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Investigation into the biologically active metabolites of Coccoloba acuminata and Minquartia guianensis Nelson, Jim 2002

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INVESTIGATION INTO THE B I O L O G I C A L L Y A C T I V E METABOLITES OF COCCOLOBA ACUMINATA A N D MINQUART1A GUIANENSIS by JIM N E L S O N B.Sc, University of British Columbia, 2000 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Chemistry) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A September 2002 ©Jim Nelson, 2002 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia Vancouver, Canada Department Date DE-6 (2/88) 11 A B S T R A C T The comprehension of cellular processes remains central to the development of novel techniques for the treatment of disease. The use of small molecule protein inhibitors can aid in better understanding these processes and potentially be used therapeutically. The G2 checkpoint allows for cells to pause and repair DNA damage. In conjunction with radiation therapy, the use of G2 checkpoint inhibitors has demonstrated (in vitro) a greater mortality for cancer cells over healthy cells. The bioassay guided fractionation of Coccoloba acuminata afforded the G2 checkpoint inhibitor enf-kaur-16-en-15-oxo-18-oic acid (7). At an optimal concentration of 12 p,g/ml, diterpene 7 exhibited a maximum release of cells from G2 -> M of 48%. A biotinylated linker was attached to 7 to form an affinity probe 14. \ H COOH 7 O Jr°- N O H IT ^ ^ o 14 Ill At an optima] concentration of 7 ug/ml, compound 14 released 39-41% of cells from G2 to M phase. The binding of Pinl to phosphorylated tau plays a role in the formation of paired helical filaments, a hallmark of Alzheimer's disease. Therefore, inhibitors of the binding of Pinl to phosphorylated tau could be used as a potential therapeutic for Alzheimer's disease. The bioassay guided fractionation of M. guianensis yielded the cytotoxic polyacetylene minquartynoic acid 8 and two inhibitors of Pinl binding 4-0-(a-rhamnopyranosyl)ellagic acid 9 and 4-0-((j-xylopyranosyl)ellagic acid 10. HQ OH O 8 OH OH 9 10 The IC50 is 0.7 |lg/mL for 4-0-(P-xylopyranosyl)ellagic acid 10 and 0.6 ug/mL for 4-0-(a-rhamnopyranosyl)ellagic acid 9 for inhibition of Pinl binding to phosphorylated tau. iv TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS iv LIST OF TABLES vi LIST OF FIGURES vii LIST OF ABBREVATIONS x ACKNOWLEDGEMENTS xiv 1. Introduction 1 1.1 Natural Products Chemistry 1 1.2 Cell cycle 2 1.3 Cell cycle checkpoints 3 1.4 G2 Checkpoint inhibition 5 1.5 Pin 1 9 1.6 Aims of this study 12 1.7 References 14 2. Coccoloba acuminata 17 2.1 enr-kaurene 18 2.1.1 Isolation of active diterpene from the cocoloba acuminata extract 18 2.1.2 Structure elucidation of active diterpene 19 2.2 Biological activity 28 2.3 Biotin-Avidin 31 2.3.1 Attachment of Biotin 31 2.3.2 Biological activity of biotinylated compounds 34 2.3.3 Isolation of target protein 35 2.4 References 38 3. Minquartia guianensis 39 3.1 Isolation of Active Metabolites from Minquartia guianensis 40 3.2 Minquartynoic acid 42 3.2.1 Structure elucidation of minquartynoic acid 42 3.2.2 Biological activity of Minquartynoic acid 47 TABLE OF CONTENTS 3.2 Minquartynoic acid (continued) 3.2.3 Semisynthesis of Minquartynoic acid analogues 48 3.2.4 Biological activity of Minquartynoic acid analogues 53 3.3 Ellagic acid glycosides and ellagic acid 56 3.3.1 Structure elucidation of 4-0-(a-rhamnopyranosyl)ellagic acid 56 3.3.2 Structure elucidation of 4-0-(p-xylopyranosyl)ellagic acid 62 3.3.3 Ellagic acid 67 3.3.4 Pin 1 inhibition of ellagic acid and ellagic acid glycosides 67 3.3.5 Therapeutic potential of Ellagic acid glycosides 70 3.4 References 71 4. Experimental 73 4.1 General procedures 73 4.2 Isolation 74 4.3 Synthesis 77 ] LIST OF T A B L E S v i Table 2.1 N M R data for enr-kaur-16-en-15-oxo-18-oic acid (7) recorded in C^D6 Table 3.1 N M R data for Minquartynoic acid 8 recorded in C D 3 O D Table 3.2 N M R data for 4-0-(a-rhamnopyranosyl)ellagic acid 9 recorded in (CD 3) 2SO Table 3.3 N M R data for 4-0-((3-xylopyranosyl)ellagic acid 10 recorded in 4:1 C D 3 C N : DMSO 22 43 59 63 V l l LIST OF FIGURES Figure 1.1 Mammilian cell cycle and Cyc/Cdk protein complexes 3 Figure 1.2 Suggested interactions of proteins in the G2 checkpoint 5 cascade Figure 1.3 Rational for G2 checkpoint inhibitors 6 Figure 1.4 Selected G2 checkpoint inhibitors 7 Figure 1.5 G2 inhibition bioassay 8 Figure 1.6 Cis-trans isomerization of prolyl peptide bonds 9 Figure 1.7 Structure of Juglone 6 10 Figure 1.8 Summary of Pinl binding to phosphorylated tau Bioassay 11 Figure 1.9 Structure of mr-kaur-16-en-15-oxo-18-oic acid (7) 12 Figure 1.10 Structure of (S)-17-hydroxy-9,l 1,13,15-octadecatetraynoic 13 acid 8 Figure 1.11 Structure of 4-0-(a-rhamnopyranosyl)ellagic acid 9 13 and 4-0-((3-xylopyranosyl)ellagic acid 10 Figure 2.1 Coccoloba acuminata 17 Figure 2.2 Isolation scheme of active diterpene from Coccoloba 19 acuminata Figure 2.3 en -^kaur-16-en-15-oxo-18-oic acid 7 20 Figure 2.4 Selected HMBC correlations of compound 7 20 Figure 2.5 Selected HMBC correlations of compound 7 21 Figure 2.6 Selected HMBC correlations of compound 7 21 Figure 2.7 'H NMR and 1 3 C NMR of m?-kaur-l 6-en-15-oxo-18-oic 24 acid (7) Figure 2.8 COSY spectrum of ent-kam-16-en-15-oxo-18-oic acid (7) 25 viii Figure 2.9 HMQC spectrum of en?-kaur-16-en-15-oxo-18-oic acid (7) 26 Figure 2.10 HMBC spectrum of ercf-kaur-16-en-15-oxo-18-oic acid (7) 27 Figure 2.11 G2 inhibition response to MCF-7 p53- cells 28 Figure 2.12 Structural formula of parinari G2 checkpoint inhibitors 29 Figure 2.13 G2 checkpoint inhibition response of 7 compared to 11 30 Figure 2.14 Structural formula of Biotin 31 Figure 2.15 Scheme for biotin attachment of 7 32 Figure 2.16 'H NMR and l 3 C NMR of biotinylated diterpene (14) 33 Figure 2.17 N-acetyl-N-biotinylhexylenediamine 34 1 -methylcyclohexanate (15) Figure 2.18 G2 response of biotinylated compounds 35 Figure 2.19 Schematic of protein target isolation 36 Figure 2.20 Gel for detection of Biotinylated proteins 37 Figure 3.1 Leaves and stem of Minquartia guianensis 39 Figure 3.2 Isolation of active metabolites from Minquartia guianensis 41 Figure 3.3 'H NMR and l 3 C NMR Spectra of minquartynoic acid (8) 45 Figure 3.4 HMBC spectrum of minquartynoic acid (8) 46 Figure 3.5 Activity of Minquartynoic acid in G2 checkpoint 47 inhibition bioassay Figure 3.6 Naturally occurring analogues of minquartynoic acid 48 Figure 3.7 Regions of minquartynoic acid 8 for structure and 48 reactivity studies Figure 3.8 Synthesis of Minquartynoic acid analogues 51 Figure 3.9 Selected *H NMR of Minquartynoic acid analogues 52 Figure 3.10 Biological activity of the semi-synthetic analogues 53 in the antimitotic bioassay Figure 3.11 Cytotoxixity of semi-synthetic analogues 55 Figure 3.12 Structure of 4-0-(a-rhamnopyranosyl)ellagic acid 56 Figure 3.13 Coupling constants in Hertz of rhamnose moiety 57 Figure 3.14 Exchange of protons with deuterium for compound 9 58 Figure 3.15 ! H and l 3 C NMR spectra of 4-(a-rhamnopyranosyl) 60 ellagic acid (9) Figure 3.16 HMBC spectrum of 4-0-(a-rhamnopyranosyl) 61 ellagic acid (9) Figure 3.17 Structure of 4-0-((3-xylopyranosyl)ellagic acid 62 Figure 3.18 Coupling constants in Hertz of xylose moiety 63 Figure 3.19 'HNMR and 1 3CNMR of 4-0-(p-xylopyranosyl) 65 ellagic acid (10) Figure 3.20 Homonuclear J resolved spectrum of 66 4-0-(P-xylopyranosyl)ellagic acid (10) Figure 3.21 Structural formula of Ellagic acid 23 67 Figure 3.22 Inhibitory Effects of ellagic acid and ellagic acid 68 glycosides on Pinl binding to phosphorylated Tau. Figure 3.23 Cytotoxicity of Ellagic acid glycosides and ellagic acid 69 X LIST O F A B B R E V I A T I O N S ABTS 3-ethylbenzthiazoline-6-sulfonic acid ATM ataxia telangiectasia mutated ATR ATM and Rad3-related B-OKA biotinylated diterpene (14) br broad BSA bovine serum albumin Cdk cyclin dependent kinase CD 3CN Acetonitrile -d3 COSY correlation spectroscopy l 3 C NMR carbon nuclear magnetic resonance Cyc cyclin 8 chemical shift in parts per million from tetramethylsilane D2O deuterium oxide d doublet DBH debromohymenialdisine dd doublet of doublets DIBAL-H diisobutylaluminum hydride DMSO dimethylsulfoxide DNA deoxyribonucleic acids 2D NMR 2 dimensional nuclear magnetic resonance ED50 Effective dose at 50% survival xi EDTA ethylenediaminetetraacetic acid EGTA Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid ELISA enzyme-linked immunosorbent ent enantiomer ESMS electrospray ionization mass spectrometry EtOAc ethyl acetate Gi Gap 1 G 2 Gap 2 Gy Grays h hour(s) AH change in enthalpy HCT116 human colon carcinoma cells HMBC heteronuclear multiple-bond correlation HMQC heteronuclear multiple-quantum correlation ' H NMR Proton nuclear magnetic resonance HREIMS High resolution electron ionization mass spectrometry HRESMS High resolution electrospray ionization mass spectrometry HTS high throughput screening Hz hertz IC50 inhibitory concentration for 50% of activity in vitro outside the cell in vivo inside the cell J coupling constant in hertz K 2 C O 3 potassium carbonate K2HPO4 potassium phosphate KJ/mol kilojoules per mole M molar m multiplet MCF-7 human breast cancer cells MeOH methanol (ig/mL micrograms per milliliter mM micromolar MTT 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazoli bromide NaCl sodium chloride NCI National Cancer Institute r|g/mL nanogram per milliliter P-388 human leukemia cells pH -log,0[H+] PHFs paired helical filaments PMSF Phenylmethanesulfonyl fluoride ppm parts per million Rf retention factor RIPA radioimmunoprecipitation factor S DNA synthesis s singlet ser serine t triplet TLC thin layer chromatography Thr threonine Tyr tyrosine UCN-01 7-hydroxystaurosporine xiv A C K N O W L E D G E M E N T S I want to gratefully acknowledge all the support and encouragement from my supervisor Dr. Raymond Andersen. I would also like to thank him for the opportunity to work in his laboratory. I want to thank Dr. Michel Roberge for all his enthusiasm and expertise in bioassays. I would also like to thank all the members of his group that ran my bioassays, Natalie Rundle, Priscilla Brastianos, Tamsin Tarling and Lianne McHardy. I think we found some exciting results together. I would especially like to thank all the members of my group for passing on their expertise in separation and structure elucidation. I was really fortunate to have the experience of working with my bench mates, Dr. Todd Barsby, Roger Linington and Kyle Craig. I want to acknowledge the expert NMR staff at UBC for all my questions about using the spectrometers. 1 Introduction: The intricate mechanisms of biochemical processes continues to be a topic of much interest in the understanding of cellular dynamics and in the development of therapeutics. The pursuit of these interests incorporates a wide range of disciplines including cellular biology, microbiology, biochemistry, and chemistry. The research described in this thesis is the product of a close collaboration between our marine natural product chemistry group and Michel Roberge's group in the department of biochemistry at the University of British Columbia. 