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Synthesis of biologically active marine natural product analogues Nodwell, Matthew B. 2008

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Synthesis of Biologically Active Marine Natural Product Analogues by Matthew B. Nodwell B.Sc., University of Victoria, 1998 M.Sc., University of Victoria, 1999 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) January 2009 © Matthew B. Nodwell, 2008 ABSTRACT Natural products have long been a source of inspiration for many drugs in human use. The Andersen lab examines compounds from marine sources that can be used as lead structures for drug discovery. Synthetic studies, structure-activity relationships (SAR) and biological findings of two such compounds are described in this thesis. The first is pelorol, a meroterpene isolated from a tropical sponge Dactylospongia elegans. Pelorol is a small molecule activator of SHIP 1, a phosphatase that is a negative regulator of the P13K pathway in hematopoetic cells. Using a synthetic route from a previous co-worker, Lu Yang, a series of SHIP 1 activating compounds based on pelorol were synthesized. These compounds were evaluated for selectivity, potency, and efficacy in a series of biological studies, leading to the discovery of 2.27 as a preclinical lead compound. Water-soluble prodrugs of the SHIP 1-activating compounds were also synthesized and their properties reported. HOCH pelorol The second compound examined is ceratamine A, an alkaloid isolated from the sponge Pseudoceratina sp. from Papua New Guinea. Ceratamines A and B are microtubule stabilizing antimitotic agents that may be useful in cancer chemotherapy. The core imidazo[4,5,d]azepine heterocycle of the ceratamines has no precedent among 2.27 11 known synthetic or natural compounds. The relatively simple structure of the ceratamines and the novel antimitotic phenotype they generate makes them attractive targets. Desbromo ceratamine A (3.44) was synthesized by an efficient and scaleable route, confirming the structure of ceratamine A and validating the biological activity of the core pharmacophore. Synthetic efforts towards ceratamine A were ultimately thwarted by the inability to install the bromine atoms present in the natural product. A significant finding is that the bromine atoms in ceratamine A contribute significantly to the antimitotic potency of the compound necessitating a bioisosteric approach to more potent antimitotic ceratamine-based agents. Br 0 ceratamine A 3.44 111 TABLE OF CONTENTS Abstract.ii Table of Contents iv List of Figures vi List of Schemes xi List of Abbreviations xiii Acknowledgements xvi Dedication xvii Chapter 1: Introduction 1 1.1: Natural Product Drug Discovery 1 1.2: Natural Product Total Synthesis 6 1.3: Function Oriented Synthesis 8 1.3.1: Halichondrin B 9 1.3.2: Bryostatins 11 1.4: Scope of Thesis 12 Chapter 2: Synthesis and Structure Activity Relationships of SHIP1 Activating Analogues of the Sponge Meroterpenoid Pelorol 15 2.1. P13K Signaling Pathways in Human Cancers and Inflammation 15 2.2. Cell Signaling Pathways as Drug Targets 17 2.3. Analogue Synthesis 21 iv 2.4. Prodrug Synthesis 35 2.5. Biological Results 39 2.5.1: Structure-Activity Relationships 46 2.5.2: Prodrugs of 2.10 51 2.6. Conclusion 55 2.7: Experimental 57 Chapter 3: Synthesis of the Ceratamine Heterocycles 90 3.1. Microtubules as a Target for Anticancer Drugs 90 3.2: Synthesis of Ceratamine Analgues 92 3.3. Biological results 120 3.4. Conclusions 122 3.5. Experimental 126 References 154 Appendix A: NMR spectra for selected compounds from Chapters 2 and 3 167 Appendix B: X-ray structure reports for compounds 2.10, 2.40 and 3.37 197 V LIST OF FIGURES Figure 1.1. Representative natural product drugs 1 Figure 1.2. Wohiers’ urea synthesis 6 Figure 1.3. Structural truncation of halichondrin B 10 Figure 1.4. Simplification of bryostatin analogues by function oriented synthesis 12 Figure 1.5. Structural analogues of pelorol 14 Figure 1.6. Ceratamine A and analogues 14 Figure 2.1. SHIP and PTEN regulated cell signaling pathways 16 Figure 2.2. Possible oxidation and Michael addition to catechol moiety 20 Figure 2.3. Possible structural modifications of pelorol 21 Figure 2.4. Lewis acid mediated ring closure 23 Figure 2.5. Crystal structure of (2.1O)-CH3CN 27 Figure 2.6. Regiochemical assignment of 2.28 and 2.29 29 Figure 2.7. 1H NMR of 2.39 recorded in CDC13 at 600 MHz 32 Figure 2.8. 1D NOE NMR of 2.39 recorded in CDC13 at 600 MHz 32 Figure 2.9. Crystal structure of 2.40-CH3CN 33 Figure 2.10. ‘H NMR of a mixture of 2.35 and 2.36 recorded in CDC13 at 300 MHz 34 Figure 2.11. ‘H NMR of a mixture of 2.37 and 2.38 recorded in CDC13 at 400 MHz 34 Figure 2.12. General prodrug concept 35 Figure 2.13. 2.4 preferentially activates SHIP1 over SHIP2 39 vi Figure 2.14. 2.4 and 2.10 (2 pM) activate SHIP1 to approximately the same extent 40 Figure 2.15. TNFo inhibition by 2.10 in wild type and SHIP 1 knockout macrophages 41 Figure 2.16. Reduction of TNFo levels in LPS stimulated mice by 2.10 42 Figure 2.17. Both Pdt(3,4)P2(20 laM) and 2.10 (3 laM) enhance activity of wild type SHIP1 but not AC2 SHIP1 43 Figure 2.18.[3H]-2.10 binds to SHIP1 C2 domain 44 Figure 2.19. SHIP 1 activating compounds evaluated for biological activity 46 Figure 2.20. Dose response curves and approximate EC50 values for selected compounds in TNFct inhibition assay 47 Figure 2.21. Dose response curves and approximate EC5O values for selected compounds in a multiple myeloma cell cytotoxicity assay 49 Figure 2.22. Enzymatic SHIP 1 modulation assay. Concentrations are given in ag/mL 51 Figure 2.23. Enzymatic SHIP1 activation assay with 2.10 and 2.41 52 Figure 2.24. Possible decomposition of prodrug 2.41 53 Figure 2.25. Possible intramolecular decomposition of prodrug 2.42 54 Figure 2.26. Evolution of SHIP 1 activating drug candidates from natural product inspiration (pelorol) 56 Figure 3.1. Confocal microscopy of cells arrested in mitosis by ceratamine A. “Li” 93 vii and “L2” are 0.32 urn thick optical sections sliced along the lines shown in “L” Figure 3.2. Crystal structure of chloroimidazole 3.37 108 Figure 3.3. ‘H NMR spectra of A) ceratamine A (4) in DMSO-d6at 500 MHz and B) 3.44 in DMSO-d6at600 MHz 113 Figure 3.4. ‘H NMR spectra of 3.44 in DMSO-d6at 400 MHz recorded at A) 298 K, B)35OKandC)400K 114 Figure 3.5. Hindered rotation about C2/N18 bond in 3.44 115 Figure 3.6. 1H NMR spectrum of 3.49 in DMSO-d6recorded at 400 MHz 117 Figure 3.7. Interconverting rotamers of 3.44 and 3.49 117 Figure 3.8. Analogues submitted for TG3 assay 120 Figure 3.9. Summary of antimitotic screen for selected compounds 121 Figure 3.10. ‘H NMR chemical shifts of selected protons 123 Figure A.1. ‘H and ‘3C NMR spectra of 2.10 recorded in CDC13 at 400 and 100 MHz respectively 168 Figure A.2. ‘H and ‘3C NMR spectra of 2.20 recorded in CDC13 at 600 and 150 MHz respectively 169 Figure A.3. 1H and ‘3C NMR spectra of 2.22 recorded in CDC13 at 400 and 100 MHz respectively 170 Figure A.4. ‘H and ‘3C NMR spectra of 2.24 recorded in CDC13 at 400 and 100 MHz respectively 171 Figure A.5. ‘H and ‘3C NMR spectra of 2.25 recorded in CDC13 at 400 and 100 MHz respectively 172 viii Figure A.6. ‘H and ‘3C NMR spectra of 2.26 recorded in CD3O at 400 and 100 MHz respectively 173 Figure A.7. ‘H and ‘3C NMR spectra of 2.27 recorded in CD3O at 600 and 150 MHz respectively 174 Figure A.8. ‘H and ‘3C NMR spectra of 2.28 recorded in CDC13 at 600 and 150 MHz respectively 175 Figure A.9. HSQC spectrum of 2.28 recorded in CDC13 at 600 MHz 176 Figure A.1O. HMBC spectrum of 2.28 recorded in CDC13 at 600 MHz 177 Figure A.11. Expanded HMBC spectrum of 2.28 recorded in CDC13 at 600 MHz 178 Figure A.12. ‘H and ‘3C NMR spectra of 2.29 recorded in CDC13 at 600 and 150 MHz respectively 179 Figure A.13. HSQC of 2.29 spectrum recorded in CDC13 at 600 MHz 180 Figure A.14. HMBC spectrum of 2.29 recorded in CDC13 at 600 MHz 181 Figure A.15. Expanded HMBC spectrum of 2.29 recorded in CDC13 at 600 MHz 182 Figure A.16. ‘H and ‘3C NMR spectra of 2.31 recorded in CD3O at 400 and 100 MHz respectively 183 Figure A.17. ‘H and ‘3C NMR spectra of 2.39 recorded in CDC13 at 600 and 150 MHz respectively 184 Figure A.18. ‘H and ‘3C NMR spectra of 2.40 recorded in CDC13 at 600 and 150 MHz respectively 185 Figure A.19. ‘H and ‘3C NMR spectra of ceratamine A recorded in DMSO-d6at 500 and 100 MHz respectively 186 ix Figure A.20. ‘H and ‘3C NMR spectra of 3.28 recorded in CD3O at 600 and 150 MHz respectively 187 Figure A.21. ‘H NMR spectrum of 3.28 recorded in DMSO-d6at 400 MHz 188 Figure A.22. 111 NMR spectrum of 3.29 recorded in CD3O at 600 MHz 188 Figure A.23. HMBC spectrum of 3.29 recorded in CD3O at 600 MHz 189 Figure A.24. Partial HMBC spectrum of 3.29 recorded in CD3O at 600 MHz 190 Figure A.25. 1H and ‘3C NMR spectra of 3.44 recorded in DMSO-d6at 600 and 150 MHz respectively 191 Figure A.26. ‘H and ‘3C NMR spectra of 3.45 recorded in DMSO-d6at 600 and 150 MHz respectively 192 Figure A.27. ‘H NMR spectrum of 3.46 recorded in CD21 at 600 MHz 193 Figure A.28. HSQC spectrum of 3.46 recorded in CD21 at 600 MHz 194 Figure A.29. HMBC spectrum of 3.46 recorded in CD21 at 600 MHz 195 Figure A.30. ‘H and ‘3C NMR spectra of 3.49 in DMSO-d6at 400 and 100 MHz respectively 196 x LIST OF SCHEMES Scheme 2.1. General synthetic route to pelorol analogues 22 Scheme 2.2. Synthesis of aldehyde 2.8 24 Scheme 2.3. Attempted synthesis of 2.10 25 Scheme 2.4. Synthesis of phenols 2.10, 2.20, 2.22, and 2.27 26 Scheme 2.5. Analogue synthesis via bromide 2.23 28 Scheme 2.6. Synthesis of 2.31 via Mitsunobu reaction 29 Scheme 2.7. Synthesis of epimers 2.39 and 2.40 via Nazarov cyclization 31 Scheme 2.8. Synthesis of amino acid-based prodrugs of 2.10 37 Scheme 2.9. Synthesis of PEGylated 2.10 38 Scheme 3.1. Proposed biosynthesis of ceratamines A and B 95 Scheme 3.2. Proposed biomimetic synthesis of ceratamine A 96 Scheme 3.3. Second retrosynthetic analysis of the ceratamines 97 Scheme 3.4. Third reterosynthetic analysis of ceratamine A 98 Scheme 3.5. Fourth reterosynthetic analysis of the ceratamines 99 Scheme 3.6. Preparation of heterocycle 3.20 100 Scheme 3.7. Alternate 7-membered ring formation 102 Scheme 3.8. Proposed Wittig cyclization to form 3.20 103 Scheme 3.9. Preparation of aminoimidazole 3.27 104 Scheme 3.10. Formation of ceratamine analogues 3.28 and 3.29 105 xi Scheme 3.11. Preparation of aminoimidazole 3.27 by Pd° catalyzed amination 107 Scheme 3.12. Possible mechanism of Michael addition of water to 3.27 109 Scheme 3.13. Scrambling of BOM regiochemistry of 3.37 110 Scheme 3.14. Formation of desbromoceratamine A by amidation of chloride 3.37 112 Scheme 3.15. Preparation of analogues 3.45 and 3.49 116 Scheme 3.16. Mechanistic rationale for formation of ceratamine analogues 119 xii LIST OF ABBREVIATIONS - degree(s) ± - racemic 1D - one-dimensional 2D - two-dimensional Ac0 - acetate AcOH - acetic acid AIBN - azobisisobutyronitrile AKT - protein kinase B amu - atomic mass unit ar - aryl Asp - aspartic acid hr - broad Bn - benzyl Boc - t-butoxycarbonyl BOM - benzyloxymethyl Bu - butyl n-BuLi - n-butyllithium t-BuLi - t-butyllithium - degrees Celsius calcd - calculated cGMP - current good manufacturing practice COSY - two-dimensional correlation spectroscopy - chemical shift in parts per million - deleted d - doublet dba - dibenzylideneacetone DCM - dichioromethane dd - doublet of doublets DEAD - diethyl azodicarboxylate DIEA - diisopropylethylamine DIPC - N,N’- diisopropylcarbodiimide DMAP - 4-dimethylaminopyridine DMF - N,N-dimethylformamide DMSO - dimethyl suiphoxide DMSO-d6 - deuterated dimethyl suiphoxide DNA - deoxyribonucleic acid dppf - 1,1 ‘-bis(diphenylphosphino)ferrocene dt - doublet of triplets EC50 - median effective concentration ELISA - enzyme linked immuno sorbant assay EPR - enhanced permeability and retention eqiv. - equivalent(s) ElMS - electron impact mass spectrometry xiii ESIMS - electrospray ionization mass spectrometry Et - ethyl Et20 - diethyl ether EtOAc - ethyl acetate EtOH - ethanol FDA - U.S. Food and Drug Administration g - gram(s) h - hour(s) HMBC - two-dimensional heteronuclear multiple bond coherence HMPA - hexamethyiphosphoramide HPLC - high-performance liquid chromatography HOAc - acetic acid HOBt - l-hydroxybenzotriazole HOSu - N-hydroxysuccinimide HRESIMS - high resolution electrospray mass spectrometry HSQC - two-dimensional heteronuclear single quantum coherence HTS - high throughput screening Hz - hertz 1C50 - median inhibitory concentration J - coupling constant in hertz K - degrees kelvin K1 - in vitro inhibition constant L - levorotatory lb - pound LiHMDS - lithium hexamethyldisilazide LPS - lipopolysaccharide Lys - lysine m - multiplet M - molar concentration m-CPBA - meta-chloroperbenzoic acid Me - methyl MeCN - acetonitrile MeOH - methanol mg - milligram(s) MHz - megahertz mm - minute mL - rnillilitre(s) mm - millimetre(s) MM - multiple myeloma mmol - millimol(s) - micromolar mlz - mass to charge ratio nM - nanomolar NBS - N-bromosuccinimide NCE - new chemical entity nm - nanometers xiv NMR - nuclear magnetic resonance NOE - nuclear Overhauser enhancement nu: - nucleophile OD 650 - optical density at 650 nm ORTEP - Oak Ridge Thermal Ellipsoid Plot PEG - poly(ethylene)glycol pH - -log10[Hj Ph - phenyl PIP3 - phosphatidylinositol-3 ,4,5-triphosphate PIP2 - phosphatidylinositol-4,5-biphosphate P13K - phosphatidylinositol-3-kinase PMB - p-methylbenzoate ppm - parts per million Ptdlns(4,5)P2 - phosphatidylinositol-4,5-biphosphate Ptdhs(3 ,4,5)P - phosphatidylinositol-3 ,4,5-triphosphate Ptdlns(3 ,4)P2 - phosphatidylinositol-3 ,4-biphosphate PTEN - phosphatase and tensin homologue q - quartet rt - room temperature s - singlet SAR - structure-activity relationship SHIP - Src Homology 2-containing Inositol 5’-Phosphatase sp. - species SPA - scintillation proximity assay t - triplet TFA - trifluoroacetic acid THF - tetrahydrofuran TLC - thin-layer chromatography TNFcL - tumor necrosis factor c VT - variable temperature XPhos - 2-dicyclohexylphosphino-2’,4’,6’-triisopropylbiphenyl xv ACKNOWLEDGEMENTS I would like to thank Dr. Raymond J. Andersen for his patience and direction throughout this project. It has been an honor working for him; one of the best bosses I have ever had. A thank you to all my lab mates, past and present, for all that you have taught me and for all the good times we have shared. I am a better person for having known all of you. I would like to thank everyone who has taken the time to answer my questions, run an assay or experiment for my project, proofread a document for me, or sent me data. None of this could have happened without your assistance. My most sincere gratitude to you all; your hard work and patience does not go unnoticed. And of course, a huge thank you to my lovely wife Nataliya for putting up with all my nonsense. ‘...But the reason I call myself by my childhood name is to remind myself that a scientist must also be absolutely like a child. If he sees a thing, he must say that he sees it, whether it was what he thought he was going to see or not. See first, think later, then test. But always see first. Otherwise you will only see what you were expecting...’ -Wonko the Sane from “So Long and Thanks for All the Fish” by Douglas Adams xvi DEDICATION To my parents, Bruce and Margarita xvii Chapter 1: Introduction. 1.1: Natural Product Drug Discovery Natural products have found use in human medicine for thousands of years. The compounds shown in Figure 1.1 are all well known examples of natural product derived drugs whose impact on human health and well-being cannot be overstated. Ry Aspirin . Morphine (acetylsalicylic acid) Quinine Perncillins(f3-Iactams) Figure 1.1: Representative natural product drugs Morphine, extracted from the opium poppy Papaver somniferum, is a very powerful analgesic and has been used to treat pain for millennia. Despite its addictive properties and hence its potential for abuse (diacetylmorphine is commonly known as heroin), there is little doubt that its benefits outweigh its potential to cause suffering.’ Most modern medicine no longer treats pain as a side effect of disease but rather as harmful in and of itself and treatable. Morphine and its synthetic offspring still form the cornerstone of modern pain management. 1 Another analgesic, acetylsalicylic acid (commonly known as Aspirin), needs no introduction. It is a semisynthetic derivative of the natural product salicylic acid which occurs in many plant tissues, most notably willow bark. Aspirin, which is used as an analgesic, an antipyretic and an anti-inflammatory medication, is one of the most widely taken medications in the world today.2 Malaria is a serious and widespread infectious disease caused by protozoan parasites of the genus Plasmodium. It has infected humans for over 50,000 years and may have been a human pathogen for the entire history of our species.3 It is currently estimated to cause one million deaths per year. The first effective treatment for malaria was the compound quinine found in the bark of the cinchona tree of South America, where it was used by aboriginal peoples as a muscle relaxant. Jesuit missionaries brought it to Europe as a treatment for malaria in the 17th century, when malaria was still endemic to Europe. It remained the antimalarial drug of choice until the 1940s, when other drugs such as Chioroquine and proguanil replaced it. Although several syntheses of quinine have been reported, isolation of the alkaloid from natural sources remains the most economical route of large scale production.4 While the above examples have had considerable impact on human medicine, it is the discovery of penicillin by Alexander Fleming in 1928 and its subsequent development for use as a medicine by Howard Walter Florey and Ernst Boris Chain, which is probably one of the greatest medical breakthroughs of modern times. From its discovery in a Penicillium mold,5 to its wartime mass production via fermentation, to the novel chemical reagents and reactions that allowed its total synthesis6,to the development of multiple 2 generations of 13-lactam antibiotics, the story of the penicillins is a microcosm of modern natural product drug discovery. Currently, approximately half of the drugs in clinical use are of natural product origin and the pharmaceutical industry has long looked towards natural products as a source of drug leads and structural inspiration. A recent summary of new chemical entities (NCE5) introduced into clinical use over the past 25 years reveals that 57% of the 1184 NCEs introduced during that time frame were derived from natural products or designed based on a natural product pharmacophore.7 However, during the 1990s, natural product based drug discovery was largely de emphasized by the pharmaceutical industry. The widespread use of high throughput screening (HTS) against defined molecular targets prompted many companies to move from natural extract libraries to more “screen-friendly” synthetic libraries.8The advent of combinatorial chemistry offered the prospect of rapidly synthesizing simpler and more drug-like libraries of wide chemical diversity.9 Additionally, there was (and still is) a declining emphasis among major pharmaceutical companies on infectious disease, a traditional area of strength for natural products)° However, in recent years, the interest in natural products as a basis for drug discovery has been rekindled. It has long been recognized that natural product structures possess high molecular diversity, biochemical specificity, and other unique molecular properties that distinguish them from libraries of synthetic compounds. Computational studies have compared the physicochemical properties of representative combinatorial, natural, and synthetic drug molecules using various 3 molecular descriptors such as number of chiral centers, ring topology, distribution of heavy atoms, number of rotatable bonds, and Lipinski-type” descriptors. These studies reveal that natural products, not surprisingly, typically have a greater number of chiral centers and increased steric complexity compared with either synthetic drugs or combinatorial libraries. Natural products were also found to possess greater molecular rigidity and a larger number of solvated hydrogen-bond donors and acceptors.12 Interestingly, the diversity of ring systems found in natural products was far greater than that found in the synthetic and combinatorial libraries, with only one-fifth of the ring systems found in natural products represented in current trade drugs.8 Considering these factors, a compelling case for the intrinsic utility of natural products as sources of drug leads can be made. Given that an infinite number of small molecule structures can exist, it is important that a molecular screening library covers a large portion of chemical space, but is also predisposed towards compounds that display biological interactivity.13Early combinatorial libraries were designed more on a principle of chemical accessibility and maximum achievable size rather than on biologically relevant structural motifs and chemical diversity. Natural product libraries (often existing as crude extracts), by virtue of their co-evolution with biological systems, can be considered validated starting points for screening-library design.’4However, most natural products are available only in very small quantities in a particular natural extract and HTS of natural source extracts has unique liabilities that must be overcome before a single active compound is obtained. Once a “hit” is uncovered by the screening platform, multiple bioassay-guided separations of the bulk natural material are required until a single bioactive compound is obtained, usually on the scale of one to tens of milligrams. 4 Elucidation of the structure of the pure compound usually follows. If the compound is to be pursued with further biological or chemical studies, a sustainable supply of it must then be secured. The supply issue for novel naturally occurring structures is a key problem in natural product drug discovery and one that must be solved before any significant preclinical work can be done on a particular compound. If the compound is of microbial origin, large-scale fermentation may be an option. For many plant-derived compounds, farming the producing organism and isolating the desired compound is usually sufficient to secure a supply. The study of marine natural products presents unique challenges to the supply issue. Because there is much humans still do not know about marine ecosystems, the cultivation of marine invertebrates (aquaculture) for natural product extraction is at present for the most part unattainable. One major complicating factor is a growing body of evidence that symbiotic bacteria, and not the animal itself, may be producing many of the natural products isolated from ‘marine animals’.15 This symbiosis is exceedingly complicated and precludes simply culturing the producing bacteria in a lab. Repeat collection of the wild organism followed by isolation is usually unacceptable for environmental, economic, and social reasons and would still likely not yield the needed quantities. With many marine natural products, synthesis is the preferred option for their production to support their biological evaluation as potential therapeutic agents. While the strength of natural products as drug leads lies in their structural complexity, this presents a challenge to their further development. The total synthesis of a particular structure often results in lengthy and unscaleable synthetic routes that seldom yield significantly more compound than could be extracted from nature.16 Although the 5 total synthesis of a complex natural product is unto itself a significant scientific accomplishment, the evaluation of the biological utility of a particular compound requires a scaleable and efficient synthesis. 