SYNTHESIS OF A-GALCERAMIDES, (-)-TREPROSTINIL, AND
`
`DESIGN AND SYNTHESIS OF ANTI-VIRAL AGENTS
`
`BY
`
`KUANQIANG GAO
`B.S., Fudan University, China, 1997
`M.S., University of Illinois at Chicago, 2002
`M.B.A., University of Illinois at Chicago, 2004
`
`THESIS
`
`Submitted as partial fulfillment of the requirements
`for the degree of Doctor of Philosophy in Chemistry
`in the Graduate College of the
`University of Illinois at Chicago, 2006
`
`Chicago, Illinois
`
`Liquidia's Exhibit 1019
`IPR2020-00770
`Page 1
`
`
`
`DESIGN AND SYNTHESIS OF ANTI-VIRAL AGENTS
`
`BY
`
`KUANQIANG GAO
`B.S., Fudan University, China, 1997
`M.S., University of Illinois at Chicago, 2002
`M.B.A., University of Illinois at Chicago, 2004
`
`THESIS
`
`Submitted as partial fulfillment of the requirements
`for the degree of Doctor of Philosophy in Chemistry
`in the Graduate College of the
`University of Illinois at Chicago, 2006
`
`Chicago, Illinois
`
`Liquidia's Exhibit 1019
`IPR2020-00770
`Page 1
`
`
`
`UMI Number: 3218919
`
`INFORMATION TO USERS
`
`The quality of this reproduction is dependent upon the quality of the copy
`
`submitted. Broken or indistinct print, colored or poor quality illustrations and
`
`photographs, print bleed-through, substandard margins, and improper
`
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`
`In the unlikely event that the author did not send a complete manuscript
`
`and there are missing pages, these will be noted. Also, if unauthorized
`
`copyright material had to be removed, a note will indicate the deletion.
`
`UMI
`
`UMI Microform 3218919
`
`Copyright 2006 by ProQuest Information and Learning Company.
`
`All rights reserved. This microform edition is protected against
`
`unauthorized copying under Title 17, United States Code.
`
`ProQuest Information and Learning Company
`300 North Zeeb Road
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`
`Liquidia's Exhibit 1019
`IPR2020-00770
`Page 2
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`
`
`THE UNIVERSITY OF ILLINOIS AT CHICAGO
`Graduate College
`CERTIFICATE OF APPROVAL
`
`March 9, 2006
`
`I hereby recommend that the thesis prepared under my supervision by
`KUANQIANG GAO
`
`SYNTHESIS OF A-GALCERAMIDES, (-)- TREPROSTINIL, AND DESIGN
`
`entitled
`
`AND SYNTHESIS OF ANTI-VIRAL AGENTS
`
`be accepted in partial fulfillment of the requirements for the degree of
`DOCTOR OF PHILOSOPHY
`
`f
`
`Adviser (Chairperson of Defense Committee)
`
`(/ department Head/Chair
`
`Members of
`
`Thesis or
`
`Dissertation
`
`Defense
`
`Committee
`
`I concur with this recommendation
`
`ion concurred in:
`
`2'"
`
`/y '
`
`I 11^^ University of Illinois
`at Chicago
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`Liquidia's Exhibit 1019
`IPR2020-00770
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`
`
`This thesis is dedicated to
`
`my parents, Xiqing Gao, Qiao'e Ma
`
`and my wife Peiying and daughter Kathyrn
`
`for their love and unwavering support
`
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`ACKNOWLEDGEMENT
`
`I would like to express my sincerest gratitude and appreciation to my advisor
`
`Professor Robert M. Moriarty for his academic and financial support over the past years.
`
`He has provided me with research environment enabling me to learn and conduct the
`
`chemical research described in this thesis and I greatly appreciate his guidance.
`
`I would like to thank the member of my thesis committee, Professor Kassner,
`
`Professor Morrison, Professor Bums, and Dr. Anderson for their helpfiil discussions.
`
`I would like to thank all current and former members of Moriarty's group for their
`
`support and friendship, particular Dr. Rajesh Naithani, Dr. Harpreet Kaur, Dr. Grubjesic
`
`Simonida, Dr. Wendy Hirschelman, Dr. Anca Hirtopeanu, and Mr. Same Idene.
`
`Thanks also goes to Patricia Ratajczyk, Rhonda Staudohar, Silvia Solis, Tayna
`
`Ray, Olyer Anderson, Brian Schwandt, Don Rippon from Chemistry Department and
`
`Helen Georgas from Science Library for their help during my stay at UIC.
