Derivatives of (R)-3-(5-Furanyl)carboxamido-2-aminopropanoic Acid as Potent NMDA Receptor Glycine Site Agonists with GluN2 Subunit-Specific Activity.

NMDA receptors mediate glutamatergic neurotransmission and are therapeutic targets due to their involvement in a variety of psychiatric and neurological disorders. Here, we describe the design and synthesis of a series of (R)-3-(5-furanyl)carboxamido-2-aminopropanoic acid analogues 8a-s as agonists at the glycine (Gly) binding site in the GluN1 subunit, but not GluN3 subunits, of NMDA receptors. These novel analogues display highly variable potencies and agonist efficacies among the NMDA receptor subtypes (GluN1/2A-D) in a manner dependent on the GluN2 subunit. Notably, compound 8p is identified as a potent partial agonist at GluN1/2C (EC50 = 0.074 µM) with an agonist efficacy of 28% relative to activation by Gly and virtually no agonist activity at GluN1/2A, GluN1/2B, and GluN1/2D. Thus, these novel agonists can modulate the activity of specific NMDA receptor subtypes by replacing the full endogenous agonists Gly or d-serine (d-Ser), thereby providing new opportunities in the development of novel therapeutic agents.

Fragment-Based Optimization of Dihydropyrazino-Benzimidazolones as Metabotropic Glutamate Receptor-2 Positive Allosteric Modulators against Migraine.

Our previous scaffold-hopping attempts resulted in dihydropyrazino-benzimidazoles as metabotropic glutamate receptor-2 (mGluR2) positive allosteric modulators (PAMs) with suboptimal drug-like profiles. Here, we report an alternative fragment-based optimization strategy applied on the new dihydropyrazino-benzimidazolone scaffold. Analyzing published high-affinity mGluR2 PAMs, we used a pharmacophore-guided approach to identify suitable growing vectors and optimize the scaffold in these directions. This strategy resulted in a new fragment like lead (34) with improved druglike properties that were translated to sufficient pharmacokinetics and validated proof-of-concept studies in migraine. Gratifyingly, compound 34 showed reasonable activity in the partial infraorbital nerve ligation, a migraine disease model that might open this indication for mGluR2 PAMs.

Use of the 4-Hydroxytriazole Moiety as a Bioisosteric Tool in the Development of Ionotropic Glutamate Receptor Ligands.

We report a series of glutamate and aspartate analogues designed using the hydroxy-1,2,3-triazole moiety as a bioisostere for the distal carboxylic acid. Compound 6b showed unprecedented selectivity among ( S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor subtypes, confirmed also by an unusual binding mode observed for the crystal structures in complex with the AMPA receptor GluA2 agonist-binding domain. Here, a methionine (Met729) was highly disordered compared to previous agonist-bound structures. This observation provides a possible explanation for the pharmacological profile. In the structure with 7a, an unusual organization of water molecules around the bioisostere arises compared to previous structures of ligands with other bioisosteres. Aspartate analogue 8 with the hydroxy-1,2,3-triazole moiety directly attached to glycine was unexpectedly able to activate both the glutamate and glycine agonist-binding sites of the N-methyl-d-aspartic acid receptor. These observations demonstrate novel features that arise when employing a hydroxytriazole moiety as a bioisostere for the distal carboxylic acid in glutamate receptor agonists.

Synthesis and Pharmacological Characterization of C4ß-Amide-Substituted 2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylates. Identification of (1 S,2 S,4 S,5 R,6 S)-2-Amino-4-[(3-methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid (LY2794193), a Highly Potent and Selective mGlu3 Receptor Agonist.

