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Graduate Theses and Dissertations Graduate School

April 2018

Synthesis of Novel Agents for the treatment ofInfectious and Neurodegenerative diseasesBenjamin Joe EdufulUniversity of South Florida, beduful@mail.usf.edu

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Scholar Commons CitationEduful, Benjamin Joe, "Synthesis of Novel Agents for the treatment of Infectious and Neurodegenerative diseases" (2018). GraduateTheses and Dissertations.http://scholarcommons.usf.edu/etd/7148

Synthesis of Novel Agents for the treatment of Infectious and Neurodegenerative diseases

by

Benjamin Joe Eduful

A dissertation submitted in partial fulfilment

of the requirements for the degree of

Doctor of Philosophy

Department of Chemistry

College of Arts and Sciences

University of South Florida

Major Professor: James W. Leahy, Ph.D.

Edward Turos, Ph.D.

Jianfeng Cai, Ph.D.

Nicholas J. Lawrence, Ph.D.

Yu Chen, Ph.D.

Date of Approval:

March 6, 2018

Keywords: Infectious diseases, Leishmania donovani, promastigotes, amastigotes, parasite,

chaperone proteins, Alzheimer’s disease, slingshot homology-1, Cofilin, protein phosphatases.

Copyright © 2017, Benjamin Joe Eduful

Dedication

To the loving memories of Emelia and Peter

Acknowledgements

Attaining this height has been nothing short of a great miracle - given the many challenges along

the way. Indeed without the help of my God, in whom I live, move and have being, I am certain I

could not have made it to this far. So I am profoundly grateful to God for bringing me to this place.

I am grateful to Prof. James W. Leahy, my mentor and advisor. I had the privilege of working on

two (2) interesting projects within the Leahy lab, which have helped me gain many versatile skills,

concepts and techniques including the critical thinking required for success as an

organic/medicinal chemist. Throughout the period of my graduate studies, Prof. Leahy provided

the much needed guidance, continuous support and encouragement to enable me to reach my full

potential. These have nurtured my growth and provided me with the confidence I need as a

medicinal and synthetic organic chemist. Indeed it has been a great honor to learn from your

profound insight - one that I will cherish for many more years to come.

I also had the privilege of having great minds serve on my academic committee: To Professors

Edward Turos, Jianfeng Cai, Nicholas J. Lawrence and Yu Chen, I say THANK YOU for helping

me progress through my academic work and research. I will forever cherish the mentorship, caring,

valuable suggestions and guidance during the course of my studies.

To my fellow graduate students in the Leahy lab: Ankush Kanwar, Linda Barbeto, Zachary Schultz,

Jeanine Yacoub, Andrea Lemus and Elena Bray - I really appreciate the wonderful ideas, fun and

jokes we shared together. I have grown in my knowledge of organic synthesis because I worked

alongside you. Thank you all so much. To my undergraduate researchers: Catherine Costa, Melissa

Chin, Arianna Rashedi, Ousman Jallow and Marylin Medina, you were astounding researchers and

I am very grateful for all your dedication, commitment and assistance in helping me reach most of

my goals in research.

I am also grateful to our collaborators: Prof. Dennis Kyle and his team (Dr. Brian Vesely, Tina

Mutka and Ala Azhari), and Prof. David Kang and his postdoctoral student, Dr. Xingyu Zhao, for

obtaining the biological data for our projects.

I remain thankful to my wife, Dr. Mrs. Sanita Boateng Eduful, for her prayers, emotional and moral

support throughout the course of my studies. Sani, your help contributed to the completion of this

manuscript, and I love you for what you do.

I am also grateful to my family: my Mum, Mrs. Akosua Foaah Eduful, my brothers, Alexander,

Michael, Thompson, James and my sisters, Joyce and Naomi Eduful for their prayers, financial,

moral and emotional support in encouraging me throughout my time as a student.

To the Boateng family, particularly Dr Ofori and Mrs Nana Boateng, words alone are not enough

to express my heartfelt gratitude to you all for your help and support. I cannot sign off without

mentioning some individuals who contributed in diverse ways to helping me finish strong. Rev.

Dr. Karl and Mrs Gifty Badu, Rev. Samuel and Mrs Joyce Koomson, Mr Yaw and Mrs Mary

Afram, Mrs. Mary Sakyi-Amfo, Mrs. Vida French and Eld. Emmanuel Robertson.

