SYNTHESIS, ANTI-HIV ACTIVITY and MOLECULAR MODELING STUDIES of CARBOCYCLIC and 4'-THIONUCLEOSIDES by HYUNAH CHOO (Under the D
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SYNTHESIS, ANTI-HIV ACTIVITY AND MOLECULAR MODELING STUDIES OF CARBOCYCLIC AND 4’-THIONUCLEOSIDES by HYUNAH CHOO (Under the Direction of CHUNG K. CHU) ABSTRACT This thesis describes the synthesis, biological evaluation and molecular modeling studies of carbocyclic and 4’-thionucleosides as potential antiviral agents. Chapter 1 reviews the activity, metabolism, toxicity and the development of resistance of antiviral nucleosides as well as their status in clinical trials. In Chapter 2, an efficient method for the synthesis of carbocyclic L-2’- deoxynucleosides using D-ribose is presented. The Mitsunobu reaction was used to condense various purine and pyrimidine bases with the carbocyclic sugar moiety. Chapter 3 describes a comparison study of solid phase synthesis versus solution phase synthesis of carbocyclic L-2’-deoxynucleosides. Solid phase synthesis gave better 2 selectivity of N1-alkylated products versus O -alkylated products in the synthesis of pyrimidine nucleosides, while only N9-alkylated products were obtained in both solid and solution phase synthesis of purine nucleosides. Chapter 4 details the synthesis and biological evaluation against HIV-1 in primary human lymphocytes of β-L-2’,3’-didehydro-2’,3’-dideoxy-2’-fluoro-4’- thionucleosides (β-L-2’-F-4’-S-d4Ns). The cytosine, 5-fluorocytosine and adenine derivatives showed potent anti-HIV activities (EC50 0.12, 0.15 and 1.74 µM, respectively) without significant cytotoxicity. The cytosine derivative (β-L-2’-F-4’-S-d4C), however, showed cross-resistance to 3TC-resistant variant (HIV-1M184V). According to our molecular modeling studies, the steric hindrance between the sugar moiety of the unnatural L-nucleoside and the side chains of Val184 of M184V RT in 3TC-resistant mutant HIV strains destabilizes the RT-nucleoside triphosphate complex, which causes the cross-resistance to 3TC (M184V mutant). Chapter 5 describes the development of an efficient synthetic method for 3’-thio analogs of oxetanocin A. The thietane ring was synthesized using a carbohydrate template and the condensation between the sugar moiety and various heterocyclic bases was conducted using the Pummerer rearrangement reaction. Chapter 6 details the synthesis of various L-thietanose nucleosides with different sugar moieties to extend the usage of the synthetic method of the thietanse ring developed in our group. Chapter 7 desicribes molecular modeling studies showing that the 2’,3’-double bond of the sugar moiety of various 2’,3’-unsaturated nucleosides interacts with the aromatic moiety of Tyr115 of HIV-1 reverse transcriptase (RT) by hydrophobic π–π interaction. In 3TC-resistant mutant (M184V) RT, 2’-fluoro-2’,3’-unsaturated nucleosides with a bulky 4’-substituent experience significant steric hindrance with the side chain of Val184. INDEX WORDS: carbocyclic nucleosides, 4’-thionucleosides, 3’-thio analogs, thietanose nucleosides, cross-resistance, 3TC-resistant mutant, M184V, HIV and HBV SYNTHESIS, ANTI-HIV ACTIVITY AND MOLECULAR MODELING STUDIES OF CARBOCYCLIC AND 4’-THIONUCLEOSIDES by HYUNAH CHOO B.S., Seoul National University, Republic of Korea, 1993 M.S., Seoul National University, Republic of Korea, 1996 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2003 © 2003 Hyunah Choo All Rights Reserved SYNTHESIS, ANTI-HIV ACTIVITY AND MOLECULAR MODELING STUDIES OF CARBOCYCLIC AND 4’-THIONUCLEOSIDES by HYUNAH CHOO Major Professor: Chung K. Chu Committee: J. Warren Beach Anthony C. Capomacchia Larry B. Hendry Vasu Nair Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia August 2003 ACKNOWLEDGEMENTS This thesis is the result of four years of work whereby I have been supported by many people. Now I have the opportunity to express my gratitude to all of them. The first person I would like to thank is my research advisor, Dr. Chung K. Chu. His enthusiasm and integral view on research have made a deep impression on me. I am also grateful for his invaluable support and encouragement. I would also like to thank the members of my PhD committee who monitored my work and took effort in reading and providing me with valuable comments: Drs J. Warren Beach, Anthony C. Capomacchia, Larry B. Hendry and Vasu Nair. Many thanks also go to my colleagues: Dr. Giuseppe Gumina and other group members. And I feel a deep sense of gratitude for Youhoon Chong who is my husband as well as my colleague. He has given me his sincere support and love throughout all my life. I am also grateful to Ms. Wendy Nix for her wonderful help. I am also very grateful to my family for their endless support and love. iv TABLE OF CONTENTS Page