Modified Nucleosides

in Biochemistry, Biotechnology and Medicine

Edited byPiet Herdewijn

Innodata9783527623129.jpg

Modified Nucleosides

Edited by

Piet Herdewijn

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Modified Nucleosides

in Biochemistry, Biotechnology and Medicine

Edited byPiet Herdewijn

The Editor

Prof. Dr. Piet HerdewijnRega InstituteKatholieke UniversiteitMinderbroedersstraat 10003000 LeuvenBelgium

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Contents

Preface XIXList of Contributors XXI

Part I Biochemistry and Biophysics 1

1 Investigations on Fluorine-Labeled Ribonucleic Acidsby 19F NMR Spectroscopy 3Christoph Kreutz and Ronald Micura

1.1 Introduction 31.1.1 NMR Spectroscopic Properties of the 19F Nucleus 31.1.1.1 General NMR Spectroscopic Properties 31.1.1.2 19F versus 1H NMR Spectroscopy 31.1.1.3 Factors Affecting the 19F Chemical Shift in Biomolecules 51.1.1.4 Fluorine Relaxation in Biological Systems 61.1.1.5 Solvent-Induced Isotope Shifts of 19F NMR Resonances 71.1.2 19F NMR Spectroscopy of Proteins 71.1.2.1 Incorporation of Fluorinated Amino Acids into Proteins 71.1.2.2 19F NMR Spectroscopic Studies of Proteins 81.2 19F NMR Spectroscopy of Nucleic Acids 131.2.1 Nucleic Acids with Fluorinated Nucleobases 141.2.1.1 Transfer RNAs 141.2.1.2 HhaI Methyltransferase in Complex with DNA Duplexes 141.2.1.3 Minimal Hammerhead Ribozyme 151.2.1.4 HIV TAR RNA 171.2.2 Nucleic Acids with Fluorinated Ribose Units 191.2.2.1 R1inv RNA 191.2.2.2 RNA Secondary Structure Equilibria 211.2.2.3 RNA Ligand Binding 221.2.3 Influence of Fluorine Modifications on Nucleic Acid Structure 221.3 Conclusions 24

References 24

V

2 8-Oxo-7,8-Dihydro-20-Deoxyguanosine: A Major DNA OxidationProduct 29Jean Cadet and Paolo Di Mascio

2.1 Introduction 292.2 Formation of 8-Oxo-7,8-Dihydroguanine 302.2.1 Single Lesion 302.2.1.1 �OH Radical 312.2.1.2 One-Electron Oxidation 312.2.1.3 Singlet Oxygen 322.2.2 Tandem Lesions 342.3 Reactivity of 8-Oxo-7,8-Dihydro-20-Deoxyguanosine 352.3.1 One-Electron Oxidation 352.3.1.1 Secondary Oxidation Products 362.3.1.2 DNA–Protein Crosslinks 372.3.2 Singlet Oxygen 372.3.2.1 Nucleoside 372.3.2.2 Oligonucleotide 382.4 Formation of 8-Oxo-7,8-Dihydro-20-Deoxyguanosine in

Cellular DNA 392.4.1 Methods of Measurement 392.4.1.1 HPLC Methods (HPLC-ECD and HPLC-MS/MS) 392.4.1.2 Enzymic Assays 402.4.2 Indirect Effects of Ionizing Radiation (�OH Radical) 412.4.3 High-Intensity UV Laser Irradiation (One-Electron Oxidation) 412.4.4 UVA Photosensitization (1O2) 422.5 Synthesis of 8-OxodGuo and Insertion into Oligonucleotides 422.6 Conclusions 43

References 44

3 Modified DNA Bases: Probing Base-Pair Recognitionby Polymerases 49Eric T. Kool

3.1 Introduction 493.1.1 The Importance of Understanding DNA Polymerases 493.1.2 The Utility of Modified Nucleobases in Probing Mechanisms 503.1.3 The Scope of this Chapter 503.2 Basic Principles and Methods in Replication 513.2.1 The Chemistry of Polymerases 513.2.2 Different Classes of Polymerases 513.2.3 Methods Used in Polymerase Studies 523.3 Alternative Hydrogen-Bonding Schemes 533.3.1 Thioguanine-Pyridone 533.3.2 Benner Hydrogen-Bonding Variants 543.4 Non-Polar Nucleoside Isosteres 563.4.1 The Concept of Nucleobase Isosteres 56

VI Contents

3.4.2 Synthesis, Structure, and Physical Properties 563.4.3 Base-Pairing Properties 573.4.4 Polymerase Behavior 583.4.5 Other Classes of Polymerases 593.4.6 Summary of Watson–Crick H-Bonding Effects in Polymerase