1.2: Natural Product Total Synthesis The field of total synthesis of natural products has a rich history. Beginning with the accidental generation of urea from silver cyanate and ammonium chloride by Friedrich Wohier in 1828 (Figure 1•2),i7 total synthesis has progressed to the point where virtually any target compound can be synthesized, given time and resources. Expected: AgOCN + NH4CI A NH4OCN + AgCI Obtained: AgOCN + NH4CI A H2NANH Figure 1.2: Wohlers’ urea synthesis A total synthesis of a novel organic compound is undertaken for a variety of reasons, the most basic of which is simply “because it’s there”. However, the pursuit of a synthetic route to a natural product often gives rise to novel transformations and reagents, as well as furthering the fundamental understandings of chemical reactivities and stabilities. Furthermore, the total synthesis of a natural product often provides absolute proof of an assigned structure and proof that the proposed structure is indeed responsible 6 for any observed biological activity. With respect to drug discovery, total synthesis is often used to produce a compound for development, as well as provide various analogues to map structure-activity relationships (SAR). One striking example of the power of modern total synthesis to provide a scarce natural product at large scale is that of (+)- discodermolide. (+)-Discodermolide is a polyketide that was isolated from a marine sponge in 1990.18 It is a microtubule stabilizing antitumor agent with a potency ten fold greater than that of paclitaxel.19(+)-Discodermolide also has strong activity against multidrug resistant tumor cells and improved aqueous solubility compared with existing antimitotic agents.2°These properties made discodermolide a strong candidate for development as an anticancer drug. (+)-discodermolide Natural sourcing was not an option because of the low yield of compound from the sponge tissue and limited supply of the producing organism. The process chemistry team at Novartis therefore undertook a total synthesis at a multigram scale. This was not a trivial undertaking as (+)-discodermolide is a very complex organic compound with 13 stereocenters, lactone and carbamate moieties, and three Z-configured alkenes, one of which is part of a terminal diene unit.21 Blending the synthetic routes developed by several research groups,22 Novartis was able to synthesize 60 g of pure (+)- discodermolide in 39 synthetic steps.23 This remarkable achievement in process HO,, OH OH 0NH2 7 chemistry provides a compelling case that no compound is beyond reach for development and it illustrates the power of modern synthetic chemistry. 1.3: Function Oriented Synthesis The complexity of many bioactive natural products often makes it extremely difficult to develop a scaleable and efficient synthesis of the compound. However, it may be possible to use the structure of the natural product as a template for the design of simpler and more synthetically tractable analogues. This approach is essentially a shift in focus from the total structure of a biologically active compound to the subset of its functionalities and their spatial orientation that is primarily responsible for its biological activity, namely the ‘pharmacophore’.24 The term “function oriented synthesis” was coined to describe this approach to natural product drug discovery. While this is not a new idea — it forms the cornerstone of modern medicinal chemistry- the term is quite apt and it can be used to describe many aspects of this thesis. Two illustrations of function oriented synthesis include the design and synthesis of simplified analogues of halichohdrin B and the bryostatins. 8 1.3.1: Halichondrin B Halichondrin B is a complex polyether marcolide isolated from various marine sponges.25 It displays very potent (sub nM) activity as a microtubule-depolymerizing agent,26 which generated significant interest in the development of halichondrin B as an anticancer therapeutic. However, the compound could not be supplied on the necessary scale despite the fact that studies suggest aquaculture may be feasible for small scale production.27 Kishi’s total synthesis of halichondrin B28, along with SAR studies29 on synthetic intermediates and fragments, revealed that the biological activity of halichondrin B resides in its macrocyclic lactone fragment. Furthermore, additional in vivo stability of the. truncated compound was realized by replacing the easily cleaved lactone linkage with a ketone.3°Thus, E7389, a simplified mimic of halichondrin B that is far more readily synthesized, shows equal in vitro potency as the natural product (Figure 1.3). This compound, under the trade name eribulin mesylate, is currently undergoing a series of clinical trials as an anticancer therapy. HO H Halichondrin B 9 HH( -Total synthesis attempts -SAR -Structural modification Figure 1.3: Structural truncation of halichondrin B HQ HQ A H Halichondrin B II E7389 10 1.3.2: Bryostatins The bryostatins are complex macrolide natural products isolated from the marine bryozoan Bugula neritina.31 They display a range of very interesting anticancer activities, including the ability to induce apoptosis,32 reverse multidrug resistance,33 and modulate the immune system. The isolation of 18 g of pure bryostatin 1 from 28,000 lb of wet organism over ten months34 demonstrated that gram amounts of pure compound can be isolated from the animal source under cGMP conditions. However, the isolation is impractical for larger amounts and access to the natural product via synthesis is limited, as current total syntheses require a very large number of synthetic steps.35 Analysis of the binding domain of the bryostatins revealed that the C-i carbonyl, the C-19 and C-26 alcohols, and a lipophilic tail are all key elements to the binding of bryostatin to its target, protein kinase C.36 With these structural requirements in mind, Wender et at. designed the simplified analogue of bryostatin 1 shown in Figure 1.4, which contains the required binding elements held in place by a rigid scaffold.37 This analogue can be synthesized in 29 steps and it was found to be more potent than bryostatin 1 itself. 11 Target binding I analYs> Required binding elements Figure 1.4: Simplification of bryostatin analogues by function oriented synthesis 1.4: Scope of Thesis The Andersen lab is involved in the isolation and structural elucidation of marine natural products. The nature of the projects undertaken in the lab range from biosynthetic studies to chemical prospecting to chemical ecology. However, in the majority of cases, the compounds isolated possess biological activity in assays designed to find lead Bryostatin 1 = 1.4nM -.70 steps Structural 0 Lead analogue K1 = 0.3nM —30 steps 12 compounds for the development of drugs for treating human diseases. Anticancer and antiinflammatory indications in particular are a common focus for the Andersen lab and their biological collaborators. As part of the investigation of these compounds, a partial or total synthesis is often undertaken to provide compound for further biological evaluation and to generate SAR to explore the pharmacophore. In this thesis, two such projects are described. The first is an SAR study of pelorol, a meroterpenoid isolated from a marine sponge. Pelorol was isolated simultaneously in the Andersen, Konig,38 and Schmitz39 laboratories. The Andersen group found that pelorol is an activator of a cell signaling phosphatase called SHIP1.4°SHIP1 is a negative regulator of the phosphatidylinositol-3- kinase (P13K) pathway and its activation amounts to functional P13K inhibition. However, unlike P13K, SHIP 1 is restricted to hematopoietic cells making it an attractive drug target. It has been proposed that activators of SHIP 1 may be used as novel therapies for hematopoetic malignancies as well as inflammatory disorders. Lu Yang from the Andersen lab accomplished the first total synthesis of pelorol and several analogues in 2OO5.° In Chapter 2 of this thesis, I describe the synthesis of several new analogues of pelorol and their water-soluble prodrugs (Figure 1.5) as part of a SAR study of the SHIP 1 activating pharmacophore. From this work we identified a new pelorol analogue that is currently undergoing preclinical evaluation.41 13 OH SAR Prodrug I__ __ Figure 1.5: Structural analogues of pelorol The second project described in this thesis is the development of a scaleable route to the ceratamine alkaloid family. Ceratamines A and B were isolated by the Andersen group in 2003 from the marine sponge Pseudoceratina sp. collected in Papua New Guinea.42 They are very interesting microtubule stabilizing antimitotic agents that bind to a different site on tubulin than the highly efficacious antimitotic compound paclitaxel and generate a novel antimitotic phenotype.43Furthermore, the core heterocyclic structure of the ceratamines is unprecedented and presents an opportunity to explore, both chemically and biologically, a novel antimitotic pharmacophore. Chapter 3 of this thesis describes the development of a scaleable route to several ceratamines analogues that are currently being evaluated to validate the in vivo efficacy of the ceratamine pharmacophore (Figure 1.6). H2N Figure 1.6: Ceratamine A and analogues Pelorol NH2 14 Chapter 2: Synthesis and Structure Activity Relationships of SHIP1 Activating Analogues of the Sponge Meroterpenoid Pelorol 2.1: P13K signaling Pathway in Human Cancers and Inflammation. Phosphatidylinositol-3-kinase (P13 K) first attracted attention in the cancer- research field in the mid-1980s when it was found that P13K activity was associated with the transforming activity of viral oncogenes.44 It is now known that the P13K pathway regulates various cellular processes such as proliferation, growth, apoptosis, and cytoskeletal rearrangement. The main function of P13K is to synthesize the second messenger phosphatidylinositol-3 ,4,5-triphosphate (Ptdlns(3 ,4,5)P or PIP3) from phosphatidylinositol-4,5-biphosphate (Ptdlns(4,5)P2 or PIP2) (Figure 2.1). The phosphatase and “tumor suppressor” phosphatase and tensin homologue (PTEN) regenerates PIP2, while the Src homology 2-containing inositol 5-phosphatases (SHIP) family of phosphatases act on PIP3 to generate phosphatidylinositol-3,4-biphosphate (Ptdlns(3 ,4)P2).‘ The generation of PIP3 on the inner leaflet of the plasma membrane results in the recruitment and activation of AKT (protein kinase B). Phosphorylation of AKT mediates the activation and inhibition of several targets resulting in cellular growth, survival, and proliferation and has been strongly implicated in a wide variety of human cancers (Figure 2.1). 15 P13K PTEN II AKT (protein kinase B) Cancer: Inflammation: Cellular growth gE induced mast cell activation Cell survival -histamine and prostaglandin release Proliferation LPS induced macrophage activation Figure 2.1: SHIP and PTEN regulated cell signaling pathways Under unstimluated growth conditions, PIP3 levels are barely detectible in mammalian cells as its concentration is rigorously controlled by the balance between its production by P13K and its destruction by the PIP3 phosphatases PTEN and SHIP1. PTEN mutation or silencing is implicated in a large proportion and variety of human malignancies, such as glioblastoma, endometrial, hepatocellular, melanoma, lung, renal- cell, thyroid, and lymphoid cancers, demonstrating that the PIP3 phosphatase most clearly SHIP1 SH I P2 sSHIP 3 Pdtlns(4,5)P2 Pdtlns(3,4,5)P3 “PIP3. 5 Pdtlns(3,4)P2 16 involved in oncogenesis is PTEN.45 While the SHIP1 product Ptdlns(3,4)P9 is also capable of stimulating AKT phosphorylation in vitro, it does not appear to be a major AKT activator in vivo.46 The differences between PTEN and SHIP 1 have been clearly demonstrated by loss-of function mouse models. Homozygous PTEN deletion causes embryonic lethality in mice, while heterozygous PTEN deletion animals are viable but develop various tumors.47 Conversely, homozygous deletion of SHIP 1 in mice generates mice that are viable and fertile but display hyperproliferation of myeloid cells.5 Futhermore, on the basis of knockout studies, SHIP 1 has also been found to be a negative regulator of IgE induced mast cell activation, ‘‘ lipopolysaccharide (LPS) induced macrophage activation,50and osteoclast formation and resorptive function. In humans there is evidence that mutated SHIP 1 genes may play a role in the development of acute myeloid leukemia and chemotherapy resistance through the deregulation of the P13K signaling pathway, indicating that modulation of SHIP1 activity may be clinically relevant.5’Diminished SHIP1 activity or expression has been observed in human inflammatory diseases as well.52 2.2: Cell Signaling Pathways as Drug Targets. Based on the successes of the small molecule kinase inhibitors Gleevec and Gefitinib and the attractiveness of P13K deregulation as a drug target, much effort has been made to discover selective P13K inhibitors. Early generation P13K inhibitors such as LY294002 (2.1) and the natural product wortmannin (2.2) have been used in vitro as chemical genetics probes for many years. However, short half-life, low therapeutic index, 17 and lack of selectivity have slowed these molecules’ development into a human therapeutic.54 2.2 worlmannin While there aie several compounds that target many kinase components of the P13K signaling pathway under preclinical evaluation,55 there are virtually no small molecules in preclinical evaluation that act as activators of the phosphatases PTEN or SHIP1. The prevalence of PTEN mutations and dysfunctions in various human cancers would seem to preclude this as a simple drug target. The hypothesis underlying the research described in this chapter is that selective SHIP 1 activators should be useful experimental tools and drug leads for modulation of P13K signaling. SHIP 1 is a particularly attractive target for development of possible therapeutics for immune and hemopoetic disorders as its hematopoietic restricted expression should limit the activity of a selective SHIP1 agonist to target cells, thus reducing off-target effects.56 Additionally, the activation of the SHIP 1 enzyme is expected to proceed via allosteric binding. A high degree of homology at the active site of many phosphatases is to be expected but this should not be the case with allosteric binding sites, thus increasing the potential for high target selectivity.57 A chromogenic enzyme assay was used to screen a library of marine invertebrate extracts in a search for small molecule agonists of SHIP 1. The MeOH extract of the 2.1 LY294002 18 sponge Dactlyospongia elegans collected in Papua New Guinea exhibited promising activity in the assay. Bioassay-guided fractionation of the extract led to the identification of pelorol58 as the sole SHIP 1-activating component of the extract. Three related sesquiterpenes, illimaquinone,59 mamanuthaquninone6°and dactyloquinone61 were also isolated from the D. elegans extract, but were not active in the assay. 0- 1 14 1213 pelorol .illimaquinone mamanuthaquninone dactyloqu inone Pelorol (2.3) displayed selective and potent SHIP 1 agonist activity and therefore a chiral synthesis of the compound was undertaken and completed4°by Lu Yang in the Andersen group. This synthesis was able to provide additional amounts of pelorol for biological evaluation and various analogues for SAR studies. Analogue 2.4, in which the C-20 methyl ester of pelorol was replaced with a methyl group, displayed more potent SHIP1 agonist activity and was easier to synthesize.4° :Q 19 02A With a scaleable route to a potent SHIP 1 agonist in hand, compound 2.4 underwent a series of biological evaluations that provided convincing evidence that selective SHIP 1 activators could be viable drug candidates for treating inflammatory diseases and cancers (Section 2.5). Although the biological activity displayed by both pelorol and 2.4 were promising, the structures suffered from liabilities as drug candidates. The catechol moieties in these structures are capable of binding metals or undergoing oxidation to orthoquinones that can then undergo a Michael addition with nucleophiles to form covalent linkages (Figure 2.2).62 The potential lack of in vivo chemical stability was a major concern for the further study and development of both pelorol and 2.4. HO OH [0] chemica’ or enzymatic Figure 2.2: Possible oxidation and Michael addition to catechol moiety In an effort to solve the stability problems of the catechol-containing compounds, as well as extend the SAR of the SHIP 1 activating pelorol mimics, we undertook a 2.3 Pelorol HO R R 20 medicinal chemistry program to try to uncover a potential development candidate (Figure 2.3). Prevent catechol oxidation Explore stereochemistry at 5-membered ring HO OH Explore aromatic ring H 0 substitution 2.3 Pelorol Figure 2.3: Possible structural modifications of pelorol 2.3 Analogue Synthesis At the onset of the project, a robust route towards the chiral pelorol system had been developed40 starting from (+)-sclareolide, a natural product isolated from Salvia sciarea, or clary sage. Attempts to source this starting material led to the donation of 1 kg of (+)-sclareolide from Avoca Inc. With a large supply of enantiopure starting material in hand, we sought to synthesize analogues of pelorol that were missing the undesirable catechol functionality using the general synthetic route as shown in Scheme 2.1. 21 MeLi Baeyer-Villiger Hydrolysis TH F (÷)-sclareolide [O] Lewisacid Deox. Ar-Li Scheme 2.1: General synthetic route to pelorol analogues. Given the nature of this synthetic route, the terpene portion of the analogues would remain fairly static, while chemical diversity could be introduced in a rapid manner to the aromatic portion of the molecule. Two of the first target molecules were the C-17 and C-18 phenol analogues, 2.9 and 2.10. 2.9 2.10 Literature precedent coupled with previous experience with the pelorol synthesis indicated that the Lewis acid-mediated ring closure to form the 5-membered “C” ring of the pelorol analogues requires an electron rich arene ring directly activated at C-21 in order to capture the C-8 carbocation.63Thus, the C-18 phenol analogue 2.10 was targeted 2.5 2.6 27 2.8 22 for synthesis because the aromatic carbon where the new bond is to be formed is para- to a strongly electron donating oxygen substituent (Figure 2.4). Figure 2.4: Lewis acid mediated ring closure The synthesis of 2.10 started from (+)-sclareolide and followed the synthetic route to pelorol (Scheme 2.1). Opening of the lactone with MeLi, followed by a Baeyer Villiger reaction of the resulting methyl ketone with in-situ generated trifluoroperacetic acid yielded acetate 2.6. Basic hydrolysis of this acetate, followed by Pyr-SO3/DM mediated oxidation of the resulting primary alcohol 2.7 yielded aldehyde 2•8M (Scheme 2.2). R Lewis acid Lewis acid R R _______ 2.10 Multiple products (rearrangements, elimination) R 23 00 MeLi OH F3C 0 CF3 — THF H20,NaHCO3 CHI/ 2.5 :60% KOH MeOH H20 Pyr-S03 DMSO DIEA CH2I 2.8 : 70 % Scheme 2.2: Synthesis of aldehyde 2.8 Bromide 2.13a was prepared by bromination of aniline 2.11, followed by diazotization and treatment of the diazonium salt with hypophosphorous acid (Scheme 2.3).65 The aryllithium reagent generated by lithium-halogen exchange of the bromide 2.13a with t-BuLi was then reacted with aldehyde 2.8 to yield benzylic alcohol 2.14a. Repeating the lithium-halogen exchange with the commercially available bromides 2.13b and 2.13c yielded diols 2.14b and 2.14c, respectively. One isomer was predominantly obtained from these reactions, presumably due to the reaction proceeding via 6- membered intermediate 2.8a as shown in Scheme 2.3. Attempts to remove the benzylic alcohol via Pd-catalyzed hydrogenolysis of diol 2.14a under a large variety of conditions produced only very small amounts of the desired product 2.15a. Attempts to make the (+)-sclareolicie 2.6 : 75 % 2.7: 95 % 24 benzylic alcohol of 2.15a into a better leaving group suitable for reduction by acetatylation or tosylation gave only the Grob fragmentation product 2.16. Scheme 2.3. Attempted synthesis of 2.10 2.14a R = Me: 89% 2.14b R = H :80% 2.14c R = OMe 61 % The inability to produce large amounts of deoxygenated compound 2.15a via catalytic hydrogenation prompted us to pursue a Barton-McCombie radical deoxygenation sequence.66 Thus, treatment of benzyl alcohols 2.14a-c with NaH, followed by CS2, and then Mel yielded xanthates 2.17a-c, respectively, as shown in Scheme 2.4. Reaction of xanthates 2.17a-c with with tributyltin hydride and a catalytic amount of A1BN gave deoxygenated intermediates 2.15a-c in good yield. Attempts to NH2 Br2 I. HOAc MeOH 2.11 -78°C 2) H3P02 2) 2.8 Br 2.12:95% 2.l3aR=Me:50% 2.13b R = H (commercial) 2.13c R = OMe (commercial) 1 2.8a 2.15a Pd/Cor Pd(OH)2/C Various solvents and pressures Ac20, TsCI, MsCI Et3SiH, TFA R H 2.14a 2.16 A = Me 25 perform this deoxygenation with various silanes67 as hydride donors failed to yield any product. Cyclization of 2.15a-c using SnC14 in CH21 gave a quantitative mixture of tetracycles 2.18a-c. In the case of 2.15a, a very small amount of regioisomer 2.19 was also produced from a large-scale ring closing reaction. Reaction of these compounds with BBr3 in CH21 yielded the analogues 2.10, 2.20, 2.22 and 2.27 in high yield (Scheme 2.4). Scheme 2.4: Synthesis of phenols 2.10, 2.20, 2.22 and 2.27. 0 2.14a A = Me 2.14b P = H 2.14c R = OMe Bu3SnH AIBN, Tol reflux 2.17a A = Me : 60% 2.17b R = H : 56% 2.17c R = OMe:76% 1) NaH, THF, rt 2) CS2, rt 3) Mel, rt major minor 2.15a R = Me : 99% 2.15b R = H : 97% 2.150 A = OMe :80% SnCI4 CH2I 0°C H R 2.10 R = Me: 93% 2.22 R = H : 68 % 2.27 R = OH : 60 % R+ BBr3 CH2I, ii ‘4 2.18a P = Me : 99% 2.18b R = H : 90% 2.18c A = OMe : 83% A + OH 2.20 R = Me 2.19 A = Me 26 Compound 2.10 was shown to be active in various enzyme, cell-based, and murine model assays (Section 2.5) and, therefore, was chosen as our “benchmark” compound.41 Its structure was verified by single crystal x-ray diffraction analysis and it is shown in Figure 2.5 as a dimer with acetonitrile. Figure 2.5: Crystal structure of (2.1O)-CH3CN. Methyl ether 2.18b could be brominated at C-19 with NBS in DMF.68 No trace of the regioisomer 2.21 could be found from this reaction (Scheme 2.5). This bromine atom could then act as a versatile synthetic handle, allowing for structural diversification at an advanced intermediate. Lithium-halogen exchange of this compound, followed by quenching with an electrophile (Mel) yielded the analogue 2.24 after deprotection of the phenol. Alternately, a series of Pd°-mediated couplings could be carried out. As a proof of this principle, Suzuki couplings were carried out on this bromide to yield 2.25 and 2.26 in 93% and 83% yield respectively (Scheme 2.5).69 The ability to rapidly create a library of biaryl analogues is usually considered advantageous to an SAR study as the biaryl C2% 2.10 27 motif is considered to be a privileged structural class.70 One can envision amidations and aminations71 being carried out on intermediate 2.23 to yield aniline analogues as well. 1) tBuLi, THE, -78°C NBS,DMF 2) Mel 3) BBr3,CH2I 2.24: 49 % 2 steps1)PdCI2(dppf) Pd° bond forming Tol, 2M Na2CO3 X NBS, DMF Ar-B(OH)2 reflux 2) BBr3,CH2I \/Ar 2.25 Ar = phenyl : 93 %, 2 steps R = Ar, NHAr, NH2, etc2.26 Ar = 4-pyridyl : 83 %, 2 steps Scheme 2.5: Analogue synthesis via bromide 2.23. The resorcinol analogue 2.27 was synthesized by carrying through the reaction sequence with 1-bromo-3,5-dimethoxybenzene as shown in Scheme 2.4. This compound was treated with one equivalent of Mel in DMF in the presence ofK2C03resulting in two compounds 2.28 and 2.29. 2D NMR established their regiochemistry and the key HMBC correlations are shown in Figure 2.6. In a variation of the bromine chemistry above, resorcinol 2.27 could be treated with one equivalent of triflic anhydride. This would, in theory, give rise to two triflates which then could undergo various Pd°- catalyzed C—C and C-N bond forming reactions. 2.21 28 Mel, K2C03 DMF + Figure 2.6: Regiochemical assignment of 2.28 and 2.29. 4.65 6.20 97.2 3.73 In another class of structural analogues, an amine group was appended to phenol 2.10. A Mitsunobu reaction72 was performed on 2.10 with Boc-protected ethanolamine to yield 2.30. Deprotection of the amine yielded 2.31 as shown in Scheme 2.6. Scheme 2.6: Synthesis of 2.31 via Mitsunobu reaction. 6.40 2.28:18% 2.27 4.66 2.29: 21 % = HMBC correlation PPh3, DEAD, THF ri 2.10 NHBOC TFA CH2I NH2 2.30 2.31 : 25 %, 2 steps 29 In order to explore the effects of the stereochemistry of the terpene portion of compound 2.10, an alternate cyclization procedure was carried out as outlined in Scheme 2.7. Allowing the oxidation of alcohol 2.7 to proceed for 3 h at rt yielded a mixture of enals (2.32). Isomerization of these compounds (KOHIMeOH) primarily yielded enal Reaction of 2.33 with the lithium species derived from 2.13a yielded allylic alcohol 2.34. A tin tetrachloride promoted Nazarov-type74cyclization of 2.34 proceeded cleanly to give a 1:1 mixture of the two C-8 epimers 2.35 and 2.36. Without separation, these two isomers were hydrogenated over a PdJC catalyst to yield compounds 2.37 and 2.38 in 95 % yield. Again, without separation, the mixture was treated with BBr3 in C112 to yield a 1:1 mixture of phenols 2.39 and 2.40, the C-9 and C-8 epimers of 2.10, respectively. The diastereomers were separated by fractional crystallization. Isomer 2.39 was crystallized from the 1:1 mixture by cooling a toluene solution of the mixture. The second isomer, 2.40, was separated from the residual mixture by crystallization from CH3N. The stereochemistry of isomer 2.39 was verified by 1D NOE experiments (Figures 2.7 and 2.8) and the stereochemistry of 2.40 was verified by single crystal x-ray diffraction analysis as shown in Figure 2.9 30 2.7 KOH MeOH Pyr-S03 iPr2NEt DMSO CH2I OH 2.32 2.33 : 60 % 2.34 : 56 % SnCI4 CH2I 2.39 1) BBr3,CH2I _ _ A. THF, -78°C Pd/C H2, MeOH 68% Scheme 2.7: Synthesis of epimers 2.39 and 2.40 via Nazarov cyclization. 