European Journal of Pharmacology, 218 (1992) 343-345
343
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 21090 Short communication
Actions of thienyl analogs of baclofen in the guinea-pig isolated ileum J e n n i f e r O n g , D a v i d I.B. K e r r , P a s c a l B e r t h e l o t a C l a u d e V a c c h e r a, N a t h a l i e F l o u q u e t a and Michel Debaert a Department of Anaesthesia and Intensive Care, University of Adelaide, Adelaide, South Australia 5000, Australia and a Laboratoire de Pharmacie Chimique, Facult~de Pharmacie, Universit~de Lille, Lille, France
Received 24 February 1992, revised MS received 6 May 1992, accepted 26 May 1992
In guinea-pig isolated ileal preparations, the 5-methylthien-2-yl (5d), 5-bromothien-2-yl (5f) and 5-chiorothien-2-yl (5h) analogs of baclofen depressed twitch responses to field stimulation in a dose-dependent manner. These actions were reversibly and competitively antagonised by 2-hydroxysaciofen but not by naloxone, phentolamine, propranolol or theophylline. The relative potencies (ECs0 values) were baclofen (10/~M) > 5h (40/~M) > 5d (80/zM) > 5f (120/~M). These analogs represent a novel class of specific GABA a receptor agonists which, like baclofen, should readily enter the brain. Baclofen; GABA B receptor agonists; Thienyl baclofen analogs; 2-Hydroxysaclofen; Ileum (guinea-pig, isolated)
1. Introduction
y-Aminobutyric acid (GABA), a classical inhibitory neurotransmitter, has been shown to activate at least two distinct classes of receptor subtypes, G A B A A and G A B A a receptors (Bowery et al., 1981). G A B A A receptors are sensitive to bicuculline and are coupled to chloride channels, whilst G A B A a receptors are bicuculline-insensitive but are activated by baclofen (Bowery et al., 1981), and antagonised by the phosphonic and sulphonic baclofen analogs, phaclofen and 2-hydroxysaclofen (Kerr et al., 1987, 1988). Activation of G A B A a receptors leads to neuronal inhibition at various synapses, through G A B A B receptor pre- and postsynaptic subtypes (Dutar and Nicoll, 1988), but heterogeneity among these receptors is still not firmly established. This is largely due to the limited range of G A B A n receptor agonists and antagonists so far available. Indeed, to date, baclofen is the only ligand commonly used to activate G A B A B receptor sites, since relatively few specific agonists have been developed, apart from the G A B A analog, 3-aminopropanephosphonic acid (Dingwall et al., 1987; Seabrook et al., 1991), and its P-methyl derivative, SKF 97541 (Seabrook et al., 1991). Baclofen (/3-4-chlorophenyl-GABA) is a well characterised specific agonist for G A B A a receptors, in which the fl-4-chlorophenyl substituent on the
Correspondence to: J. Ong, Department of Anaesthesia and Intensive Care, University of Adelaide, Adelaide, South Australia 5000, Australia. Tel. 61.8.228 5163, fax 61.8 232 3283.
G A B A backbone imparts the specificity for these receptors (Bowery et al., 1981). Few true baclofen analogs are known, especially with any heterocyclic replacement of the 4-chlorophenyl group, although the benzofuran analogs (Berthelot et al., 1987) are partial agonists/antagonists (Kerr et al., 1989). Recently, however, Berthelot et al. (1991) have synthesised a series of heterocyclic compounds structurally related to baclofen, among which the 5-methylthien-2-yl (5d), 5-bromothien-2-yl (5f) and 5-chlorothien-2-yl (5h) analogs of baclofen, are selective ligands for G A B A B receptor sites, and potent displacers of ( R ) - ( - ) [3H]baclofen in binding studies. Using isolated ileal preparations from the guinea-pig, we have investigated the pharmacological activity of these thienyl analogs of baclofen, and now show that, while weaker than baclofen, they are full agonists at G A B A B receptor sites.
