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Bente Frølund

Publications and source records attributed to Bente Frølund.

At least 19 recordsLinked to original sources
PubMed2006

Differences in kinetics of structurally related competitive GABA(A) receptor antagonists.

Previously, 4-alkyl and 4-aryl substituted analogues of the low-efficacy partial GABA(A) receptor agonist 5-(4-piperidyl)-3-isothiazole (4-PIOL) have been identified as competitive GABA(A) receptor antagonists. These structurally related competitive antagonists show marked differences in their kinetic properties. The kinetics of 20 4-alkyl and 4-aryl substituted analogues of 4-PIOL, two 4-arylalkyl substituted 3-isothiazolol analogues and the classical GABA(A) receptor antagonist SR95531 was studied in cultured cerebral cortical neurons using whole-cell patch-clamp techniques. The kinetics of the antagonists was studied indirectly by measuring the changes in the response of the full GABA(A) receptor agonist isoguvacine (IGU) induced by concurrent application of an antagonist. When added, the majority of the antagonists did not affect the rate of deactivation of the IGU-induced responses. When removed, however, the majority of the antagonists slowed the reactivation phase of IGU implying that the dissociation of the antagonist from the GABA(A) receptor is the rate-limiting step. Surprisingly, the functional off-rates of the antagonists seemed to correlate better with the lipophilicity of the compounds than with the affinity and potency. This suggests that the dissociation of the tested antagonists from the GABA(A) receptor is restricted by lipophilic interactions, perhaps with the aromatic amino acids surrounding the GABA binding site.

Animals↗
PubMed2006

A novel selective gamma-aminobutyric acid transport inhibitor demonstrates a functional role for GABA transporter subtype GAT2/BGT-1 in the CNS.

The system of GABA transporters in neural cells constitutes an efficient mechanism for terminating inhibitory GABAergic neurotransmission. This transport system is an important therapeutical target in epileptic disorders, but potentially also in other neurological disorders. Thus, selective intervention in GABA uptake has been the subject of extensive research for several decades. In a series of lipophilic diaromatic derivatives of (RS)-3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazole (exo-THPO), N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (EF1502) turned out to be an equipotent inhibitor at the mouse transporters GAT1 and GAT2 (BGT-1) but inactive at GAT3 and GAT4. This novel pharmacological profile among GABA uptake inhibitors prompted a thorough investigation of the in vivo properties of this compound. These investigations have for the first time demonstrated a functional role for GABA transporter subtype GAT2/BGT-1, which points to the therapeutic relevance of inhibiting this transporter subtype. An overview of the development and characterisation of EF1502 is presented here.

Animals↗
PubMed2006

Potent 4-arylalkyl-substituted 3-isothiazolol GABA(A) competitive/noncompetitive antagonists: synthesis and pharmacology.

The GABA(A) agonists muscimol (1), 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, gaboxadol, 3), and the partial GABA(A) agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL, 6a) and their respective 3-isothiazolol analogues thiomuscimol (2), thio-THIP (4), and thio-4-PIOL (7a) are ligands at the GABA(A) orthosteric (recognition) site. The structure-activity relationships (SARs) between these structures are key elements of a 3D-pharmacophore model for GABA(A) agonists and competitive antagonists [Frølund, B.; Jørgensen, A. T.; Tagmose, L.; Stensbøl, T. B.; Vestergaard, H. T.; Engblom, C.; Kristiansen, U.; Sanchez, C.; Krogsgaard-Larsen, P.; Liljefors, T. J. Med. Chem. 2002, 45, 2454-2468]. Prompted by this model, we now report the synthesis and SAR of a series of analogues of 7a, in which the 4-position of the 3-isothiazolol was substituted by alkyl or bulky aromatic groups such as naphthylmethyl and diphenylalkyl groups (7b-h). The compounds have been pharmacologically characterized using receptor binding assays and two-electrode voltage-clamped Xenopus oocytes expressing alpha1beta3gamma2S- and alpha4beta3delta-containing receptors. The compounds show SARs comparable with those of 6b-h but are generally 5-15 times more potent. The 2-naphthylmethyl, the 1-bromo-2-naphthylmethyl, and the 3,3-diphenylpropyl analogues, compounds 7e, 7f, and 7h, respectively, show affinity in the low-nanomolar range (K(i) 2-10 nM). Interestingly, 7e and 7h exhibited a mixed antagonist profile consisting of a noncompetitive component in the picomolar range and a competitive component at concentrations above 1 nM. This unique profile was shown not to be due to either use dependence or kinetic effects. This antagonist profile of 7e and 7h was particularly pronounced at alpha4beta3delta-containing GABA(A) receptors, which showed three- and 10-fold selectivity for 7h and 6h, respectively.