Multiple therapeutic opportunities have been suggested for compounds capable of selective activation of metabotropic glutamate 3 (mGlu3) receptors, but small molecule tools are lacking. As part of our ongoing efforts to identify potent, selective, and systemically bioavailable agonists for mGlu2 and mGlu3 receptor subtypes, a series of C4ß-N-linked variants of (1 S,2 S,5 R,6 S)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 1 (LY354740) were prepared and evaluated for both mGlu2 and mGlu3 receptor binding affinity and functional cellular responses. From this investigation we identified (1 S,2 S,4 S,5 R,6 S)-2-amino-4-[(3-methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 8p (LY2794193), a molecule that demonstrates remarkable mGlu3 receptor selectivity. Crystallization of 8p with the amino terminal domain of hmGlu3 revealed critical binding interactions for this ligand with residues adjacent to the glutamate binding site, while pharmacokinetic assessment of 8p combined with its effect in an mGlu2 receptor-dependent behavioral model provides estimates for doses of this compound that would be expected to selectively engage and activate central mGlu3 receptors in vivo.

Discovery and Kinetic Profiling of 7-Aryl-1,2,4-triazolo[4,3-a]pyridines: Positive Allosteric Modulators of the Metabotropic Glutamate Receptor 2.

We report the synthesis and biological evaluation of a series of 7-aryl-1,2,4-triazolo[4,3-a]pyridines with mGlu2 positive allosteric modulator (PAM) activity and affinity. Besides traditional in vitro parameters of potency and affinity, kinetic parameters kon, koff and residence time (RT) were determined. The PAMs showed various kinetic profiles; kon values ranged over 2 orders of magnitude, whereas RT values were within a 10-fold range. Association rate constant kon was linearly correlated to affinity. Evaluation of a short, medium, and long RT compound in a label-free assay indicated a correlation between RT and functional effect. The effects of long RT compound 9 on sleep-wake states indicated long RT was translated into sustained inhibition of rapid eye movement (REM) in vivo. These results show that affinity-only driven selection would have resulted in mGlu2 PAMs with high values for kon but not necessarily optimized RT, which is key to predicting optimal efficacy in vivo.

Characterization of l-Theanine Excitatory Actions on Hippocampal Neurons: Toward the Generation of Novel N-Methyl-d-aspartate Receptor Modulators Based on Its Backbone.

l-Theanine (or l-γ-N-ethyl-glutamine) is the major amino acid found in Camellia sinensis. It has received much attention because of its pleiotropic physiological and pharmacological activities leading to health benefits in humans, especially. We describe here a new, easy, efficient, and environmentally friendly chemical synthesis of l-theanine and l-γ-N-propyl-Gln and their corresponding d-isomers. l-Theanine, and its derivatives obtained so far, exhibited partial coagonistic action at N-methyl-d-aspartate (NMDA) receptors, with no detectable agonist effect at other glutamate receptors, on cultured hippocampal neurons. This activity was retained on NMDA receptors expressed in Xenopus oocytes. In addition, both GluN2A and GluN2B containing NMDA receptors were equally modulated by l-theanine. The stereochemical change from l-theanine to d-theanine along with the substitution of the ethyl for a propyl moiety in the γ-N position of l- and d-theanine significantly enhanced the biological efficacy, as measured on cultured hippocampal neurons. l-Theanine structure thus represents an interesting backbone to develop novel NMDA receptor modulators.

Total synthesis of biologically active natural products based on highly selective synthetic methodologies.

Total syntheses of structurally and biologically intriguing natural products relying on new synthetic methodologies are described. This article features cinchona alkaloid-catalyzed asymmetric Morita-Baylis-Hillman reactions, heterocycle syntheses based on rhodium-catalyzed C-H amination and indium-catalyzed Conia-ene reactions, and their utilization for the syntheses of the phoslactomycin family of antibiotics, glutamate receptor agonists and antagonists, and alkaloids with characteristic highly substituted pyrrolidinone core structures.

Design, synthesis, and structure-activity relationship of a novel series of GluN2C-selective potentiators.

NMDA receptors are tetrameric complexes composed of GluN1 and GluN2A-D subunits that mediate a slow Ca(2+)-permeable component of excitatory synaptic transmission. NMDA receptors have been implicated in a wide range of neurological diseases and thus represent an important therapeutic target. We herein describe a novel series of pyrrolidinones that selectively potentiate only NMDA receptors that contain the GluN2C subunit. The most active analogues tested were over 100-fold selective for recombinant GluN2C-containing receptors over GluN2A/B/D-containing NMDA receptors as well as AMPA and kainate receptors. This series represents the first class of allosteric potentiators that are selective for diheteromeric GluN2C-containing NMDA receptors.