Thank you all and God bless!

i

Table of Contents

List of Abbreviations .................................................................................................................... iv

List of Tables ................................................................................................................................ vi

List of Figures .............................................................................................................................. vii

List of Schemes ...............................................................................................................................x

List of Graphs .............................................................................................................................. xii

Abstract……. .............................................................................................................................. xiii

Chapter 1 Introduction ............................................................................................................1

1.1 The threat of infectious diseases ..............................................................................1

1.2 Leishmaniasis ...........................................................................................................2

1.2.1 Leishmaniasis life cycle ..................................................................................3

1.2.2 Forms of leishmaniasis ...................................................................................5

1.3 Current treatment and vaccine development............................................................6

1.4 Compound screening (high thoughput screening) .................................................10

1.5 Heat shock proteins ................................................................................................10

1.5.1 Heat shock protein 90 (Hsp 90) as anti-parasitic drug targets ...................... 11

1.5.2 Hsp 90 in Leishmania ...................................................................................12

1.6 Biological assays ....................................................................................................14

1.7 List of references....................................................................................................15

Chapter 2 Indazole Analogs ..................................................................................................22

2.1 Drug design and medicinal chemistry strategies ...................................................22

2.2 Synthesis of initial analog ......................................................................................25

2.2.1 Synthesis of initial test compound 2.008 ......................................................25

2.2.2 Computational modelling strategies .............................................................28

2.2.3 Synthesis of methyl-substituted test compound ...........................................30

2.3 Results and discussions ..........................................................................................33

2.4 Experimental section ..............................................................................................36

2.5 List of references....................................................................................................46

ii

Chapter 3 Tetrahydroquinoline analog ................................................................................50

3.1 Tetrahydroquinoline scaffolds in biologically active molecules ............................50

3.2 Rationale for designing tetrahydroquinoline analog ..............................................51

3.3 Synthesis of first tetrahydroquinoline analog ........................................................52

3.4 Synthesis of tetrahydroquinoline-quinazoline analog ............................................57

3.5 Results and discussions ..........................................................................................60

3.6 Experimental ..........................................................................................................61

3.7 List of references....................................................................................................66

Chapter 4 Tropane Analogs ...................................................................................................69

4.1 Tropanes .................................................................................................................69

4.2 XL-888 – potent Hsp 90 inhibitor: A novel tropane derivative .............................71

4.3 Synthesis of piperidine analogs .............................................................................72

4.3.1 Synthesis of piperidine-benzylamide analog ................................................72

4.3.2 Synthesis of piperidine-amide analog ...........................................................76

4.3.3 Synthesis of piperidine-ester analog .............................................................77

4.4 Synthesis of tropane/bridged analogs ....................................................................78

4.4.1 Alternative synthesis to tropane/bridged analog ...........................................80

4.5 Results and discussions ..........................................................................................81

4.6 Experimental ..........................................................................................................83

4.7 References ............................................................................................................100

Chapter 5 Conclusions .........................................................................................................104

5.1 Summary and conclusions ...................................................................................104

Chapter 6 Introduction to Alzheimer’s disease .................................................................108

6.1 Alzheimer’s disease (AD) ....................................................................................108

6.2 Etiology and pathology of AD .............................................................................109

6.2.1 The cholinergic hypothesis ......................................................................... 110

6.2.2 Amyloid hypothesis .................................................................................... 110

6.2.3 Tau hypothesis ............................................................................................ 112

6.3 Current treatment ................................................................................................. 113

6.4 Virtual screening of compounds .......................................................................... 116

6.5 Cofilin-Slingshot pathway in AD......................................................................... 117

6.6 In vitro phosphatase enzyme inhibitory assay ..................................................... 118

6.7 References ............................................................................................................120

Chapter 7 Pyridones analogs...............................................................................................125

7.1 Pyridones..............................................................................................................125

7.2 Pyridone alkaloids ................................................................................................127

7.3 The Huprines ........................................................................................................129

iii

7.4 Synthesis of pyridone analogs as SSH1 inhibitors ..............................................130

7.5 Masking phenol: Intended mechanistic studies ...................................................132

7.6 Derivatization at the 5 position ............................................................................134

7.6.1 Synthesis of phenyl analog via Suzuki coupling of starting material .........138

7.7 Modifications to the bottom portion of the molecule ..........................................138

7.7.1 Synthesis of iodo and boronic ester derivatives: top portion of

quinazoline analog ......................................................................................139

