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Synthesis of bioactive natural products Zheng, Zehua 2016

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 SYNTHESIS OF BIOACTIVE NATURAL PRODUCTS  by  ZEHUA ZHENG  B.Sc., Nanjing University, 2013  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  November 2016  © Zehua Zheng, 2016   ii Abstract Crude extracts of the rare macrofungus Serpula sp. collected from a wooded area in Sri Lanka showed antimicrobial activity. The novel fungal metabolite serpulanine (2.1) was isolated from the crude extract in very small amounts along with a number of additional secondary metabolites. In order to obtain sufficient quantities of serpulanine (2.1) for biological evaluation, a synthetic route was developed to the natural product and a small library of analogs that have been evaluated in a panel of bioassays. Serpulanine (2.1) inhibits the histone deacetylase I/II with a clear dose response curve.  Halitoxins (3.1) that are frequently isolated from marine sponges have a complex macrocyclic chemical structure made of different numbers of monomeric alkylpyridinium units. An unknown halitoxin-related natural product named alotau potently inhibited the dephosphorylation activity of calcineurin. With the goal to elucidate the structure of alotau, compounds of one, two and three pyridinium rings (3.10, 3.7 and 3.8) were synthesized. Though these compounds have NMR spectra similar to the natural alotau, according to bioassay results, none of them recapitulates the activity of the unknown natural product alotau.  (+)-Makassaric acid 4.1 was isolated in the Andersen Lab from the marine sponge Acanthodendrilla sp. It showed promising activity in a zebrafish screen for new drugs to treat stroke patients. The convergent synthetic scheme shown below was undertaken to conduct structure activity relationship (SAR) studies. The key intermediate 4.17 has been obtained, and further synthetic efforts will be needed to produce 4.1.      iii Preface  Chapter 2 is based on work conducted at UBC. The natural product serpulanine was isolated and characterized by Dr. David E. Williams in the Andersen lab. I was responsible for designing synthetic routes, synthesizing and characterizing all serpulanine-related analogs. My supervisor Dr. Raymond J. Andersen gave me technical suggestions. The biological data was collected by Samantha Ellis in the Prof. Wilfred Jefferies group of the Michael Smith Laboratories at UBC.  Chapter 3 is based on work conducted at UBC and University of Alberta. The natural product alotau was isolated by Dr. David E. Williams in my lab and by Phuwadet Pasarj in the Prof. Charles Holmes group of the Department of Biochemistry at University of Alberta. The alotau structure was proposed by Phuwadet Pasarj and Dr. David E. Williams. My supervisor Prof. Raymond J. Andersen gave me suggestions regarding synthetic route design and compound characterization. I was responsible for synthesizing and characterizing all alotau-related analogs. All biological experiments and data were collected by Phuwadet Pasarj.  Chapter 4 is based on work conducted at UBC. I was responsible for designing the synthetic scheme, synthesizing and characterizing all compounds. My supervisor Dr. Raymond J. Andersen provided synthetic suggestions.    iv Table of contents  Abstract .......................................................................................................................................... ii	  Preface ........................................................................................................................................... iii	  Table of contents ........................................................................................................................... iv	  List of tables ................................................................................................................................ viii	  List of figures ................................................................................................................................ ix	  List of schemes ............................................................................................................................ xiv	  List of abbreviations and symbols ............................................................................................ xvi	  Acknowledgements ..................................................................................................................... xxi	  Dedication .................................................................................................................................. xxii	  Chapter 1: Introduction ................................................................................................................ 1	  1.1	   Introduction to natural products ......................................................................................... 1	  1.2	   Classes of natural products ................................................................................................. 2	  1.2.1	   Hydroxamate-containing natural products .................................................................. 2	  1.2.2	   Halitoxins .................................................................................................................... 3	  1.2.3	   Terpenoids ................................................................................................................... 4	  1.3	   Introduction to marine natural products ............................................................................. 5	  1.4	   Importance of marine natural products .............................................................................. 6	  Chapter 2: Synthesis of analogs of serpulanine .......................................................................... 7	  2.1	   Histone deacetylase (HDAC) ............................................................................................. 7	  2.2	   Histone deacetylase and cancer .......................................................................................... 8	  2.3	   Histone deacetylase inhibitors (HDIs) ............................................................................... 8	    v 2.4	   Isolation and characterization of serpulanine ..................................................................... 9	  2.5	   Synthesis of serpulanine ..................................................................................................... 9	  2.5.1	   Retrosynthetic analysis of serpulanine ...................................................................... 10	  2.5.2	   Initial synthetic trials ................................................................................................. 10	  2.5.3	   Synthesis of serpulanine ............................................................................................ 17	  2.5.4	   Other possible synthetic routes and future work ....................................................... 25	  2.6	   Biological results .............................................................................................................. 29	  2.7	   Conclusion ........................................................................................................................ 31	  Chapter 3: Synthesis of halitoxin-related compounds ............................................................. 32	  3.1	   Calcineurin ....................................................................................................................... 32	  3.2	   Important calcineurin inhibitors ....................................................................................... 33	  3.3	   Synthesis of halitoxin-related compounds for structure elucidation ................................ 34	  3.4	   Another potential method of structure elucidation ........................................................... 37	  3.5	   Conclusion ........................................................................................................................ 38	  Chapter 4: Synthesis of intermediates of (+)-makassaric Acid ............................................... 39	  4.1	   MK2’s role in inflammation ............................................................................................. 39	  4.2	   Prior syntheses of (+)-makassaric acid and related synthetic target ................................ 39	  4.3	   Synthesis of intermediates of (+)-makassaric acid ........................................................... 41	  4.4	   Conclusions ...................................................................................................................... 45	  Chapter 5: Experimental section ............................................................................................... 46	  5.1	   General experimental procedures ..................................................................................... 46	  5.2	   Synthesis procedures ........................................................................................................ 46	  5.2.1	   Preparation of (E)-oxime 2.6 ..................................................................................... 46	    vi 5.2.2	   Preparation of methyl ester 2.7 and 2.8 ..................................................................... 51	  5.2.3	   Preparation of phenol 2.9 .......................................................................................... 55	  5.2.4	   Preparation of methyl ester 2.10 ............................................................................... 57	  5.2.5	   Preparation of carboxylic acid 2.11 ........................................................................... 59	  5.2.6	   Preparation of hydroxamic acid 2.12 ........................................................................ 61	  5.2.7	   Preparation of oxime 2.13 ......................................................................................... 63	  5.2.8	   Preparation of methyl ester 2.14 ............................................................................... 65	  5.2.9	   Preparation of carboxyl acid 2.15 ............................................................................. 67	  5.2.10	   Preparation of hydroxamic acid 2.17 and 2.46 ........................................................ 69	  5.2.11	   Preparation of PMB protected oxime 2.21 .............................................................. 73	  5.2.12	   Preparation of phenyl prenylated ether 2.22 ........................................................... 75	  5.2.13	   Preparation of hydroxamic acid 2.22.1 ................................................................... 77	  5.2.14	   Preparation of methyl ester 2.23 ............................................................................. 79	  5.2.15	   Preparation of tosylate 2.24 ..................................................................................... 81	  5.2.16	   Preparation of alcohol 2.25 ..................................................................................... 83	  5.2.17	   Preparation of oxime 2.26 ....................................................................................... 85	  5.2.18	   Preparation of methyl ester 2.29 ............................................................................. 87	  5.2.19	   Preparation of serpulanine 2.1 ................................................................................. 89	  5.2.20	   Preparation of phenol 2.31 ...................................................................................... 92	  5.2.21	   Preparation of methyl ester 2.34 ............................................................................. 94	  5.2.22	   Preparation of protected alcohol 3.5a and 3.5b ....................................................... 96	  5.2.23	   Preparation of compound 3.5.5a and 3.5.5b ............................................................ 99	  5.2.24	   Preparation of pyridine alcohol 3.6a and 3.6b ...................................................... 103	    vii 5.2.25	   Preparation of dimer and trimer 3.7 and 3.8 ......................................................... 106	  5.2.26	   Preparation of pyridinium bromide 3.9 ................................................................. 110	  5.2.27	   Preparation of monomer 3.10 ................................................................................ 112	  5.2.28	   Preparation of reduced trimer 3.11 ........................................................................ 114	  5.2.29	   Preparation of ethyl ester 4.13 ............................................................................... 116	  5.2.30	   Preparation of all E-geranylgeraniol 4.14 ............................................................. 118	  5.2.31	   Preparation of ester 4.15 ........................................................................................ 120	  5.2.32	   Preparation of bromohydrin 4.16 .......................................................................... 122	  5.2.33	   Preparation of epoxide 4.17 .................................................................................. 124	  References .................................................................................................................................. 126	     viii List of tables  Table 2.1 Attempts to obtain desired compound 2.8 in a higher yield ......................................... 14	  Table 2.2 Various oxime protection methods ............................................................................... 15	  Table 2.3 Attempts to deprotect PMB group ................................................................................ 20	  Table 2.4 Attempts to dehydrate tertiary alcohol 2.26 to 2.8 ........................................................ 22	  Table 2.5 Attempts to synthesize desired phenol prenylated ether 2.8 ......................................... 23	  Table 3.1 Attempts to reduce halitoxin and alotau ........................................................................ 38	     ix List of figures  Figure 1.1 Structures of morphine, quinine, and penicillin ............................................................. 2	  Figure 1.2 Structures of DFO-B, TSA and fosmidomycin ............................................................. 3	  Figure 1.3 The general structure of halitoxins ................................................................................ 4	  Figure 1.4 Structures of (+)-makassaric acid, (-)-menthol, retinol and cholesterol ........................ 4	  Figure 1.5 Structures of spongothymidine, ara-A, ara-C, ziconotide ............................................. 5	  Figure 1.6 Structures of ET-743 and cyanosafracin B .................................................................... 6	  Figure 2.1 The roles of HATs and HDACs in gene transcription ................................................... 7	  Figure 2.2 Structures of hydroxamic acids as HDIs ........................................................................ 9	  Figure 2.3 The structure of serpulanine 2.1 .................................................................................... 9	  Figure 2.4 HDAC assay results of compound 2.8 and 2.1 ............................................................ 29	  Figure 2.5 HDAC assay results of compound 2.46 and 2.17 ........................................................ 30	  Figure 2.6 Synthesized serpulanine analogs ................................................................................. 31	  Figure 3.1 Role of calcineurin in T-cell activation ....................................................................... 32	  Figure 3.2 Chemical structure of cyclosporine A (CsA) and FK506 ............................................ 33	  Figure 3.3 1H spectrum of the unknown natural product alotau and the structure of halitoxins ... 34	  Figure 3.4 The biological activities of samples to inhibit dephosphorylation of GST-NFAT1(1-415) ................................................................................................................................................ 37	  Figure 4.1 The structure of (+)-makassaric acid ........................................................................... 39	  Figure 5.1 1H and 13C NMR spectra of 2.6 recorded in acetone-d6 at 300 MHz and 75 MHz ..... 48	  Figure 5.2 1D NOE NMR spectrum of 2.6 (irradiating oxime proton) recorded in DMSO-d6 at 400 MHz ........................................................................................................................................ 49	    x Figure 5.3 1D NOE NMR spectrum of 2.6 (irradiating methylene) recorded in DMSO-d6 at 400 MHz ............................................................................................................................................... 50	  Figure 5.4 1H and 13C NMR spectra of 2.7 recorded in chloroform-d at 300 MHz and 75 MHz . 53	  Figure 5.5 1H and 13C NMR spectra of 2.8 recorded in chloroform-d at 400 MHz and 100 MHz ....................................................................................................................................................... 54	  Figure 5.6 1H and 13C NMR spectra of 2.9 recorded in chloroform-d at 300 MHz and 75 MHz . 56	  Figure 5.7 1H and 13C NMR spectra of 2.10 recorded in chloroform-d at 300 MHz and 75 MHz ....................................................................................................................................................... 58	  Figure 5.8 1H and 13C NMR spectra of 2.11 recorded in chloroform-d at 400 MHz and 100 MHz ....................................................................................................................................................... 60	  Figure 5.9 1H and 13C NMR spectra of 2.12 recorded in methanol-d4 at 400 MHz and 100 MHz ....................................................................................................................................................... 