Hydroxymethyl Cytidine As a Potential Inhibitor for Hepatitis C Virus

Home , Cytidine, Miravirsen

A Thesis

entitled

Synthesis of 2’- Hydroxymethyl Cytidine as a Potential Inhibitor for Hepatitis C Virus

Polymerase Enzyme

Ali Hayder Hamzah

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

Master of Science Degree in

Medicinal Chemistry

______Dr. Amanda C. Bryant-Friedrich, Committee Chair

______Dr. Hermann Von Grafenstein, Committee Member

______Dr. Caren L. Steinmiller, Committee Member

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

The University of Toledo

August 2016

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 2’- Hydroxymethyl Cytidine as a Potential Inhibitor for Hepatitis C Virus Polymerase Enzyme

Ali Hayder Hamzah

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Medicinal Chemistry

The University of Toledo

August 2016

Hepatitis C virus infection (HCV) is a major cause of liver disease. Due to the asymptomatic nature of the infection, large populations are unware of their infection and become carriers, with progression to chronic stage including liver cirrhosis and hepatocellular carcinoma.

Currently, there are seven genotypes and several subtypes of HCV; genotype 1 is the most global distributed form, acquired predominantly through illegal intravenous drug injection. HCV heterogeneity and high replication rates, lead to mutant formation and consequent reinfection. The diseases’ lack of susceptibility to antiviral agents facilitates chronic infection and as such is most challenging when searching for a cure.

For decades, the standard of care (SOC) was a combination of PEGylated interferon-α

(PEGINF-α) and ribavirin. A Sustained viral response (SVR) was achieved in only a low percentage and was limited to some genotypes and associated with serious adverse effects. It is therefore urgent to develop compounds which have pan-genotype activity, and have increased bioavailability and improved safety profiles.

iii

A better understanding of the viral life cycle and the determination of the crystal structure of HCV NS5B polymerase enzyme, led to identification of multiple points of intervention to disrupt viral protein synthesis and to interrupt the viral life cycle. Many development stages for these drugs were halted because of either low barrier to resistance or high toxicity.

The catalytic site of HCV polymerase is the most conserved motif among HCV and other polymerases and is responsible for HCV RNA replication. It is considered the primary focus in the effort to synthesize molecules targeting the inhibition of HCV life cycle.

In our project, we developed a new synthetic pathway toward synthesis of hydroxy methyl cytidine as a potential substrate of the enzyme with an inhibitory effect through working as a chain terminator for nucleotide polymerization.

Dedicated to my wife Sura and to my father

Acknowledgements

I would like to express my gratitude to my advisor Dr. Amanda C. Bryant

Friedrich for providing me an opportunity to be a part of her research group and for all of her support throughout the two years of graduate study. This work would not have been possible without her guidance, knowledge, and personal experience.

Sincere gratitude to all professors in the College of Pharmacy and Pharmaceutical

Sciences and the department of Medicinal and Biological Chemistry for their unlimited continuous support.

Special thanks to my committee members Dr. Caren L. Steinmiller and Dr.

Hermann Von Grafenstein.

Thank you to all my lab members for their support and for sharing their chemistry knowledge and experience during my research work, especially Bader.Alabdullah for his support, encouragement and help me with my research. Also I would like to thank Dr.

Fernand Mel Bedi who supported me during the research and writing.

To my friends Salam AL Maliki and Hassan Al Hadad, thank you for everything.

A great thanks to my sponsor the Higher Committee for Education Development in Iraq (HCED) for their financial support during my study.

Finally, to my family and my life, my wife Sura and my kids, I cannot do anything without their unlimited support.

Table of Contents

Abstract ...... iii

Acknowledgements ...... vi

Table of Contents ...... vii

List of Tables ...... xii

List of Figures ...... xiii

List of Schemes…………………………………………………………………………..xv

List of Abbreviations ...... xvi

List of Symbols ...... xix

1 Introduction… …………………………………………………………………….1

1.1 Hepatitis C Virus Epidemiology ...... 1

1.1.1 Prevalence and Incidence of Hepatitis C Virus (HCV) ...... 2

1.1.2 Mode of Transmission for HCV ...... 3

1.1.3 HCV Genotype Distribution……………………………………...…4

1.2 HCV Structure………………………………………………………………..6

1.2.1 Viral Genome Organization………………………………………6

1.2.2 HCV Proteins……………………………………………………..8

1.2.2.1 Structural Proteins………………………………………...9

1.2.2.2 Non-Structural Proteins…………………………………11

1.3 HCV Life Cycle……………………………………………………………...13 vii

1.3.1 HCV Attachment, Entry and Fusion…………………………….13

1.3.2 RNA Translation and Post-Translation Processing……………..16

1.3.3 HCV RNA Replication………………………………………….17

1.3.4 HCV Assembly and Release…………………………………….18

1.3.5 HCV Circulating Forms…………………………………………19

1.4 HCV Pathogenesis………………………………………………………….20

1.4.1 HCV Induced Apoptosis………………………………………..20

1.5 HCV Progression and Complication ……………………………………...22

1.6 HCV Treatment…………………………………………………………….24

1.6.1 Indirectly Acting Antiviral Agents…………………………….24

1.6.2 Direct Acting Antiviral Agents (DAAs)……………………….26

2 Background……………… ...... 29

2.1 The Structural Feature of HCV NS5B RdRp Protein………………………..29

2.2 HCV NS5B RdRp Activities and the Mode of Action………………………31

2.3 The HCV NS5B RdRp as a Potential Drug Target…………………………..35

2.4 Inhibitors of HCV NS5B Polymerase………………………………………..36

2.4.1 Nucleoside and Nucleotide inhibitors (NIs)………………….….36

2.4.1.1 Sugar Modified NIs………………………………………37

2.4.1.2 Base Modified NIs………………...... 42

2.4.2 Non-nucleoside Inhibitors of NS5B (NNIs) ………………………………..42

3 Results and Discussion…………………………………………………………45

viii

3.1 Overview on the Synthesis of Nucleosides Analogs…………..…………45

3.2 Design and Synthesis of 2-C-hydroxymethyl Ribose…………………….46

3.2.1 Synthesis of 5-((benzoyloxy) methyl)-3-oxotetrahydrofuran-2, 4- diyl dibenzoate (19)……………………………………………………………………..47

3.2.2 Synthesis of 5-((benzoyloxy) methyl)-3- methylenetetrahydrofuran-2, 4-diyl dibenzoate (20)……………………………………48

3.2.3 Synthesis of 5-((benzoyloxy) methyl)-3-hydroxy-3-

(hydroxymethyl) tetrahydrofuran-2, 4-diyl dibenzoate (21)…………………………….51

3.3 Synthesis of Glycosyl Donor……………………………………………….52

3.3.1 Synthesis of 3-acetoxy-3-(acetoxymethyl)-5-((benzoyloxy) methyl) tetrahydrofuran-2, 4-diyl dibenzoate (32)………………………………………52

3.3.2 Synthesis of 5-((benzoyloxy) methyl)-3-(((tert butyldimethylsilyl) oxy) methyl)-3-hydroxytetrahydrofuran-2, 4-diyl dibenzoate 2133……………………55

3.3.3 Synthesis of 3-acetoxy-5-((benzoyloxy) methyl)-3-(((tert butyldimethylsilyl) oxy) methyl) tetrahydrofuran-2, 4-diyl dibenzoate (34)……………57

3.4 Synthesis of Hydroxy Methyl Cytidine…………………………………....57

3.4.1 Synthesis of 5-(4-acetamido-2-oxopyrimidin-1(2H)-yl)-4- acetoxy-4-(acetoxymethyl)-2-((benzoyloxy) methyl) tetrahydrofuran-3-yl benzoate

(43a)…………………………………………………………………………………….59

4 Conclusions and Future Work…………………………………………………64

4.1 Conclusions ……………………………………………………………….64

4.2 Future Work ……………………………………………………………….65

5 Experimental procedures ……………………………………………………...67

5.1 Materials…………………………………………………………………...67

5.2 Structural Analysis………………………………………………………..67

5.2.1 NMR Analysis…………………………………………...…67

5.2.1.1 1H-NMR…………………………………….…….67

5.2.1.2 13C-NMR…………………………………………69

5.2.2 Mass Spectrometry………………………………………….69

5.2.2.1 ESI-MS……………………………………………69

5.2.2.2 High Resolution Mass Spectroscopy……………………..69

5.3 Chromatographic Methods……………………………………………………..69

5.3.1 Thin Layer Chromatography (TLC)……………………………..69

5.3.2 Flash Chromatography…………………………………………..70

5.4 Other Equipment and devices………………………………………………….70

5.5 Synthesis of 4-amino-1-(3, 4-dihydroxy-3, 5-bis (hydroxymethyl) tetrahydrofuran-2-yl) pyrimidin-2(1H)-one……………………………………………..71

5.5.1 5-((benzoyloxy) methyl)-3-oxotetrahydrofuran-2, 4-diyl dibenzoate

(19)……………………………………………………………………………………….71

5.5.2 5-((benzoyloxy) methyl)-3-methylenetetrahydrofuran- 2, 4-diyl dibenzoate 20…………………………………………………………………………….71

5.5.3 5-((benzoyloxy) methyl)-3-hydroxy-(hydroxymethyl) tetrahydrofuran-2, 4-diyl dibenzoate 21...... …...72

5.5.4 3-acetoxy-3-(acetoxymethyl)-5-((benzoyloxy) methyl) tetrahydrofuran-2, 4-diyl dibenzoate 32…………………………………………………73

5.5.5 Synthesis of 5-((benzoyloxy) methyl)-3-(((tert butyldimethylsilyl) oxy) methyl)-3-hydroxytetrahydrofuran-2,4-diyl dibenzoate (33)……………………..73

5.5.6 Synthesis of 3-acetoxy-5-((benzoyloxy) methyl)-3-(((tert- butyldimethylsilyl) oxy) methyl) tetrahydrofuran-2, 4-diyl dibenzoate (34)…………. 74

5.5.7 Synthesis of 5-(4-acetamido-2-oxopyrimidin-1(2H)-yl)-4-acetoxy-4-

(acetoxymethyl)-2-((benzoyloxy) methyl) tetrahydrofuran-3-yl benzoate (43a)……….75

References ...... 76

A Supplemental Information ...... 95

List of Tables

1 NNIs and their Corresponding Binding Sites ...... 44

xii

List of Figures

1 HCV Genotype Distribution ...... 6

2 HCV Particle ……………………………………………………………………...7

3 HCV Proteins ...... 9

4 HCV Life Cycle ...... 19

5 NS3/4A Protease Inhibitors……………………………………………………...27

6 Crystal Structure of HCV NS5B Polymerase ...... 31

7 The Mechanism of Polymerase Enzyme...... 34

8 Cytidine Derivatives with Ribose Modifications………………………………...39

9 Distereomers of Acetylated Diol ……………………………………………….55

10 MS/MS of Compound 43a………………………………………………………60

11 Numbering System and Hydrogen Atoms A: Sugar, B: Nucleoside……………68

12 H-NMR of Compound 20………………………………………………….…....96

13 ESI-MS of Compound 20………………………………………………….....97

14 HRMS of Compound 20………………………………………………………98

15 C-13 NMR of Compound 20………………………………………………….99

16 C-13 NMR of Compound 21…………………………………………………100

17 ESI-MS of Compound 21……………………………………………………..101

18 HRMS of Compound 21………………………………………………………102

19 C-13 NMR of Compound 32……………………………………...………..103 xiii

20 ESI-MS of Compound 32…………………………………………………….104

21 HRMS of Compound 32…………………………………………………….105

22 C-13 NMR of Compound 33…………………………………………………106

23 HRMS of Compound 33………………………………………………………..107

24 H-NMR of Compound 33…………………………………………………….108

25 C-13 NMR of Compound 34…………………………………………………109

26 H-NMR of Compound 34…………………………………………………….110

27 ESI-MS of Compound 34…………………………………………………….111

28 H-NMR for Compound 43a…………………………………………………..112

29 C-13 NMR of Compound 43a…………………………………………………..113

30 HRMS of Compound 43a……………………………………………………114

31 ESI-MS of Compound 43a……………………………………………………..115

32 C-13 NMR of Compound 44………………………………………………...116

33 H-NMR of Compound 44……………………………………………………117

34 ESI-MS of Compound 44……………………………………………………118

35 C-13 NMR of Compound 45………………………………………………...119

36 ESI-MS of Compound 45……………………………………………………120

37 HNMR for Compound 45……………………………………………………121

xiv

List of Schemes

Scheme 1 Synthesis of 2-C-Hydroxymethyl Ribose……………………………47

Scheme 2 Lombardo Reagent Formation……………………………………....49

Scheme 3 Wittig Reaction…………………………………………...... 50

Scheme 4 Dihydroxylation with Osmium Tetroxide……………………………52

Scheme 5 Synthesis of Glycosyl Donor………………………………………..53

Scheme 6 Acetylation Reaction………………………………………………...54

Scheme 7 Silylation Reaction with TBDMSCl…………………………………56

Scheme 8 Synthesis of Hydroxymethyl Cytidine……………………………...58

Scheme 9 Glycosylation Reaction of Compound 43a…………………………..61

Scheme 10 Glycosylation Reaction of compound 43b…………………………...62

Scheme 11 Future Modifications of Hydroxymethyl Cytidine…………………66

List of Abbreviations

1DPI……………….. DNA polymerase I AA………………….. Amino acid ADA………... ………Adenosine deaminase ApoBandApoE……. Apolipoproteins B and E ARFP ………………Alternate reading frame protein Asp…………. ………Aspartic acid ATP………………… Adenosine triphosphate

C……………. ………Core CD81………. ………Cluster of Differentiation 81 CD95……………….. Cluster of differentiation 95 CIDE-B…………….. Cell death-inducing DFFA-like effector B CLDN1……………... Claudin-1 CTP………………… Cytidine triphosphate

D……………………. Distinct domain DAAs………. ………Direct acting antiviral agents DFF45……… ………DNA-fragmentation-factor DMP………...... Dess-martin per iodate DMSO……… ………Dimethyl sulfoxide DMVs ………………Double-membrane vesicles DNA……………….. Deoxyribonucleic acid

E. coli………………. Escherichia coli EHM……………….. Extra hepatic manifestations ER………………….. Endoplasmic reticulum

F……………………. Frame shift F protein FDA………………... Food and Drug Administration G……………………. Genotype Gly…………………. Glutamic acid GT………………….. Genotype GTP………………… Guanosine triphosphate

HBV………………... Hepatitis B virus HCC... ………………Hepato cellular carcinoma HH29………………. Hamburger and Hamilton's stage 29 HIV………………… Human immunodeficiency virus xvi

