A novel class of potent 3-isoxazolol GABAA antagonists: Design, systhesis, and pharmacology [3]

Full text of DOI:10.1021/jm000371q

4930
J . Med. Chem. 2000, 43, 4930-4933
A Novel Cla ss of P oten t 3-Isoxa zolol
GABAA An ta gon ists: Design , Syn th esis,
a n d P h a r m a cology
Bente Frølund,† Lena Tagmose,† Tommy Liljefors,†
Tine Bryan Stensbøl,† Christine Engblom,‡
Uffe Kristiansen,‡ and Povl Krogsgaard-Larsen*,†
Departments of Medicinal Chemistry and Pharmacology,
Centre for Drug Design and Transport, The Royal Danish
School of Pharmacy, 2 Universitetsparken,
DK 2100 Copenhagen, Denmark
Received August 28, 2000
In tr od u ction . 4-Aminobutyric acid (GABA, 1) (Fig-
ure 1) is the major inhibitory neurotransmitter in the
central nervous system (CNS) and exerts its effect
through activation of the ionotropic GABAA and GABAC
receptors and the metabotropic GABAB receptors.1,2 The
GABAergic system, especially GABAA receptors, has
been associated with certain neurological and psychi-
atric disorders and is a potential target for therapeutic
intervention in such disorders.2,3 To pharmacologically
characterize this receptor class, a number of GABAA
ligands bioisosterically derived from GABA, such as the
selective agonists muscimol (2)4 and 4,5,6,7-tetrahydro-
isoxazolo[5,4-c]pyridin-3-ol (THIP, 3),5,6 have been de-
veloped. Gabazine (4),7 now used as a standard antago-
nist for the GABAA receptors, represents another struc-
tural class of ligand.
The nature of dysfunction of the GABAergic system
in schizophrenia is still unclear, but there is growing
indirect evidence supporting the hypothesis of GABAA
receptor hyperactivity as being a component of the
mechanisms underlying schizophrenic symptoms. On
the basis of receptor binding studies, an increase in the
density of GABAA receptors in brains from schizophrenic
patients has been observed.8-10 Furthermore, activation
of GABAA receptors has been reported to produce
psychotomimetic effects in normals and to stimulate
psychotic symptoms in schizophrenics.11 In Alzheimer’s
disease, a reduced central cholinergic neurotransmission
appears to be an important factor.12 Since these acetyl-
choline neurons are under inhibitory GABAergic control,
a stimulation of cholinergic transmission could, in
principle, be achieved by blockade of GABAA receptors.13
Therapeutic GABAergic approaches in Alzheimer’s dis-
ease seem to be supported by studies on different
ligands for the GABAA receptor in animal models
relevant to learning and memory.14,15
These observations suggest that in Alzheimer’s dis-
ease as well as schizophrenia, GABAA antagonists or
low-efficacy partial agonists may have therapeutic
interest. In a previous study we have described 5-(4-
piperidyl)-3-isoxazolol (4-PIOL, 5)16,17 and, more re-
cently, analogues of 4-PIOL as low-efficacy partial
GABAA agonists showing dominating antagonist pro-
files.18 The structure-activity relationships (SARs) of
F igu r e 1. Structures of GABA (1), the GABAA agonists
muscimol (2), THIP (3), and 7, the GABAA antagonist gabazine
(4), the low-efficacy partial GABAA agonists 4-PIOL (5) and
6, and the new 3-isoxazolols (8a -f).
