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Synthesis and Biological Evaluation of Novel Guanine-containing Compounds as Potential HIV-1 Non-nucleoside Reverse Transcriptase or Integrase Inhibitors KASSIM FOLORUNSHO ADEBAMBO Submitted for the Degree of Doctor of Philosophy Heriot – Watt University School of Engineering and Physical Sciences March 2010 The copyright in this thesis is owned by the author. Any quotation from the thesis or use of any of the information contained in it must acknowledge this thesis as the source of the quotation or information. ABSTRACT The global epidemic of HIV/AIDS has become one of the most pressing public health emergencies of this century. Various chemotherapy approaches towards the treatment of HIV-1 have been developed. These approaches were based on targets identified in the life-cycle of HIV-1. In this research work the synthesis of novel guanine compounds is described which were tested for their activities towards reverse transcriptase and integrase enzymes. Initial attempts to synthesise Boc and Cbz protected guan-9-ylacetic acids from commercially available 2-amino-6-chloropurine were inspired by the work of Dey and Garner. Their approach was extended to the synthesis N2-mono-Boc-guan-9ylacetic acid in 55% yield. Furthermore, a novel route was designed to the latter compound involving the activation of the exocyclic amino group of the purine ring with triphosgene, followed by addition of tert-butanol. product in 55% yield. This approach gave the desired Although the yields of the two strategies were similar, the time taken for completion of the second route was much shorter (3 days cf 7 days). An attempt to synthesize N2-diBoc-guan-9-ylacetic acid only afforded this compound in 20% yield. The activation of the exocyclic amino group of 2-amino-6-chloropurine with triphosgene was also explored for the synthesis of N2-Cbz protected guan-9-ylacetic acid. This gave the desired Cbz-protected N2-guanyl-9-ylacetic acid in yields ranging from 50% to 90% depending on whether ethylbromoacetate or tert-butylbromoacetate was used. The synthesis of orthogonally protected guanine-PNA monomers is described. Boc- and Fmoc-protected aminoethylglycine (PNA backbones) were efficiently prepared using established procedures. The various intermediates were screened for their biological activities and it was found out that N2-benzyloxycarbonylguan-9-ylacetic acid derivatives inhibited HIV-1 reverse transcriptase by interacting with the non-nucleoside binding pocket and they showed a reduced loss of potency towards common drug resistant HIV-1 mutant strains. Various structural modifications of N2- benzyloxycarbonylguan-9-ylacetic acid derivatives were also prepared. Finally, attempts were made to synthesize guanine-PNA dimers and guanine-rich PNA oligomers containing guanine and thymine bases. ii DEDICATION This work is dedicated to God and to over 40 million people living with HIV worldwide. iii ACKNOWLEDEMENT I seize this opportunity to thank the following people for their support during my study, Professor Brian Wherret and Professor Alan Welch for their moral and financial support (James Watt Fee Only Scholarship), Professor Dave Adam for his various useful scientific advices. The untiring support of my supervisor Dr. Nicola Howarth morally, academically and socially, gave me the necessary energy needed to complete my PhD work without any problem. I am very grateful for her honesty, hospitality, and willingness to help at anytime. I acknowledge the support of the technical staff of the department of Chemistry Heriot Watt University, namely: Dr. Alan Boyd, for NMR Services, Christina Graham, for elemental analysis, Dr. Georgina Rosair for X-ray analysis and EU-Frame-work Project 6 on Targeting Replication and Integration of HIV (TRiOH) for bench and conference travelling