Animals↗
PubMed2006

Structure-activity relationships of selective GABA uptake inhibitors.

For more than four decades there has been a search for selective inhibitors of GABA transporters. This has led to potent and selective inhibitors of the cloned GABA transporter subtype GAT1, which is responsible for a majority of neuronal GABA transport. The only clinically approved compound with this mechanism of action is Tiagabine. Other GABA transporter subtypes have not been targeted with comparable selectivity and potency. We here review a comprehensive series of competitive inhibitors that provide information about the GABA recognition site and summarise the structure-activity relations in a ligand-based pharmacophore model that suggests how future compounds could be designed. Finally, some of the recent results on subtype-characterised competitive inhibitors and recent lipophilic aromatic GABA uptake inhibitors are reviewed.

Animals↗
PubMed2005

Tetrazolyl isoxazole amino acids as ionotropic glutamate receptor antagonists: synthesis, modelling and molecular pharmacology.

Two 3-(5-tetrazolylmethoxy) analogues, 1a and 1b, of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA), a selective AMPA receptor agonist, and (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), a GluR5-preferring agonist, were synthesized. Compounds 1a and 1b were pharmacologically characterized in receptor binding assays, and electrophysiologically on homomeric AMPA receptors (GluR1-4), homomeric (GluR5 and GluR6) and heteromeric (GluR6/KA2) kainic acid receptors, using two-electrode voltage-clamped Xenopus laevis oocytes expressing these receptors. Both analogues proved to be antagonists at all AMPA receptor subtypes, showing potencies (Kb=38-161 microM) similar to that of the AMPA receptor antagonist (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA) (Kb=43-76 microM). Furthermore, the AMOA analogue, 1a, blocked two kainic acid receptor subtypes (GluR5 and GluR6/KA2), showing sevenfold preference for GluR6/KA2 (Kb=19 microM). Unlike the iGluR antagonist (S)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid [(S)-ATPO], the corresponding tetrazolyl analogue, 1b, lacks kainic acid receptor effects. On the basis of docking to a crystal structure of the isolated extracellular ligand-binding core of the AMPA receptor subunit GluR2 and a homology model of the kainic acid receptor subunit GluR5, we were able to rationalize the observed structure-activity relationships.

Animals↗
PubMed2005

Novel cyclic gamma-hydroxybutyrate (GHB) analogs with high affinity and stereoselectivity of binding to GHB sites in rat brain.

Gamma-hydroxybutyrate (GHB) is a psychotropic compound endogenous to the brain. Despite its potentially great physiological significance, its exact molecular mechanism of action is unknown. GHB is a weak agonist at GABA(B) receptors, but there is also evidence of specific GHB receptor sites, the molecular cloning of which remains a challenge. Ligands with high affinity and specificity for the reported GHB binding site are needed for pharmacological dissection of the GHB and GABA(B) effects and for mapping the structural requirements of the GHB receptor-ligand interactions. For this purpose, we have synthesized and assayed three conformationally restricted GHB analogs for binding against the GHB-specific ligand [3H]NCS-382 [(E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene-)acetic acid] in rat brain homogenate. The cyclohexene and cyclopentene analogs, 3-hydroxycyclohex-1-enecarboxylic acid [(RS)-HOCHCA] and 3-hydroxycyclopent-1-enecarboxylic acid [(RS)-HOCPCA], were found to be high-affinity GHB ligands, with IC50 values in the nanomolar range, and had 9 and 27 times, respectively, higher affinity than GHB. The stereo-selectively synthesized R,R-isomer of the trans-cyclopropyl GHB analog, HOCPrCA, proved to have 10-fold higher affinity than its enantiomer. Likewise, the R-enantiomers of HOCHCA and HOCPCA selectively inhibited [3H]NCS-382 binding. The best inhibitor of these, (R)-HOCPCA, has an affinity 39 times higher than GHB and is thus among the best GHB ligands reported to date. Neither of the cycloalkenes showed any affinity (IC50 > 1 mM) for GABA(A) or GABA(B) receptors. These compounds show excellent potential as lead structures and novel tools for studying specific GHB receptor-mediated pharmacology.

