The Pennsylvania State University
The Graduate School
Department of Chemistry
CHEMICAL SYNTHESIS OF BASE-MODIFIED RIBONUCLEOSIDES AS
NOVEL ANTIVIRAL AGENTS
A Thesis in
Chemistry
by
Daniel Allen Harki
© 2005 Daniel Allen Harki
Submitted in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
August 2005
The thesis of Daniel Allen Harki was reviewed and approved* by the following:
Blake R. Peterson Associate Professor of Chemistry Thesis Advisor Chair of Committee
Craig E. Cameron Louis Martarano Associate Professor of Biochemistry and Molecular Biology
J. Martin Bollinger, Jr. Associate Professor of Biochemistry and Molecular Biology Associate Professor of Chemistry
Ken S. Feldman Professor of Chemistry
Ayusman Sen Professor of Chemistry Head of the Department of Chemistry
*Signatures are on file in the Graduate School
ii ABSTRACT
RNA viruses exist in nature as a population of genetic variants termed a quasispecies. This inherent genomic variability permits the rapid evolution of a virus population in response to changing environmental conditions, reestablishing a new population of viruses that have adapted to their surroundings. The development of antiviral drug resistance is propagated through this mechanism.
To maintain this high degree of genomic adaptability, RNA viruses exist on the edge of “error catastrophe.” Slight increases in the relative mutation frequencies of RNA virus genomes can surpass the tolerated error threshold yielding viral inviability or “lethal mutagenesis.”
The antiviral drug ribavirin was recently demonstrated to function as an antiviral lethal mutagen. Following intracellular phosphorylation to the 5’- triphosphate, this antiviral nucleotide is misincorporated into the viral (poliovirus)
RNA genome by the promiscuous viral RNA-dependent RNA polymerase
(RdRP). Once present in the viral genome, ribavirin templates the misincorporation of the pyrimidines C and U during multiple rounds of replication.
The degenerate templating specificity of ribavirin enhances the frequency of A →
G and G → A transition mutations, forcing the virus into error castrophe and loss of viability.
Efforts towards the development of novel antiviral lethal mutagens are described throughout this thesis. Chapter one provides an overview of the lethal mutagenesis strategy, citing many of the pioneering accomplishments in the iii development of this antiviral drug discovery approach. Chapter two describes
the development of a “universal base” ribonucleoside designed to be
misincorporated opposite all four RNA bases. A structural comparison between
this analogue (3-NPN) and the antiviral drug ribavirin is presented. The third
chapter of this thesis expands on the development of universal base
ribonucleosides and examines a series of substituted indoles and azaindoles for
antiviral activity. One particular compound, termed 5-NINDN, functions as a
universal base by misincorporating opposite all four RNA bases when utilized as
a substrate for poliovirus and coxsackievirus RdRPs. Chapter four examines a family of 5-substituted cytidine derivatives, structurally inspired by 5-hydroxy-2’- deoxycytidine, a lethal mutagen for HIV. Two analogues from this series were shown to possess good antiviral activities against poliovirus and coxsackievirus, with 5-nitrocytidine surpassing the observed antiviral activity for ribavirin. The final chapter, Chapter five, is split among two studies. The first section describes basic strategies for nucleotide synthesis, focusing on efforts directed towards an efficient synthesis of ribavirin triphosphate and pyrazofurin triphosphate. The second part describes the biochemical evaluation of a 4-nitroimidazole ribonucleoside analogue. iv TABLE OF CONTENTS
LIST OF FIGURES ...... ix
LIST OF TABLES ...... xiv
ACKNOWLEDGEMENTS...... xv
Chapter 1 LETHAL MUTAGENESIS AS AN ANTIVIRAL STRATEGY...... 1
1.1 Overview ...... 1 1.2 Life Cycle of an RNA virus: Poliovirus...... 2 1.3 Known Diseases Resulting from RNA Virus Infections ...... 3 1.4 Clinically Approved Antiviral Therapeutics and Their Molecular Targets...... 4 1.5 Lethal Mutagenesis as an Antiviral Strategy ...... 11 1.5.1 Quasispecies and the Error Threshold...... 11 1.5.2 Lethal Mutagenesis of HIV ...... 13 1.5.3 Lethal Mutagenesis of Other RNA Viruses...... 15 1.5.4 Discovery of the Mutagenic Capacity of Ribavirin ...... 17 1.5.4.1 Discovery of Ribavirin and Initial Mechanism-of-Action Assignment ...... 17 1.5.4.2 Intracellular Metabolism of Ribavirin and Alternative Theories on the Molecular Mechanism of Action ...... 18 1.5.4.3 The Development by the Cameron Laboratory of an In Vitro Assay to Directly Measure Nucleotide Incorporation by a Viral Polymerase ...... 21 1.5.4.4 Ribavirin is an RNA Virus Mutagen ...... 23 1.6 Considerations for the Rational Design of Novel Lethal Mutagens ..27 1.7 Advantages for the Use of Viral Lethal Mutagens ...... 28 1.8 References...... 30
Chapter 2 CHEMICAL SYNTHESIS AND ANTIVIRAL EVALUATION OF 1-β-D-RIBOFURANOSYL-3-NITROPYRROLE...... 44
2.1 Overview ...... 44 2.2 Existing Scaffolds for Universal Bases...... 45 2.3 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole (3-NP, 47): A Universal Deoxyribonucleoside Analogue...... 46 2.3.1 Rational Design and Chemical Synthesis of 3-NP by Bergstrom and Coworkers ...... 46 2.3.2 Thermal Denaturation Studies on Duplex DNA Containing 47 ...... 47 2.3.3 Biochemical Applications of Universal Deoxyribonucleosides: 3-NP (47)...... 48 2.4 Molecular Design of a Universal Ribonucleoside Analogue ...... 49 v 2.5 Chemical Synthesis of 3-NPN (48)...... 51 2.5.1 Coupling Strategy with Chlorinated Ribose 50 ...... 51 2.5.2 Coupling Strategy with Chlorinated Ribose 54 ...... 52 2.6 Evaluation of Cellular Cytotoxicity of 3-NPN (48) ...... 56 2.7 Antiviral Evaluation of 3-NPN ...... 57 2.8 Phosphorylation of 3-NPN by Adenosine Kinase ...... 58 2.9 Kinetics of Incorporation of 3-NPNTP (61) by Poliovirus RNA- dependent RNA polymerase (3Dpol) ...... 60 2.9.1 Synthesis of 3-NPNTP (61) ...... 60 2.9.2 Kinetics of Incorporation of 3-NPNTP (61) into Sym/Sub by Poliovirus RNA-Dependent RNA Polymerase (RdRP) ...... 61 2.10 Structural Comparison of 3-NPN (48) and Ribavirin (25) ...... 63 2.11 Implications for the Design of New Universal Ribonucleosides...... 65 2.12 Experimental Section ...... 65 2.13 References...... 72
Chapter 3 DESIGN, SYNTHESIS, AND ANTIVIRAL EVALUATION OF A SERIES OF SUBSTITUTED INDOLE AND 7-AZAINDOLE RIBONUCLEOSIDES ...... 81
3.1 Overview ...... 81 3.2 Substituted Indole Ribonucleosides as Antiviral Lethal Mutagens ...82 3.2.1 Design of an Improved Universal Deoxyribonucleoside: 1-(2'- deoxy-β-D-ribofuranosyl)-5-nitroindole...... 82 3.2.2 Coupling Strategies for the Synthesis of Indole Ribonucleosides...... 86 3.2.3 Cellular Cytotoxicity Study of 5-NINDN (63)...... 91 3.2.4 Antiviral Activity of 5-NINDN against Poliovirus and Coxsackievirus B3...... 92 3.2.5 Incorporation of 5-NINDNTP into the RNA Template Mediated by Poliovirus and Coxsackievirus RNA-dependent RNA polymerases ..93 3.2.6 Efforts Towards Elucidating the Molecular Mechanism of Action of 5-NINDN (63) ...... 96 3.3 Design, Synthesis, and Preliminary Antiviral Evaluation of a Series of Substituted Indole Ribonucleosides ...... 97 3.3.1 Molecular Design of Substituted Indoles ...... 97 3.3.2 Chemical Synthesis of Substituted Indole Ribonucleosides ...... 98 3.3.3 Preliminary Cytoxicity and Antiviral Activity Studies of the Substituted Indole Ribonucleosides ...... 100 3.3.4 Conclusions and Future Directions ...... 102 3.4 Synthesis and Biochemical Evaluation of 7-azaindole Ribonucleosides as Antiviral Lethal Mutagens...... 103 3.4.1 Substituted and Unsubstituted 7-azaindole Deoxyribonucleosides for the Expansion of the Genetic Alphabet...... 103 vi 3.4.2 Design and Synthesis of 7-azaindole Ribonucleosides 65 and 67 ...... 105 3.4.3 Cellular Cytoxicity and Antiviral Activity Studies of 7-azaindole Ribonucleoside Analogues...... 106 3.4.4 Conclusions and Future Directions ...... 108 3.5 Experimental Section ...... 109 3.6 References...... 130
Chapter 4 5-SUBSTITUTED CYTIDINE DERIVATIVES: CHEMICAL SYNTHESES AND ANTIVIRAL ACTIVITIES...... 138
4.1 Overview ...... 138 4.2 Oxidative DNA Damage and the Structural Basis for the Ambiguous Base-Pairing Properties of 5-hydroxy-2'-deoxycytidine (26)...... 139 4.3 Molecular Design of 5-Substituted Cytidines...... 142 4.4 Chemical Syntheses of 5-Substituted Cytidine Ribonucleosides .....144 4.4.1 Preparation of 5-hydroxycytidine (107) and 5-bromocytidine (108)...... 144 4.4.2 Previous Syntheses of 5-nitrocytidine (109) and 5- aminocytidine (110)...... 145 4.4.3 An Improved Chemical Synthesis of 5-nitrocytidine (109) and 5- aminocytidine (110)...... 146 4.4.3.1 Vorbrüggen Coupling Chemistry ...... 146 4.4.3.2 Chemical Synthesis of 5-nitrocytidine (109) and 5- aminocytidine (110)...... 148 4.5 Cytotoxicity of 5-Substituted Cytidines ...... 150 4.6 Antiviral Activity of 5-Substituted Cytidines ...... 151 4.7 Kinetics of Incorporation of 5-nitrocytidine Triphosphate into the Viral (PV) RNA Genome...... 153 4.7.1 Synthesis of 5-nitrocytidine Triphosphate...... 153 4.7.2 Incorporation of 5-NO2CyTP (123) and 5-BrCyTP (124) by Poliovirus RNA-Dependent RNA Polymerase...... 153 4.8 Intracellular Phosphorylation of 5-nitrocytidine to the Corresponding Triphosphate (5-NO2CyTP)...... 156 4.9 Discussion and Future Directions...... 159 4.10 Experimental Section ...... 161 4.11 References...... 171
Chapter 5 MISCELLANEOUS RIBONUCLEOSIDES AND RIBONUCLEOTIDES: (I) TOWARDS AN EFFICIENT SYNTHESIS OF RIBAVIRIN AND PYRAZOFURIN TRIPHOSPHATES; (II) SYNTHESIS AND ANTIVIRAL EVALUATION OF 1-β-D-RIBOFURANOSYL-4- NITROIMIDAZOLE ...... 178
vii 5.1 Overview ...... 178 5.2 Efforts Towards an Efficient Syntheses of Ribavirin Triphosphate (41) and Pyrazofurin Triphosphate (128) ...... 178 5.2.1 Biochemical Applications of Nucleotide Triphosphates: Ribavirin Triphosphate and Pyrazofurin Triphosphate ...... 178 5.2.2 Overview of Methods for the Synthesis of Nucleotides ...... 180 5.2.3 One-Pot, Three-Step Approach for the Synthesis of Ribavirin Triphosphate (41) and Pyrazofurin Triphosphate (128)...... 184 5.2.4 Synthesis of Ribavirin Triphosphate (41) and Ribavirin Diphosphate (154) from an Activated Phosphormorpholidate...... 186 5.2.5 Efforts Towards a Chemo-Enzymatic Approach for the Synthesis of Ribavirin Triphosphate (41) and Pyrazofurin Triphosphate (128)...... 188 5.3 Synthesis and Antiviral Evaluation of 1-β-D-Ribofuranosyl-4- nitroimidazole ...... 191 5.3.1 Background ...... 191 5.3.2 Cellular Cytotoxicity and Antiviral Activity of 4-NIN (167) Against Poliovirus...... 192 5.3.3 Phosphorylation of 4-NIN by Adenosine Kinase...... 193 5.3.4 Conclusions...... 195 5.4 Experimental Section ...... 195 5.5 References...... 201
Appendix A X-RAY CRYSTALLOGRAPHIC DATA FOR 3',5'-DI-O- BENZOYL-1',2'-O-(S)-[PHENYL(3- NITROPYRROLE)METHYLIDENE]-α-D-RIBOFURANOSE (52)...... 206
A.1 General Experimental ...... 206 A.2 Funding Acknowledgement and Assignment of Credit ...... 207 A.3 References ...... 208 A.4 Thermal Ellipsoid Drawing of 52 ...... 209 A.5 Crystal Packing Diagram of 52 ...... 210 A.6 Crystallographic Information for Orthoamide 52 ...... 211
Appendix B X-RAY CRYSTALLOGRAPHIC DATA FOR 1-(β-D- RIBOFURANOSYL)-3-NITROPYRROLE (48) ...... 218
B.1 General Experimental ...... 218 B.2 Funding Acknowledgement and Assignment of Credit ...... 220 B.3 References ...... 220 B.4 Thermal Ellipsoid Drawing of 48 ...... 221 B.5 Crystal Packing Diagram of 48 ...... 222 B.6 Crystallographic Information for 48 ...... 223 viii Appendix C X-RAY CRYSTALLOGRAPHIC DATA FOR 1-(β-D- RIBOFURANOSYL)-5-NITROINDOLE (63) ...... 228
C.1 General Experimental...... 228 C.2 Funding Acknowledgement and Assignment of Credit ...... 229 C.3 Thermal Ellipsoid Drawing of 5-NINDN (63) ...... 230 C.4 Crystallographic Information for 63...... 230
Appendix D X-RAY CRYSTALLOGRAPHIC DATA FOR 1-(β-D- RIBOFURANOSYL)PYRROLO[2,3-B]PYRIDINE (65) ...... 233
D.1 General Experimental...... 233 D.2 Funding Acknowledgement and Credit...... 234 D.3 Thermal Ellipsoid Drawing of 7AI-R (65)...... 235 D.4 Crystallographic Information for 65...... 236
ix LIST OF FIGURES
Figure 1.1. Reproductive cycle of poliovirus. Poliovirus RNA-dependent RNA polymerase (RdRP) from Thompson and Peersen (pdb 1R46).2.....3
Figure 1.2. Clinically-employed therapeutics for HIV infection...... 6
Figure 1.3. HIV fusion and protease inhibitors...... 7
Figure 1.4. Antiviral drugs for the treatment of hepatitis B virus infection...... 8
Figure 1.5. Clinically-employed therapeutics effective against RNA virus infections...... 10
Figure 1.6. Compounds tested by Loeb for HIV mutagenesis.44 ...... 14
Figure 1.7. Mutagenic ribonucleosides...... 16
Figure 1.8. 1,2,4-triazole ribonucleosides synthesized by ICN pharmaceuticals...... 18
Figure 1.9. Graphical depiction of primer-extension assay developed by Cameron and coworker.82 Structure of PV 3Dpol from Hanson et al. (pdb 1RA6).2 ...... 22
Figure 1.10. Kinetics of incorporation of RTP into sym/sub (adapted from 55)...... 24
Figure 1.11. A simple mechanistic model of the antiviral activity of ribavirin against poliovirus. Poliovirus RNA-dependent RNA polymerase from Thompson and Peersen (pdb 1RA6).2 ...... 26
Figure 2.1. Popular hydrophobic heterocycles used to construct universal bases (adapted from 1)...... 45
Figure 2.2. Synthesis of the universal deoxyribonucleoside 3-NP 47.3 ...... 47
Figure 2.3. Melting denaturation studies with duplex DNA containing 47.3 ....48
Figure 2.4. Structural comparison of nucleoside analogues...... 50
Figure 2.5. Initial coupling attempts directed at the synthesis of 3-NPN 48...52
Figure 2.6. Precedented chemical synthesis of an analogous 3- cyanopyrrole ribonucleoside 57.24 ...... 53 x Figure 2.7. Proton NMR analysis (in DMSO-d6) of differences in chemical shifts of isopropylidene methyls from ribonucleosides with α and β anomeric stereochemistries (adapted from 25)...... 54
Figure 2.8. Chemical synthesis of 3-NPN (48)...... 55
Figure 2.9. Phosphorylation of 3-NPN (48) and ribavirin (25) by adenosine kinase...... 59
Figure 2.10. Quantitative comparison of phosphorylation of adenosine (●), 3-NPN (▲), and ribavirin (■) by adenosine kinase...... 59
Figure 2.11. Chemical synthesis of 3-NPNTP (61)...... 60
Figure 2.12. Incorporation of monophosphates derived from 3-NPNTP (61) opposite complementary nucleotides in symmetrical substrates...... 62
Figure 2.13. Comparison of x-ray crystal structures of 3-NPN and ribavirin. Atom labels for panels A (3-NPN) and B (ribavirin): light gray for carbon, dark gray for nitrogen, black for oxygen. (C) Structural overlay for calculation of root-mean-square (rms) deviations of atom positions (0.83 Å): solid bonds and empty circles for 3-NPN and dashed bonds and shaded circles for ribavirin...... 64
Figure 3.1. Compound Structures...... 84
Figure 3.2. Melting temperature studies of 5-nitroindole (62) and 3- nitropyrrole (47) 2'-deoxyribonucleosides in a synthetic DNA duplex (adapted from 5)...... 84
Figure 3.3. The first reported chemical synthesis of 5-NINDN (63).23 ...... 87
Figure 3.4. An alternative indoline-indole coupling for the synthesis of 5- NINDN (63)...... 88
Figure 3.5. Coupling of the sodium salt of 5-nitroindole (78) with the stereospecific α-chlorosugar 54...... 89
Figure 3.6. Chemical synthesis of 5-NINDN (63) and 5-NINDNTP (70)...... 90
Figure 3.7. Cellular cytotoxicity of 5-NINDN (63) towards HeLa S3 cells...... 91
Figure 3.8. Antiviral activity of 5-NINDN (63) and ribavirin (25) against PV and CVB3...... 93 xi Figure 3.9. Incorporation of 5-NINDNTP opposite all four RNA bases mediated by poliovirus and coxsackievirus RNA-dependent RNA polymerases...... 94
Figure 3.10. Chain termination assay for 5-NINDNTP (70). Extension of s/s-U by PV RdRP in the presence of Mn2+...... 95
Figure 3.11. Structures of substituted indole ribonucleosides...... 98
Figure 3.12. Chemical synthesis of substituted indole ribonucleosides...... 99
Figure 3.13. Cytotoxicity of substituted indole ribonucleosides towards cultured HeLa cells (7 hour exposure)...... 100
Figure 3.14. Antiviral activity of substituted indole ribonucleosides against poliovirus-infected HeLa cells...... 101
Figure 3.15. Antiviral activity of substituted indole ribonucleosides against coxsackievirus-infected HeLa cells...... 102
Figure 3.16. Kinetics of nucleotide incorporation into a synthetic DNA template by KF DNA polymerase.43 ...... 104
Figure 3.17. Synthesis of 7AI-R (65)44 and P7AI-R (67)...... 106
Figure 3.18. Antiviral activity of 7AI-R (65) and P7AI-R (67) against poliovirus-infected HeLa cells...... 107
Figure 3.19. X-ray crystal structure of 7AI-R (65) showing internal hydrogen-bond between C5'-OH and N-7...... 109
Figure 3.20. Separation of 63 and indoline ribonucleoside 77 by reversed- phase HPLC...... 116
Figure 3.21. Purity analysis of 5-NINDNTP (70)...... 120
Figure 4.1. Oxidation by hydroxide radicals of (A) cytosine in the DNA template and (B) dCTP pools...... 140
Figure 4.2. Tautomerization of 5-hydroxy-2'-deoxycytidine...... 142
Figure 4.3. Watson-Crick hydrogen bonding patterns (A & B). Mispairing of 5-hydroxycytosine with adenosine (C)...... 142
Figure 4.4. 5-Substituted cytidine nucleosides evaluated for antiviral activity...... 143 xii Figure 4.5. Synthesis of 5-OHCy (107)...... 145
Figure 4.6. Synthesis of 5-nitrocytidine and 5-aminocytidine by Fox and coworkers.17 ...... 146
Figure 4.7. Generalized Vorbrüggen coupling for pyrimidines...... 148
Figure 4.8. Chemical synthesis of 5-nitrocytidine (109) and 5- aminocytidine (110)...... 150
Figure 4.9. Cytotoxicity of cytidine analogues towards HeLa S3 cells (Cell monolayers were treated with nucleosides for 7 hours, then allowed to recover in the absence of drug for 24 hours prior to measurement of cell viability)...... 151
Figure 4.10. Antiviral activity of cytidine analogues towards poliovirus and coxsackievirus B3 infected HeLa cells...... 152
Figure 4.11: Synthesis of 5-nitrocytidine triphosphate...... 153
Figure 4.12. Incorporation of 5-nitrocytidine triphosphate and 5- bromocytidine triphosphate into symmetrical primer-templates by poliovirus RNA-dependent RNA polymerase...... 155
Figure 4.13. Reversed-phase HPLC analysis of HeLa cell extracts. (a) Trace of 5-NO2CyTP (123, 0.56 nmoles) with characteristic UV trace (inlay). (b) Separation of untreated HeLa cell extracts (90 µL injection). (c) Separation of HeLa cell extracts (90 µL injection) that were treated with 5-nitrocytidine (2 mM, 3 hr); (c, inlay) UV trace characteristic of 123. The absorbance wavelength for all HPLC traces is 295 nm...... 158
Figure 4.14. Reversed-phase HPLC analysis of HeLa cell extracts. (a) Close-up view of the 5-nitrocytidine triphosphate peak from Figure 4.13, c (90 µL of cell extracts were analyzed). (b) Close-up view of the 5-nitrocytidine triphosphate standard from Figure 4.13, a (0.56 nmoles of 123 was injected). (c) Analysis of HeLa cell extracts (90 µL) that were treated with 5-nitrocytidine (2 mM, 3 hr) doped with 0.56 nmoles of 5-nitrocytidine triphosphate. The absorbance wavelength for all HPLC traces is 295 nm...... 159
Figure 4.15. Structures of 5-nitro-2'-deoxycytidine and 5-amino-2'- deoxycytidine...... 161
Figure 4.16. Analysis of purity of triphosphate 123...... 171
Figure 5.1. Compound structures...... 179 xiii Figure 5.2. One-pot, three-step synthesis of nucleotides...... 181
Figure 5.3. Synthesis of triphosphates from activated nucleoside monophosphates 134 and 137...... 182
Figure 5.4. Synthesis of triphosphates from electrophilic and nucleophilic nucleoside diphosphates...... 183
Figure 5.5. Synthesis of nucleotides by activation of 5'-hydroxyl groups and enzymatic methods...... 184
Figure 5.6. One-pot, three-step approach towards ribavirin triphosphate (41) and pyrazofurin triphosphate (128)...... 186
Figure 5.7. Previously reported synthesis of ribavirin triphosphate (41) and ribavirin diphosphate (154)...... 187
Figure 5.8. Chemo-enzymatic synthesis of triazole ribonucleosides...... 189
Figure 5.9. Efforts towards the synthesis of ribavirin triphosphate (41)...... 190
Figure 5.10: Structural comparison: ribavirin (25), 3-NPN (48), 4-NIN (167)...... 192
Figure 5.11. Phosphorylation of 4-NIN (167), ribavirin (25) and 3-NPN (48) by adenosine kinase...... 194
Figure 5.12. Quantitative comparison of phosphorylation of adenosine (●), 3-NPN (▲),ribavirin (■), and 4-NIN (♦) by adenosine kinase...... 194
xiv LIST OF TABLES
Table 1.1. Examples of clinical conditions resulting from RNA virus infection...... 4
Table 1.2. Inhibition of HIV-1 reverse transcriptase by the 5'-triphosphates of clinically-employed nucleoside reverse transcriptase inhibitors...... 7
Table 1.3. Lethal mutagenesis of other RNA viruses...... 16
Table 2.1. Cellular cytotoxicity of 3-NPN 48 towards HeLa S3 cells (6 day treatment). Untreated cells were assigned arbitrarily a cell viability of 100%...... 56
Table 2.2. Antiviral activity of 3-NPN (48) on poliovirus-infected HeLa S3 cells...... 58
Table 2.3. Kinetics of incorporation of 3-NPNTP (61) into symmetrical substrates by poliovirus RdRPa...... 62
Table 3.1: Kinetics of incorporation of 5-NINDNTP (70) into s/s-U mediated by PV 3Dpol...... 94
Table 5.1. Cytotoxicity and antiviral activity of 4-NIN (167)...... 193
xv ACKNOWLEDGEMENTS
I’d like to begin by thanking my advisor, Professor Blake Peterson, for his mentorship and support over the past six years. I sincerely appreciate the responsibilities you entrusted in me throughout my graduate career, and the latitude you gave me to freely explore my own ideas. I would also like to recognize and thank our research collaborator, Professor Craig Cameron. The science described in this thesis stems from Craig’s research program, a truly innovative and fascinating project. I am indebted to Craig for his countless discussions on RNA virus biology, and the active advisory role he undertook in my training as a scientist. Additional thanks goes to Professor Ken Feldman and Professor Marty Bollinger for serving on my dissertation committee, and for the helpful critiques and suggestions provided throughout my graduate career.
During my time at Penn State I’ve had the pleasure to work with some very gifted scientists. Foremost, I would like to thank and recognize Jason Graci (Cameron Lab) who performed most of the antiviral evaluation experiments described throughout this thesis. Additionally, I’d like to thank my coworkers in the Peterson lab, especially Scott Martin, Dan Clark, and Mike DeGrazia, who always managed to keep lab lively and interesting. To Jocelyn Edathil and Rob Feltz, I wish you both the best of luck in continuing this project. I’d also like to thank the Cameron lab for their warm hospitality, and recognize those members who have contributed to this project: Christian Castro, Saikat Ghosh, Victoria Korneeva, and Rebecca Morgan. I’d also like to mention the undergraduates I’ve mentored while at Penn State: B.J. Chain (graduate student in chemistry, Harvard University) and Bob Rarig (graduate student in chemistry, University of Michigan). I hope the frustrations you encountered while learning and performing nucleoside chemistry prepared you well for graduate school.
xvi Although previously acknowledged for his scientific achievements, I would like to again recognize Scott Martin, my intramural golf partner, for his clutch putting and driving accuracy. We will forever be recognized as the 2003 Penn State Intramural Golf Scramble--Open Pioria Champions.
I would be remiss without mentioning many of the people who directed me towards a career in research. I would like to thank my undergraduate advisor, Professor Kay Brummond (Department of Chemistry, West Virginia University), for introducing me to organic chemistry and providing the opportunity to work in her lab. The synthesis skills I obtained as an undergraduate researcher in the Brummond lab has proven invaluable during my graduate school career. I would also like to thank my coworkers at the National Institute for Occupational Safety and Health (NIOSH, Morgantown, WV) for their mentorship, guidance, and friendship during my time as a (student) biological science laboratory technician. I’m especially grateful to Dr. Michael Luster and Dr. Petia Simeonova, my research advisors, and to Dr. Randy Gallucci and Dr. Joanna Matheson.
I would also like to acknowledge the American Heart Association (Pennsylvania/Delaware affiliates) for financial support of my research through a predoctoral fellowship (2002-2005). xvii I dedicate this thesis to those who’ve made my successes possible, my family. For my parents, Ronald & Deborah Harki, I thank you for your enduring love, support, and encouragement. I cannot express enough gratitude for everything you’ve done for me. To my brother, Michael Harki, I sincerely apologize for inflicting (indirectly) the head wound you received while learning to ride a bike. Although I can think of other “near-miss” incidents from when we used to fight as kids, I thank you for being not only a good brother but a great friend. To my grandparents, Frank Mishko and the late Gladys Mishko, and the late Albert and Twylah Garcia, thanks for a lifetime of unforgettable memories…and for spoiling me. To my remaining family and friends, thank you for being such an important part of my life.
Finally, I’d like to acknowledge my fiancé Angela Perkins. Throughout the highs and lows of graduate school you’ve always been there for me in so many ways—friend, coworker, and now fiancé. Thank you for everything you’ve done for me. You make me truly happy, and I love you very much.
Chapter 1
LETHAL MUTAGENESIS AS AN ANTIVIRAL STRATEGY
1.1 Overview
Chemical agents that enhance the frequency of mutations in viral genomes constitute a promising new class of antiviral therapeutics. By taking advantage of relatively promiscuous viral RNA-dependent RNA polymerases
(RdRPs), mimics of nucleosides with ambiguous base-pairing capabilities can become incorporated into viral genomes. Replication of viral genomic material containing such compounds can increase viral mutation frequencies to intolerable levels. By surpassing an error threshold, termed “error catastrophe,” viral genomic “meltdown” ensues, yielding a non-viable virus population and an antiviral effect. Recently, it has been demonstrated that ribavirin (1-β-D-
ribofuranosyl-1,2,4-triazole-3-carboxamide, 25), a synthetic ribonucleoside
analogue, is a lethal mutagen to multiple RNA viruses, including poliovirus and
hepatitis C virus. Using this compound as a biochemical tool, it was revealed
that enhancement of the mutation frequency of poliovirus 4-fold results in lethal
mutagenesis, rendering the infectivity of the population to 5% of untreated
populations. Therefore, chemical agents that modestly enhance mutagenesis of
viral genomes have the potential to function as antiviral lethal mutagens.
Discussed herein is a brief overview of RNA virus biology, pioneering work in the 2 field of lethal mutagenesis, and the discovery that ribavirin is an RNA virus mutagen.
1.2 Life Cycle of an RNA virus: Poliovirus
Replication of RNA viruses is a host-dependent process. The reproductive cycle of poliovirus (PV), a (+)-stranded RNA virus, begins by first binding to the poliovirus receptor on a host cell. Intracellular internalization of the virus through a pore in the cell membrane (Figure 1.1 (a)) precedes the process of viral uncoating, of which the cellular location and mechanism are poorly understood. The (+)-stranded RNA genome of PV is immediately available for protein synthesis (Figure 1.1 (b)), which utilizes the host ribosome to synthesize a long polyprotein. Individual viral proteins necessary for the synthesis, structural organization, and packaging of the viral genome are produced by cleavage of this polyprotein (Figure 1.1 (c)). Replication of the (+)-strand viral genome by the
RNA-dependent RNA polymerase (RdRP) yields progeny (-)-stranded viral genomes (Figure 1.1 (d)). These newly synthesized viral genomes serve as templates for the synthesis of additional (+)-stranded viral RNA, which is also mediated by the viral RdRP (Figure 1.1 (f)). This duplication of viral genetic material occurs in the cytoplasm on surfaces of cell-specific membrane vesicles obtained from the endoplasmic reticulum. Viral structural proteins (Figure 1.1
(g)) associate with the newly formed RNA and assemble into new virons
(Figure 1.1 (h)), that are ultimately released by cell lysis (Figure 1.1 (i)).1 3
Figure 1.1. Reproductive cycle of poliovirus. Poliovirus RNA-dependent RNA polymerase (RdRP) from Thompson and Peersen (pdb 1R46).2
1.3 Known Diseases Resulting from RNA Virus Infections
Infection by RNA viruses has been implicated in the etiology of many
human diseases (Table 1.1). Included among these ailments are the well-known
common cold, hepatitis A and C, AIDS, and diseases caused by the widely publicized SARS and Ebola viruses. Unfortunately, pandemic human disease, 4 such as polioa, is also attributed to RNA virus infections. In many cases effective
vaccines have been developed to prevent widespread outbreaks of such
diseases, although global vaccination has proved difficult in many
underdeveloped nations, preventing complete viral eradication. The continuous
burden of these persistent viruses, coupled with the emergence of new,
potentially lethal RNA viruses such as SARS and Ebola, has increased the need
for therapeutics against RNA viruses.
Table 1.1. Examples of clinical conditions resulting from RNA virus infection. Clinical Condition Virus Family Virus genus Common cold Picornaviridae rhinovirus Polio Picornaviridae enterovirus Myocarditis Picornaviridae enterovirus Hepatitis A Picornaviridae hepatovirus Foot-and-Mouth Disease Picornaviridae aphthovirus SARS Coronaviridae coronavirus Ebola Filoviridae filovirus Hepatitis C Flaviviridae hepacivirus Yellow Fever Flaviviridae flavivirus Respiratory Syncytial Virus Paramyxoviridae pneumovirus Human T-cell leukemia Retroviridae* HTLV-BLV group HIV-1 and HIV-2 Retroviridae* lentivirus *Retroviruses (utilize DNA in the replication cycle)
1.4 Clinically Approved Antiviral Therapeutics and Their Molecular Targets
As of early 2004, 37 drugs have been licensed for the clinical treatment of viral infections.4 Included among these compounds are therapeutics for RNA
aAs of May 11, 2005 155 global cases of poliovirus still exist across nine nations.3 5 viruses, DNA viruses, and RNA-DNA (retroviral) virus infections. Impressively, most of these compounds did not exist a decade ago as only five antiviral drugs were approved for clinical use in the early 1990's.5 The major impetus for this accelerated drug discovery effort was the growing human immunodeficiency virus (HIV) plague. Approximately 19 compounds currently exist for the treatment of HIV infection, with inhibitiors of HIV reverse transcriptase
(Figure 1.2) and viral protease (Figure 1.3) the most common classes of therapeutics.4 In most cases, the molecular mechanisms of antiviral activity are similar across compounds in the same class. The nucleoside/nucleotide reverse transcriptase (RT) inhibitors (1-8) are phosphorylated in vivo to the 5'- triphosphate and bind the active site of HIV RT, yielding a competitive inhibitory effect (Table 1.2).6 However, the incorporation of these phosphorylated metabolites into viral DNA (by HIV RT) inhibits further DNA elongation through chain termination (due to the lack of a 3'-alcohol), which is the predominant mechanism for the antiviral activity of these compounds.6 Additionally, abacavir
(7) must be deaminated in vivo to the corresponding guanosine analogue, whereas tenofovir disoproxil (8), a nucleotide prodrug, must be converted to tenofovir, then phosphorylated to the diphosphate and incorporated into viral
DNA. The non-nucleoside reverse transcriptase inhibitors (9-11) inhibit the action of HIV-1 reverse transcriptase by targeting an allosteric non-substrate binding site on the enzyme. The fusion inhibitor peptide 12 blocks virus-cell fusion by binding with viral glycoprotein gp41 through a coil-coil interaction. The 6 final series of compounds, protease inhibitors 13-19, are all transition-state inhibitors of HIV protease.4
Figure 1.2. Clinically-employed therapeutics for HIV infection.
7
Table 1.2. Inhibition of HIV-1 reverse transcriptase by the 5'-triphosphates of clinically-employed nucleoside reverse transcriptase inhibitors.
Nucleoside RT Inhibitor Ki (µM) Reference Zidovudine (AZT, 1) 0.0084 6 Zalcitabine (ddC, 3) 0.2 7 Stavudine (d4T, 4) 0.0083 8 Emtricitabine ((-)-FTC, 6) 0.17 9
Figure 1.3. HIV fusion and protease inhibitors. 8 The development of antiviral therapeutics for the treatment of hepatitis B
virus (HBV) infection is another area of active pharmaceutical research.
Approximately 1.25 million Americans are chronically infected with HBV, with 20
– 30% infected from childhood.10 As of early 2004, two drugs have received approval for the treatment of HBV infection. Lamivudine (5), also referred to as
3TC, Epivir®, and Zeffix®, is an approved treatment of HIV-1, HIV-2, and hepatitis
B virus (HBV) infections (Figure 1.4).4,11 The molecular mechanism of this
compound’s action against HBV involves phosphorylation in vivo to the
12 triphosphate, binding to HBV DNA polymerase (Ki = 0.2 µM), and chain termination following incorporation into the viral genome.4,11 Adefovir dipivoxil
® (20, Hepsera ) also functions as an HBV RT chain terminator (Ki = 0.1 µM for
HBV DNA polymerase)13 following in vivo unmasking to adefovir and
phosphorylation to the diphosphate, the biologically-active form of the drug
(Figure 1.4). In addition to HBV, antiviral nucleoside analogues have been
utilized for the treatment of other DNA viruses, including herpes simplex virus
(HSV), varicella-zoster virus (VZV) and cytomegalovirus (CMV).4,11
Figure 1.4. Antiviral drugs for the treatment of hepatitis B virus infection. 9 Aside from the HIV drugs and those developed for the treatment of DNA
virus infections, only a handful of chemotherapeutics exist for the treatment of
RNA viruses. Fiveb antiviral drugs are clinically approved for the treatment of
specific RNA virus-induced diseases (Figure 1.5).11 A brief summary of their
molecular mechanisms of action follows. Two structurally similar adamantane-
derived compounds, amantadine (21, Symmetrel®, Mantadix®, Amantan®) and rimantadine (22, Flumadine®) are utilized as treatments for influenza A virus
infections. These drugs block the M2 ion channel, which prevents the passage
of H+ ions into the cell, necessary for the acidic viral uncoating process.
Zanamivir (23, Relenza®) and oseltamivir (24, Tamiflu®) are structurally similar
neuraminic acid-derived compounds which inhibit influenza virus neuraminidase.
The inhibition of this enzyme prohibits progeny virus particles from being
released by infected cells.4 The final approved therapeutic for RNA virus
infections, ribavirin (25, Virazole®, Virazid®, Viramid®), is a broad-spectrum
antiviral agent that exhibits activity against a variety of RNA and DNA viruses.14,15
Ribavirin is clinically employed as a co-therapy with interferon-α for the treatment for hepatitis C virus infection,16-19 and as a monotherapy for the treatment of
lassa fever20-22 and respiratory syncytial virus.23,24 Ribavirin has also been
utilized as an experimental therapeutic for SARS-associated coronavirus
infections.25 Although ribavirin is clinically effective for the treatment of these
b From a recent 2004 survey11 10 RNA virus infections, the molecular mechanism of action of ribavirin is widely debated and further discussed in section 1.5.4.2.
Figure 1.5. Clinically-employed therapeutics effective against RNA virus infections.
The limited number of antiviral therapeutics available for treatment of existing RNA virus infections, coupled with the emergence of new pathogenic viruses, has driven many drug discovery efforts. The inhibition of viral proteins necessary for reproduction remains a classical approach towards the development of antiviral drugs. However, the relatively high mutation 11 frequencies of viruses often leads to the emergence of antiviral drug resistance, a consistent problem associated with many of these compounds. Improved antiviral drug discovery requires effective chemotherapeutics that are less susceptible to the development of viral resistance. One new approach designed to accomplish this goal is focused on the discovery of compounds that promote viral (lethal) mutagenesis.
1.5 Lethal Mutagenesis as an Antiviral Strategy
1.5.1 Quasispecies and the Error Threshold
The mutation frequencies of replicating RNA viruses tend to be extremely high.26 The first quantitative evidence for this phenomenon was reported with the
single-stranded RNA phage Qβ.27 Experiments with this system revealed the mutation frequency to be 10-3 to 10-4 at a particular site in the genome. This level
of mutagenesis was a stark contrast to the known levels of chromosomal DNA mutagenesis that range from 10-8 to 10-11 per incorporated nucleotide.28-30 This result was subsequently confirmed with other RNA viruses, including poliovirus
(10-3 to 10-4 mutation frequency at a defined genomic site),31 yielding an overall rate of approximately 1.5 mutations per genome per replication cycle (genome
size of poliovirus is ca. 7400 nucleotides) for RNA viruses.32
A lack of proofreading mechanisms associated with viral RNA
polymerases is thought to contribute to the high mutation frequencies that occur 12 during viral genomic replication.33 As a result, newly replicated viral genomes
exist as a population of closely related genetic variants, termed a quasispecies.
