SYNTHESIS AND EVALUATION OF PARTHENOLIDE ANALOGUES: CHEMICAL PROBES AND THERAPEUTIC AGENTS A Thesis SUBMITTED TO THE FACULTY OF UNIVERSITY OF MINNESOTA BY Dan Wang IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE Advisor: Dr. Daniel A. Harki MARCH 2013 © Dan Wang 2013 Acknowledgements I would like to begin by thanking my advisor, Dr. Dan Harki for his mentorship and support over these years. Your wisdom, knowledge and enthusiasm for science were a guiding light throughout my graduate school career. I wouldn’t be where I am without your vision, encouragement and advise. Additional thanks goes to Professor Rick Wagner, Professor Chengguo Xing and Professor Mark Distefano for serving on my dissertation committee, and for the helpful critiques and suggestions provided throughout my graduate career. Then, I would like to extend my sincere thanks to every past and present member of Harki group. Thank you all for the scientific help you have provided me all these years as well as being a constant source of support. In particular, Dr. Fred Meece for guiding me into the “parthenolide field” and Joe Hexum and Tim Andrew for providing some of the biological data in chapter 2. I would also like to thank and recognize my collaborators for all their assistance with regards to my projects. In particular, I would like to thank Professor David Largaespada, Sue Rathe and Zohar Sachs for their help and valuable discussion in primary AML cells; Professor John Ohlfest and Chani Becker for performing the brain tumor animal studies; Dr. Victor G. Young, Jr. and the UMN X-Ray Crystallographic Laboratory for solving several structures for me. Additionally, my sincerest thanks goes to Department of Medicinal Chemistry for giving me the chance to study in the US, and for providing the intense, but vivid research environment. I would also like to thank every individual person in the department, i especially my fellow classmates, who were amazing friends and an invaluable source of support and laughter all these years and beyond. I would also like to acknowledge the American Heart Association (Midwest affiliates) for financial support of my research through a predoctoral fellowship (2011- 2013, 11PRE7240035). Finally, I’d like to dedicate the thesis to my friends and family for their never- ending support. Thanks for being the cheerleaders throughout my journey. I would have never made it this far without your unconditional love, support and encouragement. ii Abstract Cancer stem cells (CSCs), also known as tumor-propagating cells or tumor- initiating cells, are subpopulations of undifferentiated, highly tumorigenic cells found within bulk tumors. The rapid advances of cancer research and development of relative technologies have provided more and more evidence for the existence of CSCs, as well as the important roles they play in drug resistance and disease relapse of cancer. However, because of their quiescent nature and the similarities to normal stem cells, eradicating CSCs presents a challenging task. Chapter one provides an overview of cell surface markers of CSCs. Those markers are potential diagnostic macromolecules and targets for drug delivery. Parthenolide (PTL) is a sesquiterpene lactone natural product isolated from Mexican Indian medicinal herb Tanacetum parthenum (feverfew plant), a known medical herb utilized for centuries. PTL has been extensively studied as an anticancer agent, showing significant efficacy towards a wide spectrum of human cancer cells. In 2005, the identification of PTL as the first stand-alone and selective cytotoxic agent against the acute myeloid leukemia CSCs further heightened its therapeutic potential. However, the mechanism of action of PTL’s CSC inhibitory activity is still an area of debate. Our efforts to elucidate the molecular targets of PTL is described in chapter two. The design and synthesis of two PTL affinity probes with diverse biological activity as well as their utilization in comparative and competitive protein pull-down experiments to enrich the cellular protein targets of PTL is presented. iii Although exhibiting promising anticancer and anti-CSC activities, the modest biological potency and poor water solubility prevent further development of PTL. Chapter three describes our efforts to synthesize PTL analogues, as well as our strategy to prepare water-soluble PTL prodrugs. iv Table of Contents Acknowledgements..…………………………………………………………….....……...i Abstract…………………………………………………………………………………...iii Table of Contents………………………………………………………………………….v List of Tables…………………………………………………………………………..…ix List of Schemes……………………………………………………………………….......xi List of Figures……………………………………………………………………………xii List of Abbreviations………………………………………………………………..…..xiii Preface…………..