HIV RT…………….. Human immunodeficiency virus reverse transcriptase HSPG………………. Heparan sulfate protoglycan HTAs………………. Host-targeting antiviral agents HTS………………… High Throughput Screening HVR………………... Hyper variable region

I.V………………….. Intravenous IFN- α………. ………Interferon alpha IFN‑λ3…………….. Interferon-lambda-3 IL28B………………. Interleukin 28B INF- λ………………. Interferon lambda IRES………………... Internal ribosome entry site

JFH1……………….. Japanese fulminant hepatitis1

Kb………………….. Kilo base pair

LDL………………… Low density lipoprotein LDL-R……………… Low density lipoprotein receptor LDs…………………. Lipid droplets LEL………………… Large extracellular loop miR-122……………. Micro ribonucleic acid mRNA……………… Messenger ribonucleic acid

NBM……………….. Nucleotide binding motif NHANES…………... National Health and Nutrition Examination Survey NIs…………………. Nucleotide inhibitors NLS………………… Nuclear localization signal NM…………………. Nanometer NMO……………….. N-methylmorpholine N-oxide NNIs……………….. Non Nucleotide inhibitors NS2………………… Non-structural protein 2 NS3………………… Non-structural protein3 NS4A………………. Non-structural protein 4A NS4B……………….. Non-structural protein 4B NS5A………………. Non-structural protein 5A NS5B………………. Non-structural protein 5B nt…………………… nucleotide NTPase……………... Nucleoside triphosphate hydrolase NTPi………………... Nucleotide triphosphate initial NTPs……………….. Nucleotide triphosphates

OCLN………………. Occludin ORF………………… Open reading frame P21…………………. Protein 21 xvii

P7…………………... Protein 7 PCC………………… Pyridinium chlorochromate PCR………………… polymerase chain reaction PEG-IFN α…………. PEGylated interferon alpha PI4KIII……………... Phosphatidyl-inositol-4-kinase-III PI4P………………… Phosphatidylinositol-4-phosphate PKR………………… Protein kinase RNA PNP………………… purine nucleoside phosphorylase

RdRp………………. RNA-dependent-RNA-polymerase RNA………………... Ribo nucleic acid rNTP……………….. Ribo nucleotide triphosphate

SEL………………… Small extracellular loop SNP………………… single nucleotide polymorphism SOC………………… Standard of care SR-BI………………. Scavenger receptor B type I SVR………………… Sustained virologic response

TGF-β………………. Transforming growth factor beta TNF-R1…………….. Tumor necrosis factor receptor 1 TNFα……………….. Tumor necrosis factor alpha TRAIL……………… Tumor necrosis factor related apoptosis inducing ligand

UTR………………... Untranslated region

VAP-A……………... Vesicle-associated membrane protein-associated protein A VAP-B……………... Vesicle-associated membrane protein-associated protein B

WHO………………. World health organization

xviii

List of Symbols

13C—NMR………… Carbon nuclear magnetic resonance 1H-NMR…………… Proton nuclear magnetic resonance AC2O………………. Acetic anhydride

C……………………. Cysteine CDCl3……………… Chloroform CH2Br2…………….. Dibromo methane CH2Cl2…………….. Methylene chloride CH3ONa……………. Sodium methoxide

DMAP ………………4-Dimethylaminopyridine DMF……………….. Dimethylformamide

EC50……………….. Effective concentration ESI…………………. Electrospray ionization Et3N………………...Triethyl amine

H……………………. Histidine HMDS ………………Hexamethyldisilazane HPLC………………. High Performance Liquid Chromatography hr…………………… Hour HRMS ………………High resolution mass spectrometry Hz………………….. Hertz

+ − K (CH3)3CO …...... Potassium tertiary butoxide KMnO4…………….. Potassium permanganate

L…………………….Leucine

M…………………… Methionine M…………………… Molar Mg2+………………. Magnesium MgSO4……………... Magnesium sulfate Mn2+……………….. Manganese MnO2………………. Manganese oxide MS………………….. Mass spectroscopy NaCl………………... Sodium chloride NaHCO3…………... Sodium bicarbonate xix

(CH3)3Si) 2NNa)……Sodium bis (trimethylsilyl) amide NaOH………………. Sodium hydroxide NOE………………... Nuclear over Hauser effect

OAc………………… Acetate OBz………………… Benzoyl oC…………………... Degrees Celsius OH…………………. Hydroxyl OsO4……………….. Osmium tetraoxide

P…………………… Proline Ph………………….. Phenyl rt…………………… Room temperature

S……………………. Serine SAR………………… Structural activity relationship SnCl4………………. Tin chloride

T……………………. Threonine t……………………….. Tertiary TBDMSCl………….. Tertiary-Butyldimethylsilyl chloride TBDPS……………... Tertiary-butyldiphenylsilyl THF………………… Tetra hydro furan TiCl4……………….. Titanium tetrachloride TIPS………………... Triisopropylsilyl TMS………………... Trimethylsilyl

UL……………...... Microliter UV………………….. Ultraviolet

Y……………………. Tyrosine

Zn………………… Zinc

α-F…………………. Alpha-fluoro α-OCH3…………….. Alpha-methoxy

Chapter 1

Introduction

1.1 Hepatitis C Virus Epidemiology

Since its discovery in 1989 [1], hepatitis C virus (HCV) has distinguished itself as a major cause of chronic liver disease worldwide. HCV is endemic in many countries and is an expanding burden for society and health-care systems. Liver cirrhosis and hepatocellular carcinoma (HCC) represent the late complications for HCV and are the most common indication for liver transplantation [1].

HCV was previously referred to as non-A, non-B hepatitis [1, 2]. The primary reason

HCV remained so ambiguous for many years is that infection with this virus has a silent onset and emerges asymptomatically into a chronic form of hepatitis [3].

There are two forms of HCV, acute, in which HCV RNA is detected in serum within seven to 21 days after viral transmission or chronic, a state which is marked by the persistence of HCV RNA in the blood for at least 6 months after onset of acute infection.

Acute infection is cleared rarely and leads to massive liver damage in addition to extra hepatic manifestations (EHM) approximately 20 to 30 years after infection [4].

1.1.1 Prevalence and Incidence of (HCV)

The World Health Organization (WHO) reports that 3% of the world’s population has been infected with HCV, which equates to 170 million people. These individuals are at risk of developing chronic liver diseases such as liver cirrhosis (20-30% of patients after

2-3 decades). From 1 to 4% of these patients will develop HCC per year [5-7].

The distribution of HCV infection is highly irregular, and varies substantially in different countries and in different areas within the same country [8]. Countries located in Africa, the Eastern Mediterranean, South-East Asia and the West Pacific represent the highest prevalence rates of HCV, while lower prevalence rates have been reported in the

Americas, Australia, Northern and Western Europe [9, 10].

Towards understanding the global epidemiology of HCV, seroprevalence studies, which include screening persons who exhibit positive HCV antibody, provide useful descriptive data. However, most studies mainly depended on specific populations such as blood donors, or patients with chronic liver disease—which are not typical of the community or region in which they reside. Population-based studies focusing on integrated communities are far more useful. This kind of study is not practical in most parts of the world because less data is available to validate assumptions about the burden of disease, for example in developing countries which represent the higher prevalence regions of HCV [5].

The major morbidities associated with HCV infections are due to the progression of chronic liver disease in a subset of infected people years after initial acquisition of the infection. Thus, the past and present incidence of infection represents the primary determinant of the future burden of disease [11]. Due to the generally asymptomatic nature of acute HCV, and the fact that available assays do not distinguish acute from 2

chronic or resolved infection, estimations of the incidence of newly acquired HCV infection are difficult. Most studies have relied upon mathematical models to assume incidence trends, depending on the fact that current prevalence reflects the cumulative risk of acquiring infection [12].

1.1.2 Mode of Transmission for HCV

Before the establishment of screening tests in 1990, a history of repeated direct blood transfusion or intravenous (i.v) drug abuse were the two most efficient patterns for HCV infection transmission and were present in about 60-80% of HCV cases [13-15]. The accomplishment of screening for HCV in blood donations throughout the world reduced the incidence in transfused patients to less than 1% in developed countries. Therefore,

HCV epidemiology studies have shifted toward non-transfusion settings. However, receiving contaminated blood products remains a dominant source of HCV infection in developing countries [5].

In the developed world, injection of illegal drug use is the primary mode of transmission for HCV infection. For more than 30 years, it has been the major risk for HCV infections and presently accounts for 68% and 80% in the United States and Australia, respectively

[16, 17]. After initiating injection behavior, HCV infection was thought to occur rapidly.

Epidemiologic data have shown that the environment can act a reservoir for infection which facilitates HCV transmission by cross-contamination from multiple-use medication vials and reused needles and syringes. For example, glass syringes previously were reused in the treatment of schistosomiasis, and the inappropriate cleaning and disinfection of equipment in health care buildings led to transmission [18]. Unsafe therapeutic 3

injections, which includes reuse of syringes or needles from patient to patient without sterilization appears to be the major risk factor for HCV infection in developing countries such as Egypt. Due to the limited supplies of sterile syringes and inadequate of number of health care providers, non-professionals often give injections outside the medical setting

[19].

Other sources for HCV transmission which are less likely occur are perinatal transmission when an infant is born from HCV- infected mother [20]. Sexual transmission of HCV is far less likely than other sexually transmitted viruses.

Nosocomial infection with HCV occurs primarily during hemodialysis procedures. In addition, there are other ways for procurement of HCV infection related to human activities, such as exposure to blood or blood-derived body fluids including cultural or religious practices such as tattooing, body-piercing, cosmetic procedures, circumcision, acupuncture, and cupping [12].

1.1.3 HCV Genotype Distribution

On the basis of phylogenetic and sequence analyses of whole viral genomes, it is currently believed that there are at least seven genotypes (G) of HCV. Each genotype is subdivided into 67 confirmed and 20 provisional subtypes such as (a, b, c, and so forth).

Genotypes 1, 2, and 3 involve 11, 6, 17, subtypes, respectively, while G4 and G6 includes

17 and 24 subtypes. Only 1 subtype is found in G5 and G7. Differences in nucleotide sites (30-35%) were detected in HCV strains belonging to different genotypes, while strains that belong to the same subtype differ at <15% of nucleotide sites [21, 22]. A high level of variability in HCV genotypes was attributed to the error-prone nature of the 4

nonstructural protein HCV NS5B polymerase and the aggregation of mutations in a small hypervariable region in the envelope-encoding genes. The tremendous viral heterogeneity can lead to multiple consequences including the possibility for reinfections with a different genotype because of the very limited cross-antigenicity and the higher rate of chronic infection from the development of immune-escape mutants. Due to the fact that the therapeutic response is genotype- and subtype-specific, and there exists a collection of viral-resistant strains, a need for combination therapies is obvious. [Figure 1] shows the complex and diverse global HCV genotypes distribution [22, 23].

HCV genotypes 1, 2 and 3 have a broad geographical distribution, whereas HCV genotypes 4, 5 and 6 are generally restricted to specific geographical regions. HCV genotype 1 is the most prevalent genotype worldwide and constitutes about 75% of HCV cases. It is the genotype found in most of Northern and Western Europe North and South

America, Asia and Australia. Intravenous drug abuse is the primary mode of transmission for genotype 1a, while genotype 1b is principally transmitted via blood transfusions. Ten to 30% of global HCV types are represented by genotypes 2a and 2b which are common in Japan, North America and Europe, while genotype 2c predominates in Northern Italy.

Countries located in South Asia represent the predominant regions of genotype 3.

Genotype 4 predominates in Egypt and the Middle East, while genotypes 5 and 6 are primarily found in South Africa and Hong Kong. To date, detection of first infection related to genotype 7 has been reported in Canada from a Central African immigrant [21,

22, 24, 25].

Genotypes 1a, 1b, 2a, and 3a are believed to have spread rapidly in the decades prior to the discovery of HCV by way of contaminated blood and blood products, injection drug 5

use, and other routes, therefore these are so-called epidemic subtypes. While many other

HCV subtypes are categorized as endemic strains, these are comparatively rare in their distribution [22].

Figure 1: HCV Genotype Distribution [23]. Reprinted with Permission

1.2 HCV Structure

1.2.1 Viral Genome Organization

HCV is an enveloped virus with a positive sense RNA genome that is approximately 9.5 kb long, and belongs to the Hepacivirus genus within the Flaviviridae family. The virus particle size is approximately 55-65 nm [26, 27]. The difficulties in the characterization of viral particles by Electron Microscopy can be attributed to the lack of HCV cell

cultures that produce a large number of virus particles for visualization. However, further characterization was achieved by recent work using the Japanese Fulminant Hepatitis 1

(JFH-1) cell culture [28]. Despite several biochemical and morphological studies, much information remains to be identified regarding the composition of the HCV particles. In comparison with other Flaviviridae family members which shared a number of basic structure motifs, it is believed that HCV has an icosahedral arrangement [Figure 2]. The viral envelope, derived from host membrane, consists of a lipid bilayer in which the structural glycoproteins E1 and E2 are anchored. The envelope surrounds the nucleocapsid, which is composed of multiple copies of a small basic protein (core or C), and contains the RNA genome [29]. HCV virions are associated with lipoproteins ApoE and ApoC and exist as lipoviroparticles (LVPs) and representing an unique feature of

HCV biology.

Figure 2: HCV Particle [30]. Reprinted with Permission

The viral genome consists of a single open reading frame (ORF) encoding a polyprotein of 3000 amino acids (aa) or more. The N-terminal part of the ORF is responsible for encoding the structural proteins (E1, E2, core protein C), whereas the C- terminal portion of the ORF codes for the nonstructural NS proteins (NS2, NS3, NS4A, NS4B,

NS5A and NS5B) [Figure 3] [31].

The ORF is flanked by 5’ and 3’ untranslated (UTR) regions which are crucial for replication of the genome. The HCV 5'UTR contains 341 nucleotides (nt), and is considered the most conserved region of the genome. The 5'UTR region consists of four highly organized (I –IV) domains. The interaction between domains II, III and IV together with the first 12 to 30 nt of the core coding region leads to the formation of internal ribosome entry site (IRES) that plays an important role in HCV polyprotein translation [32].

The 3'UTR contains approximately 225 nt, divided into three structured regions including a poly-(U)/polypyrimidine tract, a variable 40 nucleotide sequence, and a highly conserved 98 nucleotide sequence with stable secondary structure essential for viral replication [33, 34].

