A Dissertation Entitled Synthesis of Novel Nucleoside Analogs

Home , Entry inhibitor, Mericitabine, Miravirsen, NS5B inhibitor, Ravidasvir, Valopicitabine

A Dissertation

entitled

Synthesis of Novel Nucleoside Analogs Targeting HCV

Bader Saleh Alabdullah

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Doctor of Philosophy Degree in

Medicinal Chemistry

______Dr. Amanda Bryant-Friedrich, Committee Chair

______Dr. James Slama, Committee Member

______Dr. Viranga Tillekeratne, Committee Member

______Dr. Malathi Krishnamurthy, Committee Member

______Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies

The University of Toledo May 2018

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of

Synthesis of Novel Nucleoside Analogs Targeting HCV

Bader Saleh Alabdullah

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Medicinal Chemistry

The University of Toledo

May 2018

Despite all the advancement that has been produced in the clinic for treating HCV infection, it is nevertheless a major causal agent of liver disease due to its silent nature.

With 7 genotypes and many subtypes, the search for a pan-genotypic treatment is challenging and leads in many cases to the use of combination treatments to tackle the infection.

Ribavirin (RBV) and Pegylated Interferon alfa-2b (pegIFNα-2b) represented the standard of care (SoC) for a long period. Sustained Virologic Response (SVR) was only achieved in low percentage of patients with this SoC. Also, the SoC was associated with severe side effects that resulted in the termination of treatment in many cases. So, it is necessary to find a treatment that is pan-genotypic and has fewer side effects than that of the SoC.

Identification of multiple points to disrupt the HCV viral life cycle and to halt viral protein synthesis was enabled through determination of the crystal structure of the viral proteins and a better understanding of the viral life cycle. Most notably the determination

iii of the crystal structure of the HCV NS5B RdRp as it is the catalytic machinery for viral replication.

The conserved nature of the HCV NS5B active site across viral species, as compared to other HCV viral proteins, along with the fact that there is no human NS5B made NS5B a primary target for drug design and development.

In this project, we synthesized 2′-C-acetyl and 2′-C-(1-hydroxyethyl) uridine and started the synthesis of 2′-C-formyl and 2′-C-aminomethyl uridine analogs. Based on the approval of Sofosbuvir, which is 2′-modified uridine that act as an NS5B inhibitor, we assume that these compounds will work as non-obligate chain terminators for the synthesis of the viral RNA by the NS5B enzyme.

I dedicate this work to my mother, my wife, my son, and my two daughters

Acknowledgements

First and foremost, I would like to show my appreciation to Dr. Amanda Bryant-

Friedrich for giving me this opportunity to do this research and for supporting and encouraging me through my work, without her enlightenment and supervision none of this would have happened.

Candid thanks to the Higher Committee for Education Development in Iraq

(HCED/Iraq) for sponsoring me through my study.

Also, I would like to thank my committee members, Dr. James Slama, Dr.

Viranga Tillekeratne, and Dr. Malathi Krishnamurthy for their valuable advice.

Thank you to all the faculty members of the department of Medicinal and

Biological Chemistry at the College of Pharmacy and Pharmaceutical Sciences at the

University of Toledo for accepting me as a graduate student.

Sincere gratitude for Dr. Fernand Mel Bedi for his help during my four years in

Dr. Amanda Bryant-Friedrich’s lab.

Thank you, a lot, to my lab members for their kindness and understanding.

To all my friends especially Dr. Qasim Alahdidi and Ayad Al-Hamashi for backing me during my study, thank you.

Lastly, to the ones who bear all the troubles so I can finish this journey, my mother, my wife, my son, and my two daughters, thank you very much, God bless you-I am forever in your debt.

Table of Contents

Abstract ...... iii

Acknowledgements ...... vi

Table of Contents ...... vii

List of Tables ...... xiii

List of Figures ...... xiv

List of Schemes ...... xvii

List of Abbreviations ...... xviii

List of Symbols ...... xxii

1 Introduction ...... 1

1.1 Historical Background...... 1

1.2 Significance ...... 2

1.3 HCV Transmission ...... 3

1.4 HCV Prevalence ...... 4

1.5 Types of HCV infection ...... 4

1.6 HCV Structure ...... 4

1.7 HCV Genome ...... 6

1.8 HCV Genotypes ...... 8

1.9 HCV Genotype Distribution...... 10

1.10 HCV Proteins ...... 11

1.10.1 Structural Proteins ...... 12 vii

1.10.1.1 Core ...... 12

1.10.1.2 Envelop glycoproteins ...... 13

1.10.2 Nonstructural Proteins ...... 13

1.10.2.1 p7...... 13

1.10.2.2 NS2 ...... 14

1.10.2.3 NS3/4A complex ...... 15

1.10.2.4 NS4B ...... 16

1.10.2.5 NS5A...... 17

1.10.2.6 NS5B ...... 19

1.11 HCV Life cycle ...... 23

1.11.1 Entry and uncoating ...... 24

1.11.2 Translation and polyprotein processing ...... 24

1.11.3 HCV RNA Replication ...... 25

1.11.4 Virus assembly and release...... 26

1.13 HCV Prognosis ...... 27

2 Background ...... 28

2.1 HCV Treatment ...... 28

2.2 Indirect Acting Antivirals...... 30

2.2.1 Biologics ...... 31

viii

2.2.1.1 Interferons ...... 31

2.2.1.2 DNA vaccines ...... 32

2.2.2 Chemical Compounds...... 33

2.2.2.1 Ribavirin ...... 33

2.2.2.2 Miravirsen ...... 33

2.2.2.3 Cellular Component Inhibitors ...... 35

2.3 Direct acting antivirals ...... 38

2.3.1 E1/E2 and p7 inhibitors ...... 38

2.3.2 NS2 inhibitors ...... 41

2.3.3 NS3/4A inhibitors ...... 42

2.3.4 NS4B inhibitors ...... 45

2.3.5 NS5A inhibitors ...... 46

2.3.6 NS5B inhibitors ...... 48

2.3.6.1 NNIs ...... 49

2.3.6.2 NIs ...... 49

2.3.6.2.1 Base modified NIs ...... 51

2.3.6.2.2 Sugar modified NIs ...... 53

3 Results and Discussion ...... 58

3.1 Synthesis of nucleoside analogues ...... 58

3.2 Design and synthesis of 2′-C-acetyluridine compound 100 ...... 59

3.2.1 The convergent route ...... 59

3.2.2 The linear route ...... 62

3.2.2.1 Synthesis of key intermediate compound 98 ...... 62

3.2.2.1.1 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)uridine (97) .. 62

3.2.2.1.2 Synthesis of 3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-ketouridine

(98) ...... 63

3.2.2.2 Cyanohydrin formation ...... 64

3.2.2.2.1 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-O-

(trimethylsilyl)-2′-C-cyanouridine (99)...... 64

3.2.2.2.2 Synthesis of 2′-C-acetyluridine (100) ...... 65

3.2.2.3 Wittig olefination ...... 68

3.2.2.3.1 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-deoxy-2′-

ethylideneuridine (101) ...... 69

3.2.2.3.2 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-C-(1-

hydroxyethyl)uridine (102) ...... 71

3.2.2.3.3 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-C-

acetyluridine (103) ...... 72

3.2.2.3.4 Synthesis of 2′-C-acetyluridine (100) ...... 73

3.3 Design and synthesis of 2′-C-(1-hydroxyethyl)uridine compound 104 ...... 74

3.4 Design and synthesis of 2′-C-formyluridine compound 108 ...... 75

3.4.1 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-deoxy-2′-C-

methyleneuridine (105) ...... 75

3.4.2 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-C-

hydroxymethyluridine (106) ...... 76

3.4.3 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-C-formyluridine

(107)...... 76

3.4.4 Synthesis of 2′-C-formyluridine (108) ...... 77

4 Conclusions and Future Work ...... 78

4.1 Conclusions ...... 78

4.2 Future work ...... 79

4.2.1 Synthesis of 2′-C-formyluridine (108) ...... 79

4.2.2 Synthesis of 2′-C-aminomethyluridine (110) ...... 79

4.2.3 Design and synthesis of phosphoramidate derivatives of the modified

nucleosides...... 80

5 Experimental ...... 84

5.1 General methods ...... 84

5.2 Synthesis of 2′-C-acetyluridine ...... 85

5.2.1 Synthesis of α-D-1,3,5-tri-O-benzoyl-2-ketoribofuranose (95) ...... 85 xi

5.2.2 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)uridine (97) ...... 86

5.2.3 Synthesis of 3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-ketouridine (98) ... 87

5.2.4 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-O-(trimethylsilyl)-

2′-C-cyanouridine (99) ...... 88

5.3.5 Synthesis of 2′-C-acetyluridine (100) ...... 88

5.3.6 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-deoxy-2′-

ethylideneuridine (101) ...... 89

5.3.7 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-C-(1-

hydroxyethyl)uridine (102) ...... 91

5.3.8 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-C-acetyluridine

(103)...... 92

5.3.9 Synthesis of 2′-C-acetyluridine (100) ...... 93

5.4 Synthesis of 2′-C-(1-hydroxyethyl)uridine (104) ...... 94

5.5 Synthesis of 2′-C-formyluridine ...... 95

5.5.1 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-deoxy-2′-C-

methyleneuridine (105) ...... 95

5.5.2 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-C-

hydroxymethyluridine (106) ...... 96

5.5.4 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-C-formyluridine

(107)...... 97

References ...... 99

Appendix A ...... 129 xii

List of Tables

Table 1: HCV genomic heterogeneity terms...... 9

Table 2: Methods and reagents for silyl deprotection...... 73

Table 3: Compounds 118-121...... 83

xiii

List of Figures

Figure 1: HCV infection global reach...... 2

Figure 2: HCV transmission...... 3

Figure 3: HCV particle...... 5

Figure 4: HCV genome...... 6

Figure 5: HCV IRES...... 7

Figure 6: HCV 5′ UTR miRNA-122 interactions...... 7

Figure 7: HCV genotypes distribution...... 11

Figure 8: HCV proteins...... 12

Figure 9: HCV p7...... 14

Figure 10: HCV NS3/4A complex with inhibitor ...... 15

Figure 11: HCV NS5A...... 18

Figure 12: HCV NS5B ...... 19

Figure 13: HCV NS5B motifs...... 20

Figure 14: HCV NS5B active site...... 21

Figure 15: HCV NS5B mechanism of action...... 22

Figure 16: HCV replication by NS5B...... 22

Figure 17: HCV life cycle...... 23

Figure 18: Double membranous vesicle ...... 25

Figure 19: HCV SVR12...... 28

Figure 20: Anti-HCV approved medication ...... 29

Figure 21: Mechanisms of viral inhibition by IFNs...... 31

Figure 22: Inovio plasmid ...... 32

xiv

Figure 23: Ribavirin...... 33

Figure 24: Miravirsen locked nucleotides...... 34

Figure 25: Miravirsen’s mechanism of action...... 34

Figure 26: Ritonavir...... 35

Figure 27: Cyclophilin A inhibitors...... 36

Figure 28: Cellular proteins inhibitors...... 37

Figure 29: HCV E1/E2 and p7 inhibitors...... 40

Figure 30: Chlorcyclizine derivative 30...... 41

Figure 31: HCV NS2 inhibitor...... 41

Figure 32: Approved NS3/4A inhibitors...... 43

Figure 33: Experimental NS3/4A inhibitors ...... 44

Figure 34: HCV NS4B inhibitors...... 45

Figure 35: Approved HCV NS5A inhibitors...... 47

Figure 36: Investigational HCV NS5A inhibitor...... 48

Figure 37: HCV NS5B allosteric sites...... 49

Figure 38: HCV NS5B NNIs...... 50

Figure 39: HCV NS5B NIs mechanism of action ...... 51

Figure 40: Base and sugar modified NIs...... 52

Figure 41: Base modified NIs...... 52

Figure 42: Sugar Modified HCV NS5B NIs...... 55

Figure 43: Mechanism of action of sofosbuvir...... 56

Figure 44: HCV NS5B NIs approved and in clinical trials...... 57

Figure 45: Structures of DMP and IBX...... 60

Figure 46: 18-crown-6 complex with potassium ion...... 65

Figure 47: Postulated mechanism of phosphoramidate activation...... 81

Figure 48: Structures of compounds 111 and 112...... 82

Figure 49: H-NMR of compound 101...... 130

Figure 50: C-13 NMR of compound 101...... 131

Figure 51: ESI-MS of compound 101...... 132

Figure 52: H-NMR of compound 102...... 133

Figure 53: C-13 NMR of compound 102...... 134

Figure 54: ESI-MS of compound 102...... 135

Figure 55: H-NMR of compound 103...... 136

Figure 56: C-13 NMR of compound 103...... 137

Figure 57: ESI-MS of compound 103...... 138

Figure 58: H-NMR of compound 100...... 139

Figure 59: C-13 NMR of compound 100...... 140

Figure 60: ESI-MS of compound 100...... 141

Figure 61: H-NMR of compound 104...... 142

Figure 62: C-13 NMR of compound 104...... 143

Figure 63: ESI-MS of compound 104...... 144

Figure 64: H-NMR of compound 107...... 145

Figure 65: C-13 NMR of compound 107...... 146

Figure 66: ESI-MS of compound 107...... 147

xvi

List of Schemes

Scheme 1: Formation of compound 95 by Dess-Martin oxidation...... 61

Scheme 2: Synthesis of compound 96...... 61

Scheme 3: Synthesis of key intermediate compound 98...... 62

Scheme 4: Role of pyridine in silyl protection...... 63

Scheme 5: Mechanism of cyanohydrin reaction...... 64

Scheme 6: Synthesis of compound 100 from intermediate 98 via cyanohydrin method. . 66

Scheme 7: Mechanism of organolithium reaction with nitriles...... 67

Scheme 8: Reaction of methyllithium with water...... 67

Scheme 9: Acid catalyzed silyl deprotection...... 68

Scheme 10: The Wittig olefination ...... 69

Scheme 11: Ylide resonance structures...... 69

Scheme 12: Mechanism of the Wittig reaction...... 70

Scheme 13: Synthesis of compound 100 from compound 98 following Wittig olefination.

