The Design, Synthesis, and Biological Evaluation of Subunit-Selective Modulators of the N-Methyl-D-Aspartate Receptor Open Access
Epplin, Matthew (Spring 2019)
Abstract
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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