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Design and Syntheses of Potential Drugs Based on GABAA Receptor Pharmacophores

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Design and Syntheses of Potential Drugs Based on GABAA Receptor Pharmacophores Design and Syntheses of Potential Drugs Based on GABAA Receptor Pharmacophores Ella Chow Clement Dissertation submitted to the Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the Degree of Doctor of Philosopy in Chemistry Dr. Paul R. Carlier, Chairman Dr. Jeffrey R. Bloomquist Dr. Richard D. Gandour Dr. David G. I. Kingston Dr. James M. Tanko June 28, 2005 Blacksburg, Virginia Keywords: GABAA receptor, Partial/full agonists, Superagonist, Antagonists, Non-zwitterionic GABA amide homodimers and heterodimers, 36Cl- Flux assay, [3H]Muscimol binding, ZAPA, PEG, Memory of Chirality Copyright 2005, Ella Chow Clement Design and Syntheses of Potential Drugs Based on GABAA Receptor Pharmocophores Ella Chow Clement ABSTRACT Numerous previous studies of GABAAR ligands have suggested that GABAAR agonists must be zwitterionic and feature an intercharge separation similar to that of GABA (approx. 4.7-6.0 Å). We have demonstrated that monomeric, homodimeric and heterodimeric non-zwitterionic GABA amides are partial, full, or superagonists at the murine GABAA receptor (GABAAR). The agonism of these GABA amides is comparable to that of THIP, as shown by in vitro assay results. The assay data indicate that the agonism of GABA amides is tether length-dependent. Optimum agonism is achieved with a tether length of four methylenes in GABA amide dimers and in GABA amides bearing pendant amide or amino groups. We have further investigated the structure-activity relationship for GABA amides on the GABAAR by performing structural modifications to both the superagonist 2c and the agonist 6c. Synergism and [3H]muscimol binding experiments show that 2c binds to the same sites as GABA. Structural modification of 2c demonstrated that partial rigidification of the tether eliminated agonism and caused ligands to behave as weak competitive antagonists. We have also investigated the agonism of four ZAPA derivatives in 36Cl- uptake functional assay. Two of them are found to be as potent as GABA. In our studies of 1,4-benzodiazepines, our goal was to synthesize three different subtypes of quaternary 1,4-benzodiazepines by use of the memory of chirality (MOC) strategy. Disappointingly, most of the deprotonation/alkylations failed, due to various reasons. The failure of the reactions of (S)-alanine-derived tetrahydro-1,4- benzodiazepin-3-ones was probably due to either the unexpected side reactions or the steric hindrance of enolate alkylation. In the case of tetrahydro-1,4-benzodiazepin-2- ones, computational studies suggested that steric hindrance by both the benzo ring and N4-allyl group might retard deprotonation at C3 by bulky bases like KHMDS or LDA. Finally, (S)-serine-derived 1,4-benzodiazepin-2-ones and their elimination products (α- methylene benzodiazepines) were prepared. These proved unreactive towards deprotonation/alkylations and conjugate additions, respectively. The low reactivity of the α-methylene benzodiazepines towards nucleophiles was attributed to highly delocalized LUMOs that failed to direct nucleophiles to the β-carbons. iii Acknowledgements First of all, I would like to thank my advisor, Dr. Paul Carlier, for giving me the opportunity to come with him and study at Virginia Tech. I would also like to thank Dr. Carlier for his guidance and financial support during my Ph.D. study. Secondly, I would like to thank Dr. Jeffrey Bloomquist for his help and support in doing bioassay experiments. Thirdly, I would like to thank my committee members for their assistance in fulfilling departmental requirements. I would also like to thank Mr. Tom Glass and Mr. Bill Bebout for their analytical services, and my group members for sharing lab responsibility. I would like to thank Mrs. Kay Castagnoli and Dr. Emre Isin for their friendship and support. I also like to thank friends, especially Dr. Mary Dean Coleman, Miss Amy Fletcher, Miss Jennifer Farris, and Ms. Mary Billings from the Graduate Christian Fellowship for their prayers and support. I would like to thank my family for their love and support during my study. I would like to especially thank my father, who has gone to be with the Lord, for his great influence in my life. Finally, I would like to thank my husband Jason for his love, help, and support in every aspect of my studies and life. iv Table of Contents List of Figures vii Chapter 1 Introduction and Background of GABAA Receptors................................... 1 1.1 Background and Significance of the GABAA Receptor ......................................... 1 1.2 Classification of Three GABA Receptor Subtypes ................................................ 2 1.3 GABAA Receptor Physiology and Pharmacology.................................................. 