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Design and Synthesis of Novel Prodrugs to Modulate Gaba Receptors in Cancer Hui Zhang

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Design and Synthesis of Novel Prodrugs to Modulate Gaba Receptors in Cancer Hui Zhang DESIGN AND SYNTHESIS OF NOVEL PRODRUGS TO MODULATE GABA RECEPTORS IN CANCER HUI ZHANG Thesis submitted in partial fulfilment of the requirements of Edinburgh Napier University for the degree of Doctor of Philosophy March 2017 Declaration It is hereby declared that this thesis and the research work upon which it is based were conducted by the author, Hui Zhang. Hui Zhang Abstract of Thesis GABA (gamma-amino butanoic acid) is the main inhibitory neurotransmitter in the mammalian central nervous system. GABA has been found to play an inhibitory role in some cancers, including colon carcinoma, cholangiocarcinoma, and lung adenocarcinoma. Growing evidence shows that GABAB receptors are involved in tumour development. The expression level of GABAB receptors was found to be upregulated in some human tumours, including the pancreas, and cancer cell lines, suggesting that GABAB receptors may be potential targets for cancer therapy and diagnosis. In this research programme, several diverse series of potential anticancer prodrugs of GABA and GABA receptor-targeting agents have been rationally designed and synthesised for selective activation in the tumour microenvironment. In one approach, a series of oligopeptide conjugate prodrugs have been synthesised as protease-activatable substrates for either the extracellular matrix metalloproteinase MMP-9 or the lysosomal endoprotease legumain; each of which are overexpressed in the tumour environment and are effectors of tumour growth and metastasis. Specifically, a novel fluorogenic, oligopeptide FRET substrate prodrug of legumain HZ101 (Rho-Pro-Ala-Asn~GABA-spacer-AQ) has been characterised and shown to have theranostic potential. Proof of principle has been demonstrated using recombinant human legumain for which HZ101 is an efficient substrate and is latently quenched until cleaved. HPLC methods have been developed to monitor prodrug activation. In another approach, cyclic prodrugs of the GABA-B receptor agonist baclofen have been designed to be activated in the acidic environment of solid tumours to exert antitumour effects through modulation of the receptor response. During the oligopeptide synthetic work, novel, coloured, anthraquinone-based reagents have been designed and evaluated as new chemical tools for amine detection and monitoring in solid phase peptide synthesis (SPPS); characterisation by spectroscopic and HPLC methods have demonstrated their advantages over existing methods and their potential applications for use on solid supported resins. Contents Acknowledgements ........................................................................................................................ 1 Preface ........................................................................................................................................... 2 Abbreviations ................................................................................................................................. 7 Nomenclature ................................................................................................................................ 8 Chapter 1. MMP-9 Activated Prodrugs ........................................................................................ 10 1.1 Abstract .................................................................................................................................. 10 1.2 Hypothesis .............................................................................................................................. 10 1.3 Introduction ......................................................................................................................... 11 1.3.1 MMPs in Tumour and Neurodegenerative Disease ........................................................ 11 1.3.1.1 MMPs activation and regulation .............................................................................. 13 1.3.1.2 MMPs inhibitors ....................................................................................................... 14 1.3.1.3 The substrate preference of MMP-9 ....................................................................... 16 1.3.2 GABA and its receptors ................................................................................................... 18 1.3.2.1 Regulatory role of GABA in Tumour ......................................................................... 18 1.3.2.2 GABAA receptors in Tumour ..................................................................................... 19 1.3.2.3 GABAB receptors in Tumour ..................................................................................... 