`
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`
`
`CHAPTER
`
`PAGE
`
`TABLE OF CONTENTS
`
`1
`
`INTRODUCTION
`1.1
`Introduction: Viral Hepatitis
`1.1.1 Hepatitis B and Hepatitis B Virus
`1.1.2 Hepatitis C
`Develop New Anti-Viral Drug
`
`1.2
`
`2.2
`
`2. DEOXYNOJIRIMYCIN (DNJ) DERIVATIVES AS ANTI-HEPATITIS
`AGENTS
`2.1
`Iminosugars as Antiviral Agents
`2.1.1
`Iminosugars as Glycosidase Inhibitor
`2.1.2 Antiviral Mechanism of DNJ and its Derivatives
`Design and Synthesis of New DNJ derivatives
`2.2.1 Synthesis of N-Substituted Derivative of DNJ
`2.2.2 Synthesis of 6-0-Alkylated Derivative of DNJ
`Structure-Activity Relationship (SAR) of anti-Hepatitis Activities of
`Iminosugars
`2.3.1 Anti-Hepatitis Assay and Toxicity Assessment
`2.3.2 Anti-Hepatitis Activities of DNJ Derivatives
`2.3.3 SAR Study of anti-Hepatitis activity of DNJ Derivatives
`
`2.3
`
`3. fl//j/ifl-GALACTOSYL CERAMIDE AS anti-HEPATITIS AGENTS
`3.1
`a-Glycolipids and Its Antiviral Activity
`3.1.1 a-Galactosyl Ceramide
`3.1.2 NKT cells Activation Mechanism
`Design and Synthesis of New Glycolipids
`3.2.1 Synthesis of Enantiomers of 77jreo-Dihydrosphingosine
`3.2.2 Synthesis of a-Galactosyl Amide Derivative of DHS
`Antiviral Activity of Glycolipids
`
`3.2
`
`3.3
`
`4. EXPERIMENT SECTION
`
`CITED LITERATURE
`
`5. INTRODUCTION
`5.1
`Discovery and Biological Properties of KRN 7000
`5.2
`Previous Syntheses of KRN 7000
`5.2.1 Koezuka et al's Synthesis of KRN 7000
`5.2.2 Mori et al's Synthesis of KRN 7000
`5.2.3 Schmidt et al's Synthesis KRN 7000
`5.2.4 Wong et al's Synthesis of KRN 7000
`5.2.5 Savage et al's Synthesis of KRN 7000
`5.2.6 Synthesis of Derivatives of KRN 7000
`Summary of Previous Synthesis of KRN 7000
`
`5.3
`
`iv
`
`1
`1
`2
`10
`13
`
`16
`16
`17
`19
`26
`27
`28
`
`30
`30
`31
`36
`
`38
`39
`39
`39
`42
`43
`45
`48
`
`49
`
`82
`
`96
`96
`98
`99
`101
`103
`104
`105
`107
`113
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`CHAPTER
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`PAGE
`
`TABEL OF CONTANTS (continue)
`
`6. RESULTS AND DISCUSSION
`6.1
`Retrosynthetic Analysis
`6.2
`Synthesis of KRN 7000 starting from L-serine
`6.2.1 Preparation of Phytosphingosine by opening of Serine-derived
`115
`Epoxide
`6.2.2 Synthesis of Phytosphingosine by Addition of Dithiane to Serinal...117
`6.2.3 Preparation of Galactosyl Donor 110
`119
`Synthesis of KRN 7000 starting from o-lyxose
`120
`Summary of the Synthesis of KRN 7000
`123
`
`114
`114
`115
`
`6.3
`6.4
`
`7. SYNTHESIS OF STREOISOMER OF KRN 7000 AND ITS ANTIVIRAL
`ACTIVITY
`124
`7.1
`Introduction
`124
`7.2
`Synthesis of KGA76
`125
`7.3
`Summary of the Synthesis of KGA76
`128
`
`8. EXPERIMENT SECTION
`
`CITED LITERATURE
`
`9. INTRODUCTION
`9.1
`Biological Properties of Prostacyclin and its Analogues
`9.2
`Previous Synthesis of Prostacyclin Analogues
`9.2.1 Aristoffe? a/5 Synthesis of Prostacyclin Analogue U-60959...