these 4-PIOL analogues and the corresponding ana-
logues of muscimol (2) and THIP (3) are not straight-
forward and do not seem to fit into previously described
GABAA agonist pharmacophore models.18-20
Molecu la r Mod elin g. Previous attempts to elucidate
the structural relationships of muscimol (2), THIP (3),
and 4-PIOL (5) in their bioactive conformations at the
GABAA receptor have all been based on the assumption
that superimpositions of the amino nitrogens and the
3-isoxazolol rings are required. Compounds 2 and 3 have
been superimposed using this assumption,20 and Buur
et al.19 superimposed 3 and 4-PIOL (5) in this manner
by assuming that the 3-isoxazolol moiety in the bioactive
conformation of 5 adopts an axial position of the
piperidine ring. However, several lines of arguments
make these superimpositions less probable, and in order
to test the superimposition proposed by Buur et al.,
compound 6 was synthesized and pharmacologically
characterized. This compound was found to have an
affinity for the GABAA receptor which is lower than that
of 5 by only a factor of 3.18 Since an axial position of
the 3-isoxazolol ring is ruled out in compound 6 due to
ring unsaturation, it is not likely that the corresponding
ring system in 5 adopts an axial orientation of the
piperidine ring when binding to the receptor. Further-
* To whom correspondence should be addressed. Phone: (+45)
35306247. Fax: (+45) 35306040. E-mail: ano@dfh.dk.
† Department of Medicinal Chemistry.
‡ Department of Pharmacology.
10.1021/jm000371q CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/23/2000

Communications to the Editor
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26 4931
Sch em e 1a
a
Reagents: (a) NaOEt, RBr; (b) NH2OH‚HCl, NaOH, then
concd HCl; (c) HBr, AcOH.
binding modes of 2 and 5, it was envisaged that an
arginine residue is a suitable binding partner to the
carboxylate group of the endogenous ligand, GABA (1),
as well as to the 3-isoxazolol anions of 2 and 3. High-
level quantum chemical ab initio calculations show that
a bidentate interaction between the arginine side chain
and the ligand is compatible with observed SARs.24 The
involvement of an arginine residue in the binding of
muscimol has recently been demonstrated by site-
directed mutagenesis studies.25-27
The flexibility of the arginine side chain makes it
feasible to accommodate 2 and 5 in the same binding
pocket by assuming that the side chain adopts different
conformations in its binding to the two ligands, as
schematically illustrated in Figure 2a. A least-squares
molecular superimposition of mutually low-energy con-
formations of 2 and 5 calculated by using the MM3*
force field implemented in the MacroModel program28
is shown in Figure 2b.
In the proposed binding modes of 2 and 5, the
3-isoxazolol rings do not overlap (Figure 2). This implies
that the 4-position in 5 does not correspond to the
“forbidden” 4-position in 2. Thus, in contrast to the
muscimol (2) case, substitution in the 4-position of 5
may be allowed. Since no 4-substituted 4-PIOLs have
so far been reported, the properties at the GABAA
receptor of such compounds are of considerable interest
to explore.
We here describe the synthesis and pharmacological
evaluation of a series of 4-PIOL (5) analogues 8a -f in
which the above-mentioned 4-position of the 3-isoxazolol
ring was substituted by alkyl groups such as methyl and
ethyl, a benzyl group, and more bulky and lipophilic
aromatic groups such as naphthylmethyl, naphthyl-
ethyl, and anthracylmethyl (Figure 1).
Ch em istr y. The analogues of 5 were all synthesized
using the same route as outlined in Scheme 1 starting
from the â-oxo ester 9, which was synthesized by a
modification of the reported method.16 Alkylation of 9
was performed using the appropriate alkyl halide in the
presence of sodium ethoxide to give the compounds
10a -f. 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-isox-
F igu r e 2. Hypothesis for the binding of muscimol (2) and
4-PIOL (5) to the GABAA receptor (a) and a molecular least-
squares superimposition of the proposed bioactive conforma-
tions of 2 (green) and 5 binding to two different conformations
of an arginine residue (b).
more, the replacement of the isoxazol oxygens in 3 and
5 by a sulfur atom has markedly different effects on
GABAA receptor affinity.18,20 The affinity of the sulfur
analogue of 5 is 7 times higher than that of the parent
compound,18 whereas the sulfur analogue of 3 displays
more than 300 times lower affinity than 3 itself.21 These
observations strongly indicate that the 3-isoxazolol rings
in 3 and 5 do not have identical positions in the receptor
binding cavity.
