UCL SCHOOL OF PHARMACY BRUNSWICK SQUARE Synthesis and discovery of the putative cognitive enhancer BRS- 015: effect on glutamatergic transmission and synaptic plasticity Blanka Szulc Supervisors: Dr Stephen Hilton and Dr Arnaud Ruiz 29/07/2014 This research project is submitted in part fulfilment of the requirements for the PhD degree, UCL School of Pharmacy Department of Pharmaceutical and Biological Chemistry and the Pharmacology Department Blanka Szulc Discovery and mode of action of BRS-015 Abstract This thesis is concerned with the discovery of a novel heterocyclic compound – BRS-015, its synthesis and an analysis of its effects on excitatory synaptic transmission at a major pathway in the brain. BRS-015 is related to the natural product clausenamide, which has been shown to facilitate synaptic transmission. As such, clausenamide and related analogues may possess therapeutic potential as memory enhancing drugs, which are in urgent need of development due to the increasing numbers of patients diagnosed with memory disorders and for which there is no current effective therapy. BRS-015 was synthesized using a novel approach to the core structure of clausenamide involving an intramolecular acylal cyclisation reaction, which has not previously been reported. The first section of the thesis opens with a description of the discovery, structure and biological activity of clausenamide and discussion of previous synthetic strategies adopted by a number of research groups and attempts to classify these into the varying approaches towards the central core of clausenamide. The second section describes the structure of the rat brain and the types of processes involved in memory formation, as well as the neurophysiological assays used to investigate synaptic transmission and plasticity. The second group of chapters describes our own approach to the core of clausenamide and the synthesis of BRS-015, with a detailed discussion of the structural analysis and investigation of the intramolecular acylal cyclisation reaction used during the synthetic process. The third chapter describes the neurophysiological assays used in our investigations into the effects of BRS-015, which was tested against glutamatergic synaptic transmission and plasticity in acute rat hippocampal slices. BRS-015 was shown to reversibly enhance the amplitude of AMPA receptor mediated EPSCs recorded from CA3 pyramidal neurones and evoked by dentate stimulation. When tested in the presence of selective glutamate receptor antagonists, BRS-015 did not have this powerful enhancing effect on kainate or NMDA receptor mediated EPSCs. In addition, BRS-015 increased the amplitude of glutamate-evoked currents in CA3 pyramidal neurones and did not alter short-term synaptic plasticity but facilitated the induction of mossy fibre LTP, with little effect at associational/commissural synapses. BRS-015 has striking enhancing properties on AMPA receptor mediated synaptic transmission at mossy fibre synapses either by directly interacting with AMPA receptors or via indirect modulation, the mechanisms of which could lead to synapse strengthening. i Blanka Szulc Discovery and mode of action of BRS-015 Acknowledgements Firstly I would like to express my sincere thanks to both of my supervisors - Dr Stephen Hilton and Dr Arnaud Ruiz for all of their help, support and advice during my PhD from transitioning through to electrophysiology from chemistry. Your experience and great guidance enabled me achieve success on such a wide-ranging project. Thank you for all your time and in particular your proof- reading of this thesis! I would secondly like to thank all of my laboratory colleagues in Steve and Arnaud’s labs for all their help and support over the years. Special thanks go to Bruno, Georgina, Bhaven, Moussa, Chris, Zenobia, Raiza, Anna and Antonia for all your help, friendship and advice over my time at UCL School of Pharmacy. I would also like to thank the staff in both departments for their friendship and support who are too numerous to mention. I would like to thank Dr Gary Parkinson for the X-ray structure of the cyclised product and for patiently allowing me to watch him at work. Thank you also to Colin and Mire for your help with NMR and to Emmanual for mass spectrometry. Thanks also go to the team in the biological service unit at the School for their help and advice. I would also like to thank the Leverhulme Trust for generously funding this research, with the provision of a PhD studentship. Lastly, thanks to all of my family and friends, who are close to my heart who have always been there for me. ii Blanka Szulc Discovery and mode of action of BRS-015 Plagiarism Statement I, Blanka Szulc, hereby confirm that the work submitted in the thesis is my own. Any ideas submitted in this thesis are my own. Any