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

Epplin, Matthew (Spring 2019)

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The N-Methyl-D-Aspartate receptor (NMDAR) is an ion channel responsible for mediating the slow, Ca2+-permeable component of glutamatergic synaptic transmission in the central nervous system (CNS). NMDARs are known to play a significant role in basic neurological functions and their dysfunction has been implicated in several CNS disorders. Four isoforms of the NMDAR, GluN2A-D, exhibit unique expression patterns in the brain and display different functional properties depending on the subunit makeup. Previous molecules targeting the receptor display a wide range of neurological side effects in vivo, perhaps due to engaging NMDAR subunits irrelevant to the targeted disease. This has led to the hypothesis that developing subunit-biased modulators could maintain efficacy while limiting the observed off-target effects. For this reason, our lab previously identified CIQ, a tetrahydroisoquinoline-based GluN2C- and GluN2D-selective positive allosteric modulator (PAM) for the receptor. Structural modifications resulted in IPQ2, a GluN2B-preferring PAM, albeit with modest potency and high lipophilicity. High affinity and low lipophilicity (i.e. high lipophilic efficiency) are highly desired in drug development to achieve low-dose compounds for patients. Chapter One of this thesis outlines efforts to improve the lipophilic efficiency of the series targeting the GluN2B subunit. This work resulted in the most potent small molecule positive modulator for GluN2B known at a pEC50 of 6.5. Although CIQ has made a significant impact as a first-in-class in vitro tool compound, it exhibits several liabilities limiting its use in vivo, including modest potency, modest potentiation, low aqueous solubility, and high lipophilicity. Chapters Two and Three of this thesis outline core changes from the tetrahydroisoquinoline scaffold of CIQ to two novel scaffolds for the receptor targeting these liabilities. Chapter Two describes a change to a dihydroisoquinolinone core that improved doubling concentrations 5-fold on average. These substrates also maintained selectivity for GluN2C- and GluN2D-containing receptors across a wider range of substrates without increasing compound lipophilicity. Chapter Three of this thesis describes the discovery of a second core change to a pyrrolopyrazinone scaffold that addresses each of potency, efficacy, and lipophilicity. The prototypical compound, 1180-453, shows roughly order of magnitude improvements in doubling concentration, lipophilic efficiency, aqueous solubility, and an order of magnitude decrease in cLogP compared to CIQ. Separation of the enantiomers via chiral HPLC resulted in compounds such as R-(+)-1180-450 with doubling concentrations up to 0.4 µM and a solubility to doubling concentration ratio of >100:1. This work has resulted in second-generation GluN2C- and GluN2D-selective PAMs of the NMDAR showing improved affinity, efficacy, and ADME properties over their predecessors. These compounds are a promising step towards the first useful in vivo tools to study positive modulation of GluN2C- and GluN2D-containing NMDA receptors.

Table of Contents

Chapter 1: The Design, Synthesis, and Biological Evaluation of GluN2B-Preferring Positive Allosteric Modulators of the N-Methyl-D-Aspartate Receptor 15

1.1 Statement of Purpose 15

1.2 Introduction and Background 16

1.2.1 NMDAR structure, function and localization 16

1.2.2 Therapeutic Rationale for NMDAR Positive Modulators 22

1.2.3 Classes of NMDAR modulators 23

1.2.4 Discovery and structure-activity relationship of a tetrahydroisoquinoline class of NMDAR positive allosteric modulators 32

1.3 Synthesis, Rationale, Results, and Discussion of GluN2B-Selective Positive Modulators 35

1.4 Conclusions 40

1.5 Experimental Details 41

1.5.1 Chemistry experimental procedures 41

1.5.2 In vitro analysis of 1180 series analogs 72

1.6 References 73

Chapter 2: Discovery of a 1,4-dihydroisoquinolin-3(2H)-one core that selectively potentiates the GluN2C and GluN2D subunits of the N-methyl-ᴅ-aspartate (NMDA) receptor with improved doubling concentrations 83

2.1 Statement of Purpose 83

2.2 Introduction and Background 85

2.2.1 Therapeutic rationale for GluN2C- and GluN2D-selective positive allosteric modulators of the NMDAR  85

2.2.2 Photocrosslinking 85

2.2.3 The identification of a 1,4-dihydroisoquinolin-3(2H)-one scaffold that maintains selectivity for GluN2C- and GluN2D-containing receptors 87

2.3 Synthesis, Rationale, Results, and Discussion of GluN2C/D-Selective 1,4-dihydroisoquinolin-3(2H)-one-Based Positive Allosteric Modulators 89

2.3.1 Synthesis of 1,4-dihydroisoquinolin-3(2H)-ones 89

2.3.2 Potentiation of NMDARs by a 1,4-dihydroisoquinolin-3(2H)-one series 91

2.3.3 Effect of modifying the A-ring, A-ring linker and amide 92

2.3.4 Investigating GluN2B-preferring substitution on the dihydroisoquinolinone scaffold 94

2.3.5 Revisiting the A-ring 96

2.3.6 Enantiomers 99

2.3.7 Olefins 100

2.3.8 1,4-dihydroisoquinolin-3(2H)-one compounds show improved doubling concentrations over their TIQ counterparts 103

