Navigating the Roads to Drug Discovery: Part 1: Design, Synthesis, and Biological Evaluation of a Novel Class of Allosteric Modulators of N-Methyl-D-Aspartate Receptor Function Part 2: Discovery of Novel Tetrahydroisoquinoline (THIQ)-Based CXCR4 Antagonists and Conformational Analysis of Structurally Similar CXCR4 Antagonists Öffentlichkeit
Katzman, Brooke M. (2014)
Abstract
Part 1:
The N-methyl-d-aspartate receptor (NMDA) is an ionotropic glutamate receptor that mediates excitatory synaptic transmission. Under normal physiological conditions, NMDA receptors are involved in many neurological processes including learning and memory. NMDA receptor dysfunction has been implicated in several neuropathological conditions, such as Parkinson's disease, Alzheimer's disease, and neuronal damage during ischemia. Therapeutic agents acting at NMDA receptors have been proposed for treating disorders, however side effects have complicated the clinical evaluation. This dissertation describes a novel class of allosteric modulators that have a modest preference for GluN2C/D receptors. Several molecules in this series exhibit sub-maximal inhibition. The mechanism of action for a typifying member of this class was determined. We show that this compound is active in neurons, with potent inhibition of NMDA receptor function in cerebellar granule cells. Additionally, this compound possesses neuroprotective properties against NMDA-induced toxicity in primary neuronal cultures. We are optimistic that this series of compounds could be useful neuroprotective agents in stroke or neurodegenerative diseases, with their partial antagonism potentially lessening undesirable side effects.
Part 2:
The CXC chemokine receptor 4 (CXCR4) is a G protein-coupled receptor (GPCR) that interacts with the CXCL12 chemokine to regulate several diverse processes such as cell migration, proliferation, and angiogenesis. Additionally, it plays a pivotal role in HIV pathogenesis as this protein is utilized by T-Tropic HIV-1 variants as a co-receptor for viral entry. Several peptides and small molecules have been developed that target CXCR4 and exploit these biological roles. The second part of this dissertation describes the discovery of a novel series of small molecule CXCR4 antagonists. During the development of a structure activity relationship (SAR), several important details regarding the requirements for effective binding were revealed. A combination of computational and experimental methods was employed with our most potent compound to predict its binding interactions to CXCR4. With knowledge of its conformation in solution and a predicted bioactive pose, we were able to better understand the fundamental factors that contribute to its favorable interactions with the protein. Furthermore, we used this information to design new molecules predicted to be more potent than our lead compound.
Table of Contents
Table of Contents
List of Illustrations
Figures
Tables
Schemes
Graphs
Charts
List of Abbreviations
Part 1: Design, Synthesis, and Biological Evaluation of a Novel Class of Allosteric Modulators of N-Methyl-d-Aspartate Receptor Function
CHAPTER 1
1.1 Statement of Purpose 1
1.2 Introduction and Background 2
1.2.1 Receptor Structure, Function, and Synaptic Localization 2
1.2.2 NMDA Receptor Pharmacology 6
1.2.3 Clinical Implications of NMDA Receptor Dysfunction 11
1.2.4 NMDA Receptor as a Therapeutic Target 12
1.2.5 Rationale for 1794-Series Analogues 14
1.3 Synthesis of 1794-Series Analogues 15
1.4 Results and Discussion 23
1.4.1 Structure Activity Relationships of 1794 Series 23
1.4.2 In Vitro Analysis of 1794 Series Mechanism of Action 33
1.4.3 In Vitro Analysis of 1794 Series in Primary Cultured Neurons 35
1.4.4 In Vitro Analysis of 1794 Series in Neuroprotection Model 36
1.5 Conclusions 37
1.6 Chemistry Experimental Detail 38
1.6.1 Synthetic Chemistry Experimental Detail 39
1.6.2 Crystal Structure Data 80
1.7 Biology Experimental Detail 96
1.7.1 In Vitro Antagonism Assay 96
1.7.2 Patch Clamp Recordings from Cerebellar Granule Cells 98
1.7.3 Lactate Dehydrogenase (LDH) Assay 99
Part 2: Discovery of Novel Tetrahydroisoquinoline (THIQ)-Based CXCR4 Antagonists and Conformational Analysis of Structurally Similar CXCR4 Antagonists
CHAPTER 2
2.1 Statement of Purpose 102
2.2 Introduction and Background 103
2.2.1 Chemokines and Chemokine Receptors 103
2.2.2 CXCR4 and its Ligand CXCL12 106
2.2.3 CXCR4/CXCL12 Signaling Pathway 108
2.2.4 Therapeutic Rationale for Targeting CXCR4/CXCL12 Axis 109
2.2.5 Clinically Relevant CXCR4 Antagonists 113
2.2.6 Rationale for THIQ-Based CXCR4 Antagonists 115
2.2.7 Rationale for Masking the Basicity of the Butylamine Nitrogen 120
2.3 Synthesis of THIQ Analogues 123
2.3.1 Retrosynthetic Analysis Toward THIQ Analogues 123
2.3.2 Optimization of Synthetic Routes Toward Analogues 124
2.3.3 Forward Synthesis of THIQ Analogues 127
2.4 Results and Discussion 134
2.4.1 Structure Activity Relationship of THIQ Analogues 134
2.4.2 Interpretation of Biological Data Through Molecular Modeling 137
2.4.3 In Vitro Analysis of Drug Efflux and Permeability 139
2.5 Conclusions 142
2.6 Chemistry Experimental Detail 144
2.6.1 Synthetic Chemistry Experimental Detail 144
2.6.2 Crystal Structure Data 161
2.7 Biology Experimental Detail 167
2.7.1 MAGI Antiviral Assay with HIV-1IIIB 167
2.7.2 Calcium Flux Assay 169
2.7.3 Caco-2 Permeability Assay 170
CHAPTER 3
3.1 Statement of Purpose 172
3.2 Introduction and Background 173
3.2.1 History of Medicine and Drug Discovery 173
3.2.2 Structured-based Drug Design 175
3.2.3 Conformation and Drug Design 175
3.2.4 Tools for Conformational Analysis 177
3.2.5 NMR Analysis of Molecular Flexibility in Solution (NAMFIS) 182
3.3 Conformational Analysis of CXCR4 Antagonists 183
3.3.1 Introduction 183
3.3.2 Synthesis of CXCR4 Antagonists for NAMFIS Studies 184
3.3.3 Results and Discussion 186
3.4 Design of Constrained Analogues 200
3.4.1 Introduction 200
3.4.2 Synthesis of Constrained Analogues 202
3.4.3 Results and Discussion 208
3.5 Conclusions 210
3.6 Chemistry Experimental Detail 212
3.6.1 Synthetic Chemistry Experimental Detail 212
3.6.2 NMR Experimental Detail 234
3.6.3 Crystal Structure Data 242
3.7 Computational Methods 254
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