Ab initio quantum chemical calculations on the con-
formational properties of 2 indicate that in order for 2
to mimic the conformation of 3, a very high conforma-
tional energy (8.9 kcal/mol) is required.22 Thus, it is
highly improbable that the bioactive conformation of 2
corresponds to that of 3. Furthermore, the very low
affinity of 4-methylmuscimol (7) (IC50 > 100 µM)23
compared to that of 2 (IC50 ) 0.006 µM)6 is most
probably due to strong steric repulsions between the
4-methyl group and the receptor. This makes a super-
imposition of the 3-isoxazolol ring systems of 2 and 3
less likely as the “sterically forbidden” 4-methyl group
would then overlap with a methylene group in 3, which
is not compatible with the high GABAA receptor affinity
of 3 (IC50 ) 0.13 µM).6
To overcome the problems of the structural relation-
ships of the bioactive conformations and the receptor

4932 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26
Communications to the Editor
Ta ble 1. Receptor Binding and in Vitro Electrophysiological
Data
[3H]muscimol bindinga
electrophysiologyb
compd
Ki (µM)c
IC50 (µM)c
gabazine (4)
4-PIOL (5)
0.074 (0.059; 0.094)
9.1 (8.2; 10)
10 (10; 11)
6.3 (5.1; 7.6)
3.8 (3.3; 4.5)
0.049 (0.043; 0.057)
0.49 (0.43; 0.54)
5.9 (5.0; 7.0)
0.24 (0.22; 0.25)
110 (76; 170)
26 (22; 31)
10.3 (7.7; 13)
4.0 (3.7; 4.3)
0.37 (0.31; 0.44)
0.89 (0.83; 0.95)
1.3d (1.2; 1.5)
8a
8b
8c
8d
8e
8f
a
Standard receptor binding on rat brain synaptic membranes,
b
n ) 3. Whole-cell patch clamp recordings from cerebral cortical
neurons cultered for 7-9 days, n ) 6-17. c Mean and SEM were
calculated assuming a logarithmic distribution of the IC50 value.29
Hence, numbers in parentheses (min; max) indicate (SEM ac-
d
cording to a logarithmic distribution of IC50
.
Because of slow
onset of antagonism, the parameters for 8d were calculated using
the response magnitude after 5-s application.
F igu r e 3. Effect of the partial agonists or antagonists on the
response to 20 µM isoguvacine using whole-cell patch-clamp
recordings from cultured cerebral cortical neurons. 20 µM
isoguvacine and varying concentrations of antagonists/partial
agonist were applied simultaneously to the cells. The response
of 20 µM isoguvacine alone has been set as 100%, and the other
responses are expressed as a fraction of this. The response to
isoguvacine is progressively reduced with increasing concen-
trations of the partial agonist or antagonist. The numbers of
cells tested in this way with each compound varied from n )
6 to n ) 17.
azolols 11a -f, which were deprotected by treatment
with hydrogen bromide in glacial acetic acid to give the
target compounds 8a -f.
In Vitr o P h a r m a cology. The affinities of the com-
pounds 8a -f for GABAA and GABAB receptor sites or
GABA uptake sites in rat brain membrane preparations,
using either [3H]muscimol or [3H]GABA as the radio-
active ligand, were determined using a modified version
of the methods described previously.18 At test concen-
trations of 100 µM for 8a -c and, due to solubility
problems, of 10 µM for 8d -f, none of the compounds
showed a detectable affinity for GABAB receptors or
GABA uptake sites. Like 5, all of the tested compounds
did show affinity for GABAA receptor sites, and binding
affinities (Ki values) of compounds 8a -f for GABAA
receptor sites are listed in Table 1.
