Novel class of potent 4-arylalkyl substituted 3-isoxazolol GABAA antagonists: Synthesis, pharmacology, and molecular modeling

Article abstract of DOI:10.1021/jm020027o

A number of analogues of the low-efficacy partial GABA_A agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL, 5), in which the 4-position of the 3-isoxazolol ring was substituted by different groups, were synthesized and tested as GABA_A rece

Full text of DOI:10.1021/jm020027o

2454
J . Med. Chem. 2002, 45, 2454-2468
Novel Cla ss of P oten t 4-Ar yla lk yl Su bstitu ted 3-Isoxa zolol GABAA An ta gon ists:
Syn th esis, P h a r m a cology, a n d Molecu la r Mod elin g
Bente Frølund,*,† Anne T. J ørgensen,† Lena Tagmose,† Tine B. Stensbøl,† Henrik T. Vestergaard,‡
Christine Engblom,‡ Uffe Kristiansen,‡ Connie Sanchez,§ Povl Krogsgaard-Larsen,† and Tommy Liljefors†
Departments of Medicinal Chemistry and Pharmacology, The Royal Danish School of Pharmacy,
DK 2100 Copenhagen, Denmark, and Department of Neurology, H. Lundbeck A/ S, 9 Ottiliavej,
DK-2500 Valby, Denmark
Received J anuary 18, 2002
A number of analogues of the low-efficacy partial GABAA agonist 5-(4-piperidyl)-3-isoxazolol
(4-PIOL, 5), in which the 4-position of the 3-isoxazolol ring was substituted by different groups,
were synthesized and tested as GABAA receptor ligands. Substituents of different size and
structural flexibility such as alkyl, phenylalkyl, diphenylalkyl, and naphthylalkyl were explored.
Pharmacological characterization of the synthesized compounds was carried out using receptor
binding assays and by electrophysiological experiments using whole-cell patch-clamp tech-
niques. Whereas none of these compounds significantly affected GABAB receptor sites or GABA
uptake, they did show affinity for the GABAA receptor site. While alkyl or benzyl substitution,
compounds 7a -h , provided receptor affinities comparable with that of 5 (Ki ) 9.1 µM),
diphenylalkyl and naphthylalkyl substitution, as in compounds 7m -t, resulted in a dramatic
increase in affinity relative to 5. The 3,3-diphenylpropyl and the 2-naphthylmethyl analogues,
compounds 7s and 7m , respectively, showed the highest affinities of the series (Ki ) 0.074 µM
and Ki ) 0.049 µM). In whole-cell patch-clamp recordings from cultured cerebral cortical
neurons, all of the tested compounds were able to inhibit the effect of the specific GABAA agonist
isoguvacine (1), compounds 7m and 7s showing antagonist potency (IC50 ) 0.37 µM and IC50
) 0.02 µM) comparable with or markedly higher than that of the standard GABAA antagonist
4 (IC50 ) 0.24 µM). Highly potent convulsant activity was demonstrated in mice with compounds
7m (ED50 ) 0.024 µmol/kg) and 7s (ED50 ) 0.21 µmol/kg) after intracerebroventricular
administration, whereas no effects were found after subcutaneous administration. According
to a previously proposed pharmacophore model for GABAA receptor agonists, a receptor cavity
in the vicinity of the 4-position of the 3-isoxazolol ring in 4-PIOL exists. A molecular modeling
study, based on compounds 7o,m ,l,q,s, was performed to explore the dimensions and other
properties of the receptor cavity. This study demonstrates the importance of the arylalkyl
substituents in 7m and 7s and the considerable dimensions of this proposed receptor cavity.
In tr od u ction
yl)pyridazinium bromide (SR 95531, 4),7 now used as a
standard antagonist for the GABAA receptors, repre-
sents another structural class of ligands.
4-Aminobutyric acid (GABA), the major inhibitory
neurotransmitter in the central nervous system (CNS),
operates through three different classes of receptors
consisting of the ionotropic GABAA and GABAC recep-
tors and the metabotropic GABAB receptors.1,2 Besides
playing an important role in central transmission
processes, these receptors, especially the GABAA recep-
tors, have been associated with certain neurological and
psychiatric disorders and are therapeutic targets in
certain diseases.3,4 To pharmacologically characterize
this receptor class, a number of GABAA ligands (Figure
1), such as the highly selective GABAA agonists isogu-
vacine (1), muscimol (2),5 and 4,5,6,7-tetrahydroisox-
azolo[5,4-c]pyridin-3-ol (THIP, 3),5,6 have been devel-
oped. 2-(3′-(Carboxypropyl)-3-amino-6-(paramethoxyphen-
Hypoactivity of the GABA neuronal function seems
to be associated with neurological disorders such as
epilepsy,8 Huntingtons chorea,9 anxiety,10 sleep disor-
ders, and pain.11 In contrast, GABA receptor mediated
hyperactivity has been suggested to be an important
component of schizophrenic symptoms.12-15 Using full
GABAA receptor agonists or antagonist may therapeuti-
cally lead to either desensitization of the receptors or
severe side effects, respectively. This has focused inter-
est on partial GABAA receptor agonists as potential
therapeutics. In a previous study, we have described
5-(4-piperidyl)-3-isoxazolol (4-PIOL, 5)16,17 and, more
recently, analogues of 5 as low-efficacy partial GABAA
agonists showing dominating antagonist profiles.18
In a preliminary communication,19 we proposed a
binding mode of the bioactive conformations of 2 and 5.
In these binding modes of 2 and 5, the 3-isoxazolol rings
do not overlap. This means that the 4-position in the
3-isoxazolol ring in 2 does not correspond to the 4-posi-
tion in the 3-isoxazolol ring of 5 in the receptor-bound
conformations. To investigate this in further detail, we
* To whom correspondence should be addressed.Address: Depart-
ment of Medicinal Chemistry, The Royal Danish School of Pharmacy,
2 Universitetsparken, DK-2100 Copenhagen, Denmark. Phone: (+45)
35306495. Fax: (+45) 35306040. E-mail: bfr@dfh.dk.
† Department of Medicinal Chemistry, The Royal Danish School of
Pharmacy.
‡ Department of Pharmacology, The Royal Danish School of Phar-
macy.
§ H. Lundbeck A/S.
10.1021/jm020027o CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/04/2002

Substituted Isoxazolol GABAA Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2455
F igu r e 1. Structures of GABA, the GABAA agonists isoguvacine (1), muscimol (2), 4-methylmuscimol (2a ), and THIP (3), the
GABAA antagonist 4, the low-efficacy partial GABAA agonist 4-PIOL (5), and the new isoxazolols (6 and 7a -t).
synthesized and pharmacologically tested a small series
of 4-PIOL analogues substituted in the 4-position of the
3-isoxazolol ring of 5. The results indicated that alkyl
substituents in the 4-position of 5 are allowed, in
contrast to what has been found for the corresponding
position of 2. Introduction of large aromatic substituents
into the 4-position of the 3-isoxazolol ring of 5 markedly
enhanced the affinity for the GABAA receptors. Fur-
thermore, these structural modifications led to a change
in the pharmacological profile of the compounds from
low-efficacy partial GABAA agonist activity of 5 to potent
GABAA antagonist effect. These results supported the
hypothesis concerning the binding modes of muscimol
(2) and 5, indicating the existence of a large cavity at
the 4-PIOL (5) recognition site of the GABAA receptor.
To explore this cavity in more detail, we here expand
the structure-activity study to include a broader spec-
trum of 4-substituted analogues of 5, with substituents
that differ mainly in size and conformational flexibility.
The synthesis and pharmacological characterization of
this series of compounds are described, and the results
of a molecular modeling study exploring the dimensions
and other properties of the receptor cavity accommodat-
ing the 4-substituents of the analogues of 5 are reported.
triethylamine, using the method described by Clay et
al.21 Alkylation of 15 was performed using the appropri-
ate alkyl halide in the presence of sodium ethoxide to
give compounds 16a -f,h -p ,s,t. The halides necessary
for these reactions were either commercially available
or prepared by bromination of the corresponding com-
mercially available alcohols using either phosphorus
tribromide or aqueous hydrogen bromide. In cases
where the alcohol was not available, it was prepared
by reduction of the corresponding carboxylic acid, using
lithium aluminum hydride. Cyclization of the alkylated
â-oxo esters with hydroxylamine at -30 °C followed by
heating with concentrated hydrochloric acid at 80 °C
gave the 3-isoxazolols 17a-f,h -p ,s,t, which were depro-
tected by treatment with hydrogen bromide in glacial
acetic acid to give the target compounds 7a -f,h -p ,s,t.
Cyclization and subsequent deprotection to give com-
pound 6 was done in a similar manner. Alkylation of
the â-oxo esters represents the limiting step of the
above-mentioned route (Scheme 2) reflecting steric
hindrance and risk of elimination of hydrogen halide
from the alkylating agent under strongly basic condi-
tions. An alternative approach was set up, as shown in
Scheme 3, for the preparation of compounds 7g,q,r ,
where the substituent was introduced in the 4-position
of the 3-isoxazolol ring of 18 following cyclization of the
â-oxo ester 15 (Scheme 3). Protection of the hydroxy
group of 18 using isopropyl bromide in the presence of
potassium carbonate selectively gave the O-protected
compound 19 in high yield. The iodinated analogue of
19 underwent magnesium-iodine exchange using eth-
ylmagnesium chloride followed by reaction with the
appropriate aldehyde or ketone to give the hydroxy
compounds 21g,q,r . Attempts to remove the benzylic
hydroxy group by catalytical hydrogenation failed.
However, ionic hydrogenation using triethylsilane and
trifluoroacetic acid in dichloromethane,22 at 0 °C for the
more activated hydroxy group in 21q and at 50 °C for
compounds 21g and 21r , gave compounds 22g,q,r in
Resu lts a n d Discu ssion
Ch em istr y. The analogues of 4-PIOL were synthe-
sized using two different strategies, introducing the
substituent in the 4-position of the 3-isoxazolol ring at
either an early stage or a later stage in the synthetic
sequence. Compounds 6 and 7a -f,h -p ,s,t were all
synthesized from the appropriate â-oxo esters, 11 or 15,
as outlined in Schemes 1 and 2. The preparation of both
â-oxo esters was based on malonate. Compound 11 was
synthesized via the imidazolide of carboxylic acid 10
using the complex formed from monoethyl malonate and
magnesium in THF,20 whereas compound 15 was pre-
pared from acyl chloride 14 and potassium ethyl mal-
onate in the presence of magnesium chloride and

2456 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Frølund et al.
Sch em e 1a
Sch em e 3a
a
Reagents. (a) LDA, THF, -70 °C; CH3I. (b) 2 M NaOH, 100
°C; ClCOOCH3, 0 °C, pH ) 10. (c) N,N′-Thionyldiimidazole, THF.
(d) [3-Ethoxy-3-hydroxyacrylato(2-)-O1O3]magnesate(2+), THF.
(e) NH2OH‚HCl, NaOH, then concentrated HCl. (f) HBr in AcOH,
room temperature.
Sch em e 2a
a
Reagents: (a) isopropyl bromide, K2CO3, DMF, 60 °C; (b) ICl,
AcOH, H2O, 80 °C; (c) EtMgCl, THF, -30 °C; (d) R1COR2, THF, 0
°C; (e) CF3COOH, Et3SiH, CH2Cl2; (f) HBr, AcOH, 65 °C.
for 6 and 7a -h ,j,r and because of solubility problems
10 µM for 7i,k -q ,s, none of the compounds showed
detectable affinity for GABAB receptors or GABA uptake
sites. Like 4-PIOL (5), all of the tested compounds did
show affinity for the GABAA receptor sites, and binding
affinities (Ki values) of compounds 6 and 7a -t for
GABAA receptor sites are listed in Table 1.
Introduction of a methyl group in the 4-position of the
piperidine ring of 5 to give compound 6 resulted in a
significant reduction of affinity for the GABAA receptor
sites compared to that of 5. In contrast, unbranched
alkyl groups as substituents in the 4-position of the
3-isoxazolol ring of 5 such as methyl, ethyl, propyl,
butyl, hexyl, and octyl groups as well as a benzyl group
are tolerated, compounds 7b-f,h showing affinity for
the GABAA receptor sites comparable to or slightly
higher than that of 5. Increasing the length of the alkyl
chain joining the phenyl group and the 3-isoxazolol ring
of compound 7h gave compounds 7i and 7j, where the
2-phenylethyl compound, 7i, was equipotent with 5,
while the 3-phenylpropyl compound, 7j, showed en-
hanced affinity compared to 5. Reduction of the phenyl
group of 7h to a cyclohexyl group in 7g was tolerated,
with only a slight decrease in affinity. Affinity was
increased 10-fold relative to compound 7h when a
phenyl group was attached to the 4-position of the
phenyl group to give the biphenyl-4-methyl analogue 7l.
Addition of another phenyl group to the terminal carbon
of 7j, as in the 3,3-diphenylpropyl compound 7s, gave a
16-fold increase in affinity relative to the 3-phenylpropyl
analogue 7j. The diphenylmethyl, 2,2-diphenylethyl,
and 4,4-diphenylbutyl analogues 7q,r ,t all showed
reduced affinity compared to 7s.
a
Reagents: (a) SOCl2, DMF, 85 °C; (b) potassium ethyl mal-
onate, EtOAc, Et3N, MgCl2; (c) NaOEt, RBr or RI (R, see Figure
1), 80 °C; (d) NH2OH‚HCl, NaOH, then concentrated HCl; (e) HBr
in AcOH, room temperature.
high yields. Deprotection to give the target compounds
7g,q,r was accomplished by treatment with hydrogen
bromide in acetic acid at 65 °C.
In Vitr o P h a r m a cology. The compounds were char-
acterized in receptor binding studies using rat brain
membrane preparations and electrophysiologically using
whole-cell patch-clamp recordings from cultured cere-
bral cortical neurons. The affinities of the compounds
for GABAA and GABAB receptor sites or GABA uptake
sites, using either [3H]muscimol or [3H]GABA, were
determined using a modified version of the methods
described previously.18 At test concentrations of 100 µM
An even greater increase in affinity was observed
when the more bulky 2-naphthylmethyl was introduced