expenses. I am also grateful to my research co-workers in the Department of Chemistry (Research Associates and PhD students), for their support and love during the PhD programme. I acknowledged the contribution of Pastor Christiana Longe of Redeemed Christian Church of God (Open Heavens Edinburgh) and other ministers of God, for their financial and spiritual support during my PhD programme. Finally, I appreciate the support of my family and in-laws during this program. To my little princesses Boluwatife, Mofeoluwa, Oyinoluwabukunmi and my darling wife Victoria I say a very big thank you for your support and understanding during the laboratory work and my write-up period, thank you and God bless you all. iv ACADEMIC REGISTRY Research Thesis Submission Name: ADEBAMBO, KASSIM FOLORUNSHO School/PGI: ENGINEERING AND PHYSICAL SCIENCES Version: FINAL (i.e. First, Resubmission, Final) Degree Sought (Award and Subject area) PHD ORGANIC CHEMISTRY Declaration In accordance with the appropriate regulations I hereby submit my thesis and I declare that: 1) 2) 3) 4) 5) * the thesis embodies the results of my own work and has been composed by myself where appropriate, I have made acknowledgement of the work of others and have made reference to work carried out in collaboration with other persons the thesis is the correct version of the thesis for submission and is the same version as any electronic versions submitted*. my thesis for the award referred to, deposited in the Heriot-Watt University Library, should be made available for loan or photocopying and be available via the Institutional Repository, subject to such conditions as the Librarian may require I understand that as a student of the University I am required to abide by the Regulations of the University and to conform to its discipline. Please note that it is the responsibility of the candidate to ensure that the correct version of the thesis is submitted. Signature of Candidate: Date: 19TH AUGUST 2010 Submission Submitted By (name in capitals): ADEBAMBO, KASSIM FOLORUNSHO Signature of Individual Submitting: Date Submitted: 19TH AUGUST 2010 For Completion in Academic Registry Received in the Academic Registry by (name in capitals): v Method of Submission (Handed in to Academic Registry; posted through internal/external mail): E-thesis Submitted (mandatory for final theses from January 2009) Signature: Date: vi TABLE OF CONTENTS Pages Title page i Abstract ii Dedication iii Acknowledgement iv Declaration v Table of contents vi List of tables and figures x List of abbreviations xiii List of publications xv CHAPTER ONE: INTRODUCTION 1.0 Introduction 1 1.1 Global Epidemic of Human Immunodeficiency Syndrome 3 1.2 Viruses 6 1.3 Classification of Human Immunodeficiency Virus type 1 Strains 8 1.3.1 Origin of HIV-1 9 1.3.2 HIV-1 Life Cycle 11 1.4 Common Chemotherapy of Human Immunodeficiency Virus Type 1 Infection 21 1.4.1 Viral Entry Inhibitors 22 1.4.2 Reverse Transcriptase Inhibitors 24 1.4.2.1 Nucleoside Reverse Transcriptase Inhibitors 24 1.4.2.2 Non-nucleoside Reverse Transcriptase Inhibitors 26 1.4.3 Resistance of HIV-1 Reverse Transcriptase Enzymes vii 1.5. 1.6 1.7 to Reverse Transcriptase Inhibitors 29 1.4.4 Highly Active Anti-Retroviral Therapy 29 1.4.5 Integrase Inhibitor Compounds 30 1.4.5.1 The Diketo Aryl Compounds 30 1.4.5.2 Catechol Derivatives 36 1.4.5.3 Other Integrase Inhibitors 38 1.4.6 39 Protease Inhibitors Guanine-Derived Compounds as Novel HIV-1 Therapeutics 41 1.5.1 Guanine-Derived Compounds as RT inhibitors 41 1.5.2 Guanine-Derived Compounds as IN inhibitors 46 Peptide Nucleic Acids 48 1.6.1 Properties of Peptide Nucleic Acids 50 1.6.2 PNAs as a Potential Anti-HIV-1 Therapeutics 53 Aims and Objectives of the Research 54 CHAPTER TWO: RESULTS AND DISCUSSION (CHEMICAL SYNTHESIS OF GUANINE CONTAINING COMPOUNDS) 2.1 2.2 Established Routes for the