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G Opal P H DT Hesis Search for Pseudomonas aeruginosa Immune-modulatory but Non-inducing agents by GOPAL P. JADHAV, M. Pharm. (Pune) Thesis is submitted to the University of Nottingham for the degree of Doctor of Philosophy August 2007 I Confidential To my beloved wife Madhuri & To my parents This thesis is gratefully dedicated. Their constant encouragement and inspiration made this work possible. II Confidential TABLE OF CONTENTS Abstract………………………………………………………………………… VII Acknowledgements……………………………………………………………. IX List of abbreviations…………………………………………………………... X CHAPTER 1: INTRODUCTION…………………………………………………………….. 1 1.1. Immune System…………………………………………………………………….….. 2 1.1.1. Primary (central) lymphoid organs…………………………………………... 3 1.1.2. Secondary (peripheral) lymphoid organs…………………………………..… 4 1.1.3. Lymphatic vessels……………………………………………………………. 5 1.1.4. Products of immune cells.………...………………………………………….. 5 1.1.4.1. Lymphocytes …..……………………………………………………….... 6 1.1.4.1.1. B-lymphocytes…………………………………………..………. 6 1.1.4.1.2. T-lymphocytes……………………………………………...……. 8 1.1.4.1.3. Natural killer (NK) cells…………………………………………. 9 1.1.4.2. Phagocytes……………………………………….………………………. 9 1.1.4.3. Granulocytes……………………………………………………………… 10 1.1.4.4. Cytokines ……………………………………………………………..….. 10 1.2. How Does the Immune System Defend the Body? …………………………………… 12 1.2.1. Innate immunity…………………………………………………………….... 12 1.2.1.1. Physical and chemical barriers………………………..………………….. 13 1.2.1.2. Cellular defences………………………………………………………..... 14 1.2.2. Acquired immunity…………………………………………………………… 14 1.2.2.1. Humoral immunity……………………………………………………….. 15 1.2.2.2. Cell mediated immunity………………………………………………….. 16 1.3. Disorders of the Immune System……………………………………………................. 17 1.3.1. Hypersensitivity ……………………………………….......………………..... 17 1.3.2. Immunodeficiency disorders ………………………………………………..... 18 1.3.3. Cancers of the immune System……………………………………………..... 19 1.3.4. Autoimmune diseases……………………………………………………….... 19 1.4. Modulation of the Immune System……………………………………………………. 23 1.4.1. Immunosuppressants ………………………………………………………… 23 1.4.1.1. Helper T-cell blockers………………………………………………......... 23 1.4.1.2. Glucocorticoids (Glucocorticostroids) ……………………………......…. 27 1.4.1.3. Cytotoxic agents…………………………………………………....……. 27 1.4.1.4. Antibody reagents (cytokines therapy) ………….……………....………. 30 1.4.2. Immunostimulants……………………………………………………............ 31 1.4.2.1. Synthetic agents………………………………….……………………… 32 1.4.2.2. Natural immunostimulants……………………….………………………. 35 1.4.2.3. Androgenic steroids ……………………………………………………… 39 1.4.2.4. Intravenous immunoglobulins …………………………………………… 40 1.4.2.5. Cytokines as immunostimulants……………………………………….…. 40 1.4.3. Selective tolerogens………………………………………………..…….…… 41 1.4.4. Antirheumatic drugs as immune modulators…………………………….…… 41 1.4.5. Evasion of immune responses by microbes …………………………………. 42 1.5. Quorum Sensing and Immune Modulation……………………………………………. 45 III Confidential 1.5.1. Regulators of virulence factor producing genes……………………..……….. 45 1.5.2. The quorum-sensing paradigm………………………………….……………. 46 1.5.3. The diversity of signal molecules...................................................................... 