7.7.2 Synthesis of quinazoline bottom portion ....................................................141

7.8 Summary of synthesized 2-pyridone analogs ......................................................143

7.9 Experimental ........................................................................................................144

7.10 References ............................................................................................................161

Chapter 8 Thiazoles analogs ...............................................................................................168

8.1 Thiazoles ..............................................................................................................168

8.1.1 Thiazole based therapeutics ........................................................................168

8.2 Synthesis of thiazole analogs ...............................................................................171

8.2.1 Phenyl analogs of thiazoles .........................................................................173

8.2.2 Pyridine analogs of thiazoles ......................................................................174

8.2.3 tert-butyl analog of thiazoles ......................................................................175

8.2.4 Phosphonic acid analog...............................................................................175

8.3 Summary of synthesized compounds...................................................................177

8.4 Results and discussion .........................................................................................179

8.5 Experimental section ............................................................................................184

8.6 References ............................................................................................................196

Chapter 9 Conclusions .........................................................................................................203

9.1 Summary and conclusions .................................................................................203

Appendices Assays, spectra and copyright permission ..........................................................207

Appendix I. Biological Assays ..................................................................................208

Appendix II. Chapter 2 NMR Spectra ........................................................................212

Appendix III. Chapter 3 NMR Spectra ........................................................................226

Appendix IV. Chapter 4 NMR Spectra ........................................................................231

Appendix V. Chapter 7 NMR Spectra ........................................................................248

Appendix VI. Chapter 8 NMR Spectra .........................................................................271

Appendix VII Copyright Permission ............................................................................288

iv

List of Abbreviations

AA Axenic amastigote

Aβ Beta amyloid peptides

ACh Acetylcholine

AChE Acetylcholinesterase

AChEI Acetylcholinesterase inhibitor

AcOH Acetic acid

AD Alzheimer’s disease

ADF Actin-depolymerizing factor

APP Beta amyloid precursor protein

BACE1 β-secretase

BBB Blood brain barrier

BCG Bacille Calmette Guerin

Bn Benzyl

Boc tert-butoxycarbonyl

CL Cutaneous leishmaniasis

CNS Central nervous system

DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene

DCM Dichloromethane (methylene chloride)

DIPEA Diisopropylethylamine

DMF Dimethylformamide

DMAP Dimethylaminopyridine

DMSO Dimethyl sulfoxide

DPPA Diphenylphosphoryl azide

DPPF 1,1'-Bis(diphenylphosphino)ferrocene

ESI Electrospray ionization

Et3N Triethylamine

EtOAc Ethyl acetate

EtOH Ethanol

GA Geldanamycin

h Hour(s)

H2O2 Hydrogen peroxide

HCS High content screen

HPLC High pressure liquid chromatography

Hsp 90 Heat shock protein 90

HTS High throughput screen

v

IM Infected macrophages

IPA Isopropyl alcohol

LCMS Liquid chromatography mass spectrometry

LC/QToF-MS Liquid chromatography quadrupole time-of-flight mass

spectrometry

MeOH Methanol

min Minute(s)

ML Mucocutaneous leishmaniasis

MPLC Medium pressure liquid chromatography

MsBr Mesityl bromide

mW microwave irradiation

NaNO2 Sodium nitrite

NBS N-bromosuccinimide

NF Neurofibrillary tangles

NH4OH Ammonium hydroxide

NIS N-iodosuccinimide

NMR Nuclear magnetic spectroscopy

NMDA N-methyl-D-aspartate

NTDs Neglected tropical diseases

P2O5 Phosphorus pentoxide

Pyr Pyridine

Pd2(dba)3 Tris(dibenzylideneacetone)dipalladium(0)

pNPP para-Nitrophenyl phosphate

rt Room temperature

SAR Structure activity relationships

SN2 Nucleophilic substitution

SOCl2 Thionyl chloride

SSH1 Slingshot homology-1 protein

TBABr Tetrabutyl ammonium bromide

TBAI Tetrabutyl ammonium iodide

tBuOK Potassium tert-butoxide

TEA Triethylamine

TFA Trifluoroacetic acid

THF Tetrahydrofuran

TLC Thin Layer Chromatography

TMBA Trimethoxybenzylamine

TMSBr Trimethylsilyl bromide

UHD Ultra-high-definition

USF University of South Florida

US FDA United States food and drugs administration

VL Visceral leishmaniasis

WHO World Health Organization

vi

List of Tables

Table 1.1 Toxicity profile of drugs used to treat leishmaniasis .....................................................8

