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STRUCTURE ELUCIDATION AND STUDIES RELATING TO THE SYNTHESIS OF PLASMALOPENTAENE- 12 by Robert F. Keyes Dissertation submitted to the Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry aid Cite} D. G. I. Kingston, Chairman wom Alf bboy, C “Ite 7 H. M. Bell H. C. Dorn bY ble la Co aa * { , < (/ J. S.-Merola J. M. Tanko November 20, 1992 Blacksburg, Virginia LD S655 VES6 1992 je 4/44] C,4 STRUCTURE ELUCIDATION AND STUDIES RELATING TO THE SYNTHESIS OF PLASMALOPENTAENE- 12 by Robert F. Keyes D.G.I. Kingston, Chairman Chemistry (ABSTRACT) The glycerol enol ether, fecapentaene-12, is a direct acting fecal mutagen that is formed in the lower portion of the gastrointestional tract by anaerobic bacteria. The biological precursor to fecapentaene-12 is a natural product of mammalian origin whose role in the etiology of colon cancer is unknown. Preliminary evidence indicated that the precursor may be a plasmalogen with an intact pentaenol ether moiety. Further structural studies by means of degradative methods and chromatographic techniques enabled the structure of the precursor to be elucidated. Based on the structure of the precursor, the name plasmalopentaene-12 was coined. Synthetic methodology was developed for obtaining synthetic plasmalopentaene- 12. This was necessary in order to confirm the structure and to determine the precursor's biological role. The synthetic methodology proceeded through a novel "acyl migration" which enables the highly labile pentaenol ether to be generated late in the synthesis. Model studies indicated that this was a feasible pathway. It was also determined that this methodology may be highly adaptable to the synthesis of other plasmalogens and may also provide a new synthetic route to fecapentaene-12. To My Parents ACKNOWLEDGEMENTS The author wishes to express his gratitude to his research director, Dr. David G.I. Kingston for his ideas, support, and patience during the completion of this research project. The suggestions and assistance provided by Dr. Harold Bell, Dr. Harry Dorm, Dr. Joseph Merola, and Dr. James Tanko are also greatly appreciated. Much gratitude is also owed to Dr. Neal Castagnoli for his support. The author also expresses his thanks to the National Cancer Institute for their financial support. Many thanks are extended to Dr. Leslie Gunatilaka. It was indeed fortunate to have the opportunity to learn about chemistry and life from an outstanding scientist. It made the time spent in Blacksburg much more rewarding. The members of Dr. Kingston's group are also gratefully acknowledged for their support. The author would especially like to thank Kurt Neidigh and Dr. George Harrigan for continued moral support and for providing an outlet from the stress of graduate school. Thanks also must be given to some very special friends including Dr. Paul Engen, Dr. Mike Berg, Jim Witsch, Dr. Carl Wild, John Rimoldi, André Melendez and the members of the Blacksburg Rugby Football Club. It is fitting to acknowledge the support of loved ones when undertaking a project such as this. Therefore, the author wishes to express his sincere gratitude to his parents, Raymond and Louise Keyes and to his sister, Rae Ann Kegley. Without their love, support, patience, and most of all sacrifice, it never would have been possible to accomplish life's goals. This degree is more theirs than the author's. Finally, the author expresses his most sincere appreciation to Lisa Hamblin. The typing of this manuscript is greatly appreciated, but most of all her love and support made the completion of this project possible. Thank you. iv TABLE OF CONTENTS I. Introduction 1.1 Colon Cancer Incidence 1.2 Possible Causes of Colon Cancer 1.3. Isolation and Structural Determination of Fecapentaene-12 1.4 | Fecapentaene-12 Synthesis 11 1.5. Fecapentaene-12 Distribution 17 1.6 Biological Activity of Fecapentaene-12 19 1.7. Statement of Goals 22 II. Structure Elucidation of Plasmalopentaene-12 25 2.1 Introduction 25 2.2 Precursor Isolation and Purification 26 2.3 Preliminary Structure Determination Experiments 27 2.4 Elucidation of the Functional Groups at the sn-2 and sn-3 Positions 31 2.4.1 Determination of the Acyl Group at the s7-2 Position 31 2.4.2 Determination of the Functional Group at the sn-3 Position 39 2.5 Conclusions 41 2.6 General Experimental Procedures 42 2.7 Experimental 43 III. Synthesis of Precursor Models 45 3.1 _ Plasmalopentaene-12 Synthetic Strategy 45 3.2 Synthesis of Acyl Migration Models 54 3.2.1 Synthesis of the Saturated and Unsaturated Silyl Ether Analogs 54 3.2.2 Synthesis of the Saturated and Unsaturated