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(R)-N-[4,4-Bis(3-methyl-2-thienyl)but-3-en-1-yl]nipecotic acid (NO 328) has previously been shown to be a potent anticonvulsant in both mice and rats. Here, we report that NO 328 is a potent inhibitor of gamma-[3H]aminobutyric acid [( 3H]GABA) uptake in a rat forebrain synaptosomal preparation (IC50 = 67 nM) and in primary cultures of neurons and astrocytes. Inhibition of [3H]GABA uptake by NO 328 is apparently of a mixed type when NO 328 is preincubated before [3H]GABA uptake; the inhibition is apparently competitive without preincubation. NO 328 itself is not a substrate for the GABA uptake carrier, but NO 328 is a selective inhibitor of [3H]GABA uptake. Binding to benzodiazepine receptors, histamine H1 receptors, and 5-hydroxytryptamine1A receptors was inhibited by NO 328 at 5-30 microM, whereas several other receptors and uptake sites were unaffected. [3H]NO 328 showed saturable and reversible binding to rat brain membranes in the presence of NaCl. The specific binding of [3H]NO 328 was inhibited by known inhibitors of [3H]GABA uptake; GABA and the cyclic amino acid GABA uptake inhibitors were, however, less potent than expected. This indicates that the binding site is not identical to, but rather overlapping with, the GABA recognition site of the uptake carrier. The affinity constant for binding of [3H]NO 328 is 18 nM, and the Bmax is 669 pmol/g of original rat forebrain tissue. The regional distribution of NaCl-dependent [3H]NO 328 binding followed that of synaptosomal [3H]GABA uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

A series of different synthetic approaches to novel GABA uptake inhibitors are described, leading to examples which are derivatives of nipecotic acid and guvacine, substituted at nitrogen by 4,4-diaryl-3-butenyl or 2-(diphenylmethoxy)ethyl moieties. The in vitro value for inhibition of [3H]-GABA uptake in rat synaptosomes was determined for each compound. It was found that the most potent examples are those having a substituent in an "ortho" position in one or both aromatic/heteroaromatic groups. The majority of the compounds described are structurally related to tiagabine, (R)-1-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3- piperidinecarboxylic acid hydrochloride (NNC 05-0328) and some of the reasoning behind the selection of this compound as a drug candidate is summarized.

The synthesis and pharmacological profile of a series of neuroprotective adenosine agonists are described. Novel A(1) agonists with potent central nervous system effects and diminished influence on the cardiovascular system are reported and compared to selected reference adenosine agonists. The novel compounds featured are derived structurally from two key lead structures: 2-chloro-N-(1-phenoxy-2-propyl)adenosine (NNC 21-0041, 9) and 2-chloro-N-(1-piperidinyl)adenosine (NNC 90-1515, 4). The agonists are characterized in terms of their in vitro profiles, both binding and functional, and in vivo activity in relevant animal models. Neuroprotective properties assessed after postischemic dosing in a Mongolian gerbil severe temporary forebrain ischemia paradigm, using hippocampal CA1 damage endpoints, and the efficacy of these agonists in an A(1) functional assay show similarities to some reference adenosine agonists. However, the new compounds we describe exhibit diminished cardiovascular effects in both anesthetized and awake rats when compared to reference A(1) agonists such as (R)-phenylisopropyladenosine (R-PIA, 5), N-cyclopentyladenosine (CPA, 2), 4, N-[(1S,trans)-2-hydroxycyclopentyl]adenosine (GR 79236, 26), N-cyclohexyl-2'-O-methyladenosine (SDZ WAG 994, 27), and N-[(2-methylphenyl)methyl]adenosine (Metrifudil, 28). In mouse permanent middle cerebral artery occlusion focal ischemia, 2-chloro-N-[(R)-[(2-benzothiazolyl)thio]-2-propyl]adenosine (NNC 21-0136, 12) exhibited significant neuroprotection at the remarkably low total intraperitoneal dose of 0.1 mg/kg, a dose at which no cardiovascular effects are observed in conscious rats. The novel agonists described inhibit 6, 7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate-induced seizures, and in mouse locomotor activity higher doses are required to reach ED(50) values than for reference A(1) agonists. We conclude that two of the novel adenosine derivatives revealed herein, 12 and 5'-deoxy-5'-chloro-N-[4-(phenylthio)-1-piperidinyl]adenosine (NNC 21-0147, 13), representatives of a new series of P(1) ligands, reinforce the fact that novel selective adenosine A(1) agonists have potential in the treatment of cerebral ischemia in humans.

By bioisosteric transformations and successive optimization of known GABA uptake inhibitors, several series of novel GABA uptake inhibitors have been prepared by different synthetic approaches. These compounds are derivatives of nipecotic acid and guvacine, substituted at the nitrogen of these amino acids by various lipophilic moieties such as diarylaminoalkoxyalkyl or diarylalkoxyalkyl. The in vitro values for inhibition of [(3)H]GABA uptake in rat synaptosomes was determined for each compound, and it was found that the most potent compound from this series, (R)-1-(2-(3,3-diphenyl-1-propyloxy)ethyl)-3-piperidinecarboxyli c acid hydrochloride (29), is so far the most potent parent compound inhibiting GABA uptake into synaptosomes. Structure-activity results confirm our earlier observations, that an electronegative center in the chain connecting the amino acid and diaryl moiety is very critical in order to obtain high in vitro potency. Several of the novel compounds were also evaluated for their ability in vivo to inhibit clonic seizures induced by a 15 mg/kg (ip) dose of methyl 6, 7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). Some of the compounds tested show a high in vivo potency comparable with that of the recently launched anticonvulsant product 6 ((R)-1-(4, 4-bis(3-methyl-2-thienyl)-3-butenyl)-3-piperidinecarboxylic acid).

(3R)-1-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-piperidinecarboxylic acid 1 (tiagabine, Gabitril) is a potent and selective gamma-aminobutyric acid (GABA) uptake inhibitor with proven anticonvulsant efficacy in humans. This drug, which has a unique mechanism of action among marketed anticonvulsant agents, has been launched for add-on treatment of partial seizures with or without secondary generalization in patients >12 years of age. Using this new agent as a benchmark, we have designed two series of novel GABA uptake inhibitors of remarkable potency, using a putative new model of ligand interaction at the GABA transporter type 1 (GAT-1) uptake site. This model involves the postulated interaction of an electronegative region in the GABA uptake inhibitor with a positively charged domain in the protein structure of the GAT-1 site. These two novel series of anticonvulsant agents contain diaryloxime or diarylvinyl ether functionalities linked to cyclic amino acid moieties and were derived utilizing the new model, via a series of design steps from the known 4,4-diarylbutenyl GABA uptake inhibitors. The new compounds are potent inhibitors of [(3)H]-GABA uptake in rat brain synaptosomes in vitro, and their antiepileptic potential was demonstrated in vivo by their ability to protect against seizures induced by the benzodiazepine receptor inverse agonist methyl 4-ethyl-6,7-dimethoxy-beta-carboline-3-carboxylate (DMCM) in mice. From structure-activity studies of these new GABA uptake inhibitors, we have shown that insertion of an ether oxygen in conjugation with the double bond in tiagabine (K(i) = 67 nM) improves in vitro potency by 5-fold to 14 nM.