2) Crystallize 2.40 2.38 95% 2.36 31 cc Figure 2.7: ‘H NMR of 2.39 recorded in CDC13 at 600 MHz Figure 2.8: 1D NOESY of 2.39 recorded in CDC13 at 600 MHz 2.74 “ 2.33 1.62 0.87 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 EH U, U, U) 0, 3.0 2.5 2.0 1.5 1.0 0.5 0 32 C17 Figure 2.9. Crystal structure of 2.40-CH3CN. The substrate-controlled stereoselectivity of the hydrogenation reactions in Scheme 2.7 (2.35 to 2.37 and 2.36 to 2.38) are interesting in that each isomer (2.35 and 2.36) gives exclusively a single product (2.37 and 2.38, respectively). While mixtures of 2.35/2.36 and 2.37/2.38 were not separated, 1H NMR spectra of both these mixtures show no more than two products being formed during the hydrogenation step (Figures 2.10 and 2.11). Furthermore, upon initial inspection of 2.35 and 2.36, it would appear that hydrogen is being added to the more hindered side of the alkenes. However, manipulation of a model reveals that compound 2.35 can adopt a “bowl” like conformation in which the less hindered face of the alkene does indeed lead to the observed products. The stereoselective hydrogenation of 2.36 is less easily explained, as molecular models show no obvious preference for either face of the alkene in 2.36. 2.40 C6 C7 33 1.99191 1.03 L_J_LJI.J_LI ___________________ 7.0 6.5 Figure 2.10: ‘H NMR of a mixture of 2.35 and 2.36 recorded in CDC13 at 300 MHz Figure 2.11: ‘H NMR of a mixture of 2.37 and 2.38 recorded in CDC13 at 400 MHz + 2.36 cD (D—o) 6.12 6.12 I_I LI I 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 2.37 2.38 (0 2.00 6.17 6.12 LJ LI 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 34 2.4: Prodrug Synthesis With a number of pelorol analogues in hand, we then turned our attention to the issue of water solubility. A key aspect of any drug molecule is its water solubility, as it exerts its activity in an aqueous environment. All the pelorol analogues described thus far are extremely insoluble in water as may be expected from their hydrocarbon-like structures. This was expected to be a major problem when attempting to administer precise amounts of pelorol-based drug candidates to mice or humans. While a hydrophobic drug substance can be administered as an emulsion or suspension, this approach can cause adverse allergic responses and precipitation upon aqueous dilution, leading to erratic and poorly controlled dosing. The preferred approach is that of a solution. In the case of the pelorol analogues where the drug substance itself is poorly water soluble and an appropriate synthetic handle is available, a prodrug approach can overcome formulation liabilities. A prodrug can be defined as a bioreversible derivative of a drug that undergoes an enzymatic and/or chemical transformation in vivo to release the active parent drug as shown in Figure Enzymatic/chemical transformation —foiy — + [PromoieJ Barrier Figure 2.12. General prodrug concept. 35 In most cases, prodrugs require only one or two chemical or enzymatic transformation steps to release the active drug. The non-drug promoiety of the prodrug is usually a biologically inert compound or it can also be a second biologically active drug and such derivatives are called co-drugs.76 A prodrug strategy can accomplish many objectives, such as improved oral absorption, aqueous solubility, lipophilicity, and site- selective delivery. In the case of the pelorol analogues, we sought to improve the administration of the active drugs by appending a polar, ionizable group to the phenol in order to increase water solubility. The first obvious choice was to form amino acid esters. The ester linkage is synthetically easy to form, readily cleaved in vivo, and releases a biologically benign promoiety upon cleavage. Thus, a series of prodrugs was made by carbodiimide mediated coupling of protected amino acids with the phenol of 2.10. Deprotection then yielded the prodrug compounds 2.41 to 2.43. Both positively and negatively charged prodrugs were synthesized as shown in Scheme 2.8. 36 1) HO(N0c R NH2-HCI R DIPC, DMAP, CH2I 2) TFAICHCI 3) HCI(aq) (salt swap) 2.41 R = H 2.42 R = NH2-HCl NHB0c 0 1) OH j OBn DIPC, DMAP, CH2I 2) TEA, CH2I 3) DMAP 4) Pd/C, H2, MeOH Scheme 2.8. Synthesis of amino acid-based prodrugs of 2.10. In addition to amino acid esters, poly(ethylene)glycol (PEG) chains can be used as a promoiety. PEG chains are non-toxic, non-immunogenic, non-antigenic, highly soluble in water, and FDA approved.77 In addition to conferring water solubility to the conjugated drug, PEGylation can also alter the pharmacokinetics of the drug, resulting in a longer in vivo residence time by slowing drug clearance from the body.78 For antitumor drugs, PEGylation can passively target solid tumors by virtue of the enhanced permeability and retention (EPR) effect.79 A shortcoming of straight-chain PEG conjugation to small molecule therapeutics is the low loading observed due to the 2.10 0 2.10 2.43 37 polymer having only one or two terminal hydroxyl or thiol groups to which a drug molecule can be attached. A PEGylated version of 2.10 was synthesized as outlined in Scheme 2.9. Reaction of 2.10 with bromoacetyibromide yielded bromide 2.44. Reaction of this compound with PEG thiol (5000 amu) yielded PEGylated prodrug 2.45. Characterization of this compound by ‘H NMR was precluded by the extremely small size of the signals due to 2.10 relative to the PEG signals; however, it is freely soluble in water. The stabilities of the described prodrugs are discussed in Section 2.5.2 below. 0 Br r 0 CH2I DMF, IPr2NEt 2.45 Scheme 2.9. Synthesis of PEGylated 2.10 2.10 2.44 MW = 5000 38 2.5: Biological Results The biological activity and site of SHIP 1 binding of pelorol analogues 2.4 and 2.10 has been investigated by our collaborator, Dr. Alice Mui.41 The compounds display a high degree of target specificity for SHIP1 and in a screen of 100 other kinases and phosphatases displayed minimal off-target effects. OH 2.4 2.10 To evaluate the specificity of 2.4 for SHIP1, the ability of 2.4 to activate the most closely related phosphoinositol phosphatase, SHIP2 was examined. As shown in Figure 2.13, 2.4 preferentially activates SHIP1 over SHIP2. — 4 8 10 [2.4] pM Figure 2.13: 2.4 preferentially activates SHIP 1 over SHIP2 0 12 39 As a direct comparison of activity between 2.4 and 2.10, we turned to an enzymatic assay. In this assay, SHIP 1 was incubated with compounds (at 2 jiM) or solvent for 15 mm before the addition of inositol-1,3,4,5-tetrakisphosphate. Malachite green is then added, and the amount of inorganic phosphate released by the action of SHIP1 is measured at 650 nm. As shown in Figure 2.14, both 2.4 and 2.10 activate SHIP 1 by approximately the same amount. 10 ,..E 6 4- .•_ : 4- ‘) . o. 4 ( .C o. 2 0 -C 1 0 EtOH 2.4 2.10 Figure 2.14: 2.4 and 2.10 (2 jiM) activate SHIP1 to approximately the same extent Target specificity as well as biological activity was then evaluated for compound 2.10 by comparing its effects on P13K regulated processes in wild type (SHIP 1 +1+) versus homozygous knockout (SHIP 1 ‘) macrophages. Lipopolysaccharide (LPS) induced macrophage activation involves P13K dependent pathways which are negatively regulated by SHIP 1. The stimulation of macrophages by LPS is associated with a PIP3 dependent release of proinflammatory mediators such as tumor necrosis factor o. (TNFc). Both SHIP1 and SHIP1 ‘ bone-marrow derived macrophages were treated for 30 mm 40 with 2.10 prior to stimulation with 10 ng/mL LPS for 2 hours (Figure 2.15). Compound 2.10 was able to able to suppress TNFx production in SHIP 1 +1+ cells in a dose-dependent manner, while in SHIP 1 ‘ cells were unresponsive to 2.10. The P13K inhibitor LY294002 (2.1) inhibited both SHIP 1 +1+ and SHIP 1 “ macrophages to the same extent (data not shown). E E x E 0 z I. [2.10] iM Figure 2.15: TNFc inhibition by 2.10 in wild type and SHIP1 knockout macrophages In vivo activity for 2.10 was demonstrated by establishing their protective effects in a mouse model of endotoxic shock. In this experiment, mice were orally dosed with 2.10 or the potent anti-inflammatory drug dexamethasone 30 minutes prior to an intraperitoneal injection of bacterial LPS. A measurement of serum TNFo levels was performed 2 hours later. Compound 2.10 reduced the level of serum TNFo to the same level as dexamethasone as shown in Figure 2.16. —••— SHIPT] —S—SHIPj 0 1 2 3 4 5 6 7 41 .:/ - 2000 T - - T -l ______ E 1500 UT 0. 1000 I r_i 4ri IO-Q.!Ofl R);Th1 2.13 (0.5mg/kg) 2.13 (2.0mg/kg) Dexamethasone Figure 2.16: Reduction of TNFct levels in LPS stimulated mice by 2.10 The site of SHIP1 binding by 2.10 was also explored. The discovery of small- molecule activators of SHIP 1 led to the hypothesis that SHIP 1 may be allosterically regulated.56Initial enzyme kinetic studies suggest that SHIP 1 may in fact be allosterically activated by its end product, Pdtlns(3,4)P2(Figure 2.1). Addition of the SHIP1 product Pdtlns(3 ,4)P2 (20 jiM) to the enzymatic reaction activated wild-type SHIP 1 enzyme, as did the addition of 2.10 (3 jiM) (Figure 2.17). The SHIP 1 protein contains a C2 domain located at the carboxy-terminal end of its phosphatase domain that was hypothesized to be its allosteric binding site. To test this hypothesis, SHIP 1 lacking a C2 domain (AC2 SHIP 1) was generated. As shown in Figure 2.17, although AC2 SHIP 1 was as active as wild type SHIP 1, its activity could not be enhanced by the addition of either Pdtlns(3,4)P2(20 jaM) or by 2.10 (3 jaM). Vehicle 42 Figure 2.17: Both Pdtlns(3,4)P2(20 pM) and 2.10 (3 kiM) enhance activity of wild-type SHIP 1 but not zC2 SHIP 1 Compound 2.10 was was verified to directly bind the C2 domain using a scintillation proximity assays (SPA) in which SPA beads were coated with either the SHIP1 C2 domain or control protein (BSA) prior to incubation with[3H]-2.1O. As shown in Figure 2.18, 2.10 does indeed bind to the SHIP1 C2 domain. 100 £ o 80 £ 160 40 U WIdtpe C2 deletion 43 0. 0 0 C cJ Figure 2.18:[3H]-2.1O binds to SHIP1 C2 domain The above results provide a model for cellular SHIP 1 activation. Upon recruitment to the plasma membrane, SHIP 1 hydrolyses a small amount of PIP3 at a low rate. This generates some Pdtlns(3,4)P2,which then binds to the C2 domain, localizing SHIP 1 to the plasma membrane in addition to inducing a conformation change which enhances its catalytic activity. This positive feedback mechanism is also observed in the case of PTEN which allosterically binds its end product Pdtlns(4,5)P2,8°localizing it to the plasma membrane and enhancing its catalytic activity.8’ Regardless of the biological mechanism of SHIP 1 regulation. small molecule SHIP1 activators such as 2.10 can be exploited for therapeutic purposes. An analysis of P13K regulated cell signaling pathways shows that SHIP 1 activating compounds may be useful agents for the treatment of various inflammatory disorders. Also, these BSA C2 Domain 44 compounds may play a role in the treatment of hematological malignancies. While compounds that selectively inhibit P13K are actively being sought, functional inhibition of the P13K pathway can be achieved by the activation of SHIP1. Both drug actions, inhibition of P13K or activation of SHIP 1, would act to reduce the levels of PIP3. This reduction of cellular PIP3 levels then reduces the recruitment and activation of protein kinase B and, therefore, may be clinically relevant in the treatment of certain types of cancer. Additionally, the hematopoetic-restricted expression of SHIP 1 should limit the effects of a selective SHIP 1 activator to target cells. With this in mind, several of the compounds described above were evaluated in an anti-inflammatory assay and in a multiple myeloma tumor cell-killing assay. 45 2.5.1: Structure-Activity Relationships Figure 2.19: SHIP 1 activating compounds evaluated for biological activity In section 2.3, the synthesis of 12 novel monophenolic analogues of pelorol were described. These analogues are summarized in Figure 2.19. A preliminary SAR analysis of these compounds was performed using two assays. An anti-inflammatory assay similar to that described in section 2.5, Figure 2.15 was carried out on the following compounds; 2.10, 2.20, 2.22, 2.27, 2.28, 2.29, 2.39 and 2.40. RAW 264.7 Mouse leukaemic monocyte cell line macrophages were treated with the SHIP 1 activating compounds at the indicated concentrations for 3 h prior to the addition of 10 ng/mL LPS. Supernatants were collected and assayed for TNFot concentration by ELISA. The results are summarized in Figure 2.20. 2.10 2.20 2.39 46 TNFa production from RAW macrophages after 3 h Compound 2.10 2.20 2.22 2.27 2.28 2.29 2.39 2.40 Approx.EC50 12 11 4.5 2.5 10 4.8 4.8 11 (j.LgImL) Figure 2.20: Dose response curves and approximate EC50 values for selected compounds in TNFCL inhibition assay. Several conclusions can be drawn from the SAR analysis derived from the TNFc inhibition assay data. Comparison of 2.10 and 2.22 would seem to indicate that alkyl substituents are not preferred at the C-20 position, however, compound 2.29 (C-20 -J 0) .2 250 C 0 0 •0 0 . 200 z I- 0.1 1 10 100 Compound concentration (tgImL) 47 methoxy) is equally as potent as 2.22. More data would be needed to draw any accurate conclusions regarding the effect of substituents in the C-20 position. From comparison of analogues 2.10, 2.20, 2.28 and 2.29, it would appear that the preferred position for the phenol group is in the C-18 position, and not the C-20 position, and the activity of the resorcinol compound 2.27 indicates that two phenols in the C- 18 and C-20 gives greater potency. The series of 2.10, 2.39 and 2.40 illustrate that epimerizing the C-8 methyl (2.40) has no effect on potency, while the C-9 epimer (2.39) is approximately 2 fold more potent than its parent compound, 2.10. A cytotoxicity assay on multiple myeloma cells was also carried out on the compounds shown in Figure 2.19. Multiple myeloma (MM) cells were cultured for 40 h along with compounds at the indicated concentrations. At this point, labeling with tritiated thymidine for 8 h gave a measure of DNA synthesis to assay for cell proliferation. The results are summarized in Figure 2.21. 48 MM cytoxicity assay 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 C -1000 Compound concentration (iiglmL) Compound 2.10 2.20 2.22 2.24 2.25 2.26 2.27 2.28 2.29 2.31 2.39 2.40 Approximate 2.8 6.5 2.8 3.1 3.1 7.0 2.8 2.1 3.8 0.2 3.8 7.0 EC50 (j.tg/mL) Figure 2.21: Dose response curves and approximate EC50 values for selected compounds in a multiple myeloma cell cytotoxicity assay. The data for the MM cytotoxicity assay is much less informative than that generated by the TNFc assay. Foremost it should be noted is that target selectivity is not E 0.(3 V E I. \ \.N 10 49 addressed in this assay. From a previous publication4’we have demonstrated that compounds 2.4 and 2.10 act selectively on the cell signaling pathways via activation of SHIP1. While the selectivity of the other compounds from Figure 2.19 have not been evaluated, it seems to be a reasonable assumption that they will have some measure of selectivity for SHIP 1. However, it should not be forgotten that any number of factors could be responsible for the observed response of the MM cells to the dosed compounds. For example, compound 2.31 shows an EC50 of 0.2 ig/mL. Compound 2.31 is unique in that is has a 10 amine appended to the C-18 phenol. This unique structural feature, coupled with the massive increase in potency almost certainly indicates a general cytotoxic compound (also see compound 2.41 in Figure 2.22). The only trend that is consistent with the TNFo assay is the finding that 2.40 is approximately 2-fold less active than 2.39. The compounds in the biphenyl class, 2.25 and 2.26, appear to be tolerated, but further studies need to be carried out to complete an SAR study to select a compound for further development. In addition to the cell-based assays, the SHIP1 enzymatic assay as described for Figure 2.14 was carried out on compounds 2.10, 2.20, 2.22, 2.25, 2.26, 2.27, 2.28, 2.29, 2.31, 2.39 and 2.40. The amount of inorganic phosphate released by SHJP1 from inositol-1,3,4,5-tetrakisphosphate in the presence of each compound is plotted as a percent change compared to control. The results are sunmiarized in Figure 2.22. 50 1250% 0 - 4’ 1 -c 100.0% a) •13.2 I 75.0% 19.8 2 50.0% 1 - :29.6 P 2 -25.0% - • ioo -50.0% 2.10 2.25 2.26 2.27 2.28 2.29 2.20 2.31 2.22 2.39 2.40 Figure 2.22. Enzymatic SHIP 1 modulation assay. Concentrations are given in ig/mL From this data, it is apparent that compounds with basic amino substituents appended to the phenyl ring of 2.10 act as SHIP 1 inhibitors as shown by 2.26 and 2.31. This provides convincing evidence that the MM cytotoxicity observed for 2.31 in Figure 2.21 is not due to SHIP 1 activation. The data for 2.25 shows that a biaryl compound is well tolerated, and the data for 2.39 and 2.40 confirms the finding from the TNFo assay (Figure 2.20) that a C-9 epimer of the pelorol system confers an increase in potency. Also confirmed is the increased potency of 2.27 compared to 2.10, making resorcinol 2.27 an intriguing lead compound. 2.5.2: Prodrugs of 2.10 While there is significant work to be done on the solubilities of 2.10 and its prodrugs, some preliminary studies have been completed. Compound 2.10 is sparingly soluble in water, while its lysine ester 2.42 is freely soluble as its his-hydrochloride salt. The hydrochloride salt of glycine ester 2.41 does not appear to be freely soluble, but 51 specific measurements have not yet been taken. Bis-carboxylic acid derivative 2.43 is also freely soluble in water as its disodium salt. PEG prodrug 2.45 was also found to be freely water-soluble. The key aspect of a prodrug is that it cleaves in a cellular environment, releasing the drug to exert its effect. Ideally, the cleavage from the prodrug to the drug and promoiety will not occur until the prodrug is in the cellular environment. To test if prodrugs of 2.10 would cleave only under cellular conditions, we used the enzymatic SHTP1 assay described above (Figure 2.23).82 OD 650 EtCH 2.10 2.41 Compound (40 pg/mE) Figure 2.23: Enzymatic SHIP1 activation assay with 2.10 and 2.41 As shown in Figure 2.23, dosing of 2.10 results in a significant increase in SHIP activity, but the glycine prodrug 2.41 does not activate SHIP above baseline. This result 52 indicates that the prodrug 2.41 is not cleaving to release 2.10 under the conditions studied, nor does 241 activate SHIP1 by itself (also see 2.31 in Figure 2.21 and Figure 2.22). Precipitation of 2.10 is observed over a period of several days when 2.41 is dissolved in pH 7.4 buffer. The decomposition of lysine prodrug 2.42 is much more rapid. When a solution of 2.42-2HC1 in water is diluted with pH 7.4 phosphate buffer, an immediate precipitate of 2.10 is observed. These findings can be rationalized as shown in Figures 2.24 and 2.25. When 2.41-HC1 is freebased in a dilute solution, the phenol ester can be cleaved by the now-nucleophilic 10 amine. The first ester cleavage is most likely intramolecular to form 2.46, and in the dilute solutions (—1 mg/mL) we examined, this cleavage will be slow — on the order of days (Figure 2.24). The second possible cleavage is most likely intramolecular with 2.46 forming another equivalent of 2.10 and diketopiperazine, 2.47. NH H NO I Y IntramolecularJ +2.1O ON H 2.47 Figure 2.24: Possible decomposition of prodrug 2.41 pH 7.4 buffer H 2.41 Intermolecular +2.10 2.46 53 Prodrug 2.42 exhibits rapid decomposition as the lysine moiety is capable of purely intramolecular ester cleavage to form 2.10 and caprolactam 2.48 (Figure 2.25). As would be expected, both 2.41 and 2.42 are indefinitely stable in acidic solution. Unfortunately, neither diketopiperazine 2.47 nor caprolactam 2.48 were isolated, so for the time being, these decomposition pathways remain speculative. 0 HCI pH 7.4 buffer NH2 HCI Intramolecular 0 H2N,NH +2.10 2.48 Figure 2.25: Possible intramolecular decomposition of prodrug 2.42 When compound 2.41 was assayed in a cellular TNFc inhibtion assay, it was found to inhibit the release of TNFa to the same extent as 2.10. The timeframe of this experiment (several hours) and the high dilution of 2.41 in the cellular medium most likely rules out cleavage of the ester via the intermolecular route shown in Figure 2.24. Whether or not the cellular cleavage of 2.41 to release 2.10 is enzymatic or not has not 0 H 2.42 NH2 2.42 54 been ascertained. These findings underlie a hazard when designing an ester prodrug of a phenol. Because a phenoxide makes a good leaving group, the ester may be cleaved under non-cellular conditions if caution is not taken with their handling and design. However, carefully designed esters of 2.10 may be a viable prodrug strategy for the in vivo dosing of the hydrophobic SHIP1 activators. More studies on the solubility, composition and dosing of SHIP 1 activator prodrugs is pending. 2.6: Conclusion This chapter describes a series of SHIP 1 activating compounds based on pelorol that are missing the catechol moiety of both pelorol and 2.4. Based on 2.4, analogue 2.10 was synthesized and evaluated in a series of biological tests that demonstrated the selectivity and efficacy of 2.10 as a P13K pathway modulator via the allosteric activation of SHIP1. The activation of SHIP1 represents a novel and selective method to modulate the P13K cell-signaling pathway and, therefore, 2.10 may be useful as a therapeutic agent in hematological malignancies and inflammatory pathway dysfunctions. A library (Figure 2.19) was then generated to examine structure activity relationships of SHIP 1 activating compounds. Prodrug strategies for solubilizing these compounds in water were also examined. The biological data from these studies led to several conclusions: 1) amino groups appended to the phenyl ring of 2.10 act as SHIP1 inhibitors, 2) the resorcinol compound 2.27 is significantly more potent than any other analogue, 3) C-9 epimerization of the pelorol system gives greater SHIP1 activating potency (2.10 vs 2.39), 4) at least two charges, negative or positive, are required for water solubility and 5) the compounds presented here in are orally available and efficacious in a murine 55 endotoxemia model. These studies provide a validation of SHIP 1 activation as a drug target, and allow the evolution of small molecule SHIP 1 activators based on pelorol to be observed (Figure 2.26). From its origins with the discovery of the sponge-based natural product pelorol to the planned syntheses of compounds 2.49 and 2.50, the research described within this chapter is a good example of marine natural product based drug discovery. HOCH Figure 2.26: Evolution of SHIP 1 activating drug candidates from natural product inspiration (pelorol). OH pelorol 2.4 H 2.10 B NH ONH2 2.50 H 2.49 2.27 56 2.7: Experimental General Methods. All non-aqueous reactions were carried out in flame-dried glassware and under an Ar atmosphere unless otherwise noted. Air and moisture sensitive liquid reagents were manipulated via a dry syringe. Anhydrous tetrahydrofuran (THF) was obtained from distillation over sodium. All other solvents and reagents were used as obtained from commercial sources without further purification. All amino acids used are the natural “L” configuration. 1H and ‘3C NMR spectra were obtained on Bruker Avance 400 direct or Bruker Avance 600 cryoprobe spectrometers at room temperature unless otherwise noted. Flash chromatography was performed using Silicycle Ultra Pure silica gel (230-400 mesh) and using gradients of the indicated solvents. All biological assays were carried out by researchers in the laboratory of Dr. Alice Mui at the BC Cancer Research Agency. Preparation of diol 2.14a Procedure A: Bromomethoxytoluene 2.13a (3.64 g, 18.3 mmol) was dissolved in 35 mL dry THF under Ar. This solution was cooled to —78 °C, and t-BuLi (21.5 mL, 36.6 mmol) was added dropwise via syringe. The solution was stirred for 10 mm at —78 °C, then warmed to rt H 57 for 20 mm. The solution was re-cooled to —78 °C, and a solution of aldehyde 2.8 (1.45 g, 6.09 mmol) in 6 mL dry THF was added via syringe. The solution was stirred at —78 °C for 2 h, after which time the reaction was halted with the addition of 1 M HC1. EtOAc (100 mL) was added, and the organic phase was washed with 1 M HC1, followed by saturated NaHCO3. The organic phase was dried over MgSO4,filtered and concentrated. The crude reaction mixture was purified by column chromatography (hexanes/EtOAc) to yield diol 2.14a (1.94 g, 5.39 mmol, 89%). 111 NMR (400 MHz, CDCI3) 80.34 (td, J = 13.3, 3.6 Hz, 1H), 0.77 (s, 3H), 0.82 (s, 3H), 0.90 (m, 1H), 0.97 (td, J = 13.5, 3.6 Hz, 1H), 1.02 (s, 3H), 1.13 (m, 1H). 