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The classic muscarinic acetylcholine receptor (mAChR) agonist carbamoylcholine (carbachol) does not seem to be the most obvious lead for the development of selective ligands at nicotinic acetylcholine receptors (nAChRs). In the past, however, N-methylations of carbachol have provided N-methylcarbamoylcholine and N,N-dimethylcarbamoylcholine (DMCC), which predominantly display nicotinic activity. In this study, 12 homologous analogs of DMCC and its corresponding tertiary amine, N,N-dimethylcarbamoyl-N,N-dimethylaminoethanol, were synthesized and their binding affinities to native mAChR and nAChR sites estimated. One of the compounds in the series, 3-N,N-dimethylaminobutyl-N,N-dimethylcarbamate (7), displayed low nanomolar binding affinity to nAChRs and a 400-fold selectivity for nAChRs over mAChRs. Hence, a new series of compounds was synthesized in which alkyl and aryl groups and different ring systems were introduced in the carbamate moiety of 7. In a [3 H]epibatidine binding assay, the K i values of 7 and its analogs at rat ␣2␤2, ␣4␤2, ␣2␤4, ␣3␤4, and ␣4␤4 nAChRs, stably expressed in mammalian cell lines, ranged from low nanomolar to midmicromolar concentrations, whereas all of the compounds displayed weak binding to an ␣7/5-HT 3 chimera and to native mAChRs. Compound 7 and its analogs were determined to be agonists at the ␣3␤4 nAChR subtype. This series includes the most potent and selective nicotinic agonists structurally derived from ACh to date. Furthermore, the compounds are tertiary amines, implying some advantages in terms of bioavailability pertinent to future in vivo pharmacological studies. Finally, observations made in the study hold promising perspectives for future development of ligands selective for specific nAChR subtypes.The neurotransmitter acetylcholine (ACh) exerts its effects in the central and peripheral nervous systems through two distinct classes of receptors, the muscarinic and nicotinic acetylcholine receptors (mAChRs and nAChRs, respectively). The five cloned mAChR subtypes, m1 to m5, are members of the G-protein-coupled receptor superfamily and mediate their effects through intracellular metabolic cascades (Eglen et al., 2001). In contrast, the nAChRs belong to a superfamily of ligand-gated ion channels that also includes receptors for GABA, glycine, and serotonin (5-HT) (Corringer et al., 2000;Karlin, 2002). The nAChRs are involved in a wide array of physiological functions; over the years, nicotinic ligands have attracted considerable interest as potential therapeutics in the treatment of pain and a number of neurodegenerative and psychiatric disorders (Gotti et al., 1997;Lindstrom, 1997; Arneric and Brioni, 1999;Levin, 2002). Furthermore, nicotine replacement therapy has become the predominant treatment for smoking cessation (Arneric and Brioni, 1999;Levin, 2002).The nAChR is a transmembrane allosteric protein complex composed of five subunits. So far, 17 nAChR subunits have been cloned and divided into five muscle-type subunits (␣1, ␤1, ␥, ⑀, and ␦) and 12 neuronal subun...

In a recent study, EF1502 [N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo [d]isoxazol-3-ol], which is an N-substituted analog of the GAT1-selective GABA uptake inhibitor exo- 5,6,isoxazol-3-ol), was found to inhibit GABA transport mediated by both GAT1 and GAT2 in human embryonic kidney (HEK) cells expressing the mouse GABA transporters GAT1 to 4 (mGAT1-4). In the present study, EF1502 was found to possess a broad-spectrum anticonvulsant profile in animal models of generalized and partial epilepsy. When EF1502 was tested in combination with the clinically effective GAT1-selective inhibitor, another GAT1-selective N-substituted analog of exo-THPO, a synergistic rather than additive anticonvulsant interaction was observed in the Frings audiogenic seizure-susceptible mouse and the pentylenetetrazol seizure threshold test. In contrast, combination of the two mGAT1-selective inhibitors, tiagabine and LU-32-176B, resulted in only an additive anticonvulsant effect. Importantly, the combination of EF1502 and tiagabine did not result in a greater than additive effect in the rotarod behavioral impairment test. In subsequent in vitro studies conducted in HEK-293 cells expressing the cloned mouse GAT transporters mGAT1 and mGAT2, EF1502 was found to noncompetitively inhibit both mGAT1 and the betaine/GABA transporter mGAT2 (K i of 4 and 5 M, respectively). Furthermore, in a GABA release study conducted in neocortical neurons, EF1502 did not act as a substrate for the GABA carrier. Collectively, these findings support a functional role for mGAT2 in the control of neuronal excitability and suggest a possible utility for mGAT2-selective inhibitors in the treatment of epilepsy.Reduction of GABA-mediated inhibitory neurotransmission is associated with seizure activity and drugs that elevate synaptic GABA levels either by inhibition of GABA degradation or inhibition of high-affinity transport have been demonstrated to possess anticonvulsant activity (see Dalby, 2003;Sarup et al., 2003). For example, the GABA-transaminase inhibitor vigabatrin and the GABA-transport inhibitor tiagabine [(R)-N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]nipecotic acid] are clinically effective antiepileptic drugs (for review and references, see Ben-Menachem, 2002;Kalviainen, 2002).Since the advent of cloning of several GABA transporters from different species including mouse, rat, and human, in-

No abstract

5-(4-Piperidyl)isoxazol-3-ol (4-PIOL, 10), a structural analog of 4-aminobutanoic acid (GABA, 1) and the GABAa agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 5), is a lowefficacy partial GABAa agonist. A number of compounds bioisosterically derived from 10, including 5-(4-piperidyl)isothiazol-3-ol (11), 3-(4-piperidyl)isoxazol-5-ol (12), 5-(l,2,3,6-tetrahydropyrid-4-yl)isoxazol-3-ol (13), and 5-(l,2,3,6-tetrahydropyrid-4-yl)isothiazol-3-ol ( 14), were synthesized and tested as GABAa receptor ligands. Whereas none of these compounds significantly affected GABAb receptor binding or GABA uptake, they showed affinities for GABAa receptor sites in the low-micromolar range. Using cultured cerebral cortical neurons and whole-cell patch-clamp techniques, the efficacies of these compounds relative to that of the full GABAa agonist, isoguvacine (8) (20 µ ), were determined. The relative efficacy of 11, which has a higher receptor affinity (IC50 = 1.3 ± 0.3 µ ) than 10 (IC50 = 9.3 ± 2.6 µ ), was comparable with that of 10 (30-35%). The tetrahydropyridine analog of 10, compound 13, showed a markedly lower receptor affinity (IC50 = 32 ± 10 µ ) and apparently a lower relative efficacy than 10. The corresponding unsaturated analog of 11, compound 14, showed a slightly weaker receptor affinity (IC50 = 4.0 ± 2.0 µ ) but a significantly higher relative efficacy (50-55%) than 11. The 5-isoxazolol isomer of 10, compound 12, showed a reduced receptor affinity (IC50 = 26 ± 7 µ ) and a very low relative efficacy. Substitution of propanoic or propenoic acid moieties for the acidic heterocyclic units of these compounds gave the monocyclic amino acids 15-18, which have very little or no affinity for GABAa receptor sites.