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

University of Montana <a href="/goto?url=https://scholarworks.umt.edu/" target="_blank">ScholarWorks at University of Montana </a><a href="/goto?url=https://scholarworks.umt.edu/etd" target="_blank">Graduate Student Theses, Dissertations, & </a><a href="/goto?url=https://scholarworks.umt.edu/etd" target="_blank">Professional Papers </a> <a href="/goto?url=https://scholarworks.umt.edu/grad" target="_blank">Graduate School </a>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: <a href="/goto?url=https://scholarworks.umt.edu/etd?utm_source=scholarworks.umt.edu%2Fetd%2F9450&utm_medium=PDF&utm_campaign=PDFCoverPages" target="_blank">https:</a><a href="/goto?url=https://scholarworks.umt.edu/etd?utm_source=scholarworks.umt.edu%2Fetd%2F9450&utm_medium=PDF&utm_campaign=PDFCoverPages" target="_blank">/</a><a href="/goto?url=https://scholarworks.umt.edu/etd?utm_source=scholarworks.umt.edu%2Fetd%2F9450&utm_medium=PDF&utm_campaign=PDFCoverPages" target="_blank">/</a><a href="/goto?url=https://scholarworks.umt.edu/etd?utm_source=scholarworks.umt.edu%2Fetd%2F9450&utm_medium=PDF&utm_campaign=PDFCoverPages" target="_blank">scholarworks.umt.edu/etd </a><a href="/goto?url=https://goo.gl/forms/s2rGfXOLzz71qgsB2" target="_blank">Let us know how access to this document beneﬁts you. </a>Recommended Citation Denton, Travis Taylor, "Synthesis of conformationally constrained glutamate analogues and their preliminary evaluation as glutamate transport inhibitors" (2002). 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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 X J r P T -r Any copying for commercial purposes or financial gain may be undertaken only with the author’s explicit consent. 8/98 <ul style="display: flex;"><li style="flex:1">Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. </li><li style="flex:1">Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. </li></ul>SYNTHESIS OF CONFORMATIONALLY CONSTRAINED GLUTAMATE ANALOGUES AND THEIR PRELIMINARY EVALUATION AS GLUTAMATE TRANSPORT INHIBITORS by Travis Taylor D enTon 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 Microform3078916 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. Chemistry Denton, Travis T. Ph. D., December 2002 Synthesis of conformationally constrained glutamate analogues and their preliminary evaluation as glutamate transport inhibitors Director: Charles M. Thompson (J M / 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. 1thank 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. vReproduced 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. M ultigram synthesis o fthe key intermediate, dimethyl 4-oxoglutaconate (DOG) 2. Survey o fDOG as a dienophile in the Diels-Alder reaction 3. Ketone to amine transformations:form al 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). 5. Investigations into the synthesis o f 4-oxoglutamate 6. Synthesis o fstructurally constrained 2-oxoglutarate analogues: potential substrates o fdehydrogenases and aminotransferases 45 50 53 7. S ynthesis ofphosphono analogues o f 2-oxoglutarate, 2-oxoisocaproate, 2-oxoisovalerate and 2-oxo-3-methylvalerate 57 <ul style="display: flex;"><li style="flex:1">60 </li><li style="flex:1">III. Experimental </li></ul><ul style="display: flex;"><li style="flex:1">IV. References </li><li style="flex:1">109 </li></ul>Chapter 3: Enzymatic transformation of keto diacids to glutamic acid analogues <ul style="display: flex;"><li style="flex:1"><a href="#0_0">I. Introduction </a></li><li style="flex:1"><a href="#0_0">112 </a></li></ul>II. Results and Discussion 1. A nalysis o f Compounds 