`9.2.2 Aristoff e? a/'.s Synthesis of U-68215 as Antiulcer Agent
`9.2.3 Fuchs et al's Synthesis of Benzindene Prostacyclin
`9.2.4 Moriarty et al's Synthesis of (+)-Treprostinil
`Summary of Previous Synthesis of Treprostinil
`
`9.3
`
`10. RESULTS AND DISCUSSION
`10.1 Retrosynthesis of (-)-Treprostinil
`10.2 Synthesis of (-)-Treprostinil
`10.3 Summary of the Synthesis of (-)-Treprostinil
`
`11. EXPERIMENT SECTION
`
`CITED LITERATURE
`
`VITAE
`
`130
`
`182
`
`168
`168
`173
`173
`175
`Ill
`179
`181
`
`182
`184
`192
`
`193
`
`211
`
`217
`
`V
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`LIST OF SCHEMES
`
`SCHEME
`
`PAGE
`
`Scheme 1 Model of Transition State of Glucosidase Hydrolyzing of Glucoside and Its
`
`Inhibitor Iminosugars
`
`Scheme 2 Processing of Oligosaccharide Chains of N-linked Glycoprotein
`
`Scheme 3 Synthesis of N-alkyl-deoxynojirimycin (DNJ)
`
`Scheme 4 Protection of DNJ to 29
`
`Scheme 5 Synthesis of 6-0-nonyl-DNJ
`
`Scheme 6 Synthesis of 77?reo-Dihydrosphingosine 28
`
`Scheme 7 Preparation of Galactosyl Donor 44
`
`Scheme 8 Synthesis of a-Galactosyl Ceramides from DHS 33
`
`Scheme 9 Synthesis of D-^/zreo-dihydrosphingosine and Its a-Galactosyl Ceramides
`
`Derivatives 54
`
`Scheme 10 Koezuka's Total Synthesis of KRN 7000
`
`Scheme 11 Mori's Total Synthesis of KRN 7000
`
`Scheme 12 Schmidt's Total Synthesis of KRN 7000
`
`Scheme 13 Wong's Total Synthesis of KRN 7000
`
`Scheme 14 Savage's Synthesis of KRN 7000
`
`Scheme 15 Koezuka's Synthesis of AGL-597
`
`Scheme 16 Koezuka's Synthesis of AGL-592
`
`18
`
`21
`
`27
`
`28
`
`29
`
`43
`
`45
`
`46
`
`47
`
`100
`
`102
`
`103
`
`104
`
`106
`
`107
`
`108
`
`Scheme 17 Bonin's Synthesis of Novel Fluorescent BODIPY a-Galactosylceramide..l09
`
`Scheme 18 Savage's Synthesis of Fluorophore-Appended 6"-Amino-6'-deoxy-lactosylc~
`
`- e r a m i d e
`
`I l l
`
`vi
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`LIST OF SCHEMES (continued)
`
`SCHEME
`
`PAGE
`
`Scheme 19 Retrosynthetic Analysis of KRN7000
`
`Scheme 20 Preparation of Protected Serinal 101
`
`Scheme 21 Addition of Vinyl Grignard to Serinal 101
`
`Scheme 22 Addition of Dithiane to Serinal 101
`
`Scheme 23 Synthesis of D-Galactosyl Imidate 110
`
`Scheme 24 Chain Elongation and Azide Introduction
`
`Scheme 25 Galactosylation with Imidate to Form Galactoside 118
`
`Scheme 26 Amidation and Completion of Synthesis
`
`Scheme 27 Preparation of Protected D-lyxose
`
`Scheme 28 Chain Elongation and Azide Formation
`
`Scheme 29 Complete Synthesis of KGA 76
`
`Scheme 30 Mechanism of acid catalyzed hydrolysis of PGI2
`
`Scheme 31 Aristoff et al's Synthesis of U-60959
`
`Scheme 32 Aristoff et al's Synthesis U-68215
`
`Scheme 33 Fuchs et al's Synthesis of Benzindene Prostacyclin Analogues
`
`Scheme 34 Moriarty et al's Synthesis of (+)-Treprostinil
`
`Scheme 35 Retrosynthetic Analysis for (-)-Treprostinil
`
`Scheme 36 Asymmetric Reduction of Alknyl Ketone 166
`
`Scheme 37 Asymmetric Pauson Khand Cyclization
`
`Scheme 38 Proposed Transition State of PKC
`
`Scheme 39 Reduction of Enone and TBSO Ether Removal
`
`vii
`
`114
`
`116
`
`117
`
`118
`
`120
`
`121
`
`122
`
`123
`
`125
`
`126
`
`127
`
`169
`
`174
`
`175
`
`178
`
`179
`
`182
`
`184
`
`185
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`186
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`187
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`LIST OF SCHEMES (continued)
`
`SCHEME
`
`Scheme 40 Hydroxyl Configuration Inversion
`
`Scheme 41 Complete Synthesis of (-)-Treprostinil
`
`PAGE
`
`188
`
`190
`
`viii
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`
`
`LIST OF FIGURES
`
`FIGURE
`
`PAGE
`
`Figure 1 Geographic Distribution of Chronic HBV Infection
`
`Figure 2 Genome of Hepatitis B Virus (HBV)
`
`Figure 3 HBV Envelope Proteins with N-Glycan Attaching Sites
`
`Figure 4 Mechanism of Different Treatment Methods
`
`Figure 5 Structures of Hepatitis B and C's Drugs
`
`Figure 6 Geographic Distribution of HCV Infection
`
`Figure 7 Structure of the RNA Genome of HCV and Protein Encoded
`
`Figure 8 Structures of New Antiviral Agents
`
`Figure 9 Structures of Some Iminosugars
`
`Figure 10 Calnexin/Calreticulin Binds to Monoglucosylated Glycoprotein
`
`Figure 11 Structure of N and 0-alkyl-deoxynojirimycin
`
`2
`
`3
`
`4
`
`5
`
`8
`
`10
`
`11
`
`14
`
`17
`
`23
`
`26
`
`Figure 12 Ligand-specificity of NKT Cells Compared with Conventional T cells...