2. Materials and methods 2.1. Guinea-pig isolated ileal preparations
Male guinea-pigs, weighing between 200-400 g, were stunned by a blow on the head and bled. Segments of the terminal ileum, taken 2 cm from the ileo-caecal valve, were quickly removed and placed in modified Krebs-bicarbonate solution of the following composition (mM): Na + 151.0; K + 4.6; Mg z+ 0.6; Ca 2÷ 2.0; CI- 134.9; H C O 3 24.9; H z P O 4 1.3; SO42+ 0.6; glucose 7.7 (pH 7.4). The Krebs solution was continuously aerated with a gas mixture of 95% 0 2 and 5% CO z.
344 Ileal segments 2 cm long were m o u n t e d in a 5-ml organ bath and maintained in Krebs solution at a constant t e m p e r a t u r e of 37°C. T h e tissues were initially placed u n d e r a resting tension of 1 g and were allowed to equilibrate for 60 min in the bath. T h e effects of drug treatments on electrically evoked ileal twitch contractions were examined. Contractions were elicited t h r o u g h ring electrodes positioned a r o u n d the segments of the ileum to provide field stimulation. Pulses (duration 0.5-1 ms, repetitive frequency 0.15 Hz, just submaximal voltage) were delivered from a Grass $48 stimulator to give transmural stimulation of cholinergic intrinsic neurones. Isometric contractions of the longitudinal muscle were m e a s u r e d by m e a n s of a Grass FT03 force transducer and changes in tension were subsequently displayed on a Linear chart recorder. Agonists were applied within 30-min intervals, depending on the recovery of the tissue responses to control levels. T h e antagonist was a d d e d 5 min before agonists were tested, and control responses to agonists were constantly re-established after washing out each antagonist. T h e drug volumes used never exceeded 1% of the total bath volume. All experiments were rep e a t e d on at least six tissues from six different animals. Student's t-test for paired and u n p a i r e d samples was used to assess the significance (P < 0.05) of differences between m e a n values of the d o s e - r e s p o n s e effects. T h e concentrations of the various agonists which ind u c e d a 50% inhibitory response were calculated to give an ECs0 value for each agonist; the ECs0 value was d e t e r m i n e d by interpolation from the dose-response curves.
% max. inhibition 100
50
5
Fig. l. Dose-response curves for the depression by baclofen and the thienyl analogs of baclofen, 5-chlorothien-2-yl (5h) and 5-methylthien-2-yl (5d) of cholinergic twitch contractions in the guinea-pig isolated ileum. The dose-response curves for baclofen (11; n = 10), 5h (o; n = 8) and 5d (o; n = 8) are based on percentages of the maximal inhibition (expressed as 100%) of the twitch contractions induced by the agonists. Each point represents the mean and S.E.M.; n indicates the number of experiments performed.
s2
H3C
H2N
3. Results As shown in fig. 1, racemic baclofen and the analogs 5h and 5d elicited a d o s e - d e p e n d e n t depression of cholinergic twitch contractions in the guinea-pig isolated ileal preparations, 5h and 5d being less potent t h a n baclofen itself. C o m p o u n d 5f was the least active of all these agonists. T h e depression of the responses elicited by baclofen, 5h, 5d and 5f (see fig. 2, for chemical structures) was antagonised surmountably by 2-hydroxysaclofen (100/.tM), the latter causing a rightward shift of the d o s e - r e s p o n s e curve for each of th~ agonists (not shown). With each of these c o m p o u n d s , the depressions r e t u r n e d to the control levels on re-
Br
LmN 1 rain
CI
COOH
/l/b ii 5d
2.2. Drugs T h e thienyl analogs 5d, 5f and 5h were synthesised as racemates by Dr. Berthelot and his colleagues at Lille; 2-hydroxysaclofen was a gift from Professor R.H. Prager (Flinders University, South Australia). Baclofen was o b t a i n e d f r o m Ciba-Geigy. Theophylline, propranolol, p h e n t o l a m i n e and naloxone were f r o m Sigma.
4 -log dose
5f
BA"~"
5h
5h
5d
5f
iii ii/ Iii 5h
2-OH-SAC Fig. 2. Depression by baclofen (BAC) and the thienyl analogs of baclofen of cholinergic twitch contractions in the guinea-pig isolated ileum. The chemical structures of 5-methylthien-2-$,l (5d), 5bromothien-2-yl (50 and 5-chlorothien-2-yl (5h) analogs are shown in the upper panel, while the middle panel indicates typical responses to equipotent concentrations of the agonists in the same ileal preparation, with wash-outs between drug applications: baclofen (BAC, 10 p.M), 5d (80 ~tM), 5f (120 ~M) and 5h (40 /xM). The lower panel shows the reversible antagonism of 5h (10 p.M)-induced depression of guinea-pig ileal twitch contractions by 2-hydroxysaclofen (2-OHSAC, 100 /xM; n = 6), with recovery of the control response to 5h upon wash-out within 30 min.
