Nucleoside Analogues for Positron Emission Tomography Imaging and to Study Radiation Mediated Generation of Radicals from Azides
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Florida International University FIU Digital Commons FIU Electronic Theses and Dissertations University Graduate School 6-27-2019 Nucleoside Analogues for Positron Emission Tomography Imaging and to Study Radiation Mediated Generation of Radicals from Azides Maria E. de Cabrera Florida International University, [email protected] Follow this and additional works at: https://digitalcommons.fiu.edu/etd Part of the Nucleic Acids, Nucleotides, and Nucleosides Commons Recommended Citation de Cabrera, Maria E., "Nucleoside Analogues for Positron Emission Tomography Imaging and to Study Radiation Mediated Generation of Radicals from Azides" (2019). FIU Electronic Theses and Dissertations. 4229. https://digitalcommons.fiu.edu/etd/4229 This work is brought to you for free and open access by the University Graduate School at FIU Digital Commons. It has been accepted for inclusion in FIU Electronic Theses and Dissertations by an authorized administrator of FIU Digital Commons. For more information, please contact [email protected]. FLORIDA INTERNATIONAL UNIVERSITY Miami, Florida NUCLEOSIDE ANALOGUES FOR POSITRON EMISSION TOMOGRAPHY IMAGING AND TO STUDY RADIATION MEDIATED GENERATION OF RADICALS FROM AZIDES A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR IN PHILOSOPHY in CHEMISTRY by Maria de Cabrera 2019 To: Dean Michael R. Heithaus College of Arts, Sciences and Education This dissertation, written by Maria de Cabrera, and entitled Nucleoside Analogues for Positron Emission Tomography Imaging and to Study Radiation Mediated Generation of Radicals from Azides, having been approved in respect to style and intellectual content, is referred to you for judgment. We have read this dissertation and recommend that it be approved. __________________________________________ Raphael Raptis __________________________________________ Kathleen Rein __________________________________________ Jose Almirall __________________________________________ M. Alejandro Barbieri __________________________________________ Stanislaw F. Wnuk, Major Professor Date of Defense: June 27, 2019 The dissertation of Maria de Cabrera is approved. __________________________________________ Dean Michael R. Heithaus College of Arts, Sciences and Education __________________________________________ Andrés G. Gil Vice President of Research and Economic Development and Dean of the University Graduate School Florida International University, 2019 ii © Copyright 2019 by Maria de Cabrera All rights reserved. iii DEDICATION I would like to dedicate this dissertation to all the people in my life that have, in one way or another, helped me to grow as person over these last years. iv ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Stanislaw F. Wnuk, for his support and guidance over these last four years. I would also like to thank my committee members: Dr. Raphael Raptis, Dr. Kathleen Rein, Dr. Jose Almirall, and Dr. Alejandro Barbieri for their support and advice throughout my graduate studies. I am very thankful for your insight and novel perspectives about my presentations and projects. I would like to extend my gratitude towards everyone in the Department of Chemistry and Biochemistry at Florida International University for all their help and support. I would also like to thank all my lab mates, past and present, for all their help day to day. I would like to thank all of my collaborators, without whom this dissertation would not be possible. I would like to thank Dr. Anthony McGoron, from FIU's Department of Biomedical Engineering, and Dr. Alejandro Amor-Coarasa, from Weill Cornell Medical College, for their 68Ga radiolabeling and in vivo rat studies of 4-N-alkylgemcitabine NOTA conjugates. Also, I would like to thank Dr. Caius Radu, from University of California Los Angeles, for sharing with us his unpublished biological studies of clofarabine ProTides, which were the basis for our design of clofarabine 5'-diphosphate prodrugs. I would like to thank Dr. Michael Sevilla and Dr. Amitava Adhikary, from Oakland University, for sharing with us their unpublished EPR results of the azido-modified nucleosides prepared in my dissertation. I would like to thank Dr. Adhikary for all his time and effort in helping me understand EPR results and properly explain them in this dissertation. Finally, I would like to thank Dr. Rupak Pathak from University of Arkansas for Medical Sciences for the biological evaluation of the radiosensitizing potential of azido-modified nucleosides. v I want to express my sincere gratitude towards my family and friends. First and foremost, I would like to thank to my parents, Silvia and Carlos, who have had nearly godly patience with me