This diversity allows the virus population to rapidly adapt to changing
environmental conditions, host immune responses, and antiviral
therapeutics.30,34-38 For example, a certain fraction of a viral quasispecies will be
less susceptible to a specific antiviral chemotherapeutic than other members of
the same quasispecies. If this variant can survive the treatment and reproduce,
a new population of drug-resistant variants will be established propagating the existence of the virus.38 Conversely, a high level of mutagenesis can be
dangerous for the survival of the quasispecies.
RNA virus quasispecies possess an intrinsic limit to their genomic
variability. Hence, the accrual of genomic mutations, at some point, will surpass
the tolerated threshold for genomic variability and result in viral inviability. This
accumulation of lethal genomic mutations is termed “error catastrophe”.36,39-41 It has been postulated that RNA viruses exist on the edge of error catastrophe, allowing them maximal genetic diversity and yielding maximal adaptability.40
Chemical mutagenesis of poliovirus and vesicular stomatitis virus supported this hypothesis; as mutation frequencies increased by only 1.1 to 2.8-fold by treatment with 5-fluorouracil abolished > 99% of the viable virus at the highest concentration tested.30,42 Direct biochemical proof was recently reported by
Crotty et al.43 using ribavirin as a chemical tool. In brief, “wild-type” (wt)
poliovirus contains approximately 1.5 mutations per viral genome. Enhancement of this mutation rate with ribavirin (an RNA virus mutagen, discussed in 13 mechanistic detail in section 1.5.4.4) to approximately 2 mutations per genome resulted in a 50% decrease in infectious poliovirus genomes. Further promotion of the basal mutation frequency to 4-fold over wt lethally mutated 95% of poliovirus genomes.43 Therefore, RNA virus genomes exist near the edge of
error catastrophe, as only modest increases in basal mutation frequencies confer
antiviral effects. Hence, this element of RNA virus biology may yield a promising avenue for the development of novel antiviral therapeutics.
1.5.2 Lethal Mutagenesis of HIV
The term “lethal mutagenesis” was coined by Loeb and coworkers in a
1999 report on the chemically-induced mutagenesis of HIV-1.44 Built on previous
studies demonstrating the existence of viral quasispecies and tolerated error
thresholds, the authors hypothesized that enhancing the mutation frequency of
HIV-1 slightly may exceed the tolerated error threshold and confer a loss in
replication capacity and infectivity. Chemical mutagenesis studies previously
conducted with vesicular stomatitis virus and poliovirus supported this
hypothesis, noting that small increases in single site mutagenesis yielded
significant decreases in virus viability.42 To test their theory, Loeb and coworkers
screened a collection of nucleoside analogues for cellular cytotoxicity and
mutagenic capacity (Figure 1.6). From this initial screen, 5-hydroxy-2'-
deoxycytidine (26) fulfilled the requirements for low cytotoxicity, diminished HIV
replication, and the promotion of viral mutagenesis. Further biochemical 14 evaluation of 26, as the 5'-triphosphate, found that the nucleotide was a substrate for HIV reverse transcriptase (RT), efficiently substituting for dCTP during HIV replication. Hence, the proposed mechanism of therapeutic intervention of 26 starts with intracellular phosphorylation to the triphosphate, a substrate for HIV
RT. Replication of HIV in the presence of the triphosphate of 26 yields mutations in proviral DNA, resulting in the loss of viral genetic information and conferring an antiviral effect. Sequencing analysis of the proviral DNA found approximately 18 drug-induced mutations per genome for HIV serially passaged in the presence of
26 and isolated immediately prior to lethality. The majority of these mutations were noted by G → A substitutions, owing to the altered base-pairing capability of the 5-hydroxycytosine pseudobase.44
Figure 1.6. Compounds tested by Loeb for HIV mutagenesis.44
In addition to the mutagenic deoxyribonucleosides utilized in their initial
publication,44 Loeb and coworkers have proposed the development of mutagenic 15 ribonucleosides as antiviral therapeutics for HIV.45 Because retroviral RNA synthesis is mediated by host cell RNA polymerase II, reproduction of the viral
RNA genome in the presence of mutagenic ribonucleosides (phosphorylated to their nucleotide forms) was proposed to result in the mutagenesis of the newly formed viral RNA, conferring an antiviral effect in the latter portion of the viral reproductive cycle. An additional benefit to utilizing ribonucleoside analogues is safety. Misincorporation of a mutagenic ribonucleoside into host mRNA is relatively harmless, whereas misincorporation of deoxyribonucleosides into host cell DNA could result in serious genomic instability.45 In light of these initial,
albeit recent, studies only a handful of publications have described new
mutagenic nucleosides against HIV.46,47
1.5.3 Lethal Mutagenesis of Other RNA Viruses
Small molecules that promote lethal mutagenesis have been studied with
other RNA viruses (Table 1.3). Among these viruses is the pathogenic foot-and-
mouth disease virus (FMDV), which is effectively mutagenized by treatment with
5-fluorouracil (31) or 5-azacytidine (32, Figure 1.7).48 Viral extinction of high- fitness FMDV has been achieved by co-admininstering chemical mutagens and antiviral inhibitors.49-51 5-Fluorouracil also effectively mutagenizes lymphocytic
choriomeningitis virus (LCMV), promoting error catastrophe at a concentration of
100 µg/mL. This concentration confers a 3.6 to 10-fold increase in the mutation frequency of the three viral genes monitored.52-54 However, the most prolific 16 chemical mutagen for the promotion of lethal mutagenesis is ribavirin (25). In
2000, this broad-spectrum ribonucleoside analogue was demonstrated to be a poliovirus (PV) mutagen.55 Enhancing the relative mutation frequency of PV a
modest 4-fold by treatment with 400 µM ribavirin (25) lethally mutated 95% of the viral genomes.43 Subsequent studies have demonstrated the mutagenic capacity of ribavirin against other RNA viruses, including Hantaan virus,56,57 GB Virus B (a
model of hepatitis C virus),58 and hepatitis C virus (in a subgenomic replicon
model).59 In light of the recent discovery of the mutagenic capacity of ribavirin
(discussed in detail in section 1.5.4.4),55 a classic antiviral drug over 32 years old,14 additional examples of lethal mutagenesis promoted by 25 against other
RNA viruses will likely be demonstrated in the future.
Table 1.3. Lethal mutagenesis of other RNA viruses. RNA Virus Chemical Mutagen References Foot-and-Mouth Disease Virus 5-fluorouracil (31) 48 Lymphocytic Choriomeningitis 5-fluorouracil (31) 52-54 GB virus B (GBV-B) ribavirin (25) 58 Hantaan virus (HTNV) ribavirin (25) 56,57 Hepatitis C Virus (HCV) ribavirin (25) 59 Poliovirus (PV) ribavirin (25) 55
Figure 1.7. Mutagenic ribonucleosides. 17 1.5.4 Discovery of the Mutagenic Capacity of Ribavirin
1.5.4.1 Discovery of Ribavirin and Initial Mechanism-of-Action Assignment
Ribavirin (25) was developed at ICN Pharmaceuticals in 1972 as part of a program aimed at the discovery of broad-spectrum antiviral agents. Researchers at ICN chemically synthesized a family of structurally analogous 1,2,4-triazole containing ribonucleosides (Figure 1.8) and exhaustively evaluated the antiviral potential of one particular member, ribavirin (25).60 Ribonucleoside 25 (ICN trade name Virazole®) was found to exhibit broad-spectrum antiviral activity against a
variety of RNA and DNA viruses, possessing potent antiviral activity both in vitro and in vivo.14 Subsequent biochemical analyses suggested that the primary
mechanism of action was inhibition of inosine monophosphate dehydrogenase
(IMPDH), a key enzyme in the de novo synthesis of GTP in cells.61 This enzyme
was thought to be the target because intracellular reduction of GTP pools
impedes virus replication, which requires free GTP for genomic reproduction.
18
Figure 1.8. 1,2,4-triazole ribonucleosides synthesized by ICN pharmaceuticals.
1.5.4.2 Intracellular Metabolism of Ribavirin and Alternative Theories on the Molecular Mechanism of Action
Many biochemical studies on the metabolism and molecular mechanism of action of ribavirin (25) have appeared since the initial drug discovery reports.
From a metabolism standpoint, ribavirin can be regarded as a purine mimic.
Following clinical administration of 25 as the nucleoside, the drug is phosphorylated in vivo by adenosine kinase yielding the monophosphate (RMP,
152).62 Subsequent stepwise phosphorylations by nucleoside monophosphate and diphosphate kinases transforms RMP to the triphosphate (RTP, 41),63,64 which is the major cellular metabolite.30,62,64 RTP accumulates within hours to high micromolar concentrations in vivo (> 100 µM) at clinically-relevant dosing,
rivaling the basal concentration levels of the purine nucleotide pools.64-68
However, this short-lived metabolite is rapidly degraded in vivo, possessing a 19 half-life (excluding erythrocytes) of less than two hours in cell culture studies.62,64,65,69
Ribavirin possesses broad-spectrum antiviral activity against a large
number of RNA and DNA viruses.14,15 An assortment of theories describing the
molecular mechanism of action have been proposed.15,30,38,39,70 Among these
are inhibition of the capping of viral (Sindbis) RNA;71 inhibition of viral
polymerases, such as influenza,72 vesicular stomatitis virus73 and human
immunodeficiency virus (HIV);74 modulation of host immune responses,75,76 and
the widely-accepted mechanism of inhibition of IMPDH.61,77 An important
consideration is that many of these theories are specific to particular viruses and the system in which they are investigated (cell line or animal model). The ability of ribavirin to influence multiple viral or cellular pathways within the same system must also be considered, as synergistic antiviral effects may result from bimodal mechanisms of action.
In light of these considerations, a variety of concerns can be raised regarding the generality of the postulated mechanisms of action for ribavirin. For example, inhibition of the viral capping process appears specific to viruses such as Sindbis,71 as other RNA viruses (such as poliovirus and hepatitis C virus)
utilize a cap-independent mechanism to initiate translation.30 Inhibition of viral
polymerases by ribavirin (and it's phosphorylated metabolites)72,73,77 cannot explain the antiviral activity against poliovirus and hepatitis C virus, as ribavirin triphosphate has been shown to function as a substrate for these viral polymerases.55,78,79 The promotion of host immune responses75,76 cannot fully 20 explain the molecular mechanism of action for ribavirin since antiviral activity has been demonstrated in cell-based systems that lack immune responses.30 The most widely-accepted theory explaining the antiviral activity of ribavirin is the inhibition of IMPDH, which starves the replicating virus of the GTP it needs to reproduce.61,77 However, the observation that some IMPDH inhibitors lack antiviral activity,55,58 coupled with animal studies showing insignificant reductions
(2-fold) in IMPDH levels following ribavirin treatment weakens this argument.c
Additional evidence against this theory was obtained by studying deviations in intracellular GTP pools and the levels of virus ribonucleoprotein (RNP) synthesis with increasing concentrations of ribavirin. At 25 µM ribavirin the cellular GTP pools in L5178Y lymphoma cells are maximally reduced 35 – 45%, and the synthesis of the viral RNP is significantly inhibited (approximately 40 – 50%).
However, increasing the concentration of ribavirin up to 100 µM does not alter the intracellular GTP concentration, although ca. 95% of RNP synthesis is abolished.80 This experiment suggests a bimodal mechanism of action for
ribavirin in this application, with inhibition of IMPDH providing only part of the
antiviral activity. Further evidence against this theory was revealed using
poliovirus (PV) as a model RNA virus system. Cameron and coworkers55 demonstrated that administration of ribavirin at concentrations yielding antiviral effects had insignificant consequences on viral replication and translation. This result was paralleled in a subsequent animal model study of hepatitis C, which
c Personal communication, Dr. Craig E. Cameron (The Pennsylvania State University) 21 found that treatment with ribavirin yielded potent antiviral effects, whereas administration of mycophenolic acid, a known IMPDH inhibitor, yielded no antiviral effect.58 In summary, although ribavirin may influence a discrete
biochemical pathway in a specific virus system, the mechanism of antiviral
activity may not translate to other virus systems. Virus-specific biochemical
analyses of this drug, or any antiviral therapeutic, should also consider novel
mechanisms of viral inhibition.
1.5.4.3 The Development by the Cameron Laboratory of an In Vitro Assay to Directly Measure Nucleotide Incorporation by a Viral Polymerase
The in vivo accumulation of ribavirin triphosphate 41 to levels comparable
with native nucleotide pools raised the possibility that nucleotide 41 could
function as a substrate for a viral polymerase.55 Previous studies had failed to
detect [3H]-ribavirin incorporation into cellular RNA, suggesting that the drug
does not interfere with host cellular polymerases.81 However, converse
experiments with the more promiscuous viral polymerases were not possible due
to the lack of an effective assay. Recently, Cameron and coworkers82 described
an efficient in vitro assay utilizing poliovirus RNA-dependent RNA polymerase
(3Dpol)83 and a symmetrical primer/template which permits detailed kinetic
analyses of nucleotide incorporations by a viral polymerase (Figure 1.9). Utilizing
this new technology, the complete kinetic mechanism of nucleotide incorporation
by 3Dpol has recently been solved,84,85 resulting in the first detailed structural
model of polymerase fidelity in nucleotide incorporation.86,87 In addition to 22 poliovirus, other viral polymerases including hepatitis C virus78 and coxsackievirus polymerasesd have been studied in analogous primer-extension assays. Described below is a brief overview of the aforementioned primer-
extension assay, which will be referenced throughout this thesis.
Figure 1.9. Graphical depiction of primer-extension assay developed by Cameron and coworker.82 Structure of PV 3Dpol from Hanson et al. (pdb 1RA6).2
d Unpublished results (J.D. Graci and C.E. Cameron) 23 The kinetics and thermodynamics of nucleotide incorporation by a viral polymerase are determined by measuring polymerase-mediated elongation of a symmetrical RNA primer-template termed sym/sub. In this procedure the functional viral polymerase, such as poliovirus 3Dpol,83 is precomplexed with a
32P-labeled primer-template in the presence of necessary cofactors, such as
Mg2+ (Figure 1.9 (a & b)). Addition of an appropriate nucleotide triggers the
reaction, which can extend the 10-mer primer-template in either direction
(Figure 1.9 (c)). At various time points the reaction is quenched by addition of
EDTA either manually, or by utilization of a rapid chemical quench-flow device.
The reaction products are resolved by denaturing polyacrylamide gel electrophoresis (PAGE), and visualized on a phosphorimager (Figure 1.9 d)).
Quantitative analysis of the band intensities are performed utilizing imaging
software, and the data is fit to statistical models yielding the kinetics and
thermodynamics of the reaction (Figure 1.9 (e)).82
1.5.4.4 Ribavirin is an RNA Virus Mutagen
The ability of ribavirin triphosphate 41 to function as a substrate for the
poliovirus RNA-dependent RNA polymerase was evaluated by the Cameron
laboratory utilizing the previously described primer-extension assay.82 RTP (41)
was found to incorporate opposite both pyrimidines in the sym/sub template at a
rate similar to natural nucleotide misincorporation (Figure 1.10). However, once
present in the template the pseudobase of ribavirin efficiently templated the 24 incorporation of both CTP and UTP at rates rivaling that of natural base pairs.55
The ability of ribavirin to function as a substrate for 3Dpol, coupled with it's
degenerate templating specificity once incorporated in the viral genome, provides
strong evidence that ribavirin can function as a lethal mutagen.
Figure 1.10. Kinetics of incorporation of RTP into sym/sub (adapted from 55).
Additional evidence supporting lethal mutagenesis as the primary
mechanism of action was provided by a guanidine resistance assay. In short,
wild-type poliovirus will not multiply in media supplemented with 2 mM guanidine
due to inhibition of the 2CATPase protein.88 However, single amino acid mutations in this protein have been identified that result in guanidine resistant phenotypes
(guar).89,90 A single point mutation in the coding region for 2CATPase protein
(C4605U) yields a guanidine resistant phenotype, which normally exists at low
frequences in the poliovirus quasispecies.55 Quantifying this variant following the growth of poliovirus (in media containing 2 mM guanidine) in the presence and absence of antiviral drugs provides a rapid biological screen for mutagenesis. In 25 support of the lethal mutagenesis mechanism of ribavirin, poliovirus grown in 2 mM guanidine supplemented with ribavirin yielded a substantial increase in guar plaques, compared with control wells lacking 25.55
Sequencing of virus capsid cDNA of poliovirus grown in the presence of 1
mM ribavirin revealed a 600% increase in G → A and C → U transition
mutations, further supporting the mutagenic capacity of ribavirin (25). The C →
U transition mutations can be envisioned as arising from the misincorporation of
both pyrimidines opposite templated ribavirin (which equivalently mimics both
purines).55 Control experiments conducted by Cameron and coworkers55 confirmed lethal mutagenesis as the primary mechanism of action against PV by demonstrating that known IMPDH inhibitors lacked antiviral activity, and that ribavirin treatment did not significantly effect either virus production or translation.
Recently, a single amino acid change (G64S) in the RNA-dependent RNA polymerase of poliovirus has been identified that confers resistance to ribavirin,91 providing additional evidence that 25 functions by lethal mutagenesis.
The current mechanistic model of the antiviral activity for ribavirin against poliovirus is shown in Figure 1.11. Clinical administration of ribavirin (25) precedes in vivo phosphorylation to the triphosphate (RTP, 41), which is the major cellular metabolite and active form of the drug. Cellular accumulation of 41 to high concentrations permits competition with native nucleotide pools, yielding a substrate for poliovirus RNA-dependent RNA polymerase (3Dpol). Viral replication in the presence of RTP incorporates the purine mimic into the viral genome. Replication of viral RNA containing templated ribavirin is highly 26 mutagenic, as the pseudobase of ribavirin efficiently templates both pyrimidines during replication. This degenerate templating specificity promotes multiple transition mutations during replication, forcing the virus into error catastrophe and loss of viability.15,30,36,38-40,70
Figure 1.11. A simple mechanistic model of the antiviral activity of ribavirin against poliovirus. Poliovirus RNA-dependent RNA polymerase from Thompson and Peersen (pdb 1RA6).2 27 1.6 Considerations for the Rational Design of Novel Lethal Mutagens
Shortly following the discovery of the mutagenic capacity of ribavirin, the
Peterson and Cameron laboratories at The Pennsylvania State University initiated a program aimed at the development of novel ribonucleoside-based lethal mutagens. Described throughout this thesis are rational designs, chemical syntheses, and antiviral evaluations of many structurally similar and dissimilar analogues from our drug discovery studies. In addition to the considerations placed upon the molecular design of these compounds, attention must be directed towards the biochemical conversion of these compounds to their active metabolites in vivo. Described below is a brief summary of the discrete biochemical events that are likely to be required for the promotion of viral lethal mutagenesis by synthetic nucleosides.
Antiviral nucleosides are typically delivered into infected host cells by a family of transport proteins. For many nucleoside-based antiviral chemotherapeutics, these proteins have been identified.92 Once inside the cell,
host cellular kinases presumably would need to phosphorylate nucleosides
designed as lethal mutagens stepwise to the triphosphate, yielding the active
metabolite (see Figure 1.11). This active metabolite should both promote lethal
mutagenesis and avoid cellular cytotoxicity to function as an effective antiviral
agent.35 In clinical applications, the nucleotide should eventually be further
metabolized and cleared from the body. The inability of a candidate drug to 28 accomplish any of these objectives is likely to circumvent its ability to function as a safe and effective lethal viral mutagen.
1.7 Advantages for the Use of Viral Lethal Mutagens
The continuous emergence of antiviral drug resistance remains an enduring problem in the maintenance of effective antiviral therapeutics.
Relentless viral replication in the presence of many antiviral drugs facilitates the selection and propagation of drug-resistant variants, eventually rendering the therapeutic ineffective. Hence, the development of new antiviral strategies less susceptible to drug resistance is in high demand. A solution to this problem could result from the development of novel compounds that promote lethal mutagenesis of viral genomes. The emergence of resistance to lethal mutagens from a cellular standpoint is unlikely compared to other antiviral therapeutics that inhibit specific cellular proteins and enzymes (thus starving the reproducing virus of a critical component). For example, the clinically-approved lethal mutagen ribavirin (25) exhibits low toxicity towards host cells and the active metabolite, ribavirin triphosphate (41), has not been detected as a substrate for cellular RNA polymerases.81 This absence of cellular toxicity and evasion of interference with cellular biochemical pathways places little selection pressure on the host cell to
develop resistance. The development of drug resistance on the level of the virus
should also be disfavored with the use of lethal mutagens. As previously
described, viral RNA-dependent RNA polymerases are highly promiscuous, a 29 trait that is necessary for the maintenance for the viral quasispecies (refer 1.5.1).
Antiviral lethal mutagens take advantage of this characteristic of RNA viruses to incorporate mutagenic drugs into the viral genome (refer 1.5.4.4). Due to the lack of proofreading mechanisms associated with RNA viruses,33 the (in)fidelity of
the RdRP directly controls the ability of the drug to reach its target. The evolution
of a more faithful viral RdRP could deter the incorporation of the lethal mutagen,
providing a mechanism of drug resistance. However, a significant cost to viral
fitness would result.
The maintenance of the viral quasispecies requires a level of infidelity in
the viral polymerase. The evolution of a more faithful RdRP would significantly
reduce the amount of variation in the virus population, lowering the overall fitness
of the virus. Hence, the resulting quasispecies would be more susceptible to
external factors such as host immune responses and other antiviral drugs. The
development of resistance via this mechanism would be under a strong negative
selection pressure.93 In fact, recent studies with the high fidelity G64S poliovirus
mutant91 has supported this hypothesis, revealing the higher fidelity virus exhibits
decreased pathogenesis and restricted tissue tropism compared with wild-type
virus populations. Chemical mutagenesis of the G64S quasispecies restores the
pathogenesis and tissue tropism of the virus to wild-type levels, providing direct
evidence for the importance of genomic variation in virus populations.e
e Unpublished data (M. Vignuzzi, J.K. Stone, J.J. Arnold, C.E. Cameron, R. Andino) 30 Additionally, comparative studiesf between the picornaviruses poliovirus (PV) and
coxsackievirus B3 (CVB3) revealed an interesting relationship between
polymerase fidelity and susceptibility to lethal mutagens. CVB3 is extremely
susceptible to antiviral treatment with ribavirin (25), yielding antiviral effects
comparable as those for PV at significantly lower drug concentrations. To
explain the increase susceptibility of CVB3 to lethal mutagenesis the kinetics of
incorporation of RTP were determined with the CVB3 RdRP utilizing the
previously described primer-extension assay (refer 1.5.4.3). Surprisingly, the
efficiency of incorporation of RTP was greater for PV than CVB3, suggesting that
a more faithful polymerase may render a genome more susceptible to lethal
mutagenesis.93 Considering the relatively low risk of developing drug resistance
and the effectiveness of the existing lethal mutagens, the development of novel
lethal mutagens is of significant interest.
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Chapter 2
CHEMICAL SYNTHESIS AND ANTIVIRAL EVALUATION OF 1-β-D-RIBOFURANOSYL-3-NITROPYRROLE
2.1 Overview
Ribonucleoside analogues with degenerate templating specificity have the
potential to function as antiviral lethal mutagens. Ribavirin (25) is an example of this class of antiviral agents. Incorporation of 25 into the genome of poliovirus is lethal to the virus, as the pseudobase of ribavirin templates the pyrimidines C and U during viral replication, resulting in A → G and G → A transition mutations.
As described in Chapter 1, the accrual of these mutations forces the virus into
“error catastrophe” and loss of viral viability. Based on this understanding of the mechanism of action of ribavirin we hypothesized that ribonucleoside analogues capable of templating all four RNA bases might constitute more effective antiviral agents. These compounds, “universal bases”, were proposed to promote a higher frequency of mutagenesis per round of viral replication. Previous studies on DNA-based “universal bases” found that 1-(2'-deoxy-β-D-ribofuranosyl)-3-
nitropyrrole (47) exhibited unbiased base-pairing properties in duplex DNA.
Therefore, to target RNA viruses, we synthesized and evaluated biochemically
the RNA analogue of this compound, 1-β-D-ribofuranosyl-3-nitropyrrole (48) as
an antiviral lethal mutagen. Surprisingly, nucleoside 48 exhibited no antiviral 45 activity against either poliovirus or coxsackievirus B3 grown in culture, although it functioned as a substrate for adenosine kinase. Elucidation of the kinetics of incorporation of the triphosphate 61 by the poliovirus RNA-dependent RNA polymerase (3Dpol) revealed a slow rate of incorporation into the primer-template.
This characteristic is sufficient to explain the absence of antiviral activity
associated with 48.
2.2 Existing Scaffolds for Universal Bases
Numerous deoxyribonucleosides capable of ambiguous base-pairing have
been reported.1 The majority of these molecular designs incorporate
hydrophobic, non-hydrogen bonding heterocycles that confer stability to DNA
duplexes by aromatic π-stacking interactions between adjacent bases
(Figure 2.1). The removal of restrictive hydrogen-bonding patterns is believed to
prevent base-pairing bias with the existing H-bond donors and acceptors on the
template DNA strand. Although various ring systems have been employed in
these molecular designs, mimicry of nucleobases with pyrrole derivatives has
been the most widely studied base modification approach.1
Figure 2.1. Popular hydrophobic heterocycles used to construct universal bases (adapted from 1). 46 2.3 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole (3-NP, 47): A Universal Deoxyribonucleoside Analogue
2.3.1 Rational Design and Chemical Synthesis of 3-NP by Bergstrom and Coworkers
The deoxyribonucleoside analogue 1-(2'-deoxy-β-D-ribofuranosyl)-3- nitropyrrole (3-NP, 47) was developed in 1994 by Bergstrom and coworkers2 for
use as a universal base in oligonucleotide probes and primers. The molecular
design was centered on the hypothesis that eliminating restrictive hydrogen-
bonding interactions would permit unbiased base-pairing with all four
deoxyribonucleosides, and the resulting decrease in stability could be recovered by optimizing aromatic π-stacking interactions between adjacent bases in the
DNA duplex. The pyrrole scaffold was selected to fulfill the requirements of a hydrophobic, π-aromatic system, small enough to avoid steric interactions with the larger purines in the DNA duplex. Appending a nitro group on the pyrrole
heterocycle polarizes the aromatic system, enhancing the capability for stacking
interactions with other aromatic bases.2
Bergstrom and coworkers synthesized the universal 3-nitropyrrole
deoxyribonucleoside 47 as shown in Figure 2.2.3 The 3-nitropyrrole heterocycle
44 was prepared in two steps from commercially-available 42.4 Sodium salt
glycosylation with the toluoyl-protected 2'-deoxyribose derivative 45 yielded the
coupled intermediate 46 in 88% yield. Deprotection with ammonia in methanol at
55 ºC yielded the target deoxyribonucleoside 47 in 83% yield.3 47
Figure 2.2. Synthesis of the universal deoxyribonucleoside 3-NP 47.3
2.3.2 Thermal Denaturation Studies on Duplex DNA Containing 47
The ability of 47 to exhibit ambiguous base pairing with all four DNA bases was examined by Bergstrom using melting denaturation studies on synthetic duplex DNA (Figure 2.3).3 Incorporation of 47 opposite all four
deoxyribonucleosides yielded nearly the same melting temperature (Tm) for the
DNA duplex (3 °C variation across all samples), demonstrating the ambiguous base-pairing capability of the 3-nitropyrrole pseudobase. Duplex destabilization resulting from incorporation of this universal base paralleled the destabilization observed with a native base-pair mismatch, as incorporation of 47 across from C yielded the same melting temperature as A base-pairing with C.3 Subsequent
denaturation studies with antiparallel oligopurine•oligopyrimidine duplexes
containing 47 opposite the native deoxyribonucleosides noted similar reductions 48 in Tm values, although some bias towards A•47 and 47•T base pairs was
observed.1,5
Figure 2.3. Melting denaturation studies with duplex DNA containing 47.3
2.3.3 Biochemical Applications of Universal Deoxyribonucleosides: 3-NP (47)
Applications of universal deoxyribonucleosides have traditionally involved their use as components of primers for DNA synthesis and probes for DNA hybridization studies.1 The universal 3-nitropyrrole deoxyribonucleoside, 1-(2'-
deoxy-β-D-ribofuranosyl)-3-nitropyrrole (3-NP, 47), has been used in
hybridization of duplex DNA5-12 and in the development of primers for PCR and
DNA sequencing.3,13-15 In addition, 47 has been incorporated into peptide nucleic 49 acids,16,17 utilized as a biochemical tool in MALDI-MS analysis,18 and employed
in oligonucleotide probes for detection of single nucleotide polymorphisms.19,20
Interestingly, the 5'-triphosphate of 47 functions as a universal nucleotide with the exonuclease-free Klenow fragment of DNA polymerase I, incorporating opposite all four DNA bases.1
2.4 Molecular Design of a Universal Ribonucleoside Analogue
Ribonucleoside analogues with ambiguous base-pairing capabilities have
the potential to function as antiviral agents that promote lethal mutagenesis.
Previous studies with ribavirin 25, a known lethal mutagen, revealed that a modest increase in the mutation frequency of poliovirus (PV) can confer significant antiviral effects.21 By mimicking the purine ribonucleosides, ribavirin
incorporates into PV genomes and promotes mutagenesis by templating the
pyrimdines C and U during multiple rounds of viral replication (see section
1.5.4.4). Based on this precedent it was hypothesized that a ribonucleoside analogue capable of templating all four ribonucleosides (“a universal base”) might comprise a more effective antiviral agent by enhancing the frequency of mutagenesis per round of viral replication. The mechanism of antiviral activity by a universal ribonucleoside was proposed to be similar to that observed with ribavirin: in vivo phosphorylation to the triphosphate, incorporation into the viral
RNA genome by the RNA-dependent RNA polymerase, and misincorporation of
other RNA bases opposite the templated universal base. 50 The molecular design of a proposed universal ribonucleoside analogue was based on the universal 3-nitropyrrole deoxyribonucleoside 47 (Figure 2.4).
As shown in Figure 2.4, conversion of the 2'-deoxyribose sugar of 47 to a ribose would provide the target 3-nitropyrrole ribonucleoside analogue 48 (3-NPN). A comparison of 48 with the known lethal mutagen ribavirin (25) revealed many structural similarities. The 1,2,4-triazole of ribavirin and the pyrrole of 48 are both
5-membered heterocycles appended with a functional group at the 3-position of the ring. The major structural deviation resides in the nature of the functionality on the pseudobase; ribavirin possesses a hydrogen-bonding carboxamide whereas the target ribonucleoside 48 is substituted with an isosteric nitro group.
Based on known biochemical data obtained with the DNA analogue 47, it was hypothesized that exclusion of hydrogen-bonding moieties was necessary to achieve unbiased base pairing with native RNA bases and the resulting loss in stability could be compensated by aromatic π-stacking interactions with adjacent bases in the RNA strand.
Figure 2.4. Structural comparison of nucleoside analogues. 51 2.5 Chemical Synthesis of 3-NPN (48)
2.5.1 Coupling Strategy with Chlorinated Ribose 50
The initial strategy for the synthesis of 48 focused on coupling the readily prepared 3-nitropyrrole base 44 (Figure 2.2)4 with the chlorinated ribose sugar
50. This approach was analogous to the method reported by Bergstrom and
coworkers for the synthesis of 47, which coupled the commercially-available
chlorinated sugar 45 and the sodium salt of 3-nitropyrrole (42) in 88% yield.3
Commercially-available 49 was reacted with HCl in 1,4-dioxane at 0 ºC to yield chloro-sugar 50 as a mixture of epimers (Figure 2.5).22 Under these conditions,
the expected reaction product based on literature precedent22 was the 1-α- chlorosugar 50. However, TLC analysis of the crude reaction mixture indicated the presence of both α and β anomers. In either case, neighboring-group participation from the 2'-O-benzoyl ester should deter coupling from the bottom
(α) face of the anomeric carbon, directing the 3-nitropyrrole nucleophile to attack
from the top (β) face. Unfortunately, reaction of crude 50 with the sodium salt of
44 in acetonitrile failed to yield the desired β-substituted ribonucleoside.
Subsequent spectroscopic analysis by X-ray crystallography (refer Appendix A) of the major reaction product revealed the formation of orthoamide 52.
Presumably, neighboring-group participation from the 2'-O-benzoyl ester expels the chlorine at the anomeric carbon, yielding the carbocation intermediate 51.
Trapping this intermediate by the 3-nitropyrrole anion (from the least-hindered 52 face) resulted in the formation of 52 in 47% yield (2 steps). A similar orthoamide was isolated from reaction of deprotonated 2-cyanopyrrole with the analogous bromide of 50.23 Based on the high yield of this unwanted product, an alternative protecting group approach was explored.
Figure 2.5. Initial coupling attempts directed at the synthesis of 3-NPN 48.
2.5.2 Coupling Strategy with Chlorinated Ribose 54
A previous report24 described the chemical synthesis of the analogous 3-
cyanopyrrole ribonucleoside 57 by SN2 coupling of 3-cyanopyrrole (55) with 1-α- 53 chlorosugar 54 in good overall yield (Figure 2.6).24 It was hypothesized that this
approach might improve the synthesis of 48. Coupling of 3-nitropyrrole (44) with
1-α-chlorosugar 54 was proposed to eliminate othoamide formation, the major
reaction product from previous synthesis attempts (refer 2.5.1). Additionally, well
precedented 1H NMR correlation data for ribonucleoside analogues employing
an isopropylidene to protect the 2'- and 3'-alcohols is available.25,26 Analysis of the difference in 1H chemical shifts (δ) between the isopropylidene methyl protons in DMSO-d6 provides a rapid method to assign the anomeric
stereochemistry of these systems. As a general rule-of-thumb, differences in
chemical shifts ranging from 0.18 – 0.23 ppm are indicative of the β-configuration
of the pseudobase whereas ∆δ 0.0 – 0.10 ppm differences indicate the
pseudobase in the α-configuration (Figure 2.7).25,27 Based on these favorable
attributes and the high degree of similarity between the desired 3-nitropyrrole
analogue 48 and the previously reported synthesis of 57, this approach24 was investigated.
Figure 2.6. Precedented chemical synthesis of an analogous 3-cyanopyrrole ribonucleoside 57.24 54
Figure 2.7. Proton NMR analysis (in DMSO-d6) of differences in chemical shifts of isopropylidene methyls from ribonucleosides with α and β anomeric stereochemistries (adapted from 25).
The synthesis of 48 commenced with the preparation of the 1-α- chlorosugar 54 (Figure 2.8). D-Ribose (58) was protected as the 2',3'-O- isopropylidene by reaction with acetone in H2SO4, then converted to the 5'-silyl ether by reaction with TBDMSCl under basic conditions, providing the known intermediate 59.28-33 Stereoselective chlorination of the anomeric alcohol 59 was 55 achieved by reaction with CCl4 and hexamethylphosphorous triamide (HMPT) at
low temperatures providing the known 1-α-chlorosugar 54.34-38 Coupling of the
sodium salt of 3-nitropyrrole (44) with 54 yielded the advanced intermediate 60 in
20% yield from intermediate 59. The anomeric stereochemistry of 60 was
assigned by the difference in chemical shift between the isopropylidene methyls
(δ 0.23 ppm), which was indicative of the β-configuration (Figure 2.7).25,27 Global deprotection was achieved by reaction with 50% trifluoroacetic acid in water24,28 to yield the 3-nitropyrrole ribonucleoside 48 in 97% yield. The chemical structure of 48 was verified by small molecule x-ray crystallography (Appendix B).
Figure 2.8. Chemical synthesis of 3-NPN (48). 56 2.6 Evaluation of Cellular Cytotoxicity of 3-NPN (48)g
The cellular cytotoxicity of 3-NPN 48 towards HeLa S3 cells grown in culture was examined at 100 µM and 1000 µM concentrations and compared with ribavirin (25) following a 6 day treatment (Table 2.1). 3-NPN 48 exhibited low levels of cellular cytotoxicity across all concentrations examined. At the highest concentration tested (1000 µM) 3-NPN treated cells showed only a 36% reduction in cell viability. Conversely, ribavirin treated cells were significantly cytostatic at all concentrations tested, with 1000 µM treatment exhibiting only a slight increase in cell growth over the six day period (the level of cell growth was
3% of untreated control cells).
Table 2.1. Cellular cytotoxicity of 3-NPN 48 towards HeLa S3 cells (6 day treatment). Untreated cells were assigned arbitrarily a cell viability of 100%. Nucleoside Nucleoside Concentration Cell Viability (%) None N/A 100 3-NPN (48) 100 µM 106 3-NPN (48) 1000 µM 64 Ribavirin (25) 100 µM 28 Ribavirin (25) 1000 µM 3
g Experimental data obtained by Jason D. Graci (Cameron Lab). For experimental details refer to Harki et al. 2002.39 57 2.7 Antiviral Evaluation of 3-NPNh
The antiviral activity of 3-NPN (48) was evaluated against poliovirus (PV)
and compared with ribavirin (25), a known antiviral lethal mutagen effective
against this virus (see section 1.5.4.4).21 In this experiment, HeLa S3 cells were
treated with various concentrations of 48 and 25 then infected with 2000 PFU
(plaque forming units) of poliovirus (Mahoney strain). The time to complete
cellular lysis was recorded, as well as the fold reduction in the titer of the virus.
Ribavirin treated cells (1000 µM) delayed the onset of cytopathic effects (CPE)
by 4 days compared with untreated cells, yielding a 1900-fold reduction in the
titer of the virus (Table 2.2). Conversely, 3-NPN treated cells failed to extend the
time to CPE at all concentrations tested, and no reduction in virus titer was
observed. The ability of 48 to exert therapeutic effects on coxsackievirus B3
(CVB3), a virus similar to PV that is generally considered to be more susceptiblei to mutagenesis, was also examined (data not shown); however, no antiviral effects were observed in this system as well.
h Experimental data obtained by Jason D. Graci (Cameron Lab). For experimental details refer to Harki et al. 2002.39 i Unpublished data, Jason D. Graci and Craig E. Cameron (The Pennsylvania State University) 58
Table 2.2. Antiviral activity of 3-NPN (48) on poliovirus-infected HeLa S3 cells. Nucleoside Time to 100% Fold Reduction Nucleoside Concentration CPE (days) in PV Titer None N/A 2 -- 3-NPN (48) 100 µM 2 -- 3-NPN (48) 1000 µM 2 -- Ribavirin (25) 100 µM 3 14 Ribavirin (25) 1000 µM 6 1900
2.8 Phosphorylation of 3-NPN by Adenosine Kinasej
The 3-NPN ribonucleoside analogue 48 presumably must be
phosphorylated in vivo to the active metabolite, the 5'-triphosphate, to confer
antiviral effects. Previous studies on structurally-similar ribavirin (25) revealed
that adenosine kinase phosphorylates the nucleoside to the 5'-monophosphate,
the first step in the synthesis of RTP (41).40 In general, the rate limiting step for conversion to the triphosphate is thought to be the production of the monophosphate. To determine if poor in vivo phosphorylation of 48 contributes to its lack of antiviral activity, the ability of 48 to be phosphorylated by adenosine kinase was directly measured. In this assay, conversion of α-32P-labeled ATP to
α-32P-ADP provides an indirect measure of monophosphorylation of nucleoside
analogues mediated by this purified enzyme (Figure 2.9). Quantitative analysis
of the level of phosphorylation of both ribavirin and 3-NPN revealed that both
j Experimental data obtained by Saikat K.B. Ghosh (Cameron Lab). For experimental details refer to Harki et al. 2002.39
59 nucleosides were converted to their monophosphates equivalently (Figure 2.10).
This data suggests that the active metabolite of 3-NPN, the triphosphate 3-
NPNTP (61), should be present in vivo.
Figure 2.9. Phosphorylation of 3-NPN (48) and ribavirin (25) by adenosine kinase.
Figure 2.10. Quantitative comparison of phosphorylation of adenosine (●), 3- NPN (▲), and ribavirin (■) by adenosine kinase.