345
peated washing of the tissue with Krebs solution within 30-min intervals. The ECs0 value for baclofen in inhibiting the twitch contractile responses was 10 /zM, and the'ECs0 values for 5h, 5d and 5f were 40, 80 and 120/zM respectively, indicating that 5h, 5d and 5f were approximately 4, 8 and 12 times less potent than baclofen to depress the contractions (fig. 2). Within 5 min, the depressant effect induced by 5h (10 /~M) was reversibly and competitively antagonised by 2-hydroxysaclofen (100 #M; fig. 2), which did not affect the twitch responses on its own. Following wash-out, there was a recovery of the depressive response to compound 5h within 30 min. None of these compounds displayed any partial agonist/antagonist activity at concentrations up to 500 /zM, and the depressions elicited by baclofen, 5h, 5d and 5f were not affected by theophylline (50 /xM), naloxone (50 /.~M), propranoiol (50 /xM) or phentolamine (5 ~M). The depressions due to 5h, 5d or 5f did not show any desensitization over the 2-min period of application, while the maximal effect of each of the agonists was similar, and was not additive with a maximum baclofen response (not shown). 4. Discussion In the present study, the 5-methyl-, 5-bromo- and 5-chlorothien-2-yl analogs of baclofen (compounds 5d, 5f, 5h; Berthelot et al., 1991) all behaved in the same way as baclofen at GABA ~ receptors in the guinea-pig ileum, although they were less potent than baclofen. They each dose dependently depressed electrically stimulated ileal twitch responses, the depression being competitively antagonised by the GABA B receptor antagonist, 2-hydroxysaclofen. Furthermore, the maximal responses they caused were comparable to that to baclofen, and they displayed no additivity with a maximal response to baclofen. This indicates that all these agonists, including baclofen, act through a common receptor. In keeping with this, the responses they elicited were not affected by naloxone, phentolamine, propranolol or theophylline. Furthermore, none of the analogs displayed any partial agonist/antagonist activity; instead, they each acted as full GABA B receptor agonists in the guinea-pig isolated ileum. These thienyl analogs were all less potent than baclofen to inhibit the electrically stimulated twitch contractions, with 5h, 5d and 5f being 4, 8 and 12 times respectively less potent than baclofen. Berthelot et al. (1991) found the relative ICs0 values for baclofen, 5h, 5d and 5f, displacement of the binding of (R)-(-)[3H]baclofen to GABA B sites in the rat brain to be 0.33, 0.61, 1.34 and 1.86 /zM respectively, which is comparable to the rank order we now found for their agonist activities in the periphery. So far, apart from baclofen itself, /3-(5-chlorothien-2-yl)-GABA is the most potent analog of baclofen and, like baclofen,
exhibits no binding activity at GABA A receptors. Both baclofen and compound 5 h have a chloro-substituent on the ring attached at the /3-carbon of the GABA backbone, where the /3-(5-chlorothien) substituent of 5h appears directly comparable with the 4-chlorophenyl of baclofen. Moving the chlorine or changing the nature of this halogen substituent causes lower potency in both the thienyl analogs and baclofen. In imparting specificity at GABA B receptors, this hydrophobic substituent evidently contributes to the binding of these agonists rather than simply excluding binding at the GABA A site, since R-(-)-baclofen is more potent at GABA B receptors than is GABA itself (Bowery et al., 198l). Thus, the present study confirmed the original findings that these thienyl analogs of baclofen, which represent a novel class of specific GABA B receptor agonists that should readily enter the brain, are effective ligands at GABA B receptor sites.
Acknowledgements Dr. Jennifer Ong is the recipient of an Australian Research Council Research Fellowship. We thank Professor Prager (Flinders University, South Australia) for the gift of 2-hydroxysaclofen.
