Identification and characterization of novel NMDA receptor antagonists selective for NR2A- over NR2B-containing receptors

• Published in: The Journal of pharmacology and experimental therapeutics Vol. 335; no. 3; pp. 636 - 644
• Main Authors: Bettini, Ezio, Sava, Anna, Griffante, Cristiana, Carignani, Corrado, Buson, Alberto, Capelli, Anna Maria, Negri, Michele, Andreetta, Filippo, Senar-Sancho, Sergio A, Guiral, Lorena, Cardullo, Francesca
• Format: Journal Article
• Language: English
• Published: United States 01.12.2010
• Subjects: Animals; Binding, Competitive; Calcium Signaling - drug effects; Cell Line, Tumor; Cell Membrane - metabolism; Cerebral Cortex - cytology; Drug Evaluation, Preclinical - methods; Excitatory Amino Acid Antagonists - chemistry; Excitatory Amino Acid Antagonists - metabolism; Excitatory Amino Acid Antagonists - pharmacology; Excitatory Postsynaptic Potentials - drug effects; Fluorometry - methods; Glutamic Acid - pharmacology; Glycine - pharmacology; HEK293 Cells; Humans; Male; Molecular Structure; N-Methylaspartate - pharmacology; Neurons - cytology; Neurons - drug effects; Neurons - metabolism; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate - antagonists & inhibitors; Receptors, N-Methyl-D-Aspartate - genetics; Receptors, N-Methyl-D-Aspartate - metabolism; Transfection
• ISSN: 0022-3565; 1521-0103
• DOI: 10.1124/jpet.110.172544

NR1/NR2A is a subtype of N-methyl-d-aspartate receptors (NMDARs), which are glutamate and glycine-gated Ca(2+)-permeable channels highly expressed in the central nervous system. A high-throughput screening (HTS) campaign using human osteosarcoma (U-2 OS) cells transiently transduced with NR1/NR2A NMDAR subunits, tested in a specifically designed fluorometric imaging plate reader (FLIPR)/Ca(2+) assay, identified sulfonamide derivative series, exemplified by 3-chloro-4-fluoro-N-[(4-{[2-(phenylcarbonyl)hydrazino]carbonyl}phenyl)methyl]benzenesulfonamide (compound 1) and thiodiazole derivative N-(cyclohexylmethyl)-2-({5-[(phenylmethyl)amino]-1,3,4-thiadiazol-2-yl}thio)acetamide (compound 13) as novel NR1/NR2A receptor antagonists. Compounds 1 and 13 displayed submicromolar and micromolar potency at NR1/NR2A receptor, respectively, although they did not show activity at NR2B-containing receptor up to 50 μM concentration. Addition of 1 mM glycine, but not 1 mM l-glutamate, was able to surmount compound 1 and 13 inhibitory effects in FLIPR NR1/NR2A assay. However, compounds 1 and 13 displaced a glutamate site antagonist [(3)H]d,l-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid ([(3)H]CGP 39653) to a greater extent than the glycine site antagonist [(3)H]3-[(E)-2-carboxy-2-phenylethenyl]-4,6-dichloro-1H-indole-2-carboxylic acid ([(3)H]MDL 105,519), in rat brain cortex binding assay. Results of FLIPR cell-based, electrophysiological, and biochemical binding assays suggest that compounds 1 and 13 are the prototypes of novel classes of NMDAR ligands, which to the best of our knowledge are the first selective antagonists at NR1/NR2A over NR1/NR2B receptor, and might constitute useful tools able to elucidate the relative role of the NR2A subunit in physiological and pathological conditions.