`
`40
`
`Figure 13 Structures of Some Sphingolipids
`
`Figure 14 Transition State of Grignard Addition to Anhydride
`
`Figure 14 The Stereochemistry of NaBH4 Reduction of Ketone 39
`
`Figure 15 Structures of a-Galactosyl and a-Galactosyl Ceramide of DHSs
`
`Figure 16 Structures of Agelasphins and KRN7000
`
`Figure 17 Structural Features of KRN 7000
`
`Figure 18 Structure of KRN 7000 and Its Stereoisomer KG A 76
`
`Figure 19 Structures of PGI2 Analogues
`
`Figure 20 Structures of Benzindene Prostacyclins
`
`42
`
`44
`
`44
`
`47
`
`96
`
`98
`
`124
`
`171
`
`173
`
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`LIST OF FIGURESS (continued)
`
`FIGURE
`
`Figure 21 Proposed Transition State for CBS Reduction
`
`Figure 22 Equilibration of 180 in ethanolic sodium hydroxide
`
`Figure 23 X-ray Structure of 190
`
`PAGE
`
`184
`
`189
`
`191
`
`X
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`
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`LIST OF TABLES
`
`Table 1 N-Alkyl (with branch)-DNJ's anti-HBV and BVDV Activities
`
`Table 2 N-Cycloalkyl-DNJ's anti-HBV and BVDV Activities
`
`Table 3 N-Oxaalkyl-DNJ's anti-HBV and BVDV Activities
`
`Table 4 Antiviral activity of a-Galactosyl and a-Galactosyl Ceramides
`
`31
`
`33
`
`35
`
`48
`
`Table 5 The IH-NMR Spectrum Comparison of KRN 7000 and KGA 76
`
`129
`
`xi
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`
`
`LIST OF ABBREVIATIONS
`
`Ac
`
`Bn
`
`acetyl
`
`benzyl
`
`BOP
`
`benzotriazol-1 -yl-oxytris(dimethylaniino)phosphonium
`
`Hexafluorophosphate
`
`br
`
`broard (NMR)
`
`BVDV
`
`bovine viral diarrhea virus
`
`Bz
`
`C
`
`CC50
`
`Calcd
`
`CSA
`
`5
`
`d
`
`benzoyl
`
`concentration
`
`50% cytotoxic concentration
`
`calculated
`
`10-camphorsulphonic acid
`
`chemical shift in parts per million downfield from tetramethylsilane
`
`doublet
`
`DBU
`
`l,8-diazabicyclo[5.4.0]undec-7-ene
`
`dd
`
`ddd
`
`doublet of doublets
`
`doublet of doublets of doublets
`
`DDQ
`
`2,3-dichloro-5,6-dicyano- p-benzoquinone
`
`de
`
`diastereometric excess
`
`DEAD
`
`diethyl azodicarboxylate
`
`DIAD
`
`diisopropyl azodicarboxylate
`
`DIBAL
`
`diisopropyl aluminum hydride
`
`DMD
`
`dimethyldioxirane
`
`xii
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`
`LIST OF ABBREVIATIONS (continued)
`
`DMF
`
`dimethyl formamide
`
`DMSO
`
`dimethyl sulfoxide
`
`DNJ
`
`ER
`
`FDA
`
`1 -deoxynorj irimycin
`
`endoplasmic reticulum
`
`Food and Drug Administration
`
`aGalCer
`
`a-galactosyl ceramides
`
`HOBt
`
`A^-hydroxybenzotriazol
`
`HRMS
`
`high resolution mass spectroscopy
`
`Hz
`
`IC50
`
`IR
`
`J
`
`Hertz
`
`inhibition concentration, 50%
`
`infrared
`
`coupling constant (NMR)
`
`LAH
`
`lithium aluminum hydride
`
`m
`
`m
`
`M
`
`micro
`
`multiplet (NMR)
`
`molar
`
`m-CPBA
`
`meta-chloroperoxybenzoic acid
`
`Me
`
`MHz
`
`min
`
`methyl
`
`megahertz
`
`minute (s)
`
`MOM
`
`methoxymethyl
`
`m.p.
`
`melting point
`
`xni
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`
`LIST OF ABBREVIATIONS (continued)
`
`Ms
`
`M/z
`
`N
`
`methanesulphonyl
`
`mass to charge ratio (in mass spectrometry)
`
`nano
`
`NB-DNJ
`
`N-butyl-1 -deoxynorjirimycin
`
`NKT cells
`
`natural killer T cells
`
`HMPA
`
`hexamethylphosphorus amide
`
`NMR
`
`nuclear magnetic resonance
`
`NN-DNJ
`
`N-nonyl-l-deoxynorjirimycin
`
`NOE
`
`nuclear overhauser effect
`
`Ph
`
`Piv
`
`PMB
`
`PPA
`
`PPTS
`
`iPr
`
`q
`
`s
`
`phenyl
`
`pivoly
`
`/?ara-methoxybenzyl
`
`joara-tolunesulphonate
`
`pyridinium P-toluenesulfonates
`
`isopropyl
`
`quartet
`
`single
`
`SAR
`
`structure and activity relationship
`
`t
`
`triplet
`
`TBAF
`
`tetabutylammonium fluroride
`
`TBS (TBDMS) terf-butyldimethylsilyl
`
`TFA
`
`trifluroacetic acid
`
`XIV
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`
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`THF
`
`TIPS
`
`tetrahydrofuran
`
`triisopropylsilyl
`
`TMGN3
`
`tetramethylguanidinium azide
`
`WSC'HCl
`
`l-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride
`
`XV
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`
`
`SUMMARY
`
`This thesis describes the design and synthesis of new anti-hepatitis agents and the
`
`total synthesis of two biological active molecules, (-)-Treprostinil, KRN 7000 and its
`
`stereoisomer.
