Synthesis and sar of 2h-1,2,4-benzothiadiazine-1,1-dioxide-3- carboxylic acid derivatives as novel potent glycine antagonists of the nmda receptor-channel complex

The N-methyl-d-aspartate (NMDA) receptor is a ligand-gated ion channel that requires both glutamate and glycine for efficient activation. Here, a strategy combining cysteine scanning mutagenesis and affinity labeling was used to investigate the glycine binding site located on the NR1 subunit. Based on homology modeling to the crystal structure of the glutamate binding site of the 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)-propionic acid receptor GluR2, cysteines were introduced into the NR1 subunit as chemical sensors for three thiol-reactive derivatives of the competitive antagonist L-701,324. After coexpressing the mutant NR1 with wild-type NR2B subunits in Xenopus oocytes, agonist-induced currents were recorded to monitor irreversible receptor inactivation by the reactive antagonists. For each derivative, glycine site-specific inactivations were observed with a distinct subset of cysteine-substituted receptors. Together these inactivating substitutions identified seven NR1 residues (Ile-385, Gln-387, Glu-388, Thr-500, Asn-502, Ala-696, and Val-717) that undergo proximity-induced covalent coupling with specific regions of the bound antagonist and disclose its mode of docking in the glycine binding pocket of the NMDA receptor. Our approach may help to unravel the structural basis of distinct NMDA receptor subtype pharmacologies.

In just under 20 years the kynurenine family of compounds has developed from a group of obscure metabolites of the essential amino acid tryptophan into a source of intensive research, with postulated roles for quinolinic acid in neurodegenerative disorders, most especially the AIDS-dementia complex and Huntington's disease. One of the kynurenines, kynurenic acid, has become a standard tool for use in the identification of glutamate-releasing synapses, and has been used as the parent for several groups of compounds now being developed as drugs for the treatment of epilepsy and stroke. The kynurenines represent a major success in translating a basic discovery into a source of clinical understanding and therapeutic application, with around 3000 papers published on quinolinic acid or kynurenic acid since the discovery of their effects in 1981 and 1982. This review concentrates on some of the recent work most directly relevant to the understanding and applications of kynurenines in medicine.

Strokes (intracranial thomboses or haemorrhaging) cause death and disability, but effective treatments are lacking. The metabolism of tryptophan leads to the generation of quinolinic acid, an agonist potentially neurotoxic at glutamate receptors, and kynurenic acid, an antagonist at the same population of receptors. The commercial development of the kynurenine pathway has included the use of analogues of kynurenic acid as antagonists at glutamate receptors. A second has been to use prodrugs of kynurenic acid or its analogues. Alternatively, it is proving possible to interfere directly with the kynurenine pathway to block the synthesis of quinolinic acid and promote the formation of kynurenic acid. This change yields neuroprotectant and anticonvulsant compounds.

A number of 2-substituted-3,4-dihydro-3-oxo-6,8-dichloro-2H-1,4-benzothiazine-1,1-dioxides (1-2a-b) and -1-oxides (3-4a-b) bioisosters of RPR 104632 in which the 3-carboxylic group was replaced by a carbonyl group were synthesized. Comparative in vitro pharmacological studies on this series of RPR 104632 analogs were performed using receptor binding assays. None of these compounds showed detectable binding affinity for the glycine-NMDA receptor.

This chapter provides an overview of methodologies used to make single enantiomers to assist medicinal chemists in preparing the desired single enantiomer. In addition, it reviews methods used to manufacture single enantiomers of pharmaceutical agents and emphasize the different requirements of a synthetic procedure to make the first amounts of material, and to manufacture a drug in bulk. Methodologies for single enantiomers can be divided into three basic types: (i) Chirality pool: synthetic modification of an available single-enantiomer raw material with maintenance or transfer of stereochemical integrity throughout the synthesis makes the product. (ii) Resolution: a synthetic racemic mixture is separated into its enantiomers. (iii) Asymmetric synthesis: control is at the point where the chirality is created by asymmetric modification of a prochiral substrate. All methods for providing a single enantiomer, whether chemical or biochemical can be considered as being comprised of one or more of the above. For instance, fermentation may involve a microbial strain effecting an asymmetric synthesis, etc.

The effects of five glycine site antagonists were comparatively examined on maximal and plateau currents evoked by 200 μM N-methyl-d-aspartate (NMDA) in the presence of 1 μM glycine in cultured cerebrocortical cells of the rat using whole-cell patch-clamp technique. 5,7-Dichlorokynurenic acid, ACEA-1021 (5-nitro-6,7-dichloro-quinoxalinedione), L-695,902 (methyl 7-chloro-4-hydroxy 2(1 H)-quinolone-3-carboxylate), LY-294,619 (5,7-dichloro-3-(4-hydroxphenyl)-4-hydroxyquinolin-2(1 H)-one) and RPR-104,632 (2-(3-bromo-benzyl)-6,8-dichloro-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide-3-carboxylic acid) caused concentration-dependent inhibition of NMDA-activated currents. However, antagonists showed different relative efficacies to block peak currents and plateau currents, characterised by the following IC₅₀ ratios: L-695,902: 0.98; RPR-104,632: 1.06; ACEA-1021: 1.69; LY-294,619: 1.71; and 5,7-dichlorokynurenic acid: 3.42. Our findings indicate a heterogeneity of glycine site antagonists in affecting NMDA receptor desensitisation, and suggest potential differences in their pharmacologies.