2.41 —2.45 as Substrates o f L-Amino Acid Dehydrogenases 2. Analysis o fKeto Diacid Analogues 2.41 - 2.45 as Substrates o fLDH, GTK, AspAT, AlaAT and KGDHC 116 118 120 <ul style="display: flex;"><li style="flex:1">l</li><li style="flex:1">/</li></ul>3. Preliminary Screening o f the CloneZymeT Library 4. A bility o fAT-5 to Catalyze Amine Transfer to Compounds 2.41 —2.45 using Phe 123 vii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5. Survey o f ce-Ketoglutarate Analogues as Inhibitors o fDehydrogenasesand Aminotransferases 130 132 133 III. Summary IV. Conclusion V. Experimental 1. E nzymes and Reagents 137 139 142 143 144 2. Enzyme assays 3. Apparatus 4. a-Ketoglutaric Acid Analogues VI. References Chapter 4: Evaluation of trans-3 ,4-Conformationally Constrained Glutamate Analogues (2.31,2.32,2.33,2.34) as Excitatory Amino Acid Transport Inhibitors <ul style="display: flex;"><li style="flex:1">I. Introduction </li><li style="flex:1">146 </li></ul><ul style="display: flex;"><li style="flex:1">148 </li><li style="flex:1">II. Biological Activity </li></ul>III. Experimental 1. EAAT2 uptake 152 153 155 156 157 2. EAA T3 and system x j uptake 3. VGLUT uptake IV. Collaborators V. References Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 5: Conclusions and Future Directions <ul style="display: flex;"><li style="flex:1">I. Conclusions </li><li style="flex:1">157 </li></ul><ul style="display: flex;"><li style="flex:1">159 </li><li style="flex:1">II. Future directions </li></ul>ix Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. List of Abbreviations <ul style="display: flex;"><li style="flex:1">elemental composition; calculated value </li><li style="flex:1">anal, calc </li></ul>ACPD AMPA anhyd atm trans-1-aminocyclopentane-1,3-dicarboxylate (R,S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)proprionic acid anhydrous atmosphere ATP br adenosine 5-triphosphate broad BSA calcd CBZ CBZC1 concd cpm bovine serum albumin calculated carbobenzyloxy carbobenzyloxy chloride concentrated counts per minute methylene chloride or dichloromethane chemical shift in parts per million day(s); doublet (spectral) doublet of doublets dimethoxyethane CH2C12 5ddd DME DMF DMSO td dimethylformamide dimethylsulfoxide triplet of doublets Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. EAAT ESI excitatory amino acid transporter electron spray ionization <ul style="display: flex;"><li style="flex:1">ethyl </li><li style="flex:1">Et </li></ul>EtOH Et20 EtOAc FT ethyl alcohol diethyl ether ethyl acetate Fourier Transform gram(s) g<ul style="display: flex;"><li style="flex:1">glu </li><li style="flex:1">glutamate </li></ul>GVT hglutamate vesicular transporter hour(s) HCI HEPES Hz hydrogen chloride (N-[2-hydroxylethyl]piperazine-N hertz HRMS IR high-resolution mass spectrum infrared Jcoupling constant (NMR) in hertz <ul style="display: flex;"><li style="flex:1">kainate </li><li style="flex:1">KA </li></ul><ul style="display: flex;"><li style="flex:1">K.C1 </li><li style="flex:1">potassium chloride </li></ul>micro PmMmultiplet (spectral), milli moles per liter <ul style="display: flex;"><li style="flex:1">m-CPBA </li><li style="flex:1">m-chloroperoxybenzoic acid </li></ul>XI Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. <ul style="display: flex;"><li style="flex:1">Me </li><li style="flex:1">methyl </li></ul>MeOH mg methyl alcohol milligram(s) MHz min mL megahertz minute(s) milliliter(s) mol mp mole(s) melting point m/z Nal NMDA NMR ppm qmass to charge ratio (mass spectrometry) sodium iodide N-methyl-D-aspartate nuclear magnetic resonance parts per million quartet QDC Rf quinoline-2,4-dicarboxyiate