1.2.2 HCV Proteins

Depending on the genotype, the HCV ORF contains 9024 to 9111 nt. The ORF encodes at least 11 proteins which are translated into 3 structural proteins (C or core, E1 and E2), a small protein, (p7, whose role is poorly understood), 6 nonstructural (NS) proteins

(NS2, NS3, NS4A, NS4B, NS5A and NS5B), and (F) protein which results from a frameshift in the core coding region. 8

Figure 3: HCV Proteins [31]. Reprinted with Permission

1.2.2.1 Structural Proteins

The HCV core protein is a highly basic, RNA-binding protein, cleaved from the polyprotein via host signal peptide peptidase and released as a 191 amino acid precursor of 23-kDa (P23) which represents the immature form of core protein. Despite the presence of various sizes (17 to 23 kDa) of the protein, the 21-kDa core protein (P21) seems to be the predominant form [35]. Mature forms of core protein result from cleavage between amino acids 173 to 179 by host Signal Peptide Peptidase (SPP).

Interaction of mature core protein with genome RNA leads to formation of nucleocapsid of the virus. The core protein includes three distinct domains (D). D1 is an N-terminal hydrophilic domain which contains numerous positive charges that are involved in RNA binding. In vitro studies show detection of nuclear localization signals (NLS) in D1 domain reflecting their role in nuclear localization. In the infected cells, fusion of the core

protein within the endoplasmic reticulum (ER) membranes, lipid particles and outer mitochondria membranes is mediated by a C-terminal hydrophobic D2 domain of the core protein. The last domain consisting of 20 aa serves as a signal peptide for the downstream envelope protein E1 [36-38].

The HCV core protein exists in a dimeric or multimeric form due to the presence of a D1 tryptophan-rich sequence that allows the P21 core protein to interact with itself [36]. In addition to its role in nucleocapsid formation, the HCV core protein has other properties.

Many in vitro studies have shown other roles for the core protein in the pathogenesis of

HCV including apoptosis [39], cell life cycle, lipid metabolism[40], implications in fibrosis progression and tissue injury [41], regulation of the activity of cellular genes and modulation of the transcription of other viral promoters [42].

The E1 and E2 proteins of HCV are envelope type I transmembrane glycoproteins, with long N-terminal ectodomains, and a short C-terminal transmembrane domain. The E1 and E2 transmembrane domains are composed of two regions of hydrophobic aa distinguished by a short polar region including fully conserved charged residues. These glycoproteins play important roles in the viral entry through multiple functions including membrane anchoring, ER localization and heterodimer formation with the viral envelope

[43]. E2 contains hypervariable regions (HVR) responsible for the interaction between

E2 protein and negatively charged molecules on the surface of host cell receptors leading to initiation of viral attachment. HVRs consist of basic amino acids residues which differ in sequences (up to 80%) between HCV genotypes and between subtypes of the same genotype [44]. This frequent mutation nature of HVRs protect the HCV particles from detection by the host immune system [45]. Less information about the role of E1 is 10

available, but it is believed to be involved in intra-cytoplasmic virus-membrane fusion

[46].

The frame shift (F) protein or alternate reading frame protein (ARFP) translated with random events although it does not contain an AUG start codon. It is produced as a result from a -2/+1 ribosomal frame shift in the core-encoding region of the HCV polyprotein located at the N-terminus. In chronically infected patients, detection of antibodies to F protein indicate the production of the protein during infection [47]. Currently, the role of

F protein in the HCV life cycle still unknown, but it was thought to be involved in viral persistence [48].

1.2.2.2 Non-Structural Proteins

P7 is an integral membrane small (63 aa polypeptide) protein that is essential for HCV infectivity. Recently in vitro studies suggested that p7 is a member of the viroporin family and has calcium ion channel activity [49].

The NS2 protein is a hydrophobic short lived protease. In association with the amino- terminal domain of the NS3 protein it constitutes a zinc-dependent metalloprotease that is responsible for HCV polyprotein cleavage between NS2 and NS3. This cleavage is important for HCV replication. Following cleavage, NS2 losses its protease activity, and is degraded by the proteasome, and is then localized in the ER membrane. NS2 appears to be involved in modulation of lipid metabolism, HCV induced apoptosis, innate immunity response and cell proliferation [20, 50-52].

NS3 is located in the ER membrane and has two domains. The N-terminal domain has serine protease activity mediated by NS4A which acts as a cofactor of NS3 protease 11

activity, allowing its stabilization, as well as cleavage of the HCV polyprotein downstream of NS3 which is important for formation of the viral replication complex

[53]. The C-terminal domain contains a helicase/NTPase, capable of nucleic-acid binding and 3′ to 5′ translocation coupled to hydrolysis of ATP. During RNA replication, the

NS3 helicase/NTPase induces conformational changes in the binding of the nucleic acid substrate using the energy of NTP hydrolysis. The helicase activity of NS3 protein seems to be modulated by the NS3 protease domain and the NS5B RdRp [54]. NS3-4A has multiple functions important in the life cycle and pathogenesis of HCV infection [53, 55].

At early stages of infection, HCV could use NS3-4A protease to bypass the innate immune response. In response to viral infection, the host immune system induces production of interferon mediated by the dsRNA-dependent interferon regulatory factor 3

(IRF-3) pathways. In in vitro studies, the HCV NS3-NS4A was shown to inhibit the interferon regulatory factor-3 (IRF-3) pathway via blockade the phosphorylation of the intracellular double stranded RNA sensor protein (RIG-I) [56].

The NS4B protein is present on the ER membrane, with an N-terminal amphipathic helix that mediates membrane association and plays a critical role in viral replication [57].

After cleavage of the HCV polyprotein, the replication complex is formed and results from interaction of viral proteins with the host cellular factors. Formation of cytosolic microenvironment space for viral replication is facilitated by reorientation of the cellular membrane leading to formation of membranous webs or vesicles which represent one of the candidate sites for HCV replication and possible early assembly. The NS4B has been thought to form the scaffold of membranous vesicles due to it is ability of oligomerization [58]. In vitro studies have detected additional properties of the NS4B 12

protein such as an antagonist effect on NS5B activity and host translational mechanisms

[59, 60]. NS4B has a nucleotide binding motif (NBM) that binds and hydrolyzes GTP, specifically [58].

NS5A is also an ER-associated hydrophilic protein. It plays multiple roles in mediating viral replication, and viral pathogenesis. NS5A consists of three domains. Domain I of

NS5A is a zinc metalloprotein containing well conserved zinc-binding motifs involving four cysteine residues in the N-terminal region. This motif plays a critical role in the structural stability and function of the protein. Domains II and III are located in the C- terminal region; they contain a nuclear localization signal (NLS) that mediates the nuclear localization of NS5A. NS5A exists as multiple phospho-isoforms that play an important role in HCV pathogenesis. NS5A hyper-phosphorylation leads to decreased stability and serves as a switch point between HCV RNA replication and downstream processes, such as particle assembly or particle maturation [61]. NS5A undergoes multiple interactions with host cell proteins. NS5A shields the viral particle from innate immunity recognition through modulations of interferon-induced double stranded RNA activated protein kinase PKR [62].

1.3 HCV Life Cycle

1.3.1 HCV Attachment, Entry and Fusion

The hepatocytes represent the target cells for HCV infection during primary infection.

HCV particles cross the fenestrated endothelium of the liver sinusoids and come in contact with their target cells via the blood stream. After that the virions have direct contact with the basolateral surface of the hepatocytes. HCV entry appears to be 13

complicated, involving interactions with multiple cellular surface components that mediated HCV binding and internalization. Many details need to be known about the exact role of each cellular components in HCV binding [63]. Due to HCV particle interaction with lipoproteins, initial low-affinity cell binding of HCV is believed to be mediated by the low density lipoprotein LDL receptor and glycosaminoglycan. It was found that virus-like particles complex with LDL prior to entry into the cell via LDL-R

[64], Furthermore, the use of antibodies against LDL-R lead to inhibition of HCV adsorption from the serum of infected patients, reflecting the important role of HCV complexation with lipoproteins in the early attachment step of the viral entry [65, 66].

Through the use of a newly developed infectious cell culture system, it has been shown that in the presence of heparinase, an enzyme that disrupts the high affinity binding of the heparan sulfate proteoglycan (HSPG), a cell surface molecule to the HVR1 of the E2 envelope lipoprotein leads to inhibition of the initial attachment step of HCV [67].

Following initial attachment, HCV undergoes complex interactions mediated by coordinated actions of four cellular cofactors including: scavenger receptor B type I SR-

BI, CD81 and tight-junction proteins claudin-1 (CLDN1), and occludin (OCLN). The scavenger receptor B type I (SR-BI) represents the first candidate receptor for HCV entry. It has dual interactions with HCV glycoprotein E2 through binding to it is HVR1 with high specificity, it appears to interact with virus-associated lipoproteins and modulate the lipid composition of membranes through it is lipid transfer activity. High density lipoproteins (HDL) are a natural ligand of SR-BI. Hepatocytes and steroidogenic cells represent a higher level for expression of SR-BI. A partial blockade of HCV binding

occurs when antibodies are directed against SR-BI which explains the role of other cellular receptors in HCV entry [68-70].

CD81 is a cell surface protein that belongs to tetraspanin or transmembrane 4 superfamily and plays a major role for HCV attachment. CD81 consists of four hydrophobic transmembrane regions and two extracellular loop domains, a small extracellular loop (SEL) and a large extracellular loop (LEL) [71]. The binding of HCV amino acid residues located at the surface of the core of the envelope glycoprotein E2 has been thought to be mediated by CD81 LEL [72]. In in vitro studies, HCV infectivity was inhibited by CD81 antibodies. In addition, infection occurred by expression of CD81 in cell lines that are not permissive to HCV infection such as HepG2 and HH29 hepatoma cells, while expression of CD81 in other non-permissive cell lines does not allow HCV infection to occur indicating that involvement of other cellular factors in the HCV binding or that the CD81 molecule could act as a post-attachment entry co-receptor that mediates binding and entry of the virus into the target cells [73, 74].

Late entry steps of the virus into the cell are thought to be mediated by the interaction with two tight junction cell proteins Claudin-1(CLDN1) and Occludin (OCLN). CLDN1 has been shown to interact with CD81 to form a co-receptor complex which is important for the downstream events of HCV entry. This association is regulated by multiple signaling pathways. In addition to its potential direct interaction with the viral particle

[68, 75], the exact role of (OCLN) in HVC entry needs to be clarified. Recent studies have shown that depletion of (OCLN) has no effect on (CLDN1) expression which indicates that these two factors may act separately [69]. HCV may also use the tight junction proteins for cell to cell transfer of the virus [70]. 15

A fusion process of virus envelope with the endosome membrane, including liberation of viral nucleocapsid in to the cytoplasm of host cell, occurs following internalization of the particle into endosomes or by direct interaction of the viral particles with the plasma membrane. It has been shown that the fusion process is mediated by endocytosis in a pH- dependent and clathrin-dependent manner and controlled by viral surface glycoproteins which share structural homology with class II fusion proteins. However, the identity of the HCV fusion peptide remains questionable. In addition, knock-down of clathrin, a critical component of the endocytotic vesicles, prevents HCV infection [76]. Following fusion, the HCV genome is presumably released into the cytosol and starts the translation process.

1.3.2 RNA Translation and Post-Translation Processing

Free positive-strand genomic RNAs are released in the cellular cytoplasm following viral nucleocapsid decapsidation and acts as messenger RNAs together with newly synthesized

RNAs for the synthesis of viral proteins. The open reading frame (ORF) of the HCV

non-translated regions (NTRs) which contain RNA ׳and 3 ׳genome is flanked by 5 conserved structural elements that are important for genome translation and replication of

HCV RNA. The internal ribosomal entry site IRES, spanning domains II to IV of the

5'UTR and the first nucleotides of the core-coding region have been shown to regulate the HCV genome translation process [77, 78].

The ER membrane represents the associated site for the HCV genome translation products mentioned in section 1.2.2. Signal peptidase and signal peptide peptidase mediate cleavage of structural proteins [63]. 16

1.3.3 HCV RNA Replication

Before the formation of the replication complex, HCV produces alterations in the intracellular membrane to provide microenvironment space for RNA replication in the cytoplasm of infected host cells. The formation of this endoplasmic reticulum derived membrane or membranous web is mediated by the HCV proteins NS4B and NS5A. The membranous web serves as a scaffold for the HCV replication complex which consists of viral proteins NS3/4A, NS4B, NS5A, and NS5B which constitute the replication machinery, cellular components and nascent RNA strands. In addition, lipid rafts are involved in the formation of the replication complex [64, 79].

Compared with other positive-strand RNA viruses, the mechanism of HCV replication is still poorly understood. In the first step, the positive-strand genome RNA serves as a template for the synthesis of a negative-strand intermediate that is then used to make more positive strand copies for packaging. The viral RNA-dependent RNA polymerase

NS5B is the key enzyme of RNA synthesis [65].

Many approaches have shown a large number of host cell proteins affecting HCV replication [63]. For example, phosphatidyl-inositol-4-kinase-III (PI4KIII) interacts with

NS5A and induces aggregation of phosphatidylinositol-4-phosphate (PI4P) within the membranous web and leads to changes in its morphology. The protein–protein interactions between PI4KIII and oxysterol-binding protein lead to accumulation of cholesterol in the HCV-induced double-membrane vesicles (DMVs) which aggregate in parallel to the peak of RNA replication. This reflects the importance of lipid rafts as a potential site for viral replication. NS5A and NS5B interact with vesicle-associated membrane protein-associated protein A (VAP-A) and (VAP-B), which are necessary for 17

viral RNA replication [63]. Cyclophilin B, a peptidyl-prolyl cis-trans isomerase, interacts with NS5B and increases its RNA binding activity[66]. Lipid droplets (LDs) were also found to be surrounded by viral double-stranded RNA indicating that these organelles could play a role in RNA replication [67].

1.3.4 HCV Assembly and Release

Virus assembly and release is a highly regulated process connected to host cell lipid metabolism. Difficulties in the detection of virion assembly, budding, or egressing, reflect that these processes are either rare or rapid. There is a little information about

HCV assembly and release due to the lack of appropriate study models. It is believed that viral nonstructural proteins play an essential role in these processes. The genomic

RNA interacts with the core protein to form the nucleocapsid. The core protein then homodimerizes and interacts with cytosolic LDs via a C-terminal domain leading to a change in intracellular distribution of LDs [80, 81]. The core-LD association has been shown to be important for the enrollment of other viral components involved in virion assembly. Mutations that target this interaction strongly inhibit viral assembly [82]. Many host cellular proteins modulate the core-LD interaction [63]. The other important viral structural proteins are the E1, and E2 envelope proteins that form noncovalent heterodimers with the ER. Interactions of this complex with the viral proteins NS2 and p7 lead to formation of a functional unit that migrates close to the LDs where assembly takes place. The budding process of the HCV particle results from the essential and coordinated role of protein –protein interactions through disulfide bond formations which are assisted by the presence of disulfide bridges between the HCV envelope glycoproteins at the 18

surface of HCV particle [63]. Interactions of the C-terminal domain of NS5A with the

LD-bound core protein are also involved in the assembly process. The NS3/4A enzyme complex is also involved in HCV assembly by formation of the HCV RNA replicase.