Substituted Isoxazolol GABAA Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2457
Ta ble 1. Receptor Binding and in Vitro Electrophysiological Data
[3H]muscimol binding,a
GABAB bindinga and
electrophysiology,c
compound
R
Ki (µM)d
GABA uptake,b IC50 (µM)
IC50 (µM)d
4
6
0.074 (0.059; 0.094)
37 (20; 70)
>10e
>100
0.24 (0.22; 0.25)
nd
5 (4-PIOL)
H
9.1 (8.2; 10)
10 (10; 11)
6.3 (5.1; 7.6)
6.6 (5.9; 7.3)
7.7 (6.6; 9.0)
4.5 (3.6; 5.5)
1.8 (1.1; 1.3)
4.9 (4.4; 5.5)
3.8 (3.3; 4.5)
5.0 (4.7; 5.4)
1.1 (0.7; 1.6)
5.9 (5.0; 7.0)
0.4 (0.31; 0.43)
0.049 (0.043; 0.057)
0.49 (0.43; 0.54)
0.10 (0.095; 0.11)
1.7 (1.5; 1.9)
0.96 (0.48; 1.9)
0.36 (0.30; 0.43)
0.068 (0.061; 0.074)
0.70 (0.67; 0.74)
>100
>100
>100
>100
>100
>100
>100
>100
>100
>10
110 (76; 170)
26 (22; 31)
10.3 (7.7; 13)
4.6 (4.3; 5.0)
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
methyl
ethyl
propyl
butyl
hexyl
octyl
cyclohexylmethyl
benzyl
2-phenylethyl
3-phenylpropyl
9-anthracylmethyl
4-biphenylmethyl
2-naphthylmethyl
2-naphthylethyl
1-naphthylmethyl
1-naphthylethyl
diphenylmethyl
2,2-diphenylethyl
3,3-diphenylpropyl
4,4-diphenylbutyl
3.0 (2.6, 3.5)
1.1 (0.86; 1.3)
0.44 (0.41; 0.48)
1.1 (0.97; 1.3)
4.0 (3.7; 4.3)
4.1 (3.8; 4.5)
>100
>10
0.53 (0.48; 0.58)
1.3f (1.2; 1.5)
>10
0.71 (0.63; 0.80)
0.37 (0.31; 0.44)
0.89 (0.83; 0.95)
0.48 (0.45; 0.50)
1.50 (1.40; 1.61)
0.78f (0.71; 0.85)
0.81f (0.75; 0.86)
0.02f (0.02; 0.03)
0.05f (0.05; 0.06)
>10
>10
>10
>10
>10
>100
>10
7s
7t
>100
a
b
Standard receptor binding on rat brain synaptic membranes, n ) 3. Inhibition of GABA uptake into synaptosomes. c Whole-cell
d
patch-clamp recordings from cerebral cortical neurons cultered for 7-9 days, n ) 6-17. Mean and SEM were calculated assuming a
normal distribution of the logarithm of the IC50 and Ki values. Hence, numbers in parentheses (min; max) indicate the antilog of the
mean ( SEM of IC50 and Ki.50 e Reference 7. f Because of the slow onset of antagonism, the parameters for 7k ,q-t were calculated using
the response magnitude after 5 s of application.
in the same position of the 3-isoxazolol ring to afford
7m . This led to a 78-fold increase in binding affinity
compared to the benzyl analogue 7h . The introduction
of the isomeric 1-naphthylmethyl group to provide 7o
proved to be less favorable showing a decrease in
binding affinity compared to compound 7m . Extending
the linker joining the 2-naphthyl or the 1-naphthyl
groups and the 3-isoxazolol ring with a carbon atom to
give compounds 7n and 7p , respectively, resulted in
both cases in a 10-fold reduction in affinity for the
GABAA receptor relative to the parent compound.
Further reduction was observed for compound 7k , where
the extension of the aromatic system from a naphthyl
group to an anthracyl group to give 7k markedly
decreased the affinity for the GABAA receptor, produc-
ing a binding affinity comparable to that of 5.
The pharmacology of compounds 7a -t was studied
using whole-cell patch-clamp recordings from cultured
cerebral cortical neurons, performed as described previ-
ously.18 The compounds were tested in the absence or
presence of the specific GABAA receptor agonist isogu-
vacine (1) (20 µM). In a previous study, using cerebral
cortical neurons, we have characterized 4-PIOL (5) as
a partial agonist at GABAA receptors.18 In the present
study, all of the compounds tested were capable of
inhibiting the current induced by 1 in a dose-dependent
manner, as exemplified in Figure 2 for compounds 4, 5,
and 7a ,h ,m ,s. With only a few exceptions, the electro-
physiological data showed a fairly good correlation to
the obtained binding affinities (Table 1). The 2-naph-
thylmethyl analogue 7m was shown to have the highest
affinity of this series, showing an affinity comparable
with that of the standard GABAA antagonist SR 95531
F igu r e 2. Effect of the partial agonists or antagonists on the
response to 20 µM of the full GABAA agonist isoguvacine (1)
using whole-cell patch-clamp recordings from cultured cere-
bral cortical neurons. A total of 20 µM 1 and varying
concentrations of antagonists/partial agonist were applied
simultaneously to the cells. The response of 20 µM 1 alone
has been set as 100%, and the other responses are expressed
as a fraction of this. The response to 1 is progressively reduced
with increasing concentrations of the partial agonist or
antagonist. The number of cells tested in this way with each
compound varied from n ) 6 to n ) 17.
(4) in the GABAA receptor binding assay. Interestingly,
results from the electrophysiological model showed the
3,3-diphenylpropyl and the 4,4-diphenylbutyl analogues
7s and 7t to be 19- and 7-fold, respectively, more potent
than 7m as GABAA antagonists. As in the GABAA
receptor binding assay, 7m showed antagonist potency

2458 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Frølund et al.
In the proposed binding modes of 2 and 5 (Figure 3),
the 3-isoxazolol rings do not overlap, and consequently,
the 4-position in 5 does not correspond to the “forbidden”
4-position in 2. In fact, as previously communicated19
and further extended in the present work, high-affinity
analogues of 5 could be obtained by the introduction of
large substituents in the 4-position.
To explore the dimensions and properties of the part
of the binding cavity accommodating the 4-substituents
in the analogues of 5 included in Table 1, we have
extended the pharmacophore model in the present work.
Those compounds that display the highest affinity in
[3H]muscimol binding and at the same time have
structural features that are useful for the exploration
of the size of the binding cavity have been analyzed. The
selected compounds are 7l,m ,o,q,s. In addition, 7h and
7k will be discussed as examples of lower affinity
compounds.
F igu r e 3. Pharmacophore model for GABAA receptor agonists
showing the proposed binding modes of 2 (green bonds) and 5
(grey bonds) and their interactions with two different confor-
mations of an arginine residue. The tetrahedrons indicate
positions of methyl groups in GABAA agonists that cause
strong steric repulsions with the receptor. The arrow points
to the tetrahedron corresponding to the methyl group in the
inactive compound 4-methylmuscimol (2a ). The red spheres
indicate sites to which the ammonium group in 2 interacts
via hydrogen bonds.
The two isomeric naphthylmethyl analogues 7m and
7o were chosen as starting points for the molecular
modeling study. These compounds display high affinities
and contain only 2 degrees of torsional freedom, which
facilitates the conformational analyses. For each com-
pound, an exhaustive unconstrained conformational
analysis for the aqueous solution was performed by
using the Monte Carlo multiple minimum method
(MCMM) and the MM3* force field in conjunction with
the GB/SA hydration model as described in the Experi-
mental Section. To identify possible bioactive conforma-
tions of the 4-substituents, conformational analyses of
the 4-substituents were performed with the 4-PIOL (5)
skeleton conformationally constrained to its proposed
binding conformation (Figure 3).
comparable to that of 4. As earlier described,19 only
compounds 7a and 7b retained some ability to induce
currents themselves at concentrations of 1 mM (not
shown) while compounds 7c-t showed no detectable
agonist effect at test concentrations of 100 µM (7c-
i,k ,l,n ,p -r ), 30 µM (7j,m ,o), or 10 µM (7s,t).
In Vivo P h a r m a cology. Compounds 7m and 7s
were tested for convulsant effects in mice after intrac-
erebroventricular (icv) and subcutaneous (sc) adminis-
tration following a previously described procedure.23
After icv administration, compounds 7m and 7s showed
potent convulsant activity with ED50 ) 0.024 µmol/kg
and ED50 ) 0.21 µmol/kg, respectively. After sc admin-
istration, none of the compounds 7m and 7s showed
significant convulsant effects, ED50 > 51 µmol /kg and
ED50 > 45 µmol/kg, respectively. The lack of in vivo
convulsant effects of the two compounds after sc ad-
ministration may reflect poor blood-brain barrier per-
meability or rapid metabolism. These aspects will be the
subject of further studies.
Molecu la r Mod elin g. We have previously reported
a pharmacophore model for GABAA receptor agonists
based on conformational analyses and superimposition
studies of muscimol (2) and 4-PIOL (5).19,24 In this
model, as shown in Figure 3, the anionic parts of 2 and
5 (the deprotonated 3-isoxazolol ring) interact with two
different conformations of an arginine residue. This
makes it possible for the cationic part (the substituted
ammonium ion) of the two compounds to overlap in
space. When low or inactive methyl-substituted ana-
logues of GABA, 2, 3, and 5 are fitted to the model,
regions of repulsive steric interactions (receptor es-
sential volumes) with the receptor could be identified.24
These are shown as tetrahedrons in Figure 3. The
corners of the tetrahedrons correspond to the positions
of the carbon and the three hydrogen atoms of the
methyl groups, causing a significant decrease in receptor
affinity. Thus, the tetrahedrons indicate regions in space
that are forbidden for high-affinity GABAA ligands. For
instance, the methyl group in 4-methylmuscimol (2a )
causes a very large decrease in the affinity (IC50 > 100
µM)25 compared to that of 2 (IC50 ) 0.006 µM).26 An
arrow in Figure 3 indicates the tetrahedron correspond-
ing to the position of the methyl group in 2a when fitted
to the pharmacophore model.
The following criteria were applied in the search for
a possible bioactive conformation of a 4-substituent:
(i) no part of the substituent is allowed to come closer
than 1 Å to any of the sterically “forbidden” regions
shown as tetrahedrons in Figure 3; (ii) the calculated
conformational energy penalties for binding, i.e., the
conformational energy required for the molecule to
adopt its bioactive conformation, should be low. Further
details of the computational procedures are given in the
Experimental Section. If more than one possible bioac-
tive conformation was found by this procedure, the one
with the lowest energy was selected as the most prob-
able one.
The conformational analysis of 7o resulted in only one
possible bioactive conformation, fulfilling the criteria
described above. This conformation with a calculated
conformational energy penalty of 2.6 kcal/mol is shown
in Figure 4a. The 1-naphthylmethyl substitutent in the
proposed bioactive conformation of 7o is close to or-
thogonal to the 3-isoxazolol ring and is located on the
same side of the 3-isoxozolol ring as the piperidine
nitrogen atom. The unsubstituted naphthyl ring is
pointing away from the 3-isoxazolol ring.
For the highest affinity compound in the present work
(the 2-naphthylmethyl analogue 7m ), two possible
conformations fulfilling the criteria were found. The
conformations display very low and similar calculated
conformational energy penalties for binding (0.5 and 0.8
kcal/mol). The two conformations, shown in Figure 4b,
differ only in the orientation of the distal aromatic ring.

Substituted Isoxazolol GABAA Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2459
F igu r e 4. Proposed bioactive conformations for some high-affinity 4-substituted analogues of 5. Compound 7m is illustrated in
two forms exhibiting different conformations of the 2-naphthylmethyl substituent.
As expected, the conformational analysis of the benzyl
analogue 7h yielded a bioactive conformation with a
low-energy penalty (1.1 kcal/mol) in which the phenyl
ring closely superimposes the substituted aromatic rings
of 7m and 7o. The addition of a phenyl ring to the
methyl-substituted compound 7a to give 7h leads to an
increase in the affinity by a factor of less than 3 (Table
1). In contrast, addition of an aromatic ring to 7h to
give the naphthyl systems in 7m and 7o increases the
affinity by factors of 78 and 38, respectively. The strong
effect on the affinity of the unsubstituted aromatic rings
F igu r e 5. Proposed bioactive conformation of 7k displaying
in 7m and 7o is not due to an increase of the lipophi-
licity as expressed by log P. The increase of log P by
replacing a methyl by a benzyl substituent is 1.43 log
units, whereas the increase by substituting a naphth-
ylmethyl for a benzyl substituent is smaller, 1.0 log unit,
the log P values being calculated according to the
method of Ghose and Crippen as implemented in the
Spartan program.27 This indicates that the distal aro-
matic rings in 7m and 7o have strong specific interac-
tions with the binding cavity of the GABAA receptor and
that the difference in affinity is not merely a conse-
quence of a difference in solvation energy.
The dimensions of the cavity accommodating the
4-substituents may be extended beyond that of a
2-naphthylmethyl group as demonstrated by the high
affinity of the biphenyl-4-methyl analogue 7l. Confor-
mational analysis resulted in two conformations fulfill-
ing the criteria for a bioactive conformation. The one in
Figure 4c has a calculated conformational energy pen-
alty of 0.9 kcal/mol; the other one, which has an energy
penalty of 1.1 kcal/mol, differs only by having the
opposite sign of the inter-ring torsion in the biphenyl
moiety.
electrostatic repulsion between one of the aromatic rings and
a putative electron-rich binding site (magenta sphere).
conflict with the receptor binding site interacting with
the axial piperidyl N+H presumably via a hydrogen
bond. Thus, this binding site should be negatively
charged or at least have a high electron density. As
shown in Figure 5, this putative binding site is located
close to a line orthogonal to one of the aromatic rings
in 7k and passing through the centroid of this ring. The
negative electrostatic potential of the 9-anthracyl sys-
tem at this position in space leads to an electrostatic
attraction with cations and consequently an electrostatic
repulsion with anions or electron-rich functional groups.28
This clearly shows that the aromatic ring system of the
benzyl substituent in 7h may be extended in a direction
pointing away from the 3-isoxazolol ring, as demon-
strated by the proposed bioactive conformation of 7o
(Figure 4a), but not in the opposite direction.
The reasonably high affinity of the diphenylmethyl
compound 7q and the very high affinity of the 3,3-
diphenylpropyl compound 7s demonstrate that the
binding cavity may be extended in a direction not
exploited by any of the other substituents. The most
probable bioactive conformation of the diphenylmethyl
compound 7q, according to the conformational analysis,
is shown in Figure 4d (conformational energy penalty
of 4.6 kcal/mol). One of its phenyl rings superimposes
well on the aromatic rings in 7l,m ,o as shown by the
Extending the 1-naphthyl ring system in 7o to the
9-anthracyl system in 7k decreases the affinity by a
factor of 59 (Table 1). Conformational analysis displays
a quite high conformational energy penalty for the most
probable bioactive conformation of 7k (6.9 kcal/mol). In
addition, the added aromatic ring is in electrostatic