2.3.9 Photocrosslinking 104

2.4 Conclusions 106

2.5 Experimental Details 107

2.5.1 Chemistry experimental procedures 107

2.5.2 Crystal structure data and experimental 154

2.5.3 In vitro analysis of 1180 series analogs 155

2.6 References 157

Chapter 3: Discovery of GluN2C- and GluN2D-selective N-methyl-ᴅ-aspartate (NMDA) receptor positive allosteric modulators showing improved potency, efficacy, and decreased lipophilicity 159

3.1 Statement of Purpose 159

3.2 Introduction and Background 160

3.2.1 The absorption, distribution, metabolism, and excretion (ADME) and safety profile of a small molecule drug is impacted by its lipophilicity 160

3.2.2 Core changes to a tetrahydroisoquinoline class of GluN2C/D-selective NMDAR PAMs resulted in a second-generation pyrrolopyrazine class with improved ADME properties 163

3.3 Synthesis, Rationale, Results, and Discussion of Hydrophilic GluN2C/D-Selective Positive Allosteric Modulators 164

3.3.1 Synthesis of alternative A-, B-, and C-rings to the phenyl rings of CIQ  164

3.3.2 Potentiation of NMDARs by a novel series of PAMs 169

3.3.3 Evaluation of hydrophilic A-ring replacements 170

3.3.4 Evaluation of hydrophilic B-ring replacements 171

3.3.5 Effect of bioisosteric replacement of the C-ring 173

3.3.6 SAR of additional TIQ core changes 174

3.3.7 Pyrrolopyrazine- and pyrrolopyrazinone-based analogues 178

3.3.8 Enantiomers 179

3.3.9 Off-target activity 180

3.3.10 Conclusions 184

3.4 Experimental Details 185

3.4.1 Chemistry experimental procedures 185

3.4.2 In vitro analysis of 1180 series analogs 260

3.4.3 Solubility determination 261

3.4.4 In vitro analysis of off-target selectivity for 1180-420 and 1180-447 262

3.5 References 262

List of Illustrations

List of Figures                                                                                                                       Page

Chapter 1:

Figure 1. Prototypical compounds previously developed in 1180 series. 16

Figure 2. Synthetic ligands selective for the three main classes of glutamate receptors. 17

Figure 3. Linear glutamate receptor sequence and flat representation of a singular subunit showing the four semiautonomous domains (left).1 Cartoon depiction of NMDAR heterotetramer, binding by co-agonists glutamate and glycine, and subsequent ion flow in and out of the cell (right). Subunit makeup of several diheteromeric and triheteromeric NMDARs (bottom).35 18

Figure 4. Recent crystal structure of the NMDAR with the GluN2B-selective negative allosteric modulator ifenprodil and co-agonists glutamate and glycine bound.45 19

Figure 5. NMDAR subunit expression patterns in a mouse brain at P0, P14, and adulthood.35 21

Figure 6. Classes of NMDAR modulators. 26

Figure 7. Structures of FDA-approved uncompetitive antagonists of the NMDAR. 27

Figure 8. Negative allosteric modulators of the NMDAR. 29

Figure 9. Subunit-selective positive allosteric modulators of the NMDAR. 30

Figure 10. CIQ and ring-labeling convention. 32

Figure 11. High-throughput screening hit. 32

Figure 12. Summary of previous SAR in the 1180 series.150, 151 34

Figure 13. Structure and dose response curve comparison between trifluoromethyl substituted CIQ scaffold and 1180-55 scaffold. 37

Figure 14. Proposed derivatives based on increasing alkyl substituent size on the B-ring. Affinities are reported as pEC50s and (max). 38


Chapter 2:

Figure 1. Scaffold of GluN2C/D-selective CIQ (left), GluN2B/C/D-preferring isopropoxy derivative (middle), and GluN2C/D-selective 1,4-dihydroisoquinolin-3(2H)-one that is the subject of this work (right). 84

Figure 2. Process for photoaffinity labeling.15 86

Figure 3. Detailed NMDAR photoaffinity assay.15 87

Figure 4. Model of the steric encumbrance upon translocation of the carbonyl in the 1,4-dihydroisoquinolin-3(2H)-one series. The “cis” conformation was hypothesized to create steric clash between the carbonyl and A-ring, creating preference for the more active “trans” conformation. 88

Figure 5. Labeled 1,4-dihydroisoquinolin-3(2H)-one to distinguish between the A-, B-, and C-rings. 89

Figure 6. Crystal structure of bis-acylated R-(+)-1180-332 or 1180-434. 91

Figure 7. Expected NOEs for the E- and Z- configurations of 1180-369. 102

Figure 8. Aromatic region of the NOE spectrum of the active isomer, 1180-369. 102


Chapter 3:

Figure 1. Development of the 1180 series from the tetrahydroisoquinoline core of CIQ (left), to the dihydroisoquinolinone core (middle) discussed in Chapter 2, to the hydrophilic derivatives (right) discussed in this chapter. 160

