Design, Synthesis, and Biological Evaluation of Subunit-Selective N-Methyl-D-Aspartate Receptor Modulators Open Access

Abstract

Chapter 1: N-Methyl-D-aspartate (NMDA) receptors are members of the family of ionotropic glutamate receptors that mediate excitatory neurotransmission. The overactivation of NMDA receptors has been associated with a range of neurological conditions including Parkinson's disease (PD), Alzheimer's disease (AD), stroke, epilepsy, neuropathic pain, and traumatic brain injury (TBI). In an effort to discover a treatment for these neurological insults, a number of antagonists of the NMDA receptor have been developed.

A fluorescence-based primary screen revealed a class of antagonists selective for GluN2C- and GluN2D-containing receptors over other NMDA receptor subtypes, with selectivity greater than 500-fold. Evaluation of a series of analogs resulted in compounds with potency in the low micromolar range (IC50 =1-5 µM) and high selectivity (> 500-fold) for GluN2C- and GluN2D-containing NMDA receptors over GluN2A- and GluN2B-containing NMDA receptors. These analogs represent a novel series of allosteric inhibitors that are selective for GluN2C- and GluN2D-containing NMDA receptors.

Chapter 2: NMDA hypofunction contributes to the psychosis observed in various neuropsychiatric diseases, such as schizophrenia. Potentiators of NMDA function may therefore offer therapeutic potential for such diseases of psychosis. In addition, an increasing amount of research has indicated that potentiation of NMDA receptors may find utility towards the treatment of anxiety disorders, as well as towards the enhancement of learning and memory.

A series of novel compounds that selectively potentiate GluN2C-containing NMDA receptors were developed based on a screening hit identified in a fluorescence-based primary screen. The most active analogs tested were over 100-fold selective for recombinant GluN2C-containing receptors over GluN2A/B/D-containing NMDA receptors. These analogs represent a novel class of NMDA receptor modulators that are highly selective for one NMDA receptor subunit (GluN2C) and provide a useful tool with which to evaluate the role of GluN2C in normal and neuropathological function.

