Proc. Nad. Acad. Sci. USA Vol. 82, pp. 1832-1836, March 1985 Neurobiology An arylaminopyridazine derivative of y-aminobutyric acid (GABA) is a selective and competitive antagonist at the GABAA receptor site (amino acid neurotransmitters/bicuculline/sensory ganglia) JEAN-PIERRE CHAMBON*, PAUL FELTZt, MICHEL HEAULME*, SERGE RESTLEt§, REMY SCHLICHTERt, KATHLEEN BIZIERE*, AND CAMILLE G. WERMUTHt¶ *Centre de Recherches Clin Midy, Groupe SANOFI, 34082 Montpellier Cedex, France; tlnstitut de Physiologie et de Chimie Biologique (LA 309, Centre National de la Recherche Scientifique), Universitd Louis Pasteur, 67084 Strasbourg Cedex, France; tLaboratoire de Chimie Organique, Facultd de Pharmacie (ERA 393, Centre National de la Recherche Scientifique), Universitd Louis Pasteur, 67400 Illkirch, France Communicated by Bernhard Witkop, October 9, 1984

ABSTRACT In view of finding a new y-aminobutyric acid (GABA) receptor ligand we synthesized an arylaminopyr- idazine derivative of GABA, SR 95103 [2-(carboxy-3'-propyl)- 3-amino-4-methyl-6-phenylpyridazinium chloride]. SR 95103 NH2 displaced [3H]GABA from rat brain membranes with an apparent K1 of 2.2 #tM and a Hill number near 1.0. SR 95103 (1-100 IAM) antagonized the GABA-mediated enhancement of FIG. 1. Structure of SR 95103 (R = C6H5) and SR 95436 (R = [3H]diazepam binding in a concentration-dependent manner H). without affecting [3H]diazepam binding per se. SR 95103 competitively antagonized GABA-induced membrane depolari- nize inhibition elicited by compounds other than GABA (10). zation in rat spinal ganglia. In all these experiments, the Of the more recently described GABAA antagonists, 5,6,7,8- potency of SR 95103 was close to that of bicuculline. SR 95103 tetrahydro-4H-isoxazolo[3,4-dlazepin-3-ol (iso-THAZ) (11) (100 IAM) did not interact with a variety of central receptors- also blocks neuronal inhibition elicited by glycine (12), R in particular the GABAB, the strychnine, and the glutamate 5135 strongly interacts with benzodiazepine and glycine receptors-did not inhibit Na'-dependent synaptosomal receptors (13), and pitrazepine interacts with benzodi- GABA uptake, and did not affect GABA-transaminase and azepine receptors (14). glutamic acid decarboxylase activities. Intraperitoneally ad- This report describes the chemical synthesis of the GABA ministered SR 95103 elicited clonicotonic seizures in mice derivative SR 95103 [2-(carboxy-3'-propyl)-3-amino-4-meth- (ED50 = 180 mg/kg). On the basis of these results it is yl-6-phenylpyridazinium chloride] (Fig. 1) and summarizes postulated that St 95103 is a competitive antagonist of GABA the pharmacology of this compound, showing that it specifi- at the GABAA receptor site. In addition to being an interesting cally and competitively antagonizes GABA at the GABAA lead structure for the search of GABA ligands, SR 95103 could receptor site.11 also be a useful tool to investigate GABA receptor subtypes because it is freely soluble in water and chemically stable. MATERIALS AND METHODS Synthesis of SR 95103 and Analogs. 3-Hydrazino-4-methyl- The synthesis of new ligands for the y-aminobutyric acid 6-phenylpyridazine (0.05 mol) (15) was dissolved in 200 ml of (GABA) receptor is of particular interest from the viewpoint methanol and, after addition of 3 g of Raney nickel catalyst, of finding new therapeutic agents as well as molecular was hydrogenated [760 mm Hg (100 kPa), 20'C]. After probes to study different types of GABA receptors (1, 2). All absorption of 1 mol of hydrogen the catalyst was removed by known GABA receptor agonists contain within their struc- filtration and the solvent was evaporated. The solid residue ture a sequence that can be superimposed on the GABA was dissolved in warm isopropyl alcohol and recrystallized molecule (3). Conformationally restricted analogs of GABA by addition of small amounts of isopropyl oxide. 