Phosphate Analogs 62 3.3 Acyl Migration Studies 65 3.4 General Experimental Procedures 84 3.5 Experimental 85 IV. Synthesis of a Protected Phosphodiester 101 4.1 Introduction: Explanation of Phosphodiester Choice 101 4.2 Methods of Phosphoester Synthesis 102 4.3 Phosphodiester Synthesis via the Cyclic Enediol Phosphoryl Method 106 4.4 Initial Synthetic Attempt of the Protected Phosphorylethanolamine: An Electrophilic Synthon 109 4.4.1 Synthesis of the Protected Phosphorylethanolamine from Dichloromethoxyphosphine 110 4.4.2 Synthesis of the Protected Phosphorylethanolamine from Phosphorous Oxychloride 113 4.5 Synthesis of a Nucleophilic Protected Phosphorylethanolamine 115 4.6 Experimental 120 Synthesis of 2,4,6,8-Tetraundecenoic Acid 126 3.1 Introduction 126 5.2 Synthesis of the Tetraenoic Acid via Wittig Methodology 128 5.2.1 Synthesis of the Tetraenoic Acid using a Triphenylphosphine Ylide 129 5.2.2 Synthesis of the Tetraenoic Acid via the Wittig Horner-Emmons Reaction 134 3.3 Synthesis of the Tetraenoic Acid via the Claisen Rearrangement 138 5.4 Oxidation of a Tetraenol to a Tetraenoic Acid 143 3.5 Experimental 147 VI. Attempted Synthesis of Plasmalopentaene-12 154 6.1 Introduction 154 6.2 Attachment of the Phosphate 158 6.2.1 Ring Opening of the Oxirane with the Protected Phosphate 158 6.2.2 Coupling the Protected Phosphate with an Alcohol 166 6.2.3 Synthesis of Phosphotriester 6-3 via a Bromohydrin 167 6.2.4 Opening of Epoxides with Alcohols 173 6.3. Synthesis of a Precursor Analog 175 6.3.1 Synthesis of the Diphenethyl Phosphate Analog-- Generation of the Pentaenol Ether 175 6.3.2 Synthesis of a Precursor Analog with a Trienyl Ether Moiety 180 6.4 Synthesis of a Fecapentaene-12 Analog Via the Acyl Migration Strategy 186 6.5 Conclusions 191 6.6 Experimental 193 VII. Literature Cited 204 LIST OF FIGURES Figure 1-1. HPLC Chromatogram of Natural FP-12 Figure 1-2. Gunatilaka and Kingston's Synthesis of FP-12 13 Figure 1-3. Nicolaou's Synthesis of FP-12 14 Figure 1-4. Van der Gen's Synthesis of FP-12 15 Figure 1-5. Pfaendler's Synthesis of FP-12 16 Figure 1-6. Schematic Representation of the Possible Roles of the Human Intestinal Microflora in Colon Cancer 23 Figure 2-1. GC of the FP-12 Precursor Fatty Acid Methyl Ester 34 Figure 2-2. Expansion GC of the FP-12 Precursor Fatty Acid Methyl Ester 35 Figure 2-3. MS of Methyl Hexadecanoate 36 Figure 2-4. MS of Methyl Octadecanoate 37 Figure 2-5. Descending Paper Chromatogram of FP-12 Precursor for Phosphate Determination 40 Figure 3-1. Synthesis of Ethanolamine Plasmalogens. Method A. 46 Figure 3-2. Synthesis of Ethanolamine Plasmalogens. Method B. 47 Figure 3-3. Retrosynthetic Analysis for Plasmalopentaene-12 50 Figure 3-4. 270 MHz NMR of the Unmigrated Saturated Silyl Ether, 3-13 68 Figure 3-5. 270 MHz NMR of the Migrated Saturated Silyl Ether, 3-33 69 Figure 3-6. 270 MHz NMR of the Unmigrated Unsaturated Silyl Ether, 3-14 70 Figure 3-7. 270 MHz NMR of the Migrated Unsaturated Silyl Ether, 3-34 71 Figure 3-8. 270 MHz NMR of the Unmigrated Saturated Phosphate, 3-15 80 Figure 3-9. 270 MHz NMR of the Unmigrated Unsaturated Phosphate, 3-16 81 Figure 3-10. 270 MHz NMR of the Migrated Saturated Phosphate, 3-41 82 Figure 3-11. 270 MHz NMR of the Migrated Unsaturated Phosphate, 3-42 83 Figure 5-1. Generation of the Pentaenol Ether of Plasmalopentaene-12 127 Figure 5-2. Mechanism of the Wittig Reaction 130 Figure 6-1. 1,2-Reduction of the Precursor Analog 185 Figure 6-2. UV Spectrum of the Fecapentaene-12 Analog 190 LIST OF TABLES Table 1-1. Distribution of the Most Common Causes of Death in the United States from 1950-1977 Table 1-2. 30-Year Trends in Cancer Death Rates By Site and Sex, 1956-58 to 1986-88 Table 1-3. In Vitro Incubation of Fresh and Autoclaved Feces and and Fecal Extract Broth 21 Table 2-1. Treatment of Fecapentaene-12 and Precursor with BTZ 28 Table 2-2. Plasmalogen Composition in Mammalian Tissue 31 Table 5-1. Attempted Reaction Conditions Used for the Wittig Reaction 134 I. Introduction 1.1 Colon Cancer Incidence. Cancer is a large group of diseases characterized by uncontrolled growth and spread of abnormal cells. If the spread is not controlled or checked, it results in death. Normally, cells in the body replicate in an orderly fashion, thus bodily repairs can be made and growth will occur. In this respect the body is functioning in a normal way. Occasionally, cells malfunction and begin to grow and spread at an uncontrolled rate giving rise to the masses of abnormal cells called tumors. Initially the tumors are localized, but after time the cancer cells may metastasize, that is, invade other organs or tissues.