Deconstruction – reconstruction approach to analyze the essential structural elements of tetrahydro-3-benzazepine-based antagonists of GluN2B subunit containing NMDA receptors

doi:10.1016/j.ejmech.2017.06.068

European Journal of Medicinal Chemistry

Volume 138, 29 September 2017, Pages 552-564

https://doi.org/10.1016/j.ejmech.2017.06.068 Get rights and content

Highlights

•
Synthesis of tetrahydro-3-benzazepines as GluN2B selective NMDA receptor antagonists.
•
3-Benzazepines with high GluN2B affinity and high selectivity over related receptors.
•
Analysis of the ligand receptor interactions by docking studies.
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No antagonistic activity in two-electrode voltage clamp and cytoprotective assays.

Abstract

The role of the phenolic and benzylic OH moieties for the interaction of tetrahydro-3-benzazepine-1,7-diol 3d with GluN2B subunit containing NMDA receptors was analyzed by their stepwise removal. Elimination of trifluormethanesulfinate from 10 and 13 represent the key steps in the synthesis. Removal of phenolic OH moiety led to 5-fold reduced GluN2B affinity of 4d compared with 3d. Additional removal of the benzylic OH moiety (5d) resulted in further reduced GluN2B affinity but increased σ₁ and σ₂ affinities. Introduction of a NO₂ (6d) or NH₂ moiety (7d) decreased the GluN2B affinity. 3-Benzazepin-1-ol 4i with the N-phenylcyclohexyl side chain showed the highest GluN2B affinity of this series of compounds (K_i = 2.2 nM) and, moreover, high selectivity over the PCP binding site, σ₁ and σ₂ receptors. In docking studies 3-benzazepines (S)-4-7 adopt the same binding poses as ifenprodil and display the same crucial interactions. Unexpectedly, the high-affinity ligands (S)-4i, (S)-4j, and (S)-6i were not able to inhibit the glutamate/glycine evoked current in two-electrode voltage clamp measurements and the cytotoxic effects of glutamate/glycine on transfected cell lines.

Graphical abstract

Introduction

The ionotropic glutamate receptors are classified into three groups, which are named according to their corresponding agonists N-methyl-d-aspartate (NMDA receptor), 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA receptor) and kainic acid (kainate receptor) [1], [2]. The NMDA receptor plays an important role in central nervous system functions. It is involved in neuronal plasticity and responsible for long term changes of synaptic activity influencing higher cognitive functions [3]. Therefore, the NMDA receptor represents a key therapeutic target for drugs to treat several neurological disorders including Alzheimer's disease and Parkinson's disease [4].

The NMDA receptor is composed of four subunits which form an ion channel controlling cations such as Na⁺ and Ca²⁺ to enter the neuronal cell [5]. Cloning of the NMDA receptor subunits resulted in three major types of subunits, which are termed GluN1, GluN2 and GluN3 subunits. For the GluN1 subunit eight splice variants (GluN1a-h) were found, which are encoded by one gene. On the other hand, GluN2 and GluN3 subunits are encoded by four (GluN2A-D) and two different genes (GluN3A-B), respectively [6]. A functional NMDA receptor consists of two GluN1 and two GluN2 subunits. The subunit composition determines the gating kinetics of the NMDA receptor [7]. Simultaneous binding of two agonists, (S)-glutamate at the GluN2 subunit and glycine or d-serine at the GluN1 or GluN3 subunit, is necessary to activate and open the NMDA receptor associated ion channel. In the resting state, the NMDA receptor is blocked by Mg²⁺ ions bound within the channel pore, which have to be removed by lowering the membrane potential in the environment of the receptor to allow for ion flux.

Overstimulation of the NMDA receptor is caused by an excess of (S)-glutamate, which induces an increased influx of Ca²⁺-ions leading to excitotoxicity. Compared to unselective NMDA receptor antagonists such as (+)-MK-801 [8] and dexoxadrol [9], antagonists selectively addressing NMDA receptors with a GluN2B subunit lead to decreased side effects due to restricted expression of this subunit in only some regions of the central nervous system. Ligands addressing selectively the ifenprodil binding site at the GluN2B subunit can behave as negative allosteric modulators of the NMDA receptor. Such ligands do not affect the activity of NMDA receptors in the absence GluN2B subunit. Therefore, GluN2B selective NMDA antagonists are of major interest for the treatment of the above mentioned pathologies without having the severe side effects produced by open channel blockers interacting with the phencyclidine (PCP) binding site in the channel pore [10].

