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Synthesis of Conformationally Constrained Glutamate Analogues and Their Preliminary Evaluation As Glutamate Transport Inhibitors

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Synthesis of Conformationally Constrained Glutamate Analogues and Their Preliminary Evaluation As Glutamate Transport Inhibitors University of Montana ScholarWorks at University of Montana Graduate Student Theses, Dissertations, & Professional Papers Graduate School 2002 Synthesis of conformationally constrained glutamate analogues and their preliminary evaluation as glutamate transport inhibitors Travis Taylor Denton The University of Montana Follow this and additional works at: https://scholarworks.umt.edu/etd Let us know how access to this document benefits ou.y Recommended Citation Denton, Travis Taylor, "Synthesis of conformationally constrained glutamate analogues and their preliminary evaluation as glutamate transport inhibitors" (2002). Graduate Student Theses, Dissertations, & Professional Papers. 9450. https://scholarworks.umt.edu/etd/9450 This Dissertation is brought to you for free and open access by the Graduate School at ScholarWorks at University of Montana. It has been accepted for inclusion in Graduate Student Theses, Dissertations, & Professional Papers by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. ProQuest information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Maureen and Mike MANSFIELD LIBRARY The University of Montana Permission is granted by the author to reproduce this material in its entirety, provided that this material is used for scholarly puiposes and is properly cited in published works and reports. ♦♦Please check "Yes” or "No" and provide signature** Yes, I grant permission ______ No, I do not grant permission ___________ Author's Signature: Date; \~ls! 1 j XJrPT-r Any copying for commercial purposes or financial gain may be undertaken only with the author’s explicit consent. 8/98 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. SYNTHESIS OF CONFORMATIONALLY CONSTRAINED GLUTAMATE ANALOGUES AND THEIR PRELIMINARY EVALUATION AS GLUTAMATE TRANSPORT INHIBITORS by T ravis T a y lo r D en to n B.S Central Washington University, USA, 2002 presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy The University of Montana December 2002 Approved by: Chairperson: Dean, Graduate School: 1 1 - 2-3, — Q ~2_ Date: Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number 3078916 Copyright 2002 by Denton, Travis Taytor All rights reserved. UMI* UMI Microform 3078916 Copyright 2003 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Denton, Travis T. Ph. D., December 2002 Chemistry Synthesis of conformationally constrained glutamate analogues and their preliminary evaluation as glutamate transport inhibitors Director: Charles M. Thompson (JM/ The primary goals of the research effort were to assess the reactivity of dimethyl 4- oxoglutaconate in the normal electron demand Diels-Alder reaction. Once the Diels-Alder reaction was deemed a success, the adducts were transformed into conformationally constrained analogues of 2-oxoglutarate by saponification. The cyclic 2-oxoglutarate analogues were evaluated as substrates and inhibitors of a number of transaminases and dehydrogenases. One transaminase was found to convert the oxoglutarate analogues to glutamate analogues. The Diels-Alder adducts were also chemically transformed into conformationally constrained analogues of glutamate by preliminary formation of the corresponding N, N-dimethylhydrazones and subsequent reduction with sodium hydrosulfite and saponification. The glutamate analogues were tested at four glutamate transporters and two compounds, namely 6-(amino-carboxy-methyl)-cyclohex-3-enecarboxylic acid and 3- (amino-carboxy-methyl)-bicyclo[2.2.2]oct-5-ene-2-carboxylic acid, 38 ± 2 and 35 ± 5 percent of control, respectively, were found to be potent and selective inhibitors of the excitatory amino acid transporter 2 (EAAT2). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Copyright by Travis T. Denton, 2002 All rights reserved iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Dedication It is with extreme delight that I dedicate this thesis to the loving memory of my recently departed Grandfather Clifford Denton, my grandmother Wilda Denton, my mother Karen Denton and my father James C. Denton. Without these people my life would not be complete. Thank you!! iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Acknowledgements I have to express my extreme gratitude towards Professor Charles M. Thompson. His abilities as a synthetic medicinal chemist and a mentor are insurmountable. He has proven to be an outstanding advisor and a truly great friend and collaborator. I must thank my entire research thesis committee Professors Donald Kiely, Edward Waali, Holly Thompson, John Gerdes and Richard bridges. Their comments and criticisms of my thesis work and future goals proved to be a very valuable resource I am appreciative of the entire Thompson group for their intellectual as well as social stimulation. I would especially like to acknowledge Professor Sean Esslinger who guided me through most of my initial hands-on laboratory techniques while a postdoctoral fellow in the Thompson group. I also thank Dr. Joe Degraw for all of his insightful chemical ideas, synthetic strategies and his great golf game. 1 thank Todd Talley for whom I have shared many insightful conversations, Troy Voelker for all of his insight, chemical knowledge and astute conversations. I appreciate the input of the rest of the Thompson group for valuable help in the preparation of manuscripts, posters and seminars including: Katie George, Doug Williamson, Jason Mullins, Jean-Louis Etoga, Jennifer Saltmarsh, Christina Carrigan, and Greg Muth. I would also like to show my appreciation to the Richard J. Bridges group for all their wonderful help with the pharmacology obtained for my thesis especially Todd Seib, Fred Rhoderick, Brady Warren and Kimberly Cybulski. I must gratefully acknowledge Dr. Arthur J. L. Cooper for all his help and mentoring in every facet of the enzymology used in my thesis work. And last but by no means least, I have to thank my wife Monica for all her love and support throughout my Ph. D. pursuit. v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Contents List of Abbreviations List of Figures List of Tables List of Equations Chapter 1: Overview of the Glutamate neurosystem I. Glutamate as a Neurotransmitter II. References Chapter 2: Organic Synthesis I. Introduction II. Results and Discussion 1. Multigram synthesis o f the key intermediate, dimethyl 4-oxoglutaconate (DOG) 2. Survey o f DOG as a dienophile in the Diels-Alder reaction 3. Ketone to amine transformations: formal reductive amination 4. Synthesis o f l-dimethylamino-3-methoxalyl- 1,2,3,4-tetrahydropyridine-2-carboxylic acid vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. methyl esters. Reaction o fDOG with azadiene (a, fi-unsaturated N, N-dimethylhydrozones). 45 5. Investigations into the synthesis o f 4-oxoglutamate 50 6. Synthesis o f structurally constrained 2-oxoglutarate analogues: potential substrates o f dehydrogenases and aminotransferases 53 7. Synthesis ofphosphono analogues o f 2-oxoglutarate, 2-oxoisocaproate, 2-oxoisovalerate and 2-oxo-3-methylvalerate 57 III. Experimental 60 IV. References 109 Chapter 3: Enzymatic transformation of keto diacids to glutamic acid analogues I. Introduction 112 II. Results and Discussion 1. Analysis o f Compounds 2.41 — 2.45 as Substrates o f L-Amino Acid Dehydrogenases 116 2. Analysis o f Keto Diacid Analogues 2.41 - 2.45 as Substrates o f LDH, GTK, AspAT, AlaAT and KGDHC 118 T l / 3. Preliminary Screening of the CloneZyme Library 120 4. Ability o f AT-5 to Catalyze Amine Transfer to Compounds
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