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 ionotropic glutamate receptor family which mediate a slow Ca²⁺-permeable component of excitatory synaptic transmission within the central nervous system. Each NMDA receptor is composed of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. The GluN2 subunits are divided into four distinct subtypes labeled GluN2A-GluN2D; each subtype endows the receptor with unique pharmacological properties. Overactivation of NMDA receptors has been implicated in a number of neurodegenerative diseases including traumatic brain injury (TBI), Alzheimer's disease, stroke, and neuropathic pain. In the search for treatments for these neurological conditions, a number of NMDA receptor antagonists have been developed.

Towards this treatment goal, our lab developed a small library of ethanolamines which selectively target GluN2B-containing NMDA receptors. Unfortunately, the compounds displayed significant off-target effects. A structurally related class was developed with the goal to decrease off-target effects while maintaining potency. In Chapter 1, a subset of compounds is described which sought to increase potency and efficacy by incorporating functionality from potent compounds in both series. The compounds described have been evaluated against the GluN1/GluN2B subunit of the NMDA receptor for both potency (IC50) and pH sensitivity (fold shift).

Chapter 2: The published work around subunit-selective modulators of NMDA receptors has mostly focused on antagonists A few endogenous compounds, such as the polyamines and neurosteroids have shown some subunit-selective potentiation of the NMDA receptor complex, but no therapeutically useful subunit-selective potentiators have been described to date. Based on well-documented experimental evidence, NMDA receptor potentiators may have therapeutic potential in the treatment of schizophrenia, anxiety disorders or for enhancement of learning and memory.

Based on a screening hit from a fluorescence based high-throughput screen, a library of novel compounds has been developed. Herein, we describe the synthesis and biological evaluation of this new class of subunit-selective potentiators of NMDA receptors containing a tetrahydroisoquinoline core. Through structural modifications, the potency of the series was improved 40-fold over the original screening hit. One compound from the series was evaluated for pharmacokinetic properties as well as in an animal model.

Table of Contents

Table of Contents

List of Illustrations

Figures Tables Schemes

List of Abbreviations

Chapter 1: Design, Synthesis and Biological Evaluation of GluN1/GluN2B Selective NMDA Receptor Antagonists for the Potential Treatment of Neurodegenerative Diseases

CHAPTER 1

1.1 STATEMENT OF PURPOSE 1 1.2 INTRODUCTION AND BACKGROUND 4

1.2.1 NMDA Receptor structure, function, and localization 4

1.2.2 Classes of NMDA Receptor Antagonists 8

1.2.3 Therapeutic Rationale for NMDA Antagonists 11

1.2.4 GluN2B-Subunit Selective Antagonists 12

1.2.5 Brain pH acidification 14

1.2.6 Phenethanolamines 15

1.2.7 The human-ether-a-go-go related gene (hERG) ion channel 18

1.2.8 Previous Research/SAR Development of GluN2B-selective pH

dependent antagonists 24

1.3 SYNTHESIS OF GLUN2B-SELECTIVE ANALOGUES 27

1.4 RESULTS AND DISCUSSION 32 1.5 CONCLUSIONS 36 1.6 CHEMISTRY EXPERIMENTAL DETAIL 38

1.6.1 Chemistry Experimental Procedures 38

1.6.2 Combustion Analysis 50

1.7 BIOLOGY EXPERIMENTAL DETAIL 51

1.7.1 In vitro assay: NMDA receptor antagonism 51

1.7.2 In vitro binding studies for off-target effects 53

Chapter 2: Design, Synthesis, and Biological Evaluation of First-in-Class GluN2C/GluN2D-Selective NMDA Receptor Potentiators