...... 71

Scheme 14: Mechanism of the Upjohn reaction...... 72

+ - Scheme 15: Mechanism of silyl deprotection by NH4 F ...... 74

Scheme 16: Synthesis of compound 104 from compound 102...... 75

Scheme 17: Synthesis of compound 108...... 76

Scheme 18: Synthesis of compound 110...... 80

Scheme 19: Synthesis of phosphoramidates...... 83

xvii

List of Abbreviations

3D 3 Dimensional aa Amino Acid ALT Alanine aminotransferase ApoA-I ApolipoproteinA-I ApoB-100 ApolipoproteinB-100 ApoE ApolipoproteinE Asn Asparagine ASO Anti-Sense Oligonucleotide Asp Aspartic Acid ATP Adenosine triphosphate

Calc. Calculated CD Cluster of Differentiation CDC Centers for Disease Control and Prevention cDNA complementary Deoxyribonucleic Acid CLDN1 Claudin-1 cLDs cytoplasmic LDs COX 2 Cyclooxygenase 2 cryo-ET Cryoelectron Tomography CTL Cytotoxic T Lymphocyte Cys Cysteine

D1 Domain 1 D2 Domain 2 D3 Domain 3 DAA Direct Acting Antiviral DAPN-PD1 β-D-2′-C-methyl-2,6-diaminopurine- ribonucleotide phosphoramidate prodrug 1 DCM Dichloromethane DGAT-1 Diacylglycerol Acyltransferase-1 DMAP Dimethyl Aminopyridine DMF Dimethylformamide DMP Dess-Martin Periodinane DNA Deoxyribonucleic acid

E1 Envelope glycoprotein 1 E2 Envelope glycoprotein 2 EGFR Epidermal Growth Factor Receptor xviii

EM Electron Microscopy ER Endoplasmic Reticulum ESI-MS Electrospray Ionization Mass Spectrometry

F Fluorine FDA US Food and Drug Administration g Gram GAK cyclin G Associated Kinase GBD Global Burden of Disease GSK GlaxoSmithKline

H Hydrogen HCl Hydrochloric acid HCV Hepatitis C Virus HDL High-Density Lipoprotein His Histidine HIV-1 Human Immunodeficiency Virus-1 hr Hour Hz Hertz

IFN Interferon Ile Isoleucine IMP Inosine-5′-monophosphate IRES Internal Ribosome Entry Site kb Kilo Base kDa Kilodaltons

LDL Low-Density Lipoprotein LNA Locked Nucleic Acid luLDs luminal LDs LVP Lipoviral Protein

MAVS Mitochondrial Antiviral Signaling MeLi Methyl Lithium mg Milligram MHC Major Histocompatibility Complex miRNA-122 microRNA-122 mL Milliliter mmol Millimole

N Nitrogen NANBH Non-A Non-B Hepatitis NCR Noncoding Region NI Nucleoside Inhibitor xix nm Nanometer NMR Nuclear Magnetic Resonance NNI Non-Nucleoside Inhibitor NPC1L1 Niemann-Pick C1–Like 1 NPHV Non-Primate Hepacivirus NS2 Nonstructural protein 2 NS3 Nonstructural protein 3 NS3/4A NS3-NS4A noncovalent complex NS4A Nonstructural protein 4 A NS4B Nonstructural protein 4 B NS5A Nonstructural protein 5 A NS5B Nonstructural protein 5 B NTPase Nucleoside Triphosphatase

O Oxygen OCLN Occludin ORF Open Reading Frame

PEG Polyethylene Glycol pegIFNα-2b Pegylated Interferon alfa-2b Phe Phenylalanine

QALY Quality-Adjusted-Life-Year

RBV Ribavirin RdRp RNA-dependent RNA-polymerase RIG-I Retinoic acid Inducible Gene-I RNA Ribonucleic Acid

Sat. Saturated Ser Serine SoC Standard of Care SorA Soraphen A sp Species SPP Signal Peptide Peptidase SR-BI Scavenger Receptor class B type I SVR Sustained Virologic Response

THF Tetrahydrofuran TLC Thin Layer Chromatography TLR3 Toll-like Receptor 3 TMD Terminal Membrane Domain TMS Trimethyl Silyl TP Triphosphate

UTR Untranslated Region xx

UV Ultraviolet

VLDL Very Low-Density Lipoprotein

WHO World Health Organization

xxi

List of Symbols

% Percent (-) Negative (+) Positive ~ Approximately ′ Prime > More than

µM Micromolar µmol Micromole

13C NMR Carbon nuclear magnetic resonance 1H NMR Proton nuclear magnetic resonance

1st First

Bn Benzyl

CD3OD Deuterated methanol CDCL3 Deuterated chloroform CeCl3 Cerium chloride CeCl3.7H2O Cerium chloride heptahydrate CN- Cyanide anion CuI Copper iodide d Doublet dd Doublet of doublet DMSO dimethyl sulfoxide dt Doublet of triplet et al And others Et3N Triethyl amine EtOAc Ethyl acetate EtPh3PBr ethyltriphenylphosphonium bromide

F- Fluoride ion

HF Hydrogen fluoride HINT1 histidine triad nucleotide-binding protein 1 HMPA Hexamethylphosphoramide i-Bu Isobutyl xxii

IBX 2-iodoxybenzoic acid i-Pr Isopropyl

J Coupling constant

KCN Potassium cyanide KF Potassium fluoride

LDA Lithium diisopropyl amide m Mutiplet Me Methyl MePh3PBr methytriphenylphonphonium bromide Mg2+ Magnesium ion MgSO4 Magnesium sulfate Mn2+ Manganese ion

Na2S2O3.5H2O Sodium thiosulfate pentahydrate NaCl Sodium Chloride NaH Sodium hydride NaHCO3 Sodium bicarbonate NaHMDS sodium bis(trimethylsilyl)amide NaOH Sodium hydroxide NBS N-bromosuccinimide + NH4 ammonium ion NH4+F- Ammonium fluoride NH4Cl Ammonium chloride

OH Hydroxyl OsO4 Osmium tetroxide

P Phosphorus Ph Phenyl q Quartet s Singlet S Sulfur Si Silicone SnCl4 Tin(IV) chloride t Triplet TBAF tetrabutylammonium fluoride TIPDSCl2 1,3-Dichloro-1,1,3,3- tetraisopropyldisiloxane TMSCN Trimethyl cyanide xxiii

α Alpha β Beta δ Delta π Pi

xxiv

Chapter 1

1 Introduction

1.1 Historical Background

In 1989 M. Houghton et. al.1 described the construction of a complementary DNA

(cDNA) library from plasma that contains the uncharacterized non-A, non-B hepatitis

(NANBH) agent. They also described the isolation of a cDNA clone that encodes an antigen associated with NANBH.1 They showed that this clone was not derived from host

DNA, but from an RNA present in NANBH. Which was made of at least ten thousand nucleotides and it was positive-stranded. Their data indicate that it was similar to the

Togaviridae or Flaviviridae family of viruses. They decided to call it hepatitis C virus

(HCV).2 It is now known that HCV belongs to the genus Hepacivirus and the family

Flaviviridae.3 The immediate sources associated with its pandemic spread were diverse variants of HCV found in Central and West sub-Saharan Africa and South and South East

Asia. Those variants appeared to be there for hundreds of years. As of its origin, it is believed to be of zoonotic origin. There is no published data for infection of HCV-like

viruses in primates despite the analogy of HCV origin to origin of HIV-1 from chimpanzees in Central Africa. In fact, there are very recent findings of a non-primate hepacivirus (NPHV) in horses and dogs.4

1.2 Significance

With approximately 185 million people worldwide infected with HCV, and with an estimated 700 thousand people dying each year from HCV related liver diseases, HCV represents a notable health burden, globally. Eighty percent of infected individuals will develop chronic infection, 75% of those will develop hepatocellular carcinoma. HCV has overtaken HIV-1 as a major cause of death due to viral infection. More importantly, 95% of the patients in the world do not know their status due to the silent nature of the disease

(figure 1).3,5-6

Figure 1: HCV infection global reach. [Reprinted with permission from ref. 5]

1.3 HCV Transmission

The major route of HCV transmission is the parenteral route, as parenteral exposure leads

to infection in most cases, most intravenous drug users become infected. Other routes of

transmission may include transmission within the family, because HCV antibody

prevalence is higher in family members and sexual partners. Vertical transmission-

transmission from mother to baby- is also possible. There is also the possibility of

transmission by salivary contamination. There are a number of HCV carriers in whom

route of transmission was not identified (Figure 2).7-8

Figure 2: HCV transmission. [Reprinted with permission from ref. 7]

1.4 HCV Prevalence

Hepatitis C is a worldwide disease. The World Health Organization (WHO) stated that

Eastern Mediterranean and European regions are the most affected regions, with prevalence of 2.3% and 1.5% respectively. Prevalence of HCV infection in other regions varies from 0.5% to 1.0%. Different genotypes of HCV distribution vary by region.9

1.5 Types of HCV infection

Both acute and chronic infections can be caused by HCV. Per the Centers for Disease

Control and Prevention (CDC) and the WHO, acute HCV infection is usually asymptomatic or with mild symptoms like fatigue, abdominal pain, poor appetite, or jaundice. The incubation period for acute infection is 4-12 weeks and very rarely causes life threatening disease. On the other hand, chronic infection with HCV can lead to cirrhosis or liver cancer and death. It is insidious in nature and patients are often diagnosed after routine screening for blood donation and/or when alanine aminotransferase (ALT) levels are found elevated in standard examinations.7, 9

1.6 HCV Structure

HCV has the most irregular structure among members of the Flaviviridae family. Its viral particle is enveloped and spherical in shape with spike projections (Figure 3).10

Figure 3: HCV particle. [Reprinted with permission from ref. 10]

The viral particle is composed of an endoplasmic reticulum (ER) lipid bilayer that envelopes a nucleocapsid which contains the viral genome. Infectious HCV present in the blood stream as a hybrid lipoviral protein (LVP). HCV purification and structural analysis using electron microscopy (EM) were very challenging because of its insufficient binding to the conventional EM grid as well as poor stability and low yields.

The use of affinity tags targeting the E2 glycoprotein enabled the purification of enveloped particles, and the use of cryo-electron tomography (cryo-ET) provided valuable information to construct a 3-dimensional (3D) image of the HCV virion. The

HCV virion is associated with apolipoprotein E (ApoE), in addition, it also incorporates apolipoprotein B-100 (ApoB-100) and apolipoproteinA-I (ApoA-I). ApoE is essential for viral assembly. ApoA-I, a major high-density (HDL) structural lipoprotein, interacts with scavenger receptor class B type I (SR-BI) facilitating HCV entry and attenuating HCV neutralization. The role of ApoB-100 is still unclear. The size of an HCV particle ranges from 41-100 nanometer (nm) in diameter with a mean of 62 nm.10

1.7 HCV Genome

HCV has a 9.6-kilo base (kb) positive single strand RNA genome. The RNA genome includes 5′ and 3′ noncoding regions (NCRs) or untranslated regions (UTRs). Between these regions lies an open reading frame (ORF) which encodes a polyprotein that is cleaved into structural and nonstructural proteins. The 5′ UTR includes the internal ribosome entry site (IRES) (Figure 4).11-12

Figure 4: HCV genome. [Reprinted with permission from ref. 11]

The 5′ UTR is 340 nucleotides in length. These nucleotides are divided into 4 domains (I-

IV). This region has a conserved sequence across all HCV genotypes. The first domain binds the hepatocyte’s microRNA-122 (miRNA-122), while the other domains form the

IRES. The IRES recruits and stabilizes the cellular translation machinery and thereby directs translation of the coding sequence (Figure 5).11

Figure 5: HCV IRES. [Reprinted with permission from ref. 11]

Figure 6 shows the 2 interaction sites for miRNA-122 at the 5′ UTR. Although it is

unclear whether miRNA-122 enhances translation, replication, or protects the HCV

genome, medications directed toward preventing the interaction between miRNA-122

and HCV genome have been exploited.11, 13

Figure 6: HCV 5′ UTR miRNA-122 interactions. [Reprinted with permission from ref. 11]

Most of the ORF region is conserved yet there are some regions in the ORF that are

divergent among different HCV genotypes. Stem loops in the ORF region (Figure 4) are

required for translation of the viral genome, replication, infectivity, and avoidance of host immune defenses.11

The 3′ UTR has secondary structures important for replication (Figure 4). These sections of the 3′ UTR direct the HCV RNA-dependent RNA-polymerase (RdRp) for replication.

More importantly, the RdRp initiates replication from the negative-sense 3′ UTR better than the positive-sense 3′ UTR, which explains why there are more copies of positive- sense RNA than negative-sense RNA in infected hepatocytes.11

1.8 HCV Genotypes

Several HCV isolates were obtained and sequenced worldwide after the determination of the complete HCV genome by Choo et al. in 1991. Sequence variability arose, and several distinct types were identified. These types might differ from each other by 33% over the viral genome. This causes difficulty in the development of vaccines and/or pan- genotypic drugs. A consensus nomenclature system was proposed at the 2nd International meeting of HCV and Related Viruses in San Diego, CA 1994, that is to be used in future studies of HCV genotypes and subtypes. HCV was classified based on the similarity of the nucleotide sequence into genotypes. HCV genotypes are numbered-Arabic numerals- in the order of their discovery. The more closely related HCV strains are designated

Subtypes. Subtypes are assigned lowercase letters-alphabetical letters- based on the order of their discovery as well. Quasispecies are a complex of genetic variants found within an individual isolate, which results from the accumulation of mutations during viral replication (Table 1).14

Table 1: HCV genomic heterogeneity terms. [Reprinted with permission from ref. 14]

Terminology Definition % Nucleotide similarity

Genotypes Genetic heterogeneity among different 65.7-68.9

HCV isolates

Subtypes Closely related isolates within each of the 76.9-80.1

major genotypes

Quasispecies Complex of genetic variants within 90.8-99

individual isolates

Accordingly, HCV has 7 genotypes, many subtypes (a, b, c, etc.), and approximately 100 different strains (1, 2, 3, etc.). Genotype 1 is the most prevalent worldwide (46.2% of all

HCV cases). Genotype 3 is the second most prevalent globally (30.1%). Genotypes 2, 4, and 6 account for a total 22.8% of all cases; genotype 5 comprises the remaining <1%.

Genotype 7 has only been reported in 4 patients so far. Genotypes 1 and 3 are present in most countries irrelevant of socioeconomic status. Although genotype 1 is most common worldwide, other genotypes still comprise over half of all HCV cases.15

After acute exposure, 92% of the patients exposed to genotype 1b develop chronic infection, while only 33% to 50% of those who are exposed to the other genotypes progress to chronicity.16 Isolates that produce little genetic diversity tend to be self- limiting and resolve in the acute phase, while those who develop considerable genetic diversity tend to progress to chronic infection.17-18

Quasispecies can also be used to determine the route of transmission of HCV. Examples include, but are not limited to, mother/infant pair, sexual partners, and in nosocomial transmission of the disease.19

1.9 HCV Genotype Distribution

Hepatitis C infection is a worldwide disease but the viral genotypes are not distributed evenly across the globe. Distribution of HCV genotypes is presented based on WHO global burden of disease (GBD) regions. Genotype 1 is the most common worldwide and predominates in Northern, Southern, and Eastern Europe, in the Americas, parts of Asia and Japan. While type 2 is mainly found in Western Africa, genotype 3 is found in South

Asia, India, Pakistan, and parts of Northern Europe. Genotype 4 is the most encountered type in the Middle East, especially Egypt, and Central Africa. Genotype 5 is found almost exclusively in South Africa, and genotype 6 in South East Asia (Figure 7). Genotype 7 was found in Canada from patients originating from Central Africa. It is worth mentioning that not all the countries in the world have statistical data about the genotype of HCV found in those countries. No studies were available from the Oceania GBD region (not including Australia). The Caribbean, Central Latin America-excluding

Mexico- and parts of Africa have a poor number of studies about HCV genotypes.15, 20-22

Figure 5: HCV genotypes distribution. [Reprinted with permission from ref. 15]

1.10 HCV Proteins

HCV protein structures and functions have been elucidated. HCV proteins can be divided into structural and nonstructural proteins (Figure 8). Structural proteins include core protein, E1, and E2, while non-structural proteins include p7, NS2, NS3, NS4A, NS4B,

NS5A, and NS5B. HCV proteins may also be classified into assembly and replication modules from a functional point of view. The assembly module is comprised of core, E1,

E2, and NS2. The replication module incorporates NS3-NS5B. Non-structural proteins deploy structural activities other than their non-structural functions, such as forming protein clusters, arrays, or lattices on intracellular membranes.23-24

Figure 6: HCV proteins. [Reprinted with permission from ref. 45]

1.10.1 Structural Proteins

1.10.1.1 Core

The core protein is the first structural protein encoded by the ORF region of the HCV genome.25 It forms the nucleocapsid of the virus. An immature 191-amino-acids (aa) core protein results from the cleavage of the core-E1 signal sequence by signal peptidase. C- terminal processing by the signal peptide peptidase (SPP) yields the mature core protein of ~177 aa ~21 Kilodaltons (kDa).26 The HCV core protein is dimeric in nature and held together through disulfide bond formation at cysteine 128 (Cys128). The HCV core protein is composed of two domains. The N-terminal basic hydrophilic domain 1 (D1, aa