Active Sites 603.5 Non-Polar Steric Probes 603.5.1 Isomers of Hydrocarbons Illustrate Hydrophobic ‘‘Packing’’ Effects 603.5.2 Systematic Size Variants 623.5.3 Systematic Shape Variants 633.6 Minor Groove Hydrogen Bonds in Polymerases 643.6.1 Base Analogues Testing Minor Groove Interactions 643.7 Other Non-Polar Bases and Pairs 653.7.1 The Quest for New Base Pairs 653.7.2 A Broad Variety of Heterocycles and Hydrocarbons 663.7.3 Benzimidazoles Continue the Debate on Steric Effects 673.7.4 New Pairs of Hirao and Yokoyama 673.8 Replication of Designed Bases in Living Cells 683.8.1 Effects of Hydrogen Bonding in E. coli 683.8.2 Effects of Nucleobase Size in E. coli 693.9 Conclusions and Future Prospects 703.9.1 What We Know About Replication 703.9.2 And What Remains Unknown 713.9.3 Future Directions 71

References 71

4 20-Deoxyribose-Modified Nucleoside Triphosphates and theirRecognition by DNA Polymerases 75Karl-Heinz Jung and Andreas Marx

4.1 Introduction 754.2 Modified Nucleotides as Alternative Building Blocks to Natural

Nucleic Acids 764.2.1 Introduction 764.2.2 Nucleotides with Downsized Residues: a-L-Threose-Derived

Nucleotides 764.2.2.1 Introduction 764.2.2.2 Synthesis 774.2.2.3 DNA Polymerase Studies 804.2.3 Nucleotides with Downsized Residues: Glycerol-Derived

Nucleotides 804.2.3.1 Introduction 804.2.3.2 Synthesis 814.2.3.3 DNA Polymerase Studies 824.2.4 Nucleotides with Expanded Sugar Residues: 1,5-Anhydrohexitol

Nucleotides 82

Contents VII

4.2.4.1 Introduction 824.2.4.2 Synthesis 834.2.4.3 Functional DNA Polymerase Studies 854.2.5 NucleotideswithExpandedSugarResidues:CyclohexenylNucleotides 854.2.5.1 Introduction 854.2.5.2 Synthesis 864.2.5.3 DNA Polymerase Studies 884.3 DNA Polymerase Selectivity: 40-C-Modified Nucleotides 894.3.1 Introduction 894.3.2 Design and Synthesis 904.3.3 DNA Polymerase Studies 914.4 Concluding Remarks 93

References 94

5 Pyrimidine Dimers: UV-Induced DNA Damage 97Shigenori Iwai

5.1 Introduction 975.2 Formation of Pyrimidine Dimers 985.2.1 Cyclobutane Pyrimidine Dimers 985.2.2 The (6–4) Photoproducts and their Dewar Valence Isomers 1015.2.3 Other UV Lesions 1015.3 Chemical Synthesis of Oligonucleotides Containing Pyrimidine

Dimers 1035.3.1 Oligonucleotides Containing a CPD 1035.3.2 Oligonucleotides Containing the (6–4) or Dewar Photoproduct 1055.4 Structure and Mutagenesis of Pyrimidine Dimer-Containing DNA 1095.4.1 Tertiary Structures of Pyrimidine Dimer-Containing Duplexes 1095.4.2 Base-Pair Formation by Pyrimidine Dimers 1115.4.3 Mutations Induced by Pyrimidine Dimers 1125.5 Repair of Pyrimidine Dimers in Cells 1165.5.1 T4 Endonuclease V 1165.5.2 Photolyases 1185.5.3 Nucleotide Excision Repair (NER) 1195.5.4 UV Damage Endonuclease (UVDE) 1215.6 Bypass of Pyrimidine Dimers by DNA Polymerases 122

References 124

6 Locked Nucleic Acids: Properties, Applications, and Perspectives 133Poul Nielsen and Jesper Wengel

6.1 Introduction 1336.2 LNA in High-Affinity Hybridization: Designing Sequences 1356.3 Structural Studies 1396.4 Analogues of LNA and their Structural Impact 1406.5 LNA as Potential Therapeutics 1436.6 LNA-Probes 147

VIII Contents

6.7 Concluding Remarks 148References 149

7 Synthesis and Properties of Oligonucleotides IncorporatingModified Nucleobases Capable of Watson–Crick-Type Base-PairFormation 153Mitsuo Sekine, Akio Ohkubo, Itaru Okamoto, and Kohji Seio

7.1 Introduction 1537.2 Natural, Enzyme-Assisted Sophisticated Devices for Maintaining

Correct Base Recognition of Canonical Nucleobases 1547.3 Synthesis and Properties of Oligodeoxynucleotides Incorporating

4-N-Acylated Cytosine Derivatives 1557.4 Base-Recognition Ability of 4-N-Alkoxycarbonylcytosine Derivatives 1577.5 Synthesis and Properties of Oligonucleotides Incorporating