60 2.9 Kinetics of Incorporation of 3-NPNTP (61) by Poliovirus RNA-dependent RNA polymerase (3Dpol)
2.9.1 Synthesis of 3-NPNTP (61)
To measure the kinetics and thermodynamics of nucleotide incorporation
by the poliovirus RNA-dependent RNA polymerase, the triphosphate analogue of
3-NPN (3-NPNTP, 61) was prepared (Figure 2.11). This nucleotide was
synthesized utilizing a well-precedented three-step, one-pot method for the
preparation of nucleoside triphosphates (discussed in detail in Chapter 5).41-47 In
brief, 3-NPN (48) was converted to the phosphodichloridate by reaction with
POCl3. This unstable intermediate was immediately reacted with
tributylammonium pyrophosphate, then quenched by addition of aqueous base.
Purification of the crude salt by reverse-phase HPLC, followed by conversion to
the sodium salt via ion-exchange chromatography yielded 3-NPNTP (61) in 47%
yield.
Figure 2.11. Chemical synthesis of 3-NPNTP (61). 61 2.9.2 Kinetics of Incorporation of 3-NPNTP (61) into Sym/Sub by Poliovirus RNA-Dependent RNA Polymerase (RdRP)k
The kinetics and thermodynamics of incorporation of 3-NPNTP (61) by the
poliovirus RNA-dependent RNA polymerase (3Dpol) were measured utilizing the
primer-extension assay previously described (section 1.5.4.3).48 The incorporation of 61 mediated by 3Dpol was evaluated across each of the native
RNA bases (Figure 2.12). Surprisingly, 3-NPNTP was incorporated only
opposite the pyrimidine U and the purine A, an unexpected result for a hypothesized “universal base”. Measurement of the kinetics of incorporation
opposite sym/sub-U and sym/sub-A revealed that 3-NPNTP was incorporated
much more slowly compared with a “correct” nucleotide and approximately 100-
fold more slowly than RTP in the same context (Table 2.3).21
k Experimental data obtained by Victoria S. Korneeva (Cameron Lab). For experimental details refer to Harki et al. 2002.39 62
Figure 2.12. Incorporation of monophosphates derived from 3-NPNTP (61) opposite complementary nucleotides in symmetrical substrates.
Table 2.3. Kinetics of incorporation of 3-NPNTP (61) into symmetrical substrates by poliovirus RdRPa. s/s-U s/s-A Km (µM) 19.2 ± 7.1 46.1 ± 15.5 -1 kcat (s ) 0.000120 ± 0.000001 0.00010 ± 0.00001 IC50 (µM) 76.3 ± 18.4 26.0 ± 3.0 as/s-U is the template for incorporation opposite U. s/s-A is the template for incorporation opposite A.
63 This diminished rate of incorporation into the viral RNA genome by the
RdRP most likely precludes 3-NPNTP from functioning as an antiviral agent. At therapeutically-useful concentrations of ribavirin, only approximately three molecules are incorporated per genome per replication cycle. However, due to the relatively small enhancement of mutagenesis necessary to confer antiviral effects, this level of incorporation is sufficient to promote lethal mutagenesis of the virus.21,49 In the case of 3-NPNTP, a 100-fold reduction in the rate of
nucleotide incorporation into the viral genome implies that approximately 0.03
molecules of 3-NPNTP will be incorporated into the viral genome per replication
cycle. This observation suggests that most viral genomes grown in the presence
of 61 will escape incorporation of the nucleotide into their genomes, eliminating
the possibility of lethal mutagenesis even if the pseudobase of 61 functioned as a
universal base.
2.10 Structural Comparison of 3-NPN (48) and Ribavirin (25)
The x-ray crystal structures of 3-NPN and ribavirin50 were compared to
examine differences between these compounds (Figure 2.13). Both 3-NPN and
ribavirin project their pseudobases in the anti-conformation, placing the
functionality at the 3-position of the heterocycles into the hydrogen-bonding face
of hypothetical complementary RNA bases. The sugar pucker of 3-NPN is C2'- endo, whereas the conformation of ribavirin is C3'-endo, characteristic of A-form
RNA. However, in solution both conformations should exist in equilibrium as only 64 a small energy barrier exists for sugar pucker interconversions (ca. 5 kcal/mole).51 To measure the similarities in overall structure, the structures of 25 and 48 were overlaid and root-mean-square similarities were calculated.l An overall deviation of 0.83 Å was calculated between the two x-ray structures, primarily reflecting the differences in sugar pucker between the nucleosides.
This analysis provides strong evidence that the differences in antiviral activities between ribavirin and 3-NPN are probably not a result of differences in the overall conformation of the nucleosides, but relate to other dissimilarities between the two structures, such as hydrogen-bonding capability.
Figure 2.13. Comparison of x-ray crystal structures of 3-NPN and ribavirin. Atom labels for panels A (3-NPN) and B (ribavirin): light gray for carbon, dark gray for nitrogen, black for oxygen. (C) Structural overlay for calculation of root-mean- square (rms) deviations of atom positions (0.83 Å): solid bonds and empty circles for 3-NPN and dashed bonds and shaded circles for ribavirin.
l Data provided by Dr. Richard Koerner (deceased, The Pennsylvania State University) 65 2.11 Implications for the Design of New Universal Ribonucleosides
The data presented here suggests that hydrophobic, non-hydrogen bonding ribonucleosides similar to 48 may exhibit slow rates of incorporation into the viral genomes by RNA-dependent RNA polymerases (such as PV 3Dpol). By failing to incorporate into the viral template at frequencies similar to natural misincorporations, nucleoside analogues such as 3-NPN 48 are unable to
promote mutagenesis to levels resulting in antiviral effects. The major structural
difference between 48 and ribavirin, an antiviral drug, is the lack of hydrogen-
bonding capability on the pseudobase of 48. Therefore, the ability of universal bases to merely hybridize with all four RNA bases may be insufficient to promote the efficient incorporation into the viral genome, requiring additional stabilizing
factors with the viral polymerase or the RNA template.
2.12 Experimental Section
General synthesis information. All reactions were performed under an argon atmosphere unless otherwise noted. Commercial grade reagents (Aldrich,
Acros) were used without further purification unless specifically noted.
Tetrahydrofuran and diethyl ether were distilled from sodium benzophenone ketyl
under nitrogen. Acetonitrile was distilled from calcium hydride under nitrogen.
Column chromatography employed ICN SiliTech silica gel (32-63 µm). HPLC purification was performed on either a Hewlett Packard 1100 series instrument equipped with a Zorbax 300SB-C18 semi-preparative column (9.4 x 250 mm, 5 66 µm; Agilent Technologies). The HPLC gradient for the purification of 61 (2 mL/min flow rate) was as follows: 2 to 15% acetonitrile (MeCN) in 0.1 M triethylammonium bicarbonate (TEAB, pH = 7.5) from 0 to 30 min; 15 to 40%
MeCN in 0.1 M TEAB from 30 to 45 min; and 40 to 70% MeCN and 0 to 20% methanol in 0.1 M TEAB from 45 to 50 min. Nuclear magnetic resonance (NMR) spectroscopy employed Bruker CDPX-300, DPX-300, AMX-360, DRX-400, and
AMX-2-500 MHz spectrometers. Internal solvent peaks were referenced in each
13 31 case. Chemical shifts for C NMR and P NMR analyses performed in D2O
52 were indirectly referenced to 10% acetone in D2O (CH3 set to 30.89 ppm) and
13 85% H3PO4 (0 ppm), respectively. C chemical shifts for nucleoside diphosphates and triphosphates denoted with a (*) fail to resolve into clean singlets due to apparent conformational restrictions. Mass spectral data was obtained from either The University of Texas at Austin Mass Spectrometry
Facility (ESI and CI) or The Pennsylvania State University Mass Spectrometry
Facility (ESI). Elemental analyses were performed by Midwest Microlab, LLC
(Indianapolis, IN). Melting points are uncorrected.
3',5'-Di-O-benzoyl-1',2'-O-(S)-[phenyl(3-nitropyrrole)methylidene]-α-D-
ribofuranose (52). Chlorosugar 50 was prepared as previously described.22 67 Briefly, protected ribose 49 (7.173 g, 14.22 mmoles) was dissolved in HCl in 1,4- dioxane (4 M, 23 mL, 92 mmoles) and stirred at 0 °C for 5 d. The crude reaction was poured into CH2Cl2 (60 mL) and washed with aq. NaHCO3 (5 %, 150 mL,
1x), then distilled water (dH2O, 150 mL, 1x). The organic layer was dried with anhydrous MgSO4, concentrated in vacuo, and used directly in the coupling step.
Separately, 3-nitropyrrole4 (44, 2.980 g, 26.58 mmoles) was dissolved in MeCN
(10 mL) and cooled to 0 °C. Base 44 was deprotonated by addition of NaH
(degreased with hexanes, 519.4 mg, 21.64 mmoles) in MeCN (3 mL), yielding a
bright orange solution with liberation of H2. This material was transferred via cannula to a solution of crude 50 in MeCN (15 mL) at 23 °C using additional
MeCN to transfer (3 mL). The reaction was stirred for 2 h at 23 °C, then poured
into EtOAc (200 mL). The organic layer was washed with dH2O (ca. 200 mL, 2x)
and set aside. The small amount of product remaining in the water layer was
extracted with EtOAc and added to the original organic layer. The combined
organic layers were dried with anhydrous MgSO4 and concentrated in vacuo.
Purification through a plug of silica gel (step gradient: 10% → 25% → 50%
EtOAc in hexanes) yielded semi-pure material, which was purified finally by
dissolving in a minimal amount of EtOAc then precipitated by the addition of excess hexanes (50:1 hexanes:EtOAc). The material from two precipitations of
the semi-pure material were pooled together yielding orthoamide 52 (3.744 g,
1 48% yield) as a white powder. mp 164-166 ºC. H NMR (299.9 MHz, CDCl3): δ
8.01 (m, 4H), 7.68 (dd, J = 1.9 Hz, J = 2.4 Hz, 1H), 7.62 (m, 1H), 7.55 (m, 1H), 68 7.49-7.36 (m, 9H), 6.76 (dd, J = 2.6, J = 3.3, 1H), 6.72 (dd, J = 1.8, J = 3.4, 1H),
6.25 (d, J = 4.1 Hz, 1H), 5.26 (m, 1H), 5.15 (m, 1H), 4.72 (m, 1H), 4.54-4.45 (m,
13 2H). C NMR (75.4 MHz, CDCl3): δ 166.0, 165.4, 137.7, 135.7, 133.9, 133.4,
130.6, 129.9, 129.7, 129.3, 128.8, 128.6, 128.4, 125.6, 119.3, 119.2, 114.8,
106.2, 105.5, 78.3, 76.9, 72.5, 62.2. IR (film): 1724, 1267 cm-1. HRMS (ESI-)
- calcd. for C30H24N2O9Cl [M+Cl] 591.1170, found 591.1182.
1-(5'-O-Tert-butyldimethylsilyl-2',3'-O-isopropylidene-β-D-ribofuranosyl)-3-
nitropyrrole (60). The stereoselective chlorination of anomeric alcohol 59 was
34-38 performed as previously described. Briefly, anhydrous CCl4 (0.75 mL, 7.77
mmole) was added to a solution of 59 (1.511 g, 4.96 mmole) in THF (16 mL).
The mixture was cooled to -72 °C and hexamethylphosphorous triamide (HMPT,
1.2 mL 6.60 mmole) was added slowly via syringe. The reaction was stirred at -
70 °C for 2 hours. Periodically, the mixture was briefly warmed to 25 °C to
solubilize the formed gel phase, then recooled to -70 °C as needed to enable
stirring. After 2 h the reaction was concentrated in vacuo (25 °C) and vented to
atmospheric pressure with dry argon. This chloro-sugar was triturated with
diethyl ether (2 x 10 mL), concentrated in vacuo (25 °C), and vented to 69 atmospheric pressure with dry argon. The resulting orange/brown solid was dissolved in acetonitrile (3 mL) and employed immediately in the subsequent coupling step. To a solution of 3-nitropyrrole4 44 (1.085 g, 9.68 mmoles) in
acetonitrile (15 mL) cooled to 2 °C was added NaH (95%, 175.6 mg, 7.32
mmole). To the resulting bright yellow solution was added the chloro-sugar 54 via
cannula, with additional acetonitrile (2 mL) used in the transfer. The reaction was gradually warmed to 23 °C and stirred for 40 h. The reaction mixture was then
poured into dH2O (60 mL) and extracted with CH2Cl2 (3 x 60 mL). The organic
layers were combined, diluted with CH2Cl2 (to 300 mL) and washed with
saturated aq. NH4Cl (300 mL, 1x) and saturated aq. NaCl (300 mL, 1x). The
organic layer was dried with anhydrous MgSO4, filtered through a pad of Celite,
and concentrated in vacuo. Column chromatography (10% Et2O in hexanes)
provided the β anomer 60 (393.9 mg, 20% yield) as a light yellow oil. 1H NMR
(300.1 MHz, CDCl3): δ 7.75 (m, 1H), 6.78 (dd, J = 2.4 Hz, J = 3.3 Hz, 1H), 6.70
(dd, J = 1.8 Hz, J = 3.3 Hz, 1H), 5.59 (d, J = 3.2 Hz, 1 H), 4.79 (dd, J = 2.2 Hz, J
= 6.0 Hz, 1H), 4.64 (dd, J = 3.2 Hz, J = 6.0 Hz, 1H), 4.37 (m, 1H), 3.87-3.73 (m,
2H), 1.55 (s, 3H), 1.32 (s, 3H), 0.85 (s, 9H), 0.05 (s, 3H), 0.04 (s, 3H). 13C NMR
(75.5 MHz, CDCl3): δ 137.4, 119.2, 118.8, 114.1, 105.9, 95.2, 86.2, 86.1, 81.0,
63.4, 27.2, 25.8, 25.2, 18.2, -5.6, -5.7. IR (film): 1299, 1126, 1086 cm-1. MS
+ + (ESI ) calcd. for C18H31N2O6Si [M+H] 399.2, found 399.1. Anal. calcd. for
C18H30N2O6Si: C 54.25; H 7.59; N 7.03. Found C 54.38; H 7.62; N 6.99.
70
1-(β-D-Ribofuranosyl)-3-nitropyrrole (48). To 1-(5-O-tert-butyldimethylsilyl-2,3-O- isopropylidene-β-D-ribofuranosyl)-3-nitropyrrole (60, 331.7 mg, 0.83 mmoles) was
24,28 added a solution of trifluoroacetic acid and dH2O (1:1, 10 mL). The reaction was stirred for 40 min at 23 °C, concentrated in vacuo, and triturated with MeOH
(10 mL, 3x). Column chromatography (5% MeOH in CH2Cl2) afforded 48 (196.3
1 mg, 97% yield) as a pale-yellow solid. mp 139–141 °C H NMR (MeOH-d4,
400.1 MHz): δ 8.01 (m, 1H), 7.04 (dd, J = 2.4 Hz, J = 3.3 Hz, 1H), 6.72 (dd, J =
1.8 Hz, J = 3.3 Hz, 1H), 5.54 (d, J = 4.9 Hz, 1H), 4.20 (m, 2H), 4.06 (m, 1H),
13 3.83-3.71 (m, 2H). C NMR (MeOH-d4, 75.4 MHz): δ 138.4, 121.6, 121.1, 106.2,
94.2, 87.1, 77.7, 72.0, 62.8. IR (KBr): 3346, 1480, 1386, 1294 cm-1. MS (CI+)
+ calcd. for C9H13N2O6 [M+H] 245.1, found 245. Anal. calcd. for C9H12N2O6: C
44.27; H 4.95; N 11.47. Found: C 44.11; H 4.97; N 11.19.
1-(β-D-Ribofuranosyl)-3-nitropyrrole-5’-triphosphate sodium salt (61). This
compound was prepared via modification of a previously reported procedure.41-47 71 Briefly, a solution containing 48 (14.4 mg, 0.059 mmoles), Proton-Sponge (1,8- bis(dimethylamino)naphthalene, 21.3 mg, 0.099 mmoles), and trimethyl phosphate (0.6 mL, 5.127 mmoles) was cooled to 2 °C and POCl3 (11 µL, 0.118
mmoles) was added dropwise. The resulting solution gradually turned lavender
in color and was stirred for 2 h. Tributylamine (0.06 mL, 0.252 mmoles) was
injected into this mixture, followed by a solution of tributylammonium
pyrophosphate (147.8 mg) in DMF (0.5 mL). The reaction was stirred for 2 min
then quenched by the addition of TEAB (6 mL, 0.2 M, pH = 8). The reaction was
warmed to 23 °C, diluted with dH2O (5 mL), and lyophilized. The crude reaction
products were purified by HPLC. Triphosphate 61 eluted as the
triethylammonium salt with a retention time of 18.8 min. Excess salts were
removed by loading this material onto a Waters Light C18 Sep-Pak syringe
column and eluting dropwise with dH2O (material off the column was fractionally
collected; the first few drops contained excess salts followed by elution of 61).
Residual 61 was eluted with acetonitrile. Fractions containing the triphosphate
(identified as UV-active material at 254 nm) were pooled and lyophilized. The
triphosphate was converted from the triethylammonium salt to the sodium salt by
passing through a DOWEX 50WX8-100 ion-exchange column (Na+ form, activated by equilibration with saturated aq. NaHCO3 followed by removal of salts
with dH2O) and fractions containing 61 were combined and lyophilized. The
resulting material was then resuspended in dH2O, filtered through an Acrodisc
syringe filter (0.2 µm), and lyophilized to afford 61 (15.7 mg, 47% yield) as a 72
1 yellow-brown powder. H NMR (D2O, 500.1 MHz): δ 8.09 (m, 1H), 7.17 (m, 1H),
6.89 (dd, J = 1.7 Hz, J = 3.4 Hz, 1H), 5.69 (d, J = 6.4 Hz, 1H), 4.56-4.47 (m, 2H),
13 4.37 (m, 1H), 4.29-4.20 (m, 2H). C NMR (D2O, 125.8 MHz): δ 139.8, 125.65*,
31 123.60*, 109.5, 95.0*, 87.4*, 78.2*, 73.8, 68.7*. P NMR (D2O, 145.8 MHz): δ
- -9.94 (br s), -10.61 (d, J = 15.0 Hz), -22.08 (br s). MS (ESI , MeCN, H2O, 1%
- - CH3CO2H) calcd. for C9H11N2Na3O15P3 [M-Na] 548.9, found 548.7. MS (ESI ,
- CH3OH, H2O, Et3N) calcd. for C9H14N2O15P3 [M-4Na+3H] 483.0, found 483.
2.13 References
1. "The applications of universal DNA base analogues." Loakes, D., Nucleic
Acids Res. 2001, 29, 2437-2447.
2. "A universal nucleoside for use at ambiguous sites in DNA primers."
Nichols, R.; Andrews, P. C.; Zhang, P.; Bergstrom, D. E., Nature 1994,
369, 492-493.
3. "Synthesis, structure, and deoxyribonucleic acid sequencing with a
universal nucleoside: 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole."
Bergstrom, D. E.; Zhang, P.; Toma, P. H.; Andrews, P. C.; Nichols, R., J.
Am. Chem. Soc. 1995, 117, 1201-1209.
4. "N-(triisopropylsilyl)pyrrole. A progenitor "par excellence" of 3-substituted
pyrroles." Bray, B. L.; Mathies, P. H.; Naef, R.; Solas, D. R.; Tidwell, T. T.;
Artis, D. R.; Muchowski, J. M., J. Org. Chem. 1990, 55, 6317-6328. 73 5. "Effect of the 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole residue on
the stability of DNA duplexes and triplexes." Amosova, O.; George, J.;
Fresco, J. R., Nucleic Acids Res. 1997, 25, 1930-1934.
6. "Effect of the universal base 3-nitropyrrole on the selectivity of neighboring
natural bases." Oliver, J. S.; Parker, K. A.; Suggs, J. W., Org. Lett. 2001,
3, 1977-1980.
7. "NMR structure of a DNA duplex containing nucleoside analog 1-(2'-
deoxy-beta-D-ribofuranosyl)-3-nitropyrrole and the structure of the
unmodified control." Klewer, D. A.; Hoskins, A.; Zhang, P. M.; Davisson, V.
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4522.
8. "Triple helix formation: binding avidity of acridine-conjugated AG motif third
strands containing natural, modified and surrogate bases opposed to
pyrimidine interruptions in a polypurine target." Orson, F. M.; Klysik, J.;
Bergstrom, D. E.; Ward, B.; Glass, G. A.; Hua, P.; Kinsey, B. M., Nucleic
Acids Res. 1999, 27, 810-816.
9. "Triple helices formed at oligopyrimidine center dot oligopurine sequences
with base pair inversions: effect of a triplex-specific ligand on stability and
selectivity." Kukreti, S.; Sun, J. S.; Loakes, D.; Brown, D. M.; Nguyen, C.
H.; Bisagni, E.; Garestier, T.; Helene, C., Nucleic Acids Res. 1998, 26,
2179-2183.
10. "Comparison of the base pairing properties of a series of nitroazole
nucleobase analogs in the oligodeoxyribonucleotide sequence 5'- 74 d(CGCXAATTYGCG)-3'." Bergstrom, D. E.; Zhang, P.; Johnson, W. T.,
Nucleic Acids Res. 1997, 25, 1935-1942.
11. "An acyclic 5-nitroindazole nucleoside analog as ambiguous nucleoside."
van Aerschot, A.; Rozenski, J.; Loakes, D.; Pillet, N.; Schepers, G.;
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M., Nucleos. Nucleot. 1995, 14, 1001-1003.
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1999, 18, 2685-2695.
15. "3-nitropyrrole and 5-nitroindole as universal bases in primers for DNA-
sequencing and PCR." Loakes, D.; Brown, D. M.; Linde, S.; Hill, F.,
Nucleic Acids Res. 1995, 23, 2361-2366.
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Acids Res. 1997, 25, 5072-5076. 75 19. "Enhanced discrimination of single nucleotide polymorphisms by artificial
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20. "Improved allelic differentiation using sequence-specific oligonucleotide
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25. "Recherches sur les nucleosides de synthese. III. Sur un nouveau critere
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28. "C-glycosidation of pyridyl thioglycosides." Stewart, A. O.; Williams, R. M.,
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33. "The preparation and utility of ethyl-2-(5'-O-t-butyldimethylsilyl-2',3'-O-
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34. "Stereoselective preparations of ribofuranosyl chlorides and ribofuranosyl
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36. "Stereoselective synthesis of pyrrolo[2,3-D]pyrimidine alpha-D-
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(aminomethyl)-2,5-dihydrofuran-2-yl]imidazoles by efficient use of a PhSe
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bonding ribonucleoside analogue: 1-beta-D-ribofuranosyl-3-nitropyrrole."
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81
Chapter 3
DESIGN, SYNTHESIS, AND ANTIVIRAL EVALUATION OF A SERIES OF SUBSTITUTED INDOLE AND 7-AZAINDOLE RIBONUCLEOSIDES
3.14 Overview
Continued efforts towards the development of universal ribonucleosides as antiviral lethal mutagens are described in this chapter. We hypothesized that ribonucleoside analogues capable of templating all four RNA bases (i.e.,
“universal bases”) might accelerate the onset of antiviral lethal mutagenesis by enhancing the frequency of viral mutations per round of replication (see section
2.4). Initial efforts to develop of universal ribonucleosides were based on the 3- nitropyrrole pseudobase (3-NPN, 48). However, ribonucleotide 61 exhibited poor kinetics of incorporation into the viral genome, precluding its function as an antiviral lethal mutagen.
A second generation of universal ribonucleosides was rationally designed, chemically synthesized, and evaluated biochemically as antiviral lethal mutagens. The ribonucleosides discussed herein are based primarily on 5- nitroindole (62) and 7-azaindole (64 and 66) 2'-deoxyribonucleosides that exhibit favorable biochemical properties in their respective applications (described in sections 3.15.1 and 3.17.1). The chemical syntheses of the analogous RNA nucleosides (63, 65, and 67 respectively) were completed and each compound was evaluated for antiviral activity. Screening experiments against poliovirus 82 (PV) and coxsackievirus B3 (CVB3) revealed that 5-NINDN (63) exhibits antiviral activity against HeLa cells infected with CVB3 and functions as a universal base
(as the triphosphate 70) by incorporating opposite all four bases in the RNA template when subjected to poliovirus and coxsackievirus RNA-dependent RNA polymerases. Efforts to elucidate the mechanism of antiviral activity of 63 were pursued and are described herein. Based on these results, a series of analogous indole ribonucleosides (Figure 3.24) were prepared to examine the structure-activity relationships that contribute to antiviral activity. Antiviral screening of this series of compounds revealed that the 4-nitroindole (4-NINDN,
79) and 6-nitroindole (6-NINDN, 80) ribonucleosides exhibit promising antiviral activities. Antiviral screening of unsubstituted and substituted azaindoles 65 and
67 revealed no activity against PV or CVB3, although X-ray crystallography of 65 revealed the presence of a superfluous hydrogen-bond, providing a potential explanation for the lack of antiviral activity.
3.15 Substituted Indole Ribonucleosides as Antiviral Lethal Mutagens
3.15.1 Design of an Improved Universal Deoxyribonucleoside: 1-(2'-deoxy- β-D-ribofuranosyl)-5-nitroindole
The publication of 3-nitropyrrole deoxyribonucleoside (3-NP, 47,
Figure 3.14)1-3 as a universal base lead to the subsequent development of 1-(2'-
deoxy-β-D-ribofuranosyl)-5-nitroindole (62),4 a hydrophobic, non-hydrogen-
bonding deoxyribonucleoside analogous to 3-NP (47). This analogue (62) was 83 constructed to address the reduction in DNA duplex stability resulting from 3-NP incorporation.5 It was hypothesized that 3-nitropyrrole pseudobases were too
small to efficiently form aromatic π-stacking interactions with adjacent bases in
DNA,3 a theory that was later supported by NMR spectroscopy.6 The 5-
nitroindole deoxyribonucleoside 62 addresses this structural concern by
expanding the size of the pseudobase from 3-nitropyrrole to 5-nitroindole, a
significantly larger aromatic system with enhanced π-stacking capabilities.
Melting temperature studies of synthetic DNA containing 5-nitroindole
deoxyribonucleoside 62 compared with 3-NP (47) show a significant
enhancement in duplex stability with the larger aromatic system, as indicated by
5 higher Tm values (Figure 3.15). Separate experiments examining the effects of
incorporation of multiple units of 62 into DNA primers revealed improved stabilities in relation to 47 in the same context, as determined by melting temperature denaturation studies with a complementary oligonucleotide.7
Additionally, Kool and coworkers measured the overall stacking capability of 62 by incorporating this nucleotide into a “dangling end” DNA oligonucleotide
(dXCGCGCG).8 This self-hybridizing oligonucleotide places one nucleotide (X)
on each terminus of the DNA strand without a base-pairing partner. Aromatic
stacking interactions between oligonucleotide X and the pseudobase below
(cytosine) are reflected by increases in duplex Tm values. Results from this study
found that 5-nitroindole (Tm = 60.6 ºC) stacks more efficiently than each of the
native DNA pseudobases (Tm range = 46.2 ºC to 51.6 ºC) as well as pyrrole (Tm =
46.6 ºC). 84
Figure 3.14. Compound Structures.
Figure 3.15. Melting temperature studies of 5-nitroindole (62) and 3-nitropyrrole (47) 2'-deoxyribonucleosides in a synthetic DNA duplex (adapted from 5). 85 The 5-nitroindole 2'-deoxyribonucleoside 62 has been used in a variety of biochemical applications ranging from DNA hybridization,9-12 incorporation into
DNA hairpins,13,14 DNA sequencing15,16 the detection of single nucleotide
polymorphisms17,18 primers for PCR,7 and incorporation (of the pseudobase) into
peptide nucleic acids.19 Interestingly, studies with 5-nitroindole 2'-
deoxyribonucleotide (69) have demonstrated the ability of this compound to
function as a substrate for DNA polymerases. Both DNA polymerase α (pol α) and the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I
(KF) incorporate 69 as a universal base opposite all four DNA bases in the template.20,21 The kinetics of incorporation for 69 across each base mediated by
pol α and KF polymerases are extremely fast, rivaling rates of natural nucleotide
incorporations.20 However, once incorporated into the DNA template, extension
past the 5-nitroindole pseudobase is not observed with either polymerase, suggesting chain termination.20,21 A different study with the exonuclease-free mutant of bacteriophate T4 DNA polymerase (gp43 exo-) measured 5-nitroindole
2'-deoxyribonucleotide 69 incorporation across abasic sites in the DNA template at rates 1000-fold greater than the best deoxyribonucleotide (dATP).22 Based upon the added stability of nucleoside 62 and the favorable interactions of nucleotide 69 with genomic replication machinery, we chose to examine 5-
NINDN (63) as an RNA virus mutagen. 86 3.15.2 Coupling Strategies for the Synthesis of Indole Ribonucleosides
Two chemical syntheses of the 5-nitroindole ribonucleoside 63 have been previously reported.23,24 Both synthesis routes utilize different coupling strategies
to form the anomeric bond between the sugar moiety and the pseudobase, but
each procedure has serious limitations. Attempts to reproduce both procedures
were investigated in an effort to prepare suitable quantities of 63 for biochemical
evaluation. Additionally, we developed a novel, rapid procedure for the synthesis
of 5-nitroindole and related ribonucleosides solving many problems associated
with existing approaches.
The first synthesis approach reported for the preparation of ribonucleoside
63 is shown in Figure 3.16.23 The key reaction is an indoline-indole coupling between the trityl-protected ribose moiety 71 and 5-nitroindoline (72) in refluxing ethanol. Subsequent acylation of the 2',3'-diol followed by dehydrogenation of the indoline moiety with manganese (IV) oxide (MnO2) provides the protected
intermediate 73. Efforts to reproduce this procedure were investigated in this project. However, the formation of 73 was never detected. The reaction between 71 and indoline 72 in refluxing absolute ethanol yielded a product
resembling triphenylmethanol as the major reaction component, suggesting that
the 5'-alcohol is labile under the reaction conditions.
87
Figure 3.16. The first reported chemical synthesis of 5-NINDN (63).23
An adaptation of the indoline-indole approach shown in Figure 3.16 was explored for the preparation of 5-NINDN (63). It was hypothesized that benzoyl- protected sugar 49 would be more robust for coupling with 5-nitroindoline (72), thereby facilitating the production of intermediate 75 (Figure 3.17). Utilizing conditions developed for the coupling of indoline with 49,25,26 5-nitroindoline (72)
was reacted with 49 in refluxing EtOH containing acetic acid to provide
intermediate 75 in 34% yield. Dehydrogenation to indole 76 was achieved by reaction with MnO2 in refluxing benzene, providing an inseparable mixture of 76
and unreacted 75. Global deprotection of the semi-pure reaction products by
reaction with ammonia in methanol provided a mixture of 5-NINDN (63) and
indoline 77 (5:2 ratio) in 32% yield (two steps). Efforts to separate 63 and 77 by
standard silica gel chromatography were unsuccessful, although both
compounds can be individually isolated by reverse-phase HPLC. Unfortunately,
due to the relatively large quantities of nucleosides required for complete
biochemical evaluation (hundreds of milligrams), the chromatography problems 88 associated with this synthesis approach rendered it impractical for this application.
Figure 3.17. An alternative indoline-indole coupling for the synthesis of 5-NINDN (63).
Previous studies on the synthesis of 3-NPN (48) directly coupled the stereospecific α-chlorosugar 54 and the sodium salt of 3-nitropyrrole (44) to form the anomeric bond of 60 with the desired β-stereochemistry (see section 2.5.2).
Due to the similarities between the 3-nitropyrrole (44) and 5-nitroindole (78) pseudobases, it was hypothesized that this method could be employed to synthesize 63 (Figure 3.18). However, coupling of the sodium salt of 5- nitroindole with the previously-described chlorosugar 54 in acetonitrile yielded the coupled product 79 in 18% yield as a mixture of α and β anomers (ca. 1:1 ratio).
Unfortunately, efforts to separate these isomers by silica gel chromatography 89 were unsuccessful. Although the mechanism of erosion of stereocontrol in this reaction is unclear, a recently reported chemical synthesis of 63 utilized the same transformation, obtaining 79 in 35% yield (2 steps) as a mixture of α and β anomers (ca. 3 : 2 ratio).24 Global deprotection of 79 with aqueous TFA followed
by reprotection of the free 3' and 5'-alcohols as the tetraisopropyldisiloxane
24 (reaction with TIPDSCl2) finally yielded a method for separation of the anomers.
The inability of this synthesis approach to provide ribonucleoside 63 as the pure
β isomer prompted additional investigation of methods to prepare this nucleoside.
Figure 3.18. Coupling of the sodium salt of 5-nitroindole (78) with the stereospecific α-chlorosugar 54.
We developed a novel chemical synthesis of 5-NINDN (63) employing elements from the synthesis strategies presented in Figures 3.17 & 3.18.
Elimination of the indoline to indole transformation was attractive due to the difficulties associated with separating reaction products from unreacted starting materials. Additionally, the utilization of the benzoyl-protected sugar 49 provides an element of stereocontrol in the anomeric coupling with the pseudobase due to neighboring-group participation from the 2'-benzoyl ester. In this regard, our 90 synthesis of 63 begins by chlorination of 49 with titanium (IV) chloride, forming the 1-chlorosugar 50 as a mixture of anomers (Figure 3.19).27 Immediate
coupling of 50 with the sodium salt of 5-nitroindole (78) yields intermediate 76,
which is partially purified, then globally deprotected by reaction with ammonia in
methanol. Standard silica gel chromatography of the crude reaction products
easily separates ribonucleoside 63 from reaction impurities and achieves an 18%
overall yield for the synthesis. Transformation of 63 into the 5'-triphosphate 70
was achieved under the standard “one-pot, three-step” phosphorylating
conditions (see section 5.2.3),28-35 providing 5-NINDNTP (70) in 24% yield.
Figure 3.19. Chemical synthesis of 5-NINDN (63) and 5-NINDNTP (70). 91 3.15.3 Cellular Cytotoxicity Study of 5-NINDN (63)m
The cytotoxicity of 5-NINDN (63) towards cultured HeLa S3 cells was evaluated at various concentrations following a 7 hour treatment (Figure 3.20).
For comparison purposes, ribavirin (25) was utilized as a control in this assay. In brief, cells were treated with ribonucleoside-supplemented media at the appropriate concentration for 7 hours. The media was removed, replaced with fresh media (lacking compounds), and the cells were allowed to recover for 24 hours. Staining of dead cells with trypan blue followed by manual counting of viable cells provided the cytotoxicity values shown. Based on this data, neither
5-NINDN (63) nor ribavirin (25) exhibits significant cytotoxicity to the cellular host
during the time course of this experiment.
Figure 3.20. Cellular cytotoxicity of 5-NINDN (63) towards HeLa S3 cells.
m Experimental data obtained by Jason D. Graci (Cameron Lab). 92 3.15.4 Antiviral Activity of 5-NINDN against Poliovirus and Coxsackievirus B3n
The antiviral activity of 5-NINDN (63) was measured against poliovirus
(PV) and coxsackievirus B3 (CVB3)-infected HeLa S3 cells grown in the
presence of various concentrations of 63. The known lethal mutagen ribavirin
(25) was utilized as a control in this experiment (Figure 3.21). 5-NINDN (63)
exhibited minimal antiviral activity against poliovirus in these experiments, with
only a small decrease in virus titer observed at the highest concentration tested
(2 mM). However, 5-NINDN (63) exhibits strong antiviral effects against CVB3 at
2 mM concentrations, yielding a 2000-fold reduction in the virus titer compared
with untreated controls. Surprisingly, this activity surpassed the effects of
ribavirin (25), which only reduced CVB3 titer 200-fold at 2 mM. Interestingly, the
antiviral activity profile of ribavirin reveals similar reductions in virus titer across
different concentrations regardless of virus, whereas coxsackievirus exhibited a
unique susceptibility to 5-NINDN at high concentrations.
n Experimental data obtained by Jason D. Graci (Cameron Lab). 93
Figure 3.21. Antiviral activity of 5-NINDN (63) and ribavirin (25) against PV and CVB3.
3.15.5 Incorporation of 5-NINDNTP into the RNA Template Mediated by Poliovirus and Coxsackievirus RNA-dependent RNA polymeraseso
To investigate potential mechanisms of antiviral activity, the ability of 5-
NINDNTP (70) to function as a universal ribonucleotide was measured using the
previously-described primer-extension assay (see section 1.5.4.3). In these
experiments, the incorporation of 70 across all four RNA bases was measured
for poliovirus (3Dpol) and coxsackievirus (CVB-H3) RNA-dependent RNA polymerases (Figure 3.22). We found that the 5-nitroindole ribonucleotide 70
functions as a universal base in all contexts, incorporating opposite all four RNA
o Experimental data obtained by Jason D. Graci (Cameron Lab). 94 bases in the primer-template mediated by both viral RNA-dependent RNA polymerases. The kinetics of incorporation of 70 into an s/s-U RNA substrate mediated by PV 3Dpol were quantified to provide additional data concerning the
mechanism of action (Table 3.4). Ribonucleotide 70 was found to incorporate
approximately 10-fold slower than ribavirin triphosphate in this context opposite
36 the pyrimidines, whereas the low Kd value (9.9 µM) of 5-NINDNTP (70)
suggests strong binding of the nucleotide to either the polymerase active site or
another region capable of an allosteric interaction.
Figure 3.22. Incorporation of 5-NINDNTP opposite all four RNA bases mediated by poliovirus and coxsackievirus RNA-dependent RNA polymerases.
Table 3.4: Kinetics of incorporation of 5-NINDNTP (70) into s/s-U mediated by PV 3Dpol. -1 Nucleotide TP Kd (µM) kpol (s ) 5-NINDNTP (70) 9.9 ± 1.5 0.00136 ± 0.00006 RTP (41) 496 ± 21 0.014 ± 0.001 95 The slow rate of incorporation of 70 into the primer-template and the unusually low Kd value associated with poliovirus polymerase binding suggests
that 70 might function by a mechanism of action distinct from lethal mutagenesis.
In this regard, the capability of 70 to function as a chain terminator was
addressed. A reexamination of the primer-extension assay with PV RdRP and
s/s-U was performed, except enzyme-bound Mg2+ was replaced with Mn2+ to decrease the fidelity of the viral RdRP and overcome the slow rate of nucleotide incorporation relative to enzyme dissociation.37 Under the conditions of this assay, the primer-template was extended up to 3 nucleotides, suggesting that 70
does not function as a chain terminator in this application (Figure 3.23).
Figure 3.23. Chain termination assay for 5-NINDNTP (70). Extension of s/s-U by PV RdRP in the presence of Mn2+. 96 3.15.6 Efforts Towards Elucidating the Molecular Mechanism of Action of 5-NINDN (63)
The current focus of this project is the continued elucidation of the molecular mechanism of antiviral action for 5-NINDN (63). We are studying the hypothesis that a phosphorylated analogue of 63 inhibits the viral RNA- dependent RNA polymerase. The relatively low Kd associated with 5-NINDNTP
binding to the viral RdRP supports this idea. In addition, previously reported
studies of the 5-nitroindole 2'-deoxyribonucleotide 69 revealed this nucleotide
functions as an inhibitor of Maloney murine leukemia virus reverse
transcriptase.20 Stopped-flow kinetic analysis of ATP incorporation into the s/s-U
primer-template by RV RdRP revealed that 5-NINDNTP (70) inhibits the
incorporation of this nucleotide opposite its complementary base (data not
shown).p However, the formation of nucleotide 70 in vivo has been questioned.
Adenosine kinase phosphorylation experiments (described in 2.8) failed to demonstrate that 63 functions as an efficient enzyme substrate. However, multiple cellular kinases are known to exist for the phosphorylation of nucleosides in vivo. Recently, a series of HPLC-based metabolism studies were conducted to detect the presence of 5-NINDNTP (70) from lysed HeLa cells
treated with nucleoside (63). Although 70 was not detected in these studies,
other metabolites with similar spectroscopic characteristics but different retention
times have been observed. These results have raised the possibility that another
p Experimental data obtained by Christian Castro (Cameron Lab). 97 phosphorylated metabolite of 63 may predominate in cells that confers the observed antiviral effects. Positive identification of the metabolites present in vivo will provide targets for chemical syntheses and subsequent evaluation as inhibitors of viral RNA-dependent RNA polymerases.
