kg−1 of the test compound. At several time intervals
(0.25, 0.5, 1, 2 and 4 h) post-treatment with the test
compound, four mice at each time point were chal-
lenged with 12.5 mA of electrical current for 0.2 s via
corneal electrodes. This stimulation produced a TTE
seizure in the animals. For each time interval, results
were expressed as a ratio of the number of animals
protected over the number of animals tested. The pro-
cedure for hippocampal slices is as follows. A bipolar
stimulating electrode was stereotactically implanted in
the ventral hippocampus (AP −3.6, ML 4.9, DV −5.0
from dura, incisor bar +5) of adult male Sprague–
Dawley rats (250–300 g) under ketamine–xylazine
anesthesia. Three anchor screws were attached to the
1
powder. H-NMR (DMSO d6): l 1.06 (3H, d, J=6.0
Hz, CH3 on C6), 1.24 (3H, t, J=6.9 Hz, CH3 on
isoxazole ring), 2.55 (3H, complex m, CH2+CH of
cyclohexene ring), 2.59 (2H, q, J=6.9 Hz, CH2 of
isoxazole ring), 3.17 (1H, d, J=11.0 Hz, CH on C1),
3.67 (3H, s, OCH3), 5.54 (1H, s, ꢁCH), 7.40 (1H, s, CH
on isoxazole ring), 8.11 (1H, bs, NH). Anal.
(C14H18N2O4) C, H, N.
6.4.1.5. Ethyl 4-[(3-ethyl-5-isoxazolyl)amino]-6-methyl-
2-oxo-3-cyclohexene-1-carboxylate, (18). In like man-
ner to isoxazole 17, ketone 2 and 5-amino-3-ethyliso-
xazole (13), produced 18, m.p. 122–123 °C; yield 15%
(after chromatography and three recrystallisations from
1
2-propanol) as white crystals. H-NMR (DMSO d6): l
1.06 (3H, d, J=6.0 Hz, CH3 on C6), 1.24 (3H, t,
J=7.0 Hz, CH3 on isoxazole ring), 1.28 (3H, t, J=7.0
Hz, CH3 of ethyl group), 2.55 (3H, complex m, CH2+
CH of cyclohexene ring), 2.59 (2H, q, J=6.9 Hz, CH2
of isoxazole ring), 3.17 (1H, d, J=11.0 Hz, CH on C1),
4.20 (2H, q, J=7.0 Hz, CH2 of ethyl group), 5.54 (1H,
s, ꢁCH), 7.40 (1H, s, CH on isoxazole ring), 8.12 (1H,
bs, NH). Anal. (C15H20N2O4) C, H, N.
6.4.1.6. Tert-butyl 4-[(3-ethyl-5-isoxazolyl)amino]-6-
methyl-2-oxo-3-cyclohexene-1-carboxylate, (19). Com-
pound 19 was prepared from ester 3 and 5-amino-3-
ethylisoxazole (13), m.p. 102–104 °C; yield 14% (after
chromatography and three recrystallisations from ethyl
acetate:ether, 3:1) as light yellow crystals. 1H-NMR
(DMSO d6): l 1.06 (3H, d, J=6.0 Hz, CH3 on C6),
1.24 (3H, t, J=6.9 Hz, CH3 on isoxazole ring), 1.40
(9H, s, 3×CH3), 2.55 (3H, complex m, CH2+CH of
cyclohexene ring), 2.59 (2H, q, J=6.9 Hz, CH2 of
isoxazole ring), 3.17 (1H, d, J=11.0 Hz, CH on C1),
5.54 (1H, s, ꢁCH), 7.40 (1H, s, CH on isoxazole ring),
8.1 (1H, bs, NH). Anal. (C17H24N2O4) C, H, N.
6.5. X-ray crystal analysis
Ethyl
4-[(5-methyl-3-isoxazolyl)amino]-6-methyl-2-
oxo-3-cyclohexene-1-carboxylate (10), and methyl 4-[(5-