ACKNOWLEDGEMENTS............................................................................................... iv LIST OF TABLES............................................................................................................ vii LIST OF FIGURES ......................................................................................................... viii LIST OF SCHEMES......................................................................................................... xii CHAPTER 1 ADVANCES IN ANTIVIRAL NUCLEOSIDES .............................................1 INTRODUCTION.........................................................................................2 ANTI-HIV AGENTS ....................................................................................4 ANTI-HBV AGENTS.................................................................................47 2 SYNTHESIS AND ANTIVIRAL ACTIVITY OF CARBOCYCLIC L-2’- DEOXYNUCLEOSIDES ................................................................................66 INTRODUCTION.......................................................................................67 RESULTS AND DISCUSSION .................................................................68 EXPERIMENTAL SECTION ....................................................................73 3 SOLID PHASE SYNTHESIS OF CARBOCYCLIC L-2’- DEOXYNUCLEOSIDES ................................................................................91 INTRODUCTION.......................................................................................92 RESULTS AND DISCUSSION .................................................................93 EXPERIMENTAL SECTION ....................................................................98 v 4 SYNTHESIS, ANTI-HIV ACTIVITY AND MOLECULAR MECHANISM OF DRUG RESISTANCE OF L-2’,3’-DIDEHYDRO-2’,3’-DIDEOXY-2’- FLUORO-4’-THIONUCLEOSIDES.............................................................110 ABSTRACT ..............................................................................................111 INTRODUCTION.....................................................................................112 RESULTS AND DISCUSSION ...............................................................113 EXPERIMENTAL SECTION ..................................................................125 5 SYNTHESIS OF 3’-THIONUCLEOSIDES .................................................145 INTRODUCTION.....................................................................................146 RESULTS AND DISCUSSION ...............................................................147 EXPERIMENTAL SECTION ..................................................................156 6 SYNTHESIS OF L-3’-THIETANOSE NUCLEOSIDES .............................171 INTRODUCTION.....................................................................................172 RESULTS AND DISCUSSION ...............................................................173 EXPERIMENTAL SECTION ..................................................................178 7 THE ROLE OF 2’,3’-UNSATURATION ON THE ANTIVIRAL ACTIVITY OF ANTI-HIV NUCLEOSIDES AGAINST 3TC-RESISTANT NUTANT (M184V) ........................................................................................................194 INTRODUCTION.....................................................................................195 METHODS................................................................................................196 RESULTS AND DISCUSSION ...............................................................198 REFERENCES ................................................................................................................204 vi LIST OF TABLES Page Table 1.1: FDA-approved anti-HIV drugs in chronological order ......................................7 Table 1.2: Fold resistances in mutant HIV-1 RT associated with NRTI resistance ............8 Table 2.1: Anti-HIV activity of carbocyclic L-2’-deoxynucleosides ................................72 Table 2.2: Elemental Analysis Data...................................................................................90 Table 3.1: Comparison between solid and solution phase syntheses.................................96 Table 3.2: Optimized solid phase synthesis.......................................................................98 Table 4.1: 1H NMR Data .................................................................................................117 Table 4.2: Activity of L-2’,3’-didehydro-2’,3’-dideoxy-2’-fluoro-4’-thionucleosides...119 Table 4.3: Correlation of relative binding energy difference (∆Erel) with fold increase .121 Table 4.4: Elemental analysis data...................................................................................141 Table