References Berthelot, P., C. Vaccher, A. Musadad, N. Flouquet, M. Debaert and M. Luyckx, 1987, Synthesis and pharmacological evaluation of y-aminobutyric acid analogues. New ligand for GABA B sites, J. Med. Chem. 30, 743. Berthelot, P., C. Vaccher, N. Flouquet, M. Debaert, M. Luyckx and C. Brunet, 1991, 3-Thienyl- and 3-furylaminobutyric acids. Synthesis and binding GABA B receptor studies, J. Med. Chem. 34, 2557.
Bowery, N.G., A. Doble, D.R. Hill, A.L. Hudson, J.S. Shaw, M.J. Turnbull and R. Warrington, 1981, Bicuculline-insensitive GABA receptors on peripheral autonomic nerve terminals, Eur. J. Pharmacol. 71, 53. Dingwall, J.-G., J. Ehrenfreund, R.G. Hall and J. Jack, 1987, Synthesis of gamma-aminopropylphosphonous acid using hypophosphorous acid synthons, Phosph. Sulfur. 30, 571. Dutar, P. and R.A. Nicoll, 1988, Pre- and postsynaptic GABA B receptors have different pharmacological properties, Neuron 1, 585. Kerr, D.I.B., J. Ong, R.H. Prager, B.D. Gynther and D.R. Curtis, 1987, Phaclofen: a peripheral and central baclofen antagonist, Brain Res. 405, 150. Kerr, D.I.B., J. Ong, G.A.R. Johnston, J. Abbenante and R.H. Prager, 1988, 2-Hydroxy-saclofen: an improved antagonist at central and peripheral GABAa-receptor , Neurosci. Lett. 92, 92. Kerr, D.I.B., J. Ong, G.A.R. Johnston, P. Berthelot, M. Debaert and C. Vaccher, 1989, Benzofuran analogues of baclofen: a new class of peripheral GABAB-receptor antagonists, Eur. J. Pharmacol. 164, 361. Seabrook, G.R., W. Howson and M.G. Lacey, 1990, Electrophysiological characterization of potent agonists and antagonists at preand postsynaptic GABA a receptors on neurones in rat brain slices, Br. J. Pharmacol. 101, 949.
343
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 21090 Short communication
Actions of thienyl analogs of baclofen in the guinea-pig isolated ileum J e n n i f e r O n g , D a v i d I.B. K e r r , P a s c a l B e r t h e l o t a C l a u d e V a c c h e r a, N a t h a l i e F l o u q u e t a and Michel Debaert a Department of Anaesthesia and Intensive Care, University of Adelaide, Adelaide, South Australia 5000, Australia and a Laboratoire de Pharmacie Chimique, Facult~de Pharmacie, Universit~de Lille, Lille, France
Received 24 February 1992, revised MS received 6 May 1992, accepted 26 May 1992
In guinea-pig isolated ileal preparations, the 5-methylthien-2-yl (5d), 5-bromothien-2-yl (5f) and 5-chiorothien-2-yl (5h) analogs of baclofen depressed twitch responses to field stimulation in a dose-dependent manner. These actions were reversibly and competitively antagonised by 2-hydroxysaciofen but not by naloxone, phentolamine, propranolol or theophylline. The relative potencies (ECs0 values) were baclofen (10/~M) > 5h (40/~M) > 5d (80/zM) > 5f (120/~M). These analogs represent a novel class of specific GABA a receptor agonists which, like baclofen, should readily enter the brain. Baclofen; GABA B receptor agonists; Thienyl baclofen analogs; 2-Hydroxysaclofen; Ileum (guinea-pig, isolated)
1. Introduction
y-Aminobutyric acid (GABA), a classical inhibitory neurotransmitter, has been shown to activate at least two distinct classes of receptor subtypes, G A B A A and G A B A a receptors (Bowery et al., 1981). G A B A A receptors are sensitive to bicuculline and are coupled to chloride channels, whilst G A B A a receptors are bicuculline-insensitive but are activated by baclofen (Bowery et al., 1981), and antagonised by the phosphonic and sulphonic baclofen analogs, phaclofen and 2-hydroxysaclofen (Kerr et al., 1987, 1988). Activation of G A B A a receptors leads to neuronal inhibition at various synapses, through G A B A B receptor pre- and postsynaptic subtypes (Dutar and Nicoll, 1988), but heterogeneity among these receptors is still not firmly established. This is largely due to the limited range of G A B A n receptor agonists and antagonists so far available. Indeed, to date, baclofen is the only ligand commonly used to activate G A B A B receptor sites, since relatively few specific agonists have been developed, apart from the G A B A analog, 3-aminopropanephosphonic acid (Dingwall et al., 1987; Seabrook et al., 1991), and its P-methyl derivative, SKF 97541 (Seabrook et al., 1991). Baclofen (/3-4-chlorophenyl-GABA) is a well characterised specific agonist for G A B A a receptors, in which the fl-4-chlorophenyl substituent on the
Correspondence to: J. Ong, Department of Anaesthesia and Intensive Care, University of Adelaide, Adelaide, South Australia 5000, Australia. Tel. 61.8.228 5163, fax 61.8 232 3283.