47 1.5.4. Quorum sensing in Pseudomonas aeruginosa………………………………… 48 1.5.5. Pseudomonas aeruginosa and immune modulation………………………….. 53 1.6. Project Aims…………………………………………………………………………… 54 CHAPTER 2: SEARCH FOR IMMUNE-MODULATORY BUT NON-INDUCING AGENTS: Design and synthesis of analogues of the Pseudomonas aeruginosa quorum-sensing molecule 3O, C12-HSL.................................... 56 2.1. The Diverse Immunological Bioactivity of 3O, C12-HSL…………………………….. 58 2.1.1. Effect of 3O, C12-HSL on TH1-TH2 balance…………………………………. 58 2.1.2. Effect of 3O, C12-HSL on insulitis and diabetes…………………………….. 58 2.1.3. Effect of 3O, C12-HSL on inflammatory chemokines and cytokines….…….. 59 2.1.4. Effect of 3O, C12-HSL on Cox enzymes……………………………………... 60 2.1.5. Effect of 3O, C12-HSL on Prostaglandin E2…………………………………. 60 2.1.6. Effect of 3O, C12-HSL on biochemical signaling events of innate immunity.. 61 2.1.7. Contradictory effects of 3O, C12-HSL………………………………………. 62 2.2. Biological Targets for 3O C12-HSL…………………………………………………… 64 2.3. Is the 3O C12-HSL an ideal molecule to aim at in order to combat dysfunctional immune system? …………………………………….…………………. 66 2.4. First generation Immune Modulatory analogues of 3O C12-HSL: SAR Studies to date....................................................................................................... 67 2.4.1. Limitations in the SAR studies and conflicting reports in the immune modulatory behaviour of 3O C12-HSL……………………………………….. 70 2.5. Second generation Immune Modulatory analogues of 3O C12-HSL and its SAR Studies..……..………………………………………….………………… 72 2.5.1. Strategies for new SAR studies of 3O C12-HSL……………………………… 72 2.6. 4-Aza analogues of 3O C12-HSL………………………………………………………. 75 2.6.1. Synthesis of secondary amines……………………………………………….. 77 2.6.2. Synthesis of 3-oxo-4-azaalkanoyl-L-homoserine lactones…………………… 80 2.7. Fluorinated 4-aza-3O, C12-HSL analogues…………………………………………….. 84 2.7.1. Synthesis of 4-aza-2-F2-3O, C12-HSL……………………………………….. 88 2.7.2. Synthesis of 4-aza-2-F-3O, C12-HSL………………………………………… 89 2.8. 3-Aza analogues of 3O, C12-HSL……………………………………………………… 89 2.8.1. Synthesis of 3-OH, 3-aza-C12-HSL………………………………………….. 91 2.8.2. Synthesis of 3-amino, 3-aza C12-HSL……………………………………….. 93 2.9. Thia analogue of 3O, C12-HSL ……………………………………………………….. 94 2.9.1. 3-Thia analogues of 3O, C12-HSL…………………………………………… 95 2.9.2. Sulfonamide motif containing analogues of 3O, C12-HSL………………….. 98 2.10. Hetero ring altered analogues of 3O, C12-HSL……………………..………………… 101 2.10.1. Retro-synthesis of NOdDA and NOdDG…………………………………… 105 2.10.2. Synthesis of NOdDA, NOdDG and NOdAT……………………………….. 106 2.11. Hetero ring altered analogues of 4-Aza 3O, C12-HSL…………..……………………. 108 2.11.1. Selection of R-group for the hetero-ring replacement………………………. 109 2.12. Oxa analogues of 3O, C12-HSL………………………..……………………………... 110 2.12.1. Synthesis of 3O, 4-oxa C12-HSL……………………………………………. 111 2.12.2. Synthesis of 8-oxa and 7, 10-dioxa analogues of 3O, 4-aza C12-HSL............ 112 IV Confidential 2.13. Miscellaneous analogues of 3O, C12-HSL…………………………………………….. 114 2.13.1. Synthesis of tetramic acid derivative………………………………………… 114 2.13.2. Synthesis of 3-acetoxy, C12-HSL……………………………………………. 115 CHAPTER 3: BIOLOGICAL ASSAYS................................................................................ 