Design, Synthesis and in vitro investigations of Novel Fluorescently Labeled Steroids by Nisal U. Gajadeera B.S. in Chemistry, Northeastern University A thesis submitted to The Faculty of the College of Science of Northeastern University in partial fulfillment of the requirements for the degree of Master of Science December 2018 Thesis directed by Robert Hanson Matthews Distinguished University Professor of Chemistry and Chemical Biology 1 Acknowledgements Firstly, I would like to thank my advisor, Dr Robert Hanson for providing me the opportunity to work in his lab. His guidance and support has been incredible for me throughout my time at Northeastern. I would also like to take this opportunity to thank all the past Hanson lab members, Dr Emily Corcoran, Dr James Teh and Kelton Barnsely. I would also like to thank Dr Vladimir Torchilin and Dr Tatiana Levchenko for the opportunity to conduct cell studies in their lab. My heartfelt gratitude goes to Dr Livia Mendez for conducting all the incubation studies and competitive binding studies. It was under her guidance that I learned the techniques such as passaging cells, FACS and fluorescence microscopy. She played a huge role the in vivo data gathering for my compounds. I would also like to take this opportunity to thank Dr Michael Pollastri and the members of his group, especially Dr Dana Klug, Dr Lori Ferrins and Dr Baljinder Singh for the support they’ve shown in my research by allowing me to use their analytical instruments ( LC-MS). Without their support, my research would not have been possible. A special thanks goes to - John Bottomy and Brian D’Amico for the tremendous support they’ve given in helping me with my TA duties. Especially Brian and Dr Jason Guo for helping me with NMR. I would also like to mention Jeff Peterson and Alex Henrikson for their support in making the UV-Vis spectrometer available. Also, I would like to thank Richard Pumphry, Cara Shockley and Tara Loschiavo for their support in my graduate studies. 2 To all the professors who I met during my undergraduate and graduate studies at Northeastern University, I offer you my most sincere gratitude. I am the person who I am because of the knowledge you imparted. I would especially like to thank Dr Raymond Booth and Dr George O’Doherty for their role in reviewing my thesis. On similar note, I would like to sincerely thank Northeastern University community. I am very grateful for the opportunities given to me at this institution. Finally, I would like to thank my parents for their unconditional support in throughout their life. Without you, I wouldn’t have been able to come this far. You stood beside me during good times and bad. 3 Abstract of Thesis Breast cancer is the most common cancer among women, accounting for nearly third of all the diagnosed cancers. According to the American cancer society, over 41,000 patients died from the disease in 2018. Due to the high importance of the role played by estrogen and progesterone, agents that target the estrogen receptor (ER) and progesterone receptor (PR) play a major role in breast cancer therapy. Therapeutic strategies include the use of selective estrogen receptor modulators, selective estrogen receptor down regulators and aromatase inhibitors. Treatment success of breast cancer therapy is assessed by tumor estrogen receptor status which employs immunohistochemistry among other modalities. Although suitable for assaying ER expression in primary breast tumors, the accuracy of immunohistochemistry is lower in metastases. Fluorescent imaging methods with high ER affinity and selectivity offer the potential for noninvasive, clinical imaging for primary and metastatic tumors. My thesis describes the background, synthesis and in vitro investigations of a series of fluorescently labeled steroids. Chapter 1 of this thesis provides an extensive review of the fluorescently labeled estrogens prepared since 1995 with an emphasis on their synthesis and their efficacy as imaging agents, with our rationale for developing the ‘next generation’ fluorescent steroidal ER imaging agents. Chapter 2 provides our synthetic approach for the fluorescent steroidal imaging agents and their controls. Our approached utilized the 11β-(4-azidoethoxyphenyl) estradiol scaffold with small, neutral fluorophores appended Sharpless “click” chemistry. 4 Chapter 3 reports in vitro evaluation of our steroidal imaging agents. The results strongly suggest that our steroidal fluorophores show selectivity towards ER. Potential improvements for the steroidal-fluorophore conjugates is also discussed. 5 Table of Contents Acknowledgements .........................................................................................2 Abstract of Thesis ..........................................................................................4 Table of Contents…………………………………………..…………….………………………………6 List of Figures .................................................................................................9 List of Schemes and Tables ..........................................................................13 List of Abbreviations ....................................................................................14 Chapter 1 – A review of fluorescently labeled steroids ................................19 1.1.1 Introduction ................................................................................20 1.1.1 The target- Estrogen receptor .....................................................21 1.1.2 Estradiol- The native ligand .......................................................22 1.1.2.1 Structure-Activity Relationships ...................................................22 1.1.2.2 Evaluation of ligand-ER-LBD complexes ....................................24 1.2 Fluorescent properties .................................................................25 1.3 Conjugation properties ...............................................................27 1.3.1 Survey of fluorescent probes - representative examples..................28 1.3.2 Fluorescent probes post-1995 ..........................................................29 1.4 B-ring derivatives................................................................................30 1.5 D-Ring Derivatives -17α-Substitution ................................................35 1.6 C-Ring substitution using 11β-subsitution .........................................49 6 1.7 Proposed Approach to High Affinity Fluorescent Steroidal ER Probes - Synthesis and future directions ...............................................................55 1.8 References ...........................................................................................56 Chapter 2 - Design and synthesis of fluorescently labeled antiestrogens .....74 2.1 Introduction .........................................................................................75 2.2 Synthesis .............................................................................................79 2.3 Fluorescent properties .........................................................................86 2.4 Future work – alternative synthesis routes..........................................87 2.4 Summary .............................................................................................89 2.5 References. ..........................................................................................90 Chapter 3 – In vitro investigations of fluorescently labeled antiestrogens ...96 3.1 Introduction – previous research .........................................................97 3.2 In vivo studies introduction – Estrogen receptor ................................99 3.3 General Experimental Setup .............................................................100 3.4 Time point study – NBD-E2 (12b) and Dansyl-E2 (12a).................103 3.5 Time point study – NBD-E2 (12b) at 1µM ......................................106 3.6 NBD-E2 (12b) competitive binding study at 1µM ...........................109 3.7 NBD-E2 (12b), NBD-propargyl (9), competitive binding and temperature dependence in receptor localization at 1µM .......................113 7 3.8 Competitive binding – NBD control compound (10b) and NBD-E2 (12b) at 1µM ...........................................................................................115 3.9 Discussion of results .........................................................................118 3.10 Conclusion and future work ............................................................125 3.11 References .......................................................................................126 Appendix .....................................................................................................130 8 List of Figures Figure 1-1. Basic structure activity relationships for substituted estradiol derivatives. ............................................................................................. 23 Figure 1-2. Representative examples of parent fluorescent dyes described in the review. ......................................................................... 27 Figure 1-3 Examples of estradiol-fluorophore conjugates prior to 1995 ................................................................................................................ 29 Figure 1-4. Transformations of estrone to fluorescent derivatives ....... 31 Figure 1-5. 6-Carboxymethyloximino derivatives with fluorophores conjugated through oligoethylene glycol linkers ................................... 32 Figure 1-6. Synthesis of 6β-and 7α-fluorophore