62	  Figure 5.10 1H and 13C NMR spectra of 2.13 recorded in methanol-d4 at 600 MHz and 150 MHz ....................................................................................................................................................... 64	  Figure 5.11 1H and 13C NMR spectra of 2.14 recorded in chloroform-d at 400 MHz and 100 MHz ............................................................................................................................................... 66	  Figure 5.12 1H and 13C NMR spectra of 2.15 recorded in chloroform-d at 400 MHz and 100 MHz ............................................................................................................................................... 68	  Figure 5.13 1H and 13C NMR spectra of 2.17 recorded in chloroform-d at 400 MHz and 100 MHz ............................................................................................................................................... 71	  Figure 5.14 1H and 13C NMR spectra of 2.46 recorded in chloroform-d at 400 MHz and 100 MHz ............................................................................................................................................... 72	    xi Figure 5.15 1H and 13C NMR spectra of 2.21 recorded in chloroform-d at 400 MHz and 100 MHz ............................................................................................................................................... 74	  Figure 5.16 1H and 13C NMR spectra of 2.22 recorded in chloroform-d at 400 MHz and 100 MHz ............................................................................................................................................... 76	  Figure 5.17 1H and 13C NMR spectra of 2.22.1 recorded in chloroform-d at 400 MHz and 100 MHz ............................................................................................................................................... 78	  Figure 5.18 1H and 13C NMR spectra of 2.23 recorded in chloroform-d at 400 MHz and 100 MHz ............................................................................................................................................... 80	  Figure 5.19 1H and 13C NMR spectra of 2.24 recorded in chloroform-d at 300 MHz and 75 MHz ....................................................................................................................................................... 82	  Figure 5.20 1H and 13C NMR spectra of 2.25 recorded in chloroform-d at 400 MHz and 100 MHz ............................................................................................................................................... 84	  Figure 5.21 1H and 13C NMR spectra of 2.26 recorded in chloroform-d at 400 MHz and 100 MHz ............................................................................................................................................... 86	  Figure 5.22 1H and 13C NMR spectra of 2.29 recorded in chloroform-d at 400 MHz and 100 MHz ............................................................................................................................................... 88	  Figure 5.23 1H and 13C NMR spectra of 2.1 recorded in methanol-d4 at 400 MHz and 100 MHz ....................................................................................................................................................... 90	  Figure 5.24 1D NOE NMR spectrum of 2.1 by irradiating methylene recorded in DMSO-d6 at 600 MHz ........................................................................................................................................ 91	  Figure 5.25 1H and 13C NMR spectra of 2.31 recorded in chloroform-d at 400 MHz and 100 MHz ............................................................................................................................................... 93	    xii Figure 5.26 1H and 13C NMR spectra of 2.34 recorded in methanol-d4 at 400 MHz and 100 MHz ....................................................................................................................................................... 95	  Figure 5.27 1H and 13C NMR spectra of 3.5a recorded in chloroform-d at 300 MHz and 75 MHz ....................................................................................................................................................... 97	  Figure 5.28 1H and 13C NMR spectra of 3.5b recorded in chloroform-d at 300 MHz and 75 MHz ....................................................................................................................................................... 98	  Figure 5.29 1H and 13C NMR spectra of 3.5.5a recorded in chloroform-d at 300 MHz and 75 MHz ............................................................................................................................................. 101	  Figure 5.30 1H and 13C NMR spectra of 3.5.5b recorded in chloroform-d at 300 MHz and 75 MHz ............................................................................................................................................. 102	  Figure 5.31 1H and 13C NMR spectra of 3.6a recorded in chloroform-d at 300 MHz and 75 MHz ..................................................................................................................................................... 104	  Figure 5.32 1H and 13C NMR spectra of 3.6b recorded in chloroform-d at 400 MHz and 100 MHz ............................................................................................................................................. 105	  Figure 5.33 1H and 13C NMR spectra of 3.7 recorded in methanol-d4 at 400 MHz and 100 MHz ..................................................................................................................................................... 108	  Figure 5.34 1H and 13C NMR spectra of 3.8 recorded in methanol-d4 at 400 MHz and 100 MHz ..................................................................................................................................................... 109	  Figure 5.35 1H and 13C NMR spectra of 3.9 recorded in methanol-d4 at 400 MHz and 100 MHz ..................................................................................................................................................... 111	  Figure 5.36 1H and 13C NMR spectra of 3.10 recorded in DMSO-d6 at 400 MHz and 100 MHz ..................................................................................................................................................... 113	  Figure 5.37 1H spectrum recorded in chloroform-d at 600 MHz and MS spectrum of 3.11 ...... 115	    xiii Figure 5.38 1H spectrum of 4.13 recorded in chloroform-d at 400 MHz .................................... 117	  Figure 5.39 1H and 13C NMR spectra of 4.14 recorded in chloroform-d at 400 MHz and 100 MHz ............................................................................................................................................. 119	  Figure 5.40 1H and 13C NMR spectra of 4.15 recorded in chloroform-d at 400 MHz and 100 MHz ............................................................................................................................................. 121	  Figure 5.41 1H and 13C NMR spectra of 4.16 recorded in chloroform-d at 400 MHz and 100 MHz ............................................................................................................................................. 123	  Figure 5.42 1H and 13C NMR spectra of 4.17 recorded in chloroform-d at 400 MHz and 100 MHz ............................................................................................................................................. 125	     xiv List of schemes  Scheme 2.1 Retrosynthetic analysis of serpulanine ...................................................................... 10	  Scheme 2.2 Synthesis of (E)-oxime 2.6 and important 1D NOE correlations in 2.6 .................... 11	  Scheme 2.3 Proposed mechanism of stereoselective oxidation (adapted from Noyori et al. 18) .. 12	  Scheme 2.4 Trace amounts of desired phenol prenylated ether 2.8 synthesized from oxime 2.6 13	  Scheme 2.5 Synthesis of oxime protected compound 2.9 and phenol alkylated ether 2.10. ........ 16	  Scheme 2.6 Total synthesis of serpulanine analog 2.13. ............................................................... 16	  Scheme 2.7 Synthesis of phenol prenylated ether 2.15 ................................................................. 17	  Scheme 2.8 Failed synthesis of hydroxamic acid 2.17 ................................................................. 18	  Scheme 2.9 Direct amidation synthesis of hydroxamic acid 2.17 inspired by Spilling’s work .... 18	  Scheme 2.10 Deprotection of benzyl group leading to undesired phenol 2.20 ............................. 19	  Scheme 2.11 Synthesis of the PMB protected oxime 2.22 ........................................................... 19	  Scheme 2.12 Side reactions during PMB deprotection trials ........................................................ 21	  Scheme 2.13 Synthesis of tertiary alcohol 2.26 ............................................................................ 21	  Scheme 2.14 Synthesis of ester 2.29 ............................................................................................. 23	  Scheme 2.15 Synthesis of serpulanine 2.1 and key 1D NOE correlation in 2.1 ........................... 24	  Scheme 2.16 Synthetic route to the natural product serpulanine and its analogs. ........................ 25	  Scheme 2.17 Attempts to install prenyl group on the phenol position of L-tyrosine methyl ester ....................................................................................................................................................... 26	  Scheme 2.18 Attempts to synthesize ether 2.35 ............................................................................ 26	  Scheme 2.19 Spilling’s work and future work to synthesize serpulanine in a higher yield .......... 28	  Scheme 3.1 Synthesis of dimer 3.7 and trimer 3.8 ........................................................................ 35	    xv Scheme 3.2 Synthesis of monomer 3.10 ....................................................................................... 36	  Scheme 3.3 Reduction of trimer 3.8 .............................................................................................. 37	  Scheme 4.1 Retrosynthetic scheme of (+)-makassaric acid reported by Basabe group ................ 40	  Scheme 4.2 Synthesis of compound 4.5 reported by Basabe group ............................................. 40	  Scheme 4.3 Synthetic proposal using fragment B analog 4.9 ....................................................... 41	  Scheme 4.4 Retrosynthetic scheme of compound 4.9 ................................................................... 41	  Scheme 4.5 Synthesis of all E compound 4.13 ............................................................................. 42	  Scheme 4.6 Attempts to optimize HWE reaction ......................................................................... 42	  Scheme 4.7 Synthesis of bromohydrin 4.16 .................................................................................. 43	  Scheme 4.8 Synthesis of epoxide 4.17 .......................................................................................... 43	  Scheme 4.9 Attempt to synthesize tricyclic alkene 4.9 ................................................................. 44	  Scheme 4.10 Attempt to synthesis tricyclic alcohol 4.20 ............................................................. 44	  Scheme 4.11 Synthesis of epoxide 4.17 from farnesylacetone ..................................................... 45	     xvi List of abbreviations and symbols #  °C $  (±)  %  δ 1D-  2D-  A9 Ac ARE APL B.C.  Bn Boc  br BRSM Bu Calcd CAN d number  degrees Celsius dollar(s)  racemic  per cent  chemical shift in parts per million one dimensional-  two dimensional-  murine metastatic lung carcinoma acetyl AU-Rich Element acute promyelocytic leukemia Before Christ  benzyl t-butoxycarbonyl  broad based on recovered starting material butyl calculated ceric ammonium nitrate doublet   xvii DBN DBU DCC  DCM  dd DDQ DHP DIBAL-H  DIEA  DIPEA  DMAP  DME DMF  DMSO  DNA  EDCI  EtO2 EtOAc  FDA  g  h  HAT  1,5-diazabicyclo(4.3.0)non-5-ene 1,8-diazabicycloundec-7-ene N,N'-dicyclohexylcarbodiimide  dichloromethane  doublet of doublets 2,3-dichloro-5,6-dicyano-1,4-benzoquinone dihydropyranyl diisobutylaluminium hydride  N,N-diisopropylethylamine  N,N-diisopropylethylamine  4-dimethylaminopyridine  dimethoxyethane dimethylformamide  dimethyl sulfoxide  deoxyribonucleic acid  1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide diethyl ether  ethyl acetate  the Food and Drug Administration  gram(s)  hour(s)  histone acetyltransferase   xviii HDAC HDI HIV  HPLC  HRESIMS  Hsp27  HTS  HWE Hz  IC50  imid. IL  J  L- LDA lps m  M  Me  MeCN MeOH mg  histone deacetylase Histone deacetylase inhibitor human immunodeficiency virus  high-performance liquid chromatography  high resolution electrospray ionisation mass spectroscopy  heat-shock protein of 27 kDa  high-throughput screening Horner–Wadsworth–Emmons  hertz  half maximal inhibitory concentration imidazole  interleukin coupling constant  laevus lithium diisopropylamide lipopolysaccharide multiplet  mole  methyl acetonitrile methanol  milligram(s)    xix mg/mL MHz  min MK2  µM µg/mL mmol/L  MOM mRNA  MS  NBS NFAT nM  NMR  NOE  Nu  PG pKa  pM  PMB RNA r.t. (rt) milligram(s) per milliliter megahertz  minute(s)  mitogen-activated protein kinase-activated protein kinase 2 micromole(s)  microgram(s) per milliliter  millimole(s) per liter  methoxymethyl messenger ribonucleic acid  mass spectroscopy  N-bromosuccinimide nuclear factor of activated T-cells nanomole(s)  nuclear magnetic resonance  nuclear overhauser effect  nucleophile  protecting group -lg (acid dissociation constant)  picomole(s)  4-methoxybenzyl ribonucleic acid room temperature   xx s  S  SAR SCUBA sp. t  TBAF TBDMS TES Tf  TFA  TFAA  THF THP  TLC  TMS  TNF-α  Ts TSA TTP UBC UV  second(s)  sinister  structure-activity relationship  self-contained underwater breathing apparatus  species triplet tetrabutylammonium fluoride hydrate  tert-butyldimethylsilyl triethylsilyl Trifluoromethanesulfonyl  trifluoroacetic acid  trifluoroacetic anhydride  tetrahydrofuran  tetrahydropyranyl thin layer chromatography  trimethylsilyl  tumor necrosis factor alpha  4-toluenesulfonyl trichostatin A tristetraprolin the University of British Columbia  ultraviolet    xxi Acknowledgements  First and foremost I would like to thank my supervisor, Prof. Raymond J. Andersen, for his support in helping me explore the world of natural products and organic synthesis. His patience and encouragement helped me to go through difficulties in research, and his dedicated work and passion about science taught me what I should do both in my career and my life.   I would like to thank Dr. David E. Williams for his useful advice in compound purifications and thesis writing. I would also like to thank Mike LeBlanc for his generous help in maintaining our lab and conducting bioassays. Thanks also go to Prof. Glenn Sammis and Prof. Martin Tanner for participation in my MSc examining committee.  I would like to extend my gratitude towards my lab mates who offered me a hand whenever I needed. Special thanks go to Dr. Ryan Centko, Kalindi Morgan and Rosanne Persaud for helpful comments on thesis; Meng Wang and Lingzhi Zhang for joyful and meaningful chats. I would also thank my fellow friends in the chemistry department: Dr. Sanjia Xu, Nikita Jain and Wei Zhang for inspirational organic discussion, as well as everybody in the NMR and MS labs for collecting valuable data.  At last, I would like to thank my parents, who have supported me throughout my years of education, both morally and financially.    xxii Dedication       To my beloved parents, Chaoxiu Xiao and Pizhen Zheng 致我最爱的父母,肖朝秀和郑丕珍   1 Chapter 1: Introduction 1.1 Introduction to natural products The interchangeable terms ‘natural product’ and ‘secondary metabolite’ generally refer to metabolites produced by living organisms that are not needed for normal growth and development, but provide the producing organisms with competitive advantages during the evolutionary process.1 For thousands of years, natural product producing plants and animals have been used as medicines. For example, Epimedium species were recorded as beneficial herbs to improve muscle and bone health in Shen Nong Herbal during 200-300 B.C. in China.2 Despite this long history, natural products would not be purified, identified and synthesized until the 19th century. Morphine was the first purified natural product in 1805, and now it is still one of the most widely used painkillers.3 The antimalarial drug quinine had its structure determined in 1908. It was not until 40 years later, in 1944, when, in a landmark for organic chemistry, Robert Burns Woodward completed its total synthesis.4, 5 Arguably, the most influential natural product is penicillin whose important anti-bacterial activity was discovered by Alexander Fleming in 1928.  Subsequently mass-produced by fermentation in the early 1940’s, penicillin changed both the face of World War II and of modern medicine.6, 7 Penicillin has been estimated to have saved as many as 200 million lives since its first production. The chemical structure of penicillin was determined by Dorothy Hodgkin in 1945, and its total synthesis was developed by John C. Sheehan in 1957.8, 9     2  Figure 1.1 Structures of morphine, quinine, and penicillin  1.2 Classes of natural products In this thesis, the following chapters will discuss three synthetic projects related to three classes of natural products: hydroxamates, halitoxins and terpenoids.   