2460 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Frølund et al.
4-substituents studied in the present work, may inter-
fere with the mechanism for ion channel opening in
GABAA receptors giving the compounds antagonistic
properties.
Con clu sion
We have here described a new series of competitive
GABAA antagonists that are derived from the low-
efficacy partial GABAA agonist 4-PIOL (5). With the aim
of further investigating the previously described recep-
tor cavity in the vicinity of the 4-position of the 3-isox-
azolol ring in 5, substituents of different size were
incorporated. The effects of alkyl, phenylalkyl, diphe-
nylalkyl, and naphthylalkyl substitution were investi-
gated. The substitution of the 4-position with an alkyl
or phenylalkyl group gave compounds with affinity and
potency comparable with those of 5, while introduction
of more bulky groups such as diphenylalkyl and naph-
thylalkyl groups gave rise to a marked increase in both
affinity and potency. The two most potent analogues of
the series containing a 2-naphthylmethyl and a 3,3-
diphenylpropyl substituent, compounds 7m and 7s,
respectively, were evaluated in vivo and showed potent
convulsant effects following icv administration. Molec-
ular modeling studies of the compounds synthesized
exposed a cavity of substantial size in the vicinity of the
4-position of 5. On the basis of conformational analysis
of the high-affinity compounds 7m and 7s containing a
diphenyl ring system in the 4-position in the 3-isoxazolol
ring, there seem to be additional sites for specific
receptor interactions in the above-mentioned cavity.
This study has provided new information on the
agonist and competitive antagonist binding site in the
GABAA receptor and is going to be exploited in further
development of the pharmacophore model. In addition,
the compounds described could serve as useful tools for
studies of the GABAA receptor mechanisms. Further
studies in this area are in progress.
F igu r e 6. Superimposition (in two orientations) of compounds
7l (magenta), 7m (green), 7o (blue), 7q (red), and 7s (yellow)
in their proposed bioactive conformations illustrating the large
space spanned by the 4-substituents. Hydrogen atoms except
on nitrogen have been omitted for clarity. Only the sterically
forbidden regions closest to the 4-substituents are shown.
superimposition in Figure 6, whereas the other phenyl
ring extends in an almost orthogonal direction. This is
also shown by the calculated bioactive conformation of
7s displayed in Figure 4e and Figure 6. In 7s, one of
the phenyl ring overlaps well with the distal ring of the
high-affinity 1-naphthylmethyl compound 7o whereas
the other phenyl ring occupies an unexplored region in
space in the pharmacophore model. The observation
that one of the phenyl rings in 7s overlaps well with
the distal aromatic ring in 7o rationalizes the affinity
difference between 7s and 7q. As concluded above, this
position of an aromatic ring is highly favorable for the
receptor affinity and it is not possible for any of the
aromatic rings in 7q to be positioned in this region in
space.
The superimposition of the proposed bioactive con-
formations of 7l,m ,o,q,s shown in Figure 6 demon-
strates the large space spanned by the high-affinity
compounds in the present series of analogues of 4-PIOL
(5) and that the receptor cavity in the vicinity of the
4-position in (5) is of considerable dimensions. The
length of the biphenyl-4-methyl substituent in 7l is 9.7
Å as measured from the methyl carbon to the para
hydrogen atom of the distal ring, and one of the phenyl
rings in 7s extends 6.4 Å in a direction almost perpen-
dicular to the rings in 7l,m ,o.
As described above, the compounds studied in the
present work all display antagonistic properties. There
is strong evidence that the GABA binding site in the
GABAA receptor is located between an R- and a â-sub-
unit.29 It may be speculated that the large cavity
accommodating the 4-substituents of the compounds
studied in the present work is located in the space
between these subunits, a space that may extend into
the aqueous phase outside the receptor. GABAA recep-
tors belong to the same superfamily as nicotinic acetyl-
choline receptors. On the basis of electron microscopic
image analyses, it has been proposed that the mecha-
nism for ligand-induced channel opening in nicotinic
acetylcholine receptors involves rotations of the subunits
in the ligand binding domain.30,31 It is likely that other
receptors in the same superfamily use a similar mech-
anism for channel opening. Large substituents, as the
Exp er im en ta l Section
Ch em istr y. Gen er a l P r oced u r es. Melting points were
1
determined in capillary tubes and are uncorrected. H NMR
spectra were recorded on a Varian Gemini 2000 (300 MHz)
instrument in CDCl3 solutions using TMS as an internal
standard or in D2O solutions using 1,4-dioxane as an internal
standard. Column chromatography (CC) was performed on
Merck silica gel 60 (0.06-0.200 mm). Analytical thin-layer
chromatography (TLC) was carried out using Merck silica gel
60 F254 plates. All compounds were detected as single spots
on TLC plates and visualized using UV light and KMnO4
spraying reagent. Compounds containing amino groups were
also visualized using a ninhydrin spraying reagent. Com-
pounds containing the 3-isoxazolol unit were visualized using
a FeCl3 spraying reagent. Elemental analyses were performed
at Analytical Research Department, H. Lundbeck A/S, Den-
mark, or by Mr. J . Theiner, Department of Physical Chemistry,
University of Vienna, Austria, and are within (0.4% of the
calculated values unless otherwise stated.
Eth yl 1-Meth oxyca r bon yl-4-m eth yl-4-p ip er id in eca r -
boxyla te (9). To a solution of diisopropylamine (4.0 mL, 28
mmol) in anhydrous THF (100 mL) was added dropwise a
solution of n-butyllithium in hexane (1.6 M, 18 mL, 28 mmol).
The reaction mixture was stirred at 0 °C for 15 min. After it
was cooled to -70 °C, a solution of ethyl 1-methoxycarbonyl-
4-piperidinecarboxylate16 (5.6 g, 26 mmol) in THF (40 mL) was
added dropwise over 15 min, and the reaction mixture was
then stirred at -70 °C for 1 h. A solution of methyl iodide (2.4
mL, 39 mmol) in THF (40 mL) was added dropwise over 15
min, and the reaction mixture was stirred for 4 h, during which

Substituted Isoxazolol GABAA Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2461
time the temperature was allowed to rise to room temperature.
The reaction mixture was quenched with saturated aqueous
ammonium chloride (60 mL) and extracted with EtOAc (3 ×
100 mL). The combined extracts were evaporated, and CC
(toluene/EtOAc (1:1)) gave the product as an oil (4.9 g, 82%).
1H NMR (200 MHz, CDCl3): δ 4.17 (2H, q, J ) 7.1 Hz), 3.91-
3.70 (2H, m), 3.68 (3H, s), 3.10-2.94 (2H, m), 2.14-2.00 (2H,
m), 1.49-1.29 (2H, m), 1.27 (3H, t, J ) 7.1 Hz), 1.20 (3H, s).
Anal. (C11H19NO4) C, H, N.
1-Met h oxyca r bon yl-4-m et h yl-4-p ip er id in eca r boxylic
Acid (10). A mixture of 9 (4.9 g, 21 mmol) in 2 M sodium
hydroxide (20 mL) was stirred at 100 °C for 30 min. The
reaction mixture was cooled to 0 °C, and 2 M HCl was added
until pH 10 was attained. Methyl chloroformate (3.3 mL, 43
mmol) was added dropwise, and stirring was continued at 0
°C for 1 h followed by stirring at room temperature for 1 h.
The mixture was extracted with CH2Cl2 (3 × 50 mL), and the
combined extracts were dried, evaporated, and subjected to
CC (toluene/EtOAc (4:1)) to give the product (3.18 g, 74%). 1H
NMR (200 MHz, CDCl3): δ 3.91-3.70 (2H, m), 3.69 (3H, s),
3.24-3.00 (2H, m), 2.18-1.98 (2H, m), 1.52-1.16 (2H, m), 1.29
(3H, s). Anal. (C9H15NO4) C, H, N.
Eth yl 3-(1-Meth oxyca r bon yl-4-m eth yl-4-p ip er id yl)-3-
oxop r op ion a te (11). To a solution of N,N′-thionyldiimida-
zole20 (21 mmol) in THF (60 mL) was added dropwise a solution
of 10 (3.2 g, 16 mmol) in THF (8 mL). The reaction mixture
was protected from light, and stirring was continued for 16 h
at room temperature. The solution was added dropwise to a
suspension of [3-ethoxy-3-hydroxyacrylato(2-)-O1,O3]magnesate-
(2+)16 (48 mmol) in THF (60 mL). The mixture was stirred for
2 h and acidified with HCl (4 M), and stirring was continued
for 30 min. THF was distilled off in vacuo, and the aqueous
residue was extracted with Et2O (3 × 50 mL). The combined
extracts were washed with saturated aqueous NaHCO3 (3 ×
50 mL) and then with H2O (3 × 30 mL), dried, and evaporated
to give the crude product as an oil (2.29 g, 53%). 1H NMR (200
MHz, CDCl3): δ 4.29-4.10 (3H, m), 3.69 (3H, s), 3.50 (2H, s),
3.39-3.95 (3H, m), 2.15-1.95 (2H, m), 1.49-1.16 (8H, m).
(12.7 g, 75 mmol) in EtOAc (125 mL) cooled to 0-5 °C was
added triethylamine (26 mL, 190 mmol) followed by MgCl2 (8.6
g, 91 mmol). The mixture was slowly heated to 35 °C and then
maintained at 35 °C for 6 h. The reaction mixture was cooled
to 0 °C, and 14 (11 g, 54 mmol) was added dropwise over 30
min. Stirring was continued overnight at room temperature,
and then the mixture was cooled to 0 °C and 4 M HCl (110
mL) was added slowly (T < 25 °C). The aquous phase was
extracted with toluene (3 × 100 mL), and the combined organic
phases were washed with 2% NaHCO3 (2 × 40 mL) followed
by water (2 × 25 mL). The organic phase was dried and
evaporated. CC (toluene/EtOAc (4:1)) gave 1516 (8.9 g, 65%)
1
as an oil. H NMR (300 MHz, CDCl3): δ 4.17 (2H, q, J ) 7.2
Hz), 4.13 (2H, broad s), 3.66 (3H, s), 3.47 (2H, s), 2.88-2.98
(2H, m), 2.67-2.57 (1H, m), 1.90-1.79 (2H, m), 1.60-1.47 (2H,
m), 1.25 (3H, t, J ) 7.2 Hz).
Eth yl 3-(1-Meth oxyca r bon yl-4-p ip er id yl)-2-m eth yl-3-
oxop r op ion a te (16a ). 15 (3.0 g, 12 mmol) was added to a
solution of NaOEt (12 mmol) in EtOH (20 mL). Methyl iodide
(1.2 mL, 13 mmol) was dropwise added to the reaction mixture
at 80 °C, and stirring was continued at 80 °C for 16 h.
Filtration and evaporation followed by CC (toluene/EtOAc (3:
1)) gave 16a as a colorless oil (1.7 g, 53%). 1H NMR (300 MHz,
CDCl3): δ 4.12-4.03 (4H, m), 3.71-3.61 (1H, m), 3.68 (3H, s),
2.92-2.62 (3H, m), 1.90-1.78 (2H, m), 1.76-1.40 (2H, m), 1.32
(3H, d, J ) 7.4 Hz), 1.25 (3H, t, J ) 7.2 Hz). Anal. (C13H21
NO5) C, H, N.
-
5-(1-Met h oxyca r b on yl-4-p ip er id yl)-4-m et h yl-3-isox-
a zolol (17a ). 17a was synthesized as described for 12 by using
16a (1.6 g, 5.9 mmol) and NaOH (250 mg, 6.3 mmol) in MeOH/
water (5 mL/0.3 mL), hydroxylamine hydrochloride (820 mg,
11.8 mmol) and NaOH (472 mg, 11.8 mmol) in MeOH/water
(6 mL/6 mL), and concentrated HCl (2.6 mL). The crude
product was purified by CC (toluene/EtOAc (4:1) containing
AcOH (1%)) and gave 17a (810 mg, 57%). A sample was
1
recrystallized (toluene/light petroleum): mp 145-147 °C. H
NMR (300 MHz, CDCl3): δ 10.70 (1H, broad s), 4.21 (2H, broad
s), 3.70 (3H, s), 2.91-2.81 (3H, m), 1.87 (3H, s), 1.81-1.73 (4H,
m). Anal. (C11H16N2O4) C, H, N.
5-(1-Met h oxyca r b on yl-4-m et h yl-4-p ip er id yl)-3-isox-
a zolol (12). A solution of 11 (2.0 g, 7.4 mmol) in MeOH (0.5
mL) was added dropwise to a solution of NaOH (310 mg, 7.8
mmol) in MeOH (7 mL) and water (0.5 mL) at -30 °C. After
the mixture was stirred for 10 min, a solution of hydroxylamine
hydrochloride (1.0 g, 15 mmol) and NaOH (620 mg, 15 mmol)
in MeOH/water (7 mL/0.5 mL) was added at -30 °C. Stirring
was continued for 2 h at -30 °C, and the reaction mixture
was poured into concentrated HCl (1.5 mL) at 80 °C and heated
at 80 °C for 2 h. After the mixture was cooled, the organic
solvent was evaporated and the residue was extracted with
Et2O (3 × 50 mL). The combined extracts were dried and
evaporated. CC (toluene/EtOAc (1:1) containing AcOH (1%))
of the crude product gave the product as an oil (1.0 g, 57%).
1H NMR (200 MHz, CDCl3): δ 10.70 (1H, broad s), 5.70 (1H,
s), 3.79-3.61 (2H, m), 3.70 (3H, s), 3.31-3.14 (2H, m), 2.11-
1.97 (2H, m), 1.69-1.51 (2H, m), 1.31 (3H, s). Anal. (C11H16N2O4)
C, H, N.
5-(4-Meth yl-4-piper idyl)-3-isozaxolol Hydr obr om ide (6).
A solution of 12 (360 mg, 1.6 mmol) in a solution of HBr in
AcOH (33%, 15 mL) was stirred at room temperature for 16
h. The reaction mixture was evaporated, and the residue was
recrystallized (MeOH/Et2O) to give 6 (210 mg, 52%). 1H NMR
(200 MHz, D2O): δ 5.93 (1H, s), 3.36-3.25 (2H, m), 3.11-2.97
(2H, m), 2.31-2.23 (2H, m), 1.92-1.77 (2H, m), 1.31 (3H, s).
Anal. (C9H14N2O2‚HBr‚0.5H2O) C, H, N.
4-Met h yl-5-(4-p ip er id yl)-3-isoxa zolol H yd r ob r om id e
(7a ). A solution of 17a (1.0 g, 4.4 mmol) in a solution of HBr
in AcOH (33%, 50 mL) was stirred at room temperature for
16 h. The reaction mixture was evaporated, and the residue
was recrystallized (MeOH/Et2O) to give 7a (950 mg, 82%): mp
>250 °C. 1H NMR (200 MHz, D2O): δ 3.46-3.38 (2H, m),
3.21-2.98 (3H, m), 2.00-1.80 (4H, m), 1.74 (3H, s). Anal.
(C9H14N2O2‚HBr) H, Br, N; C, calcd, 54.97, found, 55.58.
E t h yl 2-E t h yl-3-(1-m et h oxyca r b on yl-4-p ip er id yl)-3-
oxop r op ion a te (16b). 16b was synthesized as described
above for 16a by using 9 (3.0 g, 11.7 mmol), NaOEt (11.7 mmol)
in EtOH (20 mL), and ethyl bromide (0.96 mL, 12.8 mmol).
The crude product was purified by CC (toluene/EtOAc (4:1))
1
to give the title compund as an oil (1.9 g, 58%). H NMR (300
MHz, CDCl3): δ 4.22-4.03 (4H, m), 3.70 (3H, s), 3.58 (1H, t,
J ) 7.3 Hz), 2.79-2.61 (3H, m), 1.98-1.78 (4H, m), 1.77-1.46
(2H, m), 1.25 (3H, t, J ) 7.3 Hz), 0.98 (3H, t, J ) 7.3 Hz).
Anal. (C14H23NO5) C, H, N.
4-E t h yl-5-(1-m e t h oxyca r b on yl-4-p ip e r id yl)-3-isox-
a zolol (17b). 17b was synthesized as described for 12 by using
16b (1.8 g, 6.3 mmol) and NaOH (270 mg, 6.7 mmol) in MeOH/
water (6 mL/0.3 mL), hydroxylamine hydrochloride (870 mg,
13 mmol) and NaOH (500 mg, 13 mmol) in MeOH/water (8
mL/8 mL), and concentrated HCl (2.8 mL). The crude product
was purified by CC (toluene/EtOAc (4:1) containing AcOH
1
1-Meth oxyca r bon yl-4-p ip er id in eca r boxylic Acid Ch lo-
r id e (14). A mixture of 1316 (12 g, 64 mmol), thionyl chloride
(14 mL, 190 mmol), and DMF (0.5 mL) was stirred at 85 °C
for 20 min. The reaction mixture was evaporated, and ball-
tube distillation (0.1 mmHg; oven temperature, 200 °C) of the
residue gave 14 (11 g, 83%). 1H NMR (200 MHz, CDCl3): δ
4.19-4.00 (2H, m), 3.69 (3H, s), 3.02-2.82 (3H, m), 2.16-2.00
(2H, m), 1.90-1.61 (2H, m).
(1%)) and gave 17b (900 mg, 56%): mp 129-132 °C. H NMR
(300 MHz, CDCl3): δ 10.36 (1H, broad s), 4.24 (2H, broad s),
3.71 (3H, s), 2.93-2.81 (3H, m), 2.34 (2H, q, J ) 7.5 Hz), 1.83-
1.77 (4H, m), 1.15, (3H, J ) 7.5 Hz). Anal. (C12H18N2O4) C, H,
N.
4-Eth yl-5-(4-p ip er id yl)-3-isoxa zolol (7b). 7b was syn-
thesized as described for 7a by using 17b (1.6 g, 6.3 mmol)
and HBr in AcOH (33%, 80 mL). Recrystallization (MeOH/
Et2O) gave the title compound (712 mg, 41%): mp > 250 °C.
1H NMR (300 MHz, D2O): δ 3.50-3.38 (2H, m), 3.23-2.90 (3H,
Eth yl 3-(1-Meth oxyca r bon yl-4-p ip er id yl)-3-oxop r op i-
on a te (15). To a stirred mixture of potassium ethyl malonate