4-N-Carbamoylcytosine Derivatives 1597.6 2-Thiouracil as an Improved Nucleobase in Place of Thymine 1607.7 Modified Adenine Bases Capable of Recognizing the Thymine Base 1617.8 DesignofModifiedGuanineBasesCapable ofRecognizingCytosine 1657.9 Conclusions 168

References 168

8 The Properties of 40-Thionucleosides 173Masataka Yokoyama

8.1 Introduction 1738.2 Synthesis of 40-Thionucleosides 1738.3 Synthesis of Isothionucleosides 1958.4 Synthesis of L-Thionucleosides 1968.5 Synthesis of Thioxonucleosides 1988.6 Synthesis of Miscellaneous Thionucleosides 2058.7 Biological Activity of Thionucleosides 2108.8 Conclusions 219

References 219

9 S-Adenosyl-L-methionine and Related Compounds 223Christian Dalhoff and Elmar Weinhold

9.1 Introduction 2239.2 The Biochemistry of AdoMet 2249.2.1 AdoMet as a Methyl and Methylene Group Donor 2249.2.2 AdoMet as an Aminocarboxypropyl Group Donor 2279.2.3 AdoMet as an Adenosyl Group Donor 2289.2.4 AdoMet as a Ribosyl Group Donor 2289.2.5 AdoMet as a Radical Source 2299.2.6 AdoMet as an Amino Group Donor 2299.2.7 AdoMet-Dependent Riboswitches 2309.2.8 The Biosynthesis and Metabolism of AdoMet 230

Contents IX

9.3 The Chemistry and Biochemistry of Modified AdoMet 2319.3.1 Synthetic Approaches to AdoMet Analogues 2319.3.2 Isotope-Labeled AdoMet 2329.3.3 Selenium and Tellurium Analogues of AdoMet 2349.3.4 Sulfoxide and Sulfone Analogues of AdoMet 2369.3.5 Sinefungin 2379.3.6 Nitrogen Analogues of AdoMet 2379.3.7 Aziridine Analogues of AdoMet 2389.3.8 AdoMet Analogues with Methyl Group Replacements 2389.4 AdoMet as a Pharmaceutical 2409.5 Concluding Remarks 241

References 242

Part II Biotechnology 249

10 5-Substituted Nucleosides in Biochemistry and Biotechnology 251Mohammad Ahmadian and Donald E. Bergstrom

10.1 Introduction 25110.2 Synthesis 25210.2.1 Organopalladium Coupling Reactions 25210.2.2 Strategies for Post-Oligonucleotide-Synthesis Modification

through Pyrimidine C-5 25310.3 Incorporation of C-5-Substituted Pyrimidine Nucleotides into

Nucleic Acids through Modified Nucleotide 50-Triphosphates 25510.3.1 The Early Studies 25510.3.2 Incorporation of Diverse Functionality into DNA 25710.3.3 T7 RNA Polymerase-Mediated Synthesis of Modified RNA 26210.3.4 Incorporation of C-5-Appended Fluorophores 26210.4 C-5 Substituents that Stabilize DNA Duplexes 26410.5 Photochemistry 26910.6 Conclusions 271

References 271

11 Universal Base Analogues and their Applications to Biotechnology 277Kathleen Too and David Loakes

11.1 Introduction 27711.2 General Methods of Synthesis 27811.3 Properties of Universal Bases 28211.4 Structure, Stacking, and Stabilization 28311.5 Hydrogen-Bonding Universal Base Analogues 28711.6 Applications of Universal Base Analogues 29011.7 Triphosphate Derivatives 29511.8 Therapeutic Applications 298

References 300

X Contents

Part III Medicinal Chemistry 305

12 The Properties of Locked Methanocarba Nucleosides in Biochemistry,Biotechnology, and Medicinal Chemistry 307Victor E. Marquez

12.1 Introduction 30712.2 Structural Representation 30812.2.1 The Bicyclo[3.1.0]hexane Template 30812.2.2 Pseudoboat versus Pseudochair Conformations 30912.3 Synthesis of Locked Nucleosides 30912.3.1 North (N) Conformer Mimics 31112.3.1.1 Dideoxyribonucleoside Analogues 31112.3.1.2 20-Deoxyribonucleoside Analogues 31212.3.1.3 Ribonucleoside Analogues 31812.3.2 South (S) Conformer Mimics 32012.3.2.1 20-Deoxyribonucleoside Analogues 32012.3.2.2 Ribonucleoside Analogues 32112.3.3 Synthesis of N- and S-Methanocarba AZT Analogues 32312.3.4 Synthesis of Bicyclo[3.1.0]hexene Nucleosides 32412.3.5 Reshuffling of Groups on a Bicyclo[3.1.0]hexane

