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IMAGING SERVICES NORTH Boston Spa, Wetherby West Yorkshire, LS23 7BQ www.bl.uk This PDF was created from the British Library’s microfilm copy of the original thesis. As such the images are greyscale and no colour was captured. Due to the scanning process, an area greater than the page area is recorded and extraneous details can be captured. This is the best available copy ■^- ■■■f^ i% -■ -( : WH-‘'^ v :■> 4 %ÄÄ>-i-..>>. THE STHTHESIS ASD BIOCHEBCICAL ASD FHARNAGOLOaiCAL EVALaATION OF Y^ANUHOBOTYHIC ACII) AHTAQOHISTS. by Veil James NcLau^lixii B.Tech. A thesis submitted to the Council for National Academic Awards in partial fulfilment for the Degree of Doctotr of Philosophy Department of Chemistryf Department of Chemistry« Sir John Cass School of Chemioal Defense Solenoe and Technology! Establishment« City of London Polytechnic. Port on Down« Wiltshire. December« 1981. Memorandiim Except where acknowledgement Is made hy reference« this thesis and the experiments described were the \maided work of the author. The work '>^as carried out under the supervision of Dr. J. F. Collins in the Depcurfcment of Chemistry at the City of London Polytechnic in collaboration with Professor T. D. Inch at the Department of Chemistry« Chemical Defence Establishment« Porton Down« Wiltshire« during the period October 1976 - October 1979» and was supported by a strident ship from the Science Research Coimcil. Statement of Advanced Studies Itedertaken Attendance at a course of ireuropharmacolog7 Lectures for postgraduates« run by the IMiversity of London« as a fulfillment of the requirement of this degree, AttexxLanoe at several of the meetings of the British Pharmacological Society smd departmental lectures amd seminars at the Sir John C ,se College. Acknowledgement a I would like to thank Dr. J. F. Collins for his advice and encouragement during the tenure of this study» also other members of the department for their useful discussions and advice. I wish to thauik Dr. IT. G. Bowery» Depajrtment of Pharmacology» St. ThomasHospital Medical School for invaluable help and discussions; Dr. I. W. Lawston» Department of Chemistry» Chemical Defence Establishment» Porton Down» for excellent guidance in chemical synthetic techniques» and P. Villieuns» Department of Biological Sciences» Porton Down» for the toxic evaluation of compounds synthesised. Ny thanks also to Mrs. Anne Hoddinott for the excellent typing of this thesis. Finally» I am deeply indebted to my parents for their great support and endurance throu^out my academic career. U i i ) Abbreviations Y-Aminobutyric acid Central nervous system Anhydrous magnesium sulphate Boiling point Melting point Proton nuclear magnetic resonance Dihydropicrotozinin Corey-Paul in g ^ o It urn Y- ^ (M) 3 “ aminobutyric acid ^ethylene - ^ (IT)3 ” muscimol a-(8,10 - - dihydropiorotoxinin Cyclic guanosine 3’ 5* phosphate Cyclic adenosine phosphate Qlutamic aoid deoarbosqylase bicTclic 4-eubstituted 2,6,7-trioxa-l-i>hosphabicyolo phogphate eerters (2,2,2) ootan-l-ones silatranes l-(aryl or alkyl) - 2,8.9-trioxa-5-aza-l- silabicyolo (3f3f3) uxKiecanes Tetramethylenedisulphotetramine Cis—3-aminocyolohexane oarbozylio acid 4,5,6,7-tetrahydro-isoxaaole (5»5f-C) piridin-3-ol Tetrahydrofuran Tetramethylsilioon Coupling constant between hydrogens (oyoles per second) Chemical shift from HIS in ppm (v) Abstract Y^bninobutyrio acid has bssxi shown to bs a major inhibitory nsurotransmitter in the central nervous system (CKS)« This study is oonoemed with the examination of a number of QABA anteigonists in two injjitro^ assay systems. A series of cage-structured compounds including bicyolic phosphate esters (2,6,7 f-Trioxa-l-phosphabicyclo (2,2,2,) ootan-l-oxides with suitable 4-substituents) have been shown to be potent aA'RA antagonists. These compounds do not act directly on the postsysnaptio QABA receptor site but probably at the same site as another potent QABA antagonist piorotoxinin. It has been shown that the substituent group on the bridge-head on the 4-oarbon atom in the molecule plays a very important role in the toxic potency and GABA antagonistic properties of these compounds. A very b u U ^ and branched substituent caused a great increase in potency. This property was utilised in the synthesis of a series of these compoxxnds. The binling of radiolabelled ligands (both agonists yirt antagonists) to synaptic membrane fractions can bo utilised as a convenient assay for the study of GABA receptors. Such assays tusing frozen--thawed rat brain synaptosomal membranes has been demonstrated to be stereospeoifio and saturable using (^)- GABA, the potent GABA agonist (^)-muscimol also with GABA antagonists using (^)-bicuculline methobromide and (^)- <vi) dihydropicrotoxinin, Utilifling these methods a number of agonists and antagonists have been examined to evaluate their ability to displaoe the stereospeoific binding. The synthesised QIBA antagonists have also been used to evaluate their properties on the depolarising effeot of on the rat superior cervical ganglion in comparison to results obtained by known GABA, antagonists. The bicyolio phosphate esters were shown to bo very toxio when administered to mammals causing seizures and death» this effeot being attributed to the emtagonism of Q A M in the CHS. They wore shown to bo potent inhibitors of GABA in the superior cervical ganglion. In binding studios with (%)- muscimol they did not displaoe any of the bound agonist and also were shown not to displaoe bound (^)Hiihydropiorotoxinin. The res\ilt8 obtained in this study have been discussed and compared with structure-activity relationships to other compounds of similar structure. (vii) coBTEjars Title Memorandum Acknowledgment s Publioation Abbre viat ions Abstract 1.1 Int roduot ion 1.2 Evidence for GABA as a ne\irotransmitter 1.3 Mechanism of GABA action 1.4 GABA in invertebrates 1.5 Regional variation of GABA concentrations in the CHS 1.6 Stereochemistry of GABA 1.7 Drugs which affect the actions of GABA 1.8 The role of GABA in neurologic and psychiatric disorders nff/LPTBR 2 > lEOQS WHICH MDDIFr THE ACTIOHS OP Y-AMIHO- BUTYRIC ACID 40 2.1^ Analogues of bicyclio phosphorus esters 42 2.2 Piorotoxinin 50 2.3 Biouculline 57 2.4 Action of barbiturates 63 2.5. Action of bensodiazepines 64 (viii) 3.2 Preparation of 4-(2,2, dimethyl propyl) 2,6,7-trioxa-l-phosphabioyclo (2t2»2) ootan-l-one 81 3.3 Preparation of 4-(lf2, dimethyl propel) 