1–117) which is involved in RNA binding and homo-oligomerization, and has the RNA chaperone activity required for the structural remodeling and packaging of the RNA genome. It is also involved in interactions with numerous cellular factors. Its structure includes several α-helices, and of note, a helix-loop-helix motif constituting an antigenic site and components critical for core function. The C-terminal hydrophobic domain 2

(D2, aa 118 to ~177) mediates association with lipid droplets (LDs). D2 structure consists of a central hydrophobic loop connecting two amphipathic α-helices.25-28

1.10.1.2 Envelop glycoproteins

E1 and E2 are type I transmembrane proteins composed of an N-terminal ectodomain

(~160 and ~360 aa for E1 and E2, respectively) and a short C-terminal transmembrane domain (TMD) of ~30 aa. A short polar segment contains highly conserved charged residues separating two stretches of hydrophobic aa of both TMDs.29 Membrane anchoring, ER retention, and E1–E2 noncovalent heterodimer formation are among the functions of the TMDs.30 A segment in the stem region of E2 (aa 279-331) is essential for virus entry and harbors a central amphipathic α-helix.31 Assembly of the infectious particle, virus entry, and fusion with the endosomal membrane are vital roles played by the envelope glycoproteins E1 and E2 at different steps of the HCV life cycle.24

1.10.2 Nonstructural Proteins

1.10.2.1 p7

Protein p7 belongs to the viroporin family. It has cation channel activity within its heptamer or hexamer structures and facilitates virus production (Figure 9). It is a 63-aa integral membrane polypeptide. Two transmembrane α-helices connected by a positively charged cytosolic loop comprises the structure of p7, with the N and C termini oriented toward the ER lumen. Although it is not needed for RNA replication in vitro, it is integral

to the assembly and release of infectious HCV in vitro as well as in vivo. Its precise function is unknown.32-34

Figure 7: HCV p7. [Reprinted with permission from ref. 34]

1.10.2.2 NS2

A cysteine protease encoded by NS2 cleaves the polyprotein precursor at the NS2/NS3 junction. Cleavage at the NS2/NS3 junction is crucial to forming fully functional NS3 protein. Hence, NS2 promotes viral RNA replication. The catalytic activity resides in the

C-terminal half of NS2, the N-terminal half represents a membrane domain. NS2 is a dimer. It has 2 active sites composed of residues from both monomers. Three transmembrane segments and a α-helix make the membrane domain of NS2. NS2 plays a pivotal role in HCV assembly aside from its protease activity. This role involves a complex network of interactions with structural and other nonstructural proteins (E1, E2, p7, NS3, and NS5A).35

1.10.2.3 NS3/4A complex

NS3 and cofactor NS4A make up a noncovalent complex (NS3/4A) (Figure 10).

NS3 is a 70 kDa protein, which has serine protease activity located on the N-terminal

Figure 8: HCV NS3/4A complex with inhibitor. [Reprinted with permission from ref. 34]

one-third and an NTPase/RNA helicase activity in the C-terminal two-thirds. With a

chymotrypsin-like fold and two β-barrel subdomains, the structure of the NS3/4A

protease is stabilized by a Zn2+ ion that is coordinated with Cyt97, Cyt99, Cyt145, and

histidine 149 (His149). NS4A polypeptide works as a cofactor for the NS3 serine

protease. A β-strand comprises its central portion that is incorporated into the N-terminal

β-barrel of NS3. The N-terminal hydrophobic portion of NS4A forms a transmembrane

α-helix essential for integral membrane association of NS3/4A. The C-terminal acidic

portion is composed of a negatively charged α-helix, which interacts with other replicase

components to contribute to HCV RNA replication and viral particle assembly.36-38

The NS3 nucleoside triphosphatase (NTPase)/RNA helicase activity is used for the

unwinding of double-stranded RNA or of single-stranded RNA regions with extensive 15

secondary structure coupled with adenosine triphosphate (ATP) hydrolysis. The precise function(s) of NS3 helicase in the viral life cycle remain(s) unclear. The reason why the protease domain and NTPase/RNA helicase domain are physically linked, is yet to be discovered.39-40

Of note, NS3/4A is located on membranes of the ER in replication complexes as well as, to a lesser extent, on mitochondrial or mitochondria-associated membranes, which are thought to be specialized ER sites near mitochondria. This explains how the NS3/4A plays imperative roles in replication and in pathogenesis and persistence of HCV. It does so by cleaving and hence inactivating a mitochondrial host protein, the retinoic acid inducible gene-I (RIG-I) adaptor mitochondrial antiviral signaling (MAVS) protein (a.k.a

Cardif, IPS-1, and VISA). It also cleaves the Toll-like receptor 3 (TLR3) adaptor TRIF

(a.k.a TICAM1) another crucial adaptor protein in innate immune sensing. NS3/4A also cleaves, a modulator of the epidermal growth factor receptor, T cell protein tyrosine phosphatase.37, 41

1.10.2.4 NS4B

NS4B is a poorly characterized, hydrophobic protein of ~261 aa ~27 kDa. It is a membrane protein consisting of an N-terminal part, a central part with four transmembrane passages, and a C-terminal part. It was shown by site-directed mutagenesis that the N-terminal segment plays an integral role in the formation of a functional replication complex, like other HCV nonstructural protein membrane segments. Intriguingly, the membrane topology of the N-terminal part of NS4B may be

dynamic because it is reduced and modulated by co-expression of NS5A, and by protein– protein interactions within the HCV replication complex. Therefore, membrane interaction of NS4B is mediated by transmembrane domains in its central, N- and C- terminal parts.42

NS4B has been found to bind viral RNA and interacts with other viral nonstructural proteins. In addition to that, it has NTPase activity and has been shown to play a role in viral assembly. NS4B induces the formation of a specific membrane alteration, which consists of local membranous vesicles that serves as a platform for the HCV replication complex, the membranous web.43-44

Evidence from electron microscopy studies as well as structural, genetic, and biochemical studies, all taken together, denote that NS4B is a master organizer of HCV replication complex formation. 45

1.10.2.5 NS5A

NS5A is an ~ 447-aa membrane-associated phosphoprotein. It plays an essential role in controlling HCV RNA replication and particle formation (Figure 11).12, 46

Data from limited proteolysis of recombinant NS5A and comparative sequence analyses indicated that NS5A has an N-terminal membrane anchor and 3 domains separated by low complexity sequences. Domains 1 and 2 (D1 and D2 respectively) are mainly involved in RNA replication while domain 3 (D3) is pivotal for viral assembly. D1 is also

Figure 9: HCV NS5A. [Reprinted with permission from ref. 46] involved in LDs binding and D3 is involved in the interaction with the core protein.47

NS5A adjusts HCV RNA replication by modulating interactions with replication specific host factors.47 Due to the unfolded nature of D2 and D3, NS5A has a remarkably high number of viral and cellular interactions. Hence, it is a hub for protein interactions.

However, only few of these assumed interactions of NS5A have been confirmed in the context of the entire viral life cycle in vitro and/or in vivo.24

1.10.2.6 NS5B

The positive strand RNA of the virus serves as a template for the synthesis of a negative

RNA strand. This forms a double stranded RNA molecule that is copied multiple times to form many copies of the positive strand viral RNA genome. The key protein in HCV viral replication is NS5B. It is considered to be the catalytic machinery of the replication complex, because it has RdRp activity.48 NS5B contains finger, palm, and thumb subdomains similar to the right hand analogy of other polymerases (Figure 12).49 It also contains a C-terminal membrane anchoring tail and a thumb domain β-loop insertion.50

Figure 10: HCV NS5B. [Reprinted with permission from ref. 47]

The HCV NS5B also contains six conserved motifs designated A-F like other known

RdRps (Figure 13).50

Due to the extensive interactions between the finger and thumb subdomains, NS5B has an encircled active site, which is considered to be an unusual feature among

Figure 11: HCV NS5B motifs. [Reprinted with permission from ref. 48] polymerases.51 Limited structural changes take place upon nucleotide binding as revealed by structural analysis of HCV NS5B, and that de novo initiation is the probable mode of

RNA synthesis.52 Two conformations of NS5B present, even in the absence of RNA, as indicated by structural studies of NS5B. The difference between the 2 conformations is the orientation of the thumb subdomain in relation to the palm and finger subdomains.53

The β-hairpin loop is another uncommon structural characteristic of HCV NS5B. It protrudes into the active site in the palm subdomain. The β-hairpin loop could have a role in directing the 3′ terminus of the viral RNA for correct initiation.50 The NS5B active site is highly conserved. It is located in the palm subdomain because the residues involved in nucleotidyl transfer are found in palm motifs A and C. Motif A has the metal binding residue aspartic acid 220 (Asp220), while motif C harbors the conserved metal binding and nucleotidyl transfer residues Asp318 and Asp319.54 Asp225, serine 282 (Ser282), and asparagine 291 (Asn291) in the active site of NS5B form a hydrogen bonding network with the 2ʹ-hydroxyl of the incoming ribonucleotide (Figure 14).51 The main chain of Ser282 flips, allowing its side chain to hydrogen bond with the 2ʹ-hydroxyl of

the incoming ribonucleotide and the carboxyl group of Asp225. Also, the amine side chain of Asn291 hydrogen bonds with the 2ʹ-hydroxyl of the ribonucleotide on the opposite face of ribose (Figure 14).55

NS5B can decompress stable secondary and tertiary RNA structures because recombinant

Figure 12: HCV NS5B active site. [Reprinted with permission from ref. 53]

NS5B is enough to synthesize full length RNA in vitro.50 NS5B replicates the 3′ terminal region of the negative (−)-strand RNA more efficiently than the 3′ terminal region of positive (+)-strand RNA.11 HCV NS5B does not have high substrate specificity toward

RNA. In fact, it has been discovered that an RNA with 3 stem-loop structures at the 5′ and at least 1 cytidine at the 3′ would have high affinity toward the viral polymerase.50

NS5B follows a two-metal-ion mechanism of nucleotide addition. In this mechanism, metal ion A lowers the affinity of the 3′-hydroxyl (OH) for the hydrogen (H), allowing the 3′-oxygen (O) attack at the alpha (α)-phosphate of the nucleoside triphosphate (NTP).

Metal ion B assists the leaving of the pyrophosphate, and both metal ions stabilize the structure and charge of the penta-covalent transition state. The conserved catalytic residues Asp220, Asp318, and Asp319 coordinate the two catalytic metal ions, which in 21

turn coordinate the α and beta (β) phosphates of the incoming nucleotide. The incoming nucleotide forms a Watson-Crick interaction with the pairing residue of the template strand (Figure 15).55-56

Figure 13: HCV NS5B mechanism of action. [Reprinted with permission from ref. 53]

In the apo enzyme the β-hairpin loop and the C-terminus occludes the active site. The incoming nucleotide and the 3′ end of the HCV RNA enter the NS5B active site through conformational changes, then the initial phosphoryl transfer generates a dinucleotide primer. Displacement of the β-loop happens as elongation of the primer continues. This allows the double stranded RNA to exit and transforms the enzyme to the highly processive state (Figure 16).55

Figure 14: HCV replication by NS5B. [Reprinted with permission from ref. 53]

It has been suggested that manganese (Mn2+) is the preferred metal ion for initiation and magnesium (Mg2+) is the preferred metal ion for elongation. HCV NS5B represents a good drug target due to its highly conserved active site and because it’s absence in human cells.57-58

1.11 HCV Life cycle

Understanding the HCV life cycle paves the way for more efficient antiviral therapies.

The viral life cycle can be divided into the following stages: entry and uncoating,

Figure 15: HCV life cycle. [Reprinted with permission from ref. 45]

translation and polyprotein processing, HCV RNA replication, and viral assembly and release (Figure 17).48

1.11.1 Entry and uncoating

A significant number of cellular molecules are involved in viral entry into the hepatocyte.

Low-affinity cell binding is believed to be mediated at the beginning by low-density lipoprotein (LDL) and glycosaminoglycans receptors. This happens before the interaction of E1 and E2 with co-receptors SR-BI and CD81.59 Occludin (OCLN) and claudin-1

(CLDN1) are required for entry.60 Epidermal growth factor receptor (EGFR) and ephrin receptor type A2 modulate the interactions between CD81 and CLDN1 and are required for entry into cells.61 Niemann-Pick C1–Like 1 (NPC1L1) cholesterol absorption receptor was identified as another hepatitis entry factor utilized at or before fusion at a late stage of HCV entry.62 HCV uptake depends on clathrin-mediated endocytosis. Fusion takes place in the endosomes because the endosomes have low pH and fusion of the virus requires low pH environment.63 These processes lead to the release of the virus inside the hepatocytes.

As a mean to avoid neutralization of the virus, direct cell to cell spread may also occur.64

1.11.2 Translation and polyprotein processing

The 5′ UTR, 3′ UTR, and coding region are highly structured with important RNA elements.11 The IRES situated in the 5′ UTR initiates translation. The polyprotein formed is co- and post-translationally processed by signalase, SPP, NS2, and NS3/4A proteases.

The maturation process of the core protein includes cellular SPP cleavage of a C-terminal signal peptide and cleavage from E1 by the same enzyme, which also cleaves E1, E2, and p7 from the polyprotein. The NS2-NS3 protease cleaves itself in an autocleavage mechanism. The NS3 protease assisted by its cofactor, NS4A, cleaves the remaining proteins NS3, NS4A, NS4B, NS5A and NS5B from the polyprotein. This results in 10

HCV proteins (Figure 8).48, 65 Interferon (IFN) synthesis instigated by RIG-I and TLR-3 is blocked by the effect of NS3/4A on MAVS and TRIF adaptor proteins.66 Mature NS2 and NS2-NS3 cleavage are mandatory for infectious virus production and for formation of virus replicase, respectively.35

1.11.3 HCV RNA Replication

RNA replication is considered to take place in the membranous web, which is an accumulation of ER-associated membrane vesicles formed by the actions of NS4B and

NS5A (Figure 18).67

Figure 16: Double membranous vesicle. [Reprinted with permission from ref. 65]

Displacement of RNA binding proteins, unwinding of RNA secondary structures, and separation of growing and template RNA strands all are done by helicase/NTPase

activities of NS3.40 Domains I and II of NS5A are essential for RNA replication. NS5A phosphorylation state modulates the balance between RNA replication and other processes.46

The work horse of HCV replication is NS5B. Binding, initiation, elongation, and termination are the steps involved in RNA synthesis by NS5B.68 RNA binding is a slow process. NS5B binds to single stranded RNAs of more than (>) 7 nucleotides with high affinity but with low specificity.69 De novo initiation involves binding of a single stranded template and 2 nucleotides matching the 3′ end of the template. This results in the formation of a dinucleotide primer. The advancement to elongation is inept and a rate limiting step.70 The change from primer synthesis to processive elongation requires high concentrations of the next nucleotide. This happens with a conformational change in

NS5B. The enzymatic core opens and double stranded RNA accommodates the egressing template-primer duplex.70 This conformational change generates a cavity large enough to accommodate double stranded RNA.71 It has been proven that low concentrations of the nucleotides are required for elongation and it was suggested that NS5B falls off after reaching the end of the template (Figure 16).72

1.11.4 Virus assembly and release

Assisted by diacylglycerol acyltransferase-1 (DGAT-1), the viral core protein moves from the ER to the cytoplasmic LDs (cLDs). It is poorly understood how the HCV genome is transferred to the nucleocapsid assembly site, but it is believed to be due to interactions between NS2 and E1, E2, p7, NS3, and NS5A.35, 73-74 The E1-E2 are trimmed

by glycosidases I and II after folding, heterodimer formation and the addition of N-linked sugars. HCV chooses the very low-density lipoprotein (VLDL) pathway during later stages of assembly, as a way to enhance persistence. The nucleocapsid is transferred to luminal LDs (luLDs). To form LVPs, nucleocapsid-containing luLDs fuse with Apo B- containing pre-VLDL particles, and acquire apoE and apoC and egress through the

Golgi.48

1.13 HCV Prognosis

Hepatitis C is acute and chronic. Among acutely infected individuals 80% will progress to chronicity. Seventy five percent of patients with chronic hepatitis eventually develop cirrhosis and hepatocellular carcinoma.58 Also, patients with chronic hepatitis are at risk of developing extra-hepatic disorders like cryoglobulinemia, porphyria cutanea tarda,

Type 2 diabetes, glomerulonephritis, and non-Hodgkin's lymphoma.75

Chapter 2

2 Background

2.1 HCV Treatment

HCV treatments range from biologics to chemical compounds. Success of treatment is based on achieving a sustained virologic response (SVR) at specific post treatment times.76, 77 SVR is defined as an undetectable HCV RNA level using a sensitive

Figure 17: HCV SVR12. [Reprinted with permission from ref. 79]

assay (typically with a lower limit of 25 IU/ml) for a period after completion of therapy for hepatitis C (Figure 19).78

It is worth mentioning that response to treatment depends on many factors. Examples include, but are not limited to, HCV genotype, the presence of cirrhosis, previous treatment, or resistance to anti-HCV medications.79 The first Food and Drug

Administration (FDA) approved medication against HCV was IFN alfacon-1. Ribavirin

(RBV), then pegylated IFN alpha-2b (pegIFNα-2b) followed. The first direct acting antivirals (DAA), boceprevir and telaprevir, were approved to be used in combination with IFN and RBV in 2011. Simeprevir was approved in 2013 followed by sofosbuvir a month later. In 2014 a fixed dose of ledipasvir and sofosbuvir was approved to be used with or without RBV. Ombitasvir, paritaprevir, ritonavir, and dasabuvir, were approved just 2 months after ledipasvir and sofosbuvir. In July 2015, daclatasvir was approved.