In vivo expression of a light-activatable potassium channel using unnatural amino acids.

Optical control of protein function provides excellent spatial-temporal resolution for studying proteins in situ. Although light-sensitive exogenous proteins and ligands have been used to manipulate neuronal activity, a method for optical control of neuronal proteins using unnatural amino acids (Uaa) in vivo is lacking. Here, we describe the genetic incorporation of a photoreactive Uaa into the pore of an inwardly rectifying potassium channel Kir2.1. The Uaa occluded the pore, rendering the channel nonconducting, and, on brief light illumination, was released to permit outward K(+) current. Expression of this photoinducible inwardly rectifying potassium (PIRK) channel in rat hippocampal neurons created a light-activatable PIRK switch for suppressing neuronal firing. We also expanded the genetic code of mammals to express PIRK channels in embryonic mouse neocortex in vivo and demonstrated a light-activated PIRK current in cortical neurons. These principles could be generally expanded to other proteins expressed in the brain to enable optical regulation.

Development of thiophenic analogues of benzothiadiazine dioxides as new powerful potentiators of 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors.

On the basis of the results obtained in previous series of AMPA potentiators belonging to 3,4-dihydro-2H-benzo- and 3,4-dihydro-2H-pyrido-1,2,4-thiadiazine 1,1-dioxides, the present work focuses on the design of original isosteric 3,4-dihydro-2H-thieno-1,2,4-thiadiazine 1,1-dioxides. Owing to the sulfur position, three series of compounds were developed and their activity as AMPA potentiators was characterized. In each of the developed series, potent compounds were discovered. After screening the selected active compounds on a safety in vivo test, 6-chloro-4-ethyl-3,4-dihydro-2H-thieno[2,3-e]-1,2,4-thiadiazine 1,1-dioxide (24) appeared as the most promising compound and was further evaluated. Its effects on long-term potentiation in vivo and on AMPA-mediated noradrenaline release were measured to predict its potential cognitive enhancing properties. Finally, an object recognition test performed in mice revealed that 24 was able to significantly enhance cognition, after oral administration, at doses as low as 0.3 mg/kg. This study validates the interest of the isosteric replacement of the benzene or pyridine nuclei by the thiophene nucleus in the ring-fused thiadiazine dioxides class of AMPA potentiators.

Synthesis and structure activity relationship of tetrahydroisoquinoline-based potentiators of GluN2C and GluN2D containing N-methyl-D-aspartate receptors.

We describe here the synthesis and evaluation of a series of tetrahydroisoquinolines that show subunit-selective potentiation of NMDA receptors containing the GluN2C or GluN2D subunits. Bischler-Napieralski conditions were employed in the key step for the conversion of acyclic amides to the corresponding tetrahydroisoquinoline-containing analogs. Compounds were evaluated using both two-electrode voltage clamp recordings from Xenopus laevis oocytes and imaging of mammalian BHK cells loaded with Ca(2+)-sensitive dyes. The most potent analogues had EC50 values of 300 nM and showed over 2-fold potentiation of the response to maximally effective concentrations of glutamate and glycine but had no effect on responses from NMDA receptors containing the GluN2A or GluN2B subunits AMPA, kainate, and GABA or glycine receptors or a variety of other potential targets. These compounds represent a potent class of small molecule subunit-selective potentiators of NMDA receptors.

Development of 2'-substituted (2S,1'R,2'S)-2-(carboxycyclopropyl)glycine analogues as potent N-methyl-d-aspartic acid receptor agonists.

A series of 2'-substituted analogues of the selective NMDA receptor ligand (2S,1'R,2'S)-2-(carboxycyclopropyl)glycine ((S)-CCG-IV) have been designed, synthesized, and pharmacologically characterized. The design was based on a docking study hypothesizing that substituents in the 2'-position would protrude into a region where differences among the NMDA receptor GluN2 subunits exist. Various synthetic routes were explored, and two different routes provided a series of alkyl-substituted analogues. Pharmacological characterization revealed that these compounds are NMDA receptor agonists and that potency decreases with increasing size of the alkyl groups. Variations in agonist activity are observed at the different recombinant NMDA receptor subtypes. This study demonstrates that it is possible to introduce substituents in the 2'-position of (S)-CCG-IV while maintaining agonist activity and that variation among NMDA receptor subtypes may be achieved by probing this region of the receptor.