retention factor (in chromatography) room temperature singlet (NMR); second(s) bimolecular nucleophilic substitution triplet(spectra) rt sSn2 tTEA TFA THF triethylamine trifluoroacetic acid tetrahydrofuran xu Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TLC TMSBr Torr Ts thin layer chromatography trimethylsilylbromide 1 mm Hg, 1/760 atm tosyl, p-toluenesulfonyl ultraviolet UV XU1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. List of Figures Figure 1.1. Structure of L-glutamate Figure 1.2. EAA synapse Figure 1.3. Examples of structurally constrained and unconstrained analogues of L-glutamate utilized in characterizing the glutamate receptors Figure 1.4. Structurally constrained analogues of L-glutamate utilized in characterizing the EAATs Figure 1.5. Overlay of L -CCG-II and R, R-3,4-(4,5-cyclohexenyl)- L-glutamate rendered in PC Spartan Pro Figure 1.6. Proposed target 3,4-(4,5-cyclohexenyl)-D/L- glutamate Molecules Figure 2.1. Retrosynthetic disconnection scheme towards 3,4- conformationally restricted glutamates Figure 2.2. Synthesis of DOG (2.6) Figure 2.3. Transition states for endo vs. exo regioisomers Figure 2.4. Frontier orbital interactions in [4+2] cycloaddition reactions Figure 2.5. Orbital symmetry of 2 71and 4 k systems Figure 2.6. Representative regioselective outcomes due to substituent effects xiv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2.7. Synthesis of DOG as reported by Corey (1981) Figure 2.8. Monocyclic Diels-Alder adducts from symmetrical dienes Figure 2.9. Bicyclic Diels-Alder adducts 23 24 27 27 28 31 Figure 2.10. Diels-Alder adducts from extended aromatic dienes Figure 2.11. Diels-Alder adducts derived from unsymmetrical dienes Figure 2.12. Structure of 2, 3,4, 5-tetraphenylcyclopentadienone Figure 2.13. ‘H NMR of the regioisomer mixture obtained from the <ul style="display: flex;"><li style="flex:1">Diels-Alder reaction between DOG and isoprene </li><li style="flex:1">33 </li></ul>34 Figure 2.14. lH NMR comparison of furan-DOG adduct, 2-methylfuran-DOG and N-trifluoroacetyl furfuralamine-DOG adduct Figure 2.1S. Formation of the imine intermediate in route to reductive <ul style="display: flex;"><li style="flex:1">animation </li><li style="flex:1">37 </li></ul><ul style="display: flex;"><li style="flex:1">38 </li><li style="flex:1">Figure 2.16. Ketoxime of compound 2.8 </li></ul>Figure 2.17. Synthesis of N, N-dimethylhydrazones of the <ul style="display: flex;"><li style="flex:1">Diels-Alder adducts </li><li style="flex:1">39 </li></ul>40 42 43 Figure 2.18. Hydrazone products from select Diels-Alder adducts Figure 2.19. Sodium hydrosulfate reduction of hydrazones Figure 2.20. Saponification to afford the target amino acids Figure 2.21. Proposed mechanism and reaction outcome between DOG <ul style="display: flex;"><li style="flex:1">and a, p-unsaturated N, N-dimethylhydrazones </li><li style="flex:1">45 </li></ul>Figure 2.22. Regiochemical outcomes of the reaction of methacrolein xv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. N, N-dimethylhydrazone with acrylonitrile and <ul style="display: flex;"><li style="flex:1">methylvinylketone(Serckx-Poncin, 1983) </li><li style="flex:1">46 </li></ul>47 Figure 2.23. Adducts of the reaction between the N, N-dimethylhydrazones of acrolein and methacrolein and DOG Figure 2.24. Reduction of hydrazine and olefin with zinc <ul style="display: flex;"><li style="flex:1">in acetic acid </li><li style="flex:1">48 </li></ul>50 Figure 2.25. Proposed synthesis of 4-oxoglutamate Figure 2.26. 1,4 Addition-cyclization-enamine formation of

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