University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 10-5-2016 Investigating the Antiproliferative Activity of Synthetic Troponoids Eric R. Falcone University of Connecticut, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Falcone, Eric R., "Investigating the Antiproliferative Activity of Synthetic Troponoids" (2016). Doctoral Dissertations. 1302. https://opencommons.uconn.edu/dissertations/1302 Investigating the Antiproliferative Activity of Synthetic Troponoids Eric Ryan Falcone, Ph.D. University of Connecticut, 2016 Troponoid (or more commonly, tropone/tropolone) refers to a family of naturally occurring compounds with a unique seven-membered aromatic ring. As with many secondary metabolites, many of the tropolones display a wide range of biological activity. This activity includes: antibacterial/antifungal, insecticidal, phytotoxic, antimitotic, anti-diabetic, anti- inflammatory, enzyme inhibition, anticancer, cytoprotective and reactive oxygenated species (ROS) scavenging. Most of this broad-range activity is attributed to the tropolone moiety while side chains are seen as attributing to selectivity. Therefore, tropolone may offer a unique scaffold to develop antiproliferatives since it can chelate metal ions, is relatively small and most likely promiscuous, likely resulting in several mechanisms of action to diminish the development of resistantance. This work focuses on the synthesis of a more diverse library of α-tropolones and tropones with functional groups capable of chelation other than the native hydroxyl. The library was used to assess structure activity relationship on the antiproliferative effects against cancer cell lines. Previous results with α-tropolones in the Anderson and Wright labs suggested selectivity towards hematological malignancies. As a result, α-tropolones were assessed for antiproliferative effects against a panel of malignant hematological cell lines from T-cell, B-cell and myeloid lineage. From this panel, there appears to be selectivity towards cell lines of T-cell lineage. The most susceptible cell line, Molt-4, was explored further to understand the mechanism through which α-tropolones exhibit antiproliferative properties. Eric Ryan Falcone University of Connecticut 2016 Patients with hematological malignancies are at the highest risk of potentially deadly infections typically caused by gram-negative bacteria, with E. coli, K. pneumoniae, and P. aeruginosa being the most common and most lethal. Since several natural tropolones have been found to be bacteriostatic and bactericidal against a broad range of bacteria and exhibit lead-like Gram-negative antibacterial activity, α-tropolones may be developed as antiproliferative agents effective against both hematological malignancies and bacterial infections. The library of α- tropolones was then screened for antiproliferative effects on Gram-negative bacteria. Key potent tropolones were used to explore potential targets using an unbiased target identification approach. These studies show that bacterial enolase is most likely the target through which tropolones exhibit antiproliferative properties against Gram-negative bacteria. Investigating the Antiproliferative Activity of Synthetic Troponoids By ERIC RYAN FALCONE B.S. (Chemistry), Fairfield University, 2008 M.S. (Bioorganic Chemistry), Syracuse University 2010 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy At the University of Connecticut 2016 Copyright by Eric Ryan Falcone 2016 i APPROVAL PAGE Doctor of Philosophy Investigating the Antiproliferative Activity of Synthetic Troponoids Presented by Eric Ryan Falcone, M.S. Major Advisor________________________________________________________________ Prof. Dennis L. Wright Associate Advisor_______________________________________________________________ Prof. Charles A. Giardina Associate Advisor_______________________________________________________________ Prof. Akiko Nishiyama Associate Advisor_______________________________________________________________ Prof. M. Kyle Hadden Associate Advisor_______________________________________________________________ Prof. José E. Manautou University of Connecticut 2016 ii Dedicated to my grandfathers Henry and John who taught me the values of hard work, dedication and the importance of a good education iii ACKNOWLEDGEMENTS With heart-felt gratitude I wish to extend my thanks to Dr. Amy Anderson, my advisor for the past five years, for believing in my abilities and offering me the opportunity to work in her lab based solely on my work experience and a phone interview. If it were not for her honesty, faith and impressive research, I would not have attended UConn. She was a constant source of