Table of Contents

Table of Contents List of Illustrations Figures Tables Schemes List of Abbreviations Chapter 1: Design, Synthesis, and Structure-Activity Relationship of GluN2C/GluN2D-Selective NMDA Receptor Antagonists CHAPTER 1 1.1 STATEMENT OF PURPOSE 1 1.2 INTRODUCTION AND BACKGROUND 3 1.2.1 NMDA Receptor Structure, Function, and Localization 3 1.2.2 Therapeutic Rationale for NMDA Receptor Antagonists 7 1.2.3 Classes of NMDA Receptor Antagonists 8 1.2.4 Rationale for Antagonist Design 13 1.3 SYNTHESIS OF 1063-SERIES ANALOGS 18 1.4 RESULTS AND DISCUSSION 23 1.4.1 Structure-Activity Relationship of 1063-Series Analogs 23 1.4.2 In vitro Analysis of 1063-Series Mechanism of Action 28 1.4.3 In vivo Analysis of Pharmacokinetic Properties of 1063 32 1.5 CONCLUSIONS 34 1.6 CHEMISTRY EXPERIMENTAL DATA 35 1.7 BIOLOGY EXPERIMENTAL DATA 57 1.7.1 In vitro Analysis of 1063-Series Analogs (Dr. Stephen Traynelis) 57 1.7.2 In vivo Analysis of 1063-Series Analogs for Pharmacokinetic Properties 59 Chapter 2: Design, Synthesis, and Structure-Activity Relationship of Novel GluN1/GluN2C-Selective NMDA Receptor Positive Allosteric Modulators CHAPTER 2 2.1 STATEMENT OF PURPOSE 67 2.2 INTRODUCTION AND BACKGROUND 69 2.2.1 Subunit-Selective Modulators of NMDA Receptor Function 69 2.2.2 Therapeutic Rationale for GluN2C-Selective Agonists 71 2.2.3 Glutamate Hypofunction Hypothesis 72 2.2.4 Enhancement of Learning and Memory 74 2.2.5 Extinction of Fear 74 2.2.6 Biological Screening Hit and Rationale for 1616-Series Analogs 77 2.3 SYNTHESIS OF 1616-SERIES ANALOGS 83 2.3.1 Synthesis of Pyrrolidinone Analogs 83 2.3.2 Synthesis of Pyruvate Analogs 96 2.3.3 Synthesis of Benzaldehyde Analogs 99 2.3.4 Synthesis of B-Ring Modifications 105 2.3.5 Synthesis of R¹¹ Modifications 106 2.3.6 Separation of 1616-Series Enantiomers 107 2.3.7 Synthesis of β-Lactam Analog 118 2.4 RESULTS AND DISCUSSION 119 2.4.1 Structure-Activity Relationship of 1616-Series - R¹ Modifications 119 2.4.2 Structure-Activity Relationship of 1616-Series - A-Ring Modifications 124 2.4.3 Structure-Activity Relationship of 1616-Series - B-Ring Modifications 130 2.4.4 Structure-Activity Relationship of 1616-Series - R¹¹ Modifications 136 2.4.5 Structure-Activity Relationship of 1616-Series - Linker Modifications 138 2.4.6 Rationale and Results for β-Lactam Analog 139 2.4.7 Off-Target Effects of 1616-Series Analogs 141 2.4.8 In vitro Analysis of 1616-Series Mechanism of Action and Structural Determinants of Activity 143 2.4.9 In vivo Analysis of 1616-Series Analogs 147 2.5 CONCLUSIONS 148 2.6 CHEMISTRY EXPERIMENTAL DETAIL 151 2.6.1 Chemistry Experimental Detail for 1616-Series 151 2.6.2 Separation of Enantiomers of 1616-19 244 2.7 BIOLOGY EXPERIMENTAL DETAIL 245 2.7.1 In vitro Analysis of 1616-Series Analogs (Dr. Stephen Traynelis) 245 List of Illustrations List of Figures Page Chapter 1: Figure 1. Structure of Ifenprodil 1 Figure 2. Screening hits identified in a fluorescence-based assay 2 Figure 3. Synthetic agonists which selectively bind to iGluRs 3 Figure 4. NMDA receptor subunit structure 4 Figure 5. Domains of a single NMDA receptor subunit 5 Figure 6. Cartoon representation of a single NMDA receptor subunit 5 Figure 7. Initial screening hit and generic structure for SAR development 13 Figure 8. Proposed analogs for exploration of conformational flexibility and stability of the carbamate 16 Figure 9. Proposed asymmetrical carbamate 16 Figure 10. Proposed R¹ substitutions 17 Figure 11. Dose response curve of 1063-2 at various splice variants of GluN1 and GluN2D-containing NMDA receptors 29 Figure 12. Schematic diagram of GluN1/GluN2A and GluN1/GluN2D chimeras that were prepared by Dr. Katie Vance 30 Figure 13. Mutagenesis studies evaluating the effect of 1063-2 at GluN1/GluN2D receptors 30 Figure 14. Site directed mutagenesis evaluating the effect of 1063-2 at GluN1/GluN2D receptors 31 Figure 15. Structural determinants of activity for the 1063-series 32 Figure 16. Concentration of 1063-2 in rat plasma and brain over time 34 Chapter 2: Figure 1. Screening hit