I. ISOLATION AND CHARACTERIZATION OF BIOACTIVE COMPOUNDS FROM SURINAME AND MADAGASCAR FLORA. II. A SYNTHETIC APPROACH TO LUCILACTAENE Eba Adou Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirement for the degree of Doctor of Philosophy In Chemistry Dr. David G. I. Kingston, Chairman Dr. James Tanko Dr. Felicia Etzkorn Dr. Timothy E. Long Dr. Paul Deck November 28, 2005 Blacksburg, Virginia Keywords: Anticancer agents, Cytotoxicity assay, Indole alkaloids, Diterpenoids, Cardenolide glycosides, Physalins, Cucurbitacins, Cell cycle, Cell cycle inhibitor, p53 Tumor suppressor Gene, Lucilactaene Copyright 2005, Eba Adou ISOLATION AND CHARACTERIZATION OF BIOACTIVE COMPOUNDS FROM SURINAME AND MADAGASCAR FLORA AND A SYNTHETIC APPROACH TO LUCILACTAENE ABSTRACT Eba Adou As part of an International Cooperative Biodiversity Group (ICBG), extracts of plants from Suriname and Madagascar were bioassayed for cytotoxicity and antimalarial activity. Six cytotoxic extracts and one potential antimalarial were selected for fractionation, and yielded a number of bioactive compounds which were characterized by spectroscopy methods. Craspidospermum verticillatum (Apocynaceae) yielded four known indole alkoids. Casimirella sp (Icacinaceae) gave three new and five known diterpenoids. Pentopetia androsaemifolia (Apocynaceae) afforded one new and three known cardenolide glycosides. Physalis angulata (Solanaceae) yielded seven known physalins. Roupellina boivinnii (Apocynaceae) yielded four known and three new cardenolide glycosides, and three known cucurbitacins were isolated from Octolepis aff. dioica (Thymelaeaceae). In addition to these structural studies, a synthetic approach to lucilactaene, a cell cycle inhibitor was developed. ACKNOWLEDGMENTS I dedicate this to my family in Côte d’Ivoire (Ivory Coast) for giving me the opportunity to come to the U.S.A. I have been fortunate to come from a family who gave me all the opportunities and love to succeed in life. I would like to thank my beloved wife, Dr. Nan Chi Wan, for her emotional support and her unconditional love. I would like to thank Dr. David G.I. Kingston for his kindness, tolerance and support he has shown during my time in his research group. I would also like to thank my committee members: Dr. James Tanko, Dr. Felicia Etzkorn, Dr. Timothy E. Long and Dr. Paul Deck. I would also like to thank Dr. Cao Shugeng for his help and friendship, without forgetting his wife, Xiaohua Wu and his son, Longji for their kindness. Additional thanks to Jennifer Schilling and Tom Glass for technical assistance with bioassays and NMR spectroscopy. III TABLE OF CONTENTS PART I I. GENERAL INTRODUCTION 1 1.1 Cancer 1 1.2 Natural Products in Drug Discovery 1 1.3 Natural Products as Anticancer Drugs 2 1.4 Biodiversity loss and the ICBG Program 5 1.5 Bioassays in the discovery of antitumor agents 6 1.5.1 Role of assays 6 1.5.2 Antitumor assays 7 1.6 A2780 Cytotoxicity Assay 9 II CRASPIDOSPERMUM VERTICILLATUM (APOCYNACEAE) 11 2.1 Introduction 11 2.2 Results and Discussion 11 2.3 Experimental Section 15 2.3.1 General Experimental Procedures 15 2.3.2 Cytotoxicity Bioassays 16 2.3.3 Plant Material 17 2.3.4 Extraction and Isolation 17 III. CASIMIRELLA SP. (ICACINACEAE) 21 3.1 Introduction 21 3.2 Results and Discussion 21 IV 3.3 Experimental Section 30 3.3.1 General Experimental Procedure 30 3.3.2 Cytotoxicity Bioassays 31 3.3.3 Plant Material 31 3.3.4 Extraction and Isolation 31 IV. PENTOPETIA ANDROSAEMIFOLIA (APOCYNACEAE) 35 4.1 Introduction 35 4.2 Results and Discussion 35 4.3 Experimental Section 44 4.3.1 General Experimental Procedure 44 4.3.2 Cytotoxicity Bioassays 44 4.3.3 Plant Material 44 4.3.4 Extraction and Isolation 44 V. PHYSALINS FROM PHYSALIS ANGULATA (SOLANACEAE)) 48 5.1 Introduction 48 5.2 Results and Discussion 48 5.3 Experimental Section 59 5.3.1 General Experimental Procedure 59 5.3.2 Cytotoxicity Bioassays 59 5.3.3 Plant Material 59 5.3.4 Extraction and Isolation 59 VI. ROUPELLINA BOIVINII (APOCYNACEAE) 65 6.1 Introduction 65 V 6.2 Results and Discussion 69 6.3 Experimental Section 78 6.3.1 General Experimental Procedure 78 6.3.2 Cytotoxicity Bioassays 78 6.3.3 Plant Material 78 6.3.4 Extraction and Isolation 79 VII. CUCURBITACINS FROM OCTOLEPIS AFF. DIOICA (THYMELAEACEAE) 82 7.1 Introduction 82 7.2 Results and Discussion 82 7.3 Experimental Section 88 7.3.1 General Experimental Procedure 88 7.3.2 Cytotoxicity Bioassays 88 7.3.3 Plant Material 88 7.3.4 Extraction and Isolation 88 VIII. OTHER PLANTS STUDIED, BUT DROPPED 93 PART II