Chemical Diversity of Eremophila Species and Screening Library Generation Author Barnes, Emma Catherine Published 2012 Thesis Type Thesis (PhD Doctorate) School School of Biomolecular and Physical Sciences DOI https://doi.org/10.25904/1912/908 Copyright Statement The author owns the copyright in this thesis, unless stated otherwise. Downloaded from http://hdl.handle.net/10072/366931 Griffith Research Online https://research-repository.griffith.edu.au Chemical Diversity of Eremophila species and Screening Library Generation Emma Catherine Barnes B.Sc. (Hons.) School of Biomolecular and Physical Sciences Science, Environment, Engineering and Technology Griffith University Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy October 2012 Abstract This thesis explores two aspects of natural product (NP) chemistry. In part A, the use of NPs as scaffolds in the generation of screening libraries was explored as a valuable way to produce structurally diverse compounds with lead- or drug-like physicochemical parameters. Part B describes the chemical investigations of several species from the under-studied Australian endemic plant genus Eremophila, which was selected for examination as it had high potential to be a source of both new chemistry and of unique scaffolds for screening library production. Part A. The known plant NP 14-hydroxy-6,12-muuroloadien-15-oic acid (62) was identified from the Nature Bank compound repository as a unique scaffold that could be chemically elaborated to generate lead- or drug-like screening libraries. Scaffold 62 was isolated from the leaves of Eremophila sturtii, then utilised in the parallel solution-phase synthesis of two series of analogues. The first library consisted of six semi-synthetic amide derivatives (82-84 and 91-93), whilst the second contained six carbamate analogues (103-108). Prior to synthesis a virtual library was generated and prioritised based on drug-like physicochemical parameters such as Log P, Log D5.5, hydrogen bond donors/acceptors, and molecular weight. These semi-synthetic libraries have been evaluated for their antimalarial activity against a chloroquine- sensitive Plasmodium falciparum line (3D7). Several compounds displayed moderate activity in this screen with IC50 values ranging from 14 to 33 μM. I Further chemical investigations of the E. sturtii extract resulted in the isolation of three novel tetracyclic sesquiterpene lactones, mitchellenes A-C (68-70), two new sesquiterpene carboxylic acids, mitchellenes D and E (71 and 72), and the known compounds casticin (73), and centaureidin (74). A proposed biosynthetic pathway from mitchellene D to mitchellenes A-C is described. During attempts to re-isolate scaffold 62 in order to generate further library members, it was found that the original plant source of NP 62 had been taxonomically misidentified. A number of Eremophila species were examined by analytical HPLC and off-line (+)-LRESIMS in order to assist in the identification of the sample, and after consultation with the Queensland Herbarium the specimen was re-classified from E. mitchellii to E. sturtii. Part B. While researching the Eremophila genus from which scaffold 62 had been obtained, it was observed that while this genus has been a source of structurally diverse compounds, a large number of its > 215 species remain chemically uninvestigated. This observation lead to a number of Eremophila species being chosen for chemical examination in this project. Forty Eremophila specimens labelled as either 'leaf' or 'aerial parts' in the Nature Bank biota library were examined by analytical HPLC. Three of these species with no reported chemical investigations in the literature were then selected for analysis. II E. eriocalyx and E. linsmithii were both found to predominantly contain the previously reported compounds verbascoside (110), mannitol (179), and geniposidic acid (155). E. eriocalyx additionally contained the known NPs mussaenoside (181), ladroside (182), 5,19-dihydroxy-3,14-viscidadien-20-oic acid (186) and the new cembrane 3,15-epoxy-19-hydroxycembra-7,11-dien-18-oic acid (188). The NPs from E. linsmithii also included the known NP 3,15-epoxycembra-7,11-dien-18-oic acid (191) and the new hemiterpene glycoside 3-methylbut-3-enyl O-α-L-rhamnopyranosyl- (1→6)-O-β-D-glucopyranoside (193). Extraction and purification of a leaf sample of E. microtheca collected from south east Queensland led to the isolation of three new serrulatane type NPs, 3-acetoxy- 7,8-dihydroxyserrulat-14-ene (198), 3,7,8-trihydroxyserrulat-14-en-19-oic acid (199), and 3,19-diacetoxy-8-hydroxyserrulat-14-ene (200). The known compounds verbascoside (110) and jaceosidin (201) were also isolated. NP 199 was used in acetylation and methylation reactions to generate analogues for crystallisation and biological studies (205-207). The NPs and semi-synthetic analogues were analysed against a panel of nine Gram-positive and one Gram-negative bacterial strains. The serrulatanes were found to be moderately active (MICs 32-128 μg/mL) against Streptococcus pyogenes (ATCC 12344). NP 198 demonstrated activity at MIC 128 μg/mL against the majority of bacterial strains. The flavonoid jaceosidin (201) had the greatest potency (MICs 8-32 μg/mL) against most Staphylococcus aureus strains. In all, three novel, seven new, and ten known NPs were isolated from several Eremophila species, and twelve screening library analogues were generated from the Eremophila derived scaffold 62. All of these compounds were fully characterised using a combination of MS, IR, UV, [α]D, and 1D/2D NMR spectroscopic data analyses. III Statement of Originality This work has not previously been submitted for a degree or diploma in any university. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself. _____________________ ________________ Emma Catherine Barnes Date IV Table of Contents Abstract .............................................................................................................................. I Statement of Originality ................................................................................................. IV Acknowledgements ....................................................................................................... VII List of Figures ................................................................................................................. IX List of Schemes .............................................................................................................. XI List of Tables ................................................................................................................. XII Abbreviations ............................................................................................................... XIII Publications Arising from this Thesis ......................................................................... XVI Chapter 1. Introduction Part A ........................................................................................ 1 1.1. Strategies for the generation of compound libraries in the search for new lead and/or drug molecules .................................................................................................. 1 1.2. PhD research aims ........................................................................................... 21 Chapter 2. Selection and isolation of the muurolane scaffold 62 along with the cyclic sesquiterpenes mitchellenes A-E from Eremophila sturtii ............................................. 22 2.1. NP scaffold selection process .............................................................................. 22 2.2. E. sturtii ............................................................................................................... 25 2.3. Isolation of scaffold 62 from E. sturtii ................................................................ 26 2.4. Isolation of mitchellenes A-E from E. sturtii ...................................................... 33 2.5. Proposed biosynthesis of mitchellenes A-E ........................................................ 50 2.6. Conclusion ........................................................................................................... 53 Chapter 3. Screening library generation using scaffold 62 ........................................... 54 3.1. Introduction ......................................................................................................... 54 3.2. Selection of reaction partners .............................................................................. 55 3.3. Amide library generation ..................................................................................... 55 3.4. Carbamate library generation .............................................................................. 64 3.5. Biological activity of the muurolane libraries ..................................................... 67 3.6. Synthesis of a di-substituted analogue of scaffold 62 ......................................... 70 3.7. Conclusions ......................................................................................................... 72 Chapter 4. Where's my compound? Analytical strategies for the determination of taxonomy ........................................................................................................................ 73 4.1. Introduction ........................................................................................................