Introduction of alkyl groups such as methyl and ethyl
as well as a benzyl group in the 4-position of the
3-isoxazolol ring of 5 is tolerated, compounds 8a -c
showing affinity for the GABAA receptor sites compa-
rable to that of 5. Remarkably, introduction of the more
bulky 2-naphthylmethyl group in the same position to
afford 8d not only was tolerated but led to a 70-fold
increase in binding affinity as compared to the benzyl
analogue 8c. Extension of the linker joining the 2-naph-
thylmethyl group and the 3-isoxazolol ring to give
compound 8e resulted in a 10-fold reduction in affinity
for the GABAA receptor relative to 8d . Further reduction
in GABAA receptor affinity was found for compound 8f,
where the 2-naphthylmethyl group of 8d has been
replaced by a 9-anthracylmethyl group.
Using whole-cell patch-clamp recordings from cul-
tured cerebral cortical neurons, performed as described
previously,18 the functional properties of compounds
8a -f in the absence or presence of the specific GABAA
receptor agonist isoguvacine5,6 (20 µM) were studied. In
a previous study, using cerebral cortical neurons, we
have characterized 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 isoguvacine in a dose-dependent manner, as il-
lustrated in Figure 3, showing a good correlation to the
obtained binding affinities (Table 1). As shown in the
GABAA receptor binding assay, the 2-naphthylmethyl
analogue 8d was the most active member of this series
showing an antagonist potency comparable with that
of the standard GABAA antagonist gabazine (4). Inter-
estingly, only compounds 8a ,b retained some ability to
induce currents themselves at a concentration of 1 mM
(not shown), while compounds 8c-f showed no detect-
able agonist effect at test concentrations of 100 µM (8c)
or 30 µM (8d -f).
The present results indicate that substitution in the
4-position of the 3-isoxazolol ring of 5 is indeed allowed
in contrast to substitution in the corresponding position
in 2 and support the hypothesis mentioned above
concerning the binding modes of 2 and 5 illustrated in
Figure 2, where the 4-positions of the 3-isoxazolol rings
of 2 and 5 are placed differently. A tendency for larger
substituents to show higher affinity for the GABAA
receptor can be seen for the methyl, ethyl, and benzyl
analogues (compounds 8a -c). Extension of the aromatic
system from a phenyl to a naphthyl group to give 8d
markedly increases the affinity, with a corresponding
increase in potency. The naphthylmethyl group in
compound 8d , seems to be optimal, at least in this
series, as regards the sterical and/or conformational
requirements for interaction with the antagonist con-
formation of the GABAA receptor. Although the affinity
of 8f is 100-fold lower than that of 8d , it still has affinity
for the receptor, which suggests a large cavity at the
4-PIOL recognition site of the GABAA receptor, capable
of accommodating not only alkyl substituents but also
aromatic systems of larger size in this type of com-
pounds. Occupancy of this suggested GABAA receptor
cavity by an appropriately sized group may contribute
to the stabilization of the antagonist conformation or,
alternatively, destabilization of the agonist conformation
of the GABAA receptor.
In conclusion, the modest GABAA receptor affinity of
5 could be markedly enhanced by introducing large
aromatic substituents into the 4-position of the 3-isox-
azolol ring of 5, supporting the hypothesis concerning
the binding mode of 5 and muscimol (2). Furthermore

Communications to the Editor
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26 4933
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pharmacological profile of the compounds from low-
efficacy partial GABAA receptor agonist activity of 5 to
potent and selective antagonist effect of 8d as the key
compound. This and related compounds could be useful
tools for studies of the GABAA receptor mechanisms,
and further studies in this area are in progress.
Ack n ow led gm en t. This work was supported by
grants from the Lundbeck Foundation, the Danish
Medical Research Council, the Academy of Finland, and
the Danish State Biotechnology Programme. The tech-
nical assistance of Lærke Andersen, Tina Lindgreen,
and Annette Kristensen and the preparation of cell
cultures by Gunilla Steven are gratefully acknowledged.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails. This material is avaible free of charge via the Internet
at http://pubs.acs.org.
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