1.1 Natural products chemistry The isolation and characterization of the chemical constituents of a living organism remain a key focus of natural products chemistry. The extensive array of structurally diverse metabolites identified are an excellent source of potential therapeutics; nearly half of all pharmaceuticals available on the market today are derived from natural products.1 For example, the well known anticancer agent paclitaxel (1) was first identified from the Pacific yew tree.2 1 Apart from their role as drug lead compounds, biologically active natural products are also being used to probe the inner processes of the cell. Active compounds can be used as affinity probes 2 for studying localization within the cell or as a ligand for purification of target proteins. An example of this technique, is the labeling of okadaic acid (2) - isolated from the marine sponge Halichondria okadai - with biotin to identify the protein phosphatases that it binds to. ' 2 A traditional approach for the isolation of biologically active natural products involves working with extracts that have been used by traditional healers and believed to possess some medicinal properties. A more recent approach, known as high throughput screening (HTS), involves the screening of thousands of extracts for a very specific biological responses. An advantage to this method is that each step of the isolation can be monitored for biological activity and the separation is carried out with a clear rationale in mind. 1.2 Cell cycle The most fundamental cellular process is the ability of a cell to propagate and pass on an exact genetic replica. The model developed to describe the cell cycle divides it into the following four major phases: M phase (mitosis), Gi (Gap 1), S phase (DNA synthesis) and G2 (Gap 2).4 During M phase, the duplicated chromosomes are separated into two nuclei and cytokinesis occurs. The Gap 1 phase occurs between mitosis and the commencement of DNA synthesis. In S phase, the cell replicates its DNA. The Gap 2 phase occurs between the end of DNA synthesis and the start of mitosis. Before progressing into either mitosis or DNA synthesis the integrity of the genome is assessed by control mechanisms. If any abnormalities are detected, normal cells have the ability to pause and allow time for DNA repair at either the Gl checkpoint or the G2 checkpoint. The cell cycle is regulated by the phosphorylation and dephosphorylation of protein complexes composed of cyclin (Cyc) and cyclin dependent kinases (Cdk). Different Cyc/Cdk complexes appear periodically throughout the cell cycle and each performs a specific type of kinase activity.5 For example, the progression of the cell cycle from G2 to M phase is mediated by the kinase activity of cyclin B/Cdc2. G2 checkpoint 6-8 M G2 G1 G1 checkpoint Figure 1.1 Mammilian cell cycle and Cyc/Cdk protein complexes 1.3 Cell cycle checkpoints In multicellular organisms, cells are naturally asynchronous with each other and can be at any stage of the cell cycle when DNA damage occurs. If cells are in the G l phase, the cycle pauses at the G l checkpoint to allow time for repair; if cells are in the G2 phase, the cycle pauses at the G2 checkpoint to allow time for repair. The Gl checkpoint is regulated by the presence of the transcription factor p53; in normal cells p53 is rapidly degraded, but in damaged cells p53 is stabilized. 9 - 1 0 This stabilization is believed to be achieved by the phosphorylation of the serine-15 and serine-20 residues on p53 by the activity of two key enzymes, ataxia telangiectasia mutated (ATM) kinase and Chk2 kinase. In response to DNA damage, ATM kinase is thought to provide the following two functions: directly mediate the phosphorylation of the serine-15 residue" and activate the Chk 2 kinase which subsequently phosphorylates the serine-20 residue.12 Once stabilized, p53 increases the transcription of p21 1 3 , an enzyme which binds directly to cyclin/Cdk complexes and acts as a Cdk inhibitor.14 p21 inhibits the activity of the cyclin E/cdk2 complex and prevents the cell from progressing from Gl phase to S phase;15 hence arresting the cells at the Gl checkpoint. The G2 checkpoint cascade involves an entirely different mechanism than the Gl checkpoint cascade. As mentioned previously, the entry of a cell into mitosis is determined by the kinase activity of cyclin B/Cdc2. The activity of this kinase is inhibited if two residues on the Cdc2 subunit, Tyr 15 and Thr 14, are phosphorylated.16 The phosphorylation at these sites is primarily carried out by the Weel protein kinase and secondly by the kinases Miki and Mytl. 1 7 These inhibitory kinases are opposed by the activity of Cdc25c protein phosphatase and diphospho Cdc25c protein phosphatase which reactivate Cdc2 by dephosphorylating Tyr 15.18 Recently, the prolyl isomerase Pin 1 was found to bind to phophorylated Cdc25c and prevent the interaction of Cdc25c and Cdc2/cyclinB.19-21 In undamaged cells, the activities of Cdc25c are greater than the inhibitory kinases and cells proceed into mitosis. In DNA damaged cells, the activity of Cdc25c is reduced and cells remain in G2 arrest. DNA damage induces the activity of ATM kinase which is believed to phosphorylate the gene product Chkl . 2 2 - 2 3 Activated Chkl is involved in the following inhibitory functions: Chkl increases the activity of the inhibitory kinase Weel 2 4 and phosphorylates Cdc25c at the serine-216 residue25. Phosphorylated Cdc25c binds to the protein 14-3-3 and is sequestered from the nucleus into the cytoplasm.26 The reduction of activity of Cdc25c causes the cells to pause in G2 arrest. 5 CHK1 (Sequestered) ATM T C H K l ) (inactive) G2 arrest (inactive) (Active) Mitosis Figure 1.2 Suggested interactions of proteins in the G2 checkpoint cascade 1.4 G2 Checkpoint inhibition The progression of cells directly into either M phase or S phase with DNA damage can result in 97 mitotic arrest or apoptosis." Therefore, cells lacking a Gl or G2 checkpoint would be more susceptible to radiation therapy. One of the differentiating characteristics between certain types of human cancer cells and normal cells is the lack of a G l checkpoint. Over 60% of human cancers completely lack a G l checkpoint due to a mutation in the p53 tumour suppressor gene product." A potential therapeutic approach to further sensitize tumour cells is to apply agents 6 that inhibit the G2 checkpoint. Upon D N A damage, tumour cells would be forced to enter either M phase or S phase without pausing for repair, causing cell death. Alternatively, normal cells would have the opportunity to repair at the G l checkpoint. (Figure 1.3) A. p53" tumour cells G1 checkpoint G2 checkpoint G1 S G2 M Cell Death B. p53 + norma] cells G1 checkpoint G2 checkpoint G1 S G2 M Cells Arrested Figure 1.3: Rational for G2 checkpoint inhibitors A. p53- tumour cells are unable to repair at the Gl or G2 checkpoint resulting in cell death. B. p53+ normal cells have the ability to repair at the Gl checkpoint resulting in cellular arrest. In vitro, most G2 checkpoint inhibitors have been found to sensitize cancer cells to the effects of D N A damage.29"31 This has led to a search for G2 checkpoint inhibitors that are adjuvants to radiation therapy and can increase the survival of normal cells relative to tumour cells. 7 The few G2 checkpoint inhibitors that have been identified act upon either checkpoint kinases ( caffeine 3, D B H , UCN-01 4) 3 2" 3 4, Ser/Thr protein phosphatases (fostriecin, okadaic acid 3 5" 3 6 2) or 3 7 unknown modes of action (13-Hydroxy-15-oxozoapatlin 5). H I ^ N H 3 4 5 Figure 1.4 Selected G2 checkpoint inhibitors A high throughput screening process was developed for detecting G2 checkpoint inhibitors by the Roberge laboratory at the University of British Columbia. 3 8 In this bioassay, monolayers of human breast cancer cells MCF-7 p53- are seeded on a 96 well plate and grown for 24 hours. They are then exposed to 6.5 Gy of D N A damaging y-irradiation. After 16 hours, extracts and the antimitotic agent nocodazole are added. Cells containing G2 checkpoint inhibitors arrest in mitosis; cells not containing a G2 checkpoint inhibitor remain in G2 arrest. The remaining steps of the assay involve detecting cells arrested in mitosis by the ELISA (enzyme-linked immunosorbent) assay protocol using the TG-3 antibody and the HRP- conjugated secondary antibody. MCF-7 p53- cells grown on a 96 well plate 6.5 Gy of y-irridation Extracts and Nocodazole added G2 checkpoint inhibitor present Cells arrest in Mitosis G2 checkpoint inhibitor absent Cells remain arrested in G2 Quantitate Mitosis by ELISA protocol using TG-3 antibody and HRP-conjugated secondary antibody Positive Signal Negative Signal Figure 1.5: G2 inhibition bioassay 9 1.5 P i n l Pin 1 is a negative regulator of the G2 to M phase transition (Figure 1.2); overexpression of Pinl causes G2 arrest.39 In addition, Pinl function has been associated with the pathogenesis of Alzheimer's disease. 4 0 Pinl belongs to a belongs to a family of proteins, the peptidyl-prolyl cis-trans isomerases, that catalyze the cis-trans isomerization of the bonds preceding prolyl bonds. These conformational changes play an important role in protein folding, regulation of the cell cycle and signal transduction.41 Figure 1.6 Cis-trans isomerization of prolyl peptide bonds Pinl is unique amongst the prolyl isomerases as it has a high degree of specificity for phosphorylated Serine-Proline bonds or phosphorylated Threonine-Proline bonds.42 The formation of neurofibrillary tangles, which contain paired helical filaments (PHFs), is a characteristic of all studied cases of Alzheimer's disease. PHFs are composed of a hyperphosphorylated adduct of the microtubule associated protein tau.4 3 In normal brains, tau plays a crucial role in establishing the neuronal microtubules network and in inducing microtubule assembly44; in Alzheimer's brains, tau is hyperphosphorylated and unable to bind to microtubules and promote microtubule assembly 4 5 Pinl has a high affinity for PHFs and binds Cis Trans 10 to phophorylated tau 4 0 In vitro results have demonstrated that Pinl binding to phosphorylated tau can restore the binding of tau to microtubules.40 However, hyperphosphorylation of tau (as observed in PHFs of Alzheimer's disease) sequesters Pinl from the nucleus into the cytoplasm, effectively reducing the concentration of Pinl in the nucleus. Depletion of Pinl from the nucleus is believed to initiate the following deleterious effects: activate kinases which further hyperphosphorylate tau, accelerate the formation of PHFs 4 3 and abnormally activate mitotic events in neuron cells.40 All of these effects can lead to neuronal degradation and accelerate the onset of Alzheimer's disease. There is much interest in compounds which inhibit the binding of Pin 1 to phosphorylated tau as a potential therapeutic for Alzheimer's disease. The only compound known to inhibit the activity of Pin 1 is 5-hydroxy-l,4-napthoquinone (Juglone) 6;41 it binds covalently to two cysteine residues on Pinl. 4 6 O OH O Figure 1.7 Structure of Juglone 6 However, juglone shows a broad range of activity and inhibits other enzymes such as pyruvate decarboxylase limiting its usefulness as a potential therapeutic. In response to the need to develop an assay for compounds which inhibited the binding of Pinl to phosphorylated tau, the laboratory of Michel Roberge developed a novel screening process. In this assay, streptavidin was immobilized onto a 96-well plate and given two hours to bind to the plate. The wells were then incubated with Alzheimer buffer (20 mM K2HPO4 pH 7.2, 0.12 M NaCl, 0.5 mM EDTA, 0.5 mM EGTA and 0.1 [M PMSF) and 2% BSA for at least 24 hours. 