Template 32612.3.6 Bicyclo[3.1.0]hexane Pseudosugars as Surrogates of Abasic

Nucleosides 32712.4 Synthesis of Oligodeoxynucleotides (ODNs) Containing Locked

Nucleosides 32812.4.1 The Dickerson–Drew (DD) Dodecamer 33012.5 Molecular Targets, Ligand Properties, and Binding

Modes 33112.5.1 Kinases and Polymerases 33112.5.2 HIV Reverse Transcriptase 33512.5.3 DNA Methyltransferase 33712.6 Concluding Remarks 339

References 339

13 Synthesis, Chemical Properties and Biological Activities of CyclicBis(30–50)diguanylic Acid (c-di-GMP) and its Analogues 343Mamoru Hyodo and Yoshihiro Hayakawa

13.1 Introduction 34313.2 Synthesis of c-di-GMP and its Analogues 34513.2.1 Synthesis of c-di-GMP 34513.2.2 Synthesis of Artificial Analogues of c-di-GMP 34713.3 Chemical Properties of c-di-GMP and its Analogues 34813.3.1 Stability and Chemical Properties of c-di-GMP under Acidic, Basic,

and Physiological Conditions 34813.3.2 Polymorphism of c-di-GMP in Aqueous Solutions 349

Contents XI

13.4 Bioactivities of c-di-GMP and its Analogues 35113.4.1 Activity of c-di-GMP on Biofilm Formation 35213.4.1.1 Inhibition of Biofilm Formation and Prevention of Bacterial

Infection of S. aureus in vitro 35213.4.1.2 Activity as an Immunostimulatory Molecule 35313.4.1.3 Activity on Biofilm Formation and Virulence Emergence of

P. aeruginosa 35713.4.2 Inhibition of Proliferation of Human Colon Cancer Cells with

c-di-GMP 35713.4.3 Biological Activity of c-dGpGp 35813.5 Conclusions 359

References 361

14 Siderophore Biosynthesis Inhibitors 365Courtney C. Aldrich and Ravindranadh V. Somu

14.1 Introduction 36514.2 Synthesis, Physico-Chemical Properties, Metabolism, Mechanism

of Action, and Biological Activity 36514.2.1 Synthesis 36514.2.2 Physico-Chemical Properties 36614.2.3 Metabolism 36714.2.4 Toxicity 36714.2.5 Biochemical Target 36814.2.6 Mechanism of Action 36914.2.7 Biological Activity 36914.3 Background of Siderophores: Molecular Target and Rationale

for Inhibitor Design 37014.4 Ligand Properties/Binding Mode 37414.4.1 Nature of the Linker 37514.4.2 Importance of the Aryl Ring 37914.4.3 Role of the Ribose 38214.4.4 Impact of the Nucleobase 38514.5 Conclusions 388

References 388

15 Synthesis and Biological Activity of Selected CarbocyclicNucleosides 393Adam Mieczkowski and Luigi A. Agrofoglio

15.1 Introduction 39315.2 A-5021, Synguanol, and Cyclopropane Derivatives 39515.3 Lobucavir and Cyclobutane Nucleoside Derivatives 39815.4 Carbovir and 20,30-Unsaturated Nucleoside Derivatives 40515.5 Locked Nucleosides 41315.6 Conclusions 420

References 420

XII Contents

16 40-C-Ethynyl-20-Deoxynucleosides 425Hiroshi Ohrui

16.1 Introduction 42516.2 Murine Toxicity of Purine 40EdNs 42616.3 40EdA Derivatives Stable to Adenosine Deaminase, and their

Biological Properties 42716.4 40-C-Ethynylnucleosides without 30-OH 42916.4.1 20,30-Dideoxy-40-C-ethynylnucleoside 42916.4.2 20,30-Didehydrodideoxy-40-C-ethynylnucleosides 42916.4.3 Carbocyclic and Other Heterocyclic Analogues of

40-C-ethynylnucleoside 431References 431

17 Modified Nucleosides as Selective Modulators of Adenosine Receptorsfor Therapeutic Use 433Kenneth A. Jacobson, Bhalchandra V. Joshi, Ben Wang, Athena Klutz,Yoonkyung Kim, Andrei A. Ivanov, Artem Melman, and Zhan-Guo Gao

17.1 Introduction 43317.2 Molecular Targets and Binding Modes 43417.3 AR Agonists as Clinical Candidates 43417.3.1 Modified Nucleosides as A1 AR Agonists 43517.3.2 Modified Nucleosides as A2A AR Agonists 43717.3.3 Modified Nucleosides as A2B AR Agonists 43917.3.4 Modified nucleosides as A3 AR Ligands 44117.4 Summary 444

References 444

18 The Design of Forodesine HCl and Other Purine NucleosidePhosphorylase Inhibitors 451Philip E. Morris and Vivekanand P. Kamath