`
`Part one of this thesis presents the design, synthesis and SAR study of two classes
`
`of new anti-hepatitis agents, DNJ derivatives and alpha-galactosyl ceramides. Naturally
`
`occurring iminosugar DNJ was modified by alkylation at 1-N and 6-0 positions to
`
`generate a library of new DNJ derivatives, which were subjected to anti-viral assay. The
`
`biological data showed that antiviral efficacy and toxicity of our new N-alkylated DNJ
`
`derivatives are better than their leading counterparts reported in literature. Further
`
`variation the substituents on nitrogen led to the discovery of the lead compound 8-
`
`methoxyoctyl-DNJ as a potential drug. Four glycolipids have also been prepared and
`
`their antiviral activity has been evaluated. SAR studies have determined several key
`
`structural features that are responsible for the antiviral activities.
`
`Part two of the thesis describes the total synthesis of a biologically important a-
`
`galactosyl ceramide (KRN 7000). D-lyxose has been employed as a starting material and
`
`the synthesis has been accomplished in 10 steps with 14.9% total yield. A stereoisomer of
`
`KRN 7000, named KGA 76, with the galactosyl group on the adjacent secondary
`
`hydroxyl, has also been prepared using a similar route. The antiviral assay shows that
`
`KGA 76 possesses very weak activity against hepatitis B/C virus.
`
`Part three of this thesis describes the sjTithesis of (-)-Treprostinil. The synthesis
`
`uses commercially available intermediate and employs stereoselective Pauson Khand
`
`cyclization to generate the tricyclic core. The stereo-directing TBDMSO group has been
`
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`selectively removed by hydrogenation. Inversion of the stereochemistry of hydroxyl on
`
`side chain was achieved with Mitsunobu reaction.
`
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`
`
`PART ONE: DESIGN, SYNTHESIS AND SAR STUDIES OF
`ANTI-HEPATITIS AGENTS
`
`CHAPTER 1
`
`INTRODUCTION
`
`1.1 Introduction: Viral Hepatitis
`
`Hepatitis is a liver disease caused by bacterial or viral infection, parasitic
`
`infestation, alcohol, drugs, toxins, or transfiasion of incompatible blood. Since the liver is
`
`the largest organ in the human body performing many vital functions, damage to liver
`
`cells can impair these functions and even lead to death. Hepatitis varies in severity from a
`
`self-limited condition with total recovery to a life-threatening or life-long disease. No
`
`matter what the cause of hepatitis, it can take either an acute (short term) or chronic form.
`
`In some cases, acute hepatitis develops into a chronic condition, but most cases of
`
`hepatitis are caused by viruses that infect liver cells and begin replicating. The different
`
`forms of hepatitis are designated by the letters A through G: ^
`
`• Hepatitis A, B, and C are the most common types of viral hepatitis. Among them,
`
`hepatitis A spreads through contaminated food and water and does not cause
`
`chronic liver disease, while hepatitis B and C infect via blood and may lead to
`
`long-term, persistent infections and chronic hver diseases that have lethal
`
`consequences.
`
`• Like hepatitis A, hepatitis E is caused by contact with contaminated food or water,
`
`and it is not serious except in pregnant women when it can be life threatening.
`
`Hepatitis G is always chronic and shares the same modes of transmission as
`
`hepatitis C, but to date, it does not appear to have serious effects.