NS4B and NS5B have also been shown to be involved in virus assembly [62]. Following the budding process of the HCV particle into the lumen of the ER, it is believed that the virus is released from the cell after transit through the Golgi apparatus and the secretory pathway [83]. The hypothetical pathways for HCV life cycle are show in [Figure 4].

Figure 4: HCV Life Cycle [31]. Reprinted with Permission

1.3.5 HCV Circulating Forms

There are many forms of HCV that have been detected during acute and chronic HCV infection with different sizes and densities. The lower density particles contain (besides 19

HCV RNA), high amounts of triglycerides, core protein and apolipoproteins B and E

(ApoB and ApoE). These appear to be more infectious than the higher density particles that form the immune complexes in association with immunoglobulins when tested in animal models [84]. In addition, non-enveloped nucleocapsids have been detected in patient serum and in hepatocytes which exhibit Fcγ receptor-like activity and bind non- immune IgG, but the infectivity of these forms remains to be understood [85-87].

1.4 HCV Pathogenesis

1.4.1 HCV Induced Apoptosis

Apoptosis, or programmed cell death, plays an essential role in eradication of infected cells. Many cells develop pathways to modulate or inhibit host mechanisms in order to ensure their survival. During chronic HCV infection, an increased amount of apoptosis through the presence of typical pathomorphological features of apoptosis (e.g., nuclear fragmentation, cell shrinkage) is detected in the liver although it is unknown whether uninfected hepatocytes or HCV infected cells are undergoing apoptosis [88]. Death receptor ligands including CD95 Ligand, Tumor Necrosis Factor α (TNFα) and Tumor

Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL), which are secreted by the immune cells (e.g., macrophages) may be membrane-bound, and are considered major players in the formation of a death-inducing signaling complex which leads to activation of the caspases that represent the proteases involved in the apoptosis signaling cascade.

These ligand receptors are upregulated during HCV infection [89]. In normal liver cells

CD95 Ligand and TNFα were determined to induce apoptosis. Furthermore, CD95

Ligand-induced apoptosis was detected by killing of bystander cells of non-HCV infected 20

hepatocytes, whereas infected or malignantly transformed hepatocytes/hepatoma cells undergo apoptosis in response to TRAIL [88].

Depending on the types and the conditions of the experimental system used, studies to explain the influence of viral proteins on hepatocyte apoptosis show all HCV proteins have both pro-apoptotic and anti-apoptotic effects and are highly variable due to HCV heterogeneity [90]. Many studies detected the complex implications of HCV proteins in mediating cell survival and apoptosis, and remain to be fully understood. In a hepatoma cell line, HCV core protein exhibited inhibitory effects on TNF-α and CD95 Ligand- induced apoptosis, while in other studies, it did not prevent CD95 Ligand-induced apoptosis in hepatoma cells [91-93]. Direct interaction of core proteins with several pro- apoptotic proteins including CD95, TNF-R1 and lymphotoxin-β can reflect the essential role of HCV in the induction of apoptosis in chronic infection [94, 95]. The anti - apoptotic activity of the core protein was also detected through its direct interaction with the DNA-binding domain of Smad3, which is the apoptosis mediator of the fibrogenic transforming growth factor (TGF)-beta 1. In addition to the apoptotic effect of (TGF)- beta 1, it has an essential role in fibrosis development in chronic HCV infection [96].

HCV core proteins also modulate the mitochondrial functions through induction of oxidative stress, making cells more susceptible to apoptosis [97].

TRAIL-induced apoptosis in hepatoma cell lines are inhibited by E2 protein, while E1 has no effect [98]. NS2 protein inhibits CIDE-B-induced apoptosis (cell death-inducing

DFF45 (DNA-fragmentation-factor)-like effector) [99]. NS3 induces caspase-8 dependent apoptosis in hepatocytes with an unknown mechanism [100]. The role of other nonstructural HCV proteins is not yet well defined. 21

1.5 HCV Progression and Complications

The majority of patients do not become aware of their disease due to the asymptomatic nature of the acute phase of the disease. The incubation period may vary according to viral transmission patterns. Mild and nonspecific symptoms are detected in 15–30% of infected patients within 5–12 weeks of HCV exposure [101].

Viral clearance (undetectable levels of HCV RNA in the blood) occurs in 25% of cases with acute HCV infections by unclear mechanisms. The complex interactions between host and virus have been thought to be associated with viral clearance. The nucleotide polymorphisms (SNP) close to the interleukin 28B (IL28B) gene that encode for interferon-lambda-3 (IFN‑λ3), which has an unknown role in viral control, is considered the most important host factor included in viral clearance [102]. Patients with nonfavourable IL28B genotypes are less able to clear HCV infection compared with those who have favorable genotypes [103]. In contrast, 75% of patients fail to clear the virus in

6 months and develop chronic hepatitis which further progress to liver fibrosis. There are many host factor effects on the rate of chronic HCV infection such as, age, gender, race, and the development of jaundice during the acute infection.

Age is one of the most important risk factors for fibrosis development in chronic HCV infection. Many study methodologies have shown that the rate of fibrosis progression increases with patients older than 40 years although the exact mechanisms are unclear[104]. Fibrosis rates in men are faster than in women, possibly due to hormonal factors which may be important in the regulation of liver fibrosis [105]. Ethnicity and other genetic factors may also be included in disease progression. Based on National

Health and Nutrition Examination Survey NHANES studies, a higher rate of chronic 22

infection is seen among African Americans (86%), compared to Caucasians (68%) [106].

During acute onset of HCV infection, symptomatic patients who developed jaundice have a lower rate of chronic infection than those who are anicteric [107]. The immune response plays a significant role in the development of chronic hepatitis C and its subsequent progression. Other significant risk factors for liver fibrosis include HCV coinfections with HIV or HBV. In addition, different HCV genotypes are also associated with differential chronic HCV progression. For example, recent studies have shown accelerated disease progression in patients infected with genotype 3 [108].

Liver cirrhosis and hepatocellular carcinoma (HCC) represent the long term complications of chronic HCV infections. Cirrhosis develops in approximately 10% to

15% of individuals after 20 years with chronic HCV infection [108]. Development of ascites, upper gastrointestinal bleeding secondary to varices or portal hypertensive gastropathy, hepatorenal syndrome, and hepatic encephalopathy represent the main sequelae of liver cirrhosis, and constitute the major cause of chronic HCV infection related death in the United States [109]. Besides the host factors that are mentioned above, chronic alcohol use (more than 50 g per day) is considered a major external risk factor for the progression of chronic hepatitis C to cirrhosis and HCC, while increases in coffee consumption has beneficial effects on the overall mortality of HCV infections

[110]. The rate of HCC development among people with chronic HCV infection has been estimated at 1–3% after 30 years [111]. Numerous extra hepatic manifestations have been reported in chronic HCV infections and may involve multiple organs and up to 40–74% of HCV-infected individuals will develop at least one extra hepatic manifestation [108].

1.6 HCV Treatment

Prevention of the complications of chronic HCV infection such as cirrhosis, hepatocellular carcinoma, and liver transplant represent the primary goals of treatment.

Sustained virologic response (SVR), which is defined as absence of HCV RNA by polymerase chain reaction PCR after cessation of antiviral therapy, is considered a good indicator for the success of HCV therapy. Classically, SVR was measured 24 weeks after stopping therapy, but recently the Food and Drug Administration (FDA) approved an endpoint of an SVR at 12 weeks as being applicable for HCV trials [112].

According to their targets, HCV therapies can be divided into indirectly acting antivirals, directly acting antiviral agents (DAAs), and host-targeting antiviral agents (HTAs). The responses to these agents differ, depending on the HCV genotype, host factors, and baseline of the viral loads. Patient’s response to antiviral therapies of HCV can be divided into three groups, sustained virological response (SVR), relapse or end of treatment response which occurs in 10–25% of patients with optimal regimens, and non- response which occurs in about one-third of patients with chronic hepatitis C. The mechanisms behind relapse or non-response groups are unknown [113].

1.6.1 Indirectly Acting Antiviral Agents

Until 2011, the standard regimen of care for HCV relied on the combination therapy of interferon and ribavirin. These are still used in combination with other new regimens.

This combination is associated with low SVR as well as serious side effects. The mechanism of antiviral effects exerted by this regimen is incompletely understood.

Interferons are host proteins including three major types based on their receptors: type I 24

(interferons α and β), type II (interferon γ), and type III (interferon λ). All these types of interferons have antiviral, antiproliferative and immunomodulatory activities but differ in their relative potencies. During HCV infection, interferons exhibit antiviral effects through induction of IFN-stimulated genes (ISGs), which stimulate a non-virus-specific antiviral state within the cell [114]. In 1992, IFN-α was approved in the United States for the treatment of hepatitis C. Approval of PEGylated IFN-α (PEG-IFN-α) to be given subcutaneously occurred in 2001, leading to improved pharmacokinetic properties and reduction the dose frequencies.

Ribavirin, which was approved for the treatment of chronic HCV in 1998, is an oral guanosine analog with broad antiviral activity against several RNA and DNA viruses. It has a complex and unclear mode of action. The possible related mechanisms include direct inhibition of HCV in which ribavirin acts as a chain terminator for HCV polymerase after misincorporation of triphosphate form of ribavirin. This mechanism has only a small effect on HCV replication. Other mechanisms involve depletion of the GTP necessary for viral RNA synthesis by competitive inhibition of inosine monophosphate dehydrogenase (IMPDH) by ribavirin monophosphate RMP. Ribavirin may also act as a viral mutagen and leads to increased rates of mutagenesis resulting in a so called “error catastrophe”. Immunomodulatory effects of ribavirin include enhanced induction of interferon-stimulated genes in response to IFN-α [113]. Response to this regimen is genotype dependent; SVR is 45% in patients infected with GT1 (treated for 48 weeks) while it is 66-80% in patients infected with GT2 or GT3 (treated for 24 weeks) [115,

116]. The difficulties in tolerance and associated adverse effects such as flu-like symptoms, anemia, neutropenia, thrombocytopenia, fatigue, depression, autoimmunity, 25

thyroid dysfunction, hemolysis, skin rash, and teratogenicity, are considered the major causes for discontinuation of this regimen [117].

1.6.2 Direct Acting Antiviral Agents (DAAs)

A better understanding of the HCV life cycle through the availability of robust in vitro systems to culture HCV and the resolution of the crystal structures of viral proteins are considered the primary evolutionary steps in the discovery of new drugs that act directly on non-structural viral proteins. During the last years, some DAAs were approved by the

FDA and others are being examined in ongoing clinical trials; these drugs used alone or in combination with INF, with or without ribavirin, showed more SVR comparable to the standard care regimen. Currently, there are three major classes of DAAs: NS3/4A protease inhibitors; NS5B polymerase inhibitors; and NS5A inhibitors.

NS3/4A protease inhibitors target NS3 (serine protease) and its cofactor, NS4A, which mediates the proteolytic cleavage of the polyprotein and leads to block non-structural viral protein maturation and hence stop viral replication. Ciluprevir (BILN 2061) was the first in this class but due to cardiotoxicity, its development was stopped [118]. To date we have two generations of NS3/4A protease inhibitors according to their genotype covering and degree of genetic barrier to resistant HCV. First generation protease inhibitors include teleprevir 1 and boceprevir 2 [Figure 5], they were first approved in 2011, and are used in conjunction with peginterferon-ribavirin for the treatment of genotype 1 infection.

Simeprevir was licensed in late 2013. Other drugs such as faldaprevir, asunaprevir, vaniprevir, and ritonavir boosted ABT-450, and are currently in phase II or phase III trials. The first generation drugs have been shown to display potent anti-viral activity, 26

increase SVR rate up to 89% in patients infected with GT1, but have been associated with low barrier to genotyping resistance as well as serious side effects and drug-drug interactions [119].

Figure 5: NS3/4A Protease Inhibitors.

The second generations of NS3/4A protease inhibitors are drugs with modified pharmacokinetic properties and better safety profiles, in addition to broader genotypic activities. Examples include MK-5172 and ACH-2684 which are in various stages of clinical development [118-120].

NS5A inhibitors target the NS5A protein which is essential for both viral assembly and replication. Examples include potent antiviral drugs that act within picomolar concentrations, have excellent safety profiles, and fewer side effects, but the genetic barrier to resistance is low. These features have brought some drugs such as Daclatasvir,

ledipasvir, and ABT-267 which are in advanced clinical stages, to be used alone or in combination, with standardized regimens in the treatment of HCV infections [140-142].

NS5B inhibitors target the NS5B RNA dependent RNA polymerase which is responsible for replication of HCV RNA. As they are the subject of this thesis they will be discussed in detail in chapter two.

Extensive studies that focus on the role of host factors, such as enzymes and receptors, on the HCV life cycle led to the discovery of new drugs called host-targeting antiviral agents

(HTAs) which can be used in conjugation with other HCV treatment regimens and are considered promising candidates to improve SVR rates among various genotypes.

Alisporivir (Debio-025) acts as an inhibitor of Cyclophilin A which enhances HCV replication by unclear mechanisms. It is effective against all HCV genotypes and displays a very high barrier to resistance development, but detection of pancreatitis during combination therapy with PEG-IFN-α-2a resulted in halting development of this drug

[118]. Another important host factor includes miR-122, a liver-specific micro RNA. The

HCV RNA genome binds two conserved miR-122 seed sites near the 5′ end of viral untranslated region. This interaction protects the 5′ end of the HCV genome from recognition by the host innate immunity. Miravirsen, an antisense oligonucleotide acts against the miR-122, and can achieve a significant decline in the HCV RNA serum level

[63].