identified in a GluN1/GluN2C screening effort 68 Figure 2. Subunit-selective potentiators of NMDA receptor function 70 Figure 3. Initial screening hit (1616) and generic structure for SAR development 77 Figure 4. Concentration-effect curves of 1616 at GluN1/GluN2A, GluN1/GluN2B, GluN1/GluN2C, and GluN1/GluN2D 78 Figure 5. Proposed R¹ modifications 79 Figure 6. Benzoic acid 1616-01, prepared by Dr. Ethel Garnier 79 Figure 7. Proposed ester and isostere analogs 80 Figure 8. Proposed A-ring substitutions 80 Figure 9. Proposed heteroaryl A-ring replacements 81 Figure 10. Proposed B-ring replacement analogs 81 Figure 11. Proposed analogs containing substituted indoles 82 Figure 12. Proposed modifications at R¹¹ 82 Figure 13. Proposed linker modifications 83 Figure 14. Overlaid structures of 1616 (green) and 1616-92 (blue) 139 Figure 15. Points of structural diversity between 1616 and 1616-92 141 Figure 16. Off-target responses to 1616 142 Figure 17. Off-target responses to 1616-19 142 Figure 18. In vitro analysis of 1616 analogs 143 Figure 19. Schematic diagram of GluN1/GluN2A and GluN1/GluN2C chimeras that were prepared 144 Figure 20. Chimeric receptors indicate that the ATD and S1 regions of the GluN2C subunit are essential for activity of 1616 145 Figure 21. Point mutations in the ATD, L0 and S1 domains indicate two residues in the S2 domain that are essential for activity of 1616 145 Figure 22. Residues K470, S472, S393 and R401 alter potentiation of 1616 146 Figure 23. Homology model of GluN1/GluN2C subunits 147 Figure 24. SAR summary of 1616-series 149 List of Tables Page Chapter 1: Table 1. Classes of NMDA receptor antagonists with exemplary analogs shown 9 Table 2. Lead analogs to come out of previous SAR efforts 14 Table 3. Optimization of R¹ 24 Table 4. Asymmetric evaluation of R^2a and R^2b 25 Table 5. Optimization of A-ring substituent position and identity 26 Table 6. Optimization of the linker 27 Table 7. Optimization of the linker 27 Table 8. In vivo pharmacokinetic properties of 1063 analogs 33 Chapter 2: Table 1. Heat catalyzed Biginelli-like conditions attempted 84 Table 2. Acid catalyzed Biginelli-like conditions attempted 85 Table 3. Summary of pyrrolidinone analogs with modifications at R¹ 86 Table 4. Summary of pyrrolidinone analogs with A-ring substitutions 88 Table 5. Summary of pyrrolidinone analogs containing modifications at A-ring positions R³ and R⁴ 90 Table 6. Summary of pyrrolidinone analogs containing A-ring replacements 91 Table 7. Summary of pyrrolidinone analogs containing B-ring replacements 92 Table 8. Summary of pyrrolidinone analogs containing B-ring substitutions 93 Table 9. Summary of pyrrolidinone analogs containing modifications at R¹¹ 95 Table 10. Summary of pyrrolidinone analogs containing modified linkers 96 Table 11. Summary of pyruvate analogs 97 Table 12. Amide coupling conditions 99 Table 13. Di-alkylation of benzoic acid derivatives 101 Table 14. Initial attempts towards benzaldehyde derivatives 39 and 40 102 Table 15. Attempted formylation conditions of bromine analogs 104 Table 16. Summary of benzaldehyde derivatives prepared 104 Table 17. Conditions for preparation of protected enols 1616-14 and 1616-20 106 Table 18. Attempts at diastereomeric separation via normal phase column chromatography 108 Table 19. Attempts at separation as diastereomeric salts 109 Table 20. Attempted enantioselective synthesis of 1616 and 1616-19 112 Table 21. Synthesis of ester derivatives for enzymatic resolution 113 Table 22. Attempts at enzymatic resolution of 1616-19 114 Table 23. HPLC and biological data from 1616-19 enantiomers 118 Table 24. Optimization of potency though evaluation of keto-linked substituents 120 Table 25. Effect of aryl substituent position and identity at R¹ 121