2462 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Frølund et al.
m), 1.99 (2H, q, J ) 7.6 Hz), 2.10-1.79 (4H, m), 0.95 (3H, t, J
) 7.6 Hz). Anal. (C10H16N2O2‚HBr‚0.25H2O) C, H, Br, N.
Eth yl 3-(1-Meth oxyca r bon yl-4-p ip er id yl)-2-p r op yl-3-
oxop r op ion a te (16c). 16c was synthesized as described above
for 16a by using 15 (3.0 g, 12 mmol), NaOEt (12 mmol) in
EtOH (20 mL), and propyl bromide (1.2 mL, 12 mmol). The
crude product was purified by CC (toluene/EtOAc (6:1)) to give
the title compound as an oil (2.64 g, 75%). 1H NMR (200 MHz,
CDCl3): δ 4.19 (2H, q, J ) 7.2 Hz), 4.23-4.07 (2H, broad s),
3.69 (3H, s), 3.60 (1H, t, J ) 7.2 Hz), 2.99-2.58 (3H, m), 1.98-
1.40 (6H, m), 1.28 (5H, m), 1.25 (3H, t, J ) 7.2 Hz), 0.98 (3H,
t, J ) 7.2 Hz).
16e (2.2 g, 6.6 mmol), NaOH (280 mg, 7.0 mmol) in MeOH/
water (7 mL/0.35 mL), hydroxylamine hydrochloride (920 mg,
13 mmol) and NaOH (530 mg, 13.2 mmol) in MeOH/water (10
mL/10 mL), and concentrated HCl (3.0 mL). The crude product
was purified by CC (toluene/EtOAc (4:1) containing AcOH
(1%)) and gave the pure product (690 mg, 30%): mp 95-97
°C. 1H NMR (300 MHz, CDCl3): δ 10.41 (1H, broad s), 4.21
(2H, broad s), 3.71 (3H, s), 2.92-2.79 (3H, m), 2.28 (2H, t, J )
7.2 Hz), 1.81-1.74 (4H, m), 1.52-1.46 (2H, m), 1.32-1.25 (6H,
m), 0.87 (3H, J ) 6.9 Hz). Anal. (C16H26N2O4) C, H, N.
4-Hexyl-5-(4-piper idyl)-3-isoxazolol Hydr obr om ide (7e).
7e was synthesized as described above for 7a by using 17e
(620 mg, 2.0 mmol) in HBr in AcOH (33%, 30 mL). Recrystal-
lization (MeOH/Et2O) gave the title compound (290 mg, 43%):
mp 228-232 °C. 1H NMR (300 MHz, D2O): δ 3.4-3.35 (2H,
m), 3.10-2.94 (3H, m), 2.17 (2H, t, J ) 7.2 Hz), 1.93-1.85 (4H,
m), 1.34-1.29 (2H, m), 1.1-1.08 (6H, m), 0.67 (3H, t, J ) 7.2
Hz). Anal. (C14H24N2O2‚HBr) C, H, Br, N.
5-(1-Me t h oxyca r b on yl-4-p ip e r id yl)-4-p r op yl-3-isox-
a zolol (17c). 17c was synthesized as described for 12 by using
16c (1.5 g, 5.1 mmol) and NaOH (210 mg, 5.3 mmol) in MeOH/
water (6 mL/0.3 mL), hydroxylamine hydrochloride (700 mg,
10 mmol) and NaOH in MeOH/water (7 mL/7 mL), and
concentrated HCl (2.0 mL). The crude product was purified
by CC (toluene/EtOAc (4:1) containing AcOH (1%)) and gave
Eth yl 3-(1-Meth oxyca r bon yl-4-p ip er id yl)-2-octyl-3-ox-
op r op ion a te (16f). 16f was synthesized as described above
for 16a using 15 (3.0 g, 11.7 mmol), NaOEt (11.7 mmol) in
EtOH (20 mL), and 1-bromooctane (2.2 mL, 12.8 mmol). The
crude product was purified by CC (toluene/EtOAc (8:1)) to give
1
17c (560 mg, 40%). H NMR (300 MHz, CDCl3): δ 8.52 (1H,
broad s), 4.22 (2H, broad s), 3.70 (3H, s), 2.90-2.70 (3H, m),
2.28 (2H, q, J ) 7.5 Hz), 1.85-1.68 (4H, m), 1.56 (2H, q, J )
7.3 Hz), 1.15-0.70, (3H, m). Anal. (C13H20N2O4) C, H, N.
5-(4-P ip er id yl)-4-p r op yl-3-isoxa zolol H yd r ob r om id e
(7c). 7c was synthesized as described for 7a by using 17c (0.6
g, 2.2 mmol) and HBr in AcOH (33%, 20 mL). Recrystallization
(MeOH/Et2O) gave the title compound (400 mg, 61%): mp >
200 °C. 1H NMR (200 MHz, D2O): δ 3.48-3.38 (2H, m), 3.10-
2.90 (3H, m), 2.28-1.88 (6H, m), 1.51 (2H, sex, J ) 7.4 Hz),
0.91 (3H, t, J ) 7.4 Hz). Anal: (C11H18N2O2‚HBr‚0.5H2O) C,
H, N.
1
the title compund as an oil (2.5 g, 58%). H NMR (300 MHz,
CDCl3): δ 4.17 (2H, q, J ) 7.2 Hz), 4.21-4.09 (2H, m), 3.68
(3H, s), 3.58 (1H, t, J ) 7.2 Hz), 2.89-2.78 (2H, m), 2.68 (1H,
dt, J )11.1 and 3.6 Hz), 1.84-1.78 (4H, m), 1.70-1.48 (3H,
m), 1.4-1.25 (14H, m), 0.87 (3H, t, J ) 7.2 Hz).
5-(1-Me t h o x y c a r b o n y l-4-p ip e r id y l)-4-o c t y l-3-is o x -
a zolol (17f). 17f was synthesized as described for 12 by using
16f (2.5 g, 6.8 mmol) and NaOH (300 mg, 7.5 mmol) in MeOH/
water (12 mL/0.7 mL), hydroxylamine hydrochloride (945 mg,
13.6 mmol) and NaOH (544 mg, 13.6 mmol) in MeOH/water
(15 mL/5 mL), and concentrated HCl (3.0 mL). The crude
product was purified by CC (toluene/EtOAc (8:1) containing
E t h yl 2-Bu t yl-3-(1-m et h oxyca r b on yl-4-p ip er id yl)-3-
oxop r op ion a te (16d ). 16d was synthesized as described for
16a using 15 (3.0 g, 12 mmol), NaOEt (12 mmol) in EtOH (20
mL), and butyl bromide (1.4 mL, 13 mmol). The crude product
was purified by CC (toluene/EtOAc (8:1)) to give the title
1
AcOH (1%)) and gave the pure product (1.0 g, 44%). H NMR
1
(300 MHz, CDCl3): δ 9.94 (1H, broad s), 4.15 (2H, broad s),
3.65 (3H, s), 2.83-2.73 (3H, m), 2.22 (2H, t, J ) 7.2 Hz), 1.76-
1.68 (4H, m), 1.50-1.38 (2H, m), 1.26-1.08 (10H, m), 0.80 (3H,
t, J ) 7.2 Hz).
compound as an oil (2.4 g, 65%). H NMR (300 MHz, CDCl3):
δ 4.18 (2H, q, J ) 7.2 Hz), 4.21-4.13 (2H, m), 3.69 (3H, s),
3.58 (1H, t, J ) 7.2 Hz), 2.91-2.79 (2H, m), 2.74-2.61 (1H,
m), 1.92-1.75 (4H, m), 1.70-1.43 (2H, m), 1.34-1.18 (4H, m),
1.26 (3H, t, J ) 6.9 Hz), 0.89 (3H, t J ) 7.2 Hz). Anal. (C16H27
NO5) C, H, N.
-
5-(4-P ip er id yl)-4-octyl-3-isoxa zolol Hyd r obr om id e (7f).
7f was synthesized as described for 7a by using 17f (500 mg,
1.4 mmol) in HBr in AcOH (33%, 15 mL). Recrystallization
(MeOH/Et2O) gave the title compound (225 mg, 42%): mp
4-Bu t yl-5-(1-m e t h oxyca r b on yl-4-p ip e r id yl)-3-isox-
a zolol (17d ). 17d was synthesized as described for 10 by using
16a (1.8 g, 5.7 mmol), NaOH (240 mg, 6.1 mmol) in MeOH/
water (6 mL/0.3 mL), hydroxylamine hydrochloride (790 mg,
11 mmol) and NaOH (460 mg, 11 mmol) in MeOH/water (9
mL/9 mL), and concentrated HCl (2.5 mL). The crude product
was purified by CC (toluene/EtOAc (4:1) containing AcOH
1
226-234 °C. H NMR (300 MHz, CD3OD): δ 3.49-3.45 (2H,
m), 3.19-3.10 (3H, m), 2.32 (2H, t, J ) 7.2 Hz), 2.07-2.04 (4H,
m), 1.60-1.42 (2H, m), 1.39-1.22 (10H, m), 0.89 (3H, t, J )
6.6 Hz) Anal. (C16H28N2O2‚HBr) C, H, Br, N.
Meth yl 2-Ben zyl-3-(1-m eth oxyca r bon yl-4-p ip er id yl)-3-
oxop r op ion a te (16h ). 16h was synthesized as described
above for 16a using 15 (3.0 g, 11.7 mmol), NaOMe (11.7 mmol)
in MeOH (20 mL), and benzyl bromide (1.5 mL, 12.8 mmol).
The crude product was purified by CC (toluene/EtOAc (3:1))
1
(1%)) and gave the pure product (640 mg, 40%) as an oil. H
NMR (300 MHz, CDCl3): δ 10.41 (1H, broad s), 4.19 (2H, broad
s), 3.68 (3H, s), 2.88-2.79 (3H, m), 2.27 (2H, t, J ) 7.2 Hz),
1.80-1.74 (4H, m), 1.48 (2H, q, J ) 7.5 Hz), 1.31 (2H, sex, J
) 7.2 Hz), 0.89 (3H, J ) 7.5 Hz). Anal. (C14H22N2O4) C, H, N.
4-Bu tyl-5-(4-piper idyl)-3-isoxazolol Hydr obr om ide (7d).
7d was synthesized as described above for 7a by using 17d
(440 mg, 1.6 mmol) in HBr in AcOH (33%, 22 mL). Recrystal-
lization (MeOH/Et2O) gave the title compound (220 mg, 73%):
1
to give the title compund as an oil (2.4 g, 60%). H NMR (300
MHz, CDCl3): δ 7.32-7.02 (5H, m), 4.03 (2H, broad s), 3.98
(2H, t, J ) 7.2 Hz), 3.70 (3H, s), 3.68 (3H, s), 3.19 (2H, d, J )
7.2 Hz), 2.82-2.60 (2H, m), 2.50-2.41 (1H, m), 1.78-1.63 (1H,
m), 1.60-1.41 (2H, m), 1.24-1.50 (1H, m). Anal. (C18H23NO5)
C, H, N.
1
mp 218-221 °C. H NMR (300 MHz, D2O): δ 3.45-3.39 (2H,
m), 3.16-2.98 (3H, m), 2.21 (2H, t, J ) 7.2 Hz), 2.01-1.89 (4H,
m), 1.35 (2H, q, J ) 7,2 Hz), 1.18 (2H, sex, J ) 7.2 Hz), 0.77
(3H, t, J ) 7.2 Hz). Anal. (C12H20N2O2‚HBr) C, H, Br, N.
E t h yl 2-H exyl-3-(1-m et h oxyca r b on yl-4-p ip er id yl)-3-
oxop r op ion a te (16e). 16e was synthesized as described
above for 16a using 15 (3.0 g, 11.7 mmol), NaOEt (11.7 mmol)
in EtOH (20 mL), and 1-bromohexane (1.8 mL, 12.8 mmol).
The crude product was purified by CC (toluene/EtOAc (10:1))
4-Be n zyl-5-(1-m e t h oxyca r b on yl-4-p ip e r id yl)-3-isox-
a zolol (17h ). 17h was synthesized as described for 12 by using
16h (2.4 g, 7.1 mmol) and NaOH (300 mg, 7.5 mmol) in MeOH/
water (7 mL/0.35 mL), hydroxylamine hydrochloride (990 mg,
14.2 mmol) and NaOH (570 mg, 14.2 mmol) in MeOH/water
(10 mL/10 mL), and concentrated HCl (3.0 mL). The crude
product was purified by CC (toluene/EtOAc (4:1) containing
AcOH (1%)) and gave the pure product (970 mg, 43%): mp
1
1
to give the title compund as an oil (2.3 g, 60%). H NMR (300
195-196 °C. H NMR (300 MHz, CDCl3): δ 10.41 (1H, broad
MHz, CDCl3): 4.20-4.10 (4H, m), 3.68 (3H, s), 3.57 (1H, t, J
) 7.2 Hz), 2.87-2.78 (2H, m), 2.70-2.62 (1H, m), 1.84-1.77
(4H, m), 1.66-1.47 (2H, m), 1.27-1.24 (11H, m), 0.85 (3H, t,
J ) 7.2 Hz). Anal. (C18H31NO5) C, H, N.
4-H e xyl-5-(1-m e t h oxyca r b on yl-4-p ip e r id yl)-3-isox-
a zolol (17e). 17e was synthesized as described for 10 by using
s), 7.34-7.10 (5H, m), 4.20-4.00 (2H, m), 3.68 (5H, s), 2.93-
2.69 (3H, m), 1.93-1.40 (4H, m). Anal. (C17H20N2O4) C, H, N.
4-Ben zyl-5-(4-p ip er id yl)-3-isoxa zolol H yd r ob r om id e
(7h ). 7h was synthesized as described above for 7a by using
17h (600 mg, 1.9 mmol) in HBr in AcOH (33%, 30 mL).
Recrystallization (MeOH/Et2O) gave the title compound (340

