Structural Determinants of Activity, Mechanism and Structure Activity Relationships of Novel GluN2C/D Subunit Selective Antagonists of the N-methyl-D-Aspartate Receptor Público

Abstract

The N-methyl-D-Aspartate (NMDA) receptors are ionotropic glutamate receptors whose family members, identified by sequence homology and pharmacology, comprise the 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA), kainate and delta receptors. The NMDA receptors respond to the co-agonists glycine and glutamate and mediate the slow component of excitatory neurotransmission throughout the central nervous system (CNS). The functional NMDA receptor is a hetero-tetramer consisting of two GluN1 subunits which bind to glycine and two GluN2 subunits (GluN2A-D). Both the Glun1 subunits, which have eight different splice variants, and the GluN2 subunits, which are encoded by four distinct genes, can impart various unique functional and pharmacological properties to the functional receptor, with the GluN2 subunits having a greater impact on the various different properties. While the receptors have been known and studied intensely for several decades, until recently, subunit-selective pharmacological tools remained elusive since the 1980s when the first selective agent targeting GluN2B receptors was discovered and characterized. This dissertation describes novel subunit-selective allosteric modulators which target the GluN2C- and GluN2D-containing NMDA receptors. The findings include the identification of key structural determinants of activity for one of the classes described, the identification of highly potent and selective congeners within the same class, the stereochemical preference of one of the more potent and selective members of the small molecules, the beginning of the physicochemical property optimization of the molecules and data and hypotheses suggesting that distinct classes of molecules bind to a shared, or overlapping site at the GluN2D containing receptors.

Table of Contents

Chapter 1: Introduction

1

1.1. Abstract

1

1.2. Introduction

1

1.3. NMDA receptor topology

4

a. Subunit arrangement and stoichiometry

4

b. The amino-terminal domain

6

c. The ligand-binding domain

d. The trans-membrane linker and spanning domains

e. The carboxy-terminus domain

9

11

13

1.4. NMDA receptor pharmacology

13

a. NMDA receptor agonists

13

b. Competitive antagonists of the NMDA receptor

18

c. Noncompetitive modulators of the NMDA receptor

21

d. Uncompetitive antagonists of the NMDA receptor

27

1.5. Anatomical location, physiological function and therapeutic rationale

30

a. Anatomical location and physiological function

30

b. Therapeutic rationale

33

1.6. Structure activity relationship rationale

35

Chapter 2: Materials and Methods

39

2.1. Molecular biology

39

2.2. Two-electrode voltage-clamp recording from Xenopus laevis oocytes

39

2.3. Compound solubility

41

2.4. MDR-MDCK1 permeability assay

41

2.5. Human liver microsomal stability

43

2.6. Reagents

43

2.7. Chemistry experimental

44

2.8. Computational analysis

45

2.9. Data analysis

45

Chapter 3: Mechanism and structural determinants of activity for DQP-1105

47

3.1. Abstract

47

3.2. Introduction

47

3.3. Results

49

a. Subunit selectivity of DQP-1105 inhibition

49

b. Mechanism of action of DQP-1105

52

c. Structural determinants of DQP-1105 activity

55

3.4. Discussion

58

Chapter 4: Structure activity relationship of DQP-1105 class of compounds

63

4.1. Abstract

63

4.2. Introduction

63

4.3. Results

66

a. Chemistry

66

b. Evaluation of off-target effects

92

c. Aqueous solubility, BBB penetration and human liver microsomal stability

94

4.4. Discussion

98

4.5. Chemistry experimental

103

a. Evaluation of enantiomers

103

b. Synthetic procedures

105

Chapter 5: QSAR and ROCS computational modeling

271

5.1. Abstract

271

5.2. Introduction

271

5.3. Results

274

a. Tanimoto comparison of distinct classes and synthesis of hybrid compounds

274

b. QSAR Modeling

283

5.4. Discussion

295

5.5. Chemistry experimental

296

a. Synthesis of hybrid molecules

296

b. Synthesis of QNZ analogs

301

Chapter 6: 1063 Series of Antagonists

316

6.1. Abstract

317

6.2. Introduction

317

6.3. Results

322

6.4. Discussion

335

6.5. Chemistry experimental

336

Chapter 7: Discussion and Conclusion

355

7.1. Summary

365

7.2. DQP-1105 as a representative member of the class of compounds

366

7.3. Optimizing the DQP-class of compounds through synthetic chemistry

368

7.4. QSAR and ROCS computational modeling

372

7.5. 1063 Series of Antagonists

374

7.6. Conclusion

375

Chapter 8: References

377

Figures and Tables

Fig. 1.1. Linear amino acid sequence and structural homology model of an NMDA

receptor

5

Table 1.1. Sequence identity and conservation between NMDA receptor subunits

7

Fig. 1.2. GluN1/GluN2D ligand binding domain interface and transmembrane linker regions.