Figure 18: Anti-HCV approved medications. [reprinted with permission from ref. 36]

Grazoprevir and elbasvir were approved by FDA in 2016, five months later velpatasvir was approved (Figure 20). Asunaprevir and vaniprevir were also approved in Japan during the same time period.36 For structures see (Figure 23), (Figure 26), (Figure 32),

(Figure 35), (Figure 38), and (Figure 44).

Despite the approval of all the above medications, access to those medications is still limited due mainly to the high cost of treatment. The cost of treatment ranges from

$31,452 to $410,548 per Quality-Adjusted-Life-Year (QALY) gained owing to individual patient characteristics such as fibrosis stage, comorbidities, estimated life expectancy, and HCV genotype.80

2.2 Indirect Acting Antivirals

Anti-HCV medications can be divided into direct and indirect acting depending on their mode of action. As the name implies, indirect acting antivirals exert their effect by acting on a target other than the virus itself, such as inducing an immune response or blocking entry of the virus into cells. Many biologics and chemical compounds have been tried and/or used against HCV and proved to be useful in treating the infection.

2.2.1 Biologics

2.2.1.1 Interferons

Interferons are one of the natural defenses of human cells against viruses and other

microbes. Interferons when released from the infected cell, cause neighboring cells to

increase their defenses (Figure 21).81

Figure 19: Mechanisms of viral inhibition by IFNs. [Reprinted with permission from ref. 82] IFN alfacon-1 was the first medication approved to be used to combat HCV. It is a

recombinant synthetic IFN with 166 aa. It upregulates the expression of major

histocompatibility complex (MHC) proteins, which increases the presentation of peptides

derived from viral antigens. This enhances the activation of the immune response against

82 the virus. IFNα-2b was approved thereafter, which differs from IFN alfacon-1 at 20/166

aa (~88% homology). pegIFNα-2b then joined the fight against HCV. Polyethylene glycol

(PEG) was added to the IFN molecule to increase the duration of action.83 IFN based

treatments are often associated with side effects ranging from mild flu like symptoms to

serious low blood cell count. Thus, an IFN free regimen was a necessity.84

2.2.1.2 DNA vaccines

It is apparent- from patients who acutely resolve HCV infection- that a broad and strong cytotoxic T lymphocyte (CTL) response is important in HCV clearance. One way to elicit this response is by using DNA vaccines. To induce pathogen specific immunity, a plasmid DNA molecule that encodes pathogen antigens is used as a vaccine. These segments are delivered either as naked DNA or within a viral vector.85-86 INO-8000 is a naked DNA vaccine developed by Inovio. It covers HCV 1a and 1b and targets antigens

NS3/4A, NS4B, and NS5A. It is in phase I clinical trials (Figure 22).86

Figure 20: Inovio plasmid. [Reprinted with permission from ref 87]

AdCh3NSmut1 is another vaccine, in phase II clinical trials, being developed by

GlaxoSmithKline (GSK). It is also a DNA plasmid, but it is delivered in an adenovirus vector.85

2.2.2 Chemical Compounds

2.2.2.1 Ribavirin

Along with IFNα-2b, RBV constituted the standard of care (SoC) for HCV until 2011.

RBV is considered to be a nucleoside analog having a ribose moiety and 1,2,4-triazole-3- carboxamide base (Figure 23). Researchers believed that RBV acts by inhibition of inosine-5′-monophosphate (IMP) dehydrogenase.87 Others suggested the mechanism of lethal mutagenesis is the one by which RBV exerts it effect.88

Figure 21: Ribavirin.

2.2.2.2 Miravirsen

Miravirsen is a locked nucleic acid (LNA) antisense oligonucleotide (ASO). It is a 15- mer that was designed to target the 5′ end of miRNA-122, excluding the nucleotide complementary to the first nucleotide in miRNA-122. It has LNA modifications at the site of passenger strand cleavage. It mediates robust inhibition of miRNA-122 function in liver cells. It showed markedly improved efficiency in antagonizing miRNA-122 in mice compared with animals that were treated with either cholesterol-conjugated anti-miRNA or with other unconjugated anti-miRNA oligonucleotides (Figure 24) and (Figure 25).13 33

NH2 N

5' N O O O NH2

O O N P O S N O O O

O O 3'

Figure 22: Miravirsen locked nucleotides.

Figure 23: Miravirsen’s mechanism of action. [Reprinted with permission from ref. 13]

2.2.2.3 Cellular Component Inhibitors

These are newly discovered compounds that inhibit cellular components (e.g. cyclophilin

A, cyclin G associated kinase (GAK), SR-BI, etc.).36

Ritonavir inhibits human gene Cytochrome P450, family 3, subfamily A (CYP3A). This gene encodes important enzymes in drug metabolism. As an example, inhibition of paritaprevir CYP3A -mediated metabolism increases paritaprevir plasma concentration

(Figure 26).89

Figure 24: Ritonavir. [Reprinted with permission from ref. 90]

Alisporivir targets the host protein cyclophilin A. It is a non-immunosuppressive compound. It inhibits the interaction between cyclophilin A and NS5A in a dose- dependent manner. Of note, it acts synergistically with NS5A inhibitors. It also furthers activation of CD8+ T cells. Alisporivir act as a stimulus for antigen presentation. Hence, it has notable antiviral activity. Due to severe adverse effects, alisporivir was discontinued in phase III clinical trials (Figure 27).90-91

A novel bis-amide derivative called compound 25 (Figure 27), was discovered to pursue cyclophilin inhibition. It efficiently restored host immune responses and inhibited HCV replication without acute toxicity in vitro and in vivo.92

Figure 25: Cyclophilin A inhibitors. [Reprinted with permission from ref. 36, 91-95]

NIM258 is a modified cyclosporine analog that acts as a non-immunosuppressive cyclophilin A inhibitor (Figure 27). It has a promising pharmacokinetic profile against

HCV infection. NIM258 decreased transporter inhibition, but maintained comparable efficacy to Alisporivir against cyclophilin A.93

Using nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography and structure-based compound optimization, Ahmed-Belkacem et al. were able to discover phenyl pyrrolidine derivative 31 (Figure 27). It was found to inhibit cyclophilin A with broad spectrum and high barrier to resistance.94

GAK is essential for HCV entry and assembly, so compounds with inhibitory effects towards GAK should display inhibitory actions against HCV. Isothiazolo[5,4-b] pyridines were developed to target GAK. Lead derivatives showed potent anti-HCV activity in nanomolar binding affinity (Figure 28).95 ITX-5061 is an inhibitor of SR-BI.

It could be combined with DAAs without conferring cross-resistance in vitro. ITX-5061

Figure 26: Cellular proteins inhibitors. [Reprinted with permission from ref. 36, 96-99] was safe and well tolerated in a phase 1b clinical trial (Figure 28).96 A lipo- cyclodepsipeptide-called MA026-isolated from the fermentation broth of Pseudomonas species (sp) effectively inhibits HCV entry (Figure 28). The antiviral mechanism can be conferred to an interaction between MA026 and CLDN-1.97 Soraphen A (SorA) inhibits the acetyl-CoA carboxylase, a key enzyme in lipid biosynthesis. It has been identified as an effective inhibitor for HCV (Figure 28).98 Aspirin (acetyl salicylic acid) demonstrated

activity against HCV. Researchers from Mexico proved that aspirin inhibits HCV RNA replication and protein expression by its effect on the cyclooxygenase 2 (COX 2) signaling pathway.99 In 2016, Yin et al. discovered that aspirin also prevents the entry of

HCV into hepatocytes by downregulating CLDN1 receptor.60

2.3 Direct acting antivirals

The FDA defines DAAs as “drugs that interfere with specific steps in the HCV replication cycle through a direct interaction with the HCV genome, polyprotein, or its polyprotein cleavage products”.100

2.3.1 E1/E2 and p7 inhibitors

No E1/E2 inhibitors have been approved by the FDA. E1/E2 inhibitors neutralize E1/E2 glycoproteins, which inhibits viral attachment and prevents viral entry. These inhibitors provide another way to clear the virus.101

In a dose dependent and pan-genotypic manner, a new benzimidazole derivative B5 inhibited HCV infections in primary hepatocytes. Although benzimidazole B5 inhibited

HCV entry, a single mutation phenylalanine (Phe)291isoleucine (Ile) in E1 can confer the virus resistant to the effect of B5 (Figure 29).102 Two compounds from the sesquiterpene lactone group of compounds, cynaropicrin and grosheimol, were extracted from the wild

Egyptian artichoke. These natural products inhibited HCV efficiently. Their mechanism of action is still unknown, but it is believed that they impede viral entry (Figure 29).103

Saikosaponin b2 inhibits HCV in early stages by acting on E2. It is extracted from

Bupleurum kaoi root. It has a pan-genotypic effect at noncytotoxic concentrations.

Despite its activity, saikosaponin b2 clinical use needs more investigation (Figure 29).104

Flunarizine- a calcium antagonist that efficiently combats migraines-was identified as an

HCV inhibitor in a whole life cycle screen of a compound library including clinically approved drugs. This compound targeted the E1 glycoprotein and inhibited HCV

membrane fusion in a submicromolar concentration. Its anti-HCV activity is preferable for genotype 2 infections (Figure 29).105

Figure 27: HCV E1/E2 and p7 inhibitors. [Reprinted with permission from ref. 36]

Crystallization of HCV p7 enabled in-silico compound identification. Promising agents were selected (e.g. adamantane and rimantadine) that interfered with p7 according to preliminary analysis (Figure 29).106 Human monoclonal antibodies targeting E1/E2

Figure 28: Chlorcyclizine derivative 30. [Reprinted with permission from ref. 109] envelope glycoproteins were explored for the inhibition of HCV infection. MBL-HCV1-a human monoclonal antibody-passes phase I clinical trials.107 Using structure-activity analysis, a piperazine derivative named compound 30 was optimized from the antihistamine chlorcyclizine HCI. As a chlorcyclizine derivative, it may act by interfering with viral entry into hepatocytes (Figure 30).108

2.3.2 NS2 inhibitors

A structure-guided virtual high-throughput screening approach was used to identify a lead small molecule inhibitor of the NS2. An epoxide-containing compound, later called

Figure 29: HCV NS2 inhibitor. [Reprinted with permission from ref. 110] compound 160, inhibited NS2-mediated proteolysis in vitro and possessed antiviral activity in cell culture (Figure 31).109 DNA aptamers against NS2 protein were made and

antiviral effects of the aptamers were studied. From those, the most effective aptamer was identified. Data suggest that an aptamer against NS2 protein exerts its antiviral effects via binding to the N-terminus of NS2 and disrupting the interaction of NS2 with NS5A. The resistance profile of the aptamer was attributed to a single mutation Ile861Threonine

(Thr) in HCV NS2.110

2.3.3 NS3/4A inhibitors

For structures of approved or investigational NS3/4A inhibitors see (Figure 32), (Figure

33).

Using structure based drug design approaches, boceprevir was derived from a α- ketoamide after the failure of screening of over 4,000,000 compounds. This was made possible after the discovery of the crystal structure of NS3/4A. As a Ser protease inhibitor, it blocks the Ser hydroxyl of the NS3/4A.111 It was approved, in May 2011, by the FDA to be used with pegIFNα and RBV for the treatment of HCV genotype 1 infection. In 2015, Merck announces its voluntarily discontinuation.112

Telaprevir was approved at the same time as boceprevir, by the FDA to be used with pegIFNα and RBV for the treatment of HCV genotype 1 infection. It is a reversible noncovalent inhibitor of NS3. It was also discovered by structure based drug design from tetra-aldehyde peptide. The fact that a protease can be inhibited by its cleavage product was the principle behind the discovery of telaprevir.113 Its discontinuation was announced by Vertex Pharmaceuticals in 2014.

Simeprevir is a potent inhibitor of NS3/4A. Based on the earlier discovered Medivir peptide inhibitor, Simeprevir was developed by structure based drug design as a cyclopentane macrocyclic derivative. What paved its way to clinical trials was its excellent antiviral activity, in vitro and in vivo biological, safety, and pharmacology profiles. It was approved by the FDA in 2013.114 ABT-450-a. k. a. Paritaprevir-is another

NS3/4A inhibitor. It shows potent activity against different HCV genotypes.115 The fixed- dose combination of 100 mg grazoprevir plus 50 mg elbasvir was approved by the FDA in 2016 with or without RBV to treat HCV genotype 1 and 4 infections. Grazoprevir was

Figure 30: Approved NS3/4A inhibitors. [Reprinted with permission from ref. 90] developed using a molecular modeling derived strategy. This method optimized the 43

contact of the protease inhibitor to the active site of the enzyme.116 Even though

Asunaprevir was declined by the FDA, its combination with daclatasvir was approved in

Japan. Vaniprevir was also approved in Japan but in combination with pegIFNα and

RBV.89

The combination of voxilaprevir with velpatasvir and sofosbuvir in now in phase III clinical trials. Being a reversible noncovalent inhibitor, vedroprevir efficiently binds the active site of NS3/4A. It is in phase II clinical trials now. Optimization of drug metabolism and pharmacokinetics, investigation of structure activity relationships (SAR), and structure based drug design all led to the discovery of danoprevir. Danoprevir is in phase II clinical trials as well. Glecaprevir (ABT-493) in combination with pibrentasvir

Figure 31: Experimental NS3/4A inhibitors. [Reprinted with permission from ref. 36]

(ABT-530) from AbbVie is in phase III clinical trials. Novel spiro-proline macrocycle,

MK-8831 offers pan-genotypic activity and good coverage of NS3/4A resistant strains.

