European Journal of Pharmacology, 164 (1989) 361-364 361

Elsevier

EJP 20373

Short communication

Benzofuran analogues of baclofen: a new class of central

and peripheral GABA a-receptor antagonists
D a v i d I.B. K e r r 1,., J e n n i f e r O n g 1, G r a h a m A.R. J o h n s t o n 1, Pascal B e r t h e l o t 2
Michel D e b a e r t 2 a n d C l a u d e V a c c h e r 2
1 Department of Pharmacology, The University of Sydney, New South Wales 2006, Australia,
and 2 Laboratoires de Pharmacie Chimique et de Pharmacodynamie, Facultd de Pharmacie, 59045 Lille Cedex, France

Received 12 January 1989, accepted 21 March 1989

Two novel fl-(benzo[b]furan) analogues of baclofen (4-amino-3-benzo[b]furan-2-ylbutanoic acid, 9G, and 4-amino-
3-(5-methoxybenzo[b]furan-2-yl)butanoic acid, 9H) antagonised the baclofen-induced depression of twitch contrac-
tions in the guinea-pig isolated ileum (estimated apparent pA 2 3.9 and 4.1 respectively); both 9G and 9H also
antagonised in a dose-dependent manner the baclofen-induced reduction of repetitive paroxysmal discharges in rat
neo-cortical slice preparations maintained in Mg2+-free Krebs solution. These benzofurans evidently represents a new
class of GABAB-receptor antagonist.

GABA B receptor antagonists; Benzofuran-baclofen analogues; Baclofen; Ileum (guinea-pig); Neocortex (rat)