11 Biotinylated Tau was then added to the plates and unbound peptide was washed away with Tris buffer. Pinl was incubated with extracts for 20-30 minutes and then added into the 96 well plates; unbound Pin 1 was washed with Tris buffer after one hour. The remaining steps of the bioassay involve a qualitative method to detect Pinl bound to phosphorylated tau. A mouse monoclonal antibody which binds to the 6-His residue of Pinl was added and after 1.5 hours any unbound antibody was washed away with Tris buffer. A secondary antibody, goat horseradish peroxidase-conjugated anti-mouse IgM, was added and again the unbound antibody was washed with Tris buffer after one hour. ABTS was then added and the absorbance was measured at 405 nm using a microwell plated reader after 10-15 minutes of colour development. Upon Pinl binding to phosphorylated tau, a green colour resulted because of the reaction with of ABTS with goat horseradish peroxidase. Wells that contained extracts which inhibited the binding of Pinl to phosphorylated tau were colourless. Secondary ABTS Coloured Product Figure 1.8: Summary of Pinl binding to phosphorylated tau Bioassay 12 1.6 Aims of this study The goals of this study were to isolate the biologically active metabolites in Coccoloba acuminata and Minquartia guianensis using bioassay guided fractionations, elucidate the structures, and perform structural modifications in attempts to further study intracellular events. The C. acuminata crude extract gave a strong positive response in the G2 inhibition bioassay and, as discussed in Chapter 2, the active compound was determined to be e/7/-kaur-16-en-15-oxo-18-oic acid 7. Figure 1.9 Structure of ent-kaur-J6-en-15-oxo-18-oic acid (7) In methods directed at identifying the target protein of this G2 inhibitor, this diterpene was coupled with a biotin linker and the cellular effects were studied by Natalie Rundle of the Roberge group. In order to investigate the possible relationship between the G2 checkpoint cascade and Pin 1 inhibition, extracts from NCI were first screened for G2 inhibition and then for Pin 1 inhibition. Minquartia guianensis gave a strong positive response to both the G2 inhibition bioassay and the Pin 1 bioasssay. As described in chapter 3, three different metabolites were responsible for the observed activity; (S)-17-hydroxy-9,l 1,13,15-octadecatetraynoic acid 8 (minquartynoic acid) caused a positive response in the G2 inhibition bioassay and 4-0-(a-rhamnopyranosyl)ellagic 13 acid 9 and 4-0-(P-xylopyranosyl)ellagic acid 10 caused a positive response in the Pinl inhibition bioassay. HO 8 Figure 1.10: Structure of (S)-l7-hydroxy-9,l 1,13,15-octadecatetraynoic acid 8 9 10 Figure 1.11: Structure of 4-0-(a-rhamnopyranosyl)ellagic acid 9 and 4-0-(/5-xylopyranosyl)ellagic acid 10 A series of structural modifications were made to minquartynoic acid 8 to try to determine the regions of the molecule responsible for the observed activity. The aglycon of compounds 9 and 10 was also tested for activity in the Pin 1 inhibition bioassay. 14 1.7 References 1. Clark, A . M . Pharmaceut. Res. 1996, 13, 8:1133-1141 2. Wal l , M .E . ; Wani , M . C . Cancer Res. 1995 55:753 3. 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Curman, D.; Cinel , B.; Wil l iams, D.; Rundle, N. ; Block, W. ; Goodarzi, A . ; Gutchins, J . ; Clarke, P.; Zhou, B.; Lees-Mil ler, S.; Andersen, R.; and Roberge, M . J. Biol. Chem. 2001 276: 17914-17919 34. Sarkaria, J . ; Busby, E.; Tibbets, R.; Roos, P.; Taya, Y . ; Karnitz, L. Cancer Res. 1999 59: 4375-4382 16 35. Yamashita, K.; Yasuda, H.; Pines, J . ; Yasumoto, K.; Nishitani, H.; Ohtsubo, M . ; Hunter, T.; Sugimura, T.; Nishimoto, T. EMBO J. 1990 9: 4331-4338 36. Guo, X . ; Th 'ng, J . ; Swank, R.; Anderson, H.; Tudan, C ; Bradbury, E.; Roberge, M . EMBO J. 1995 14: 976-985 37. Rundle, N.T.; X u , L.; Andersen, R.J. ; Roberge, M . J Bio Chem-2001 276, 51: 48231-48236 38. Berlinck R.; Britton, R.; Piers, E.; L i m , L.; Roberge, M . ; da Rocha, R.; Andersen, R. J. Org. Chem. 1998 63 (26): 9850-9856 39. Lu , K. P.; Hanes, S.D.; Hunter, T. Science 1996 380: 544-547 40. Lu , P.; Wulf , G . ; Zhou, X . ; Davies, P.; L u , K.P. Nature 1999 399: 784-788 41. Gothel, S.F.; Marahiel. M .A . Cell. Mol. Life Sci. 1999 55: 423-436 42. Yaffe, M . B . Science 1997 278: 1957-1960 43. Spillantini, M . ; Goedert, M . Trends Neurosci. 1998 21: 428-433 44. Caceres, A . ; Kosik, K. Nature 1990, 343: 461-463 45. Bramblett, G.T. Neuron, 1993 10: 1089-1099 46. Henning, L.; Christner, C ; Kipping, M . ; Schelbert, B.; Rucknagel, K.; Grabley, S.; Kullertz, G. ; Fischer, G . Biochem. 1998 37: 5953-5960 Chapter 2: Coccoloba acuminata 17 Coccoloba acuminata is a seed bearing tree found in the Amazon region of South America. An extract of C. acuminata gave a positive response in the G 2 inhibition bioassay during high throughput screening of thousands of marine and terrestrial extracts from the National Cancer Institute's open repository. The biological activity of the crude extract coupled with no previous reports on the chemical constituents of C. acuminata in the literature made it an ideal candidate for further study. Figure 2.1: Coccoloba acuminata 18 2.1 enf-kaurene 2.1.1 Isolation of active diterpene from the cocoloba acuminata extract A concentrated pale brown methanol extract of C. acuminata was received from N C I and stored in a freezer. This solid extract was dissolved in methanol and loaded directly onto a Sephadex L H 20 column and eluted in 100% M e O H . The fractions obtained from this size-exclusion separation were collected and submitted for G2 checkpoint inhibition bioassay. A single peak of activity was observed in fractions 53-67. In a similar manner, the active fractions were combined and loaded directly onto a Sephadex LH20 column and eluted in 20:5:1 E tOAc :MeOH:H20 . The fractions obtained from this size-exclusion/polarity separation were collected and submitted for the G2 checkpoint inhibition bioassay. A single peak of activity was observed in fractions 24-26. Thin layer chromatography (40% EtOAc: 60% Hexanes) of fractions 22-28 and spraying with a vanillin spray revealed a variety of compounds with different R F values. The active fractions (24-26) contained a compound that was unique for these fractions and became a target for the next separation step. The active fractions were preloaded onto sil ica and separated using normal phase column chromatography (5% EtOAc : 95% Hexanes -> 15% E tOAc : 85% Hexanes). The purified compounds were submitted for bioassay and enr-kaur-16-en-15-oxo-18-oic acid (7) was determined to be a G2 checkpoint inhibitor. 19 Crude Coccoloba acuminata 1-52 Inactive in G2 inhibition bioassay 100% M e O H LH20 68-127 Inactive in G2 inhibition bioassay 20:5:1 E tOAc : M e O H : H 2 0 LH20 1-23 Inactive in G2 inhibition bioassay 27-68 Inactive in G2 inhibition bioassay 5-7.5-10-15% EtOAc:Hexane gradient flash column Figure 2.2: Isolation scheme of active diterpene from Coccoloba acuminata 2.1.2 Structure elucidation of active diterpene The structure of en?-kaur-l 6-en-15-oxo-18-oic acid was determined by analysis of the ' H N M R , l 3 C N M R , 2D N M R and high resolution mass spectrometery; the structure was confirmed by comparison of the data to literature values. 4 9 Figure 2.3 ent-kaur-16-en-l5-oxo-18-oic acidl The H R E I M S had a very intense base peak at [M + ] m/z 316.20416 corresponding to a molecular formula of C 2 o H 2 8 0 3 , consistent with a diterpene. The calculated degree of unsaturation for this molecular formula is seven. The molecular connectivity was determined based on the following 2D N M R experiments: correlation spectroscopy (COSY) , heteronuclear multiple-quantum correlation ( H M Q C ) and heteronuclear multiple-bond correlation ( H M B C ) . 4 8 The ' H N M R spectrum had two singlets at 8 6.05 ppm and 8 4.89 ppm corresponding to H-17' and H-17. The downfield shift suggested that the signal is caused by a conjugated terminal alkene. This is further supported by the three bond H M B C correlations from H-17 and H-17' to the carbonyl carbon, C-15 (5 208.9 ppm), consistent with an a , (3 unsaturated ketone. The H-17 and H-17' protons also showed a H M B C correlation to the methine carbon C-13 at 8 38.6 ppm. Figure 2.4: Selected HMBC correlations of compound 7 21 The relative positions of the two methyl groups (C-19 and C-20) and the carboxylic acid (C-18) were determined from the H M B C data. The H-5 proton (8 0.91 ppm) shows H M B C correlations to C-20 (8 16.0 ppm), C-19 (8 29.0 ppm), and C-18 (8 184.2 ppm); however, the H-20 protons (8 0.93 ppm) do not show a H M B C correlation to neither C-18 nor C-19. The H-19 proton (8 1.06 ppm) does show a H M B C correlation to C-18, suggesting a three bond separation. H-5 H-19 H-20 Figure 2.5: Selected HMBC correlations of compound 7 The connectivity between C-5 and the a , (3 unsaturated carbonyl C-15 can be observed through the H M B C correlations of the protons attached to C-7. The H - 7 ' (8 2.05 ppm) proton shows a H M B C correlation to C-15 at 8 208.9 ppm; the H-7 (8 1.12 ppm) proton shows a H M B C correlation to C-5 at 8 56.2 ppm. 20 _ 11 10 H .3 5 o 19 COOH 18 17 Figure 2.6: Selected HMBC correlations of compound 7 22 Based on this spectral data, a structure for compound 7 was proposed and the structure was confirmed by comparing spectral data to literature values. 