BMS-890068 is an optimized derivative of asunaprevir. It is an acyclic tripeptidic sulfonamide. It has enhanced potency, safety, metabolic, and pharmacokinetic profiles, as compared to earlier derivatives. The investigation of faldaprevir was discontinued in

2014 because the SVR12 rate for it was lower than that of the other inhibitors. Derived from boceprevir, narlaprevir is a second generation NS3/4A inhibitor. Phase III clinical trials were discontinued because of post marketing commitments. BMS-605339 is an acyl sulfonamide based tripeptide inhibitor of NS3/4A. It was terminated due to potential cardiovascular liabilities in clinical trials.117

2.3.4 NS4B inhibitors

Figure 32: HCV NS4B inhibitors. [Reprinted with permission from ref. 36]

There is no NS4B inhibitors approved. PCT-725 is a 6-(indol-2-yl) pyridine-3- sulfonamide derivative. Although it offers an advantageous pharmacokinetic profiles, its clinical use needs more investigation (Figure 34).118 A 2-oxadiazoloquinoline compound 45

showed good anti-HCV activity in vitro. It is still in the preliminary stages of development (Figure 34).119 Though synergistic responses from Semiprevir, daclatasvir, and sofosbuvir were accomplished when used with imidazo[2,1-b]thiazole containing compounds, more clinical studies are warranted to establish their clinical effects (Figure

34).120

2.3.5 NS5A inhibitors

The fixed dose combination of ledipasvir and sofosbuvir was approved by the FDA in

2014 to be used with or without RBV to treat HCV genotypes 1 and 4-6. As a potent

NS5A inhibitor, Ledipasvir was developed via modifications in an asymmetric benzimidazole difluorofluorene-imidazole core and distal [2.2.1] azabicyclic ring system

(Figure 35).121 Ombitasvir is another NS5A inhibitor. It was developed from asymmetric

N-phenyl pyrrolidine based compounds with chiral pyrrolidine cores. Along with paritaprevir and dasabuvir, it represents the 1st co-formulated triple DAAs therapy,

Viekira PakTM (Figure 35).122 Daclatasvir was discovered by optimizing an iminothiazolidinone lead compound, which was obtained from high throughput phenotypic screening (Figure 35). It interferes with protein-protein interactions of NS5A by binding at positions 31 and 93 of the enzyme.123 Daclatasvir was approved by the

FDA in July 2015. Elbasvir was designed by introducing a tetracyclic indole into an earlier clinical candidate. This modification significantly enhanced its virologic profile

(Figure 35).124 Epclusa®, in 2016, became the first FDA approved treatment against all

HCV genotypes. Velpatasvir, the key component of Epclusa®, was developed as a 2nd generation NS5A inhibitor (Figure 35).125

Figure 33: Approved HCV NS5A inhibitors. [Reprinted with permission from ref 90]

Other NS5A inhibitors like samatasvir, ravidasvir, GSK-2336805, EDP-239, ruzasvir, biaryl imidazole chemotype 50b, AV-4025, pibrentasvir, and disulfiram are at different stages of development (Figure 36).36

Figure 34: Investigational HCV NS5A inhibitor. [Reprinted with permission from ref. 36]

2.3.6 NS5B inhibitors

FDA approval of combination DAAs therapies shows that it is possible to eliminate the

126 use of pegIFNα, thus decreasing side effects and increasing patient compliance. HCV

NS5B RdRp is the most important machine in the replication module of the virus.68 Since

NS5B is highly conserved and is not present in human cells, NS5B inhibitors provide advantages over other anti-HCV medications. Among these advantages are activity against different viral genotypes, and a high barrier to the development of resistance.55

NS5B has been pursued by researchers as a drug target for many years.127 These collective efforts led to the development of nucleoside or nucleotide inhibitors (NIs) and non-nucleoside inhibitors (NNIs).128-129

2.3.6.1 NNIs

NNIs bind to one of the NS5B allosteric sites, resulting in the inhibition of the conformational changes required for enzyme function (Figure 37).58, 127, 130

As shown above, these allosteric sites are named thumb site I, thumb site II, palm site I,

Figure 35: HCV NS5B allosteric sites. [Reprinted with permission from ref. 130] and palm site II.130 To date, the only NNI approved by FDA is dasabuvir. It binds to the palm site to hinder conformational changes in the enzyme.131-132 Figure 38 shows the structures of dasabuvir along with other NS5B NNIs under investigation.36

2.3.6.2 NIs

NIs are analogs of the naturally occurring ribonucleotides needed to synthesize viral

RNA. They compete with the natural ribonucleotides for incorporation into the growing viral RNA chain. NIs need to be phosphorylated to give 5′-triphosphates before they bind the enzyme. However, once the NIs bind to NS5B, incorporation of the next ribonucleotide is prevented by a steric clash with the incoming nucleotide. Hence the

Figure 36: HCV NS5B NNIs. [Reprinted with permission from ref. 36] name non-obligate chain terminators.133 Identification of an in vivo effective nucleoside 50

polymerase inhibitor can be challenging, because of the multiple substrate requirements

associated with nucleoside phosphorylation and the requirement that the polymerase

recognizes the nucleoside triphosphate as a substrate.3 Figure 39 illustrates the

mechanism of action of HCV NS5B NIs.134

Figure 37: HCV NS5B NIs mechanism of action. [Reprinted with permission from ref. 133]

The exploration of both sugar and base modifications led to the discovery of nucleoside

inhibitors of HCV NS5B and to the identification of both unique and potent agents.

Hence NIs can be divided into base modified NIs and sugar modified NIs.58

2.3.6.2.1 Base modified NIs

Modifying base moieties and leaving the sugar untouched led to the development of

several active inhibitors of HCV. N4-Hydroxycytidine was shown to be a good inhibitor

135 with 5µM EC90 (Figure 40). Compound 67 was developed from a series of 7-deaza-

6,7-disubstituted purines derived from toyocamycin. It was identified as having potent activity and showed submicromolar activity in the replicon assay (Figure 40).136

Modification of the sugar and the base at the same time was pursued as well. This

Figure 39: Base modified NIs. [Reprinted with permission from ref. 135-136] resulted in the identification of a number of active inhibitors (Figure 41). The least toxic and the most active one was compound 69 with a 7-deaza-7-fluoro modification at the adenosine base and a 2′-β-C-methyl modification at the ribose.137

Figure 38: Base and sugar modified NIs. [Reprinted with permission from ref 137]

2.3.6.2.2 Sugar modified NIs

Several sugar modifications were explored, like mono-substitutions at the 2′ position.

These substitutions led to the development of compounds active in isolated enzyme assays but not in the replicon system. Adding a 2′-β-C-methyl while keeping the 2′-α-OH resulted in the discovery of a class of inhibitors active in enzyme assays and in replicon systems. To overcome the poor bioavailability of these compounds, 3′-O-valinyl ester prodrug valopicitabine (NM283) was investigated. Also, replacing the 2′-α-OH with fluorine (F) produced a group of derivatives with interesting clinical properties. A 2′- deoxy-2′-spirocyclopropyl derivative of cytidine was shown to be a non-obligate chain terminator along with its ester prodrugs. Modifications at the 4′ position with azido substituent were screened against HCV as well. As with other inhibitors, these derivatives had poor bioavailability which led to the development of a triisobutyrate ester prodrug

(R1626). Making conformationally constrained nucleosides was thought to lead to possible NS5B inhibitors. Unfortunately, those compounds only show mediocre results

(Figure 42).55, 58, 128, 138-141 The only FDA approved NI is sofosbuvir, which is a modified nucleotide. It acts by interrupting the hydrogen bonding network in the active site of

NS5B. When the inhibitor is bound at the active site Asp225 is oriented away and Ser282 is oriented in the same direction as in the apo enzyme. This results in the deprivation of the hydrogen bonding network. Recognition of the 2′-F of sofosbuvir by Asn291 and

Watson-Crick base pairing with the template allows it to form the required conformation necessary for incorporation into the growing chain (Figure 43) and (Figure 44).55

Mericitabine is the prodrug of 2′-deoxy-2′-β-Me-2′-α-F-cytidine. It shows good activity 53

against HCV and mutation L159F and L320F only give low-level resistance and shows cross-resistance to sofosbuvir.142 Due to the low level of participation in clinical trials,

Roche discontinued mericitabine clinical trials in 2016 (Figure 42). β-D-2′-C-methyl-

2,6-diaminopurine-ribonucleotide phosphoramidate prodrug 1 (DAPN-PD1) is a novel

NS5B inhibitor. It is metabolized into 2 distinct bioactive nucleoside triphosphate (TP) analogs. These analogs efficiently inhibit RNA replication by NS5B (Figure 44).143 A C- nucleoside monophosphate prodrug, GS-6620, was discovered during a series optimization of a 1′-cyano-2′-C-Me-4-aza-7,9-dideaza adenosine analog. It exhibited pan- genotypic activity and high barrier to drug resistance.144 Since October 2016, GS-6620 was absent from the Gilead clinical trials pipeline (Figure 44). JNJ-54257099 is a cyclic phosphate ester derivative in the class of 2′-deoxy-2′-spiro-oxetane uridine nucleotide prodrugs (Figure 44). As an NS5B inhibitor, this compound decreased HCV RNA levels in mouse models. A phase I clinical trial of JNJ-54257099 was terminated in 2016.145

SAR studies of NS5B inhibitors indicate that the 3′-OH and the 2ʹ-substituent on the ribose are crucial for activity, along with the 2′-OH group.146 The 3ʹ-OH group of the ribonucleoside analog is important for activity because inhibitors lacking this group showed inefficient phosphorylation in cells.133 The 2′-OH allows for recognition of the inhibitor by viral NS5B through H-bonding with Asn291 along with Watson-Crick base pairing. The 2′ substituent is required to disrupt the H-bonding network via steric effects.55 Based on previously synthesized NIs of HCV polymerase and the recently published crystal structure of the RdRp with its inhibitor sofosbuvir in the

Figure 40: Sugar Modified HCV NS5B NIs.

Figure 41: Mechanism of action of sofosbuvir. [Reprinted with permission from ref. 55] active site, new uridine analogs modified at the 2ʹ-carbon can be developed to act as chain terminators in the treatment of HCV infection.

Our design is based on the only FDA approved NI Sofosbuvir. We chose uridine as sofosbuvir is uridine analog. Our analogs retain the 2′-OH which provides the H-bonding acceptor needed in all active NIs (F in sofosbuvir). Maintaining the natural 2′-OH simplify the chemical synthesis. Our analogs sport 2′-substituents with the ability to form

H-bonding. These substituents have extra carbons-as compared to methyl group (Me) in sofosbuvir- to increase steric effect required to disrupt the H-bonding network at the active site of the enzyme. The fact that these analogs have 2′ substituents capable of H- bonding eliminates the need for the specific stereochemical orientation in sofosbuvir, because both orientations (α and β) can make H-bonding with Asn291 and disrupting the

H-bonding network via steric effect. 56

Figure 42: HCV NS5B NIs approved and in clinical trials. [Reprinted with permission from ref. 36]

Chapter 3

3 Results and Discussion

3.1 Synthesis of nucleoside analogues

Generally, there are two routes for the synthesis of modified nucleosides: (1) a convergent route, and (2) a divergent or linear route. Each route has its advantages and disadvantages, and both routes could be used in a synthetic scheme to get the final compound.147 In the convergent route, the nucleobase, protected or not, is conjugated to the appropriately modified sugar.148 The linear route starts from the commercially available unmodified nucleosides.149 The convergent approach is more flexible because a variety of nucleobases can be coupled to the sugar. However, the convergent strategy is not stereoselective, low yielding, relatively lengthy, and not acquiescent to the addition of alkyl groups other than Me.150 The linear approach offers a shorter route toward the formation of the modified nucleosides. This route is only limited by the availability of the natural nucleosides.151

3.2 Design and synthesis of 2′-C-acetyluridine compound 100

For the synthesis of this compound, we explored both the convergent and the divergent routes.

3.2.1 The convergent route

In the ribose donor route, the synthesis begins with commercially available 1,3,5-tri-O- benzoyl-α-D-ribofuranose. Oxidation of the starting material using Dess-Martin

Periodinane (DMP) and treatment of the resulting ketone with vinyl magnesium bromide/cerium chloride will provide the tribenzoylated vinyl derivative. The product will be converted to the tetrabenzoylated vinyl compound upon treatment with benzoyl chloride and dimethylamino pyridine (DMAP) in the presence of triethylamine (Et3N).

After purification, the tetrabenzoylated vinyl product will be coupled with uracil under persilylation conditions. Coupling will be accomplished using tin(IV)chloride (SnCl4) as

Lewis acid in refluxing acetonitrile.150 Through the use of the Lemieux–Johnson oxidation, the newly formed alkene will be converted to an aldehyde, which will be converted to the 1-hydroxyethyl derivative through reaction with methyl magnesium bromide. Oxidation with DMP and deprotection with methanolic ammonia will yield the desired product.

DMP has many advantages over other oxidants. Among those advantages are mild conditions, higher yield, and simple workup. No heteroatom oxidation or over oxidation has been observed with DMP. DMP is based on 2-iodoxybenzoic acid (IBX), though the

presence of the acetate groups on the iodine make it more reactive and more soluble in organic solvents as compared to IBX (Figure 45).152-154

O O

O O O I O I OH O O O

O O DMP IBX

Figure 43: Structures of DMP and IBX.

Formation of the ketone at the 2-position allows the addition of the vinyl group at the same position using a Grignard reagent. Cerium chloride (CeCl3) significantly enhances the addition of Grignard reagents to ketones by remarkably suppressing enolization.155

Following the first approach, compound 95 was synthesized from commercially available

1,3,5-tri-O-benzoyl-α-D-ribofuranose via a method developed by Harry-O’kuru et al through oxidation using DMP.150 Desired ketone 95 was obtained in 74% yield (Scheme

1).

Scheme 1: Formation of compound 95 by Dess-Martin oxidation.

The addition of vinylmagnesium bromide to ketone 95 was unsuccessful despite all efforts made. A plausible reason for the failure of this reaction is the presence of water in

CeCl3. Since CeCl3 comes in the form of cerium chloride heptahydrate (CeCl3.7H2O), it is crucial to get rid of this water of crystallization and convert it to anhydrous CeCl3 before proceeding with the Grignard reaction. Changing the temperature and time of the drying process as well as changing the solvents of the reaction were all unsuccessful.

Even buying and using new reagents did not help change the outcomes of this reaction

(Scheme 2).

Scheme 2: Synthesis of compound 96. 61

For this reason and to synthesize the required compound, we moved to the linear route.

3.2.2 The linear route

In the second approach, we decided to synthesize key intermediate compound 98, followed by either cyanohydrin formation or Wittig olefination.

3.2.2.1 Synthesis of key intermediate compound 98

Synthesis will begin from commercially available uridine.156 In order to oxidize the 2′-

OH, one should protect the 3′, 5′-OH groups.156

3.2.2.1.1 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)uridine (97)

The 3′, 5ʹ-hydroxyl groups will be protected with a 1,1,3,3-tetraisopropyldisiloxane group

(TIPDS) before the 2ʹ-OH is oxidized to ketone 98 using DMP (Scheme 3).

Scheme 3: Synthesis of key intermediate compound 98.

Silyl ether protection of hydroxyl groups is used widely in organic synthesis. Silyl ethers are adequately stable, can be prepared easily, enhance compound solubility in organic solvents, and can be deprotected under mild conditions.157 In this method, we eliminated

the need to use imidazole in dimethylformamide (DMF)-the later has a very high boiling point and is not easy to remove from the reaction mixture. Pyridine was used as it acts as a solvent and as a catalyst for the protection reaction (Scheme 4).

Scheme 4: Role of pyridine in silyl protection.

We chose the TIPDS protecting group because it provides protection to both 3′ and 5′-OH groups in one step and avoids the formation of mono protected by products.