G A B A backbone imparts the specificity for these receptors (Bowery et al., 1981). Few true baclofen analogs are known, especially with any heterocyclic replacement of the 4-chlorophenyl group, although the benzofuran analogs (Berthelot et al., 1987) are partial agonists/antagonists (Kerr et al., 1989). Recently, however, Berthelot et al. (1991) have synthesised a series of heterocyclic compounds structurally related to baclofen, among which the 5-methylthien-2-yl (5d), 5-bromothien-2-yl (5f) and 5-chlorothien-2-yl (5h) analogs of baclofen, are selective ligands for G A B A B receptor sites, and potent displacers of ( R ) - ( - ) [3H]baclofen in binding studies. Using isolated ileal preparations from the guinea-pig, we have investigated the pharmacological activity of these thienyl analogs of baclofen, and now show that, while weaker than baclofen, they are full agonists at G A B A B receptor sites.
2. Materials and methods 2.1. Guinea-pig isolated ileal preparations
Male guinea-pigs, weighing between 200-400 g, were stunned by a blow on the head and bled. Segments of the terminal ileum, taken 2 cm from the ileo-caecal valve, were quickly removed and placed in modified Krebs-bicarbonate solution of the following composition (mM): Na + 151.0; K + 4.6; Mg z+ 0.6; Ca 2÷ 2.0; CI- 134.9; H C O 3 24.9; H z P O 4 1.3; SO42+ 0.6; glucose 7.7 (pH 7.4). The Krebs solution was continuously aerated with a gas mixture of 95% 0 2 and 5% CO z.
344 Ileal segments 2 cm long were m o u n t e d in a 5-ml organ bath and maintained in Krebs solution at a constant t e m p e r a t u r e of 37°C. T h e tissues were initially placed u n d e r a resting tension of 1 g and were allowed to equilibrate for 60 min in the bath. T h e effects of drug treatments on electrically evoked ileal twitch contractions were examined. Contractions were elicited t h r o u g h ring electrodes positioned a r o u n d the segments of the ileum to provide field stimulation. Pulses (duration 0.5-1 ms, repetitive frequency 0.15 Hz, just submaximal voltage) were delivered from a Grass $48 stimulator to give transmural stimulation of cholinergic intrinsic neurones. Isometric contractions of the longitudinal muscle were m e a s u r e d by m e a n s of a Grass FT03 force transducer and changes in tension were subsequently displayed on a Linear chart recorder. Agonists were applied within 30-min intervals, depending on the recovery of the tissue responses to control levels. T h e antagonist was a d d e d 5 min before agonists were tested, and control responses to agonists were constantly re-established after washing out each antagonist. T h e drug volumes used never exceeded 1% of the total bath volume. All experiments were rep e a t e d on at least six tissues from six different animals. Student's t-test for paired and u n p a i r e d samples was used to assess the significance (P < 0.05) of differences between m e a n values of the d o s e - r e s p o n s e effects. T h e concentrations of the various agonists which ind u c e d a 50% inhibitory response were calculated to give an ECs0 value for each agonist; the ECs0 value was d e t e r m i n e d by interpolation from the dose-response curves.