3-Amino- such as trans-aminocrotonic acid (4), muscimol (5), 4,5,6,7- 4-methyl-6-phenylpyridazine (mp 131-1320C) was obtained tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) (5), and with a 60% yield. isoguvacine (6) are potent GABA agonists at the GABAA, The above 3-aminopyridazine (0.02 mol) and ethyl-4- bicuculline-sensitive, receptor site (7). A second category of bromobutyrate (0.03 mol) were dissolved in 10 ml of GABA analogs includes compounds such as baclofen and SL dimethylformamide and heated 2 hr at 80'C. Cooling led to a 75102, in which the conformational mobility of GA3A is to a precipitate, which was collected by filtration and washed certain extent preserved. Baclofen has been shown to be a with anhydrous diethyl ether. The obtained hydrobromide selective ligand of the GABAB, bicuculline-insensitive, re- was then converted to the corresponding hydrochloride. ceptor site (8), whereas SL 75102 is active at both the GABAA and the GABAB receptor site The structural Abbreviations: GABA, t-aminobutyric acid; THIP, 4,5,6,7-tetra- (9). hydroisoxazolo[5,4-c]pyridin-3-ol; iso-THAZ, 5,6,7,8-tetrahydro- requirements that differentiate agonist from antagonist activ- 4H-isoxazolo[3,4-dlazepin-3-ol; SR 95103, 2-(carboxy-3'-propyl)-3- ity at the GABAA receptor site are still unclear (2, 10). amino-4-methyl-6-phenylpyridazinium chloride; DRG, dorsal root Bicuculline is considered as the most specific GABAA ganglia; NG, nodose ganglion. antagonist, but it has nevertheless been reported to antago- §Present address: Centre de Recherches L'Oreal, 93600 Aulnay- sous-Bois, France. ITo whom reprint requests should be addressed. The publication costs of this article were defrayed in part by page charge IIA preliminary report of some of these findings was presented at the payment. This article must therefore be hereby marked "advertisement" 14th Meeting of the Groupe d'Etudes Structure-Activitd (GESA), in accordance with 18 U.S.C. §1734 solely to indicate this fact. Les Embiez, France, May 15-19, 1984. 1832 Neurobiology: Chambon et al. Proc. Natl. Acad. Sci. USA 82 (1985) 1833

After dissolution in water, alkalinization (pH 9-10) with 2 M Na'-dependent synaptosomal GABA uptake was measured sodium hydroxide, and extraction with a 1:1 (vol/vol) mix- as described by Ramsay et al. (27). ture of ether and ethyl acetate, the combined organic layers Electrophysiological Studies. The effects of SR 95103 on were dried and concentrated under reduced pressure. The the electrical activity of rat primary afferent sensory residue was dissolved in absolute ethanol and treated with neurones were studied in vitro by intracellular recordings. dry hydrogen chloride. The hydrochloride was precipitated Either group A and C neurones from L4-L5 dorsal root by addition of anhydrous ether and recrystallized in absolute ganglia (DRG) or cranial sensory cells (NG, nodose ethanol. The yield of 2-(carboxy-3'-propyl)-3-amino-4- ganglion) were used as described previously (28-33). Ganglia methyl-6-phenylpyridazinium chloride (mp 248-2490C) was were superfused with a conventional Ringer solution (30), 72%. and 7.5 mM tetraethylammonium was added when studying To the above ethyl ester chloride (0.01 mol) were added 10 drug effects on Ca2+ spikes (31, 33). Neurones were charac- ml of concentrated hydrochloric acid and 60 ml of glacial terized on the basis of their conduction velocity as Ap, >25 acetic acid and the mixture was heated at 100'C for 2.5 hr. m/s; A8, 25-2.5 m/s; and C, <1.5 m/s (28, 33). Ion-sensitive The solvents were removed under reduced pressure and the microelectrodes were used to monitor the concentration of residue was recrystallized in glacial acetic acid (with addi- trimethylammonium in the superfusing medium, thus provid- tion of anhydrous ether, to complete crystallization). The ing a direct