1.2.1 Hydroxamate-containing natural products Of the various hydroxamate-containing natural products that have been isolated, many have demonstrated important biological activities. It is thought that this is due to the potent metal chelating property of the hydroxamic acid moiety. As such, hydroxamates are commonly found in siderophores, the small iron chelating compounds produced by microorganisms for acquiring ferric ions from the environment. One such siderophore, DFO-B (desferrioxamine B, deferoxamine), was isolated by Keller-Schierlein et al. from the bacterium Streptomyces pilosus in 1965.10 DFO-B is an important medication used to treat patients with acute or chronic iron poisoning. Another hydroxamate compound, trichostatin A (TSA), was isolated from the bacterial species Streptomyces hygroscopicus by Tsuji, et al. in 1976.11 TSA is used as a histone deacetylase inhibitor to regulate gene expression, and this will be discussed in greater detail in Chapter 2.12 Fosmidomycin is a hydroxamate antibiotic isolated by Iguchi E. et al. from a Streptomyces sp. in 1979.13 Fosmidomycin also exhibits antimalarial activity.  The mode of action for this activity was shown to be due to the inhibition of an enzyme in the mevalonate-independent terpenoid biosynthetic pathway present in the malaria parasite, Plasmodium HOHOO HN1.1  MorphineOHONN1.2  QuinineHNORNSCOOHO1.3  Penicillin  3 falciparum. 14  The crystal structure of this target, DXR (1-deoxy-D-xylulose 5-phosphate reductoisomerase), showed the hydroxamate group binding directly to the active site Mg2+ ion.15  Figure 1.2 Structures of DFO-B, TSA and fosmidomycin  1.2.2 Halitoxins Halitoxins are a family of compounds characterized by complex macrocyclic structures consisting of different numbers of monomeric 1,3 alkyl-pyridinium units. 16 Each kind of halitoxin is a complex mixture with a molecular weight range, showing the same NMR and MS spectra and biological activities. Halitoxins are produced as toxins by different species of marine sponges and were first characterized by Francis J. Schmitz et al. in 1978.17 One of the halitoxins, HvTX, isolated by Carlos Sevcik et al. from the sponge Haliclona viridis. HvTX was shown to block potassium permeability in resting muscle fibers, leading to adepolarizing effect on muscle membrane.18 The halitoxin isolated by Roberto G. S. Berlinck et al. showed moderate lethal activity characterized by the death of half the tested mice population after around 35 minutes following the injection of 1.4 mg/kg of halitoxin complex.19 Halitoxins have also been used to cause cell membranes lesions, allowing transfection of plasmid cDNA (complementary DNA) into intracellular environment for gene expression.20  H2NHNONHONOHO HNONOHONHO OOHN1.5  TSA1.4  DFO-BP NOHOOOHHO1.6 Fosmidomycin  4  Figure 1.3 The general structure of halitoxins 1.2.3 Terpenoids The hydrocarbon skeletons of terpenoids, which are the most common natural products, are constructed of repeating five carbon isoprene units. Many terpenoid natural products contain additional functional groups that influence their bioactivity. In this thesis, chapter 4 will discuss (+)-makassaric acid. It is a meroterpenoid, being partially derived from a terpenoid fragment and a polyketide fragment.21 Terpenoids are categorized by the number of isoprene units involved in their biosynthesis. For example, monoterpenoid, diterpenoid and triterpenoids are common classes of terpenoids made of two, four, and six isoprene units, respectively. (-)-Menthol is a monoterpenoid widely used in the food industry. Phytol is a diterpenoid as the industrial precursor of the vitamin E.22 Cholesterol is a degraded triterpenoid produced by all animal cells to maintain the structural integrity and fluidity of cell membranes.      Figure 1.4 Structures of (+)-makassaric acid, (-)-menthol, retinol and cholesterol NCH2 nNCH2 nN3.1 Halitoxins ( n = 2, 3, 4, 5)HHOHCOOH4.1 (+)-Makassaric acidOH1.7 (-)-MentholHOHHH1.9  Cholesterol1.8 PhytolHO  5  1.3 Introduction to marine natural products In two out of the three synthetic projects described in the following chapters, the target molecules originated from marine habitats. The beginning of marine natural products started in the1950s with the improvement of scuba diving technology.23, 24 The first reported bioactive marine natural product, spongothymidine was isolated from marine sponge by Werner Bergman in 1950.25 Spongothymidine is a nucleoside analogue which contains D-arabinose instead of D-ribose. This inspired the development of antiviral drug ara-A (vidarabine, Vira-A®) and anticancer drug ara-C (cytarabine, Cytosar-U®).26 In the 1970s, the emergence of advanced analytical techniques such as high-performance liquid chromatography (HPLC), mass spectrometry (MS) and nuclear magnetic resonance (NMR) set the stage for systematic investigation of marine natural products. For example, the venom secreted from sea snail was isolated and characterized by Baldomero Olivera in 1977 as a complex mixture of small peptides.27 One toxin produced by Conus magus, ziconotide (SNX-111; Prialt®) was developed as a potent painkiller that was approved by the Food and Drug Administration (FDA) in 2004 for treatment of severe chronic pain.28   Figure 1.5 Structures of spongothymidine, ara-A, ara-C, ziconotide OHOHOHONNNNNH21.10  Ara-AOHOHOHONNNH2O1.11  Ara-CCys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-amide1.12  Ziconotide  6 1.4 Importance of marine natural products Marine organisms provide structurally diverse secondary metabolites with therapeutic potential.29 As of April 2016, there are seven marine-derived drugs approved by the FDA, with five of the seven approved in the past 12 years.30 ET-743 (Trabectedin, Yondelis®) was the latest approval in 2015, used for unresectable or metastatic soft tissue sarcomas. The development of ET-743 showed the value of marine drug discovery that facilitated innovative methods such as aquaculture production, high-efficiency total synthesis and semi-synthesis aided by genetic engineering.31 ET-743 was originally isolated in 1992 from the sea squirt Ecteinascidia turbinata by Kenneth L. Rinehart et al. in low yields.32 It took 1 ton of sea squirts to collect 1 gram of ET-743, and the first attempt of mariculture provided about 5 grams for clinical evaluation.33 Later, the total synthesis of ET-743 was reported by Elias James Corey et al. in an industrially impracticable 24 steps. 34  In 2000, the semisynthesis starting from cyanosafracin B was developed by PharmaMar to obtain multi grams of ET-743 in 20 steps.35   Figure 1.6 Structures of ET-743 and cyanosafracin B     AcOMeOONNMeOHHOMeHO MeSOONHHOMeONNOOMeMeOHNHONH2OMeHO MeCNMe1.13 ET-743 1.14  Cyanosafracin B  7 Chapter 2: Synthesis of analogs of serpulanine 2.1 Histone deacetylase (HDAC) Histones are proteins surrounded by double-stranded DNA to form nucleosomes, the basic unit of chromatin.36 Post-translational modifications of histones, such as acetylation can regulate gene transcription.37, 38 There are two opposing enzymes controlling acetylation of histones: histone deacetylases (HDACs) and histone acetyltransferases (HATs).39, 40 The roles of HDAC and HAT in gene transcription are shown in Figure 2.1.41   Figure 2.1 The roles of HATs and HDACs in gene transcription In Figure 2.1, highly charged lysine side chain amino groups in non-acetylated histones lead to the tight binding to the DNA phosphate backbone, preventing transcription proteins and complexes from approaching DNA.42 Once histone lysine side chain amino groups become acetylated by HATs, the reduced charges on the histones allow a more open DNA conformation that promotes gene expression. Conversely, HDACs deacetylate histones, leading to more compact DNA and repression of gene transcription.    8 2.2 Histone deacetylase and cancer HDACs have been identified as druggable anticancer targets due to their interaction with oncoproteins to over-repress transcription.43  In the disease of acute promyelocytic leukemia (APL), the retinoid acid receptor mutant proteins repress the retinoid acid target gene through the corepressor-HDAC complex, resulting in the blockage of myeloid differentiation.44 However, dissociation of corepressor-HDAC complexes leaves a positive effect on APL patients. Robson et al. also reported the overexpression of HDAC in the development of prostate caner. The large contribution of HDAC to prostate caner tumor cells’ aggressive proliferation and metastatic potential allowed HDAC to be a therapeutic target.45   2.3 Histone deacetylase inhibitors (HDIs) Most of HDIs have functional groups that bond to the zinc-containing active domain of HDAC leading to inhibition.46 HDIs are classified to different groups according to the zinc-binding functional group: Short-chain fatty acid (butyrate), hydroxamate (Trichostatin A), cyclic tetrapeptide (Romidepsin, approved by the FDA for clinical use in 2009) etc.47  Several important HDIs in the hydroxamate class are shown in Figure 2.2. Trichostatin A (TSA) inhibits HDACs with IC50 values in the low nM range.48 Vorinostat (SAHA, Zolinza®), developed by Merck for the treatment of patients with manifestations of cutaneous T-cell lymphoma, was approved for clinical use by the FDA in 2006. 49  Belinostat (PXD101, Beleodaq®), developed by the company TopoTarget, was approved by the FDA in 2007 for treating patients with peripheral T-cell lymphoma.50 Panobinostat (LBH-589, Farydak®) was approved by the FDA in 2015 as a pan-HDAC inhibitor for treating patients with multiple myeloma.51   9  Figure 2.2 Structures of hydroxamic acids as HDIs   2.4 Isolation and characterization of serpulanine  Serpulanine (2.1), a novel hydroxamic acid, was isolated from dried fruiting bodies of the macrofungus Serpula sp. collected in Sri Lanka. The structural proposal for serpulanine 2.1 was based upon analysis of its NMR and HRESIMS data. X-ray diffraction analysis of crystals grown from methanol confirmed the proposed structure, including the configuration of the oxime.   Figure 2.3 The structure of serpulanine 2.1  2.5 Synthesis of serpulanine Serpulanine is a novel natural product having the hydroxamic acid functional group, with the potential to be a histone deacetylase inhibitor. The vicinal oxime hydroxamic acid moiety is also unusual and no synthesis of serpulanine has been reported. Therefore, we decided to develop a synthetic scheme to serpulanine in order to generate more material for biological evaluation.  NHO OOHNHNONHOOHNHSNHOOHO O NHOHNHNOH2.2 Trichostatin A 2.3 Vorinostat2.4 Belinostat 2.5 PanobinostatNONHHOOOH  10  2.5.1 Retrosynthetic analysis of serpulanine Serpulanine 2.1 has prenylated phenol, (E)-oxime and hydroxamic acid functionalities (Scheme 2.1). Our proposed synthetic route to serpulanine 2.1 shown in Scheme 2.1 was designed to install the hydroxamic acid group at the end of the synthesis due to its complexity. Prenylation of the phenol group was to take place after the introduction of the (E)-oxime using methodology reported by Spilling et al. through stereoselective oxidation of the amino group present in the methyl ester of tyrosine.52   Scheme 2.1 Retrosynthetic analysis of serpulanine  2.5.2 Initial synthetic trials Following the procedure described by Spilling, treatment of L-tyrosine methyl ester with a stoichiometric amount of sodium tungstate afforded oxime 2.6 in 82% yield (Scheme 2.2). In order to determine the configuration of oxime 2.6, a 1D NOE experiment was performed (spectra are in Figure 5.2, 5.3 of the experimental section). The E configuration was supported by correlations observed between δH 7.01 (The aromatic protons meta to the phenol group) and δH 12.39 (=N-OH), and between δH 3.72 (protons on the benzyl methylene) and δH 12.39 (=N-OH).  NONHHOOOHNOOMeHOONH2OMeOOH2.1 2.8L-tyrosine methyl ester  11  Scheme 2.2 Synthesis of (E)-oxime 2.6 and important 1D NOE correlations in 2.6  Prof. Noyori has studied the tungsten-catalyzed hydrogen peroxide oxidation of alcohols to carbonyl compounds.53 Based on his work, the primary amine oxidization mechanism shown below is proposed to explain the stereoselectivity of oxime formation in 2.6. (Scheme 2.3).    NH2OMeONOMeOHOOH OHNa2WO4 1.0 equiv. 30% H2O2 aq.H2O, EtOHL-tyrosine methyl ester2.6NOMeOHOOH2.6NOE  12   Scheme 2.3 Proposed mechanism of stereoselective oxidation (adapted from Noyori et al. 18) In Scheme 2.3, the primary amine is oxidized by hydrogen peroxide to an amine oxide. Sodium tungstate is oxidized to bisperoxotungstate compound A, in equilibrium with the more reactive mono-protonated species B. Water-amine oxide ligand exchange with B gives bisperoxo complex C, which undergoes proton transfer to produce the reactive compound D. In the chair conformation transition state of D, the amine oxide is further oxidized to the (E)-oxime by the adjacent hydroperoxy ligand, because the positively polarized oxygen atom induces the hydridic NR2R1HCHHO OHHNR2R1HCHHOHOHNR2R1HCHOH+ H2ONa2WO4 + 2 H2O2 Na2[WO(O2)2(OH)2] + H2O+6 +8AW OOOHO OO-NHHO CHR1 R2W OOOHO OO-NCHR1R2HO HW OOOHO OO-OH HWOOHOO-OOH HNR2R1HCHOHH2OH2O2H2ONHO R1R2R2 = Bulky groupE-OximeWOHOOOHO OO2-W OOOHO OO-OH HH+- H+A BNot Active ActiveBCEDHOOWNHOR2HR1OOO OH-D  13 hydrogen migration and the bulky group R2 stays in the equatorial position. Along with (E)-oxime, the monoperoxo tungstate ion E is generated, which is oxidized back to active species B by hydrogen peroxide in the catalytic cycle. Reducing the amount of sodium tungstate from 1.00 to 0.05 equivalent, the same product (E)-oxime 2.6 was obtained with a similar yield of 81%, confirming the catalytic role of sodium tungstate and reducing the environmental risk of using heavy metals.  In the next step, we attempted to prenylate (E)-oxime 2.6 to obtain compound 2.8. Unfortunately, as shown in Scheme 2.4, the oxime prenylated ether 2.7 was the major product obtained in this reaction, with the desired phenyl prenylated ether 2.8 being produced only in trace amounts.   Scheme 2.4 Trace amounts of desired phenol prenylated ether 2.8 synthesized from oxime 2.6  In order to find optimal conditions for the formation of desired phenyl ether 2.8, a series of screening experiments were conducted that involved changing bases, solvents, and reaction temperature as described in Table 2.1. TLC analysis showed that only the conditions in entry 5 provided limited amounts of the desired compound 2.8. The strong nucleophilicity of the oxime can be explained by two factors: first, the pKa of the oxime group (pKa=20) is higher than phenol group (pKa=18) in aprotic solvents, which means that the conjugate base of the oxime is NOOMeNOMeOHO OOH OHBr 1.0 equiv.NOOMeHOO+2.8 Trace amounts2.7 Major productTHFNaH 1.2 equiv.2.6  14 a stronger base and is therefore expected to be more nucleophilic; second, the presence of the adjacent nitrogen atom with lone pair electrons increases the nucleophilicity of oxygen by the ‘alpha effect’.54  Table 2.1 Attempts to obtain desired compound 2.8 in a higher yield Entry Base Solvent T (°C) Time (h) Result 1 NaH 1.2 equiv. THF r.t. 24 Trace amounts of 2.8 2 NaH 2.0 equiv. DMF r.t. 24 Trace amounts of 2.8 3 K2CO3 2.0 equiv. DMF -42 to r.t. 24 Trace amounts of 2.8 4 K2CO3 2.0 equiv. DMF 75 12 Trace amounts of 2.8 5 K2CO3 2.0 equiv. DMF r.t. 24 Minor amounts of 2.8  Due to our inability to overcome the problem of the strong oxime nucleophilicity, it became clear that protection of the oxime was required to efficiently prenylate the phenol group. Different protection strategies were explored as shown in Table 2.2.    NOMeOHOOHBrbase, solvent1 equiv.NOOMeOOHNOOMeHOO+2.6 2.7 Major product 2.8 Trace amounts  15  Table 2.2 Various oxime protection methods Entry Protecting reagent Acid or Base T (°C) Time (h) Solvent Result 1 DHP TsOH cat. r.t. 48 Dioxane No reaction 2 TBDMS-OTf K2CO3 1.2 equiv. r.t. 24 DMF No reaction 3 TBDMS-OTf K2CO3 1.2 equiv. 75 24 DMF No reaction 4 TES-Cl NaOMe 2.0 equiv. r.t. 24 DMF No reaction 5 TES-Cl NaH 2.0 equiv. r.t. 12 DMF No reaction  As seen in Table 2.2, THP and silyl protection all failed to protect the oxime functionality. The difficulty of installation of bulky groups could be caused by steric effect of the adjacent phenyl group. Changing protecting reagent to benzyl bromide led to successful protection by the less bulky benzyl group (Scheme 2.5) to give the oxime 2.9.  As shown in Scheme 2.5, when isopentyl bromide was used as the alkylating reagent, phenol ether 2.10 was directly synthesized from tyrosine oxime 2.6 in one pot. Two reasons accounted for using isopentyl bromide instead of prenyl bromide: first, ordinary benzyl deprotection step by hydrogenolysis would reduce the prenyl group; second, serpulanine analogs with an isopentyl moiety could be obtained quickly to conduct an SAR study. NOMeOHOOHbase, solventprotecting reagent1.0 equiv.NOMeOPGOOH  16  Scheme 2.5 Synthesis of oxime protected compound 2.9 and phenol alkylated ether 2.10. With phenol alkylated ether 2.10 in hand, base hydrolysis gave carboxylic acid 2.11 (Scheme 2.6). Carboxylic acid 2.11 was then activated to the acid chloride intermediate by SOCl2, observed using NMR in CDCl3. Excess SOCl2 was evaporated under reduced pressure, and immediate addition of hydroxylamine hydrochloride and triethylamine provided hydroxamic acid 2.12. Removal of the benzyl group by hydrogenolysis and purification of the crude product by HPLC gave desired analog 2.13, the saturated version of the natural product serpulanine.  Scheme 2.6 Total synthesis of serpulanine analog 2.13. NOMeOBnOOBnBr, K2CO3, DMFthenBrNOMeOHOOHNOMeOBnOOHBnBr, K2CO3DMF2.6 2.92.10NH2OH•HCl, NEt3, DCM, 44%MeOH, H2O, 87%NaOH, pH=12NOHOBnOOSOCl2, CDCl3, then evaporatedNNHOBnOOOHPd/C, H2MeOH, 59%NNHOHOOOHNH2OMeONOMeOHOOH OHNa2WO4, H2O2 aq.H2O, EtOH, 81%NOMeOBnOOBnBr, K2CO3, DMFthen isopentyl bromide60%79% BRSML-tyrosine methyl ester2.102.62.112.122.13  17 2.5.3 Synthesis of serpulanine Following the synthetic route to analog 2.6, phenyl prenylated ether 2.14 was produced from benzyl protected oxime 2.9 (Scheme 2.7). Basic hydrolysis provided carboxylic acid 2.15. After stirring with SOCl2 at 40 °C in CDCl3 for 40 minutes, NMR showed the cleavage of the prenyl group, suggesting that 2.15 was easily decomposed by the hydrochloric acid generated from reaction of SOCl2 with the carboxylic acid.   