Targeting glutamate synapses in schizophrenia.

Although early clinical observations implicated dopamine dysfunction in the neuropathology of schizophrenia, accumulating evidence suggests that multiple neurotransmitter pathways are dysregulated. The psychotomimetic actions of NMDA receptor antagonists point to an imbalance of glutamatergic signaling. Encouragingly, numerous preclinical and clinical studies have elucidated several potential targets for increasing NMDA receptor function and equilibrating glutamatergic tone, including the metabotropic glutamate receptors 2, 3 and 5, the muscarinic acetylcholine receptors M(1) and M(4), and the glycine transporter GlyT1. Highly specific allosteric and orthosteric ligands have been developed that modify the activity of these novel target proteins, and in this review we summarize both the glutamatergic mechanisms and the novel compounds that are increasing the promise for a multifaceted pharmacological approach to treat schizophrenia.

Glutamate receptor agonists: stereochemical aspects.

The neurotransmitter (S)-glutamate [(S)-Glu] is responsible for most of the excitatory neurotransmission in the central nervous system. The effect of (S)-Glu is mediated by both ionotropic and metabotropic receptors. Glutamate receptor agonists are generally α-amino acids with one or more stereogenic centers due to strict requirements in the agonist binding pocket of the activated state of the receptor. By contrast, there are many examples of achiral competitive antagonists. The present review addresses how stereochemistry affects the activity of glutamate receptor ligands. The review focuses mainly on agonists and discusses stereochemical and conformational considerations as well as biostructural knowledge of the agonist binding pockets, which is useful in the design of glutamate receptor agonists. Examples are chosen to demonstrate how stereochemistry not only determines how the agonist binding pocket is filled, but also how it affects the conformational space of the ligand and in this way restricts the recognition of various glutamate receptors, ultimately leading to selectivity.

Group II metabotropic glutamate receptor agonists as a potential drug for schizophrenia.

Metabotropic glutamate receptors (mGlu receptors), with their unique signaling systems and pharmacological characteristics, have emerged as a new topic in excitatory amino acid research. Among them, the unique distribution of group II mGlu receptors, such as mGlu(2) and mGlu(3) receptors, and the involvement of these receptors in the regulation of neurotransmission are particularly interesting. Recently, potent agonists for mGlu(2/3) receptor have been synthesized, and their pharmacological roles have been intensively investigated using animal models. mGlu(2/3) receptors clearly have crucial roles in the central nervous system, and accumulating evidence in both rodents and human studies has suggested that agonists for mGlu(2/3) receptors may be beneficial for the treatment of psychiatric disorders such as schizophrenia. Possible neuronal circuits through which mGlu(2/3) receptor agonists exert their pharmacological effects have also been investigated.

4-hydroxy-1,2,5-oxadiazol-3-yl moiety as bioisoster of the carboxy function. Synthesis, ionization constants, and molecular pharmacological characterization at ionotropic glutamate receptors of compounds related to glutamate and its homologues.

In order to investigate the 4-hydroxy-1,2,5-oxadiazol-3-yl moiety as a carboxylic acid bioisoster at ionotropic glutamate receptors (iGluRs), a series of acidic alpha-aminocarboxylic acids in which the distal carboxy group was replaced by the 4-hydroxy-1,2,5-oxadiazol-3-yl group was synthesized. Ionization constants were determined. All target compounds, except the Asp analogue 12, were resolved using chiral HPLC. Whereas 12 showed good affinity exclusively at NMDA receptors, the Glu analogue, (+)-10, was an unselective, though potent AMPA receptor preferring agonist (EC(50) = 10 microM at iGluR2) showing only low stereoselectivity. The two higher Glu homologues, (+)-15 and (+)-18, turned out to be weak agonists at iGluR2 as well as weak antagonists at NR1/NR2A, whereas the corresponding (-)-isomers were selective NR1/NR2A antagonists with somewhat higher potency. The results proved the 4-hydroxy-1,2,5-oxadiazol-3-yl moiety to be a useful bioisoster at all three classes of iGluRs, capable of being integrated into agonists as well as antagonists.