display of drug dilution at the cell surface ("TMA compound SR 95103 was obtained in an 84% yield as a freely method"; for details see refs. 34 and 35). Drugs were applied water-soluble white crystalline powder, mp 238-240'C; 1H upstream from the neurones under study by releasing a 5-til NMR (in deuterated dimethyl sulfoxide) 8 1.8-2.6 (4H, m, 2' drop of a concentrated drug solution. To avoid the problem and 3' -CHz-), 2.3 (3H, s, CH3), 4.4 (2H, t, 1' -CH2-, of receptor desensitization (Fig. 4B; refs. 29 and 32) all tests JH1'-JH2' = 6.0 Hz), 7.37-8.54 (6H, m, C6H5), 8.40 (1H, s, were performed with more than 1 min between each applica- H5), 9.75 (2H, s, NH2). C, H, and N analysis was compatible tion. In certain cases SR 95103 was applied at steady-state with C14H18N302C1. concentrations (1-100 ,uM) in the superfusate and GABA The analogs bearing a glycine, a /3-alanine, and a 3-ami- was still administered by fast rising pulses (for details see novaleric acid side chain as well as the 6-desphenyl com- ref. 30). Membrane potential and currents were measured by pound, SR 95436 (Fig. 1), were prepared by similar proce- means of a single-electrode voltage-clamp technique (for dures. details see refs. 28 and 33). Stability assays were performed on 1% solutions of the in 0.1 M and 0.1 M NaOH. No Convulsant Effects. The convulsant effects of SR 95103 compounds aqueous HCl and chemical changes were observed (TLC, UV) after 2 hr at (+)-bicuculline (Sigma) were examined after intraperi- 250C. toneal administration in groups of 10 female Swiss albino Biochemical Assays. Tritiated derivatives were purchased CD1 mice (Charles River Breeding Laboratories). from New England Nuclear. Bicuculline methiodide was purchased from Pierce Eurochemie (Rotterdam, The Neth- RESULTS erlands). Strychnine sulfate, L-glutamic acid, atropine sul- Biochemical Results. SR 95103 exhibited an affinity in the fate, and serotonin creatinine sulfate were purchased from micromolar range for the GABAA receptor. The Eadie- Sigma; GABA and muscimol were from Fluka; (+)-butaclam- Hoftsee plot of SR 95103 displacement revealed a mass- ol-HCl was from Research Biochemicals (Wayland, MA). action type of kinetics of inhibition with an apparent inhibi- Diazepam was provided by Hoffmann-La Roche; (-)- tion constant, K1, of 2.2 AM; the slope of the Hill plot was baclofen and phentolamine methanesulfonate, by CIBA- not significantly different from 1.0 (Fig. 2). As shown in Geigy; SL 75102, by Synthelabo (Paris); and haloperidol, by Table 1, the GABA antagonists bicuculline and iso-THAZ Janssen Pharmaceutica. iso-THAZ and THIP were kindly were weaker inhibitors of [3H]GABA binding than SR 95103, provided by P. Krogsgaard-Larsen (Royal Danish School of whereas R 5135 revealed an approximately 50-fold greater Pharmacy, Copenhagen, Denmark) and R 5135 was provided affinity for the GABA receptor than SR 95103 did. The by P. Hunt (Roussel-UCLAF, France). GABA agonists muscimol and THIP were considerably [3H]GABA binding (2.9 nM; 83 Ci/mmol; 1 Ci = 37 GBq) more potent and SL 75102 was approximately as potent as was measured with Triton X-100-treated rat whole brain membranes prepared according to Enna and Snyder (16). SR 95103. The effects of SR 95103 and bicuculline on the GABA- elicited enhancement of [3H]diazepam (1.9 nM; 72 Ci/mmol) binding in rat brain membranes was evaluated according to 100. Fujimoto and Okabayashi (17). We also examined the possible effects of SR 95103 on the r80 \ 0O specific binding of [3H]baclofen (30 nM; 37 Ci/mmol) to GABAB rat whole brain receptors (8), [3H]strychnine (6 nM; 15 Ci/mmol) to glycine rat pons medulla receptors (18), L-[3H]glutamate (3 nM; 53 Ci/mmol) to rat glutamate frontal cortex receptors (19), [3H]diazepam (1.9 nM; 72 Ci/mmol) to rat brain benzodiazepine receptors (20), [3H]quinuclidinyl 40 I benzilate (0.2 nM; 42 Ci/mmol) to rat cortical muscarinic -7 - 5 receptors (21), [3H]WB 4101 (0.2 nM; 26 Ci/mmol) to rat i 20j lg1iMi brain a1-adrenergic receptors (22), [3H]serotonin (2 nM; 30 Ci/mmol) to rat hippocampal serotonin receptors (23), and [3H]spiroperidol (0.4 nM; 24 Ci/mmol) to rat striatal dopa- L _____ 7 6_ mine 2 (D2) receptors and cortical serotonin 2 (5HT2) recep- -8 -7 -6 -5 -4 tors (24). Rat brain L-glutamic acid decarboxylase (EC log[drug (M)] 4.1.1.15) activity was measured according to Tappaz et al. FIG. 2. Displacement curves for SR 95103 (o) and bicuculline (o) (25). GABA:a-ketoglutarate transaminase (EC 2.6.1.19) ac- versus [3H]GABA binding in rat brain membranes. (Inset) Hill plot tivity was measured according to Jung et al. (26). In vitro of displacement curve of SR 95103. 1834 Neurobiology: Chambon et al. Proc. Natl. Acad. Sci. USA 82 (1985) Table 1. GABA receptor affinities of SR 95103 and reference compounds SR 95103 Bicuculline Ki for inhibition of ci 200 Compound [3H]GABA binding, uM ._ SR 95103 2.2 D GABA antagonists E 150 Bicuculline 38 CZ a. iso-THAZ 50 N R 5135 0.047 CZ C GABA agonists 100 .-0,{-O~--Oo -0--ov,=0 GABA 0.027 P' Muscimol 0.002 THIP 0.48 50 SL 75102 1.2 -6 -5 -4 -6 -5 -4 log[drug (M)] Dose-inhibition curves were generated with four to eight concen- trations of drug in triplicate incubations. IC50 values were calculated FIG. 3. Antagonism by SR 95103 and bicuculline of the GABA- by log-probit analysis. Ki values were calculated with the equation mediated enhancement of [3H]diazepam binding in vitro. Specific = + in which C = concentration of [3H]GABA Ki IC50/[1 (C/Kd)], [3H]diazepam binding, defined as the difference between total (2.9 nM) and Kd = dissociation constant for dissociation of GABA from the high-affinity receptor site (30 nM). binding and binding in the presence of 100 ,uM diazepam, was measured in the presence (o) or absence (o) of 10 ,uM GABA. The left-most point on each curve represents binding in the absence of It has previously been reported that GABA (10 ,M) drug. Plots represent the results obtained from one experiment; enhances [3H]diazepam binding in rat synaptosomal mem- individual data points were determined in triplicate. Identical results branes in vitro and that this effect can be antagonized by were obtained from three different experiments. GABA antagonists (17, 36). Like bicuculline, SR 95103 (1-100 AM) antagonized the GABA-elicited enhancement of activities (results not shown). [3H]diazepam binding in a dose-dependent fashion without The structural analogs of SR 95103 in which the GABA affecting [3H]diazepam binding per se (Fig. 3). This result moiety was lengthened to 8-aminovaleric acid or shortened strongly suggests an antagonist-like interaction of SR 95103 to 3-alanine or glycine were all weaker displacers of specific with the GABAA receptor site. [3H]GABA binding with apparent K1 values of 10.5 ,uM, 6.8 SR 95103 (100 ,uM) did not interact with binding sites for ,uM, and >100 ,M, respectively. The 8-aminovaleric acid [3H]baclofen, [3H]strychnine, L-[3H]glutamate, [3H]diaze- and the f3-alanine derivatives antagonized GABA-mediated pam, [3H]quinuclidinyl benzilate, [3H]WB 4101, [3H]sero- enhancement of [3H]diazepam binding but were weaker than tonin, or [3H]spiroperidol (D2 and 5HT2) (results not shown). SR 95103 (results not shown). The 6-desphenyl analog, SR Thus SR 95103 appears to be a specific ligand of the GABAA 95436 (Fig. 1) did not displace [3H]GABA from its receptor receptor. Furthermore SR 95103 (100 ,M) did not inhibit site (apparent K1 > 100 ,uM), yet this compound carries a Na+-dependent synaptosomal GABA uptake and did not GABA side chain. affect GABA transaminase and glutamate decarboxylase Electrophysiological Results. Dose-dependent responses to

GABA 0.05 nmol 0.5 nmol 5 nmol 50 nmol 50 nmol Vw Vw IV . .