Animals↗
PubMed2005

Selective inhibitors of GABA uptake: synthesis and molecular pharmacology of 4-N-methylamino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol analogues.

A series of lipophilic diaromatic derivatives of the glia-selective GABA uptake inhibitor (R)-4-amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol [(R)-exo-THPO, 4] were synthesized via reductive amination of 3-ethoxy-4,5,6,7-tetrahydrobenzo[d]isoxazol-4-one (9) or via N-alkylation of O-alkylatedracemic 4. The effects of the target compounds on GABA uptake mechanisms in vitro were measured using a rat brain synaptosomal preparation or primary cultures of mouse cortical neurons and glia cells (astrocytes), as well as HEK cells transfected with cloned mouse GABA transporter subtypes (GAT1-4). The activity against isoniazid-induced convulsions in mice after subcutaneous administration of the compounds was determined. All of the compounds were potent inhibitors of synaptosomal uptake the most potent compound being (RS)-4-[N-(1,1-diphenylbut-1-en-4-yl)amino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (17a, IC50 = 0.14 microM). The majority of the compounds showed a weak preference for glial, as compared to neuronal, GABA uptake. The highest degree of selectivity was 10-fold corresponding to the glia selectivity of (R)-N-methyl-exo-THPO (5). All derivatives showed a preference for the GAT1 transporter, as compared with GAT2-4, with the exception of (RS)-4-[N-[1,1-bis(3-methyl-2-thienyl)but-1-en-4-yl]-N-methylamino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (28d), which quite surprisingly turned out to be more potent than GABA at both GAT1 and GAT2 subtypes. The GAT1 activity was shown to reside in (R)-28d whereas (R)-28d and (S)-28d contributed equally to GAT2 activity. This makes (S)-28d a GAT2 selective compound, and (R)-28d equally effective in inhibition of GAT1 and GAT2 mediated GABA transport. All compounds tested were effective as anticonvulsant reflecting that these compounds have blood-brain barrier permeating ability.

Animals↗
PubMed2005

Potent 4-aryl- or 4-arylalkyl-substituted 3-isoxazolol GABA(A) antagonists: synthesis, pharmacology, and molecular modeling.

We have previously described a series of competitive GABA(A) antagonists derived from the low-efficacy partial agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL, 4). The 2-naphthylmethyl analogue, 4-(2-naphthylmethyl)-5-(4-piperidyl)-3-isoxazolol (5), provided affinity for the GABA(A) receptor site higher than that of the standard GABA(A) receptor antagonist, SR 95531 (3). Molecular modeling studies of these compounds exposed a cavity at the receptor recognition site capable of accommodating aromatic groups of substantial size in the 4-position in the 3-isoxazolol ring. Here we present a series of analogues of 5, with various substituents in different positions in the naphthyl ring system (6a-k), and compounds with aromatic substituents directly attached to the 4-position of the 3-isoxazolol ring (7l-n). The compounds have been pharmacologically characterized using receptor-binding assays and electrophysiological whole-cell patch-clamp techniques. All of the tested compounds show affinity for the GABA(A) receptor site. While the 5-, 7-, and 8-bromo analogues, 6b-d, showed receptor affinities (K(i) = 45, 109, and 80 nM, respectively) comparable with that of 5 (K(i) = 49 nM), the 1-bromo analogue, 6a, provided the highest receptor affinity of the series (K(i) = 10 nM). Introduction of a series of different substituents in the 1-position in the 2-naphthyl ring system led to compounds, 6e-k, with retained high affinity for the GABA(A) receptor (K(i) = 16-250 nM). Introduction of a phenyl ring directly into the 4-position on the 3-isoxazolol ring gave a 41-fold increase in affinity relative to that of 4-PIOL. In whole-cell patch-clamp recordings from cultured cerebral cortical neurons, all of the tested compounds were able to inhibit the effect of the specific GABA(A) agonist isoguvacine, 6a showing antagonist potency (IC(50) = 42 nM) markedly higher than that of 3 (IC(50) = 240 nM). Molecular modeling studies, based on the compounds described, emphasized the importance of the distal ring in 5 for receptor affinity and the considerable dimensions of the proposed receptor cavity. Furthermore, the phenyl rings in 7l and in 6k were shown to represent highly favorable positions for an aromatic ring in previously unexplored receptor regions in terms of a pharmacophore model.