18.1 Introduction 45118.2 Purine Nucleoside Phosphorylase Enzyme Structure 45218.2.1 Purine-Binding Site 45318.2.2 Phosphate-Binding Site 45418.2.3 Sugar-Binding Site 45418.3 First-Generation PNP Inhibitors: Substrate Analogues 45418.3.1 Chemistry of First-Generation PNP Inhibitors 45818.4 Second-Generation PNP Inhibitors: Transition-State

Inhibitors 46018.4.1 Chemistry of Second-Generation PNP Inhibitors 46118.4.2 Convergent Synthesis of Forodesine HCl 46318.5 Third-Generation PNP Inhibitors: Transition-State

Inhibitors 46718.5.1 Chemistry of BCX-4208 467

References 469

Contents XIII

19 Formycins and their Analogues: Purine NucleosidePhosphorylase Inhibitors and their Potential Applicationin Immunosuppression and Cancer 473Agnieszka Bzowska

19.1 Introduction 47319.2 Chemical Structure of Formycins and their Analogues 47519.2.1 Formycin A and B, Oxoformycin B 47519.2.2 Structural Modifications of Formycins 47619.2.2.1 N-Methyl and N-Substituted Analogues 47619.2.2.2 Other Base-Modified Analogues 47819.2.2.3 Sugar-Modified Analogues 47819.2.3 Formycin Phosphates and Polyformycin Phosphates 47819.3 Spectral Properties of Formycins 47919.4 Sources of Formycins 48319.4.1 Natural Sources and Biosynthesis 48319.4.2 Synthesis 48319.5 The Biological Activity of Formycins: A Brief Summary 48519.6 Formycins and Analogues as Purine Nucleoside Phosphorylase

Inhibitors 48819.6.1 Molecular Target of Formycins: Purine Nucleoside

Phosphorylase (PNP) 48819.6.2 Formycins and Analogues in Studies of the Molecular Mechanism

of Catalysis: The 3-D Structure of PNPs 48919.6.2.1 Low-Molecular-Mass PNPs 49019.6.2.2 High-Molecular-Mass PNPs 49219.6.3 PNP Deficiency and the Potential Role of PNP Inhibitors 49519.6.4 Formycins and Analogues as Inhibitors of Mammalian PNPs 49619.7 Formycins as Inhibitors of Parasitic PNPs and Hydrolases 50019.8 Actual and Potential Applications of Formycins 50119.8.1 Formycin and Analogues in Assays of Enzyme Activity 50119.8.2 Formycin and Analogues as Protein Ligands for X-Ray Structural

Studies 50219.8.3 Formycins and Analogues as Molecular Probes 50219.8.4 Formycins as Ligands in Affinity Chromatography 50319.8.5 Formycin B as a Tool to Study Nucleoside Transport 504

References 504

20 1-(3-C-Ethynyl-b-D-ribo-pentofuranosyl)cytosine (ECyd) 511Akira Matsuda

20.1 Introduction 51120.2 Synthesis of ECyd and its Analogues 51120.3 Cytotoxic Activity and Structure–Activity Relationships of ECyd

Analogues In Vitro, and In Vitro Antitumor Activity 51320.4 Structural Features of ECyd and 40-Thio-ECyd 51320.5 Metabolism and Mechanism of Action 517

XIV Contents

20.6 An Apoptotic Pathway Involving the Action of ECyd 51920.7 Combination of ECyd with Low-Dose X-Irradiation 51920.8 ECyd is Effective against Gemcitabine-Resistant Human Pancreatic

Cancer Cells 52020.9 Conclusions 521

References 521

21 Syntheses and Biological Activity of Neplanocin and Analogues 525Dilip K. Tosh, Hea Ok Kim, Shantanu Pal, Jeong A. Lee,and Lak Shin Jeong

21.1 Introduction 52521.2 New Methodologies in the Synthesis of Neplanocin A 52721.3 Modifications on Neplanocin A and Aristeromycin 53221.3.1 C20 Modification 53321.3.2 C30 Modification 53621.3.3 C40 Modification 53821.3.4 C50 Modification 54021.3.5 C60 Modification 55021.3.6 Base Modification 55221.3.7 Miscellaneous 55921.4 Conclusions 563

References 563

22 Clitocine and Its Analogues 567Hyunik Shin and Changhee Min

22.1 Clitocine: Isolation, Synthesis, and Biological Activity 56722.1.1 Isolation 56722.1.2 Synthesis 56722.1.3 Biological Activity 56922.2 Clitocine Analogues 57122.2.1 Aglycone Modifications 57122.2.2 Carbocyclic Analogues 57322.2.3 Acyclic Analogues 57622.2.4 50-Amino Analogues 578

References 583

Part IV Antitumorals and Antivirals 585

23 Capecitabine Preclinical Studies: From Discovery to TranslationalResearch 587Hideo Ishitsuka and Nobuo Shimma

23.1 Introduction 58723.2 Drug Design and Discovery of Capecitabine 58823.2.1 50-DFUR as a Lead Compound of Capecitabine 588