Table 2.1 Biological data for indazole series of analogs .............................................................34

Table 3.1 Biological data for tetrahydroquinoline analog ...........................................................60

Table 4.1 Biological data for piperidine and tropane analogs .....................................................81

vii

List of Figures

Figure 1.1 Female phlebotomine sandfly .....................................................................................2

Figure 1.2 Leishmania life cycle ..................................................................................................3

Figure 1.3 L. major promastigotes and amastigote infected tissue (VL) .....................................4

Figure 1.4 Cutaneous, mucocutaneous and visceral leishmaniasis ..............................................6

Figure 1.5 Current drugs used for the treatment of leishmaniasis ...............................................7

Figure 1.6 Structures of sitamaquine and fexinidazole ................................................................9

Figure 1.7 SNX-2112 and its prodrug analog, SNX-5422 .........................................................10

Figure 1.8 Radicicol and geldanamycin ..................................................................................... 11

Figure 1.9 Analogs of geldanamycin .........................................................................................12

Figure 1.10 VER-52296 ..............................................................................................................12

Figure 1.11 L donovani promastigotes incubation with GA ......................................................13

Figure 1.12 96 Well plate of Leishmania ...................................................................................14

Figure 2.1 Initial hit compound – SNX-2112 ...........................................................................22

Figure 2.2 Indazole based biologically active molecules .........................................................23

Figure 2.3 Docking studies of analogs of 1.001 .......................................................................24

Figure 2.4 Acid by-product of initial analog .............................................................................27

Figure 2.5 Initial analog 2.008 and quinazoline analog 2.010 ..................................................28

Figure 2.6 Docking studies of quinazoline analog ....................................................................29

Figure 2.7 Methyl substituted analog 2.016 and XL-888 .........................................................30

Figure 2.8 Test compounds .......................................................................................................33

viii

Figure 3.1 Selected tetrahydroquinoline compounds and their biological activities ................51

Figure 3.2 Interaction of 1.001 and 3.013 with Hsp 90 ............................................................52

Figure 4.1 Examples of some natural esters of tropanes ...........................................................69

Figure 4.2 Major applications of tropane moiety and annual global revenue ...........................70

Figure 4.3 Compound 1.001 and XL-888 with proposed binding interactions ........................71

Figure 4.4 Piperidine analogs ....................................................................................................72

Figure 4.5 4.013 diacid .............................................................................................................75

Figure 4.6 Tropane ester acid-by-product .................................................................................79

Figure 5.1 Hsp 90 Fluorescence polarization assay ................................................................106

Figure 6.1 Cross section of the brain caused by AD ...............................................................108

Figure 6.2 Formation of Aβ- and Aβ fibrils from APP by sequential cleavage by sequential

cleavage by β- and γ-secretase ............................................................................... 111

Figure 6.3 Signalling from Aβ via tau drives AD progression ................................................ 113

Figure 6.4 Current drugs used to treat AD .............................................................................. 114

Figure 6.5 Agents in clinical trials for the treatment of AD in 2017 ...................................... 115

Figure 6.6 Lead compounds from virtual screen .................................................................... 116

Figure 6.7 Progressive pathogenic cycle of AD ...................................................................... 118

Figure 7.1 Biologically active compounds containing 2-pyridones .......................................125

Figure 7.2 Pyridones as treatment for AD ...............................................................................126

Figure 7.3 H3 receptor antagonists in clinical trials ................................................................127

Figure 7.4 Naturally occurring pyridone alkaloids .................................................................128

Figure 7.5 The huprines as AChEIs ........................................................................................129

Figure 7.6 3-Formylchromone and its various reactive sites ..................................................131

ix

Figure 7.7 Compound 7.005A and its binding orientation in SSH2 active site ......................132

Figure 7.8 Compound 7.005A alkylated with proposed linkers for mechanistic studies .......134

Figure 7.9 Hartwig-Buchwald amination with XANTPHOS and Pd2(dba)3 ..........................136

Figure 7.10 Modification to bottom portion of molecule .........................................................139

Figure 7.11 Retrosynthetic scheme for the synthesis of 7.039 ..................................................139

Figure 7.12 Summary of test pyridone test compounds............................................................143

Figure 8.1 Structure of thiazole...............................................................................................168

Figure 8.2 Some thiazole based drugs in clinical use .............................................................169