Scheme 2.7 Synthesis of phenol prenylated ether 2.15  Scheme 2.8 shows that carboxylic acid 2.15 failed to be converted to the acid chloride using oxalyl chloride and only the starting material was recovered. In the second trial, carboxylic acid 2.15 was reacted with EDCI under neutral conditions as confirmed by NMR changes. However, after the addition of hydroxylamine hydrochloride and triethylamine, only trace amounts of hydroxamic acid 2.17 were isolated.  NOMeOBnOOMeOH, H2ONaOH, pH=12NOHOBnOOSOCl2, CDCl3decomposeNOMeOBnOOHK2CO3, DMFBr2.9 2.14 2.15  18  Scheme 2.8 Failed synthesis of hydroxamic acid 2.17  Spilling et al. has shown that hydroxamic acids can be formed by direct amidation from a methyl ester at 60 °C in MeOH.55 Inspired by this work, the hydroxamic acid group was successfully installed under the optimized basic conditions using excess triethylamine (Scheme 2.9).  Scheme 2.9 Direct amidation synthesis of hydroxamic acid 2.17 inspired by Spilling’s work   NOHOBnOO(COCl)2 1.2 equiv.,DMF cat.NClOBnOOCDCl3, r.t., 24 hEDCI 1.5 equiv.,DMAP 1.5 equiv., CDCl3, r.t., 18 hNNHOBnOOOH2.17 Trace amountsthenNH2OH•HCl 5 equiv.,  NEt3, 5 equiv.,r.t., 24 h2.15 2.16NH2OH•HCl, NEt3, MeOH, 60 °C, 3 days58%NOOMeBnOONONHBnOOOH2.14 2.17BrOMeNHOOOMeBrOMeNHOOH2N NNHMeOH, 60 °C, 98%NHNNHSpilling's work:My work:2.18 2.19  19 Removal of the benzyl protecting group on the oxime in 2.17 was required to complete the synthesis of the natural product serpulanine. Hydrogenolysis was not attempted, because it was not compatible with the double bond. As shown in Scheme 2.10, Lewis acid BCl3·SMe2 catalyzed removal of the benzyl group was attempted but only undesired phenol 2.20 was obtained, confirming the sensitivity of the prenylated phenol ether to acid.  Scheme 2.10 Deprotection of benzyl group leading to undesired phenol 2.20  Due to the intractability of benzyl deprotection, a PMB (4-methoxybenzyl) group was introduced at the oxime (Scheme 2.11). PMB group offers a more labile alternative to the benzyl group and can be deprotected using various non-acidic methods.   Scheme 2.11 Synthesis of the PMB protected oxime 2.22   BCl3•SMe2 7 equiv.DCM, r.t., 30 minNONHBnOONONHBnOOHOHOH2.17 2.20NOOMePMB-Cl, K2CO3NOMeOHO PMBOOH OHDMFBrNOOMe, K2CO3PMBOODMF2.6 2.21 2.22  20   Table 2.3 Attempts to deprotect PMB group Entry Type of reagent Deprotecting reagent T (°C) Time (h) Solvent Result 1 Oxidative DDQ 2 equiv. 60 24 CDCl3 No reaction 2 Oxidative CAN 3 equiv. 60 24 MeCN No reaction 3 Oxidative I2 4 equiv. r.t. 20 MeOH Side reaction 4 Reductive NaBH3(CN) 2 equiv./ BF3·OEt2 3 equiv. 60 24 THF Side reaction 5 Acid AlCl3 and anisole 4 equiv. r.t. 0.2 DCM Decompose  Various reagents were screened for PMB group removal (Table 2.3). In entry 1 and 2, the standard deprotection reagents DDQ and CAN failed to give desired product 2.8. In entry 3, pure side product 2.23 was obtained (Scheme 2.12). In entry 4, undesired phenol 2.21 was produced along with the starting material (Scheme 2.12). In entry 5, NMR showed the loss of the PMB and prenyl groups indicating the formation of undesired side products. Due to the intractability of deprotection for this group the route was abandoned. NOOMePMBOONOOMeHOO2.22 2.8  21  Scheme 2.12 Side reactions during PMB deprotection trials  Facing the difficulty of deprotecting the PMB group, we decided to revisit benzyl protection since the benzyl group could be successfully deprotected by hydrogenolysis. The 3-hydroxy-3-methylbutyl ether was introduced on phenol 2.9 to give 2.25 as shown in Scheme 2.13. The plan was to use the 3-hydroxy-3-methylbutyl ether as a prenyl group precursor after removal of the benzyl protecting group in 2.25 by hydrogenolysis to give 2.26.  Scheme 2.13 Synthesis of tertiary alcohol 2.26  Different dehydration methods were tried to convert 2.26 to desired alkene 2.8 (Table 2.4). The first five entries all failed to give desired product 2.8. In the entry 6, Burgess reagent NOOMeNOMeOPMBO PMBOO OMeOHOMeINOOMeNaBH3(CN)BCl3•SMe2PMBOOHTHFI22.22 2.232.21NOOMeBnOOHOTsNOOMe, K2CO3BnOODMFOHOHPd/C, H2MeOHNOOMeHOOOH2.92.242.25 2.26  22 conditions afforded only trace amounts of product 2.8 shown in the NMR spectrum of the crude product, along with its terminal alkene side product 2.27.   Table 2.4 Attempts to dehydrate tertiary alcohol 2.26 to 2.8 Entry Reagent Solvent T (°C) Time (h) Result 1 Phosphoryl chloride 3 equiv. Pyrdine r.t. 48 No reaction 2 Phosphoryl chloride 3 equiv. Pyrdine 60 24 Decomposition 3 Thionyl chloride 3 equiv. Pyrdine 60 20 Decomposition 4 DMSO-d6  DMSO-d6 140 48 No reaction 5 Martin sulfurane 1.2 equiv. CDCl3 60 12 No reaction 6 Burgess reagent 1.2 equiv. CDCl3 60 5 Trace amounts  To facilitate dehydration of 2.26, the tertiary alcohol was converted to a better leaving group (Scheme 2.14). The tosylate group could not be installed on the bulky tertiary alcohol position, but tertiary alcohol 2.26 was converted to ester 2.29 using trifluoroacetic anhydride (TFAA).  NOOMeHOOOHNOOMeHOONOOMeHOO+2.27 Side product2.26 2.8  23  Scheme 2.14 Synthesis of ester 2.29  Though trifluoroacetate is a good leaving group, all three trials described in Table 2.5 failed to produce desired prenyl ether 2.8.   Table 2.5 Attempts to synthesize desired phenol prenylated ether 2.8 Entry Base Solvent T (°C) Time (h) Result 1 Triethylamine 10 equiv. MeOH r.t. 1.5 Tertiary alcohol 2.26 was obtained. 2 DBN 5 equiv. DCM 40 2 No reaction 3 Pyridine 100 equiv. Toluene 110 15 No Reaction  NOOMeBnOOOH TsCl 1.5 equiv.Et3N 3.0 equiv.DMF, 60 °C, 24 hNOOMeBnOO O CF3OTFAA 2 equiv.py 4 equiv.DCM, r.t., 4.5 hNOOMeBnOOOTs2.26 2.282.29NOOMeBnOO O CF3ONOOMeHOO2.29 2.8  24 Due to the difficulty of deprotecting the benzyl and PMB groups in 2.17 and 2.22, respectively, of dehydrating the tertiary alcohol in 2.26, and eliminating the trifluoroacetate group in 2.29, we turned back to direct prenylation from tyrosine oxime 2.6 (Scheme 2.15). Though the yield was low, starting material tyrosine oxime 2.6 could easily be made in large quantities, and product 2.8 could be readily purified by simple flash chromatography. Following the basic amidation conditions, oxime 2.8 could be converted in low yield to serpulanine 2.1, which was found to match the natural product by 1H and 13C spectra. A 1D NOE experiment was performed to confirm the E configuration (spectra are in Figure 5.24 of the experimental section), supported by the correlation observed between δH 3.73 (protons on the benzyl methylene) and δH 11.67 (=N-OH).   Scheme 2.15 Synthesis of serpulanine 2.1 and key 1D NOE correlation in 2.1  The total synthetic scheme is shown below (Scheme 2.16). Serpulanine 2.1 can be synthesized in three steps from L-tyrosine methyl ester with a yield of 1.1%. This synthetic route could be used to build a small library of serpulanine analogs for structure activity relationship (SAR) evaluation. NOMeOHOOHNOOMeHOODMF, 3%NONHHOOOHNH2OH•HCl 10 equiv.NEt3 20 equiv., 60 °CMeOH, 47%(70% BRSM)prenyl bromide 1 equiv.K2CO3 2 equiv.2.6 2.8 2.1NONHHOOOH2.1NOE  25  Scheme 2.16 Synthetic route to the natural product serpulanine and its analogs.  2.5.4 Other possible synthetic routes and future work Serpulanine was synthesized with a low yield likely resulting from the strong nucleophilicity of the oxime. In order to increase the yield, several reactions shown below were tried. As shown in Scheme 2.17, the desired product was the prenylated phenol ether 2.30, but prenylated amine 2.31 was obtained instead. Though there were amine protection methods to install prenyl group on the phenol position, the sodium tungstate condition was likely to oxidize the prenyl group. So this synthetic route was not further studied. NH2OMeONOMeOHOOH OHNa2WO4, H2O2 aq.H2O, EtOH, 81%NOOMeHOOBr , K2CO3DMF, 3%NONHHOOOHNH2OH•HCl,NEt3,  60 °CMeOH, 47%(70% BRSM)R1-XNOMeOR1OOHR2-XNOMeOR1OOR2NR3OR1OOR2L-tyrosine methyl esterCAD $78 / 5g2.6 2.8 2.1  26  Scheme 2.17 Attempts to install prenyl group on the phenol position of L-tyrosine methyl ester   In Scheme 2.18, 4-hydroxyphenylpyruvic acid 2.33 was considered as a starting material since the oxime could be synthesized from the ketone group. According to the NMR data collected for 2.33, ketone exists mainly as the enol form in conjugation with the aromatic ring. Methyl 4-hydroxyphenylpyruvic acetate 2.34 was synthesized with TMSCl in MeOH, which readily decomposed within two days. Freshly made ester 2.34 was reacted with prenyl bromide, but desired phenol prenylated ether 2.35 was not obtained.   Scheme 2.18 Attempts to synthesize ether 2.35   NH2OMeONHOMeOOH OHL-tyrosine methyl esterDMFBr , K2CO3NH2OMeOONOMeOHOO2) Na2WO4, H2O21) deprotectionDMF, 45 min, 59%Br , K2CO31) Protection of amine2) prenyl bromide, baseHNOMeOOPG2.30 2.312.32 2.8OOHOOHOHOHOOHMeOH, 24 h, 89%TMSCl 3 equiv.OHOMeOOHOHOMeOODMFPrenyl bromide1 equiv.K2CO3 2 equiv.2.34 Not stable2.33 2.33 2.35  27 In Scheme 2.19, TMS chloride failed to protect enol 2.34. In Spilling’s work, silylenolether 2.39 was synthesized in quantitative yield. Immediate addition of hydroxylamine hydrochloride and triethylamine reduced the unnecessary homo-aldol condensation side reaction, and oxime stereoisomers 2.40 were synthesized in an excellent yield.52 This work shed a light on future work to synthesize serpulanine in a higher yield. After prenylation, aldehyde 2.42 could undergo the HWE reaction condition to produce silylenolether 2.43. Right after silyl deprotection of 2.43, hydroxylamine hydrochloride and triethylamine could be added to give oxime stereoisomers 2.44. Basic transesterification and HPLC purification could provide natural product serpulanine 2.1.      28  Scheme 2.19 Spilling’s work and future work to synthesize serpulanine in a higher yield   OHOMeOOHDMFMe3Si-Cl, K2CO3OTMSOMeOOHOMeMOMOCHOPOMeOMeOOTBSCO2Me, LDATHF, 99%OTBSOMeOOMeMOMOHF•Et3N, MeOHthen NH2OH•HCl, Et3N, 99%NOHOMeOOMeMOMOOCHOPOMeOMeOOTBSCO2Me, LDATHFOTBSOMeOOTBAFthen NH2OH•HCl, Et3NNOHOMeOOOHCHODMFBr , K2CO3NH2OH•HCl, NEt3heatedNOHNHOOOHHPLCNNHOOOHHOSpilling's work:Future work:2.34 2.362.372.382.39 2.402.41 2.422.432.442.452.12.38  29 2.6 Biological results  To assess the inhibitory activity of serpulanine analogs, our collaborator Samantha Ellis at UBC conducted a general class I/II HDAC assay. The assay was preformed on a murine metastatic lung carcinoma (A9), at a concentration of 30,000 cells/well, and all treatments were completed in triplicates. In these results, the Y-Axis indicates the relative HDAC enzyme activity relative to that of the untreated A9 cells. The X-Axis indicates the treatment at varying concentrations along with the positive control TSA (trichostatin A), which is a known HDAC inhibitor in the A9 cell line.     Figure 2.4 HDAC assay results of compound 2.8 and 2.1 In Figure 2.4, compound 2.8 caused no significant changes in class I/II HDAC enzymatic activity; while 2.1 showed HDAC inhibition ability that half inhibition concentration was 4 µg/mL (14 µM). The difference in structure of the two compounds shows the importance of NOOMeHOONONHHOOOH2.8 2.1                    A9  TSA (0.15 µg /ml)              2.8 (1 µg /ml)                 2.8 (0.5 µg /ml)                    2.8 (0.25 µg /ml)                         2.8 (0.12 µg /ml)                              2.8 (0.06 µg /ml)                                   2.8 (0.03 µg /ml)                      A9     TSA (7.6 ng/ml)               2.1 (8 µg /ml)                    2.1 (4 µg /ml)                         2.1 (2 µg /ml)                              2.1 (1 µg /ml)                              2.1 (0.05 µg /ml)                                   2.1 (0.25 µg /ml)    30 hydroxamic acid moiety, present only on 2.1, to inhibit HDAC by binding to the zinc-containing active domain.     Figure 2.5 HDAC assay results of compound 2.46 and 2.17 In Figure 2.5, compound 2.46 and 2.17 enhanced the activity of class I/II HDAC enzymes. The activity of class I/II HDAC enzymes was found to double upon treatment with either compound, 2.46 or 2.17, at a concentration of 2 µg/mL. By structural analysis of these four compounds, we concluded that bulky substituents present on the oxime group of both 2.46 and 2.17 prevent them from binding in the narrow active domain of HDAC enzymes to cause inhibition. As for HDAC activation, this mechanism is yet to be understood, and further work is undergoing in this field.56  NONHOOOH2.46NONHBnOOOH2.17                    A9     TSA (7.6 ng/ml)              2.46 (8 µg /ml)                   2.46 (4 µg /ml)                        2.46 (2 µg /ml)                             2.46 (1 µg /ml)                              2.46 (0.05 µg /ml)                                   2.46 (0.25 µg /ml)                      A9     TSA (7.6 ng/ml)              2.17 (8 µg /ml)                   2.17 (4 µg /ml)                        2.17 (2 µg /ml)                             2.17 (1 µg /ml)                              2.17 (0.05 µg /ml)                                   2.17 (0.25 µg /ml)    31 2.7 Conclusion In this project, natural product serpulanine 2.1 was synthesized in three steps with an overall yield of 1.1%. The synthetic route was used to build a small library of serpulanine analogs shown in Figure 2.5 to exhibit effects on HDAC enzymes. From the biological results, natural product serpulanine 2.1 showed its potential as a HDAC inhibitor. Bulky substituents on the oxime moiety led to the loss of HDAC inhibiting ability, and intact oxime and hydroxamic acid groups are required for HDAC inhibition. Future work includes synthesizing serpulanine in a higher yield as discussed in Scheme 2.19.  Figure 2.6 Synthesized serpulanine analogs  NR3OR1OOR2R1 = H, Me, PMBR2 = prenyl group,         3-methylbutyl group        3-methyl-3-hydroxylbutyl groupR3 = NHOH, OMe, OH  Serpulanine analogs  32 Chapter 3: Synthesis of halitoxin-related compounds 3.1 Calcineurin Calcineurin is a serine/threonine protein phosphatase, playing a role in various signal transduction pathways.57, 58 The name calcineurin was given by Klee et al. due to its calcium- and calmodulin-binding properties.59 Calcineurin has two subunits: Calcineurin A binds Ca2+ activated calmodulin; Calcineurin B interacts with Ca2+. One of the most critical roles for calcineurin is its signaling role in T-cell activation, shown in figure 3.1.60   Figure 3.1 Role of calcineurin in T-cell activation On the T-cell surface, the initial recognition of antigen leads to an increase in the intracellular Ca2+ concentrations. As well binding calmodulin with resulting activation of calcineurin,61 activation can also result when Ca2+ binds directly to the calcineurin subunit. Activated calcineurin dephosphorylates NFAT (nuclear factor of activated T-cells), allowing it to enter the nucleus. Kinases will rephosphorylate NFAT to export this molecule back to the   33 cytosol. In the nucleus dephosphorylated NFAT works with other transcription factors for gene expression,62 e.g. IL-2 (interleukin), leading to T-cell development and differentiation.63, 64  3.2 Important calcineurin inhibitors Two important calcineurin inhibitors, cyclosporine A (CsA) and FK506, are used as immunosuppressive drugs (Figure 3.2).65 CsA and FK506 inhibit calcineurin by forming the complexes cyclophilin-CsA and FKBP-FK506 respectively. These have been validated by the crystal structure of a FKBP-FK506-calcineurin complex.66  Figure 3.2 Chemical structure of cyclosporine A (CsA) and FK506  Cyclosporine A is a cyclic peptide isolated from cultures of Fusarium sp.67 FK-506 is a 23-membered macrocyclic polyketide isolated from fermentation broths of Streptomyces tsukubaensis.68 These two drugs are widely used to prevent organ rejection after allogenic organ transplantation. Both drugs have severe side effects including long term depression, gout, nephrotoxicity, and cardiovascular disease.69, 70, 71, 72  NNHNOONOOHNOHNONONONNOOHNOOHOO OHOONOOOHOOHOO3.2 Cyclosporine A (CsA) 3.3 FK506  34 3.3 Synthesis of halitoxin-related compounds for structure elucidation An unknown natural product named alotau inhibited calcineurin dephosphorylation of GST-NFAT1(1-415) protein. The 1H spectrum of alotau (Figure 3.3) matches the characteristic signals that are observed for the polymeric alkyl pyridinium alkaloids known as the halitoxins. The pyridinium rings result in the halitoxins being multiply-charged, one positive charge for each monomeric unit of the polymer, and this made the MS analysis of alotau and the determination of its molecular weight ambiguous.  Comparison of alotau with a high molecular weight halitoxin showed that they had different HPLC retention times. In addition, the high MW polymeric halitoxin did not show the calcineurin-inhibiting properties of alotau. These results led to the hypotheses that alotau might be a low molecular weight monomer or oligomeric form of halitoxin. In an attempt to elucidate the structure of alotau, halitoxin monomer, dimer and trimer analogs were synthesized following literature procedures (Scheme 3.1) and compared with the natural product by bioassay and HPLC analyses.73   Figure 3.3 1H spectrum of the unknown natural product alotau and the structure of halitoxins   35   Scheme 3.1 Synthesis of dimer 3.7 and trimer 3.8 In scheme 3.1, bromo alcohol 3.4a was protected to give TBDMS-protected alcohol 3.5a. Treating 3-picoline with LDA gave the lithiated product which after the reaction with TBDMS-protected primary bromide 3.5a and the subsequent TBAF deprotection step afforded desired pyridine alcohol 3.6a. Treatment of 3.6a with triflic anhydride gave a mixture of dimer 3.7 and trimer 3.8. The mixture was separated by Silica Gel flash chromatography using 7:3 20% saturated KNO3/CH3CN as the eluent. Portions of fractions containing dimer 3.7 and trimer 3.8 were evaporated in vacuo, triturated, and sonicated with 1:10 CH2Cl2/MeOH, and then filtered to remove excess KNO3 solids. The filtrate was evaporated to give 3.7 and 3.8 as nitrate salts  To conduct an SAR study along with dimer 3.7 and trimer 3.8, monomer 3.10 was also prepared for biological evaluation (Scheme 3.2).  