`
`1
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`Liquidia's Exhibit 1019
`IPR2020-00770
`Page 20
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`2
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`1.1.1 Hepatitis B and Hepatitis B Virus
`
`Hepatitis virus (HBV), which was discovered in 1966, infects more than 400
`
`million people worldwide and an estimated 1.25 million persons in the United States are
`
`currently infected.^ Although people chronically infected with HBV live in all parts of the
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`world, HBV infection is especially endemic in Asia, the South Pacific Region, sub-
`
`Saharan Africa, in certain indigenous population groups residing in the Arctic (Alaska,
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`Greenland, and Northern Canada), Australia, New Zealand, and populations in South
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`Africa and Mid-East Asia.^ The prevalence of hepatitis C is also considerably higher in
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`developing countries, such as parts of Africa and the Mid-East of Asia.^ (Figure 1)
`
`HBsAg Prevalence
`• ^8%-High
`2-7% • Mermedlate
`S <2%-Low
`
`Figure 1. Geographic Distribution of Chronic HBV Infection'^
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`Liquidia's Exhibit 1019
`IPR2020-00770
`Page 21
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`3
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`Virologic Characteristics
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`Hepatitis B virus belongs to a family of DNA viruses called hepadnaviruses. It is
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`a partially double stranded circular DNA that is approximately 3200 base pairs in length
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`and has four overlapping reading frames. These encode several viral products: S for
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`surface gene, C for core, X for the x gene, and P for DNA polymerase. ^ The S and C
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`genes have upstream regions termedpreS andpreC. (Figure 2)
`
`n
`
`^
`
`i
`
`1^
`
`Figure 2. Genome of Hepatitis B Virus (HBV) (The EcoRI restriction-enzyme binding
`
`site is included as a reference point and the size of each segment is shown in parentheses,
`
`aa = amino acids)^
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`The DNA polymerase is a large reading frame of approximately 2500 base pairs
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`that acts as a conventional DNA polymerase but also serves a reverse transcription
`
`function for RNA intermediates. When HBV enters the hepatocytes, the genome moves
`
`to the nucleus and is converted to covalently closed circular DNA (cccDNA), which is
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`transcribed and translated to form an RNA intermediate. This is RNA intermediate can
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`Liquidia's Exhibit 1019
`IPR2020-00770
`Page 22
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`4
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`move to the cytoplasm, where the virus polymerase uses reverse transcription to convert
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`it to a new circular DNA. The virus polymerase is the site of action of the new reverse
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`transcriptase inhibitors that are used to treat chronic HBV infection.^
`
`The viral envelope encoded by the S gene contains three distinct configurations
`
`synthesized in all persons infected with hepatitis B, termed the large, middle, and small
`
`proteins, which are produced by beginning transcription with, respectively,
`
`or the S gene alone. The L protein consists of three domains: the preSl domain, preSl
`
`domain, and the S domain. M contains the preS2 and S domains while the S protein
`
`contains only the S domain. All three proteins have a common N-linked glycosylation
`
`site at Asn-146 of the S domain with the M protein containing an additional site at Asn-4
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`of the preS2 domain. The Asn-146 glycan site is partially occupied in all three envelope
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`proteins while the preS2 site is fully occupied in M but unoccupied in the L protein. The
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`Asn-4 site on M has been shown to interact with calnexin of endoplasmic reticulum
`
`(ER).^ (Figure 3)
`
`pmSl
`
`pr«S2
`
`L HB« ^
`
`MHBS
`SHBt
`
`»
`
`J
`
`I
`
`Figure 3. HBV Envelope Proteins with N-Glycan Attaching Sites^
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`Liquidia's Exhibit 1019
`IPR2020-00770
`Page 23
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`Current Treatment of Chronic Hepatitis B
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`Acute hepatitis needs no treatment other than careful monitoring of liver function
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`by measuring serum transaminases and prothrombin time.^ However, chronic hepatitis B
`
`5
`
`requires extensive medical treatment.
`
`frifcctums
`HBV virion
`
`L^mivuiJine I
`
`Pse^'cnoiiiic
`mRNAf+1
`
`truerfimi
`
`]
`
`mRNA y
`
`VW
`
`cccDKA
`
`Figure 4. Mechanism of Different Treatment Methods.^"
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`1. Interferon alpha
`
`Interferon alpha-2b (IFN-a-2b) was approved in the United States (1992) as a
`
`therapy for chronic HBV infection. IFN exerts an antiviral effect on infection with HBV
`
`through two mechanisms.'^ First, EFN has a direct antiviral effect by inhibiting synthesis
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`of viral DNA and by activating hepatocellular mechanism(s) that prevent the formation of
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`replication-competent pregenomic RNA-containing capsids (Figure 4), thus, inhibit HBV
`
`replication antiviral enzymes. Second, IFN enhances the cellular immune response
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`against hepatocytes infected with HBV by increasing the expression of class I
`
`histocompatibility antigens and by stimulating the activity of helper T lymphocytes and
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`natural killer lymphocytes.
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`12
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`Interferons are effective for the treatment of chronic HBV
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`Liquidia's Exhibit 1019
`IPR2020-00770
`Page 24
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`
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`Current Treatment of Chronic Hepatitis B
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`Acute hepatitis needs no treatment other than careful monitoring of liver function
`
`by measuring serum transaminases and prothrombin time.^ However, chronic hepatitis B
`
`5
`
`requires extensive medical treatment.
`
`MBV virion
`
`i^mivudina j
`
`PregcmMriic
`uiRNA[+l
`
`mRNA ^
`
`WV
`
`4
`
`intetfcmn }
`
`cecDNA
`
`Figure 4. Mechanism of Different Treatment Methods/"
`
`1. Interferon alpha
`
`Interferon alpha-2b (IFN-a-2b) was approved in the United States (1992) as a
`
`therapy for chronic HBV infection. IFN exerts an antiviral effect on infection with HBV
`
`through two mechanisms.^' First, IFN has a direct antiviral effect by inhibiting synthesis
`
`of viral DNA and by activating hepatocellular mechanism(s) that prevent the formation of
`
`replication-competent pregenomic RNA-containing capsids (Figure 4), thus, inhibit HBV
`
`replication antiviral enzymes. Second, IFN enhances the cellular immune response
`
`against hepatocytes infected with HBV by increasing the expression of class I
`
`histocompatibility antigens and by stimulating the activity of helper T lymphocytes and
`
`natural killer lymphocytes.