3.16 Design, Synthesis, and Preliminary Antiviral Evaluation of a Series of Substituted Indole Ribonucleosides
3.16.1 Molecular Design of Substituted Indoles
After our initial discovery of antiviral activity for 5-NINDN (63), a series of analogous indole ribonucleosides were designed to probe structure activity relationships (Figure 3.24). The nitroindole regioisomers 4-NINDN (79) and 6-
NINDN (80) maintain the hydrophobic π-stacking capabilities of 63, but position the large nitro group at different sites on the indole ring. The 5-aminoindole ribonucleoside 81 introduces an electron rich hydrogen-bonding group onto the heterocycle, potentially affecting the π-stacking capabilities of the indole ring system. The unsubstituted indole 82 was prepared to measure the importance of the nitro functionality on antiviral activity. Previous studies comparing the rates of incorporation of 2'-deoxyribonucleotides containing 5-nitroindole and indole pseudobases opposite abasic sites in DNA revealed large decreases in catalytic efficiencies associated with removal of the nitro functionality.38 The carboxamide
indole 83 was synthesized to examine the effects of this hydrogen-bonding
donor/acceptor on the ring system. 98
Figure 3.24. Structures of substituted indole ribonucleosides.
3.16.2 Chemical Synthesis of Substituted Indole Ribonucleosides
The chemical synthesis of the 4-NINDN (79) and 6-NINDN (80) ribonucleosides are based upon the rapid chemical synthesis developed for preparation of 63 (see Figure 3.19). Commercially-available sugar 49 is chlorinated with TiCl4 to yield the chlorosugar 50 as a mixture of anomers
(Figure 3.25).27 Coupling of this crude material with the sodium salt of 84 or 85,
followed by deprotection with ammonia in methanol, yields 4-NINDN (79) and 6-
NINDN (80) in 11% and 25% overall yields, respectively. The 5-aminoindole ribonucleoside 81 is prepared from 5-NINDN (63) by hydrogenation with Raney
2800 Ni (1 atm) in good yields.21 Other catalyst sources examined, including
Pd/C, resulted in epimerization at the anomeric carbon. The final two 99 ribonucleoside analogues are derived from the common intermediate 88.
Adaption of a published procedure25 was used to prepare 88, which was derived
from an indoline-indole coupling39 with 49 and 86 followed by dehydrogenation
with manganese (IV) oxide.40 Global deprotection with ammonia in methanol
yielded the unsubstituted analogue 82. The carboxamide derivative 83 was
prepared by reaction of 88 with the electrophile chlorosulfonyl isocyanate,
followed by alkaline hydrolysis to provide 83 in 35% yield.41
Figure 3.25. Chemical synthesis of substituted indole ribonucleosides. 100 3.16.3 Preliminary Cytoxicity and Antiviral Activity Studies of the Substituted Indole Ribonucleosides
The newly synthesized indole ribonucleosides have been subjected to
preliminary screenings for cellular cytotoxicity and antiviral activity using methods
employed for the testing of 5-NINDN (see sections 3.15.3 & 3.15.4). Although
this data set is still incomplete, some interesting structure-activity relationships
have been observed. In general, the substituted indole ribonucleosides exhibit low cellular cytotoxicity (Figure 3.26). A reduction in cell viability to
approximately 50% was observed with the 2 mM treatment of 4-NINDN, the
highest level of toxicity observed with this series of compounds.
60 0.1 mM 0.5 mM 50 1.0 mM 2.0 mM
40 ) 4
30
cells/well (10 cells/well 20
10
0 INDN 4-NINDN 6-NINDN 3-AMINDN 5-AINDN No Cmpd
Figure 3.26. Cytotoxicity of substituted indole ribonucleosides towards cultured HeLa cells (7 hour exposure). 101 Antiviral evaluation of these ribonucleosides against poliovirus
(Figure 3.27) and coxsackievirus B3 (Figure 3.28) revealed that 4-NINDN (79)
and 6-NINDN (80) exhibit the best antiviral activities in the series. At 1 mM
concentration, 4-NINDN (79) substantially decreases virus titer compared with
untreated controls. The 6-NINDN (80) regioisomer exhibits modest activity as
well, whereas the remaining ribonucleosides (INDN, 3-AMINDN, and 5-AINDN)
fail to exhibit activity against this virus. Similar trends in antiviral activities are
observed against coxsackievirus B3, as both 79 and 80 exhibit the greatest
reduction in virus titer.
Figure 3.27. Antiviral activity of substituted indole ribonucleosides against poliovirus-infected HeLa cells. 102
Figure 3.28. Antiviral activity of substituted indole ribonucleosides against coxsackievirus-infected HeLa cells.
3.16.4 Conclusions and Future Directions
Future research on this family of ribonucleoside analogues will focus initially on completing the biochemical analyses of all the nucleosides. Once the biochemical profiles for nucleosides 79-83 are completed, the most active nucleosides will be phosphorylated to generate the nucleoside triphosphates for study with viral polymerases. The preliminary data presented herein suggest that hydrophobic, aromatic nucleobases capable of forming stable π-stacking 103 interactions exhibit the most promise as antiviral agents. Future biochemical
studies elucidating the precise mechanisms of action of these compounds are
highly warranted.
3.17 Synthesis and Biochemical Evaluation of 7-azaindole Ribonucleosides as Antiviral Lethal Mutagens
3.17.1 Substituted and Unsubstituted 7-azaindole Deoxyribonucleosides for the Expansion of the Genetic Alphabet
In an effort to develop non-natural “coding” base-pairs of nonnatural
deoxyribonucleotides,42 synthesis and biochemical properties of the 7-azaindole
2'-deoxyribonucleosides 64 and 66 have been reported.43 These compounds
were designed to form stable base-pairs with other non-natural
deoxyribonucleotides in duplex DNA driven by shape-complementary
hydrophobic interactions. Melting temperature studies of duplex DNA containing
64 and 66 base-paired with each of the native DNA bases resulted in significantly
lower Tm values, whereas base-pairing with other non-natural hydrophobic
pseudobases only slightly decreased the stability of the duplex. Self-
complementary base pairing was also favored as duplex DNA containing 64:64
and 66:66 base pairs melted at 55.5 °C and 56.3 °C, respectively, compared with
43 a native A:T base-pair (Tm = 59.2 °C) in the same context.
The kinetics of incorporation of 64 and 66 (as the 5'-triphosphates 89 and
90) into a synthetic primer-template mediated by the exonuclease-free fragment 104 of Klenow DNA polymerase has been measured (Figure 3.29).43 Both the
unsubstituted 7-azaindole triphosphate 89 and the propyne-substituted 7- azaindole nucleotide 90 exhibited fast rates of incorporation opposite all four
DNA bases. In some cases, the kinetics of incorporation rivaled that of the natural A-T base pair, incorporating only 10-fold slower than the correct base.
Based upon this data, the deoxyribonucleoside 64 and 66, if phosphorylated in vivo, have the potential to compete with the natural nucleotides and become incorporated into DNA.
Figure 3.29. Kinetics of nucleotide incorporation into a synthetic DNA template by KF DNA polymerase.43 105 3.17.2 Design and Synthesis of 7-azaindole Ribonucleosides 65 and 67
Previous studies with the hydrophobic ribonucleoside analogues 3-NPN
(48) and 5-NINDN (63) indicated that these compounds are poor substrates for
poliovirus RNA-dependent RNA polymerase (as the triphosphates), precluding
their incorporation into viral genomes (see sections 2.9.2 & 3.15.5). Based on
the favorable kinetics of incorporation of 7AI(TP) (89) and P7AI(TP) (90) into a
DNA template mediated by DNA polymerases, it was hypothesized that the RNA analogues 7AI-R (65) and P7AI-R (67) might be incorporated into viral genomes
(as the triphosphates) with better efficiencies as well. Additionally, the
hydrophobic scaffolds of 65 and 67 fit the structural requirements for ambiguous base-pairing universal bases, a characteristic reflected in the unbiased hybridization properties previously observed for 64 and 66 across other
hydrophobic bases in duplex DNA.43
The unsubstitued 7-azaindole ribonucleoside analogue 65 was chemically synthesized as previously described (Figure 3.30).44 7-azaindole (91) was hydrogenated to 7-azaindoline (92) by reaction with palladium on carbon at
elevated pressure. A melt-fusion coupling between 92 and the tetra-acetylated
ribose 93 yielded the coupled indoline intermediate 94. Dehydrogenation of 94 with DDQ yielded the protected 7-azaindole ribonucleoside 95. Reaction of this common intermediate with ammonia in methanol yielded 7AI-R (65). To prepare the propynyl-substituted analogue 67, intermediate 95 was subjected to electrophilic iodination conditions by reaction with ICl, then immediately 106 converted to the propynyl analogue 96 by reaction with propyne under
Sonogashira coupling conditions.43 Intermediate 96 was partially purified, then globally deprotected with ammonia in methanol to provide P7AI-R (67) in 26% overall yield from 95.
Figure 3.30. Synthesis of 7AI-R (65)44 and P7AI-R (67).
3.17.3 Cellular Cytoxicity and Antiviral Activity Studies of 7-azaindole Ribonucleoside Analogues
The 7-azaindole ribonucleoside analogues were evaluated for cellular cytotoxicity against HeLa S3 cells grown in culture utilizing the procedure previously described in section 3.15.3. Under the conditions of this experiment neither 65 nor 67 exhibited cellular cytotoxicity compared with untreated controls 107 (data not shown). Although these studies were encouraging, both compounds
(65 and 67) failed to exhibit significant antiviral activity against poliovirus-infected
HeLa cells (Figure 3.31). No reduction in virus titer was observed with P7AI-R, a
surprising result due to the favorable kinetics of incorporation of the 2'-deoxy
analogue 66. The unsubstituted azaindole, 7AI-R (65) showed a slight reduction in virus titer (less than one log unit), although this is insignificant in comparison with the antiviral activity of the drug ribavirin (RBV, 25).
Figure 3.31. Antiviral activity of 7AI-R (65) and P7AI-R (67) against poliovirus- infected HeLa cells. 108 3.17.4 Conclusions and Future Directions
The lack of antiviral activity of 7-azaindole ribonucleosides 65 and 67 was disappointing considering the promise the 2'-deoxy analogues 64 and 66
exhibited with DNA polymerases. Additionally, studies from others working on
this project (data not presented) have revealed good antiviral activity from
isocarbostyril-based pseudobases (see Figure 2.1), hydrophobic scaffolds known
to exhibit good kinetics of incorporation into DNA templates by DNA
polymerases.43 To help explain the lack of antiviral activities for 65 and 67, an X-
ray crystal structure of 7AI-R (65) was obtained (Figure 3.32). In the crystalline
state, the pseudobase of 7AI-R exists in the syn conformation via an internal
hydrogen bond between the C5'-OH of ribose and N-7 of the 7-azaindole
pseudobase. Although no direct experimental evidence is available, it is possible
that bias towards this conformation may hinder the accessibility of the C5'-OH to
phosphorylation to the monophosphate, a critical step in metabolism to the active
triphosphate. Once phosphorylated, the bias towards this conformation would be
eliminated. A biochemical comparison of phosphorylation capabilities (such as
the adenosine kinase phosphorylation experiment, 2.8) between 65 and INDN
(82, unsubstituted indole ribonucleoside) could test this hypothesis. 109
Figure 3.32. X-ray crystal structure of 7AI-R (65) showing internal hydrogen- bond between C5'-OH and N-7.
3.18 Experimental Section
Cells and viruses. HeLa S3 cells were maintained in DMEM/F-12 supplemented with 2% dialyzed fetal bovine serum and penicillin/streptomycin (1X, Invitrogen).
Nucleosides were freshly suspended in 100% DMSO (200 mM) immediately prior
to use. Ribavirin (a gift of Zhi Hong, Valeant Pharmaceuticals) was suspended in
deionized water. For cytotoxicity studies, HeLa S3 cells (1 X 105) were plated the
day before in 24-well plates. Cells were incubated with ribonucleosides at
various concentrations for 7 hours at 37 °C. All wells were adjusted to a final
concentration of 1% DMSO. Media was removed and cells were washed with
PBS (0.5 mL). Cells were allowed to grow for an additional 24 h in the absence
of either compound. Cell monolayers were washed in PBS (0.5 mL), dissociated 110 by treatment with trypsin (1X, Invitrogen), and viable cells were counted by trypan blue exclusion using a hemacytometer.
Infection with poliovirus (PV) and coxsackievirus B3 (CVB3/0) employed
HeLa S3 host cells (1 X 105) plated 1 day prior to treatment in 24-well plates.
Cells were pretreated by addition of nucleoside at the specified concentration in
fresh media adjusted to a final concentration of 1% DMSO. After a 1-hour
incubation at 37 °C, media was removed and cells were infected with PV or
CVB3/0 (1 X 106 PFU) in phosphate-buffered saline (PBS, total volume = 0.1
mL). Plates were incubated for 15 min at 23 °C, PBS was removed by
aspiration, and fresh, prewarmed (37 °C) media containing the specified amount
of nucleoside was added. The infection was allowed to proceed at 37 °C for 6
hours. Cells were washed with PBS and collected after treatment with trypsin.
Cells were pelleted by centrifugation, resuspended in PBS (0.5 mL), and subjected to 3 freeze-thaw cycles. Cell debris was removed by centrifugation and the supernatant containing the cell-associated virus was saved. Titer was determined by applying serial dilutions of supernatant to HeLa S3 monolayers
(plated in 6-well plates 1 day before at 5 X 105 cells/well) and overlaying with growth media containing low melting point agarose (1% for PV, 0.5% for
CVB3/0). Plates were incubated for 2 (PV) or 3 (CVB3/0) days at 37 °C, at which time the agar was removed and plaques were visualized by staining with crystal violet (1%) in aqueous ethanol (20%).
111 Nucleotide incorporation by poliovirus and coxsackievirus B3 RNA-dependent
RNA polymerases in vitro. PV 3Dpol was expressed and purified as previously
described.45 Purified CVB3-H3 RNA-dependent RNA polymerase was utilized for coxsackievirus primer-extension assays.q Extension assays utilizing symmetrical
primer-template substrates (s/s) were performed as described.46 s/s RNAs were
synthesized by Dharmacon, Inc. In brief, PV 3Dpol was incubated with the
appropriate s/s duplex for 90 s at 30 °C to allow formation of preinitiation
enzyme-RNA complexes. Extension reactions were initiated by the addition of
nucleotide and reactions were incubated at 30 °C. The initiated reaction
contained 3Dpol (5 µM), s/s RNA (1 µM), HEPES (50 mM, pH 7.5), 2- mercaptoethanol (10 mM), MgCl2 (5 mM) and NTP. Reactions were quenched
by addition of EDTA (final conc. = 50 mM, pH 8.0). For the experiment shown in
Figure 3.23, MgCl2 was replaced with MnCl2. For all experiments, s/s “trap” (100
µM) was added along with initiating nucleotide to prevent reinitiation of
dissociated enzyme. Product was added to an equal volume of loading buffer
(90% formamide, 0.025% bromphenol blue, and 0.025% xylene cyanol) and
heated to 65 °C prior to loading on a denaturing polyacrylamide gel containing
23% acrylamide, TBE buffer (1X, 89 mM Tris base, 89 mM boric acid, and 2 mM
EDTA), and urea (6 M). Electrophoresis was performed in TBE buffer (1X) at 80
W for ca. 2 h. Products were visualized using a PhosphorImager (Molecular
Dynamics). Quantitation was performed using ImageQuant software (Molecular
q Unpublished results (C. Castro, Q. Wang, and C.E. Cameron) 112 Dynamics) and fit by non-linear regression using KaleidaGraph 3.5 software
(Synergy Software, Reading, PA).
General synthesis information. All reactions were performed under an argon atmosphere unless otherwise noted. Commercial grade reagents (Aldrich,
Acros) were used without further purification unless specifically noted.
Triethylammonium acetate (TEAA, pH = 7 at 1 M) and triethylammonium
bicarbonate (TEAB, pH = 8.5 at 1 M) buffers were from Fluka. Tetrahydrofuran,
dichloromethane, acetonitrile, and N,N-dimethylformamide were rendered
anhydrous by passing through the resin column of a solvent purification system
(GlassContour; Laguna Beach, CA). Column chromatography employed ICN
SiliTech silica gel (32-63 µm). HPLC analyses were performed on a Hewlett
Packard 1100 series instrument equipped with an Aquasil C18 analytical column
(4.6 x 250 mm, 5 µm) or a Hamilton PRP-1 analytical column (4.6 x 250 mm, 7
µm). HPLC purifications were performed on an Agilent 1100 series instrument
equipped with an Aquasil C18 preparative column (21.2 x 250 mm, 5 µm;
Thermo Electron Corporation) or a Hamilton PRP-1 preparative column (21.5 x
250 mm, 7 µm; Hamilton Company). MPLC purification was performed on a
Büchi Sepacore preparative system (equipped with an isocratic pump module
and fraction collecter) utilizing ICN SiliTech silica gel (32-63 µm). Two columns
with dimensions of 100 mm x 460 mm (Column A) and 49 mm x 460 mm
(Column B) were employed during MPLC purifications. Nuclear magnetic 113 resonance (NMR) spectroscopy employed Bruker CDPX-300, DPX-300, AMX-
360, DRX-400, or AMX-2-500 MHz spectrometers. Internal solvent peaks were referenced in each case. Chemical shifts for 13C NMR and 31P NMR analyses
performed in D2O were indirectly referenced to 10% acetone in D2O (CH3 set to
47 13 30.89 ppm) and 85% H3PO4 (0 ppm), respectively. C chemical shifts for
nucleoside diphosphates and triphosphates denoted with a (*) fail to resolve into
clean singlets due to apparent conformational restrictions. Mass spectral data
was obtained from either The University of Texas at Austin Mass Spectrometry
Facility (FAB, ESI and CI) or The Pennsylvania State University Mass
Spectrometry Facility (ESI and APCI). Elemental analyses were performed by
Midwest Microlab, LLC (Indianapolis, IN). Melting points are uncorrected.
1-(2',3',5'-Tri-O-benzoyl-β-D-ribofuranosyl)-5-nitroindoline (75). A solution of β-D-
ribofuranose-1-acetate-2,3,5-tribenzoate (49, 10.420 g, 20.65 mmoles) and 5-
nitroindoline (72, 10.192 g, 62.09 mmoles) in abs. EtOH (100 mL) containing
glacial AcOH (12 mL) was refluxed for 3 h. The reaction was quenched by
pouring into saturated aq. NaHCO3 (250 mL), then extracted with CHCl3 (150 mL,
3x). The combined organic layers were washed with saturated aq. NaCl (100
mL, 1x), dried over anhydrous MgSO4, and concentrated in vacuo. The crude 114 material was MPLC purified (column A; eluant: 60% hexanes, 35% benzene, 5%
Et3N; flow rate: 150 mL/min) yielding 75 (4.325 g, 34% yield) as a bright yellow
1 foam. H NMR (CDCl3, 400.1 MHz): δ 8.10 (m, 2H), 8.02 (dd, J = 2.3 Hz, J = 8.9
Hz, 1H), 7.99 (m, 2H), 7.93 (m, 2H), 7.90 (m, 1H), 7.61-7.32 (m, 9H), 6.69 (d, J =
8.9 Hz, 1H), 5.88 (m, 2H), 5.79 (dd, J = 3.3 Hz, J = 5.2 Hz, 1H), 4.76 (m, 1H),
13 4.60 (m, 1H), 4.54 (m, 1H), 3.76 (m, 2H), 3.02 (m, 2H). C NMR (CDCl3, 75.4
MHz): δ 166.1, 165.4, 165.2, 155.3, 140.4, 133.7, 133.5, 130.8, 129.82, 129.76,
129.65, 129.4, 128.9, 128.7, 128.6, 128.5, 125.6, 121.1, 106.0, 87.2, 78.9, 71.8,
70.4, 64.1, 46.1, 27.0. IR (film): 1725, 1266 cm-1. MS (ESI+) calcd. for
+ C34H29N2O9 [M+H] 609.2, found 609.3. Anal. calcd. for C34H28N2O9: C 67.10; H
4.64; N 4.60. Found: C 66.74; H 4.81; N 4.38.
1-(β-D-Ribofuranosyl)-5-nitroindole (63).23,24 This compound was prepared by
two methods.
Method A. A solution of 5-nitroindoline ribonucleoside 75 (4.098 g, 6.734 mmoles) and manganese (IV) oxide (22.513 g, 258.9 mmoles) in PhH (150 mL) was refluxed through a Dean-Stark trap for 6 h. The reaction was then filtered through a pad of Celite, which was washed with excess benzene, and the filtrate 115 concentrated in vacuo. The crude material was MPLC purified (column B; eluant:
60% hexanes, 35% benzene, 5% Et3N; flow rate: 40 mL/min) yielding an
inseparable mixture of the indole product and unreacted starting material (ca. 2:1
ratio favoring the indole) as a bright yellow foam (yield: 2.568 g). This mixture
was then loaded into a pressure tube that was subsequently charged with NH3 in
MeOH (75 mL, ca. 7 N). The closed system was heated to 55 °C for 15 h. After
cooling for ca. 1 h, the pressure tube was vented and the crude material
concentrated in vacuo. Column chromatography (step gradient: 5% MeOH in
CH2Cl2 to 10% MeOH in CH2Cl2) followed by recrystallization from warm abs.
EtOH yielded a mixture of 63 and indoline derivative 77 (ca. 2.5:1 ratio favoring
63) separable by RP-HPLC (638.7 mg, ca. 32% yield over 2 steps).
The separation of 63 and indoline derivative 77 was performed by analytical HPLC utilizing a PRP-1 (analytical) column running the following linear gradient (flow rate = 0.8 mL/min). The mobile phase comprised 1 to 50% CH3CN
(containing 0.1% trifluoroacetic acid [TFA]) in double distilled water (ddH2O, 0.1%
TFA, 0 to 30 min). As shown in Figure 3.33, 63 eluted at 23.5 min. 116
Figure 3.33. Separation of 63 and indoline ribonucleoside 77 by reversed-phase HPLC.
Method B. To a degassed solution of β-D-ribofuranose-1-acetate-2,3,5-
tribenzoate (49, 5.91 g, 11.71 mmol) in CH2Cl2 (70 mL) was added a solution of
27 TiCl4 in CH2Cl2 (13 mL, 1.0 M). The reaction was stirred at 23 °C for 2 h, then stopped by addition of deionized H2O (75 mL). The organic layer was separated,
washed with deionized H2O (75 mL, 1x), dried over anhydrous MgSO4, and concentrated in vacuo. The crude material was dissolved in CH3CN (20 mL) and
used without further purification in the subsequent coupling step. Separately, a
suspension of 5-nitroindole (78, 6.17 g, 38.05 mmol) in CH3CN (20 mL) was
added to a suspension of NaH (95% dry, 556.3 mg, 23.18 mmol) in CH3CN (80 mL) at 2 ºC. To this solution was added crude chlorosugar 50 via cannula followed by additional CH3CN (10 mL). The reaction was allowed to warm to 23
°C and stirred for 22 h. The crude product was then poured into saturated aq.
NH4Cl (250 mL) and EtOAc (250 mL) was added. The organic layer was 117 separated, washed with saturated aq. NaCl (250 mL), dried over anhydrous
MgSO4, and concentrated in vacuo. The crude material was partially purified by
column chromatography (20% EtOAc in hexanes) and used directly in the next
step. A pressure tube was charged with the reaction products and NH3 in MeOH
(80 mL, ca. 7 N). The tube was sealed and heated to 50 ºC for 12 h. The tube
was cooled, vented, and the contents concentrated in vacuo. The crude material was purified first by column chromatography (step gradient: 5% MeOH in CH2Cl2 to 10% MeOH in CH2Cl2) and subsequently by recrystallization (compound was
dissolved in warm acetone [75 mL] and Et2O [75 mL] was added). The light
yellow precipitate was collected, washed with excess Et2O, and dried in vacuo to
yield ribonucleoside 63 (630.5 mg, 18% yield over 3 steps). mp 197–199 °C. 1H
NMR (360.1 MHz, DMSO-d6): δ 8.58 (d, J = 2.2 Hz, 1H), 8.04 (dd, J = 2.3 Hz, J =
9.1 Hz, 1H), 7.89 (d, J = 3.4 Hz, 1H), 7.81 (d, J = 9.2 Hz, 1H), 6.83 (d, J = 3.4 Hz,
1H), 5.98 (d, J = 6.2 Hz, 1H), 5.43 (d, J = 6.7 Hz, 1H), 5.22 (d, J = 4.8 Hz, 1H),
5.08 (t, J = 5.2 Hz, 1H), 4.28 (m, 1H), 4.10 (ddd [app dt], J = 3.2 Hz, J = 5.0 Hz, J
13 = 5.0 Hz, 1H), 3.97 (m, 1H), 3.67-3.58 (m, 2H). C NMR (90.6 MHz, DMSO-d6):
δ 141.1, 138.7, 129.3, 127.9, 117.4, 116.7, 110.9, 104.6, 89.0, 85.4, 74.5, 70.3,
-1 61.3. IR (KBr): 3426, 3364, 1510, 1347, 1324, 1086 cm . UV λmax (MeOH) 267
-1 -1 + + nm (ε = 18,700 M cm ). HRMS (CI ) calcd. for C13H15N2O6 [M+H] 295.0930, found 295.0936. Anal. calcd. for C13H14N2O6: C 53.06; H 4.80; N 9.52. Found:
C 53.13; H 4.78; N 9.29.
118
1-(β-D-Ribofuranosyl)-5-nitroindole-5’-triphosphate triethylammonium salt (70).
This compound was prepared utilizing well-precedented methodology for the synthesis of nucleoside triphosphates.28-35 A solution of 63 (62.5 mg, 0.21 mmol), Proton-Sponge (1,8-bis(dimethylamino)naphthalene, 85.6 mg, 0.40 mmol), and trimethylphosphate (2.2 mL) was cooled to 2 °C. POCl3 (40 µL, 0.43 mmol) was added dropwise, and the solution was stirred for 2 h. Anhydrous
Bu3N (240 µL, 1.01 mmol) was injected followed by a solution of tributylammonium pyrophosphate (538.5 mg) in DMF (2 mL). The reaction was stirred for 2 minutes then hydrolyzed by addition of triethylammonium bicarbonate (TEAB, 1.0 M, 5 mL). The crude reaction products were purified by preparative HPLC (Aquasil C-18 preparative column) to yield triphosphate 70 as the triethylammonium salt (tR = 11-13.5 min.). The purification of 70 by HPLC utilized the following linear gradient (flow rate = 20 mL/min). The mobile phase comprised 10% to 20% CH3CN in TEAA buffer (0 to 20 min, 20 mM TEAA, pH =
6) followed by 20% to 90% CH3CN in TEAA buffer (20 to 30 min, 20 mM TEAA, pH = 6). The purified material was concentrated in vacuo, redissolved in TEAB
(100 mM, 10 mL) and lyophilized to dryness. The material was redissolved in deionized H2O (3 mL) and lyophilized again to dryness. Triphosphate 70 was 119
1 isolated as a yellow solid (49.1 mg, 24% yield). H NMR (360.1 MHz, D2O): δ
8.37 (d, J = 2.2 Hz, 1H), 7.90 (dd, J = 2.2 Hz, J = 9.2 Hz, 1H), 7.63 (d, J = 3.4 Hz,
1H), 7.49 (d, J = 9.2 Hz, 1H), 6.69 (d, J = 3.3 Hz, 1H), 5.96 (d, J = 7.0 Hz, 1H),
4.54 (m, 1H), 4.38 (m, 1H), 4.18 (m, 1H), 4.09-3.99 (m, 2H), 2.97 (q, J = 7.3 Hz, ca. 27H, TEAA salt), 1.73 (s, ca. 3.5H, TEAA salt), 1.06 (t, J = 7.3 Hz, ca. 40.5H,
13 TEAA salt). C NMR (90.6 MHz, D2O): δ 182.0 (TEAA salt), 142.3, 140.5, 129.4,
129.0, 119.1, 118.5, 110.9, 106.8, 88.8, 84.5, 84.4, 74.4, 71.4, 66.4*, 47.4 (TEAA
31 salt), 24.0 (TEAA salt), 9.0 (TEAA salt). P NMR (145.8 MHz, D2O): δ -9.07 (br
− − s), -10.69 (m), -21.92 (br s). HRMS (FAB ) calcd. for C13H16N2O15P3 [M–
TEAA+3H]− 532.9764, found 532.9775.
The chemical purity of 70 was further analyzed by analytical HPLC
running the following linear gradient (flow rate = 1 mL/min). The mobile phase
comprised 1 to 80% CH3CN in KH2PO4 (0 to 40 min, 100 mM KH2PO4, pH = 6).
As shown below (Figure 3.34), triphosphate 70 eluted at 9.4 min in greater than
93% purity. 120
Figure 3.34. Purity analysis of 5-NINDNTP (70).
1-(2',3',5'-Tri-O-benzoyl-β-D-ribofuranosyl)indoline (87). This known compound was prepared as previously described.25 Briefly, a solution of β-D-ribofuranose-
1-acetate-2,3,5-tribenzoate (49, 13.688 g, 27.13 mmoles) and indoline (86, 9.2 mL, 82.06 mmoles) in abs. EtOH (140 mL) containing glacial AcOH (9.5 mL) was refluxed for 6.5 h. The reaction was concentrated in vacuo, then redissolved in
CHCl3 (300 mL). The organic layer was washed with saturated aq. NaHCO3 (300 mL, 1x) and saturated aq. NaCl (300 mL, 1x), then dried over anhydrous MgSO4 and concentrated in vacuo. The crude material was MPLC purified (column A; step eluant: 100% benzene, followed by 5% EtOAc in benzene; flow rate: 150 mL/min) yielding a mixture of product 87 and a lower Rf (TLC) by-product. 121 Column chromatography (2% EtOAc in PhH) yielded product 87 (5.604 g, 37%
1 yield) as an off-white foam. H NMR (400.1 MHz, CDCl3): δ 8.11 (m, 2H), 7.97
(m, 4H), 7.60-7.33 (m, 9H), 7.06 (m, 2H), 6.78 (d. J = 7.8 Hz, 1H), 6.72 (m, 1H),
5.91 (m, 2H), 5.80 (m, 1H), 4.70 (m, 1H), 4.55 (m, 2H), 3.62 (m, 2H), 2.96 (m,
13 2H). C NMR (100.6 MHz, CDCl3): δ 166.2, 165.5, 165.3, 149.5, 133.5, 133.4,
133.3, 130.3, 129.8, 129.73, 129.68, 129.5, 129.1, 129.0, 128.6, 128.5, 128.4,
127.2, 125.0, 119.6, 107.9, 88.1, 78.3, 72.0, 70.6, 64.5, 45.4, 28.1. IR (film):
-1 + + 1724, 1265 cm . MS (ESI ) calcd. for C34H30NO7 [M+H] 564.2, found 564.2.
Anal. calcd. for C34H29NO7: C 72.46; H 5.19; N 2.49. Found C 72.52; H 5.46; N
2.76.
1-(2',3',5'-Tri-O-benzoyl-β-D-ribofuranosyl)indole (88). This known compound
was prepared by an adaptation of the previously reported procedure.25 In brief, a
solution of indoline ribonucleoside 87 (5.383 g, 9.55 mmoles) and manganese
(IV) oxide (31.877 g, 366 mmoles) in 200 mL of benzene was refluxed through a
Dean-Stark trap for 5 hours. The reaction was then filtered through a pad of
Celite, which was washed with excess EtOAc, and the filtrate concentrated in
vacuo. The crude material was purified stepwise, first via MPLC (column B;
eluant: 65% hexanes, 30% benzene, 5% Et3N; flow rate: 40 mL/min) then by 122
column chromatography (step gradient: 1:1 CH2Cl2:hexanes to 100% CH2Cl2)
yielding indole ribonucleoside 88 (2.928 g, 55% yield) as a white foam. 1H NMR
(400.1 MHz, CDCl3): δ 8.14 (m, 2H), 7.95 (m, 4H), 7.62-7.46 (m, 7H), 7.40-7.34
(m, 5H), 7.13 (m, 2H), 6.55 (d. J = 3.4 Hz, 1H), 6.49 (d, J = 5.8 Hz, 1H), 6.01 (m,
13 2H), 4.86-4.69 (m, 3H). C NMR (90.6 MHz, CDCl3): δ 166.2, 165.3, 165.0,
136.0, 133.61, 133.58, 133.36, 129.8, 129.7, 129.5, 129.4, 128.9, 128.7, 128.6,
128.5, 128.4, 124.1, 122.4, 121.2, 120.6, 109.9, 104.3, 87.5, 79.8, 73.9, 71.4,
-1 + + 63.9. IR (film): 1726, 1265 cm . MS (ESI ) calcd. for C34H27KNO7 [M+K]
600.1, found 600.0. Anal. calcd. for C34H27NO7: C 72.72; H 4.85; N 2.49. Found
C 72.59; H 4.90; N 2.61.
1-(β-D-Ribofuranosyl)-4-nitroindole (79). To a degassed solution of β-D-
ribofuranose-1-acetate-2,3,5-tribenzoate (49, 2.072 g, 4.11 mmol) in CH2Cl2 (15
27 mL) was added a solution of TiCl4 in CH2Cl2 (4.8 mL, 1.0 M). The reaction was
stirred at 23 °C for 2 h, then stopped by addition of deionized H2O (20 mL). The
organic layer was separated, and the water layer was re-extracted with CH2Cl2
(30 mL). The organic layers were combined, dried over anhydrous MgSO4, and
concentrated in vacuo. The crude material was dissolved in CH3CN (10 mL) and
used without further purification in the subsequent coupling step. Separately, a 123
suspension of 4-nitroindole (84, 2.213 g, 13.65 mmol) in CH3CN (40 mL) was
added to a suspension of NaH (95% dry, 196 mg, 8.17 mmol) in CH3CN (20 mL)
at 0 ºC. To the resulting dark red solution was added crude chlorosugar 50,
followed by additional CH3CN (5 mL). The reaction was allowed to warm to 23
°C and stirred for 22 h. The crude products were then poured into aq. citric acid
(1M, 100 mL) and EtOAc (100 mL) was added. The organic layer was
separated, washed with saturated aq. NaCl (100 mL), dried over anhydrous
MgSO4, and concentrated in vacuo. The crude material was partially purified by
column chromatography (20% EtOAc in hexanes) and used directly in the next
step. A pressure tube was charged with the reaction products and NH3 in MeOH
(40 mL, ca. 7 N). The tube was sealed and heated to 55 ºC for 25 h. The tube
was cooled, vented, and the contents filtered through a pad of Celite (washed with excess MeOH) and concentrated in vacuo. The crude material was purified by column chromatography (5% MeOH in CH2Cl2), then dried at 40 ºC over P2O5 yielding 79 (131 mg, 11% yield over 3 steps) as a bright yellow, glassy solid. mp
1 155-157 °C. H NMR (299.9 MHz, DMSO-d6): δ 8.16 (d, J = 8.3 Hz, 1H), 8.10
(dd, J = 0.6 Hz, J = 8.0 Hz, 1H), 8.03 (d, J = 3.3, 1H), 7.36 (app t, J = 8.1, 1H),
7.12 (d, J = 3.2 Hz, 1H), 6.00 (d, J = 6.1 Hz, 1H), 5.44 (d, J = 6.6 Hz, 1H), 5.23
(d, J = 4.8 Hz, 1H), 5.10 (t, J = 5.2 Hz, 1H), 4.28 (m, 1H), 4.09 (m, 1H), 3.97 (m,
13 1H), 3.70-3.56 (m, 2H). C NMR (90.6 MHz, DMSO-d6): δ 139.3, 137.9, 131.0,
122.3, 120.9, 118.3, 117.6, 101.8, 89.1, 85.4, 74.6, 70.3, 61.3. IR (KBr): 3379,
-1 -1 -1 1317, 1088 cm . UV (MeOH) 207 nm (λmax, ε = 33,100 M cm ), 371 nm (ε = 124 -1 -1 + + 6,100 M cm ). HRMS (ESI ) calcd. for C13H14N2O6Na [M+Na] 317.0750, found
317.0751.
1-(β-D-Ribofuranosyl)-6-nitroindole (80).23 To a degassed solution of β-D- ribofuranose-1-acetate-2,3,5-tribenzoate (49, 2.082 g, 4.13 mmol) in CH2Cl2 (15
27 mL) was added a solution of TiCl4 in CH2Cl2 (4.8 mL, 1.0 M). The reaction was
stirred at 23 °C for 2 h, then stopped by addition of deionized H2O (20 mL). The
organic layer was separated, and the water layer was re-extracted with CH2Cl2
(30 mL). The organic layers were combined, dried over anhydrous MgSO4, and
concentrated in vacuo. The crude material was dissolved in CH3CN (10 mL) and
used without further purification in the subsequent coupling step. Separately, a
solution of 6-nitroindole (85, 2.016 g, 12.43 mmol) in CH3CN (15 mL) was added
to a suspension of NaH (95% dry, 197 mg, 8.21 mmol) in CH3CN (20 mL) at 0
ºC. To the resulting dark red solution was added crude chlorosugar 50, followed by additional CH3CN (5 mL). The reaction was allowed to warm to 23 °C and
stirred for 22 h. The crude products were then poured into aq. citric acid (1M,
100 mL) and EtOAc (100 mL) was added. The organic layer was separated, washed with saturated aq. NaCl (100 mL), dried over anhydrous MgSO4, and concentrated in vacuo. The crude material was partially purified by column chromatography (20% EtOAc in hexanes) and used directly in the next step. A 125
pressure tube was charged with the reaction products and NH3 in MeOH (40 mL,
ca. 7 N). The tube was sealed and heated to 55 ºC for 25 h. The tube was
cooled, vented, and the contents filtered through a pad of Celite (washed with
excess MeOH) and concentrated in vacuo. The crude material was purified by
column chromatography (5% MeOH in CH2Cl2), then dried at 40 ºC over P2O5 yielding 80 (299 mg, 25% yield over 3 steps) as a yellow solid. mp 167–168 °C.
1 H NMR (300.1 MHz, DMSO-d6): δ 8.63 (d, J = 2.0 Hz, 1H), 8.05 (d, J = 3.3 Hz,
1H), 7.94 (dd, J = 2.1 Hz, J = 8.8 Hz, 1H), 7.75 (d, J = 8.8 Hz, 1H), 6.74 (d, J =
3.2 Hz, 1H), 6.03 (d, J = 6.2 Hz, 1H), 5.46 (d, J = 6.9 Hz, 1H), 5.24 (d, J = 5.0 Hz,
1H), 5.10 (t, J = 5.2 Hz, 1H), 4.27 (m, 1H), 4.09 (ddd [app dt], J = 3.2 Hz, J = 5.0
Hz, J = 5.0 Hz, 1H), 3.98 (m, 1H), 3.66-3.60 (m, 2H). 13C NMR (75.5 MHz,
DMSO-d6): δ 142.3, 134.2, 133.6, 132.2, 120.8, 114.8, 107.5, 103.2, 88.9, 85.4,
74.6, 70.4, 61.3. IR (KBr): 3328, 1322, 1120, 1036 cm-1. UV (MeOH) 212 nm
-1 -1 -1 -1 + (λmax, ε = 26,600 M cm ), 366 nm (ε = 6,200 M cm ). HRMS (ESI ) calcd. for
+ C13H14N2O6Na [M+Na] 317.0750, found 317.0755.