OHBr BrOTBDMSOHTBDMS-Cl, Et3N, DMAP, 0 °C to r.t.n n Nn3.6a n = 3, 76% over 2 steps3.5a n = 3, 72%3.4a n = 3Et2O,46 hii. KNO3i. (CF3SO2)2O, DIPEA,      -42 °C to r.t., DCM, 21 hN(CH2)4N(CH2)4•2NO3-N(CH2)4(CH2)4NN(CH2)3•3NO3-+Dimer 3.7Trimer 3.828%16%-78 °C to r.t.,THF, 14 hNLi,1)2) TBAF, THF, 2 h  36  Scheme 3.2 Synthesis of monomer 3.10 Following a procedure that was similar to that used to prepare alcohol 3.6a, pyridine alcohol 3.6b was obtained. Treatment of alcohol 3.6b with hydrobromic acid gave pyridinium bromide 3.9,74 which then, as shown in Scheme 3.2, under high dilution afforded monomer 3.10 in a 46% yield.  The resulting inhibition of calcineurin dephosphorylation by the synthetic samples is shown in Figure 3.4. Unfortunately, none of these compounds, monomer 3.10, dimer 3,7, trimer 3.8, or the higher oligomers (personal communication) exhibited phosphatase inhibition activity at 1 µg comparable to alotau. Therefore, the problem of identifying the alotau structure was not resolved by the trial of synthesizing halitoxin-related oligomers. OHBr BrOTES-78 °C to r.t.,THF, 12 hNLiOHTES-Cl, Et3N, DMAP, 0 °C to r.t.n n N,1)2) TBAF, THF, 2 hn3.6b n = 9, 75% over 2 steps3.5b n = 9, 93%3.4b n = 9Et2O, 3 h48% HBr aq., 90 °C, 15 hBrNHBri. Na2CO3ii. NaI, Butanone,    reflux, over 4 daysNInn3.9 n = 9, 91%3.10 Monomer, n = 7, 46% over two steps  37  Figure 3.4 The biological activities of samples to inhibit dephosphorylation of GST-NFAT1(1-415)  3.4 Another potential method of structure elucidation One difficulty in elucidating the structure of alotau was to determine the molecular weight. Molecular weight can be obtained from mass spectrometry by analysis of mass/charge ratio, based on the fact that charge is usually 1. Alotau has multi-charged pyridinium rings, that complicated this situation. In order to solve the problem, all pyridinium rings could be reduced to saturated piperidines, avoiding multi-charges. In scheme 3.3, trimer 3.8 was reduced under hydrogen to give fully reduced 3.11 as a model reaction.   Scheme 3.3 Reduction of trimer 3.8 N(CH2)4(CH2)4NN(CH2)3•3NO3-3.8 TrimerN(CH2)4(CH2)4NN(CH2)33.11 Reduced trimerPtO2, H2MeOH, 4 h    38 The structure of 3.11 was confirmed by analysis of its 1H NMR spectra and observation of MS peaks with m/z at 544.8, 273.2, and 182.6 that could be assigned as [M+H]+, [M+2H]2+/2 and [M+3H]3+/3 peaks, respectively (calculated MS peaks: M=543.5, [M+H]+=544.5, [M+2H]2+/2 =272.7, [M+3H]3+/3=182.2). This model reaction provided a possible solution to the structure elucidation of halitoxins and alotau. Following the model reaction reduction of both a halitoxin and alotau were attempted (Table 3.1). In entry 1, according to NMR and TLC analysis the halitoxin was not fully reduced and none of the desired product was obtained after flash chromatography. For entry 2, TLC analysis showed the disappearance of alotau under UV light, but NMR and MS analysis did not provide unambiguous evidence for structural determination. Table 3.1 Attempts to reduce halitoxin and alotau Entry Reactant Catalyst H2 Solvent T (°C) Time (h) 1 Halitoxin PtO2 5 bar MeOH r.t. 72 2 Alotau PtO2 10 bar MeOH r.t. 1  3.5 Conclusion To elucidate the structure of the unknown natural product alotau, monomer 3.10, dimer 3.7 and trimer 3.8 were synthesized. A method for reducing pyridinium to piperidine rings was developed. The synthesized halitoxin oligomers have similar NMR spectra to alotau, however, none of these compounds exhibited the potent phosphatase activity shown by alotau, and the structure of alotau remains elusive.     39 Chapter 4: Synthesis of intermediates of (+)-makassaric Acid 4.1 MK2’s role in inflammation In 2004, Dr. D. Williams in our lab isolated and characterized natural product (+)-makassaric acid 4.1 from the marine sponge Acanthodendrilla sp. Makassaric acid showed MK2 inhibition with an IC50 of 20 µM.75 More recently, our collaborator Dr. Xiao-Yan from Saint Michael’s hospital in Toronto found that makassaric acid showed inhibition of hemmorrhagic lesion formation in a zebrafish model of stroke with an IC50 of 2 µM.  This promising finding prompted us to undertake an SAR study of makassaric acids’ activity in this assay.  Figure 4.1 The structure of (+)-makassaric acid  4.2 Prior syntheses of (+)-makassaric acid and related synthetic target A total synthesis of (+)-makassaric acid has been published by the Basabe group. 76 Their retrosynthetic scheme is shown below (Scheme 4.1). (+)-Makassaric acid is obtained from alcohol 4.2 after Barton-MacCombie radical deoxygenation and deprotection. Alcohol 4.2 is constructed through a coupling reaction between fragment A lithium derivative 4.3 and fragment B diterpene aldehyde 4.4. Fragment A 4.3 is lithiated from bromoderivative 4.5, and fragment B 4.4 is a key intermediate synthesized from (-)-sclareol 4.6 in 10 steps. HHOHCOOH4.1 (+)-Makassaric acidH  40  Scheme 4.1 Retrosynthetic scheme of (+)-makassaric acid reported by Basabe group  The reported synthesis of bromoderivative 4.5 is shown below (Scheme 4.2). 77 Bromination and protection steps provided methoxymethyl ether 4.8, which was followed by DIBAL-H reduction and protection steps to give THP derivative 4.5 in excellent yields.  Scheme 4.2 Synthesis of compound 4.5 reported by Basabe group  Fragment B analog 4.9, which had been synthesized by Cuerva et al., was chosen as the synthetic target in this project.78 Our initial objective was to use analog 4.9 is to introduce an alcohol group on the tricyclic skeleton of (+)-makassaric acid analog 4.12 following similar synthetic steps. This alcohol group could be used to reduce the partition coefficient log P to improve water solubility and druglikeness. Furthermore, this alcohol group could also be used as an anchoring position for C-H activation on the terminal methyl group nearby. HHOHCOOH4.1 (+)-Makassaric acidCH2OTHPO OBrHOHOHHHOMOMCH2OTHPHOHHCHOHHHCH2OTHPOMOMLi4.3 Fragment A 4.4 Fragment B+4.24.5 4.6O OOHO OOMOMi. Br2, DCM, 95%ii. DMM, P2O5, CHCl3, 99%i. DIBAL-H, DCM, 99%ii. DHP, HCl/dioxane, 99%CH2OTHPOMOMBr Br4.7 4.8 4.5  41  Scheme 4.3 Synthetic proposal using fragment B analog 4.9  4.3 Synthesis of intermediates of (+)-makassaric acid A retrosynthetic scheme is shown below for our route towards fragment B analog 4.9. The tricyclic skeleton of 4.9 is to be constructed by a biomimetic polyene cascade cyclization from terminal alkene epoxide product 4.17. All E-alkene acetate 4.15 is to be obtained after HWE (Horner–Wadsworth–Emmons) reaction, reduction and esterification of the starting material farnesylacetone.  Scheme 4.4 Retrosynthetic scheme of compound 4.9  CHOPGOHHH4.10CHOHHH4.4 Fragment BHOHHHOAcHHOMOMCH2OTHPHOHPGOHHOHCOOH4.1 (+)-Makassaric acidH HHOHCOOHHHO4.94.114.12HOHHHOAcOOAc AcOHOAcOH2EtOH2OO24.9 4.174.174.154.13Farnesylacetone  42 As shown in Scheme 4.5, commercially available farnesylacetone underwent HWE olefination using triethyl phosphonoacetate to afford the all E ester 4.13 as the major product.79 Side product 4.18 could be separated by careful flash chromatography. Distillation could be used for the purification of large quantities of compound 4.13 in the future in order to avoid the challenging flash chromatography purification used in the current work.  Scheme 4.5 Synthesis of all E compound 4.13  To improve stereoselectivity, the HWE conditions were optimized as below (Scheme 4.6). Using diisopropyl compound 4.19 failed to provide product 4.13. Lithium salts did not give significant improvements on stereoselectivity. Both desired product 4.13 and side product 4.18 were found according to TLC analysis. Since all E ester 4.13 could be obtained in grams following Scheme 4.5, the HWE reaction optimization was not further studied.   Scheme 4.6 Attempts to optimize HWE reaction  POEtOEtOOOEt , NaHTHF, 21 hFarnesylacetoneO2EtOH2OH2OOEt+4.18 Side product4.13 44%, 60% BRSMOFarnesylacetonePOi-PrOi-PrOOOEt , NaHOEtOH4.134.19DME, reflux, 20 hOFarnesylacetonePOEtOEtOOOEt , LiCl, DBUOEtOH4.13MeCN, reflux, 16 h  43 In scheme 4.7, all E ester 4.13 underwent DIBAL-H reduction to provide all E-geranylgeraniol 4.14 in an excellent yield.79 Treatment geranylgeraniol 4.14 with acetic anhydride provided acetate 4.15 in a yield of 86%.80 Terminal hydrobromination of acetate 4.15 was ensured by slow addition of the THF solution of NBS to afford bromohydrin 4.16.81  Scheme 4.7 Synthesis of bromohydrin 4.16  The ring closure of bromohydrin 4.16 using NaH gave epoxide 4.17 with a yield of 31% following the procedures reported by the Spielmann group (Scheme 4.8).82 Weak base potassium carbonate was subsequently used to optimize this reaction with an increased yield of 63%.    Scheme 4.8 Synthesis of epoxide 4.17  With epoxide 4.17 in hand, next step was cascade cyclization to obtain tricyclic alkene 4.9, which had been synthesized by the Cuerva group.83 Following published procedures, DIBAL-H, 0 °C to rtTHF, 4.5 h, 70%EtOH2OHOH2AcOH2Ac2Opy, 14h, 86%AcOH2NBS,  0 °C45%, 64% BRSM BrOH4.13 4.144.15 4.16THF/H2O, 2hAcOH2BrOH MeOH, 63%K2CO3, 0 °C, 40 min AcOH2 OAcOH2BrOH THF, 31%NaH, r.t., 3 h AcOH2 O4.16 4.174.16 4.17  44 tricyclic alkene 4.9 was not found after flash chromatography. Use of THF that was not strictly deoxygenated might account for the failure of this reaction.   Scheme 4.9 Attempt to synthesize tricyclic alkene 4.9 To avoid the strict degassed solvent requirement, cationic polyene cyclization was tried using indium bromide.84 However, there were different stereoisomers in the crude, and pure product 4.20 was not isolated via flash chromatography. This trial also failed.   Scheme 4.10 Attempt to synthesis tricyclic alcohol 4.20   OOAc[Cp2TiCl2] (20 mol %)Mn, Me3SiCl, Collidine OAcHOHH4.17 4.9AcOO4.17THF, 22hOOAcOHOAcHOHH4.17 4.20DCM, r.t., 4 hInBr3 2 equiv.  45 4.4 Conclusions To conduct an SAR study of (+)-makassaric acid, intermediate triene alkene 4.17 was synthesized in 5 steps with a yield of 14.6%. Using alkene 4.17 would introduce an alcohol group on the (+)-makassaric acid analog for useful structure modifications. Unfortunately, this project was not finished, and lots of meaningful work remains to be done in the future.  Scheme 4.11 Synthesis of epoxide 4.17 from farnesylacetone   OFarnesylacetone OOAc4.17AcOO4.175 steps14.6%$225 / 25g  46 Chapter 5: Experimental section 5.1 General experimental procedures All water and air sensitive reactions were performed in over-dried glassware and under an argon atmosphere unless otherwise noted. Water and air sensitive liquid reagents were handled via a dry syringe. Anhydrous THF was obtained from distillation with sodium. All other solvents and reagents were obtained from commercial sources without further purification. Flash chromatography was performed using Silicycle Ultra-Pure silica gel (230-400 mesh) with the solvent system indicated. Thin-layer chromatography (TLC) was carried out on aluminum-backed ultrapure silica gel 250 µm plates. Electrospray ionization mass spectrometry (ESI-MS) was recorded with Bruker Esquire-LC or Micromass LCT mass spectrometers. 1H and 13C NMR spectra were recorded on Bruker Avance 600 CryoProbe, 400 direct, 400 inverse, or 300 direct spectrometers.  5.2 Synthesis procedures 5.2.1 Preparation of (E)-oxime 2.6  L-Tyrosine methyl ester (2.00 g, 10.2 mmol) was added in EtOH (30 mL) at 0 °C to form a suspension. Na2WO4·2H2O (169 mg, 0.512mmol), hydrogen peroxide solution (30% w/w in water 10 mL) and H2O (20 mL) were added in sequence to form a light yellow suspension. After stirring at 0 °C for 45 minutes and at room temperature for 30 hours, this reaction suspension was extracted with EtOAc (50×3 mL). The EtOAc extracts were combined, washed with aqueous Na2S2O3·5H2O (10% w/w, 60×3 mL), brine (60 mL), dried with MgSO4, filtered and NOMeOHOOH  47 then concentrated in vacuo.  Product 2.6 (1.73 g, 81%) was a yellow solid, which was pure enough to be used without any further purification. Following a similar procedure, the same product was obtained in 82% yield using 1 equiv. of Na2WO4·2H2O in 4.5 h at r.t. 1H NMR (300 MHz, ACETONE-d6) δ 3.12 (br. s., 1 H), 3.76 (s, 3 H), 3.88 (s, 2 H), 6.77 (d, J=8.5 Hz, 2 H), 7.15 (d, J=8.5 Hz, 2 H); 13C NMR (75 MHz, ACETONE-d6) δ 52.4, 116.0, 128.1, 130.9, 151.9, 156.8, 165.2, 206.4; HRESIMS [M+Na]+ calcd for C10H11NO4Na 232.0586, found 232.0583.     48   Figure 5.1 1H and 13C NMR spectra of 2.6 recorded in acetone-d6 at 300 MHz and 75 MHz   7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5ChemicalShift(ppm)1.073.052.111.982.003.12053.76293.87786.75626.78447.13907.1672208 200 192 184 176 168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24ChemicalShift(ppm)52.3641115.9768128.0688130.8558151.8722156.7912165.2130206.3778  49  Figure 5.2 1D NOE NMR spectrum of 2.6 (irradiating oxime proton) recorded in DMSO-d6 at 400 MHz   12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0ChemicalShift(ppm)2.831.961.991.990.960.922.50003.37353.70023.72836.66186.68316.99617.01759.237012.388812.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0ChemicalShift(ppm)3.37263.72746.99617.01669.236912.3887Irradiate=N-OH  50  Figure 5.3 1D NOE NMR spectrum of 2.6 (irradiating methylene) recorded in DMSO-d6 at 400 MHz  12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0ChemicalShift(ppm)3.022.032.022.000.960.932.49662.50003.37353.70023.72836.66186.68316.99617.01759.237012.388812.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5ChemicalShift(ppm)3.37353.72926.68486.99366.99967.020912.3897Irradiatemethylene  51 5.2.2 Preparation of methyl ester 2.7 and 2.8  Oxime 2.6 (2.35 g, 11.23 mmol) and K2CO3 (1.86 g, 13.48 mmol) were added in dry DMF (40 mL) to provide a white suspension. Prenyl bromide (1.37 mL, 11.23 mmol) was added via syringe to give a brown suspension. After stirring at room temperature for 4 hours, the reaction was ceased by adding H2O (60 mL). The aqueous layer was acidified (pH < 1) by adding concentrated HCl, then EtOAc (60×4 mL) was used for extraction. Combined EtOAc layers were washed by H2O (100 mL), brine (100 mL), dried over MgSO4, filtered and evaporated in vacuo to give the crude. The crude was purified by silica gel chromatography (hexane/EtOAc = 4:1) to give product 2.7 (1.64 g, 53%) as a yellow oil, and product 2.8 (88.0 mg, 3%) as a yellow oil. 2.7 1H NMR (300 MHz, CHLOROFORM-d) δ 1.71 (s, 3 H), 1.78 (s, 3 H), 3.81 (s, 3 H), 3.85 (s, 2 H), 4.79 (d, J=7.1 Hz, 2 H), 5.03 (s, 1 H), 5.45 (t, J=7.1 Hz, 1 H), 6.71 (d, J=8.5 Hz, 2 H), 7.13 (d, J=8.5 Hz, 2 H); 13C NMR (75 MHz, CHLOROFORM-d) δ 18.4, 25.9, 30.5, 52.9, 72.6, 115.4, 119.1, 128.2, 130.5, 139.5, 150.7, 154.4, 164.2; HRESIMS [M+Na]+ calcd for C15H19NO4Na 300.1212, found 300.1215. 2.8 1H NMR (400 MHz, CHLOROFORM-d) δ 1.73 (s, 3 H), 1.78 (s, 3 H), 3.83 (s, 3 H), 3.92 (s, 2 H), 4.47 (d, J=6.5 Hz, 2 H), 5.48 (t, J=6.1 Hz, 1 H), 6.82 (d, J=8.5 Hz, 2 H), 7.22 (d, J=8.5 Hz, 2 H); 13C NMR (100 MHz, CHLOROFORM-d) δ 18.3, 26.0, 29.8, 52.9, 64.9, 114.8, NOOMeOOHNOOMeHOO  52 119.8, 127.6, 130.3, 138.3, 152.1, 157.8, 164.0; HRESIMS [M+Na]+ calcd for C15H19NO4Na 300.1212, found 300.1204.     53   Figure 5.4 1H and 13C NMR spectra of 2.7 recorded in chloroform-d at 300 MHz and 75 MHz 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5ChemicalShift(ppm)3.113.133.022.062.030.910.951.982.001.70861.78093.81003.85114.77664.80025.03005.42815.45175.47536.69386.72197.11547.14367.2600168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16ChemicalShift(ppm)18.368925.938630.523152.882072.606076.733577.160077.5712115.4047119.0601128.1529130.4832139.5456150.6793154.4109164.2348  54  Figure 5.5 1H and 13C NMR spectra of 2.8 recorded in chloroform-d at 400 MHz and 100 MHz   7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5ChemicalShift(ppm)3.053.252.951.811.730.942.001.801.62511.72581.78463.81563.82843.85063.91634.45964.47585.45935.47645.49006.81396.83527.21147.2600168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16ChemicalShift(ppm)18.303925.969629.779552.913564.861576.840077.160077.4648114.8480119.8466127.5884130.3164138.3020152.0788157.8394163.9658  55 5.2.3 Preparation of phenol 2.9  Oxime 2.6 (500 mg, 2.39 mmol) and K2CO3 (363 mg, 2.63 mmol) were added in dry DMF (20 mL). Benzyl bromide (0.28 mL, 2.39 mmol) was added via syringe to give a brown suspension. After stirring at room temperature for 2 hours, the reaction was ceased by adding H2O (30 mL). EtOAc (40×3 mL) was used for extraction, and combined EtOAc layers were washed by H2O (60 mL), brine (60 mL), dried over MgSO4, filtered and evaporated in vacuo to give the crude. The crude was purified by silica gel chromatography (hexane/EtOAc = 3:1) to give product 2.9 (0.48 g, 67%) as a light yellow oil.  1H NMR (300 MHz, CHLOROFORM-d) δ 3.84 (s, 3 H), 3.98 (s, 2 H), 5.52 (br. s., 1 H), 5.67 (s, 2 H), 6.64 (d, J=8.7 Hz, 2 H), 7.09 (d, J=8.5 Hz, 2 H), 7.29 - 7.48 (m, 5 H); 13C NMR (75 MHz, CHLOROFORM-d) δ 33.2, 53.0, 67.9, 115.5, 128.0, 128.7, 128.7, 129.0, 130.3, 134.0, 140.8, 154.7, 163.0; HRESIMS [M+Na]+ calcd for C17H18NO4 300.1236, found 300.1235.   NOOMeBnOOH  56   Figure 5.6 1H and 13C NMR spectra of 2.9 recorded in chloroform-d at 300 MHz and 75 MHz 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5ChemicalShift(ppm)3.112.060.832.062.012.005.043.84273.98205.51565.67166.62536.65427.09417.12227.26007.31187.32017.33317.42217.42827.4404168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24ChemicalShift(ppm)33.162652.991167.854976.733677.160077.5712115.4921127.9801128.6806128.7416128.9548130.2950133.9652140.7879154.7379162.9770  57 5.2.4 Preparation of methyl ester 2.10  Oxime 2.6 (347 mg, 1.66 mmol) and Cs2CO3 (1136 mg, 3.49 mmol) were added in dry DMF (15 mL) to give a yellow suspension. Benzyl bromide (198 µl, 1.66 mmol) was added via syringe without causing any significant changes. After stirring at room temperature for 1.5 hours, isopentyl bromide (398 µl, 3.32 mmol) was added with a syringe. After stirring for another 6 hours, the reaction was ceased by adding H2O (15 mL). EtOAc (30×3 mL) was used for extraction, and combined EtOAc layers were washed by H2O (90 mL), brine (90 mL), dried over MgSO4, filtered and evaporated in vacuo to give a crude yellow oil. The crude was purified by silica gel chromatography (hexane/EtOAc = 9:1) to give product 2.10 (365 mg, 60%) as a yellow oil. 1H NMR (300 MHz, CHLOROFORM-d) δ 0.95 (d, J=6.6 Hz, 6 H), 1.66 (dt, J=6.6, 6.6 Hz, 2 H), 1.83 (m, 1 H), 3.83 (s, 3 H), 3.88 (s, 2 H), 3.94 (t, J=6.6 Hz, 2 H), 5.33 (s, 2 H), 6.77 (d, J=8.45 Hz, 2 H), 7.14 (d, J=8.45 Hz, 2 H), 7.31 - 7.40 (m, 5 H); 13C NMR (75 MHz, CHLOROFORM-d) δ 22.7, 25.2, 30.8, 38.2, 52.9, 66.5, 78.0, 114.6, 127.7, 128.4, 128.4, 128.6, 130.3, 136.6, 151.4, 158.0, 164.1; HRESIMS [M+Na]+ calcd for C22H27NO4Na 392.1838, found 392.1833. NOOMeBnOO  58  Figure 5.7 1H and 13C NMR spectra of 2.10 recorded in chloroform-d at 300 MHz and 75 MHz   7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5ChemicalShift(ppm)2.952.971.960.953.032.051.942.001.851.994.970.94300.96511.62191.64401.66681.68891.82511.84791.87003.82603.88463.94243.96455.33376.75246.78057.12767.15577.26007.3506168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16ChemicalShift(ppm)22.745825.197730.756438.173152.915066.453877.9519114.5936127.6755128.4370130.2950136.5542151.4180158.0122164.1039  59 5.2.5 Preparation of carboxylic acid 2.11  Methyl ester 2.10 (300 mg, 0.81 mmol) was dissolved in MeOH/H2O (30 mL/4 mL) to form a yellow solution. 