21 1.3.3 Tumour activated prodrugs ............................................................................................ 22 1.4 Results and discussion ......................................................................................................... 24 1.4.1 The design strategy of MMP-9 activated prodrugs ........................................................ 24 1.4.2 Synthesis of Model compounds ...................................................................................... 25 1.4.2.1 Synthesis of Fmoc-Ala-[Boc-Spacer]-AQ (HZ26) ...................................................... 26 1.4.2.2 Synthesis of H-Ala-Spacer-AQ TFA salt (HZ27) ......................................................... 28 1.4.2.3 Synthesis of H-Ala-[Boc-Spacer]-AQ (HZ28) ............................................................. 28 1.4.2.4 Synthesis of Fmoc-Ala-Propranolol (HZ29) .............................................................. 29 1.4.3 Synthesis of Prodrug 1: Fmoc-Pro-Ala-Gly-Leu-Ala-Ala-Propranolol (HZ32) .................. 31 1.4.3.1 Resin swelling ........................................................................................................... 31 1.4.3.2 Deprotection of Fmoc protecting group .................................................................. 32 1.4.3.3 Colour Test ............................................................................................................... 33 1.4.3.4 Peptide coupling ...................................................................................................... 34 1.4.3.5 Hexapeptide-resin cleavage ..................................................................................... 35 1.4.4 Synthesis of Prodrug 2: AQ-Spacer-Pro-Ala-Gly-Leu-Ala-Ala-Propranolol (HZ34) .......... 39 1.4.5 Synthesis of Prodrug 3: FITC-Pro-Ala-Gly-Leu-Pro-GABA-[Propyl-spacer]-AQ (HZ43) .... 43 1.4.6 Synthesis of Prodrug 4: AQ-[Propyl-spacer]-Pro-Ala-Gly-Leu-Ala-ethylpiperidine-3-carboxylate (HZ45) ..................... 47 I 1.4.7 Synthesis of Prodrug 5: Podophyllotoxin-Pro-Ala-Gly-Leu-Pro-GABA-[Propyl-spacer]-AQ (HZ 47) ...................................................................................................................................... 50 1.4.8 Synthesis of Prodrug 6: AQ-Spacer-Pro-Ala-Gly-Nva-Pro-Baclofen (HZ54) .................... 53 1.4.9 Synthesis of Prodrug 7: 5(6)-Carboxyfluorescein-Pro-Ala-Gly-Leu-Pro-GABA-[Propyl-spacer]-AQ (HZ57) ................... 58 1.4.10 Synthesis of Prodrug 8: Podophyllotoxin-Succinyl-Pro-Ala-Gly-Leu-Ala-Ala-Piperazinyl-AQ (HZ16) ............................. 60 1.4.10.1 Synthesis of Boc-Ala-Ala-Piperazinyl-AQ (HZ7) ...................................................... 60 1.4.10.2 Synthesis of H-Ala-Ala-Piperazinyl-AQ TFA salt (HZ8) ............................................ 62 1.4.10.3 Synthesis of Boc-Leu-Ala-Ala-Piperazinyl-AQ (HZ9) ............................................... 64 1.4.10.4 Synthesis of H-Leu-Ala-Ala-Piperazinyl-AQ TFA salt (HZ10) ................................... 64 1.4.10.5 Synthesis of Boc-Ala-Gly-Leu-Ala-Ala-Piperazinyl-AQ (HZ11) ................................ 65 1.4.10.6 Synthesis of H-Ala-Gly-Leu-Ala-Ala-Piperazinyl-AQ TFA salt (HZ12) ...................... 67 1.4.10.7 Synthesis of Boc-Pro-Ala-Gly-Leu-Ala-Ala-Piperazinyl-AQ (HZ13) ......................... 68 1.4.10.8 Synthesis of H-Pro-Ala-Gly-Leu-Ala-Ala-Piperazinyl-AQ TFA salt (HZ14) ............... 69 1.4.10.9 Synthesis of Succinyl-Pro-Ala-Gly-Leu-Ala-Ala-Piperazinyl-AQ (HZ15) .................. 70 1.4.10.10 Synthesis of Prodrug 8: Podophyllotoxin-Succinyl-Pro-Ala-Gly-Leu-Ala-Ala-Piperazinyl-AQ (HZ16) ......................... 71 1.4.11 HT1080 Cell Culture ...................................................................................................... 76 1.4.11.1 Materials for HT1080 cell culture .......................................................................... 76 1.4.11.2 Method for HT1080 cell culture ............................................................................. 77 1.4.12 Fluorescence Studies of Prodrug 3 (HZ43) and Prodrug 7 (HZ57) ................................ 77 1.5 Conclusion ............................................................................................................................ 80 1.6 Structure Library .................................................................................................................. 82 1.7 Experimental ........................................................................................................................ 90 1.7.1 General methods ............................................................................................................ 90 1.7.1.1 Thin layer chromatography (TLC) ............................................................................. 90 1.7.1.2 Thick TLC plate ........................................................................................................
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