Figure 8.3 Some thiazole based compounds as anti-AD agent in development .....................170

Figure 8.4 Retrosynthetic scheme for thiazole synthesis ........................................................171

Figure 8.5 Phenyl and substituted phenyl analogs of thiazoles ..............................................174

Figure 8.6 Pyridine analogs of thiazoles .................................................................................175

Figure 8.7 tert-butyl analogs of thiazoles ...............................................................................175

Figure 8.8 Summary of thiazole test compounds ....................................................................177

Figure 8.9 Pyridone analogs....................................................................................................178

x

List of Schemes

Scheme 2.1 Synthesis of initial analog ......................................................................................26

Scheme 2.2 Synthesis of desmethyl analog ...............................................................................29

Scheme 2.3 Synthesis of methyl substituted analog ..................................................................31

Scheme 3.1 Synthesis of 7,7-dimethyl-5,6,7,8-tetrahydroquinolin-5-amine ............................53

Scheme 3.2 Preparation of advanced intermediate 3.008 via azide formation .........................54

Scheme 3.3 Preparation of advanced intermediate 3.008 via tosylation ...................................55

Scheme 3.4 Synthesis of tetrahydroquinoline analog ................................................................56

Scheme 3.5 Synthesis of tetrahydroquinoline-quinazoline analog ............................................57

Scheme 3.6 Synthesis of quinazolin-4,7-diamine for tetrahydroquinoline/quinazoline

analog .....................................................................................................................58

Scheme 4.1 Synthesis of Boc-protected piperidine ...................................................................72

Scheme 4.2 Synthesis of 6-(4-aminopiperidin-1-yl)N-benzylnicotinamide .............................73

Scheme 4.3 Synthesis of 4-carbamoyl-3-((3-methoxypropyl)amino)benzoic acid ...................74

Scheme 4.4 Synthesis of piperidine-benzylamide analog .........................................................75

Scheme 4.5 Synthesis of piperidine-amide analog ....................................................................76

Scheme 4.6 Synthesis of piperidine-ester analog ......................................................................77

Scheme 4.7 Synthesis of tropane/bridged analogs ....................................................................78

Scheme 4.8 Alternative synthesis to tropane/bridged analogs ..................................................80

Scheme 7.1 Synthesis of 2-pyridone analogs ..........................................................................130

Scheme 7.2 O-alkylation of the phenolic functionality ...........................................................133

xi

Scheme 7.3 Synthesis of bromo-substituted analog ................................................................135

Scheme 7.4 Hartwig-Buchwald reaction .................................................................................136

Scheme 7.5 Synthesis of nitro-analog .....................................................................................137

Scheme 7.6 Synthesis of phenyl analog ..................................................................................138

Scheme 7.7 Synthesis of iodo and boronic-ester derivatives ..................................................140

Scheme 7.8 Synthesis of phenyl analog: modification to bottom portion ...............................141

Scheme 7.9 Synthesis of quinazoline derivatives ....................................................................141

Scheme 7.10 Synthesis of quinazoline analog...........................................................................142

Scheme 8.1 Synthesis of thiazole analogs ...............................................................................172

Scheme 8.2 Synthesis of phosphonic acid analog ...................................................................176

xii

List of Graphs

Graph 1 SSH1 inhibitory activity of analogs 8.013A, 7.005A-D ............................................179

Graph 2 SSH1 inhibitory activity of analogs 8.013A, 7.007, 7.011, 7.021 .............................180

Graph 3 Comparison of 8.013A to E1-series of analogs .........................................................181

Graph 4 Comparison of 8.013A to C2-series of analogs .........................................................181

Graph 5 Comparison of 8.013A to E-series of analogs ...........................................................182

Graph 6 SAR studies of thiazole analogs ................................................................................183

xiii

Abstract

Infectious and neurodegenerative diseases continue to be a major concern worldwide. In

spite of the great advances in drug therapy for treating various infectious and neurodegenerative

diseases, there is still an urgent need for new and improved drugs due to increasing drug resistance

among pathogens, emergence of new pathogens, ease of transmission of infections, ineffective

available treatments, toxicity associated with current standard of care, aging populations and the

lack of better alternative treatment options.