inspiration and encouragement. Without her I may never have had the opportunity or confidence to take on such a variety of biological techniques. I believe I have become a better scientist by working with her. I wish to thank Dr. Dennis Wright, my other major advisor, for his constant support, advice and overall enthusiasm for my projects. His knowledge of chemistry never ceases to amaze me and I strongly believe I have become a better chemist through our numerous discussions, the classes he taught and the many highly skilled chemists he helped develop in his lab. I would also like to thank the members of my committee, Dr. Charles Giardina, Dr. Akiko Nishiyama, Dr. M. Kyle Hadden and Dr. José Manautou, for making time in their busy schedules to serve as members of my committee and for their advice towards my research and the preparation of this thesis. I especially wish to thank Dr. Andrew Wiemer for providing the Molt-4 and hPBMC cells, allowing me to work in his lab and to learn several different techniques from his graduate student, Jin Li. Thanks to all the current and former members of the Anderson and Wright research groups, who have contributed in many different ways towards my time in the lab. I especially wish to thank Dr. Michael van Heyst for his guidance and mentoring as well as his tireless efforts iv towards broadening the synthetic techniques that could be used to develop new tropolone derivatives. Thanks Jin Li from the Wiemer lab for donating her time to show me the proper techniques for culturing Molt-4 and hPBMCs, Western blot analysis and RNA extraction. I also wish to thank Jolanta Krucinska for her incredible efforts to push the enolase project forward. Thanks to all the faculty and staff members at the University of Connecticut and other institutions who have contributed in many different ways to my education and research experience. I especially would like to thank Dr. Matthew Kubasik for his continued support and mentoring. I am forever grateful for the undergraduate summer research opportunity and academic advice he provided me as well as his insistence that I declare my undergraduate major as chemistry. Through this I found a world of opportunities and a love for research. Finally and most importantly, I would like to give my thanks to my family who has supported me throughout my life. Without their constant source of love and encouragement it would have been difficult to accomplish everything I have to date. I would like to especially thank my parents who made sure I was able to have a great education, encouraged and supported my desire to further my graduate education and have always been there for me. I would also like to thank my in-laws for their support and for allowing me to take over their dining room table in order to write part of this thesis. Finally, I would like to thank my wife, Kelly, for her love, support, encouragement, motivation and for making sure I made time to have fun along the way. She helped make this whole experience possible and I am forever indebted to her. v TABLE OF CONTENTS Chapter 1 Page Inhibition of Metalloenzymes: Lessons from Clinically Used Inhibitors and Future Directions 1.1 Introduction 1 1.2 Clinical Inhibitors of Zinc Metalloenzymes 2 1.2.1 Carbonic Anhydrase 3 1.2.2 Angiotensin Converting Enzyme 5 1.2.3 Matrix Metalloproteinase 7 1.2.4 Tumor Necrosis Factor-α Converting Enzyme 10 1.2.5 Histone Deacetylase 13 1.2.6 Farnesyltransferase 16 1.2.7 Leukotriene-A4 Hydrolase 19 1.2.8 Uridine Disphophate-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine 22 Deacetylase 1.3 Clinical Inhibitors of Iron Metalloenzymes 25 1.3.1 Hypoxia-Inducible Factor Prolyl Hydroxylase 26 1.3.2 Peptide Deformylase 29 1.3.3 5-Lipoxygenase 32 1.3.4 Cytochrome P450 35 1.4 Clinical Inhibitors of Magnesium Metalloenzymes 38 1.4.1 Human Immunodeficiency Virus-Integrase 39 1.4.2 DNA Gyrase 42 1.5 Clinical Inhibitors of Manganese Metalloenzymes 46 1.5.1 1-deoxy-D-xylulose 5-phosphate Reductoisomerase 47 1.5.2 Arginase 49 1.6 Development of Inhibitors Towards A Copper Metalloenzyme 53 1.5.1 Tyrosinase 54 1.7 Properties of Common Metal Binding Groups 60 1.7.1 Hydroxamates 61 1.7.2 Carboxylates 62 1.7.3 Phosphonates/Phosphates 64 1.7.4 Sulfonamides/Sulfamides/Sulfamates 65 1.7.5 Thiols 65 1.7.6 Other Metal Binding Groups 66 vi 1.8 Tropolone as a Metal Binding Group 74 1.9 Conclusions 77 1.10 References 78 Chapter 2 Development of Lead-like Troponoids Based on the Natural Product α-Thujaplicin 2.1 Background and Significance 115 2.1.1 Discovery of Natural Troponoids 115 2.1.2 Biological Activity of Natural Tropolones 118 2.1.3 Synthesis of the Tropolone Core and the Thujaplicins 125 2.1.4 α-Tropolones