Knud Erik Andersen

(R)-N-[4,4-Bis(3-Methyl-2-Thienyl)but-3-en-1-yl]Nipecotic Acid Binds with High Affinity to the Brain γ-Aminobutyric Acid Uptake Carrier

(R)‐N‐[4,4‐Bis(3‐methyl‐2‐thienyl)but‐3‐en‐l‐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 γ‐[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‐hydroxytryptaminelA receptors was inhibited by NO 328 at 5—30 μM, 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 NaCI. 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. It is concluded that NO 328 is a potent and selective inhibitor of neuronal and glial GABA uptake and that [3H]NO 328 is a useful radioligand for labeling the GABA uptake carrier in brain membranes. In the mouse brain in vivo, [3H]NO 328 likewise showed saturable and reversible binding that could be displaced by analogues of NO 328. Further studies are needed to demonstrate whether the uptake carrier is indeed labeled by [3H]NO 328 in vivo.

Characterization of tiagabine (NO-328), a new potent and selective GABA uptake inhibitor.

Tiagabine (NO-328) (R(-)-N-[4,4-bis(3-methylthien-2-yl)but-3-enyl]nipecotic acid, hydrochloride) is a new centrally acting GABA uptake inhibitor. The anticonvulsant activity of tiagabine was evaluated against seizures induced by methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), pentylenetetrazol, bicuculline, maximal electrostimulation (MES), or high intensity sound. The sedative actions of tiagabine were evaluated in tests for traction, rotarod performance and exploratory behavior. Finally, interoceptive properties of tiagabine were assessed using diazepam-, CGS 9896-, pentylenetetrazol-, or amphetamine-discriminating rats. Tiagabine was an effective anticonvulsant in doses which did not produce sedation or motor debilitation, although it was not potent against MES. In a manner similar to other anti-epileptic drugs, tiagabine potentiated dopaminergic function (methylphenidate-induced gnawing in mice) although it did not substitute for amphetamine in amphetamine-trained animals. Furthermore, although tiagabine antagonized DMCM-induced convulsions, it exhibited neither CGS 9896 or diazepam-like interoceptive effects, nor did it block (or potentiate) pentylenetetrazol-discrimination. Thus, GABA uptake inhibition represents a novel rationale for a valproate-like anticonvulsant drug therapy.

NNC-711, a novel potent and selective γ-aminobutyric acid uptake inhibitor: pharmacological characterization

NNC-711 (1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3- pyridinecarboxylic acid hydrochloride) is a novel, potent and selective gamma-aminobutyric acid (GABA) uptake inhibitor. NNC-711 inhibited synaptosomal (IC50 = 47 nM), neuronal (IC50 = 1238 nM) and glial (IC50 = 636 nM) GABA uptake in vitro NNC-711 lacked affinity for other neurotransmitter receptor binding sites, uptake sites and ion channels examined in vitro. In vivo, NNC-711 was a potent anticonvulsant compound against rodent seizures induced by methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) (ED50 (clonic) = 1.2 mg/kg i.p.), pentylenetetrazole (PTZ) (ED50 (tonic) = 0.72 mg/kg i.p., mouse; and ED50 (tonic) = 1.7 mg/kg, rat), or audiogenic (ED50 (clonic and tonic) = 0.23 mg/kg i.p.). At higher doses NNC-711 produced behavioral side effects characterized by inhibition of traction (ED50 = 23 mg/kg i.p.), rotarod (ED50 = 10 mg/kg i.p.) and exploratory locomotor activity (ED50 = 45 mg/kg i.p.) in the mouse. Following acute (3-h) in vivo pretreatment with NNC-711, behavioral tolerance developed to its motor impairing side effects (inhibition of traction, rotarod or exploratory locomotor activity) without corresponding tolerance to the anticonvulsant effects. These data suggest that NNC-711 will be useful for future in vitro and in vivo experiments to elucidate the role of the GABA uptake carrier in the central nervous system.

Oxadiazoles as bioisosteric transformations of carboxylic functionalities. II

Summary In order to improve the in vivo efficacy of a series of known benzodiazepine receptor (BZR) ligands, 1 -(2-phenyl-4-quinolinyl)-4-piperidinecarboxamides, a series of analogs has been prepared in which the amide group of these ligands has been replaced by a 1,2,4-oxadiazole moiety or converted to other carboxylic isosters such as esters or nitriles. An increase in the in vivo efficacy was observed for some of the compounds prepared in this investigation compared to the parent carboxamide derivatives.

