Design, Synthesis, Pharmacological Evaluation and Docking Studies of GluN2B‐Selective NMDA Receptor Antagonists with a Benzo[7]annulen‐7‐amine Scaffold

Abstract

Antagonists that selectively target GluN2B-subunit-containing N-methyl-d-aspartate (NMDA) receptors are of major interest for the treatment of various neurological disorders. In this study, relationships between variously substituted benzo[7]annulen-7-amines and their GluN2B affinity were investigated. 2-Nitro-5,6,8,9-tetrahydrobenzo[7]annulen-7-one (8) represents the central building block for the introduction of various substituents at the 2-position and various 7-amino moieties. N-(3-Phenylpropyl)-6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-amines with a 2-NO (7 c), 2-Cl (15 c), or 2-OBn group (22 c) show very high GluN2B affinity (K =1.6-3.6 nm). Docking studies revealed the same binding poses for benzo[7]annulen-7-amines and ifenprodil at the interface of GluN1b and GluN2B subunits. The large 2-OBn moiety of 22 c occupies a previously unrecognized subpocket, which explains its high GluN2B affinity (K =3.6 nm). In two-electrode voltage clamp experiments and cytoprotection assays, the high-affinity GluN2B ligands 7 c, 15 c, and 22 c could not inhibit the glutamate-/glycine-evoked current and cytotoxic effects. However, the analogous phenols 16 c ((3-phenylpropyl)amino moiety) and 16 d ((4-phenylbutyl)amino moiety) with 10-fold lower GluN2B affinity (K =28 and 21 nm, respectively) showed promising inhibition of glutamate-/glycine-evoked effects in both assays. The presence of a phenolic hydroxy group seems to be essential for inducing conformational changes of the receptor protein, which finally results in closure of the ion conduction pathway.

Comparative Pharmacological Study of Common NMDA Receptor Open Channel Blockers Regarding Their Affinity and Functional Activity toward GluN2A and GluN2B NMDA Receptors

“…The K d value of ifenprodil is 10 nm. [38] Cytoprotection assays [39] Mouse L(tkÀ)c ells stably transfected with the dexamethasone-inducible eukaryotic expression vectors pMSG NR1-1a, pMSG NR2A (1:5 ratio) or pMSG NR1-1a, pMSG NR2B (1:5 ratio) for human NMDA receptor subunits were grown in sterile 96-well plates (Nunclon Delta Surface, Thermo Fisher Scientific, Langenselbold, Germany) in Dulbecco'sm odified Eagle's medium (DMEM) containing 10 %s tandardized fetal calf serum (FCS;B iochrom AG, Berlin, Germany). When the cell density of the adherent growing cells had reached~90 %o fc onfluency,t he medium was carefully removed, and the cells were incubated with 200 mLd examethasone/ketamine solution (8 mm dexamethasone and 100 mm (S)-ketamine) for 20 h. The assays were based on work reported by Te wes et al [40] After careful removal of the medium, the plate was blocked twice with 200 mLD MEM containing 1% bovine serum albumin (BSA) and rinsed once with 200 mLD MEM.…”

Synthesis and pharmacological evaluation of fluorinated benzo[7]annulen‐7‐amines as GluN2B‐selective NMDA receptor antagonists

“…By a conformational restriction approach on the prototypical ligand ifenprodil ( 1 ), we obtained tetrahydro‐3‐benzazepines (including the recently reported [ 11 C]Me‐NB1 [ 11 C] 2 ) as well as benzo[7]annulen‐7‐amines ( 4 ) derived from Ro 25‐6981 ( 3 ) in a similar manner (Figure ) . Ligands of both compound classes possess high binding affinity towards the GluN2B subunit …”

“…Stimulated by the promising GluN2B affinity of reported benzo[7]annulen‐7‐amines, a series of fluorinated GluN2B ligands of type 4 was designed. The aim was to find a fluorinated ligand with high GluN2B affinity and high selectivity over other targets allowing the development of a fluorinated PET tracer to label GluN2B subunit containing NMDARs.…”

Replacement of the Benzylpiperidine Moiety with Fluorinated Phenylalkyl Side Chains for the Development of GluN2B Receptor Ligands

“…In a preliminary study we have demonstrated that a fluorine atom in the phenylalkyl side chain is well tolerated by GluN2B‐NMDA receptors: the benzo[7]annulenamines 12 a and 12 b with fluorinated phenylalkyl side chains show high GluN2B affinity . Because compounds with a phenylpropyl side chain attached to 3‐benzazepines or benzo[7]annulenamines exhibited GluN2B affinity in the low nanomolar range, fluorinated building block 7 a with a shorter phenylpropyl side chain was taken into account as well . To evaluate the design concept a scaffold hopping approach based on known ligands was envisaged.…”

“…[19] Because compounds with ap henylpropyl side chain attached to 3-benzazepines or benzo [7]annulenamines exhibitedG luN2B affinity in the low nanomolar range, fluorinated building block 7a with as horter phenylpropyls ide chain was taken into account as well. [20][21][22] To evaluate the design concept as caffold hopping approach based on known ligandsw as envisaged.H erein, the synthetic implementation of an ew ligand design and the pharmacological evaluation of the synthesized compounds are reported.…”