throughout all my studies and have always supported me in every way they could. I would also like to thank by little brother, Carlos Jr., for always listening to me; you know our conversations are therapeutic for me. I would like to thank my boyfriend, Santiago, for always being there to support me over these last years; you are the kindest person I know. Finally, I would like to thank all my friends in the program and outside for all their help day to day; especially Vanessa for always listening to me and helping me in any way she could. vi ABSTRACT OF THE DISSERTATION NUCLEOSIDE ANALOGUES FOR POSITRON EMISSION TOMOGRAPHY IMAGING AND TO STUDY RADIATION MEDIATED GENERATION OF RADICALS FROM AZIDES by Maria de Cabrera Florida International University, 2019 Miami, Florida Professor Stanislaw F. Wnuk, Major Professor Gemcitabine is a potent anticancer cytidine analogue used to treat solid tumors. Its efficacy is diminished by rapid deamination to a toxic uridine derivative by cytidine deaminase. To overcome this limitation and add radioactive nuclei (18F or 68Ga) for PET imaging, I synthesized two 4-N-alkylgemcitabine analogues i) bearing β-keto tosylate moiety for subsequent 18F-fluorination and ii) having SCN-Bn-NOTA chelator to complex 68Ga. The first was synthesized by replacement of tosylamide in 4-N-tosylgemcitabine with 1-amino-10-undecene, followed by elaboration of terminal alkene through dihydroxylation, regioselective tosylation and oxidation. Subsequent fluorination using KF in presence of 18-Crown-6 at 75°C for 1 hr gave 4-N-alkylgemcitabine fluoromethyl ketone. The second was synthesized by analogous replacement of tosylamide with N-Boc- 1,3-propanediamine, followed by deprotection with TFA. The reactive terminal amine was condensed with SCN-Bn-NOTA, giving 4-N-alkylgemcitabine-SCN-Bn-NOTA ligand, which efficiently complexed Ga or 68Ga for in vivo PET studies in rats. vii Clofarabine is a highly effective chemotherapeutic adenosine analogue used for treatment of acute lymphoblastic leukemia. Clofarabine undergoes rate limiting phosphorylation from its 5'-monophosphate to 5'-diphosphate by purine monophosphate kinase, and possible dephosphorylation of its respective 5'-monophosphate by 5'- nucleotidases. Synthesis of clofarabine diphosphate prodrugs, and potentially their 18F- radiolabeled analogues, were undertaken to overcome these limitations. Successful synthesis of model adenosine diphosphate prodrug, by coupling adenosine monophosphate with bis(benzoyloxybenzyl) phosphoramidite in presence of 5-phenyl-1-H-tetrazole activator was achieved. The aminyl radical generated from azide moiety in 3'-azido-3'-deoxythymidine (3'- AZT) or 5-azidomethyl-2'-deoxyuridine (AmdU), upon addition of radiation-produced electrons, is thought to be the source of their radiosensitizing effects. Herein, I report synthesis of azido-modified purine and pyrimidine analogues for EPR study of formation of reactive aminyl radical in guanine, adenine and cytidine bases. The EPR studies of electron addition to 2-azidoguanosine (i.e. 2-azidoinsoine), protected 4-azidocytidine and 4-tetrazolocytidine analogues clearly establish that the position of the azide in base moiety dictates reactivity. The azide directly attached to nucleobases at ortho/para position to ring - nitrogens produce stable RN3• that does not rapidly convert to aminyl radical, except in the excited state. Hence, these did not display much radiosensitizing effects in in vivo biological studies in MDA-MB-231, MCF7 and U87 cell lines. viii TABLE OF CONTENT CHAPTER PAGE 1 INTRODUCTION ........................................................................................................... 1 Anti-cancer properties of nucleoside analogues........................................................ 1 1.1.1 Gemcitabine: biological activity ......................................................................... 1 1.1.2 Clofarabine: biological activity .......................................................................... 3 Nucleoside prodrugs .................................................................................................. 5 Positron Emission Tomography (PET) imaging ....................................................... 9 1.3.1 Overview of PET imaging agents ..................................................................... 11 Nucleoside- and carbohydrate-based 18F-PET imaging agents ................. 11 68Ga based radiopharmaceuticals ............................................................... 13 Radical induced DNA damage in radiotherapy ....................................................... 14 1.4.1 Radical formation on the guanine base caused by DNA oxidation .................. 18 1.4.2 Studies on the formation of guanyl and adenyl aminyl radical ........................ 19 1.4.3 Radical formation in DNA oxidation