6 1.3.1 Current Models for the Structure and Function of the GABAA Receptors. 6 1.3.2 Benzodiazepine Binding Sites of The GABAA Receptors ....................... 14 1.3.3 Barbiturates, Picrotoxinin and Steroid Anesthetic Binding Sites in the GABAAR................................................................................................... 16 1.4 GABAA Receptor-Agonists and Partial Agonists................................................. 20 1.4.1 Structure and Conformation of Active GABA Analogs (Exogenous GABAA agonists)...................................................................................... 20 1.4.2 Endogenous Agonists................................................................................24 1.5 GABAA Agonist-Receptor Interactions................................................................ 25 1.5.1 Electronic Factors ..................................................................................... 26 1.5.2 Structural and Conformational Factors..................................................... 26 1.5.3 Stereochemical Factors............................................................................. 27 1.5.4 Current Agonist Binding model of the GABAA Receptor........................ 28 1.6 Traditional Assays for Agonists............................................................................ 32 1.7 Pharmacokinetic Aspects and Prodrugs................................................................ 34 References for chapter 1 ................................................................................................... 38 Chapter 2 Syntheses of GABAA receptor analogs...................................................... 47 2.1 Syntheses and Evaluation of homodimeric GABAA receptor analogs ................. 48 2.1.1 Bioassay Results of New GABA amide homodimers .............................. 49 2.1.2 Synthesis of Derivatives of ‘Superagonist’ Dimer 2c (4a-h).................... 53 2.1.3 Bioassay Results of the Derivatives (4a-h)............................................... 57 2.1.4 [3H]Muscimol Binding Experiments ........................................................ 59 2.2 Syntheses and Evaluation of heteromeric GABAA receptor analogs ................... 62 2.2.1 Bioassay Results of the Amide Heterodimers .......................................... 63 2.3 Syntheses and Evaluation of ZAPA and its derivatives........................................ 68 2.3.1 Synthesis of ZAPA and its derivatives ..................................................... 68 2.3.2 Bioassay Results of ZAPA and its derivatives ......................................... 72 2.4 Syntheses and Evaluation of PEG-linked GABAA receptor analogs.................... 76 2.4.1 Synthesis of PEG-linked GABA amide dimers........................................ 76 2.4.2 Bioassay Results of PEG-linked GABA amide dimers ............................ 85 2.4.3 Synthesis of PEG-linked ZAPA analogs .................................................. 87 2.5 HPLC Analysis of GABA amides ........................................................................ 88 2.6 Conclusions........................................................................................................... 93 References and notes for chapter 2 ................................................................................... 95 Chapter 3 Experimental Section of GABA Project .................................................... 99 3.1 Chemistry.............................................................................................................. 99 3.1.1 General Methods....................................................................................... 99 3.1.2 Procedures............................................................................................... 100 3.2 Biological Assays................................................................................................128 v References for chapter 3 ................................................................................................. 132 Chaper 4 Introduction and Background of 1,4-benzodiazopines ........................... 133 4.1 Importance of 1,4-benzodiazopine scaffolds in medical chemistry.................... 133 4.2 Importance of exploring enantiopure quaternary 1,4-benzodiazepines.............. 136 4.3 Overview of Pioneering Work in Carlier’s Research Group.............................. 139 Reference for chapter 4................................................................................................... 143 Chapter 5 Syntheses of 1,4-Benzodiazepine Analogs .............................................. 150 5.1 Investigation of Enantioselective Synthesis of Quaternary (S)-alanine Derived Tetrahydro-1,4-benzodiazepin-3-ones...............................................................
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