1.16 (m, 1H), 1.23 (m, ill) 1.33 (m, 1H), 1.40 (m, 1H), 1.54 (s, 3H), 1.56 (m, 1H), 1.63 (m, 1H), 1.84 (dt, J= 12.2, 3.3 Hz, 1H), 2.12 (d, 8.1Hz, 1H), 2.33 (s, 3H), 3.79 (s, 3H), 4.79 (d, 8.1Hz, 1H), 6.61 (s, 1H), 6.78 (s, 1H), 6.85 (s, 1H); ‘3c NMR (100 MHz, CDC13)815.9, 18.3, 19.8, 21.50, 21.53, 26.1, 33.2, 33.5, 38.6, 40.8, 41.3, 44.0, 55.1, 55.8, 62.84, 62.85, 76.0, 110.5, 113.6, 120.7, 139.8, 149.0, 159.7; ESIMS [M+Na] calcd forC23H36ONa383.2562, found 383.2563. Preparation of xanthate 2.17a Procedure B: Diol 2.14a (1.94 g, 5.39 mmol) was dissolved in 20 mL dry THF under an Ar. To this solution was added NaH (237 mg, 60 % in oil, 5.93 nmiol). The reaction mixture was H 58 then heated to 50 °C until the solution was clear orange. The reaction mixture was cooled to 0 °C, and CS2 (1.0 mL, 17 mmol) was added. The solution was stirred for 20 mm at 0 °C, then warmed to rt for an additional 20 mm, after which time Mel (1.0 mL, 17 mmol) was added. The reaction mixture was stirred at rt for 1 h, then concentrated to dryness. The crude mixture was dissolved in EtOAc, and washed with 3 x H20. The organic solution was dried over MgSO4,filtered and concentrated to yield a mixture of xanthate 2.17a and fragmentation product, ketone 2.16 (approx 4:1). A portion of this mixture was purified by flash chromatography (hexanes/EtOAc) for characterization, however, this product mixture could be used in the next step without further purification. ‘H NMR (400 MHz, CDC13) 50.56 (td, J = 12.9, 3.5Hz, 1H), 0.77 (s, 311), 0.80 (s, 3H), 0.87 (dd, J = 12.2, 2.4 Hz, 111), 0.99 (dt, J = 13.6, 3.8 Hz, lH), 1.02 (s, 3H), 1.28 (m, 1H), 1.31 (m, 1H), 1.34 (m, 111), 1.45 (m, 111), 1.50 (s, 3H), 1.55 (m, 1H), 1.65 (m, 1H), 1.75 (m, 111), 1.78 (m, 1H), 1.81 (m, 111), 2.18 (d, J = 5.2 Hz 1H), 2.28 (s, 3H), 2.38 (s, 3H), 3.75 (s, 3H), 5.18 (d, J = 5.2Hz, 1H), 6.5 (s, 1H), 6.7 (s, 111), 6.8 (s, 111); 13C NMR (100 MHz, CDC13) 8 13.0, 15.9, 18.3, 20.2, 21.3, 21.6, 26.3, 33.26, 33.30, 40.2, 41.0, 41.3, 46.0, 46.8, 55.0, 55.9, 65.1, 74.2, 110.9, 112.3, 120.9, 139.5, 149.9, 159.4, 189.7; ESIMS [M+Na] calcd forC25H38OSNa473.2160, found 473.2159. 59 Preparation of akohol 2.15a Procedure C: The mixture of xanthate 2.17a and ketone 2.16 from the previous procedure was dissolved in 50 mL toluene under Ar. Bu3SnH (2.90 mL, 10.8 mmol) was added, and the solution was heated. Once at reflux, a catalytic amount of AIBN (-‘-50 mg) was added through the top of the condenser. The solution was refluxed for 1 h, then an additional amount of AIBN was added (—50 mg). The solution was refluxed for another 45 mm, after which time TLC analysis (20% EtOAc:hexanes) indicated the reaction to be complete. The reaction mixture was cooled, then concentrated to dryness. Flash chromatography (hexanes/EtOAc) of the crude product yielded alcohol 2.15a (1.12 g, 3.23 mmol, 60%, 2 steps) as a white foam. ‘H NMR (400 MHz, CDC13) öO.78 (s, 3H), 0.85 (s, 3H), 0.87 (s, 3H), 0.90 (m, 1H), 0.93 (m, 1H), 0.96 (m, 1H), 1.09 (td, J = 13.3, 3.9 Hz, lI-I), 1.25 (s, 3H), 1.31 (m, 1H), 1.35 (m, 1H), 1.39 (m, 1H), 1.43 (m, 1H), 1.54 (m, 1H), 1.64 (m, 1H), 1.70 (m, 1H), 1.84 (dt, J = 12.4, 3.1 Hz, 1H), 2.27 (s, 3H), 2.60 (dd, J = 14.7, 4.5Hz, 1H), 2.70 (dd, J = 14.7, 5.9 Hz, 1H), 3.75 (s, 3H), 6.49 (s, 1H), 6.63 (s, 1H), 6.68 (s, 1H); 13C NMR (100 MHz, CDC13) S 15.4, 18.4, 20.2, 21.4, 21.5, 24.5, 60 31.2, 33.2, 33.3, 39.1, 40.3, 41.7, 44.0, 55.0, 56.0, 63.0, 74.1, 111.3, 111.9, 122.1, 139.2, 145.9, 159.5; ESIMS [M+Nar calcd forC23H36ONa367.2613, found 367.2615. Preparation of tetracycle 2.18a Procedure D: Alcohol 2.15a (1.12 g, 3.23 mmol) was dissolved in 10 mL CH21 and cooled to 0 °C. To this solution was added neat SnC14 (1 mL). The orange solution was then stirred for 1 h at 0 °C, followed by quenching with MeOH. The reaction mixture was extracted into EtOAc, and washed with 2 x satd NaHCO3. The organic phase was dried over MgSO4, filtered and concentrated to yield tetracycle 2.18a (1.05 g, 3.20 mmol, 99%). A portion of this mixture was purified by flash chromatography (hexanes/EtOAc) for characterization; however, this product mixture could be used in the next step without further purification. ‘H NMR (400 MHz, CDCI3) 8 0.86 (s, 6H), 0.98 (m, 1H), 1.02 (s, 3H), 1.06 (s, 3H), 1.17 (td, J = 13.5, 4.2Hz, 1H), 1.24 (m, 111), 1.40 (m, 2H), 1.54 (m, 2H), 1.71 (m, 4H), 2.27 (s, 3H), 2.34 (m, 1H), 2.49 (dd, J = 14.5, 6.2Hz, 1H), 2.60 (m, 1H), 3.74 (s, 3H), 6.41 (s, 1H), 6.62 (s, 1H); ‘3C NMR (100 MHz, CDC13) 8 15.7, 17.9, 18.6, 19.1, 19.9, 20.7, 28.6, 32.6, 32.9, 36.5, 37.9, 38.5, 39.7, 42.1, 54.8, 56.7, 64.2, 107.9, 113.4, 117.9, 132.5, 143.8, 157.3; ESIMS [M+H] calcd for C23H350327.2688, found 327.2685. 61 Preparation of phenol 2.10 Procedure E: Tetracycle 2.18a (1.05 g, 3.20 mmol) was dissolved in 15 mL CH21. To this solution was added a solution of BBr3 (3.2 mL, 1.0 M in CH21,3.2 mmol). The solution was stirred at ii for 2 h, then concentrated to dryness. The brown residue was dissolved in EtOAc, and washed with H20 until the pH of the aqueous layer was neutral. The crude product was purified by flash chromatography (hexanes/EtOAc) to yield 2.10 (931 mg, 2.98 mmol, 93%) as a white solid. ‘H NMR (400 MHz, CDCI3)80.88 (s, 6H), 0.97 (m, 111), 1.00 (m, 1H), 1.04 (s, 3H), 1.07 (s, 3H), 1.18 (td, J = 13.2, 4.2 Hz, 1H), 1.42 (m, 1H), 1.43 (m, 1H), 1.53 (m, 1H), 1.58 (m, 1H), 1.71 (m, 111), 1.73 (m, 1H), 1.74 (m, 1H), 1.75 (m, 1H), 2.26 (s, 3H), 2.35 (dt, J = 11.7, 3.0 Hz, 1H), 2.48 (dd, J = 14.35, 6.44 Hz, 1H), 2.59 (m, 1H), 6.36 (d, J = 1.9 Hz, 1H), 6.55 (d, J = 1.9 Hz, 1H); 13C NMR (100 MHz, CDC13)816.1, 18.3, 18.8, 19.6, 20.4, 21.1, 29.0, 33.1, 33.4, 37.0, 39.0, 40.1, 42.6, 47.1, 57.1, 64.5, 109.9, 115.1, 133.1, 144.2, 144.7, 153.5; ESIMS [M+Hf’ calcd for C22H330313.2531, found 313.2526. 62 Isolation of phenol 2.20 OH Regioisomer 2.20 was isolated by flash chromatography (hexanes/EtOAc) as a byproduct of a large scale ring-closure/deprotection of alcohol 2.15a. ‘H NMR (600 MHz, CDC13) 80.86 (s, 6H), 0.98 (m, 1H), 1.00 (m, 1H), 1.02 (s, 3H), 1.12 (s, 3H), 1.18 (td, J = 13.3, 4.1 Hz, 1H), 1.41 (m, 211), 1.51-1.83 (m, 6H), 2.24 (m, 311), 2.34 (dt, J = 11.9, 3.0 Hz, 1H), 2.50 (dd, J = 14.4, 6.1 Hz, 111), 2.60 (m, 1H), 4.48 (s, 1H), 6.37 (s, 1H), 6.63 (s, 1H); ‘3C NMR (150 MHz, CDC13) 316.2, 18.3, 19.5, 20.1, 21.1, 28.9, 30.9, 33.1, 33.4, 36.9, 38.5, 40.1, 42.5, 46.0, 57.3, 64.5, 114.5, 118.6, 135.7, 137.1, 145.0, 150.6; ESIMS [M+H1ca1cd forC2211330313.2531, found 313.2535. Preparation of diol 2.14b Following procedure A, the following amounts were used: 3-bromoanisole (5.0 mL, 40 mmol) in 60 mL dry THF, t-BuLi (46.4 mL, 78.9 mmol), aldehyde 2.8 (3.10 g, 13.2 H 63 mmol) in 20 mL dry THF. Yields 2.14b (3.68 g, 10.6 mmol, 80%) as a white foam. ‘H NMR (400 MHz, CDC13) 50.30 (td, J = 13.3, 3.8 Hz, 1H), 0.76 (s, 3H), 0.81 (s, 3H), 0.87 (m, 1H), 0.94 (td, J= 13.4, 3.5 Hz, 1H), 1.01 (s, 3H), 1.11 (m, 2H), 1.21 (m, 1H), 1.31 (m, 1H), 1.37 (m, 1H), 1.52 (s, 3H), 1.58 (m, 1H), 1.62 (m, 1H), 1.82 (m, 111), 2.10 (d, J = 7.8 Hz, 1H), 3.37 (s, br, 211), 3.79 (s, 3H), 4.81 (d, J 8.0 Hz, 1H), 6.77 (dd, J = 8.2, 2.2 Hz, 1H), 6.99 (m, 2H), 7.22 (t, J = 7.8 Hz, 1H); ‘3C NMR (100 MHz, CDC13) S 15.9, 18.3, 19.8, 21.5, 26.1, 33.1, 33.5, 38.6, 40.8, 42.3, 44.1, 55.2, 55.8, 63.1, 74.3, 75.8, 112.7, 113.4, 120.1, 129.6, 149.3, 159.7; ESIMS [M+Na] calcd for C22H43O3Na 369.2406, found 369.2402. Preparation of xanthate 2.17b Following procedure B, the following amounts were used: diol 2.14b (2.92 g, 8.44 mmol) in 30 mL dry THF. NaH (371 mg, 60% in oil, 9.28 mmol), CS2 (5mL, 84.4 mmol), Mel (5.25 mL, 84.4 mmol). The crude product dissolved in 100 mL hexanes, and cooled to 0 °C for 12 h. The resulting crystals were filtered, and washed with cold hexanes to yield 1.82 g 2.17b. Another 264 mg was recovered following flash chromatography (hexanes/EtOAc) to yield 2.17b (2.08 g, 4.76 mmol, 56% total) as a white solid. ‘H NMR (400 MHz, CDC13)50.54 (td, J = 13.0, 3.3 Hz, 1H), 0.77 (s, 3H), 0.80 (s, 3H), 0.86 (m, 1H), 0.98 (td, J = 13.3, 4.0 Hz, 111), 1.02 (s, 3H), 1.26 (m, 1H), 64 c9 ‘6Th11 ‘E011 ‘0IL ‘E9 ‘09c ‘occ ‘Ii7fr ‘9J1‘EOV‘6E‘liE‘ci‘viz ‘101 ‘t’8T ‘tci9(13UD ‘ZHN 001) NNN DEl (HT ‘ZN 6L I ‘i) çrL‘(Hl ‘iu) c89 ‘(HI ‘ZH l•l ‘F8 1‘PP) L99 ‘(HE ‘s) LLE ‘(HI ‘ZR vc‘9171I‘pp)9LZ‘(HI ‘zH 8Th ‘9Th1 = r‘pp)l9‘(RI ‘ZH £E ‘IlI=I ‘IP) cwi ‘(HI ‘m) 891 ‘(Hl ‘ui) I9i ‘(rn ‘w) ici‘(HI ‘m) ‘(Hl ‘Ui) cci‘(Hl ‘LU) 611 ‘(HE ‘s) frI‘(HI ‘Zil ETh ‘cEI =r ‘ps) 801 ‘(HI ‘m) E60 ‘(HE ‘s) 8W0 ‘(HE ‘s) t’WO ‘(HE ‘s) 8L09 (EIDUD ‘ZHN 00t)MV1N H1 UJI?OJ !L1M 1E S1 (%L6 ‘10mw c017 ‘17E1)quzPP!Awoc)NHIVjoiunouw uonJpp‘(guioc)NHIV‘(jowwE8“1w1Zl)HUSEnH‘unJo1‘1woc‘(10mw LIt’‘i81)qLUz 11?qiunx:psnaIMsiunouIuTMoJJojqinpo.iduiMo11oJ qrzjoqoujoUO!WJUdUd Looi6ct’ punoj ‘t00i6cV JOj PT’° +[N+I’1 swIsJ‘t’6cT‘zoc‘V611‘101I‘6E11‘ElIl‘1ThL ‘1c9‘6cc‘vcc‘L917‘i9I‘El17‘0lt’‘10t’‘L1EE‘czcc‘V91‘Eli‘101‘E81‘6c1 ‘6119 (EIDUD ‘zHN 001)1NN(Hi ‘ZH 18[‘)91L‘(Hi ‘m) 669 ‘(HI ‘zil 81 ‘c8 =r‘pp)99‘(HI ‘zH ic=r‘p)zzc‘(HE ‘s) LLE ‘(HE ‘s) sci‘(HI ‘zil rc=r‘p) 611‘(Hi ‘m) 8L1 ‘(Hi ‘m) 99i ‘(HI ‘w) cci‘(HE ‘s) ici‘(HI ‘w) Lt’l‘(HI ‘w) 121.3, 129.2, 146.3, 159.5; ESIMS [M+Na] calcd forC22H34ONa 353.2457, found 353.2458. Preparation of tetracycle 2.18b Following procedure D the following amounts were used: alcohol 2.15b (1.34 g, 4.05 mmol) in 50 mL CH21,1.5 mL neat SnC14.Yields 2.18b (1.14 g, 3.65 mmol, 90%) as a colorless oil. A portion of the compound was purified for characterization; otherwise this compound was used without further purification. ‘H NMR (400 MHz, CDC13) 60.85 (s, 6H), 0.97 (m, 1H), 1.01 (s, 311), 1.02 (s, 311), 1.17 (td, J = 13.5, 4.2 Hz, 1H), 1.24 (m, 1H), 1.39 (m, 211), 1.54 (m, 2H), 1.71 (m, 4H), 2.10, (m, 1H), 2.50 (dd, J= 14.6, 6.1 Hz, 1H), 2.63 (m, 1H), 3.75 (s, 3H), 6.65 (dd, J = 8.1, 2.2 Hz, 1H), 6.78 (d, J = 2.2 Hz, 111), 6.90 (d, J = 8.3 Hz, 1H); ‘3C NMR (100 MHz, CDC13) 6 16.0, 18.2, 19.4, 21.0, 23.2, 28.7, 33.1, 33.3, 36.9, 37.4, 40.1, 42.5, 45.2, 55.2, 57.4, 64.7, 111.1 (2C), 120.4, 144.1, 147.8, 157.8; ESIMS [M+Hf’ calcd forC22H330313.2531, found 313.2532. Preparation of phenol 2.22 66 Following procedure E, the following amounts were used: ether 2.18b (1.14 g, 3.65 mmol) in 40 mL CH21,BBr3 (7.3 mL, 1.0 M in CH21,7.3 mmol). Yields 2.22 (737 mg, 2.47 mmol, 68%) as a white solid. ‘H NMR (400 MHz, CDC13)30.85 (s, 6H), 0.93 (m, 1H), 0.97 (m, 1H), 1.00 (s, 3H), 1.01 (s, 3H), 1.16 (td, J= 13.3, 4.2 Hz, 1H), 1.38 (m, 1H), 1.41 (m, 111), 1.50 (m, 1H), 1.53 (m, 1H), 1.67 (m, 111), 1.70 (m, 1H), 1.72 (m, 1H), 2.09 (m, 1H), 2.48 (dd, J = 14.3, 6.1 Hz, 1H), 2.61 (m, 1H), 6.57 (dd, J = 8.1, 2.2 Hz, 1H), 6.69 (d, 2Hz), 6.84 (d, J = 7.9 Hz, 111); ‘3C NMR (100 MHz, CDC13) 616.0, 18.2, 19.4, 21.0, 23.2, 28.6, 33.1, 33.3, 36.8, 37.4, 40.1, 42.5, 45.2, 57.4, 64.6, 112.2, 112.5, 120.5, 144.4, 147.9, 153.4; ES]MS [M+Hf calcd forC21H30299.2375, found 299.2376. Preparation of bromide 2.23 Tetracycle 2.18b (50 mg, 0.16 mmol) was dissolved in 2 mL DMF. NBS (31.3 mg, 0.176 mmol) was added, and the solution was stirred at rt for 16 h. EtOAc was added, and the solution was washed with 2 x H20. The organic phase was dried over MgSO4, filtered and concentrated. The crude product was purified by flash chromatography (hexanes/EtOAc) to yield 2.23 (58.7 mg, 0.15 mmol, 93%) as a white solid. ‘H NMR (400 MHz, CDC13) (300 MHz) 80.87 (s, 6H), 0.96 (m, 111), 1.02 (s, 6H), 1.05 (m, 1H), 1.18 (td, J = 13.8, 4.0 Hz, 1H), 1.26 (m, 1H), 1.42 (m, 1H), 1.51 (m, 1H), 1.56 (m, 1H), 1.60 (m, 1H), 1.69 (m, 1H), 1.73 (m, 2H), 2.08 (m, 1H), 2.50 (dd, J= 14.5, 6.3 Hz, ill), Br 67 2.62 (m, 111), 3.85 (s, 311), 6.82 (s, 1H), 7.16 (s, 111); ‘3C NMR (100 MHz, CDCI3) ö 16.0, 18.2, 19.3, 21.0, 23.1, 28.7, 33.1, 33.3, 36.9, 37.2, 40.1, 42.5, 45.8, 56.3, 57.4, 64.7, 108.8, 109.7, 124.8, 143.2, 149.0, 153.7; ESIMS [M+Hr calcd for C22H379BrO 391.1637, found 391.1647. Preparation of phenol 2.24 OH Bromide 2.23 (46.8 mg, 0.119 mmol) was dissolved in 5 mL dry THF under Ar and cooled to —78 °C. t-BuLi (0.14 mL, 0.24 mmol) was added via syringe, and the solution was stirred cold for 10 mm. The solution was allowed to warm to ii and stirred for an additional 1 h. The solution was recooled to —78 °C, and Mel (0.5 mL) was added. The solution was stirred for 1 h, then allowed to warm to ii, and stirred for an additional 12 h. EtOAc was added, and the solution was washed with 2 x 1 M HC1 and 1 x satd NaHCO3. The organic phase was dried over MgSO4, filtered and concentrated. The residue was then resissolved in 3 mL CH2I and BBr3 (0.5 mL, 1.0 M in CH21,0.5 mmol) was added. The solution was stirred at rt for 1 h, then concentrated to dryness. The residue was dissolved in EtOAc and washed with 1120 until the pH of the aqueous layer was neutral. The organic phase was dried over MgSO4,filtered and concentrated. The crude product was purified by flash chromatography (hexanes/EtOAc) to yield 2.24 (18.2 mg, 0.58 mmol, 49%, 2 steps) as white solid. 111 NMR (400 MHz, CDC13) öO.86 (s, 6H), 0.95 (s, 1H), 1.00 (s, 311), 1.01 (s, 311), 1.16 (m, 1H), 1.25 (m, 1H), 1.40 (m, 2H), 1.51 68 69 ‘ITt ‘c6T ‘E81 ‘Z9I9 (IDuD ‘ZHN 001) TIAIN tQ-jj 7 ‘m) LVL ‘(HI ‘w) 9EL ‘(HI ‘s) 89 ‘(HI ‘s) 989 ‘(HI ‘tu) 89‘(HI ‘Z1119 ‘j7j7J =1‘pp) 9l‘(HI ‘w) II‘(He ‘w) 17L1 ‘(He ‘w) Lçl ‘(Hr ‘ui) VI‘(HI ‘w) 0Z1 ‘(He ‘s) LO1‘(He ‘s) coi‘(Hr ‘w) J0j ‘(H9 ‘s) 8W09 (E13U3 ‘ZHN oot’) ii”iu1(%c6 ‘loaltu oo‘ucit)szzpi’ oi (ov1]Jsulxq) i(qdiuoitwoqoqsij Aq pgund s pnpoid pn.i qjssuip 01J1UDUO3 uqi HON qlJM pquonb ‘unu oc ioj paunssarnlxiuiuonai pppn si(jomw01‘i,iOj“irnoI) EJgcj pm? 1IYHD UT PAI0S5Puq Jn1x!m uoiprai pruo qj ssui(ip oj pu pl?d 1TpJ i qnoqi p’ig ‘1 01 P100D uqi S1?M Jfl1XTW UO!1O1?aIqjq ioj pxnjpi S1?M uoilnlos q1 prn ‘pppi siM (w)£O3ZiNuno1]Wutpuiqwo(w)zI3 1HD-(Jddp){3p pun (I0UIUIp(j‘u9)pionoiuooqji(uqd‘Qouiuioo‘wi)rzpiwoiq rz jo uoptuduj çEIE P10J ‘IEcEIEOH1D joj puo +[H+N1 SWISEI cci‘8L171 ‘91171 ‘I‘L0I‘6111‘W179‘17Lc‘Ect7‘ci ‘F017 ‘VLE ‘69E‘VEE ‘FEE‘E8‘E‘01‘V61‘81‘F91‘Lc1(I3UD‘HN001) aNN(rn ‘s) çL9 ‘(HI ‘s) 1799 ‘(HI ‘iq ‘s) cvt ‘(Hr ‘m) ‘(HI ‘ZH F9‘VfrI= r‘pp)cv‘(HE ‘s) 6I ‘(HI ‘w) 01 ‘(Hi ‘w) OLI ‘(Hr ‘w) 991 ‘(HI ‘w) Lç1‘(Hr ‘w) 23.4, 28.7, 33.2, 33.5, 37.0, 37.5, 40.2, 42.6, 45.5, 57.5, 64.8, 112.6, 121.4, 125.6, 127.5, 129.16, 129.18, 137.9, 144.1, 148.1, 150.3; ESIMS [M+Na] calcd for C27H34ONa 397.2507, found 397.2499. Preparation of biarylphenol 2.26 Bromide 2.23 (85 mg, 0.22 mmol), 4-pyridyl boronic acid (41 mg, 0.33 mmol) and PdCJ2(dppf)- CH21 (—2 mg) were combined in 2 mL toluene. 2 M Na2CO3(2 mL) was added, and the solution was refluxed for 3 h. The solution was cooled, filtered through a Celite pad, and concentrated to dryness. The crude product was purified by flash chromatography (hexanes/EtOAc) to yield the methyl ether of 2.26 (70 mg, 0.18 mmol, 83%). ‘H NMR (400 MHz, CDC13) 60.87 (s, 6H), 1.00 (m, 1H), 1.05 (s, 3H), 1.06 (s, 311), 1.19 (m, 2H), 1.42 (m, 3H), 1.57 (m, 3H), 1.75 (m, 211), 2.14 (m, 1H), 2.59 (dd, J= 14.7, 6.1Hz, 1H), 2.71 (m, 111), 3.78 (s, 311), 6.90 (s, 1H), 6.97 (s, 1H), 7.46 (s, br, 2H), 8.58 (s, br, 2H); ‘3C NMR (100 MHz, CDCI3) 816.1, 18.3, 19.4, 21.1, 23.3, 29.0, 33.1, 33.4, 36.9, 37.4, 40.2, 42.5, 45.6, 55.7, 57.5, 64.7, 108.9, 121.8, 124.3, 125.3, 145.1, 147.2, 148.1, 149.2, 154.8; ESIMS [M+Hf’ calcd for C27H36N04 390.2797, found 390.2783. The methyl ether of 2.26 (50 mg, 0.129 mmol) was dissolved in 1 mL CH21. BBr3 (0.3 ml, 1.0 M in CH21,0.3 mmol) was added, and the solution was stirred for 1 h. The reaction mixture was quenched with MeOH, and the solution was concentrated to dryness. The crude residue was redissolved in 5 mL CH21,upon which a crystalline 70 precipitate was formed. This was filtered, washed with CH2I arid dried to yield 2.26 (30 mg, 0.7 mmol, 54%) as the hydrobromide salt. ‘H NMR (400 MHz, CD3O ) S0.82 (s, 3H), 0.84 (s, 3H), 0.95 (m, 2H), 1.01 (s, 6H), 1.14 (m, 1H), 1.34 (m, 1H), 1.37 (m, 1H), 1.48 (m, 3H), 1.62 (m, 2H), 1.70 (m, 1H), 2.18 (m, 1H), 2.49 (dd, J = 14.9, 5.7 Hz, 1H), 2.63 (m, 111), 6.86 (s, 1H), 7.19 (s, 1H), 8.35 (d, J = 6.8 Hz, 211), 8.69 (d, J = 6.8 Hz, 2H); 13C NMR (100 MHz, CD3O ) ö16.7, 19.3, 20.5, 21.5, 23.8, 29.8, 33.9, 34.1, 38.1, 38.6, 41.3, 43.7, 46.7, 49.9, 58.8, 65.8, 114.9, 120.2, 122.5, 127.3, 141.3, 149.4, 150.9, 156.2, 159.3; ES1MS [M+Hj calcd forC26H34N0 376.2640, found 376.2653. Preparation of diol 2.14c Following procedure A, the following amounts were used: 1-Bromo-3,5- dimethoxybenzene (4.84 g, 22.3 mmol) in 40 mL dry THF, t-BuLi (26.2 ml, 1.7 M in pentane, 44.6 mmol), aldehyde 2.8 (1.77 g, 7.44 mmol) in 20 mL dry THF. Yields 2.14c (1.7 g, 4.5 mmol, 61%) as a white foam. ‘H NMR (400 MHz, CDC13) 80.36 (m, 1H), 0.75 (s, 311), 0.81 (s, 3H), 0.86 (dd,J= 12.1, 1.8Hz, 1H), 0.94 (m, 111), 1.99 (s, 3H), 1.15 (m, 2H), 1.23 (m, 1H), 1.25-1.42 (m, 2H), 1.50 (s, 3H), 1.51-1.68 (m, 2H), 1.80 (dt, J = 12.2, 3.0Hz, 1H), 2.06 (d, J = 8.1Hz, 1H), 3.75 (s, 6H), 4.74 (d, J = 8.3Hz, 1H), 6.32 (t, J = 2.2Hz, 1H), 6.57 (d, J = 2.4Hz, 2H); 13C NMR (100 MHz, CDC13) 8 15.9, 18.3, 19.8, 71 21.6, 26.0, 33.2, 33.5, 38.6, 40.6, 41.3, 44.1, 55.2, 55.3, 55.8, 62.8, 74.4, 75.8, 99.1, 105.8, 150.1, 160.8; ESIMS [M+Naj calcd forC23H36O4Na399.2511, found 399.2510. Preparation of xanthate 2.17c Following procedure B, the following amounts were used: diol 2.14c (1.60 g, 4.25 mmol) in 20 mL dry THF, Nail (187 mg, 60% in oil, 4.67 mmol), CS2 (280 iL, 4.67 mmol), Mel (293 jiL, 4.67 mmol). Yields 2.17c (1.22 g, 3.25 mmol, 76%). ‘H NMR (400 MHz, CDC13) S0.60 (td, J = 12.9, 3.5 Hz, 1H), 0.77 (s, 3H), 0.81 (s, 311), 0.87 (m, 1H), 0.99 (m, 111), 1.01 (s, 3H), 1.32 (m, 2H), 1.46 (m, 1H), 1.51 (s, 3H), 1.56 (dt, J = 13.7, 3.5 Hz, 111), 1.66 (m, 111), 1.79 (m, 3H), 2.19 (d, J= 5.2 Hz, 1H), 2.38 (s, 3H), 3.75 (s, 611), 5.18 (d, J = 5.0 Hz, 1H), 6.25 (t, J = 2.2 Hz, 1H), 6.60 (d, J = 2.2 Hz, 211); ‘3C NMR (100 MHz, CDC13)513.0, 14.1, 15.9, 18.4, 20.3, 21.3, 26.4, 33.3, 33.4, 40.2, 41.0, 41.4, 46.2, 46.9, 55.2, 55.9, 65.0, 74.2, 97.9, 106.2, 151.0, 160.6, 189.9; ESIMS [M+Na] calcd forC25H38O4SNa 489.2109, found 489.2112. 72 Preparation of alcohol 2.lSc Following procedure C, the following amounts were used: xanthate 2.17c (1.10 g, 2.36 mmol), Bu3SnH (1.25 mL, 4.72 mmol) in 6 mL toluene, AIBN (—10 mg) added every h until reaction was complete. Yields 2.15c (681 mg, 1.88 mmol, 80%). ‘H NMR (400 MHz, CDC13) 50.79 (s, 3H), 0.85 (s, 311), 0.88 (s, 3H), 0.95 (dd, J = 12.1, 2.0 Hz, 1H), 1.10 (td, J = 13.5, 4.1 Hz, 1H), 1.25 (s, 3H), 1.26 (m, 1H), 1.38 (m, 3H), 1.45 (m, 1H), 1.56 (dt, J = 13.5, 3.1 Hz, 1H), 1.61-1.73 (m, 3H), 1.85 (dt, J = 12.4, 3.1 Hz, 111), 2.59 (dd, J = 14.6, 4.6 Hz, 1H), 2.73 (dd, J = 14.6, 5.5 Hz, 1H), 3.76 (s, 6H), 6.25 (t, J = 2.2 Hz, 1H), 6.45 (d, J = 2.2 Hz, 2H); ‘3C NMR (100 MHz, CDC13)515.4, 18.5, 20.3, 21.5, 24.5, 31.6, 33.2, 33.4, 39.2, 40.3, 41.7, 44.1, 55.2 (2C), 56.1, 63.0, 74.0, 97.1, 107.1, 147.2, 160.6; ESIMS [M+Naf1 calcd forC23H36ONa 383.2562, found 383.2567. Preparation of tetracycle 2.18c Following procedure D, the following amount were used: 2.15c (681 mg, 1.88 mmol) in 20 mL CH2I,SnC14 (—2 mL). Yields 2.18c (536 mg, 1.56 mmol, 83% yield). ‘H NMR 0- 73 17L punoj‘çTOHhzDiojPDIT?D+[H+N1SWISEIFLçT ‘TicI‘I9t’I‘Z1‘LtOI‘8101‘P99‘68c‘FLfr‘Wt‘V1‘0017‘r‘ri‘om ‘V0‘9I‘0TZ‘L0‘c61‘W912(uouD‘Zllh’\I001)NNN(rn‘s)çp9‘(HI‘s) 009‘(HI‘ai)oc‘(HI‘w)cv‘(III‘ZilP9‘viII=I’‘PP)LE‘(HI‘ui)LT‘(Hi‘iii) 691-c91‘(Hr‘m)91-Ic1‘(HI‘w)oci‘(Hi‘w)Ot1‘(HI‘Zfl0I‘cET=I‘pi)611 ‘(- ‘s) coT‘(He‘s)zoi‘(i-it‘m)oo‘(He‘s)LWO‘(He‘s)90g(uou3‘zi-IN0017) IlAINH1•(%o9‘IOW1760‘“c6)LVZppiiC01ZTYHDWOJJpZffl11Si(J3SMnpisai ta•(lounu0L‘EYHD UT N01“PUL)‘H‘z1DHDiuocrn(10mw9J ‘guiç)gj•punodmoD:psnäIMsiunowiuiMoTToJqi‘.Inpo.IduTMouoJ HO LVZI0U!3J0SJ JO UOfl1.Wd.Id punoj‘LE9E17EzocHJ0JpDp3+[H-’-NlSIAIISJ6c1‘8cc1‘c17T‘LECT ‘6101‘L96‘9179‘cLc‘Pcc‘Fcc‘E917‘917‘017‘c8E‘OLE‘PEE‘FEE‘c6‘1I ‘POZ‘6T‘P81‘‘919(iDcI3‘HN001)INN(Hi‘s)0179‘(HI‘s)ç‘(HE‘s) LLE‘(HE‘s)çLE‘(Hr‘ai)I9z‘(HI‘ZHE9‘VtT=r‘pp)Ic‘(HI‘w)‘(HI‘ZH E9‘911=I‘PP)9L1‘(HE‘w)L1-6c1‘(HZ‘w)t’ci‘(Hi‘w)I‘(HI‘zilci‘cET j’ ‘pi)611‘(HE‘s)801‘(HE‘s)E01‘(Hr‘ui)860‘(H9‘s)L802(Du‘zHN0017) Preparation of phenol 228 and phenol 2.29 Resorcinol 2.27 (77.6 mg, 0.25 mmol) was dissolved in 1 mL DMF. K2C03 (69.1 mg, 0.50 mmol) was added, and the solution was stirred for 30 mm at rt. Mel (15.7 jaL, 0.25 mmol) was added, and the solution was stirred for 18 h. The reaction mixture was then extracted into EtOAc, washed with 1 x 1 M HCI, then with 3 x H20. The organic phase was dried over MgSO4,filtered and concentrated. The crude compounds were purified by flash chromatography (hexanes/EtOAc) to yield 2.28 (15.0 mg, 0.045 mmol, 18%) ‘H NMR (600 MHz, CDC13) öO.87 (s, 6H), 0.99 (m, 111), 1.01 (m, 1H), 1.02 (s, 3H), 1.12 (s, 3H), 1.18 (m, 111), 1.41 (m, 2H), 1.51 (m, 1H), 1.61 (m, 111), 1.69 (m, 1H), 1.71- 1.83 (m, 3H), 2.81 (m, 111), 2.51 (dd, J = 14.4, 6.0 Hz, 1H), 2.60 (m, 1H), 3.73 (s, 3H), 4.66 (s, IH), 6.14 (s, 1H), 6.40 (s, 1H); ‘3C NMR (150 MHz, CDCI3) ö16.1, 18.3, 19.5, 20.8, 21.1, 29.4, 33.1, 33.4, 36.9, 38.6, 40.1, 42.5, 45.8, 55.4, 57.3, 64.5, 99.8, 103.8, 131.1, 145.8, 151.3, 159.2; ESIMS [M+H] calcd for C22H330 329.2481, found 329.2483. Compound 229 (17 mg, 0.052 mmol, 21%) was eluted from the same column. OH 75 1H NMR (600 MHz, CDC13) öO.85 (s, 6H), 0.95 (d, J = 2.2 Hz, 1H), 0.97 (d, J = 2.7 Hz, 1H), 1.00 (s, 3H), 1.05 (s, 311), 1.17 (td, J = 13.6, 4.4 Hz, 1H), 1.39 (m, 2H), 1.49 (m, 1H), 1.55 (td, J = 12.4, 3.0 Hz, 1H), 1.61-1.69 (m, 3H), 1.74 (dd, J = 12.9, 6.2 Hz, 1H), 2.40 (dt, J = 12.2, 3.0 Hz, 1H), 2.46 (dd, J = 14.5, 6.2 Hz, 1H), 2.56 (m, 1H), 3.72 (s, 3H), 4.63 (s, 1H), 6.20 (s, 1H), 6.31 (s, 1H); ‘3C NMR (150 MHz, CDC13)ö 16.2, 18.4, 19.5, 20.5, 21.1, 29.3, 33.1, 33.4, 36.9, 38.4, 40.2, 42.6, 46.3, 55.1, 57.5, 64.6, 97.0, 104.4, 133.7, 145.5, 154.9, 155.9; ESIMS [M+H] calcd forC22H330329.2481, found 329.2487. Preparation of amine 2.31 Phenol 2.10 (108.8 mg, 0.35 mmol), PPh3 (275.4 mg, 1.05 mmol) and Boc-ethanolamine (169.1 mg, 1.05 mmol) were dissolved in 5 mL THF. Neat diethyl azodicarboxylate (165 tL, 1.05 mmol) was added, and the solution was stirred at rt for 18 h. Another 100 jiL portion of DEAD was added at this time and the solution was stirred at rt for another 48 h. The reaction mixture was concentrated to dryness, and the crude product was purified by flash chromatography (hexanes/EtOAc). Compound 2.30 could not be separated from residual 2.10, so the mixture was used in the deprotection. A mixture of compound 2.30 and 2.10 was dissolved in 2 mL CH21 and TFA (3 mL) was added. The solution was stirred at rt for 1 h, then concentrated. The residue was redissolved in toluene and 76 concentrated to dryness. The residue was dissolved in 10 mL Et20 and, upon cooling, white crystals formed. The crystals were filtered, washed with 25 mL Et20 and air dried. This product was slurried in 5 mL H20 and 0.5 mL 1.0 M HCI was added. CH3N was added to the slurry until the product was completely dissolved. The solution was filtered through a 0.45 im syringe filter and lyophilized to yield 2.31 (30.7 mg, 0.08 mmol, 23%). 1H NMR (400 MHz, CD3O ) 80.83 (s, 3H), 0.84 (s, 3H), 0.94 (m, 1H), 0.97 (m, 1H), 1.00 (s, 3H), 1.01 (s, 311), 1.16 (td, J = 13.0, 4.5 Hz, 1H), 1.36 (m, 2H), 1.49 (m, 111), 1.55-1.72 (m, 5H), 2.21 (m, 3H), 2.33 (dd, J = 8.6, 2.7 Hz, IH), 2.43 (dd, J = 14.4, 6.1 Hz, 1H), 2.55 (m, 1H), 3.27 (t, J = 5.0 Hz, 2H), 4.10 (t, J = 5.0 Hz, 211), 6.45 (s, 111), 6.62 (s, 1H); 13C NMR (100 MHz, CD3O ) 5 16.7, 19.3, 19.4, 20.7, 20.8, 21.5, 29.9, 33.9, 34.0, 38.2, 40.4,40.5,41.4,43.7, 58.5, 65.3, 66.2, 110.3, 115.9, 134.0, 145.5, 146.4, 157.7; ESIMS [M+Hy calcd forC24H38N0 356.2953, found 356.2959. Preparation of alcohol 2.58 Bromide 2.13 (1.41 g, 7.09 mmol) was dissolved in 30 ml dry THF under an argon atmosphere and cooled to —78 °C. t-BuLi (8.30 ml, 1.7 M in pentane, 14.2 mmol) was added over a period of 10 mm and the solution was warmed to rt. After 15 mm, the solution was recooled to —78 °C and stirred for an additional 30 mm. A solution of enal 2.32 (521 mg, 2.36 mmol) in 8 mL dry THF was then added to the cold solution and the 77 reaction mixture was stirred at —78 °C for 30 mm. 1 M HCJ was then added and the reaction mixture was warmed to rt. The crude product was extracted into EtOAc and washed with satd. NaHCO3 The organic phase was dried over MgSO4, filtered and concentrated. The crude compound was purified by flash chromatography (hexanes/EtOAc) to yield alcohol 2.34 (451 mg, 1.32 mmol, 56%). 