646
N.D. Eddington et al. / European Journal of Medicinal Chemistry 37 (2002) 635–648
skull and the electrode assembly anchored to the skull
with dental acrylic cement. After the incision is closed
with sutures, the animal received a single dose of bi-
cillin (60 000 U, im) and returned to the home cage to
recover. Animals were kindled according to the proce-
dure of Lothman and Williamson [34]. 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 are fully kindled.
One week later the effect of a single dose of test
substance (50 mg kg−1, ip) on the behavioural seizure
score (BSS) and after-discharge was assessed in a single
group of kindled rats (n=6–8) 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 are scored according to the following
criteria: Stage 1, mouth and facial clonus; Stage 2, stage
1 plus head nodding; Stage 3, stage 2 plus forelimb
clonus; Stage 4, stage 3 plus rearing; Stage 5, stage 4
plus repeated rearing and falling [35]. When a drug
treatment is observed to significantly lower seizure
score and decrease afterdischarge, a dose–response
study will be initiated. Compound 11 was studied, but
did not significantly score well to qualify, however.
brains were obtained and the minced gray matter of the
cortex was collected and placed in a sterile vessel,
suspended to a final volume of 500 mL in serum free
minimum essential medium (MEM, pH 9.0) containing
dispase. The final concentration of the suspension was
0.5% (w/v). This suspension was placed in a shaking
water bath (100 oscillations per min) for 3 h at 37 °C.
The crude capillary pellets remaining after two succes-
sive centrifugations was resuspended in 20 mL of MEM
containing 1 mg mL−1 of collagenase/dispase and
placed on a shaking water bath for 3 h at 37 °C. The
capillary suspension was diluted to 50 mL in MEM and
centrifuged. The pellet was resuspended in 8–10 mL of
MEM. Aliquots (2–3 mL) of the suspension were lay-
ered over 45 mL of a 50% Percol gradient in each of
four 50-mL centrifuge tubes and centrifuged for 10 min
at 4 °C. The second band was removed via pipette,
placed into four separate centrifuge tubes, and diluted
in MEM up to 50 mL. Final capillary pellets were
obtained by centrifugation for 10 min. The primary
bovine microvessel fragments were then seeded onto 12
well Costar inserts (Transwell). Filter membranes (AP
surface) of inserts were treated with rat tail collagen
type 1 (ca. Two to three drops), and allowed to incu-
bate for 30 min in NH3 fumes. Fibronectin (0.5 mL)
was then added to each insert, removed after 40 min
and allowed to dry. BBMECs were then seeded onto
the collagen/fibronectin-coated surfaces at a density of
ca. 25×106 cells per cm2. Upper (AP) compartments
received 0.5 mL of plating medium and the lower (BL)
received 1.5 mL for the first 3 days of growth and every
other day thereafter. Cells were placed under standard
culture conditions (37 °C, 5% CO2), and 95% humidity
until confluence was reached within 14–15 days.
6.7. Sodium channel binding
The sodium channel binding assay on isoxazole com-
pound 10 was performed by NovaScreen, Hanover,
MD USA. [3H]Batrachotoxinin A 20a-benzoate was
prepared as described and assayed to contain a final
ligand concentration of 2.0 nM [20]. The sodium chan-
nel binding assay was similar to reported methods [20].
Reactions were carried out in 50 nM HEPES (pH 7.4 at
25 °C) containing 130 nM choline chloride at 37 °C
for 45 min. The reaction was terminated by rapid
vacuum filtration of the reaction mixture onto glass
fibre filters. Radioactivity trapped onto the filters was
determined and compared with control values in order
to ascertain any interaction of test compounds with the
sodium site 2 binding site. The IC50 value (concentra-
tion of compound required to inhibit 50% of specific
neurotoxin binding) were determined from a dose–res-
ponse curve generated by plotting the log of anticonvul-
sant concentration (over a range of 10–800 mM) versus
percent of specifically bound [3H]BTX-B. Isoxazole
compound 10 was inactive in this test.
6.8.2. Monolayer integrity
Monolayer integrity of both BBMECs was assessed
by noting the flux of radiolabelled markers [14C]sucrose
(paracellular) and [3H]propranolol (transcellular) for
each 12 well Costar plate before the transport
experiments.
6.8.3. Transport studies
Enaminone solutions were prepared in DMSO (B
2%) and PBS reaching a final concentration of ca.
1×10−4 M. Upon confluency of both BBMECs, com-
plete culture medium was removed from both the AP
and BL sides and washed twice with sterile PBS. Tran-
swell inserts were placed into tissue culture plates con-
taining 1.5 mL of prewarmed PBS for permeability
studies. The appropriate enaminone solution [isoxazole
10 or prototype 20a (R3=4-Cl)] radiolabelled marker,
or blank buffer was added to either the AP or BL side.
All transport experiments were performed at 37 °C in
PBS. BBMECs were continuously agitated on a plate
during transport experiments (100 cycles per min). For
6.8. Pharmacokinetics
6.8.1. BBMECs—isolation and seeding
BBMEC were isolated manually from the gray mat-
ter of cerebral cortices as described by Audus and
Borchardt [27,36]. Approximately two to three bovine

N.D. Eddington et al. / European Journal of Medicinal Chemistry 37 (2002) 635–648
647
examination of transport in the AP“BL direction, the
Transwell inserts were moved into wells with fresh PBS.
At time (t=0), 400 mL of drug was added to the AP
side of the monolayer. For examination in the BL“AP
direction, drug (1500 mL) was initially added to the
donor side at time (t=0) and at times between 5 and
120 min. Complete amounts of sample from the AP
side were removed and replaced with fresh prewarmed
PBS.
where DQ/Dt is equal to the linear appearance rate of
mass in the receiver solution; A, the cross sectional area
and C(0) is the initial enaminone concentration in the
donor compartment [37]. All values are represented as
mean and standard deviation of the values from three
monolayer Transwell inserts prepared under identical
conditions and from the same preparation of cells. The
results of the evaluations of isoxazole 10 and the
anilino prototype, 20a (R3=4-Cl) are shown on Table
5.
6.8.4. Inhibition studies
Inhibition studies using either verapamil (50 mM),
probenecid (100 mM) or indomethacin (10 mM) were
performed to examine the possible interaction of the
enaminones with possible efflux mechanisms on the
BBMEC monolayers (i.e. Pgp and MDR). Transport
studies were performed as previously indicated. After
removal of the medium and washing with PBS, Tran-
swell inserts were placed in fresh buffer and preincu-
bated with inhibitor on both the AP or BL side at
37 °C for 30–45 min. Drug was then added to the
donor side at time (t=0), and Transwell inserts were
removed after 120 min. All samples were removed after
120 min and kept frozen (−70 °C) until HPLC
analysis.
7. Supplementary material
Crystal structures have been deposited at the Cam-
bridge Crystallographic Data Centre and allocated the
deposition number CCDC number 182109 and 182110.
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DQ
Dt AC(0)
1
Papp
=

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