% max. inhibition 100
50
5
Fig. l. Dose-response curves for the depression by baclofen and the thienyl analogs of baclofen, 5-chlorothien-2-yl (5h) and 5-methylthien-2-yl (5d) of cholinergic twitch contractions in the guinea-pig isolated ileum. The dose-response curves for baclofen (11; n = 10), 5h (o; n = 8) and 5d (o; n = 8) are based on percentages of the maximal inhibition (expressed as 100%) of the twitch contractions induced by the agonists. Each point represents the mean and S.E.M.; n indicates the number of experiments performed.
s2
H3C
H2N
3. Results As shown in fig. 1, racemic baclofen and the analogs 5h and 5d elicited a d o s e - d e p e n d e n t depression of cholinergic twitch contractions in the guinea-pig isolated ileal preparations, 5h and 5d being less potent t h a n baclofen itself. C o m p o u n d 5f was the least active of all these agonists. T h e depression of the responses elicited by baclofen, 5h, 5d and 5f (see fig. 2, for chemical structures) was antagonised surmountably by 2-hydroxysaclofen (100/.tM), the latter causing a rightward shift of the d o s e - r e s p o n s e curve for each of th~ agonists (not shown). With each of these c o m p o u n d s , the depressions r e t u r n e d to the control levels on re-
Br
LmN 1 rain
CI
COOH
/l/b ii 5d
2.2. Drugs T h e thienyl analogs 5d, 5f and 5h were synthesised as racemates by Dr. Berthelot and his colleagues at Lille; 2-hydroxysaclofen was a gift from Professor R.H. Prager (Flinders University, South Australia). Baclofen was o b t a i n e d f r o m Ciba-Geigy. Theophylline, propranolol, p h e n t o l a m i n e and naloxone were f r o m Sigma.
4 -log dose
5f
BA"~"
5h
5h
5d
5f
iii ii/ Iii 5h
2-OH-SAC Fig. 2. Depression by baclofen (BAC) and the thienyl analogs of baclofen of cholinergic twitch contractions in the guinea-pig isolated ileum. The chemical structures of 5-methylthien-2-$,l (5d), 5bromothien-2-yl (50 and 5-chlorothien-2-yl (5h) analogs are shown in the upper panel, while the middle panel indicates typical responses to equipotent concentrations of the agonists in the same ileal preparation, with wash-outs between drug applications: baclofen (BAC, 10 p.M), 5d (80 ~tM), 5f (120 ~M) and 5h (40 /xM). The lower panel shows the reversible antagonism of 5h (10 p.M)-induced depression of guinea-pig ileal twitch contractions by 2-hydroxysaclofen (2-OHSAC, 100 /xM; n = 6), with recovery of the control response to 5h upon wash-out within 30 min.
345
peated washing of the tissue with Krebs solution within 30-min intervals. The ECs0 value for baclofen in inhibiting the twitch contractile responses was 10 /zM, and the'ECs0 values for 5h, 5d and 5f were 40, 80 and 120/zM respectively, indicating that 5h, 5d and 5f were approximately 4, 8 and 12 times less potent than baclofen to depress the contractions (fig. 2). Within 5 min, the depressant effect induced by 5h (10 /~M) was reversibly and competitively antagonised by 2-hydroxysaclofen (100 #M; fig. 2), which did not affect the twitch responses on its own. Following wash-out, there was a recovery of the depressive response to compound 5h within 30 min. None of these compounds displayed any partial agonist/antagonist activity at concentrations up to 500 /zM, and the depressions elicited by baclofen, 5h, 5d and 5f were not affected by theophylline (50 /xM), naloxone (50 /.~M), propranoiol (50 /xM) or phentolamine (5 ~M). The depressions due to 5h, 5d or 5f did not show any desensitization over the 2-min period of application, while the maximal effect of each of the agonists was similar, and was not additive with a maximum baclofen response (not shown). 