Contents XV

23.2.2 50-DFCR Derivatives 58923.2.3 N4-Acyl-50-DFCR Derivatives 58923.2.4 N4-Alkoxycarbonyl-50-DFCR Derivatives 59023.3 Preclinical Studies 59023.3.1 Tumor-Selective Delivery of the Active 5-FU 59023.3.2 Anti-Tumor Activities 59223.3.3 Dose Fractionation and Schedule 59223.3.4 Safety (Dose Range and Mild Myelotoxicity) 59323.4 Translational Research for Optimizing Capecitabine Efficacy 59323.4.1 Factors that Influence Capecitabine Efficacy 59323.4.2 Combination Therapy with Rational Partners 59423.4.2.1 Combination with TP Up-Regulators 59423.4.2.2 Combination with DPD Down-Regulators 59423.4.3 Personalized Therapy of Rational Patient Populations 59623.5 Conclusions 597

References 598

24 Tenofovir and Adefovir as Antiviral Agents 601Tomas Cihlar, William E. Delaney IV, and Richard Mackman

24.1 Introduction 60124.2 Synthesis 60224.2.1 Adefovir and Adefovir Dipivoxil 60324.2.2 Tenofovir and Tenofovir Disoproxil Fumarate 60524.3 Mechanism of Action 60624.3.1 Membrane Transport and Intracellular Metabolism 60624.3.2 Inhibition of Viral Polymerases 60824.3.3 Spectrum of Antiviral Activity 60924.4 Activity in Animal Models 61224.4.1 Models for Retroviral Infections 61224.4.2 Models for Hepadnavirus Infections 61324.4.3 Herpes Models 61424.5 Clinical Experience 61424.5.1 Tenofovir Disoproxil Fumarate (Viread1) 61424.5.2 Adefovir Dipivoxil (Hepsera1) 61624.6 Drug Resistance 61824.6.1 HIV Resistance 61824.6.2 HBV Resistance 61924.7 Novel Antiviral Nucleotides and Nucleotide Prodrugs 61924.8 Conclusions 621

References 622

XVI Contents

25 Clofarabine: From Design to Approval 631John A. Secrist III, Jaideep V. Thottassery, and William B. Parker

25.1 Introduction 63125.2 Clofarabine: The Background 63225.3 The Beginnings 63225.4 The Next Generation of Compounds 63525.5 Mechanism of Action of Clofarabine 63925.5.1 Transport and Metabolism to Active Metabolites 63925.5.2 Inhibition of DNA Synthesis 64025.5.3 Induction of Apoptosis 64125.5.4 Activity against Non-Proliferating Cancer Cells 64225.6 Clofarabine to the Clinic 64325.6.1 Clinical Trials and Approval 64325.7 Summary and Comments 644

References 644

Index 647

Contents XVII

Preface

The chemical modification of nucleosides has been – and will continue to remain – amajor research topic in bioorganic and medicinal chemistry. The reason for this isobvious: nucleosides, oligonucleotides and nucleic acids are involved in all aspects ofcellular life – from the storage of genetic information to metabolic regulation,catalysis, and energy supply. Indeed, research investigations in this area have ledto the provision of many life-saving drugs in cancer and infectious disease, as well asinvaluable diagnostic tools.The study of the biological effect of modified nucleosides and oligonucleotides has

provided us with a deeper insight into cellular functions. Their potential applicationsare continuing to explore new horizons, amongst others in the field of nanotechno-logy, microarrays, RNA interference, and small-molecule therapeutics.Although, from the 1960s until the 1980s, the discovery of new biological active

nucleosides and oligonucleotides was largely a matter of serendipity, this is nolonger the case. Today, fundamental insights into the conformational behavior ofnucleosides, enzyme mechanisms, the physico-chemistry of nucleosides and oligo-nucleotides, and the availability of increasing amounts of structural data on nucleo-sides and oligonucleotides has simplified the design process of biological activenucleosides and oligonucleotides. Likewise, whilst between the 1950s and 1970s thesynthesis of a modified nucleoside was a difficult undertaking, the evolution oforganic synthesis in general – and of nucleoside chemistry in particular – has led tomodified nucleosides and oligonucleotides becoming much more easily available.In this book, we have assembled different aspects of the chemistry and biology of

modified nucleosides. In order to demonstrate the broad field of research that iscovered bymodifiednucleosides,pure ‘‘chemical’’chaptersaswellasmore ‘‘biological’’and even ‘‘clinically oriented’’ chapters are included. However, the major focus ison modified nucleosides, as it in this region that the applications are most clear,though new directions in the nucleotide and oligonucleotide fields have alsobriefly been discussed.Clearly, it would be impossible to cover such as vast research field within a single

volume, and consequently the contents of this book must be considered as a generalintroduction to the fascinating world of modified nucleosides.