In 1971, ifenprodil (1) was developed as a α₁ adrenoceptor antagonist [12]. Unfortunately, 1 interacted additionally with some other receptors and ion channels including 5-HT_1A, 5HT₂, σ₁, σ₂ and NMDA receptors, which led to termination of its further development [13]. However, the high GluN2B affinity rendered 1 the lead compound for the development of GluN2B selective NMDA antagonists. With the aim of increasing the selectivity of 1 without loosing GluN2B affinity, tetrahydro-3-benzazepines 2 and 3 with reduced conformational freedom were designed, synthesized and evaluated pharmacologically as novel GluN2B-specific antagonists [14], [15] (Fig. 1). The GluN2B affinity of the methylated 3-benzazepines 2 depends on their configuration of the two centers of chirality. The GluN2B affinity of the (S,S)-configured stereoisomer of 2 is given in Fig. 1 (K_i = 31 nM). Removal of the 2-methyl moiety of 2 led to the very promising GluN2B antagonist 3d with a binding affinity of 14 nM and an antagonistic activity of 18 nM.

The aim of this project is the analysis of the structural elements of the 3-benzazepine-based GluN2B antagonists 2 and 3, which are crucial for high GluN2B affinity and receptor selectivity. For this purpose, the 3-benzazepine 3 will be deconstructed by successive removal of the OH moieties leading to compounds 4 and 5 (Fig. 1). Very recently, we have reported the synthesis and GluN2B affinity of 3-benzazepin-1-ol 4d (R = (CH₂)₄Ph) lacking the phenolic OH moiety. The slightly reduced GluN2B affinity of 4d (K_i = 73 nM) indicates that the phenolic OH moiety is not essential, but increases the GluN2B affinity [16]. In order to detect further relationships between structure and GluN2B affinity, additional substituents will be attached to the N-atom of 4. Furthermore, the benzylic OH moiety will be removed to obtain 3-benzazepines 5 with only one substituent at the N-atom. Finally, in a reconstruction approach, an additional substituent will be introduced into 3-benzazepineols 4 to afford 8-substituted 3-benzazepinols 6 and 7 (Fig. 1). The contribution of the different structural elements to GluN2B binding will be analyzed by docking studies.

Section snippets

Synthesis of various tetrahydro-3-benzazepines

The aminoalcohol 11 served as the central intermediate for the introduction of various substituents at the N-atom. As described recently, the ketone 10 was prepared by the condensation of phenylethanol 9 with a glycine derivative. K₂CO₃ induced elimination of F₃CSO₂⁻ and subsequent NaBH₄ reduction provided the aminoalcohol 11 [16] (Scheme 1). Alkylation of 11 with alkyl halides or reductive alkylation with aldehydes or ketones in the presence of NaBH(OAc)₃ afforded N-substituted

Conclusion

Stepwise removal of the phenolic and benzylic OH moiety of the lead compound 3d (K_i = 14 nM) led to 4d (K_i = 73 nM) and 5d (K_i = 227 nM) with 5-fold and 15-fold reduced GluN2B affinity, respectively. However the moderate GluN2B affinity indicates that the 3-benzazepines 4d and 5d can still interact with the ifenprodil binding site of GluN2B subunit containing NMDA receptors even if one or two OH moieties are missing. The interactions of the lead compound 3d and the dehydroxylated 3-benzazepines

Chemistry, general

Unless otherwise noted, the moisture sensitive reactions were carried out under dry nitrogen. Dry CH₃CN was bought from Acros and was used without further purification. CH₂Cl₂was distilled over CaH₂. THF was dried and distilled over sodium/benzophenone prior to use. Thin layer chromatography (tlc): Silica gel 60, F254 plates (Merck). Flash chromatography (fc): Silica gel 60, 40–64 μm (Merck); parentheses include: diameter of the column (d), length (l), fraction size (V), eluent, R_f value.

Acknowledgement

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) which is gratefully acknowledged. The authors thank the NRW Graduate School of Chemistry for the PhD scholarship to SD, which is funded by the Government of the state of Nordrhein-Westfalen and the Westfälische Wilhelms-Universität Münster.