2,6y7~trioza-l-idi08phabioyclo (2f2t2) octan-l-one 3.4 Preparation of 4-oyolopentyl 2.6,7- trioxa—l-jdioephabioyclo (2,2,2) octan—1-^ne 3.5 Attempted prepeiration of 4-(l»lf2,trimetlyrl- propyl) 2,6,7-trioxa-l-phos^abioyolo (2,2,2) octan-l-one 89 3.6 Attempted preparation of 3»4 dimethyl pentanal 93 3.7 Attempted preparation of 3f3i4 trimethyl pentanal 97 3.8 Attempted preparation of 1,1,2 trimethyl ethoxy propane 3.9 Acute toxicity of bicyclic phosphate esters 3.10 Discussion of synthetic routes 3.11 Conclusions qTAPTTO A Î PHABMACQLDQICAL MALDATION OF QABA AlüTAGOlSnSTS ilTD SYiyPHESISED BICTCLIC PHOSPHATES OU TEÏE ACTIOas nw flABA AHD CARBACHPL 0^ THE I30LATO snPWRTOR GERTtCAL OAHOUOH OP TOE RAT 114 116 4.1 Introduction 4.2 Methods and materials 117 4.3 Results and conclusions 119 (ix) nTTAPTOR s ; A STUDY OF THE GABA HECEFTOR DSISQ BDIDIIIO fPPnnTTlS Oli A STUAPTOSOllAL PHACTIOIT OP RAT BRADJ 145 148 5.1 Introduction 5.2 •Rinding studies with brain tissues on the opiate receptor 151 155 5.3 The glycine receptor 157 5.4 The GABA receptor 163 5.5 The use of ( % ) niuscimol in GABA binding 168 5.6 The use of (^)-IHP binding • 5.7 The binding assay 175 5.8 Binding studies using (^)Hnusoimol on rat brain synaptosomal membranes 180 5.9 Binding studies using (^)-IHP on rat b m i n syxiaptosomal membranes 207 DISCUSSION ASH COHCLUSIONS 214 6.1 £otreduction 215 6.2 The receptor model 216 6.3 The two-state receptor model 217 6.4 The active conformation of GABA 221 6.5 The anion channel 226 6.6 Prospects 230 6.7 Conolxieion 231 rcES 233 (x) CHAPTER 1 : Y-AMUTOBOTraiC ACID AS A IJEDROTRAITSMI'PIIIR 1.1 Introduct ion 1.2 Evidence to support QABA as a neurotransmitter 1.2.1 Synthesis 1.2.2 Storage 1.2.3 Release 1.2.4 Physiological action 1.2.3 Uiptake and inactivation 1.2.6 Antagonism. 1.3 Mechanism of QABA action 1.3.1 Postsynaptic inhibition 1.3.2 Presynaptic inhibition 1.3.3 Excitatory effects of GABA 1.4 GABA action in invertebrates 1.5 Regional variation of QABA concentrations in the CBS 1.5.1 Cerebral cortex 1.5.2 Cerebellum 1.5.3 Hippocampus 1.5.4 Basal ganglia 1.5.5 Olfactory bulb 1 .5.6 Retina 1*5»7 Spinal cord 1.5.8 Other areas Stereochemistry of GABA. Dru^ which affect the actions of GABA 1.7.1 GABA mimetics 1.7.2 Summary of the structure-activity requirements of GABA aigonists 1.7.3 OABA "inhibitors" 1.8 The role of GABA in neurologic and psychiatric disorders 1.8.1 Huntington’s Chorea 1.8.2 Parkinson * s Disease 1.8.3 Schizophrenia 1.8.4 Vitamin Bg Introduction^^jf—jiininobutyrio acid (GASA) (Pig»l»l) was first discovsrsd in the mammalian central nervous system (CBS) as one of the amino acids in mouse hrain extracts (Roberts and Prankeli 1950a). Later these brain extracts were shown to have a Factor I which had inhibitory effects on the synapses in the crayfish stretch receptor (Bazemore, Elliott and florey, 1957). This inhibitory Factor I was found to be GABA. Further research on GABA eventually established it as a ma.jor inhibitory neurotransmitter (Curtis and Watkins, I960, Em.ievic and Phillis 1963, BmjeviS a«d Schwartz 1966, Emjevi’6 197A, Curtis 1975)» Since then a great deal of evidence has been accumulated which supports the proposal that GABA is a naturally occurin« inhibitory neurotransmitter.