Figure 2. A series of core changes to the TIQ-based CIQ lead to a pyrrolopyrazine-based scaffold. 164

List of Tables

Chapter 1:

Table 1. Structures and activities of 1180-55, 1180-87, and 1180-169. 33

Table 2. SAR of 1180-55 derivatives with modifications to the A- and B-rings. 37

Table 3. SAR of compounds with increased alkyl bulk on the B-ring. 40


Chapter 2:

Table 1. Potentiation of GluN2A-D upon co-application of compound 323 and subsaturating concentrations of glutamate and glycine. 88

Table 2. Optimization of potency based on identity of A-ring and A-ring linker. 94

Table 3. Optimization of potency and selectivity based on identity of C- and B-rings. 96

Table 4. Potentiation of GluN2A and GluN2B-containing receptors. 97

Table 5. Optimization of potency and selectivity based on identity of A-ring and amide carbonyl. 98

Table 6. Stereodependence of 1,4-dihydroisoquinolin-3(2H)-one series. 100

Table 7. Activity and selectivity profile of olefinic 1,4-dihydroisoquinolin-3(2H)-one precursors. 101

Table 8. Doubling concentrations of 1,4-dihydroisoquinolin-3(2H)-one compounds and their TIQ counterparts. 104

Table 9. Crystal data and structure refinement for 1180-434. 154


Chapter 3:

Table 1. Hydrophilic A-ring substitutions. 171

Table 2. Hydrophilic B-ring substitutions. 173

Table 3. Optimization of A-ring functionality for thiophene core. 175

Table 4. Optimization of the heterocyclic core. 177

Table 5. Optimization of potency and solubility for pyrrolopyrazine-and pyrrolopyrazinone-based derivatives. 179

Table 6. Stereodependence of select pyrrolopyrazine cores. 180

Table 7. Off-target actions of 1180-447 and 1180-420 at ligand-gated ion channels. 181

Table 8. Off-target actions of compound 1180-447. 182

Table 9. Off-target actions of compound 1180-420. 183

List of Schemes

Chapter 1:

Scheme 1. Synthesis of 1180-55 scaffold starting material. 35

Scheme 2. Synthesis of 1180-55 scaffold derivatives. 36

Scheme 3. Synthesis of alkylated phenols. 38

Scheme 4. Synthesis of 1180-55 derivatives with increased alkyl steric bulk on the B-ring. 39

Scheme 5. Synthesis of thioamide compounds. 39


Chapter 2:

Scheme 1. Synthesis of dihydroisoquinolinones. 90

Scheme 2. Synthesis of 1180-292 for photoaffinity linking. 105


Chapter 3:

Scheme 1. Synthesis of dihydroisoquinolinones. 165

Scheme 2. Synthesis of A-ring triazole intermediate. 165

Scheme 3. Synthesis of B-ring derivatives. 166

Scheme 4. Synthesis of heterocyclic core scaffolds. 168

Scheme 5. Synthesis of phenyl-based 1180-436. 168

Scheme 6. Synthesis of dihydropyrrolopyrazinones. 169


List of Abbreviations

ABD: agonist binding domain

ACN: acetonitrile

ADHD: attention deficit hyperactivity disorder

ADME: absorption, distribution, metabolism, and excretion

ATD: amino terminal domain

AMPA: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

AMPAR: AMPA receptor

BHK: baby hamster kidney cells

Bn: benzyl

CI: confidence interval

CIQ: (3-chlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone

cHex: cyclohexyl

CNS: central nervous system

CO2: carbon dioxide

cPent: cyclopentyl

Cryo-EM: single-particle electron cryomicroscopy

CTD: carboxy terminal domain

DC: doubling concentration

DCM: dichloromethane

DMAP: dimethylamino pyridine

DMSO: dimethylsulfoxide

EC50: half-maximal effective concentration

EDCI: N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride

ee: enantiomeric excess

FDA: Food and Drug Administration

GPCR: G-protein-coupled receptor

HPLC: high-performance liquid chromatography

iBu: isobutyl

ID: indeterminable

iPr: isopropyl

iGluR: ionotropic glutamate receptor

IP: intraperitoneal

IV: intravenous

LCMS: liquid chromatography-mass spectrometry

Me: methyl

MDD: major depressive disorder

mGluR: metabotropic glutamate receptor

µw: microwave

NAM: negative allosteric modulator

NHP5G: N-hydroxypyrazol-5-yl glycine

NMDA: N-methyl-d-aspartic acid

NMDAR: NMDA receptor

NMR: nuclear magnetic resonance

NRG1: neuregulin-1

OCD: obsessive-compulsive disorder

PAM: positive allosteric modulator

PAS: 20-oxo-5β-pregnan-3α-yl sulfate

PFC: pre-frontal cortex

PS: pregnenolone sulfate

PTSD: post-traumatic stress disorder

PYD: pyrrolidinone

SAR: structure activity relationship

SEM: standard error of the mean

TEVC: two-electrode voltage clamp

THF: tetrahydrofuran

TIQ: tetrahydroisoquinoline

TMD: transmembrane domain

TMS: trimethylsilyl ether

TRD: treatment-resistant depression

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