XIX

The editor cordially thanks all authors who have contributed to the book, andhopes that it will inspire scientists not only to study the chemistry and biology ofmodified nucleosides but also to explore new opportunities in this area.

Leuven, May 2008 Piet Herdewijn

XX Preface

List of Contributors

XXI

Luigi A. AgrofoglioUniversité dOrléansUFR SciencesInstitut de Chimie Organiqueet AnalytiqueICOA UMR CNRS 6005BP 675945067 Orléans Cedex 2France

Mohammad AhmadianMDRNA Inc.3350 Monte Villa ParkwayBothell, WA 98021USA

Courtney C. AldrichUniversity of MinnesotaAcademic Health CenterCenter for Drug Design516 Delaware St. S.E., 7–169 PWBMinneapolis, MN 55455USA

Donald E. BergstromPurdue UniversityBirck Nanotechnology CenterDepartment of Medicinal Chemistryand Molecular Pharmacology1205 West State StreetWest Lafayette, IN 47907-2057USA

Agnieszka BzowskaUniversity of WarsawInstitute of Experimental PhysicsDepartment of BiophysicsZ·wirki i Wigury 93

02089 WarsawPoland

Jean CadetDépartement de RechercheLaboratoire Lésions des AcidesNucléiquesLCIB-UMR-E no. 3 CEA–UJFFondamentale sur laMatière CondenséeCEA/Grenoble38054 Grenoble Cedex 9France

Tomas CihlarGilead Sciences, Inc.Department of Biology362 Lakeside DriveFoster City, CA 94404USA

Christian DalhoffCaprotec Bioanalytic GmbHVolmerstrasse 512489 BerlinGermany

William E. Delaney, IVGilead Sciences, Inc.Department of Biology342 Lakeside DriveFoster City, CA 94404USA

Paolo Di MascioUniversidade de São PauloInstituto de QuímicaDepartamento de BioquímicaCP 26077CEP 05513-970São Paulo, SPBrazil

Zhan-Guo GaoNational Institutes of HealthNational Institute of Diabetes,Digestive and Kidney DiseasesLaboratory of Bioorganic ChemistryMolecular Recognition SectionBethesda, MD 20892-0810USA

Yoshihiro HayakawaNagoya UniversityGraduate School of InformationScienceFuro-cho, ChikusaNagoyaJapan

Mamoru HyodoNagoya UniversityGraduate School of InformationScienceFuro-cho, ChikusaNagoyaJapan

Hideo IshitsukaRoche Diagnostics K.K.Shiba 2-6-1Minato cityTokyo 105-0014Japan

Andrei A. IvanovNational Institutes of HealthNational Institute of Diabetes,Digestive and Kidney DiseasesLaboratory of Bioorganic ChemistryMolecular Recognition SectionBethesda, MD 20892-0810USA

Shigenori IwaiOsaka UniversityGraduate School of Engineering ScienceDivision of Chemistry1-3 MachikaneyamaToyonakaOsaka 560-8531Japan

Kenneth A. JacobsonNational Institutes of HealthNational Institute of Diabetes,Digestive and Kidney DiseasesLaboratory of Bioorganic ChemistryMolecular Recognition SectionBethesda, MD 20892-0810USA

Lak Shin JeongEwha Womans UniversityCollege of Pharmacy11-1 Seodaemun-gu, Dae hyun-dongSeoul 120-750Korea

XXII List of Contributors

Bhalchandra V. JoshiNational Institutes of HealthNational Institute of Diabetes,Digestive and Kidney DiseasesLaboratory of Bioorganic ChemistryMolecular Recognition SectionBethesda, MD 20892-0810USA

Karl-Heinz JungUniversity of KonstanzDepartment of ChemistryUniversitätsstrasse 1078457 KonstanzGermany

Vivekanand P. KamathBioCryst Pharmaceuticals2190 Parkway Lake DriveBirmingham, AL 35244USA

Hea Ok KimEwha Womans UniversityCollege of Pharmacy11-1 Seodaemun-gu, Dae hyun-dongSeoul 120-750Korea

Yoonkyung KimEwha Womans UniversityCollege of Pharmacy11-1 Seodaemun-gu, Dae hyun-dongSeoul 120-750Korea

Athena KlutzNational Institutes of HealthNational Institute of Diabetes,Digestive and Kidney DiseasesLaboratory of Bioorganic ChemistryMolecular Recognition SectionBethesda, MD 20892-0810USA

Eric T. KoolStanford UniversityDepartment of ChemistryStanford, CA 94305-5080USA

Christoph KreutzUniversity of ViennaDepartment of BiomolecularStructural ChemistryCampus Vienna Biocenter 51030 ViennaAustria