CHAPTER 2

2.1 STATEMENT OF PURPOSE 58 2.2 INTRODUCTION AND BACKGROUND 59

2.2.1 Subunit-Selective Modulators of NMDA function 59

2.2.2 Therapeutic Rationale for GluN2D-selective potentiators 60

2.2.3 Glutamate Hypofunction Hypothesis 61

2.2.4 Enhancement of Learning and Memory 64

2.2.5 Extinction of Fear 65

2.2.6 Rationale for 1180-Series Analogues 68

2.2.7 Cysteine Reactive Groups 70

2.2.8 Literature Precedent for Chiral Synthesis 73

2.3 SYNTHESIS OF 1180-SERIES ANALOGUES 74

2.3.1 Synthesis of tetrahydroisoquinoline analogues 74

2.3.2 Synthesis of potential Cysteine-reactive analogues 91

2.3.3 Synthesis of indole-containing compound 93

2.3.4 Synthesis of the enantiomers of 1390 93

2.4 RESULTS AND DISCUSSION 102

2.4.1 Structure Activity Relationship of 1180-series - A-Ring Modifications 102

2.4.2 Structure Activity Relationship of 1180-series - Linker A modifications 107

2.4.3 Structure Activity Relationship of 1180-series - B-Ring Modifications 109

2.4.4 Structure Activity Relationship of 1180-series - Linker B Modifications 110

2.4.5 Structure Activity Relationship of 1180-series - C-Ring Modifications 112

2.4.6 Rationale and results for indole-containing compound 117

2.4.7 In vitro analysis of 1180-series mechanism of action and binding

interactions 119

2.4.8 Cysteine Mutants 123

2.4.9 Results of cysteine-reactive mutants 124

2.4.10 In vivo analysis for pharmacokinetic properties of 1390 126

2.4.11 In vitro analysis of selectivity of 1390 127

2.4.12 Evaluation of 1390 in fear extinction 128

2.5 CONCLUSIONS 131 2.6 CHEMISTRY EXPERIMENTAL DETAIL 134

2.6.1 Chemistry Experimental Detail for 1180-series 134

2.6.2 Combustion Analysis 256

2.7 BIOLOGY EXPERIMENTAL DETAIL 258

2.7.1 in vitro analysis of 1180 series 258

2.7.2 Separation of Enantiomers of 1390 261

2.7.3 In vitro analysis of selectivity for 1390 262

2.7.4 In vivo analysis of 1390 for pharmacokinetic properties 262

2.7.5 Evaluation of 1390 in a fear extinction animal model 263

List of Illustrations

List of Figures Page Chapter 1:

Figure 1. Structures of example GluN2B-selective NMDA antagonists, ifenprodil and traxoprodil (CP-101,606)

3

Figure 2. Structure of screening hit on which 93-series of ethanolamines was based

4

Figure 3. Structure of screening hit on which the 96-series of NMDAR antagonists was based

4

Figure 4. Synthetic glutamate mimics which bind selectively to respective iGluRs

6 Figure 5. NMDA Receptor Subunit Arrangement 7 Figure 6. Cartoon Depiction of NMDA Receptor Subunit Architechture 8

Figure 7. NMDA subunit expression in rat brain from post-natal day 1 to adult generated via in situ hybridization

9

Figure 8. The GluN2B-selective antagonist pharmacophore

15 Figure 9. Structure of Traxoprodil (CP-101,606), a GluN2B-selective antagonist 16 Figure 10. Structures of known GluN2B-selective NMDA receptor antagonists 18

Figure 11. Structure of screening hit on which the 93-series was based

19

Figure 12. Interactions of MK-499 with the hERG channel based on homology model

21

Figure 13. Structures of screening hit, most potent compound from 93-series and the pharmacophore describing their relationship

27 Figure 14. Structures of 96-series compounds chosen for further modification 29

Chapter 2:

Figure 1. Structure of ifenprodil, the classical GluN2B-selective antagonist and 1180, the screening hit on which this work is based

58

Figure 2. Generic structure illustrating the planned modifications to the screening hit (1180) for SAR development

68

Figure 3. Commonly used functional groups for the covalent modification of nucleophilic residues in proteins

72

Figure 4. Proposed rationale for observed stereoselectivity in the reduction of the iminium ion (56)

102

Figure 5. Rotational isomers of 1180-series compounds depicting steric interactions which may be the cause of observed potency decrease

114

Figure 6. Structure of synthesized indole-containing compound based on the generic structure 57

119

Figure 7. In vitro analysis of 1180-series

120

Figure 8. Schematic representation of the GluN2 subunit polypeptide

121

Figure 9. Schematic representation of the GluN2 subunit polypeptide with chimeras shown between the GluN2D ATD-M1 region and the corresponding region in GluN2A

122

Figure 10. Homology model of a GluN1/GluN2 heterodimer of the tetrameric NMDA receptor based on GluR2 receptor

122

Figure 11. Graphs depicting the current elicited by glutamate and glycine application both before and after administration of 1180-66

125

Figure 12. Graphs depicting the current elicited by application of CIQ (1390) both before and after 1180-66 administration

126

Figure 13. Graph depicting the current elicited by application of CIQ (1390) to both cysteine-mutant GluN2D receptors

126

Figure 14. Pharmacokinetics of 1390 (CIQ).

127

Figure 15. Percent freezing in mice treated with 1390 (CIQ) or vehicle for the tone-alone trials

128

Figure 16. Evaluation of percent freezing in mice treated with 1390 (CIQ) or vehicle during the tone-alone trials

129

Figure 17. Percent freezing in tone-alone trials following fear extinction training

130

Figure 18. Evaluation of percent freezing in response to conditioned stimuli (tone) for mice treated with CIQ (1390) and vehicle

130 List of Tables Page

Chapter 1:

Table 1. Classes of NMDA receptor antagonists with example compounds from each class

10

Table 2. Correlation between chain length and fold shift for the 93-series

18

Table 3. Medicinal chemistry optimizations on structural classes for hERG binding

21 Table 4. Medicinal chemistry efforts to optimize hERG binding 23 Table 5. Summary of 96-series compounds and associated biological data 33

Table 6. Off-target data for a selection of 96-series compounds

35 Table 7. MDR1-MDCK Permeability 36 Chapter 2:

Table 1. Effect of A-ring substituent placement on potency of 1180-series

69

Table 2. Effect of B-ring substituent type on potency and potentiation of 1180-series