10

Table 1.2. Glycine site agonists

15

Table 1.3. Glutamate site agonists

16

Table 1.4. Competitive antagonsits of the NMDA receptor

19

Fig. 1.3. GluN2A and GluN2B subunit-selective modulators.

22

Table 1.5. IC50 values for noncompetitive GluN2B-selective NMDA receptor antagonists

23

Fig. 1.4. Negative allosteric modulators of GluN2C- and Glun2D-containing receptors.

26

Fig. 1.5. Clinically relevant NMDA receptor antagonists

28

Table 1.6. Uncompetitive antagonists of the NMDA receptor

29

Fig. 1.6. In Situ hybridization of the NMDAR subunit mRNA throughout rat development

31

Fig. 3.1. DQP-1105 inhibition and subunit-selectivity

50

Table. 3.1. Concentration-response data for DQP-1105 at ionotropic glutamate receptors

51

Fig. 3.2. DQP-1105 inhibits recombinant GluN1/GluN2D receptors through a non-competitive and voltage-independent mechanism

54

Fig. 3.4. Chimeric receptor data using DQP-1105

56

Fig. 3.5. Cartoon illustration of structural determinants of selectivity for DQP-1105

61

Fig. 4.1. General structure of DQP-class of compounds

67

Scheme 4.1. Synthesis of dihydro-quinolone-pyrazoline derivatives

68

Scheme 4.2. Synthesis of unsaturated chain containing compounds

69

Table 4.1. Evaluation of A-Ring para substitutions

71

Table 4.2. Evaluation of A-Ring ortho- and meta- substitutions

72

Scheme 4.3. Synthesis of primary alcohol and amide derivatives

73

Figure 4.2. Correlation between C-ring σ and π paramaters to potency

74

Table 4.3. A and B Ring Modifications

76

Fig. 4.3. Evaluation of substituent effects of B-ring modifications

77

Table 4.4. B Ring Modifications

79

Table 4.5. B-Ring Di-substitutions

80

Table 4.6. C-Ring Modifications

81

Table 4.7. Acyl chain perturbations

83

Scheme 4.3. Synthesis of alky derivatives

85

Scheme 4.4. Synthesis of mono-fluoro isostere of the alcohol derivative

86

Scheme 4.5. Scaffold-hopping synthesis

88

Fig. 4.3 Enantiomeric resolution of 997-23

90

Table 4.8. Stereochemistry preference of purified enantiomers

91

Table 4.9. Off-target responses for compounds 997-23 and 997-33

93

Table 4.10. MDR1-MDCK permeability assay

95

Table. 4.11. Human liver microsomal stability

96

Fig. 4.5. Improvements in selectivity and potency

99

Fig. 4.6. Pharmacophore features elucidated through SAR

100

Fig. 5.1. ROCS overlay of energy minimized conformation of 1179 S with the most similar 1936 conformation found in ROCS

276

Fig. 5.2. ROCS overlay of energy minimized conformation of 1179 R with most similar conformation of 1936

278

Fig. 5.3. ROCS overlay of energy minimized compound 1936 and most similar 1179 S conformation

277

Scheme 5.1. Synthesis of hybrid compound

281

Table 5.1. Potency of novel hydroxy-napthyl containing compounds at recombinant NMDA receptors.

282

Table 5.2. Structural modifications made to the QNZ class of molecules

284

Fig.5.4. Schematic representation of QSAR model

286

Table 5.3. Analytical data for QSAR models derived from QNZ data set

288

Table 5.4. Ligand data set used to derive QSAR models

289

Table 5.5. QSAR model ANRRRR.36 site measurements and angles

292

Fig. 5.5. Compounds identified as active in the initial screening using QNZ-QSAR

294

Fig. 6.1. Screening hit and general structure for 1063-series SAR

318

Scheme 6.1. General synthetic scheme for 1063-analogs

320

Scheme 6.2. Retro-synthetic route to differentially substituted indoles

321

Table 6.1. Substitutions to the terminal aryl ring

323

Scheme 6.3 Sonagashira methodology

326

Scheme 6.4. Larock indole synthesis of 3, 5-dimethyl substituted indole containing

327

Scheme 6.5. Larock indole synthesis of 3-methyl, 5-caroxylate derivative

328

Scheme 6.6. Larock methodology for mono-substituted indole compounds

329

Scheme 6.7. Sonagashira method access to 2-substituted indoles

330

Scheme 6.8. Substitution to the phenyl linker

331

Scheme 6.9. Further substitution to the phenyl linker

332

Scheme 6.10. Orientation of the amide linkage

333

Table 6.2. Potency of 1063 compounds against NMDA receptors

334

About this Dissertation

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• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Molecular & Systems Pharmacology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, General Biology, General
Palabra Clave	GluN2D GluN2C Antagonist
Committee Chair / Thesis Advisor	Liotta, Dennis C, Emory University
Committee Members	Hall, Randy A, Emory University Morgan, Edward T, Emory University Traynelis, Stephen, Emory University Ortlund, Eric, Emory University

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