T100 g&M

**M50

A B

* *~~~~~~~~___ I** GABA SR 95103 15 mVL 25 nmol 25 nmol v V lOs

FIG. 4. Effects of GABA and SR 95103 on membrane potential and membrane conductance of A,9-DRG neurones. Drugs were applied by fast microperfusion as described in Materials and Methods; arrowheads indicate time of release of 5-,ul drop and amount of drug in the drop. Penwriter traces display input resistance of the membrane as intermittently monitored by hyperpolarizing current pulses (1 nA, 60 ms). Resting potential was -75 mV. Upper traces, membrane resistance and potential as a function of time. Lower traces, drug concentration at the cell surface as a function of time ("TMA method"); 5 nmol of trimethylammonium was present in the drops released at times indicated by * and the concentration scale is given to the left of B. (A) GABA dose-response; (B) desensitization to GABA; (C) effects of SR 95103 (v). Neurobiology: Chambon et al. Proc. Natl. Acad. Sci. USA 82 (1985) 1835 100 (GABA 50 0 nmol rnnol nmol IQ v 0 t._~ Control C_ 1t ct I,..... la ,I 11W 1'1 ,...:,; N

I _,.. 0 z

v 0 m SR 95103 C -O .. q 100

80 I- c 60 v 0 9 Wash t4 -oC 40 nA. i 1' . . . .~~~~~~~~~a 0

20 s z 20

.40 a M n-- l -7 -6 -5 -4 -3 -2 log[GABA (M)] FIG. 5. Quantitative study of antagonism potency. (Left) Penwriter traces of the effects of SR 95103 (10 1xM) on responses to GABA in voltage-clamp recording; holding potential was -80 mV. (Lower Right) Dose-response curves as obtained in voltage clamp (As cells). Currents are normalized to the maximum, 1.2 nA. (Upper Right) GABA-induced depolarizations in a sample of 20 An cells. Note clear-cut inhibition with the standard dose of SR 95103 (10 uM, continuously present in superfusate) (control ED50 = 13 tLM GABA). GABA was applied in the concentration jump mode (Fig. 4A); values on the abscissa were calculated either according to ref. 30 (dilution of K+) or from trimethylammonium calibrations (see scale in Fig. 4B). Voltage changes are normalizedOf to the maximum, 15.8 ± 1.3 mV. Dose-response curves were determined by the least-squares method.

