Strong Inhibitory Effect, Low Cytotoxicity and High Plasma Stability of Steroidal Inhibitors of N-Methyl-D-Aspartate Receptors With C-3 Amide Structural Motif.

Santosh Kumar Adla,Tereza Smejkalova,Michaela Nekardova,Pavla Hubalkova,Marek Ladislav,Lenka Monincova,Marketa Smidkova,Hana Chodounska,Eva Kudova,Barbora Krausova,Barbora Slavikova,Vojtech Vyklicky,Radko Soucek,Ladislav Vyklicky

doi:10.3389/fphar.2018.01299

Abstract

Herein, we report the synthesis, structure-activity relationship study, and biological evaluation of neurosteroid inhibitors of N-methyl-D-aspartate receptors (NMDARs) receptors that employ an amide structural motif, relative to pregnanolone glutamate (PAG) – a compound with neuroprotective properties. All compounds were found to be more potent NMDAR inhibitors (IC50 values varying from 1.4 to 21.7 μM) than PAG (IC50 = 51.7 μM). Selected compound 6 was evaluated for its NMDAR subtype selectivity and its ability to inhibit AMPAR/GABAR responses. Compound 6 inhibits the NMDARs (8.3 receptors (8.3 ± 2.1 μM) more strongly than it does at the GABAR and AMPARs (17.0 receptors (17.0 ± 0.2 μM and 276.4 ± 178.7 μM, respectively). In addition, compound 6 (10 μM) decreases the frequency of action potentials recorded in cultured hippocampal neurons. Next, compounds 3, 5–7, 9, and 10 were not associated with mitotoxicity, hepatotoxicity nor ROS induction. Lastly, we were able to show that all compounds have improved rat and human plasma stability over PAG.

Highlights

N-Methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels, involved in excitatory synaptic transmission and synaptic plasticity (Citri and Malenka, 2008)
We were able to identify lead compound 6, which we subsequently evaluated on recombinant GluN1/GluN2A-D receptors and native NMDARs α-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid (AMPA)/kainate receptors (AMPAR), and gamma-aminobutyric acid (GABA) receptors (GABARs) expressed in hippocampal neurons
Compound 13 was prepared according to the literature: (Adla et al, 2017) in brief, commercially available 3β-hydroxy-5β-androstane was treated with phthalimide and triphenylphosphine, followed by deprotection of the amino group in hydrazine hydrate, to give 3α-amino derivative 13

Summary

Introduction

N-Methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels, involved in excitatory synaptic transmission and synaptic plasticity (Citri and Malenka, 2008). As a part of our continuing interest in the structure-activity relationship (SAR) study of neurosteroid modulators of NMDARs, we have recently reported on several new structural modifications that generate potent inhibitors of NMDARs: (i) the negatively charged C-3 substituent can be substituted by a positively charged moiety or zwitterion group (e.g., glutamic acid ester); (Borovska et al, 2012) (ii) the C-17 acetyl moiety of the pregnane skeleton is not essential for the inhibitory effect and can be substituted by non-polar substituents (methyl, ethyl, etc.), including full elimination of the C-17 moiety altogether, e.g., androstane 3-sulfate (Figure 1B); (Kudova et al, 2015) (iii) the steroidal D-ring is not essential for inhibitory effect and can be fully or partially degraded (Slavikova et al, 2016)

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Results

Conclusion