Design and synthesis of anti-cancer agents that inhibit cysteine proteases, limit oxidative stress or terminate proliferation of BCR- ABL expressing cells. A dissertation submitted to the Graduate School of the University of Cincinnati in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY (Ph.D.) In the Department of Chemistry of McMicken College of Arts and Sciences by Purujit N. Gurjar Bachelor of Technology, Institute of Chemical Technology, Mumbai, India, 2012 Dissertation Advisor: Edward J. Merino, Ph.D. Abstract Malignant diseases or in common terms Cancer is one of the major causes of death in the world. Cancer patients are treated using several advanced techniques including surgery, radiation and chemotherapy. Chemotherapeutic treatment involves the use of anticancer agents or drug to fight against cancer. It is used to decrease the tumor burden and to eliminate malignant cells. However, in most cases, resistance against chemotherapy develops. Therefore, there is a permanent need for new additional treatment strategies and chemotherapeutic agents. In this thesis I have explained my work on three different projects targeting different mechanisms to stop the growth of cancer cells. The first project is focused on inhibition of cysteine proteases which are overexpressed in many types of cancers. By inhibiting these enzymes, we believe that the malignant capacity of a cancer tumor will be arrested. We have tried to impart a solution to a common problem in enzyme inhibition world: can a reversible inhibitor be converted to an irreversible inhibitor? Apart from cysteine proteases, there are another molecular species which are found expressed in large amounts in cancerous tumors. They are known as Reactive Oxygen Species (ROS). There are several types of ROS and each can have lethal effects on humans depending upon their concentrations. In the second chapter we have tried to selectively inhibit one of these ROS species: hydroxyl radical. Hydroxyl radicals are known to form DNA lesions and protein degradation inside human cells and their inhibition can be part of the solution to stop malignancy. In the third project I got an opportunity to work on a di-pyridine derivative which shows in vivo against many cancer models in mice. This molecule was obtained from NCI in an impure form. We were able to purify it and synthesize it on large scale. Upon testing the pure product, we i achieved more potency and further experiments are still ongoing. Furthermore, we immobilized this active molecule on agarose-based microspheres. These microspheres will be used to investigate protein and enzyme targets of the active compound inside the cell. Overall, I worked on 3 different strategies towards development of new chemotherapeutic drugs. Although targets and mechanism of these molecules are different, they all works towards a common goal of making chemotherapy more efficient than it is today. ii iii Acknowledgements Firstly, I would like to express my sincere gratitude to my advisor Prof. Edward Merino for the continuous support of my Ph.D. study and related research, for his patience, motivation, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis. I could not have imagined having a better advisor and mentor for my Ph.D. study. Besides my advisor, I would like to thank the rest of my thesis committee: Prof. David Smithrud and Prof. In- Kwon Kim, for their insightful comments and encouragement, but also for the hard question which incented me to widen my research from various perspectives. Thank you, University of Cincinnati department of Chemistry, for giving me a chance to pursue my doctoral studies here. My sincere thanks also goes to Dr. Stephen Macha, Dr. Larry Sallans, and Dr. Keyang Ding, who gave access to the laboratory and research facilities. Without they precious support it would not be possible to conduct this research. I was extremely fortunate to work in collaboration with Dr. Nicolas Nassar at Cincinnati children’s hospital. I am thankful to him and his entire team for giving me the opportunity to work on a fantastic research project. I thank my present and past labmates; Anish Vadukoot, Shazna Nusair, Safnas Abdulsalam, Kaylin Earnest, Haizhou Zhu, Jing Liu. Gurdat Premnauth and Priyangika Senevirathne for the stimulating discussions, for the sleepless nights we were working together before deadlines, and for all the fun we have had in the last six years. Also, I thank all my friends from University of Cincinnati, in particular, I am grateful to Jessica Ringo and Kayla Borland for always motivating me when I needed it the most. Last but not the least I thank my family: my father Dr. Narendra and my mother Mrs. Anita and my sister Poorva for supporting me spiritually all my life. You have been pillars of support, iv guidance and love in my life since the day I was born. I am eternally grateful for all the sacrifices you have made and all the love you have always bestowed upon me. v Table of Contents Chapter 1: Role of anticancer agents in cancer therapy .................................................................. 1 1.1 History of anticancer agents .................................................................................................. 2 1.2 Classification of anticancer agents ........................................................................................ 3 1.2.1 Alkylating agents ............................................................................................................ 3 1.2.2 Antimetabolites ............................................................................................................... 3 1.2.3 Microtubule targeting agents .......................................................................................... 4 1.2.4 Topoisomerase inhibitors ............................................................................................... 4 1.2.5 Anthracyclines ................................................................................................................ 5 1.2.6 Monoclonal antibodies ................................................................................................... 5 1.2.7 ROS-activated anticancer prodrugs ................................................................................ 5 1.2.8 Protease inhibitors: Aid to an antineoplastic agent ........................................................ 6 1.2.9 Kinase inhibitors ............................................................................................................. 7 1.3 Goals of dissertation .............................................................................................................. 8 1.4 Overview of Chapter 2 .......................................................................................................... 9 1.4.1 Cysteine Protease Inhibitors: Research Strategy .......................................................... 10 1.4.2 Hypothesis .................................................................................................................... 11 1.4.3 Methods to Synthesize Cyclopropene moiety .............................................................. 13 1.4.4 Example of application ................................................................................................. 14 vi 1.5 Overview of Chapter 3 ........................................................................................................ 16 1.5.1 Free radicals: introduction ............................................................................................ 16 1.5.2 How are free radicals generated? .................................................................................. 16 1.5.3 How do free radicals damage the cells: ........................................................................ 20 1.5.4 ROS-targeted anticancer agents .................................................................................... 23 1.6 Overview of Chapter 4 ........................................................................................................ 24 Chapter 2: Cyclopropenes as Potential Warheads for Inhibitors of Cysteine Proteases ............... 25 2.1 Introduction ......................................................................................................................... 26 2.2 Experimental ....................................................................................................................... 27 2.2.1 Materials ....................................................................................................................... 27 2.2.2 Synthetic Methods ........................................................................................................ 28 2.3 Results and discussion ......................................................................................................... 37 2.3.1 Synthesis of cyclopropenes .......................................................................................... 37 2.3.2. Stability studies............................................................................................................ 38 2.3.3 Kinetic studies .............................................................................................................. 39 2.4 Conclusion ........................................................................................................................... 41 Chapter 3: Synthesis and Characterization of Self-Cyclizing Antioxidants as Scavengers of Hydroxyl