The first part of this manuscript (chapters 1 - 5) describes the synthesis of novel agents active

against Leishmania donovani. According to the World Health Organization (WHO), a significant

number of deaths worldwide can be attributed to infectious diseases – particularly neglected

tropical diseases (NTDs), one of which is leishmaniasis - a complex and clinically diverse disease

transmitted through the bite of an infected female phlebotomine sand-fly. The pathogen that causes

leishmaniasis develops through a complex life cycle via different morphological changes. Its

clinical presentations range from the less severe (cutaneous) to lethal/fatal (visceral) forms

depending upon the level of systemic involvement, infecting species and the endemic environment.

Treatments (and vaccines) must be species-specific to be particularly effective since sensitivity to

commonly used drugs is largely species-specific. Heat shock protein 90 (Hsp 90) has been shown

to promote the differentiation of the protozoan parasite that causes leishmaniasis from the

promastigote stage to the amastigote pathogenic stages. To this end a series of compounds were

prepared based on known Hsp 90 inhibitors, SNX2112 and XL888. The synthetic approach allows

xiv

the probing of a hydrophobic pocket and rapid access to a collection of anti-leishmanial

compounds. The most active compound, was found to be more than twice as active as the clinically

used drug, miltefosine, in an infected J774 macrophage at IC50 = 0.65 µM.

The second part of this manuscript (chapters 6 - 9) describes the synthesis of novel anti-

Alzheimer’s agents. Alzheimer’s disease is a progressive neurodegenerative disease believed to be

caused by tau hyperphosphorylation and plaque aggregation in the brain. It is known to affect about

44 million people worldwide and it is marked as the 6th leading cause of death in the United States.

Slingshot homology-1 (SSH1) proteins, important protein phosphatases, are promising targets for

the discovery of a new generation of small molecule inhibitors as treatment for Alzheimer’s

disease, since SSH1 is believed to contribute to both tau hyperphosphorylation and plaque

aggregation in the brain. Through structure and activity relationships (SAR) studies, two (2) series

of compounds were synthesized, thiazoles and pyridones, bearing a carboxylic acid or phosphonic

acid functionality as inhibitors of SSH1 enzymes. In the preliminary screening efforts against

SSH1 phosphatase activity, the thiazole series were found to be more potent at inhibiting the

phosphatase activity than the pyridone series. Among the active thiazole series, eight (8) analogs

exhibited significant inhibitory activity over the initial hit compound, observed via phosphatase

inhibition curves (using a pNPP phosphatase assay). Further investigations into the molecular

target (SSH1) are currently underway.

1

Chapter 1

Introduction

1.1 The threat of infectious diseases

Chemotherapy continues to revolutionize medical practice. The discovery and

development of different classes of medicaments have led to the transformation of deadly

infectious diseases into curable ones – saving lives and relieving patient suffering.1 However,

notwithstanding the great advances in drug therapy, infectious diseases continue to be a major

concern worldwide2 with increasing threat to human health.3 The development of resistant strains

of pathogens to available drugs, the emergence or the discovery of new pathogens, and the ease of

transmission due to globalization,2,4,5 including the increasing numbers of immune-compromised

patients6 and elderly populations,7 suggest that mankind may be fast approaching a post-antibiotic

era.8

The vast majority of infectious diseases affect the poorest of the poor and are prevalent in the

tropical regions of the world,9 where there is limited access to both diagnosis and treatment.10 The

lack of profitability in the discovery and development of essential medicaments,1,11 particularly

the ones designated by the World Health Organization (WHO) as neglected tropical diseases

(NTDs),9 has dried up the infectious disease pharmaceutical pipeline. There are twenty (20) NTDs9

that threaten human health12,13 and are currently affecting over 1 billion people in the tropics and

subtropics, costing developing economies billions of dollars annually.9 Leishmaniasis, a zoonotic

2

parasitic infection caused by protozoa of the genus Leishmania, has been identified as one of the

many NTDs.

1.2 Leishmaniasis

Leishmaniasis is a complex and clinically diverse disease transmitted through the bite of

an infected female phlebotomine sand-fly, Figure 1. It is ranked among the most important

poverty-related diseases14,15 and a major health problem in larger parts of the world, mainly in

developing countries, causing enormous disability, death and economic loss.2,16

Figure 1.1: Female Phlebotomine sand fly

The World Health Organization (WHO) has designated leishmaniasis as one of the world’s most

neglected tropical diseases (NTDs).17 About twenty (20) different protozoan Leishmania species

cause the disease,2,16 which is particularly worrying from the standpoint of vaccine development

and drug discovery. It is estimated that about 12 million people are afflicted with the disease

worldwide; 350 million people are considered at risk, with an estimated 2 million new cases