1-(β-D-Ribofuranosyl)-5-aminoindole (81). To a fine suspension of 5-nitroindole
63 (118 mg, 0.403 mmoles) in MeOH (30 mL) and glacial AcOH (0.5 mL) was
added Raney 2800 Ni (slurry in H2O, 0.3 mL). The solution was degassed with 126
N2, then charged with H2 (1 atm). The reaction was stirred at 23 °C under H2 (1
atm) for 23 h, then filtered through a pad of Celite (washed with excess MeOH)
and the filtrate concentrated in vacuo. The crude material was purified by
column chromatography (10% MeOH in CH2Cl2), then dissolved in dH2O (10 mL) and lyophilized to dryness (repeated 1x). Drying in vacuo at 23 °C over P2O5 yielded 81 (86 mg, 76% yield) as a light pink solid. mp 124-125 °C. 1H NMR
(299.9 MHz, DMSO-d6): δ 7.36 (d, J = 3.2 Hz, 1H), 7.21 (d, J = 8.7 Hz, 1H), 6.66
(d, J = 2.0 Hz, 1H), 6.51 (dd, J = 2.1 Hz, J = 8.6 Hz, 1H), 6.19 (d, J = 3.2 Hz, 1H),
5.70 (d, J = 6.0 Hz, 1H), 5.26 (d, J = 6.5 Hz, 1H), 5.08 (d, J = 5.0 Hz, 1H), 4.94 (t,
J = 5.3 Hz, 1H), 4.60 (br s, 2H), 4.21 (m, 1H), 4.02 (m, 1H), 3.84 (m, 1H), 3.58-
13 3.49 (m, 2H). C NMR (75.4 MHz, DMSO-d6): δ 141.8, 129.8, 129.6, 125.4,
111.9, 110.4, 103.6, 100.8, 88.7, 84.3, 73.5, 70.2, 61.6. IR (KBr): 3430, 3319,
-1 + + 3270, 3220 cm . HRMS (ESI ) calcd. for C13H17N2O4 [M+H] 265.1188, found
265.1190.
1-(β-D-Ribofuranosyl)indole (82).25,39 This compound was prepared by
modification of the previously reported procedure.25 A pressure tube was
charged with 88 (515 mg, 0.917 mmoles) and NH3 in MeOH (15 mL, ca. 7N),
then sealed and heated to 55 ºC for 16 h. The tube was cooled, vented, and the 127 contents concentrated in vacuo. The crude material was purified by column chromatography (5% MeOH in CH2Cl2), then recrystallized from hot dH2O (10
mL). The resulting white solid was collected and dried at 23 ºC over P2O5 yielding 82 (130 mg, 57% yield). mp 141-142 °C. 1H NMR (300.1 MHz, DMSO-
d6): δ 7.60 (d, J = 3.3 Hz, 1H), 7.55 (m, 2H), 7.13 (m, 1H), 7.04 (m, 1H), 6.50 (d,
J = 3.2 Hz, 1H), 5.87 (d, J = 6.0 Hz 1H), 5.33 (d, J = 6.6 Hz, 1H), 5.15 (d, J = 5.0
Hz, 1H), 5.00 (t, J = 5.3 Hz, 1H), 4.27 (m, 1H), 4.06 (m, 1H), 3.90 (m, 1H), 3.66-
13 3.52 (m, 2H). C NMR (75.5 MHz, DMSO-d6): δ 135.9, 128.6, 125.7, 121.4,
120.4, 119.7, 110.3, 102.1, 88.6, 84.7, 73.9, 70.3, 61.5. IR (KBr): 3431, 3374,
-1 + + 1083 cm . HRMS (ESI ) calcd. for C13H16NO4 [M+H] 250.1079, found
250.1088. Anal. calcd. for C13H15NO4: C 62.64; H 6.07; N 5.62. Found C 62.35;
H 6.08; N 5.62.
1-(β-D-Ribofuranosyl)indole-3-carboxamide (83). To a solution of 88 (289 mg,
41 0.515 mmoles) in MeCN (5 mL) at 3 ºC was added ClSO2NCO (49 µL, 0.563
mmoles) dropwise. The solution was stirred at 3 ºC for 5 min, then allowed to
gradually warm to 23 ºC. After 30 min, aq. NaOH was added (2 N, 10 mL), and
the reaction was stirred vigorously for 1 h. The solution was neutralized by
addition of DOWEX 50WX8-100 (H+) resin, filtered, and the filtrate concentrated 128 in vacuo. The crude material was purified by column chromatography (10 %
MeOH in CH2Cl2), redissolved in dH2O (10 mL) and lyophilized to dryness yielding 83 (53 mg, 35 % yield) as a white solid. mp 70-74 °C. 1H NMR (300.1
MHz, DMSO-d6): δ 8.23 (s, 1H), 8.16 (m, 1H), 7.62 (m, 1H), 7.44 (br s, 1H), 7.18
(m, 2H), 6.91 (br s, 1H), 5.88 (d, J = 6.0 Hz, 1H), 5.47 (d, J = 6.5 Hz, 1H), 5.24
(d, J = 5.1 Hz, 1H), 4.99 (t, J = 5.6 Hz, 1H), 4.26 (m, 1H), 4.08 (m, 1H), 3.95 (m,
13 1H), 3.69-3.56 (m, 2H). C NMR (75.5 MHz, DMSO-d6): δ 166.0, 136.2, 128.4,
127.0, 122.2, 121.4, 121.1, 111.0, 110.8, 88.8, 85.1, 74.1, 70.3, 61.6. IR (KBr):
-1 + + 3354, 1646, 1593 cm . HRMS (ESI ) calcd. for C14H16N2O5Na [M+Na]
315.0957, found 315.0960.
1-(β-D-Ribofuranosyl)pyrrolo[2,3-b]pyridine (65). This known compound was
prepared by the published method.44 mp 154-155 °C. 1H NMR (360.1 MHz,
DMSO-d6): δ 8.23 (dd, J = 1.5 Hz, J = 4.7 Hz, 1H), 7.99 (dd, J = 1.6 Hz, J = 7.8
Hz, 1H), 7.74 (d, J = 3.7 Hz, 1H), 7.13 (dd, J = 4.7 Hz, J = 7.8 Hz, 1H), 6.54 (d, J
= 3.6 Hz, 1H), 6.21 (d, J = 6.3 Hz, 1H), 5.28 (d, J = 6.5 Hz, 1H), 5.19 (m, 1H),
5.12 (d, J = 4.8 Hz, 1H), 4.46 (m, 1H), 4.10 (m, 1H), 3.90 (m, 1H), 3.65-3.50 (m,
13 2H). C NMR (90.6 MHz, DMSO-d6): δ 147.4, 142.3, 128.8, 126.9, 120.9, 116.4, 129 100.6, 87.1, 84.8, 73.6, 70.7, 61.8. IR (KBr): 3330, 1437, 1121 cm-1. HRMS
+ + (ESI ) calcd. for C12H15N2O4 [M+H] 251.1032, found 251.1021.
1-(β-D-Ribofuranosyl)-3-(prop-1-ynyl)-pyrrolo[2,3-b]pyridine (67). This ribonucleoside was prepared utilizing chemistry developed for the synthesis of
43 66. To a solution of 95 (554 mg, 1.47 mmoles) in CH2Cl2 (10 mL) was added
ICl (1.8 mL, 1.0 M solution in CH2Cl2). The reaction was stirred at 23 °C for 20
min, then poured into saturated aq. Na2S2O3 (50 mL). The solution was
extracted with CH2Cl2 (40 mL, 2x), dried over anhydrous MgSO4, and concentrated in vacuo into a pressure tube. The crude material was then suspended in Et3N (50 mL) and Pd(PPh3)2Cl2 (104 mg, 0.148 mmoles) and CuI
(42 mg, 0.220 mmoles) were added. The solvent was purged with dry N2 for 15 min, then cooled to ca. -78 °C. Liquified propyne (ca. 5 mL) was added to the pressure tube, which was sealed and allowed to gradually warm to 23 °C. The reaction was stirred for 4 hr at 23 °C, then cooled and vented to atmospheric pressure. The crude reaction products were poured into distilled water (75 mL), and extracted with CH2Cl2 (75 mL, 2x). The organic layer was dried over
anhydrous MgSO4, concentrated in vacuo, and partially purified by column 130 chromatography (15% EtOAc in PhH). The semi-pure material was then added
to a pressure tube and dissolved in NH3 in MeOH (15 mL, ca. 7N). The sealed
tube was heated to 55 °C for 23 hr, then cooled and filtered through a pad of
Celite (washed with excess MeOH). The filtrate was concentrated in vacuo, then
purified stepwise, beginning with column chromatography (5% MeOH in CH2Cl2) followed by preparative HPLC (PRP-1 column). The purification of 67 by HPLC utilized the following linear gradient (flow rate = 20 mL/min). The mobile phase comprised 10% to 70% CH3CN (containing 0.1% TFA) in ddH2O (containing
0.1% TFA) over 20 min. Ribonucleoside 67 eluted at 12.6 min. This isolated material was resubjected to column chromatography (5% MeOH in CH2Cl2), concentrated, then dried in vacuo at 40 °C over P2O5 yielding 67 as an off-white
solid (110 mg, 14% yield over 3 steps). mp 147-148 °C. 1H NMR (299.9 MHz,
DMSO-d6): δ 8.29 (dd, J = 1.6 Hz, J = 4.7 Hz, 1H), 8.00 (s, 1H), 7.97 (dd, J = 1.6
Hz, J = 7.8 Hz, 1H), 7.21 (dd, J = 4.7 Hz, J = 7.9 Hz, 1H), 6.20 (d, J = 6.1 Hz,
1H), 4.42 (m, 1H), 4.10 (m, 1H), 3.89 (m, 1H), 3.65-3.50 (m, 2H), 2.08 (s, 3H).
13 C NMR (75.5 MHz, DMSO-d6): δ 146.7, 143.5, 129.6, 127.8, 121.2, 117.1, 96.7,
87.6, 86.9, 85.0, 73.9, 72.0, 70.6, 61.5, 4.2. IR (KBr): 3421, 2938, 1446 cm-1.
+ + HRMS (ESI ) calcd. for C15H17N2O4 [M+H] 289.1188, found 289.1184.
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35. "Synthesis and antiviral evaluation of a mutagenic and non-hydrogen
bonding ribonucleoside analogue: 1-beta-D-ribofuranosyl-3-nitropyrrole." 136 Harki, D. A.; Graci, J. D.; Korneeva, V. S.; Ghosh, S. K. B.; Hong, Z.;
Cameron, C. E.; Peterson, B. R., Biochemistry 2002, 41, 9026-9033.
36. "The broad-spectrum antiviral ribonucleoside ribavirin is an RNA virus
mutagen." Crotty, S.; Maag, D.; Arnold, J. J.; Zhong, W.; Lau, J. Y. N.;
Hong, Z.; Andino, R.; Cameron, C. E., Nat. Med. 2000, 6, 1375-1379.
37. "Poliovirus RNA-dependent RNA polymerase (3D(pol)): Pre-steady-state
kinetic analysis of ribonucleotide incorporation in the presence of Mn2+."
Arnold, J. J.; Gohara, D. W.; Cameron, C. E., Biochemistry 2004, 43,
5138-5148.
38. "Evaluating the contributions of desolvation and base-stacking during
translesion DNA synthesis." Zhang, X. M.; Lee, I.; Berdis, A. J., Org.
Biomol. Chem. 2004, 2, 1703-1711.
39. "Glycosylindoles-VII. Synthesis of 1-(D-beta-ribofuranosyl)indole."
Preobrazhenskaya, M. N.; Vigdorchik, M. M.; Suvorov, N. N., Tetrahedron
1967, 23, 4653-4660.
40. "1-beta-D-ribofuranosylindole. The "indoline-indole" method applied to
indole nucleosides." Preobrazhenskaya, M. N.; Yartseva, I. V.; Ektova, L.
V., Nucleic Acid Chem. 1978, 2, 721-723.
41. "Reaction of indoles with chlorosulfonyl isocyanate; a versatile route to 3-
substituted indoles." Mehta, G.; Dhar, D. N.; Suri, S. C., Synthesis 1978,
374-376.
42. "Beyond A, C, G and T: augmenting nature's alphabet." Henry, A. A.;
Romesberg, F. E., Curr. Opin. Chem. Biol. 2003, 7, 727-733. 137 43. "Efforts toward expansion of the genetic alphabet: Optimization of
interbase hydrophobic interactions." Wu, Y. Q.; Ogawa, A. K.; Berger, M.;
McMinn, D. L.; Schultz, P. G.; Romesberg, F. E., J. Am. Chem. Soc. 2000,
122, 7621-7632.
44. "Inhibition of adenosine deaminase from several sources by deaza
derivatives of adenosine and EHNA." Lupidi, G.; Cristalli, G.; Marmocchi,
F.; Riva, F.; Grifantini, M., J. Enzyme Inhib. 1985, 1, 67-75.
45. "Production of "authentic" poliovirus RNA-dependent RNA polymerase
(3D(pol)) by ubiquitin-protease-mediated cleavage in Escherichia coli."
Gohara, D. W.; Ha, C. S.; Ghosh, S. K. B.; Arnold, J. J.; Wisniewski, T. J.;
Cameron, C. E., Protein Expr. Purif. 1999, 17, 128-138.
46. "Poliovirus RNA-dependent RNA polymerase (3D(pol)). Assembly of
stable, elongation-competent complexes by using a symmetrical primer-
template substrate (sym/sub)." Arnold, J. J.; Cameron, C. E., J. Biol.
Chem. 2000, 275, 5329-5336.
47. "NMR chemical shifts of common laboratory solvents as trace impurities."
Gottlieb, H. E.; Kotlyar, V.; Nudelman, A., J. Org. Chem. 1997, 62, 7512-
7515.
Chapter 4
5-SUBSTITUTED CYTIDINE DERIVATIVES: CHEMICAL SYNTHESES AND ANTIVIRAL ACTIVITIES
4.1 Overview
A series of 5-substituted cytidine ribonucleosides was rationally designed, chemically synthesized, and evaluated as antiviral lethal mutagens. The molecular design of these compounds was based on the known lethal mutagen,
5-hydroxy-2'-deoxycytidine (26, see section 1.5.2).1 Deoxyribonucleoside 26
enhances the frequency of G → A substitution mutations in HIV genomes by
mispairing with adenosine during viral replication. The resulting accumulation of
26-induced mutations forces HIV into error catastrophe and viral viability is lost.
The structural basis for miscoding mediated by 26 is the addition of the hydroxyl group to the pseudobase. This functionality directly alters the amino-imino tautomerization of the cytosine base, providing a new face for hydrogen-bonding with adenine. Based upon these known mutagenic effects, the RNA analogue, 5- hydroxycytidine (107), was chemically synthesized and evaluated as an RNA virus lethal mutagen. In addition to 107, a series of 5-substituted cytidine derivatives was synthesized to probe structure-activity relationships necessary for antiviral activity. One particular analogue, 5-nitrocytidine (109), exhibited potent antiviral activity against both poliovirus and coxsackievirus, surpassing the antiviral activity of the clinically-employed drug, ribavirin (25). 139 4.2 Oxidative DNA Damage and the Structural Basis for the Ambiguous Base-Pairing Properties of 5-hydroxy-2'-deoxycytidine (26)r
Hydroxylated cytosines are major hallmarks of oxidative DNA damage.
Hydroxyl radicals, produced from cellular oxidative damage, add into the 5-6
olefin of cytosine to form products such as cytosine glycol, a highly unstable
species in aqueous solution (Figure 4.1). Dehydration of the glycol yields 5-
hydroxycytosine, the pseudobase for the mutagenic deoxyribonucleoside 26.2,3
This form of oxidative damage can occur to cytidine residues located in the DNA template or to nucleotide pools of CTP, resulting in the formation of 5-OHdCyTP
(101). DNA damage resulting from CTP oxidation can be conferred by incorporation of 101 into the DNA genome by DNA polymerases (e.g., E. coli
Klenow fragment, exonuclease free).2,4 By either mechanism of induction, the
basal occurrence of hydroxycytosine in DNA has been estimated at
approximately 10 fmole/µg of DNA (in calf thymus DNA).3 Fortunately, multiple
repair enzymes exist for the removal of 5-hydroxy-2'-deoxycytidine residues from
DNA providing a mechanism for maintaining the integrity of the DNA genome.5
r The discovery of the mutagenic capacity of 5-hydroxy-2'-deoxycytidine (26) towards HIV is presented in section 1.5.2. 140
Figure 4.1. Oxidation by hydroxide radicals of (A) cytosine in the DNA template and (B) dCTP pools.
Lethal mutagenesis of HIV by 5-hydroxy-2'-deoxycytidine 26 exploits the infidelity of HIV reverse transcriptase (RT) to incorporate this mutagenic deoxyribonucleoside into viral DNA during first strand DNA synthesis.1 HIV-1 RT
catalyzes the incorporation of 5-OHdCyTP 101 opposite G and A in the DNA
template equivalently,6 whereas Klenow DNA polymerase favors the
incorporation of 101 opposite G (the correct base) over A by a factor of 1000.4
The enhanced base-pairing ambiguity of HIV RT-mediated DNA synthesis promotes lethal transition mutations during replication. Unlike cellular DNA, the repair of viral DNA containing mutations is highly disfavored. DNA repair enzymes are located in the nucleus of cells, whereas the synthesis of (-)- stranded viral DNA occurs in the cytoplasm. Additionally, DNA repair enzymes recognize mutations in double-stranded DNA, a complex whose overall structure 141 is vastly different from the RNA•DNA duplex utilized for viral DNA synthesis.1
The resulting non-repaired accumulation of mutations in the HIV genome forces the virus into error catastrophe and viral viability is lost.
The structural basis for the ambiguous base-pairing capabilities of 5-
OHdCy 26 relates to the amino-imino tautomerization of the 5-hydroxycytosine pseudobase (Figure 4.2). Introduction of the 5-hydroxy moiety places an acidic alcohol (pKa = 7.37) directly on cytosine, which influences the tautomerization
7 between N-3 and the exocyclic NH2 (on carbon 4) of cytosine. At physiological
pH, the 5-hydroxy substituent exists in both the protonated and anionic forms (ca.
1:1 ratio), with each having distinct contributions to the electronics and H-bonding
patterns of the pseudobase. The electron-withdrawing 5-hydroxy group lowers the pKb of N-3 from 4.31 to 3.75, diminishing the strength of the H-bonding
interaction with N-1 on guanosine (104).7 Additionally, when ionized to the anion,
the inductive effect of the 5'-OH functionality is reversed, favoring the formation
of the imino tautomer.8 This reversal in H-bonding patterns alters the base-
pairing properties of the cytosine pseudobase, permitting H-bonding with adenosine (102, Figure 4.3), which is hypothesized to contribute to the mutagenic activities of 5-hydroxy-2'-deoxycytidine (26). The presence of the imino tautomer of 26 has been detected by Raman spectroscopy and is estimated to exist at a frequency of approximately 0.3 – 0.7%,8 which has been
roughly correlated to the frequencies of mutations induced by 5-OHdCy 26.8,9
142
Figure 4.2. Tautomerization of 5-hydroxy-2'-deoxycytidine.
Figure 4.3. Watson-Crick hydrogen bonding patterns (A & B). Mispairing of 5- hydroxycytosine with adenosine (C).
4.3 Molecular Design of 5-Substituted Cytidines
We hypothesized that 5-hydroxycytidine 107 could accelerate the rate of
mutagenesis in RNA viruses by mispairing with adenosine during viral replication.
This mechanism of antiviral activity would parallel that observed with the 2'-deoxy
predecessor 26: phosphorylation in vivo to the 5'-triphosphate, misincorporation
into the viral RNA genome opposite A, and enhancement of viral genomic
mutations. In addition to the target ribonucleoside 107, a series of related 5-
substituted ribonucleosides was rationally designed, chemically synthesized, and
evaluated biochemically as antiviral lethal mutagens (Figure 4.4). 5- 143 Bromocytidine 108 was examined to probe the effects of large, aprotic, electronegative substituents at the 5-position of cytidine. In a similar manner, 5-
nitrocytidine 109 possesses aprotic, electron-withdrawing properties, but lacks
the inductive effects of the aryl halide. The amino analogue, 5-aminocytidine
110, was designed with an analogous hydrogen-bonding functionality at the 5-
position (compared with 107), but with a higher pKa. In addition to the
ribonucleoside analogues, two well-known deoxyribonucleosides 5-OHdCy 26
and 5-BrdCy 106 were screened for antiviral activity against RNA viruses. The 5- bromo-2'-deoxycytidine (106) is a known antiviral therapeutic, selectively inhibiting both varicella-zoster virus10,11 and herpes simplex viruses 1 and 2.12,13
Figure 4.4. 5-Substituted cytidine nucleosides evaluated for antiviral activity. 144 4.4 Chemical Syntheses of 5-Substituted Cytidine Ribonucleosides
4.4.1 Preparation of 5-hydroxycytidines (107) and 5-bromocytidine (108)
The target ribonucleoside 5-hydroxycytidine 105 was originally synthesized in the early 1960's for use as an antimicrobial agent and exhibited inhibitory properties towards the growth of E. coli K-12. The previously reported synthesis of 107 utilized aqueous bromine to brominate the 5-6 olefin of cytosine followed by passage through a column of strongly-basic resin (–OH) to yield the vinyl hydroxide.14 This procedure was reproduced to generate the material described herein, with the only deviation being the substitution of DOWEX 550A
(–OH) resin in place of the Amberlite IRA-4B (–OH) resin utilized previously. The regiochemistry of the hydroxylation was verified by 2-dimensional NMR spectroscopy (NOESY) by observing the presence of nuclear Overhauser effects
(nOe) between H-6 on cytosine and H-1' (anomeric proton) on ribose
(Figure 4.5). The 5-bromocytidine (108) that was evaluated for antiviral activity in this study was purchased from Sigma (however, the manufacture of 108 was recently discontinued). As an alternative, multi-gram quantities of 108 can be synthesized by reaction of cytidine (dissolved in pyridine) with bromine in carbon tetrachloride as previously described.15,16
s William J. Chain (undergraduate, Peterson Laboratory) contributed to this synthesis. 145
Figure 4.5. Synthesis of 5-OHCy (107).
4.4.2 Previous Syntheses of 5-nitrocytidine (109) and 5-aminocytidine (110)
Only one synthesis of 5-nitrocytidine (109) has been reported to date in the literature, and no studies regarding biochemical applications of 109 have
been described (Figure 4.6). The previously published synthesis of 109 began
with 5-nitrocytosine 112, which was converted to the mercury salt 113 by
deprotonation with sodium hydroxide followed by reaction with mercury (II) chloride. This intermediate was isolated, dried, and then coupled with chlorosugar 50 in refluxing toluene in 82% yield. Saponification of the benzoyl esters yielded 5-nitrocytidine in good yield. The related 5-aminocytidine 110 was prepared in one step from 109 by hydrogenation of the nitro functionality with 5% palladium on carbon (Pd/C).17
146
Figure 4.6. Synthesis of 5-nitrocytidine and 5-aminocytidine by Fox and coworkers.17
Similar to the related 5-nitrocytidine nucleoside 109, the 5-aminocytidine
nucleoside 110 is essentially unexplored. Excluding the synthesis described in
Figure 4.6, three additional reports describing syntheses of 5-aminocytidine (110)
have been published. In each case, ribonucleoside 110 was prepared in one
step from 5-bromocytidine 108 by reaction with ammonia at elevated
temperatures in a pressure bomb.15,16,18 Additionally, only one report on the
biological activity of 110 has been described, providing qualitative evidence that
110 inhibits the growth of the fungus Neurospora.18
4.4.3 An Improved Chemical Synthesis of 5-nitrocytidine (109) and 5- aminocytidine (110)
4.4.3.1 Vorbrüggen Coupling Chemistry
A major advance in the field of nucleoside chemistry was the development
of the Vorbrüggen coupling reaction.19 In this methodology, a persilylated base, 147 such as 116, is reacted directly with a fully protected sugar, such as 49, in the presence of Friedel-Crafts catalysts to form the anomeric bond (Figure 4.7). The scope and utility of this procedure has been exploited for the syntheses of diverse nucleoside analogues, yielding a vast literature for tailoring of reaction
conditions to specific substrates.20 For pyrimidine synthesis, the Vorbrüggen
coupling eliminates the necessity for pre-forming the unstable 1-chlorosugar 50
and obviates the use of mercury salts, which are toxic and frequently difficult to
remove.19 Additionally, high levels of stereocontrol are maintained in this
strategy, which is amenable to multi-gram scale-up reactions. Based upon these
favorable attributes, 5-nitro and 5-aminocytidine were prepared using a
Vorbrüggen coupling to form the anomeric bond. 148
Figure 4.7. Generalized Vorbrüggen coupling for pyrimidines.
4.4.3.2 Chemical Synthesis of 5-nitrocytidine (109) and 5-aminocytidine (110)
The chemical synthesis of 5-nitrocytidine (Figure 4.8) began with
preparation of the 5-nitrocytosine base (112). Utilizing the known literature
procedure, cytosine (115) was nitrated with a mixture of concentrated sulfuric
acid and fuming nitric acid at 80 °C to arrive at 5-nitrocytosine (112).21 This material was meticulously dried by heating to ca. 75-80 °C over P2O5 under high
vacuum (ca. 0.5 Torr), then silylated by reaction with refluxing 149 hexamethyldisilazane (HMDS) containing a catalytic amount of
chlorotrimethylsilane (TMSCl).20,22 The crude reaction product (compound 121)
was concentrated in vacuo under high vacuum (ca. 0.4 Torr) for 36 hours, then
subjected to Vorbrüggen coupling with commercially-available sugar 49 utilizing
20,22 tin (IV) chloride (SnCl4) as the Friedel-Crafts catalyst. Upon completion of the
reaction, water was added to hydrolyze the SnCl4, which facilitated the
precipitation of product 114 with continued stirring. Filtration of the precipitated
solid followed by a series of basic liquid-liquid extractions, chloroform triturations,
then drying yielded 114 in 78% yield (from 5-nitrocytosine 112) without the need
for silica gel chromatography. The benzoyl-protected 5-aminocytidine 122 was
synthesized by hydrogenation17 of 114 with 10% Pd/C in a mixture of glacial
acetic acid (AcOH) and tetrahydrofuran (THF) in 70% yield. Saponification of the
protected intermediates 114 and 122 with aqueous sodium hydroxide in ethanol17 followed by purification yielded the target 5-nitrocytidine ribonucleoside 109 (52% yield) and 5-aminocytidine 110 (48% yield). 150
Figure 4.8. Chemical synthesis of 5-nitrocytidine (109) and 5-aminocytidine (110).
4.5 Cytotoxicity of 5-Substituted Cytidinestu
The six cytidine analogues shown in Figure 4.4 were evaluated for cytotoxicity in HeLa S3 cells grown in culture (Figure 4.9). The most cytotoxic nucleoside analogue under the conditions of this experiment was 5- hydroxycytidine (107), which yielded cell viabilities ranging from 31 - 40% across the four concentrations tested. Interestingly, 5-hydroxy-2'-deoxycytidine (26) was significantly less cytotoxic with > 73% cell viability observed for all treatments.
The least cytotoxic ribonucleoside tested in this experiment was 5-nitrocytidine
t Experimental data from Jason D. Graci (Cameron Lab, unpublished results). Experimental conditions described in Chapter 3. u 5-OHdCy (26) and 5-BrdCy (106) were purchased from Berry & Associates (Dexter, MI) 151 (109) yielding cell viabilities ranging from 76 – 87% for the concentrations
examined.
Figure 4.9. Cytotoxicity of cytidine analogues towards HeLa S3 cells (Cell monolayers were treated with nucleosides for 7 hours, then allowed to recover in the absence of drug for 24 hours prior to measurement of cell viability).
4.6 Antiviral Activity of 5-Substituted Cytidinesv
The ribonucleoside analogues shown in Figure 4.4 were evaluated for
antiviral activity against HeLa cells infected with poliovirus and coxsackievirus B3
v Experimental data from Jason D. Graci (Cameron Lab, unpublished results). Experimental conditions described in chapter 3. 152 (Figure 4.10). As a control, the known antiviral drug ribavirin (25) was assayed in
this experiment. Both deoxyribonucleosides 5-OHdCy 26 and 5-BrdCy 106
(shown in blue and purple) were completely inactive against either RNA virus.
The RNA analogue of the known lethal mutagen, 5-OHCy 107, exhibited modest antiviral activity, but was inferior to the activity of ribavirin. 5-Aminocytidine (110)
exhibited good activity against both poliovirus and coxsackievirus B3, paralleling
the activity observed with ribavirin. The best compound in this assay was 5-
nitrocytidine (109). Remarkably, this ribonucleoside analogue exhibited better
antiviral activity than ribavirin (25), yielding ca. 30-fold enhanced reduction in PV
titer and ca. 10-fold reduction in CVB3 titer compared with 25 at the highest
concentration tested.
Figure 4.10. Antiviral activity of cytidine analogues towards poliovirus and coxsackievirus B3 infected HeLa cells. 153 4.7 Kinetics of Incorporation of 5-nitrocytidine Triphosphate into the Viral (PV) RNA Genome
4.7.1 Synthesis of 5-nitrocytidine Triphosphate
The triphosphate of 5-nitrocytidine was synthesized to determine if this
nucleotide could function as a substrate for viral RNA-dependent RNA
polymerases. Utilizing the one-pot, three-step phosphorylation procedure23-30 discussed in detail in Chapter 5, 5-nitrocytidine triphosphate (123) was prepared in 12% overall yield (Figure 4.11).
Figure 4.11: Synthesis of 5-nitrocytidine triphosphate.
4.7.2 Incorporation of 5-NO2CyTP (123) and 5-BrCyTP (124) by Poliovirus RNA-Dependent RNA Polymerasew
The ability of 5-nitrocytidine triphosphate (123) to function as a substrate
for RNA-dependent RNA polymerases was measured with the primer-extension
w Experimental data from Jason D. Graci (Cameron Lab, unpublished results). Experimental conditions described in chapter 3. 154 assay previously described (see section 1.5.4.3). As a comparison, the
incorporation characteristics of 5-bromocytidine triphosphatex 124 were
measured as well (Figure 4.12). Both 5-nitrocytidine and 5-bromocytidine
triphosphates incorporated opposite guanosine in the RNA template as expected
for a cytidine analogue. Additionally, both compounds misincorporated opposite templated adenosine, supporting the hypothesis that 5-substituted analogues such as 109 and 108 can adopt hydrogen-bonding tautomers that base-pair with adenosine. Neither 123 nor 124 incorporated opposite the pyrimidines C and U.
Although the kinetics of incorporation have not been calculated, qualitative analysis of the gel images suggests that 5-BrCyTP is incorporated more efficiently than 5-NO2CyTP by the RNA-dependent RNA polymerase of
poliovirus.
x Purchased from TriLink BioTechnologies (San Diego, CA) 155
Figure 4.12. Incorporation of 5-nitrocytidine triphosphate and 5-bromocytidine triphosphate into symmetrical primer-templates by poliovirus RNA-dependent RNA polymerase.
156 4.8 Intracellular Phosphorylation of 5-nitrocytidine to the Corresponding Triphosphate (5-NO2CyTP)
Although biochemical studies in vitro have demonstrated that 5-NO2CyTP
(123) functions as a substrate for poliovirus RNA-dependent RNA polymerases, multiple enzymatic phosphorylations of intracellular 5-nitrocytidine (109) must occur successfully to render nucleotide 123 in vivo. To determine if 5- nitrocytidine (109) is phosphorylated in cells to the 5'-triphosphate 123, HeLa S3 cells were incubatedy with 5-nitrocytidine and the composition of intracellular
nucleotide pools were evaluated by reversed-phase HPLC (see experimental
section 4.10 for details). Analysis of 5-nitrocytidine treated cells (Figure 4.13, c)
revealed the presence of a new peak, characteristic in retention time and UV
properties to the triphosphate standard (123, Figure 4.13, a). Analysis of
untreated (DMSO) cells under identical conditions lacked this peak (Figure 4.13,
b). To verify the identity of the 5-nitrocytidine triphosphate peak observed in
Figure 4.13 (c, dashed box), an identical aliquot of this material was doped with
5-NO2CyTP 123 and subjected to the same analysis conditions (Figure 4.14, c).
If the 5-nitrocytidine triphosphate (123) peak from the HeLa cell extracts is correctly assigned, doping the sample with additional 123 will increase the area
under the peak. Separately, standard integration of the 5-NO2CyTP peak from
HeLa cell extracts yielded a value of 226 mAU•sec (Figure 4.14, a), whereas
0.56 nmoles of the 5-NO2CyTP standard integrated for 120 mAU•sec
y The cellular component of this experiment was performed by Jason D. Graci (Cameron Lab). 157
(Figure 4.14, b). Integration of the 5-NO2CyTP peak following HPLC analysis of
premixed HeLa cell extract (Figure 4.14, a) and nucleotide standard (Figure 4.14,
b) yielded a value of 342 mAU•sec (Figure 4.14, c). This experimental value
(342 mAU•sec) agrees almost perfectly with the theoretical value of 346 mAU•sec, calculated by addition of the individual components shown in
Figure 4.14 a & b. The results from this doping experiment, coupled with the characteristic retention times and UV properties shown in Figure 4.13, confirm that 5-nitrocytidine (109) is phosphorylated in vivo to the triphosphate 5-
NO2CyTP (123). 158
Figure 4.13. Reversed-phase HPLC analysis of HeLa cell extracts. (a) Trace of 5-NO2CyTP (123, 0.56 nmoles) with characteristic UV trace (inlay). (b) Separation of untreated HeLa cell extracts (90 µL injection). (c) Separation of HeLa cell extracts (90 µL injection) that were treated with 5-nitrocytidine (2 mM, 3 hr); (c, inlay) UV trace characteristic of 123. The absorbance wavelength for all HPLC traces is 295 nm. 159
Figure 4.14. Reversed-phase HPLC analysis of HeLa cell extracts. (a) Close-up view of the 5-nitrocytidine triphosphate peak from Figure 4.13, c (90 µL of cell extracts were analyzed). (b) Close-up view of the 5-nitrocytidine triphosphate standard from Figure 4.13, a (0.56 nmoles of 123 was injected). (c) Analysis of HeLa cell extracts (90 µL) that were treated with 5-nitrocytidine (2 mM, 3 hr) doped with 0.56 nmoles of 5-nitrocytidine triphosphate. The absorbance wavelength for all HPLC traces is 295 nm.
4.9 Discussion and Future Directions
Despite the numerous syntheses of base-modified ribonucleosides, little biochemical data has been obtained with the relatively simple 5-nitrocytidine and
5-aminocytidine analogues. Both compounds exhibit strong antiviral activity against poliovirus and coxsackievirus B3, with nitro analogue 109 surprisingly surpassing the antiviral activity of the drug ribavirin (25). The therapeutic potential of 5-nitrocytidine is supplemented by the low cellular cytotoxicity observed in cell culture experiments, warranting additional studies to elucidate its mechanism of action. 160 The triphosphate of 5-nitrocytidine (123) is a substrate for the viral RNA-
dependent RNA polymerase of poliovirus, misincorporating opposite G and A in
the (in vitro) primer-extension assay. This incorporation profile supports the
hypothesis that 5-substituted ribonucleosides with electron-withdrawing
functionalities can alter the amino-imino base-pairing properties of the cytosine base, thereby permitting hydrogen-bonding with adenosine (see section 4.2).
However, the inefficient incorporation of 123 into the primer-template suggests that lethal mutagenesis of viral genomes may not be the primary mechanism of viral inhibition. This hypothesis is supported by the lack of guanidine resistant variants obtained when 5-nitrocytidine was subjected to the guanidine resistance assayz (see section 1.5.4.4), which has been used to demonstrate the mutagenic
capacity of ribavirin. Although it has been demonstrated that 5-nitrocytidine is
phosphorylated in vivo to the 5'-triphosphate 123, additional studies are
necessary to (1) determine if the triphosphate is the active metabolite, and (2)
identify the molecular target(s) that confers antiviral activity.
Previous biochemical studies with 5-nitro-2'-deoxycytidine (125,
Figure 4.15) have revealed antiviral activities against herpes simplex virus-1
(MIC50 = 0.2-0.3 µg/mL) and herpes simplex virus-2 (MIC50 = 0.2-0.3 µg/mL), as well as inhibitory effects on the growth of mouse leukemia L1210 cells (ID50 = 2.7
µg/mL).31 A recent study examined both 5-nitro (125) and 5-amino-2'-
deoxycytidines (126) as antiviral therapeutics against HIV-1 and hepatitis B virus,
z Unpublished results, Jason D. Graci (Cameron lab) 161 but found no activity against either virus for 5-amino-2'-deoxycytidine, whereas 5-
nitro-2'-deoxycytidine inhibited HIV-1 growth in MT-4 cells with an CC50 of 30
µM.32 Unfortunately, no attempts were made to elucidate the antiviral molecular
mechanisms of action of 125 in either study. Nonetheless, these earlier studies,
coupled with our findings, have revealed that nucleoside analogues possessing
5-nitrocytosine pseudobases are promising lead for the development of novel
antiviral therapeutics.
Figure 4.15. Structures of 5-nitro-2'-deoxycytidine and 5-amino-2'-deoxycytidine.
4.10 Experimental Section
Cell treatment and preparation of cell extracts. This procedure is a modification
of the method described by Pogolotti and Santi.33 HeLa S3 cells were
maintained in DMEM/F-12 supplemented with 2% dialyzed fetal bovine serum and penicillin/streptomycin (1X, Invitrogen). Prior to initiation of the experiment,
HeLa cells (7.5 x 106) were plated in 100 mm dishes (50% confluency) and
grown for 24 hours at 37 °C. The media was removed, replaced with fresh media 162 (5 mL) containing actinomycin D (2.5 µg/mL, inhibits cellular transcription), and incubated for 15 min at 37 °C. The cells were then treated with either 5- nitrocytidine (109, 2 mM in DMSO) or DMSO (control) and incubated for an additional 3 hr at 37 °C. The final concentration of DMSO in the media following treatment was 1%. The media was again removed, and the cells were washed with PBS (1 mL) and dissociated by treatment with trypsin (1X, Invitrogen). The dissociated cells were centrifuged to a pellet (ca. 5,000 g for 2 min), then resuspended in aqueous trichloroacetic acid (0.6 M, 200 µL), and incubated on ice for 10 minutes. Important: the following steps must be performed on ice or in a cold room. The resulting supernatant was collected and 1,1,2- trichlorotrifluoroethane (200 µL) containing tri-n-octylamine (0.5 M) was added.
The solution was mixed by vortexing, then centrifuged at 12,000 g for 30 sec.
The aqueous upper phase of this extraction was collected, frozen, and stored at
-80 °C until immediately prior to analysis.
Analysis of cell extracts by reversed-phase HPLC. HeLa cell extracts were analyzed on a Hewlett Packard 1100 series instrument equipped with an Aquasil
C18 analytical column (4.6 x 250 mm, 5 µm; Keystone Scientific Inc., [Thermo
Electron Corp]) running the following mobile phase (flow rate = 1 mL/min): isocratic 1% CH3CN in KH2PO4 buffer (0 to 5 min, 100 mM KH2PO4, pH = 6), gradient 1% - 15% CH3CN in KH2PO4 buffer (5 to 20 min), 15% - 80% CH3CN in
KH2PO4 buffer (20 to 25 min). The phosphate buffer was prepared by dissolving
KH2PO4 in double distilled water at 100 mM and adjusting the pH to 6.0 by 163 addition of aqueous KOH (10% solution). The integration of peak areas and
generation of 3D UV plots were obtained from the ChemStation for LC 3D
software (Rev.A.09.03, Agilent Technologies).
General synthesis information. All reactions were performed under a nitrogen
atmosphere unless otherwise noted. Commercial grade reagents (Aldrich,
Acros) were used without further purification unless specifically noted.