GABA were recorded in more than 60 neurones in 18 95103 was slightly less great than that previously observed preparations (12 DRG and 6 NG). Half-maximal responses of with bicuculline in this preparation (30, 37). As shown in Fig. 7.6 ± 0.4 mV for a membrane potential at rest of -63.7 ± 1.9 S Lower Right, SR 95103 (10 ktM and 100 4M) caused a mV (n = 32; input resistance = 15.3 ± 2.1 Mfl) were parallel shift of the GABA dose-response curve, strongly observed (Fig. 4A). As shown in Fig. 4C, SR 95103 alone suggesting a competitive inhibition. Mean values of maximal never modified either the membrane potential or the mem- responses are indicated in the legend of Fig. 5; this figure brane conductance even at high millimolar (n = 16). Under exemplifies inward Cl- currents recorded in Ail cells (1.2 nA voltage-clamp conditions (Fig. 5 Left) no additional mem- for the example shown in Lower Right; for a sample of eight brane current was observed with SR 95103 when the holding cells, 1.83 ± 0.34 nA) and shows membrane depolarization potential at rest was stepped in either the hyperpolarizing or of Ap cells (20 cells; Upper Right). depolarizing direction (within a limited range of ± 40 mV On DRG and NG group C neurones SR 95103 did not studied at steady state). Thus SR 95103 is devoid of agonist- mimic the effects of (-)-baclofen (shortening of Ca2+ type activity at the GABAA receptor site. spikes), thus confirming selectivity at the GABAA receptor SR 95103 constantly inhibited GABAA-induced responses; site. Bicuculline (10 AuM) has previously been shown to inhibition was usually greater than 70% (Fig. 4C) and was induce nonspecific effects such as prolonging the plateau reversible within 1 min. Interestingly, in NGs no evidence phase of Ca2+-dependent potentials (31, 33); this was not was found for a possible noncompetitive depression of observed with SR 95103 for concentrations as high as 100 serotonin-induced responses, which would have indicated a JIM. nonspecific channel block at receptor-operated cationic Convulsant Effects of SR 95103. In mice, intraperitoneal channels (results not shown). These results confirm that SR SR 95103 elicited clonicotonic seizures comparable to those 95103 antagonizes GABA at the GABAA receptor site and, together with those of competition experiments (see below), observed with bicuculline: ED50 values were 180 mg/kg indicate that inhibition does not result from supplementary (150-240) and 4.6 mg/kg (4.1-5.5), respectively. binding at the level of Cl- channels. When equimolar DISCUSSION amounts of SR 95103 and GABA were applied on a sample of Ad neurones (DRG) the magnitude of the inhibition of the The present study shows that the phenylaminopyridazine GABA response induced by SR 95103 was dependent on derivative of GABA, SR 95103, is a selective and competi- timing between concentration jumps. Tests were performed tive antagonist of GABA at the GABAA receptor site. SR on the falling phase of the concentration jump of SR 95103: 95103 is approximately 10 times more potent than bicuculline inhibition was 74% ± 6% and 53% ± 5% for drug application in displacing tritiated GABA from its specific binding sites in intervals of 3 and 5 sec, respectively (number of cells: 15 and rat brain membranes and is as potent as bicuculline in 23). These experiments also showed that the potency of SR antagonizing GABA-mediated enhancement of diazepam 1836 Neurobiology: Chambon et al. Proc. Natl. Acad. Sci. USA 82 (1985) binding. Electrical recordings from primary sensory chemical and Pharmacological Aspects, eds. Krogsgaard-Larsen, neurones confirm that SR 95103 competitively antagonizes P., Scheel-Kruger, J. & Kofod, H. (Munksgaard, Copenhagen), pp. GABA-induced responses; in this preparation SR 95103 was 149-164. 