1H NMR (400 MHz, CDC13) öO.88 (s, 3H), 0.91 (s, 311), 1.12 (s, 3H), 1.17 (m, 1H), 1.20 (m, IH), 1.27 (s, 3H), 1.34 (td, J = 12.9, 3.5 Hz, 1H), 1.41-1.75 (m, 6H), 2.01 (m, 2H), 2.31 (s, 3H), 3.77 (s, 3H), 5.33 (s, 111), 6.55 (s, 111), 6.75 (s, 1H), 6.83 (s, 1H); ‘3C NMR (100 MHz, CDC13) ö18.9, 19.1, 20.4, 21.5, 21.7, 21.8, 33.3, 33.4, 34.8, 37.1, 38.9, 41.5, 52.5, 55.1, 69.6, 108.4, 111.7, 118.5, 133.3, 138.9, 143.4, 147.6, 159.6; ESIMS [M+Na] calcd for C23H34ONa365.2457, found 365.2458. Preparation of tetracycles 2.35 and 2.36 Alcohol 2.34 (450 mg, 1.32 mmol) was dissolved in 10 mL CH21 under an argon atmosphere and cooled to —78 °C. SnCI4 (1 mL) was added and the resulting yellow solution was stirred for 15 mm. 1 M HC1 was added to the cold solution and the mixture was allowed to warm to rt. The layers were separated and the organic phase was washed with 2 x H20, dried over MgSO4, filtered and concentrated. The crude product was purified by flash chromatography (hexanes/EtOAc) to yield 2.35 and 2.36 (284 mg, 0.88 + 78 mmol, 67%) as a 1:1 mixture of epimers. ESIMS [M+Hf1calcd for C23H30325.2531, found 321.2536. Preparation of tetracycles 2.37 and 2.38 A 1:1 mixture of epimers 2.35 and 2.36 (84 mg, 0.25 mmol) was dissolved in 5 mL 1:1 MeOH:DMF. 10% Pd/C (32 mg) was added, and the slurry was saturated with 112. The solution was stirred for 16 h under an 112 atmosphere, after which the solid catalyst was filtered off and washed with EtOAc. The organic phase was washed with 3 x H20, dried over MgSO4,filtered and concentrated to yield 2.37 and 2.38 (80 mg, 0.24 mmol, 95%) as a 1:1 mixture of diastereomers. ESIMS [M+HV calcd forC23H350327.2688, found 327.2685. Preparation of phenols 2.39 and 2.40 The mixture of diastereomers 2.37 and 2.38 (80 mg, 0.24 mmol) was dissolved in 0.5 mL CH21. BBr3 (2 mL, 1 M in CH21,2.0 mmol) was added and the solution was stirred at rt for 15 mm. The reaction was halted by slow addition of MeOH, and the crude reaction mixture was concentrated under vacuum. The crude product was purified by flash chromatography (hexanes/EtOAc) to yield 2.39 and 2.40 (69 mg, 0.22 mmol, 90%) + 79 as a 1:1 mixture of diastereomers. Phenol 2.39 was crystallized from the mixture by cooling from toluene. OH ‘H NMR (600 MHz, CDCI3) ö 0.87 (s, 3H), 0.89 (s, 3H), 1.20 (m, 2H), 1.25 (s, 3H), 1.30-1.45 (m, 7H), 1.62 (s, 3H), 1.70 (m, 111), 1.85 (dd, J = 12.0, 8.4 Hz, 1H), 2.01 (m, 1H), 2.33 (s, 311), 2.73 (dd, J = 15.5, 8.3 Hz, 1H), 2.78 (m, 111), 4.51 (s, 111), 6.39 (s, 111), 6.50 (s, 1H); ‘3C NMR (150 MHz, CDC13)c517.9 (2 C), 19.7, 21.5, 24.1, 25.8, 32.5, 33.1, 33.5, 36.1, 36.2, 37.9, 41.9, 46.4, 47.5, 61.9, 108.4, 115.8, 133.9, 142.4, 143.3, 153.2; ESIMS [M+H] calcd forC22H330313.2531, found 313.2533. Phenol 2.40 was crystallized from the enriched remainder from CH3N. A single crystal suitable for X-ray analysis was grown by slow infusion of iPrOH into a toluene solution of 2.40 111 NMR (600 MHz, CDC13)80.47 (s, 311), 0.80 (s, 311), 0.89 (s, 3H), 0.90 (m, 111), 1.00 (dd, J= 11.3, 4.4 Hz, 1H), 1.17 (m, 1H), 1.18 (s, 311), 1.29 (m, 111), 1.40 (m, 2H), 1.52 (m, 111), 1.62 (m, 1H), 1.70 (m, 211), 2.33 (s, 3H), 2.52 (dt, J = 14.4, 5.5Hz, 1H), 2.62 (d, J = 16.9Hz, 1H), 2.97 (dd, J = 16.9, 8.0Hz, 111), 4.52 (s, 1H), 6.35 (s, 1H), 6.47 (s, 111); 80 13C NMR (150 MHz, CDC13) 515.2, 18.0, 19.1, 19.5, 21.4, 30.5, 31.7, 32.8, 32.9, 34.3, 36.9, 40.7, 41.7, 47.7, 52.0, 62.1, 108.3, 115.3, 133.2, 140.7, 145.6, 153.4; ESTMS [M+H] calcd forC2211330313.2531, found 313.2533. Preparation of glycine ester 2.41 Phenol 2.10 (36.1 mg, 0.116 mmol), Boc-Gly-OH (30.5 mg, 0.174 mmol), DMAP (-2 mg) were combined in 1 mL CH2I. 1,3-Diisopropylcarbodiimide (27 iL, 0.174 mmol) was added, and the solution was stirred at 11 for 2 h. The reaction mixture was then directly submitted to flash chromatography (hexanes/EtOAc) to yield a white foam. ‘H NMR (400 MHz, CDC13) 50.85 (s, 6H), 0.93 (m, 1H), 0.96 (m, 1H), 1.01 (s, 3H), 1.05 (s, 3H), 1.16 (m, 1H), 1.39 (m, 1H), 1.45 (s, 9H), 1.56 (m, 311), 1.70 (m, 411), 2.26 (s, 3H), 2.32 (m, 1H), 2.49 (dd, J = 14.6, 6.1 Hz, 1H), 2.60 (m, 1H), 4.10 (d, J = 5.0 Hz, 2H), 5.09 (s, br, 1H), 6.55 (s, 1H), 6.74 (s, 1H); ‘3C NMR (100 MHz, CDC13) 5 15.9, 18.2, 18.8, 19.4, 20.1, 21.0, 28.2, 28.8, 33.0, 33.2, 36.9, 38.5, 40.0, 42.4, 42.5, 47.3, 56.9, 64.2, 80.0, 115.4, 120.8, 133.0, 144.3, 147.9, 149.5, 155.5, 169.3; ESIMS [M+Na] calcd forC29H43NOa 492.3090, found 492.3088. This compound was dissolved in 50% TFA/ CH21 for 1 h. The solution was concentrated, redissolved in toluene, and concentrated to dryness. Et20 (15 mL) was added, and the compound was triturated until the precipate appeared as a uniform solid. Centrifugation of the mixture, followed by 81 washing of the solid with Et20 yielded 2.41 (44.5 mg, 0.092 mmol, 80%) as the TFA salt. 111 NMR (400 MHz, CD3O ) (50.87 (s, 3H), 0.88 (s, 311), 1.02 (m, 211), 1.06 (s, 3H), 1.08 (s, 3H), 1.19 (m, 111), 1.41 (m, 2H), 1.53 (m, 1H), 1.67 (m, 1H), 1.72 (m, 411), 2.28 (s, 3H), 2.39 (m, 1H), 2.50 (dd, J= 14.6, 6.1 Hz, 1H), 2.64 (m, 111), 4.07 (s, 2H), 4.87 (s, br, 3H), 6.64 (s, 111), 6.81 (s, 111); ‘3C NMR (100 MHz, CD3O ) (516.7, 19.1, 19.4, 20.5, 20.6, 21.5, 29.8, 33.9, 34.0, 38.2, 40.0, 41.22, 41.25, 43.7, 48.7, 58.4, 66.0, 116.6, 122.0, 134.4, 145.7, 149.3, 151.1, 167.7; ESIMS [M-i-H] calcd forC24H36N0 370.2746, found 370.2743. Preparation of lysine ester 2.42 H2N H2N Phenol 2.10 (41.7 mg, 0.133 mmol) was dissolved in 4 mL DMF. K2C03 (37 mg, 0.266 mmol) was added, and the solution was stirred for 10 mm. Boc-L-Lys(Boc)-OSu (115.3 mg, 0.266 mmol) was added, and the solution was stirred for 18 h at rt. The reaction mixture was extracted into EtOAc, and washed with 3 x 1120. The organic phase was dried, filtered and concentrated. The crude product was purified by flash chromatography to yield the bis-protected ester as a white foam. This foam was dissolved in 2 mL CH21 and TFA (2 mL) was added. The solution was stirred at it for 2 h, then concentrated to dryness. Toluene (3 mL) was added, and the solution was concentrated 82 to dryness again. The resulting residue was dissolved in 5 mL H20 and 100 jiL 1 M HCI was added. The aqueous solution was then filtered through a 0.22 jim syringe filter, and lyophilized to yield 2.42 as the bis-HC1 salt. (56 mg, 0.11 mmol, 85%, 2 steps) as a white powder. ‘H NMR (400 MHz, CD3O ) 80.82 (s, 3H), 0.83 (s, 3H), 0.96 (m, 2H), 1.01 (s, 3H), 1.04 (s, 3H), 1.16 (td, J = 13.5, 4.5 Hz, 111), 1.36 (m, 2H), 1.50 (m, 111), 1.59 (m, 3H), 1.69 (m, 511), 2.04 (m, 2H), 2.24 (s, 3H), 2.36 (m, 1H), 2.48 (dd, J = 14.7, 6.2 Hz, 1H), 2.60 (m, 1H), 2.92 (m, 2H), 3.23 (m, 111), 4.23 (m, 1H), 6.58 (s, 111), 6.76 (s, 1H); ‘3C NMR (100 MHz, CD3O ) ö 16.7, 19.0, 19.4, 20.5, 20.7, 21.5, 23.3, 28.1, 29.8, 31.1, 33.8, 34.0, 38.3, 40.1,40.3,41.4,43.7,53.9,58.4,66.1, 116.5, 121.9, 134.5, 145.9, 149.3, 151.4, 169.4; ESIMS [M+H] calcd forC28H45N0441.3481, found 441.3484. Preparation of aspartate ester 2.A H2N 0 OBn Phenol 2.10 (68.7 mg, 0.21 mmol) and Boc-L-Asp(OBn)-OH (107 mg, 0.33 mmol) were dissolved in 2 mL CH2I. DMAP (-1 mg) was added, followed by DIPC (52 jil, 0.33 mmol). The resulting solution was stirred at rt for 3 h. The urea byproduct was filtered off and the solution was concentrated. The crude product was purified by flash chromatography (hexanes/EtOAc), concentrated and redissolved in CH21 (2 mL). TFA (4 mL) was added, and the solution was stirred for 1.5 h. The reaction mixture was concentrated, extracted into EtOAc and washed with 2 x satd NaHCO3. The organic 83 phase was dried over Na2SO4,filtered and concentrated to dryness to yield 2.A (103 mg, 0.19 mmol, 87%). 1H NMR (400 MHz, CDC13) 80.87 (s, 611), 0.99 (m, 2H), 1.03 (s, 3H), 1.07 (s, 3H), 1.19 (m, 1H), 1.42 (m, 211), 1.52 (m, 1H), 1.60 (m, 111), 1.73 (m, 4H), 2.15 (s, br, 2H), 2.27 (s, 3H), 2.34 (m, 1H), 2.50 (dd, J= 14.6, 6.1 Hz, 1H), 2.60 (m, 1H), 2.96 (m, 2H), 4.03 (s, br, 1H), 5.18 (s, 2H), 6.53 (s, 1H), 6.70 (s, 1H), 7.35 (m, 5H); ‘3C NMR (100MHz, CDC13)616.0, 18.2, 18.8, 19.5, 20.1, 21.0, 28.8, 33.0, 33.3, 36.9, 38.5, 38.8, 40.0, 42.4, 47.3, 51.3, 56.9, 64.3, 66.7, 115.4, 120.8, 128.2, 128.3, 128.5, 133.0, 135.5, 144.4, 148.1, 149.4, 170.9; ESIMS [M+Na] calcd forC33H4NOa540.3, found 540.4 Preparation of acid 2.B Compound 2.A (89.6 rng, 0.173 mol) and succinic anhydride (51.9 mg, 0.519 mmol) were combined in 2 mL DMF. DMAP (-1 mg) was added, and the solution was stirred for 18 h. The reaction mixture was then extracted into EtOAc, washed with 1 x 1 M HC1, then with 3 x H20. The organic layer was dried over Na2SO4,filtered and concentrated. The crude product was purified by flash chromatography (hexanes/EtOAc) to yield 2.B (40 mg, 0.64 mmol, 37%). 111 NMR (400 MHz, CDC13)60.86 (s, 611), 0.91 (dd, J = 6.6, 84 cg ‘(He ‘s) ‘(118 ‘w) çLi-‘(Hr ‘ui) çTj ‘(He ‘s) ‘(He ‘s) 860 ‘(Hi ‘w) i60 ‘(119 ‘s) çgog (1DuD ‘zHN 00t) INN H1(%o8 ‘iouw LVO oci) £rz PP!’ o:i p1luiU3UO3 pU1E P1I!J ‘vOSN JAO P!P ssiqd 3iu1iJo puiqwocl qjDV1 ‘iu 01 ‘ii”piux pu DIIuo1p!ApgipTaTMsiiCisnonbipuJquioo qj UOiitiOS ODH’N p11S ‘jW O X OIUT p1wJu pui VO1E1 UT PAI0SS!P1 npTsaJ qj ssu&ip o poiuuuopuiijijuijswilcvoqnoiqip’Tj“ JflXTUJ UOfl3flJ qj 14 J JOJpauns prn ‘H q1M p11U1wS S1M UOflflOS T4 ‘ppp1 S1M (w oc) 3/Pd %(JJ HON 1Wj UT PAI0SSPsi(Ioww6ç0‘u69E)wzpunodulo3 H 0 H0(N0 £t’Z PP’!P JO UO!JflJtd.IJ c.o9 Pt0J ‘0ciE0I9 PNLONHD .ioj PI÷[N+N]SWISJ99L1‘LiLT‘60L1 ‘9691‘L6t’I ‘08V1‘V17171 ‘ccci‘TI‘9811‘V8i1‘8iT‘L0i1‘tcii‘0L9£179 ‘69c‘W817 ‘VLI7‘czt‘00v ‘98‘0L ‘t’9‘0E ‘170‘U61 ‘W81‘Iii‘FOi‘c61 ‘881‘E8I‘F91 9 (E13U3 ‘ZHN ooi)INN (iic ‘s) £L‘(HT ‘ZH 6L=f‘p) i89 ‘(HI ‘s) 999 ‘(HI ‘s) 6V9 ‘(Hi ‘zH i =I‘P) cTc ‘(HI ‘ar) coc ‘(HI ‘zH i17‘iLT =r ‘Pp) oi ‘(HI ‘ZH i17‘ELI =r‘pp)oo‘(Hz ‘m) 99i‘(Hi ‘w) Tçi ‘(HI ‘tu) ‘s) czz ‘(Ht’ ‘ui) ILl ‘(HI ‘ui) ‘(HI ‘m) ci‘(HI ‘w) oci‘(HI ‘w) iVI‘(HI ‘u) 8I ‘(HI ‘m) gli‘(Hc ‘s) çj‘(He ‘s) ‘(HI ‘w) L60 ‘(HI ‘m) 1760 ‘(HI ‘Zil Li 98 ‘EçII ‘V9 ‘OLçt’LI ‘czi ‘F017 ‘98 ‘0L‘IE ‘68 ‘Lc ‘11‘FOZ‘c61 ‘681‘81 ‘T912 (IDuD ‘ZHN 001)NNN ,Qn ‘s) 8L9‘(HI ‘s) 6c9‘(Hi ‘s) oom ‘(HI ‘ui) ‘(Hr ‘Zil F9‘9m1 =r‘pp)zc‘(Hr ‘ai) c‘(Hg ‘s) 6‘(Hi ‘w) LU ‘(Hi ‘w) ILl‘(HI ‘uj) 091‘(HI ‘Ui) cJ ‘(Hr ‘iii) wT‘(HT ‘s) c1 ‘(HI ‘zil cm ‘cg =1 ‘P1) 811‘(Hg ‘s) L01‘(Hg ‘s) OI‘(HT ‘w) L60‘(H9 ‘s) 9802 (ETDU3 ‘zHw 0017) NIAN H, (%LL ‘10mw ogoo ‘s” 6I) vrz Pi iqdi1o1rnuonp qsjj iCq pijund si IDnpoid pruo qi pui ‘piiJiuuoo siainxauuonoiai qj iqiuaAo pauns si ainxiwuoiai qi pui ‘(10mw 6c0o “ul i ç) pauoiq JA1D1ouIoJq q paooj ‘pppn (w i-) JVIAIU ID1H3 1W 1 UTpAJossipS1M (J0i11 6E00 ‘‘‘ ITT) 01Z H /\ 0 VYZ pUIOqjoUOJILWdJJ l8L0çç punoj ‘T8Locc flNLONtHbD J0Jp3J ÷[flN+IAI]SJAIJS1 cLU1‘SmLI‘81L1 ‘L691‘6t’1‘08lI ‘mtT‘6gI‘LOZI‘ccII‘ZI9‘69c ‘W81 ‘gLI7 ‘II7‘66g ‘çgg ‘9g‘o9g ‘gg ‘6zg ‘tog‘g6‘L8‘01‘00 ‘V61‘L81‘8I‘091(DUD ‘ZHJAI 001) II4IN ‘q‘s) E60I ‘(HI ‘q‘s) 1VU‘(HI ‘s) 1L9 ‘(HI ‘s) Z9 ‘(HI ‘q‘s) coc ‘(HI ‘zil 9Th1 =I ‘P) 9T‘(HI ‘zil oci=I’‘P)66z‘(Hc ‘w) 8L-LV ‘(HI ‘w) IV‘(HI ‘m) ogz 120.6, 133.2, 144.6, 148.1, 149.8, 166.2; ESIMS [M+Nai calcd forC24H3379BrONa 455.1562, found 455.1550. Preparation of PEGylated prodrug 2.45 Bromide 2.44 (10.8 mg, 0.025 mmol) and HS-PEG (150 mg, 5000 amu, 0.030 mmol) were dissolved in 200 jiL CH3N. DIEA (5.2 mL, 0.030 mmol) was added and the solution was stirred at ii for 24 h. i-PrOH (5.0 mL) was added and the resulting precipitate was isolated by centrifugation. The solid was washed with 3 x 5 mL iPrOH and dried under vacuum to yield a white powder. This powder was dissolved in 2 mL H20, filtered through a 0.25 jim millex membrane and lyophilized to yield 2.45, possibly contaminated with excess HS-PEG. In vitro SHIP enzyme assay. SHIP 1/SHIP2 enzyme assays were performed in 96-well microtiter plates with 10 ng enzyme/well in a total volume of 25 jiL of 20 mM Tris HC1 (pH 7.5) and 10 mlvi MgCl2. SHIP1/SHIP2 enzyme was incubated with test compounds provided in EtOH for 15 mm at 23 °C before the addition of 100 jiM inositol-1,3,4,5- tetrakisphosphate (1P4; Echelon Biosciences, Salt Lake City, UT). After 20 mm at 37 °C, the amount of inorganic phosphate released was assessed by the addition of Malachite Green reagent, and absorbance measurement was at 650 nm. For enzyme kinetics determinations enzyme reaction progress was measured every minute for 15 minutes at concentrations of 1P4 ranging from 10 to 100 jiM. Initial reaction velocities were determined from the slope of the linear portion of the resulting time courses and plotted against P4 concentration. Experiments were performed at least 3 times. 87 LPS stimulation of RAW macrophages. For the analysis of lipopolysaccharide (LPS) stimulated tumor necrosis factor c (TNFc) production, 2 x i0 RAW 264.7 (Mouse leukaemic monocyte macrophage cell line) cells were plated the night before in 24 well plates in macrophage medium. The next day, the medium was changed, and the compounds were added to the cells at the indicated concentrations for 3 h prior to the addition of 10 ng/mL LPS. Supernatants were collected for TNFa determination by enzyme-linked immunosorbent assay (ELISA; BD Biosciences, Mississauga, ON). Mouse endotoxemia model. C57B 16 mice aged 6 to 8 weeks were orally administered the indicated dose of 2.10, dexamethasone or vehicle 30 minutes prior to an intraperitoneal injection of 2 mg/kg LPS (E coli serotype 011 1:B4). Blood was drawn 2 hours later for determination of plasma TNFcx by ELISA. Construction of SHIP1 zC2 mutant and isolated C2 domain. A His6-tagged SHIP 1 AC2 deletion mutant (deleting residues 725 to 863) in the mammalian expression vector pME18S was generated by a standard polymerase chain reaction (PCR) based methodology. An N-terminal His6 C2 domain construct was also generated by PCR inserted into the pET28C bacterial expression vector using EcoRI an NdeI restriction sites. Scintillation proximity assays. 2.10 was radiolabeled with tritium by GE Healthcare (Piscataway, NJ) to a specific activity of 155.4 x 1010 Bq (42 Ci/mmol). Copper chelate 88 (His-Tag) Ysi SPA Scintillation Beads were diluted in 0.25 % BSAJTBS to 1.5 mglmL, and recombinant, His6-tagged protein was added at the indicated concentrations; wild type (1 pM), zC2 SHIP1 enzyme (1 pM) or C2 domain (10 nM). Protein was allowed to bind for 1 hour at 23 °C, and 250 mg of beads were aliquoted per well of a 96-well plate. A total of 0.185 MBq (5 siC) of[3H]-10 was added per well, the plate was gently agitated for 30 minutes, and the amount of bead-associated radioactivity was quantified by counting in a Wallac BetaPlate plate scintillation counter. 89 Chapter 3: Synthesis of the Ceratamine Heterocycles 3.1: Microtubules as a Target for Anticancer Drugs Microtubules are highly dynamic components of the cytoskeleton and are essential in all eukaryotic cells. These long protein polymers consist of x-tubu1in and 13- tubulin heterodimers that are arranged in the form of filamentous tubes that can be many micrometers long and as such are readily observable under a light microscope. They play crucial roles in development and maintenance of cell shape, as well as the transport of vesicles, mitochondria, and other components throughout the cell. In the context of cell division, they play a key role as the entire microtubule framework rearranges to form the mitotic spindle in the M phase of the cell cycle. The formation of the mitotic spindle provides the scaffold for the separation of the duplicated chromosomes before cleavage of the cell into two daughter cells. The correct movements of the chromosomes and their segregation into separate cells requires extremely rapid microtubule dynamics, making cells in mitosis very sensitive to microtubule targeting drugs.83 Since cancer cells divide more rapidly then normal cells, they pass through mitosis more frequently and, therefore, are more vulnerable to microtubule-targeted drugs than normal cells. The key action of an antimitotic drug is the suppression of microtubule dynamics that leads to blockage of the cell cycle in mitosis. For example, the presence of a single chromosome that is unable to attach to the mitotic spindle is enough to prevent a cell from exiting mitosis. The cell remains blocked in a mitotic state and eventually undergoes apoptosis.84 90 Antimitotic drugs represent a wide variety of compounds, most of which are natural products. This class of compounds has been so successful in the treatment of cancer that their targets, soluble tubulin and microtubules, are often considered to be the best single target identified to date for the treatment of cancer.85 Among the most successful microtubule-targeted chemotherapeutic drugs are paclitaxel and the Vinca alkaloids. 3.1 Vinblastine R = Me 3.2 Vincristine R = CHO The Vinca alkaloids, exemplified by their naturally occurring members, vinbiastine (3.1) and vincristine (3.2) have been used in clinical settings for the treatment of haematological and solid malignancies for over 40 years. Their clinical efficacy has prompted the development of semisynthetic analogues, several of which are currently in clinical use.86 The taxanes, as exemplified by the natural compound paclitaxel (3.3) and its semisynthetic analogue, docetaxel, are some of the most important anticancer drugs and are currently used for the treatement of ovarian, breast, and non-small cell lung cancers as well as AIDS-related Kaposi’s sarcoma. Microtubule targeting drugs are effective in a broad variety of malignancies and display interesting synergies with each other, even when their molecular target is the same.86 0 NHO OH 3.3 91 Due to the multiple validations of microtubules as an efficacious anticancer target, there is considerable interest in the discovery and development of novel antimitotic compounds for use as single agents or in synergy with existing treatments.87A novel cell- based assay developed by Roberge et al. has provided a robust and sensitive method to screen extract libraries for compounds that arrest cells in mitosis.88 Use of this assay led to the isolation and structural elucidation of ceratamines A and B. The attempted synthesis of these compounds and the biological evaluation of their close analogues is described in this chapter. 3.2: Synthesis of Ceratamine Analogues Ceratamines A (3.4) and B (3.5) are antimitotic alkaloids isolated from the marine sponge Pseudoceratina sp. collected in Papua New Guinea.42 The imidazo[4,5-d]azepine core heterocycle at the oxidation state found in the ceratamines appears to have no precedent among known natural products or synthetic compounds. 1 9 HN 7NR 3.4 A = Me Ceratamine A 19 / N Br 3.5 R= H Ceratamine B Br 15 O21 Ceratamines stabilize microtubules but do not appear to bind to the taxol-binding site as shown by an assay in which microtubules assembled in the presence of [3H]- labeled paclitaxel were exposed to other microtubule stabilizing agents. Agents that 92 compete for the paclitaxel binding site should result in a release of[3Hjpaclitaxel into solution. Addition of 4 imolfL eleutherobin, a microtubule-stabilizing agent which binds to the paclitaxel site, caused the release of a significant amount of radiolabeled paclitaxel from the microtubules in the assay. By contrast, ceratamine A added at 20 or 50 imoL/L caused no statistically significant release of radiolabeled paclitaxel. Furthermore, the effect of ceratamine A on mitotic microtubules gave rise to a cellular phenotype that was different from that typically observed with other microtubule stabilizing agents. Examination of cells arrested in mitosis by ceratamine A with confocal microscopy shows the formation of pillar-like tubulin structures that extend vertically from the basal cell surface and span the entire thickness of the arrested cells (Figure 3.1). Figure 3.1: Confocal microscopy of cells arrested in mitosis by ceratamine A. “Li” and “L2” are 0.32 tm thick optical sections sliced along the lines shown in “L” L 93 This unusual activity, combined with their structural uniqueness and relative simplicity, make the ceratamines an intriguing synthetic target. Goals of our scientific efforts were to ascertain any possible in i’ivo efficacy of this novel pharmacophore, discover structure-activity relationships for the ceratamine family, and confirm the structure of the natural product.89 Ceratamines A (3.4) and B (3.5) appear to be biogenetically derived from a histidine-tyrosine dipeptide (Scheme 3.1).42 Support for this proposal comes from the obvious biogenetic relationship between the ceratamines and the known compounds 5- bromoverngamide (3.6) and ianthelline (3•7)•90 The 2-aminimidazole moiety is known to be nucleophilic at the 4- and 5- positions,9’so a condensation from this position to either an imine, oxime, or ketone would have good chemical precedent. This proposed biosynthesis was the basis of our first reterosynthetic analysis of ceratamine A. 94 HO H >rrO <N]NH N H2N— OH HO _____ NH ______ \> 1 N 0 H2 Br Br HF / HO H /O R-fNH HO’ R = H, NH2 Scheme 3.1: Proposed biosynthesis of ceratamines A and B Attempts by several members of our lab to implement a biomimetic synthesis of the ceratamines failed. Difficulties in synthesizing and handling 2-aminohistamine923.8 and the tyrosine derivative 3.9 quickly established that the chemistry outlined in Scheme 3.2, while simple on paper, was anything but trivial in practice. 3.4 R = Me 3.5 R= H Br - Br R = H, NH2, NHMe x =0, NOH 3.6 R=H 3.7 R=NH2 95 N—- NH2 H2N-K 3.8 1) Couple 2) deprotect ketone H2N- 3.10 cyclize \ N HN—<\ N— NN 0 Br Br 1) Oxidize 2) Methylate H2N NH / \ Br 3.11 / 0- Br Scheme 3.2: Proposed biomirnetic synthesis of ceratamine A. The 2-aminoimidazole structural motif is known to impart processing and handling difficulties to many compounds.93 Solubility, purification, and unwanted reactivities are all issues when dealing with certain aminoimidazole systems. Therefore, we decided to introduce the 2-amino group at one of the last stages in the synthesis for relative ease of handling, solubility, and purification of intermediates. The bromines and N-18-methyl present in the natural product were also to be installed in the later stages of the synthesis,94 as we recognized that both desmethylamine and desbromoceratamines would be useful for structure confirmation and biological evaluation. Our second approach to the imidazo[4,5-d]azepine ring system was to fuse the 5- membered 2-aminoimidazole ring onto a suitable 7-membered lactam ring (3.B), thus installing 3 of the 4 nitrogen atoms of the ceratamines in one operation (Scheme 3.3). Br R ketone or protected equivalent 96 We envisioned assembling the imidazo{4,5-d]azepine ring system at various lower oxidation states, then bringing the system to the level found in the natural product, relying on the aromaticity of the ring system as a driving force (3.A to 3•4)95 H N HN—4 N— N 0 / \ Br 3.A 0 Br / Scheme 3.3: Second retrosynthetic analysis of the ceratamines. A co-worker in the Andersen lab, Alban Pereira, examined this approach extensively and his efforts towards this synthetic route are detailed in his thesis. Unfortunately, this approach did not lead to the successful synthesis of the ceratamine heterocyclic family and was abandoned. The failure to graft the 5-membered 2-aminoimidazole ring onto the 7-membered lactam then prompted us to approach this synthetic problem a different way. Annulation of the 7-membered lactam onto a preexisting imidazole ring was to be the central feature of the next synthetic approach to the ceratamines. A third reterosynthetic scheme is shown in Scheme 3.4. Once again, we expected that the heterocyclic system could be assembled at a lower oxidation state then oxidized to the level of the natural product (3.F to 3.E). An obvious first disconnection was the amide bond, and a second disconnection gives a functionalized histamine (3.1) and a cinnamic acid stannane (3.H). 3.4 97 Ri Scheme 3.4: Third reterosynthetic analysis of ceratamine A. We envisioned that 3.H and 3.1 could be coupled in a transition metal catalyzed carbon-carbon bond forming reaction. Implicit in our synthetic plan was the expectation that the deprotected intermediate 3.D would spontaneously rearrange to the presumably more stable aromatic isomer 3.C. Attempting to carry out this route using a Stille reaction to couple 3.H and 3.1 (where R1 = H and P = trityl or H) ultimately failed. This was most likely due to the steric bulk surrounding the two coupling partners.96 Other approaches using histamine as a synthon ultimately failed as well. The second strategy of building the ceratamine ring system at a lower oxidation state with the expectation that it would spontaneously oxidize and aromatize was realized to be potentially problematic. Should the expected oxidation not occur, the synthesis would be halted at a late stage in the game. We therefore decided to pursue a synthetic route in which the oxidation state of the intermediates rises as the synthesis proceeds, N NN N N HN—<. N— Ri—<. N— Ri—<’ N— (0 o_ 34! \ Br o 3.