70

Table 3. Results of model study in the synthesis of chiral tetrahydroisoquinolines using methylbenzylamine as a directing group

74

Table 4. Oxidation conditions attempted for the conversion of the primary alcohol to the desired aldehyde building block

75 Table 5. Summary of phenethylamines subjected to acylation conditions 80

Table 6. Summary of α-chloroamides subjected to alkylation conditions

81

Table 7. Summary of phenethylamines subjected to EDCI coupling conditions

81

Table 8. Summary of acyclic amides subjected to Bischler-Napieralski conditions to form the tetrahydroisoquinoline core

83

Table 9. Summary of 1180-series analogues with modified A-ring substitution

85

Table 10. Summary of 1180-series analogues with modified A-ring

86

Table 11. Summary of 1180-series analogues with modified substitution around B-ring

87

Table 12. Summary of 1180-series analogues with modifications to C-ring substitution

88

Table 13. Conditions attempted for the Bischler-Napieralski onto the 4-methoxy substituted ring

89

Table 14. Comparison of biological activity between 1180-series compounds with and without B ring and linker B

95

Table 15. HPLC and biological data from enantiomers of 1390 as separated by supercritical fluid chromatography (SFC)

96

Table 16. Attempted crystallization conditions for the resolution of the racemic tetrahydroisoquinoline intermediate

97

Table 17. Conditions attempted for the reduction of amide 51

100 Table 18. HPLC and biological data for synthesized enantiomers of 1390 101

Table 19. Effect of A ring substituent identity on potency and max potentiation of 1180 analogues

104

Table 20. Effect of A ring disubstitution on potency and max potentiation of 1180 series analogues

105

Table 21. Effect of replacing A-ring on potency and max potentiation

106

Table 22. Effect of increasing length of linker A on potency of 1180-series

108

Table 23. Effect of linker A on potency of 1180-series

109

Table 24. Effect of varied B-ring substitution on potency and max potentiation

110 Table 25. Effect of modifications to Linker B on potency and max potentiation 111 Table 26. Effect of varied C ring substitution on potency and potentiation 113

Table 27. Effect of varied C ring single substitution on potency and max potentiation of 1180 series

116

Table 28. Comparison between the potency and max effect of isomeric 1180 analogues with varied C ring substitution pattern

118

Table 29. Comparison between most potent compounds in the series with original screening hit

132 List of Schemes Page

Chapter 1:

Scheme 1. Retrosynthesis of 96-series GluN2B-selective antagonists

28 Scheme 2. Retrosynthetic analysis of 96-49, a 96-series analogue 29 Scheme 3. Construction of the fragment, 46, towards the synthesis of 96-49 29

Scheme 4. Completion of the synthesis of 96-49

30

Scheme 5. Construction of the diamine building block required for the synthesis of four 96-series analogues

30

Scheme 6. Completion of the synthesis of 96-61 and 96-66 using the previously synthesized diamine building block

31

Scheme 7. Completion of the synthesis of 96-65 and 96-70

32

Chapter 2:

Scheme 1. Retrosynthetic analysis of 1180-series with Pictet-Spengler reaction employed as key step

74

Scheme 2. Cleavage of the epoxide to the benzofuran instead of the desired aldehyde building block

75

Scheme 3. Use of a masked aldehyde to attempt the formation of desired aldehyde

76

Scheme 4. Attempted one-pot Pictet-Spengler from masked aldehyde

77

Scheme 5. Retrosynthetic analysis incorporating a Bischler-Napieralski cyclization as the key step

77

Scheme 6. Generalized synthesis of amide with an ether (22) or thioether (23) linker

78

Scheme 7. Generalized synthesis of amide 25 with a saturated or unsaturated carbon linker

79 Scheme 8. Synthesis of phenethylamines 79

Scheme 9. Synthesis of the tetrahydroisoquinoline core

82

Scheme 10. Final step in the synthesis of 1180-series analogues

84

Scheme 11. Formation of 1180-65 from a debenzylation of 1180-22

84

Scheme 12. Retrosynthesis of 1180-analogue with single methoxy on C-ring

90

Scheme 13. Attempted Bischler-Napieralski onto chlorine-substituted aromatic ring

91 Scheme 14. Synthesis of potential Cys-reactive analogue 1180-68 92 Scheme 15. Synthesis of potential Cys-reactive analogue, 1180-66 92 Scheme 16. Synthesis of indole-containing analogue of 1180-series 93 Scheme 17. Synthesis of compounds without the B-ring and linker B 94

Scheme 18. Synthesis of nitrotartanilic acid for chiral resolution

98 Scheme 19. Synthesis of enantiomers of 1390 99

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Organic Chemistry, General
Keyword	synthetic chemistry N-Methyl-D-Aspartate NMDA Allosteric modulators
Committee Chair / Thesis Advisor	Liotta, Dennis C, Emory University
Committee Members	Lutz, Stefan, Emory University Blakey, Simon, Emory University

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