Tetrahydrofuran, acetonitrile, and N,N-dimethylformamide were rendered
anhydrous by passing through the resin column of a solvent purification system
(GlassContour; Laguna Beach, CA). Column chromatography employed ICN
SiliTech silica gel (32-63 µm). HPLC purification was performed an Agilent 1100
series instrument (preparative scale) equipped with an Aquasil C18 preparative
column (21.2 x 250 mm, 5 µm; Thermo Electron Corporation). Analysis of nucleotide purity was conducted on a Hewlett Packard 1100 series instrument
(analytical scale) equipped with an Aquasil C18 analytical column (4.6 x 250 mm,
5 µm; Keystone Scientific Inc., [Thermo Electron Corp]) running the following
mobile phase (flow rate = 1 mL/min): gradient 1 to 80% CH3CN in KH2PO4 (0 to
40 min, 100 mM KH2PO4, pH = 6). Nuclear magnetic resonance (NMR)
spectroscopy employed Bruker CDPX-300, DPX-300, AMX-360, DRX-400, or
AMX-2-500 MHz spectrometers. Internal solvent peaks were referenced in each
13 31 case. Chemical shifts for C NMR and P NMR analyses performed in D2O
34 were indirectly referenced to 10% acetone in D2O (CH3 set to 30.89 ppm) and
13 85% H3PO4 (0 ppm), respectively. C chemical shifts for nucleoside diphosphates and triphosphates denoted with a (*) fail to resolve into clean 164 singlets due to apparent conformational restrictions. Mass spectral data was
obtained from either The University of Texas at Austin Mass Spectrometry
Facility (FAB, ESI and CI) or The Pennsylvania State University Mass
Spectrometry Facility (ESI and APCI). Elemental analyses were performed by
Midwest Microlab, LLC (Indianapolis, IN). Melting points are uncorrected.
5-hydroxycytidine hydrate (107). This is a known compound prepared according
14 1 to the method of Fukuhara and Visser. H NMR (DMSO-d6, 400.1 MHz): δ 8.91
(br s, 1H), 7.36 (br s, 1H), 7.23 (s, 1H), 6.77 (s, 1H), 5.78 (d, J = 4.5 Hz, 1H),
5.23 (br s, 1H), 5.00 (m, 2H), 3.90 (m, 2H), 3.78 (m, 1H), 3.61-3.51 (m, 2H). 13C
NMR (DMSO-d6, 100.6 MHz): δ 160.9, 154.2, 126.6, 122.3, 88.8, 84.2, 73.8,
+ + 70.0, 61.2. MS (APCI ) calcd. for C9H14N3O6 [M+H] 260.1, found 260.1. Anal.
calcd. for C9H15N3O7: C 38.99; H 5.45; N 15.16. Found: C 38.79; H 5.45; N
14.77. 165
1-(2',3',5'-Tri-O-benzoyl-β-D-ribofuranosyl)-5-nitrocytidine (114).17
5-nitrocytosine21 (112, 3.009 g, 19.28 mmoles) was suspended in HMDS (40 mL,
191.8 mmoles) containing TMSCl (1.0 mL, 7.82 mmoles).20,22 The mixture was
refluxed for 36 h then concentrated in vacuo under high vacuum (ca. 0.4 Torr).
To this crude material was added β-D-ribofuranose-1-acetate-2,3,5-tribenzoate
(49, 9.737 g, 19.30 mmoles) and the material was suspended in MeCN (125 mL).
The suspension was degassed with N2, then SnCl4 (23 mL, 1.0 M solution in
20,22 CH2Cl2) was added. The suspension quickly clarified and the solution was
stirred at 23 °C for 3 h. Distilled water (dH2O, 100 mL) was added with vigorous
stirring to hydrolyze the remaining SnCl4, which facilitated the precipitation of the
product. The solid was collected, suspended in EtOAc (500 mL) and washed with saturated aq. K2CO3 (100 mL, 4x). The organic layer was diluted with
EtOAc (to 3.2 L) and washed with saturated aq. NaCl (800 mL, 1x) and dH2O
(800 mL, 2x). The organic layer was dried over anhydrous MgSO4 and
concentrated in vacuo. The resulting solid was triturated with warm CHCl3 (100 mL, 2x) and dried in vacuo yielding 114 (9.008 g, 78% yield over 2 steps) as a
1 cream colored solid. mp 211-213 °C (blackened) H NMR (360.1 MHz, CDCl3):
δ 9.07 (s, 1H), 8.44 (br s, 1H), 8.07 (m, 2H), 7.91 (m, 5H), 7.52 (m, 3H), 7.42 (m,
2H), 7.33 (m, 4H), 6.34 (d, J = 3.6 Hz, 1H), 5.94 (m, 2H), 4.87-4.74 (m, 3H). 13C 166
NMR (90.6 MHz, CDCl3): δ 166.2, 165.21, 165.19, 157.8, 152.0, 146.4, 133.73,
133.68, 133.5, 129.9, 129.8, 129.7, 129.1, 128.6, 128.54, 128.49, 128.42, 120.2,
90.5, 81.1, 74.9, 70.9, 63.4. IR (film): 1727, 1646, 1267 cm-1. HRMS (ESI+)
+ calcd. for C30H24N4O10Na [M+Na] 623.1390, found 623.1387.
1-(2',3',5'-Tri-O-benzoyl-β-D-ribofuranosyl)-5-aminocytidine (122). To a
degassed (N2) solution of 114 (1.035 g, 1.72 mmoles) in THF (65 mL) and glacial
AcOH (5 mL) was added Pd/C (10%, 372 mg).17 The solution was degassed
again, charged with H2 (1 atm), and stirred for 7 h at 23 ºC. The reaction was
filtered through a pad of Celite, washed with excess MeOH, and concentrated in
vacuo. The crude material was purified by column chromatography (10% MeOH
in EtOAc) yielding 122 (624 mg, 63% yield) as a beige, glassy solid. mp 153-157
1 °C H NMR (299.9 MHz, CDCl3): δ 8.10 (m, 2H), 7.91 (m, 4H), 7.53-7.29 (m,
9H), 6.98 (s, 1H), 6.35 (d, J = 4.5 Hz, 1H), 5.91 (m, 1H), 5.81 (m, 1H), 4.87-4.58
13 (m, 3H), 2.81 (br s, 2H, D2O exchangeable). C NMR (75.4 MHz, CDCl3): δ
166.1, 165.44, 165.35, 163.0, 155.0, 133.6, 133.5, 129.9, 129.8, 129.7, 129.4,
128.8, 128.68, 128.67, 128.4, 126.5, 115.3, 89.0, 79.7, 74.2, 71.2, 63.8. IR
-1 + (film): 3342, 3196, 3068, 1726, 1268 cm . HRMS (ESI ) calcd. for C30H27N4O8
[M+H]+ 571.1829, found 571.1833. 167
5-Nitrocytidine (109). Protected ribonucleoside 114 (1.27 g, 2.12 mmoles) was
o suspended in aq. EtOH (50 mL, 4:1 absolute EtOH:distilled H2O) at 23 C. Three
portions of NaOH (2.1 mL each, 1 N solution, 6.3 mmoles total) was added at 20
min intervals and the solution was stirred for 3 h.17 The reaction was then
acidified to pH ca. 2 with aqueous HCl (1 N) and the EtOH was removed in
vacuo. The resulting material was diluted to a total volume of ca. 75 mL by addition of distilled H2O and warmed to resolubilize the material. The aqueous
layer was extracted with CHCl3 (50 mL, 3x) then neutralized to pH ca. 8 with aq.
NH4OH (10%). The material was concentrated in vacuo until a small amount of
precipitate was observed, then cooled to 4 °C. The white solid was collected,
washed with a minimal amount of water (ca. 10 mL), then redissolved in hot distilled H2O (50 mL). The solution was quickly frozen and lyophilized to dryness.
This step was repeated one additional time. The lyophilized material was dried in
vacuo at 23 °C over P2O5 yielding 109 (318 mg, 52% yield) as a white solid. mp
1 129-131 °C (shrank at ca. 100 °C) H NMR (360.1 MHz, DMSO-d6): δ 9.72 (s,
1H), 8.48 (s, 1H), 8.03 (s, 1H), 5.69 (s, 1H), 3.97 (m, 3H), 3.82-3.59 (m, 2H). 13C
NMR (90.6 MHz, DMSO-d6): δ 157.2, 151.8, 147.4, 119.4, 91.1, 83.8, 74.7, 67.6,
-1 -1 -1 58.8. IR (KBr): 3323, 1648 cm . UV (H2O) 227 nm (λmax, ε = 21,900 M cm ), 168
-1 -1 + + 321 nm (ε = 10,900 M cm ). HRMS (ESI ) calcd. for C9H13N4O7 [M+H]
289.0784, found 289.0778.
5-Aminocytidine (110). Protected ribonucleoside 122 (1.12 g, 1.97 mmoles) was
suspended in aq. EtOH (50 mL, 4:1 absolute EtOH:distilled H2O) at 23 °C. Three
portions of NaOH (2.0 mL each, 1 N solution, 6.0 mmoles total) was added at 20
min intervals and the solution was stirred for 3 h.17 The reaction was then
acidified to pH ca. 2 with HCl (1 N) and the EtOH was removed in vacuo. The
residual water layer was warmed to resolubilize the material then extracted with
CHCl3 (40 mL, 3x). The aqueous layer was neutralized to pH ca. 8 with aq.
NH4OH (10%) then concentrated in vacuo. The crude material was dissolved in
distilled H2O (5 mL) followed by addition of absolute EtOH (50 mL) which yielded
a cloudy solution. The solution was cooled to -20 °C and the material was
allowed to precipitate. The resulting solid was collected, washed with excess
Et2O and set aside. Recrystallization of the mother liquor (2 additional crops)
was achieved by redissolving the concentrated mother liquor in distilled H2O (3
mL) followed by addition of absolute EtOH (50 mL), then cooling to -20 ºC. The
three recrystallization batches were pooled together, dissolved in distilled H2O
(15 mL), and lyophilized to dryness. This step was repeated one additional time. 169
The lyophilized material was dried in vacuo at 23 °C over P2O5 yielding (242 mg,
48% yield) of 110 as a light yellow solid. mp 191-192 °C 1H NMR (360.1 MHz,
DMSO-d6): δ 8.43 (br s, 2H, D2O exchangeable), 7.39 (s, 1H), 6.22 (br s, 2H,
D2O exchangeable), 5.78 (d, J = 4.9 Hz, 1H), 5.27 (m, 3H, D2O exchangeable),
13 3.94 (m, 2H), 3.82 (m, 1H), 3.63-3.49 (m, 2H). C NMR (75.4 MHz, DMSO-d6):
δ 158.1, 150.7, 122.8, 116.8, 88.5, 84.8, 73.8, 70.0, 61.1. IR (KBr): 3336 cm-1.
+ + HRMS (ESI ) calcd. for C9H15N4O5 [M+H] 259.1042, found 259.1037.
5-Nitrocytidine-5'-triphosphate triethylammonium salt (123). 5-nitrocytidine (109,
66.3 mg, 0.23 mmoles) was suspended in anhydrous pyridine (5 mL) and
concentrated in vacuo (3x) to render the nucleoside anhydrous. To the dried material was added Proton-Sponge (100 mg, 0.47 mmoles) and the material was
dissolved in trimethyl phosphate (2.2 mL). 23-30 The solution was cooled to 0 °C
and POCl3 (43 µL, 0.46 moles) was added dropwise. The resultant dark purple
solution was stirred for 2 h at 0 °C, after which time Bu3N (270 µL, 1.13 mmoles) was added followed by a solution of tributylammonium pyrophosphate (560 mg) in DMF (2 mL). The solution was stirred for 2 min, then quenched by the addition of triethylammonium bicarbonate (TEAB, 5 mL, 1.0 M solution). The reaction components were frozen and lyophilized to dryness. The crude material was 170 purified by stepwise preparative-scale HPLC purifications. The initial purification
of 123 employed the following linear gradient (flow rate = 20 mL/min): the mobile
phase comprised 1% to 25% CH3CN in triethylammonium acetate (TEAA) buffer
(0 to 30 min, 20 mM TEAA, pH = 6) followed by 25% to 90% CH3CN in TEAA
buffer (30 to 35 min) and isocratic 90% MeCN in TEAA buffer (35 to 40 min).
The material eluting broadly from 11 – 13 minutes was collected and
concentrated in vacuo. This crude material was purified an additional time by
preparative-scale HPLC utilizing the following linear gradient (flow rate = 20
mL/min): the mobile phase comprised isocratic 1% MeCN in TEAA buffer (0 to 5
min, 20 mM TEAA, pH = 6), 1% to 10% CH3CN in TEAA buffer (5 to 20 min),
10% to 90% CH3CN in TEAA buffer (20 to 25 min), isocratic 90% MeCN in TEAA
buffer (25 to 30 min). The material eluting broadly from 17.5 – 19 minutes was
collected and concentrated in vacuo. The material was redissolved in double
distilled water (ddH2O, 10 mL), frozen, then lyophilized to dryness. This step was
repeated one additional time using ddH2O (5 mL), providing 5-nitrocytidine
triphosphate 123 (triethylammonium salt) as an oily solid (27.2 mg, 12% yield).
1 H NMR (300.1 MHz, D2O): δ 9.13 (s, 1H), 5.66 (m, 1H), 4.19 (m, 5), 2.99 (q, J =
7.3 Hz, ca. 29 H, TEAA salt), 1.07 (t, J = 7.3 Hz, ca. 44 H, TEAA salt). 13C NMR
(75.5 MHz, D2O): δ 163.4, 155.2, 147.6, 118.9, 92.1, 83.7*, 75.4, 68.7, 64.7*,
31 47.3 (TEAA salt), 8.9 (TEAA salt). P NMR (145.8 MHz, D2O): δ -8.99 (m),
− − -10.92 (d, J = 21.8 Hz), -22.42 (br s). HRMS (FAB ) calcd. for C9H14N4O16P3
[M–TEAA+3H]− 526.9618, found 526.9613. 171 The chemical purity of 123 was further analyzed by analytical HPLC
running the following linear gradient (flow rate = 1 mL/min). The mobile phase
comprised 1% CH3CN in KH2PO4 (0 to 5 min, 100 mM KH2PO4, pH = 6), 1% to
15% CH3CN in KH2PO4 (5 to 20 min), and 15% to 80% CH3CN in KH2PO4 (20 to
25 min). As shown in Figure 4.16, triphosphate 123 eluted at 4.3 min in 90%
purity.
Figure 4.16. Analysis of purity of triphosphate 123.
4.11 References
1. "Lethal mutagenesis of HIV with mutagenic nucleoside analogs." Loeb, L.
A.; Essigmann, J. M.; Kazazi, F.; Zhang, J.; Rose, K. D.; Mullins, J. I.,
Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 1492-1497.
2. "Major Oxidative Products of Cytosine, 5-Hydroxycytosine and 5-
Hydroxyuracil, Exhibit Sequence Context-Dependent Mispairing in-Vitro." 172 Purmal, A. A.; Kow, Y. W.; Wallace, S. S., Nucleic Acids Res. 1994, 22,
72-78.
3. "Endogenous oxidative damage of deoxycytidine in DNA." Wagner, J. R.;
Hu, C. C.; Ames, B. N., Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 3380-
3384.
4. "5-Hydroxypyrimidine deoxynucleoside triphosphates are more efficiently
incorporated into DNA by exonuclease-free Klenow fragment than 8-
oxopurine deoxynucleoside triphosphates." Purmal, A. A.; Kow, Y. W.;
Wallace, S. S., Nucleic Acids Res. 1994, 22, 3930-3935.
5. "New substrates for old enzymes - 5-hydroxy-2'-deoxycytidine and 5-
hydroxy-2'-deoxyuridine are substrates for Escherichia coli endonuclease
III and formamidopyrimidine DNA N-glycosylase, while 5-hydroxy-2'-
deoxyuridine is a substrate for uracil DNA N-glycosylase." Hatahet, Z.;
Kow, Y. W.; Purmal, A. A.; Cunningham, R. P.; Wallace, S. S., J. Biol.
Chem. 1994, 269, 18814-18820.
6. "Incorporation of oxidatively modified 2'-deoxynucleotide triphosphates by
HIV-1 RT on RNA and DNA templates." Wuenschell, G. E.; Valentine, M.
R.; Termini, J., Chem. Res. Toxicol. 2002, 15, 654-661.
7. "Conformation and proton configuration of pyrimidine deoxynucleoside
oxidation damage products in water." La Francois, C. J.; Jang, Y. H.;
Cagin, T.; Goddard, W. A.; Sowers, L. C., Chem. Res. Toxicol. 2000, 13,
462-470. 173 8. "Identification by UV resonance Raman spectroscopy of an imino tautomer
of 5-hydroxy-2'-deoxycytidine, a powerful base analog transition mutagen
with a much higher unfavored tautomer frequency than that of the natural
residue 2'-deoxycytidine." Suen, W.; Spiro, T. G.; Sowers, L. C.; Fresco, J.
R., Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 4500-4505.
9. "Reverse Chemical Mutagenesis - Identification Of The Mutagenic Lesions
Resulting From Reactive Oxygen Species-Mediated Damage To DNA."
Feig, D. I.; Sowers, L. C.; Loeb, L. A., Proc. Natl. Acad. Sci. U. S. A. 1994,
91, 6609-6613.
10. "Selective inhibition of the replication of varicella-zoster virus by 5-
halogenated analogs of deoxycytidine." Jerkofsky, M. A.; Dobersen, M. J.;
Greer, S., Ann. NY Acad. Sci. 1977, 284, 389-395.
11. "Enzymic basis for the selective inhibition of varicella-zoster virus by 5-
halogenated analogs of deoxycytidine." Dobersen, M. J.; Jerkofsky, M.;
Greer, S., J. Virol. 1976, 20, 478-486.
12. "Herpes simplex virus type 2 induced pyrimidine nucleoside kinase:
enzymic basis for the selective antiherpetic effect of 5-halogenated
analogs of deoxycytidine." Dobersen, M. J.; Greer, S., Biochemistry 1978,
17, 920-928.
13. "Incorporation of 5-substituted analogs of deoxycytidine into DNA of
herpes simplex virus-infected or -transformed cells without deamination to
the thymidine analog." Fox, L.; Dobersen, M. J.; Greer, S., Antimicrob.
Agents Chemother. 1983, 23, 465-476. 174 14. "Uridine, cytidine, and deoxyuridine derivatives." Fukuhara, T. K.; Visser,
D. W., Biochemistry 1962, 1, 563-568.
15. "Synthesis and structure determination of a nucleoside-derived new
heterocyclic system: 8H,10H,15b(S)-2,3,6,7-tetrahydro-1,5,3-
dioxazepino[3,2-c]indolo[3,2-g]pteridine-7-one." Ge, P.; Voronin, G. O.;
Kalman, T. I., Nucleos. Nucleot. 1996, 15, 1701-1710.
16. "Formation of 5- and 6-aminocytosine nucleosides and nucleotides from
the corresponding 5-bromocytosine derivatives: synthesis and reaction
mechanism." Goldman, D.; Kalman, T. I., Nucleos. Nucleot. 1983, 2, 175-
87.
17. "Pyrimidine nucleosides. VIII. Synthesis of 5-nitrocytidine and related
nucleosides." Fox, J. J.; Van Praag, D., J. Org. Chem. 1961, 26, 526-532.
18. "Cytidine derivatives." Fukuhara, T. K.; Visser, D. W., J. Am. Chem. Soc.
1955, 77, 2393-2395.
19. "A general synthesis of pyrimidine nucleosides." Niedballa, U.;
Vorbruggen, H., Angew. Chem. Int. Edit. 1970, 9, 461-462.
20. Vorbruggen, H.; Ruh-Pohlenz, C., Synthesis of nucleosides. In Organic
Reactions, Paquette, L. A., Ed. Wiley-Interscience: 2000; Vol. 55, pp 1-
631.
21. "Regioselective addition of Grignard reagents to a 2-oxopurinium salt."
Andresen, G.; Gundersen, L. L.; Lundmark, M.; Rise, F.; Sundell, S.,
Tetrahedron 1995, 51, 3655-3664. 175 22. "5,6-Diaminocytidine, a versatile synthon for pyrimidine-based bicyclic
nucleosides." Rajeev, K. G.; Broom, A. D., Org. Lett. 2000, 2, 3595-3598.
23. "Dual specificity of the pyrimidine analogue, 4-methylpyridin-2-one, in DNA
replication." Hirao, I.; Ohtsuki, T.; Mitsui, T.; Yokoyama, S., J. Am. Chem.
Soc. 2000, 122, 6118-6119.
24. "Efforts toward the expansion of the genetic alphabet: Information storage
and replication with unnatural hydrophobic base pairs." Ogawa, A. K.; Wu,
Y.; McMinn, D. L.; Liu, J.; Schultz, P. G.; Romesberg, F. E., J. Am. Chem.
Soc. 2000, 122, 3274-3287.
25. "The synthesis of cyclonucleotides with fixed glycosidic bond linkages as
putative agonists for P2-purinergic receptors." Tusa, G.; Reed, J. K.,
Nucleos. Nucleot. Nucl. 2000, 19, 805-813.
26. "Enzymatic synthesis of unlabeled and beta-P-32-labeled beta-L-2',3'-
dideoxyadenosine-5'-triphosphate as a potent inhibitor of adenylyl
cyclases and its use as reversible binding ligand." Shoshani, I.; Boudou,
V.; Pierra, C.; Gosselin, G.; Johnson, R. A., J. Biol. Chem. 1999, 274,
34735-34741.
27. "New N-2-labelled fluorescent derivatives of guanosine nucleotides and
their interaction with GTP-binding proteins." Ostermann, N.; Ahmadian, M.
R.; Wittinghofer, A.; Goody, R. S., Nucleos. Nucleot. 1999, 18, 245-262.
28. "4-substituted uridine 5'-triphosphates as agonists of the P-2Y2 purinergic
receptor." Shaver, S. R.; Pendergast, W.; Siddiqi, S. M.; Yerxa, B. R.; 176 Croom, D. K.; Dougherty, R. W.; James, M. K.; Jones, A. N.; Rideout, J.
L., Nucleos. Nucleot. 1997, 16, 1099-1102.
29. "Identification of potent, selective P-2y-purinoceptor agonists- structure-
activity-relationships for 2-thioether derivatives of adenosine 5'-
triphosphate." Fischer, B.; Boyer, J. L.; Hoyle, C. H. V.; Ziganshin, A. U.;
Brizzolara, A. L.; Knight, G. E.; Zimmet, J.; Burnstock, G.; Harden, T. K.;
Jacobson, K. A., J. Med. Chem. 1993, 36, 3937-3946.
30. "Synthesis and antiviral evaluation of a mutagenic and non-hydrogen
bonding ribonucleoside analogue: 1-beta-D-ribofuranosyl-3-nitropyrrole."
Harki, D. A.; Graci, J. D.; Korneeva, V. S.; Ghosh, S. K. B.; Hong, Z.;
Cameron, C. E.; Peterson, B. R., Biochemistry 2002, 41, 9026-9033.
31. "Antiviral, antimetabolic, and cytotoxic activities of 5-substituted 2'-
deoxycytidines." De Clercq, E.; Balzarini, J.; Descamps, J.; Huang, G. F.;
Torrence, P. F.; Bergstrom, D. E.; Jones, A. S.; Serafinowski, P.; Verhelst,
G.; Walker, R. T., Mol. Pharmacol. 1982, 21, 217-223.
32. "Synthesis and biological evaluation of some 5-nitro- and 5-amino
derivatives of 2'-deoxycytidine, 2',3'-dideoxyuridine, and 2',3'-
dideoxycytidine." Colacino, E.; Sindona, G.; Gosselin, G.; Mathe, C.,
Nucleos. Nucleot. Nucl. 2003, 22, 2013-2026.
33. "High-pressure liquid chromatography-ultraviolet analysis of intracellular
nucleotides." Pogolotti, A. L., Jr.; Santi, D. V., Anal. Biochem. 1982, 126,
335-345. 177 34. "NMR chemical shifts of common laboratory solvents as trace impurities."
Gottlieb, H. E.; Kotlyar, V.; Nudelman, A., J. Org. Chem. 1997, 62, 7512-
7515.
178
Chapter 5
MISCELLANEOUS RIBONUCLEOSIDES AND RIBONUCLEOTIDES: (I) TOWARDS AN EFFICIENT SYNTHESIS OF RIBAVIRIN AND PYRAZOFURIN TRIPHOSPHATES; (II) SYNTHESIS AND ANTIVIRAL EVALUATION OF 1-β-D-RIBOFURANOSYL-4-NITROIMIDAZOLE
5.12 Overview
Presented in this chapter is (I) a discussion of synthesis efforts towards
ribavirin triphosphate (41) and pyrazofurin triphosphate (128), and (II) the
synthesis and antiviral evaluation of 1-β-D-ribofuranosyl-4-nitroimidazole (167).
General strategies and principles for nucleotide synthesis are highlighted.
5.13 Efforts Towards an Efficient Syntheses of Ribavirin Triphosphate (41) and Pyrazofurin Triphosphate (128)
5.13.1 Biochemical Applications of Nucleotide Triphosphates: Ribavirin Triphosphate and Pyrazofurin Triphosphate
The ribonucleoside analogue ribavirin (25, Figure 5.17) was recently demonstrated to function as an RNA-virus mutagen (see section 1.5.4.4).1 As the triphosphate, ribavirin serves as a substrate of the RNA-dependent RNA polymerase (RdRP) of poliovirus and incorporates into the viral genome opposite the pyrimidines C and U. The elucidation of this molecular mechanism-of-action was based on studies with a primer-extension assay, which measures the 179 kinetics and thermodynamics on nucleotide incorporation into a symmetrical primer-template mediated by a viral RdRP.2 To perform this in vitro assay, purified ribavirin triphosphate was required. Other related biochemical assays, such as viral polymerase inhibition studies (Chapter 3) and HPLC-based metabolism experiments (Chapter 3) also rely on synthetic nucleotides to help answer important biological questions.
Figure 5.17. Compound structures.
An efficient procedure for the synthesis of ribavirin triphosphate (41) was desired since this compound has been used widely throughout this project.
Additionally, the synthesis of structurally-analogous pyrazofurin triphosphate
(128) was also sought. Pyrazofurin (127) is a C-nucleoside natural product that exhibits potent antiviral activity against multiple viruses.3 It has been hypothesized that pyrazofurin (127) might function as an antiviral lethal mutagen 180 similarly to ribavirin (25, see section 1.5.4.4). To investigate whether 127 (as the
triphosphate 128) functions as a substrate for viral polymerases, and exhibits ambiguous base-pairing properties, studies using the aforementioned primer- extension assay were desired (see section 1.5.4.3).2 However, a synthesis of
pyrazofurin triphosphate (128) has not been published. To generate ribavirin
triphosphate (RTP, 41), multiple chemical syntheses have been presented,
although most are lengthy or marred by low reaction yields.4-6 An enzymatic
approach for the synthesis of RTP has shown promise, although ribavirin
monophosphate (152) must be synthesized, purified, then subjected to multiple
enzymatic transformations.7 Therefore, the development of a modular approach
to preparing both RTP 41 and PyTP 128 in reasonable yields and few steps
would be highly attractive.
5.13.2 Overview of Methods for the Synthesis of Nucleotides
Although a variety of methods have been published for the synthesis of
nucleotides,8 the “one-pot, three-step” approach is one of the most widely used
(Figure 5.18).5 In this procedure the 5'-OH of a nucleoside is selectively
converted to the phosphodichloridate 130 by reaction with phosphorous
oxychloride and trimethyl phosphate utilizing conditions developed by Yoshikawa
and coworkers.9 Subsequent studies by Ludwig10 and others11 found that
immediate reaction of the unstable intermediate 130 with pyrophosphate
facilitates the formation of cyclic phosphate 131 which upon hydrolysis yields 181 nucleoside 5'-triphosphates 132. It was determined that addition of an amine
base greatly increases the yields of cyclic phosphate formation, which takes only
minutes to form.8,10 Hydrophobic substrates, such as 3-NPN (48) and 5-NINDN
(63), are relatively efficiently phosphorylated by this procedure.
Figure 5.18. One-pot, three-step synthesis of nucleotides.
An array of other synthetic procedures have been developed for
nucleotide synthesis, although most methods are longer in sequence or
specialized for particular substrates. A general method is the activation of
nucleoside monophosphates by formation of phosphormorpholidates12 or
phosphoroimidazolidates.13 These electrophilic species are suitable for
displacement by pyrophosphate to yield triphosphates 135 and 138 (Figure 5.19).
Nucleoside monophosphates are readily prepared by hydrolysis of the previously
described phosphorodichloridates9 (such as 130). 182
Figure 5.19. Synthesis of triphosphates from activated nucleoside monophosphates 134 and 137.
Nucleoside diphosphates have been utilized as both electrophiles and
nucleophiles in reactions with phosphate moieties to arrive at unique nucleotide
structures (Figure 5.20). Conversion of adenosine diphosphate (139) to the
activated imidazolidate, followed by nucleophilic addition of 32P-radiolabeled
tributylammonium phosphate yields γ-32P-labeled ATP (140).14 Conversely,
nucleophilic addition of guanosine diphosphates (141) into the activated
phosphate species 142 yields γ-methylated (capped) guanosine triphosphate
143.15
183
Figure 5.20. Synthesis of triphosphates from electrophilic and nucleophilic nucleoside diphosphates.
Activation of the 5'-OH of nucleosides has also been utilized for the
preparation of triphosphates (Figure 5.21). Activated nucleoside phosphites,
such as 146, have been reacted with pyrophosphate yielding cyclic structures such as 147, which upon hydrolysis with water results in triphosphate formation.
A disadvantage to this approach is the necessity to protect the 3'-OH of the nucleoside.16 Conversion of the 5'-alcohol of nucleosides into good leaving
groups, such as a tosylate 148, has permitted displacement by triphosphate
nucleophile to yield adenosine triphosphate (135).17 Unfortunately, the generality
of this simple method has been questioned.8 The final method for nucleotide preparation to be highlighted is the enzymatic synthesis of ATP (135) starting from adenosine (149). In this work a large-scale production of ATP was executed starting from adenosine (40 grams, 150 mmoles), utilizing adenosine
kinase, adenylate kinase, acetate kinase, and acetyl phosphate as the major 184 reaction components. Following purification, 54 mmoles of ATP (135) was obtained by enzymatic assay.18
Figure 5.21. Synthesis of nucleotides by activation of 5'-hydroxyl groups and enzymatic methods.
5.13.3 One-Pot, Three-Step Approach for the Synthesis of Ribavirin Triphosphate (41) and Pyrazofurin Triphosphate (128)
Initial trials towards the synthesis of RTP (41) and PyTP (128) examined the feasibility of the one-pot, three-step coupling methodology9-11 to rapidly 185 synthesize both triphosphates. The conditions studied for this transformation are
described elsewhere in this thesis (e.g., synthesis of 3-NPN (48), Chapter 2).
Reaction of both ribavirin and pyrazofurin under these reaction conditions failed to produce the corresponding nucleotides 41 and 128 in appreciable yields
(Figure 5.22). In multiple reaction attempts small quantities of nucleotide 41 was isolated, however yields were erratic and irreproducible. A recent study utilizing similar chemistry to prepare RTP supported these results, as only a 30% reaction yield was achieved for their synthesis.6 In our hands, the direct phosphorylation
of pyrazofurin 127 was equally troublesome as triphosphate 128 was never
observed. It was hypothesized that the acidic alcohol on 127 might be interfering
with the initial phosphorylation of the 5'-alcohol. To address this concern, the aryl
alcohol was protected selectively as the benzyl ether 150 using a known
procedure.3 Attempts to install a more labile protecting group, such as a
methoxy methyl ether (MOM), were unsuccessful. Regardless, phosphorylation
of 150 utilizing the same methodology was unsuccessful, as only trace amounts of triphosphate 151 was isolated, yielding almost complete recovery of unreacted
150.
186
Figure 5.22. One-pot, three-step approach towards ribavirin triphosphate (41) and pyrazofurin triphosphate (128).
5.13.4 Synthesis of Ribavirin Triphosphate (41) and Ribavirin Diphosphate (154) from an Activated Phosphormorpholidate
Both ribavirin triphosphate (41) and ribavirin diphosphate (154) have been
chemically synthesized from a common phosphormorpholidate 153
(Figure 5.23).4 The synthesis begins by preparing ribavirin monophosphate by
reaction of 25 with phosphorous oxychloride in trimethylphosphate. Purification of the crude material yields 152 as the ammonium salt, which was then converted to the free acid by passage through an acidic resin.19 Coupling of the 187 free acid (not shown) with morpholine mediated by DCC
(dicyclohexylcarbodiimide) yields the phosphormorpholidate 153 in good yield.4
Reaction of 153 with tributylammonium pyrophosphate provided ribavirin triphosphate (41) in low yield, whereas reaction with tributylammonium phosphate resulted in formation of ribavirin diphosphate (154) with slightly better efficiency.4
Figure 5.23. Previously reported synthesis of ribavirin triphosphate (41) and ribavirin diphosphate (154).
To produce ribavirin triphosphate (41) and ribavirin diphosphate (155) for
biochemical studies related to this project, the previously reported synthetic route shown in Figure 5.23 was utilized. The preparation of phosphormorpholidate 153 proceeded smoothly yielding substantial quantities of 153. From this intermediate, the synthesis of ribavirin diphosphate was performed by an 188 adaptation of the published method.4 Reaction of 153 with tributylammonium
phosphate, derived from crystalline H3PO4 instead of 85% aqueous solution to
avoid unwanted hydrolysis, yielded crude 155 as an oil. This material was
purified by reverse-phase HPLC (instead of ion-exchange chromatography)
yielding the diphosphate as the triethylammonium salt, which was converted to
the sodium salt by reaction with sodium perchlorate.20 Lyophilization from water
gave ribavirin diphosphate (155) in 19% yield. Unfortunately, multiple efforts to
synthesize ribavirin triphosphate by reaction of 41 with tributylammonium
pyrophosphate were unsuccessful (mostly starting material was recovered); a
testament to the low published reaction yield (14%).4
5.13.5 Efforts Towards a Chemo-Enzymatic Approach for the Synthesis of Ribavirin Triphosphate (41) and Pyrazofurin Triphosphate (128)
An efficient phosphorylation procedure for triazole-containing ribonucleosides has recently been reported (Figure 5.24).21 This approach
utilizes enzymatic phosphorylation of chemically-synthesized diphosphates (160,
161) to arrive at nucleoside triphosphates (162,163) in good yields. Pyruvate
kinase functions as the phosphorylating enzyme, utilizing phosphenol pyruvate
as the phosphate donor to transform 160 and 161 into triphosphates 162 and 163
in 72% and 67% yields, respectively.21 The diphosphate substrates 160 and 161
can be prepared in good overall yields by displacement of an activated (tosyl) 5'- alcohol with pyrophosphate.21 Surprisingly, the regioisomer of these analogues,
ribavirin triphosphate (41), was not prepared in this publication. Due to the high 189 reaction yields and level of similarity with ribavirin triphosphate this methodology
was applied in an effort to synthesize RTP (41). If proven successful, this methodology might be useful for the synthesis of pyrazofurin triphosphate (128) due to the similarities between the two nucleotides.
Figure 5.24. Chemo-enzymatic synthesis of triazole ribonucleosides.
The initial steps towards the synthesis of ribavirin diphosphate (166) began by protection of ribavirin as the orthoester 164 (Figure 5.25). Reaction of ribavirin with trimethyl orthoformate in 1,4-dioxane utilizing pyridine hydrochloride as the acid source22 yielded the previously synthesized5 164 in 66% yield.
Conversion of the 5'-alcohol to the activated toluenesulfonic acid 165 was accomplished in 59% yield by reaction of 164 with p-toluenesulfonyl chloride in 190 pyridine.21 An initial attempt to convert 165 into the corresponding diphosphate was performed under the previously described conditions (tributylammonium pyrophosphate in acetonitrile);21 however, poor solubility of the reaction components was encountered. Future synthesis efforts should focus initially on solvent studies for this transformation, examining polar reagents such as N,N- dimethylformamide or dimethyl sulfoxide. If proven successful, this enzymatic synthesis for the preparation of RTP (41) could serve as a blueprint for the first synthesis of pyrazofurin triphosphate (128). Minor adaptations to the synthetic scheme, such as finding a suitable protecting group for the aryl alcohol of 127, may yield an efficient synthesis of this nucleotide for biochemical evaluation.
Figure 5.25. Efforts towards the synthesis of ribavirin triphosphate (41). 191
5.14 Synthesis and Antiviral Evaluation of 1-β-D-Ribofuranosyl-4- nitroimidazole
5.14.1 Background
In conjunction with the previously described efforts to develop “universal bases” as antiviral lethal mutagens (see section 2.4) an isostere of 3-NPN (48) was synthesized and screened for antiviral activity. This ribonucleoside analogue, 1-β-D-ribofuranosyl-4-nitroimidazole (4-NIN, 167, Figure 5.26) was designed to function similarly to 3-NPN: phosphorylation in vivo to the triphosphate, incorporation into viral genomes, and promotion viral genomic mutations resulting in error catastrophe. The initial impetus for screening this ribonucleoside analogue was two-fold: (I) short chemical syntheses of 4-NIN
(167) had been previously reported23-25 and (II) the similar overall structures between 167 and 48 suggested this isostere might function as a universal RNA base, as hypothesized for 3-NPN (48). Recently, this hypothesis was strengthened by melting temperature studies of duplex DNA containing 4- nitroimidazole acyclic “sugars” base-pairing with little bias opposite all four DNA bases.26 Additionally, ribonucleoside 167 could provide additional data concerning the biological consequences of reintroducing hydrogen-bonding heteroatoms on this primarily hydrophobic pseudobase. The 4-nitroimidazole ribonucleoside (167) was synthesized as previously described.23 192
Figure 5.26: Structural comparison: ribavirin (25), 3-NPN (48), 4-NIN (167).
5.14.2 Cellular Cytotoxicity and Antiviral Activity of 4-NIN (167) Against Poliovirusaa
The cellular cytotoxicity of 4-NIN (167) towards HeLa S3 cells grown in
culture for 6 days was examined at two concentrations (100 µM and 1000 µM) and compared with the levels of cellular cytotoxicity observed for ribavirin (25) and 3-NPN (48) treatments (Table 5.1). 4-NIN (167) exhibited the least cytotoxicity of the three ribonucleoside analogues, showing no reduction in cellular growth after 6 days. A small amount of cytotoxicity was observed with 3-
NPN (48) at the highest concentration tested (1000 µM), whereas ribavirin (25)
possessed significant cytotoxicity at all concentrations examined. Unfortunately,
4-NIN (167) exhibited no antiviral activity against poliovirus-infected HeLa S3 cells. At both concentrations examined 4-NIN failed to extend the amount of time for the onset of cytopathic effects (CPE) following virus infection, nor was the
aa Experimental data from Jason D. Graci (Cameron Lab). For experimental details refer to Harki et al. 2002.27 193 final virus titer significantly reduced. These results were consistent with the
activity profile for the analogous 3-NPN (48).
Table 5.1. Cytotoxicity and antiviral activity of 4-NIN (167). Time to 100% Fold Reduction in Compound Cell Viability CPE (days) Virus Titer none 100% 2 days -- 100 µM 3-NPN (48) 106% 2 days -- 1000 µM 3-NPN (48) 64% 2 days -- 100 µM 4-NIN (169) 114% 2 days -- 1000 µM 4-NIN (169) 101% 2 days -- 100 µM Ribavirin (25) 28% 3 days 14 1000 µM Ribavirin (25) 3% 6 days 1900
5.14.3 Phosphorylation of 4-NIN by Adenosine Kinasebb
The ability of 4-NIN (167) to be phosphorylated in vivo by adenosine
kinase was measured utilizing the assay described in Section 2.8. This assay
monitors the conversion of α-32P-labeled ATP to α-32P-ADP as an indirect
measure of the monophosphorylation of nucleoside analogues mediated by
purified adenosine kinase (Figure 5.27). The in vivo transformation of nucleoside
analogues to their corresponding 5'-monophosphates is a critical step in
phosphorylation to the active metabolite, the nucleoside triphosphate. For
comparison, the phosphorylation data obtained with ribavirin (25) and 3-NPN (48)
are provided (Figure 5.28). Quantitative analysis of the level of phosphorylation
bb Experimental data from Saikat K.B. Ghosh (Cameron Lab). For experimental details refer to Harki et al. 2002.27 194 of 4-NIN reveals that this nucleoside is phosphorylated less efficiently than both
ribavirin (25) and 3-NPN (48), although some conversion to the monophosphate
is observed.