\c . / 0 0 0Br / / / NPhth + R1r 3.H NH2 Ri</jJ F // R2 98 ending at the state of the ceratamines, eliminating the need for an unprecedented late stage oxidation. A fourth reterosynthetic analysis is outlined in Scheme 3.5. Once again, the plan was to convert R1 to an amino substituent and install the bromine atoms near the end of the synthesis (3.E to 3.4). The cornerstone of this approach to the ceratamines was to be the use of an intramolecular Buchwald vinyl amidation to create the lactam ring (3.J to 3.E). RiN ÷ Ri 3M 31 3K 3J —0 0I / Scheme 3.5: Fourth retrosynthetic analysis of the ceratamines This approach made use of the fact that imidazoles can be selectively and sequentially metallated.97 The synthesis began with BOM-protected tribromoimidazole 3.12 (Scheme 3.6). In a one-pot procedure, metallation of 3.12 with one equivalent of n BuLi, followed by quenching with MeSSMe introduced the 2-thiomethyl moiety. Without workup, further metallations and quenchings installed the tributyistannane and aldehyde group respectively to form 3.13 in 48% overall yield after chromatography. Cinnamic ester 3.15 was synthesized via Cr(II) mediated condensation of anisaldehyde with tribromoester 3.14.98 Stille coupling between 3.13 and 3.15 in toluene yielded 3.16 99 in high yield.99 Z-vinyl bromide 3.17 was synthesized by reaction of the aldehyde 3.16 with (bromomethyl)triphenylphosphoniumbromide and lithium hexamethyldisilazide in the presence of HMPA.10° Saponification of the methyl ester, activation of the acid as the HOBt ester, followed by displacement of the HOBt group with excess methylamine yielded the 2° amide 3.19 in 54% overall yield (3 steps). The key step in the synthesis was a copper(I) catalyzed ring closure of 3.19 to form the enamide 3.20 following a procedure developed by Buchwald and co-workers.’°’ N Br Br Br BOM 3.12 1)nBuLi, -78°C 2) MeSSMe, -78°C - rt 3) nBuLI, -78°C 4) Bu3SnCI, -78°C-rt 5) nBuLi, -78°C 6) DMF, -78°C - rt CHO N— BOjJ 3.16:90% L1HMDS, THF Bre HMPA, -78°C Ph3Br Br N 0 N OMe BOM 3.17: 71 % Scheme 3.6: Preparation of heterocycle 3.20 Br3CAOH 0 p-AnisaldehydeMeOH Br3COH2S04(cat) CrCI2, THF, rt 3.14: 95 % 3.15: 85 % \ N CHO sX N SnBu3 BOM 3.13: 48% 3.15, Pd(PPh3)4 Cul, PhMe, retlux Bi MeNH2 NHMe 4 THE Cul, Cs2O3 0 1) L1OH-H20\\ ii THF/H N BOM 2) DIPC, HOBt DMAP, CH2I 3.18: 86 %3.19: 90% MeHN NHMe 3.20: 92 % 100 A dilute THF solution of 3.19 was treated with Cul, Cs2O3 and N,N’ dimethylethylenediamine at elevated temperatures to yield the 5,7-bicyclic system 3.20. Our initial attempts at conversion of 3.19 to 3.20 via a Cu(I) catalyzed enamide formation were met with mixed results. Yields of this conversion could range from 40 % to 90 %, and the reaction had a tendency to “stall”. To complicate matters, 3.19 and 3.20 run extremely close to each other on silica under all solvent systems attempted. Using stoichiometric amounts of Cul in the transformation seemed to be the only way to push the reaction to completion; however, this caused the reaction yield to suffer. It should be noted that as in many metal catalyzed bond-forming reactions, there are multiple combinations of metal source, base, solvent, and ligand that may improve the yield of this particular reaction.102 A full optimization of this reaction was not pursued in our research. In an offshoot of the reaction scheme shown in Scheme 3.6, we wanted to explore an alternate method of forming the 7-membered ring. We reasoned that a condensation to the aldehyde in 3.16 would form the desired ring system without need for a metal catalyzed multicomponent reaction. The alternate reaction scheme is shown in Scheme 3.7. Saponification of the ester in 3.16 yields the acid 3.21. Without isolation, this compound was coupled with the methyl ester of sarcosine. Treatment of the crude coupling product 3.22 with NaH and DMSO in dry THF for 24 h yields cyclized product 3.23 in good yield. The presence of an ester on C-8 could be problematic from a total synthesis perspective as a late-stage decarboxylation seemed a risky 103 however, from an SAR perspective, this cyclization method is advantageous. Saponification of the ester 101 to yield the acid then provides an excellent synthetic handle for rapid diversification of compounds, including possible ceratamine dimers.’°4 CHO LiOHH2 BOM THF/H20 3.16 CHO OH BOM 3.21 - Scheme 3.7: Alternate 7-membered ring formation. The proof-of principle provided by Scheme 3.7 may also allow different methods of cyclization to the aldehyde to form 3.20; for example, a Wittig-type reaction105 with 3.24, or a variant thereof as shown in Scheme 3.8. These chemistries will be followed up on in due course; however, for the purposes of total synthesis, we chose to generate 3.20 via the Cu(I) catalyzed enamide formation (Scheme 3.6). DIPC, DMAP CH2I V CHO S NaH DMSO THE rt 0 3.23: 76 %, 3 steps 3.22 102 ,CHO \ N—/N—( NaH N— DMSO BOM ‘IC THF 3.24 3.20 Scheme 3.8: Proposed Wittig cyclization to form 3.20 Enamide 3.20 is already at the oxidation state of the ceratamines, requiring 1) installation of the 2-amino functionality, 2) removal of the BOM protecting group, and 3) tautomerization to form the fully aromtic ceratamine heterocyclic system. Unfortunately, the 2-methylthio group in 3.20 proved to be resistant to substitution by a variety of nitrogen nucleophiles. Oxidation of the thiomethyl group to the sulfoxide/sulfone 3.25 could be carried out, albeit in very poor yield (<10%); however, subsititution of the oxidized 2-methylthio group failed as well. In retrospect, this result was not surprising considering the documented need for electron withdrawing substituents at C-4 or C-5 to promote nucleophilic aromatic substitution in the 2-position of an imidazole.’°6With direct substitution to install the 2-amino group failing, we aimed towards an azido transfer protocol using electrophilic azide, 107 requiring the formation of a C-2 carbanion. 103 fflN— 1)MeL1,THF H2NN BOf’O :::, H2, MeOH 3.26: 31 % 3.27: 72 % Scheme 3.9: Preparation of aminoimidazole 3.27 Compound 3.20 was reductively desulfurized with Raney nickel in poor yield to form protio-compound 3.26 (Scheme 3.9), the desired precursor to a C-2 carbanion. This procedure was unsatisfactory for a number of reasons. The use of flammable Raney nickel, large solvent volumes, adsorption of the compound to the metal surface, and over reduction leading to low yields were all problems that plagued this transformation. Extensive experimentation with different conditions and other desulfurizing agents108 failed to improve the yield or the ease of this transformation. Nevertheless, a workable amount of 3.26 was generated and following deprotonation with MeLi, quenching with multiple equivalents of TsN3, and catalytic reduction of the azido group with H2 the desired primary amine 3.27 was obtained relatively cleanly. Curiously, the use of n-BuLi or t-BuLi to carry out the deprotonation of 3.26 resulted in significantly more side products and decreased yields of 3.27. The reasons for this base selectivity are unknown. 3.20 N/ mCPBA, CH2I —S(O)n----’ N— BOM ji ° 3.25 NaN3, DMSO MeNHU,THF No reaction NH3IMeOH 104 Attempted deprotection of the BOM group in 3.27 by hydrogenation and acid catalyzed hydrolysis either failed or resulted in decomposition; however, treatment of 3.27 with A1C13, followed by an aqueous workup’°9gave two ceratamine analogues 3.28 and 3.29 (Scheme 3.10). 1) AId3,CH2I 3.27 2) H20 N H2N—<. N— NN 0 - OH / 3.28:62% -0 + H2N—<, N— 3.29:15% —0 1) AId3,CH2I 3.20 2) H20 1) AId3,CH2I 3.26 2) H20 Scheme 3.10: Formation of ceratamine analogues 3.28 and 3.29 The addition of an oxygen atom in the C-il position of the ceratamine system was puzzling, especially considering BOM-deprotection of the 2-methylthio compound 3.20 and 2-protio compound 3.26 failed to bring about either isomerization to the fully 3.31:71 % 105 aromatic system or oxidation at the benzylic position, yielding instead exocyclic alkenes 3.30 and 3.31, respectively (Scheme 3.10). Clearly, further investigation into the mechanism of what is a formal Michael addition of water to C-li followed by an air oxidation would have to be undertaken. However, given the limitations involved in producing 1° amine 3.27 via Scheme 3.9 a more reliable synthetic path would have to be developed to secure enough material for study. With this in mind, a slightly altered synthetic path was undertaken (Scheme 3.11). This modified pathway would ultimately yield chloroimidazole 3.37, which was expected to undergo Pd(0) catalyzed amination in order to form the 2-aminoimidazole compound.11°Chioroimidazole 3.37 was recognized as a key intermediate from an SAR standpoint, given the synthetic flexibility imparted by the 2-chioro atom allowing for rapid diversification at an advanced stage of the synthesis. Metallation of the BOM protected tribromoimidazole 3.12, followed by quenching with hexachioroethane yielded crystalline chloride 3.32 in 94% yield.” Further metallations and quenchings installed the tributyistannyl and aldehyde group respectively, to afford stannane 3.33 in 62% yield. The cinnamic amide 3.34 was prepared in 95% yield via saponification of methyl ester 3.15, in situ activation of the acid, and displacement with excess methylamine. This bromide was coupled with stannane 3.33 in THF at ii in 75% yield to yield aldehyde 3.35. Reaction in toluene at elevated temperatures scrambled the exocyclic alkene geometry of 3.35 h12 as evidenced by TLC and NMR spectroscopy of the reaction mixtures. This result was not observed during the Stille reaction between 3.13 and 3.15. 106 3 12 1) n-BuLl, THE, -78°C 2) C216,THF, -78°C -> rt N Br 1) nBuLi, THE, -78°C ci—K’ X 2) Bu3SnCI, -78°C -> rt Br 3) n-BuLl, THF, -78°C BOM 4) DMF, -78°C -> rt 3.32:94% N CHO / N SnBu3 BOM 3.33: 62 % 1) LiOH-H20 3.15 2) DIPC, HOBT DMAP, DCM Pd(PPh3)4,Cul THE, rt N CI—K N— BOO 85% -0 Scheme 3.11: Preparation of aminoimidazole 3.27 by Pd° catalyzed amination Wittig olefination with (bromomethyl)triphenylphosphonium bromide and potassium tert-butoxide in THF yielded the surprisingly insoluble Z-vinyl bromide 3.36 in 80% yield after precipitation from CH21 followed by chromatography of the jOBt 0 MeNH2, BriNzTHE/DCM H 3.34: 95 % 3.33 + 3.34 0 N7 H 3.36: 80 % /\ MeHN NHMe Br0 Ph3Br H KOt-Bu THE, -78°C 3.35: 73 % N—/ 1) Ph3S1NH2,L1HMDS, H2N ji__ Pd(dba) XPhos BOM 0 Tol, 100°C 2) H30 0/3.27: 69 io Cul, Cs2O3 THF, 75°C 107 remaining product. Ring closure of 3.36 via a Cu(I) catalyzed Buchwald enamide reaction yielded 2-chloroimidazo[4,5-d]azepine 3.37 in 85% yield with none of the issues observed in the transformation of 3.19 to 3.20. The structure of this compound was confirmed by single crystal x-ray diffraction analysis (Figure 3.2). Figure 3.2: Crystal structure of chloroimidazole 3.37 A palladium-mediated amination was then carried out on 3.37 using an ammonia surrogate113 in an attempt to form 3.27. Thus, heating chloroimidazole 3.37, triphenylsilylamine, and LiHMDS in the presence of Pd2(dba)3and the Buchwald ligand 2-dicyclohexylphosphino-2’,4’,6’-triisopropylbiphenyl (XPhos)”4in toluene, followed by acidic hydrolysis of the triphenylsilyl group smoothly yielded 10 amine 3.27 in 69% yield (Scheme 3.11). With a scaleable route to 3.27 in hand, we began to try to manipulate the formation of 3.28 and 3.29 enroute towards the synthesis of the ceratamines. It would C’s 03 3.37 CZ7 N’ 108 appear that oxidation at the C-il position of the ceratamine system occurs only upon A1C13 mediated N-i deprotection of a system with an amino substituent at the 2-position of the imidazole ring. Less effective electron donating substituents (MeS- and H-) do not display this reactivity as illustrated in Scheme 3.10. The formal Michael addition of water to the C-il position of 327 upon N-i deprotection could perhaps be rationalized by the increased reactivity of the 11 position upon ipso- protonation available only to a 2- aminoimidazole containing compound in acidic media (Scheme 3.12). Oxidation of the Michael addition product would then yield 3.28 and a further oxidation would yield 3.29. H2N—JN-— 1) AId3 BOM j ‘ö 2) H20Q 3.27 Scheme 3.12: Possible mechanism of Michael addition of water to 3.27 However, upon deprotection of 3.27 with A1C13, followed by quenching with MeOH, only alcohol 3.28 and ketone 3.29 were formed. Additionally, quenching the deprotection of 3.27 with 180 labeled water gave no indication of any incorporation of ‘0 into 3.28 or 3.29 as observed by ESI-MS or MALDI-MS. These results indicate that a simple Michael addition mechanism is not in operation and that the oxygen atoms present at C-il in 3.28 and 3.29 must have an alternate origin. Another possible source of the C-i i oxygen atom in 3.28 and 3.29 was from the BOM protecting group via some intramolecular process that was occurring during the A1C13 catalyzed deprotection sequence. While there was no obvious mechanistic rationale 109 for such a process, we nevertheless investigated this possibility by scrambling the regiochemistry of the BOM group as outlined in Scheme 3.13. Compound 3.37 was subjected to standard deprotection with A1C13 in CH2I followed by hydrolysis. Without isolation, the resulting imidazole 3.38 was treated with BOM-Ci and K2C03 in DMF to yield a 1:1 mixture of compounds 3.37 and its regioisomer 3.39. The regioisomers were separated and 3.39 was aminated as described above to give 3.40. When 3.40 was deprotected with A1C13, only compounds 3.28 and 3.29 were isolated, eliminating the possibility that C-il oxygenation resulted from some intramolecular process involving the BOM oxygen atoms. At this point it seemed most likely that the C 11 oxygen atoms in 3.28 and 3.29 were coming from atmospheric oxygen.115 N/\ NNr_ 1) AICI3ICH2I BOM 2)HO 0 3.37 + 37 3.38 BOM CIN DMF/K2C03 0 3.39:35% 1) Ph3SiNH2 LiHMDS Pd2(dba)3 Tol, reflux 2) H30 BOM H2NN 3.40 1) AICI3/CH2I 3.28 + 3.29 2) H20 Scheme 3.13: Scrambling of BOM regiochemistry of 3.37 110 Multiple attempts to deoxygenate 3.28 via standard procedures such as hydrogenolysis, Barton deoxygenation and TFAJEt3SiH, resulted only in decomposition. Additionally, attempts to carry out a BuchwaldlHartwig amination113 on the deprotected chloride 3.38 with an ammonia surrogate failed as well, yielding no isolable products. A synthesis of ceratamine A passing through intermediate 3.28 now seemed unlikely, so we attempted to deactivate the 2-amino substituent in order to forestall the oxidation of C-il upon removal of the BOM group. The nitrogen-deactivating substituent could then be removed under conditions in which atmospheric oxygen was excluded, hopefully resulting in clean isomerization and aromatization to the ceratamine system. Using a catalytic amidation procedure recently described by Buchwald and co workers,116 we reacted 3.37 with N-methylformamide in the presence of Cs2O3, Pd2(dba)3, using 2-di-tert-butylphosphino-3 ,4,5 ,6-tetramethyl-2’ ,4’ ,6 ‘ -triisopropyl- 1,1 - biphenyl 3.41 as a ligand. The N-methyl formyl compound 3.42 was formed in 50% yield upon refluxing in toluene for 24 h (Scheme 3.14). Exposure of 3.42 to anhydrous A1C13 in CH21 cleanly yielded 3.43 as evidenced by TLC and HRESIMS. The ‘H NMR spectrum of 3.43 revealed a complicated dynamic system, possibly due to rotamers and/or isomers; however it was clear that tautomerization/oxidation had not occurred to the compound and it likely exists as drawn. Attempts to remove the formyl group in 3.43 by basic hydrolysis failed. However, hydrolysis of 3.43 in 1 ,4-dioxane and 6 N HC1 gave the C-il alcohol 3.45 as the major product after brief heating and for the first time minor amounts of the desired desbromoceratamine 3.44 were also formed. After much experimentation, it was found that passage of dry HC1(g) through a solution of 3.43 in ill 50% 1 ,4-dioxane and water gave clean transformation of 3.43 to desbromoceratamine A 3.44 (Scheme 3.14) with the formation of only small amounts of 3.45. The stream of HCl gas employed in this reaction presumably purged atmospheric oxygen resulting in a dramatic reduction in the formation of the C-li hydroxy analogue 3.45 and formation of desbromoceratamine (3.44) as the major product, consistent with atmospheric oxygen being the source of C-il oxygenation. HCI(g) 1 ,4-dioxane/H20 3.44: 25 %, 2 steps 3.45 major minor Scheme 3.14: Formation of desbromoceratamine A by amidation of chloride 3.37 NMR analysis of 3.44 in DMSO-d6revealed the striking similarities between 3.44 and ceratamine A (Figure 3•3)•42 The spectra reveal two interconverting forms for both 3.4 and 3.44 in about a 3:1 ratio. 0N-/ H—4’ NcI H’N N N 1) AId3,CH2I BO / H / BOO 2) H20Pd2(dba)3,Cs2O3, 3.41 Tol reflux .37 P(t-Bu)2 ,Q”.42: 72 % 3.41 =i-Pr i-Pr NBS/DMF or 8r2/HOAc 112 H2 I Figure 3.3: ‘H NMR spectra of A) ceratamine A in DMSO-d6at 500 MHz and B) 3.44 in DMSO-d6at 600 MHz A 1 9 20HN 19 I b Br 13 /150 21 Br H13 H17 Ceratamine A HiS Hil H9 (ppm) B \ N HN—<, N— NN 0 IN 3.44 / 0 N. Q 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 113 VT-’H NMR experiments on 3.44 in DMSO-d6 show a coalescence of the two forms at 400 K (Figure 3.4). A B C Figure 3.4: 1H NMR spectra of 3.44 in DMSO-d6at 400 MHz recorded at A) 298 K, B) 35OKand C) 400K 114 COSY data showed that the C-19 methyl group in ceratamine A is scalar coupled to the NH-18 proton in both forms,42 suggesting that in each case they are slowly interconverting rotamers about the C-21N-18 bonds, consistent with the doubly vinylogous amide nature of the 2-N-methylamino substituents and the C-6 carbonyl in 3.4 and 3.44 (Figure 3.5) which provides a barrier to free rotation. 3.44 3.44 Figure 3.5: Hindered rotation about C21N18 bond in 3.44 To complete the synthesis of ceratamine A, the bromination of 3.44 was undertaken. To our disappointment, bromination of 3.44 using 2 eq. N bromosuccinimide in DMF or 2 eq. Br2 in HOAc yielded only the 9-monobrominated product 3.46 (Scheme 3.14). Use of excess amounts of bromine led to very complex mixtures from which nothing could be isolated. In an attempt to circumvent the long reaction times and low yields involved in the transformation of 3.37 to 3.42, we attempted to introduce the deactivating formyl group onto the more readily formed compound 3.27. Reaction of 3.27 with acetic formic anhydride in THF for 24 h afforded 3.47 in good yield (Scheme 3.15). 117 Formamide 3.47 can be readily alkylated with Mel and K2C03 in DMF to from 3.42, which represents an improved method to make this critical desbromoceratamine precursor. 115 Removal of the BOM group from 3.47 then yielded 3.48. Reduction of the N-formyl group with BH3-THF, followed by an aqueous acidic workup yielded alcohol 3.45 exclusively; however, desmethyldesbromoceratamine 3.49 was cleanly formed when a stream of dry HC1(g) was bubbled through a solution of formamide 3.48 in 50% 1,4- dioxane and water. MeI,K2C03 / DMF Scheme 3.15: Preparation of analogues 3.45 and 3.49 Examination of the 1H NMR spectrum of 3.49 (Figure 3.6) provides additional evidence that the two interconverting species observed in ii NMR spectra of 3.4 and 3.44 are rotamers about the C-21N-18 bonds in these compounds. With no N-methyl group, each rotamer is now equal and only one species is thus observed in the Ill NMR spectrum (Figure 3.7). N H2N— N— N BOM 0 3.27 \/ -0 HA0 THE H/ N HN—<’ J N— 1)AICI3,CH2I N N_ç BOM jj “o 2) H20 3.47: 80 % 0 3.48 3.42: 90 % 1) BH3-T E THF 2) H30 * N/\ HN—<. N 3.45: 33/o o N H2N—<. N— NN 0 3.49:15% / 116 ‘1 N H2N—. N— NN 0 3.49- Figure 3.6: ‘H NMR spectrum of 3.49 in DMSO-d6recorded at 400 MHz N H N\ HN”_K -\JNN Unequal rotamers I Hindered / “b Two species observed C-N bond in NMR spectrum / \ rotation / \3.44 — 3.44 - ________ Equal rotamers H N Hindered H N__- One species observed 0 C-N bond /\ 0 in NMR spectrum rotation 3.49”i? 3.49 - Figure 3.7: Interconverting rotamers of 3.44 and 3.49 117 Scheme 3.16 presents a mechanistic rationalization for the formation of C-il oxygenated species during the synthesis of desbromoceratamines. When intermediates of general structure 3.N bearing a 2-amino substituent are exposed to strong acid they can undergo ipso protonation at C-4 of the imidazole ring to give 3.0. Analogues of 3.N with methylthio, proton, chioro, or N-formyl substituents at C-2 are less basic and not expected to as readily undergo this type of C-4 protonation. Loss of a proton from the C- 3 NH in 3.0 gives the neutral species 3.P. Reaction of 3.P with atmospheric oxygen generates the radical 3.Q, which is greatly stabilized by the aromatic imidazo[4,5- djazepine core resonance structure 3.R. Reaction of 3.R with the hydroperoxide radical formed during the hydrogen abstraction step (3.P to 3.Q), can lead to the hydroperoxide 3.S that can decompose to give the C-li hydroxy (3.28 or 3.45) or keto (3.29 or 3.50) derivatives formed as the major products under most conditions. Alternatively, radical 3.R can abstract a hydrogen atom from some source to give the desired non-oxygenated analogues 3.44 and 3.49. The formation of 3.44 and 3.49 may in fact result from a radical chain reaction where the proton source is the intermediate 3.P. This mechanistic proposal provides an explanation for the highly variable relative yields of 3.44 and 3.45 that are formed when a dioxane/H20solution of 3.43 is treated with dry HC1 gas. The relative amounts of oxygen and compound 3.43 present in the reaction mixture should determine whether pathway A to C-il oxygenated products or pathway B to non oxygenated products will dominate. 118 R, N R N ______ N— I N— I N— H’ NIN H NNN_H N H’ N — 0 00 0/0 /0 / /0 3.N 3.0 3 N/ R, N/ R N/ — R, Nz7\ ,N—< I N N\ N— ;N— I N N— .0-OH H N” N H ________ H’ Nk_ + H N ____ 0 00 Path A 0H 0QH /0 0 0 // / 3.R 3.S3.29R=H 3.28R=H 3.50 R = Me 3.45 A = Me Path B XH N— I H’ /0 3.49 R = H 3.44 R = Me Scheme 3.16: Mechanistic rationale for formation of ceratamine analogues It should be noted that compound 3.50 has not been isolated; rather its existence is predicted based on the presence of 3.29. 119 3.3 Biological Results: Synthetic analogues 3.28, 3.29, 3.30, 3.31, 3.45, 3.44, and 3.49 as well as ceratamine A (3.4) (Figure 3.8) were evaluated for their ability to arrest cells in mitosis in a TG3 cell-based assay.88 The results are summarized in Figure 3.9. Not shown is the data for analogues 3.30 and 3.31 which were both completely inactive at all concentrations tested. \ N-/ N HN H2N N H2NN NNN N H N Br 0 0 /0 3.28 / 3.29 / 3.30 ceratamine A (3.4) \ N/’ \ N N4N N HN N HN N H2N NNN \/ /0 3.31 3.45 7 3.44 / 3.49 7 Figure 3.8: Analogues submitted for TG3 assay. 120 —+--- Ceratamine A 2 E 3.29C Izz_____ Figure 3.9: Summary of antimitotic screen for selected compounds The C-il hydroxy analogue 3.28 shows antimitotic activity at 10 j.tg/mL as previously reported89 but it does not generate a large percentage of cells arrested in mitosis compared with ceratamine A (3.4) and its activity falls off at higher concentrations. Desbromoceratamine A (3.44) shows the most promising activity of the synthetic analogues tested. At the highest concentration evaluated (50 jiglmL), 3.44 shows significant mitotic arrest ( 20%), while 3.49, which differs from desbromoceratamine A (3.44) simply by loss of the methyl substituent on the 2-amino group, showed no activity at the concentrations tested. The data in Figure 3.9 demonstrates that while the C-14 and C-16 bromine substituents in ceratamine A (3.4) are 0.1 1 10 100 [compound] ig/mL 121 not essential for antimitotic activity they make significant contributions to maximum potency and efficacy (3.4 versus 3.44). Similarly, it is apparent that methylation of the 2- amino functionality is required for effective antimitotic activity (3.44 versus 3.49). 3.4 Conclusions The functional inhibition of cellular microtubules is one of the most attractive and established approaches to improved cytotoxic anticancer drugs, either alone or in combination therapies. The clinical utility of the taxanes and the Vinca alkaloids has stimulated enormous interest in novel pharmacophores that disrupt microtubule function. To date, there are a number of distinct chemotypes of natural origin that display potent microtubule stabilizing activity. Some prominent members of this group include the taxanes,118 discodermolide,119 the eleutherobin structural family,120 and the epothilones.121 However, these chemical classes are often very structurally complex, creating considerable challenges to their preclinical development related to supply of compound. The structure of the ceratamines is significantly less elaborate than those mentioned above and their unique mode of action warrants further biological study. While our synthetic efforts have not provided us with additional amounts of ceratamine A, we have created scaleable routes to close analogues which display antimitotic activity. Important findings from the assay data shown in Figure 3.9 will serve to guide SAR efforts in order to design and synthesize novel analogues which capture both the potency and efficacy of the natural product, ceratamine A. 122 The ‘H NMR data for compounds 3•442, 3.30 and 3.44 provide insight into the potential aromaticity of the ceratamine analogues. Of particular interest were the resonances assigned to the enamide protons H-8 and H-9 and the proton(s) on the benzylic carbon C-il (Figure 3.10). The H-8 and H-9 enamide protons in 3.30 have chemical shifts (CD2C1)of 6 6.23 and 6.05 respectively, and the H-il proton appears as a singlet at 6 7.10. The 11-8 and H-9 resonances in 3.44 (DMSO-d6)are shifted significantly downfield to 6 7.70 and 6.40 respectively, very close to the corresponding shifts in ceratamine A [DMSO-d66 7.73 (H-8). 