4 9 Table 2.1 N M R data for ent-kaur-16-en-15-oxo-18-oic acid (7) recorded in C 6 D 6 Carbon No. 1 3 C 5 (ppm) a J H 8 (ppm) a b H M B C 1 40.0 HI 0.48, H I ' 1.54 H20, H2' 2 19.4 H2 1.34, H2' 1.94 3 37.9 H3 0.77, H3' 2.19 4 44.1 — H18 5 56.2 0.90 H18,H20, H3 ' ,H6 , H7 6 20.6 1.85 H5 7 36.9 H7 1.12, H7' 2.05 H14' 8 52.6 — H14',H7 9 51.8 = 1.20 H20, H11,H14 10 40.6 -- H20 11 18.8 1.38 H12' 12 32.6 H12 1.09, H12' 1.47 H14 13 38.6 2.64 H17,H17' 14 34.3 H14 2.07, H14' 1.34 15 208.9 -- H17,H17',H14, H7' 16 150.3 -- H17,H14, H12' 17 113.9 H17 6.05, H17'4.89 23 18 184.2 — H18,H3 19 29.0 1.06 H5 20 16.0 0.94 H5, HI a. recorded at 100 mHz b. assignment based on H M Q C 25 Figure 2.8: COSY spectrum of ent-kaur-16-en-15-oxo-l8-oic acid (7) 26 Figure 2.9: HMQC spectrum of ent-kaur-16-en-15-oxo-18-oic acid (7) 27 Figure 2.10: HMBC spectrum of ent-kaur-16-en-15-oxo-18-oic acid (7) 28 2.2 Biological activity There are no previous reports in the literature of either the crude C. acuminata extract or the diterpenoid enr-kaur-16-en-15-oxo-18-oic acid 7 exhibiting any biological activity. However, we found that 7 demonstrated two main cell cycle effects: it inhibited the G2 D N A damage checkpoint response and arrested cells in mitosis. In the G2 checkpoint inhibition bioassay using the human breast cancer cells MCF-7 p53-, 7 was found to have an IC50 of 9 ug/mL. (Figure 2.11) 0.1 1 10 100 Concentration ug/ml_ Figure 2.11 G2 inhibition response to MCF-7p53- cells Fortuitously, compound 7 is structurally related and has similar biological activity to other diterpene G2 checkpoint inhibitors that had been previously identified by my colleague Lin Xu. 13-Hydroxy-15-oxozoapatlin 5 and the diterpene ester 12 were both isolated from the African tree Parinari curatellifolia by bioassay guided fractionation and found to be G2 checkpoint inhibitors and antimitotic agents.50 29 Figure 2.12 Structural formula ofparinari G2 checkpoint inhibitors We compared the activity of 7 with 5 by studying the effects of the compounds on cells arrested at the G2 checkpoint after ionizing radiation. Human colon carcinoma cells deficient in p53 (HCT116 p53-/-) were irradiated with 10 grays of radiation and after 16 hours about 90% of the cells were arrested at the G2 checkpoint. Cells were then treated with a range of concentrations of compounds 7 and 5. Cells that were not treated with any compounds remained arrested at the G2 checkpoint, but cells with optimal treatment conditions proceeded into mitosis. The lactone diterpene 5 exhibited a maximum release into mitosis of 29% at a concentration of 10 (Ig/ml; the carboxylic acid diterpene 7 exhibited a maximum release into mitosis of 48% at a concentration of 12 ug/ml. (Figure 2.13) 30 100 w o o 0 1 10 [concentration] L I M 100 Figure 2.13: G2 checkpoint inhibition response of 7 compared to 5 The mechanism of action of a, P- unsaturated carbonyl diterpenes is of much interest because it is believed to be unique amongst G2 checkpoint inhibitors. Caffeine is believed to target A T M and A T R 5 1 , UCN-01 selects multiple targets Chkl and W e e l 5 2 ' 5 3 and D B H interacts with Chkl and Chk2 5 3 . However, none of these compounds contain an a, (3- unsaturated carbonyl functionality that make it reactive to nucleophiles. This gives 7 and 5 the potential to covalently bond to cysteine residues in proteins through Michael-type addition. The protein target of these a, P- unsaturated carbonyl diterpenes is yet to be identified. However, the carboxylic acid residue at C-19 on compound 7 invites the possibility of attaching a label via an ester linkage. 31 2.3 Biotin- Avidin Biotin is a water soluble vitamin that has a very high affinity constant of 1015 M " 1 for the egg white protein avidin or its bacterial counterpart streptavidin. The change of enthalpy, AH, for biotin bound to avidin is -86 kJ/mol and for biotin bound to streptavidin is -98.9 kJ/mol. 5 4 O x HN NH HOOC Figure 2.14: Structural formula of Biotin These extraordinary binding properties have allowed the development of avidin-biotin technologies for the separation of biotinylated compounds (low or high molecular mass) by avidin or streptavidin.55 A particularly effective aspect of this technology is the attachment of biotin to a biologically active molecule via an inert linker to create a biotinylated probe. Providing the biotinylation does not cause a loss of activity, the protein target can be separated by the biotin-avidin (streptavidin) binding; for example, the biotinylation of Okadaic acid to label protein phoshatases.56 2.3.1 Attachment of Biotin In steps directed towards identifying the protein target of a, p- unsaturated carbonyl diterpenes G2 checkpoint inhibitors, compound 7 was coupled to a biotin linker. This was achieved by reacting compound 7 with AModoacetyl-AM^iotinylhexylenediamine 13 in basic refluxing acetone. (Figure 2.15) 32 14 Figure 2.15: Scheme for biotin attachment of 7 The non-crystalline biotinylated product 14 showed the expected l 3 C N M R upfield shift of ~8 ppm (8 184^176 ppm) as C-19 changed from acarboxylic acid to an ester. As well, ESMS gave an intense peak for the parent ion [M] + at m/z 697.4, corresponding with the predicted molecular formula of C38O6N4SH58. 33 34 As a benchmark for comparison designed for biological studies, a model biotinylated compound was synthesized. 1 -Methylcyclohexanoic acid was reacted with AModoacetyl-/V-biotinylhexylenediamine in basic refluxing acetone to form a biologically inactive biotinylated probe. Figure 2.17: N-acetyl-N-biotinylhexylenediamine 1 -methylcyclohexanate (15) 2.3.2 Biological activity of biotinylated compounds Natalie Rundle of the Roberge laboratory performed the biological examinations of the biotinylated compounds and the key results are discussed below. The biotinylated compounds, 14 and 15, were tested for G2 inhibition against HCT116 p53-/-cells. At the optimum concentration for 14, 5 u M , it released 39-41% of cells from G2 arrest into mitosis. (Figure 2.18) The efficacy of 14 is slightly lower than for 7 (no biotin attachment) which releases 48% of cells from G2 arrest, but it is still greater than the lactone diterpene 5 which releases 29% of cells from G2 arrest. As expected, the model biotinylated compound (15) did not show the release of cells into mitosis. 35 Figure 2.18: G2 response of biotinylated compounds 2.3.3 Isolation of Target Protein After ensuring that the biological activity was retained with biotinylation, steps were taken to isolate the proteins that were bound to the biotin complex. HCT116 p53-/- cells were treated with nocodazole or 12 Gy of ionizing radiation. After 16 h, cells were treated with the biotinylated compounds for 4 h. The cells were resuspended and lysed by addition of RIPA lysis buffer. Lysates were filtered, incubated on ice for 1 h, centerfuged and loaded onto 50 % sterptavidin agarose bead slurry. The samples were nutated overnight at 4 °C. The unbound proteins were washed off with RIPA buffer and one wash with 20mM Tris-HCl. The proteins bound to the streptavidin were eluted by denaturing the proteins by boiling in Laemmli electrophoresis sample buffer with agitation for 10 min. (Figure 2.19) Inside cell Biotinylated probes added to cells -SH Inside cell Linker Biotin Streptavidin Bead Slurry Cells lysed S ^ 2 3 \ Linker Biotinf Streptavidin Protein denatured, Biotin released from Streptavidin beads Figure 2.19: Schematic of protein target isolation The proteins purified from the streptavidin agarose were detected by the use of protein gels as shown in Figure 2.20. In this figure, compound 14 is labeled B - O K A and compound 15 is labeled bio-model. Proteins are represented on the gel plate by thin black bands and the molecular weight increases towards the top of the plate. Sample Buffer GELS or Mass Spec 37 Figure 2.20: Gel for detection of Biotinylated proteins. The first set of columns labeled- lysis S/N- represents all the proteins in the cell. The second set of columns labeled -eluted-represents the proteins that bound to the streptavidin beads. As Figure 2.20 illustrates, the a, P- unsaturated carbonyl diterpene G 2 checkpoint inhibitor binds to a range of cellular targets. As the biotinylated model compound 15 does not appear to bind to any cellular proteins, the possibility of the biotinylated linker interfering with the results by binding to cellular proteins can be discounted. The proteins eluted from the streptavidin bead were sent for mass spectrometry. The identification of the target protein for a, p- unsaturated carbonyl diterpenes G 2 checkpoint inhibitors is ongoing by the Roberge laboratory. 38 2.4 References 47. Photograph from the Missouri Botanical Garden 48. Claridge, T.W. High-Resolution N M R techniques in Organic Chemistry 1999; C O S Y pp. 153-160; H M Q C pp. 224-229; H M B C 244-251 49. Monte, F.J. ; Dantas, E . M . ; Braz, R.; Phytochemistry 1988, 27, 10: 3209-3212 50. Rundle, N.T.; X u , L.; Andersen, R.J; Roberge, M . J. Biol. Chem. 2001 ,276, 51: 48231-48236 51. Sarkaria, J . ; Busby, E.; Tibbets, R.; Roos, P.; Taya, Y . ; Karnitz, L.; and Abraham, R. Cancer Res. 1999 59: 4375-4382 52. Graves, P.; Y u , L.; Gales, J . ; Sausville, E.; O'Connor, P.; Piwnica-Worms,H.; / . Biol. Chem. 2000 275: 5600-5605 53. Busby, E.C. ; Leistritz, D.; Abraham, R.; Karnitz, L.; Sarkaria, J .N. 2000 Cancer Res. 60: 2108-2112 54. Green, N . M . Biochem. J. 1966 ,101:74 55. Wilchek, M . ; Bayer, E .A. Methods Enzymol. 1990 184 56. Konoki , K.; Sugiyama, N. ; Murata,k M . ; Tachibana. K.; Hatanaka, Y . Tetrahedron 2000, 56: 9003-9014 Chapter 3: Minquartia guianensis Minquartia guianensis is a seed bearing tree native to tropical lowland forests in Central and South America. The characteristic hard lumber is harvested locally for heavy construction, 57 railway ties and bridges." Traditionally, an infusion of the outer and inner bark was used by medicinal healers as treatment for parasitic infections. 5 8 Figure 3.1 Leaves and stem of Minquartia guianensis59 Thousands of marine and terrestrial extracts from the National Cancer Institute's open repository were screened using the G2 checkpoint inhibition assay and several extracts were identified as 40 being active. 95 of these active extracts were further screened using the Pinl inhibition assay. Minquartia guianensis was active in both assays and selected as a candidate for further studies directed towards identifying the active metabolites. 3.1 Isolation of Active Metabolites from Minquartia guianensis A concentrated methanol extract of Minquartia guianensis was received from the National Cancer Institutes open repository and stored in the freezer. The first separation step of the crude extract involved a modified Kupchan partitioning scheme.60 Firstly, the crude M. guianensis extract was partitioned between EtOAc and H 20. Secondly, the EtOAc fraction was further partitioned by dissolving in a 9:2 H20/MeOH mixture and extracting with hexane. Finally, the 9:2 H20/MeOH fraction was further separated by dissolving in 6:4 H20/MeOH mixture and extracting with CH2C12. The collected fractions obtained from this solvent partitioning scheme were sent for both the G2 and Pinl Bioassay. The H 2 0 fraction was found to be active in the Pinl inhibition bioassay and the CH2C12 fraction was found to be active in the G2 checkpoint inhibition bioassay. The CH2C12 fraction was loaded onto a Sephadex LH20 column and eluted in 100% MeOH. The obtained fractions from the separation were submitted for G2 bioassay. The active fraction was further purified by normal phase column chromatography (30% EtOac: 70% hexanes + 0.1% acetic acid) and the active compound was found to be the known cytotoxic polyacetylene minquartynoic acid (8).58 Similarly, the H 2 0 fraction was loaded onto a Sephadex LH20 column and eluted in 100% MeOH. The collected fractions were submitted for the Pinl inhibition bioassay and two 41 biologically active ellagic acid glycosides were identified as 4-0-(a-rhamnopyranosyl)eIIagic acid 9 (eschweilenol C ) 6 1 and 4-0-((3-xylopyranosyl)ellagic acid 10.62 Crude Minquartia guianensis EtOAc/H 2 0 H ,0 E tOAc 100% MeOH LH20 c r ^cr J ^ ^ O H OH / O H 0/ o~^--r--oH HO Hexane Hexane/9:2 HnO:MeOH 9:2 H 2 0 :MeOH CH2Cl 2/6:4 H 2 0 : M e O h 1' CH,C1 2 6:4 H 2 0 :MeOH 100% MeOH LH20 HO 8 Figure 3.2 Isolation of active metabolites from Minquartia guianensis r 0 H o 42 3.2 Minquartynoic acid Minquartynoic acid was first identified from Minquartia guianensis as a tetraacetylenic compound that exhibited potent cytotoxicity against the P-388 (leukemia) cell line with an ED50 of 0.18 ug/ml . 