Figure 5.27. Phosphorylation of 4-NIN (167), ribavirin (25) and 3-NPN (48) by adenosine kinase.
Figure 5.28. Quantitative comparison of phosphorylation of adenosine (●), 3- NPN (▲),ribavirin (■), and 4-NIN (♦) by adenosine kinase. 195 5.14.4 Conclusions
The 4-nitroimidazole ribonucleoside (167) was chemically synthesized and biochemically evaluated as an antiviral lethal mutagen. Compound 167 exhibited
no cytotoxicity towards cultured HeLa S3 cells, and antiviral activity was not
detected when 4-NIN treated HeLa cells were infected with poliovirus. The ability
of 4-NIN to be phosphorylated by adenosine kinase was also measured to help
gauge whether the monophosphate might be formed in vivo. 4-NIN (167) was phosphorylated by adenosine kinase less efficiently than ribavirin (25) or 3-NPN
(48), a surprising result due to the structural similarities between the three compounds. As a result of these biochemical findings, further analyses of 4-NIN as an antiviral lethal mutagen is not warranted.
5.15 Experimental Section
General synthesis information. All reactions were performed under an argon or nitrogen atmosphere unless otherwise noted. Commercial grade reagents
(Sigma-Aldrich, Acros) were used without further purification unless specifically noted. Pyridine was distilled from calcium hydride (CaH2) and stored over 4 Å
molecular sieves under nitrogen. Column chromatography employed ICN
SiliTech silica gel (32-63 µm). HPLC purification was performed on an Agilent
1100 series instrument (preparative scale) equipped with a PRP-1 preparative
column (21.5 x 250 mm, 7 µm; Hamilton Company). Nuclear magnetic
resonance (NMR) spectroscopy employed Bruker CDPX-300, DPX-300, AMX- 196 360, DRX-400, or AMX-2-500 MHz spectrometers. Internal solvent peaks were referenced in each case. Chemical shifts for 13C NMR and 31P NMR analyses
performed in D2O were indirectly referenced to 10% acetone in D2O (CH3 set to
28 13 30.89 ppm) and 85% H3PO4 (0 ppm), respectively. C chemical shifts for
nucleoside diphosphates denoted with a (*) fail to resolve into clean singlets due
to apparent conformational restrictions. Mass spectral data was obtained from
either The University of Texas at Austin Mass Spectrometry Facility (FAB, ESI
and CI) or The Pennsylvania State University Mass Spectrometry Facility (ESI and APCI). Elemental analyses were performed by Midwest Microlab, LLC
(Indianapolis, IN). Melting points are uncorrected.
4-(Benzyloxy)-3-(β-D-ribofuranosyl)pyrazole-5-carboxamide hydrate (150). This
compound was prepared according to the published method3 and purified by RP-
HPLC utilizing the following gradient method: (20 mL/min flow rate): 0 to 20 min,
1% to 20% CH3CN (MeCN, containing 0.1% TFA) in double distilled water
(ddH2O, containing 0.1% TFA); 20 to 40 min, 20% to 99% MeCN (0.1% TFA) in ddH2O (0.1% TFA). Compound 150 eluted at 20.9 minutes. Lyophilization of the purified material yielded the monohydrate 150 as a white solid. 1H NMR (DMSO-
d6, 400.1 MHz): δ 7.48 to 7.15 (m, 7H), 5.04 (s, 2H), 4.68 (d, J = 6.6 Hz, 1H), 197 4.15 (m, 1H), 3.91 (m, 1H), 3.76 (m, 1H), 3.57 to 3.44 (m, 2H). 13C NMR
(DMSO-d6, 100.6 MHz): δ 140.8, 137.3, 128.3, 128.2, 128.0, 84.9, 76.7, 75.2,
74.0, 71.0, 61.8. Anal. calcd. for C16H21N3O7: C 52.31; H 5.76; N 11.44. Found:
C 52.54; H 5.56; N 11.29.
1-(β-D-Ribofuranosyl)-1,2,4-triazole-3-carboxamide-5'-diphosphate trisodium salt
(155). This compound was prepared by modification of the published synthesis
that yielded lithium salt 154.4 Briefly, ribavirin 5'-phosphoromorpholidate
triethylammonium salt4 (153, 700.2 mg; approximately 2.3 molar equivalents of
salt per mole of phosphormorpholidate) was triturated with anhydrous pyridine to
ensure the material was anhydrous, then redissolved in anhydrous pyridine (9
mL). Separately, Bu3N (anhydrous, 0.98 mL, 4.1 mmoles) was added to a
suspension of H3PO4 (98% crystalline, 401.7 mg, 4.1 mmoles) in pyridine (3 mL)
yielding an almost completely solubilized solution. An aliquot of this phosphate solution (2.6 mL) was added to the solution of 153 in pyridine and stirred for 72
hours at 23 °C. The reaction was then treated with distilled water (5 mL) and
concentrated in vacuo. The crude diphosphate was redissolved in
triethylammonium bicarbonate (TEAB, 1 mL, 1 M solution) and HPLC purified
utilizing the following gradient method: (20 mL/min flow rate): 0 to 21 min, 0.1% 198 to 99% MeCN in 0.1 M TEAB (pH = 7.4-7.6). The diphosphate eluted broadly
from 9 to 17 minutes. This material was frozen and lyophilized yielding the
triethylammonium salt (270 mg) as a white solid. Conversion to the sodium salt
was performed via a previously reported procedure.20 To a solution of the
triethylammonium salt (~267 mg) in double distilled water (10 mL) was added
NaClO4 (1.3355 g, 10.907 mmoles). The mixture was stirred for 2 h in an open
atmosphere then precipitated by the addition of acetone (30 mL). The material
was concentrated in vacuo yielding a colorless oil, then reprecipitated by the
addition of excess acetone. The resulting white solid was filtered, washed with
excess acetone, and then frozen in ddH2O (10 mL). Lyophilization yielded 155
(102.5 mg, ~19% yield from phosphormorpholidate) as a white solid. 1H NMR
(D2O, 400.1 MHz): δ 8.85 (s, 1H), 6.07 (d, J = 3.7 Hz, 1H), 4.71 (m, 1H), 4.64 (m,
13 1H), 4.40 (m, 1H), 4.23 (m, 2H). C NMR (D2O, 100.6 MHz): δ 163.5*, 156.9*,
31 146.4*, 92.7*, 84.4*, 75.3*, 70.4, 70.3, 65.0*. P NMR (D2O, 145.8 MHz): δ -
+ + 6.00 (m), -9.90 (m). HRMS (CI ) calcd. for C8H14N4O11NaP2 [M-2Na+3H]
427.0032, found 427.0044. 199
1-(β-D-Ribofuranosyl-2',3'-O-methoxymethylidene)-1,2,4-triazole-3-carboxamide
(164). This compound was prepared by an adaptation of the previously reported
procedure.5 To a suspension of ribavirin (25, 457.1 mg, 1.87 mmoles) in
anhydrous 1,4-dioxane (20 mL) was added pyridine hydrochloride (257.3 mg,
2.23 mmoles) and triethyl orthoformate (1.0 mL, 9.14 mmoles). The suspension
was stirred at 23 °C for 10 d then concentrated in vacuo onto silica gel. Column
chromatography (5% MeOH in CH2Cl2) yielded 164 (352.7 mg, 66% yield) as a
waxy solid. 1H NMR characterization data for this compound matches that previously reported.5
1-(β-D-Ribofuranosyl-2',3'-O-methoxymethylidene-5'-O-tosyl)-1,2,4-triazole-3-
carboxamide (165). To a solution of orthoester 165 (682 mg, 2.38 mmoles) and
DMAP (507 mg, 4.15 mmoles) in anhydrous pyridine (25 mL) was added p- 200 toluenesulfonyl chloride (695 mg, 3.64 mmoles). The solution was stirred at 23
ºC for 20 h, concentrated onto silica gel, then flushed through a plug of silica gel
(eluant: 5% MeOH in CH2Cl2). The semi-pure material was concentrated in
vacuo and further purified by preparative HPLC utilizing the following gradient:
the mobile phase comprised isocratic 1% MeCN in double distilled water (ddH2O) with a gradient flow rate of 10 mL/min to 20 mL/min (0 to 2 min), followed by 1% to 90% CH3CN in ddH2O with a constant flow rate of 20 mL/min (2 to 40 min).
Compound 165 eluted broadly from 29-35 minutes and was concentrated in
vacuo. Further drying in vacuo at 23 °C over P2O5 yielded 165 (619 mg, 59 %
1 yield) as a white solid. mp 138-140 °C H NMR (299.9 MHz, DMSO-d6,
diastereomers present: major isomer = maj, minor isomer = min): δ 8.75 (s, 1H,
min), 8.71 (s, 1H, maj), 7.84 (m, 1H, maj,min), 7.63 (m, 3H, maj,min), 7.33 (m,
2H, maj,min), 6.37 (m, 1H, maj,min), 6.12 (s, 1H, maj), 6.01 (s, 1H, min), 5.07 (m,
2H, maj,min), 4.44 (m, 1H, maj,min), 4.30 (m, 1H, maj,min), 4.08 (m, 1H,
maj,min), 3.28 (s, 3H, min), 3.19 (s, 3H, maj), 2.38 (s, 3H, maj,min). 13C NMR
(75.4 MHz, DMSO-d6, diastereomers present): δ 160.0, 159.9, 157.7, 157.6,
145.9, 145.8, 145.2, 145.1, 131.7, 131.6, 130.1, 127.45, 127.42, 117.9, 116.6,
91.7, 90.6, 85.2, 84.6, 84.1, 82.8, 80.3, 80.2, 70.2, 70.1, 51.7, 50.3, 21.2. IR
-1 + + (film): 1692, 1357, 1176 cm . HRMS (ESI ) calcd. for C17H20N4O8NaS [M+Na]
463.0900, found 463.0896.
201
1-(β-D-Ribofuranosyl)-4-nitroimidazole (167). This compound was prepared
23 1 according to the published method. H NMR (MeOH-d4, 299.9 MHz): δ 8.40 (d,
J = 1.5 Hz, 1H), 8.02 (d, J = 1.5 Hz, 1H), 5.71 (d, J = 5.4 Hz, 1H), 4.29 (m, 1H),
13 4.22 (m, 1H), 4.11 (m, 1H), 3.81-3.74 (m, 2H) C NMR (MeOH-d4, 75.5 MHz): δ
+ 148.9, 136.8, 119.2, 92.7, 87.8, 78.0, 72.0, 62.5. MS (ESI ) calcd. for C8H12N3O6
+ [M+H] 246.1, found 246.1. Anal. calcd. for C8H11N3O6•1/8 H2O: C 38.83; H
4.58; N 16.98. Found: C 38.83; H 4.70; N 16.71.
5.16 References
1. "The broad-spectrum antiviral ribonucleoside ribavirin is an RNA virus
mutagen." Crotty, S.; Maag, D.; Arnold, J. J.; Zhong, W.; Lau, J. Y. N.;
Hong, Z.; Andino, R.; Cameron, C. E., Nat. Med. 2000, 6, 1375-1379.
2. "Poliovirus RNA-dependent RNA polymerase (3D(pol)). Assembly of
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3. "Synthesis and Biological-Activity of Certain Nucleoside and Nucleotide
Derivatives of Pyrazofurin." Petrie, C. R.; Revankar, G. R.; Dalley, N. K.; 202 George, R. D.; McKernan, P. A.; Hamill, R. L.; Robins, R. K., J. Med.
Chem. 1986, 29, 268-278.
4. "Synthesis and antiviral activity of some phosphates of the broad-spectrum
antiviral nucleoside, 1-.beta.-D-ribofuranosyl-1,2,4-triazole-3-carboxamide
(ribavirin)." Allen, L. B.; Boswell, K. H.; Khwaja, T. A.; Meyer, R. B., Jr.;
Sidwell, R. W.; Witkowski, J. T.; Christensen, L. F.; Robins, R. K., J. Med.
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5. "A novel method for the preparation of nucleoside triphosphates from
activated nucleoside phosphoramidates." Wu, W. D.; Meyers, C. L. F.;
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6. "ATP-binding domain of NTPase/helicase as a target for hepatitis C
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7. "Enzyme-catalyzed synthesis of nucleoside triphosphates from nucleoside
monophosphates. ATP from AMP and ribavirin 5'-triphosphate from
ribavirin 5'-monophosphate." Kim, M. J.; Whitesides, G. M., Appl.
Biochem. Biotech. 1987, 16, 95-108.
8. "Syntheses of nucleoside triphosphates." Burgess, K.; Cook, D., Chem.
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9. "A novel method for phosphorylation of nucleosides to 5'-nucleotides."
Yoshikawa, M.; Kato, T.; Takenishi, T., Tetrahedron Lett. 1967, 50, 5065-
5068. 203 10. "A new route to nucleoside 5'-triphosphates." Ludwig, J., Acta Biochim.
Biophys. Acad. Sci. Hung. 1981, 16, 131-133.
11. "Nucleoside analogues with clinical potential in antivirus chemotherapy.
The effect of several thymidine and 2'-deoxycytidine analogue 5'-
triphosphates on purified human (alpha, beta) and herpes simplex virus
(types 1, 2) DNA polymerases." Ruth, J. L.; Cheng, Y. C., Mol. Pharmacol.
1981, 20, 415-422.
12. "Nucleoside polyphosphates. X. The synthesis and some reactions of
nucleoside-5'-phosphoromorpholidates and related compounds. Improved
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G.; Khorana, H. G., J. Am. Chem. Soc. 1961, 83, 649-658.
13. "Conversion of mono- and oligodeoxyribonucleotides to 5'-triphosphates."
Hoard, D. E.; Ott, D. G., J. Am. Chem. Soc. 1965, 87, 1785-1788.
14. "A chemical synthesis of adenosine 5'-(gamma-32P)triphosphate." Hecht,
S. M.; Kozarich, J. W., Biochim. Biophys. Acta 1973, 331, 307-309.
15. "Efficient synthesis of gamma-methyl-capped guanosine 5'-triphosphate as
a 5'-terminal unique structure of U6 RNA via a new triphosphate bond
formation involving activation of methyl phosphorimidazolidate using ZnCl2
as a catalyst in DMF under anhydrous conditions." Kadokura, M.; Wada,
T.; Urashima, C.; Sekine, M., Tetrahedron Lett. 1997, 38, 8359-8362.
16. "A convenient and general-approach to the synthesis of properly protected
D-nucleoside-3'-hydrogenphosphonates via phosphite intermediates." 204 Marugg, J. E.; Tromp, M.; Kuylyeheskiely, E.; Vandermarel, G. A.;
Vanboom, J. H., Tetrahedron Lett. 1986, 27, 2661-2664.
17. "Convenient syntheses of adenosine 5'-diphosphate, adenosine 5'-
methylenediphosphonate, and adenosine 5'-triphosphate." Dixit, V. M.;
Poulter, C. D., Tetrahedron Lett. 1984, 25, 4055-4058.
18. "Large-scale enzyme-catalyzed synthesis of ATP from adenosine and
acetyl phosphate. Regeneration of ATP from AMP." Baughn, R. L.;
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19. "Mechanism of action of 1-beta-D-ribofuranosyl-1,2,4-triazole-3-
carboxamide (Virazole), a new broad-spectrum antiviral agent." Streeter,
D. G.; Witkowski, J. T.; Khare, G. P.; Sidwell, R. W.; Bauer, R. J.; Robins,
R. K.; Simon, L. N., Proc. Natl. Acad. Sci. U. S. A. 1973, 70, 1174-1178.
20. "Syntheses and structure-activity relationships of nonnatural beta-C-
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hydroxy groups: inhibitory activities against DNA polymerases." Aketani,
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azole carboxamide nucleoside triphosphate." Wu, W. D.; Bergstrom, D. E.;
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23. "Nucleosides de synthese. V. Sur les ribofuranoses de quelques nitro-
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24. "Imidazole nucleosides. I. Nucleosides of 4(5)-nitro- and 4(5)-
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25. "Synthesis of nucleosides. XVII: Synthesis of 1-glycosyl-5-nitro
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26. "Synthesis of acyclic nitroazole nucleosides and their incorporation into
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27. "Synthesis and antiviral evaluation of a mutagenic and non-hydrogen
bonding ribonucleoside analogue: 1-beta-D-ribofuranosyl-3-nitropyrrole."
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Appendix A
X-RAY CRYSTALLOGRAPHIC DATA FOR 3',5'-DI-O-BENZOYL-1',2'-O-(S)- [PHENYL(3-NITROPYRROLE)METHYLIDENE]-α-D-RIBOFURANOSE (52)
A.1 General Experimental
The absolute structure was determined from the known configuration of a reactant. The displacement ellipsoids were drawn at the 50% probability level. A colorless needle-shaped crystal of dimensions 0.46 x 0.12 x 0.07 mm was selected for structural analysis. Intensity data for this compound were collected using a
Bruker SMART APEX ccd area detector(1) mounted on a Bruker D8 goniometer using graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). The data were collected at 100(2) K. The intensity data were measured as a series of ω oscillation frames each of 0.25 ° for 30 sec / frame. The detector was operated in 512 x 512 mode and was positioned 5.04 cm from the sample. Coverage of unique data was
99.9% complete to 26.00 degrees in θ. Cell parameters were determined from a least squares fit of 4541 peaks in the range 2.33 < θ < 30.44°. From 135 peaks that were measured both at the beginning and end of data collection, the crystal showed a decay of -0.04%. The data were corrected for absorption by the semi-empirical method (2) from equivalent reflections giving minimum and maximum transmission factors of 0.9533 and 0.9927. Lorentz and polarization corrections were applied.
The data were merged to form a set of 7014 independent data with R(int) = 0.0204. 207
The monoclinic space group P21 was determined by systematic absences and
statistical tests and verified by subsequent refinement. The structure was solved by
direct methods and refined by full-matrix least-squares methods on F2 (3).
Hydrogen atom positions were initially determined by geometry and refined by a riding model. Non-hydrogen atoms were refined with anisotropic displacement parameters. A total of 370 parameters were refined against 1 space group restraint and 7014 data to give wR(F2) = 0.1189 and S = 1.025 for weights of w= 1/[σ2 (F2) +
2 2 2 (? P) + ? P], where P = [Fo + 2Fc ] / 3. The final R(F) was 0.0477 for the 6432
observed, [F > 4σ(F)], data. The largest shift/s.u. was 0.001 in the final refinement
cycle. The final difference map had maxima and minima of 0.401 and -0.245 e/Å3,
respectively. The absolute structure, as determined by refinement of the Flack
parameter(4), cannot be determined reliably. The polar axis restraint was taken
from Flack and Schwarzenbach (5).
A.2 Funding Acknowledgement and Assignment of Credit
The authors thank the National Science Foundation (grant CHE-0079282)
and the University of Kansas for funds to purchase of the X-ray instrument and
computers. This structure was determined by Douglas R. Powell.
208
A.3 References
(1) (a) Data Collection: SMART Software Reference Manual (1994). Bruker-AXS,
6300 Enterprise Dr., Madison, WI 53719-1173, USA. (b) Data Reduction:
SAINT Software Reference Manual (1995). Bruker-AXS, 6300 Enterprise Dr.,
Madison, WI 53719-1173, USA.
(2) G. M. Sheldrick (1996). SADABS. Program for Empirical Absorption Correction
of Area Detector Data. University of Göttingen, Germany.
(3) (a) G. M. Sheldrick (1994). SHELXTL Version 5 Reference Manual. Bruker-AXS,
5465 E. Cheryl Parkway, Madison, WI 53711-5373, USA. (b) International
Tables for Crystallography, Vol C, Tables 6.1.1.4, 4.2.6.8, and 4.2.4.2,
Kluwer: Boston (1995).
(4) H. D. Flack, Acta Cryst. A39, 876-881 (1983).
(5) H. D. Flack and D. Schwarzenbach (1988). On the use of least-squares restraints
for origin fixing in polar space groups. Acta Cryst. A44, 499-506.
209
A.4 Thermal Ellipsoid Drawing of 52
210
A.5 Crystal Packing Diagram of 52
211
A.6 Crystallographic Information for Orthoamide 52
Table 1. Crystal data and structure refinement.
Empirical formula C30H24N2O9 Formula weight 556.51 Crystal system Monoclinic Space group P21 Unit cell dimensions a = 10.0128(5) Å α = 90° b = 10.5951(6) Å β = 103.481(2)° c = 12.7830(7) Å γ = 90° Volume 1318.74(12) Å3 Z, Z’ 2, 1 Density (calculated) 1.402 Mg/m3 Wavelength 0.71073 Å Temperature 100(2) K F(000) 580 Absorption coefficient 0.105 mm-1 Absorption correction Semi-empirical from equivalents Max. and min. transmission 0.9927 and 0.9533 Theta range for data collection 2.09 to 30.50° Reflections collected 11286 Independent reflections 7014 [R(int) = 0.0204] Data / restraints / parameters 7014 / 1 / 370 wR(F2all data) wR2 = 0.1189 R(F obsd data) R1 = 0.0477 Goodness-of-fit on F2 1.025 Observed data [I > 2σ(I)] 6432 Absolute structure parameter -0.1(7) Largest and mean shift / s.u. 0.001and 0.000 Largest diff. peak and hole 0.401 and -0.245 e/Å3 ------wR2 = { Σ [w(Fo2 - Fc2)2] / Σ [w(Fo 2)2] }1/2 R1 = Σ ||Fo| - |Fc|| / Σ |Fo|
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Table 2. Atomic coordinates and equivalent isotropic displacement parameters. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. ______x y z U(eq) ______
C(1) 0.64926(17) 0.47155(16) 0.98626(13) 0.0165(3) O(2) 0.56171(12) 0.50746(13) 0.88505(9) 0.0185(2) C(3) 0.64158(17) 0.51298(16) 0.80748(13) 0.0166(3) O(4) 0.76144(12) 0.44503(12) 0.84789(9) 0.0170(2) C(5) 0.79240(16) 0.46269(16) 0.96302(12) 0.0157(3) C(6) 0.84770(16) 0.33779(16) 1.01528(12) 0.0158(3) C(7) 0.71683(15) 0.26011(16) 1.00453(12) 0.0156(3) O(8) 0.61488(13) 0.35088(12) 1.01763(10) 0.0196(2) N(9) 0.68137(16) 0.64650(15) 0.79745(12) 0.0191(3) C(10) 0.61782(17) 0.75282(18) 0.82157(13) 0.0204(3) C(11) 0.69103(18) 0.85211(17) 0.79277(14) 0.0198(3) C(12) 0.80027(19) 0.80740(18) 0.75100(14) 0.0219(3) C(13) 0.79232(19) 0.67897(19) 0.75459(14) 0.0229(3) N(14) 0.66066(16) 0.98193(16) 0.80767(13) 0.0249(3) O(15) 0.72741(15) 1.06188(15) 0.77068(13) 0.0324(3) O(16) 0.57049(16) 1.00812(15) 0.85551(15) 0.0365(4) C(17) 0.56246(17) 0.45797(17) 0.70196(13) 0.0190(3) C(18) 0.4587(2) 0.5282(2) 0.63549(17) 0.0320(4) C(19) 0.3772(3) 0.4719(3) 0.54405(19) 0.0429(6) C(20) 0.3989(3) 0.3481(3) 0.51967(17) 0.0407(6) C(21) 0.5029(2) 0.2787(2) 0.58493(16) 0.0330(5) C(22) 0.5857(2) 0.33379(19) 0.67703(14) 0.0235(3) O(23) 0.93479(12) 0.27206(12) 0.95970(10) 0.0177(2) C(24) 1.05344(16) 0.33142(17) 0.95566(13) 0.0170(3) O(25) 1.09117(13) 0.42773(13) 1.00266(10) 0.0215(3) C(26) 1.12803(16) 0.26098(17) 0.88559(13) 0.0179(3) C(27) 1.26163(18) 0.29976(17) 0.88579(15) 0.0212(3) C(28) 1.33307(18) 0.23890(19) 0.81947(15) 0.0245(4) C(29) 1.2726(2) 0.1403(2) 0.75389(15) 0.0253(4) C(30) 1.1399(2) 0.1007(2) 0.75423(16) 0.0258(4) C(31) 1.06667(18) 0.16128(18) 0.82005(14) 0.0211(3) C(32) 0.72360(19) 0.15678(17) 1.08696(14) 0.0200(3) O(33) 0.74136(14) 0.21534(13) 1.19151(10) 0.0226(3) C(34) 0.84359(18) 0.17338(18) 1.27173(14) 0.0219(3) O(35) 0.91733(15) 0.08580(17) 1.26341(12) 0.0335(3) C(36) 0.85497(19) 0.25122(19) 1.37095(13) 0.0224(3) C(37) 0.9744(2) 0.2410(2) 1.45288(16) 0.0322(4)
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Table 2 (continued). ______x y z U(eq) ______
C(38) 0.9897(3) 0.3132(3) 1.54511(18) 0.0381(5) C(39) 0.8855(3) 0.3935(2) 1.55755(18) 0.0374(5) C(40) 0.7659(2) 0.4039(2) 1.47686(17) 0.0318(4) C(41) 0.7515(2) 0.3333(2) 1.38294(15) 0.0253(4) ______
Table 3. Bond lengths [Å]. ______
C(1)-O(8) 1.406(2) C(18)-C(19) 1.394(3) C(1)-O(2) 1.4347(19) C(19)-C(20) 1.377(4) C(1)-C(5) 1.533(2) C(20)-C(21) 1.384(4) O(2)-C(3) 1.4127(19) C(21)-C(22) 1.399(3) C(3)-O(4) 1.392(2) O(23)-C(24) 1.356(2) C(3)-N(9) 1.483(2) C(24)-O(25) 1.200(2) C(3)-C(17) 1.513(2) C(24)-C(26) 1.493(2) O(4)-C(5) 1.4437(19) C(26)-C(31) 1.398(3) C(5)-C(6) 1.527(2) C(26)-C(27) 1.399(2) C(6)-O(23) 1.4279(19) C(27)-C(28) 1.389(3) C(6)-C(7) 1.526(2) C(28)-C(29) 1.387(3) C(7)-O(8) 1.440(2) C(29)-C(30) 1.394(3) C(7)-C(32) 1.510(2) C(30)-C(31) 1.395(3) N(9)-C(10) 1.364(2) C(32)-O(33) 1.446(2) N(9)-C(13) 1.392(2) O(33)-C(34) 1.344(2) C(10)-C(11) 1.380(2) C(34)-O(35) 1.206(2) C(11)-C(12) 1.406(3) C(34)-C(36) 1.495(3) C(11)-N(14) 1.431(2) C(36)-C(41) 1.388(3) C(12)-C(13) 1.364(3) C(36)-C(37) 1.398(3) N(14)-O(16) 1.234(2) C(37)-C(38) 1.384(3) N(14)-O(15) 1.239(2) C(38)-C(39) 1.384(4) C(17)-C(22) 1.386(3) C(39)-C(40) 1.391(3) C(17)-C(18) 1.394(3) C(40)-C(41) 1.393(3)
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Table 4. Bond angles [°]. ______
O(8)-C(1)-O(2) 111.07(13) C(22)-C(17)-C(18) 120.51(17) O(8)-C(1)-C(5) 107.56(13) C(22)-C(17)-C(3) 119.32(16) O(2)-C(1)-C(5) 104.11(12) C(18)-C(17)-C(3) 119.92(17) C(3)-O(2)-C(1) 108.49(12) C(17)-C(18)-C(19) 119.4(2) O(4)-C(3)-O(2) 107.36(13) C(20)-C(19)-C(18) 120.3(2) O(4)-C(3)-N(9) 107.44(13) C(19)-C(20)-C(21) 120.4(2) O(2)-C(3)-N(9) 107.91(13) C(20)-C(21)-C(22) 120.0(2) O(4)-C(3)-C(17) 111.02(14) C(17)-C(22)-C(21) 119.40(19) O(2)-C(3)-C(17) 110.21(14) C(24)-O(23)-C(6) 115.78(13) N(9)-C(3)-C(17) 112.69(14) O(25)-C(24)-O(23) 123.49(15) C(3)-O(4)-C(5) 105.84(12) O(25)-C(24)-C(26) 125.63(15) O(4)-C(5)-C(6) 107.66(13) O(23)-C(24)-C(26) 110.88(14) O(4)-C(5)-C(1) 102.54(12) C(31)-C(26)-C(27) 120.62(16) C(6)-C(5)-C(1) 103.20(13) C(31)-C(26)-C(24) 121.72(15) O(23)-C(6)-C(7) 107.66(13) C(27)-C(26)-C(24) 117.65(16) O(23)-C(6)-C(5) 113.97(13) C(28)-C(27)-C(26) 119.40(17) C(7)-C(6)-C(5) 102.22(13) C(29)-C(28)-C(27) 120.29(17) O(8)-C(7)-C(32) 108.70(12) C(28)-C(29)-C(30) 120.45(17) O(8)-C(7)-C(6) 104.26(13) C(29)-C(30)-C(31) 119.92(18) C(32)-C(7)-C(6) 115.48(14) C(30)-C(31)-C(26) 119.31(16) C(1)-O(8)-C(7) 110.28(12) O(33)-C(32)-C(7) 108.04(14) C(10)-N(9)-C(13) 110.01(15) C(34)-O(33)-C(32) 118.08(14) C(10)-N(9)-C(3) 128.26(15) O(35)-C(34)-O(33) 124.06(17) C(13)-N(9)-C(3) 121.66(15) O(35)-C(34)-C(36) 124.91(17) N(9)-C(10)-C(11) 105.33(15) O(33)-C(34)-C(36) 111.02(16) C(10)-C(11)-C(12) 110.67(16) C(41)-C(36)-C(37) 119.75(18) C(10)-C(11)-N(14) 123.68(17) C(41)-C(36)-C(34) 121.87(16) C(12)-C(11)-N(14) 125.63(17) C(37)-C(36)-C(34) 118.38(18) C(13)-C(12)-C(11) 105.46(16) C(38)-C(37)-C(36) 120.0(2) C(12)-C(13)-N(9) 108.54(17) C(37)-C(38)-C(39) 120.1(2) O(16)-N(14)-O(15) 123.87(18) C(38)-C(39)-C(40) 120.3(2) O(16)-N(14)-C(11) 118.99(16) C(39)-C(40)-C(41) 119.7(2) O(15)-N(14)-C(11) 117.14(17) C(36)-C(41)-C(40) 120.08(19) ______
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Table 5. Anisotropic displacement parameters (Å2x103 ). The anisotropic displacement factor exponent takes the form: 2 2 2 -2 π [ h a* U11 + ... + 2 h k a* b* U12 ]. ______U11 U22 U33 U23 U13 U12 ______
C(1) 17(1) 15(1) 17(1) -1(1) 4(1) 2(1) O(2) 17(1) 21(1) 18(1) 3(1) 6(1) 4(1) C(3) 18(1) 13(1) 19(1) 1(1) 5(1) 0(1) O(4) 17(1) 18(1) 16(1) 1(1) 4(1) 3(1) C(5) 17(1) 13(1) 16(1) -2(1) 3(1) -1(1) C(6) 16(1) 14(1) 17(1) 0(1) 5(1) 1(1) C(7) 16(1) 14(1) 17(1) 1(1) 5(1) 0(1) O(8) 18(1) 19(1) 23(1) 5(1) 8(1) 3(1) N(9) 22(1) 14(1) 22(1) 0(1) 5(1) 0(1) C(10) 21(1) 17(1) 23(1) 0(1) 4(1) 2(1) C(11) 20(1) 14(1) 23(1) 2(1) -1(1) -1(1) C(12) 21(1) 21(1) 24(1) 0(1) 5(1) -4(1) C(13) 24(1) 22(1) 24(1) -1(1) 8(1) -2(1) N(14) 22(1) 18(1) 32(1) -1(1) 1(1) 1(1) O(15) 32(1) 18(1) 45(1) 5(1) 5(1) -4(1) O(16) 30(1) 21(1) 62(1) -6(1) 18(1) 2(1) C(17) 21(1) 18(1) 17(1) 0(1) 5(1) -4(1) C(18) 33(1) 26(1) 30(1) 2(1) -6(1) 0(1) C(19) 40(1) 43(2) 35(1) 6(1) -13(1) -6(1) C(20) 49(1) 45(1) 23(1) -4(1) -2(1) -20(1) C(21) 47(1) 29(1) 25(1) -7(1) 10(1) -13(1) C(22) 31(1) 20(1) 21(1) -1(1) 7(1) -3(1) O(23) 15(1) 15(1) 24(1) -2(1) 7(1) 0(1) C(24) 15(1) 17(1) 18(1) 4(1) 2(1) 1(1) O(25) 19(1) 20(1) 24(1) -1(1) 4(1) -3(1) C(26) 17(1) 17(1) 20(1) 4(1) 6(1) 2(1) C(27) 20(1) 17(1) 27(1) 4(1) 7(1) 1(1) C(28) 21(1) 23(1) 33(1) 5(1) 12(1) 0(1) C(29) 27(1) 27(1) 25(1) 3(1) 12(1) 5(1) C(30) 27(1) 26(1) 25(1) -3(1) 6(1) 0(1) C(31) 18(1) 24(1) 22(1) 1(1) 5(1) -1(1) C(32) 26(1) 15(1) 21(1) 1(1) 8(1) 0(1) O(33) 28(1) 23(1) 17(1) 2(1) 7(1) 7(1) C(34) 21(1) 23(1) 23(1) 4(1) 8(1) 0(1) O(35) 28(1) 38(1) 32(1) -1(1) 3(1) 13(1) C(36) 27(1) 21(1) 19(1) 2(1) 6(1) -3(1) C(37) 31(1) 37(1) 27(1) 4(1) 4(1) 3(1)
216
Table 5 (continued). ______U11 U22 U33 U23 U13 U12 ______
C(38) 41(1) 40(1) 27(1) 1(1) -5(1) -2(1) C(39) 53(1) 34(1) 25(1) -3(1) 9(1) -7(1) C(40) 41(1) 28(1) 29(1) -3(1) 13(1) -3(1) C(41) 28(1) 26(1) 23(1) 1(1) 9(1) -2(1) ______
Table 6. Hydrogen coordinates and isotropic displacement parameters. ______x y z U(eq) ______
H(1) 0.6470 0.5358 1.0432 0.020 H(5) 0.8527 0.5370 0.9888 0.019 H(6) 0.8946 0.3503 1.0927 0.019 H(7) 0.6902 0.2230 0.9307 0.019 H(10) 0.5398 0.7575 0.8518 0.024 H(12) 0.8658 0.8563 0.7256 0.026 H(13) 0.8524 0.6214 0.7317 0.027 H(18) 0.4437 0.6136 0.6523 0.038 H(19) 0.3064 0.5192 0.4983 0.052 H(20) 0.3421 0.3101 0.4577 0.049 H(21) 0.5181 0.1937 0.5672 0.040 H(22) 0.6571 0.2865 0.7221 0.028 H(27) 1.3031 0.3671 0.9309 0.025 H(28) 1.4237 0.2649 0.8190 0.029 H(29) 1.3220 0.0995 0.7084 0.030 H(30) 1.0995 0.0325 0.7097 0.031 H(31) 0.9760 0.1351 0.8203 0.025 H(32A) 0.6379 0.1066 1.0703 0.024 H(32B) 0.8016 0.0997 1.0862 0.024 H(37) 1.0452 0.1844 1.4452 0.039 H(38) 1.0718 0.3076 1.6000 0.046 H(39) 0.8957 0.4418 1.6215 0.045 H(40) 0.6944 0.4588 1.4857 0.038 H(41) 0.6707 0.3413 1.3270 0.030 ______
217
Table 7. Torsion angles [°]. ______
O(8)-C(1)-O(2)-C(3) -112.59(15) N(9)-C(3)-C(17)-C(22) 141.98(16) C(5)-C(1)-O(2)-C(3) 2.89(17) O(4)-C(3)-C(17)-C(18) -164.37(16) C(1)-O(2)-C(3)-O(4) 18.31(17) O(2)-C(3)-C(17)-C(18) 76.8(2) C(1)-O(2)-C(3)-N(9) -97.22(15) N(9)-C(3)-C(17)-C(18) -43.8(2) C(1)-O(2)-C(3)-C(17) 139.35(14) C(22)-C(17)-C(18)-C(19) 0.7(3) O(2)-C(3)-O(4)-C(5) -33.07(16) C(3)-C(17)-C(18)-C(19) -173.4(2) N(9)-C(3)-O(4)-C(5) 82.77(15) C(17)-C(18)-C(19)-C(20) 0.0(4) C(17)-C(3)-O(4)-C(5) -153.60(13) C(18)-C(19)-C(20)-C(21) -0.8(4) C(3)-O(4)-C(5)-C(6) 141.90(13) C(19)-C(20)-C(21)-C(22) 0.8(4) C(3)-O(4)-C(5)-C(1) 33.46(16) C(18)-C(17)-C(22)-C(21) -0.7(3) O(8)-C(1)-C(5)-O(4) 96.08(14) C(3)-C(17)-C(22)-C(21) 173.47(17) O(2)-C(1)-C(5)-O(4) -21.84(16) C(20)-C(21)-C(22)-C(17) 0.0(3) O(8)-C(1)-C(5)-C(6) -15.72(16) C(7)-C(6)-O(23)-C(24) 176.39(13) O(2)-C(1)-C(5)-C(6) -133.65(13) C(5)-C(6)-O(23)-C(24) 63.76(17) O(4)-C(5)-C(6)-O(23) 38.36(17) C(6)-O(23)-C(24)-O(25) 6.2(2) C(1)-C(5)-C(6)-O(23) 146.35(13) C(6)-O(23)-C(24)-C(26) -173.68(13) O(4)-C(5)-C(6)-C(7) -77.49(14) O(25)-C(24)-C(26)-C(31) -169.34(17) C(1)-C(5)-C(6)-C(7) 30.50(15) O(23)-C(24)-C(26)-C(31) 10.5(2) O(23)-C(6)-C(7)-O(8) -155.49(12) O(25)-C(24)-C(26)-C(27) 9.4(3) C(5)-C(6)-C(7)-O(8) -35.15(15) O(23)-C(24)-C(26)-C(27) -170.75(14) O(23)-C(6)-C(7)-C(32) 85.32(16) C(31)-C(26)-C(27)-C(28) 0.5(3) C(5)-C(6)-C(7)-C(32) -154.33(14) C(24)-C(26)-C(27)-C(28) -178.24(16) O(2)-C(1)-O(8)-C(7) 106.47(14) C(26)-C(27)-C(28)-C(29) -0.2(3) C(5)-C(1)-O(8)-C(7) -6.85(17) C(27)-C(28)-C(29)-C(30) -0.4(3) C(32)-C(7)-O(8)-C(1) 150.42(13) C(28)-C(29)-C(30)-C(31) 0.7(3) C(6)-C(7)-O(8)-C(1) 26.73(16) C(29)-C(30)-C(31)-C(26) -0.4(3) O(4)-C(3)-N(9)-C(10) -138.99(17) C(27)-C(26)-C(31)-C(30) -0.2(3) O(2)-C(3)-N(9)-C(10) -23.5(2) C(24)-C(26)-C(31)-C(30) 178.47(16) C(17)-C(3)-N(9)-C(10) 98.4(2) O(8)-C(7)-C(32)-O(33) -51.93(18) O(4)-C(3)-N(9)-C(13) 44.2(2) C(6)-C(7)-C(32)-O(33) 64.78(18) O(2)-C(3)-N(9)-C(13) 159.71(14) C(7)-C(32)-O(33)-C(34) -129.05(16) C(17)-C(3)-N(9)-C(13) -78.4(2) C(32)-O(33)-C(34)-O(35) -3.8(3) C(13)-N(9)-C(10)-C(11) 0.11(19) C(32)-O(33)-C(34)-C(36) 175.05(15) C(3)-N(9)-C(10)-C(11) -176.98(16) O(35)-C(34)-C(36)-C(41) -166.7(2) N(9)-C(10)-C(11)-C(12) -0.14(19) O(33)-C(34)-C(36)-C(41) 14.5(2) N(9)-C(10)-C(11)-N(14) -178.16(16) O(35)-C(34)-C(36)-C(37) 14.0(3) C(10)-C(11)-C(12)-C(13) 0.1(2) O(33)-C(34)-C(36)-C(37) -164.85(17) N(14)-C(11)-C(12)-C(13) 178.09(17) C(41)-C(36)-C(37)-C(38) -0.5(3) C(11)-C(12)-C(13)-N(9) -0.1(2) C(34)-C(36)-C(37)-C(38) 178.9(2) C(10)-N(9)-C(13)-C(12) 0.0(2) C(36)-C(37)-C(38)-C(39) 1.5(4) C(3)-N(9)-C(13)-C(12) 177.28(16) C(37)-C(38)-C(39)-C(40) -1.1(4) C(10)-C(11)-N(14)-O(16) 5.2(3) C(38)-C(39)-C(40)-C(41) -0.3(4) C(12)-C(11)-N(14)-O(16) -172.48(18) C(37)-C(36)-C(41)-C(40) -0.8(3) C(10)-C(11)-N(14)-O(15) -174.48(17) C(34)-C(36)-C(41)-C(40) 179.79(18) C(12)-C(11)-N(14)-O(15) 7.8(3) C(39)-C(40)-C(41)-C(36) 1.2(3) O(4)-C(3)-C(17)-C(22) 21.4(2) O(2)-C(3)-C(17)-C(22) -97.42(18)
218
Appendix B
X-RAY CRYSTALLOGRAPHIC DATA FOR 1-(β-D-RIBOFURANOSYL)-3- NITROPYRROLE (48)
B.1 General Experimental
The absolute structure was determined from the known configuration of a
reactant. The displacement ellipsoids were drawn at the 50% probability level. A
colorless prism-shaped crystal of dimensions 0.54 x 0.47 x 0.44 mm was selected
for structural analysis. Intensity data for this compound were collected using a
Bruker APEX ccd area detector(1) mounted on a Bruker D8 goniometer using
graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). The data were collected
at 100(2) K. The intensity data were measured as a series of ω oscillation frames
each of 0.25 ° for 5 sec / frame. The detector was operated in 512 x 512 mode and
was positioned 5.04 cm from the sample. Coverage of unique data was 99.9%
complete to 26.00 degrees in θ. Cell parameters were determined from a least
squares fit of 7286 peaks in the range 2.34 < θ < 30.52°. From 196 peaks that were
measured both at the beginning and end of data collection, the crystal showed a
decay of -0.21%. The data were corrected for absorption by the semi-empirical
method (2) from equivalent reflections giving minimum and maximum transmission
factors of 0.9354 and 0.9469. Lorentz and polarization corrections were applied.