6.42 (H-9)1. The benzylic (H-il) protons in 3.44 and ceratamine A appear at 6 4.26 and 4.23 respectively, demonstrating the lack of the exocyclic alkene present in 3.30. Significant downfield shifts of the 11-8, H-9 and H-20 resonances in 3.4 and 3.44 compared with their counterparts in 3.30 can be attributed to a ring current, suggesting that the aminoimadzoazepine core heterocycles in the ceratamines and their synthetic counterparts (3.28, 3.29, 3.45, 3.44 and 3.49) have aromatic character. 6.42 773 6.40(H-9) (H-8) 6.05 6.23 7.70 (H0) 3.12 3.53 (H-i1)(-Br ç/)7•10 4.26 DMSO-d5 CD2I DMSO-d6 Figure 3.10: ‘H NMR chemical shifts of selected protons 123 l3 NMR assignments for the aminoimidazoazepine ring of 3.44 were in excellent agreement with the corresponding assignments for ceratamine A. The similarity between the ‘H and ‘3C NMR assignments for the synthetic analogue 3.44 and the natural product 3.4 provides strong support for the structure assigned to the natural product. In addition, the antimitotic activity observed for 3.44 (Figure 3.9) provides further evidence that the ceratamine pharmacophore, and not an undetected compound, is responsible for observed antimitiotic activity. One of the more intriguing findings of this synthetic effort is the discovery that the bromine atoms in ceratamine A contribute significantly to its potency as a microtubule stabilizer. One would expect that the pharmacophore of the ceratarnines would be based mainly on the imidazo[4,5-d]azepine ring system, and not the bromine atoms. Molecular modeling of the both ceratmine A and 3.44 shows that the addition of bromine atoms in ceratamine A does not force the molecule to adopt a different three- dimensional conformation than that of 3.44. Therefore, the bromines either add to potency by altering the steric bulk or the electronic nature of the phenyl ring allowing it to interact with its target (tubulin) more effectively, but by a mechanism which is currently obscure. Marine natural products typically contain more halogen atoms than those of terrestrial origin. The halide rich seawater environment (19,000 mg/L in Cl, 65 mg/L in Br) allows marine organisms to incorporate bromine, chlorine and iodine into covalent organic structures.122 As the above research and others researchers in this field 124 would be wise to view halogen atoms not only as structural oddities, but as key features in a particular marine based pharmacophore. In conclusion, we have synthesized the first example of a imidazo[4,5-d]azepine ring system via a robust and scaleable route. The analogues synthesized allowed us to confirm the structure of the natural product and give valuable insight into the ceratamine A pharmacophore. 125 3.5: Experimental: General Methods. All non-aqueous reactions were carried out in flame-dried glassware and under an Ar atmosphere unless otherwise noted. Air and moisture sensitive liquid reagents were manipulated via a dry syringe. Anhydrous tetrahydrofuran (THF) was obtained from distillation over sodium. All other solvents and reagents were used as obtained from commercial sources without further purification. 1H and ‘3C spectra were obtained on Bruker Avance 400 direct or Bruker Avance 600 cryoprobe spectrometers at room temperature unless otherwise noted. Flash chromatography was performed using Silicycle Ultra Pure silica gel (230-400 mesh) and using the following solvent gradients; (hexanes/EtOAc), (CH2C1/MeOH) or (EtOAc/MeOH). All biological assays were carried out by researchers in the laboratory of Dr. Michel Roberge at UBC. X-ray crystallography was performed by Dr. Brian 0. Patrick at UBC on a Bruker X8 APEX CCD single crystal X-ray diffraction instrument. Preparation of aldehyde 3.13 N CHO s N BOM SnBu3 BOM protected tribromoimidazole 3.12 (6.82 g, 16.1 mmol) was dissolved in 8OmL dry THF, and the solution was cooled to —78 °C. n-BuLi (10.0 mL, 1.6 M, 16.1 mmol) was added over a period of 7 mm, and the solution was stirred for 20 mm. MeSSMe (1.42 mL, 16.1 mmol) was then added, and the solution was stirred cold for 6 mm. The cold bath was removed and replaced with a water bath, and the reaction mixture was stirred 126 for 10 mm. The reaction mixture was re-cooled to —78 °C and n-BuLi (10.OmL) was slowly added. After 30 mi Bu3SnCl (4.33 mL, 16.1 mmol) was added, and the solution was stirred cold for 5 mm. The cryobath was replaced with a waterbath, and the reaction mixture was stirred for an additional 15 mm. The reaction mixture was cooled to —78 °C, and n-BuLi (10.0 mL) was slowly added. The reaction mixture was stirred cold for 30 mm, then DMF (3 mL) was very slowly added. The reaction mixture was stirred for lh at —78 °C, and then allowed to slowly warm to rt. After 10 mm at rt, the reaction was halted by the addition of H20. The crude reaction mixture was extracted into EtOAc, and washed with 3 x 1120. The organic phase was dried over Na2SO4, filtered and concentrated. The crude product was purified by column chromatography (hexanes/EtOAc) to yield 3.13 (4.2 g, 7.6 mmol, 48%) as a pale yellow oil. ‘H NMR (400 MHz, CDC13) S0.85 (t, J = 7.2 Hz, 9H), 1.16 (m, 6H), 1.28 (m, 6H), 1.46 (m, 6H), 2.71 (s, 3H), 4.48 (s, 2H), 5.34 (s, 2H), 7.31 (m, 5H), 9.84 (s, 1H); ‘3C NMR (100 MHz, CDC13)ö12.3, 14.6, 17.0, 28.2, 29.9, 71.5, 76.5, 128.5, 129.0, 129.4, 137.5, 145.4, 151.2, 151.9, 187.7; ESIMS [M+Naf’ calcd forC25H40NOSSnNa 575.1730, found 575.1740. Preparation of methyl ester 3.16 Stannane 3.13 (3.8 g, 6.9 mmol), bromide 3.15 (2.15 g, 7.58 mmol), Pd(PPh34(875 mg, 0.76 mmol) and CuT (1.31 g, 6.89 mmol) were combined under Ar. Dry toluene (60 mL) was added, and the solution was refluxed for 2.5 h. The solution was cooled and I0 127 concentrated to dryness. The residue was redissolved in CH21,filtered through Celite and concentrated. The crude product was purified by column chromatography (hexanes/EtOAc) to yield 3.16 (2.77 g, 6.12 mmol, 90%) as a pale yellow solid. ‘H NMR (400 MHz, CDC13) 82.79 (s, 3H), 3.71 (s, 3H), 3.76 (s, 3H), 4.37 (s, 2H), 5.00 (d, J 10.9 Hz, 1H), 5.08 (d, J = 10.9 Hz, 1H), 6.76 (d, J = 8.7 Hz, 2H), 7.02 (d, J = 8.7 Hz, 2H), 7.12 (m, 2H), 7.27 (m, 3H), 8.10 (s, 1H), 9.73 (s, 1H); ‘3C NMR (100 MHz, CDC13) 6 16.4, 53.6, 56.4, 71.8, 74.2, 115.6, 126.5, 128.5, 129.0, 129.4, 133.4, 135.3, 137.2, 137.7, 139.9, 148.1, 149.6, 162.8, 167.3, 184.9; ESIMS [M+Naf’ calcd for C24HNO5SNa475.1304, found 475.1307. Preparation of vinyl bromide 3.17 Br \ F’J OMe BOM (Bromomethyl)triphenylphosphonium bromide (502 mg, 1.15 mmol) was suspended under Ar in lOmL dry THF. LiHMDS (1.06 mL, 1.0 M in THF, 1.06 mmol) was added and the resulting bright yellow solution was stirred at ii for 30 mm. The solution was cooled to —78 °C and 1.2 mL HMPA was added. After 5 mm, a solution of aldehyde 3.16 (372 mg, 0.822 mmol) in 3 mL dry THF was added over a period of 5 mm. The reaction mixture was stirred for 2 h at —78 °C, after which time satd. NaHCO3was added to the cold reaction mixture. The reaction mixture was allowed to warm to rt, and the crude product was extracted with EtOAc. The organic phase was washed with 2 x H20, dried 128 over Na2SO4,filtered and concentrated. The crude compound was purified by column chromatography (hexanes/EtOAc) to yield bromide 3.17 (312 mg, 0.59 mmol, 71%) as a bright yellow solid. ‘H NMR (600 MHz, CD21) 82.75 (s, 3H), 3.68 (s, 3H), 3.77 (s, 3H), 4.37 (s, 2H), 5.08 (d, J= 11.1 Hz, 1H), 5.12 (d, J= 11.1 Hz, 1H), 6.16 (d, J= 8.3 Hz, 1H), 6.66 (d, J = 8.3 Hz, 1H), 7.12 (m, 4H), 7.25 (m, 3H), 8.03 (s, 1H); ‘3C NMR (150 MHz, CD21) 816.4, 52.9, 55.9, 70.9, 74.0, 104.7, 114.8, 116.2, 123.5, 126.6, 128.0, 128.3, 128.8, 129.3, 133.0, 136.5, 137.4, 146.2, 147.0, 162.2, 167.5; ESIMS [M+H] calcd forC25H679BrN204S 529.0797, found 529.0782. Preparation of HOBt ester 3.18 Bromide 3.17 (312 mg, 0.59 mmol) was dissolved in 2 mL THF. LiOH-H20(75 mg, 1.78 mmol) was added, followed by 0.5 mL H20. The solution was stined rapidly for 24 h at rt. The reaction mixture was then acidified, extracted into EtOAc, and the organic phase was washed with 3 x H20, dried over Na2SO4, filtered and concentrated. The residue was then redissolved in 2 mL CH21, and HOBt (118 mg, 0.88 mmol) and DMAP (-1 mg) were added. The solution was stirred for 10 mm, and then DIPC (136 tL, 0.88 mmol) was added. The reaction mixture was stirred rapidly at ii for 1 h, concentrated and purified by column chromatography (hexanes/EtOAc). The HOBt ester 3.18 was isolated as a bright yellow solid (322 mg, 0.51 mmol, 86%). ‘H NMR (400 B 129 oI ‘8LI ‘VLT ‘0LZI‘09Z’1 ‘8II ‘9L1I ‘WEll ‘WI7OI‘LL ‘t’OL ‘wvc‘9‘ccI g (1IDu ‘ZHN oci) INN , (HT ‘s) ‘(HE ‘w) 6L ‘(Hi ‘ui) cit. ‘(Hi ‘ZH 68=I ‘p)Z0L ‘(Hi ‘ZH 68 =I‘P) 8L9 ‘(HI ‘ZH 08 =I ‘P)17L9‘(fJJ ‘Zil 08 =I‘P) LZ9 ‘(HT ‘iq ‘s) Lç‘(HI ‘ZH WOt =I‘P) L0c‘(HI ‘ZH WOI =I‘P) E6i‘(Hr ‘s) ovt ‘(HE ‘s) 9LE ‘(HE ‘s) 8L‘(HI ‘HLi =r ‘p) 1LZ2 (ZIYuD ‘ZHN 009) 1NNH1 iU0JMOJp( iCqqs isi (°bo6 ‘10U1 EE0 ‘W cU) 61E PP!’ 01 j3O1tU1UDU0C) pUTt P11!J ‘OSN JA0 poup sis1?qdDJUflJ0qjODH’NP1SX‘OHXI‘IDH141coXqlJM pqsisn snqd iuiJo qj orjj 01W ppiujx uq pui‘unu jojpauis si uorinios qj panddisip JOJOD MOJpiC iqiJq qi inun (‘pu E xoiddi) ppp s1’ (iHi ui iAi oz) HNN 1DHD ‘1W 1 UTpAf0SSTp S1M (10mw LEO ‘uJ oi) 8fl IHOH Xfl v’Jo VJHN o 6U P!WP JOUO!JU.ItdIJ 8L60E9 punoj ‘L960E9 SOcNJH 6LLHoQ J0J 3J+[H+141] S1AllSL t19l‘FI9l‘czci ‘WL171‘VfrI7I‘8ET ‘9LE1‘LVEI‘L6zT ‘96z1 ‘E6z1 ‘681I‘L8z1‘L9z1 ‘V9ZT‘LczT ‘Omzi ‘IZI‘WcI[‘FIJI ‘V601‘0L01 ‘cL‘WIL ‘c9c ‘L91 g (1DuD ‘ZHIAJ 001) 1INN z, (Hj ‘s) 9E8‘(HI ‘ZH YLI ‘P) 908‘(Hz ‘W) cL ‘(H9 ‘m) 8zL‘(Hz ‘m) IZL‘(HI ‘Zfl E8=f ‘p) c69 ‘(Hz ‘Zil 68=r‘p) 8W9 ‘(HI ‘ZH E8=I‘P)Ot’9 ‘(HI ‘ZH 601=I ‘p)‘Ec‘(HI ‘ZH 601 =I ‘P)ozc ‘(HI ‘Zil WII=I ‘P)‘(HI ‘ZR WTI=r‘p) oci7 ‘(HE ‘s) tE‘(HE ‘s) owzc (ZIYUD ‘zHN 128.1, 131.4, 136.1, 136.2, 142.2, 146.6, 160.6, 165.3; ESIIv1S [M+Na] calcd for C25H679BrN3OSNa550.0776, found 550.0757. Preparation of bicycle 3.20 Amide 3.19 (47.7 mg, 0.09 1 mmol), Cul (17.3 mg, 0.09 1 mmol) and Cs2O3 (59.2 mg, 0.18 mmol) were combined under Ar in 3 mL dry THF. N,N’-Dimethylethylenediamine (20 IlL, 0.18 mmol) was added, and the solution was stirred at rt for 24 h. The reaction mixture was diluted with EtOAc and filtered through Celite. The crude product was purified by column chromatography (hexanes/EtOAc) to yield 3.20 (37.6 mg, 92%) as a yellow solid. ‘H NMR (600 MHz, CD21) 82.65 (s, 3H), 3.26 (s, 3H), 3.76 (s, 311), 4.28 (d, J= 11.9 Hz, 1H), 4.31 (d, J= 11.9 Hz, 1H), 4.55 (d, J= 11.3 Hz, 1H), 4.83 (d, J = 11.3 Hz, 1H), 6.10 (d, J = 9.1 Hz, 1H), 6.22 (d, J = 9.1 Hz, 1H), 6.76 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 7.21-7.3 1 (m, 5H); ‘3C NMR (150 MHz, CD21)815.2, 37.5, 54.8, 70.0, 72.4, 107.6, 113.6, 121.3, 123.3, 126.6, 127.3, 127.4, 127.7, 127.8, 129.9, 133.1, 136.5, 136.6, 148.2, 159.7, 168.9; HRESIMS {M+Naf calcd for C25HN3OSNa470.1514, found 470.1495. \ N s N BOM 0 131 Preparation of methyl ester 3.23 Aldehyde 3.16 (54.3 mg, 0.12 mmol) was dissolved in 5 mL THF. LiOH-H20(10.1 mg, 0.24 mmol) was added followed by 1 mL H20 and the resulting mixture was stirred rapidly for 24 h. The reaction mixture was acidified by the addition of 1 M HC1 and extracted into EtOAc. The organic phase was dried over Na2SO4, filtered and concentrated. The resulting solid was redissolved in 10 mL CH21 and N methylsarcosine hydrochloride (19 mg, 0.13 mmol) followed by DIEA (23 mL, 0.13 mmol) and a catalytic amount of DMAP. The solution was stirred for 10 mm, then DIPC (20.4 mL, 0.13 mmol) was added. The solution was stirred for 1 h at rt, then concentrated to dryness. The residue was redissolved in EtOAc and the organic phase was washed with 3 x 1 M HC1, followed by satd NaHCO3. The organic phase was dried over Na2SO4,filtered and concentrated. This residue was dissolved in 15 mL dry THF. NaH (10 mg, 60 % in oil, 0.25 mmol) was added, followed by 3 drops of DMSO. The solution was then stirred rapdly at rt for 24 h. The reaction was then halted by addition of water, extracted into EtOAc and washed with 3 x satd NaHCO3. The organic phase was dried over Na2SO4,filtered and concentrated. The crude product was purified by flash chromatography (hexanes/EtOAc) to yield 3.23 (50.5 mg, 0.1 mmol, 76 %) as a bright yellow solid. ‘H NMR (600 MHz, CD21)82.69 (s, 3H), 3.15 (s, 3H), 3.76 (s, 3H), 3.83 BOM 132 EEl ‘cgi ‘t’xT ‘E81‘9LZ1‘cii ‘LThTT‘V1711 ‘9801 ‘6t’L‘IL ‘wcc ‘98E9(1EYUD ‘zHT,\ oci) u’iN ,(Hi ‘s) 9LL‘(i-it’ ‘w) 8L‘(HI ‘s) 6FL‘(HI ‘s) 8FL ‘(Hr ‘zH 88 =I ‘P) 9 ‘(Hz ‘ZH W8=f ‘1’)cL9 ‘(HI ‘ZH 06=I‘P) LZ9‘(HI ‘ZH 06 =I ‘I’)EF9 ‘(HI ‘Zil liT=1‘P) OL’17‘(HI ‘ZH Iii=1‘P)z917‘(HI ‘Zil 6’ll =1‘P) Ot7‘(HI ‘ZH 6J r =r‘p)9z17 ‘(HE ‘s) 9LE ‘(HE ‘s) 9Z9(j3Zq‘ZHN009)NlANH,P110sM0Jpi isi (%IE ‘10mw gzo ‘u FElT)9Z7 pii’ °(3yojsuxq) lqd1JovmoJqD uuinoo iCqpijund si irnpoid pn.io qj ssui(ip oipiuouo ar SUOflfl[Os CflU1?JO puiqmoc) qj DVo1[ ‘rn’001 pui UOM ‘P’001‘HOV1 m 001 ql’M pqsipui paijij suonnjos qjrnitwuiiJns qi jo uoiidmnsuoo IduJo3 poqs (3yoJsu1xq) sisicjnui ‘1j I!un uonioiuos qiiunjoi uIufl{Sisn pppr SflMp)jDiU iCUt?)J ‘1uI 01 UT pAJOSS1p S11’ (JOUIUJ 6W0 ‘m oot’) oz pijng N N /> —N 9Z I0ZUP!W! JOUO!JUJtd.IJ Lccr8zc punoj ‘69cF8zc NgzcoEN 6lHLD ioj ÷[N+I’I] SIAIISI LiL1 ‘Fc91‘FI9T‘cocl ‘ELEI‘W9ET‘FIEI ‘W8z1‘t’8z1 ‘69z1‘cizi‘6izl ‘8t’I1 ‘LEL‘zJL‘gcc‘6zc‘LLE ‘6c1 2 (TYuD ‘zHI,% ocl) II’\IN , (Hi ‘s) 9cL ‘(H9 ‘at) czL‘(Hz ‘zH 68=r‘p)069 ‘(Hz ‘zH 68=r‘p)çL9‘(HI ‘zil cii=1‘p) ct’‘(HI ‘zH Eli=1‘P)Lçt’‘(Hz ‘s) 6V‘(HE ‘s) 128.9, 129.2, 130.9, 134.9, 137.2, 140.8, 160.7, 170.0; ESIMS [M+Na] calcd for C24H3N3Oa424.1637, found 424.1647. Preparation of amine 3.27 N H2N—<’ N— N BOM 0 Compound 3.26 (11.7 mg, 0.029 mmol) was dissolved in 2 mL dry THF under Ar and cooled to —78 °C. MeLi (50 uL, 1.6 M, 0.080 mmol) was added and the resulting bright yellow solution was stirred for 10 mm at —78 °C. A solution of TsN3 (70 mg, 0.35 mmol) in 2 mL dry THF was added slowly, and the resulting yellow solution was stirred for 10 mm at —78 °C. H20 was added, and the solution was allowed to warm to rt. The reaction mixture was partitioned between EtOAc and satd NaHCO3 and the organic phase was dried over Na2SO4,filtered and concentrated. The crude residue was then redissolved in MeOH, and 10% Pd/C (20 mg) was added. The solution was saturated with H2 and stirred for 30 mm at rt. The reaction mixture was filtered through a 0.45 im Millex membrane, washed with MeOH and concentrated. The crude product was purified by column chromatography (EtOAc/MeOH) to yield 3.27 (9.0 mg, 0.02 1 mmol, 72%) as a bright yellow solid. 1H NMR (600 MHz, CD21)83.26 (s, 3H), 3.76 (s, 3H), 4.19 (d, J = 11.5Hz, 1H), 4.23 (d, J = 11.5 Hz, 1H), 4.45 (s, br, 2H), 4.52 (s, 2H), 6.03 (d, J = 8.9 Hz, 1H), 6.07 (d, J = 8.9 Hz, 1H), 6.77 (d, J = 8.9 Hz, 2H), 6.97 (d, J = 8.9 Hz, 2H), 7.18 (m, 3H), 7.28 (m, 3H); 13C NMR (150 MHz, CD21) 838.7, 55.8, 70.8, 72.6, 109.1, 134 114.6, 119.2, 122.5, 127.8, 128.1, 128.5, 128.7, 128.9, 130.9, 131.9, 134.0, 137.2, 152.3, 160.3, 170.0; HRESIMS [M+Naf’ calcd forC24HN4O3a439.1746, found 439.1735. Preparation of amino alcohol 3.28 H2N Compound 3.27 (9.0 mg, 0.02 1 mmol) was dissolved in CH21 (1 mL). Anhydrous A1C13 (10 mg, 0.075 mmol) was added and the resulting blood red slurry was stirred at rt for 10 mm. The solution was cooled to 0 °C and water was slowly added until all solids had dissolved. The resulting colorless biphasic system was then stirred rapidly with mixing for 5 mm, after which a bright yellow color results. Satd NaHCO3 was added, and the reaction mixture was extracted into EtOAc. The aqueous phase was saturated with NaC1 and extracted a second time with EtOAc. The combined organic phases were dried over Na2SO4,filtered and concentrated. The crude product was purified by column chromatography (CH2C1/MeOH) to yield 3.28 (4.0 mg, 0.0 13 mmol, 62%) as a bright yellow solid. 1H NMR (600 MHz, CD3O ) 63.65 (s, 3H), 3.74 (s, 3H), 6.34 (s, 1H), 6.59 (d, J = 10.0 Hz, 111), 6.80 (d, J = 8.8 Hz, 2H), 7.42 (d, J = 8.8 Hz, 2H), 7.81 (d, J = 10.0 Hz, 1H); ‘3C NMR (150 MHz, CD3O ) 645.1, 55.7, 75.3, 102.3, 114.6, 127.0, 129.2, 137.1, 145.6, 160.4, 160.5, 165.7, 173.2, 176.4; ESIMS [M+Naf’ calcd for C16HN4O3a335.1120, found 335.1115. 135 Preparation of amino ketone 3.29 H2N Ketone 3.29 (1 mg, 0.0032 mmol, 15%) was also isolated from the crude as a yellow solid. ‘H NMR (600 MHz, CD3O ) 83.66 (s, 3H), 3.86 (s, 3H), 6.65 (d, J = 10.2 Hz, 1H), 6.98 (d, J = 8.8 Hz, 2H), 7.85 (d, J = 8.8 Hz, 2H), 7.89 (d, J 10.2 Hz, 1H); ‘3C NMR (150 MHz, CD3O ) 644.3, 56.2, 102.4, 115.1, 129.9, 133.3, 146.2, 159.5, 164.6, 165.8, 173.5, 193.6; ES]MS [M+Naf’ calcd for C,6H14N4O3a 333.0964, found 333.0953. Preparation of suiphide 3.30 Compound 3.20 (10 mg, 0.022 mmol) was dissolved in 1 mL CH21. Anhydrous A1C13 (5 mg, 0.038 mmol) was added and the blood red slurry was stirred at ‘1 for 10 mm. The reaction mixture was then cooled to 0 °C and water was added until all solids had dissolved. EtOAc (5 mL) was added, and the biphasic solution was stirred rapidly for 30 136 mm. Satd. NaHCO3 was added to the solution and the mixture was partitioned. The organic phase was dried over Na2SO4,filtered and concentrated. The crude product was purified by column chromatography (hexanes/EtOAc) to yield 3.30 (5 mg, 0.0 15 mmol, 68%) as a yellow solid. ‘H NMR (600 MHz, CD71)32.60 (s, 3H), 3.12 (s, 311), 3.78 (s, 3H), 6.05 (d, J = 9.1 Hz, 1H), 6.23 (d, br, J = 9.1 Hz, 1H), 6.74 (d, J = 8.8 Hz, 2H), 6.88 (s, br, 2H), 7.10 (s, 1H); ‘3C NMR (150 MHz, CD21) 6 16.4, 39.5, 55.8, 104.4, 114.0, 114.2, 127.3, 128.6, 131.2, 132.1, 143.2, 145.9, 160.6, 169.6; ESIMS [M+Hf’ calcd forC17H,8N302S328.1120, found 328.1110. Preparation of imidazole 3.31 Compound 3.26 (10.0 mg, 0.025 mmol) was dissolved in CH21. Aid3 (20 mg, 0.15 mmoi) was added and the deep red slurry was stirred at rt for 15 mm. The reaction was halted with the addition of H20. Satd. NaHCO3was added and the crude compound was extracted with EtOAc. The organic phase was dried over Na2SO4, filtered and concentrated. The crude product was purified by column chromatography (hexanes/EtOAc) to yield 3.31 (5.0 mg, 0.017 mmoi, 71%) as a yellow solid. ‘H NMR (600 MHz, CD21)63.06 (s, 311), 3.75 (s, 3H), 6.03 (d, J = 9.1 Hz, 1H), 6.27 (d, J = 9.1 Hz, 1H), 6.69 (d, J = 8.8 Hz, 2H), 6.72 (d, J = 8.8 Hz, 211), 7.05 (s, 1H), 7.68 (s, 111); ‘3C 137 NMR (150 MHz, CD21) ö39.3, 55.8, 109.2, 114.1, 122.4, 127.4, 127.9, 128.3, 130.8, 131.1, 134.3, 137.9, 160.4, 170.3; ElMS calcd for C16H5N302 281.11643, found 281.11651 Preparation of chloride 3.32 N Br cI N Br BOM Tribromide 3.12 (6.97 g, 16.4 mmol) was dissolved in 60 mL dry THF. The solution was cooled to —78 °C, and n-BuLi (10.3 ml, 1.6 M in hexanes, 16.4 mmol) was added slowly. The solution was stirred cold for 20 mm, then a solution ofC216 (4.27 g, 18.04 mmol) in 10 mL dry THF was added over a period of 6 mm. The reaction mixture was stirred at — 78 °C for 10 mm, then warmed to rt for an additional 20 mm. The reaction was halted by addition of H20 and the product was extracted into EtOAc. The organic layer was dried over Na2SO4,filtered and concentrated. 4.64 g (12.1 mmol, 74%) of chloride 3.32 could be recovered by crystallization from cold hexanes, and flash chromatography (hexanes/EtOAc) of the remaining residue yielded an additional 1.24 g (3.3 mmol, 20%) of 3.32 (94% yield overall). 138 Preparation of aldehyde 3.33 N CHO ciX N BOM SnBu3 Chloride 3.32 (3.60 g, 9.46 mmol) was dissolved in 36 mL dry THF and cooled to —78 °C. n-BuLi (5.91 mL, 1.6 M in hexanes, 9.46 mmol) was slowly added and the solution was stirred cold for 20 mm. Bu3SnCI (2.55 mL, 9.46 mmol) was then added, the solution was stirred at —78 °C for 10 mm, then placed in a cold-water bath. This solution was stirred for 10 mm, then re-cooled to —78 °C. n-BuLi (5.91 mL, 1.6 M in hexanes, 9.46 mmol) was slowly added and the solution was stirred for 20 mm. DMF (2.0 mL) was then slowly added, the solution stirred at —78 °C for 10 mm, then the reaction mixture was allowed to slowly warm to rt. The reaction mixture was stirred for 10 mm at rt, then quenched by addition of water. The reaction mixture was extracted into EtOAc, and the organic phase dried over Na2SO4, filtered and concentrated. The crude product was purified by column chromatography (hexanes/EtOAc) to yield stannane 3.33 (3.16 g, 5.85 mmol, 62%) as a colorless oil. 111 NMR (400 MHz, CD21)öO.89 (t, J = 7.3 Hz, 9H), 1.24 (m, 5H), 1.33 (m, 7H), 1.53 (m, 6H), 4.54 (s, 2H), 5.43 (s, 2H), 7.35 (m, 511), 9.81 (s, 1H); ‘3C NMR (100 MHz, CD21) ö 12.2, 14.4, 28.2, 29.9, 71.5, 76.6, 128.5, 129.0, 129.4, 137.5, 137.8, 144.8, 150.4, 187.5; ESIMS [M+Na] calcd for C24H3751NO1 6SnNa559.1459, found 559.1447. 139 Preparation of amide 3.34 Br)N oxx Ester 3.15 (10.9 g, 40.4 mmol) was dissolved in 30 mL THF, 5 mL H20, and 10 mL MeOH. LiOH-H20(3.40 g, 80.8 mmol) was added, and the reaction mixture was stirred vigorously at rt for 2 h. The reaction mixture was then acidified with 1 M HC1 and extracted into EtOAc. The organic layer was dried over Na2SO4, filtered and concentrated to dryness. The resulting solid was redissolved in 70 mL CH21. HOBt (8.2 g, 60.6 mmol) and DMAP (-10 mg) were added and the solution was cooled to 0 °C. DIPC (9.38 mL, 60.6 mmol) was slowly added and the solution was stirred for 10 mm at 0 °C. The reaction mixture was warmed to rt, and stirred for an additional 1 h. The reaction mixture was then concentrated to dryness. The residue was then redissolved in THF and treated with MeNH2 (50 mL, 2.0 M in THF, 100 mmol) for 10 mm. The reaction mixture was then extracted into EtOAc, and washed with 2 x 1 M HC1 and 2 x satd. NaHCO3 The organic layer was dried over Na2SO4,filtered and concentrated. The crude product was purified by column chromatography (hexanes/EtOAc) to yield 3.34 (10.3 g, 38.4 mmol, 95%) as a colorless oil which crystallized upon standing. ‘H NMR (400 MHz, CDC13)82.96 (d, J = 4.8 Hz, 3H), 3.85 (s, 3H), 6.83 (s, br, 1H), 6.94 (d, J = 8.7 Hz, 2H), 7.80 (d, J = 8.7 Hz, 2H), 8.27 (s, 111); 13C NMR (100 MHz, CDC13) 828.4, 56.3, 113.3, 114.8, 127.5, 132.9, 137.9, 161.7, 164.2; ESIMS [M+Na] calcd for C11H279BrNO2Na291.9949, found 291.9940. 140 Preparation of amide 3.35 CHO H Stannane 3.33 (2.86 g, 5.30 mmol), amide 3.34 (1.58 g, 5.83 mmol), Pd(PPh34(673 mg, 0.58 mmol) and Cul (544 mg, 2.86 mmol) were combined in 50 mL dry THF and stirred at ri for 20 h. The reaction mixture was then concentrated to dryness, redissolved in CH21 and filtered through Celite. The resulting solution was then concentrated and the crude product was purified by column chromatography (hexanes/EtOAc) to yield 3.35 (1.70 g, 3.86 mmol, 73%) as a slightly yellow solid. ‘H NMR (400 MHz, CD21)c52.82 (d, J = 4.8 Hz, 3H), 3.77 (s, 3H), 4.51 (s, 2H), 5.08 (d, J = 10.7 Hz, 1H), 5.22 (d, J = 10.7 Hz, 111), 6.24 (s, br, 111), 6.78 (d, J= 8.8 Hz, 2H), 6.98 (d, J= 8.8 Hz, 2H), 7.18 (m, 2H), 7.81 (m, 2H), 8.07 (s, 1H), 9.67 (s, 1H); 13C NMR (100 MHz, CD21)827.8, 56.3, 72.4, 74.5, 115.4, 118.3, 126.8, 128.6, 129.1, 129.4, 132.7, 137.2, 137.6, 137.9, 138.9, 144.8, 162.3, 166.1, 184.5; ESIMS [M+Na] calcd forC23H2351NO4Na 462.1197, found 462.1182. 141 Preparation of vinyl bromide 3.36 N H (Bromomethyl)triphenylphosphonium bromide (1.85 g, 4.24 mmol) was slurried in 50 mL dry THF. Potassium tert-butoxide (476 mg, 4.24 mmol) was added, and the resulting yellow solution was stirred at rt for 10 mm. The ylide solution was then cooled to —78 °C and a 10 mL THF solution of aldehyde 3.35 (933 mg, 2.12 mmol) was added slowly. The reaction mixture was stirred at —78 °C for 30 mm, then allowed to warm to ii for an additional 20 mm. The reaction was halted by addition of water, and the product was extracted with EtOAc, then with 2 x CH21. The combined organic phases were dried over Na2SO4,filtered and concentrated. The resulting crude residue was dissolved in 20 mL CH21 and allowed to sit for 10 mm. The resulting ppt was filtered off and washed with 5 mL CH21 to yield 3.36 (0.50 g, 0.97 mmol, 46%). An additional 370 mg (0.72 mmol. 34%) could be isolated from the remaining residue by column chromatography (hexanes/EtOAc) (80% yield overall). 1H NMR (400 MHz, CD21) ö2.74 (d, J = 5.0 Hz, 3H), 3.78 (s, 3H), 4.46 (s, 2H), 5.00 (d, J = 10.8 Hz, 111), 5.16 (d, J = 10.8 Hz, 1H), 5.72 (s, br, 1H), 6.31 (d, J = 8.3 Hz, 111), 6.73 (d, J = 8.3 Hz, 1H), 6.80 (d, J = 8.9 Hz, 2H), 7.04 (d, J = 8.9 Hz, 2H), 7.16 (m, 2H), 7.80 (m, 3H), 8.07 (s, 1H); ‘3C NMR (100 MHz, CD21) 527.6, 56.3, 72.0, 74.2, 107.1, 115.3, 118.5, 122.8, 127.1, 128.4, 128.9, 142 129.4, 129.7, 132.8, 135.5, 137.0, 137.4, 144.6, 162.2, 166.4; ESIMS [M+Naf’ calcd for C24H379Br351NONa538.0509, found 538.0499. Preparation of chioroimidazole 3.37 N CI—< N— Bromide 3.36 (682 mg, 1.31 mmol), CuT (50 mg, 0.262 mmol) and Cs2O3(853 mg, 2.62 mmol) were combined in 40 mL dry THF. N,N’-Dimethylethylenediamine (57.0 iL, 0.524 mmol) was then added, and the solution was heated to 70 °C for 20 h. The reaction mixture was cooled to rt and concentrated to dryness. The residue was redissolved in CH21 and filtered through Celite. The resulting solution was concentrated to dryness again, and purified by column chromatography (hexanes/EtOAc) to yield 3.37 (486 mg, 1.11 mmol, 85%) as a pale yellow solid. A single crystal suitable for X-ray diffraction analysis was grown by infusion of MeOH into a solution of 3.37 in toluene. 