Synthesis, structural activity-relationships, and biological evaluation of novel amide-based allosteric binding site antagonists in NR1A/NR2B N-methyl-d-aspartate receptors☆
Graphical abstract
The synthesis and biological activity of a novel series of amide-based NR2B-selective NMDA receptor antagonists is detailed. The compounds herein represent a family of compounds with excellent on-target potency and decreased off-target effects when compared with previously described antagonists.
Introduction
Ionotropic glutamate receptors mediate excitatory synaptic transmission in the central nervous system, and can be divided into three categories based on pharmacology and amino acid sequence, which include N-methyl-d-aspartate (NMDA), kainate, and amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors.1, 2, 3 NMDA receptors are involved in neuronal development, learning, motor function, and pain transmission. Excessive activation of NMDA receptors can lead to neuronal death, and thus NMDA receptor over-activation has been proposed to occur in conditions in which extracellular glutamate is elevated, such as ischemic stroke and traumatic brain injury.4, 5, 6, 7, 8 In addition, it has also been proposed that NMDA receptors play a role in Alzheimer’s disease,9, 10, 11, 12 Huntington’s disease,13, 14, 15, 16 Parkinson’s disease,17, 18, 19 depression,20 and neuropathic pain.21, 22, 23, 24
Structurally, NMDA receptors are heterooligomeric ligand-gated cation channels containing two glycine-binding NR1 subunits, which exist as eight different RNA splice variants, and two glutamate-binding NR2 subunits. Simultaneous binding of the co-agonists glutamate and glycine leads to opening of the receptor channel, and subsequent influx of cations, including Ca2+. The resulting membrane depolarization contributes to the propagation of the excitatory signal, and the influx of Ca2+ can trigger intracellular signaling pathways.
There are numerous sites on the protein that could be pharmacologically exploited to modulate NMDA receptor function. For example, organic cations have been developed as selective blockers that bind within the ion conduction path in NMDA receptors (e.g., MK-801, 1, Fig. 1).25 In addition, competitive antagonists that bind to the co-agonist glycine site (Licostinel, 2)26 or glutamate site have been developed (Selfotel, 3).27, 28 A number of subtype-selective non-competitive antagonists that bind to the amino terminal domain have been developed and potently inhibit receptors containing the NR2B subunit such as the negative allosteric modulators ifenprodil29, 30 (4) and CP-101,606 (5).31, 32, 33, 34, 35, 36 Initial clinical trials of competitive NMDA receptor blockers and channel blockers as neuroprotectants failed for several reasons including unfavorable side effects, which led to lowering of the dose below efficacious levels. In addition, the rapidly developing neurodegeneration after cerebral ischemia made it problematic to administer the compounds soon enough after the insult to prevent NMDA receptor-mediated cell death.37 To date only one NMDA receptor antagonist, the channel blocker memantine hydrochloride (6, Namenda ©), has been approved by the FDA for the treatment of Alzheimer’s disease.38, 39
A number of lines of evidence suggest that NR2B subtype-selective blockade of NMDA receptors can be beneficial in cerebral ischemia, epilepsy, Parkinson’s disease, depression, and perhaps Alzheimer’s disease.20, 40, 41, 42 In addition, mice over-expressing NR2B-containing receptors show an increased sensitivity to pain, and NR2B-selective antagonists are antinociceptive in certain models of pain.24, 43 While NR1 is ubiquitously expressed in the CNS, NR2B-containing receptors are localized in the forebrain, dorsal root ganglion, striatum, and the spinal cord.44, 45, 46 The differential distribution of NR2 receptor subtypes17 has supported the hypothesis that targeting specific NMDA receptor subunits, such as NR2B-containing receptors, will decrease untoward side-effects previously observed in clinical trials of non-selective NMDA receptor antagonists.