3.2.2.1.2 Synthesis of 3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-ketouridine (98)

With this approach and through the protection of the 3′ and the 5′-OH groups as tetraisopropyldisilyl ethers, the formation of the ketone at the C2′-OH was accomplished with DMP in dichloromethane (DCM). The desired ketone 98 was obtained in 80% yield from alcohol 97 (Scheme 3). With the key intermediate (98) in hand, we started the synthesis of the desired compound by following either cyanohydrin formation or Wittig olefination.

3.2.2.2 Cyanohydrin formation

Cyanohydrin intermediates are known to be useful in the synthesis of modified nucleosides.158-159 In this method, the ketone will be converted to a cyanohydrin protected as a trimethyl silyl (TMS) ether. The cyano group will be converted to an imine by reaction with methyl lithium (MeLi) followed by hydrolysis with acid to afford the desired ketone 100.

3.2.2.2.1 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-O-

(trimethylsilyl)-2′-C-cyanouridine (99)

In the cyanohydrin reaction, the nucleophilic cyanide anion (CN-) from potassium cyanide (KCN) attacks the electrophilic carbonyl carbon at the 2′-ketone creating an intermediate alkoxide, that attacks the silicon in the TMS group from trimethyl cyanide

(TMSCN) forming the protected cyanohydrin and regenerating the nucleophilic CN-

(Scheme 5).

Scheme 5: Mechanism of cyanohydrin reaction.

18-Crown-6 works to trap potassium cation (Figure 46), thus the CN- becomes a naked nucleophile (Scheme 5).160

Figure 44: 18-crown-6 complex with potassium ion.

Conversion of ketone 98 to protected cyanohydrin 99 was accomplished in 32% yield.

Attempts to increase the yield of the reaction by increasing the concentration of TMSCN as well as the concentration of KCN and 18-crown-6 and letting the reaction run longer did not deliver satisfactory results.

3.2.2.2.2 Synthesis of 2′-C-acetyluridine (100)

Treatment of compound 99 with MeLi in tetrahydrofuran (THF) at -78 ⁰C in the presence of copper iodide (CuI) as a catalyst for five hours, followed by hydrolysis of the imine intermediate with 1N HCl furnished the desired ketone 100 (Scheme 6).

Scheme 6: Synthesis of compound 100 from intermediate 98 via cyanohydrin method.

Organolithiums-in our case MeLi-react with nitriles through nucleophilic addition to give ketones. This reaction proceeds via an imine intermediate which is then hydrolyzed to form the ketone. The nucleophilic carbon in the organometallic reagent adds to the electrophilic carbon in the polar nitrile. Electrons from the carbon nitrogen triple bond move to the electronegative nitrogen creating an intermediate iminium salt. Upon the addition of aqueous acid, the intermediate salt is protonated giving the imine. The imine is protonated by acid in order to undergo nucleophilic addition. Now the nucleophilic oxygen of a water molecule attacks the electrophilic carbon with the pi (π) bond breaking to neutralize the charge on the nitrogen. Before the nitrogen leaves as a neutral molecule of ammonia, it needs to be made into a better leaving group by protonation. After that, deprotonation reveals the carbonyl group of ketone 100 (Scheme 7).161

N Li H NH R N Li R R

Iminium salt Imine

NH2 NH2 NH2 R R R OH O H H

O O H H H H

Acid catalyzed deprotection of the silyl groups O NH3 O H R R R NH3 OH

Scheme 7: Mechanism of organolithium reaction with nitriles.

Care and caution should be exercised when doing this reaction to avoid any contact with water as organolithium reagents are very reactive with water and form the corresponding hydrocarbon (Scheme 8).162 That is why so much care is required to ensure dry glassware and solvents when working with organometallic reagents.

H H H C Li + H OH H C H + Li OH H H

Scheme 8: Reaction of methyllithium with water.

Acid catalyzed deprotection of the silyl protecting groups furnished the required modified nucleoside (Scheme 9). 67

Scheme 9: Acid catalyzed silyl deprotection.

Excess acid in the last step requires neutralization with sodium hydroxide (NaOH), which yielded the final compound along with sodium chloride (NaCl). Attempts to desalt compound 100 either by dissolving in nonpolar organic solvents and then filtration or by dissolving in polar protic solvents and filtration all failed, hence we moved to using

Wittig olefination.

3.2.2.3 Wittig olefination

We envisioned the synthesis of the required compound through this method by forming an alkene at the 2′ position. Then by using Upjohn dihydroxylation, a diol would be formed, which would be oxidized to the 2′-C-acetyl uridine derivative (103) by DMP.163

+ - Silyl protecting groups would be removed using ammonium fluoride (NH4 F ) in methanol (MeOH).

3.2.2.3.1 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-deoxy-2′- ethylideneuridine (101)

Wittig reaction is one of the most important reactions to convert carbonyl groups to alkenes. In this reaction, a phosphonium ylide, which acts as the nucleophile, adds to a carbonyl compound to ultimately give an olefin and the phosphine oxide (Scheme 10).164

Scheme 10: The Wittig olefination.

Carey and Sundberg define a ylide as “a molecule that has a contributing resonance structure with opposite charges on adjacent atoms, each of which has an octet of electrons”. Phosphonium ylides are stable but reactive and can be represented by the following resonance structures (Scheme 11).164

H H P C P C H H

ylene ylide

Scheme 11: Ylide resonance structures.

The original mechanism proposes the addition of the nucleophilic ylide carbon to the carbonyl group to form a betaine (a dipolar intermediate), followed by the formation of an oxaphosphetane (a four-membered intermediate). Elimination of a phosphine oxide is presumed to follow (as shown in path A in Scheme 12). Path B in Scheme 12 shows an alternative mechanism, which proposes direct formation of the oxaphosphetane by a cycloaddition reaction.164

Scheme 12: Mechanism of the Wittig reaction.

Phosphonium ylides are formed by deprotonation of phosphonium salts, usually alkyltriphenylphosphonium halides. These are weakly acidic and requires strong bases for deprotonation. Examples of bases used are lithium diisopropylamide (LDA), sodium bis(trimethylsilyl)amide (NaHMDS), potassium tert-butoxide, and organolithium reagents. The stereoselectivity of the Wittig reaction depends on reactant structure, the base used, solvent, temperature, and the presence of other ions.165

Alkene 101 was made from ketone 98 using ethyltriphenylphosphonium ylide made by the treatment of ethyltriphenylphosphonium bromide (EtPh3PBr) with NaHMDS in dry ether (Scheme 13). The alkene was formed in moderate yield due to the hindered nature of the 2′-ketone. 70

Scheme 13: Synthesis of compound 100 from compound 98 following Wittig olefination.

3.2.2.3.2 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-C-(1- hydroxyethyl)uridine (102)

Osmium tetroxide (OsO4) is the most widely used reagent for conversion of olefins to glycols. OsO4 is toxic and expensive, which makes its use stoichiometrically barring.

Using catalytic amounts of OsO4 overcomes these disadvantages, but this requires the use of a co-oxidant like N-methymorpholine-N-oxide (NMO), t-butyl hydroperoxide, barium chlorate, or potassium ferricyanide. The use of peroxide or chlorate reagents led to further oxidation to an α-ketol causing reduced yield and difficulty in separation. The use of NMO allows the use of catalytic amounts of OsO4 without the formation of an α- ketol.163, 166-167 The mechanism of the Upjohn reaction was determined through

computational studies and NMR experiments proved that the mechanism happen via

[3+2] cycloaddition (Scheme 14).168

Scheme 14: Mechanism of the Upjohn reaction.

Osmylation of alkene 101 yielded compound 102 in 58% yield (Scheme 13). The moderate yield is attributed to the steric hindrance posed by the nucleobase and the large silyl protecting group.

3.2.2.3.3 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-C-acetyluridine

(103)

Oxidation of the newly formed secondary alcohol with DMP furnished the desired ketone in 60% yield (Scheme 13).

3.2.2.3.4 Synthesis of 2′-C-acetyluridine (100)

Removal of the silyl protection yielded the final compound. Table 2 shows methods and reagents employed for cleavage of silyl ethers.169

Table 2: Methods and reagents for silyl deprotection.

No. Reagents and/or methods

1 Hydrogen fluoride (HF)

2 Potassium fluoride (KF) with crown

ethers

3 Aqueous acetic acid with solvents

4 Pyridine hydrofluoride

5 Boron trifluoride etherate

6 Aqueous mineral acids with solvents

7 N-bromosuccinimide (NBS) in dimethyl

sulfoxide (DMSO)

8 Fluoroborate salts

9 Sodium azide in DMF

10 Sodium hydride (NaH) in

hexamethylphosphoramide (HMPA)

11 catalytic transfer hydrogenation

Most of the above-mentioned methods use strongly nucleophilic, basic, reductive conditions, carcinogenic, and/or high-boiling solvents. Hence, tetrabutylammonium 73

fluoride (TBAF) is the most commonly used reagent for deprotection of silyl ethers.

Nevertheless, TBAF is inimical to substances that are base sensitive. Also, the tetrabutylammonium cation causes workup and purification difficulties.169 We used

+ - NH4 F in MeOH because it is considered as an economical alternative to TBAF in THF.

+ - The low boiling point of MeOH and its good solvating properties for NH4 F and nucleosides silyl ethers makes it advantageous for our synthesis. The ammonium ion

+ (NH4 ) facilitates the deprotection of the silyl protecting group through hydrogen bonding with the oxygen of the ether during the nucleophilic attack of the fluoride ion (F-

) on the silicone (Scheme 15).

+ - Scheme 15: Mechanism of silyl deprotection by NH4 F .

Deprotection of compound 108 was accomplished in 90% yield (Scheme 13).

3.3 Design and synthesis of 2′-C-(1-hydroxyethyl)uridine compound 104

With compound 102 in hand, and through deprotection of the TIPDS ether, compound

+ - 104 could be synthesized. Compound 104 was synthesized from 102 using NH4 F

/MeOH in 90% yield (Scheme 16).

Scheme 16: Synthesis of compound 104 from compound 102.

3.4 Design and synthesis of 2′-C-formyluridine compound 108

Starting from compound 98, through Wittig olefination, a methylene derivative would be synthesized. This methylene compound would undergo Upjohn dihydroxylation using the same conditions that have been used before to make a hydroxymethyl derivative at the 2′ carbon. Oxidation of the primary OH by DMP and deprotection of the resultant compound will furnish the required product.

3.4.1 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-deoxy-2′-C- methyleneuridine (105)

With key intermediate compound 98 in hand, compound 105 was synthesized in 60% yield using methytriphenylphonphonium bromide (MePh3PBr) instead of EtPh3PBr. All the other reaction conditions were the same as those used before in the synthesis of compound 101 (Scheme 17).

O O O

NH NH NH

N O N O N O O MePh PBr, NaHMDS O OsO , NMO, t-BuOH O Si O 3 Si O 4 Si O o o o O Ether, rt--10 C, 72hr, 60% O THF, H2O, 0 C-4 C, 120hr, 40% O Si O O Si O Si O OH OH

98 105 106 DCM, o 0 C-rt, DMP 18hr, 60% O

O NH

NH N O O NH F N O 4 Si O HO O MeOH, rt, 48 O O O Si O OH OH OH 108 107

Scheme 17: Synthesis of compound 108.

3.4.2 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-C- hydroxymethyluridine (106)

Dihydroxylation of alkene 105 with OsO4 and the same reaction conditions for compound

101 yielded hydroxymethyl 106 in 40% yield (Scheme 17).

3.4.3 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-C-formyluridine

(107)

Oxidation of the primary OH in compound 106 with DMP in dry DCM formed the required aldehyde in 60% yield (Scheme 17).

3.4.4 Synthesis of 2′-C-formyluridine (108)

Unlike deprotection of compounds 102 and 103, attempts to deprotect aldehyde 107

+ - using NH4 F /MeOH failed. One possible reason is the increased reactivity of the aldehyde carbonyl carbon as compared to the ketone carbonyl carbon in (103) and the secondary alcohol carbon in derivative 102. This increased reactivity might have led to side reactions and loss of compound (Scheme 17).

Chapter 4

4 Conclusions and Future Work

4.1 Conclusions

DAAs have been proved to be good replacements for IFN+RBV treatment for

HCV infection. Modifying the natural nucleosides to target the active site of the HCV

NS5B RdRp resulted in many active anti-HCV compounds. This also led to the introduction of the only FDA approved NI sofosbuvir.

The role of 2′ position modification in chain termination of viral RNA synthesis and blocking of the HCV life cycle was emphasized in SAR studies of nucleoside analogs and from site specific mutations of the HCV NS5B RdRp active site.

We synthesized novel nucleoside analogs starting from uridine in few steps. These modified derivatives have the potential to work as antivirals based on the fact that the only FDA approved NI anti-HCV medication is a uridine analog modified at the 2′ position.

We hypothesized that these compounds would have good activity as anti-viral polymerase inhibitors just like other compounds that have undergone clinical trials before. It is believed that these derivatives will have activities across all the different

HCV genotypes. In addition, these analogs eliminate the need for the stereospecific constraints present in sofosbuvir.

Development of these drug candidates will eliminate the need for IFN based therapies.

Those therapies are associated with many side effects, some of which are life threatening.

Thus, IFN free regimens should decrease side effect, increase patients’ compliance, and hence increase the cure rates.

4.2 Future work

4.2.1 Synthesis of 2′-C-formyluridine (108)

Synthesis of compound 108 (Scheme 17) will be revisited. Deprotection of compound

+ - 107 using NH4 F /MeOH or other reagents will be attempted.

4.2.2 Synthesis of 2′-C-aminomethyluridine (110)

Since we have successfully synthesized compound 99, compound 110 will be synthesized by deprotection of the silyl ether from the amine 109 formed by reducing nitrile 99

(Scheme 18).

Scheme 18: Synthesis of compound 110.

4.2.3 Design and synthesis of phosphoramidate derivatives of the modified nucleosides

Synthesis of phosphoramidate derivatives of the modified nucleosides will be performed to deliver substrates which do not require the first phosphorylation step for activity.

Nucleoside analogs need to be converted to their 5′-triphosphate derivatives to be active against NS5B. Unfortunately, many nucleoside analogs fail to show activity in whole cell assays, because they are poor substrates for kinases in the phosphorylation pathway, especially the first phosphorylation step.3 So nucleoside 5′-monophosphates were prepared to overcome this problem, but the instability and the ionic nature of the phosphate group make them undesirable as drug candidates. Therefore, to overcome this problem 5′-monophosphate prodrugs were developed.133 One of these prodrugs is the 5′- phosphoramidate. First described by McGuigan, these phosphoramidates have good stability and permeation through membranes and after metabolism provide the monophosphate group required to bypass the first phosphorylation step.170 Conversion of the phosphoramidate prodrugs to the 5′-monophosphate nucleotide relies on a series of

enzymatic and chemical steps, these steps initiated by either carboxyesterase or cathepsin

A remove the ester group of the amino acid moiety. The nucleotide 5′-monophosphate is revealed after a chemical cyclization step which is followed by the removal of the amino acid by either phosphoramidase or histidine triad nucleotide-binding protein 1 (HINT1)171

(Figure 47).

O O

NH NH

O N O O N O O P O hydrolysis O P O NH O (Cathepsin A, NH O other esterases)

O O O O A B O

NH attack 2 spontaneous O N O ring H2O cyclization O P O opening O NH O attack 1

H2O C O O

NH NH

O N O phosphoramidase O N O O P O -type enzyme HO P O NH O OH O

O O D d4T-monophosphate

Figure 45: Postulated mechanism of phosphoramidate activation. [Reprinted with permission from ref. 170] 81

These phosphoramidate derivatives demonstrated improved activity against HCV.170 One example is 2ʹ-C-methyl guanosine phosphoramidate 111 (Figure 48) which is 20 fold more active than parent compound 112.146 Hence we will synthesize phosphoramidate derivatives of our analogs.