5 8 Subsequent to this study, minquartynoic acid was isolated from Ochanostachys amentacea and found to exhibit anti-HIV activity. 6 3 Recently, a total synthesis of minquartynoic acid was reported. 6 4 3.2.1 Structure elucidation of minquartynoic acid 1 13 The structure of the polyacetylene 8 was solved by analysis of its H and C N M R data, H M Q C and H M B C spectra, and mass spectrometric data and confirmed by comparison with literature values. 5 8 ' 6 3 The molecular formula for 8 was determined from the parent ion in the electrospray M S . It gave a very intense peak [M + Na + ] at m/z 306.75 corresponding to a molecular formula of C 1 8 H 2 0 O 3 . The degree of unsaturation for this molecular formula is nine. HQ The l 3 C N M R spectra gave signals typical of a conjugated alkyne with eight peaks (C-9 to C-16) between 8 60-83 ppm. A carbonyl signal consistent with a carboxylic acid was observed at 8 177.38 ppm (C- l ) The *H N M R spectra shows a methine proton as a quartet at 8 4.51 ppm with a coupling constant of 7 / 7 ,7° = 6.7 Hz . The chemical shift is too far downfield to be in the typical range for a proton deshielded by only a hydroxyl group and suggests that the conjugated alkynes are contributing to the deshielding. The quartet multiplicity is consistent with the methine proton being split by the three methyl protons on C-18. This is further supported by the terminal methyl signal at 8 1.39 ppm that appears as a doublet with a coupling constant of JJS.U = 6.7 Hz. The assignment of the two methylene triplet signals at 8 2.36 ppm and 8 2.28 ppm as either H-8 or H-2 could not be done based on the ' H N M R spectra alone. However, this was readily achieved by analyzing a H M B C spectra which showed a two bond coupling from the proton at 8 2.28 ppm (H-2) to the carbonyl carbon C - l 8 177.4 ppm. Table 3.1 N M R data for Minquartynoic acid 8 recorded in C D 3 O D Carbon No. 1 3 C 8 (ppm) a * H 8 (ppm)(mult, J (Hz)) b c H M B C " 1 117.38 — H2, H3 2 34.97 2.28 (t ( J 2 > 3 , 7.6 Hz)) H3 3 25.96 1.60 (m) H2 4 29.98 1.35 (m) H2, H3 5 29.75 1.43 (m) 6 29.74 1.35 (m) 7 28.96 .1.55 (m) H8 8 20.00 2.34 (t ( J 8 i 7 , 7.0 Hz)) H7 9 82.91 — H8, H7 10 68.57 — H8 11 64.33 — H8 12 60.97 — H8 13 60.50 — 14 63.88 — H17 44 15 66.01 — H17 16 81.57 — H17.H18 17 58.79 4.51 (q (J,7,,8, 6.7 Hz)) H18 18 24.03 1.39 (d (J,8,,7, 6.7 Hz)) H17 a. recorded at 100 mHz b. recorded at 500 mHz c. assignment based on HMQC 45 Figure 3.4: HMBC spectrum of minquartynoic acid (8) 3.2.2 Biological activity of Minquartynoic acid Minquartynoic acid was found to elicit a positive response in Roberge's G2 checkpoint inhibition bioassay and antimitotic bioassay; 6 5 minquartynoic acid had an IC50 of 9 Ug/ml in the G2 checkpoint inhibition bioassay (Figure 3.5) and an IC50 of 10 Ug/ml in the antimitotic bioassay (not shown). 2 . 5 2 in 1.5 o Q O 1 0.5 \ ^ ^L* \ ^ \ ^ 0.01 0.1 1 10 100 1000 Concentration (ug/mL) - • — M . A . •X - - Caffeine Figure 3.5 Activity of Minquartynoic acid in G2 checkpoint inhibition bioassay The potent cytotoxicity of minquartynoic acid and the broad spectrum of activity limits its application as a useful pharmaceutical candidate; however, its high biological activity at low concentrations is intrinsically interesting. Previously, two naturally occurring analogues of minquartynoic acid were isolated from O. amentacea and identified as having similar biologically activity. 6 6 (S)-17, 18-dihydroxy-9,l 1,13,15-octadecatetraynoic acid 16 and (S) -17 -hydroxy-15£'-octadecen-9,l 1,13-triynoic acid 17 both exhibit a similar cellular toxicity as minquartynoic acid. 48 HO HOH2C- — — — — HQ OH O OH O 16 17 Figure 3.6: Naturally occurring analogues of minquartynoic acid Based on these reports and an interest in further studying the biological activity, a series of semisynthetic analogues were synthesized to try to develop a less cytotoxic analogue and determine the regions of the molecule important for biological activity. 3.2.3 Semisynthesis of Minquartynoic acid analogues Minquartynoic acid has the following three functionalities suitable for modifications: the hydroxyl group at C-17, the conjugated alkyne region and the carboxylic acid group at C- l . Figure 3.7: Regions of minquartynoic acid Sfor structure and reactivity studies A variety of synthetic modifications to these regions were attempted and the results are discussed below. HQ 8 49 The methyl ester of 8 was made by reacting with trimethylsilyl diazomethane in 1:4 MeOH:Benzene . 6 7 ' 6 8 The ! H N M R of methyl (S)-17-hydroxy-9,l 1,13,15-octadecatetraynate (18) showed the typical methyl ester singlet at 8 3.64 ppm. The hydroxyl group at C-17 of 8 was acetylated by reacting with acetic anhydride under basic conditions to form the acetylated adduct (S)-17-acetyl-9,l 1,13,15-octadecatetraynoic acid (19). The ! H N M R of 19 showed the expected downfield shift of ~ 1 ppm (4.51 ppm -> 5.44 ppm) for H-17 and the additional methyl singlet at 8 2.05 ppm. In a similar manner to 18, the methyl ester of 19 was made by reacting with trimethylsilyl diazomethane in 1:4 MeOH:Benzene. Again, the ' H N M R of methyl (S)-17-acetyl-9,l 1,13,15-octadecatetraynate (20) showed the typical methyl ester singlet at 8 3.64 ppm. The conjugated alkyne region of 8 was reduced down to single bonds by hydrogenation in the presence of charcoal on palladium catalyst to afford 17-hydroxystearic acid (21). Numerous attempts were made at producing a partially reduced product using Lindlar's method for the reduction of triple bonds to cis-alkenes. 6 9 " 7 2 However, these attempts were largely unsuccessful; the reaction continued to predominantly produce the fully reduced product (21), despite variation in temperature, reaction time and reagent quantities. A reaction time of one minute yielded 21 and a minor product containing a single double bond, 17-hydroxy-octadecaenoic acid (22). Analysis of the *H N M R of 22 shows signal broadening and poor resolution, possibly because more than one isomer was present. As this minor product 22 was biologically inactive, it was not purified further. 50 Another reaction that was attempted that did not produce the desired product was for the conversion of the methyl ester 18 into an aldehyde using D I B A L - H and -78°C.73 Work up yielded a partial recovery of the starting material. As well, the formation of the primary amide by bubbling ammonia through a solution containing the methyl ester 18 proved unsuccessful. One of the challenges of performing semi-synthetic modifications on natural products is the small scale required for mill igram amounts of starting material. The reactions that are generally successful necessitate high yields and optimum reaction conditions. The greatest difficulty encountered in performing synthetic modifications on minquartynoic acid was that conjugated alkynes are inherently unstable. Pure minquartynoic acid is reported as being unstable to light, air and temperatures over 100°C. 5 8 This made it very difficult to remove solvents (H2O, MeOH) as use of the rotary evaporator or vacuum line resulted in decomposition into blue crystals. A possible future approach for generating minquartynoic acid analogues of wi l l involve the use of the recently reported total synthesis. 8 T M S C H N 2 , 4:1 C 6 H 6 : M e O H HO 8 Acet ic Anhydride, Pyridine O 18 o 19 T M S C H N 2 > 4:1 C 6 H 6 : M e O H O A 19 o 20 8 H 2 P d / C E t O A c HQ 21 8 H 2 Lindlar's catalyst quinoline, M e O H Figure 3.8: Synthesis of Minquartynoic acid analogues Figure 3.9: Selected 'H NMR of Minquartynoic acid analogues 53 3.2.4 Biological activity of Minquartynoic acid analogues Tamsin Tarling of the Roberge lab performed the biological analysis of the minquartynoic acid analogues and the key results are discussed below. The semi-synthetic analogues containing the four conjugated triple bonds (18,19,20) were found to exhibit a similar biological response as the natural product minquartynoic acid; however, the semi-synthetic analogues with the conjugated triple bonds fully reduced (21) or partially reduced (22) did not exhibit any biological response. (Figure 3.10) 0.5 0 . 4 5 0.4 0 . 3 5 § 0 .3 0 . 2 5 0.2 0 . 1 5 0.1 0.01 0.1 1 cone (ug/mL) 1 0 1 0 0 - 21 X 1 8 - - - - 1 9 - -o- - 2 2 - -•- 2 0 Figure 3.10: Biological activity of the semi-synthetic analogues in the antimitotic bioassay. The methylated adduct 18 has an IC50 of 2 jlg/ml; the acetylated adduct 19 has an IC50 of 4 flg/ml; the methylated and acetylated adduct 20 has an IC50 of 15 fig/ml. 54 This biological data indicates that the preservation of the conjugated alkyne region is more important for the observed activities than the hydroxyl group at C17 or the carboxylic acid group at C- l . The cytotoxicity of minquartynoic acid and the semi-synthetic analogues was measured by a cytotoxicity assay. In this assay, one thousand MCF-7 p53- cells were plated into each well of a 96 well plate and the cells were grown overnight. Minquartynoic acid 8 and the semi-synthetic analogues were solubilized in DMSO, diluted in media, and then added to the cells in various concentrations. Wells that did not have any compounds added to them served as controls. The plate was grown for one week and each day the medium was removed and replaced with 100 uL of fresh media. After one week, an MTT assay was performed. MTT (3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) is a pale yellow substrate that is cleaved by living cells-requires active mitochondria- to yield a dark blue formazan product. For the MTT assay, 2.5 mL MTT was diluted in lOmL medium. After removing the medium from the plate, 125 uL of MTT was added, and incubated for 2 hours at 37°C. One hundred microlitres of lysis solution and MTT extraction buffer was added, and left overnight at 37°C. The plate was read at 570 nm and a graph of the results is shown in Figure 3.11. 