2. Krogsgaard-Larsen, P. (1981) J. Med. Chem. 24, 1377-1383. slightly less potent than bicuculline, but this could be due to 3. Schlewer, G., Wermuth, C. G. & Chambon, J. P. (1984) Eur. J. fast dissociation kinetics. In this respect SR 95103 markedly Med. Chem. 19, 181-186. differs from pitrazepine, which has persistent electrophysi- 4. Beart, P. M., Johnston, G. A. R. & Uhr, M. L. (1972) J. ological effects (14). When compared to other GABAA Neurochem. 19, 1855-1861. receptor antagonists, SR 95103 appears to be remarkably 5. Krogsgaard-Larsen, P. & Johnston, G. A. R. (1978) J. Neurochem. selective and notably does not interact 30, 1377-1382. with the GABAB, the 6. Krogsgaard-Larsen, P., Johnston, G. A. R., Lodge, D. & Curtis, benzodiazepine, the glutamate, or the strychnine binding D. R. (1977) Nature (London) 268, 53-55. sites and does not seem to affect directly the Cl- channels 7. Simmonds, M. A. (1983) Trends Neurosci. 6, 279-281. associated to the GABAA receptor. The relatively low 8. Hill, D. R. & Bowery, N. G. (1981) Nature (London) 290, 149-152. potency of SR 95103 in eliciting clonicotonic seizures sug- 9. Lloyd, K. G., Arbilla, S., Beaumont, K., Briley, M., De Montis, gests that the compound does not readily cross the G., Scatton, B., Langer, S. Z. & Bartholini, G. (1982) J. Pharmacol. Exp. Ther. 220, 672-677. blood-brain barrier; this could be due to the quaternary 10. Andrews, P. R. & Johnston, G. A. R. (1979) Biochem. Pharmacol. nature of the nitrogen in the 2 position of the pyridazine ring. 28, 2697-2702. Structure-activity relationship studies have shown that in 11. Arnt, J. & Krogsgaard-Larsen, P. (1979) Brain Res. 177, 395-400. GABAA receptor agonists the distance between the acidic 12. Krogsgaard-Larsen, P., Hjeds, H., Curtis, D. R., Leah, J. D. & and the basic functions must be approximately 5 A to ensure Peet, M. J. (1982) J. Neurochem. 39, 1319-1324. maximal affinity (3, 5, 38-40). This appears to be 13. Hunt, P. & Clements-Jewery, S. (1981) Neuropharmacology 20, also true 357-361. for GABAA receptor antagonists, since lengthening or short- 14. Gahwiller, B. H., Maurer, R. & Wuthrich, H. J. (1984) Neurosci. ening the side chain of SR 95103 consistently led to a Lett. 45, 311-316. decrease in the affinity for the GABAA receptor site. In the 15. Leclerc, G., Wermuth, C. G., Miesch, F. & Schwartz, J. (1976) compound SR 95103, the positive charge is delocalized over Eur. J. Med. Chem. 11, 107-113. an endoexocyclic amidinic system; this is also the case for R 16. Enna, S. J. & Snyder, S. H. (1975) Brain Res. 100, 81-97. 5135. However, this cannot be considered as a structural 17. Fujimoto, M. & Okabayashi, T. (1981) Life Sci. 28, 895-901. prerequisite warranting antagonistic properties since compa- 18. Young, A. B. & Snyder, S. H. (1973) Proc. Natl. Acad. Sci. USA rable charge delocalizations are found in partial agonists 70, 2832-2836. such as p-guanidinopropionic N 19. Biziere, K., Thompson, H. & Coyle, J. T. (1979) Brain Res. 183, acid. substitution of GABA 421-433. by simple alkyl compounds has been described as detrimen- 20. Squires, R. & Braestrup, C. (1977) Nature (London) 266, 732-734. tal (5); however, substitution through an amidinic or 21. Korn, S. E. J., Martin, M. W. & Harden, T. K. (1983) J. guanidinic charge delocalized system appears to selectively Pharmacol. Exp. Ther. 226, 118-126. restore the affinity for the GABAA receptor site. The GABA 22. U'Pritchard, D. C., Greenberg, D. A. & Snyder, S. H. (1977) Mol. side chain of SR 95103 has a considerable degree of Pharmacol. 