O O

N NH N NH O N N NH N N NH O P O 2 HO 2 NH O O

OH OH OH OH O O

111 112

Figure 46: Structures of compounds 111 and 112.

Since the enzymatic hydrolysis of the ester group of the amino acid is known to be the first step in the conversion of the phosphoramidate derivatives to the parent compounds, four phosphoramidate derivatives with various ester groups will be prepared.144 The phosphoramidate derivatives will be prepared following literature methods starting from modified nucleosides.141, 144, 172 The required phosphoramidates will be produced by reacting each of the modified nucleosides in the presence of N-methylimidazole with phosphorochloridates 113-116 (Table 3), which will be prepared by reacting the requisite

L-alanine esters with phenyl phosphorous dichloride (Scheme 19).

Table 3: Compounds 118-121.

Compound R1 R2 R3 R4

113 Ph Me H Me

114 Ph Me H i-Pr

115 Ph Me H i-Bu

116 Ph Me H Bn

O R O R R 4 3 2 Cl P Cl O O NH2.HCl R1

DCM o -78 C-rt Et3N 16hr O O R4 O R3 R2 O NH NH O HN P Cl O N O O R1 N O HO R1 O P O O 113-116 R O 2 O R R3 R OH OH OH OH O R4

Scheme 19: Synthesis of phosphoramidates.

Chapter 5

5 Experimental

5.1 General methods

Unless otherwise mentioned, reactions were done under standard laboratory conditions.

Chemical Reagents and solvents were used as obtained from the following commercial suppliers without further purification: Sigma-Aldrich, Fisher Scientific, Acros Organics,

Chem-Impex International Inc., and Oakwood Chemical.

Deuterated solvents used for NMR experiments were obtained from Cambridge Isotope

Laboratories Inc.

Varian Innova 600 and Bruker Avance 600 NMR spectrometers were used to record 1H

13 NMR and C NMR experiments in CD3OD and CDCl3, reporting the chemical shifts as ppm relative to CD3OD and CDCl3 as internal standards. Coupling constants (J) were

reported in Hertz (Hz) and multiplicities as follows: s=singlet, d=doublet, t=triplet, q=quartet, dd=doublet of doublet, dt=doublet of triplet, and m=mutiplet.

An Esquire Ion Trap mass spectrometer was used for ESI-MS experiments in methanol as solvent. Masses were reported in positive ion mode, [M+H]+, [M+Na]+, [M+H+Na]+, and

[M+Na+MeOH]+ adducts were obtained.

Aluminum backed and glass backed silica gel plates were used for monitoring of the progress of reactions and flash chromatographic separations. An ultraviolet (UV) lamp at

254 nm was used to visualize thin layer chromatography (TLC) spots. the p-

Anisaldehyde dip was used to develop TLC spots utilizing the presence of carbonyl groups in our compounds to ease visualization.

Flash chromatography was done using regular glass chromatography columns.

SiliaFlash® G60 chromatographic silica was used to pack the chromatography columns.

Other equipment and devices used, including but are not limited to, a high vacuum pump

(Edwards RV3), the rotary evaporator (Heidolph Collegiate Brinkmann), and vortex mixer (Fisher scientific).

5.2 Synthesis of 2′-C-acetyluridine

5.2.1 Synthesis of α-D-1,3,5-tri-O-benzoyl-2-ketoribofuranose (95)

Ketone 95 was prepared according to previously published work173 with some modifications. α-D-1,3,5-tri-O-benzoylribofuranose (1 gram (g), 2.16 millimole (mmol)) 85

was added to a solution of DMP (1.1 g, 2.6 mmol) in (10 milliliter (mL)) of DCM at 0

°C. The mixture was allowed to warm to room temperature and stirred for 12 hours (hr).

The solvent was removed in vacuo and the residue triturated with diethyl ether (20 mL).

Following filtration through a pad of magnesium sulfate (MgSO4), the organic solvent was stirred with an equal volume of sodium thiosulfate pentahydrate (Na2S2O3.5H2O)

(12.5%) in saturated (sat.) sodium bicarbonate (NaHCO3) until the organic layer became clear (~10 min). The organic layer was separated, washed with brine, and dried over

MgSO4 prior to removing the solvent in vacuo. The resulted solid was stirred with

MgSO4 in DCM overnight. Ketone 95 (0.74g, 74% yield) was separated as a white powder. Spectral data were identical to published work.173

5.2.2 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)uridine (97)

To a solution of uridine (1.0 g, 4.1 mmol) in 10 mL of dry pyridine cooled to 0 °C

TIPDSCl2 (1.31 mL, 1.29 g, 4.1 mmol) was added dropwise. The reaction mixture was allowed to warm to room temperature and stirred for 12 hr. and evaporated to dryness.

The residue was dissolved in 50 mL of DCM, washed with sat. NaHCO3 (3 x 50 mL), dried over MgSO4 and evaporated to dryness. The residue was purified by column

chromatography using 5-70% ethyl acetate (EtOAc) in DCM as eluent to produce 1.69 g

(85%) of 97 as a white foam. Spectral data are identical with published values.156

5.2.3 Synthesis of 3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-ketouridine (98)

Compound 97 (1.0 g, 2.06 mmol) was added to a solution of DMP (1.13 g, 2.67 mmol) in

(10 mL) of DCM at 0°C. The mixture was allowed to warm to room temperature and stirred for 18 hr. The solvent was removed in vacuo and the residue triturated with diethyl ether (20 mL). Following filtration through a pad of MgSO4, the organic solvent was stirred with an equal volume of Na2S2O3.5H2O (12.5%) in sat. NaHCO3 until the organic layer became clear (~10 min). The organic layer was separated, washed with brine, and dried over MgSO4 prior to removing the solvent in vacuo. The resulted solid was stirred with MgSO4 in DCM overnight. Ketone 98 (0.8g, 80% yield) was separated as a white foam. Spectral data were identical to published work.174

5.2.4 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-O-(trimethylsilyl)-2′-

C-cyanouridine (99)

This procedure was modified from Giese et al.158 After 18-crown-6 (1.08 mg, 4.08 micromole (µmol), KCN (1.77 mg, 0.03 mmol), and ketone 98 (0.8 g, 1.65 mmol) were co-evaporated twice with DCM, the mixture was dissolved in 15 mL of dry DCM. To the solution was slowly added TMSCN (1.03 mL, 8.25 mmol) at room temperature. After

120 hr. of stirring, the reaction was quenched by the addition of brine 15 mL, and the mixture was extracted with DCM (3 x 15 mL). The combined organic phases were washed with brine and dried over MgSO4, and the solvent was evaporated. Flash chromatography (EtOAc/DCM, 5-70%) gave 0.31 g (32%) of 99 as a mixture of diastereoisomers. Spectral data were identical to publish data.175

5.3.5 Synthesis of 2′-C-acetyluridine (100)

Cyanohydrin 99 was co-evaporated with toluene. MeLi (1.7 mL, 2.66 mmol, 1.6M in diethyl ether) was added to a solution of cyanohydrin 99 (0.31 g, 0.53 mmol) in anhydrous THF (10 mL) at -78°C. The resulting solution was stirred for 5 hr. and quenched by the addition of water (10mL). The mixture was then allowed to warm to room temperature and extracted with EtOAc (3 x 10 mL). The combined organic layers were washed with brine. The EtOAc was removed in vacuo. The resulted imine intermediate was immediately subjected to hydrolysis by the addition of 2N HCl (20 mL). The solution was allowed to stir at room temperature for 48 hr., followed by neutralization with NaOH (20 mL). Finally, the solvent was removed in vacuo to give a crude off white solid. ESI-MS [M+Na]: C11H14N2O7Na calculated (calc.) 309.07, found

309.23.

5.3.6 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-deoxy-2′- ethylideneuridine (101)

Compound 101 was synthesized per the procedure described in Wang et. al.176 with some modifications. To a stirred suspension of EtPh3PBr (1.92 g, 5.17 mmol) in anhydrous ether (120 mL) at room temperature under argon was added a solution of NaHMDS (0.95 g, 2.58 mL, 5.17 mmol, 2M in ether). The resulting orange mixture was stirred at room

temperature for 6 hr and cooled to -10°C, then a solution of 98 (1.25 g, 2.58 mmol) in ether (5 mL) was added. The reaction mixture was stirred at -10 °C for 1 hr, then at 4°C for 48 hr, and then at room temperature for 24 hr. Sat. ammonium chloride (NH4Cl) solution (125 mL) was added to quench the reaction. The organic phase was washed with brine twice, and the combined aqueous phases were extracted with diethyl ether. The combined ether dried over Na2SO4 and concentrated. Chromatography on silica with 5-

70% EtOAc in DCM gave 0.68 g (53%) of 101 as a white foam. 1H NMR (600 MHz,

CDCl3) δ ppm 1.03 - 1.13 (m, 56 H) 1.70 (d, J=7.08 Hz, 3 H) 1.91 (d, J=7.03 Hz, 3 H)

3.60 (dt, J=8.67, 2.75 Hz, 1 H) 3.92 (d, J=8.30 Hz, 1 H) 4.05 - 4.10 (m, 4 H) 5.72 (d,

J=8.06 Hz, 1 H) 5.81 (q, J=7.24 Hz, 1 H) 5.91 (q, J=7.16 Hz, 1 H) 6.68 (s, 1 H) 7.17 (d,

J=8.06 Hz, 1 H) 8.17 (br. s., 2 H). 13C NMR (151 MHz, CDCL3) δ ppm 12.86 (s, 1 C)

13.02 (s, 1 C) 13.21 (s, 1 C) 13.29 (s, 1 C) 13.94 (s, 1 C) 17.02 (s, 1 C) 17.05 (s, 1 C)

17.21 (s, 1 C) 17.26 (s, 1 C) 17.34 (s, 1 C) 17.49 (s, 1 C) 17.54 (s, 1 C) 17.68 (s, 1 C)

62.49 (s, 1 C) 71.62 (s, 1 C) 84.79 (s, 1 C) 91.05 (s, 1 C) 102.98 (s, 1 C) 124.21 (s, 1 C)

126.85 (s, 1 C) 137.39 (s, 1 C) 140.55 (s, 1 C) 150.58 (s, 1 C) 162.71 (s, 1 C). ESI-MS

[M+Na+H]: C23H41N2NaO6Si2 calc. 520.24, found 520.00.

5.3.7 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-C-(1- hydroxyethyl)uridine (102)

Compound 101 (0.5 g, 1.01 mmol) was dissolved in a mixture of THF (3 mL), t-butanol

(3 mL), and water (1 mL); NMO (0.15 g, 1.26 mmol) and OsO4 (0.1 mL of a 2.5% w/v solution in t-butanol, 0.01 mmol) were added under ice cooling. The mixture was stirred for 120 hr at 4°C and then quenched with 1M aqueous sodium bisulfite (NaHSO3) (5 mL) and extracted with EtOAc (3 x 10). The organic layer was washed with brine, dried with

Na2SO4 and evaporated. Flash chromatography of the crude product on silica gel with 5-

70% EtOAc/DCM yielded (0.31 g) (58%) of the required compound 102 as a white foam.

1H NMR (600 MHz, CDCL3) δ ppm 1.03 - 1.13 (m, 75 H) 1.23 (d, J=6.59 Hz, 3 H) 1.28

(d, J=6.59 Hz, 3 H) 1.41 (d, J=6.59 Hz, 1 H) 1.45 (d, J=6.59 Hz, 1 H) 3.71 (q, J=6.51 Hz,

1 H) 3.93 - 4.11 (m, 5 H) 4.17 - 4.23 (m, 2 H) 4.64 (d, J=8.30 Hz, 1 H) 5.66 (dd, J=8.30,

1.71 Hz, 1 H) 5.69 (dd, J=8.18, 2.08 Hz, 1 H) 5.98 (s, 2 H) 6.04 (s, 1 H) 6.09 (s, 1 H)

7.73 (d, J=8.30 Hz, 1 H) 7.82 (d, J=8.55 Hz, 1 H) 7.86 (d, J=8.06 Hz, 1 H) 8.71 (br. s., 1

H) 8.81 (br. s., 1 H). 13C NMR (151 MHz, CDCL3) δ ppm 13.18 (s, 1 C) 13.20 (s, 1 C)

13.21 (s, 1 C) 13.30 (s, 1 C) 17.35 (s, 1 C) 17.38 (s, 1 C) 17.41 (s, 1 C) 17.42 (s, 1 C)

17.43 (s, 1 C) 17.46 (s, 1 C) 18.59 (s, 1 C) 60.64 (s, 1 C) 67.95 (s, 1 C) 68.36 (s, 1 C)

69.30 (s, 1 C) 81.39 (s, 1 C) 81.92 (s, 1 C) 86.29 (s, 1 C) 90.79 (s, 1 C) 101.65 (s, 1 C)

102.14 (s, 1 C) 141.99 (s, 1 C) 150.92 (s, 1 C) 151.01 (s, 1 C) 163.03 (s, 1 C) 163.30 (s, 1

C). ESI-MS [M+Na]: C23H42N2NaO8Si2 calc. 553.24, found 553.10.

5.3.8 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-C-acetyluridine (103)

Compound 102 (0.3 g, 0.57 mmol) was added to a solution of DMP (0.31 g, 0.74 mmol) in (10 mL) of DCM at 0°C. The mixture was allowed to warm to room temperature and stirred for 18 hr. The solvent was removed in vacuo and the residue triturated with diethyl ether (20 mL). Following filtration through a pad of MgSO4, the organic solvent was stirred with an equal volume of Na2S2O3.5H2O (12.5%) in sat. NaHCO3 until the organic layer became clear (~10 min). The organic layer was separated, washed with brine, and dried over MgSO4 prior to removing the solvent in vacuo. The resulted solid was stirred with MgSO4 in DCM overnight. Ketone 103 (0.18g, 60% yield) was obtained as a white foam. 1H NMR (600 MHz, CD3OD) δ ppm 0.88 - 1.17 (m, 28 H) 2.34 (s, 3 H) 4.05 (dd,

J=13.75, 2.38 Hz, 1 H) 4.10 (q, J=6.97 Hz, 1 H) 4.15 (dd, J=9.54, 1.83 Hz, 1 H) 4.26 (d,

J=13.57 Hz, 1 H) 5.62 (d, J=8.44 Hz, 1 H) 5.69 (s, 1 H) 7.91 (d, J=8.07 Hz, 1 H). 13C

NMR (151 MHz, CDCL3) δ ppm 12.59 (s, 1 C) 12.80 (s, 1 C) 13.17 (s, 1 C) 13.79 (s, 1

C) 16.97 (s, 1 C) 17.03 (s, 1 C) 17.24 (s, 1 C) 17.34 (s, 1 C) 17.43 (s, 1 C) 17.46 (s, 1 C)

17.49 (s, 1 C) 17.62 (s, 1 C) 28.34 (s, 1 C) 59.67 (s, 1 C) 72.34 (s, 1 C) 81.55 (s, 1 C)

86.75 (s, 1 C) 90.11 (s, 1 C) 102.03 (s, 1 C) 139.96 (s, 1 C) 150.19 (s, 1 C) 162.85 (s, 1

C) 209.75 (s, 1 C). ESI-MS [M+Na+H]: C23H41N2NaO8Si2 calc. 552.23, found 552.00.

5.3.9 Synthesis of 2′-C-acetyluridine (100)

+ - A solution of 103 (61.54 mg, 0.12 mmol) and NH 4F (43.12 mg, 1.17 mmol) in reagent grade MeOH (10 mL) was stirred at room temperature for 48 hr. Silica gel was added, the mixture evaporated, and the dry powder added onto a silica column. The column was eluted (0-10%) MeOH/EtOAc and appropriately pooled fractions were combined and evaporated. The white foam residue was dried to give compound 100 (30 mg, 90% yield).