`
`Interferons are effective for the treatment of chronic HBV
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`Liquidia's Exhibit 1019
`IPR2020-00770
`Page 25
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`infection, particularly when patients are carefully selected/^ Long-term follow-up studies
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`to demonstrate improvement in survival or prevention of cirrhosis have been limited.'"^
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`Interferons have significant side effects, including flu-like symptoms; fever; myalgia;
`
`mild bone marrow suppression; thyroid abnormalities in 2% to 5% of patients; and
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`psychiatric side effects, such as depression, in approximately 15% of patients.'^
`
`2. Pegylated Interferon
`
`More recently, the efficacy of IFN has improved with the replacement of standard
`
`interferon by IFN conjugated with polyethylene glycol (PEG IFN). This new form of IFN
`
`reduces elimination of interferon by the kidneys, thus significantly increasing its half-life
`
`and resulting in more stable plasma concentrations of interferon. Finally the number of
`
`injections has been reduced from thrice to once weekly, thanks to improved
`
`pharmacokinetics, which is obviously more comfortable for the patient. Side effects
`
`associated with PEG IFN were comparable to those observed with standard interferon
`
`and the safety profile of PEG IFN was comparable to that of conventional interferon with
`
`the same frequency of adverse events.'^
`
`3. Lamivudine
`
`Lamivudine (1), an enantiomer of 3'-thiacytidine, was first used to treat HIV
`
`infection and was approved for the treatment of HBV infection in 1999. Since HBV
`
`replicates through an RNA template, the DNA polymerase resembles retroviral reverse
`
`transcripases. Lamivudine, along with other nucleotide analogues, competitively inhibits
`
`viral reverse transcriptase and terminates proviral DNA chain extension (Figure 4).'^
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`Liquidia's Exhibit 1019
`IPR2020-00770
`Page 26
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`Unlike interferon, lamivudine and the other newer nucleoside analogues do not affect the
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`1 8
`host immune response. Another advantage of lamivudine over interferon alpha-2b
`
`includes oral administration, high degree of tolerability, and safety in patients with
`
`decompensated cirrhosis. Lamivudine can be used as first-line therapy or following
`
`interferon failure. A more recent study showed that lamivudine also is effective in
`
`children with chronic HBV infection.
`
`4. Adefovir Dipivoxil
`
`Adefovir dipivoxil (2) has been recently registered for the treatment of chronic
`
`hepatitis B. Adefovir dipivoxil is the oral prodrug of adefovir. Adefovir is a nucleotide
`
`analogue of adenosine monophosphate. In vivo, adefovir dipivoxil is converted to the
`
`parent compound, adefovir, and through two phosphorylation reactions to adefovir
`
`diphosphate, the active intracellular metabolite that interacts with HBV polymerase.
`
`Adefovir diphosphate acts as a competitive inhibitor and chain terminator of viral
`
`replication. Because of its tolerability and oral route of administration, it is likely that
`
`adefovir dipivoxil will supplant interferon alpha-2b (IFN-a-2b) in most patients.^^
`
`5. Entecavir
`
`Entecavir (3) was approved by the FDA for therapy of chronic hepatitis B in early
`
`2005. Entecavir is a cyclopentyl guanosine analogue and is phosporylated to its
`
`triphosphate, the active compound, by cellular kinases. Entecavir inhibits HBV
`
`replication more effectively than lamivudine or adefovir.
`
`In addition to mterfenng with
`
`HBV polymerase-transcriptase chain prolongation, entecavir also inhibits the base
`
`Liquidia's Exhibit 1019
`IPR2020-00770
`Page 27
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`8
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`priming of HBV polymerase. In head-to-head studies comparing lamivudine to entecavir,
`
`the latter was superior in normalization of aminotransferase (ALT) and in improvement
`
`of histology and suppression of HBV DNA in both hepatitis B e antigen (HBeAg) -
`
`positive and HBeAg-negative patients with chronic hepatitis
`
`X
`N
`
`N
`
`J~^)
`O
`
`H,N
`
`NH2
`
`o
`
`Lamivudine (1)
`
`Adefovir Dipivoxil (2)
`
`N
`! >
`N
`
`HoN
`
`N
`H
`
`HO
`Entecavir (3)
`
`H0^^\^^ N T
`
`V/OH
`
`Hd
`
`'oh
`
`Ribavirin (4)
`
`Figure 5. Structures of Antiviral Agents and Nucleoside and Nucleotide Analogues
`
`for Hepatitis B and C Treatment
`
`6. Combination Therapy
`
`In the past few years, a number of excellent clinical trials have been carried out
`
`using various combinations of these agents. Although much has been learned, but
`
`significant challenges still remain. To date, the results of combinations of interferon and
`
`Liquidia's Exhibit 1019
`IPR2020-00770
`Page 28
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`9
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`nucleoside/nucleotide analogues have been disappointing.^^ The combination of adefovir
`
`with lamivudine was also carried out based on the hypothesis that the combination would
`
`maximize the viral suppression and would decrease the occurrence of viral resistance.