1M aqueous NaOH solution was added to adjust the pH to 12. After the reaction mixture was heated at 65 °C for 3.5 hours, it was concentrated to about 10 mL by rotary evaporator, and H2O (20 mL) was added. Aqueous 1M HCl solution was added to adjust the pH to 3. The reaction solution was extracted using EtOAc (20×3 mL), and combined EtOAc layers were washed by H2O (20 mL), brine (20 mL), dried over MgSO4, filtered and evaporated in vacuo to give pure product 2.11 (252 mg, 87%) as a yellow solid without any further purification. 1H NMR (400 MHz, CHLOROFORM-d) δ 0.95 (d, J=6.5 Hz, 6 H), 1.26 (s, 1 H), 1.66 (dt, J=6.7, 6.7 Hz, 2 H), 1.75 - 1.89 (m, 1 H), 3.86 (s, 2 H), 3.94 (t, J=6.7 Hz, 2 H), 5.30 (s, 2 H), 6.77 (d, J=8.5 Hz, 2 H), 7.17 (d, J=8.5 Hz, 2 H), 7.28 - 7.41 (m, 5 H); 13C NMR (100 MHz, CHLOROFORM-d) δ 22.7, 25.2, 29.6, 38.1, 66.5, 78.4, 114.7, 126.9, 128.5, 128.7, 128.8, 130.5, 136.0, 150.6, 158.2, 162.9; HRESIMS [M+Na]+ calcd for C21H25NO4Na 378.1681, found 378.1685.    NOOHBnOO  60   Figure 5.8 1H and 13C NMR spectra of 2.11 recorded in chloroform-d at 400 MHz and 100 MHz  7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ChemicalShift(ppm)6.201.012.110.962.022.021.942.002.005.000.94610.96231.25921.63111.64811.66521.68141.82391.84093.85743.94443.96155.30076.76366.78497.15597.17737.26007.32147.36757.3845160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16ChemicalShift(ppm)22.738725.177129.627138.146166.492176.840077.160077.480078.4249114.6956126.8722128.4571128.7314130.4688135.9703150.6310158.2052162.9143  61 5.2.6 Preparation of hydroxamic acid 2.12  Carboxylic acid 2.11 (153 mg, 0.43 mmol) was dissolved in CDCl3 (6 mL), and SOCl2 (0.2 mL, 2.74 mmol) was added via syringe. This reaction was monitored by NMR, and after stirring at 65 °C for 21 hours, the reaction solution was cooled and evaporated by rotary evaporator to remove excess SOCl2. Then NH2OH·HCl (35.8 mg, 0.52 mmol), NaHCO3 (43.3 mg, 0.52 mmol) and dry DCM (8 mL) were added into the reaction flask to form a suspension. After stirring at room temperature for 17 hours, the suspension was diluted by adding dry DCM (20 mL). The reaction mixture was washed by aqueous 1M HCl (10×2 mL), brine (10×2 mL), dried over MgSO4, filtered and evaporated in vacuo to give a crude yellow oil. The crude was purified by silica gel chromatography (hexane/EtOAC = 7:3) to give product 2.12 (70 mg, 44%) as a yellow solid. 1H NMR (400 MHz, METHANOL-d4) δ 0.96 (d, J=6.5 Hz, 6 H), 1.63 (dt, J=6.5, 6.5 Hz, 2 H), 1.82 (m, 1 H), 3.83 (s, 2 H), 3.95 (t, J=6.5 Hz, 2 H), 5.22 (s, 2 H), 6.75 (d, J=8.5 Hz, 2 H), 7.11 (d, J=8.5 Hz, 2 H), 7.26 - 7.38 (m, 5 H); 13C NMR (100 MHz, METHANOL-d4) δ 22.9, 26.2, 30.4, 39.2, 67.3, 78.2, 115.4, 129.0, 129.1, 129.3, 129.4, 131.2, 138.5, 153.8, 159.3, 163.2; HRESIMS [M+Na]+ calcd for C21H26N2O4Na 393.1790, found 393.1797.    NONHBnOOOH  62   Figure 5.9 1H and 13C NMR spectra of 2.12 recorded in methanol-d4 at 400 MHz and 100 MHz  7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5ChemicalShift(ppm)2.932.972.020.982.002.011.551.932.015.040.95400.97021.60831.62451.64151.65771.82411.84111.85823.83203.93273.94893.96515.21646.74256.76387.09737.11867.3208160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24ChemicalShift(ppm)22.940026.247030.361739.216167.348778.1994115.4302128.9936129.1156129.2832131.1577138.4880153.8345159.2598163.2070  63 5.2.7 Preparation of oxime 2.13  Hydroxamic acid 2.12 (28 mg, 0.0763 mmol) was dissolved in MeOH (2 mL), and Pd/C (10% wt, 2 mg, 0.0188 mmol) was added to form a black suspension. After stirring at room temperature under H2 (100 psi) for 6 days, the reaction was stopped by filtering and evaporation in vacuo. The crude was purified by silica gel chromatography (hexane/EtOAC = 1:4) to recover the starting material (8.0 mg) and to give product 2.13 (12.0 mg, 57%, 79% BRSM) as a white solid. Since the product was polar and tended to adhere strongly to the silica gel, EtOAc washing was critical to increase the yield. The product was further purified using reversed phase HPLC (eluting with 60% MeCN/H2O) to give pure product 2.13 (retention time = 12.0 min) 1H NMR (600 MHz, METHANOL-d4) δ 0.96 (d, J=6.6 Hz, 6 H), 1.63 (dt, J=6.4, 6.4 Hz, 2 H), 1.78 - 1.86 (m, 1 H), 3.84 (s, 2 H), 3.95 (t, J=6.4 Hz, 2 H), 6.77 (d, J=8.2 Hz, 2 H), 7.16 (d, J=8.2 Hz, 2 H); 13C NMR (150 MHz, METHANOL-d4) δ 23.0, 26.3, 29.3, 39.3, 67.3, 115.4, 129.6, 131.1, 153.2, 159.1, 164.2; HRESIMS [M+Na]+ calcd for C14H20N2O4Na 303.1321, found 303.1319.   NONHHOOOH  64   Figure 5.10 1H and 13C NMR spectra of 2.13 recorded in methanol-d4 at 600 MHz and 150 MHz  7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ChemicalShift(ppm)3.543.172.171.042.102.142.051.990.95440.96541.61561.63691.64801.82201.83311.84423.84323.93973.94993.96106.76716.78087.15797.1715168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16ChemicalShift(ppm)22.956426.255629.341939.254667.3354115.3632129.5785131.1292153.1895159.1493164.1816  65 5.2.8 Preparation of methyl ester 2.14  Benzyl protected oxime 2.9 (213 mg, 0.71 mmol) and K2CO3 (118 mg, 0.85 mmol) were added in dry DMF (10 mL). Prenyl bromide (0.17 mL, 1.42 mmol) was added via syringe. After stirring at room temperature for 4 hours, the reaction was quenched by adding H2O (10 mL). EtOAc (15×3 mL) was used for extraction, and combined EtOAc layers were washed by H2O (30 mL), brine (30 mL), dried over MgSO4, filtered and evaporated in vacuo to give the crude. The crude was purified by silica gel chromatography (hexane/EtOAc = 6:1) to recover the starting material (63 mg), and product 2.14 (172 mg, 66%, 93% BRSM) was a yellow oil.  1H NMR (400 MHz, CHLOROFORM-d) δ 1.73 (s, 3 H), 1.79 (s, 3 H), 3.82 (s, 3 H), 3.88 (s, 2 H), 4.46 (d, J=6.8 Hz, 2 H), 5.33 (s, 2 H), 5.48 (tm, J=6.1 Hz, 1 H), 6.78 (d, J=8.9 Hz, 2 H), 7.14 (d, J=8.5 Hz, 2 H), 7.30 - 7.40 (m, 5 H); 13C NMR (100 MHz, CHLOROFORM-d) δ 18.3, 26.0, 30.8, 52.9, 64.9, 78.0, 114.7, 119.9, 127.8, 128.4, 128.4, 128.6, 130.3, 136.6, 138.3, 151.4, 157.7, 164.1; HRESIMS [M+Na]+ calcd for C22H25NO4Na 390.1681, found 390.1682. NOOMeBnOO  66   Figure 5.11 1H and 13C NMR spectra of 2.14 recorded in chloroform-d at 400 MHz and 100 MHz  7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ChemicalShift(ppm)2.933.092.962.082.012.010.951.972.004.821.25831.55091.73171.79153.82503.88384.45544.47245.33055.47725.48075.49435.49776.77296.79517.13037.15177.26007.34447.35137.3641168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16ChemicalShift(ppm)18.325125.974730.759552.931264.862776.840077.160077.480077.9676114.7375119.8881127.8120128.3910128.4368130.2958136.5587138.2502151.4008157.7399164.0943  67 5.2.9 Preparation of carboxyl acid 2.15  Methyl ester 2.14 (55 mg, 0.15 mmol) was dissolved in the combined solution of MeOH (10 mL) and H2O (3 mL). 1M aqueous NaOH solution was added to adjust pH to 13. After heating at 60 °C for 3.5 hours, the reaction solution was concentrated to 5 mL by rotary evaporator. H2O (10 mL) was added afterwards, and aqueous 1M HCl solution was added to adjust pH to 1. EtOAc (10×3 mL) was used for extraction, and combined EtOAc layers were washed by H2O (20 mL), brine (20 mL), dried over MgSO4, filtered and evaporated in vacuo to give pure product 2.15 (42 mg, 79%) as a white solid without any further purification. 1H NMR (400 MHz, CHLOROFORM-d) δ 1.73 (s, 3 H), 1.79 (s, 3 H), 3.86 (s, 2 H), 4.47 (d, J=6.7 Hz, 2 H), 5.30 (s, 2 H), 5.48 (t, J=6.7 Hz, 1 H), 6.79 (d, J=8.5 Hz, 2 H), 7.17 (d, J=8.5 Hz, 2 H), 7.28 - 7.42 (m, 5 H); 13C NMR (100 MHz, CHLOROFORM-d) δ 18.3, 26.0, 29.6, 64.9, 78.4, 114.9, 119.8, 127.0, 128.4, 128.8, 128.8, 130.5, 136.0, 138.3, 150.6, 157.9, 162.5; HRESIMS [M+Na]+ calcd for C21H23NO4Na 376.1525, found 376.1527.   NOOHBnOO  68   Figure 5.12 1H and 13C NMR spectra of 2.15 recorded in chloroform-d at 400 MHz and 100 MHz  7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5ChemicalShift(ppm)2.933.002.102.012.080.932.012.004.751.73321.79183.85914.45974.47645.29855.46145.47815.49486.78436.80567.16037.18167.26007.32397.36737.3833168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16 8ChemicalShift(ppm)18.322325.974329.574964.889176.840077.160077.480078.4329114.8528119.8208126.9709128.4475128.8112130.4914135.9541138.3326150.5671157.9427162.5252  69 5.2.10 Preparation of hydroxamic acid 2.17 and 2.46  Methyl ester 2.14 (132 mg, 0.36 mmol) was dissolved in MeOH (12 mL) to form a colorless solution, then NH2OH·HCl (249 mg, 3.6 mmol) and NEt3 (0.10 mL, 7.2 mmol) were added without causing any significant changes. After stirring at 60 °C for 3 days, the reaction mixture was worked up by H2O (15 mL). The aqueous layer was acidified (pH < 1) by adding concentrated HCl, then EtOAc (20×3 mL) was used for extraction. Combined EtOAc layers were washed by H2O (30 mL), brine (30 mL), dried over MgSO4, filtered and evaporated in vacuo to give a crude dark yellow oil. The crude was purified by silica gel chromatography (hexane/EtOAc = 1:1), and product 2.17 (76 mg, 58%) was found as a colorless oil. Compound 2.46 could be synthesized similarly with a yield of 52%. 2.17: 1H NMR (400 MHz, CHLOROFORM-d) δ 1.73 (s, 3 H), 1.79 (s, 3 H), 3.87 (s, 2 H), 4.46 (d, J=6.8 Hz, 2 H), 5.20 (s, 2 H), 5.47 (t, J=6.7 Hz, 1 H), 6.78 (d, J=8.5 Hz, 2 H), 7.15 (d, J=8.5 Hz, 2 H), 7.27 - 7.42 (m, 5 H), 8.95 (br. s., 1 H); 13C NMR (100 MHz, CHLOROFORM-d) δ 18.3, 26.0, 29.4, 64.9, 77.6, 114.8, 119.9, 127.4, 128.3, 128.5, 128.7, 130.4, 136.7, 138.3, 151.5, 157.8, 161.5; HRESIMS [M+Na]+ calcd for C21H24N2O4Na 391.1634, found 391.1633. 2.46: 1H NMR (400 MHz, CHLOROFORM-d) δ 1.72 (br. s., 6 H), 1.79 (s, 6 H), 3.85 (s, 2 H), 4.46 (d, J=6.70 Hz, 2 H), 4.67 (d, J=7.01 Hz, 2 H), 5.40 (t, J=6.85 Hz, 1 H), 5.47 (t, J=5.48 Hz, 1 H), 6.79 (s, J=8.52 Hz, 2 H), 7.19 (d, J=8.22 Hz, 2 H); 13C NMR (100 MHz, NONHBnOOOH  70 CHLOROFORM-d) δ 18.3, 18.4, 26.0, 29.3, 29.3, 64.9, 72.3, 114.8, 119.3, 119.9, 127.7, 130.4, 135.5, 139.4, 150.7, 157.8, 161.6; HRESIMS [M+Na]+ calcd for C19H26N2O4Na 369.1790, found 369.1791.     71   Figure 5.13 1H and 13C NMR spectra of 2.17 recorded in chloroform-d at 400 MHz and 100 MHz   9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5ChemicalShift(ppm)2.993.122.002.031.950.982.002.055.710.691.72751.78723.86764.44774.46475.19925.45855.47475.49176.76446.78577.13977.16117.26007.34447.36247.37698.9523168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16ChemicalShift(ppm)18.319225.969629.444264.861576.840077.160077.480077.6324114.7718119.8619127.4208128.2742128.6857130.4078136.6561138.2563151.4692157.7937161.4665  72  Figure 5.14 1H and 13C NMR spectra of 2.46 recorded in chloroform-d at 400 MHz and 100 MHz   7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5ChemicalShift(ppm)5.946.092.012.051.910.880.952.012.031.25441.56421.72181.78573.85304.45134.46804.65684.67435.38755.40505.47356.78666.80797.17937.19997.2600160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16 8ChemicalShift(ppm)18.320918.393625.979429.296064.854072.250776.840077.160077.363677.4800114.7545119.3365119.8965127.7005130.4061135.5045139.4102150.6543157.7601161.6003  73 5.2.11 Preparation of PMB protected oxime 2.21  Oxime 2.6 (1.00 g, 4.78 mmol) and K2CO3 (1.32 g, 9.56 mmol) were added in dry DMF (15 mL) to form a white suspension. 4-Methoxybenzoyl chloride (0.65 mL, 4.78 mmol) was added via syringe without causing any significant changes. After stirring at room temperature for 9.5 hours, the reaction was ceased by adding H2O (20 mL). EtOAc (20×3 mL) was used for extraction, and combined EtOAc layers were washed by H2O (40×2 mL), brine (30 mL), dried over MgSO4, filtered and evaporated in vacuo to give the crude. The crude was purified by silica gel chromatography (hexane/EtOAc = 2:1) to give product 2.21 (1.20 g, 76%) as a light yellow oil.  1H NMR (400 MHz, CHLOROFORM-d) δ 3.82 (s, 3H), 3.82 (s, 3H), 3.84 (s, 2 H), 4.67 (br. s., 1 H), 5.25 (s, 2H), 6.68 (d, J=8.5 Hz, 2 H), 6.89 (d, J=8.9 Hz, 2 H), 7.08 (d, J=8.2 Hz, 2 H), 7.28 (d, J=8.5 Hz, 2 H); 13C NMR (100 MHz, CHLOROFORM-d) δ 30.7, 52.9, 55.4, 77.8, 114.0, 115.4, 128.2, 128.6, 130.3, 130.5, 151.1, 154.3, 159.8, 164.1; HRESIMS [M+Na]+ calcd for C18H19NO5Na 352.1161, found 352.1161. NOMeOPMBOOH  74   Figure 5.15 1H and 13C NMR spectra of 2.21 recorded in chloroform-d at 400 MHz and 100 MHz  7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5ChemicalShift(ppm)6.072.140.682.072.002.041.992.003.82243.84214.67465.24866.67316.69456.88306.90527.07237.09287.26007.27197.2933168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24ChemicalShift(ppm)30.678752.913555.443376.840077.160077.480077.7696114.0098115.3814128.1523128.5638130.3011130.5450151.1339154.3190159.8358164.1487  75 5.2.12 Preparation of phenyl prenylated ether 2.22  PMB protected oxime 2.21 (1.00 g, 3.04 mmol) and K2CO3 (0.84 g, 6.08 mmol) were added in dry DMF (15 mL) to form a white suspension. Prenyl bromide (0.70 mL, 6.08 mmol) was added via syringe without causing any significant changes. After stirring at room temperature for 17 hours, the reaction was ceased by adding H2O (20 mL). EtOAc (30×3 mL) was used for extraction, and combined EtOAc layers were washed by H2O (60 mL), brine (60 mL), dried over MgSO4, filtered and evaporated in vacuo to give a crude yellow oil. The crude was purified by silica gel chromatography (hexane/EtOAc = 3:1) to recover the starting material (0.18 g), and product 2.22 (0.81 g, 67%, 82% BRSM) was found as a light yellow oil.  1H NMR (400 MHz, CHLOROFORM-d) δ 1.73 (s, 3 H), 1.79 (s, 3 H), 3.82 (s, 3 H), 3.82 (s, 3 H), 3.85 (s, 2 H), 4.46 (d, J=6.7 Hz, 2 H), 5.25 (s, 2 H), 5.48 (t, J=6.1 Hz, 1 H), 6.78 (d, J=8.5 Hz, 2 H), 6.89 (d, J=8.2 Hz, 2 H), 7.12 (d, J=8.2 Hz, 2 H), 7.29 (d, J=8.2 Hz, 2 H); 13C NMR (100 MHz, CHLOROFORM-d) δ 18.3, 26.0, 30.7, 52.9, 55.4, 64.8, 77.7, 114.0, 114.7, 119.9, 127.9, 128.6, 130.3, 130.3, 138.2, 151.2, 157.7, 159.8, 164.1; HRESIMS [M+Na]+ calcd for C23H27NO5Na 420.1787, found 420.1787. NOOMePMBOO  76   Figure 5.16 1H and 13C NMR spectra of 2.22 recorded in chloroform-d at 400 MHz and 100 MHz  7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5ChemicalShift(ppm)2.923.066.032.162.062.020.912.011.922.021.891.73011.78953.82183.85454.45204.46885.25365.46295.47735.49336.76606.78736.88406.90457.11397.13447.26007.27837.2988160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16ChemicalShift(ppm)18.307825.967130.702352.880155.418664.845576.840077.160077.472877.7419114.0018114.7000119.8863127.8874128.6075130.3023138.2380151.1927157.7173159.8339164.1400  77 5.2.13 Preparation of hydroxamic acid 2.22.1  Methyl ester 2.22 (276 mg, 0.69 mmol) was dissolved in MeOH (15 mL) to form a colorless solution, then NH2OH·HCl (479 mg, 6.9 mmol) and NEt3 (1.92 mL, 13.8 mmol) were added without causing any significant changes. After stirring at 60 °C for 2 days, the reaction mixture was worked up by H2O (15 mL). The aqueous layer was acidified (pH < 1) by adding concentrated HCl, then EtOAc (30×3 mL) was used for extraction. Combined EtOAc layers were washed by H2O (90 mL), brine (90 mL), dried over MgSO4, filtered and evaporated in vacuo to a crude oil. The crude was purified by silica gel chromatography (hexane/EtOAc = 1:1), and product 2.22 (165 mg, 60%) was found as a colorless oil.  1H NMR (400 MHz, CHLOROFORM-d) δ 1.72 (s, 3 H), 1.78 (s, 3 H), 3.82 (s, 2 H), 3.82 (s, 3 H), 4.44 (d, J=6.4 Hz, 2H), 5.12 (s, 2 H), 5.47 (t, J=6.5 Hz, 1 H), 6.75 (d, J=8.5 Hz, 2 H), 6.88 (d, J=8.5 Hz, 2 H), 7.12 (d, J=8.5 Hz, 2 H), 7.23 (d, J=8.5 Hz, 2 H), 8.98 (br. s., 1 H); 13C NMR (100 MHz, CHLOROFORM-d) δ 18.3, 26.0, 29.4, 55.4, 64.8, 77.4, 114.1, 114.7, 119.9, 127.5, 128.7, 130.1, 130.4, 138.2, 151.3, 157.7, 159.8, 161.7; HRESIMS [M+Na]+ calcd for C22H26N2O5Na 420.1739, found 421.1746.   NONHPMBOOOH  78   Figure 5.17 1H and 13C NMR spectra of 2.22.1 recorded in chloroform-d at 400 MHz and 100 MHz  9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5ChemicalShift(ppm)3.123.225.152.071.960.951.992.002.041.890.671.72251.78492.04293.81724.43614.45205.11505.45455.47135.48726.74166.76306.87416.89547.10627.12767.22197.24337.26008.9772160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16ChemicalShift(ppm)18.307825.959829.356655.425964.845577.4146114.0600114.7146119.8935127.4946128.6584130.1423130.3969138.2089151.3018157.7318159.8485161.7324  79 5.2.14 Preparation of methyl ester 2.23  Phenyl prenylated ether 2.22 (14 mg, 35 µmol) was dissolved in MeOH (3 mL), then I2 (35 mg, 138 µmol) was added, turning the colorless solution into a dark brown solution. After stirring at room temperature for 20 hours, this reaction was quenched by aqueous Na2S2O3·5H2O solution (20% w/v in water, 10mL), then EtOAc (10×3 mL) was used for extraction. Combined EtOAc layers were washed by H2O (30 mL), brine (30 mL), dried over MgSO4, filtered and evaporated in vacuo to give pure product 2.23 (16 mg, 82%) as a light yellow oil without any further purification. 