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  • Gabaergic Signaling Linked to Autophagy Enhances Host Protection Against Intracellular Bacterial Infections

    Gabaergic Signaling Linked to Autophagy Enhances Host Protection Against Intracellular Bacterial Infections

    ARTICLE DOI: 10.1038/s41467-018-06487-5 OPEN GABAergic signaling linked to autophagy enhances host protection against intracellular bacterial infections Jin Kyung Kim1,2,3, Yi Sak Kim1,2,3, Hye-Mi Lee1,3, Hyo Sun Jin4, Chiranjivi Neupane 2,5, Sup Kim1,2,3, Sang-Hee Lee6, Jung-Joon Min7, Miwa Sasai8, Jae-Ho Jeong 9,10, Seong-Kyu Choe11, Jin-Man Kim12, Masahiro Yamamoto8, Hyon E. Choy 9,10, Jin Bong Park 2,5 & Eun-Kyeong Jo1,2,3 1234567890():,; Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the brain; however, the roles of GABA in antimicrobial host defenses are largely unknown. Here we demonstrate that GABAergic activation enhances antimicrobial responses against intracel- lular bacterial infection. Intracellular bacterial infection decreases GABA levels in vitro in macrophages and in vivo in sera. Treatment of macrophages with GABA or GABAergic drugs promotes autophagy activation, enhances phagosomal maturation and antimicrobial responses against mycobacterial infection. In macrophages, the GABAergic defense is mediated via macrophage type A GABA receptor (GABAAR), intracellular calcium release, and the GABA type A receptor-associated protein-like 1 (GABARAPL1; an Atg8 homolog). Finally, GABAergic inhibition increases bacterial loads in mice and zebrafish in vivo, sug- gesting that the GABAergic defense plays an essential function in metazoan host defenses. Our study identified a previously unappreciated role for GABAergic signaling in linking antibacterial autophagy to enhance host innate defense against intracellular bacterial infection. 1 Department of Microbiology, Chungnam National University School of Medicine, Daejeon 35015, Korea. 2 Department of Medical Science, Chungnam National University School of Medicine, Daejeon 35015, Korea.
  • Preparation of N-‐(Boc)-‐Allylglycine Methyl Ester

    Preparation of N-‐(Boc)-‐Allylglycine Methyl Ester

    Preparation of N-(Boc)-Allylglycine Methyl Ester Using a Zinc-mediated, Palladium-catalyzed Cross-coupling Reaction N. D. Prasad Atmuri and William D. Lubell* Département de Chimie, Université de Montréal, P.O. Box 6128, Station Centre-ville, Montréal, QC H3C 3J7, Canada Checked by Jean-Nicolas Desrosiers, Nizar Haddad, and Chris H. Senanayake Iodine (1.3 equiv), Ph3P (1.3 equiv), OH Imidazole (1.3 equiv) I A. DCM, 0 °C - rt BocHN CO2Me 82% BocHN CO2Me 1 2 1. Zn (6 equiv), DMF Br Br (0.6 equiv), 60 °C I TMSCl (0.2 equiv), rt - 35 °C B. BocHN CO Me BocHN CO2Me 2 2. Br (1 M in THF) 2 3 Pd2(dba)3 (0.02 equiv) P(o-tol)3 (0.1 equiv) –78 °C - rt, 12 h 65% Procedure A. tert-Butyl (R)-1-(methoxycarbonyl)-2-iodoethylcarbamate (2). An oven- dried 1000-mL, three-necked, round-bottomed flask containing an egg- shaped Teflon®-coated magnetic stir bar (7 cm long) is equipped with a rubber septum with a thermometer, a 125 mL addition funnel and an argon inlet adaptor. The apparatus is purged with argon (Note 1). Keeping a Org. Synth. 2015, 92, 103-116 103 Published on the Web 4/6/2015 DOI: 10.15227/orgsyn.092.0103 © 2015 Organic Syntheses, Inc. positive flow of argon, the septum is removed temporarily and the flask is charged with triphenylphosphine (Note 2) (32.66 g, 124.5 mmol, 1.3 equiv) and 400 mL of dichloromethane (Note 3). The solution is stirred at room temperature and imidazole (8.47 g, 124.5 mmol, 1.3 equiv) (Note 2) is added in one portion.