………………………………………………………………………xiv Chapter 1. Cell Surface Markers of Cancer Stem Cells: Diagnostic Macromolecules and Targets for Drug Delivery……………………………………………………….1 - 45 1.1 Introduction………………………………………………………………………...1 1.2 Compendium of cell surface markers for identification of cancer stem cells……...4 1.2.1 Leukemia……………………………………………………………………..5 1.2.1.1 Acute myelogenous leukemia (AML)…………………………………….5 1.2.1.2 Acute lymphoblastic leukemia (ALL)…………………………………….8 1.2.1.3 Chronic myelogenous leukemia (CML)…………………………………..9 1.2.2 Breast cancer………………………………………………………………..11 1.2.3 Colorectal cancer……………………………………………………………13 1.2.4 Liver cancer…………………………………………………………………14 1.2.5 Pancreatic cancer……………………………………………………………16 v 1.2.6 Melanoma…………………………………………………………………...17 1.2.7 Brain cancer…………………………………………………………………18 1.2.8 Lung cancer…………………………………………………………………21 1.2.9 Bladder cancer………………………………………………………………22 1.2.10 Prostate cancer……………………………………………………………..24 1.2.11 Ovarian cancer……………………………………………………………..25 1.3 The chemical biology and drug targeting of cell surface markers on cancer stem cells……………………………….…………………………………………29 1.4 Summery and conclusions………………………………………...………………45 Chapter 2 Elucidating the Cellular Targets of Anticancer Natural Product Parthenolide……………………...………………………………………46-106 2.1 Introduction……………………………………………………………….………46 2.2 Project Design………………………………………….…………………………53 2.3 Results and Discussion………………………………….………………………...55 2.3.1 Kinase Screening Assay of PTL against NF-κB Related Kinases………….55 2.3.2 Synthesis of Functional and Non-Functional PTL-Alkyne Probes………....56 2.3.3 Synthesis of Fluorescent Probes………………………………….………....58 2.3.4 Proof of Concept Experiments of Protein Pull-Down Assays……………...62 2.3.4.1 Template reaction of click chemistry………………………………..62 2.3.4.2 In gel fluorescent labeling assay of cell lysates….…………………..64 2.3.5 Protein Pull-Down Experiments………………………………….…………66 2.3.6 Annotation of Protein Pull-Down Results…………………………………..73 vi 2.4 Conclusions and Future Direction………………………………….…………….77 2.5 Experimentle………………………………….…………………………………..78 2.5.1 General………………………………….…………………………………..78 2.5.2 Synthesis of Parthenolide Affinity Probes………………………………….79 2.5.3 Synthesis of Fluorescent Probes………….…………………………………82 2.5.4 Procedures for Biochemical Experiments…………………………………..87 2.5.4.1 Kinase screening assays…………………………………………….87 2.5.4.2 Protocol for cell culture cytotoxicity assays………………………..87 2.5.4.3 Template reaction of click chemistry…………………………….....89 2.5.4.4 General protocol for RIPA lysis of HL-60 cells…………………....89 2.5.4.5 In gel fluorescent labeling and time course assay…………………..90 2.5.4.6 Streptavidin affinity purification……………………………………91 2.5.4.7 Stability assay………….……………………………………………92 2.5.4.8 Modified affinity separation strategy………….……………………92 2.6 NMR Spectrum………….………………………………………………………...97 Chapter 3 Semi-Synthesis and Biological Evaluation of Parthenolide Analogues..106-161 3.1 Introduction………….…………………………………………………………..106 3.2 Project Design………….………………………………………………………..108 3.3 Results and Discussion………….……………………………………………….109 3.3.1 Synthesis and Biological Evaluation of Cyclopropyl Parthenolide Analogues.………….………………………………..109 3.3.2 Synthesis and Biological Evaluation of vii Rearranged Parthenolide Analogues.………….………………………………..115 3.3.3 Synthesis and Biological Evaluation of Phosphate Parthenolide Prodrugs.………….……………………………………118 3.4 Conclusions and Future Direction………….……………………………………122 3.5 Experimentle………….…………………………………………………………124 3.5.1 General………….…………………………………………………………124 3.5.2 Synthesis of Parthenolide Analogues………….…………………………..127 3.5.3 Protocol for Mammalian Cell Culture……………………………………..137 3.5.4 Protocol for Cell Culture Cytotoxicity Assays ……………………………138 3.6 NMR Spectrum……….…………………………..……….……………………..140 References………………...…………………………………………………………….162 Appendix A. X-ray crystallographic data for cyclopropyl PTL 4...................................185 Appendix B. X-ray crystallographic data for rearranged PTL 6……………………….200 viii List of Tables Table 1.1. Previously reported CSC markers for human leukemias…………………….10 Table 1.2. Previously reported CSC markers for human solid tumors………………….28 Table 1.3: Targeting of CSC populations by antibodies and small molecules………….42 Table 1.4. Alternative names of CSC surface markers that appear in this chapter……...43 Table 2.1: Conditions and results of the stability assay of 27…………………………...69 Table 2.2: Number of proteins identified in each protein pull-down assay……………..73 Table 2.3: Identified protein targets by mass spectrometry..……………………………74 Table 3.1. Half maximal inhibitory concentration (IC50 in μM) of PTL (1) and MelB (3) in nine different cell lines.…………………………………..…………...109 Table 3.2. Half maximal inhibitory concentration (IC50 in μM) of PTL (1), LC-1 (2)