Substituted Isoxazolol GABAA Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2463
mg, 53%): mp 226-234 °C. 1H NMR (300 MHz, D2O): δ 7.27-
7.13 (5H, m), 3.61 (2H, s), 3.38-3.31 (2H, m), 3.04-2.87 (3H,
m), 1.89-1.82 (4H, m). Anal. (C15H18N2O2‚HBr) C, H, Br, N.
E t h yl 3-(1-Met h oxyca r b on yl-4-p ip er id yl)-3-oxo-2-(2-
p h en yleth yl)p r op ion a te (16i). 16i was synthesized as de-
scribed for 16a using 15 (3.0 g, 12 mmol), NaOEt (12 mmol)
in EtOH (20 mL), and 2-phenylethyl bromide (1.7 mL, 13
mmol). The crude product was purified by CC (toluene/EtOAc
(4:1)) to give the title compund as an oil (2.7 g, 64%). 1H NMR
(300 MHz, CDCl3): δ 7.28-7.15 (5H, m), 4.20-4.00 (4H, m),
3.68 (3H, s), 3.60 (1H, t, J ) 7.2 Hz), 2.94-2.72 (2H, m), 2.64-
2.56 (2H, m), 2.24-2.09 (2H, m), 1.92-1.40 (5H, m), 1.25 (3H,
t, J ) 7.2 Hz).
5-(1-Meth oxyca r bon yl-4-p ip er id yl)-4-(2-p h en yleth yl)-
3-isoxa zolol (17i). 17i was synthesized as described for 12
using 16i (2.3 g, 6.4 mmol) and NaOH (270 mg, 6.7 mmol) in
MeOH/water (7 mL/0.35 mL), hydroxylamine hydrochloride
(890 mg, 13 mmol) and NaOH (510 mg, 13 mmol) in MeOH/
water (9 mL/9 mL), and concentrated HCl (3.0 mL). The crude
product was purified by CC (toluene/EtOAc (2:1) containing
AcOH (1%)) and gave the pure product (1.0 g, 49%): mp 159-
161 °C. 1H NMR (300 MHz, CDCl3): δ 10.79 (1H, broad s),
7.29-7.10 (5H, m), 4.08 (2H, broad s), 3.68 (3H, s), 2.88-2.68
(2H, m), 2.64-2.60 (4H, m), 2.38-2.30 (1H, m), 1.60-1.47 (2H,
m), 1.30-1.26 (2H, m). Anal. (C18H22N2O4) C, H, N.
4-(9-An t h r a cylm et h yl)-5-(1-m et h oxyca r b on yl-4-p ip -
er id yl)-3-isoxa zolol (17k ). 17k was synthesized as described
for 12 by using 16k (1.8 g, 4.0 mmol) and NaOH (160 mg, 4.0
mmol) in MeOH/water (6 mL/0.3 mL), hydroxylamine hydro-
chloride (550 mg, 7.9 mmol) and NaOH (320 mg, 7.9 mmol) in
MeOH/water (7 mL/7 mL), and concentrated HCl (2.5 mL). The
crude product was purified by CC (toluene/EtOAc (4:1) con-
taining AcOH (1%)) and gave the pure product (250 mg,
15%): mp 213-215 °C. 1H NMR (300 MHz, CDCl3): δ 8.43 (1H,
s), 8.24-8.21 (2H, m), 8.03-8.00 (2H, m), 7.53-7.44 (4H, m),
4.66 (2H, s), 3.70-3.55 (2H, m), 3.54 (3H, s), 1.60-1.41 (2H,
m), 1.24-1.02 (3H, m), 0.60-0.31 (2H, m).
4-(9-An th r a cylm eth yl)-5-(4-p ip er id yl)-3-isoxa zolol Hy-
d r obr om id e (7k ). 7k was synthesized as described for 7a by
using 17k (100 mg, 0.24 mmol) in HBr in AcOH (33%, 10 mL).
Recrystallization (MeOH/Et2O) gave the title compound (61
mg, 58%): mp >250 °C. 1H NMR (300 MHz, D2O): δ 8.44 (1H,
s), 8.22 (2H, d, J ) 8.4 Hz), 8.05 (2H, d, J ) 8.4 Hz), 4.52 (2H,
s), 2.96-2.82 (2H, m), 2.01-1.76 (3H, m), 1.47-1.28 (2H, m),
0.8-0.75 (2H, m). Anal. (C23H22N2O2‚HBr‚1H2O) C, H, N.
E t h yl 3-(1-Met h oxyca r b on yl-4-p ip er id yl)-3-oxo-2-(4-
p h en ylben zyl)p r op ion a te (16l). 16l was synthesized as
described for 16a using 15 (3.0 g, 12 mmol), NaOEt (12 mmol)
in EtOH (20 mL), and 4-phenylbenzyl bromide32 (3.2 g, 13
mmol). The crude product was purified by CC (toluene/EtOAc
(10:1)) to give the title compund as an oil (2.95 g, 60%). 1H
NMR (300 MHz, CDCl3): δ 7.56 (2H, d, J ) 7.2 Hz), 7.50 (2H,
d, J ) 7.2 Hz), 7.42 (2H, t, J ) 8.1 Hz), 7.34 (1H, d, J ) 7.2
Hz), 7.24 (2H, d, J ) 8.4 Hz), 4.16 (2H, q, J ) 7.5 Hz), 4.09
(2H, broad s), 3.97 (1H, t, J ) 7.5 Hz), 3.65 (3H, s), 3.20 (2H,
d, J ) 7.2 Hz), 2.95-2.63 (2H, m), 2.58-2.41 (1H, m), 1.94-
1.45 (3H, m), 1.35-1.10 (1H, m), 1.21 (3H, t, J ) 7.2 Hz).
5-(1-Meth oxyca r bon yl-4-p ip er id yl)-4-(4-p h en ylben zyl)-
3-isoxa zolol (17l). 17l was synthesized as described for 12
by using 16l (2.6 g, 6.0 mmol) and NaOH (260 mg, 6.4 mmol)
in MeOH/water (8 mL/0.8 mL), hydroxylamine hydrochloride
(830 mg, 12 mmol) and NaOH (480 mg, 12 mmol) in MeOH/
water (9 mL/9 mL), and concentrated HCl (2.8 mL). The crude
product was purified by CC (toluene/EtOAc (4:1) containing
AcOH (1%)) and gave the product (1.3 g, 55%). An analytical
sample was recrystallized (toluene/light petroleum): mp 169-
170 °C. 1H NMR (300 MHz, CDCl3): δ 7.58-7.26 (9H, m), 4.17
(2H, broad s), 3.73 (2H, s), 3.69 (3H, s), 2.85-2.75 (3H, m),
1.79-1.71 (4H, m). Anal. (C23H24N2O4) C, H, N.
4-(4-P h en ylben zyl)-5-(4-p ip er id yl)-3-isoxa zolol Hyd r o-
br om id e (7l). 7l was synthesized as described for 7a by using
17l (600 mg, 1.5 mmol) in HBr in AcOH (33%, 30 mL).
Recrystallization (MeOH/Et2O) gave the title compound (400
mg, 63%): mp >240 °C. 1H NMR (300 MHz, D2O): δ 7.37-
7.29 (4H, m), 7.17-7.00 (5H, m), 3.49 (2H, s), 2.66 (2H, d, J )
12.3 Hz), 2.41-2.23 (1H, m), 2.06 (2H, t, J ) 12.3 Hz), 1.44-
1.29 (4H, m). Anal. (C21H22N2O2‚HBr) C, H, Br, N.
Eth yl 3-(1-Meth oxyca r bon yl-4-p ip er id yl)-2-(2-n a p h th -
ylm eth yl)-3-oxop r op ion a te (16m ). 16m was synthesized as
described above for 16a using 15 (15 g, 59 mmol), NaOEt (59
mmol) in EtOH (100 mL), and 2-(bromomethyl)naphthalene
(14 g, 64 mmol). The crude product was purified by CC
(toluene/EtOAc (8:1)) to give the title compound as an oil (15
g, 65%). 1H NMR (300 MHz, CDCl3): δ 7.81-7.74 (3H m), 7.61
(1H, s), 7.46-7.41 (2H, m), 7.29 (1H, d, J ) 8.4 Hz), 4.21-
3.82 (5H, m), 3.63 (3H, s), 3.36-3.32 (2H, m), 2.81-2.59 (2H,
m), 2.57-2.42 (1H, m), 1.80-1.64 (1H, m), 1.62-1.42 (2H, m),
1.30-1.18 (1H, m), 1.19 (3H, t, J ) 7.2 Hz). Anal. (C23H27NO5)
C, H, N.
4-(2-P h en yleth yl)-5-(4-p ip er id yl)-3-isoxa zolol Hyd r o-
br om id e (7i). 7i was synthesized as described for 7a using
17i (400 mg, 1.2 mmol) in HBr in AcOH (33%, 20 mL).
Recrystallization (MeOH/Et2O) gave the title compound (250
mg, 59%): mp 246-249 °C. 1H NMR (300 MHz, D2O): δ 7.29-
7.10 (5H, m), 3.30-3.26 (2H, m), 2.85-2.76 (4H, m), 2.64-
2.59 (2H, m), 2.51-2.44 (1H, m), 1.70-1.56 (2H, m), 1.31-
1.27 (2H, m). Anal. (C16H20N2O2‚HBr) C, H, Br, N
E t h yl 3-(1-Met h oxyca r b on yl-4-p ip er id yl)-3-oxo-2-(3-
p h en ylp r op yl)p r op ion a te (16j). 16j was synthesized as
described for 16a using 15 (3.0 g, 12 mmol), NaOEt (12 mmol)
in EtOH (20 mL), and 3-phenylpropyl bromide (1.9 mL, 13
mmol). The crude product was purified by CC (toluene/EtOAc
(5:1)) to give the title compund as an oil (3.0 g, 68%). 1H NMR
(300 MHz, CDCl3): δ 7.29-7.13 (5H, m), 4.19-4.00 (4H, m),
3.68 (3H, s), 3.58 (1H, t, J ) 7.2 Hz), 2.86-2.76 (2H, m), 2.66-
2.59 (3H, m), 1.92-1.44 (8H, m), 1.25 (3H, t, J ) 7.2 Hz).
5-(1-Meth oxyca r bon yl-4-p ip er id yl)-4-(3-p h en ylp r op yl)-
3-isoxa zolol (17j). 17j was synthesized as described for 12
by using 16j (2.5 g, 6.7 mmol) and NaOH (290 mg, 7.2 mmol)
in MeOH/water (10 mL/0.7 mL), hydroxylamine hydrochloride
(931 mg, 13.4 mmol) and NaOH (540 mg, 13 mmol) in MeOH/
water (9 mL/9 mL), and concentrated HCl (4.0 mL). The crude
product was purified by CC (toluene/EtOAc (5:1) containing
AcOH (1%)) and gave the pure product (1.3 g, 49%): mp 127-
128 °C. 1H NMR (300 MHz, CDCl3): δ 9.88 (1H, broad s), 7.32-
7.16 (5H, m), 4.19 (2H, broad s), 3.71 (3H, s), 2.86-2.63 (5H,
m), 2.37-2.31 (2H, m), 1.93-1.70 (6H, m). Anal. (C19H24N2O4)
C, H, N.
4-(3-P h en ylp r op yl)-5-(4-p ip er id yl)-3-isoxa zolol Hyd r o-
br om id e (7j). 7j was synthesized as described for 7a by using
17j (500 mg, 1.5 mmol) in HBr in AcOH (33%, 30 mL).
Recrystallization (MeOH/Et2O) gave the title compound (360
mg, 68%): mp 222-223 °C. 1H NMR (300 MHz, D2O): δ 7.38-
7.25 (5H, m), 3.50-3.44 (2H, m), 3.09-2.98 (3H, m), 2.64 (2H,
t, J ) 7.5 Hz), 2.35 (2H, t, J ) 7.5 Hz), 2.02-1.92 (4H, m),
1.84 (2H, pent, J ) 7.5 Hz). Anal. (C17H22N2O2‚HBr) C, H, Br,
N.
Eth yl 2-(9-An th r a cylm eth yl)-3-(1-m eth oxyca r bon yl-4-
p ip er id yl)-3-oxop r op ion a te (16k ). 16k was synthesized as
described above for 16a using 15 (3.0 g, 12 mmol), NaOEt (12
mmol) in EtOH (20 mL), and 9-chloromethylanthracene (2.9
g, 13 mmol). The crude product was purified by CC (toluene/
EtOAc (6:1)) to give the title compound as an oil (2.3 g, 43%).
1H NMR (300 MHz, CDCl3): δ 8.38 (1H, s), 8.18 (2H, d, J )
8.4 Hz), 8.00 (2H, d, J ) 8.4 Hz), 7.54-7.46 (4H m), 4.16-
3.81 (5H, m), 3.68 (2H, d, J ) 9 Hz), 3.60 (3H, s), 2.92-2.70
(2H, m), 2.62-2.50 (1H, m), 1.72-1.48 (2H, m), 1.42-1.21 (2H,
m), 1.10 (3H, t, J ) 7.2 Hz).
5-(1-Met h oxyca r b on yl-4-p ip er id yl)-4-(2-n a p h t h ylm e-
th yl)-3-isoxa zolol (17m ). 17m was synthesized as described
for 12 by using 16m (2.4 g, 5.9 mmol) and NaOH (250 mg, 6.3
mmol) in MeOH/water (7 mL/0.36 mL), hydroxylamine hydro-
chloride (820 mg, 12 mmol) and NaOH (470 mg, 12 mmol) in
MeOH/water (9 mL/9 mL), and concentrated HCl (2.5 mL). The
crude product was purified by CC (toluene/EtOAc (8:1) con-
taining AcOH (1%)) and gave the pure product (850 mg,
1
39%): mp 152-153 °C. H NMR (300 MHz, CDCl3): δ 10.05
(1H, broad s), 7.80-7.73 (3H, m), 7.59 (1H, s), 7.43-7.41 (2H,