Animals↗
PubMed2004

First demonstration of a functional role for central nervous system betaine/{gamma}-aminobutyric acid transporter (mGAT2) based on synergistic anticonvulsant action among inhibitors of mGAT1 and mGAT2.

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-THPO (4-amino-4,5,6,7-tetrahydrobenzo[d]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 tiagabine [(R)-N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]nipecotic acid] or LU-32-176B [N-[4,4-bis(4-fluorophenyl)-butyl]-3-hydroxy-4-amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol], 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 muM, 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.

Acoustic Stimulation↗
PubMed2004

GABA(A) agonists and partial agonists: THIP (Gaboxadol) as a non-opioid analgesic and a novel type of hypnotic.

The GABA(A) receptor system is implicated in a number of central nervous system (CNS) disorders, making GABA(A) receptor ligands interesting as potential therapeutic agents. Only a few different classes of structures are currently known as ligands for the GABA recognition site on the hetero-pentameric GABA(A) receptor complex, reflecting the very strict structural requirements for GABA(A) receptor recognition and activation. A large number of the compounds showing agonist activity at the GABA(A) receptor site are structurally derived from the GABA(A) agonists muscimol, THIP (Gaboxadol), or isoguvacine, which we developed at the initial stage of the project. Using recombinant GABA(A) receptors, functional selectivity has been shown for a number of compounds, including THIP, showing subunit-dependent potency and maximal response. The pharmacological and clinical activities of THIP probably reflect its potent effects at extrasynaptic GABA(A) receptors insensitive to benzodiazepines and containing alpha(4)beta(3)delta subunits. The results of ongoing clinical studies on the effect of the partial GABA(A) agonist THIP on human sleep pattern show that the functional consequences of a directly acting agonist are distinctly different from those seen after administration of GABA(A) receptor modulators, such as benzodiazepines. In the light of the interest in partial GABA(A) receptor agonists as potential therapeutics, structure-activity studies of a number of analogues of 4-PIOL, a low-efficacy partial GABA(A) agonist derived from THIP, have been performed. In this connection, a series of GABA(A) ligands has been developed showing pharmacological profiles ranging from low-efficacy partial GABA(A) agonist activity to selective antagonist effect.

Analgesics, Non-Narcotic↗
PubMed2004

Carbamoylcholine homologs: synthesis and pharmacology at nicotinic acetylcholine receptors.

In a recent study, racemic 3-(N,N-dimethylamino)butyl-N,N-dimethylcarbamate (1) was shown to be a potent agonist at neuronal nicotinic acetylcholine receptors with a high selectivity for nicotinic over muscarinic acetylcholine receptors [Mol. Pharmacol. 64 (2003) 865-875]. Here we present the synthesis and pharmacological characterization of a series of analogs of, where the methyl group at C-3 has been replaced by different alkyl substituents. Ring systems have been incorporated into the carbon backbone of some of the molecules, or the amino group has been build into ring systems. Furthermore, the (+)- and (-)-enantiomers of have been separated, and X-ray crystallography has revealed that (-)-1 possesses (S)-configuration. The compounds have been characterized pharmacologically at recombinant nicotinic receptor subtypes. The structure-activity relationship study has provided valuable insight into the mode of interactions of and its analogs with neuronal nicotinic acetylcholine receptors.

Animals↗
PubMed2004

Activation of single heteromeric GABA(A) receptor ion channels by full and partial agonists.