ideas, quotations, and paraphrasing from other peoples work and publications have been appropriately referenced. I have not violated UCL School of Pharmacy’s policy on plagiarism. Signature ....................................... Date ............................... iii Blanka Szulc Discovery and mode of action of BRS-015 Abbreviations ABSA p-Acetamidobenzenesulfonyl azide ACSF Artificial cerebrospinal fluid A/C Associational-commisural fibres AD Alzheimer’s disease ADA 5’-Nucleotidase adenosine deaminase ADHD Attention deficit hyperactivity disorder ADME Absorption, distribution, metabolism, elimination AGP α1-Acid glycoprotein AMPA α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid app Apparent BOC tert-Butoxycarbonyl br Broad Bu Butyl °C Degree Celcius CAMKIIα Calcium/calmodulin-dependent protein kinase IIα Cat Catalytic CNS Central nervous system CREB cAMP response element-binding protein d Doublet DABCO Diazabicyclo[2.2.2]octane DBU Diazabicyclo[5.4.0]undec-7-ene DCG-IV (2S,2'R,3'R)–2–(2',3'–Dicarboxycyclopropyl)glycine DCM Dichloromethane DMAP 4-N,N-Dimethylaminopyridine DMF N,N-Dimethylformamide DMSO Dimethylsulfoxide EDTA Ethylenediaminetetraacetic acid ee Enantiomeric excess EPSCs Excitatory postsynaptic currents iv Blanka Szulc Discovery and mode of action of BRS-015 EPSPs Excitatory postsynaptic potentials Et Ethyl EtOAc Ethyl acetate f-EPSPs Field excitatory postsynaptic potentials g Grammes GC Granule cells GCL Granule cell layer h Hours HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid HFS High frequency stimulation HPLC High performance liquid chromatography IAC Intramolecular acylal cyclisation IL-1β Interleukin 1 beta IR Infra red KAR Kainate receptor LDA Lithium diisopropylamide LTD Long-term depression LTP Long term potentiation M Molar m Multiplet mCPBA meta-Chloroperoxybenzoic acid Mes Mesityl Me Methyl MF-LTP Mossy fibre LTP mg Milligrams mGluRs Metabotropic glutamate receptors MFBs Mossy fibre boutons MHz Mega Hertz mL Millilitre ML Molecular layer mmol Millimoles v Blanka Szulc Discovery and mode of action of BRS-015 mp Melting point MS Multiple sclerosis m/z Mass to charge ratio NADPH Nicotinamide adenine dinucleotide phosphate NBS N-Bromosuccinimide NCS N-Chlorosuccinimide NHC N-Heterocyclic carbene NMDA N-Methyl-D-aspartate NMR Nuclear magnetic resonance nOe Nuclear Overhauser effect PG Protecting group PhTx Polyamine toxins PIFA [Bis(trifluoroacetoxy)iodo]benzene PPF Paired pulse facilitation ppm Parts per million PS Population spike PTP Post-tetanic potentiation q Quartet quin Quintet Rf Retention factor ROS Reactive oxygen species rt Room temperature s Secondary s Singlet SAR Structure activity relationship SG Stratum granulosum t Tertiary t Triplet TBDMSCl tert-Butyldimethylsilyl chloride TFA Trifluoroacetic acid THF Tetrahydrofuran vi Blanka Szulc Discovery and mode of action of BRS-015 TLC Thin layer chromatography TMS Tetramethylsilane TMSCl Trimethylsilyl chloride TMSOTf Trimethylsilyl trifluoromethanesulfonate TNF-α Tumor necrosis factor UV Ultra violet μL Microlitre VDCC Voltage-dependent calcium channels vii Blanka Szulc Discovery and mode of action of BRS-015 Contents 1.0. Introduction ................................................................................................ 1 1.1. General Introduction ............................................................................... 2 1.1.1. Memory Deficit Disorders ........................................................................... 2 1.1.2. Nootropic drugs ......................................................................................... 3 1.1.3. AMPA modulators in a clinical setting .......................................................... 9 1.2. Clausenamide ................................................................................................. 11 1.2.1. Mode of action of clausenamide................................................................. 12 1.2.2. Previous synthetic approaches to clausenamide ........................................ 14 1.3. Biological introduction .................................................................................. 24 1.4. Glutamatergic neurotransmission ................................................................ 24 1.5. Ionotropic Glutamate Receptors ................................................................... 25 1.5.1. AMPA receptors ......................................................................................... 26 1.5.2. NMDA receptors ........................................................................................ 27 1.5.3. Kainate receptors ....................................................................................... 28 1.6. Anatomy of the hippocampus ......................................................................