Synthesis of PNA Monomers 58 2.1.1 Traditional Routes to all PNA Monomers 58 2.1.2 Guan-9-ylacetic Acid Synthetic Routes 60 An Attempt to Synthesise N2-Carbamate Protected Guan-9-ylacetic Acids 71 2.2.1 Alternative Routes Investigated towards the Synthesis of Boc-Protected N9-Guanylacetic Acid 74 2.2.2 Synthesis of N2-Boc Guan-9-ylacetic Acid 76 2.2.3 Optimization of the Route to the Synthesis of Compound 95 78 2.3 Alternative Route Independent of Dey and Garner’s Approach 78 2.4 Synthesis of N2-Benzyloxycarbonyl Guanylacetic Acid 82 2.4.1 Synthesis of N2-Cbz Protected Guanylacetic Acid via the Isocyanate Intermediate 2.4.2 Effect of Ring Deactivation on the Rate of Reaction of viii 84 Benzyl Alcohol with an Isocyanate 2.4.3 89 Effect of Increasing in Lipophilicity of the Ester at the N9- Position on the Purification and Yield of the desired N2-Benzyloxycarbonyl Protected Guan-9-ylacetic Acid 2.5 2.6 90 Synthesis of PNA Backbone 93 2.5.1 Synthesis of Fmoc-PNA Backbone 93 2.5.2 Synthesis of Boc-PNA Backbone 95 Synthesis of Guanine PNA Monomers 2.6.1 97 Synthesis of Guanine PNA Monomers for Fmoc Solid Phase or Solution Phase Strategy 2.6.2 97 Synthesis of Guanine PNA Monomer for Boc Solid Phase or Solution Phase Strategy 98 2.7 Solution Phase Synthesis of Guanine PNA Dimer 100 2.8 Synthesis of Thymine PNA Monomer 104 2.9 Solid Phase Synthesis of Guanine-rich PNA Oligomers: 2.10 H-TGGG-Lysine-NH2 106 Conclusion 108 CHAPTER THREE: EXPLORATION OF GUANINE-CONTAINING COMPOUNDS FOR ANTI-REVERSE TRANSCRIPTASE AND ANTI-INTEGRASE ACTIVITIES 3.1 HIV-1 Therapeutic Studies of the Purine Compounds 109 3.1.2 Non-nucleoside Reverse Transcriptase Inhibitors 110 3.1.3 Integrase Inhibitors 113 3.2 Evaluation of Guanyl Compounds as Potential NNRTIs 113 3.3 Evaluation of Guanyl Compounds as Potential IN Inhibitors 118 ix CHAPTER FOUR: STRUCTURAL ACTIVITY STUDIES OF THE BIOACTIVE COMPOUNDS CHAPTER FIVE: 119 CONCLUSION AND FUTURE WORK 5.1 Conclusion 128 5.2 Recommendation 131 CHAPTER SIX: 6.1 6.2 EXPERIMENTAL Materials and Methods 6.1.1 Reagents and Solvents 132 6.1.2 Chromatography 132 6.1.3 Instrumentation 133 Experimental Procedures 133 Appendix: X-ray data of Compound 103 190 References 201 x LIST OF TABLES AND FIGURES LIST OF TABLES Pages Table 1: Global HIV/AIDS Estimates, end of 2006 3 Table 2: HIV-1 Infections Reported in UK per annum 5 Table 3: Guanine Compounds that Exhibit NNRTIs Properties 114 Table 4: Kinetic Parameters for N2-Cbz Guanylacetic Acid Derivatives Binding to HIV-1 RT Wild Type 115 Table 5: Anti-viral Assays of the Guanine Scaffolds 118 Table 6: Crystal Data and Structure Refinement for x80265prev 190 Table 7: Table 8: Table 9: Bond Lengths [Å] and Angles [°] for x80265prev Torsion Angles [°] for x80265prev Hydrogen Bonds for x80265prev [Å and °] 191 197 200 LIST OF FIGURES Pages Figure 1: Global Trend of HIV-1 4 Figure 2: General Structure of Viruses 6 Figure 3: Diagram Illustrating the Different Levels of HIV Classifications 8 Figure 4: Structure of HIV-1 11 Figure 5: HIV Life Cycle 11 Figure 6: HIV-1 Entry Mechanisms 12 Figure 7: HIV-1 Reverse Transcriptase Structure 13 Figure 8: Three Domains of HIV-1 IN 15 Figure 9: Schematic Illustration of IN-Mediated 3'- End Processing of Viral DNA and Nucleophilic Attack on host DNA Figure 10: 17 The Nomenclature P1…Pn, P1’…Pn’ Designate Amino Acids Residues of Peptide Substrates. The Corresponding Binding Sites xi on the Protease are referred to as S1…..Sn; S1’……Sn’ 18 Figure 11: Proposed Catalytic Mechanism for Aspartic Protease 19 Figure 12: Proposed Concerted Catalytic Mechanism for HIV-1 Protease Enzyme 20 Figure 13: HIV-1 Viral Entry Inhibitors 22 Figure 14: The Amino Acid Sequence of Fuzeon 23 Figure 15: Inhibition Process of Fuzeon 23 Figure 16: Structure of Some Current NRTIs 24 Figure 17: Structures of Some NNRTIs 27 Figure 18: Structures of Some