2464 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Frølund et al.
m), 7.33 (1H, d, J ) 8.4 Hz), 4.10 (2H, broad s), 3.84 (2H, s),
3.66 (3H, s), 2.80-2.61 (3H, m), 1.79-1.58 (4H, m). Anal.
(C21H22N2O4) C, H, N.
2.36 (3H, m), 1.64-1.53 (2H, m), 1.39-1.34 (2H, m). Anal.
(C21H22N2O4) C, H, N.
5-(4-P ip er id yl)-4-(1-n a p h th ylm eth yl)-3-isoxa zolol Hy-
d r obr om id e (7o). 7o was synthesized as described for 7a by
using 17o (630 mg, 1.8 mmol) in HBr in AcOH (33%, 30 mL).
Recrystallization (MeOH/Et2O) gave the title compound (560
mg, 80%): mp 253-255 °C. 1H NMR (300 MHz, D2O): δ 8.00-
7.97 (1H, m), 7.90-7.87 (1H, m), 7.79 (1H, d, J ) 8.1 Hz),
7.54-7.46 (2H, m), 7.38 (1H, t, J ) 6.9 Hz), 7.26-7.24 (1H,
m), 4.02 (2H, s), 3.23-3.19 (2H, m), 2.70-2.58 (3H, m), 1.78-
1.64 (2H, m), 1.53-1.50 (2H, m). Anal. (C19H20N2O2‚HBr) C,
H, N.
4-(2-Na p h th ylm eth yl)-5-(4-p ip er id yl)-3-isoxa zolol Hy-
d r obr om id e (7m ). 7m was synthesized as described for 7a
by using 17m (510 mg, 1.4 mmol) in HBr in AcOH (33%, 20
mL). Recrystallization (MeOH/Et2O) gave the title compound
1
(370 mg, 68%): mp 223-224 °C. H NMR (300 MHz, D2O): δ
7.78-7.62 (3H, m), 7.47 (1H, s), 7.39-7.30 (2H, m), 7.21 (1H,
d, J ) 8.4 Hz), 3.67 (2H, s), 3.28-3.17 (2H, m), 2.98-2.85 (1H,
m), 2.81-2.69 (2H, m), 1.87-1.68 (4H, m). Anal. (C19H20N2O2‚
HBr) C, H, Br, N.
Eth yl 3-(1-Meth oxyca r bon yl-4-p ip er id yl)-2-[2-(2-n a p h -
th yl)eth yl]-3-oxop r op ion a te (16n ). 16n was synthesized as
described above for 16a using 15 (3.0 g, 12 mmol), NaOEt (12
mmol) in EtOH (20 mL), and 2-(2-bromoethyl)naphthalene33,34
(3.0 g, 13 mmol). The crude product was purified by CC
(toluene/EtOAc (5:1)) to give the title compund as an oil (2.3
g, 48%). 1H NMR (300 MHz, CDCl3): δ 7.80-7.74 (3H m), 7.58
(1H, s), 7.47-7.38 (2H, m), 7.30 (1H, dd, J ) 8.4 and 1.8 Hz),
4.16 (2H, q, J ) 7.2 Hz), 4.11 (2H, broad s), 3.67 (3H, s), 3.61
(1H, t, J ) 7.2 Hz), 2.81-2.63 (4H, m), 2.61-2.54 (1H, m),
2.33-2.20 (2H, m), 1.80-1.42 (4H, m), 1.20 (3H, t, J ) 7.2
Hz).
5-(1-Met h oxyca r bon yl-4-p ip er id yl)-4-[2-(2-n a p h t h yl)-
eth yl]-3-isoxa zolol (17n ). 17n was synthesized as described
for 12 by using 16n (2.0 g, 4.9 mmol) and NaOH (210 mg, 5.2
mmol) in MeOH/water (8 mL/0.6 mL), hydroxylamine hydro-
chloride (820 mg, 12 mmol) and NaOH (472 mg, 12 mmol) in
MeOH/water (7 mL/7 mL), and concentrated HCl (3.0 mL). The
crude product was purified by CC (toluene/EtOAc (5:1) con-
taining AcOH (1%)) and gave the product (1.4 g, 52%). A
sample was recrystallized (toluene/light petroleum) to give the
product: mp 143-145 °C. 1H NMR (300 MHz, CDCl3): δ 10.05
(1H, broad s), 7.81-7.74 (3H, m), 7.56 (1H, s), 7.48-7.41 (2H,
m), 7.33 (1H, d, J ) 8.4 Hz), 3.94 (2H, broad s), 3.65 (3H, s),
3.02 (2H, t, J ) 6.9 Hz), 2.71 (2H, t, J ) 6.9 Hz), 2.43-2.36
(2H, m), 2.21-2.14 (1H, m), 1.55-1.38 (2H, m), 1.14-1.09 (2H,
m). Anal. (C22H24N2O4) C, H, N.
5-(4-P ip er id yl)-4-[2-(2-n a p h th yl)eth yl]-3-isoxa zolol Hy-
d r obr om id e (7n ). 7n was synthesized as described above for
7a by using 17n (400 mg, 1.1 mmol) in HBr in AcOH (33%, 30
mL). Recrystallization (MeOH/Et2O) gave the title compound
(340 mg, 80%): mp >255 °C. 1H NMR (300 MHz, D2O): δ
7.68-7.60 (3H, m), 7.39 (1H, s), 7.34-7.24 (2H, m), 7.17 (1H,
dd, J ) 8.4 Hz, J ) 1.5 Hz), 2.77-2.72 (2H, m), 2.44-2.34
(4H, m), 1.78-1.60 (3H, m), 1.02-0.86 (2H, m), 0.53-0.49 (2H,
m). Anal. (C20H22N2O2‚HBr‚1.75H2O) C, H, N.
Eth yl 3-(1-Meth oxyca r bon yl-4-p ip er id yl)-2-(1-n a p h th -
ylm eth yl)-3-oxop r op ion a te (16o). 16o was synthesized as
described for 16a using 15 (3.0 g, 12 mmol), NaOEt (12 mmol)
in EtOH (20 mL), and 1-(bromomethyl)naphthalene35 (2.8 g,
13 mmol). The crude product was purified by CC (toluene/
EtOAc (4:1)) to give the title compund as an oil (3.2 g, 70%).
1H NMR (300 MHz, CDCl3): δ 7.98 (1H, d, J ) 8.4 Hz), 7.86
(1H, d, J ) 8.1 Hz), 7.71 (1H, d, J ) 8.1 Hz), 7.57-7.46 (2H,
m), 7.34 (1H, t, J ) 6.9 Hz), 7.25 (1H, d, J ) 6.0 Hz), 4.21-
4.13 (3H, m), 4.00 (1H, broad s), 3.81 (1H, broad s), 3.71-3.64
(2H, m), 3.62 (3H, s), 2.74-2.52 (3H, m), 2.38-2.23 (1H, m),
1.70-1.64 (1H, m), 1.57-1.43 (1H, m), 1.19 (3H, t, J ) 7.2
Hz), 1.27-1.17 (1H, m).
Eth yl 3-(1-Meth oxyca r bon yl-4-p ip er id yl)-2-[2-(1-n a p h -
th yl)eth yl]-3-oxop r op ion a te (16p ). 16p was synthesized as
described for 16a using 15 (3.0 g, 12 mmol), NaOEt (12 mmol)
in EtOH (20 mL), and 1-(2-bromoethyl)naphthalene36,37 (2.9
g, 12 mmol). The crude product was purified by CC (toluene/
EtOAc (4:1)) to give the title compound as an oil (2.5 g, 52%).
1H NMR (300 MHz, CDCl3): δ 8.08 (1H, d, J ) 8.4 Hz), 7.86
(1H, d, J ) 7.5 Hz), 7.74 (1H, d, J ) 8.1 Hz), 7.58-7.32 (2H,
m), 7.27 (1H, t, J ) 8.1 Hz), 7.18 (1H, d, J ) 8.1 Hz), 4.21
(2H, q, J ) 7.5 Hz), 4.14 (2H, broad s), 3.69 (3H, s), 3.72-3.13
(1H, m), 3.11-3.02 (2H, m), 2.8-2.75 (2H, m), 2.67-2.60 (1H,
m), 2.33-2.25 (2H, m), 1.8-1.73 (2H, m), 1.63-1.39 (2H, m),
1.27 (3H, t, J ) 6.9 Hz).
5-(1-Met h oxyca r bon yl-4-p ip er id yl)-4-[2-(1-n a p h t h yl)-
eth yl]-3-isoxa zolol (17p ). 17p was synthesized as described
for 12 by using 16p (2.1 g, 5.1 mmol) and NaOH (200 mg, 5.1
mmol) in MeOH/water (16 mL/1.6 mL), hydroxylamine hydro-
chloride (700 mg, 10 mmol) and NaOH (400 mg, 10 mmol) in
MeOH/water (13 mL/1.3 mL), and concentrated HCl (3.0 mL).
The crude product was purified by CC (toluene/EtOAc (4:1)
containing AcOH (1%)) and gave the product (730 mg, 38%).
An analytical sample was recrystallized (toluene/light petro-
leum): mp 160-163 °C. 1H NMR (300 MHz, CDCl3): δ 8.09
(1H, d, J ) 8.1 Hz), 7.85 (1H, d, J ) 8.6 Hz), 7.70 (1H, d, J )
8.1 Hz), 7.57-7.46 (2H, m), 7.33 (1H, t, J ) 7.5 Hz), 7.19 (1H,
d, J ) 6.0 Hz), 3.96 (2H, broad s), 3.67 (3H, s), 3.39-3.33 (2H,
m), 2.87-2.75 (2H, m), 2.35-2.19 (2H, m), 1.96-1.82 (1H, m),
1.47-1.29 (2H, m), 1.04-0.90 (2H, m). Anal. (C22H24N2O4) C,
H, N.
4-[2-(1-Na p h th yl)eth yl]-5-(4-p ip er id yl)-3-isoxa zolol Hy-
d r obr om id e (7p ). 7p was synthesized as described for 7a by
using 17p (250 mg, 0.65 mmol) in HBr in AcOH (33%, 15 mL).
Recrystallization (MeOH/Et2O) gave the title compound (194
mg, 75%): mp >250 °C. 1H NMR (300 MHz, D2O): δ 8.06 (1H,
d, J ) 8.1 Hz), 7.89 (1H, d, J ) 7.8 Hz), 7.74 (1H, d, J ) 8.1
Hz), 7.57-7.48 (2H, m), 7.35 (1H, t, J ) 8.1 Hz), 7.17 (1H, d,
J ) 6.9 Hz), 3.29-3.25 (2H, m), 3.06 (2H, m), 2.80-2.75 (2H,
m), 2.30 (2H, m), 1.89-1.79 (1H, m), 1.34 (2H, m), 0.84-0.78
(2H, m). Anal. (C20H22N2O2‚HBr‚H2O) C, H, Br, N.
Eth yl 2-(3,3-Dip h en ylp r op yl)-3-(1-m eth oxyca r bon yl-4-
p ip er id yl)-3-oxop r op ion a te (16s). 16s was synthesized as
described for 16a using 15 (15.7 g, 61.0 mmol), NaOEt (61.0
mmol) in EtOH (100 mL), and 3,3-diphenylpropyl bromide38
(18.4 g, 67.0 mmol). The crude product was purified by CC
(toluene/EtOAc (4:1)) to give the title compound as an oil (13.4
g, 49%). 1H NMR (300 MHz, CDCl3): δ 7.30-7.15 (10H, m),
4.14 (2H, q, J ) 7.2 Hz), 4.11 (2H, broad s), 3.89 (1H, t, J )
8.1 Hz), 3.67 (3H, s), 3.58 (1H, t, J ) 7.2 Hz), 2.85-2.70 (2H,
m), 2.62-2.50 (1H, m), 2.10-2.90 (2H, m), 1.90-1.62 (4H, m),
1.61-1.39 (2H, m), 1.22 (3H, t, J ) 7.2 Hz).
4-(3,3-Dip h en ylp r op yl)-5-(1-m et h oxyca r b on yl-4-p ip -
er id yl)-3-isoxa zolol (17s). 17s was synthesized as described
for 12 by using 16s (1.4 g, 3.1 mmol) and NaOH (133 mg, 3.3
mmol) in MeOH/water (6 mL/0.4 mL), hydroxylamine hydro-
chloride (430 mg, 6.2 mmol) and NaOH (248 mg, 6.2 mmol) in
MeOH/water (5 mL/5 mL), and concentrated HCl (2.0 mL). The
crude product was purified by CC (toluene/EtOAc (5:1) con-
taining AcOH (1%)) and gave the product (600 mg, 46%). An
analytical sample was recrystallized (toluene/light petro-
leum): mp 150-151 °C. 1H NMR (300 MHz, CDCl3): δ 9.55
(1H, broad s), 7.32-7.15 (10H, m), 4.14 (2H, broad s), 3.92-
5-(1-Met h oxyca r b on yl-4-p ip er id yl)-4-(1-n a p h t h ylm e-
th yl)-3-isoxa zolol (17o). 17o was synthesized as described
for 12 by using 16o (3.0 g, 7.5 mmol) and NaOH (320 mg, 8.0
mmol) in MeOH/water (9 mL/0.6 mL), hydroxylamine hydro-
chloride (1.0 g, 15 mmol) and NaOH (600 mg, 15 mmol) in
MeOH/water (10 mL/10 mL), and concentrated HCl (3.0 mL).
The crude product was purified by CC (toluene/EtOAc (5:1)
containing AcOH (1%)) and recrystallized (toluene/light pe-
troleum) to give the product (560 mg, 31%): mp 166-167 °C.
1H NMR (300 MHz, CDCl3): δ 10.75 (1H, broad s), 8.07 (1H,
d, J ) 8.1 Hz), 7.89 (1H, d, J ) 7.5 Hz), 7.78 (1H, d, J ) 8.4
Hz), 7.58-7.42 (2H, m), 7.40 (1H, t, J ) 7.5 Hz), 7.28 (1H, d,
J ) 8.4 Hz), 4.16 (2H, s), 4.30 (2H, broad s), 3.67 (3H, s), 2.54-

