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

University of Montana

Graduate Student Theses, Dissertations, & Professional Papers
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2002

Synthesis of conformationally constrained glutamate analogues and their preliminary evaluation as glutamate transport inhibitors

Travis Taylor Denton

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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:

UMI Number 3078916

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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).

Copyright by Travis T. Denton, 2002
All rights reserved

iii

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

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.

v

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

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

60

III. Experimental

IV. References

109

Chapter 3: Enzymatic transformation of keto diacids to glutamic acid analogues

I. Introduction

112

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

l

/

3. Preliminary Screening o f the CloneZyme^TLibrary 4. A bility o fAT-5 to Catalyze Amine Transfer to
Compounds 2.41 —2.45 using Phe

123 vii

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

I. Introduction

146

148

II. Biological Activity

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

Chapter 5: Conclusions and Future Directions

I. Conclusions

157

159

II. Future directions

ix

List of Abbreviations

elemental composition; calculated value

anal, calc

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

EAAT ESI excitatory amino acid transporter electron spray ionization

ethyl

Et

EtOH Et20 EtOAc FT ethyl alcohol diethyl ether ethyl acetate Fourier Transform gram(s) g

glu

glutamate

GVT hglutamate vesicular transporter hour(s)
HCI HEPES Hz hydrogen chloride (N-[2-hydroxylethyl]piperazine-N hertz
HRMS IR high-resolution mass spectrum infrared

J

coupling constant (NMR) in hertz

kainate

KA

K.C1

potassium chloride

micro
P

mMmultiplet (spectral), milli moles per liter

m-CPBA

m-chloroperoxybenzoic acid

XI

Me

methyl

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

TLC TMSBr Torr Ts thin layer chromatography trimethylsilylbromide 1 mm Hg, 1/760 atm tosyl, p-toluenesulfonyl ultraviolet
UV

XU1

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

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

Diels-Alder reaction between DOG and isoprene

33

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

animation

37

38

Figure 2.16. Ketoxime of compound 2.8

Figure 2.17. Synthesis of N, N-dimethylhydrazones of the

Diels-Alder adducts

39

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

and a, p-unsaturated N, N-dimethylhydrazones

45

Figure 2.22. Regiochemical outcomes of the reaction of methacrolein

xv

N, N-dimethylhydrazone with acrylonitrile and

methylvinylketone(Serckx-Poncin, 1983)

46

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

in acetic acid