111 NMR (300 MHz, CD21)ö3.27 (s, 3H), 3.77 (s, 3H), 4.35 (s, 211), 4.57 (d, J= 11.5 Hz, 1H), 4.90 (d, J= 11.5 Hz, 1H), 6.13 (d, J= 9.0 Hz, 1H), 6.19 (d, J= 9.0 Hz, 1H), 6.77 (d, J= 8.6 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H), 7.28 (m, 611); ‘3C NMR (75 MHz, CD21)838.2, 55.4, 70.9, 73.2, 107.9, 114.4, 121.5, 123.5, 126.9, 127.9, 128.0, 128.4, 129.1, 130.5, 134.9, 136.1, 136.2, 136.9, 160.4, 169.3; ESIMS [M+Naj calcd forC24H2351NONa 458.1247, found 458.1242. 143 Preparation of amine 3.27 from chloride 3.37 N H2N—K” N— Chloride 3.37 (60 mg, 0.14 mmol), triphenylsilylamine (46.3 mg, 0.17 mmol), Pd2(dba)3 (13.0 mg, 0.014 mmol) and XPhos (16.2 mg, 0.034 mmol) were combined under Ar in 4.0 mL dry toluene. LiHMDS (182 j.tL, 1.0 M in toluene, 0.182 mmol) was added and the solution was heated to 100 °C for 1 h. The reaction mixture was cooled to rt and diluted with EtOAc. 1.0 M HC1 was added, and the biphasic mixture was stirred rapidly for 10 mill. The aqueous layer was basified with NaHCO3 and extracted into EtOAc. The organic layer was dried over Na2SO4,filtered and concentrated. The crude product was purified by column chromatography (EtOAcIIVIeOH) to yield 3.27 (40 mg, 0.098 mmol, 69%) as a yellow solid. ‘H NMR (600 MHz, CD21)ö3.26 (s, 3H), 3.76 (s, 3H), 4.19 (d, J = 11.5 Hz, 1H), 4.23 (d, J = 11.5 Hz, 1H), 4.45 (s, br, 2H), 4.52 (s, 2H), 6.03 (d, J = 8.9 Hz, 1H), 6.07 (d, J = 8.9 Hz, 1H), 6.77 (d, J = 8.9 Hz, 2H), 6.97 (d, J = 8.9 Hz, 2H), 7.18 (m, 3H), 7.28 (m, 3H); ‘3C NMR (150 MHz, CD21)838.7, 55.8, 70.8, 72.6, 109.1, 114.6, 119.2, 122.5, 127.8, 128.1, 128.5, 128.7, 128.9, 130.9, 131.9, 134.0, 137.2, 152.3, 160.3, 170.0; ESIMS [M+NaT’ calcd for C24HN4O3a 439.1746, found 439.1735. 144 Preparation of chloroimidazole 3.39 Chloride 3.37 (30 mg, 0.069 mmol) was dissolved in 2 mL CH21. A1C13 (92 mg, 0.69 mmol) was added, and the blood red slurry was stirred for 10 mm at rt. The reaction mixture was quenched with H20, basified with satd NaHCO3 and extracted into EtOAc. The organic phase was dried over Na2SO4,filtered and concentrated to yield deprotected chloride 3.38. Without further purification, this compound was redissolved in DMF. K2C03 (18 mg, 0.13 mmol) was added, followed by BOM-Cl (16.2 1iL, 0.07 mmol), and the solution was stirred for 30 mm at rt. The reaction mixture was extracted into EtOAc and washed with 3 x H20. The organic phase was dried over Na2SO4, filtered and concentrated. TLC analysis showed a 1:1 mixture of regenerated 3.37 and regioisomer 3.39. This compound was purified by column chromatography (hexanes/EtOAc) to yield 50 (10.3 mg, 0.024 mmol, 35%, 2 steps) as as yellow solid. ‘H NMR (600 MHz, CD21) S3.25 (s, 3H), 3.79 (s, 3H), 4.59 (s, 2H), 5.40 (s, 2H), 6.04 (d, J= 9.1 Hz, 1H), 6.17 (d, J = 9.1 Hz, 1H), 6.78 (d, J = 8.9 Hz, 211), 7.31 (m, 8H); ‘3C NMR (150 MHz, CD21)ö 39.2, 55.8, 71.4, 73.7, 100.6, 113.9, 125.7, 128.3, 128.7, 128.9, 129.0, 130.8, 132.0, 133.1, 134.3, 136.0, 137.0, 160.1, 170.6; ESIMS [M+Na] calcd forC24H235INONa 458.1247, found 458.1242. 145 Preparation of N-metliyl-formamide 3.42 HN N Chloride 3.37 (208 mg, 0.48 mmol), Cs2O3 (234 mg, 0.72 mmol), Pd2(dba)3 (22 mg, 0.024 mmol) and 3.41 (58 mg, 0.12 mmol) were combined in 4.5 mL dry toluene. N methylformamide (43 mL, 0.72 mmol) was added and the slurry was refluxed for 24 h. The reaction mixture was then cooled to rt and concentrated to dryness. The residue was redissolved in CH21, filtered through Celite, and concentrated to give an orange oil. The crude product was purified by column chromatography (hexanes/EtOAc) to yield 3.42 (159 mg, 0.34 mmol, 72%) as a slightly yellow oil. 1H NMR (600 MHz, CD21) 83.17 (s, 3H), 3.29 (s, 3H), 3.76 (s, 311), 4.28 (s, 2H), 4.53 (d, J= 11.3 Hz, 1H), 4.59 (d, J= 11.3 Hz, 1H), 6.16 (d, J= 9.1 Hz, 1H), 6.21 (d, J= 9.1 Hz), 6.75 (d, J= 8.3 Hz, 2H), 6.88 (d, J = 8.3 Hz, 2H), 7.18 (m, 2H), 7.30 (m, 4H), 8.31 (s, 111); 13C NMR (150 MHz, CD21) 631.9, 37.6, 54.8, 70.4, 72.1, 107.5, 113.6, 121.1, 121.8, 126.3, 127.4, 127.5, 127.9, 128.7, 129.9, 133.9, 136.0, 145.6, 159.9, 161.7, 168.9; ESIMS [M+K] calcd for C26HN4039K497.1591, found 497.1601. 146 Preparation of des-bromoceratamine A 3.44 Formamide 3.42 (90 mg, 0.19 mmol) was dissolved in 10 mL CH21. Anhydrous Aid3 (25 mg, 1.9 mmol) was added, and the slurry was stirred rapidly for 30 mm. The solution was quenched by careful addition of satd NaHCO3,and the crude reaction mixture was extracted into EtOAc. The organic phase was dried over Na2SO4, filtered and concentrated to yield 3.43. ESIMS [M+Na] calcd forC18HN4O3a 361.1277, found 361.1275. The resulting product was used without further purification. The crude 3.43 was then dissolved in 25 mL 1 ,4-dioxane and diluted with 25 mL deionized water. Ar was then bubbled through the solution for 30 minutes. The solution was cooled to 0 °C and anhydrous HC1(g) was bubbled through the solution for 1 mm. The solution was then allowed to sit for an additional 2 mm. The pH of the solution was then brought to neutral with addition of 5 M NaOH, then further basified with NaHCO3. The reaction mixture was extracted into EtOAc and the bright yellow organic phase was dried over Na2SO4 and concentrated. The crude product was purified by flash chromatography (CH2C1IMeOH) to yield 3.44 (15 mg, 0.048 mmol, 25%, 2 steps) as a bright yellow solid. Compound 3.45 was also isolated from the product mixture (see below). The 1H NMR spectrum showed a 3:1 mixture of isomers. Isomer 1 (major): 1H NMR (600 MHz, DMSO) S3.07 (s, 3H), 3.53 (s, 3H), 3.66 (s, 3H), 4.26 (s, 2H), 6.39 (d, J = 10.0 Hz, 1H), 6.76 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 10.0 Hz, 111), 8.51 (m, 147 1H); 13C NMR (150 MHz, DMSO) 829.2, 35.2, 43.7, 54.9, 100.3, 113.3, 123.8, 129.9, 132.7, 142.4, 157.4, 160.0, 164.1, 169.7, 175.4. Isomer 2 (minor): ‘H NMR (600 MHz, DMSO) 63.06 (s, 3H), 3.56 (s, 3H), 3.66 (s, 3H), 4.21 (s, 2H), 6.52 (d, J = 9.7 Hz, 1H), 6.75 (d, J = 8.6 Hz, 2H), 7.22 (d, J = 8.6 Hz, 2H), 7.83 (d, J = 9.7 Hz, 1H), 8.59 (m, 1H); ‘3C NMR (150 MHz, DMSO) 629.3, 35.4, 43.9, 54.9, 100.5, 113.3, 123.1, 129.8, 132.4, 143.2, 157.3, 160.6, 163.9, 170.0, 176.1; ESIMS [M+NaJ calcd for C,7H18N4O2a; 333.1327, found 333.1319. Preparation of bromide 3.46 To a solution of 3.44 (1.8 mg, 0.006 mmol) in 0.5 ml HOAc was added a solution of Br2 (0.0116 mmol, 1.0 M in HOAc, 11.6 IlL). The solution was stirred at rt for 1 h, and then the solvent was removed under vacuum. The crude compound was purified by column chromatography (CH2C1/MeOH) to yield 3.46 (1.0 mg, 0.0026 mmol, 43%) as a bright yellow solid. The NMR showed a 3:4 mixture of rotamers. Isomer 1: ‘H NMR (600 MHz, CD21)63.25 (d, J = 5.3 Hz, 3H), 3.62 (s, 3H), 3.73 (s, 311), 4.23 (s, 2H), 6.74 (d, J = 8.6 Hz, 2H), 7.36 (d, J = 8.6 Hz, 211), 7.92 (s, 111); ‘3C NMR (150 MHz, CD21)6 30.1, 36.4, 45.1, 55.2, 97.4, 113.7, 126.4, 129.1, 130.8, 143.9, 158.5, 163.2, 164.0, 167.8, 148 174.9. Isomer 2: 1H NMR (600 MHz, CD21)8 3.38 (s, 3H), 3.73 (s, 3H), 3.74 (s, 3H), 4.38 (s, 2H), 6.79 (d, J = 8.9 Hz, 211), 7.41 (d, J = 8.9 Hz, 211), 8.22 (s, 1H); ‘3C NMR (150 MHz, CD21) 830.5, 35.9, 46.3, 55.2, 99.1, 114.2, 126.8, 130.9, 148.1, 158.5, 162.8, 163.0, 163.8, 166.9, 167.6; ESIMS [M+Na] calcd forC17H79BrN4O2Na; 411.0433, found 411.0429. Preparation of formamide 3.47 To a slurry of sodium formate (215 mg, 3.16 mmol) in 3 mL dry THF was added acetyl chloride (150 mL, 2.1 mmol). The slurry was then heated to 45 °C for 4 h. The slurry was cooled to rt and a solution of 3.27 (40 mg, 0.1 mmol) in 1 mL THF was added, followed by a catalytic amount of DMAP. The solution was then stirred at ft for 24 h. 1 M HC1 was added and the solution was then stirred for 10 mm. The solution was basified with the addition of satd NaHCO3 and the crude reaction mixture was extracted into EtOAc. The organic phase was dried over Na2SO4,filtered and concentrated. The crude product was purified by column chromatography to yield 3.47 (36 mg, 0.08 mmol, 80%) as a light yellow solid. 111 NMR (600 MHz, DMSO) 83.22 (s, 3H), 3.73 (s, 3H), 4.26 (s, 2H), 4.31 (d, J= 11.5 Hz, 111), 5.03 (d, J= 11.5 Hz, 1H), 6.14 (d, J= 9.1 Hz, 1H), 6.30 (d, J= 9.1 Hz, 1H), 6.85 (d, J= 8.0 Hz, 2H), 6.91 (d, J= 8.0 Hz, 211), 7.17 (m, 311), 7.26 149 (m, 3H), 8.82 (s, ill), 10.88 (s, 111); ‘3C NMR (150 MHz, DMSO) ö37.8, 55.2, 69.9, 71.2, 107.6, 114.3, 120.9, 126.6, 127.6, 127.7, 128.2, 129.0, 130.2, 132.0, 134.0, 136.9, 144.3, 159.6, 162.9, 168.6; ESIMS [M+Na] calcd forC25H4N4Oa; 467.1695, found 467.1694. Methylation of 3.47 to yield 3.42. H,N N A solution of 3.47 (4.6 mg, C.01 mmol), K2C03 (2.7 mg, 0.02 mmol) and Mel (1.0 ml, 0.0 16 mmol) in 0.5 mL DMF was stirred at rt for 24 h. The solution was extracted into EtOAc and washed with 3 x H20. The organic phase was dried over Na2SO4,filtered and concentrated. The crude product was purified by column chromatography (hexanes/EtOAc) to yield 3.42 (4.0 mg, 0.009 mmol, 90%) as a dull yellow solid. 150 Preparation of alcohol 3.45 Compound 3.47 (20 mg, 0.045 mmol) was dissolved in 1 mL CH21. Aid3 (60 mg, 0.45 mmol) was added and the resulting blood red slurry was stirred rapidly at rt for 30 mm. The reaction mixture was quenched by careful addition of satd NaHCO3 and extracted into EtOAc. The organic phase was dried over Na2SO4, filtered and concentrated to yield 3.48. ES1MS [M+Na] calcd forC17H6N4O3a 347.1120, found 347.1128. This product was used without further purification. The crude compound 3.48 was then dissolved in 2.0 mL dry THF. BH3-T F (175 iL, 1.0 M in THF, 0.175 mmol) was added and the solution was stirred for 1 h at ft. The reaction mixture was then carefully quenched with the addition of 1.0 M HC1. The solution was basified with the addition of satd NaHCO3 and extracted into EtOAc. The organic phase was dried over Na2SO4, filtered and concentrated. The crude product was purified by column chromatography (CH2C1IMeOH) to yield 3.45 (5.0 mg, 0.015 mmol, 33%) as a bright yellow solid. The ‘H NMR spectrum showed a 3:1 ratio of rotamers. Only the major isomer is described. 1H NMR (600 MHz, DMSO) 83.06 (d, J = 5.0 Hz, 3H), 3.55 (s, 3H), 3.69 (s, 3H), 6.26 (d, J = 11.3 Hz, 1H), 6.48 (d, J = 9.9 Hz, 1H), 6.80 (d, J = 8.6 Hz, 2H), 7.16 (s, br, 1H), 7.38 (d, J = 8.6 Hz, 211), 7.80 (d, J = 9.9 Hz, 1H), 8.91 (d, J = 5.0Hz, 1H); 13C NMR (150 MHz, DMSO) 629.3, 43.7, 54.9, 72.6, 100.6, 113.2, 124.4, 151 127.3, 136.8, 143.6, 158.0, 163.8, 170.4, 174.5; ESIMS [M+Na] calcd for C17H8N4O3a349.1277, found 349.1279. Preparation of amine 3.49 H2N Compound 3.47 (25 mg, 0.056 mmol) was dissolved in 1 mL CH21. AId3 (74 mg, 0.56 mmol) was added and the resulting blood red slurry was stirred rapidly at it for 30 mm. The reaction was halted by careful addition of satd NaHCO3 and extracted into EtOAc. The organic phase was dried over Na2SO4,filtered and concentrated to yield 3.48. ESIMS [M+Na] calcd forC17H6N4O3a347.1120, found 347.1128. This product was used without further purification. Crude 3.48 was dissolved in 5 mL 1 ,4-dioxane and diluted with 5 mL deionized water. HC1(g) was bubbled through the solution for 1 mm, and the solution was allowed to sit for an additional 2 mm. The pH of the solution was brought to neutral with the addition of 5 M NaOH, then further basified by the addition of NaHCO3. The reaction mixture was extracted with EtOAc, the aqueous phase was saturated with NaCl and extracted again with EtOAc. The combined organic phases were dried over Na2SO4,filtered and concentrated. The crude product was purified by column chromatography (CH2C1/MeOH) to yield 3.49 (2.5 mg, 0.0084 mmol, 15%) as a bright yellow solid. 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Chem. 2005,48, 3684-3687. 166 Appendix A: NMR spectra for selected compounds from Chapters 2 and 3 167 Figure A.1: ‘H and ‘3C NMR spectra of 2.10 recorded in CDC13 at 400 and 100 MHz respectively 168 H 2.10 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 N. C,) 1521441361281201121049 888072 645648 40322416 0OH H 2.20 IL 0 cD CJ ,-r Q — 7.5 7:0 6.5 6.0 5.5 5.0 C’, L) — o CC U) i0) — C’) o 4.5 4.0 3.5 3.0 2:5 2.0 1.5 1.0 0.5 —0 C) c’jqr-. rrc6 0) q 1? C0 C.) CC C’) C’)Q I CC ._ .1 1. liii ...LI.II- JJI UPU I ,IPJJ1flfl, . M. I ... 152 144 136 128 120 112 1Ô4 96 88 80 72 64 56 48 40 32 24 16 Figure A.2: ‘H and ‘3C NMR spectra of 2.20 recorded in CDC13 at 600 and 150 MHz respectively 169 DC 0 CD c0c N- N-CD Figure A.3: ‘H and ‘3C NMR spectra of 2.22 recorded in CDC13 at 400 and 100 MHz respectively H 2.22 • -• 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 CD N- 0 cCCC2 — DC 0 152144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 170 Figure A.4: ‘U and 13C NMR spectra of 2.24 recorded in CDC13 at 400 and 100 MHz respectively 171 Co 0 c 2.24 N N cc • 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 q N N 0 CD COCD N N CD 0 ci ..J.LJL.If..JAi. CD Nq 0 N N N 0 N Co CD C) — CD — CD N Co (C N (C C)? CD ci CD Co N N Co Co 0 Co Co iL.á .1 CD CD CD S CD I,.’ (C C?(C Co) Ii ..jj .-.•. IL 152 144 136 128 120 112 1049688 80726456484032 2416 II I... I. CU CU La N: . U? T r— I La C La Figure A.5: ‘H and ‘3C NMR spectra of 2.25 recorded in CDC13 at 400 and 100 MHz respectively 172 0) q 2.25 0 N- I 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 La aS C’) La 0) CO La p Co0 Pc — 152 144 136 128 120 N U) La C’) La U? C’) 112 1O4 96 88 80 72 64 56484032 24 16 Figure A.6: ‘H and ‘3C NMR spectra of 2.26 recorded in CD3O at 400 and 100 MHz respectively 173 OH H 2.26 2 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 05 2 r—. c’J Cl —0°, Cd 0 F t-iJ C) N e to oSaS TI U, to oSco I •c.) C\t rn — jC’J Ccó o,tn _- i:i ..i [.. IF to to to c’JC.) — CJcj — Ta lCd / .1 a to(p Ct to Id) ièo iEo i4o iáo io iio iOo oh irflWL NC lnHfIimrltflt$flpflfl$fl .0 WW. aW*flH , -. - .1. 80 o oh sb ‘o oh io ib 4*111. C C r i — ó . .C — C) — ru, 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16 Figure A.7: ‘H and 13C NMR spectra of 2.27 recorded in CD3O at 600 and 150 MHz respectively 174 OH 2.27 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 C cc C., C C cc 0) 0) Figure A.8: 1H and 13C NMR spectra of 2.28 recorded in CDC13 at 600 and 150 MHz respectively 175 OH H 2.28 p C.) CD 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) IC) Cc, • . .jAUII . 1.1 fl CD C.) IC) UCa3 Ccc C’CCC.) CD g CCC 0) cc bC’) C.)C IC) h CI) .1 iuø . . 160 150 140 130 120 110 100 90 80 70 60 50 3 2 ib IFigure A.9: HSQC spectrum of 2.28 recorded in CDC13 at 600 MHz -— ---t..... .. - 8 16ii ....- .. . - — . 40 55.57.314 . 48 .4. .t——--- — ——4 —- 72 80 I 88 .. 112 120 75 7.0 6.5 6.0 5.5 5.0 4.5 4.0 ppm 3.5 10 2.5 20 1.5 10 128 176 L. I I _____JLL Figure A.1O: HMBC spectrum of 2.28 recorded in CDC13 at 600 MHz 177 I 0 - -— -- -b-, 16 !L4 - — — I -- —*------- ---“ -——— — --——---- —.-. .---——— —.——— 72 ——----- 88 99.82 4.67 96 ... . . 112 . . 120 a ‘ a — •.: 136 . 144 16938,6.4 a 159.25j13 . ::. 160 . 168 -... r I E ppm 2 176 7.0 6.5 6.0 5.5 I, 5.0 4.5 4.0 3.5 3.0 ppm Figure A.11: Expanded HMBC spectrum of 2.28 recorded in CDC13 at 600 MHz 178 8 16 — sA___--.-- . 24 32 H 99.82. 4.87 L . -- - - -..——.i...-—------ . -....- . - - .-—,—--—--- —--.-. — 120 • . 128 z-z..._ C 168 Figure A.12: ‘H and ‘3C NMR spectra of 2.29 recorded in CDC13 at 600 and 150 MHz respectively q 2.29 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 C U) () U) U) 179 I ii j ppm Figure A.13: HSQC spectrum of 2.29 recorded in CDC13 at 600 MHz 180 Figure A.14: HMBC spectrum of 2.29 recorded in CDC13 at 600 MHz I ii ppm 181 IFigure A.15: Expanded HMBC spectrum of 2.29 recorded in CDC13 at 600 MHz B 16 ‘)Q ‘‘ 24 ...............-.......... - .-..........-- ,...,..,.. 32 ....,-- -----...-1—----- -....- -- 48 : 56 — —-- -H 72a - 1O4 4,4,6 S 96 .- . , 112 . 120 128 —- -___ .— — 136 . 144 . 156.01,3.74 6 156.166.2 “ . iQ 7.0 6.5 6.0 55 50 ppm 168 116 4.5 4.0 . 35 3.0 182 I}6 ito ito iio io io Ho ioo So So io So 50 40 30 20 10 Figure A.16: 1H and 13C NMR spectra of 2.31 recorded in CD3O at 400 and 100 MHz respectively 2.31 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 Co—co CLI ON oSco5aS an CD Cqe, D)a5 It LDCD O fl0C(Q tfl i_i ; (‘C CLICo Co — d 0 Co (‘C Co C) — uS CD - L,I Co LI) CC) -: CC) In I. na r’w ‘MM. RPfl i_I.. I.. . I. La., Lta.. - .L.[L.a..J !WJMLISIJ!PSi.PI!N 183 a:, Figure A.17: 1H and 13C NMR spectra of 2.39 recorded in CDC13 at 600 and 150 MHz respectively H 2.39 75 70 65 60 55 50 45 40 35 30 25 20 15 10 05 C CD — c) — CD C c’o — C — r CD U, — C CU CD CU CD — D) c CDCD CD c CD QT D (I CU o CU a 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 184 — r— - crI c’J(p a c’ C(J I—c’o a r t.I(’)e) a 1r a C” 2.40 F.. 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 h(C’ — F.. h a (C) a(0 (1) 0) 0) UD(C (o ••.I I .$w (0Q a : 152 144 136 128 120 112 1Ô4 96 88 80 72 64 56 48 40 32 24 16 Figure A.18: ‘H and 13C NMR spectra of 2.40 recorded in CDC13 at 600 and 150 MHz respectively b 185 H2 1 C13 C17 I • I I:’ I I 220 200 180 10 140 120 100 80 (ppm) C2 1 CII U Figure A.19: 111 and 13C NMR spectra of ceratamine A recorded in DMSO-d6at 500 and 100 MHz respectively 1 9 N 20HN / N_j Br 19 / 15 O21Br H13 Ceratamine A H18 (ppm) C14 C19 C20 C12 do (‘6 C8 C2 C15 C$ i,,JInII, j u.k I • I 60 40 20 0 186 H2N HO 3.28 0) CD 0) r- C’) DC’) Li III 1 •18.0 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 r. 0) 0) U) C’i oSoScx5 0 0) C’) L.j- I.. - I_.. . —- . tJ j 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 Figure A.20: 1H and ‘3C NMR spectra of 3.28 recorded in CD3O at 600 and 150 MHz respectively 187 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 Figure A.22: ‘H NMR spectrum of 3.29 recorded in CD3O at 600 MHz 188 0)(0 C) 0 rr1.... 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 Figure A.21: ‘H NMR spectrum of 3.28 recorded in DMSO-d6at 400 MHz (0 N. CO N C’, 0 (0 CO 0 CCC’) (C) 3.29 0(0 ir J Figure A.23: HMBC spectrum of 3.29 recorded in CD3O at 600 MHz 189 4 32 - ._1 -— __ — .. ..__ — S — - -- - -: 40 ,. ., . r z: ,. - 72 --- —. .---- .. r. ‘ I ,,,.. - . - 1.04 I . . . II - 136 7 .. -.,. . . . 152 .. .. . b01% . I a . ‘16 84 a E 0 80 7:5 1.0 6.5 6.0 5.5 5.0 ppm ‘I’ 4.5 4.0 35 64 72 8a 88 96 , zzzHzt* 128 . . 136 , 144 1 152 7.9 7.8 7.7 76 75 7.4 7.3 7.2 1.1 7.0 6.9 6.8 6.7 6,6 Figure A.24: Partial HMBC spectrum of 3.29 recorded in CD3O at 600 MHz 190 4U 48 56 .__1 t 160 168 176 ppm 184 j192 Figure A.25: ‘H and ‘3C NMR spectra of 3.44 recorded in DMSO-d6at 600 and 150 MHz respectively 3.44 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 C 0) a) C 0) 0) C 0 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 191 C,, C,) C,) I HO 3.45 CaN CCC CpCp U 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 C ) — N cS C,, 0) C’) C,, C-. N CD 01 CD C’) C,) CD 0) C,) N C C” aS(0 C N N 0) II.N 0) — I (0 C’) 2 T I, 01 - 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 Figure A.26: 1H and ‘3C NMR spectra of 3.45 recorded in DMSO-d6at 600 and 150 MHz respectively 192 5.5 5.0 4.5 4.0 3.5 3.0 Figure A.27: ‘H NMR spectrum of 3.46 recorded in CD21 at 600 MHz 193 c’J c’J 3.46 c’J (0 (0 8.0 7.5 7.0 6.5 6.0 2.5 0Figure A.28: HSQC spectrum of 3.46 recorded in CD2I at 600 MHz 194 10 —- - — ...... - z___1Z —I 60 70 80 . 90 1(10 110 120 — . -.-- —— — - -f ---. — I 150 . 160 170 180 ppm 190 2 0Figure A.29: HMBC spectrum of 3.46 recorded in CD21 at 600 MHz 195 4 4 e I p0 30 40 . . , ,,. .. .. . 50 . . 60 . . 70 .. 80 .. . ----.-.- .... . . . ., . 100 110 , . 120 — . . - . 150 , *.. .,. S •..., —.c 50 . 160I_ -— — . 180 .. . .. 10 7 1 2 E ppm •1 c’J CDO,U, 180 170 160 150 140 130 120 110 100 90 80 70 60 50 • 40 30 • 20 Figure A.30: ‘H and 13C NMR spectra of 3.49 in DMSO-d6at 400 and 100 MHz respectively H2N 3.49 c) C,) 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 2.0 196 Appendix B: X-ray structure reports for compounds 2.10, 2.40 and 3.37 197 Sample: raO2OfMN-100 2.10 X-ray Structure Report for Prof. Raymond Andersen Department of Chemistry UBC Compound 2.10 June 5, 2006 198 C4 1 C44 C4: C43 01 C3 8 C17 C28 C35 Cli C22 C2 C9 Cl 34 C2 C3 C13 C12 199 EXPERIMENTAL DETAILS: Compound 2.10 A. Crystal Data Empirical Formula C46H67N02 Formula Weight 666.01 Crystal Color, Habit colourless, plate Crystal Dimensions 0.05 X 0.15 X 0.50mm Crystal System monoclinic Lattice Type C-centered Lattice Parameters a = 47.4 13(3) A b = 7.2967(4) A c = 11.0660(2) A U = 90.0 ° 13 = 95.115(1)0 7 90.0° V = 382 1.2(3) A3 Space Group C 2 (#5) Z value 4 Dcalc 1.158 g/cm3 1464.00 I(MoKU) 0.69 cm’ 200 B. Intensity Measurements Diffractometer Bruker X8 APEX Radiation MoKc (2k. = 0.7 1073 A) graphite monochromated Data Images 1602 exposures @ 5.0 seconds Detector Position 38.94 mm 20max 56.0° No. of Reflections Measured Total: 30662 Unique: 9116 (Rint = 0.038) Corrections Absorption (Tmin = 0.878, Tniax 0.9) Lorentz-polarization 201 C. Structure Solution and Refinement Structure Solution Direct Methods (S1R97) Refinement Full-matrix least-squares on F2 Function Minimized w (Fo - Fc2) Least Squares Weights w=1/(a2(Fo)+(0.0634P) 2 0.OOP) Anomalous Dispersion All non-hydrogen atoms No. Observations (J>O.00c(I)) 9116 No. Variables 461 Reflection/Parameter Ratio 19.77 Residuals (refined on F2, all data): Ri; wR2 0.057; 0.111 Goodness of Fit Indicator 1.05 No. Observations (I>2.OOG(I)) 7490 Residuals (refined on F): Ri; wR2 0.043; 0.104 Max Shift/Error in Final Cycle 0.00 Maximum peak in Final Diff. Map 0.22 e/A3 Minimum peak in Final Diff. Map -0.21 e/A3 202 Sample: ra023/03-176-12 2.40 X-ray Structure Report for Prof. Raymond Andersen Department of Chemistry UBC Compound 2.40 January 18, 2007 203 111 Cl C3 C 17 Cli Ci C12 C23 C6 C7 15 204 EXPERIMENTAL DETAILS: Compound 2.40 A. Crystal Data Empirical Formula C240H35N Formula Weight 353.53 Crystal Color, Habit colourless, plate Crystal Dimensions 0.12 X 0.40 X 0.40 mm Crystal System triclinic Lattice Type primitive Lattice Parameters a = 6.4440(6) A b=7.3343(7)A c= ll.9761(12)A = 94.696(5)° 93.240(5) ° y= 113.562(5)0 V = 5 14.61(9) A3 Space Group P 1 (#1) Zvalue 1 Dcalc 1.141 g/cm3 F000 194.00 !I(MoK) 0.68 cm_i 205 B. Intensity Measurements Diffractometer Bruker X8 APEX II Radiation MoK (? = 0.7 1073 A) graphite monochromated Data Images 3248 exposures @ 10.0 seconds Detector Position 36.00 mm 20max 55.0° No. of Reflections Measured Total: 15134 Unique: 4660 (Rint = 0.035) Corrections Absorption (Tmin = 0.873, TrnaxO.92) Lorentz-polarization 206 C. Structure Solution and Refinement Structure Solution Direct Methods (S1R97) Refinement Full-matrix least-squares on F2 Function Minimized w (Fo2 - Fc2) Least Squares Weights w=1/(G(Fo)+(0.0595P) 2 O.019P) Anomalous Dispersion All non-hydrogen atoms No. Observations (I>O.OOc(I)) 4660 No. Variables 245 Reflection/Parameter Ratio 19.02 Residuals (refined on F2, all data): Ri; wR2 0.043; 0.097 Goodness of Fit Indicator 1.03 No. Observations (b’2.OOcy(I)) 4213 Residuals (refined on F): Ri; wR2 0.037; 0.093 Max Shift/Error in Final Cycle 0.00 Maximum peak in Final Diff. Map 0.19 e/A3 Minimum peak in Final Diff. Map -0.14 e/A 207 Sample: ra032/06-1 11-01 3.37 X-ray Structure Report for Prof. Raymond Andersen Department of Chemistry UBC Compound 3.37 May 9, 2008 208 C23 C24 C25 CiS C16 C17 03 C26 C27 Ni 209 EXPERIMENTAL DETAILS: Compound 3.37 A. Crystal Data Empirical Formula C24H2N301 Formula Weight 435.90 Crystal Color, Habit colourless, rod Crystal Dimensions 0.18 X 0.20 X 0.50 mm Crystal System triclinic Lattice Type primitive Lattice Parameters a = 8.8404(14) A b = 9.4642(16) A c = 13.031(2) A = 94.678(7) 0 13 = 103.672(8) ° = 92.025(8) ° V = 1054.1(3) A3 Space Group P -1 (#2) Zvalue 2 Dcalc 1.373 g/cm3 F000 456.00 i(MoKo) 2.13 cm’ 210 B. Intensity Measurements Diffractometer Bruker X8 APEX II Radiation MoKa (?. = 0.7 1073 A) graphite monochromated Data Images 2582 exposures @ 5.0 seconds Detector Position 36.00 mm 20max 56.2° No. of Reflections Measured Total: 26684 Unique: 4994 (Rjnt = 0.027) Corrections Absorption (Tmin = 0.875, Tmax 0.9) Lorentz-polarization 211 C. Structure Solution and Refinement Structure Solution Direct Methods (51R97) Refinement Full-matrix least-squares on F2 Function Minimized w (Fo2 - Fc2) Least Squares Weights w=1/(G(Fo)+(0.0398P) 2 O.314P) Anomalous Dispersion All non-hydrogen atoms No. Observations (I>0.OOo(I)) 4994 No. Variables 338 Reflection/Parameter Ratio 14.78 Residuals (refined on F2, all data): Ri; wR2 0.043; 0.092 Goodness of Fit Indicator 1.04 No. Observations (I>2.OOcy(J)) 4184 Residuals (refined on F): Ri; wR2 0.035; 0.087 Max Shift/Error in Final Cycle 0.00 Maximum peak in Final Diff. Map 0.25 e/A3 Minimum peak in Final Diff. Map -0.28 e/A3 212

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