55 Cytotoxicity Concentration | i g / m l Figure 3.11: Cytotoxixity of semi-synthetic analogues Unfortunately, the biologically active semi-synthetic analogues (18,19 and 20) exhibited a cellular toxicity similar to minquartynoic acid (8); conversely, the biologically inactive semi-synthetic analogues did not show any toxicity up to 50ug/ml. The following IC50 values were recorded for the toxicity assay: minquartynoic acid had an I C 5 0 of 3 ug/ml, methylated adduct 18 had an I C 5 0 of 4 ug/ml, the acetylated adduct 19 had an IC50 of 2 ug/ml, and the methylated and acetylated adduct 20 had an I C 5 0 of 4 ug/ml. The strong cytotoxicity of the analogues is a additional example of how they responded in a similar manner to minquartynoic acid. However, it also limits the analogues potential use as a pharmaceutical candidate. 56 3.3 Ellagic acid glycosides and ellagic acid Previous studies directed towards the identification of biologically active metabolites in minquartia guianensis have reported minquartynoic acid as the only biologically active metabolite. 5 8 ' 7 4 However, the use of the Pin 1 inhibitition bioassay allowed for two more biologically active metabolites to be identified, 4-0-(a-rhamnopyranosyl)ellagic acid 9 and 4 - 0 -((3-xylopyranosyl)ellagic acid 10. 3.3.1 Structure elucidation of 4-0-(oc-rhamnopyranosyl)ellagic acid 1 13 The structure of glycoside 9 was determined by extensive analysis of the H and C N M R , A P T , 2D N M R and mass spectrometry and confirmed by comparing to literature values. 6 1 Figure 3.12: Structure of 4-0-((X-rhamnopyranosyl)ellagic acid Using H R E S M S with negative ion detection, the molecular formula of 9 was determined from the intense peak at [ M - H ] " m/z '447.0551 to be C 2 0 H 1 6 O 1 2 . This molecular formula corresponds to a degree of unsaturation of thirteen. The ' H and 1 3 C N M R data was consistent with a hexose attached to an unsaturated macrocycle. (Figure 3.15) The relative configuration of the sugar moiety was determined by analyzing the vicinal coupling constants in the ' H N M R spectrum and applying the Karplus relationship which 57 approximates the dependence of the coupling on the dihedral angle. A poorly resolved doublet (Ji",2" 1 -2 Hz) at 8 5.45 ppm, a typical shift for an acetal proton, corresponded to the anomeric proton at H - l " . The small coupling constant suggests an equatorial-equatorial or equatorial-axial coupling. The C O S Y spectrum shows that H - l " is correlated to the proton at 8 3.99 ppm, H-2". This signal is a partially resolved doublet of doublets with coupling constants of Jx'x 1 -2 Hz and T2-.r of 3.4 Hz , the latter corresponding to an axial-equatorial coupling. The doublet of doublet proton signal at 8 3.83 ppm corresponds to H-3" with coupling constants of J3"j2- 3.4 Hz and J 3 " i 4 " 9.1 Hz. The large 9.1 Hz coupling suggests an axial-axial interaction between H-3" and H-4". The signal for H-4" at 8 3.32 ppm is split into a "triplet" caused by two strong axial-axial interactions with coupling constants of J ^ - 9.1 Hz and J ^ s - 9.1 Hz. A multiplet at 8 3.54 ppm, H-5", corresponds to a strong axial-axial coupling (h"A" 9.1 Hz) and coupling (Js-^" 6.1 Hz) to the methyl group at C-6" . The relative configuration is consistent with a-rhamnose. Figure 3.13 Coupling constants in Hertz of rhamnose moiety The structure of the aglycon portion of the molecule proved to be much more difficult to elucidate than the sugar moiety. The ' H N M R spectra showed only two proton singlets at 8 7.73 and 7.46 ppm, H-5 and H-5 ' , that were not part of the sugar; however, the l 3 C N M R spectra 58 showed fourteen signals between 107.3 and 159.1 ppm that were not part of the sugar. The H M B C did not give very much information about the connectivity of the aglycon due to the low number of protons relative to the number of carbons; however, the regular pattern of the signals suggested a high degree of symmetry. As the sugar moiety only accounts for one degree of unsaturation, the aglycon must have a degree of unsaturation of twelve. To determine the number of hydroxy! groups in the compound, the hydrogens were exchanged with deuterium and the change in the mass of the parent ion was determined. M.W. 447 M.W. 452 Figure 3.14: Exchange of protons with deuterium for compound 9 The parent ion for the deuterated compound was at m/z [M - H]" 452, corresponding to a change of 5 units and an exchange of 5 hydrogens. However, the total number of hydroxyl groups is 6 because the mass spectrometer detects a negative ion caused by the loss of a proton (Figure 3.14). As the sugar moiety contains 3 hydroxyl groups, the aglycon must contain the remaining 3 hydroxyl groups. A structure for glycoside 9 was proposed based on a combination of the information above and confirmed by comparing spectral data to literature values. 59 Table 3.2 N M R data for 4-0-(a-rhamnopyranosyl)ellagic acid 9 recorded in (CD 3) 2SO Carbon No. 1 3 C 8 (ppm) a *H 8 (ppm)(mult, J (Hz))b c 1 107.88 — 2 136.38 — 3 141.32 — 4 146.36 — 5 111.59 7.73 (s) 6 114.51 — 7 159.10 — r 107.33 — 2' 136.41 — 3' 139.88 — 4' 148.72 — 5' 110.24 7.46 (s) 6' 111.79 — 7' 158.9 — 1" 100.16 5.45 (d ( J,-r-1.2 Hz)) 2" 69.84 3.99 (dd ( J2» r 1.2 Hz and h",r 3.4 Hz)) 3" 70.05 3.83 (dd ( Jr,2- 3.4 Hz and J3»,4" 9.1 Hz)) 4" 71.75 3.32 (t ( J4",3- 9.1 Hz and J4",5" 9.1 Hz)) 5" 69.90 3.54 (m ( J5",4" 9.1 Hz and J5-,6" 6.1 Hz)) 6" 17.83 1.13 (d( J 6 »5" 6.1 Hz)) a. recorded at 100 mHz b. recorded at 500 mHz c. assignment based on H M Q C 60 Figure 3.15: 'H and 13C NMR spectra of 4-{a-rhamnopyranosyl)ellagic acid (9) 61 Figure 3.16: HMBC spectrum of 4-0-(a-rhamnopyranosyl)ellagic acid (9) 3.3.2 Structure elucidation of 4-0-(p-xylopyranosyl)ellagic acid (10) 62 The structure of 10 was determined by detailed analysis of the ' H N M R , 1 3 C N M R and J Resolved spectral data, mass spectrometry, and comparison to the previously determined ellagic acid glycoside 9; the structure was confirmed by comparison to literature values.62 Figure 3.17 Structure of 4-0-(ji-xylopyranosyl)ellagic acid The molecular formula of 10, C 1 9 H 1 4 O 1 2 , was determined from the intense peak at [M +] m/z 433 in the electrospray mass spectrum. This is a mass difference of 14 units (compared to 9), consistent with a loss of one carbon and two hydrogens. Analysis of the *H N M R and l 3 C N M R revealed that 10 was structurally related to the previously identified ellagic acid glycoside 9; the aglycon is the same but the sugar moiety is different. In the sugar region of the 1 3 C N M R spectrum, the anomeric carbon is at 8 103.05 ppm and four signals are between 8 65.82- 75.54 ppm. Upfield of this region, no C signals are present, discounting the possibility of a deoxy hexose as the sugar moiety; however, the data is consistent with a pentose. The relative configuration of the pentose was determined by analyzing the coupling constants. Despite trying various different solvents (DMSO, MeOH, D 2 0 , C D 3 C N ) for the ' H N M R spectrum, the signals for the sugar protons were too close together (overlapped) to measure the 63 coupling constants. To get the approximate coupling constants, a homonuclear J-resolved spectrum was collected where multiplet splittings are in the F l dimension and the decoupled proton spectrum is in the F2 dimension.75 The anomeric proton, H - l " , is a sharp doublet at 8 4.92 ppm with a coupling constant of J\",i" 7.4 Hz. The large scalar coupling suggests an axial-axial interaction with H-2". H-2" is a multiplet at 8 3.48 ppm with coupling constants of h",r 7.4 Hz and J2",3- 9.3 Hz, the latter corresponding to an axial-axial interaction with H-3". The proton at 8 3.38 ppm, H-3", has two large scalar coupling constants of J3",2" 9.3 Hz and Jyy 8.8 Hz, consistent with two axial-axial interactions. The proton at 3.54, H-4", is a multiplet and has two large axial-axial coupling constants of J4">3" 8.8 Hz and J4",5" 9.5 Hz and one smaller scaler coupling of J4",5" 5.1 Hz suggesting a axial-equatorial relationship. The relative configuration is consistent with xylose. Figure 3.18 Coupling constants in Hertz of xylose moiety Table 3.3 N M R data for 4-0-(p-xylopyranosyl)ellagic acid 10 recorded in 4:1 C D 3 C N : DMSO H Carbon No. 13, C 8 (ppm) H 8 (ppmHmult, J (Hz)) b,c 1 108.18 64 2 136.23 — 3 141.84 — 4 146.99 — 5 111.52 7.75 (s) 6 114.90 — 7 158.98 — r 107.43 — 2' 136.70 — 3' 139.81 — 4' 148.71 — 5' 110.15 7.55 (s) 6' 111,79 — 7' 158.92 — 1" 103.04 4.92 (d ( J r , r 7 . 4 H z ) ) 2" 72.96 3.48 (m ( J2» r . 7.4 Hz and J r , r 9.3 Hz)) 3" 75.53 3.38 (m ( J 3 » r 9.3 Hz and J r , 4 - 8.8 Hz)) 4" 69.23 3.32 (m ( J4",5- 9.5 Hz, J 4 " , r 8.8 Hz and J 4 - . 5 - 5.1 Hz)) 5" 65.82 3.42 (m ( J5»5.» 11.1 Hz and J5..>4» 9.5 Hz,)), 3.96 (dd (Jy»,5» 11.1 Hz and J 5 - , 4 - 5.1 Hz)) a. recorded at 100 mHz b. recorded at 500 mHz c. assignment based on H M Q C 65 66 OH H O ^ U ^ O - ^ O OH HO (ppm) 5.2 4.8 4.4 4.0 3.6 3.2 2.8 Figure 3.20: Homonuclear J resolved spectrum of 4-0-( ji-xylopyranosyl)ellagic acid (10) 67 3.3.3 Ellagic acid Ellagic acid glycosides 9 and 10 have previously been reported in the literature as not showing any significant biological activi ty. 6 1 ' 6 2 However, ellagic acid (the aglycon moiety) 23 is a well known commercially available anti-cancer agent; it has been demonstrated in animal models to inhibit tumor growth 7 6 " 7 8 . As ellagic acid has shown a broad range of activity, it was also include in the P in l inhibition bioassay. O H Figure 3.21: Structural formula of Ellagic acid 23 3.3.4 Pin 1 inhibition of ellagic acid and ellagic acid glycosides Priscil la Brastianos of the Roberge lab performed the examination of the biological activities of the ellagic acid glycosides and the key findings are discussed below. The two natural products, 4-0-(a-rhamnopyranosyl)ellagic acid 9 and 4-0-(P-xylopyranosyl)ellagic acid 10, and the aglycon ellagic acid 23 were tested in triplicate at a range of concentrations from 0.001 r)g/mL to 100 ug/mL. (see Figure 3.22) in the P in l inhibition bioassay. As described in the introduction, (see Figure 1.8 ) a colored tag is observed if the binding between P in l and phosphorylated tau is not inhibited resulting in a high absorbance 68 reading from the colorimeter. If a colored tag is not observed, the binding between Pinl and phosphorylated tau is inhibited resulting in a low absorbance reading from the colorimeter. 