Chapter 2

Background

2.1 The Structural Feature of HCV NS5B RdRp Protein

The biochemical characterization of HCV NS5B was facilitated by the expression of recombinant NS5B in insect and bacterial cells. NS5B is a nonstructural viral protein encoded by the open reading frame of HCV; it is an RNA-dependent RNA polymerase

(RdRp) and belongs to a class of membrane proteins named tail-anchored proteins, consisting of 591 amino acids. The C-terminus includes 21 amino acid residues and represents the hydrophobic α-helix region responsible for NS5B membrane association

[121]. Like other known RdRps, HCV NS5B includes six conserved motifs designated A-

F. In 1999, the three dimensional crystal structure of the NS5B (in the absence of bound substrate or template (apoenzyme) was determined by three independent research groups

(Figure 6). In addition, studies of template binding and the mechanism of the de-novo initiation reaction by HCV RdRp were facilitated by solving the crystal structure of the

NS5B polymerase bound to a short RNA-template and to nucleotide triphosphates

(NTPs). They revealed that NS5B adopts a structure similar to other classes of polymerases which maintained right hand shapes. The catalytic domain formed at the N- terminus of 530 aa residues, includes three classical subdomains: the finger, thumb and palm in which the active site of NS5B is located [122]. HCV RdRp has two elongated 29

loops extending from the β-fingers through the thumb subdomain that form the rNTP tunnel and are considered a common structural feature. This extensive interaction decreases the flexibility of the subdomains and thus the HCV RdRp has an encircled catalytic site which limits the structural changes upon binding of its nucleotide substrate and facilitates the first steps of RNA synthesis leading to the formation of the primer strand. Therefore in the elongation steps, an opening of the thumb and the fingers are required for primer extension. The α-fingers, located near the palm subdomain, act as the exit route for the double stranded RNA product. Another unusual feature of the HCV

RdRp is a β-hairpin loop also referred to as a β-flap (12 amino acid loop) that bulges into the active site located at the base of the palm subdomain. This loop, located within the thumb subdomain (the most variable subdomain among the various polymerases), is suggested to act as a gate to direct the correct binding of the 3′-terminus of the RNA, maintaining correct initiation and replication from the terminus of the HCV genome.

Until now, all structural studies about the HCV RdRp reflect the fact that it has a closed conformation encircled on one side by the fingertips and on the other side by the β- hairpin, while the other polymerase are open handed as observed in the Klenow fragment from E. coli DNA polymerase 1 (1DPI) [121, 123, 124].

The palm subdomain represents the most conserved structural feature among different polymerases, contains the catalytic pocket of HCV RdRp, and includes A-E motifs as well as the invariant catalytic traid GDD (Gly317-Asp318-Asp319) in the C motif. The presence of an acidic side chain (aspartate in the A motif, whose geometry is conserved in all polymerases) facilitates the coordination of the divalent metal ions (Mn2+ or Mg2+) responsible for the nucleotidyl transfer reaction. Besides the active site, four allosteric 30

binding sites, including “Thumb” Pockets I and II and “Palm” Pockets I and II/III were discovered through recent crystallization of NS5B complexed with ribonucleotides

(NTPs). These sites were believed to be potential targets for anti-HCV drugs [123].

Figure 6: Crystal Structure of HCV NS5B Polymerase [124]. Reprinted with Permission

2.2 HCV NS5B RdRp Activities and the Mode of Action

NS5B is a key enzyme essential both for the transcription of viral mRNA and the replication step of the viral genome. The biochemical activity of the NS5B RdRp is unique to viruses and not present in human cells, making NS5B an attractive target for 31

antiviral drug development. Recombinant NS5B has the ability to synthesize full length

HCV RNA, requiring only divalent metals (Mn2+ or Mg2+) as cofactors. NS5B can catalyze RNA synthesis by two possible biochemical activities. Firstly, a primer dependent manner in which the extended RNA product is formed by extension at the 3ꞌ- end of an RNA molecule in the presence of primer-template duplex. NS5B can also initiate RNA synthesis through primer independent or de novo synthesis from a single- stranded template. In this mechanism a dinucleotide molecule is formed at the 3’-end of the template, which could then be used as a primer; as a result the whole genome is copied to the daughter nucleic acid. This mechanism was proposed for the initiation RNA synthesis in vivo [124-126].

Besides determination of the crystal structure, in vitro assays have provided useful information about the mechanistic view and the important motifs included in the enzymatic activities of NS5B RdRp. These studies revealed that NS5B shares the same catalytic machinery found in DNA-polymerases and HIV RT.

Catalysis by the NS5B polymerase can be simplified into temporal progression steps: template harboring and binding of the nucleotide; initiation; elongation; termination and template release. In de- novo synthesis, NS5B polymerase uses one nucleotide as a primer. Based on in vitro analysis of HCV NS5B enzymatic activity, it seems to accept

-׳GTP as the initiation nucleotide for (+)-strand RNA synthesis because cytidylate is the 3 terminal nucleotide present at (-)-strand RNAs in HCV. NS5B can also use ATP to

- (+) termini of -׳initiate (-)-strand RNA synthesis in case of uridylate present at the 3 strand RNAs. In the de-novo initiation assay of HCV, NS5B reflects asymmetric replication of positive strand RNA, and thus in infected cells the positive strand RNA is 32

more abundant than negative strand RNA. This explains the preference for GTP over

ATP in imitation of RNA synthesis. In addition, the identification of an allosteric guanosine-5ꞌ-triphosphate (GTP)-binding pocket in the interface between the fingers and thumb subdomains, after the binding of GTP to this site, the conformational changes required for a processive RdRp can occur [126-128].

׳ The 5 -untranslated region of the HCV genome accommodates an internal ribosomal

end of the viral genome, preceding the duplication ׳entry site, forcing replication at the 3 of the (–) strand into a (+) strand. The 3′-terminus of the single strand RNA and the incoming nucleotides entering the polymerase catalytic site for terminal initiation and thumb domain β-loop insertion plays an essential role in positioning of this terminus.

During the initiation step, the C-terminal membrane–anchoring linker and β-loop stay in conformation deep within the active site to be adaptable with binding to the RNA template and incoming nucleotides [126].

During the primed assemblies, the β-loop and the C-terminal domain become disordered, and generate space for the incoming nucleotide. These movements accommodate only two Watson-Crick pairs upstream of the incoming nucleotide. Additional conformational changes, such as the opening of the thumb subdomains, are required to include more nucleotides.

The presence of the divalent metal ions is important for activity of NS5B polymerase and accompanies the nucleotide by binding to its phosphates and to the two aspartic acid residues in the polymerase. As show in (Figure 7), the function of metal ion A is to

hydroxyl group at the primer for the hydrogen, facilitating- ׳reduce the affinity of the 3 attack on the α-phosphate to form a phosphodiester bond between the NTPi and the 33

second NTP generating a dinucleotide primer which precedes the primed initiation assembly. Metal ion B facilitates the leaving of pyrophosphate. The expected pentacovalent transition state is stabilized by these ions. It has been shown that the preferred cation for the initiation is Mn2+, whereas Mg2+ is the preferred cation for elongation.

Figure 7: The Mechanism of Polymerase Enzyme [128]

From the point of view of steric hindrance, the cytosine base provides more specificity for binding within the active site. Electrostatic interaction is not favored by the entrance of the uracil base, after the binding of the nucleotide, the RNA strand is ratcheted back by one nucleotide and turns the terminal cytidine for initiation [126-128]. 34

After the formation of the dinucleotide primer, the elongation step begins, which is very progressive and rapid. Serious conformational changes, including expelling of both the β- hairpin loop and the C-terminus from the active-site cavity, prevent overlap with the template strand. Opening of the thumb and the fingers occurs in this step, resulting in a slightly more relaxed state of the polymerase, facilitates movement of the template and exit of the nascent RNA product through the active site [126].

Despite the fact that the de-novo mechanism is preferred for initiation, primer extension seems to be more potent than de-novo synthesis in in vitro studies. A number of structural features have been shown to play a crucial role in suppression of primer extension such as the β-hairpin loop and the C-terminal tail that lines the catalytic pocket. It has been thought that the make this same as other de-novo mechanism is accompanied by the closed conformation of the RdRp while the primer extension activity corresponds to the open conformation [125-127].

2.3 The HCV NS5B RdRp as a Potential Drug Target

The advantages of high throughput screening (HTS), enzyme and cell based replicon assays, and animal models (chronically infected chimp, KMT mouse and HCV-trimera) have accelerated the development of HCV therapeutics. NS5B has been a primary therapeutic target for the past decade. This is due to its vital role in genomic replication; the conserved structural features which do not exist within the uninfected host cell, and the availability of several crystal structures. As a result several compounds have undergone clinical trials.

Drug resistance represents the main challenge in the treatment of chronic HCV infections. This resistance is the result of the high rate of viral replication, lack of proofreading activity and the error-prone nature of the polymerase. The mutation rate for

HCV is believed to be between 10-4 and 10-3 base substitutions per genome site per year, and single and double mutants are generated several times a day depending on the estimated number of virions produced per day. A combination therapy is usually used to overcome this problem and improve viral response [123, 124, 129].

2.4 Inhibitors of HCV NS5B Polymerase

Polymerase inhibitors can be assigned to two broad categories related to their chemical nature and mechanism of action: nucleoside and nucleotide analog inhibitors (NI) and non-nucleoside inhibitors (NNI). Nucleoside and nucleotide analog inhibitors (NI) or direct-acting agents (DAAs) target the conserved catalytic site of the NS5B polymerase, while NNIs bind to one of several allosteric sites on the enzyme. A number of HCV

NS5B inhibitors have proceeded through the clinical phases of development and have exhibited proof of concept by reducing viral loads in HCV infected patients representing a promising future free of interferon regimens for treatment of HCV patients [123, 124,

129].

2.4.1 Nucleoside and Nucleotide Inhibitors (NI)

Most nucleoside and nucleotide inhibitors are prodrugs and are converted to their respective triphosphate (NTP) by host kinase enzymes in the cytoplasm of infected cells.

These substrates must be accepted as ligands by the HCV NS5B polymerase and then

incorporated into the nascent RNA chain by successful competition with the natural ligand of the polymerase. These inhibitors hinder the further addition of subsequent bases resulting in a lack of elongation of the growing polymer chain. Due to this mode of action, these inhibitors are called chain terminators. Many factors, such as efficiency of conversion to the active form by cellular kinases, should be considered in the development of this class of inhibitors, and optimized in polymerase enzymatic assays and in-cell based replicon assays. The first phosphorylation step is the rate limiting step.

Nucleotide analogues often achieve higher cellular concentrations because they bypass this step, have better bioavailability, and do not undergo metabolic deamination and cleavage of the glyosidic bond. All of which should be considered in the development of this class of inhibitors and optimized in polymerase enzymatic assays and in-cell based replicon assays.

NIs can be subdivided into two groups: sugar modified and heterobase modified. We will

sugar modifications and cytosine-׳focus on the nucleosides and nucleotides that have 2 base modifications [123, 124, 129].

2.4.1.1 Sugar Modified NIs

In the development and design of NS5B inhibitors, there are three reported positions:

positions for nucleotide sugar modification. Structural modifications ׳ and 4׳ 3 ,׳namely 2

-׳position of the nucleoside ribose core reflects the importance of maintaining a 3 ׳at the 3

OH in the α-orientation as essential for achieving whole cell replicon activity without the occurrence of cytotoxicity.

α-OCH3 (4)) [Figure 8], have been׳α-F (3), or 2׳Different monosubstitutions like (2

-α׳tolerated in the presence of an adenine, guanine, or cytosine base. However, only the 2

F analog (3) showed activity against the NS5B polymerase in an enzyme assay.

Compound (3) showed high enzyme affinity towards some host cell polymerases. In replicon assays, large micromolar activity reflects weak antiviral activity of these analogues; this can be rationalized to their poor cellular penetration or low capacity for host cell phosphorylation. To overcome this problem, bis (tBu-S-acyl-2-thioethyl)

monophosphate ester (5) was developed. Compound (5) had 40 to 155-fold-׳nucleoside 5 more antiviral efficiency compared with the parent nucleoside [123].

α-OH (6) led to-׳β-C-methyl and a 2-׳The combined modification including a 2

׳ identification of a broad class of potent nucleoside inhibitors. It is notable that the 2 - position with base β-orientation is important for the anti-HCV activity. Changing the

-β-C-׳α- dramatically reduces the antiviral activity. The 2-׳or to 2 -׳methyl group to 3 methylated nucleotides cannot be utilized by human polymerase, which provides a higher degree of selectivity towards the viral protein. Due to poor bioavailability of compound

׳ (6), 3 -O-L-valinyl ester analogue (valopicitabine, 7) was developed with the hope of taking advantage of peptide transporter systems. The pharmacokinetic properties of valopicitabine were not affected when it was used in combination with interferon [123].

Figure 8: Cytidine Derivatives with Ribose Modifications

In vitro studies showed the antagonist effect of ribavirin on the anti–HCV activity of valopicitabine by restraining the valopicitabine phosphorylation process. However, clinical trials were ultimately discontinued because of significant gastrointestinal toxicity.

α-׳C-methyl nucleosides, replacement of the α-hydroxyl group with a 2-׳Regarding 2 fluoro group produced unique HCV nucleoside inhibitors. Base variations were tested with this modified sugar and showed that the cytosine derivative (PSI-6130 (8)) had more potent activity than uridine or guanosine derivatives which were weak or in-active due to poor phosphorylation in the first step toward triphosphate formation. Again PSI-6130 (8) had poor oral bioavailability with a significant amount of the drug being metabolized to

-di-O-׳5 ,׳the inactive uridine metabolite. A prodrug (RG7128, mericitabine 9) with a 3 isobutyrate ester group was then developed. This oral prodrug exhibited clinical proof of concept when administered as monotherapy or in combination with the standard regimen.

It had broad genotype coverage, which was anticipated by preclinical results with a high barrier to resistance [123].

C-methyluridine monophosphate prodrug (deaminated form of-׳F-2-׳Sofosbuvir is a 2

(PSI 6130, 8) and is currently approved for the treatment of chronic HCV infection.

,deoxy-2 spirocyclopropylcytidine-׳position included 2-׳Another modification at the 2 compound 10 (TMC647078), as a nonobligate chain terminator of HCV NS5B

diisobutyrate -׳5 ,׳isobutyrate ester (11) and 3-׳polymerase with no cytotoxicity. The 3 ester (12) prodrugs were prepared to improve the low plasma concentration of (10).

The serine to threonine mutation (S282T), which occurs near the enzyme active site, led

modified nucleotides by reduced affinity of the-׳to reduction of the antiviral activity of 2 mutant polymerase for the drug and restored its ability for RNA synthesis from expansion 40

of the incorporated nucleosides, representing the main challenge during the designing of

-׳NIs. However, the affinity of cytosine derivatives toward this mutation was reported, 2

C-methylcytidine established a 36-fold lower affinity to the S282T mutant NS5B genotype than the wild type due to the presence of steric interaction between the methyl group of T287 during the elongation step. S282T was shown to be only 3-fold resistant to

(6).