118 3.1. Immunology…………………………………………………………………………… 118 3.1.1. Structural modifications in 3O, C12-HSL…………………………………….. 119 3.1.2. SAR studies to develop second generation analogues of 3O, C12-HSL…….... 121 3.1.2.1. Insertion of N at position 4 in the side chain A of 3O, C12-HSL………… 121 2 3.1.2.2. Replacement of C hydrogen(s) of 4-aza 3O, C12-HSL by fluorine(s) …. 123 3 3.1.2.3. Insertion of N in 3O, C12-HSL………………………………………….. 124 3.1.2.4. Insertion of S in 3O, C12-HSL …………………………………………... 126 3.1.2.4.1. 3-Thia analogues……………………………………………….. 126 3.1.2.4.2. Sulphonamide motif containing analogues…………………….. 127 3.1.2.5. Replacement of L-HSL by other hetero ring……………………………. 128 3.1.2.5.1. Hetero ring altered analogues………………………………….. 128 3.1.2.5.2. Hetero ring altered analogues of 3O, 4-aza-C12-HSL………….. 130 3.1.2.6. Insertion of O in 3O, C12-HSL: Oxa analogues………….………………. 133 3.1.2.7. Miscellaneous analogues: Tetramic acid and 3-acetoxy analogues……… 134 3.2. Cytotoxicity Assay…………………………………………………………………….. 136 3.3. Microbiology (Quorum-Sensing-induction and antagonism) ………………………… 139 3.3.1. Screening of analogues for auto-induction (agonist) activity……………..…. 140 3.3.2. Antagonist assays…………………………………………………………….. 142 3.3.2.1. Antagonistic behavoir of 4-aza, fluorinated 4-aza and 3-aza analogues .. 142 3.3.2.2. Antagonistic behavoir of 3-thia and sulfonamide analogues……………. 143 3.3.2.3. Antagonism by hetero ring altered analogues…………………………… 144 3.3.2.4. Antagonisic behaviour of oxa-analogues and miscellaneous analogues… 145 CHAPTER 4: SUMMARY, DISCUSSION, CONCLUSIONS AND FUTURE DIRECTIONS…………………………………………………… 146 4.1. Summary……………………………………………………………………………….. 146 4.1.1. 4-aza, fluorinated, 7,10-dioxa, 8-oxa-4aza and hetero ring altered 4-aza analogues……………………………………………………………………... 147 4.1.2. 4-Oxa analogue……………………………………………………………….. 148 4.1.3. 3-Aza derivatives: ……………………………………………………………. 149 4.1.4. 1-Thia and 3-thia derivatives…………………………………………………. 150 4.1.5. Hetero ring altered derivatives of 3O, C12-HSL……………………………… 153 4.1.6. Tetramic acid derivative ……………………………………………………… 153 4.2. Discussion……………………………………………………………………………… 155 4.3. Conclusions…………………………………………………………………………….. 160 4.4. Future Directions………………………………………………………………………. 161 4.4.1. Further biological investigations of active analogues in higher animals……… 161 4.4.2. Synthesis of new series of 3-aza analogues of 3O, C12-HSL…………………. 161 4.4.3. Synthesis of bivalent ligands………………………………………………….. 162 4.4.4. Isolation and identification of target proteins (immunophillins) …………….. 164 V Confidential CHAPTER 5: EXPERIMENTAL 166 5.1. Chemicals and reagents ……………………………………………………………. 166 5.2. General analytical methods………………………………………………………… 166 5.3. Synthesis of analogues of 3O, C12-HSL……………………………………………. 168 5.4. Synthesis of 4-aza analogues of 3O, C12-HSL……………………………………… 170 5.5. Synthesis of fluorinated 4-aza analogues of 3O, C12-HSL…………………………. 182 5.6. Synthesis of 3-aza analogues of 3O, C12-HSL……………………………………… 187 5.7. Synthesis of thia analogue of 3O, C12-HSL ………………………………………… 196 5.8. Synthesis of sulphonamide analogues of 3O, C12-HSL ……………………………. 203 5.9.
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