I. Introduction for distinguishing GABAB-receptor-mediated ac-

GABA receptors are now subdivided into
GABA A- and GABAB-subtypes with differing
agonist and antagonist profiles. Of these,
GABAA-receptors have been well characterised
and are known to be involved in bicuculline-sensi-
tive central inhibition, whereas GABAB-receptors
and their functions are less well understood, al-
though baclofen is a specific agonist at these bi-
cuculline-insensitive receptors (Bowery et al.,
1981). So far, only phaclofen, the phosphono-ana-
logue of baclofen, and 2-OH-saclofen, a sulphonic
analogue, have been shown to be specific GABA ~-
receptor antagonists, where 2-OH-saclofen is more
potent (Kerr et al., 1988) than phaclofen (Kerr et
al., 1987), although the latter has been widely used
* To whom all correspondenceshould be addressed: Depart-
ment of Pharmacology, The University of Sydney, New
South Wales 2006, Australia.
0014-2999/89/$03.50 © 1989 ElsevierSciencePublishers B.V. (BiomedicalDivision)
tions in the central nervous system (Dutar and
Nicoll, 1988). In addition, we have recently seen
that alteration of the p-chlorophenyl on the r -
carbon of baclofen, as in fl-phenyl-GABA, can
result in a partial agonist/antagonist activity at
GABAB-receptors (Ong et al., 1987), and here
report that two novel fl-(benzo[b]furan) analogues
of baclofen (Berthelot et al., 1987) are also
antagonists at both central and peripheral
GABAB-receptors.
2. Materials and methods
Rat cortical slice preparations were set up for
recording spontaneous paroxysmal depolarisations
in Mg2+-free medium, according to the method
previously described (Kerr et al., 1988). Male
Sprague-Dawley rats, 150-250 g, were decapitated,
their brains rapidly removed and immersed for at
least 20 min in ice-cold Krebs solution oxygenated
362
with 95% Oz-5% C O 2. Coronal sections of cerebral
cortical slices, each approximately 500 /~m thick,
were prepared using a vibraslice microtome, and a
radial wedge was cut from each side of the dorsal
mid-line to yield slices of cingulate cortex and
corpus callosum 1.5-2 mm wide. Using a method
adapted from H o m e et al. (1986), based on a
grease-gap system (Wheatley, 1986), the neocorti-
cal slices were placed between layers of absorbent
paper supported on an inclined block at room
temperature, and arranged such that the corpus
callosum side was raised on to a non-polarizable
recording wick electrode and surrounded with a
paraffin grease barrier to provide insulation and
prevent tissue dessication, the reference electrode
and earth being positioned adjacent to the tissue.
The cortical slice was initially superfused with
Mg 2+ (1.3 mM)-containing Krebs medium
delivered by a peristaltic pump at 1 ml per min
and allowed to equilibrate for at least 1 h, after
which the solution was switched to MgZ+-free
Krebs medium and left until paroxysmal dis-
charges developed, usually after a period of 30
min. Drugs were then added to the Mg2+-free
medium and subsequently superfused over the grey
matter of the slice for 5 min, usually at 30-60 min
intervals depending on the recovery of the
responses to control level. Where antagonists were
tested, they were co-superfused with the agonist.
DC potentials between the cingulate cortex and
the corpus callosum were monitored by Ag/AgC1
electrodes via agar/saline bridges and a high in-
put impedance d.c. amplier, responses being dis-
played on a chart recorder. In this recording
arrangement, depolarising agents such as KC1 (5
mM) or N-methyl-D-aspartate (10 /~M), when
superfused over the cortical slice, caused an up-
ward deflection, as did the spontaneous paro-
xysmal depolarisations in MgZ+-free medium.
Each experiment was repeated on six slices from
six different animals. The composition of the Krebs
medium used was identical with that previously
reported (Kerr et al., 1988).
For the ileal preparations, male guinea-pigs
(200-400 g) were stunned by a blow on the head
and bled. Segments of distal ileum, 3-4 cm in
length, and taken 2-3 cm from the ileo-caecal
valve, were quickly removed and mounted in 20
ml organ baths containing modified Krebs-bi-
carbonate solution gassed with a mixture of 95%
O 2 and 5% CO 2 at 37°C, pH 7.4, as previously
described (Ong and Kerr, 1983). The tissues were
initially placed under a resting tension of 1 g and
were allowed to equilibrate for 60 min in the bath.
Isometric contractions of the longitudinal muscle
were measured by a Grass polygraph recorder. A
pair of ring electrodes was used to deliver trans-
mural stimulation using pulses at 0.5 ms duration,
0.1-0.2 Hz, and a just maximal voltage in the
isolated ileal preparations. Drugs were added at
20-30 rain intervals, depending on the recovery of
the tissue responses to control level, and the
antagonists added at least 5-10 min before the
agonist was challenged. Student's t-test for paired
and unpaired samples was used to assess the sig-
nificance (P < 0.05) of differences between mean
values of the dose-response effects. The number of
experiments performed was n = 8.
Drugs used were baclofen (Ciba-Geigy), 2-
OH-saclofen (courtesy of Professor R.H. Prager,
Flinders University, South Australia), phaclofen
(courtesy of Dr. K. Mewett, University of Sydney),
acetylcholine chloride (Sigma), and both 9G and
9H were prepared as already described (Berthelot
et al., 1987).
3. Results
Repetitive cholinergic twitch contractions in
guinea-pig isolated ileal tissues are depressed by
baclofen in a dose-dependent manner (Ong et al.,
1987). As seen in fig. la, the latter effect of
baclofen (5 /~M) was effectively antagonised by
compounds 9G and 9H (each at 0.5 raM; n = 8).
The concentration of 9G and 9H employed in fig.
l a was the threshold for blocking the ECs0 of
baclofen (5 /~M), and both 9G and 9H at the
lower concentration of 0.25 mM only antagonised
baclofen (5 /~M) by 45%. Phaclofen (0.5 mM)
similarly antagonised baclofen (5 /~M) in two of
the preparations also used with 9G and 9H,
whereas 2-OH-saclofen was effective at 50 ~tM.
An approximation of the pA 2 values for the two
benzofurans was obtained using the 'single dose'
method of Kosterlitz and Watt (1968), yielding an