The data were merged to form a set of 3326 independent data with R(int) = 0.0185. 219
The orthorhombic space group P212121 was determined by systematic absences and statistical tests and verified by subsequent refinement. The structure was solved by direct methods and refined by full-matrix least-squares methods on F2 (3).
Hydrogen atom positions were initially determined by geometry and refined by a riding model. Non-hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atom displacement parameters were set to 1.2 times the displacement parameters of the bonded atoms. A total of 154 parameters were refined against 3326 data to give wR(F2) = 0.0883 and S = 1.029 for weights of w=
2 2 2 2 2 1/[σ (F ) + (0.0640 P) + 0.1375 P], where P = [Fo + 2Fc ] / 3. The final R(F) was
0.0328 for the 3264 observed, [F > 4σ(F)], data. The largest shift/s.u. was 0.000 in
the final refinement cycle. The final difference map had maxima and minima of
0.365 and -0.209 e/Å3, respectively. The absolute structure was determined by
refinement of the Flack parameter(4).
220
B.2 Funding Acknowledgement and Assignment of Credit
The authors thank the National Science Foundation (grant CHE-0079282) and the University of Kansas for funds to purchase of the X-ray instrument and computers. This structure was determined by Douglas R. Powell.
B.3 References
(1) (a) Data Collection: SMART Software Reference Manual (1994). Bruker-AXS,
6300 Enterprise Dr., Madison, WI 53719-1173, USA. (b) Data Reduction:
SAINT Software Reference Manual (1995). Bruker-AXS, 6300 Enterprise Dr.,
Madison, WI 53719-1173, USA.
(2) G. M. Sheldrick (1996). SADABS. Program for Empirical Absorption Correction
of Area Detector Data. University of Göttingen, Germany.
(3) (a) G. M. Sheldrick (1994). SHELXTL Version 5 Reference Manual. Bruker-AXS,
5465 E. Cheryl Parkway, Madison, WI 53711-5373, USA. (b) International
Tables for Crystallography, Vol C, Tables 6.1.1.4, 4.2.6.8, and 4.2.4.2,
Kluwer: Boston (1995).
(4) H. D. Flack (1983). On enantiomorph-polarity estimation. Acta Cryst. A39, 876-
881.
221
B.4 Thermal Ellipsoid Drawing of 48
222
B.5 Crystal Packing Diagram of 48
223
B.6 Crystallographic Information for 48
Table 1. Crystal data and structure refinement.
Empirical formula C9H12N2O6 Formula weight 244.21 Crystal system Orthorhombic
Space group P212121 Unit cell dimensions a = 6.6218(4) Å α = 90° b = 9.8971(6) Å β = 90° c = 16.8035(10) Å γ = 90° Volume 1101.24(11) Å3 Z, Z’ 4, 1 Density (calculated) 1.473 Mg/m3 Wavelength 0.71073 Å Temperature 100(2) K F(000) 512 Absorption coefficient 0.125 mm-1 Absorption correction Semi-empirical from equivalents Max. and min. transmission 0.9469 and 0.9354 Theta range for data collection 2.39 to 30.53° Reflections collected 9332 Independent reflections 3326 [R(int) = 0.0185] Data / restraints / parameters 3326 / 0 / 154 wR(F2all data) wR2 = 0.0883 R(F obsd data) R1 = 0.0328 Goodness-of-fit on F2 1.029 Observed data [I > 2σ(I)] 3264 Absolute structure parameter -0.1(6) Largest and mean shift / s.u. 0.000and 0.000 Largest diff. peak and hole 0.365 and -0.209 e/Å3 ------2 2 2 2 2 1/2 wR2 = { Σ [w(Fo - Fc ) ] / Σ [w(Fo ) ] } R1 = Σ ||Fo| - |Fc|| / Σ |Fo|
224
Table 2. Atomic coordinates and equivalent isotropic displacement parameters. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. ______x y z U(eq) ______O(1) 0.29874(11) 0.73026(7) 0.42715(4) 0.01456(14) C(2) 0.20283(14) 0.76903(9) 0.35268(5) 0.01342(16) C(3) 0.31696(15) 0.69532(9) 0.28663(6) 0.01345(16) C(4) 0.41088(14) 0.57475(9) 0.32972(5) 0.01199(16) C(5) 0.45159(14) 0.63408(9) 0.41268(5) 0.01247(16) N(6) 0.44471(13) 0.53165(8) 0.47481(5) 0.01401(15) C(7) 0.60966(15) 0.48412(10) 0.51335(5) 0.01453(17) C(8) 0.54059(15) 0.38379(10) 0.56362(6) 0.01553(18) C(9) 0.32783(16) 0.36944(11) 0.55532(6) 0.01818(19) C(10) 0.27232(15) 0.46318(11) 0.49929(6) 0.01778(19) C(11) -0.01953(14) 0.73481(10) 0.35718(6) 0.01663(18) O(12) -0.04049(12) 0.59205(8) 0.36414(5) 0.02118(16) O(13) 0.47378(12) 0.78204(8) 0.25796(5) 0.01907(16) O(14) 0.59294(10) 0.53030(7) 0.29448(4) 0.01513(14) N(15) 0.66568(15) 0.31046(9) 0.61644(5) 0.01868(18) O(16) 0.84923(13) 0.33799(10) 0.61862(5) 0.02600(18) O(17) 0.58914(14) 0.22195(9) 0.65847(5) 0.02465(17) ______
Table 3. Bond lengths [Å]. ______O(1)-C(5) 1.4106(11) C(7)-C(8) 1.3816(14) O(1)-C(2) 1.4549(11) C(7)-H(7) 0.9500 C(2)-C(11) 1.5128(13) C(8)-N(15) 1.4144(12) C(2)-C(3) 1.5281(13) C(8)-C(9) 1.4228(14) C(2)-H(2) 1.0000 C(9)-C(10) 1.3720(14) C(3)-O(13) 1.4308(12) C(9)-H(9) 0.9500 C(3)-C(4) 1.5281(13) C(10)-H(10) 0.9500 C(3)-H(3) 1.0000 C(11)-O(12) 1.4245(12) C(4)-O(14) 1.4134(11) C(11)-H(11A) 0.9900 C(4)-C(5) 1.5364(13) C(11)-H(11B) 0.9900 C(4)-H(4) 1.0000 O(12)-H(12) 0.8942 C(5)-N(6) 1.4560(12) O(13)-H(13) 0.8948 C(5)-H(5) 1.0000 O(14)-H(14) 0.8470 N(6)-C(7) 1.3542(12) N(15)-O(17) 1.2341(12) N(6)-C(10) 1.3898(12) N(15)-O(16) 1.2461(13)
225
Table 4. Bond angles [°]. ______C(5)-O(1)-C(2) 110.05(7) C(7)-N(6)-C(5) 124.03(8) O(1)-C(2)-C(11) 108.83(8) C(10)-N(6)-C(5) 125.27(8) O(1)-C(2)-C(3) 106.44(7) N(6)-C(7)-C(8) 105.96(9) C(11)-C(2)-C(3) 114.25(8) N(6)-C(7)-H(7) 127.0 O(1)-C(2)-H(2) 109.1 C(8)-C(7)-H(7) 127.0 C(11)-C(2)-H(2) 109.1 C(7)-C(8)-N(15) 123.95(9) C(3)-C(2)-H(2) 109.1 C(7)-C(8)-C(9) 109.86(9) O(13)-C(3)-C(4) 109.43(8) N(15)-C(8)-C(9) 126.17(9) O(13)-C(3)-C(2) 108.49(7) C(10)-C(9)-C(8) 105.37(9) C(4)-C(3)-C(2) 103.29(7) C(10)-C(9)-H(9) 127.3 O(13)-C(3)-H(3) 111.8 C(8)-C(9)-H(9) 127.3 C(4)-C(3)-H(3) 111.8 C(9)-C(10)-N(6) 108.23(9) C(2)-C(3)-H(3) 111.8 C(9)-C(10)-H(10) 125.9 O(14)-C(4)-C(3) 113.06(7) N(6)-C(10)-H(10) 125.9 O(14)-C(4)-C(5) 110.45(8) O(12)-C(11)-C(2) 108.72(8) C(3)-C(4)-C(5) 101.71(7) O(12)-C(11)-H(11A)109.9 O(14)-C(4)-H(4) 110.4 C(2)-C(11)-H(11A) 109.9 C(3)-C(4)-H(4) 110.4 O(12)-C(11)-H(11B)109.9 C(5)-C(4)-H(4) 110.4 C(2)-C(11)-H(11B) 109.9 O(1)-C(5)-N(6) 108.89(7) H(11A)-C(11)-H(11B)108.3 O(1)-C(5)-C(4) 106.77(7) C(11)-O(12)-H(12) 106.7 N(6)-C(5)-C(4) 112.27(7) C(3)-O(13)-H(13) 106.8 O(1)-C(5)-H(5) 109.6 C(4)-O(14)-H(14) 108.1 N(6)-C(5)-H(5) 109.6 O(17)-N(15)-O(16) 122.61(10) C(4)-C(5)-H(5) 109.6 O(17)-N(15)-C(8) 118.87(10) C(7)-N(6)-C(10) 110.59(8) O(16)-N(15)-C(8) 118.52(9)
226
Table 4. Anisotropic displacement parameters (Å2x 103 ). The anisotropic displacement factor exponent takes the form: 2 2 2 -2 π [ h a* U11 + ... + 2 h k a* b* U12 ]. ______U11 U22 U33 U23 U13 U12 ______O(1) 14(1) 16(1) 13(1) -2(1) -1(1) 4(1) C(2) 13(1) 14(1) 13(1) 0(1) 0(1) 3(1) C(3) 14(1) 14(1) 13(1) 0(1) 1(1) 2(1) C(4) 11(1) 12(1) 13(1) -1(1) 1(1) 1(1) C(5) 12(1) 12(1) 13(1) 1(1) 0(1) 1(1) N(6) 13(1) 16(1) 14(1) 1(1) -1(1) -1(1) C(7) 13(1) 16(1) 14(1) -1(1) -1(1) 1(1) C(8) 17(1) 17(1) 13(1) 0(1) -2(1) 2(1) C(9) 17(1) 22(1) 16(1) 4(1) 0(1) -1(1) C(10) 13(1) 23(1) 18(1) 3(1) -1(1) -2(1) C(11) 12(1) 19(1) 19(1) -2(1) 0(1) 2(1) O(12) 14(1) 19(1) 30(1) -1(1) -3(1) -2(1) O(13) 21(1) 14(1) 22(1) 4(1) 10(1) 2(1) O(14) 12(1) 14(1) 20(1) -4(1) 2(1) 1(1) N(15) 23(1) 20(1) 13(1) 0(1) -3(1) 5(1) O(16) 20(1) 34(1) 24(1) 4(1) -7(1) 3(1) O(17) 31(1) 25(1) 19(1) 7(1) 1(1) 5(1) ______
Table 5. Hydrogen coordinates and isotropic displacement parameters. ______x y z U(eq) ______H(2) 0.2182 0.8687 0.3450 0.016 H(3) 0.2246 0.6660 0.2428 0.016 H(4) 0.3119 0.4987 0.3332 0.014 H(5) 0.5864 0.6796 0.4131 0.015 H(7) 0.7452 0.5137 0.5071 0.017 H(9) 0.2424 0.3080 0.5827 0.022 H(10) 0.1390 0.4786 0.4805 0.021 H(11A) -0.0813 0.7798 0.4038 0.020 H(11B) -0.0893 0.7670 0.3086 0.020 H(12) -0.1573 0.5702 0.3408 0.025 H(13) 0.5418 0.7354 0.2211 0.023 H(14) 0.5750 0.4506 0.2775 0.018 ______
227
Table 6. Torsion angles [°]. ______C(5)-O(1)-C(2)-C(11) 120.05(8) O(1)-C(5)-N(6)-C(10) 49.83(11) C(5)-O(1)-C(2)-C(3) -3.53(10) C(4)-C(5)-N(6)-C(10) -68.18(11) O(1)-C(2)-C(3)-O(13) -92.60(9) C(10)-N(6)-C(7)-C(8) -0.26(11) C(11)-C(2)-C(3)-O(13) 147.26(8) C(5)-N(6)-C(7)-C(8) -176.50(8) O(1)-C(2)-C(3)-C(4) 23.46(9) N(6)-C(7)-C(8)-N(15) -178.23(9) C(11)-C(2)-C(3)-C(4) -96.68(9) N(6)-C(7)-C(8)-C(9) 0.20(11) O(13)-C(3)-C(4)-O(14) -35.81(10) C(7)-C(8)-C(9)-C(10) -0.05(12) C(2)-C(3)-C(4)-O(14) -151.20(8) N(15)-C(8)-C(9)-C(10) 178.33(9) O(13)-C(3)-C(4)-C(5) 82.62(8) C(8)-C(9)-C(10)-N(6) -0.11(12) C(2)-C(3)-C(4)-C(5) -32.77(9) C(7)-N(6)-C(10)-C(9) 0.24(12) C(2)-O(1)-C(5)-N(6) -139.50(8) C(5)-N(6)-C(10)-C(9) 176.41(9) C(2)-O(1)-C(5)-C(4) -18.06(9) O(1)-C(2)-C(11)-O(12) -63.97(10) O(14)-C(4)-C(5)-O(1) 152.20(7) C(3)-C(2)-C(11)-O(12) 54.82(11) C(3)-C(4)-C(5)-O(1) 31.92(9) C(7)-C(8)-N(15)-O(17) -179.44(9) O(14)-C(4)-C(5)-N(6) -88.54(9) C(9)-C(8)-N(15)-O(17) 2.40(15) C(3)-C(4)-C(5)-N(6) 151.18(8) C(7)-C(8)-N(15)-O(16) 0.57(15) O(1)-C(5)-N(6)-C(7) -134.49(9) C(9)-C(8)-N(15)-O(16) -177.59(10) C(4)-C(5)-N(6)-C(7) 107.50(10) ______
Table 7. Hydrogen bonds [Å and °]. ______D-H...A d(D-H) d(H...A) d(D...A) <(DHA) ______O(12)-H(12)...O(14)#1 0.89 1.87 2.7633(11) 177.6 O(13)-H(13)...O(16)#2 0.89 2.00 2.8752(11) 164.6 O(14)-H(14)...O(13)#3 0.85 1.80 2.6473(10) 177.7 ______Symmetry transformations used to generate equivalent atoms: #1 x-1, y, z #2 -x+3/2, -y+1, z-1/2 #3 -x+1, y-1/2, -z+1/2
228
Appendix C
X-RAY CRYSTALLOGRAPHIC DATA FOR 1-(β-D-RIBOFURANOSYL)-5- NITROINDOLE (63)
C.1 General Experimental
A clear needle shaped crystal (C13H14N2O6) with approximate dimensions
0.10 x 0.16 x 0.50 mm, was used for the X-ray crystallographic analysis. The X-
ray intensity data were measured at 95(2) K, cooled by Rigaku-MSC X-Stream
2000, on a Bruker SMART APEX CCD area detector system equipped with a
graphite monochromator and a MoKα fine-focus sealed tube (λ = 0.71073Å)
operated at 1600 watts power (50 kV, 32 mA). The detector was placed at a
distance of 5.8 cm from the crystal.
A total of 1850 frames were collected with a scan width of 0.3º in ω and an
exposure time of 10 seconds/frame. The total data collection time was about 8
hours. The frames were integrated with the Bruker SAINT software package
using a narrow-frame integration algorithm. The integration of the data using a
Orthorhombic unit cell yielded a total of 7707 reflections to a maximum θ angle of
28.28° (0.90 Å resolution), of which 2920 were independent, completeness =
97.4%, Rint = 0.0401, Rsig = 0.0454 and 2601 were greater than 2σ(I). The
final cell constants: a = 5.7856(14)Å, b = 10.077(2)Å, c = 21.067(5)Å, α = 90°, β
= 90°, γ = 90°, volume = 1228.2(5)Å3, are based upon the refinement of the XYZ- 229 centroids of 2710 reflections above 20σ(I) with 2.240° <θ <28.231° . Analysis of
the data showed negligible decay during data collection. Data were corrected for
absorption effects using the multiscan technique (SADABS). The ratio of
minimum to maximum apparent transmission was 0.732661.
The structure was solved and refined using the Bruker SHELXTL (Version
6.1) Software Package, using the space group P2(1)2(1)2(1), with Z = 4 for the
formula unit, C13H14N2O6. The final anisotropic full-matrix least-squares
refinement on F2 with 193 variables converged at R1 = 4.20%, for the observed
data and wR2 = 10.05% for all data. The goodness-of-fit was 1.037. The largest
peak on the final difference map was 0.376 e-/Å3 and the largest hole was -0.190
e-/Å3. Based on the final model, the calculated density of the crystal is 1.591
g/cm3 and F(000) amounts to 616 electrons.
C.2 Funding Acknowledgement and Assignment of Credit
The small molecule facility was established using funds from an NSF
Chemistry Research Instrumentation and Facilities grant (CHE-0131112). This
structure was solved by Hemant Yennawar.
230 C.3 Thermal Ellipsoid Drawing of 5-NINDN (63)
C.4 Crystallographic Information for 63
Table 1. Sample and crystal data.
Empirical formula C13H14N2O6 Formula weight 294.26 Temperature 95(2) K Wavelength 0.71073 Å Crystal size 0.50 x 0.16 x 0.10 mm Crystal habit clear needle Crystal system Orthorhombic Space group P2(1)2(1)2(1) Unit cell dimensions a = 5.7856(14) Å α= 90° b = 10.077(2) Å β= 90° c = 21.067(5) Å γ = 90° Volume 1228.2(5) Å3 Z 4 Density (calculated) 1.591 g/cm3 Absorption coefficient 0.128 mm-1 F(000) 616
231
Table 2. Data collection and structure refinement.
Diffractometer CCD area detector Radiation source fine-focus sealed tube, MoK Generator power 1600 watts (50 kV, 32mA) Detector distance 5.8 cm Data collection method omega scans Theta range for data collection 1.93 to 28.28° Index ranges -7 ≤ h ≤ 7, -12 ≤ k ≤ 13, -23 ≤ l ≤ 27
Table 3. Atomic coordinates and equivalent isotropic atomic displacement parameters (Å2). U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. ______x yz U(eq) ______O1 0.2744(2) 0.27470(12) 0.75777(6) 0.0159(3) O4 -0.1916(2) 0.24677(13) 0.71551(7) 0.0211(3) O2 0.5869(2) 0.03432(13) 0.77551(7) 0.0183(3) O3 0.2322(2) -0.05523(13) 0.69804(6) 0.0191(3) C11 0.1865(3) 0.04881(18) 0.74064(8) 0.0154(4) N2 0.1770(3) 0.23128(15) 0.86491(7) 0.0149(3) O6 0.2341(3) 0.72535(16) 1.04392(8) 0.0318(4) N1 0.0872(3) 0.63744(17) 1.04010(8) 0.0244(4) C1 0.1135(4) 0.53636(19) 0.99112(9) 0.0194(4) C10 0.3557(3) 0.05831(19) 0.79617(9) 0.0156(4) O5 -0.0806(3) 0.63137(16) 1.07570(8) 0.0379(4) C5 -0.0215(3) 0.34175(19) 0.94054(8) 0.0163(4) C2 0.3157(3) 0.53658(19) 0.95479(9) 0.0199(4) C6 -0.0581(3) 0.4416(2) 0.98524(9) 0.0196(4) C12 0.2143(3) 0.18253(18) 0.70751(9) 0.0160(4) C7 -0.1513(3) 0.2275(2) 0.92209(9) 0.0190(4) C13 -0.0002(3) 0.2307(2) 0.67378(9) 0.0185(4) C9 0.3400(3) 0.20482(18) 0.81447(8) 0.0145(4) C3 0.3514(3) 0.43954(18) 0.90964(9) 0.0171(4) C8 -0.0293(3) 0.1643(2) 0.87675(9) 0.0172(4) C4 0.1827(3) 0.34174(18) 0.90378(8) 0.0154(4) ______
232 Table 4. Bond lengths (Å). ______O1-C9 1.438(2) O1-C12 1.451(2) O4-C13 1.423(2) O2-C10 1.427(2) O3-C11 1.405(2) C11-C12 1.526(3) C11-C10 1.528(3) N2-C4 1.382(2) N2-C8 1.394(2) N2-C9 1.445(2) O6-N1 1.230(2) N1-O5 1.228(3) N1-C1 1.458(2) C1-C6 1.383(3) C1-C2 1.398(3) C10-C9 1.529(3) C5-C6 1.394(3) C5-C4 1.413(3) C5-C7 1.428(3) C2-C3 1.380(3) C12-C13 1.510(3) C7-C8 1.347(3) C3-C4 1.392(3) ______
Table 5. Bond angles (°). ______C9-O1-C12 110.86(13) O3-C11-C12 110.30(14) O3-C11-C10 114.53(15) C12-C11-C10 103.13(15) C4-N2-C8 107.71(15) C4-N2-C9 124.65(15) C8-N2-C9 126.94(15) O5-N1-O6 122.82(18) O5-N1-C1 118.68(18) O6-N1-C1 118.50(18) C6-C1-C2 123.55(18) C6-C1-N1 118.11(18) C2-C1-N1 118.27(18) O2-C10-C11 110.87(15) O2-C10-C9 107.25(15) C11-C10-C9 102.43(14) C6-C5-C4 119.81(17) C6-C5-C7 133.30(18) C4-C5-C7 106.88(16) C3-C2-C1 120.09(18) C1-C6-C5 116.75(18) O1-C12-C13 109.55(15) O1-C12-C11 104.88(14) C13-C12-C11 114.34(16) C8-C7-C5 107.36(17) O4-C13-C12 112.67(15) O1-C9-N2 110.39(14) O1-C9-C10 106.22(14) N2-C9-C10 113.74(15) C2-C3-C4 117.26(17) C7-C8-N2 110.29(17) N2-C4-C3 129.73(17) N2-C4-C5 107.75(16) C3-C4-C5 122.51(17) ______
233
Appendix D
X-RAY CRYSTALLOGRAPHIC DATA FOR 1-(β-D- RIBOFURANOSYL)PYRROLO[2,3-B]PYRIDINE (65)
D.1 General Experimental
A colourless needle shaped crystal (C12H14N2O4) with approximate dimensions 0.10 x 0.11 x 0.39 mm, was used for the X-ray crystallographic analysis. The X-ray intensity data were measured at 103(2) K, cooled by Rigaku-
MSC X-Stream 2000, on a Bruker SMART APEX CCD area detector system equipped with a graphite monochromator and a MoKα fine-focus sealed tube (λ
= 0.71073Å) operated at 1600 watts power (50 kV, 32 mA). The detector was placed at a distance of 5.8 cm from the crystal.
A total of 1850 frames were collected with a scan width of 0.3° in ω and an exposure time of 10 seconds/frame. The total data collection time was about 8 hours. The frames were integrated with the Bruker SAINT software package using a narrow-frame integration algorithm. The integration of the data using a
Orthorhombic unit cell yielded a total of 7378 reflections to a maximum θ angle of
28.24° (0.90 Å resolution), of which 2773 were independent, completeness =
98.2%, Rint = 0.0289, Rsig = 0.0375 and 2551 were greater than 2σ(I). The final cell constants: a = 6.8301(17)Å, b = 8.376(2)Å, c = 20.365(5)Å, α = 90°, β =
90°, γ= 90°, volume = 1165.1(5)Å3, are based upon the refinement of the XYZ- 234 centroids of 2237 reflections above 20σ(I) with 2.629° <θ <24.385°. Analysis of
the data showed negligible decay during data collection. Data were corrected for
absorption effects using the multiscan technique (SADABS). The ratio of
minimum to maximum apparent transmission was 0.896834.
The structure was solved and refined using the Bruker SHELXTL (Version
6.1) Software Package, using the space group P2(1)2(1)2(1), with Z = 4 for the
formula unit, C12H14N2O4. The final anisotropic full-matrix least-squares
refinement on F2 with 166 variables converged at R1 = 3.84%, for the observed
data and wR2 = 9.02% for all data. The goodness-of-fit was 1.033. The largest
peak on the final difference map was 0.281 e-/Å3 and the largest hole was -0.224
e-/Å3. Based on the final model, the calculated density of the crystal is 1.427
g/cm3 and F(000) amounts to 528 electrons.
D.2 Funding Acknowledgement and Credit
The small molecule facility was established using funds from an NSF
Chemistry Research Instrumentation and Facilities grant (CHE-0131112). This
structure was solved by Hemant Yennawar.
235 D.3 Thermal Ellipsoid Drawing of 7AI-R (65)
236
D.4 Crystallographic Information for 65
Table 1. Sample and crystal data.
Empirical formula C12H14N2O4 Formula weight 250.25 Temperature 103(2) K Wavelength 0.71073 Å Crystal size 0.39 x 0.11 x 0.10 mm Crystal habit colourless needle Crystal system Orthorhombic Space group P2(1)2(1)2(1) Unit cell dimensions a = 6.8301(17) Å α= 90° b = 8.376(2) Å β= 90° c = 20.365(5) Å γ = 90° Volume 1165.1(5) Å3 Z 4 Density (calculated) 1.427 g/cm3 Absorption coefficient 0.108 mm-1 F(000) 528 ______
237
Table 2. Data collection and structure refinement.
Diffractometer CCD area detector Radiation source fine-focus sealed tube, MoK Generator power 1600 watts (50 kV, 32mA) Detector distance 5.8 cm Data collection method phi and omega scans Theta range for data collection 2.00 to 28.24° Index ranges -5 ≤ h ≤ 8, -10 ≤ k ≤ 10, -27 ≤ l ≤ 26
Table 3. Atomic coordinates and equivalent isotropic atomic displacement parameters (Å2). U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. ______x yz U(eq) ______C1 0.9288(2) 0.11921(19) 1.00572(8) 0.0226(3) C2 0.8407(3) 0.0673(2) 1.06139(8) 0.0259(4) C3 0.6571(2) -0.00094(19) 1.04278(7) 0.0211(3) C4 0.4980(3) -0.0701(2) 1.07500(8) 0.0263(4) C5 0.3426(3) -0.1215(2) 1.03711(8) 0.0274(4) C6 0.3455(2) -0.1011(2) 0.96920(8) 0.0235(3) C7 0.6438(2) 0.01312(18) 0.97352(7) 0.0184(3) C8 0.8560(2) 0.13687(18) 0.88530(7) 0.0176(3) C9 0.8643(2) 0.00390(18) 0.83322(7) 0.0169(3) C10 0.8239(2) 0.0980(2) 0.77058(7) 0.0191(3) C11 0.6759(2) 0.22393(19) 0.79329(7) 0.0200(3) C12 0.4629(2) 0.1818(2) 0.78097(8) 0.0226(3) N1 0.81261(19) 0.08648(15) 0.95146(6) 0.0180(3) N2 0.4937(2) -0.03296(15) 0.93629(6) 0.0203(3) O1 0.70614(16) 0.24157(12) 0.86369(5) 0.0205(3) O2 1.04246(16) -0.08237(13) 0.83364(5) 0.0196(2) O3 1.00578(18) 0.16524(15) 0.75159(6) 0.0250(3) O4 0.40899(16) 0.03136(14) 0.80691(6) 0.0227(3) ______
238
Table 4. Bond lengths (Å). ______C1-C2 1.355(2) C1-N1 1.3879(19) C1-H1 0.9500 C2-C3 1.430(2) C2-H2A 0.9500 C3-C4 1.396(2) C3-C7 1.418(2) C4-C5 1.381(3) C4-H4A 0.9500 C5-C6 1.393(2) C5-H5 0.9500 C6-N2 1.341(2) C6-H6 0.9500 C7-N2 1.333(2) C7-N1 1.381(2) C8-O1 1.4178(18) C8-N1 1.4426(19) C8-C9 1.539(2) C8-H8 1.0000 C9-O2 1.4154(18) C9-C10 1.524(2) C9-H9 1.0000 C10-O3 1.4181(19) C10-C11 1.533(2) C10-H10 1.0000 C11-O1 1.4558(19) C11-C12 1.518(2) C11-H11 1.0000 C12-O4 1.415(2) C12-H12A 0.9900 C12-H12B 0.9900 O2-H2 0.8400 O3-H3 0.8400 O4-H4 0.8400 ______
239 Table 5. Bond angles (°). ______C2-C1-N1 110.41(15) C2-C1-H1 124.8 N1-C1-H1 124.8 C1-C2-C3 107.22(14) C1-C2-H2A 126.4 C3-C2-H2A 126.4 C4-C3-C7 116.90(15) C4-C3-C2 136.44(15) C7-C3-C2 106.65(14) C5-C4-C3 117.71(15) C5-C4-H4A 121.1 C3-C4-H4A 121.1 C4-C5-C6 120.37(16) C4-C5-H5 119.8 C6-C5-H5 119.8 N2-C6-C5 123.97(16) N2-C6-H6 118.0 C5-C6-H6 118.0 N2-C7-N1 125.88(13) N2-C7-C3 126.22(15) N1-C7-C3 107.88(13) O1-C8-N1 108.83(12) O1-C8-C9 105.09(12) N1-C8-C9 116.07(13) O1-C8-H8 108.9 N1-C8-H8 108.9 C9-C8-H8 108.9 O2-C9-C10 115.13(12) O2-C9-C8 113.39(12) C10-C9-C8 101.30(13) O2-C9-H9 108.9 C10-C9-H9 108.9 C8-C9-H9 108.9 O3-C10-C9 105.96(12) O3-C10-C11 112.73(13) C9-C10-C11 102.87(12) O3-C10-H10 111.6 C9-C10-H10 111.6 C11-C10-H10 111.6 O1-C11-C12 108.81(12) O1-C11-C10 105.87(12) C12-C11-C10 114.96(13) O1-C11-H11 109.0 C12-C11-H11 109.0 C10-C11-H11 109.0 O4-C12-C11 113.23(13) O4-C12-H12A 108.9 C11-C12-H12A 108.9 O4-C12-H12B 108.9 C11-C12-H12B 108.9 H12A-C12-H12B 107.7 C7-N1-C1 107.84(12) C7-N1-C8 127.24(12) C1-N1-C8 124.63(13) C7-N2-C6 114.81(14) C8-O1-C11 110.21(11) C9-O2-H2 109.5 C10-O3-H3 109.5 C12-O4-H4 109.5 ______
240 Table 6. Torsion angles (°). ______
N1-C1-C2-C3 -0.70(19) C1-C2-C3-C4 -178.10(18) C1-C2-C3-C7 0.36(18) C7-C3-C4-C5 1.0(2) C2-C3-C4-C5 179.35(18) C3-C4-C5-C6 -1.2(2) C4-C5-C6-N2 0.1(3) C4-C3-C7-N2 0.2(2) C2-C3-C7-N2 -178.57(15) C4-C3-C7-N1 178.92(13) C2-C3-C7-N1 0.10(17) O1-C8-C9-O2 160.43(12) N1-C8-C9-O2 -79.30(17) O1-C8-C9-C10 36.49(14) N1-C8-C9-C10 156.76(13) O2-C9-C10-O3 -40.13(17) C8-C9-C10-O3 82.62(14) O2-C9-C10-C11 -158.66(12) C8-C9-C10-C11 -35.91(15) O3-C10-C11-O1 -89.89(14) C9-C10-C11-O1 23.78(15) O3-C10-C11-C12 149.97(13) C9-C10-C11-C12 -96.36(15) O1-C11-C12-O4 -65.00(16) C10-C11-C12-O4 53.51(17) N2-C7-N1-C1 178.16(15) C3-C7-N1-C1 -0.52(17) N2-C7-N1-C8 4.2(2) C3-C7-N1-C8 -174.51(14) C2-C1-N1-C7 0.77(18) C2-C1-N1-C8 174.96(14) O1-C8-N1-C7 55.98(18) C9-C8-N1-C7 -62.25(19) O1-C8-N1-C1 -117.07(16) C9-C8-N1-C1 124.70(15) N1-C7-N2-C6 -179.72(14) C3-C7-N2-C6 -1.3(2) C5-C6-N2-C7 1.1(2) N1-C8-O1-C11 -147.53(12) C9-C8-O1-C11 -22.58(15) C12-C11-O1-C8 123.32(14) C10-C11-O1-C8 -0.76(16) ______
241 Table 7. Anisotropic atomic displacement parameters (Å2). The anisotropic atomic displacement factor exponent takes the form: 2 2 -2π [h a*U11+... +2hka* b* U12]. ______U11 U22 U33 U23 U13 U12 ______C1 0.0209(8) 0.0220(8) 0.0251(8) -0.0054(6) -0.0059(6) 0.0004(7) C2 0.0286(9) 0.0293(9) 0.0198(8) -0.0038(7) -0.0057(7) 0.0013(8) C3 0.0240(8) 0.0187(7) 0.0206(7) -0.0007(6) -0.0011(6) 0.0050(7) C4 0.0318(10) 0.0243(8) 0.0227(8) 0.0057(7) 0.0039(7) 0.0031(8) C5 0.0247(9) 0.0257(9) 0.0318(9) 0.0086(7) 0.0065(7) 0.0000(7) C6 0.0179(8) 0.0224(8) 0.0304(8) 0.0026(7) -0.0010(7) -0.0024(7) C7 0.0183(7) 0.0156(7) 0.0213(7) -0.0005(6) 0.0018(6) 0.0033(7) C8 0.0133(7) 0.0166(7) 0.0231(8) -0.0001(6) -0.0003(6) -0.0008(6) C9 0.0120(7) 0.0181(7) 0.0206(7) -0.0017(6) 0.0002(6) -0.0008(6) C10 0.0130(7) 0.0237(7) 0.0207(7) 0.0015(6) 0.0000(6) -0.0020(7) C11 0.0178(8) 0.0205(7) 0.0218(8) 0.0032(6) 0.0005(6) -0.0005(7) C12 0.0164(8) 0.0270(8) 0.0242(8) 0.0040(7) -0.0018(7) 0.0034(7) N1 0.0153(6) 0.0198(6) 0.0190(6) -0.0028(5) -0.0004(5) -0.0013(5) N2 0.0174(7) 0.0198(6) 0.0236(7) 0.0002(5) 0.0002(5) -0.0009(6) O1 0.0184(5) 0.0198(5) 0.0233(6) -0.0009(4) 0.0003(5) 0.0036(5) O2 0.0139(5) 0.0208(5) 0.0240(5) 0.0004(5) 0.0025(4) 0.0015(4) O3 0.0157(5) 0.0311(6) 0.0282(6) 0.0100(5) 0.0027(4) -0.0024(5) O4 0.0175(6) 0.0287(6) 0.0218(5) 0.0007(5) -0.0032(5) -0.0031(5) ______
Table 8. Hydrogen atom coordinates and isotropic atomic displacement parameters (Å2). ______x/a y/b z/c U ______H1 1.0527 0.1707 1.0041 0.027 H2A 0.8915 0.0749 1.1047 0.031 H4A 0.4966 -0.0816 1.1214 0.032 H5 0.2330 -0.1709 1.0574 0.033 H6 0.2360 -0.1378 0.9447 0.028 H8 0.9834 0.1956 0.8853 0.021 H9 0.7537 -0.0720 0.8410 0.020 H10 0.7683 0.0283 0.7353 0.023 H11 0.7062 0.3276 0.7712 0.024 H12A 0.3788 0.2651 0.8008 0.027 H12B 0.4385 0.1818 0.7330 0.027 H2 1.1325 -0.0246 0.8187 0.029 H3 0.9861 0.2410 0.7254 0.038 H4 0.4395 0.0273 0.8468 0.034
242
VITA
Daniel Allen Harki
Educational Background Ph.D. The Pennsylvania State University, Ph.D. in Chemistry, 1999-2005. (Degree conferred 8/2005).
B.A. West Virginia University, Double Major in Chemistry and Biology (1995-1999).
Research Experience Postdoctoral Scholar, California Institute of Technology (7/2005- ). Research Advisor: Professor Peter B. Dervan
Graduate Student in Chemistry, The Pennsylvania State University (1999- 2005, American Heart Association predoctoral fellow 2002-2005). Research Advisor: Professor Blake R. Peterson
Biological Science Laboratory Technician, Centers for Disease Control and Prevention (CDC), National Institute for Occupational Safety and Health (NIOSH, 1997-1999). Research Advisors: Dr. Michael I. Luster (Team Leader) & Dr. Petia P. Simeonova (Project Officer)
Undergraduate Research, West Virginia University, Department of Chemistry (1998-1999). Research Advisor: Professor Kay M. Brummond
Publications “Arsenic exposure accelerates atherogenesis in apolipoprotein E-/- mice” Simeonova PP, Hulderman T, Harki D, Luster MI Environmental Health Perspectives 2003, 111(14), 1744-1748
“Synthesis and antiviral evaluation of a mutagenic and non-hydrogen bonding ribonucleoside analogue: 1-β-D-ribofuranosyl-3-nitropyrrole” Harki DA, Graci JD, Korneeva VS, Ghosh SKB, Hong Z, Cameron CE, and Peterson BR Biochemistry 2002, 41(29), 9026-9033
“Arsenic mediates cell proliferation and gene expression in the bladder epithelium: association with activating protein-1 transactivation.” Simeonova PP, Wang S, Toriuma W, Kommineni V, Matheson J, Unimye N, Kayama F, Harki D, Ding M, Vallyathan V, Luster MI Cancer Research, 2000, 60, 3445-3453
Date of Birth: November 27, 1976 (Morgantown, West Virginia)