Comprehensive SAR studies of NR2B amino terminal domain (ATD) allosteric binding site antagonists suggest that one family of compounds (7, Fig. 2) contains a non-polar A ring connected via a basic amine linker of 9–11 Å to the B ring, which traditionally contains a hydrogen bond donor substituent.47, 48, 49 Previous studies in our lab involving enantiomeric propanolamines indicated the most potent compounds contain a 3,4-dichlorophenyl A ring and para-methanesulfonamide B ring.50 The optimal linker in the series was seven atoms, and a basic amine was required for activity. Compound 8 in that study showed excellent selectivity for NR2B-containing heterodimeric NR1/NR2 NMDA receptors and high potency in vitro and neuroprotective properties in an in vivo model of transient focal ischemia. Compound 8 did exhibit off-target monoamine receptor activity including the α1-adrenergic receptor and the human-ether-a-go-go (hERG) encoded cardiac potassium channel. The hERG channel is important in ventricular repolarization, and channel blockage can lead to an increased QT interval and potentially deadly ventricular arrhythmias and represents an off-target limitation addressed through SAR studies for a diverse set of chemical structures.51, 52 Eliminating hERG activity, and interactions with other polyamine binding receptors including α1 and sigma receptors, has been a significant hurdle in the development of NR2B-selective NMDA receptor antagonists.
Recently we explored the structural features of a novel NR2B-selective antagonist that was identified while screening a focused library of biaryl compounds (compound 9). This biaryl compound contains a thiosemicarbazide functionality within a seven-atom linker that would be non-ionizable under physiological conditions. We hypothesized that this structural feature would lead to decreased activity at off-target receptors compared to the propanolamines on which we previously worked. Here we describe the synthesis and SAR evaluation of compounds with various non-ionizable linker regions between the A and B rings of scaffold 7. Our goal in this study was to achieve selective, potent, and safe compounds through manipulation of the seven-atom linker and incorporation of various amides.
Section snippets
Chemistry
A series of hydrazide derivatives were prepared from the common starting material fragment propane hydrazide 12. Originating from para-aminophenylpropionic acid (10, Scheme 1), initial conversion of the carboxylic acid to methyl ester 11 was achieved using methanol and thionyl chloride. The ester was treated with methanesulfonyl chloride to yield the para-arylmethanesulfonamide, and finally converted to 13 via hydrazinolysis.53 Thiosemicarbazide analogue 14 was formed by reaction of 13 with
Structure–activity-relationships
The potency and selectivity of all new compounds synthesized were evaluated using two electrode voltage clamp analysis of the effect of compounds on recombinant NMDA receptor function. We first screened the effect of 3 μM of each compound against current responses produced by maximally effective concentrations of the co-agonists glutamate and glycine at rat NR1/NR2A, NR1/NR2B, NR1/NR2C, NR1/NR2D as well as representative members of the AMPA-selective class of glutamate receptors (GluR1) and the
Conclusions
A novel series of amide-based NR2B-selective NMDA receptor antagonists was developed with high on-target potency and attractive off-target profiles. Compounds 52 and 71 in particular show an increased drug-like characteristics over the previous enantiomeric propanolamine series, including concomitant high potency with minimal hERG and α1 binding. In addition, 52 has an acceptable half-life in vivo while 71 is expected to cross the blood–brain barrier. These amide-based analogues provide new
Expression of glutamate receptors in Xenopus laevis oocytes
All protocols involving the use of animals were approved by the Emory University IACUC. cRNA was synthesized from linearized template cDNA for rat glutamate receptor subunits according to manufacturer specifications (Ambion). Quality of synthesized cRNA was assessed by gel electrophoresis, and quantity was estimated by spectroscopy and gel electrophoresis. Stage V and VI oocytes were surgically removed from the ovaries of large, well-fed and healthy Xenopus laevis anesthetized with
General experimental procedures
All reagents were obtained from commercial suppliers and used without further purification. Reaction progress was monitored by thin layer chromatography (TLC) on precoated glass plates (Silica Gel 60 F254, 0.25 mm). Proton and carbon NMR spectra were recorded on an INOVA-400 (400 MHz), VNMRS 400 (400 MHz), INOVA-600 (600 MHz), or Mercury 300 Vx (300 MHz). The spectra obtained were referenced to the residual solvent peak. Mass spectra were performed by the Emory University Mass Spectroscopy Center on
Acknowledgements
The authors are grateful to Drs. S. Nakanishi, P. Seeburg, and S. F. Heinemann for cDNAs encoding the glutamate receptor subunits. We thank Kimberly Vellano for excellent technical assistance. This work was supported in part by the NINDS (NS036654, ST; NS036604, RD).
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Several of the authors (C.A.M., Y.A.T., L.J.W., S.J.M., R.D., S.F.T., D.C.L.) are inventors of Emory University owned patent-pending technology associated with these compounds, or have an equity position in companies actively seeking to license these compounds (D.C.L., S.F.T., R.D.).