Substituted Isoxazolol GABAA Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2465
3.88 (1H, m), 3.71 (3H, s), 3.74-2.66 (2H, m), 2.50-2.42 (1H,
m), 2.34 (4H, s), 1.70-1.63 (4H, m). Anal. (C25H28N2O4) C, H,
N.
(700 mg, 1.8 mmol) in THF (1 mL) was added dropwise to a
solution of EtMgCl in THF (2.8 M, 640 mM, 1.78 mmol) at
-30 °C. The reaction mixture was allowed to warm to 0 °C,
and stirring was continued for 1 h. A solution of cyclohexan-
ecarboxaldehyde (210 mg, 1.8 mmol) in THF (1 mL) was added
at 0 °C, and stirring was continued for 18 h at room temper-
ature. The reaction mixture was quenched with saturated
ammonium chloride (2 mL), and Et2O (10 mL) was added. The
aqueous phase was extracted with Et2O (2 × 10 mL), and then
the combined extracts were dried and evaporated. CC (toluene/
EtOAc (2:1)) of the residue gave the product as a viscous oil
4-(3,3-Dip h en ylp r op yl)-5-(4-p ip er id yl)-3-isoxa zolol Hy-
d r obr om id e (7s). 7s was synthesized as described for 7a by
using 17s (150 mg, 0.36 mmol) in HBr in AcOH (33%, 10 mL).
Recrystallization (MeOH/Et2O) gave the title compound (110
mg, 69%): mp >250 °C. 1H NMR (300 MHz, D2O): δ 7.50-
7.25 (10H, m), 4.14-4.10 (1H, m), 3.62-3.58 (2H, m), 3.50-
3.05 (2H, m), 3.01-2.86 (1H, m), 2.56 (4H, s), 2.20-2.00 (4H,
m). Anal. (C23H26N2O2‚HBr) C, H, N.
1
(510 mg, 76%). H NMR (300 MHz, CDCl3): δ 4.91 (1H, hep,
Eth yl 2-(4,4-Dip h en ylbu tyl)-3-(1-m eth oxyca r bon yl-4-
p ip er id yl)-3-oxop r op ion a te (16t). 16t was synthesized as
described for 16a using 15 (3.0 g, 11.7 mmol), NaOEt (11.7
mmol) in EtOH (20 mL), and 4,4-diphenylbutyl bromide39 (3.7
g, 12.8 mmol). The crude product was purified by CC (toluene/
EtOAc (4:1)) to give the title compound as an oil (3.3 g, 61%).
1H NMR (300 MHz, CDCl3): δ 7.26-7.15 (10H, m), 4.11 (2H,
q, J ) 7.2 Hz), 4.10 (2H, broad s), 3.88 (1H, t, J ) 7.2 Hz),
3.67 (3H, s), 3.51 (1H, t, J ) 7.2 Hz), 2.83-2.72 (2H, m), 2.62-
2.52 (1H, m), 2.05 (2H, q, J ) 7.5 Hz), 1.92-1.80 (2H, m), 1.78-
1.34 (4H, m), 1.23-1.15 (2H, m), 1.18 (3H, t, J ) 7.2 Hz).
4-(4,4-Dip h e n ylb u t yl)-5-(1-m e t h oxyca r b on yl-4-p ip -
er id yl)-3-isoxa zolol (17t). 17t was synthesized as described
for 12 by using 16t (3.3 g, 7.1 mmol) and NaOH (303 mg, 7.6
mmol) in MeOH/water (10 mL/0.6 mL), hydroxylamine hydro-
chloride (985 mg, 14.2 mmol) and NaOH (568 mg, 14.2 mmol)
in MeOH/water (12 mL/12 mL), and concentrated HCl (3.0
mL). The crude product was purified by CC (toluene/EtOAc
(4:1) containing AcOH (1%)) and gave the product (750 mg,
24%). An analytical sample was recrystallized (toluene/light
J ) 6.0 Hz), 4.20 (2H, broad s), 4.18 (1H, d, J ) 8.4 Hz), 3.70
(3H, s), 3.02-2.80 (3H, m), 2.10-2.04 (1H, m), 1.86-1.64 (8H,
m), 1.46-0.86 (7H, m), 1.37 (6H, d, J ) 6.0 Hz).
4-(Cycloh exylm eth yl)-3-isop r op oxy-5-(1-m eth oxyca r -
bon yl-4-p ip er id yl)isoxa zole (22g). To a solution of 21g (200
mg, 0.5 mmol) and triethylsilane (150 mL, 0.9 mmol) in CH2-
Cl2 (1 mL) was added dropwise trifluoroacetic acid (1.1 mL)
at room temperature, and the mixture was then stirred at 50
°C for 2 h. The mixture was cooled, water (2 mL) was added,
and the organic phase was extracted with Et2O (3 × 10 mL).
The combined organic extracts were dried and evaporated. CC
(toluene/EtOAc (2:1)) afforded the title compound (150 mg,
78%) as a viscous oil. 1H NMR (300 MHz, CDCl3): δ 4.88 (1H,
hep, J ) 6.0 Hz), 4.20 (2H, broad s), 4.18 (1H, d, J ) 8.4 Hz),
3.72 (3H, s), 2.98-2.73 (3H, m), 2.11 (1H, m), 1.87-1.63 (9 H,
m), 1.48-1.40 (1H, m), 1.36 (6H, d, J ) 6.0 Hz), 1.23-1.09
(3H, m), 0.96-0.81 (2H, m).
4-(Cycloh exylm eth yl)-5-(4-p ip er id yl)-3-isoxa zolol Hy-
d r obr om id e (7g). A solution of 22g (150 mg, 0.5 mmol) in a
solution of HBr in AcOH (33%, 4 mL) was stirred at 65 °C for
18 h. The reaction mixture was evaporated, and the residue
was recrystallized (MeOH/Et2O) to give the title compound (79
mg, 49%): mp >220 °C. 1H NMR (300 MHz, D2O): δ 3.55-
3.48 (2H, m), 3.27-3.05 (3H, m), 2.20 (2H, d, J ) 7.2 Hz),
2.11-2.00 (4H, m), 1.70-1.58 (5H, m), 1.50-1.39 (1H, m),
1.25-1.10 (3H, m), 0.90-0.83 (2H, m). Anal. (C15H24N2O2‚HBr‚
H2O) C, H, N.
4-(Dip h en ylh yd r oxym et h yl)-3-isop r op oxy-5-(1-m et h -
oxyca r bon yl-4-p ip er id yl)isoxa zole (21q). 21q was synthe-
sized as described for 21g using 20 (700 mg, 1.8 mmol) in THF
(1 mL), EtMgCl in THF (2.8 M, 640 µL, 1.8 mmol), and
benzophenone (320 mg, 1.8 mmol) in THF (1 mL). CC (toluene/
EtOAc (6:1)) gave the product (500 mg, 63%): mp 135-137
°C. 1H NMR (300 MHz, CDCl3): δ 7.31 (10H, s), 4.86 (1H, hep,
J ) 6.0 Hz), 4.05 (3H, m), 3.63 (3H, s), 2.23-2.10 (2H, m),
2.70-1.56 (2H, m), 1.40-1.32 (2 H, m), 1.27-1.21 (1H, m),
1.17 (6H, d, J ) 6.0 Hz). Anal. (C26H30N2O5) C, H, N.
1
petroleum): mp 139-140 °C. H NMR (300 MHz, CDCl3): δ
9.55 (1H, broad s), 7.30-7.14 (10H, m), 4.17 (2H, broad s), 3.92
(1H, t, J ) 7.8 Hz), 3.73 (3H, s), 2.82-2.65 (3H, m), 2.32 (2H,
t, J ) 7.8 Hz), 2.12-2.04 (2H, m), 1.76-1.61 (4H, m), 1.54-
1.44 (2H, m). Anal. (C26H30N2O4) C, H, N.
4-(4,4-Dip h en ylbu tyl)-5-(4-p ip er id yl)-3-isoxa zolol Hy-
d r obr om id e (7t). 7t was synthesized as described for 7a by
using 17t (400 mg, 0.9 mmol) in HBr in AcOH (33%, 20 mL).
Recrystallization (MeOH/Et2O) gave the title compound (330
mg, 78%): mp >225 °C. 1H NMR (300 MHz, D2O): δ 6.81-
6.58 (10H, m), 3.55-3.50 (1H, m), 2.47-2.44 (2H, m), 2.02-
1.70 (7H, m), 1.23-1.05 (6H, m). Anal. (C24H28N2O‚HBr) C,
H, N.
3-Isop r op oxy-5-(1-m eth oxyca r bon yl-4-p ip er id yl)isox-
a zole (19). A mixture of 5-(1-methoxycarbonyl-4-piperidyl)-
3-isoxazolol (18)16 (3.8 g, 17 mmol) and potassium carbonate
(3.2 g, 24 mmol) in DMF (38 mL) was stirred at 60 °C for 30
min. Isopropyl bromide (4.4 mL, 50 mmol) was added dropwise
to the reaction mixture, and stirring was continued for 2 days
at 60 °C. After the mixture was cooled, water was added and
the aqueous phase was extracted with light petroleum (5 ×
25 mL). The combined extracts were washed with water (2 ×
30 mL), dried, and evaporated. CC (toluene/EtOAc (3:1)) of the
crude product gave the product as an oil (3.36 g, 82%). 1H NMR
(300 MHz, CDCl3): δ 5.57 (1H, s), 4.87 (1H, hep, J ) 4.8 Hz),
4.17 (2H, broad s), 3.73 (3H, s), 2.97-2.78 (3H, m), 2.07-1.80
(2H, m), 1.68-1.54 (2H, m), 1.37 (6H, d, J ) 4.8 Hz).
4-Iodo-3-isopr opoxy-5-(1-m eth oxycar bon yl-4-piper idyl)-
isoxa zole (20). To a solution of 19 (7.4 g, 28 mmol) in AcOH
(50 mL), a solution of iodomonochloride (5.4 g, 33 mmol) in
AcOH (40 mL) was added followed by water (125 mL). The
reaction mixture was stirred for 18 h at 85 °C. After the
mixture was cooled, sodium thiosulfate was added and the
mixture was evaporated. Water (100 mL) was added to the
residue, and the mixture was extracted with Et2O (3 × 200
mL). The organic extracts were dried and evaporated. CC
(toluene/EtOAc (5:1)) gave the product (9.8 g, 90%): mp 118-
4-(Dip h en ylm eth yl)-3-isop r op oxy-5-(1-m eth oxyca r bo-
n yl-4-p ip er id yl)isoxa zole (22q). 22q was synthesized as
described above for 21g using 21q (250 mg, 0.6 mmol),
triethylsilane (150 mL, 0.9 mmol) in CH2Cl2 (1 mL), and
trifluoroacetic acid (1.1 mL) at 0 °C. CC (toluene/EtOAc (8:1))
1
afforded the title compound (187 mg, 78%) as an oil. H NMR
(300 MHz, CDCl3): δ 7.32-7.21 (6H, m), 7.14-7.10 (4H, m),
5.30 (1H, s), 4.80 (1H, hep, J ) 6.0 Hz), 4.09 (2H, broad s),
3.67 (3H, s), 2.54-2.40 (2H, m), 2.26-2.17 (1H, m), 1.79-1.61
(2 H, m), 1.47-1.42 (2H, m), 1.19 (6H, d, J ) 6.0 Hz).
4-(Dip h en ylm et h yl)-5-(4-p ip er id yl)-3-isoxa zolol H y-
d r obr om id e (7q). 7q was synthesized as described above for
7g using 22q (190 mg, 0.4 mmol) in HBr in AcOH (33%, 3
mL). Recrystallization (EtOH/Et2O) gave the title compound
(44 mg, 24%): mp >220 °C. 1H NMR (300 MHz, D2O): δ 7.43-
7.32 (6H, m), 7.25-7.21 (4H, m), 5.48 (1H, m), 3.42-3.32 (2H,
m), 2.76-2.63 (2H, m), 2.42-2.30 (1H, m), 1.98-1.82 (2H, m),
1.78-1.68 (2H, m). Anal. (C21H22N2O2‚HBr‚0.25H2O) C, H, N.
4-(2,2-Dip h e n yl-1-h yd r oxye t h yl)-3-isop r op oxy-5-(1-
m eth oxyca r bon yl-4-p ip er id yl)isoxa zole (21r ). 21r was
synthesized as described for 21g using 20 (420 mg, 1.1 mmol)
in THF (1 mL), EtMgCl in THF (2.8 M, 380 mL, 1.8 mmol),
and diphenylacetaldehyde (190 mL, 1.1 mmol) in THF (1 mL).
CC (toluene/EtOAc (4:1)) gave the product (360 mg, 73%) as
an oil. 1H NMR (300 MHz, CDCl3): δ 7.44 (2H, d, J ) 7.2 Hz),
1
120 °C. H NMR (300 MHz, CDCl3): δ 4.89 (1H, hep, J ) 6.0
Hz), 4.22 (2H, broad s), 3.70 (3H, s), 3.00-2.81 (3H, m), 1.90-
1.77 (4H, m), 1.39 (6H, d, J ) 6.0 Hz). Anal. (C13H19IN2O4) C,
H, N.
4-(Cycloh exylh yd r oxym eth yl)-3-isop r op oxy-5-(1-m eth -
oxyca r bon yl-4-p ip er id yl)isoxa zole (21g). A solution of 20