To be active as inhibitors for HCV polymerase, the nucleoside derivatives must be

triphosphates) by the cellular kinases. The first step of-׳converted to their active forms (5 phosphorylation is problematic because many nucleosides are poor substrates for one or more of the kinases in the phosphorylation cascade. Therefore, in many cases, neglect of

monophosphate can lead to increases in-׳the first phosphorylation step by delivering the 5

monophosphates do-׳the intracellular levels of active triphosphate. However, nucleoside 5 not enter cells and are unstable (negatively charged) due to enzymatic dephosphorylation making them undesirable as drug candidates. For this purpose, prodrug strategies had been developed to deliver the nucleoside monophosphate. To be successful these prodrugs must bypass the first phosphorylation step and reach the liver intact after which

monophosphate group, facilitating further-׳intracellular hepatic enzymes expose the 5 metabolism to the active triphosphate.

One of the most extensive strategies in this field is the phosphoramidate prodrugs

-׳compounds 13, 14) which increase the cellular permeability of nucleotide 5) monophosphates by masking the phosphate group and its lipophilicity through attaching an aryloxy group and an amino acid ester.

׳ ׳ Other sugar modifications include 3 - deoxy nucleosides (compound 15) and 4 -azido

substituted nucleoside analog-׳nucleoside analogues. R-1479 (16) is an example of a 4 which acts as a cytidine triphosphate (CTP)-competitive inhibitor. After incorporation of

(16) as a ligand for NS5B polymerase, the azido group interferes with the chemical

OH group, and blocked further elongation of nascent RNA. In vitro studies-׳reaction of 3 with compound (16) showed more barriers toward the S282T mutant genotype. Since the azido group is long and can reach to S96 residue making compound (16) more susceptible to loss of antiviral activity with S96T mutation. R-1626 (17) an ester prodrug of (16), was developed to increase cellular permeability and oral bioavailability [123,

124, 129].

2.4.1.2 Base Modified NIs

Examination of novel base moieties within the simple furanose class led to the recognition of several active inhibitors of HCV. The advantages of heterobase modified nucleotide HCV inhibitors are the higher oral bioavailability and their superior in vivo pharmacokinetic profiles because of resistance to phosphorolysis by purine nucleoside phosphorylase (PNP). Deamination by adenosine deaminase (ADA), which is involved in purine metabolism, has an important role in decreasing the activity of other unmodified nucleotides inhibitors [129].

2.4.2 Non-nucleoside Inhibitors of NS5B (NNIs)

NNIs are non-competitive inhibitors for HCV polymerase. The binding of these compounds to the RdRp prevents conformational changes essential for polymerase activity. To date, five allosteric binding sites (I-V) have been identified and represent the target of NNIs. The I and II binding sites are located in the thumb subdomain (T1 lies close to the GTP non-catalytic binding site between thumb and fingertip regions, and T2 is located at the thumb-palm domain borders). The III and IV binding sites are located in the palm subdomain (P1 located at the top border of palm domain and P2 close to the active site) and site V or (P-β) site located in the palm subdomain involving a unique interaction with the β-hairpin extending from the thumb domain. However, NNIs are considered less potent than NIs due to their higher probability to loose their activity through mutations. For this reason, clinical development of most of NNIs has stopped

[129].

Dasabuvir is the first P1 HCV NNI to be approved for the treatment of patients with GT1 infection. Greater than 95% SVR rates were observed after 12 weeks with a combined regimen containing dasabuvir.

Table 1: NNIS and Their Corresponding Binding Sites [129]

Binding Sites Compound class Mutation

T1 a-Benzo imidazole P495L

b-Indole-6-carboxylate

T2 a-Thiophene L419M

b- 4-Quinolone

P1 a-Benzothiadiazine Y448H

b-Pyrolidine carboxylate M414T

c-Indole-2-carboxylate

d- 2-Quinolone

e-Rhodanine

P2 Benzofuran S365T

C316N

P-β Imidazopyridines C316Y

Y448H

Chapter 3

Results and Discussion

3.1 Overview on the Synthesis of Nucleoside Analogs

-β-C׳Several synthetic methodologies have been distinguished for the synthesis of 2 branched ribonucleosides. These can be separated into two broad strategic categories: the divergent strategy, which includes modification of the intact nucleosides by changing the carbohydrate portion; and the convergent method in which coupling of the nucleoside base with the carbohydrate derivative occurs at a suitable stage in the synthesis. Not all methods can be classified as either wholey divergent or convergent. Both methods can be used in combination to deliver the title compound [130]. Each method has advantages and limitations. The primary advantage of the divergent strategy is the ability to regulate of glyosidic stereochemistry. For example, it is either established by nature or can be

substituent. Naturally -׳controlled synthetically via the presence of an appropriate 2 occurring nucleosides (such as thymidine, cytosine, uridine, adenosine, and guanosine) which are limited in their availability, represent the starting substrates of the divergent approach and is considered the main disadvantage of this strategy [131].

On the other hand the convergent method of synthesis of nucleoside derivatives represents a more convenient and adaptable route due to the flexibility in the alterations

of both sugar and nucleoside base substrates. Additionally, since the nucleoside β anomers are believed to have greater biological activity against NS5B this strategy helps us in further assays and studies for antiviral activity. For these reasons, we chose the convergent approach. It has been shown that this approach facilitates the delivery of the nucleobase to the β-face of the sugar during N-glycosylation reactions with higher stereoselectivity [131].

Our synthetic plan can be divided into three main steps. (1) Synthesis of hydroxylmethyl ribose 21, (2) Synthesis of glycosyl donors 32, 34, and (3) Synthesis of hydroxymethyl cytidine 45 through N-glycosylation reaction and protecting group removal.

3.2 Design and Synthesis of 2-C-hydroxymethyl Ribose

As shown in Scheme 1, the synthesis of 2-C-hydroxymethyl ribose was performed starting from commercially available protected ribose sugar (18). This modified sugar

C-branched-׳was used previously as the starting sugar for the synthesis of analogous 2 nucleosides [132, 133]. Since it contains benzoyl ester groups at the 1, 3, 5 positions in the sugar ring, these protecting groups gave the desired compatibility, as well as the electronic and steric effects required in subsequent reactions in addition to a deprotection strategy that did not required harsh conditions [134].

The first step in our synthesis was the oxidation of the 2-hydroxyl group of modified sugar (18) with the Dess-Martin periodinane (DMP) derivative to the corresponding ketone (19) [135]. The alkene was synthesized via a Wittig reaction [136]. The resulting alkene (20) was converted to vicinal-diol (21) as a mixture of diastereoisomers. This was

achieved by reaction of (20) with catalytic amounts of osmium tetroxide (OsO4) in the presence of N-methylmorpholine N-oxide (NMO) and water [137].

Scheme 1: Synthesis of 2-C-Hydroxymethyl Ribose

3.2.1 Synthesis of 5-((benzoyloxy) methyl)-3-oxotetrahydrofuran-2, 4-diyl dibenzoate (19)

The first step of our synthesis was the oxidation of the secondary hydroxyl group at the 2- position of the modified sugar (18) to the corresponding ketone (19). There are different methods used for the oxidation of secondary alcohols to ketones. Some of these methods necessitate use of aqueous bases such as trimethylamine (Et3N) in the Swern oxidation

[138], or salts such as aluminum salts and silver carbonate used in the Oppenauer oxidation and Fétizon oxidation respectively [139, 140]. Other methods use acidified dichromate ions, agents such as potassium dichromate and pyridinium chlorochromate (PCC). These methods require use of harsh reaction conditions and result in the formation of undesired toxic byproducts [141]. 47

Oxidation using the Dess-Martin reagent involves milder reaction conditions, shorter reaction times and simplified work-up procedures. For these reasons, we chose this method in the oxidation of (18) to (19) [135].

We synthesized the Dess-Martin reagent in more than 95% yield in our laboratory using a two-step procedure according to published literature [135].

The oxidation step was performed according to previous work [132, 133], the yield of

(19) varied from 80-95% depending upon the age of the Dess Martin reagent.

3.2.2 Synthesis of 5-((benzoyloxy) methyl)-3-methylenetetrahydrofuran-2, 4-diyl dibenzoate (20)

After completion of the oxidation of (18) to (19), our next step involved conversion of 2- ketone (19) to alkene (20). Generally, two methods are usually used for the formation of terminal olefins. These are Wittig and Lombardo reactions. Each one is considered an alternative to the other in providing the desired alkenes depending on the nature of the substrates included in the reactions.

The Lombardo reaction involves the use of an organotitanium reagent (Scheme 2).

Synthesized by the addition of titanium tetrachloride (TiCl4) to a mixture of activated

o zinc dusts with dibromomethane (CH2Br2) in dry tetrahydrofuran (THF) at -40 C with stirring, followed by warming to 5 oC and continued stirring at this temperature for 3 days until formation of the active species (as a thick grey suspension) is achieved [142]. To ensure its reactivity, the reagent should be stored at -20 oC otherwise it undergoes decomposition.

Scheme 2: Lombardo Reagent Formation [142]

Despite several advantages to this reaction, including compatibility with different functional groups such as ethers, acetals, esters, carboxylic acids, alcohols [143, 144], and high reactivity with ketones at room temperature with subsets of ketone that enolize and epimerize at an adjacent chiral center, this method failed with our ketone. This can be attributed to two reasons. The optimum inert conditions required for the synthesis of the organometallic active species may not have been obtained. The preparation was repeated three times with the same result. Upon utilization in the olefination, all starting materials underwent decomposition and no alkene was collected. This led us to believe another explanation related to long reaction times with prolonged exposure to TiCl4 may be detrimental. Furthermore, (19) has multiple ester groups and may undergo hydrolysis with the Lombardo reagent, as a result this method was not suitable in our synthesis.

On the other hand, the Wittig reaction is one of the primary methods used for the synthesis of alkenes. The Wittig reagent was prepared by the action of base with easily accessible triphenylphosphonium halides (25) leading to the formation of alkylidene phosphoranes (26) (Scheme 3) [145]. This is a reversible reaction that necessitates inert and anhydrous conditions with aldehydes or ketones at low temperature. The geometry of

the resulting olefins (28) depends on the nature of the Wittig reagent and the experimental conditions [146].

Alkylidene phosphoranes ylide (Scheme 3) can be divided into three types: stabilized ylides, non-stabilized and semi-stabilized ylides, leading to the formation of E , Z and E/

Z mixture of alkenes, respectively [145].

Scheme 3: Wittig Reaction [145]

The choice of the base used in deprotonation of the organophosphorus salts and liberation of the Wittig reagent is a very important factor for the success of the reaction. Three bases were investigated in our synthesis, sodium methoxide CH3ONa, potassium tertiary

+ − butoxide K (CH3)3CO , and sodium bis (trimethylsilyl) amide NAHMDS

(CH3)3Si)2NNa. From the point of view of basicity and steric hindrance, we found that the best yield was obtained by using NAHMDS as the base. Decomposition of the sugar moiety occurred with CH3ONa due to basic hydrolysis of protecting ester groups.

An important advantage of the Wittig reaction is the fact that the carbonyl group is replaced by a carbon-carbon double bond, specifically, without formation of isomeric mixture in contrast to elimination reactions of alcohols and milder reaction conditions

[145].

3.2.3 Synthesis of 5-((benzoyloxy) methyl)-3-hydroxy-3- (hydroxymethyl) tetrahydrofuran-2, 4-diyl dibenzoate (21)

Our next step was the formation of hydroxymethyl protected ribose (21) by conversion of the resulting alkene to the vicinal diol. Primarily there are two methods used for this purpose. The first involves the use of potassium permanganate (KMnO4) with sodium hydroxide NaOH at low temperature [147]. This reaction is often used for identification

- of a double bond in a molecule by simple color change. Permanganate ion (MnO4 ) has a high oxidative potential and can lead to over-oxidation and oxidative cleavage [147, 148].

In addition, KMnO4 in stoichiometric amounts can lead to the formation of MnO2 byproducts that have to be removed by complicated work-up procedures [149].

The other reaction method involves osmium tetroxide (OsO4) as an oxidizing agent. This is considered more convenient for alkene dihydroxylation with high yield and few side products. OsO4 can be in used either in stoichiometric or catalytic amounts.

From the point of economy, toxicity and volatility of OsO4, we chose to use it in catalytic amounts in the presence of a secondary oxidizing agent such as: hydrogen peroxide, tert- butylhydroperoxide, N-methylmorpholine N-oxide (NMO), oxygen, sodium periodate, and sodium hypochlorite. The role of these co-oxidants is to hydrolyze the intermediate

osmium (VI) ester complex oxidatively and regenerate the osmium tetraoxide which can undergo further reduction by the substrate.

NMO is considered the preferred co-oxidant in this reaction since over oxidation problems have been shown to occur with other agents [150].

Alkene (20) (Scheme 4) was dissolved in pyridine, water, and t-butanol. OsO4 and NMO were added and the mixture reflux for 20 hrs at 76 oC.

There are two possible routes that the OsO4 can take for reaction with the sugar moiety.

OsO4 addition can take place on either the α-face or β-face of the molecule to deliver diastereomeres. This was the case with the synthesis of (21) which was obtained in 45% overall yield after purification with column chromatography as white foam.

The structure of the compound was confirmed by 1H NOE experiments.

Scheme 4: Dihydroxylation with Osmium Tetroxide[150]

3.3 Synthesis of Glycosyl Donors

3.3.1 Synthesis of 3-acetoxy-3-(acetoxymethyl)-5-((benzoyloxy) methyl) tetrahydrofuran-2, 4-diyl dibenzoate (32)

Upon synthesis of diol (21), it was necessary to protect both hydroxyl groups with suitable temporary protecting groups for subsequent glycosylation. These groups must provide the electronic and steric properties required for the formation of β-nucleoside stereoselectively. In addition, they should be compatible with existing protecting groups.

To act as a glycosyl donor, ester groups provide the desired properties to direct the formation of β-nucleosides. For this reason 1, 2 diol (21) was protected with acetyl ester groups. We found the best available method for the introduction of acetyl groups by addition of acetic anhydride with pyridine under inert conditions in the presence of 4-

Dimethylaminopyridine (DMAP) as a catalyst to diol (21) at room temperature (Scheme

5) [151].

Scheme 5: Synthesis of Glycosyl Donor 53

The time to completion of this reaction (Scheme 6) depends on the steric effects and number of equivalents of acetic anhydride and reacting diol (21). The primary hydroxyl group reacts quickly, as expected. Upon quenching the reaction, after 6 hrs, with methanol, a mixture of mono and diacetylated product was detected by mass spectrometry. By increasing the reaction time to 42 hrs, the tertiary hydroxyl group was fully protected. Purification with column chromatography (20% ethyl acetate in hexane) afforded the desired acetyl ester (32) as a mixture of diastereomeres with a 92% overall yield.

Scheme 6: Acetylation Reaction [151].