4. Discussion In the present study, the 5-methyl-, 5-bromo- and 5-chlorothien-2-yl analogs of baclofen (compounds 5d, 5f, 5h; Berthelot et al., 1991) all behaved in the same way as baclofen at GABA ~ receptors in the guinea-pig ileum, although they were less potent than baclofen. They each dose dependently depressed electrically stimulated ileal twitch responses, the depression being competitively antagonised by the GABA B receptor antagonist, 2-hydroxysaclofen. Furthermore, the maximal responses they caused were comparable to that to baclofen, and they displayed no additivity with a maximal response to baclofen. This indicates that all these agonists, including baclofen, act through a common receptor. In keeping with this, the responses they elicited were not affected by naloxone, phentolamine, propranolol or theophylline. Furthermore, none of the analogs displayed any partial agonist/antagonist activity; instead, they each acted as full GABA B receptor agonists in the guinea-pig isolated ileum. These thienyl analogs were all less potent than baclofen to inhibit the electrically stimulated twitch contractions, with 5h, 5d and 5f being 4, 8 and 12 times respectively less potent than baclofen. Berthelot et al. (1991) found the relative ICs0 values for baclofen, 5h, 5d and 5f, displacement of the binding of (R)-(-)[3H]baclofen to GABA B sites in the rat brain to be 0.33, 0.61, 1.34 and 1.86 /zM respectively, which is comparable to the rank order we now found for their agonist activities in the periphery. So far, apart from baclofen itself, /3-(5-chlorothien-2-yl)-GABA is the most potent analog of baclofen and, like baclofen,
exhibits no binding activity at GABA A receptors. Both baclofen and compound 5 h have a chloro-substituent on the ring attached at the /3-carbon of the GABA backbone, where the /3-(5-chlorothien) substituent of 5h appears directly comparable with the 4-chlorophenyl of baclofen. Moving the chlorine or changing the nature of this halogen substituent causes lower potency in both the thienyl analogs and baclofen. In imparting specificity at GABA B receptors, this hydrophobic substituent evidently contributes to the binding of these agonists rather than simply excluding binding at the GABA A site, since R-(-)-baclofen is more potent at GABA B receptors than is GABA itself (Bowery et al., 198l). Thus, the present study confirmed the original findings that these thienyl analogs of baclofen, which represent a novel class of specific GABA B receptor agonists that should readily enter the brain, are effective ligands at GABA B receptor sites.
Acknowledgements Dr. Jennifer Ong is the recipient of an Australian Research Council Research Fellowship. We thank Professor Prager (Flinders University, South Australia) for the gift of 2-hydroxysaclofen.
References Berthelot, P., C. Vaccher, A. Musadad, N. Flouquet, M. Debaert and M. Luyckx, 1987, Synthesis and pharmacological evaluation of y-aminobutyric acid analogues. New ligand for GABA B sites, J. Med. Chem. 30, 743. Berthelot, P., C. Vaccher, N. Flouquet, M. Debaert, M. Luyckx and C. Brunet, 1991, 3-Thienyl- and 3-furylaminobutyric acids. Synthesis and binding GABA B receptor studies, J. Med. Chem. 34, 2557.
Bowery, N.G., A. Doble, D.R. Hill, A.L. Hudson, J.S. Shaw, M.J. Turnbull and R. Warrington, 1981, Bicuculline-insensitive GABA receptors on peripheral autonomic nerve terminals, Eur. J. Pharmacol. 71, 53. Dingwall, J.-G., J. Ehrenfreund, R.G. Hall and J. Jack, 1987, Synthesis of gamma-aminopropylphosphonous acid using hypophosphorous acid synthons, Phosph. Sulfur. 30, 571. Dutar, P. and R.A. Nicoll, 1988, Pre- and postsynaptic GABA B receptors have different pharmacological properties, Neuron 1, 585. Kerr, D.I.B., J. Ong, R.H. Prager, B.D. Gynther and D.R. Curtis, 1987, Phaclofen: a peripheral and central baclofen antagonist, Brain Res. 405, 150. Kerr, D.I.B., J. Ong, G.A.R. Johnston, J. Abbenante and R.H. Prager, 1988, 2-Hydroxy-saclofen: an improved antagonist at central and peripheral GABAa-receptor , Neurosci. Lett. 92, 92. Kerr, D.I.B., J. Ong, G.A.R. Johnston, P. Berthelot, M. Debaert and C. Vaccher, 1989, Benzofuran analogues of baclofen: a new class of peripheral GABAB-receptor antagonists, Eur. J. Pharmacol. 164, 361. Seabrook, G.R., W. Howson and M.G. Lacey, 1990, Electrophysiological characterization of potent agonists and antagonists at preand postsynaptic GABA a receptors on neurones in rat brain slices, Br. J. Pharmacol. 101, 949.