Table 26. Effect of heteroaromatic substitution at R¹ 123 Table 27. Effect of ester isosteres at R² 124 Table 28. Effect of A-ring replacement 126 Table 29. Effect of A-ring modifications 127 Table 30. Optimization of A-ring substituents 129 Table 31. Effect of replacing the B-ring 131 Table 32. Optimization of B-ring substituents R⁵, R⁶, and R⁷ 134 Table 33. Optimization of B-ring substituents R⁸, R⁹, and R¹⁰ 135 Table 34. Optimization of potency though modification of R¹¹ substitutions 137 Table 35. Effects of linker modifications 136 Table 36. Biological data from scaffold hopping analog, 1616-92 140 Table 37. Comparison of the most potent 1616 analogs with the initial screening hit 150 List of Schemes Page Chapter 1: Scheme 1. General synthetic route towards 1063 analogs developed by Dr. Cara Mosley 14 Scheme 2. Failed synthesis of thiazole derivatives using basic conditions 18 Scheme 3. Optimized thiazole conditions 18 Scheme 4. Synthesis of thiazole analogs 19 Scheme 5. Synthesis of oxazolidinone analogs 20 Scheme 6. Synthesis of ether analog 1063-33 21 Scheme 7. Preparation of amide 1063-27 22 Scheme 8. Failed synthesis of carbamate 47 22 Scheme 9. Synthesis of extended linker analogs 23 Chapter 2: Scheme 1. Retrosynthetic analysis of 1616-series using Biginelli-like reaction 83 Scheme 2. Synthetic route to access pyrrolidinone analogs 86 Scheme 3. Generalized synthesis of pyruvate derivatives 97 Scheme 4. Synthesis of pyruvate analog 29 97 Scheme 5. Synthesis of para-ester benzaldehydes 30-32 99 Scheme 6. Synthesis of primary amide 33 100 Scheme 7. Synthesis of phenols 36 and 37 101 Scheme 8. Synthesis of benzaldehyde derivatives 39 and 40 103 Scheme 9. Synthesis of benzaldehyde 41 103 Scheme 10. Synthesis of naphthalene 129 106 Scheme 11. Synthesis of amine 1616-21 107 Scheme 12. Synthesis of chiral phosphoric acid catalyst 143 111 Scheme 13. Proposed mechanism of hydrolysis in aqueous conditions 116 Scheme 14. Alternative mechanism of hydrolysis in aqueous acidic medium 117 Scheme 15. Synthesis of β-lactam 1616-92 119
List of Abbreviations AD: Alzheimer's Disease ACN: Acetonitrile AMPA: α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ATD: Amino Terminal Domain BBB: Blood-Brain-Barrier CNS: Central Nervous System CS: Conditioned Stimulus CTD: Carboxy Terminal Domain DCC: N,N'-Dicyclohexylcarbodiimide DCM: Dichloromethane DCS: D-Cycloserine DLB: Dementia with Lewy bodies DMAP: 4-Dimethylaminopyridine DMF: Dimethylformamide DMSO: Dimethylsulfoxide ee: Enantiomeric Excess EC50: Half-Maximal Excitatory Concentration EDCI: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EPSP: Excitatory Postsynaptic Potential EtOAc: Ethyl Acetate FDA: Food and Drug Administration GABA: Gamma-Aminobutyric Acid GPCR: G-Protein Coupled Receptor GoF: Gain of Function HD: Huntington's Disease h(s): hour(s) HPLC: High Performance Liquid Chromatography HRMS: High Resolution Mass Spectrometry IC50: Half-Maximal Inhibitory Concentration IR: Infrared Radiation iGluR: Ionotropic Glutamate Receptor LBD: Ligand Binding Domain LCMS: Liquid Chromatography-Mass Spectrometry LoF: Loss of Function MeOH: Methanol mGluR: Metabotropic Glutamate Receptor min.: minutes mRNA: Messenger Ribonucleic Acid M.W.: Microwave NAAG: N-Acetylaspartylglutamate nACh: Nicotinic Acetylcholine Receptor NBS: N-Bromosuccinimide NMDA: N-Methyl-D-aspartate NMR: Nuclear Magnetic Resonance PCP: Phencyclidene PD: Parkinson's Disease PDD: Parkinson's Disease Dementia PPTS: Pyridinium p-Toluene Sulfonic Acid PTSD: Post-Traumatic Stress Disorder rt: room temperature SAR: Structure Activity Relationship SEM: Standard Error of the Mean SSRI: selective serotonin reuptake inhibitor t1/2: Half-life TBI: Traumatic Brain Injury t-BuOH: tert-Butanol TEA: Triethylamine TFA: Trifluoroacetic Acid TIPSCl: Triisopropylsilyl Chloride TLC: Thin Layer Chromatography TMD: Transmembrane Domain US: Unconditioned Stimulus

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School	Laney Graduate School
Department	Chemistry
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Organic
Keyword	ionotropic glutamate receptor NMDA
Committee Chair / Thesis Advisor	Liotta, Dennis C, Emory University
Committee Members	Lutz, Stefan, Emory University Liebeskind, Lanny S, Emory University Traynelis, Stephen, Emory University

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