3

annually. It is considered to be endemic in 88 countries2,16,17,18 with 59,000 deaths per year from

visceral leishmaniasis, despite the fact that leishmaniasis was among NTDs selected by the WHO

for elimination by 2015.19

1.2.1 Leishmania Life Cycle

The genus Leishmania belongs to the family Trypanosomatidae, order Kinetoplastida and

the group Euglenozoa.14 The Leishmania life cycle is dimorphic - human stage and insect stage –

with a similar mode of transmission for all species,14 as depicted in Figure 1.2.20

Figure 1.2: Leishmania life cycle20

4

During the human stage, an infected female phlebotomine sand-fly, genus Phlebotomus (Asia,

Africa and Europe) or of the genus Lutzomyia (Latin America), goes for a blood meal, and in the

process releases the slender, flagellated promastigote stages into the blood stream (Figure 1.3a).

Upon transmission, the parasites are phagocytosed by tissue macrophages and neutrophilic

granulocytes where they differentiate within the parasitophorous vacuoles of macrophages.2,15

The parasite’s ability to block the fusion of phagosomes and lysosomes ensures their survival, and

they establish themselves as round afflagelated non-motile amastigote stages as seen in Figure

1.3b, where they proliferate, leading to disruption of the macrophages. The released protozoan

parasites then re-infect other macrophages, dendritic cells and blood monocytes.15,21

Figure 1.3: (a) L. major promastigote from culture, Giemsa staining, 100X, (b) amastigotes (arrows) in

infected spleen tissue (VL), Giemsa staining.14

5

During the insect stage, a sandfly goes for a blood meal from an infected human, and takes up

amastigote-infected monocytes, which disrupt and release the parasites into the lumen of the

insect’s gut. The amastigotes then undergo stage differentiation into the flagellated promastigotes

and attach themselves to the epithelial gut until they reach stationary growth stage.14 The

promastigotes then detach from the epithelial gut and spread into the mouth parts and can be

transmitted to healthy persons following another blood meal.

1.2.2 Forms of Leishmaniasis

The disease manifests itself in three main forms/types with a wide range of complex

devastating clinical manifestations, depending upon the infecting species, endemic region and the

level of systemic involvement:14,15,17,22

i. Cutaneous Leishmaniasis (CL): the most common and least fatal localized and self-

healing lesions. However sores can take months to years to heal as seen in Figure 1.4a.

Symptoms include sores, papules or nodules on the skin, with or without swollen lymph

nodes. Sores can leave significant permanent scars and can be disfiguring, especially

for facial sores. These are caused by L. major, L. tropica, L. mexicana, L. amazonensis,

L. panamensis, L. braziliensis, L. guyanensis and L. peruviana.

ii. Mucocutaneous Leishmaniasis (ML): the most destructive form. Ocurs when

untreated cutaneous lesions on the face spread to involve the mouth and nose as seen

in Figure 1.4b. ML causes complete or partial destruction of mucus membranes in the

nose, mouth and throat and can be very disfiguring. These are caused by L. brazilensis

and L. panamensis.

6

iii. Visceral Leishmaniasis (VL): also known as black fever or Kala Azar, occurs when

the parasite disseminates into the entire reticuloendothelial system (spleen, liver and

bone marrow) as seen in Figure 1.4c. VL can be fatal if left untreated. Symptoms

include weight loss, persistent fever, anemia, massive hepatosplenomegaly (swelling

of the liver and spleen) and deterioration of the host. In later stages of the disease,

hemorrhages and edema are likely to develop. Mortality can reach >90% if efficient

chemotherapy is not administered. These generalised infections are caused by L.

donovani and L. infantum.

Figure 1.4: (a) Cutaneous Leishmaniasis, (b) Mucocutaneous Leishmaniasis, (c) Visceral Leishmaniasis

1.3 Current Treatment and Vaccine Development

New drug discovery efforts for leishmaniasis, particularly visceral leishmaniasis, come

with a plethora of challenges which include: lack of sufficient investment, validated targets, in vivo

models and very low hit rates from HTS23,24,25 due to the concealed location of the parasites within

the acidic parasitophorous vacuoles of macrophages.26 Because of these challenges, there is a

general lack of inexpensive and effective therapeutics for leishmaniasis.18 Current treatments for

leishmaniasis, shown in Figure 1.5, are not ideal due to their high cost, toxicity, long duration of

7

treatment, complicated mode of administration and severe adverse reactions, which often lead to

non-compliance and treatment abandonment.11

Figure 1.5: Current drugs used for the treatment of Leishmaniasis

Moreover, these medications often do not completely eliminate the parasite. The past 15

years have witnessed novel formulations of conventional anti-leishmanial agents, such as

liposomal amphotericin B, however these are too costly for practical use in developing countries.