Jeong A. LeeEwha Womans UniversityCollege of Pharmacy11-1 Seodaemun-gu, Dae hyun-dongSeoul 120-750Korea

David LoakesMedical Research CouncilLaboratory of Molecular BiologyHills RoadCambridge CB2 2QHUnited Kingdom

Richard MackmanGilead Sciences, Inc.Department of Biology342 Lakeside DriveFoster City, CA 94404USA

Victor E. MarquezNational Institutes of HealthNational Cancer InstituteFrederick Cancer Research andDevelopment CenterLaboratory of Medicinal Chemistry376 Boyles St.Frederick, MD 21702-1201USA

List of Contributors XXIII

Andreas MarxUniversity of KonstanzDepartment of ChemistryUniversitätsstrasse 1078457 KonstanzGermany

Akira MatsudaHokkaido UniversityFaculty of Pharmaceutical SciencesKita-12, Nishi-6Kita-kuSapporo 060-0812Japan

Artem MelmanNational Institutes of HealthNational Institute of Diabetes,Digestive and Kidney DiseasesLaboratory of Bioorganic ChemistryMolecular Recognition SectionBethesda, MD 20892-0810USA

Ronald MicuraLeopold-Franzens-UniversityInstitute of Organic ChemistryCenter for Molecular BiosciencesInnsbruck (CMBI)Innrain 52a6020 InnsbruckAustria

Adam MieczkowskiUniversité dOrléansUFR SciencesInstitut de Chimie Organiqueet AnalytiqueICOA UMR CNRS 6005BP 675945067 Orléans Cedex 2France

Changhee MinLG Life Sciences Ltd./R&DDrug Discovery Division104-1, Moonji-dongYusong-guDaejeon 305-380Korea

Philip E. Morris, Jr.BioCryst Pharmaceuticals2190 Parkway Lake DriveBirmingham, AL 35244USA

Poul NielsenUniversity of Southern DenmarkDepartment of Chemistry and PhysicsCampusvej 555230 Odense MDenmark

Akio OhkuboTokyo Institute of TechnologyDepartment of Life Science4259 NagatsutaMidorikuYokohama 226-8501Japan

Hiroshi OhruiYokohama College of PharmacyDepartment of Kampo PharmaceuticalSciences601 Matanocho, Totsukaku246-0066 YokohamaJapan

Itaru OkamotoTokyo Institute of TechnologyDepartment of Life Science4259 NagatsutaMidorikuYokohama 226-8501Japan

XXIV List of Contributors

Shantanu PalEwha Womans UniversityCollege of Pharmacy11-1 Seodaemun-gu, Dae hyun-dongSeoul 120-750Korea

William B. ParkerSouthern Research InstituteP.O. Box 55305Birmingham, AL 35255-5305USA

John A. Secrist IIISouthern Research InstituteP.O. Box 55305Birmingham, AL 35255-5305USA

Kohji SeioTokyo Institute of TechnologyDepartment of Life Science4259 NagatsutaMidorikuYokohama 226-8501Japan

Mitsuo SekineTokyo Institute of TechnologyDepartment of Life Science4259 NagatsutaMidorikuYokohama 226-8501Japan

Nobuo ShimmaChugai Pharmaceutical Co., LtdKamakura Research InstituteKajiwara, KamakuraKanagawa 247-8530Japan

Hyunik ShinLG Life Sciences Ltd./R&DChemical Development Division104-1, Moonji-dongYusong-guDaejeon 305-380Korea

Ravindranadh V. SomuUniversity of MinnesotaAcademic Health CenterCenter for Drug Design516 Delaware St. S.E., 7-169 PWBMinneapolis, MN 55455USA

Jaideep V. ThottasserySouthern Research InstituteP.O. Box 55305Birmingham, AL 35255-5305USA

Kathleen TooMedical Research CouncilLaboratory of Molecular BiologyHills RoadCambridge CB2 2QHUnited Kingdom

Dilip K. ToshEwha Womans UniversityCollege of Pharmacy11-1 Seodaemun-gu, Dae hyun-dongSeoul 120-750Korea

Ben WangNational Institutes of HealthNational Institute of Diabetes,Digestive and Kidney DiseasesLaboratory of Bioorganic ChemistryMolecular Recognition SectionBethesda, MD 20892-0810USA

List of Contributors XXV

Elmar WeinholdRWTH Aachen UniversityInstitute of Organic ChemistryLandoltweg 152056 AachenGermany

Jesper WengelUniversity of Southern DenmarkDepartment of Chemistry and PhysicsCampusvej 555230 Odense MDenmark

Masataka YokoyamaEmeritus Professor, Chiba UniversityGodo Shigen Sangyo Co. Ltd.1365 NanaidoChosei-mura, Chosei-gunChiba PrefectureJapan

XXVI List of Contributors

Part IBiochemistry and Biophysics