I. A SYNTHETIC APPROACH TO LUCILACTAENE 95 1. Introduction 95 1.1 The Cell Cycle as a Target for Anticancer Drugs 96 1.2. Cell Cycle Inhibitors 96 1.3 The p53 Tumor Suppressor Gene 97 1.4 Lucilactaene, a New Cell Cycle Inhibitor 98 VI 2. Results and Discussion 101 2.1 Retrosynthesis 101 2.2 Synthetic Aproach 102 2.2.1 Fragment II 102 2.2.2. Fragment IV 105 2.2.3 Fragment V 107 2.2.4 Fragment III 108 2.2.5. Coupling fragment III and V 112 2.2.6. Suggested work: Enzymatic lactonization 114 3. Conclusion 115 4. Experimental Section 115 VII List of Figures Figure 1.1 Sample loading in a typical microtiter plate cytotoxicity assay 10 Figure 2.1 Biossay guided fractionation of Craspidospermum verticillatum 20 Figure 3.1 Structure and HMBC correlations of compound 3.1 22 Figure 3.2 Structure and HMBC correlations of compound 3.2 24 Figure 3.3 Bioassay guided fractionation of Casimirella sp. 32 Figure 4.1 HMBC correlation compound 4.3 40 Figure 4.2 Bioassay guided fractionation of Pentopetia androsaemifolia Decne 41 Figure 5.1 Bioassay guided fractionation of Physalis angulata L 64 Figure 6.1 Structure, ROESY and HMBC correlations of compound 6.1 67 Figure 6.2 Structure, ROESY and HMBC correlations of compound 6.2 69 Figure 6.3 Structure, ROESY and HMBC correlations of compound 6.3 71 Figure 6.4 Bioassay guided fractionation of Roupellina boivinii 80 Figure 7.1 Bioassay guided fractionation of Octolepis aff. Dioica Capuron (MG 985) 91 Figure 7.2 Bioassay guided fractionation of Octolepis aff. Dioica Capuron (MG 988) 92 Figure II.1 Cell cycle 96 VIII List of Schemes Scheme 1 Retrosynthesis of lucilactaene 101 Scheme 2 Synthesis of compound 5 102 Scheme 3 Synthesis of α,β-unsaturated ester 6 103 Scheme 4 Synthesis of fragment II 104 Scheme 5 Synthesis of fragment IV 106 Scheme 6 Synthesis of fragment V 108 Scheme 7.1 Coupling Fragment V and IV 109 Scheme 7.2 Removal of the silyl group 111 Scheme 8 Synthesis of tetraenes 30a and 30b 112 Scheme 9 Trityl deprotection 113 IX List of Tables 13 Table 2.1 C NMR Data of 2.1, 2.3, and 2.4 in CDCl3 15 1 13 Table 3.1 H and C NMR data in C5D5N for Compounds 3.1-3 29 13 Table 3.2 C NMR data in DMSO-d6 for compound 3.4 -3.8 30 Table 4.1 1N NMR data for the sugar portion of compound 4.3 40 Table 4.2 13C NMR data of compounds 4.1, 4.2, 4.4 42 Table 4.3 13C NMR data of compounds 4.3 43 Table 5.1 13C NMR data of compounds 5.1-5.4 57 Table 5.2 13C NMR data of compounds 5.5-5.7 58 Table 6.1 13C NMR data of compounds 6.1-6.3 76 Table 6.2 13C NMR data of compounds 6.4-6.6 77 Table 7.1 13C NMR data of compounds 7.1-7.3 87 X PART I I. INTRODUCTION 1.1 Cancer Since 1990, more than 17 million new cancer cases have been detected in the US alone, not including carcinoma, basal cell and skin cancers.1 With all the efforts to fight this deadly disease, the number of people diagnosed has not decreased. The National Cancer Institute (NCI) predicts that 1,372,910 new cancer cases will be diagnosed in the US in 2005 and that 570,280 Americans are expected to die of the disease.1 The following estimated numbers in selected cancers are expected in 2005: breast cancer (211,240 new cases and 40,870 expected deaths), lung and bronchial cancer (172,570 new cases and 163,510 deaths expected); these figures represent about 13% of all cancer diagnoses and account for 28% of all cancer deaths, and prostate cancer (232,090 new cases and 30,350 deaths expected).1 The National Institutes of Health (NIH) estimated that the overall costs for cancer in 2004 were $189.8 billion: $60.4 billion for direct medical costs, $16.9 billion for indirect morbidity costs and $95.2 billion for indirect mortality costs.1 These numbers are a clear indication that cancer affects all aspects of society. The statistics above are for the U.S.A alone. The main goal of the American Cancer Society is to eradicate the disease, and the society predicts a reduction in the number of cancer cases by one half by the year 2015. 1.2 Importance of natural products to drug discovery For many decades, synthetic chemicals as drugs have been effective in the treatment of most diseases. The pharmaceutical industry has synthesized over 3 million new chemicals in 1 Cancer Fact and Figures 2005 www.americancancersociety.org ; Visited on June 14, 2005 1 their effort to produce new drugs. Despite their success in developing drugs to treat or cure many diseases, the treatment of certain diseases such as cancer, AIDS, heart disease and diabetes has not been a complete success due to the complexity of these diseases.