The glutamate receptor GluR5 agonist (S)-2-amino-3-(3-hydroxy-7,8-dihydro-6H-cyclohepta[d]isoxazol-4-yl)propionic acid and the 8-methyl analogue: synthesis, molecular pharmacology, and biostructural characterization.

The design, synthesis, and pharmacological characterization of a highly potent and selective glutamate GluR5 agonist is reported. (S)-2-Amino-3-((RS)-3-hydroxy-8-methyl-7,8-dihydro-6H-cyclohepta[d]isoxazol-4-yl)propionic acid (5) is the 8-methyl analogue of (S)-2-amino-3-(3-hydroxy-7,8-dihydro-6H-cyclohepta[d]isoxazol-4-yl)propionic acid ((S)-4-AHCP, 4). Compound 5 displays an improved selectivity profile compared to 4. A versatile stereoselective synthetic route for this class of compounds is presented along with the characterization of the binding affinity of 5 to ionotropic glutamate receptors (iGluRs). Functional characterization of 5 at cloned iGluRs using a calcium imaging assay and voltage-clamp recordings show a different activation of GluR5 compared to (S)-glutamic acid (Glu), kainic acid (KA, 1), and (S)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acid ((S)-ATPA, 3) as previously demonstrated for 4. An X-ray crystallographic analysis of 4 and computational analyses of 4 and 5 bound to the GluR5 agonist binding domain (ABD) are presented, including a watermap analysis, which suggests that water molecules in the agonist binding site are important selectivity determinants.

Asymmetric synthesis and receptor pharmacology of the group II mGlu receptor ligand (1S,2R,3R,5R,6S)-2-amino-3-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid-HYDIA.

The asymmetric synthesis and receptor pharmacology of (1S,2R,3R,5R,6S)-2-amino-3-Hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid (+)-9 (HYDIA) and a few of its O-alkylated derivatives are described. The key step of the synthesis utilizes Sharpless' asymmetric dihydroxylation (AD-beta) for the kinetic resolution of a bicyclic racemic precursor olefin. In contrast to the bicyclic glutamate analogue LY354740, which is a potent and selective agonist for the group II metabotropic glutamate receptors (mGluRs), these new conformationally restricted and also hydroxylated or alkoxylated glutamate analogues are potent and selective antagonists for the group II mGluRs.

Novel 5-substituted 1-pyrazolol analogues of ibotenic acid: synthesis and pharmacology at glutamate receptors.

5-Substituted 1-pyrazolol analogues of ibotenic acid have been synthesized and pharmacologically characterized on ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs). The syntheses involved introduction of bromide, alkyls, phenyl and arylalkyls in the 5-position of 1-benzyloxypyrazole leading to 5-substituted (RS)-2-amino-(1-hydroxy-4-pyrazolyl)acetic acids (5a-l). The pharmacological activities of the synthesized analogues ranged from the 5-cyclopropylmethyl analogue (5f) with weak but selective affinity for NMDA receptors (IC(50)=35 microM), over the 5-n-propyl analogue (5c), which was a selective mGluR2 agonist (EC(50)=72 microM), to the 5-cyclohexylmethyl analogue (5g), which was a selective mGluR2 antagonist (K(i)=32 microM), and the 5-phenylethyl analogue (5j), which was a weak but apparently selective mGluR1 antagonist (K(i)=230 microM). This series of compounds afforded GluR ligands with a broad spectrum of pharmacological profiles, and showing potential for development of new compounds with subtype-selective activities at various GluRs.

Investigation of a route to ibotenic acid analogues via a reduced pyroglutamate template.

Two alternative "ring switch" based syntheses have been shown to give access to the reduced protected homochiral analogues, 27, 28 and 36, of the CNS active compound ibotenic acid.