Like the diol, this mixture of diacetylated isomers (figure 9) could be not separated by normal column chromatography. In the subsequent coupling reaction with nucleobase

(47), the β-isomer (32b) is converted to a different oxonium ion intermediate that leads to the formation of an undesired product in which the cytosine base is attached to the methyl group at the 2-position. The required compound that results from α-isomer (32a) was also obtained. This compound has the same polarity as the by-product making their separation a challenge.

Figure 9: Diastereomeres of Acetylated Diol

3.3.2 Synthesis of 5-((benzoyloxy) methyl)-3-(((tert butyldimethylsilyl) oxy) methyl)-

3-hydroxytetrahydrofuran-2, 4-diyl dibenzoate (33)

Another method to synthesize the glycosyl donor involves the selective protection of the primary hydroxyl group of (21) and then protection of the tertiary hydroxyl with an ester group. A silyl ether is the preferred method for protection of groups for carbohydrates and is usually used in the protection of primary alcohols in the presence of secondary or tertiary hydroxyl groups. Silyl ethers have the general formula R1R2R3-Si-O-R4, the 55

silicone atom is covalently bound to an alkoxy group and thus more resistant toward hydrolysis than other groups and silyl ethers are conveniently stable in alkaline media.

The four classical forms of silyl ethers are triisopropylsilyl (TIPS), trimethylsilyl (TMS), tert-butyldiphenylsilyl (TBDPS) and tert-butyldimethylsilyl (TBDMS) [152]. We chose

TBDMS because of its higher selectivity for protection of primary hydroxyl groups, especially at low temperatures, in the presence of dry DMF and imidazole (Scheme 7)

[153]. Purification of the crude material on silica gel resulted in separation of the 1:1 diastereomeres and gave 70% total yield as white foam.

Further characterization of compound (33) by 2D-NOESY experiment and comparing the cross peaks between the protons at the 1 and 3 positions with protons of the methyl group at the 2 position, revealed the existence of a nuclear overhouser effect between 3 and the methyl protons with small a crosscoupling at the 1-position.

Scheme 7: Silylation Reaction with TBDMSCl [153]

3.3.3 Synthesis of 3-acetoxy-5-((benzoyloxy) methyl)-3-(((tert butyldimethylsilyl) oxy) methyl) tetrahydrofuran-2, 4-diyl dibenzoate (34)

The protection of the tertiary hydroxyl group of (33) was performed using the same procedure used for synthesis of compound (32). Compound (34) was obtained as white foam after purification with 10% ethyl acetate in hexane in an excellent yield of 95%.

3.4 Synthesis of Hydroxy Methyl Cytidine

The final step in our synthesis (Scheme 8) is the coupling of nucleobase (47) (N4-acetyl cytosine) with glycosyl donor (32) and (34). The most practical method uses

Vorbruggen-type persilylation conditions, in which the silylated nucleobase is formed from reaction with a suitable silylating agent like [1, 1, 3, 3-hexa-methyl disilazane

(HMDS)] to produce the desired nitrogen (N1) substitution product (49).

Glyosidic bond stereochemistry is the main challenge in the coupling reaction. To increase β-isomer selectivity, several factors should be considered and play determining roles for the success of this reaction. These include, along with the experience of the experimentalist, the reactivity of the glycosyl donor and acceptor, promotors, solvent, and reaction conditions [154].

The reactivity of the anomeric center depends, to a large degree, on the presence of the protecting groups especially those on C-2. Glycosyl donors can be divided in two main groups: armed donors with ester or ether protection on C-2 (as in the case of 32 and 34); and the less reactive disarmed donors without protecting groups on C-2.

Promotors are usually used in these reactions to activate the anomeric center of the glycosyl donor to facilitate nucleophilic displacement of the leaving group at the C-1 57

position by the silylated base. Promoters can be used in catalytic or stoichiometric amounts. The most popular promotors are Lewis acids with moderate acidity such as tin salts (SnCl4) and Trimethylsilyl trifluoromethanesulfonate (TMSOTF).

Due to water competition for hydrolysis of the silylated base, anhydrous solvents are required for use under inert conditions. However, in disarmed glycosyl donors, the solvents play an essential role in stereochemistry selectivity of the product through complex formation at C-2 and thus enhancing β-isomer production [154].

Scheme 8: Synthesis of Hydroxymethyl Cytidine [132] 58

3.4.1 Synthesis of 5-(4-acetamido-2-oxopyrimidin-1(2H)-yl)-4-acetoxy-4-

(acetoxymethyl)-2-((benzoyloxy) methyl) tetrahydrofuran-3-yl benzoate (43a)

The first step in the glycosylation reaction involves silylation of the oxygen atom at the

2-position of the protected cytosine base (47) through reaction with HMDS under high temperature for three hours. The diastereomeres of glycosyl donor (32) were dissolved in dry acetonitrile and transferred to the protected base (49) whereupon the promotor

(SnCl4) was added drop wise. After purification with column chromatography (43a and b) was afforded in 57% total yield as white solid.

Low and high resolution mass spectrometry revealed two major compounds (43a and b).

MS/MS fragmentation of compound (43a) (Figure 10) showed fragmentation at the glyosidic linkage indicating undesired compound (43b) along with our compound (43a).

Theoretically there are two possibilities in the coupling reaction, either α or β attack of the nucleobase. In our experience, we get selective β-isomer formation and these results can be explained according to the mechanistic pathway of the reaction.

As shown in (Scheme 9), Lewis acid (SnCl4) activation of the anomeric carbon in isomer

(32a) facilitates loss of the benzoyl substituent at C-1 leading to formation of the oxocarbenium ion intermediate (51a). The neighboring group effect of the acetate ester group at C-2 stabilizes this intermediate and bridges the oxonium ion (52a). The concave nature of (52a) blocks the bottom face of the ribose ring forcing nucleobase (49) to attack from the top face of C-1 leading to formation of β-compound (43a). However, compound

(34) undergoes the same mechanistic pathway as (32a) resulting in the formation of (44).

Figure 10: MS/MS of Compound 43a

Scheme 9: Glycosylation Reaction of Compound 43a

In case of the β-isomer (32b) (Scheme 10), SnCl4 complexes with the acetate group at C-

2 leading to formation of carbocation (51b) which is stabilized by participation of the second acetate group leading to formation of oxonium ion (52b). Following the

nucleophilic attack of the silylated base (49) at the CH2 of the oxonium ion, formation of

(43b) occurs.

Scheme 10: Glycosylation Reaction of Compound 43b

The last step involves removing all protecting groups in a single step. The best method involves the stirring of our protected cytidine compounds with saturated methanolic ammonia solution overnight at room temperature. The resulting compounds (45) and (46)

(Scheme 8) are very polar and hard to purify with normal column chromatography and will be used without purification.

Chapter 4

Conclusions and Future Work

4.1 Conclusions

Direct acting antiviral agents (DAAs) provide the more convenient results in clinical trials, in terms of effective inhibitory concentration and mutational analysis making them promising future therapies for HCV infection. Targeting the conserved active site of the polymerase enzyme responsible for viral RNA synthesis has opened the door to development of several nucleoside analogues with potent inhibitory effects.

Determination of structure-activity relationships (SAR) and mutational analysis with different amino acid substitutions in the HCV polymerase as well as the binding behavior

position substitution in-׳pattern of substrate compounds reflects the essential role of 2 chain termination of viral protein synthesis and breakdown of the viral life cycle.

hydroxymethyl-׳We developed a new synthetic pathway toward the synthesis of 2 cytidine as a potential antiviral compound in satisfactory yield taking advantage of the

-׳cytosine base specificity to the viral enzyme, while the presence of the steric group at 2 position will disrupt the hydrogen bond network essential for enzymatic activity.

This compound is hypothesized to have powerful activity similar to other molecules that have undergone clinical trials with nano-inhibitory concentrations and pan genotyping 64

activity. In addition, these drug candidates can be used in interferon and ribavirin free regimens, due to the fact that use of combination therapies are associated with serious side effects and drug-drug interactions related to multiple tissue damage which occur in chronic infection.

4.2 Future Work

Conditions will be optimized for the synthetic steps with low yield such as the pathway to alkene (20) and diol (21). Changing the temperature and solvent may be options. We will proceed with the remainder of the synthetic route to obtain a compound suitable for oligonucleotide synthesis, and testing that compound in available replicon assays.

The remainder synthetic pathway will be planned on the deprotected hydroxymethyl cytidine (45). We will seek to increase the cellular concentration of the nucleotides through tri-phosphate use. This active form escapes the first rate-limiting step of phosphorylation by cellular kinases. Therefore, the protection of 5ꞌ-hydroxyl group will be done in the presence of phosphoramidate precursor’s compound (57 and 58) to afford compound (59 and 60). Further modifications to enhance the steric requirements will be

position with suitable-׳done including oxidation of the primary hydroxyl group at the 2 oxidizing agent. Suitable steric bulky groups will be added at 2ꞌ to give as our desired compound. We are confident that the prodrugs (63, 64, 65, and 66) will be successfully synthesized (Scheme 11).

Scheme 11: Future Modifications of Hydroxymethyl Cytidine

Chapter 5

Experimental procedures

Unless indicated, the organic reactions requiring inert conditions were performed under argon in oven dried glassware using anhydrous solvents.

5.1 Materials

All reagents and anhydrous solvents were purchased from the following suppliers: Sigma

Aldrich, Arcos Organic, Fisher Scientific and CHEM-IMPEX INT’L INC. The reagents were used as obtained, without further purification unless otherwise noted.

NMR deterurated solvents were purchased from Cambridge Isotope Laboratories and

HPLC grade solvents were used for chromatographic separations.

5.2 Structural Analysis

We characterized our synthesized compounds by NMR spectroscopy and mass spectroscopy.

5.2.1 NMR Analysis

5.2.1.1 1H-NMR

Figure 11: Numbering System and Hydrogen Atoms

A: Sugar, B: Nucleoside

1H-NMR spectra were recorded on Varian VXRs 400, Varian Inova 600 or Bruker

Avance 600 NMR spectrometers in DMSO and CDCl3. Chemical shifts were reported in ppm relative to DMSO or CDCl3 as (internal references). Coupling constants (J) are reported in hertz (Hz). Multiplicity is as follows: s = singlet, d = doublet, dd = doublet of doublet, t = triplet, q = quartet, m = multiplet. The protons of the carbons of the furanose ring are designated as shown in Figure 8.

5.2.1.2 13C-NMR

13C-NMR spectra were recorded onVarian Inova 600 or Bruker Avance 600 NMR spectrometers in DMSO and CDCl3. Chemical shifts were reported in ppm using DMSO and CDCl3 as internal references.

5.2.2 Mass Spectrometry

5.2.2.1 ESI-MS

Electrospray Ionization Mass spectrometry was performed using an Esquire Ion Trap

Mass Spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a quadrupole ion-trap mass analyzer. The mass spectrometer was reported in positive ion mode with methanol used as a solvent for most analytes. The [M+H]+ or [M+Na]+ ions signals were obtained.

5.2.2.2 High Resolution Mass Spectroscopy

The high resolution mass data was obtained through the Department of Chemistry, in the

University of Toledo, by using Micromass Q ToF II mass spectrometer.

5.3 Chromatographic Methods

5.3.1 Thin Layer Chromatography (TLC)

We used both glass backed and aluminum backed silica gel plates to monitor the progress of our reactions and the content of our flash chromatography fractions. TLC spots were visualized using a UV lamp at 254 nm. Taking advantage of the presence of carbonyl

groups in our compounds, the plates were stained with a p-anisaldehyde dip and heated to facilitate spot visualization. The p-anisaldehyde dip was made of absolute ethanol, concentrated sulfuric acid and glacial acetic acid in a ratio of 18:1:0.2 with a few drops of p-anisaldehyde.

5.3.2 Flash Chromatography

For steps requiring chromatographic purification, flash chromatography was carried out using a regular glass column packed with chromatographic silica gel (200-425 MESH).

The solvents used for the purification were either ethyl acetate in hexane or methanol in

DCM with different gradients. Before fractions were concentrated, TLC was performed to confirm purity.

5.4 Other Equipment and devices

High vacuum pump – Edwards RV3

Rotary evaporator – Heidolph Collegiate Brinkmann rotary evaporator

Thermal mixer – Eppendorf Thermomixer

Vortex mixer – Fisher Scientific

Pipettes- Eppendorf Series 2100

5.5 Synthesis of 4-amino-1-(3, 4-dihydroxy-3, 5-bis (hydroxymethyl) tetrahydrofuran-2-yl) pyrimidin-2(1H)-one

5.5.1 5-((benzoyloxy) methyl)-3-oxotetrahydrofuran-2, 4-diyl dibenzoate (19)

Ketone (19) was prepared according to previous published work [132], yield 1.81 g,90% as a white foam.

5.5.2 5-((benzoyloxy) methyl)-3-methylenetetrahydrofuran- 2,4-diyl dibenzoate 20

To a stirred suspension of methyltriphenylphosphonium bromide (1.54 g,4.2 mmol) in anhydrous ether (96 ml) at room temperature under argon was added (1.88 ml,3.7 mmol) of sodium bis(trimethylsilyl)amide(NAHMDS) 2M solution in THF, the resulting light- yellow mixture was stirred for 6 h at room temperature, then a solution of (19) (1.0 g,2.1 mmol) in un-hydrous ether (3 ml) was added to a cooled reaction mixture at -10 OC and allowed to stirred at this temperature for 2 hrs, washed with equal volume with brine three times, dried with MgSO4 prior to removal of the solvent and concentrated, the crude material was purified with column chromatography (10% ethyl acetate in hexane), and gave (0.62g, 62%) of (20) as off-white syrup. 1H NMR (DMSO): δ 7.99 (m, 6H), 7.60 (m,

3H), 7.54 (m, 6H), 6.87 (s,1H, H-1), 5.97 (m, 1H, H-3), 5.84 (d, 2H, J=24 Hz,

13 methylene), 4.90 (q,1H, J1=7.2 Hz, J2=12 Hz, H-4), 4.57 (m, 2H, H-5). C NMR δ 64.22,

74.18, 82.62, 98.21, 118.74, 128.7, 129.57, 129.74, 129.95, 130.03, 130.06, 130.09,

133.43, 133.59, 133.75, 143.84, 165.88, 166.27, 166.34. HRMS (M+Na): C27H22O7Na calc. 481.1263, found 481.1267.