1H NMR (600 MHz, CD3OD) δ ppm 2.30 (s, 3 H) 3.78 (dd, J=12.50, 2.20 Hz, 1 H) 3.98

(dt, J=12.50, 9.20 Hz, 1 H) 4.00 (dd, J=12.50, 2.20 Hz, 1 H) 4.53 (d, J=9.20 Hz, 1 H)

5.64 (d, J=8.07 Hz, 1 H) 5.77 (s, 1 H) 8.23 (d, J=8.07 Hz, 1 H). 13C NMR (151 MHz,

CD3OD) δ ppm 20.36 (s, 1 C) 28.42 (s, 1 C) 60.00 (s, 1 C) 61.58 (s, 1 C) 73.45 (s, 1 C)

83.42 (s, 1 C) 88.34 (s, 1 C) 92.29 (s, 1 C) 101.43 (s, 1 C) 142.69 (s, 1 C) 152.11 (s, 1 C)

166.24 (s, 1 C) 212.54 (s, 1 C). ESI-MS [M+H]: C11H15N2O7 calc. 287.09, found 287.10. 93

5.4 Synthesis of 2′-C-(1-hydroxyethyl)uridine (104)

+ - A solution of 102 (20.45 mg, 0.04 mmol) and NH 4F (14.27 mg, 0.39 mmol) in reagent grade MeOH (5 mL) was stirred at room temperature for 48 hr. Silica gel was added, the mixture evaporated, and the dry powder added onto a silica column. The column was eluted (0-10%) MeOH/EtOAc and appropriately pooled fractions were combined and evaporated. The white foam residue was dried to give compound 104 (10 mg, 90% yield).

1 H NMR (600 MHz, CD3OD) δ ppm 1.20 (s, 3 H) 1.21 (s, 3 H) 1.23 (s, 3 H) 1.24 (s, 3 H)

3.56 - 3.98 (m, 20 H) 5.61 (d, J=8.07 Hz, 1 H) 5.65 (d, J=8.07 Hz, 1 H) 5.69 (d, J=8.44

Hz, 1 H) 5.76 (s, 1 H) 5.97 (s, 1 H) 6.31 (s, 1 H) 7.85 (d, J=8.07 Hz, 1 H) 7.98 (d, J=8.44

Hz, 1 H). 13C NMR (151 MHz, CD3OD) δ ppm 13.16 (s, 1 C) 17.61 (s, 1 C) 18.54 (s, 1

C) 37.32 (s, 1 C) 62.51 (s, 1 C) 63.96 (s, 1 C) 67.15 (s, 1 C) 68.97 (s, 1 C) 69.66 (s, 1 C)

70.90 (s, 1 C) 73.45 (s, 1 C) 75.05 (s, 1 C) 79.19 (s, 1 C) 82.07 (s, 1 C) 83.20 (s, 1 C)

83.67 (s, 1 C) 85.54 (s, 1 C) 101.21 (s, 1 C) 101.87 (s, 1 C) 153.88 (s, 1 C) 166.35 (s, 1

C) 169.27 (s, 1 C) . ESI-MS [M+Na]: C11H16N2NaO7 calc. 311.09, found 311.90.

5.5 Synthesis of 2′-C-formyluridine

5.5.1 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-deoxy-2′-C- methyleneuridine (105)

Compound 105 was synthesized per the procedure described in Wang et. al.176 with some modifications. To a stirred suspension of MePh3PBr (0.12 g, 0.34 mmol) in anhydrous ether (8 mL) at room temperature under argon was added a solution of NaHMDS (0.2 mL, 0.34 mmol, 2M in ether). The resulting orange mixture was stirred at room temperature for 6 hr and cooled to -10°C, then a solution of 102 (83.33 mg, 0.17 mmol) in ether (4 mL) was added. The reaction mixture was stirred at -10°C for 1 hr, then at 4°C for 48 hr, and then at room temperature for 24 hr. Sat. NH4Cl solution (12 mL) was added to quench the reaction. The organic phase was washed with brine twice, and the combined aqueous phases were extracted with diethyl ether. The combined ether dried over Na2SO4 and concentrated. Chromatography on silica with 5-70% EtOAc in DCM gave 50 mg (60% yield) of 105 as a white foam. Spectral data comply with the previously published data.177

5.5.2 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-C- hydroxymethyluridine (106)

Compound 105 (50 mg, 0.1 mmol) was dissolved in a mixture of THF (1 mL), t-butanol

(1 mL), and water (0.1 mL); NMO (14.56 mg, 0.12 mmol) and OsO4 (10 µL of a 2.5% w/v solution in t-butanol, 0.001 mmol) were added under ice cooling. The mixture was stirred for 120 hr at 4°C and then quenched with 1M aqueous sodium bisulfite (NaHSO3)

(2 mL) and extracted with EtOAc (3 x 5). The organic layer was washed with brine, dried with Na2SO4 and evaporated. Flash chromatography of the crude product on silica gel with 5-70% EtOAc/DCM yielded (21.41 mg) (40% yield) of the required compound 106 as a white foam. Spectral data comply with the published values for 106.178

5.5.4 Synthesis of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-C-formyluridine

(107)

Compound 106 (20 mg, 0.04 mmol) was added to a solution of DMP (21.36 mg, 0.05 mmol) in (5 mL) of DCM at 0°C. The mixture was allowed to warm to room temperature and stirred for 18 hr. The solvent was removed in vacuo and the residue triturated with diethyl ether (10 mL). Following filtration through a pad of MgSO4, the organic solvent was stirred with an equal volume of Na2S2O3.5H2O (12.5%) in sat. NaHCO3 until the organic layer became clear (~10 min). The organic layer was separated, washed with brine, and dried over MgSO4 prior to removing the solvent in vacuo. The resulted solid was stirred with MgSO4 in DCM overnight. Ketone 107 (11.95 mg, 60% yield) was obtained as a white foam. 1H NMR (600 MHz, CD3OD) δ ppm 1.06 - 1.17 (m, 28 H)

4.06 (dd, J=13.76, 2.38 Hz, 1 H) 4.22 (dd, J=9.54, 2.20 Hz, 1 H) 4.28 (d, J=13.57 Hz, 1

H) 4.58 (d, J=9.54 Hz, 1 H) 5.59 (s, 1 H) 5.65 (d, J=8.44 Hz, 1 H) 7.95 (d, J=8.07 Hz, 1

H) 9.58 (s, 1 H). 13C NMR (151 MHz, CD3OD) δ ppm 13.57 (s, 1 C) 13.89 (s, 1 C) 14.22

(s, 1 C) 14.71 (s, 1 C) 17.13 (s, 1 C) 17.43 (s, 1 C) 17.53 (s, 1 C) 17.63 (s, 1 C) 17.71 (s,

1 C) 17.85 (s, 1 C) 17.88 (s, 1 C) 17.95 (s, 1 C) 60.57 (s, 1 C) 72.33 (s, 1 C) 82.76 (s, 1

C) 87.35 (s, 1 C) 92.11 (s, 1 C) 102.07 (s, 1 C) 141.01 (s, 1 C) 151.81 (s, 1 C) 165.91 (s,

1 C) 202.86 (s, 1 C). ESI-MS [M+Na]: C22H38N2NaO8Si2 calc. 537.21, found 537.00.

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178. Gallo, M.; Monteagudo, E.; Cicero, D. O.; Torres, H. N.; Iribarren, A. M., 2′- C -

Methyluridine phosphoramidite: a new building block for the preparation of RNA analogues carrying the 2′-hydroxyl grou. Tetrahedron 2001, 57 (27), 5707-5713.

128

Appendix A

Supplemental information

Mass spectra and NMR spectra.

129

Figure 47: H-NMR of compound 101. 130

BSA-Uri-006-pure.C13.esp 0.060

0.055

0.050

0.045

0.040

0.035 17.260 0.030

0.025 17.493 13.206 Normalized Intensity Normalized

0.020 17.537 17.675 0.015 13.016 12.863 140.547 102.975

0.010 84.791 71.623 162.712 62.494 126.854 124.215 150.580 137.390

0.005 91.054

220 200 180 160 140 120 100 80 60 40 20 0 -20 Chemical Shift (ppm)

Figure 48: C-13 NMR of compound 101. 131

Figure 49: ESI-MS of compound 101. 132

BSA-Uri-007-6-20-16-FD 1.100 1.0 1.106 1.108 0.9

0.8 1.089 1.056 0.7 1.049

0.6 1.115

0.5

0.4 Normalized Intensity

0.3 1.224 1.235 1.274 1.285 4.081 0.2 1.033 5.978 7.725 4.060 1.026 7.739 4.069 4.189 1.015 4.056 3.993 4.633 1.021 4.198 4.647 3.701 3.712 5.669 4.208 5.666

0.1 5.685 5.655 5.695 8.813 8.710 6.040 1.404 1.453 1.442 6.089 7.868 7.855 7.817 7.831 0 0.89 0.89 0.12 0.07 0.99 0.11 0.17 1.190.92 1.10 0.911.935.41 0.89 0.26 0.25 3.02 3.04 64.60

8 7 6 5 4 3 2 1 0 Chemical Shift (ppm)

Figure 50: H-NMR of compound 102. 133

BSA-Uri-007-6-20-16-C13-FD.001.esp 0.12

0.11

0.10

0.09

0.08

0.07

0.06 17.435

0.05 Normalized Intensity Normalized

0.04 17.537

0.03 17.384 13.199 13.301 18.594

0.02 81.393 102.137 101.648 81.918 163.033 141.991 68.357 163.303 151.010 67.948 60.642 90.792 0.01 69.304 86.293

220 200 180 160 140 120 100 80 60 40 20 0 -20 Chemical Shift (ppm)

Figure 51: C-13 NMR of compound 102. 134

Intens. All, 0.0-1.2min (#1-#106) x107

553.1

2 571.1

587.1 605.1 539.1 441.1

0 300 350 400 450 500 550 600 650 m/z Figure 52: ESI-MS of compound 102.

135

BSA-Uri-008-11-24-16-MeOH-1H.001.esp Methanol Methanol 2.338 0.12

0.11

0.10

0.09

0.08 1.141 1.070

0.07 1.145

0.06 0.999 0.892

0.05 Normalized Intensity 5.689 1.153 0.04 1.158

0.03 7.901 7.915 5.611 5.625 2.350 0.02 4.247 4.269 4.062 4.067 4.155 4.043 4.139 4.040

0.01 6.245 7.612 7.625

0 1.000.10 0.09 0.970.17 1.07 1.03 1.06 0.46 1.16 0.30 2.86 29.46

8 7 6 5 4 3 2 1 0 Chemical Shift (ppm)

Figure 53: H-NMR of compound 103. 136

BSA-Uri-008-9-1-16.001.esp CDCL3

0.25

0.20

0.15 Normalized Intensity Normalized 0.10 17.427 17.624 13.789 17.026 12.593 0.05 17.238 86.752 102.035 81.554 59.673 72.338 90.106 28.335 139.956 162.851 150.193 209.748

220 200 180 160 140 120 100 80 60 40 20 0 -20 Chemical Shift (ppm)

Figure 54: C-13 NMR of compound 103. 137

x107 All, 0.0-1.2min (#1-#106)

552.0

2.5

2.0

1.5

1.0

0.5

490.0

573.1

587.1

0.0 400 450 500 550 600 650 700 750 m/z

Figure 55: ESI-MS of compound 103.

138

BSA-Uri-004-4-15-17-A.003.esp CD3OD

0.45 2.301

0.40

0.35

0.30

0.25

0.20 Normalized Intensity Normalized 0.15 5.774 2.115

0.10 8.224 8.238 5.643 5.629 4.523 4.538 3.989 3.968 3.785 4.010 3.768 3.983 0.05 3.765 4.013

1.00 1.01 0.99 0.99 1.06 1.12 1.07 3.06 0.63

8 7 6 5 4 3 2 1 0 Chemical Shift (ppm)

Figure 56: H-NMR of compound 100. 139

BSA-Uri-004-4-15-17-A.001.esp CD3OD

0.030

0.025

0.020 28.424

0.015 Normalized Intensity Normalized 73.447

0.010 142.692 92.288 101.431 59.995 212.542 83.422 88.336

0.005 152.105 20.360 166.243 61.585

200 180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)

Figure 57: C-13 NMR of compound 100. 140

Intens. All, 0.0-0.5min (#1-#46) x106

287.1

363.1 267.1

346.9 329.0 303.0 371.0 165.4 253.2 313.0 357.0 385.0 283.0 375.0 338.9 181.4 242.4 325.0 308.9 317.0 353.0 217.2 223.2 246.1 295.0 139.7 231.2

0 100 150 200 250 300 350 m/z

Figure 58: ESI-MS of compound 100.

141

BSA-Uri-4-4-17-AP-1.003.esp Methanol Methanol 1.225 1.214

0.060

0.055

0.050 1.237 0.045

0.040 1.204 0.035

0.030

Normalized Intensity Normalized 0.025

0.020 3.779 5.617 5.644 5.657 3.576 3.858 5.603 0.015 3.569 3.769 3.947 3.869 3.951 7.968 0.010 7.982 3.619 5.970 5.684 3.968 6.309 3.972 7.844 7.858 0.005 5.997

0 0.97 0.61 0.15 0.84 0.10 0.41 1.48 1.04 18.09 11.75

8 7 6 5 4 3 2 1 0 Chemical Shift (ppm)

Figure 59: H-NMR of compound 104. 142

BSA-Uri-11-4-4-17-AP-1.13C.esp CD3OD

0.008

0.007

0.006

0.005

0.004 Normalized Intensity Normalized 0.003

0.002 17.611 69.656 70.903 37.319 85.544 79.186 73.455 62.511 18.545 13.156 75.052 101.212 0.001 101.869 166.352 63.962 153.884 169.269

220 200 180 160 140 120 100 80 60 40 20 0 -20 Chemical Shift (ppm)

Figure 60: C-13 NMR of compound 104. 143

Intens. +MS, 0.0min (#1) x106

1.50

311.9

1.25

1.00

0.75

0.50

301.0

0.25

279.2

242.4

330.9 199.4

0.00 100 150 200 250 300 350 400 450 m/z

Figure 61: ESI-MS of compound 104.

144

BSA-Uri-010-5-6-17.003.espO CD3OD NH 1.137

N O 0.35 O Si O O O

0.30 Si O OH 1.130 1.147 1.068 0.25 1.002 1.149 9.575 0.20 5.594

0.15 Normalized Intensity 7.939 5.652 7.952

0.10 4.573 4.589 4.265 4.287 4.073 4.076 4.054 4.222

0.05 0.998 6.242 9.767 5.664 5.678 7.614 7.627

0.16 1.01 1.16 0.24 0.180.311.10 0.95 1.22 1.33 1.26 1.46 28.55

9 8 7 6 5 4 3 2 1 Chemical Shift (ppm)

Figure 62: H-NMR of compound 107. 145

BSA-Uri-010-5-6-17.13C.esp CD3OD

0.060

0.055

0.050

0.045

0.040

0.035

0.030 Normalized Intensity Normalized 0.025

0.020 17.852 17.429 0.015 17.626 17.130 14.709 87.352

0.010 82.758 102.066 17.881 60.571 13.893 72.332 141.008 92.106 165.915 202.859 0.005 151.814

200 180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)

Figure 63: C-13 NMR of compound 107. 146

x106 All, 0.0-1.3min (#1-#73) O 2.0 NH

N O 569.2 O Si O O O 1.5 Si O OH

[M+Na]+=537 [M+Na+MeOH]+=569

1.0

537.0

0.5

361.0 646.1

0.0 300 350 400 450 500 550 600 650 m/z

Figure 64: ESI-MS of compound 107.

147