Second Generation NNRTIs 28 Figure 19: Structure of a DKA 31 Figure 20: Chemical Structure of Antiviral Integrase Inhibitors 32 Figure 21 Possible Binding Mode of a Selected Integrase Inhibitor 33 Figure 22: Proposed Mechanism of Action of DKAs 34 Figure 23: Structure of Raltegravir 36 Figure 24: Structures of Some Catechol Derivatives 37 Figure 25: Structures of Other IN Inhibitors 38 Figure 26: Structures of Other Potential IN Inhibitors 38 Figure 27: FDA Approved Protease Inhibitors 40 Figure 28: Other FDA Approved Protease Inhibitors 41 Figure 29: Structure of Carbovir 42 Figure 30: Structure of Analogues of Carbovir 43 Figure 31: Structure of Cyclobutyl G 43 Figure 32: Structure of Guanine Prodrugs 44 Figure 33: Structure of Acyclovir 45 Figure 34: Structures of Other Antivaral Purine Compounds 46 Figure 35: Four G-Bases Associated through Hoogsteen Hydrogen Bonding to Form a Cyclic Structure Figure 36: Figure 37: 48 Assembly of Four Guanine Nucleobases into a G-Tetrad Stabilized by a Central Potassium Ion 48 Structure of Peptide Nucleic Acid 49 xii Figure 38: Structure of DNA and RNA 50 Figure 39: Binding of PNA to DNA using both Watson-Crick and Hoogsteen Base Pairing in 2:1 Stoichiometry 52 Figure 40: Complexes of PNA with dsDNA 53 Figure 41: Guanine PNA Monomers 56 Figure 42: X-ray Crystallographic Structure of 103 84 Figure 43: Guanine PNA Dimers 102 Figure 44: O6-Nitrophenoxy Guanine PNA Dimer 104 Figure 45: Guanine Compounds Submitted for Bioassay 109 Figure 46: NNRTIs which are Structurally Similar to our Guanine Scaffolds 112 Figure 47: Guanine Scaffolds that Exhibits NNRTIs Properties 114 Figure 48: Enzyme- Ligand Binding Association 114 Figure 49: Effect of Dilution on the Inhibition of HIV-1 RT by Compound 116 Figure 50: Resistance Profiles for Compounds 124, 108, Nevirapine and Efavirenz 117 xiii LIST OF ABBREVIATIONS ACN = Acetonitrile AZT = Azidothymidine Boc = tert-Butoxycarbonyl Boc2O = Di-tert-butyl dicarbonate BOP Benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluoro = phosphate Cbz = Benzyloxycarbonyl CCD = Catalytic core domain d4T 2’-3’-didehydro-3’-deoxythymidine = DABCO = 1,4-Diazabicyclo[2,2,2]octane DBU = 1,8-Diazabicyclo[5.4.0]undec-7-ene DCC = 1,3-Dicyclohexylcarbodimide DCM = Dichloromethane ddC = Dideoxycytosine DIPEA = N,N-Diisopropylethylamine DKAs = Diketo Acid DMAP = 4-Dimethylaminopyridine DMF = N,N-Dimethylaminoformaldehyde DNA = Deoxyribonucleic acid EC50 = 50% Effective concentration FDA = Food Drug Administration HAART = Highly Active Anti-Retroviral Therapy HBTU = O-Benzotriazol-1yl-N,N,N’,N’-tetramethyluroniumhexafluoro phosphate HOBT = 1-Hydroxybenzotriazole hydrate IC50 = 50% Inhibitory concentration IN = Integrase MBHA = 4-Methylbenzhydrylamine hydrochloride Mmt Monomethoxytrityl = xiv NMR = Nuclear Magnetic Resonance NNBS = Non-nucleoside binding site NNRTI = Non-nucleoside Reverse Transcriptase Inhibitors NRTI = Nucleoside Reverse Transcriptase Inhibitors PI Protease Inhibitors = PMEA = 9-[-2(phosphonomethoxy)-ethyl]adenine PMEG = 9-[-2(phosphonomethoxy)-ethyl]guanine PNA = Peptide Nucleic Acids r.t= Room Temperature(RT) RNA = Ribonucleic acids RT = Reverse Transcriptase TFA = Trifluoroacetic acid TFMSA= Trifluoromethanesulphonic acid THF Tetrahydrofuran = TLC = UK Thin Layer Chromatography = UNAIDS = United Kingdom Joint United Nations Programme on HIV/AIDS xv LIST OF PUBLICATIONS 1. Adebambo, K.F, Howarth, N.M and Rosair, G.M (2005): Benzyl 2-amino-6chloro-9H-purine-9-carboxylate. Acta Crystallographica, E61, 0486-0488. 2. Adebambo, K.F; Zanal, S; Thomas, M; Cancio, R; Howarth, N.M; and Maga, G (2007): N2-Benzyloxycarbonylguan-9-ylacetic acid derivatives inhibit HIV-1 reverse transcriptase by interacting with the non-nucleoside inhibitors binding pocket and show reduced loss of potency by common drug resistance. ChemMedChem 2 (10), 1405-1409 xvi