1.4 1.2 1 0.8 Q O 0.6 0.4 0.2 0 ' ' i 11111 i ,„ i 1 1 1 1 1 9 --10 23 0.000001 0.0001 0.01 Lig /mL 100 Figure 3.22: Inhibitory Effects of ellagic acid and ellagic acid glycosides on Pinl binding to phosphorylated Tau. From this Pin 1 bioassay data the following IC50 values were calculated: 0.09 Ug/mL for ellagic acid 23 , 0.7 ug/mL for 4-0-(p-xylopyranosyl)ellagic acid 10 and 0.6 ug/mL for 4-0-(a-rhamnopyranosyl)ellagic acid 9. The aglycon 23 is the most potent inhibitor of Pin 1 binding to phosphorylated tau, but the two natural products 9 and 10 are also effective Pin 1 inhibitors at a low concentration ( <1 ug/mL). From this assay, it is not possible to deduce if the compounds are binding to the phosphorylated tau protein and causing inhibition or binding to the Pinl protein and causing inhibition. To answer this, the Roberge lab performed a PhosphoTau peptide GF-31 binding assay79 -determines binding to phosphorylated tau- and found that the ellagic 69 acid glycosides did not bind to phosphorylated tau. This suggest that the mechanism of inhibition of these ellagic acid glycosides may be through binding to Pin 1. As the Pin 1 inhibition bioassay is performed in vitro, it is impossible to infer if the observed inhibition of Pin 1 would occur in a cell or even if ellagic acid and the ellagic acid glycosides will cross the cell membrane. In response to this, a number of cellular observations were obtained. The cytotoxicity of the two ellagic acid glycosides and ellagic acid was determined using the same MTT detection cytotoxicity assay described in section 3.3.4. The results of this assay are graphed in Figure 3.23. Cytotoxicity 1 T — — ! U) [EA] u.g/ml_ Figure 3.23: Cytotoxicity of Ellagic acid glycosides and ellagic acid From this data the following IC50 values were calculated: 7 Ug/mL for ellagic acid 23 , 17 ug/mL for 4-0-(P-xylopyranosyl)ellagic acid 10 and 70 ug/mL for 4-0-(a-rhamnopyranosyl)ellagic acid 9. The importance of the sugar moiety is illustrated by this cytotoxicity assay as the data suggests that the absence of a sugar or the type of sugar present can change the cellular toxicity by a factor of ten. 70 The effects of compounds 9,10 and 23 on subcellular localization of Pin 1 were also studied qualitatively under the microscope. In normal cells, Pin 1 characteristically appears as distinct dots in the nucleus. For this procedure, cells were treated with various concentrations of the compounds and after 5 hours the effects on subcellular localization were observed. For the cells treated with ellagic acid 23, no observable effects on Pinl localization were identified at concentrations below the toxicity limits (7 Ug/mL) and at toxic concentrations the cells lysed. The ellagic acid glycoside, 4-0-((3-xylopyranosyl)ellagic acid 10, did not appear to have any observable effect on Pin 1 localization; however, 4-0-(a-rhamnopyranosyl)ellagic acid 9 had a noticeable effect on Pin 1 localization. At a concentration of 50 Ug/mL, Pin 1 no longer appeared as distinct dots in the nucleus, but was dispersed throughout the nucleus. This is further support for the suggestion that 4-0-(a-rhamnopyranosyl)ellagic acid directly interacts with P in l . 3.3.5. Therapeutic potential of Ellagic acid glycosides As mentioned in the introduction, Pin 1 is a negative regulator of the G2 -> M transition. Based on this postulate, we were interested to find out if a compound which inhibited the binding of Pinl to phosphorylated tau would also be a G2 checkpoint inhibitor. We found that neither the ellagic acid glycosides nor ellagic acid exhibited any response in the G2 checkpoint inhibitor bioassay. However, the identification of compounds which are Pin 1 inhibitors will continue to be a topic of interest in the development of a possible therapeutic for Alzheimer's disease. 4-0-(a-rhamnopyranosyl)ellagic acid and 4-0-((3-xylopyranosyl)ellagic acid are the first Pin 1 inhibitors to be identified by a rational screening process. 71 References: 57. Van der Sloten, H. E T al. 1971. Properties and uses of one hundred thirteen maderables species of Panama. The United Nations for Agriculture and the Feeding (the F A O ) pp. 750 58. Maries, R.J. ; Farnsworth, N.R.; Ne i l l , D. J. Nat. Prod. 1989, 52, 2: 261-266 59. Photograph by D. L. Nickrent at the R. & C. Wilson Botanical Garden, San Vi to, Costa Rica. 60. Kupchan, S . M . ; Britton, R.W.; Laccadie, J . ; Ziegler, M.F. ; Siegel, C .W. J. Org. Chem. 1975, 40, 5: 648-654 61. Yang, S.; Zhou, B.; Wisse, J . ; Evans, R.; van der Werff, H.; Mi l ler , S.; Kingston, D. J. Nat. Prod. 1998, 61: 901-906 62. Tanaka, T.; Jiang, Z. ; Kound, I.; Phytochemistry 1998, 47,5: 851-854 63. Rashid, M .A . ; Gustafson, K.R. ; Cardellina, J . ; Boyd, M.R. Nat. Prod. Lett. 2001, 15, 1: 21-26 64. Gung, B.W.; Dickson, H Org. Lett, in press 65. Roberge, M . ; Cinel , B.; Anderson, H.; L im , L.; Jiang, X . ; X u , L.; B igg , C ; Kel ly , M . ; Andersen, R. Cancer Res. 2001 60 66. Ito, A . ; Cu i , B.; Chavez, D.; Chai , H.; Shin, Y . ; Kawanishi, K.; Kardono, L.; Riswn, S.; Farnsworth, N. ; Cordell , G . ; Pezzuto, J . ; Kinghorn, D. J. Nat. Prod. 2001, 64: 246-248 67. Aoyama, T.; Shiroiri, T. Tetrahedron Lett. 1990, 31: 5507 68. Aoyama, T.; Tersawa, S.; Sudo, K.; Shioir i , T. Chem. Pharm. Bull. 1984, 32: 3759 69. Lindlar, H. Helv. Chim. Acta. 1952 35: 446 70. Lindlar, H. ; Dubuis, R. Org. Syn. 1966, 46: 89 71. Marvel l , E .N . ; L i , T. Syn. 1973, 457 72. McEwen, A . ; Guttieri, M . ; Maier, W. ; Laine, R.; Shyo, Y . J Org Chem 1983, 48: 4436 73. Zakharkin, L.; Khorl ina, I. Tetrahedron Lett. 1962, 14: 619-620 72 74. Rasmussen, H.; Christensen, S.; Kvist, L.; Kharazmi, A.; Huansi, A. J. Nat. Prod. 2000, 63: 1295-1296 75. Claridge, T. D; High-Resolution NMR techniques in Organic Chemistry 1999, pp267-273 76. Barch, D.H.; Rundhaugen, L.M; Stoner, G.D.; Pillay, N.S.; Rosche, W.A.; Carcinogenesis 1996, 17: 265-269 77. Stoner, G.D.; Morse, A.M.; Cancer Lett. 1991, 37, 1997 78. Dow, L.R.; Chou, T.T.; Bechle, M.B.; Goddard, C ; Larson, R.E.; /. Med. Chem. 1994, 37: 2224-2231 79. procedure used by the Michel Roberge laboratory 73 Chapter 4 Experimental 4.1 General Procedures Reversed-phase and Normal-phase thin layer chromatography were carried out on Whatman M K C 1 8 F and Kieselgel 20 F254 plates. Compounds were detected by viewing under ultraviolet radiation (k =254 nm) and/or heating after spraying with a vanillin reagent. Column chromatography was performed using glass columns containing Sephadex L H 2 0 (bead size 25-100 \i) or Si l ica Gel 60 (230-400 mesh). The solvent mixtures used for the separations involving thin layer chromatography and column chromatography are described in the subsequent procedures. A l l solvents used for extraction and for column chromatography were Fisher reagent grade unless otherwise stated. A l l other reagents and standards were reagent or commercial grade and were used without further purification. ' H N M R spectra and 2D N M R were collected on either a Bruker A M X - 5 0 0 (500 M H z ) or a Bruker AV-400 (400 M H z ) spectrometer equipped with a 5 mm probe. 1 3 C N M R spectra were recorded on a Bruker A M - 4 0 0 (100.5 M H z ) spectrometer equipped with a 5 mm probe. N M R spectra were recorded using hexadeutereobenzene (CQD6), deuterated methanol (CD3OD), hexadeuteriodimethyl sulfoxide ( D M S O -do) and deuterated acetonitrile (CD3CN). Chemical shifts (5) are given in parts per mil l ion from tetramethylsilane (8 0) and spectral data was calibrated to the solvent used ( C 6 D 6 : 8 7.15 ' H N M R , 8 128.00 1 3 C N M R ; C D 3 O D : 8 3.30 ' H N M R , 8 49.5 l 3 C N M R ; D M S O - ^ j : 8 2.49 ' H N M R , 8 39.5 1 3 C N M R ; C D 3 C N : 8 1.98 ' H N M R ) . Low and high resolution electron impact (EI) mass spectra were recorded on either a' Kratos M S 5 0 or a M S 8 0 mass spectrometer at 70 eV. Low and high resolution electron spray ionization (ES) mass spectra were recorded on either a Bruker/HP Esquire or a Kratos Concept 74 IIHQ mass spectrometer. The low and high resolution EI and high resolution ES mass spectrometric analyses were performed by the U B C Mass Spectrometry Laboratory. 4.2 Isolation: ent-kaur-16-en-15-oxo-18-oic acid (7) A concentrated pale brown methanol extract of C. acuminata was received from N C I and stored in a freezer. 200 mg of this solid extract was dissolved in 3 mL methanol and loaded directly onto a Sephadex L H 20 column in 100% M e O H . The fractions obtained from this size-exclusion separation were collected and submitted for the G2 bioassay. A single peak of activity was observed in fractions 53-67. The active fractions were combined and the solvent was removed by evaporation under reduced pressure (87 mg). The crude extract was redissolved in 3 m L of a solvent system containing 20:5:1 E t O A c : M e O H : H 2 0 and loaded onto a Sephadex L H 2 0 column containing the same solvent system. The fractions obtained from this size-exclusion/polarity separation were collected and submitted for the G2 bioassay. A single peak of activity was observed in fractions 24-26. Thin layer chromatography (40% E tOAc : 60% Hexanes) of fractions 22-28 and spraying with a vanillin spray revealed a variety of compounds with different R F values. The active fractions (24-26) contained a compound that was unique for these fractions and became a target for the next separation step. The active fractions were combined and the solvent was removed under reduced pressure (16.4 mg). The crude residue was preloaded onto sil ica and separated using column chromatography (5% E tOAc : 95% Hexanes -> 15% E t O A c : 85% Hexanes). The purified compound 7 (5 mg) formed white crystals. Mass Spec EI [M + ] m/z at 316.20416 corresponds to C2oH 2 80 3 . 1 H N M R (400 M H z ) (solvent: C 6 D 6 , Reference 5 7.15 ppm): 5 6.05 (s 75 1H, H-17), 4.89 (s 1H, H-17'), 2.63 (s 1H, H-13), 1.20 (s 1H, H-9), 1.06 (s 3H, H-18), 0.93 (s 3H, H-20) 0.91 (s 1H, H-5). 1 3 C N M R (100 M H z ) (solvent: C 6 D 6 , Reference 8 128.00 ppm): 8 208.9 (C15), 184.2 (C19), 150.3 (C16), 113.9 (C17), 56.2 (C5), 52.6 (C8), 51.8 (C9), 44.1 (C4), 40.6 (CIO), 40.0 (CI) , 38.6 (C13) , 37.9 (C3), 36.9 (C7), 34.3 (C14), 32.6 (C12), 29.0 (C18), 20.6 (C6), 19.4 (C2), 16(C20) (S)-J 7-hydroxy-9,11,13,15-octadecatetraynoic acid (8) A concentrated methanol extract of Minquartia guianensis was received from the N C I open repository and stored in the freezer. The first separation step of the crude extract involved a modified Kupchan partitioning scheme. 423 mg of the crude m. guianensis was partitioned between 200 mL of E t O A c and 200 m L of H 2 0 . The solvents were removed by evaporation under reduced pressure. 235 mg of the E tOAc fraction was further partitioned by dissolving in a 200 m L 9:2 H 2 0 / M e O H mixture and extracting with 100 mL of hexane. The solvents were removed by evaporation under reduced pressure. The 9:2 H 2 0 / M e O H fraction was further separated by dissolving in 6:4 H 2 0 / M e O H mixture and extracting with C H 2 C 1 2 . The collected fractions obtained from this solvent partitioning scheme were sent for both the G2 and P in l Bioassay. The H 2 0 fraction was found to be active in the P in l Bioassay (discussed below) and the C H 2 C 1 2 fraction was found to be active in the G2 checkpoint assay. The C H 2 C 1 2 fraction (194.5 mg) was loaded onto a Sephadex L H 2 0 column and eluted in 100% M e O H . The obtained fractions from the separation were submitted for G2 bioassay. Normal phase T L C of the active fractions (30% EtOAc/Hexane + 0.1% C H 3 C O O H ) revealed a single U V active spot off the base line. The crude product was further purified by column chromatography (30% EtOAc/Hexane + 0.1 % C H 3 C O O H ) to yield pure yellow crystals of 8 (86.5 mg, 27%). Mass Spec Electrospray [M + Na + ] m/z at 306.75 corresponds to C 1 8 H 2 o 0 3 . *H N M R (400 M H z ) (solvent: C D 3 O D , Reference 8 3.30 ppm): 84.51 (q 1H, J 1 7 , i 8 , 6.7 Hz , H-17), 76 2.36 (t 2H, Jgj, 7.0 Hz, H-8, H-8'), 2.28 (t 2H, J 2, 3, 7.6 Hz, H-2, H-2'), 1.39 (d 3H, J 1 8 , 1 7 , 6.7 Hz, H-18), 1.26-1.66 (m 10H, H-7, H-7', H-6, H-6\ H-5, H-5', H-4, H-4', H-3, H-3'). 