The linkage between agonist binding and the activation of a GABA(A) receptor ion channel is yet to be resolved. This aspect was examined on human recombinant alpha1beta2gamma2S GABA(A) receptors expressed in human embryonic kidney cells using the following series of receptor agonists: GABA, isoguvacine, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), isonipecotic acid, piperidine-4-sulphonic acid (P4S), imidazole-4-acetic acid (IAA), 5-(4-piperidyl)-3-isothiazolol (thio-4-PIOL) and 5-(4-piperidyl)-3-isoxazolol (4-PIOL). Whole-cell concentration-response curves enabled the agonists to be categorized into four classes based upon their maximum responses. Single channel analyses revealed that the channel conductance of 25-27 pS was unaffected by the agonists. However, two open states were resolved from the open period distributions with mean open times reduced 5-fold by the weakest partial agonists. Using saturating agonist concentrations, estimates of the channel shutting rate, alpha, ranged from 200 to 600 s(-1). The shut period distributions were described by three or four components and for the weakest partial agonists, the interburst shut periods increased whilst the mean burst durations and longest burst lengths were reduced relative to the full agonists. From the burst analyses, the opening rates for channel activation, beta, and the total dissociation rates, k(-1), for the agonists leaving the receptor were estimated. The agonist efficacies were larger for the full agonists (E approximately 7-9) compared to the weak partial agonists ( approximately 0.4-0.6). Overall, changes in agonist efficacy largely determined the different agonist profiles with contributions from the agonist affinities and the degree of receptor desensitization. From this we conclude that GABA(A) receptor activation does not occur in a switch-like manner since the agonist recognition sites are flexible, accommodating diverse agonist structures which differentially influence the opening and shutting rates of the ion channel.

Cells, Cultured↗
PubMed2003

Aza-THIP and related analogues of THIP as GABA C antagonists.

The potency of a series of eight compounds structurally related with 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), a potent GABA(A) partial agonist exhibiting GABA(C) rho(1) antagonist effect (K(i)=25 microM), was determined electrophysiologically using homomeric human GABA(C) rho(1) receptors expressed in Xenopus oocytes. Protolytic properties (pK(a) values for the acidic bioisosteric groups) and the presence of steric bulk in the molecules appear to be structural parameters of importance for blockade of the GABA(C) rho(1) receptor. Within this series of moderately potent GABA(C) antagonists, only 4,5,6,7-tetrahydropyrazolo[5,4-c]pyridin-3-ol (Aza-THIP) does not interact detectably with GABA(A) receptors, and Aza-THIP has the potential of being a useful tool for molecular and behavioural pharmacological studies.

Animals↗
PubMed2003

Phosphinic, phosphonic and seleninic acid bioisosteres of isonipecotic acid as novel and selective GABA(C) receptor antagonists.

A number of amino acids bioisosterically derived from the specific GABA(A) agonist, isonipecotic acid, were electrophysiologically characterized as antagonists at GABA(C) rho(1) receptors expressed in Xenopus oocytes. The phosphinic acid analogue of isonipecotic acid, piperidin-4-ylphosphinic acid (2), was comparable with the standard GABA(C) antagonist, (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), in terms of potency and GABA(C) versus GABA(A) receptor selectivity. Whereas the phosphonic acid analogue, piperidin-4-ylphosphonic acid (4), was at least an order of magnitude weaker than piperidin-4-ylphosphinic acid as a GABA(C) antagonist, the seleninic acid analogue, piperidin-4-ylseleninic acid (SEPI, 6), was the most potent and selective GABA(C) antagonist within the group of isonipecotic acid derived amino acids studied.

Animals↗
PubMed2003

Design and synthesis of a new series of 4-alkylated 3-isoxazolol GABA A antagonists.

A number of analogues of the low-efficacy partial GABA(A) agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL), in which the 4-position of the 3-isoxazolol ring is substituted by different groups, were synthesized and tested as GABA(A) receptor ligands. While alkyl and benzyl substitution provided affinities and antagonist potencies comparable to those of 4-PIOL, diphenylalkyl and naphthylalkyl substitution resulted in marked increase in both affinity and potency. The 2-naphthylmethyl and the 3,3-diphenylpropyl analogues showed antagonist potencies comparable or markedly higher than that of the standard antagonist SR 95531. Molecular modeling studies exposed a large cavity in the vicinity of the 4-position of 4-PIOL, in which there seems to be additional sites for specific receptor interactions.

Alkylation↗