Novel Isosteviol Derivatives as Potential Anticancer Agents A dissertation presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Rumita Laha May 2018 ©2018 Rumita Laha. All Rights Reserved. 2 This dissertation titled Novel Isosteviol Derivatives as Potential Anticancer Agents by RUMITA LAHA has been approved for the Department of Chemistry and Biochemistry and the College of Arts and Sciences by Stephen C. Bergmeier Professor of Chemistry and Biochemistry Robert Frank Dean, College of Arts and Sciences 3 ABSTRACT LAHA, RUMITA, Ph.D., May 2018, Chemistry Novel Isosteviol Derivatives as Potential Anticancer Agents Director of Dissertation: Stephen C. Bergmeier Diterpenes are natural products that provide an interesting lead for anticancer drug discovery. For our studies the readily available diterpene Steviol was used. Our research group has an interest in making diterpene analogs using stevioside as starting material and tests them for cytotoxic activity against various cancer cell lines. The tetracyclic-diterpenoid isosteviol (ent-16-ketobeyeran-19-oic acid) was obtained in large quantity from the acid hydrolysis of steviol glycosides. We used isosteviol to prepare novel structural derivatives in parallel fashion. These analogs were assayed in a number of cancer cell lines in order to identify new anticancer agents. Our prime goal in this dissertation is to show how natural product with pre-existing chiral centers can be used for the synthesis of unique small molecule libraries. We also discussed how these libraries can be further optimized for the discovery of new therapeutics. 4 ACKNOWLEDGMENTS First and foremost, I would like to express my deepest appreciation to my academic advisor Prof. Stephen C. Bergmeier for his mentorship, guidance, and encouragements during my study and research at Ohio University. Without his consistent support, the dissertation would not have been possible. I would also like to thank the members of my dissertation committee, Prof. Jennifer Hines, Prof. Michael Held and Prof. Douglas Goetz for their time and advice. It is my great pleasure to work with such wonderful research group members. I would like to acknowledge both former members and present members for their suggestions and help. They are Dr. Fang Fang, Dr. Susann Krake, Dr. Gregg Wells, Dr. Alicia Frantz, Dr. Dennis Roberts, Dr. Zilong Zheng, Ian Armstrong, Eric Parsons, Liyi Wang and Reaz Uddin. I would also like to thank Prof. John Kopchick and his group member Dr. Reetobrata Basu for the collaboration in testing the cytotoxic activities of isosteviol analogs. Again, I would like to express my special thanks to Prof. Jennifer Hines for giving me so many opportunities to learn computer modeling in her lab. Finally, I would like to dedicate this dissertation to my parents and my dear husband for their love, continuous support and encouragement, and to my brother for his unconditional love and support. Last but not the least, I would love to mention my daughter little Rani whose dazzling smile and innocent face help me keep walking towards the exciting end of this long journey. 5 TABLE OF CONTENTS Page Abstract ................................................................................................................................3 Acknowledgments ...............................................................................................................4 List of Tables .......................................................................................................................9 List of Figures ....................................................................................................................11 List of Schemes ..................................................................................................................12 Chapter 1: Introduction ......................................................................................................15 1.1 Background and Significance ................................................................................. 15 1.1.1 Discovery of Natural Products as Anticancer Agents ...................................... 15 1.2 Overview of Natural Products Showing Anticancer Activity ................................ 17 1.2.1 Diterpenes as Anticancer Agents ..................................................................... 21 1.2.2 Stevioside ......................................................................................................... 23 1.2.3 Steviol – The Aglycon of Steviol Glycosides .................................................. 25 1.2.4 Structurally Modified Different Steviol Derivatives Induced Anticancer Activity in Various Cancer Cell Lines ...................................................................... 25 1.2.5 Isosteviol and Its Different Structural Motif Induced Cytotoxicity ................. 26 Chapter 2: Synthesis of Different Scaffolds from Isosteviol .............................................28 2.1 Introduction ............................................................................................................. 28 2.2 Isosteviol Synthesis ................................................................................................. 28 2.3 Different Scaffolds from Isosteviol ........................................................................ 30 2.3.1 Synthesis of MOM-protected Diol ................................................................... 32 6 2.3.2 Synthesis of Amino Acid ................................................................................. 33 2.3.3 Synthesis of Lactam ......................................................................................... 38 2.3.4 Synthesis of Ketal ............................................................................................ 44 2.3.5 Synthesis of Lactone and Derivatives .............................................................. 44 2.4 Summary ................................................................................................................. 57 Chapter 3: Synthesis and Biological Assay of Isosteviol Derivatives via Functional Group Modification ......................................................................................................................59 3.1 Introduction ............................................................................................................. 59 3.2 Ester and Carbamate Synthesis ............................................................................... 59 3.3 Bioactivity Results for 1st Generation Compounds in Cancer Cell Lines .............. 61 3.3.1 In Human Lung Cancer Cell Lines (H1299) .................................................... 61 3.3.2 In Human Liver Cancer Cell Lines (HepG2) ................................................... 66 3.4 Second Generation Library Synthesis ..................................................................... 68 3.4.1 Several Examples of Ether Derivatives ........................................................... 72 3.4.2 Additional Examples of Modified Ester and Carbamate Derivatives .............. 75 3.5 Bioactivity Results for 2nd Generation Ester Derivatives in H1299 Cell Lines ...... 76 3.6 Bioactivity Results for 2nd Generation Carbamate Derivatives in H1299 Cell Lines ...................................................................................................................................... 78 3.7 Bioactivity Results for all Ester Derivatives in Melanoma and Melanocytes ........ 81 3.8 Bioactivity Results for all Carbamate Derivatives in Melanoma and Melanocytes 85 3.9 Synthesis of Amide Derivatives ............................................................................. 87 3.9.1 Introduction ...................................................................................................... 87 7 3.9.2 Chemical Synthetic Route ................................................................................ 87 3.10 Bioassays for 1st Generation Amide Derivatives in Melanoma and Melanocytes 89 3.11 Synthesis of 2nd Generation Amide Derivatives ................................................... 90 3.12 Bioassays for 2nd Generation Amide Derivatives in Melanoma and Melanocytes92 3.13 Bioactivity Results for all Amide Derivatives (1st & 2nd generations) in H1299 .. 94 3.14 Summary ............................................................................................................... 96 Chapter 4: Peptidomimetics ...............................................................................................98 4.1 Introduction ............................................................................................................. 98 4.2 Generating Diversity at C-19 of Isosteviol by Means of Ugi Reaction .................. 98 4.3 Synthesis of Peptide Analogs ............................................................................... 100 4.3.1 Model Reactions Tested on Isosteviol ........................................................... 102 4.3.2 Addition of Amino Acid to 4.2 ...................................................................... 103 4.3.3 Synthesis of Dipeptide Analog 4.5 in Single Step ......................................... 103 4.4 Synthesis of Analogs with Three Amino Acid Units ........................................... 105 4.4.1 In Search of Ideal Condition for Synthesis of Analogs with Three Amino Acids ......................................................................................................................