1 3 C N M R (100 MHz) (solvent: C D 3 O D , Reference 5 49.5 ppm): 5 177.38 (CI), 82.91 (C9), 81.57 (C16), 68.57 (CIO), 66.01 (C15), 64.33 (CI 1), 63.88 (C14), 60.97 (C12), 60.50 (C13), 58.79 (C17), 34.97 (C2), 29.98 (C4), 29.75 (C5), 29.74 (C6), 28.96 (C7), 25.96 (C3), 24.03 (C18), 20.00 (C8) 4-0-((X-rhamnopyranosyl)ellagic acid (9) and 4-0-(j3-xylopyranosyl)ellagic acid (10) The H 2 0 fraction from above (143 mg) was loaded onto a Sephadex LH20 column and eluted with 100% MeOH. The fractions obtained from this separation were submitted for the Pin 1 bioassay. Two different fractions came back active. Reversed Phase T L C in 70% MeOH/H 2 0 revealed a single U V active spot that turned white after extensive charring and vanillin reagent in each active fraction. Evaporation of the solvent under reduced pressure yielded pure white crystals of 9 (6 mg) and pure white crystals of 10 (4 mg). For 9, HRESMS [M - H]" at m/z 447.0551 corresponds to C 2 0 H i 6 O | 2 . *H N M R (400 MHz) (solvent: DMSO, Reference 8 2.49 ppm): 8 7.73 (s 1H, H-5), 7.46 (s 1H, H-5'), 5.45 (d 1H, J,»2» 1.2 Hz, H - l " ) , 3.99 (dd 1H, J r , r 1.2 Hz and J2",3" 3.4 Hz, H-2"), 3.83 (dd 1H, J 3» 2.. 3.4 Hz and J r , 4 - 9.1 Hz, H-3"), 3.54 (m 1H, J5»,4" 9.1 Hz and J5",6" 6.1 Hz, H-5"), 3.32 (t 1H, J4",3" 9.1 Hz and J 4 " , r 9.1 Hz, H-4"), 1.13 (d 3H, J6-,5" 6.1 Hz, H-6"). , 3 C N M R (100 MHz) (solvent: DMSO, Reference 8 39.5 ppm): 8 159.10 (C7), 158.9 (C7'), 148.72 (C4'), 146.36 (C4), 141.32 (C3), 139.88 (C3'), 136.38 (C2), 136.41 (C2"), 114.51 (C6), 111.79 (C6'), 111.59 (C5), 110.24 (C5'), 107.88 (CI), 107.33 (CI'), 100.16 (CI"), 71.75 (C4"), 70.05 (C3"), 69.90 (C5"), 69.84 (C2"), 17.83 (C6"). For 10, Mass Spec Electrospray [M - H]" at m/z 432.45 corresponds to C i 9 H | 4 0 1 2 . *H N M R (400 MHz) (solvent: 4:1 C D 3 C N : DMSO, Reference 8 2.49 ppm): 8 7.75 (s 1H, H-5), 7.55 (s 1H, H-5'), 4.92 (d 1H, Ji-,2- 7.4 Hz, H - 1"), 3.96 (dd 1H, J 5-, 5" 11.1 Hz and J5.",4-5.1 Hz, H-5"'), 3.54 (m 1H, J4„ i 5., 9.5 77 Hz, J 4"3» 8.8 Hz and J4»5.» 5.1 Hz, H-4"), 3.48 (m IH, J 2 » , r 7.4 Hz and J r , 3 " 9.3 Hz, H-2"), 3.42 (m IH, J 5", 5- 11.1 Hz and J5.,4- 9.5 Hz, H-5"), 3.38 (m IH, J r , r 9.3 Hz and J3-,4" 8.8 Hz, H-3"). I 3 C N M R (100 MHz) (solvent: DMSO, Reference 5 39.5 ppm): 8 158.98 (CJ), 158.92 (C7'), 148.71 (C4'), 146.99 (C4), 141.84 (C3), 139.81 (C3'), 136.23 (C2), 136.70 (C2"), 114.90 (C6), 111.79 (C6'), 111.52 (C5), 110.15 (C5'), 108.18 (CI), 107.43 (CI'), 103.04 (CI"), 75.53 (C3"), 72.96 (C2"), 69.23 (C4"), 65.82 (C5") 4.3 Synthesis N-acetyl-N-biotinylhexylenediamine ent-kaur-16-en-15-oxo-18-ate (14) A heterogeneous solution of AModoacetyl-/V-biotinylhexylenediamine (13) (6.5 mg, 0.0127mmol) (Pierce Chemical Company) in dry acetone (0.5 mL) was warmed to 50°C and added dropwise to a solution of compound 7 (2.0 mg, 0.006mmol) and K 2 C 0 3 (~4 mg) dissolved in dry acetone (0.5 mL). The reaction vial was sealed with Teflon tape and left stirring overnight at 57°C. Thin layer chromatography (10% MeOH/CH 2 Cl 2 ) showed a single U V active compound. The reaction mixture was filtered through a syringe filter and the solvent was removed under reduced pressure. The reaction product was purified by column chromatography (5% MeOH: 95% CH 2 C1 2 -> 10% MeOH: 90% CH 2 C1 2 ) to yield pure non-crystalline 14 (3.0 mg, 0.004 mmol, 72%). For 14, mass spec electrospray [M +] at m/z 697.4 corresponds to C 3 8 0 6 N 4 S H 5 8 . ' H N M R (400 MHz) (solvent: DMSO, Reference 8 2.49 ppm): 8 7.86 (t IH, J = 5.5 Hz), 7.71 (t IH, J = 5.5 Hz), 6.40 (s IH), 6.34 (s IH), 5.77 (s IH), 5.33 (s IH), 4.40 (dd 2H, J = 14.0 Hz, J = 7.3 Hz), 4.29 (m IH), 4.10 (m 2H), 3.16 (d IH, J = 5.2 Hz), 3.1-2.95 (m 5H), 2.80 (dd IH, J = 4 . 9 H z J = 12.5 Hz), 2.56 (d 1HJ= 12.5 Hz), 2.30 (d 1HJ= 11.9 Hz), 2.09 (s IH), 2.02 (t 2H, J = 7.6 Hz), 1.84-1.56 (m 9H), 1.53-1.43 (m 3H), 1.42-1.20 (m 14H), 1.18 (s, 3H) 78 1.15-0.96 (m 3H), 0.86 (s, 3H) l 3 C N M R (100 M H z ) (solvent: D M S O , Reference 5 39.5 ppm): 8 209.2, 175.8, 171.6, 166.1, 162.5, 149.2, 114.1,62.0, 60.9,59.1,55.3,51.0,43.1,38.2,38.0, 37.16, 35.7, 35.1, 33.2, 31.6, 29.0, 28.9, 28.1, 28.1, 27.9, 26.0, 25.9, 25.2, 19.5, 18.3, 17.9, 15.1 N-acetyl-N-biotinylhexylenediamine I-methylcyclohexanate (15) A heterogeneous solution of AModoacetyl-A^biotinylhexylenediamine (13) (7.2 mg, 0.014mmol) in dry acetone (0.5 mL) was warmed to 50°C and added dropwise to a solution of 1-mthylcyclohexanoic acid (2.0 mg, 0.014mmol) and K2CO3 (~4 mg) dissolved in dry acetone (0.5 mL). The reaction vial was sealed with Teflon tape and left stirring overnight at 57°C. Normal phase thin layer chromatography (10% MeOH/CH 2 C l 2 ) showed a single U V active compound. The reaction mixture was filtered through a syringe filter and the solvent was removed under reduced pressure. The reaction product was purified by column chromatography (5% M e O H : 95% CH2CI2 10% M e O H : 90% C H 2 C 1 2 ) to yield pure non-crystalline 15 (3.2 mg, 0.006 mmol, 44%). For 15, mass spec electrospray [M + ] at m/z 523.3 corresponds to C26O5N4SH44. N M R (400 M H z ) (solvent: C D 3 O D , Reference 8 3.30 ppm): 8 7.83 (t IH , J = 5.5 Hz), 7.70 (t IH , J = 5.5 Hz) , 6.39 (s IH), 6.33 (s IH), 4.28 (m IH), 4.09 (m IH), 3.11-2.95 (m 5H), 2.79 (dd IH , J = 4.9 Hz , J = 12.2 Hz), 2.55 (d 1H, J = 12.5 Hz) 2.47 (m 2H), 2.00 (t 2H, J = 7.3 Hz) 1.94 (m 2H), 1.62-1.14 (m 22H), 1.10 (s 3H) (S)-17-acetyl-9, J1,13,15-octadecatetraynoic acid (19) A solution of compound 8(15 mg, 0.053 mmol), acetic anhydride (0.1 mL) and anhydrous pyridine (3 mL) was left stirring at room temperature overnight under an Argon atmosphere. Normal phase thin layer chromatography (20% EtOAc/Hexane + 0.1% Acetic Acid) showed three compounds (not the starting material) when sprayed with a vanillin reagent. The solvent was removed by evaporation under reduced pressure. The crude product was purified by column 79 chromatography (5% EtOAc: 95% Hexane + 0.1% C H 3 C O O H -> 15% EtOAc: 85% Hexane + 0.1 % C H 3 C O O H ) to yield the pure pale yellow crystals of 19 (12 mg, 0.037 mmol, 70%). Mass Spec electrospray [M + Na +] m/z 348.75 corresponds to C20H22O4. N M R (400 MHz) (solvent: CD 3 OD, Reference 5 3.30 ppm): 8 5.44 (q 1H, J 1 7 , , 8 , 6.7 Hz, H-17), 2.37 (t 2H, J 8, 7, 7.0 Hz, H-8, H-8'), 2.28 (t 2H, J 2, 3, 7.3 Hz, H-2, H-2'), 2.05 (s 3H, H-20), 1.48 (d 3H, J 1 8 , , 7 > 6.7 Hz, H-l8), 1.28-1.66 (m 10H, H-7, H-7', H-6, H-6', H-5, H-5' , H-4, H-4', H-3, H-3'). 1 3 C N M R (100 MHz) (solvent: C D 3 O D , Reference 8 49.5 ppm): 8 177.38 (CI), 172.98 (C19), 82.91 (C9), 81.57 (C16), 68.57 (C10), 66.01 (C15), 64.33 (CI 1), 63.88 (C14), 60.97 (C12), 60.50 (C13), 58.79 (C17), 34.97 (C2), 29.98 (C4), 29.75 (C5), 29.74 (C6), 28.96 (C7), 25.96 (C3), 24.03 (C18), 20.00 (C8) Methyl (S)-l 7-hydroxy-9,11,13,15-octadecatetraynate (18) A solution of compound 8 (27.4 mg, 0.096 mmol), T M S - C H 2 N H 2 (300uL), dry benzene (8 mL) and MeOH (2 mL) was left stirring at room temperature overnight. Normal phase thin layer chromatography of the reaction products (20% EtOAc/Hexane) showed a single U V active compound of the baseline. The reaction products were purified by column chromatography (5% EtOAc: 95% Hexane-> 15% EtOAc: 85% Hexane) to yield pure 18 (25.0 mg, 0.083mmol, 86%). Mass Spec electrospray [M + Na +] m/z 320.75 corresponds to C 1 9 H 2 2 O 3 . N M R (500 MHz) (solvent: C D 3 O D , Reference 8 3.30 ppm): 8 4.51 (q 1H, J , 7 , i8 , 6.5 Hz, H-17), 3.64 (s 3H, H-19) 2.36 (t 2H, J 8, 7, 7.4 Hz, H-8, H-8'), 2.31 (t 2H, J 2, 3, 7.4 Hz, H-2, H-2'), 1.39 (d 3H, J , 8 i l 7 j 6.5 Hz, H-18), 1.26-1.66 (m 10H, H-7, H-7', H-6, H-6', H-5, H-5' , H-4, H-4', H-3, H-3'). Methyl (S)-17-acetyl-9,l 1,13,15-octadecatetraynate (20) A solution of compound 19 (8.0 mg, 0.024 mmol), TMS-diazamethane (300uL), dry benzene (8 mL) and MeOH (2 mL) was left stirring at room temperature overnight. Normal phase thin layer 80 chromatography of the reaction products (20% EtOAc/Hexane) showed a single UV active compound of the baseline. The reaction products were purified by column chromatography (5% EtOAc: 95% Hexane-* 15% EtOAc: 85% Hexane) to yield pure 20 (6.0 mg, 0.018mmol, 74%) Mass Spec electrospray [M + Na +] m/z 362.75 corresponds to C 2 1 H 2 4 O 4 . N M R (400 MHz) (solvent: C D 3 O D , Reference 8 3.30 ppm): 8 5.44 (q IH, J 1 7 > 1 8 , 6.7 Hz, H-17), 3.64 (s 3H, H-19), 2.37 (t 2H, J 8, 7, 7.0 Hz, H-8, H-8'), 2.31 (t 2H, J 2, 3, 7.3 Hz, H-2, H-2'), 2.05 (s 3H, H-20), 1.48 (d 3H, J 1 8 , ,7, 6.7 Hz, H-18), 1.28-1.66 (m 10H, H-7, H-7', H-6, H-6', H-5, H-5' , H-4, H-4', H-3, H -3'). 17-hydroxystearic acid (21) Compound 8 (30.3 mg, 0.11 mmol) was dissolved in EtOAc (20 mL). 10% Pd/ charcoal catalyst (5 mg) was added to the rapidly stirring mixture. The flask was then flushed with H 2 gas and left to react for 2 V* hour. The heterogeneous reaction mixture was filtered through Celite and the solvent was removed by evaporation under reduced pressure. Normal phase thin layer chromatography (20% EtOAc/Hexane + 0.1 % Acetic acid) revealed a single spot off the baseline when sprayed with vanillin reagent. The reaction product was purified by column chromatography (5% EtOAc: 95% Hexane + 0.1% C H 3 C O O H 10% EtOAc: 90% Hexane + 0.1% CH 3 COOH) to yield pure white crystals of 21 (27 mg, 0.09mmol, 82%). Mass Spec electrospray [M + Na +] m/z 322.85 corresponds to C 1 8 H 3 6 O 3 . N M R (400 MHz) (solvent: CD3OD, Reference 8 3.30 ppm): 8 3.69 (m IH), 2.27 (t 2H, J = 7.4 Hz), 1.59 (m 2H), 1.29 (m 28H), 1.13 (d 3H, J = 6.0 Hz) (S)-17-hydroxy-octadecaenoic acid (22) Compound 8 (32 mg, 0.11 mmol) was dissolved in MeOH (5 ml). Lindlar catalyst (5 mg) and quinoline (0.15 mL) were added to the rapidly stirring mixture. The flask was then flushed with 81 H 2 gas and left to react for 1 minutes. Normal phase thin layer chromatography (40% EtOAc/Hexane + 0.1 % Acetic acid) of the reaction mixture showed 4 compounds off the base line. The heterogeneous mixture was filtered through celite and the solvent was removed by evaporation under reduced pressure. The reaction mixture was purified by column chromatography (5% EtOAc: 95% Hexane + 0.1% C H j C O O H -» 30% E t O A c : 90% Hexane + 0.1 % C H ^ C O O H ) to yield 21 and clear non-crystalline 22 (2 mg, 0.007 mmol, 6%) Mass Spec electrospray [ M + Na + ] m/z 320.85 corresponds to C 1 K H 3 4 O 3 . N M R (500 M H z ) (solvent: CD3OD, Reference 5 3.30 ppm): 8 5.39 (br s IH), 5.34 (br s IH), 3.69 (br s IH), 2.28 (br s 2H), 2.09-1.92 (m 4H), 1.59 (m 2H), 1.29 (br m 20H), 1.12 (d 3H, J = 6.3 Hz) 

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