Synthesis of novel GABA uptake inhibitors. Part 6 : Preparation and evaluation of N-Ω asymmetrically substituted nipecotic acid derivatives

In a previous series of potent GABA uptake inhibitors published from this laboratory, we noticed that asymmetry in the substitution pattern of the bis-aromatic moiety in known GABA uptake inhibitors such as 4 [1-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid] and 5 [(R)-1-(4,4-bis(3-methyl-2-thienyl)-3-butenyl)-3-piperidinecarboxylic acid] was beneficial for high affinity. This led us to investigate asymmetric analogues of known symmetric GABA uptake inhibitors in which one of the aryl groups has been exchanged with an alkyl, alkylene or cycloalkylene moiety as well as other modifications in the lipophilic part. The in vitro values for inhibition of [(3)H]-GABA uptake in rat synaptosomes was determined for each compound, and it was found that several of the novel compounds inhibit GABA uptake as potently as their known symmetrical reference analogues. Several of the novel compounds were also evaluated for their ability to inhibit clonic seizures induced by a 15 mg/kg (ip) dose of methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) in vivo. Some of the compounds, for example 18 [(R)-1-(2-(((1,2-bis(2-fluorophenyl)ethylidene)amino)oxy)ethyl)-3-piperidinecarboxylic acid], show a high in vivo potency and protective index comparable with that of our recently launched anticonvulsant product, 5 [(R)-1-(4,4-bis(3-methyl-2-thienyl)-3-butenyl)-3-piperidinecarboxylic acid], and may therefore serve as second-generation drug candidates.

The synthesis of novel GABA uptake inhibitors. Part 2. Synthesis of 5-hydroxytiagabine, a human metabolite of the GABA reuptake inhibitor tiagabine

Abstract (R)-1-(4-(2,5-Dihydro-3-methyl-5-oxothien-2-ylidene)-4-(3-methyl-2-thienyl)butyl)-3-piperidinecarboxylic acid (5-hydroxytiagabine) 13, has been prepared in 8 steps from 2-bromo-3-methylthiophene 3. Key steps are Grignard reactions, displacement of heteroaromatic chlorine with methoxy, and simultaneously demethylation and opening of a hydroxymethylcyclopropane with bromotrimethylsilane. An alternative approach involving acylation of 2-lithio-3-methylthiophene 17a was found less satisfying. A metalloporphyrin assisted hydroxylation of tiagabine 1 also yielded the target metabolite. The structure of 5-hydroxytiagabine was confirmed by NMR-data including COSY, ROESY, HMQC and HMBC experiments.

Histamine h3 receptor antagonists

The SAR of H3 ligands has been difficult to evaluate because of species differences, multiple isoforms and constitutive activity, among other complicating factors. A review is given of the sometimes-conflicting affinity, activity and efficacy data of H3 agonists that has been described in literature to date.

In vivo labeling of the central GABA uptake carrier with 3H-Tiagabine

The in vivo binding of 3H-Tiagabine to the central GABA uptake carrier in mouse brain was characterized. 3H-Tiagabine in vivo bound to a single class of binding sites with a Kd = 72.5 nM and a Bmax = 640 pmol/g tissue. 3H-Tiagabine binding in vivo was regionally distributed within the CNS, and showed a good correlation with 3H-Tiagabine binding in vitro. Pharmacological characterization of 3H-Tiagabine binding in vivo revealed a binding site exhibiting specificity for GABA uptake inhibitors. Experiments examining the in vivo receptor occupancy of the GABA uptake carrier for a series of GABA uptake inhibitors revealed that 20-30% of the GABA uptake sites were occupied at the ED50 for inhibiting DMCM-induced clonic convulsions, while a 50-62% receptor occupancy in vivo was needed to inhibit rotarod performance. These data suggest that 3H-Tiagabine in vivo binding may be a useful method for assessing GABA uptake inhibitor penetration into the CNS, and may be a useful tool for studying the physiological regulation of the GABA uptake carrier.