Total Synthesis of CPI-2081, Breitfussin B and Synthetic Studies Towards Myriastramide C and Goadsporin Ke Liu This thesis is submitted in partial fulfilment of the requirement for the award of Doctor of Philosophy School of Pharmacy August 2015 © This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. ABSTRACT Natural products have been the source and inspiration of numerous drugs. However, as they need to be isolated from natural sources, they are often obtained in minute quantities. Hence, total syntheses of natural products and their analogues give us a better chance to look into their biological activities and perform SAR studies for drug discovery. During my doctoral tenure I have synthesised a number of natural products and also some fragments. CPI-2081, a 2:1 mixture of two pentapeptides 1.8 and 1.9, were isolated from Streptomyces species NCIM 2081. These peptides are potent inhibitors (IC50 36.9 ± 1.8 nM) of the cysteine protease papain and inhibit cancer cell migration. However it was not clear which peptide is responsible for the observed biological activities. Both peptides have been prepared synthetically in our lab and they are being tested. Myriastramide C (2.23) is a modified cyclic octapeptide isolated from M. clavosa. The complete series of myriastramides A-C represent the first peptide metabolites isolated from M. clavosa. They were assumed to be cytotoxic, however myriastramide A was found inactive against 10 different human cancer cell lines. Myriastramides B and C were isolated in insufficient quantities so similar tests cannot be performed. Furthermore, the stereochemistry of the tryptophan residue in myriastramide C was not confirmed due to insufficient material isolated. Myriastramide C has been synthesised in our laboratory using L-tryptophan, however a mixture of products were obtained due to the two proline residues in the peptide. A pure product was purified by our collaborator and further NMR and biological studies is underway. Goadsporin (3.14) is a linear oligopeptide that was isolated in 2001. Its unique structure contains 19 amino acids, six of which are cyclised to form four oxazole and two thiazole rings. Goadsporin was found to be active specially in streptomycetes, with the ability to promote morphogenesis and secondary metabolism. It can also promote antibiotic production. The synthesis of this natural product was challenging, and I unfortunately with the limited time of my PhD study, we did not complete the total synthesis. However, we have synthesised several fragments of it, and they are going to be tested in the future. Breitfussin B (4.35) was first isolated in 2007 from the sample of Thuiaria breitfussi collected from Bear Island. Its highly unsaturated nature prevented its structure elucidation until 2012: Hanssen et al. proposed a structure using a combined atomic force microscopy and computational approach. However no biological activity was reported. The total synthesis of breitfussin B was completed in our lab, and tested for its antimicrobial ability. Further biological studies are underway. (All the structures of above natural products are shown in Figure A.) OH O O H O O N N H N N N N H H N H N N O O H N N H S O O O O HN O HN O Goadsporin (3.14) NH O NH O N N O S O O NH HN OH O H H N N O HN Br S O N N O HN O N NH O O O O HN N N O Br N H CPI-2081 1.8: R = H Myriastramide C (2.23) Breitfussin B (4.35) 1.9: R = CH2C6H4OH Figure A: Structures of CPI-2081 peptides 1.8 and 1.9, myriastramide C (2.23), goadsporin (3.14) and breitfussin B (4.35) II CONTENTS ABSTRACT ......................................................................................................................................................... I LIST OF SCHEMES ......................................................................................................................................... VI LIST OF FIGURES ........................................................................................................................................ VIII LIST OF TABLES .............................................................................................................................................. X ACKNOWLEDGEMENTS ............................................................................................................................... XI ABBREVIATIONS ......................................................................................................................................... XII Chapter 1: Total Synthesis of CPI-2081 ............................................................................. 1 1.1 Introduction ................................................................................................................................. 2 1.1.1 Natural products .................................................................................................................. 2 1.1.2 Cysteine protease and role of proteases in cancer ............................................................... 4 1.1.3 Examples of anticancer protease inhibitors ......................................................................... 6 1.1.3.1 Anticancer activity of cathepsin inhibitor VBY-825 ........................................................................ 6 1.1.3.2 Anticarcinogenic Bowman-Birk protease inhibitor on breast cancer cells ................................ 7 1.1.3.3 HIV protease inhibitor saquinavir induces apoptosis in ovarian cancer cells ........................... 8 1.1.4 CPI-2081 .............................................................................................................................. 9 1.1.4.1 Extraction and isolation of CPI-2081 ..................................................................................................... 9 1.1.4.2 Papain and cancer cell migration inhibition of CPI-2081 ............................................................. 10 1.1.5 Solid-Phase Peptide Synthesis (SPPS) .............................................................................. 11 1.1.6 Trp-containing peptide side-reaction in SPPS ................................................................... 13 1.1.7 Aims .................................................................................................................................. 14 1.2 Results and Discussion ............................................................................................................ 15 1.2.1 Strategy .............................................................................................................................. 15 1.2.2 Synthesis of Ac-Leu-Cys(tBu)-Trp-Ala-Phe-Wang (1.12) ................................................ 15 1.2.3 Wang resin cleavage and tryptophan modification ........................................................... 16 1.3 Conclusions and Future Work .............................................................................................. 22 1.4 Experimental ............................................................................................................................ 22 1.4.1 General Experimental Methods ......................................................................................... 22 1.4.2 Experimental Details for Chapter 1 ................................................................................... 23 1.4.2.1 Attachment of the First Amino Acid to Wang Resin ...................................................................... 23 1.4.2.2 Fmoc Deprotection ..................................................................................................................................... 23 1.4.2.3 Peptide Coupling ......................................................................................................................................... 23 1.4.2.4 Synthesis of Ac-Leu-Cys(tBu)-Trp-Ala-Phe-Wang (1.12) ........................................................... 24 1.4.2.5 Wang resin cleavage towards 1.9 .......................................................................................................... 24 Chapter 2: Synthetic Studies of Myriastramide C ........................................................ 26 2.1 Introduction .............................................................................................................................. 27 2.1.1 Natural products from marine sponge Myriastra clavosa ................................................. 27 2.1.1.1 Clavosines A-C ............................................................................................................................................ 28 2.1.1.2 Clavosolides A and B ................................................................................................................................ 28 2.1.1.3 Homologous series of polymethoxydienes ......................................................................................... 29 2.1.2 Proline-containing cyclic peptides .................................................................................... 30 2.1.2.1 Special feature of proline-X amide bond ............................................................................................ 30 III A) Amide bond ........................................................................................................................