Synthesis, Structure-Activity Relationship, and Biological Evaluation of Subunit-Selective NMDA Receptor Modulators Restricted; Files Only

Summer, Samantha (Spring 2020)

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The N-methyl-D-aspartate receptor (NMDAR) is a unique member of the ionotropic glutamate receptor family, responsible for mediating a slow and Ca2+ permeable component of synaptic transmission. These receptors play important roles in a variety of neurological functions and have a role in various central nervous system disorders. There are seven different subunits of the NMDAR with different properties and expression profiles. A major focus of NMDAR drug-development is focused on subunit-selective modulators. In the past, pan-modulators showed a variety of side-effects in vivo and in clinical trials. This is proposed to be due to targeting all NDMARs, even those irrelevant to the disease in question. The first GluN1/GluN2A selective antagonist was reported in 2010. Part of the work presented herein is focused on the structure-activity-relationship of these GluN1/GluN2A and their pH sensitivity. This work resulted in GluN1/GluN2A-selective antagonists that are 10-times more potent at acidic pH compared to physiological pH. We hypothesize that compounds of this nature may be effective at treating cerebral ischemia. This family of compounds had a unique scaffold that enabled a unique pi-stacking configuration. Another embodiment of this work incorporated this pi-stacking configuration in an already established series of compounds that target GluN1/GluN2C and GluN1/GluN2D NMDARs. This pi-stacking moiety was tolerated in this series and in some instances increased potency. This work also focuses on preparing modulators of the more recently discovered and lesser studied GluN3 subunits. Following a screen of a large and targeted NMDAR library, a positive-allosteric modulator (PAM) was found. Through a short structure-activity relationship, the first GluN1/GluN3A selective PAM is presented EU-1180-490. EU-1180-490 is a potent, selective, soluble and blood-brain barrier penetrant PAM. This compound can be used to study the role of GluN3 in normal and diseased central nervous system CNS function. From the screen, a potent negative-allosteric modulator (NAM) was also found, EU-1180-438. EU-1180-438 is potent and selective for GluN1/GluN3 NMDARs. The structural determinants of EU-1180-438 have been determined. This compound is active in native neurons. A preliminary structure-activity relationship has been studied in an attempt to elucidate the series pharmacophore. Ultimately, this work presents novel subunit-selective modulators of the NMDA receptor. These compounds can be used as tool compounds to further study the receptors and their specific roles in various neurological disorders. 

Table of Contents

Chapter 1: Introduction to the NMDA Receptor.. 17

1.1     NMDA Receptor Introduction. 17

1.2 Subunit Structure. 19

1.2.1 Subunit Assembly. 19

1.2.2 Subunit Domains. 19

1.4 Therapeutic Rationale. 23

1.4.1 Neuroprotection. 23

1.4.2 Neurodegenerative Disorders. 24 Alzheimer’s Disease. 24 Parkinson’s Disease. 25

1.4.3 Pain. 26

1.4.4 Depression. 27

1.5 NMDA Receptor Modulators. 28

1.5.1 Endogenous Modulators. 28

1.5.2 Non-Selective Modulators. 29

1.5.3 GluN2A Selective Modulators. 30

1.5.4 GluN2B Selective Modulators. 32

1.5.5 GluN2C/D Selective Modulators. 33

1.6 GluN3-Containing NMDARs. 35

1.6.1 Therapeutic Rationale for GluN3-Containing NMDARs. 37

1.6.2 GluN3 Selective Modulators. 39

1.7 Conclusion. 40

1.8 References. 41

Chapter 2: pH-Sensitive Negative Allosteric Modulators of GluN2A-containing NMDA receptors. 68

2.1 Statement of Purpose. 68

2.2 Introduction. 68

2.3 Results and Discussion. 71

2.3.1 Synthesis. 72

2.3.2 The effect of linker length. 76

2.3.3 The effect of linker group. 77

2.3.4 A-Ring substituents. 78

2.3.5 EU-TCN201-9 active pose in pocket 81

2.4 Discussion. 81

2.5 Conclusion. 82

2.6 Methods. 82

2.6.1 Biological Evaluation. 82

2.6.3 Synthetic Chemistry Methods. 83

2.7 References. 105

Chapter 3. The Sulfonamide Motif in GluN2C/GluN2D Selective Antagonists. 111

3.1 Statement of Purpose. 111

3.2 Introduction. 112

3.3 Results and Discussion. 117

3.3.1 Synthesis and Structure-Activity Relationship. 117

3.3.2 Selectivity and Off-Target Effects. 123

3.4 Conclusion. 125

3.5 Experimental Detail: 125

3.5.1 Synthetic Chemistry Methods. 125

3.5.2 In vitro Evaluation of the 1063-Sufonamide Series. 150

3.5.3 Computational Chemistry. 151

3.6 References. 152

Chapter 4: Selective and Potent Class of GluN1/GluN3A NMDA Receptor Potentiators. 155

4.1 Statement of Purpose. 155

4.2 Introduction to GluN3A.. 155

4.3 Results and Discussion. 158

4.3.1 Screening of CIQ-Class at GluN1-F484/GluN3A.. 158

4.3.2 SAR and Synthesis. 159

4.3.3 Drug-like Properties of EU-1180-490. 166

4.3.4 Pharmacokinetic Properties of EU-1180-490. 166

4.4 Conclusion. 167

4.5 Experimental Details. 168

4.5.1 Synthetic Chemistry Methods. 168

4.5.2 In vitro analysis of 1180 series analogs. 219

4.5.3 In vivo analysis of Pharmacokinetic Properties. 221

4.5.4 Solubility Determination. 222

4.6 References. 223

Chapter 5. Novel Scaffold for Selective Inhibitors of the GluN1/GluN3A Receptor.. 227

5.1 Statement of Purpose. 227

5.2 Introduction to GluN3A.. 227

5.3 Results and Discussion. 229

5.3.1 Screening of the 1180-Library. 229

5.3.2 Evaluation of EU-1180-438 as a Pharmacological Tool 230

5.3.3 Pharmacokinetic Properties of EU-1180-438. 234

5.3.4 EU-1180-438 in Native Neurons. 234

5.3.5 Synthesis of EU-1180-438 and Derivatives. 236

5.3.6 Inhibition of GluN3A by EU-1180-438 Derivatives. 241

5.4 Conclusion. 242

5.5 Experimental Details. 244

5.5.1 Synthetic Chemistry Methods. 244

5.5.2 Enantiomeric Separation of EU-1180-438. 267

5.5.3 In Vitro Analysis. 269

5.5.4 In Vivo Analysis of Pharmacokinetic Properties. 271

5.5.5 Solubility Experiment 271

5.6 References. 272

Chapter 6: Conclusions and Future Directions. 276

6.1 pH-Sensitive NMDAR Antagonists. 276

6.2 GluN2C-2D Negative-Allosteric Modulators. 276

6.3 GluN3A Potentiators. 277

6.4 GluN3A Negative-Allosteric Modulators 278

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