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The impact of the OH moieties in 1- and 7-position of WMS-1410 (2) was analyzed by their stepwise removal. The 3-benzazepine without phenolic OH moiety showed a Ki value of 73 nM15 and even the 3-benzazepine 3d (R = 4-phenylbutyl) without both OH moieties displayed a GluN2B affinity of 230 nM [15,16]. Obviously, the contribution of the phenolic and benzylic OH moieties to the interaction with the ifenprodil binding site is limited.
A set of GluN2B NMDA receptor antagonists with conformationally restricted phenylethylamine substructure was prepared and pharmacologically evaluated. The phenylethylamine substructure was embedded in ring expanded 3-benzazocines 4 as well as ring-contracted tetralinamines 6 and indanamines 7. The ligands 4, 6 and 7 were synthesized by reductive alkylation of secondary amine 11, reductive amination of ketones 12 and 16 and nucleophilic substitution of nosylates 14 and 17. The moderate GluN2B affinity of 3-benzazocine 4d (K_i = 32 nM) translated into moderate cytoprotective activity (IC₅₀ = 890 nM) and moderate ion channel inhibition (60% at 10 μM) in two-electrode voltage clamp experiments with GluN1a/GluN2B expressing oocytes. Although some of the tetralinamines 6 and indanamines 7 showed very high GluN2B affinity (e.g. K_i (7f) = 3.2 nM), they could not inhibit glutamate/glycine inducted cytotoxicity. The low cytoprotective activity of 3-benzazocines 4, tetralinamines 6 and indanamines 7 was attributed to the missing OH moiety at the benzene ring and/or in benzylic position. Docking studies showed that the novel GluN2B ligands adopt similar binding poses as Ro 25–6981 with the central H-bond interaction between the protonated amino moiety of the ligands and the carbamoyl moiety of Gln110. However, due to the lack of a second H-bond forming group, the ligands can adopt two binding poses within the ifenprodil binding pocket.
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Antagonists addressing selectively NMDA receptors containing the GluN2B subunit are of particular interest for the treatment of various neurological disorders including neurodegenerative diseases. With the aim to bioisosterically replace the metabolically labile phenol of 7-amino-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-ols, several analogs were docked into the ifenprodil binding site leading to the hydroxymethyl derivatives 4 as promising candidates. They display the same binding pose as Ro 25-6981 and the same H-bond interactions with Gln110 and Glu236 within the GluN2B subunit. The phenylalkyl moieties occupy the hydrophobic pocket formed predominantly by Pro78 (GluN2B), Phe114 (GluN2B), and Tyr109 (GluN1b). Starting from o-phthalaldehyde, the hydroxymethyl derivatives 4 were prepared in a 7-step synthesis with a haloform reaction of trichloroacetophenone 7 as key step. In receptor binding studies, the phenylpropyl derivative 4a shows promising GluN2B affinity (K_i = 101 nM) and high selectivity over the PCP binding site and both σ receptor subtypes. 4a was able to inhibit the glutamate/glycine induced cytotoxicity at mouse fibroblasts with an IC₅₀ value of 5.2 μM. It is assumed that the hydroxymethyl moiety of 4a stabilizes the closed channel conformation by an H-bond with Glu236 as does the phenolic OH moiety of 3, Ro 25-6981 and ifenprodil.
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Research paperDeconstruction – reconstruction approach to analyze the essential structural elements of tetrahydro-3-benzazepine-based antagonists of GluN2B subunit containing NMDA receptors

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Synthesis of various tetrahydro-3-benzazepines

Conclusion

Chemistry, general

Acknowledgement

Trends Pharmacol. Sci.

Curr. Opin. Pharmacol.

Curr. Opin. Neurobiol.

Neuropharmacology

Eur. J. Pharamcol

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem.

J. Pharm.Biomed. Anal.

J. Neurosci. Methods

Bioorg. Med. Chem.

Bioorg. Med. Chem.

Eur. J. Med. Chem.

Eur. J. Med. Chem.

J. Pharm. Biomed. Anal.

J. Pharm. Biomed. Anal.

Research paper
Deconstruction – reconstruction approach to analyze the essential structural elements of tetrahydro-3-benzazepine-based antagonists of GluN2B subunit containing NMDA receptors