OCH3
H2N CO2H H2N CO2H
9G 9H
b I rain 9G 9H , tion for 9G was 0.5 mM. However, neither com-
bac l
913
50,uV bac [ which retain the carboxylate of the parent baclo-
9H
5rnin
Fig. 1. Reversible antagonism of GABAB-receptor-mediated
actions by benzofuran analogues of baclofen, 9G (4-amino-3-
benzo[b]furan-2-ylbutanoic acid) and 9H (4-amino-3-(5-
methoxybenzo[b]furan-2-yl)-butanoicacid). (a) Baclofen (bac;
5 #M) depressed the ileal twitch response to transmural stimu-
lation, antagonised by compounds 9G (0.5 mM) and 9H (0.5
mM) with recovery of the response to baclofen after washout
of the benzofuran derivatives (n = 8). (b) Paroxysmal dis-
charges in the rat isolated neo-cortical slice mounted in Mg 2+-
free Krebs solution; baclofen (5/~M) inhibited the discharges,
antagonised by compound 9G (0.5 mM), with recovery of the
baclofen effect after washout. (c) Similar discharges inhibited
by baclofen (5 /LM) and antagonised by compound 9H (0.5
mM) showing recovery of the baclofen effect after washout
(n = 6 for each).
apparent pA z of 3.9 for 9G and 4.1 for 9H. Both
9G and 9H (0.5 mM, respectively) did not affect
contractile responses to exogenous ACh (10 nM).
Baclofen also inhibits spontaneous paroxysmal
depolarisations in rat neo-cortical slices main-
tained in Mg2+-free Krebs medium, an effect
363
mediated through GABAB-receptors and sensitive
to 2-OH-saclofen (Kerr et al., 1988). In such pre-
parations, a concentration of baclofen (5/~M) can
be found that just halts the discharges (fig. lb,c),
and co-administration of compound 9G (0.5 mM;
fig lb) or of 9H (0.5 mM; fig. lc) effectively
prevented this depressant action of baclofen (n =
6), whereas 2-OH-saclofen was effective at 50 # M
on the same slices (not shown). 9G was some
2.5-fold less potent than 9H in antagonising the
baclofen (5 /~M)-induced depression of the paro-
xysmal discharges, the threshold concentration of
9H being 0.2 m M whilst the threshold concentra-
pound 9G nor 9H was effective at 0.1 m M in
preventing the depressive action of baclofen in
any of the slices tested.
4. Discussion
Previously, GABA~-antagonists have been de-
veloped by alterations of the acidic function of
baclofen, respectively to phosphonate in phaclo-
fen, and to sulphonate in 2-OH-saclofen. It was
thus not anticipated that compounds 9G and 9H,
fen, would be GABAB-receptor antagonists as
found here, although the related fl-phenyl-GABA
is a partial agonist that exhibits some antagonism
at these receptors (Ong et al., 1987); it is possible,
however, that the antagonism seen here is simi-
larly due to partial agonist/antagonist actions in
compounds 9G and 9H, imparted by the aromatic
nature of the benzofuran ring they contain. The
apparent pA 2 values, 3.9 and 4.1 obtained for 9G
and 9H, respectively, are close to that of phaclo-
fen (apparent pA2 = 4.0), although the IC50 for
the latter in binding studies under conditions for
GABAB-receptor binding is 50 /~M (C.A. Drew,
personal communication), considerably higher
than for either 9G (18/~M) or 9H (5.6/~M) under
similar conditions (Berthelot et al., 1987); we have
no explanation for this discrepancy. Nevertheless,
the higher potency of 9H was also reflected in its
lower threshold concentration for antagonising the
baclofen-induced depression of spontaneous
paroxysmal discharges in neocortical slice pre-
364
p a r a t i o n s . T h e p r e s e n t results t a k e n t o g e t h e r w i t h
t h e G A B A B-receptor b i n d i n g studies ( B e r t h e l o t et
al., 1987), p r o v i d e s o m e i n s i g h t i n t o t h e s t r u c t u r a l
r e q u i r e m e n t s for a n t a g o n i s m b y s u c h b a c l o f e n
a n a l o g u e s , since e f f e c t i v e i n h i b i t i o n o f [ 3 H ] b a c l o -
f e n b i n d i n g is o n l y f o u n d w i t h p l a n a r r i n g struc-
tures that are in t h e s a m e p l a n e as t h e f l - c a r b o n o f
t h e G A B A chain, as in the b e n z o f u r a n s r a t h e r
t h a n their d i h y d r o - d e r i v a t i v e s . W i t h a p o t e n c y
comparable with that of phaclofen, these benzo-
furan analogues of baclofen evidently represent a
n e w class of G A B A B-receptor a n t a g o n i s t s .
Acknowledgements
We thank the Australian National Research Fellowship
Advisory Committee for the award of a Queen Elizabeth II
Fellowship to Jennifer Ong, and the National Health and
Medical Research Council of Australia for financial support.
We also thank Professor R.H. Prager and Dr. K. Mewett for
the gift of 2-hydroxy-saclofen and phaclofen, respectively.
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