2466 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Frølund et al.
7.36 (2H, J ) 7.2 Hz), 7.28-7.08 (6H, m), 5.22 (1H, d, J ) 9.6
Hz), 4.93 (1H, hep, J ) 6.3 Hz), 4.41 (1H, d, J ) 9.6 Hz), 4.06
(3H, broad s), 3.67 (3H, s), 2.79-2.56 (3H, m), 2.46 (1H, broad
s), 1.72-1.52 (2H, m), 1.51-1.41 (1 H, m), 1.44 (3H, d, J )
6.0 Hz), 1.36 (3H, d, J ) 6.0 Hz), 0.96-0.91 (1H, m).
4-(2,2-Dip h en ylet h yl)-3-isop r op oxy-5-(1-m et h oxyca r -
bon yl-4-p ip er id yl)isoxa zole (22r ). 22r was synthesized as
described above for 21g using 21r (270 mg, 0.6 mmol),
triethylsilane (150 mL, 0.9 mmol) in CH2Cl2 (1 mL), and
trifluoroacetic acid (1.1 mL). CC (toluene/EtOAc (4:1)) afforded
the title compound (245 mg, 93%) as an oil. 1H NMR (300 MHz,
CDCl3): δ 7.50-7.09 (10H, m), 4.94 (1H, hep, J ) 6.0 Hz),
4.16 (1H, t, J ) 7.8 Hz), 4.08 (2H, broad s), 3.68 (3H, s), 2.99
(2H, J ) 7.8 Hz), 2.92-2.57 (3H, m), 2.32-1.98 (1H, m), 1.56-
1.46 (2 H, m), 1.40 (6H, d, J ) 6.0 Hz), 1.28-1.02 (1H, m).
[inhibitor]
bound ) Bmax
IC50 + [inhibitor]
using the nonlinear curve-fitting program Grafit (Leatherbar-
row, 1992). Ki values were calculated from the Cheng-Prusoff
equation
IC50
Ki )
[radioligand]
1 +
KD
Electr op h ysiology in Vitr o. Cerebral cortical neurons
were cultured essentially as described by Herts et al.43 from
15-day-old mouse embryos. Whole-cell patch-clamp recordings
were made from cerebral cortical neurons cultured for 7-9
days. The culture dish was placed on the stage of a Zeiss
Axiovert 10 inverted-phase contrast microscope (Zeiss, Ger-
many), where the individual neurons were viewed at ×200.
The culture medium in the 35 mm Petri dish was replaced
with about 4 mL of artificial balanced salt solution (ABSS),
which was continuously renewed by constant perfusion at 0.5
mL/min at room temperature (20-22 °C). The composition of
ABSS was as follows (in mM): NaCl 140, KCl 3.5, Na2HPO4
1.25, MgSO4 2, CaCl2 2, glucose 10, and HEPES 10; pH was
7.35 at 22 °C.
4-(2,2-Dip h en yleth yl)-5-(4-p ip er id yl)-3-isoxa zolol Hy-
d r obr om id e (7r ). 7r was synthesized as described above for
7g using 22r (240 mg, 0.54 mmol) in HBr in AcOH (33%, 3
mL). Recrystallization (MeOH/Et2O) gave the title compound
1
(90 mg, 39%): mp >220 °C. H NMR (300 MHz, CD3OD): δ
7.31-7.10 (10H, m), 4.20 (1H, t, J ) 7.8 Hz), 3.31-3.29 (2H,
m), 3.09 (2H, d, J ) 7.8 Hz), 2.91 (2H, m), 2.73-2.64 (1H, m),
1.76-1.60 (2H, m), 1.26-1.20 (2H, m). Anal. (C22H24N2O2‚HBr‚
1.5 H2O) C, H, N.
Recep tor Bin d in g a n d Up ta k e Assa ys. GABAA and
GABAB receptor binding assays were performed using rat
brain synaptic membranes from male Sprague Dawley rats,
and tissue preparation was performed as described by Ransom
and Stec.40 On the day of the assay, the membrane preparation
was quickly thawed, suspended in 50 volumes (w/v) of 50 mM
Tris-HCl buffer (pH 7.4) using an Ultra-Turrax homogenizer,
and centrifuged at 48000g for 10 min at 4 °C. This step was
repeated four times. The final pellet was resuspended in
incubation buffer for the relevant binding assay.
Standard patch-clamp techniques44 were used to record from
the neurons in the whole-cell configuration using an EPC-9
patch-clamp amplifier (HEKA, Germany). The patch electrodes
were pulled from 1.5 mm o.d. glass (World Precision Instru-
ments) on a BB-CH-PC electrode puller (Mecanex, Switzer-
land) and had resistances of 2-5 MΩ. The medium in the
patch electrodes had the following composition (in mM): KCl
140, MgCl2 1, CaCl2 1, EGTA 10, MgATP 2, and HEPES 10;
pH was 7.35 at 22 °C. A holding potential of -60 mV was used.
Current signals were recorded to disk on a computer and
analyzed subsequently.
The compounds used were premixed at the required con-
centrations in ABSS. When necessary, the compounds were
initially dissolved in DMSO and then diluted with ABSS to
final concentrations of DMSO of less than 0.2%. This concen-
tration of DMSO was in itself without effect on membrane
currents. The solutions were applied in the vicinity (about 100
µm) of the recorded neuron from a multibarreled perfusion
pipet, with the multiple barrels ending in a single cap with
an opening of about 100 µm.45 When applied, solutions
emerged rapidly from the cap and surrounded the neuron
completely. Drugs were applied for 5 s every 1 min. Within 5
s of drug application, the responses always peaked or reached
a stable maximum plateau. Between ligand applications,
ligand-free ABSS was applied from one of the barrels in order
to quickly remove the drug from the cell. For all drugs except
7k ,q-t, current responses were quantified by measuring the
maximum currents recorded during application of drugs. For
7k ,q-t, the current responses were quantified using the
response magnitude after 5 s of application.
GABAA binding was studied using a modified version of the
method described by Ebert et al.41 The assay was carried out
in triplicate and in total volumes of 1 mL by incubation of
synaptic membranes (15-20 mg of original tissue per aliqout)
in 0.8 mL of Tris-HCl buffer (50 mM, pH 7.4), 0.1 mL of [3H]-
muscimol (final concentration 3-5 nM), and 0.1 mL of test
substance in various concentrations. Following incubation at
0 °C for 45 min, the samples were filtered through Whatman
GF/B filters and filters were washed with 3 × 3 mL of ice-
cooled buffer. Nonspecific binding in the presence of 0.1 mM
GABA was subtracted.
GABAB binding was carried out in triplicate by incubation
of membranes (15-20 mg of original tissue per aliqout) in 0.75
mL of Tris-HCl buffer (50 mM + 2.5 mM CaCl2, pH 7.4), 0.1
mL isoguvacine (200 µM), 0.05 mL of [3H]GABA (final con-
centration 3-5 nM), and 0.1 mL of the test substances in
various concentrations. Following incubation at 25 °C for 45
min, the bound ligand was isolated as described for GABAA
receptor binding. Nonspecific binding in the presence of 0.1
mM baclofen was subtracted.
The compounds’ effects on GABA uptake were studied using
a crude synaptosomal preparation, prepared from rat brains
as described elsewhere in detail.42 The whole brains were
homogenized in 10 volumes of ice-cold 0.32 M sucrose, and the
homogenate was centrifuged at 600g at 4 °C for 10 min. The
pellet was discarded, and the supernatant was centrifuged at
25000g at 4 °C for 55 min. The pellet fraction was resuspended
in 40 volumes of oxygenated phosphate medium at 0 °C. The
synaptosome suspensions (2000 µL) were preincubated for 10
min at 25 °C with 0.4 mL of phosphate medium containing
the inhibitor. Then [3H]GABA (100 µL) was added to give a
final GABA concentration of 50 nM (isotope dilution). Samples
were vortexed, and the incubation was continued for an
additional 10 min. The synaptosomes were isolated by rapid
filtration through Whatman GF/C glass fiber filters, and the
filters were washed with 3 × 2 mL of buffer.
For the antagonists, an equation
I0
I )
1 + antilog{(log [B] - log IC50)nH}
was fitted to the experimental concentration response data. I
is the current, I0 is the current induced by 20 mM isoguvacine
(1) alone, [B] is the concentration of antagonist, IC50 is the
concentration of antagonist that reduces the peak current to
50% of I0, and nH is the Hill coefficient. For the partial agonists,
another equation
I0 - I∞
I ) I∞
+
1 + antilog{(log [B] - log IC50)nH}
IC50 values were estimated by measuring the inhibition of
at least five different test concentrations and were estimated
from the function
was used. Here, I∞ is the current in the presence of 20 mM 1
and an infinitely high concentration of partial agonist, which

Substituted Isoxazolol GABAA Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2467
should theoretically be equal to the peak current in the
presence of an infinitely high concentration of partial agonist
alone, and IC50 is the concentration of partial agonist that
reduces the peak current to I∞ + 0.5(I0 - I∞), i.e., the
concentration, which gives rise to 50% of the maximum
inhibition.
Con for m a tion a l An a lysis. All calculations were per-
formed with the MM3* force field as implemented in Macro-
Model, version 6.5.46 To obtain an accurate geometry for the
3-isoxazolol moiety, some force-field parameters were added
and others were modified.24
Medical Research Council, the Academy of Finland, the
Centre for Drug Design and Transport, the Neuro-
Science PharmaBiotec Centre, and the Danish State
Biotechnology Programme. The technical assistance of
Lærke Andersen, Tina Lindgreen, and Annette Kris-
tensen and the preparation of cell cultures by Gunilla
Steven are gratefully acknowledged.
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Two conformational searches were performed for each
molecule, both for aqueous solution using the generalized Born/
solvent accessible surface area (GB/SA) dielectric continuum
solvation model.48 One analysis was performed to search the
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pharmacophore model. The conformational energy penalties
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energy minimum conformation in water from the internal
energy of the constrained conformations.49 The conformations
were fitted to the GABAA pharmacophore model using the
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piperidine ring, and the piperidine nitrogen as fitting points.
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above 1 Å between all parts of the 4-substituent and the
sterical hindrances indicated by tetrahedrons in Figure 3. This
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between the methyl group in 7a and the corners of the
tetrahedrons. In addition, a bioactive conformation should
have low conformational energy penalties, preferably below 3
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conformational energy penalty was recalculated. The confor-
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if no distance between the 4-substituent and the sterical
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