A University of Sussex DPhil thesis Available online via Sussex Research Online: http://eprints.sussex.ac.uk/ This thesis is protected by copyright which belongs to the author. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Please visit Sussex Research Online for more information and further details 1 A CONVENIENT SYNTHESIS OF BIOACTIVE CYCLOHEXENEPHOSPHONATES Benoît Carbain October 2005 - January 2009 University of Sussex 1 Contents Acknowledgements ........................................................................................................... 5 Abbreviations .................................................................................................................... 6 Abstract ............................................................................................................................. 9 I. Towards a convenient synthesis of bioactive cyclohexenephosphonates ............... 11 I.1. Theoretical part ..................................................................................................... 11 I.1.1. Previous work ................................................................................................ 11 I.1.2. Barton-Hunsdiecker’s iododecarboxylation and Hirao’s coupling reaction.. 13 I.1.2.1. The halodecarboxylation reaction ........................................................... 13 I.1.2.2. Hirao’s palladium-promoted coupling reaction ...................................... 16 I.1.3. Shikimic acid and the shikimate pathway...................................................... 20 I.1.3.1. Shikimic acid .......................................................................................... 20 I.1.3.2. Shikimate pathway .................................................................................. 21 I.1.3.3. Shikimate dehydrogenase ....................................................................... 22 I.1.3.4. Towards Shikimic acid mimetics ............................................................ 25 I.2. Results and discussion - Synthesis of phospha-isosteres of shikimic acid ........... 27 I.2.1. Retrosynthetic analysis and synthetic strategies towards phospha-shikimic acids and derivatives ............................................................................................... 27 I.2.2. Route A. ......................................................................................................... 28 I.2.2.1. Synthesis of 1-iodo-(3R,4S,5R)-tri-acetoxy-1-cyclohexene 6 from shikimic acid. ...................................................................................................... 28 I.2.2.2. Introduction of the phosphonate group: Synthesis of Dimethyl (3R,4S,5R)-tri-acetoxy-1-cyclohexene-1-phosphonate 9 and deprotection........ 32 I.2.3. A alternate protecting group strategy towards shikimic acid derivatives. ..... 34 I.2.3.1. A new protecting group strategy. ............................................................ 34 2 I.2.3.2. Iododecarboxylation and phosphonate introduction: key steps towards 3- dehydro-‘phospha’-shikimic acid derivatives. .................................................... 36 I.2.4. Route B. ......................................................................................................... 38 I.2.5. Dehydroquinase assay.................................................................................... 40 I.3. Conclusion. ........................................................................................................... 41 II. Efficient synthesis of novel, highly active phospha-isosteres of the Influenza neuraminidase inhibitor Oseltamivir. .............................................................................. 41 II.1. Theoretical part. ................................................................................................... 41 II.1.1. Influenza. ...................................................................................................... 41 II.1.1.1. Introduction to influenza viruses ........................................................... 41 II.1.1.2. Influenza A virus H5N1 ........................................................................ 42 II.1.1.3. The role of neuraminidase and hemagglutinin in influenza virus replication ............................................................................................................ 44 II.1.1.4. The influenza virus haemagglutinin membrane glycoprotein ............... 46 II.1.1.5. The influenza virus neuraminidase membrane glycoprotein................. 49 II.1.2. Oseltamivir. .................................................................................................. 51 II.1.2.1. A neuraminidase inhibitor. .................................................................... 51 II.1.2.2. Oseltamivir syntheses. ........................................................................... 53 II.1.3. ‘Phospha’-Tamiflu syntheses. ...................................................................... 56 II.2. Results and discussion. ........................................................................................ 58 II.2.1. Strategy towards ‘phospha’-Tamiflu and its derivatives. ............................. 58 II.2.2. Key steps towards the key intermediate 34. ................................................. 58 II.2.2.1. A first approach. .................................................................................... 58 II.2.2.2. Protected amino derivatives of Tamiflu’s precursor. ............................ 60 II.2.2.3. Optimised route leading to key monoalkyl phosphonate intermediate 34. ............................................................................................................................. 63 II.2.3. Synthesis of ‘phospha’-Tamiflu derivatives ................................................. 66 II.2.3.1. Synthesis of ‘phospha’-Tamiflu 35 and its methyl ester 36 .................. 66 3 II.2.3.2. Introduction of an hydrophobic moiety by alkylation: synthesis of the hexyl ester 40 of ‘phospha’-Tamiflu ................................................................... 67 II.2.3.3. Introduction of a sugar moiety by alkylation: Synthesis of α(2-6)- sialoglycoconjugate mimetic 46 .......................................................................... 69 II.2.4. An alternative approach to mixed diesters of ‘phospha’-Tamiflu ................ 71 II.2.4.1. Introduction of a sugar moiety by phosphonate coupling: Synthesis of α(2,3)-sialoglycoconjugate mimetic 54............................................................... 71 II.2.4.2. Introduction of a variable ω-thioacetyl linker for immobilisation and oligomerisation of the ‘phospha’-Tamiflu motif ................................................ 75 II.2.5. Inhibition of neuraminidase activity ............................................................. 77 II.2.6. Future scope.................................................................................................. 78 II.3. Conclusion ........................................................................................................... 79 III. Experimental section ........................................................................................... 80 III.1. General procedures............................................................................................. 80 III.2. Synthetic procedures and compound characterization ....................................... 81 III.2.1. ‘Phospha’-shikimic derivatives ................................................................... 81 III.2.2. ‘Phospha’-Tamiflu derivatives .................................................................... 97 III.2.3. Inhibition of neuraminidase activity of influenza viruses from allantoic fluid from infected eggs ........................................................................................ 124 IV. References ......................................................................................................... 126 V. List of publications and posters............................................................................. 133 Appendix 1 : Crystal structures ..................................................................................... 134 ORTEP-generated structure of (3R,4S,5R)-tri-acetoxy-1 cyclohexene-1-phosphonate 9 ................................................................................................................................. 134 ORTEP-generated structure of (3R,4S,5R)-3-[(tert-butyldimethylsilyl)oxy]-4,5-(2,3- dimethoxy-butan-2,3-dioxy)-cyclohex-1-ene-1-carboxylic acid 13 ......................... 138 ORTEP-generated structure of Dimethyl (3R,4S,5R)- 3-hydroxy-4,5-(2,3-dimethoxy- butan-2,3-dioxy)-cyclohex-1-ene-1-phosphonate 16 ................................................ 146 4 ORTEP-generated structure of (3R,4R,5S)-4-acetylamino-5-azido-3-(1- ethylpropoxy)-1-iodocyclohexene 25 ....................................................................... 151 ORTEP-generated structure of (3R,4R,5S)-4-acetylamino-5-N-tert-Butoxycarbonyl- amino-3-(1-ethylpropoxy)-1-iodocyclohexene 32 .................................................... 157 Appendix 2 : 1H NMR Spectra.....................................................................................