`
`Two studies have been performed and no combination therapy demonstrated a benefit as
`
`compared with monotherapy, respectively.
`
`Summary of Current Treatment of Chronic Hepatitis B
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`haterferon and nucleotide analogues have been successfully applied to chronic
`
`hepatitis B management, the majority of patients being treated with these therapies still
`
`do not reach a satisfactory clinical end-point, at least in the short term. For interferons,
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`high cost and numerous side effects, and tolerance are problems for many patients,
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`hiterferon is not effective in all patients. Literferon therapy is successful only in patients
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`with active immune responses, but the results are still unpredictable. Despite patients
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`selection, only 30% to 40 % have achieved a sustained response. Unlike interferon,
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`these nucleoside analogues do not affect the host immune response and can be
`
`administered orally. However, the drug resistance occurs after a period of treatment with
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`nucleoside analogues and the virus recurs in almost all cases, although this recurring
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`mutation may be weaker than the original strain.'^
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`Liquidia's Exhibit 1019
`IPR2020-00770
`Page 29
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`10
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`1.1.2 Hepatitis C
`
`Hepatitis C is caused by hepatitis C virus (HCV), which was identified as the
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`major etiological agent responsible for post-transfusion non-A and non-B hepatitis in
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`1989.^' There are more than 4 million people in the United States have been infected,
`
`making it the Nation's most common blood-borne disease, resulting in the deaths of
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`between 10,000 and 12,000 people each year. Approximately 170 million people
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`worldwide are infected with the hepatitis C virus (HCV), and are epidemic in countries of
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`Asia and Europe and South Afiica. Patients of HCV infection are at risk of progressive
`
`hepatic fibrosis,
`
`cirrhosis and death fi"om
`
`liver failure, as well as the advent of
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`hepatocellular carcinoma. The adverse consequences of chronic hepatitis C are usually
`
`not evident for at least 20 years following infection, leaving large numbers of patients at
`
`particularly high risk for the life threatening consequences to this disease.^"^
`
`-/.j
`
`' 1 *
`
`5".
`
`• >»%
`•: Mo ijaia awilaMe
`
`HCV PfevaJesnce, I9S7
`
`Figure 6. Geographic Distribution of HCV Infection
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`Liquidia's Exhibit 1019
`IPR2020-00770
`Page 30
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`11
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`Nature of Hepatitis C Virus
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`HCV is a spherical enveloped virus of approximately 50 nm in diameter and
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`belongs to the family Flaviviridae, which consists of three genera: flaviviruses,
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`pestiviruses, and hepaciviruses.^^ Hepatitis C virus (HCV) genetic material - a single
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`stranded linear RNA of positive sense of 9.5 kb in size^^ - is surrounded by a protective
`
`shell of protein, and further encased in a lipid (fatty) envelope of cellular material. The
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`fact that the genetic information of the virus is stored in RNA, not DNA, has important
`
`consequences in the life cycle of the virus, and gives hepatitis C its dangerous ability to
`
`mutate. The HCV viral RNA itself can be directly read by the host cell's ribosome,
`
`functioning like the normal rtiRNA present in the cell. This means hepatitis C requires
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`only a small amount of RNA to encode its core information, and thus has lots of room for
`
`genetic variation within the non-essential portions of its RNA. This also gives it fewer
`
`common characteristics that can be readily identified by the immune system or exploited
`
`by scientists working to create a treatment.
`
`Core
`\
`
`Envelope-
`
`NS4A
`
`E2
`E1
`-- Structural Proteins --
`
`P7 NS2
`
`NS4B NS5A
`NS3
`Non-structural proteins
`
`NS5B
`
`Figure 7. Structure of the RNA Genome of HCV and Protein Encoded^^
`
`The genome of HCV is directly translated after uncoating of the particle and
`
`encodes a single polyprotein precursor of approximately 3,000 amino acids. This
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`polyprotein is cleaved post-translationally into multiple structural and nonstructural (NS)
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`peptides: structural components consisting of a nucleocapsid core (C) and 2 envelope
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`Liquidia's Exhibit 1019
`IPR2020-00770
`Page 31
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`glycoproteins (El and E2) and the NS proteins being labeled NS2 through NS5.^^
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`(Figure 7) The specific functions of the individual NS proteins have not been completely
`
`elucidated. NS3 has both helicase and protease activities, and the NS5 region contains the
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`RNA dependent polypeptides.^^ (Figure 7) Like HBV, the HCV surface envelope
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`proteins (El and E2) are highly modified by N-linked glycans dur
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