5.5.3 5-((benzoyloxy)methyl)-3-hydroxy-(hydroxymethyl)tetrahydrofuran-2,4-diyl dibenzoate 21

To a mixture of (20) (0.4 g ,0.87 mmol), pyridine (0.9 ml), H2 O (0.9 ml) and (17 ml) of t- butanol was added N-methylmorpholine N-oxide (706 mg,6.02 mmol) and osmium tetra oxide OsO4 (50 uL of a 2.5% solution in t-butanol,3.86 umol), the reaction mixture was stirred at 76O C for 20 hrs then allowed to cooled to r.t , the reaction was quenched with

20% aqueous solution of sodium bisulfite (1 ml). The mixture was concentrated under reduced pressure prior to dilution with H2O (5 ml) and a saturated aqueous solution of sodium chloride (5 ml) and extracted with ethyl acetate (3× 20 ml). The combined organic phase was dried with (MgSO4) and evaporated under reduced pressure. The resulting crude material was purified by column chromatography with ethyl acetate in hexane (0-25% gradients), and gave (21) as a mixture of (1:1) diastereomeres (0.18 g,

45% as overall yield) as a white crystals. 13C NMR δ 63.73, 64.16, 64.75, 64.80, 64.82,

65.41, 78.06, 79.30, 79.93, 80.35, 82.60, 82.92, 83.12, 86.53, 99.16, 101.75, 102.75,

128.61, 128.66, 128.67, 128.71, 128.8, 128.82, 129.14, 129.18, 129.36, 129.48, 129.53,

129.71, 129.80, 129.86, 129.88, 129.93, 129.95, 129.98, 130.04, 130.07, 130.11, 130.18,

133.40, 133.43, 133.52, 133.58, 133.64, 133.90, 133.94, 134.10, 165.23, 165.95, 166.47,

166.56, 166.62, 166.75, 166.89, 167.12, 167.70. HRMS (M+Na): C27H24O9Na calc.

515.1318, found 515.1317.

5.5.4 3-acetoxy-3-(acetoxymethyl)-5-((benzoyloxy) methyl) tetrahydrofuran-2, 4- diyl dibenzoate 32

To a solution of (21) (0.13g, 0.26 mmol) in dichloromethane (DCM) (2 ml) under argon, pyridine (0.05ml, 0.624 mmol), acetic anhydride (Ac2O) (0.054 ml, 0.58 mmol) and 4-

Dimethylaminopyridine (DMAP) (0.16 mg, 1.3 ×10-3 mmol) was added, the solution was stirred at r.t. for 48 hrs, upon completion the mixture was diluted with methanol (4 ml) prior to evaporation under reduced pressure. The crude product passed through silica gel with 20% ethyl acetate in hexane, and gave (1:1) diastereomeres mixture of (32) as a white foam (0.119g, 92% yield). 13C NMR δ 20.94, 21.15, 21.31, 21.79, 59.40, 59.57,

61.10, 63.98, 64.00, 64.99, 75.83, 76.28, 79.04, 83.74, 83.91, 84.62, 89.17, 89.24, 96.08,

98.12, 98.33, 128.54, 128.59, 128.71, 128.81, 128.87, 128.89, 128.93, 129.31, 129.33,

129.35, 129.39, 129.80, 128.83, 129.85, 129.89, 129.96, 130.03, 130.08, 130.12, 133.40,

133.42, 133.44, 133.58, 133.64, 133.67, 133.97, 134.05, 164.41, 165.14, 165.42, 165.94,

165.97, 166.15, 166.36, 168.90, 168.92, 169.32, 169.44, 169.93, 169.98. HRMS (M+Na):

C31H28O11Na calc. 599.1529, found 599.1546.

5.5.5 Synthesis of 5-((benzoyloxy)methyl)-3-(((tert butyldimethylsilyl)oxy)methyl)-3- hydroxytetrahydrofuran-2,4-diyl dibenzoate 33

Compound (21) (1 gm, 2003 mmole) was dissolved in dry DMF (5 ml) in three neck flask purged with argon atmosphere. To this solution, imidazole (350 mg, 5.075 mmole) and TBDMSCl (370 mg, 2.43 mmole) were added quickly at 0 oC and stirring overnight.

After completion, 1N ammonia solution (5 ml) was added and the organic layer was extracted with 3×20 ml diethyl ether and then washed with brine solution prior to filtration with MgSO4 and removal of the solvent in rotovap.

The pure material was obtained through column chromatography purification (10% ethyl acetate in hexane) to afford (0.8 gm, 1.32 mmole, 65% yield) of (33) as a 1:1

1 diastereomere as a white foam. H NMR (CDCl3) δ: 8.08 (m, 6H), 7.56(m, 3H), 7.42 (m,

6H), 5.48 (s, 1H, H-1), 5.43 (d, 1H, J=3Hz, H-3), 4.83 (d, 1H, J=12Hz, -CH2), 4.77 (d,

1H, J=12Hz, -CH2), 4.75 (m, 2H, H-5), 4.58 (m, 1H, H-4), 3.45 (s, 1H, OH), 0.93 (s, 9H),

0.17 (s, 6H). 13C NMR δ -5.32, -4.10, 18.01, 25.74, 25.82, 64.39, 64.76, 79.26, 82.84,

83.19, 102.98, 128.50, 128.58, 128.65, 128.70, 129.20, 129.68, 129.81, 129.88, 128.92,

129.95, 129.99, 130.02, 130.17, 133.31, 133.40, 133.78, 165.86, 166.49, 166.90. HRMS

(M+Na): C33H38O9Na Si calc. 629.2183, found 629.2173.

5.5.6 Synthesis of 3-acetoxy-5-((benzoyloxy)methyl)-3-(((tert- butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2,4-diyl dibenzoate 34

Compound (34) was synthesized with the same procedure used for synthesis of (32).

Column chromatography purification of crude material with 10% ethyl acetate in hexane

1 afforded 95% yield of (34) as white foam. H NMR (CDCl3) δ: 7.99 (m, 6H), 7.56 (m,

3H), 7.42 (m, 6H), 5.92 (s, 1H, H-1), 5.74 (d, 1H, J=4.2Hz, H-3), 5.29 (d, 1H,J=12.6Hz, -

CH2), 5.03 (d, 1H, J=12.6 Hz, -CH2), 4.78 (m, 1H, H-4), 4.62 (m, 1H, H-5), 4.54 (m, 1H,

13 H-5), 2.07 (s, 3H), 0.95 (s, 9H), 0.19 (s, 6H). C NMR δ -5.42, -4.51, 17.85, 21.56,

25.56, 60.25, 64.33, 81.70, 90.29, 99.89, 128.26, 128.28, 128.44, 129.12, 129.49, 129.74,

129.76, 130.07, 132.94, 133.44, 165.12, 165.76, 166.13,169.86. ESI-MS (M+Na):

C35H40O10 Na Si calc. 671.24, found 671.23.

5.5.7 Synthesis of 5-(4-acetamido-2-oxopyrimidin-1(2H)-yl)-4-acetoxy-4-

(acetoxymethyl)-2-((benzoyloxy) methyl) tetrahydrofuran-3-yl benzoate 43a

To 4N-acetyl cytosine (330 mg, 2.15 mmole) in 3-neck flask purged with argon, was added 6 ml of HMDS (28.6 mmole) and 2 mg of ammonium sulphate. The suspension was brought to reflux for 3 hrs at 135 oC until all solid dissolved. Excess HMDS was removed in rotovap while the system was evacuated with argon three times. Compound

(32) (0.5 gm, 0.86 mmole) was dissolved in anhydrous acetonitrile (10 ml) prior to addition to the protected cytosine base. SnCl4 (0.5 ml, 4.423 mmole) was added dropwise with stirring at room temperature. After 44 hrs, the reaction was diluted with 50 ml ethyl acetate. NaHCO3 (50ml) was added with continuous stirring until effervescence ceased.

The organic layer was extracted with ethyl acetate, washed with brine and dried over

MgSO4 and concentrated.

The crude material was purified with column chromatography (10-80% gradient of ethyl acetate in hexane) to afford (0.3 gm, 57% total yield) of mixture from (43a and b) as white solid.

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Appendix A

Supplemental Information

5.60 5.95 1.00 1.97 1.03 2.01

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

5.60 2.84 5.95 1.00

8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8

0.99 1.97 1.03 2.01

6.1 6.0 5.9 5.8 5.7 5.6 5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1

Figure 12: H-NMR of Compound 20

Figure 13: ESI-MS of Compound 20

KR_AHH02_03212016_2 68 (1.173) AM (Cen,4, 80.00, Ar,8000.0,578.26,0.70) TOF MS ES+ 481.1267

100 1.24e4 %

* 578.2591

550.6269

482.1300

497.1031

551.6348 579.2607

483.1376 498.1042 552.6390 481.0010 484.0896 499.1061 522.5992 580.2728 550.5178 557.2758 480.1322 497.0089 500.9888 515.0254 523.5985 530.9763 564.6536 577.2562 581.1653 471.2029 542.9644 587.1818 0 m/z 470 475 480 485 490 495 500 505 510 515 520 525 530 535 540 545 550 555 560 565 570 575 580 585 590

Figure 14: HRMS of Compound 20

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 -20

Figure 15: C-13 NMR of Ccompound 20

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 -20

Figure 16: C-13 NMR of Compound 21

100

Figure 17: ESI-MS of Compound 21

101

KR_AHH03_03212016 55 (0.952) AM (Cen,4, 80.00, Ar,8000.0,578.26,0.70); Cm (44:56) TOF MS ES+ 515.1317 6.70e4 100 *

578.2591 %

579.2637

516.1376

550.6299

500.9958 551.6339 580.2661 514.9072 518.2062 531.1064 537.1165 556.2788 577.9261 519.1654 531.0063 582.9970 597.0074 501.9996 547.1171 568.9827 594.2308 509.0378 538.1162 557.2806 571.8521 584.9116 0 m/z 500 505 510 515 520 525 530 535 540 545 550 555 560 565 570 575 580 585 590 595 600

Figure 18: HRMS of Compound 21

102

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0

Figure 19: C-13 NMR of Compound 32

103

Figure 20: ESI-MS of Compound 32

104

KR_AHH04_03212016 54 (0.935) AM (Cen,4, 80.00, Ar,8000.0,578.26,0.70); Cm (48:66) TOF MS ES+ 599.1546

100 3.73e3 %

600.1641

* 578.2591

601.1841

579.2628

599.0216 602.1628 580.2606 594.2399 569.3499 571.3784 577.3481 591.0436 595.2489 598.7469 603.2783 606.3810 575.4672 581.3694581.9441 585.2549 587.2936 590.4041 593.3358 605.3751 0 m/z 570 572 574 576 578 580 582 584 586 588 590 592 594 596 598 600 602 604 606

Figure 21: HRMS of Compound 32

105

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 -20

Figure 22: C-13 NMR of Compound 33

106

KR_AHH05_06202016_2 19 (0.340) AM (Cen,4, 80.00, Ar,8000.0,556.28,0.70); Cm (1:19) TOF MS ES+ * 3.11e3

100 556.2771 % 629.2173

557.2782

630.2200

624.2617

558.2788 631.2278 556.1949 578.2531

541.2283 559.1833 589.2219 594.2234 618.2031 579.2440 601.2427 632.2464 556.1328 610.1943 613.3030 638.2544 543.3052 560.1498 571.2526 583.3672 0 m/z 540 545 550 555 560 565 570 575 580 585 590 595 600 605 610 615 620 625 630 635 640

Figure 23: HRMS of Compound 33

107

6.97 6.71 1.07 2.10 1.07 9.59 6.68 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

6.97 3.67 6.71

8.15 8.10 8.05 8.00 7.95 7.90 7.85 7.80 7.75 7.70 7.65 7.60 7.55 7.50 7.45 7.40 7.35

1.00 0.99 1.07 0.98 2.13 1.24

5.50 5.45 5.40 5.35 5.30 5.25 5.20 5.15 5.10 5.05 5.00 4.95 4.90 4.85 4.80 4.75 4.70 4.65 4.60 4.55 4.50

Figure 24: H-NMR of Compound 33

108

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 -20

Figure 25: C-13 NMR of Compound 34

109

6.31 6.26 1.001.02 0.90 1.03 1.10 3.21 9.35 5.83

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

6.31 3.15 6.26

8.20 8.15 8.10 8.05 8.00 7.95 7.90 7.85 7.80 7.75 7.70 7.65 7.60 7.55 7.50 7.45 7.40 7.35

1.00 1.02 0.90 1.03 1.08 1.10 1.08

6.3 6.2 6.1 6.0 5.9 5.8 5.7 5.6 5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2

Figure 26: H-NMR of Compound 34

110

Figure 27: ESI-MS of Compound 34

111

2.83 6.8412.78 21.51 1.89 2.27 1.00 2.92 4.80 7.02

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5

2.83 6.84 12.78 9.48 21.51 1.89 9.5 9.0 8.5 8.0 7.5 7.0

2.92 1.33 4.80 2.21 5.32 7.02 3.15

5.1 5.0 4.9 4.8 4.7 4.6 2.25 2.20 2.15 2.10 2.05

Figure 28: H-NMR for Compound 43a

112

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0

Figure 29: C-13 NMR of Compound 43a

113

KR_AHH07_06202016 107 (1.836) AM (Cen,4, 80.00, Ar,8000.0,556.28,0.70); Cm (95:108) TOF MS ES+ * 7.93e4

100 556.2771 %

557.2826

608.1886

609.1935 558.2855

555.9186 610.1940 550.6312 559.7404 613.2996 540.4675 565.1985 570.2819 578.2633 581.3661 594.2352 608.0260 620.2480 555.5322 587.5843 596.3090 601.2794 615.3019 0 m/z 540 545 550 555 560 565 570 575 580 585 590 595 600 605 610 615 620 625

Figure 30: HRMS of Compound 43a

114

Figure 31: ESI-MS of Compound 43a

115

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10

Figure 32: C-13 NMR of Compound 44

116

1.23 11.55 21.03 2.44 1.59 0.92 1.00 1.241.31 3.00 3.34 1.32 6.72 10.03 5.83

9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

1.23 11.55 21.03 2.44 1.59 0.92 1.00

8.5 8.0 7.5 7.0 6.5 6.0

1.24 1.31 3.00 3.34 1.32 6.72 3.54

5.0 4.5 4.0 3.5 3.0 2.5 2.0

Figure 33: H-NMR of Compound 44

117

Figure 34: ESI-MS of Compound 44

118

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0

Figure 35: C-13 NMR of Compound 45

119

Figure 36: ESI-MS of Compound 45

120

1.01 1.06 3.21 1.12 1.001.05 1.07 1.10 1.06 0.37 1.01 2.24 0.98 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0

1.01 0.36 0.49 0.98 1.06 0.39 3.21 1.12

8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6

1.00 1.05 0.96 1.07 1.10 1.06 0.97 0.37 1.01 2.24 0.98

5.5 5.0 4.5 4.0 3.5 Figure 37: HNMR for Compound 45

121