1H NMR (400 MHz, CHLOROFORM-d) δ 1.42 (s, 6 H), 3.24 (s, 2 H), 3.82 (s, 3 H), 3.82 (s, 3 H), 3.86 (s, 2 H), 4.23 (dd, J=10.9, 7.9 Hz, 1 H), 4.34 (dd, J=10.9, 4.4 Hz, 1 H), 4.44 (dd, J=7.7, 4.3 Hz, 1 H), 5.25 (s, 2 H), 6.78 (d, J=8.5 Hz, 2 H), 6.90 (d, J=8.5 Hz, 2 H), 7.14 (d, J=8.5 Hz, 2 H), 7.29 (d, J=8.5 Hz, 2 H); 13C NMR (100 MHz, CHLOROFORM-d) δ 23.6, 25.0, 30.7, 41.4, 50.0, 52.9, 55.4, 71.2, 75.9, 77.8, 114.0, 115.0, 128.5, 128.7, 130.3, 130.4, 151.1, 156.9, 159.9, 164.1; HRESIMS [M+Na]+ calcd for C24H30NO6NaI 578.1016, found 578.1016.  NOOMePMBOOOMeI  80   Figure 5.18 1H and 13C NMR spectra of 2.23 recorded in chloroform-d at 400 MHz and 100 MHz  7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ChemicalShift(ppm)5.242.486.402.180.850.871.122.132.012.161.912.131.42303.24413.82503.85664.22254.23024.24984.31804.32834.42814.43834.44684.45795.25216.76536.78666.88896.91037.12447.14577.26007.27547.2967160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24ChemicalShift(ppm)23.561724.979030.709141.361750.002752.913555.443371.170875.895176.840077.160077.480077.7848114.0250114.9699128.5486130.3011130.4383151.0577156.9403159.8511164.1182  81 5.2.15 Preparation of tosylate 2.24  3-Methyl-1,3-butanediol (2.00 mL, 16.5 mmol) was dissolved in DCM (30 mL), and TsCl (4.29 g,  22.5 mmol) was added to form a light yellow solution. Triethylamine (13.06 mL, 93.7 mmol) was added by pressure-equalizing dropping funnel within 30 minute without causing any significant changes. After stirring at room temperature for 28 hours, the reaction mixture turned to a yellow suspension, and it was washed by H2O (25×2 mL), brine (25 mL), dried over MgSO4, filtered and evaporated in vacuo to give a crude yellow oil. The crude was purified by silica gel chromatography (hexane/EtOAc = 3:1) to give product 2.24 (4.15 g, 86%) as a light yellow oil. 1H NMR (300 MHz, CHLOROFORM-d) δ 1.21 (s, 6 H), 1.86 (t, J=6.9 Hz, 2 H), 2.45 (s, 3 H), 4.20 (t, J=6.9 Hz, 2 H), 7.34 (d, J=8.0 Hz, 2 H), 7.79 (d, J=8.2 Hz, 2 H); 13C NMR (75 MHz, CHLOROFORM-d) δ 21.8, 29.8, 41.8, 67.6, 69.9, 128.0, 130.0, 133.1, 145.0; HRESIMS [M+Na]+ calcd for C12H18O4NaS 281.0824, found 281.0821.   OTsOH  82   Figure 5.19 1H and 13C NMR spectra of 2.24 recorded in chloroform-d at 300 MHz and 75 MHz  8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5ChemicalShift(ppm)6.371.702.113.082.052.002.001.21021.70411.83271.85561.87842.44544.17994.20274.22567.26007.33087.35747.77687.8042150 145 140 135 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10ChemicalShift(ppm)21.786429.827441.782467.641769.880476.733677.160077.5864128.0258130.0056133.0971144.9607  83 5.2.16 Preparation of alcohol 2.25  Benzyl protected oxime 2.9 (2.01 g, 6.72 mmol) and K2CO3 (0.67 g, 4.88 mmol) were added in dry DMF (30 mL) to form a yellow suspension. Tosylate 2.24 (1.2 g, 4.65 mmol) was added to give a golden suspension. After stirring at room temperature for 13 hours, the reaction was heated at 60 °C for 4 hours, and quenched by H2O (70 mL). EtOAc (100×3 mL) was used for extraction, and combined EtOAc layers were washed by H2O (200 mL), brine (200 mL), dried over MgSO4, filtered and evaporated in vacuo to give the crude. The crude was purified by silica gel chromatography (hexane/EtOAc = 3:1) to recover the starting material (0.96 g), and product 2.25 (1.21 g, 47%, 89% BRSM) was obtained as a yellow oil. 1H NMR (400 MHz, CHLOROFORM-d) δ 1.30 (s, 6 H), 1.98 (t, J=6.1 Hz, 2 H), 3.82 (s, 3 H), 3.89 (s, 2 H), 4.14 (t, J=6.2 Hz, 2 H), 5.33 (s, 2 H), 6.78 (d, J=8.5 Hz, 2 H), 7.15 (d, J=8.5 Hz, 2 H), 7.31 - 7.40 (m, 5 H); 13C NMR (101 MHz, CHLOROFORM-d) δ 29.7, 30.7, 41.7, 52.9, 65.3, 70.6, 78.0, 114.6, 128.3, 128.4, 128.4, 128.6, 130.4, 136.5, 151.3, 157.4, 164.1; HRESIMS [M+Na]+ calcd for C22H27NO5Na 408.1787, found 408.1782. NOOMeBnOOOH  84   Figure 5.20 1H and 13C NMR spectra of 2.25 recorded in chloroform-d at 400 MHz and 100 MHz 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5ChemicalShift(ppm)6.312.313.012.092.111.982.032.004.671.30161.96001.97521.99043.82483.88574.12704.14234.15825.32976.76986.79117.13977.16107.26007.34607.35367.3658160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24ChemicalShift(ppm)29.698530.731441.722152.923765.332870.555476.840077.160077.472877.9819114.6128128.3311128.4475130.3751136.5069151.2945157.3754164.0746  85 5.2.17 Preparation of oxime 2.26  Benzyl protected oxime 2.25 (200 mg, 0.52 mmol) was dissolved in MeOH (4 mL), and Pd/C (10% wt, 40 mg, 0.38 mmol) was added to form a black suspension. After stirring at room temperature under H2 (100 psi) for 24 hours, the reaction was stopped by filtering and evaporation in vacuo. The crude was purified by silica gel chromatography (hexane/EtOAc = 1:1) to give product 2.26 (72 mg, 47%) as a white solid.  1H NMR (400 MHz, CHLOROFORM-d) δ 1.30 (s, 6 H), 1.98 (t, J=6.3 Hz, 2 H), 2.40 (br. s., 1 H), 3.82 (s, 3 H), 3.92 (s, 2 H), 4.15 (t, J=6.3 Hz, 2 H), 6.82 (d, J=8.5 Hz, 2 H), 7.23 (d, J=8.5 Hz, 2 H); 13C NMR (100 MHz, CHLOROFORM-d) δ 29.7, 29.8, 41.7, 52.9, 65.3, 70.7, 114.7, 128.2, 130.4, 151.8, 157.4, 164.0; HRESIMS [M+Na]+ calcd for C15H21NO5Na 318.1317, found 318.1315.   NOOMeHOOOH  86   Figure 5.21 1H and 13C NMR spectra of 2.26 recorded in chloroform-d at 400 MHz and 100 MHz  7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ChemicalShift(ppm)6.112.010.412.961.962.022.002.021.30101.96121.97741.99282.40483.82413.91634.13044.14664.16196.80716.82847.22087.2600168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24ChemicalShift(ppm)29.672829.779541.697052.898365.333970.713676.840077.160077.4800114.7108128.1980130.4078151.7740157.4432163.9811  87 5.2.18 Preparation of methyl ester 2.29  Tertiary alcohol 2.26 (35 mg, 91 µmol) was dissolved in DCM (3 mL) to form a colorless solution. Pyridine (29 µl, 362 µmol) and trifluoroacetic anhydride (26 µl, 181 µmol) were added without causing any significant changes. After stirring at room temperature for 4.5 hours, this reaction was quenched by 1M HCl (10 mL), then EtOAc (10×2 mL) was used for extraction. Combined EtOAc layers were washed by brine (10 mL), dried over MgSO4, filtered and evaporated in vacuo to give a crude oil. The crude was purified by silica gel chromatography (hexane/EtOAc = 3:1) to give product 2.29 (32 mg, 73%) as a light yellow oil. 1H NMR (400 MHz, CHLOROFORM-d) δ 1.64 (s, 6 H), 2.34 (t, J=6.1 Hz, 2 H), 3.82 (s, 3 H), 3.88 (s, 2 H), 4.05 (t, J=6.1 Hz, 2 H), 5.33 (s, 2 H), 6.74 (d, J=8.5 Hz, 2H), 7.15 (d, J=8.5 Hz, 2 H), 7.30 - 7.38 (m, 5 H); 13C NMR (100 MHz, CHLOROFORM-d) δ 26.2, 30.8, 39.5, 52.9, 63.4, 78.0, 87.9, 114.5, 128.3, 128.4, 128.5, 128.6, 130.4, 136.5, 151.3, 157.3, 164.1, 191.8; HRESIMS [M+Na]+ calcd for C24H26NO6F3Na 504.1610, found 504.1613.  NOOMeBnOO O CF3O  88   Figure 5.22 1H and 13C NMR spectra of 2.29 recorded in chloroform-d at 400 MHz and 100 MHz   7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5ChemicalShift(ppm)3.226.082.022.931.972.011.982.002.004.861.54491.64392.32202.33742.35273.82413.88134.03404.04934.06475.32806.72696.74827.13557.15687.26007.34367.3632192 184 176 168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24ChemicalShift(ppm)26.167730.754939.456852.944063.383276.855277.160077.480078.013487.8888114.4975128.2895128.4724130.4230136.5342151.3168157.3213164.1030191.7937  89 5.2.19 Preparation of serpulanine 2.1  Methyl ester 2.8 (38.0 mg, 0.14 mmol) was dissolved in MeOH (5 mL) to form a colorless solution, then NH2OH·HCl (95.2 mg, 1.37 mmol), NEt3 (0.38 mL, 2.74 mmol) were added without causing any significant changes. After stirring at 60 °C for 2 days, it was worked up by H2O (10 mL). The aqueous layer was acidified (pH < 1) by adding concentrated HCl, then EtOAc (10×3 mL) was used for extraction. Combined EtOAc layers were washed by brine (20 mL), dried over MgSO4, filtered and evaporated in vacuo. The crude was purified by silica gel chromatography (hexane/EtOAc = 1:1) to recover the starting material (12.5 mg), and product 2.1 (17.9 mg, 47%, 70% BRSM) was found as a white solid. Product 2.1 was further purified using reversed phase HPLC (eluting with 40% ACN/H2O, retention time = 25.0 min). 1H NMR (400 MHz, METHANOL-d4) δ 1.73 (s, 3 H), 1.77 (s, 3 H), 3.84 (s, 2 H), 4.48 (d, J=6.5 Hz, 2 H), 5.43 (t, J=6.5 Hz, 1 H), 6.78 (d, J=8.5 Hz, 2 H), 7.16 (d, J=8.5 Hz, 2 H); 13C NMR (100 MHz, METHANOL-d4) δ 18.2, 25.8, 29.4, 65.8, 115.6, 121.4, 129.7, 131.1, 138.4, 153.2, 158.8, 164.2; HRESIMS [M+Na]+ calcd for C14H18N2O4Na 301.1164, found 301.1161.   NONHHOOOH  90  Figure 5.23 1H and 13C NMR spectra of 2.1 recorded in methanol-d4 at 400 MHz and 100 MHz   7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5ChemicalShift(ppm)3.053.112.102.100.982.092.001.73111.77293.31003.84484.46754.48374.85145.41435.43055.44686.76636.78777.15367.1749160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16ChemicalShift(ppm)18.154725.835529.355949.000065.8247115.6436121.3737129.7099131.0967138.3813153.1792158.8484164.1823  91   Figure 5.24 1D NOE NMR spectrum of 2.1 by irradiating methylene recorded in DMSO-d6 at 600 MHz    92 5.2.20 Preparation of phenol 2.31  L-Tyrosine methyl ester (176 mg, 0.902 mmol) was dissolved in DMF (5 mL), and K2CO3 (130.8 mg, 0.946 mmol) was added to form a white suspension. After stirring at room temperature for 15 minutes, prenyl bromide (115 µl, 0.946 mmol) was added via syringe to form a yellow suspension. After stirring at room temperature for 45 minutes, the reaction was ceased by adding H2O (10 mL). EtOAc (15×3 mL) was used for extraction, and combined EtOAc layers were washed by brine (30 mL), dried over MgSO4, filtered and evaporated in vacuo to give a crude yellow oil. The crude was purified by silica gel chromatography (hexane/EtOAc = 4:1) to give product 2.31 (141 mg, 59%) as a light yellow oil. 1H NMR (400 MHz, CHLOROFORM-d) δ 1.58 (s, 3 H), 1.69 (s, 3 H), 2.93 (d, J=6.5 Hz, 2 H), 3.14 (dd, J=13.0, 7.2 Hz, 1 H), 3.24 (dd, J=12.8, 7.0 Hz, 1 H), 3.55 (t, J=6.7 Hz, 1 H), 3.67 (s, 3 H), 5.19 (t, J=7.0 Hz, 1 H), 6.69 (d, J=8.5 Hz, 2 H), 7.00 (d, J=8.5 Hz, 2 H); 13C NMR (100 MHz, CHLOROFORM-d) δ 18.0, 25.9, 38.6, 45.6, 51.9, 62.2, 115.7, 121.4, 128.4, 130.4, 136.3, 155.0, 174.8; HRESIMS [M+Na]+ calcd for C15H22NO3 264.1600, found 264.1605.   NHOMeOOH  93   Figure 5.25 1H and 13C NMR spectra of 2.31 recorded in chloroform-d at 400 MHz and 100 MHz  7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5ChemicalShift(ppm)3.003.051.930.991.010.982.980.982.022.001.58081.68572.91832.93453.12813.14263.16053.21083.22793.24243.26033.53673.55293.56993.66635.16855.18555.20346.67666.69796.98627.00757.2600184 176 168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16ChemicalShift(ppm)17.959425.883338.607245.571151.940762.241776.840077.160077.4800115.6518121.3510128.3605130.4025136.2845155.0427174.8372  94 5.2.21 Preparation of methyl ester 2.34  4-Hydroxyphenylpyruvic acid (500 mg, 2.78 mmol) was dissolved in MeOH (60 mL) to form a colorless solution. TMS-Cl (1.05 mL, 8.34 mmol) was added via syringe without causing any significant changes. After stirring at r.t. for 24 hours, this reaction was quenched by rotary evaporation and concentrated in vacuo. The crude was purified by silica gel chromatography (hexane/EtOAc = 2:1) to give product 2.34 (481 mg, 89%) as an orange-red oil.  1H NMR (400 MHz, METHANOL-d4) δ 3.83 (s, 3 H), 6.43 (s, 1 H), 6.75 (d, J=8.9 Hz, 2 H), 7.63 (d, J=8.5 Hz, 2 H); 13C NMR (100 MHz, METHANOL-d4) δ 52.9, 112.9, 116.1, 127.8, 132.5, 139.8, 158.4, 167.6. OHOOMeOH  95   Figure 5.26 1H and 13C NMR spectra of 2.34 recorded in methanol-d4 at 400 MHz and 100 MHz 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0ChemicalShift(ppm)3.000.972.132.003.30663.31003.31433.83374.85056.42686.74076.76297.62367.6449170 165 160 155 150 145 140 135 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 55 50 45ChemicalShift(ppm)49.000052.9014112.9309116.1313127.7745132.5293139.7682158.3607167.5960  96 5.2.22 Preparation of protected alcohol 3.5a and 3.5b   6-bromohexanol (0.38 mL, 2.76 mmol) and TBDMS-Cl (686 mg, 4.55 mmol) were added in ether (2.5 mL) to form a colorless solution at 0 °C. Triethylamine (0.63 mL, 4.55 mmol) was added dropwise over 2 minutes to convert the colorless solution to a white suspension. Then DMAP (10.1 mg, 0.083 mmol) was added. The reaction was stirred at 0 °C for 20 minutes and stirred at room temperature for 46 hours. The white suspension was diluted by ether (10 mL), and the ether was washed by 10% aqueous citric acid (10×2 mL), H2O (10 mL), brine (10 mL), dried over MgSO4, filtered and evaporated in vacuo to give the crude product as a thick colorless oil. The crude was purified by silica gel chromatography (hexane/EtOAc = 1:100 to 5:100) to give pure colorless oil product 3.5a (0.59 g, 72%). Product 3.5b was synthesized in a similar way with a yield of 93%. 3.5a: 1H NMR (300 MHz, CHLOROFORM-d) δ 0.05 (s, 6 H), 0.89 (s, 9 H), 1.24 - 1.67 (m, 6 H), 1.87 (q, J=7.1 Hz ,2 H), 3.60 (t, J=6.4 Hz, 2 H); 13C NMR (75 MHz, CHLOROFORM-d) δ -5.1, 18.5, 25.2, 26.1, 28.1, 32.8, 33.0, 34.1, 63.2; HRESIMS [M+Na]+ calcd for C12H27ONaSiBr 317.0912, found 317.0911. 3.5b: 1H NMR (300 MHz, CHLOROFORM-d) δ 0.51 (q, J=7.9 Hz, 6 H), 0.93 (t, J=8.0 Hz, 9 H), 1.24 - 1.43 (m, 16 H), 1.56 (q, J=6.7 Hz, 2 H), 1.85 (q, J=7.1 Hz, 2 H), 3.41 (t, J=6.9 Hz, 2 H), 3.64 (t, J=6.6 Hz, 2 H); 13C NMR (75 MHz, CHLOROFORM-d) δ 6.6, 7.0, 25.9, 28.3, 28.9, 29.6, 29.7, 29.7, 29.7, 32.9, 33.0, 34.2, 63.2. BrOTBDMSBrOTES  97   Figure 5.27 1H and 13C NMR spectra of 3.5a recorded in chloroform-d at 300 MHz and 75 MHz    7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0ChemicalShift(ppm)5.618.816.451.991.952.000.04570.89121.35321.37451.40271.45061.47881.50091.52451.54801.84181.86621.88983.38453.40743.43023.58323.60453.62587.260080 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5ChemicalShift(ppm)-5.117118.521225.177126.121428.131932.762132.960134.071963.178176.748877.160077.5865  98  Figure 5.28 1H and 13C NMR spectra of 3.5b recorded in chloroform-d at 300 MHz and 75 MHz   7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0ChemicalShift(ppm)3.995.9217.462.132.052.022.000.47420.50080.52750.55330.90120.92780.95441.27481.37301.41791.56481.58771.82661.85181.87533.38303.40593.42873.61753.63953.66167.260080 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0ChemicalShift(ppm)6.55716.953025.883028.319728.913729.568529.659929.720832.979934.228763.240576.733677.160077.5864  99 5.2.23 Preparation of compound 3.5.5a and 3.5.5b   Diisopropylamine (0.44 mL, 3.12 mmol) was dissolved in THF (5 mL) at -78 °C, n-butyllithium solution (1.95 mL, 3.12 mmol, 1.6 M in hexane) was added to give a light yellow solution. After stirring at -78 °C for 45 minutes, the freshly made LDA solution was transferred to the solution of 3-picoline (0.29 mL, 2.98 mmol) in THF (5 mL) at -78 °C to form a yellow transparent solution. After stirring at -78 °C for 15 minutes, compound 3.5a in THF (1 mL) was added in without causing any significant changes. The reaction mixture gradually turned back to room temperature within 60 minutes to form a yellow suspension. After stirring at room temperature for 14 hours, it became a dark yellow solution, and the reaction was quenched by adding saturated aqueous ammonium chloride solution (4 mL). EtOAc (10 mL) was added for extraction, and the organic layer was washed by H2O (10 mL), brine (10 mL), dried over MgSO4, filtered and evaporated in vacuo to give the crude product. The crude was purified by silica gel chromatography (hexane/EtOAc = 10:100 to 20:100) to give pure product 3.5.5a (285 mg, 78%) as a thick colorless oil. Compound 3.5.5b was synthesized in a similar way with a yield of 81%. 3.5.5a: 1H NMR (300 MHz, CHLOROFORM-d) δ 0.04 (s, 6 H), 0.89 (s, 9 H), 1.33 (m, 6 H), 1.50 (m, 2 H), 1.61 (m, 2 H), 2.60 (t, J=7.7 Hz, 2 H), 3.59 (t, J=6.5 Hz, 2 H), 7.19 (dd, J=7.5, 4.8 Hz, 1 H), 7.48 (d, J=7.8 Hz, 1 H), 8.42 (s, 1H), 8.43 (s, 1H); 13C NMR (75 MHz, CHLOROFORM-d) δ -5.1, 18.5, 25.8, 26.1, 29.3, 29.3, 31.2, 32.9, 33.1, 63.4, 123.4, 135.9, 138.1, 147.3, 150.1; HRESIMS [M+Na]+ calcd for C18H34NOSi 308.2410, found 308.2417. OTBDMSNOTESN  100 3.5.5b: 1H NMR (300 MHz, CHLOROFORM-d) δ 0.59 (q, J=7.9 Hz, 6 H), 0.97 (t, J=7.9 Hz, 3 H), 1.21 - 1.38 (m, 18 H), 1.49 - 1.69 (m, 4 H), 2.62 (t, J=7.7 Hz, 2 H), 3.64 (t, J=6.6 Hz, 2 H), 7.23 – 7.30 (m, 1 H), 7.57 (d, J=7.8 Hz, 2 H), 8.43 (s, 1H), 8.45 (s, 1H); 13C NMR (75 MHz, CHLOROFORM-d) δ 5.9, 6.7, 25.9, 29.2, 29.5, 29.6, 29.6, 29.7, 29.7, 29.7, 31.2, 33.0, 33.1, 63.2, 123.7, 137.0, 138.7, 146.2, 149.0.   101   Figure 5.29 1H and 13C NMR spectra of 3.5.5a recorded in chloroform-d at 300 MHz and 75 MHz   8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0ChemicalShift(ppm)6.009.186.032.071.781.902.050.890.900.741.080.03800.88591.32961.47571.49711.61201.63482.57022.59612.62123.56573.58703.60917.17327.18927.19847.21437.26007.46707.49298.41918.4336152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16 8 0 -8ChemicalShift(ppm)-5.117118.506025.847326.121429.259029.335131.223832.944833.142863.376176.733577.160077.5865123.3704135.9359138.0834147.2828150.0701  102   Figure 5.30 1H and 13C NMR spectra of 3.5.5b recorded in chloroform-d at 300 MHz and 75 MHz   8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0ChemicalShift(ppm)5.057.6819.984.122.061.870.430.550.980.981.020.55330.58000.60660.63250.92780.94450.97040.99711.25581.29691.30911.53971.56331.58921.61811.64252.59612.62202.64713.61523.63723.65937.24487.26007.28667.55237.57818.43138.4480144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16 8ChemicalShift(ppm)5.94796.739825.883029.203029.477129.553329.705631.167633.132263.194876.733677.160077.5864123.7311137.0111138.7320146.2248148.9813  103 5.2.24 Preparation of pyridine alcohol 3.6a and 3.6b   Compound 3.5.5a (223 mg, 0.726 mmol) was dissolved in THF (5 mL) to form a light yellow solution. TBAF (1.45 mL, 1.45 mmol, 1 M in THF) was added without causing any significant changes. After stirring at room temperature for 2 hours, this solution was diluted by EtOAc (10 mL). The organic layer was washed by water (10 mL) and brine (10 mL), dried over MgSO4, filtered and evaporated in vacuo. The crude was purified by silica gel chromatography (hexane/EtOAc = 20:100) to give pure product 3.6a (136 mg, 97%) as a light yellow oil. Compound 3.6b was synthesized in a similar way with a yield of 92%. 