13, 454-473. conformational flexibility. In GABAA receptor agonists, this 23. Nelson, D. L., Herbert, A., Bourgouin, S., Glowinski, J. & flexibility is usually associated with a loss of GABAA selec- Hamon, M. (1978) Mol. Pharmacol. 14, 983-995. tivity, as is the case for SL 75102, baclofen, or 8-hydroxy- 24. Creese, I. & Snyder, S. H. (1978) Eur. J. Pharmacol. 49, 201-202. GABA (41). All 25. Tappaz, M. L., Brownstein, M. J. & Palkovits, M. (1976) Brain known GABAA receptor antagonists are Res. 108, 371-379. relatively rigid molecules, and SR 95103 is the first example 26. Jung, M. J., Lippert, B., Metcalf, B. W., Schechter, P. J., Bohlen, of an antagonist in which the flexibility of the GABA moiety P. & Sjoerdsma, A. (1977) J. Neurochem. 28, 717-723. is preserved. Thus the relationship between restricted con- 27. Ramsay, P. B., Krigman, M. R. & Morell, P. (1980) Brain Res. 187, formation and GABAA selectivity that has been observed 383-402. with agonists does not seem to hold with antagonists. 28. Desarmenien, M., Santangelo, F., Loeffler, J. Ph. & Feltz, P. Currently available structure-activity data are too incom- (1984) Exp. Brain Res. 54, 521-528. plete to define the prerequisite structural features for a 29. Hattori, K., Akaike, N., Oomura, Y. & Kuraoka, S. (1984) Am. J. GABAA antagonist. It has been suggested that antagonist- Physiol. 246, C259-265. 30. Desarmenien, M., Feltz, P., Headley, P. M. & Santangelo, F. like activity could be due to additional interactions involving (1981) Br. J. Pharmacol. 72, 355-364. aromatic or cycloaliphatic rings (10). The inactivity of the 31. Desarmenien, M., Feltz, P., Occhipinti, G., Santangelo, F. & desphenyl analog of SR 95103 is consistent with this hypoth- Schlichter, R. (1984) Br. J. Pharmacol. 81, 327-333. esis. 32. Desarmenien, M., Feltz, P. & Headley, P. M. (1980) J. Physiol. In conclusion, SR 95103 is a selective and competitive (London) 307, 163-182. antagonist of GABA at the GABAA receptor site; this 33. Schlichter, R., Bossu, J. L., Feltz, A., Desarmenien, M. & Feltz, compound also has the advantage of being freely soluble in P. (1984) Neuropharmacology 23, 869-872. water and chemically stable in aqueous solutions and could 34. Loeffler, J. Ph., Desaulles, E., Demeneix, B. A. & Feltz, P. (1984) thus be a useful tool to further investigate the pharmacology in Ion Measurements in Physiology and Medicine, eds. Kessler, M., Harrison, D. K. & Horper, J. (Springer, Berlin), in press. of GABA receptor subtypes. Moreover, SR 95103 is chemi- 35. Nicholson, C. & Phillips, J. M. (1981) J. Physiol. (London) 321, cally different from other known GABAA receptor ligands 225-257. and could prove to be a promising lead structure in the 36. Karobath, M. & Sperk, G. (1979) Proc. Natl. Acad. Sci. USA 76, continuing search for new GABA ligands. 1004-1006. 37. Gallagher, J. P., Nakamura, J. & Shinnick-Gallagher, P. (1983) J. The authors acknowledge the skillful technical assistance of Miss Pharmacol. Exp. Ther. 226, 876-884. Anghle Schoenfelder and Mr. Roger Leyris. This research was 38. Nicholson, S. H., Suckling, C. J. & Iversen, L. L. (1979) J. supported in part by a grant of the Institut National de la Sante et de Neurochem. 32, 249-252. la Recherche Mddicale (INSERM, Programme de Recherches 39. Galli, A., Zilletti, L., Scotton, M., Adembri, G. & Giotti, A. (1980) Coordonndes no. 121032). Pharmacol. Res. Commun. 12, 266-272. 40. Humblet, C. & Marshall, G. R. (1981) Drug Dev. Res. 1, 409-434. 1. Johnston, G. A. R., Allan, R. D., Kennedy, S. M. E. & Twitchin, 41. Bowery, N. J., Hill, D. R. & Hudson, A. L. (1983) Br. J. B. (1978) in GABA-Neurotransmitters, Pharmacochemical, Bio- Pharmacol. 78, 191-206.