Structural Determinants of Activity, Mechanism and Structure Activity Relationships of Novel GluN2C/D Subunit Selective Antagonists of the N-methyl-D-Aspartate Receptor Público
Acker, Timothy Michael (2013)
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
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