3.6a: 1H NMR (300 MHz, CHLOROFORM-d) δ 1.35 (br. s., 6 H), 1.51 - 1.66 (m, 4 H), 2.60 (t, J=7.7 Hz, 2 H), 3.63 (t, J=6.5 Hz, 2 H), 7.19 (dd, J=7.7, 4.9 Hz, 1 H), 7.48 (d, J=7.7 Hz, 1 H), 8.40 – 8.44 (m, 2H); 13C NMR (75 MHz, CHLOROFORM-d) δ 25.8, 29.2, 29.3, 31.2, 32.9, 33.1, 63.1, 123.4, 135.9, 138.0, 147.3, 150.1. 3.6b: 1H NMR (400 MHz, CHLOROFORM-d) δ 1.22 - 1.37 (m, 1 H), 1.52 - 1.65 (m, 4 H), 2.60 (t, J=7.6 Hz, 2 H), 3.63 (t, J=6.7 Hz, 2 H), 7.22 (dd, J=7.6, 4.9 Hz, 1 H), 7.51 (d, J=7.9 Hz, 1 H), 8.42 (s, 1H), 8.43 (s, 1H); 13C NMR (100 MHz, CHLOROFORM-d) δ 25.9, 29.2, 29.5, 29.6, 29.6, 29.6, 29.7, 29.7, 29.7, 31.2, 33.0, 33.1, 63.2, 123.5, 136.4, 138.4, 146.8, 149.6.    OHNOHN  104   Figure 5.31 1H and 13C NMR spectra of 3.6a recorded in chloroform-d at 300 MHz and 75 MHz    8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ChemicalShift(ppm)6.305.112.112.050.981.000.701.271.34801.53681.55651.57871.61801.64192.57332.59982.62453.61143.63373.65507.17377.18997.19937.21567.26007.46687.49248.41788.4298150 145 140 135 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25ChemicalShift(ppm)25.799529.220629.330531.183932.868833.132563.087376.735177.160077.5849123.3706135.9048138.0000147.3403150.0947  105  Figure 5.32 1H and 13C NMR spectra of 3.6b recorded in chloroform-d at 400 MHz and 100 MHz   8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ChemicalShift(ppm)20.014.272.122.000.980.980.811.201.25211.29861.30851.52541.54211.56041.61061.62812.58492.60402.62303.61713.63383.65067.20677.21897.22577.23797.26007.50517.52498.42008.4345155 150 145 140 135 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20ChemicalShift(ppm)25.892229.223229.485129.557829.659629.703332.968933.136163.152176.840077.160077.4800123.5112136.3846138.3774146.8214149.6070  106 5.2.25 Preparation of dimer and trimer 3.7 and 3.8  Pyridine alcohol 3.6a (65.1 mg, 0.337 mmol) was dissolved in DCM (3 mL) at -42 °C, then trifluoromethanesulfonic anhydride (61 µl, 0.371 mmol) and diisopropylethylamine (63 µl, 0.371 mmol) were added to form a red solution. After stirring at -42 °C for 1.5 hours and room temperature for 21 hours, it turned yellow, and the reaction was quenched by rotatory evaporation to give a crude yellow oil. The crude was dissolved in minimum amount of MeOH, and purified by careful silica gel chromatography (20% saturated aqueous KNO3 solution: acetonitrile= 9:1 to 7:3) to obtain the dimer and trimer fractions. These fractions were evaporated in vacuo to remove water, and the resulting solids were triturated and sonicated by the solution of 10% DCM in methanol. After filtering to remove the excess KNO3, the filtrate was evaporated in vacuo to give pure dimer 3.7 (18.2 mg, 28%) and trimer 3.8 (10.7 mg, 16%) as yellow solids. Dimer 3.7: 1H NMR (300 MHz, METHANOL-d4) δ 1.03 - 1.21 (m, 8 H), 1.25 - 1.47 (m, 8 H), 1.66 - 1.80 (m, 4 H), 1.91 – 2.03 (m, 4 H), 2.89 (t, J=6.5 Hz, 4 H), 4.62 (t, J=6.3 Hz, 4 H), 8.00 (dd, J=7.7, 6.3 Hz, 2 H), 8.43 (d, J=8.0 Hz, 2 H), 8.79 (d, J=6.2 Hz, 2 H), 8.85 (s, 2 H); 13C NMR (75 MHz, METHANOL-d4) δ 26.9, 29.0, 30.5, 30.8, 32.0, 33.1, 63.1, 129.3, 143.3, 145.0, 145.4, 146.9; HRESIMS [M2++NO3-]+ calcd for C24H36N3O3 414.2757, found 414.2765. Trimer 3.8: 1H NMR (400 MHz, METHANOL-d4) δ 1.35 - 1.50 (m, 18 H), 1.68 - 1.80 (m, 6 H), 1.97 - 2.10 (m, 6 H), 2.89 (t, J=7.7 Hz, 6 H), 4.62 (t, J=7.5 Hz, 6 H), 7.99 (dd, J=7.5, 6.1 Hz, 3 H), 8.43 (d, J=7.9 Hz, 3 H), 8.80 (d, J = 6.1 Hz, 3 H), 9.03 (s, 3H); 13C NMR (100 N(CH2)4N(CH2)4•2NO3-N(CH2)4(CH2)4NN(CH2)3•3NO3-  107 MHz, METHANOL-d4) δ 26.7, 29.3, 29.3, 31.2, 32.4, 33.3, 62.9, 128.9, 143.2, 145.5, 145.7, 146.6; HRESIMS [M3++ 2NO3-]+ calcd for C36H54N5O6 652.4074, found 652.4072.    108   Figure 5.33 1H and 13C NMR spectra of 3.7 recorded in methanol-d4 at 400 MHz and 100 MHz    9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ChemicalShift(ppm)8.348.144.404.284.384.342.032.002.011.941.08761.11431.13861.16601.28931.30381.32891.35481.69951.72091.74831.94611.96671.97731.99792.87012.89222.91353.30543.31003.31534.59934.61984.64114.85207.97167.99297.99758.01818.41538.44208.77618.79668.8522145 140 135 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20ChemicalShift(ppm)26.948029.004030.466030.831531.973733.100648.147248.436548.710649.000049.289449.563549.852863.0718129.2887143.3149144.9901145.4318146.9242  109  Figure 5.34 1H and 13C NMR spectra of 3.8 recorded in methanol-d4 at 400 MHz and 100 MHz   9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ChemicalShift(ppm)21.626.465.926.035.753.143.002.872.351.28931.40781.42831.44111.46841.72771.74481.76272.01692.03392.05102.86992.88952.90823.30233.30663.31003.31434.60144.62114.63984.84974.90607.97507.99388.00918.42378.44338.79488.81019.0293150 145 140 135 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20ChemicalShift(ppm)26.673729.279729.325431.169532.373433.303048.359948.573348.786649.000049.213449.426749.640162.8530128.9479143.2124145.5288145.7269146.5956  110 5.2.26 Preparation of pyridinium bromide 3.9  Pyridine alcohol 3.6b (843 mg, 3.04 mmol) and hydrobromic acid (14 mL, 48% aqueous solution) were combined and heated at 90 °C for 15 hours. Then the mixture was poured into water (50 mL) at 0 °C to quench the reaction. The resulted grey precipitate was filtered and washed by cooled mother liquid to give product 3.9 (1.16 g, 91%) as grey powders. 1H NMR (400 MHz, METHANOL-d4) δ 1.26 - 1.48 (m, 20 H), 1.67 - 1.78 (m, 2 H), 1.79 - 1.89 (m, 2 H), 2.90 (t, J=7.9 Hz, 2 H), 3.44 (t, J=6.7 Hz, 2 H), 8.04 (dd, J=7.9, 5.8 Hz, 1 H), 8.56 (d, J=7.9 Hz, 1 H), 8.72 (d, J=5.8 Hz, 1 H), 8.78 (s, 1 H); 13C NMR (100 MHz, METHANOL-d4) δ 29.1, 29.8, 30.2, 30.4, 30.6, 30.6, 30.6, 30.7, 30.7, 31.6, 33.5, 34.0, 34.5, 128.4, 140.3, 142.1, 145.0, 148.2. NHBrBr  111   Figure 5.35 1H and 13C NMR spectra of 3.9 recorded in methanol-d4 at 400 MHz and 100 MHz   9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ChemicalShift(ppm)19.732.172.122.112.060.981.000.940.971.30661.38351.39951.42001.43751.45501.71841.73741.81581.83491.85242.87612.89592.91573.30623.31003.31383.41883.43563.45234.85978.02548.03998.04528.05978.54688.71508.72958.7805150 145 140 135 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20ChemicalShift(ppm)29.826330.153630.408230.553730.597330.677331.622933.994234.459748.367248.578148.789149.000049.218249.429249.6401128.3571140.2715142.1118145.0432148.2145  112 5.2.27 Preparation of monomer 3.10  Pyridinium bromide 3.9 (500 mg, 1.19 mmol) was dissolved in DCM (20 mL), and neutralized by washing with 10% aqueous K2CO3 (10×3 mL). Then the DCM layer was dried over MgSO4, filtered and evaporated to reduce the volume to about 1 mL. The 1 mL DCM solution was diluted by butanone (11 mL), and the combined solution was added via syringe pump within 23 hours to the light yellow solution of NaI (0.21 g, 1.40 mmol) in butanone (200 mL) at 72 °C. After stirring at 72 °C for another 4 days, the reaction was quenched by rotatory evaporation. Then ether (30 mL) was added in for trituration, and after evaporation in vacuo, the yellow solid crude was obtained. Recrystallization from water provided pure monomer 3.10 (0.21 g, 46%) as light yellow solids. 1H NMR (400 MHz, DMSO-d6) δ 1.00 - 1.29 (m, 20 H), 1.70 - 1.80 (m, 2 H), 1.93 - 2.03 (m, 2 H), 2.87 (t, J=6.2 Hz, 2 H), 4.66 (t, J=5.9 Hz, 2 H), 8.13 (dd, J=7.6, 6.4 Hz, 1 H), 8.54 (d, J=7.9 Hz, 1 H), 9.02 (d, J=6.1 Hz, 1 H), 9.18 (s, 1 H); 13C NMR (100 MHz, DMSO-d6) δ 23.5, 25.6, 25.7, 25.9, 25.9, 26.3, 26.3, 26.5, 26.7, 28.2, 29.5, 30.6, 60.3, 127.7, 142.5, 142.6, 143.9, 145.6.  NI n=7  113   Figure 5.36 1H and 13C NMR spectra of 3.10 recorded in DMSO-d6 at 400 MHz and 100 MHz   9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ChemicalShift(ppm)22.252.012.031.921.941.041.001.040.951.03251.04921.06141.06821.10101.14741.21971.73581.75031.76621.96261.97861.99462.50002.85322.86922.88443.33964.64274.65794.67248.10838.12428.12738.14338.52618.54599.01109.02629.1845150 145 140 135 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15ChemicalShift(ppm)23.482925.744825.948526.261226.508526.653928.217730.581438.887239.098239.309139.520039.723639.934640.145560.3428127.7277142.4921142.6375143.9467145.5686  114 5.2.28 Preparation of reduced trimer 3.11  Trimer 3.8 (3.0 mg) was dissolved in MeOH (2 mL). Adam’s catalyst (PtO2, 1.5 mg) was added and the dark suspension was stirred at room temperature under H2 (5 bar) for 3.5 hour. The reaction was monitored using TLC, and the disappearance of the starting material spot under UV indicated the reaction completion. Aqueous 10% NaOH (2 mL) was added, and EtOAc (3×2 mL) was used for extraction. The combined EtOAc layers were dried over MgSO4, filtered and rotary evaporated to give crude product 3.11 as a colorless oil (3.4 mg). This product was too volatile to be dried using oil pump. The volatility also made the product hard to be purified. 1H NMR (600 MHz, CHLOROFORM-d) δ 0.05 – 0.09 (m, 6 H), 0.78 – 0.96 (m, 6 H), 1.08 - 1.43 (m, 36 H), 1.43 - 1.96 (m, 18 H), 1.96 – 2.68 (m, 18 H), 2.97 – 3.19 (m, 3 H).  N(CH2)4(CH2)4NN(CH2)3  115   Figure 5.37 1H spectrum recorded in chloroform-d at 600 MHz and MS spectrum of 3.11   8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 -0.5ChemicalShift(ppm)6.775.7436.1118.0017.613.061.160.06680.07530.87651.20491.20751.24931.30731.37561.60591.70321.98562.24843.06153.10673.6425  116 5.2.29 Preparation of ethyl ester 4.13  Triethyl phosphonoacetate (4.40 mL, 22.2 mmol) was added via syringe into the white suspension of NaH (951 mg, 39.6 mmol, 60% in mineral oil) in THF (48 mL) within 9 minutes. The white suspension became warmer and clearer, with some bubbles forming. Then catalytic EtOH (3 drops) was added without causing any significant changes. After stirring at r.t. for 30 minutes, farnesylacetone (4.74 mL, 15.9 mmol) dissolved in THF (6 mL) was added via syringe to form a yellow solution. After stirring at r.t. for another 21 hours, the reaction was quenched by adding brine (5 mL). Then rotary evaporation was used to reduce the solution volume to about 25 mL. Later, brine (15 mL) and water (20 mL) were added, and EtOAc (50×3 mL) was used for extraction. The combined EtOAc layers were washed by brine (100 mL), dried over MgSO4, filtered and evaporated in vacuo to give a crude oil (5.83 g). The crude was purified by careful silica gel chromatography (hexane/EtOAc = 1:200 to 1:100) to recover the starting material (1.10 g) and to give pure light yellow oil product 4.13 (2.32 g, 43%, 60% BRSM). The structure of 4.13 was confirmed by comparing 1H NMR spectrum to reported data. 1H NMR (400 MHz, CHLOROFORM-d) δ 1.27 (t, J=7.0 Hz, 3 H), 1.60 (br. s., 9 H), 1.68 (s, 3 H), 1.91 - 2.24 (m, 15 H), 4.14 (q, J=7.2 Hz, 2 H), 5.10 (br. s., 3 H), 5.66 (s, 1 H)  OEtOH  117  Figure 5.38 1H spectrum of 4.13 recorded in chloroform-d at 400 MHz    7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ChemicalShift(ppm)3.658.823.0815.882.172.941.001.25661.27451.29161.56031.60121.67891.96801.98762.04312.06362.08152.15914.11674.13464.15174.16965.09685.66157.2600  118 5.2.30 Preparation of all E-geranylgeraniol 4.14  Ester 4.13 (980 mg, 2.95 mmol) was dissolved in THF (15 mL) at 0°C, and DIBAL-H (7.37 mL, 1 M in methylene chloride) was added via syringe to form a grey solution with some bubbles forming. After gradually turning back to room temperature, the solution was stirred for 4.5 hours and quenched by adding water (10 mL). Aqueous saturated Rochelle salt solution (50 mL) was added to break the emulsion, and EtOAc (40×4 mL) was used for extraction. The combined EtOAc layers were washed by brine (100 mL), dried over MgSO4, filtered and evaporated in vacuo to give a crude oil. The crude was purified by silica gel chromatography (hexane/EtOAc = 1:100 to 1:10) to give colorless oil pure product 4.14 (601 mg, 70%). 1H NMR (400 MHz, CHLOROFORM-d) δ 1.60 (s, 9 H), 1.68 (s, 6 H), 1.87 - 2.22 (m, 12 H), 4.15 (d, J=6.8 Hz, 2 H), 5.10 (m, 3 H), 5.42 (t, J=6.8 Hz, 1 H); 13C NMR (100 MHz, CHLOROFORM-d) δ 16.2, 16.2, 16.4, 17.8, 25.8, 26.5, 26.8, 26.9, 39.7, 39.8, 39.9, 59.6, 123.5, 123.9, 124.3, 124.5, 131.4, 135.1, 135.5, 140.0 HOH  119   Figure 5.39 1H and 13C NMR spectra of 4.14 recorded in chloroform-d at 400 MHz and 100 MHz   7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ChemicalShift(ppm)9.206.1412.581.963.000.961.39571.60211.68231.95271.96891.98852.05672.08742.10542.12754.14404.16115.09605.11055.40305.42015.43727.2600140 135 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15ChemicalShift(ppm)16.170417.831525.847626.457226.777326.914439.700639.868259.573376.840077.160077.4648123.4585123.9156124.5252131.4289135.1169135.5436140.0241  120 5.2.31 Preparation of ester 4.15  All E-geranylgeraniol 4.14 (866 mg, 2.98 mmol) was dissolved in pyridine (10 mL) to form a colorless solution. Acetic anhydride (8.45 mL, 76.6 mmol) was added via syringe without causing any significant changes. After stirring at r.t. for 14 hours, the reaction was quenched by rotary evaporator. Then water (15 mL) was added, and EtOAc (15×4 mL) was used for extraction. The combined EtOAc layers were washed by water (40×2 mL), brine (40 mL), dried over MgSO4, filtered and evaporated in vacuo to give a crude oil. The crude was purified by silica gel chromatography (hexane/EtOAc = 3:100 to 1:5) to give colorless oil pure product 4.15 (856 mg, 86%). 1H NMR (400 MHz, CHLOROFORM-d) δ 1.60 (s, 9 H), 1.68 (s, 3 H), 1.70 (s, 3 H), 1.92 - 2.17 (m, 15 H), 4.59 (d, J=7.2 Hz, 2 H), 5.10 (br. s., 3H), 5.34 (t, J=6.7 Hz, 1 H); 13C NMR (100 MHz, CHLOROFORM-d) δ 16.1, 16.2, 16.6, 17.8, 21.2, 25.9, 26.4, 26.8, 26.9, 39.7, 39.8, 39.9, 61.6, 118.4, 123.8, 124.3, 124.5, 131.4, 135.1, 135.6, 142.4, 171.3; HRESIMS [M+Na]+ calcd for C22H36O2Na 355.2613, found 355.2616.   AcOH  121   Figure 5.40 1H and 13C NMR spectra of 4.15 recorded in chloroform-d at 400 MHz and 100 MHz   7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5ChemicalShift(ppm)9.316.2216.172.063.031.001.55941.59871.67971.70441.95181.96801.98852.05162.06872.08742.10622.12584.57734.59525.10285.32635.34425.35957.2600176 168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16 8ChemicalShift(ppm)16.146016.618417.837521.205125.852826.355626.782326.919539.689139.872061.556076.840077.160077.4648118.3795123.7586124.5205131.4082135.1111135.6444142.4407171.2715  122 5.2.32 Preparation of bromohydrin 4.16  Ester 4.15 (800 mg, 2.41 mmol) was added in water (6 mL) and THF (6 mL) to form a suspension at 0 °C. NBS (471 mg, 2.65 mmol) dissolved in THF (6 mL) was added via syringe pump within 60 minutes. After stirring at 0 °C for another 1 hour 40 minutes, the reaction was quenched by water (9 mL). EtOAc (20×3 mL) was used for extraction. The combined EtOAc layers were washed by brine (50 mL), dried over MgSO4, filtered and evaporated in vacuo to give a crude yellow oil. The crude was purified by silica gel chromatography (hexane/EtOAc = 1:20 to 2:5) to recover the starting material (240 mg) and to give pure product 4.16 (461 mg, 45%, 64% BRSM) as a light yellow oil. 1H NMR (400 MHz, CHLOROFORM-d) δ 1.33 (s, 3 H), 1.34 (s, 7 H), 1.59 (s, 6 H), 1.70 (s, 3 H), 1.72 - 1.84 (m, 2 H), 1.92 - 2.15 (m, 11 H), 2.27 - 2.37 (m, 2 H), 3.97 (dd, J=11.3, 1.7 Hz, 1 H), 4.58 (d, J=7.2 Hz, 2H), 5.10 (t, J=6.3 Hz, 1 H), 5.19 (t, J=6.5 Hz, 1 H), 5.34 (t, J=6.7 Hz, 1 H); 13C NMR (100 MHz, CHLOROFORM-d) δ 16.0, 16.1, 16.6, 21.2, 26.0, 26.3, 26.7, 26.8, 32.3, 38.3, 39.7, 39.7, 61.6, 71.1, 72.6, 118.4, 123.9, 126.1, 133.2, 135.4, 142.4, 171.3.  AcOHBrOH  123   Figure 5.41 1H and 13C NMR spectra of 4.16 recorded in chloroform-d at 400 MHz and 100 MHz   7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5ChemicalShift(ppm)2.882.916.023.211.4815.361.151.032.141.021.011.001.25321.30351.32661.33941.59271.62091.70101.95181.97141.98852.00902.03972.04732.06872.09002.10542.11992.14032.29472.31522.32883.95553.95983.98363.98794.57394.59185.08235.09775.19155.32375.33915.35707.2600168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16ChemicalShift(ppm)15.978416.146016.618421.220425.959426.721426.767132.268138.302439.673939.719661.556071.064772.588576.840077.160077.4800118.4100123.9415126.0748133.1606135.4006142.4102171.2715  124 5.2.33 Preparation of epoxide 4.17  Bromohydrin 4.16 (354 mg, 0.824 mmol) was dissolved in MeOH (15 mL) at 0 °C to form a colorless solution. K2CO3 (124mg, 0.899 mmol) was added to form a yellow solution with some white precipitants. After stirring at 0 °C for 40 minutes, this reaction was quenched by water (50 mL). Then EtOAc (50×2 mL) was used for extraction. The combined EtOAc layers were washed by brine (80 mL), dried over MgSO4, filtered and evaporated in vacuo to give a crude yellow oil. The crude was purified by silica gel chromatography (hexane/EtOAc = 1:15) to give pure product 4.17 (182 mg, 63%) as a colorless oil. 1H NMR (400 MHz, CHLOROFORM-d) δ 1.25 (s, 3 H), 1.30 (s, 3 H), 1.59 (s, 3 H), 1.61 (s, 3 H), 1.70 (s, 3 H), 1.95 - 2.22 (m, 15 H), 2.70 (t, J=6.1 Hz, 1 H), 4.58 (d, J=7.2 Hz, 2 H), 5.09 (t, J=6.3 Hz, 1 H), 5.15 (t, J=6.3 Hz, 1 H), 5.34 (t, J=6.8 Hz, 1 H); 13C NMR (100 MHz, CHLOROFORM-d) δ 16.1, 16.1, 16.6, 18.9, 21.2, 25.0, 26.3, 26.8, 27.6, 36.5, 39.7, 39.7, 58.4, 61.5, 64.3, 118.4, 123.9, 125.0, 134.2, 135.5, 142.4, 171.3.     AcOHO  125   Figure 5.42 1H and 13C NMR spectra of 4.17 recorded in chloroform-d at 400 MHz and 100 MHz  7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ChemicalShift(ppm)2.892.853.443.352.7813.230.912.000.980.940.951.25411.29501.57561.59441.61231.64131.70021.72151.96211.97911.99872.04732.06362.08572.10112.14972.68112.69652.71184.57314.59105.07725.09345.14975.32035.33655.35447.2600168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16 8ChemicalShift(ppm)16.139916.612318.883121.199525.039926.335327.615536.454539.670139.746358.445561.539264.328176.840077.160077.4800118.3989123.8547124.9672134.2025135.5131142.4015171.2657  126 References  1 Maplestone, R. A., Stone, M. J., & Williams, D. H. The evolutionary role of secondary 2 Wu, H., Lien, E. J., & Lien, L. L. (2003). Chemical and pharmacological investigations of Epimedium species: a survey. Progress in Drug Research 2003, 1-57. 3 Schmitz, R. Friedrich Wilhelm Sertürner and the discovery of morphine. 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