The clinical value of these medications have also been compromised by their associated

irreversible toxicity and the rapid development of resistance.2,22 Table 1.1 below summarizes the

toxicity profile of the commonly used drugs.11

8

Number Drugs Mode of

administration

Main limitations and Toxicity

1 Pentavalent

antimonials

Parenteral Prolonged treatment, poor

compliance, severe cardiotoxicity,

pancreatitis, nephrotoxicity,

hepatotoxicity

2 Amphotericin B Parenteral Prolonged hospitalization, severe

nephrotoxicity, infusion-related

reactions, hypokalemia, metabolic

acidosis, cardiotoxicity, high fever

3 Liposomal

amphotericin B

Parenteral Mild rigors and chills during

infusion, mild nephrotoxicity

(infrequent and mild)

4 Miltefosine Oral Vomiting and diarrhea,

nephrotoxicity, hepatotoxicity,

teratogenicity, ease of resistance

5 Aminosidine Parenteral Prolonged treatment, severe

nephrotoxicity, ototoxicity,

hepatotoxicity

6 Pentamidine Parenteral Prolonged treatment, high rate of

hypoglycemia, as a result of

pancreatic damage; hypotension,

tachycardia, electrocardiographic

changes, diabetes, poor response rate

Table 1.1: Toxicity profile of drugs used to treat leishmaniasis11

Owing to the phase II clinical trials failure of sitamaquine,27,28 a promising 8-aminoquinoline

(Figure 1.6) for the treatment of VL via oral administration, miltefosine remains the only orally

applicable drug. The rest have complicated modes of administration. Another promising drug

candidate for visceral leishmaniasis to fail in the clinic during its phase II clinical trial in Sudan29

is fexinidazole (Figure 1.6) - thus the clinical pipeline for visceral leishmaniasis is now empty.

9

Figure 1.6: Structures of sitamaquine and fexinidazole

Again, although synthetic vaccines have been developed against different protozoan parasites,30

there are currently no effective vaccines as well as no prophylactic treatments available for

leishmaniasis.2,17 However, there is strong hope for developing vaccines because recovery from

the disease is usually accompanied by strong immunity. Additionally, it is also possible to protect

experimental animals against a live challenge.17 Thus, first generation vaccine candidates for

leishmaniasis have been attempted with whole killed parasites/extracts, and among those tested

include a L. amazonensis-based vaccine, L. mexicana (given with Bacille Calmette Guerin, BCG)

and L. major (also given with BCG). Although results have been encouraging, they are largely

inconclusive or negative for prophylaxis. Second generation vaccines on the other hand have been

made with recombinant proteins and genetic vaccines. Leish-IIIf+MPL-SE is the only vaccine

candidate that has made it to clinical trials.17

Given the fact that developing protozoan vaccines is inherently difficult (based on results from

available vaccines), chances of developing prophylactic vaccines within the next few years from

any form of leishmaniasis are very slim.17 Hence chemotherapy is still the mainstay of both clinical

management and disease control.30 Therefore pursuing, designing and developing new

medications with different modes of action and that are highly effective with a good toxicity profile

can lead to the development of novel anti-leishmanial compounds.

10

1.4 Compound Screening/High throughput screening

In an effort to identify new compounds with anti-leishmanial activity, our laboratory in

collaboration with the laboratory of Prof. Dennis Kyle, formerly in the USF Department of Public

Health, conducted a high throughput screening (HTS) campaign of a library of biologically active

molecules for their antiprotozoal activity against the parasite that causes leishmaniasis using the

Leishmania donovani axenic amastigote assay.31 Compound 1.001, shown in Figure 1.6, was

found to exhibit reasonable activity in the assay (IC50 = 420 nM) and was identified as SNX-

2112.32,33 This compound and its related analog (SNX-5422) are currently being studied in clinical

trials for different cancers,34,35 targeting the 90 KDa family of heat shock proteins (Hsp 90).

Figure 1.7: SNX-2112 (initial hit) and its prodrug analog, SNX-5422

1.5 Heat Shock Proteins 90 (Hsp 90)

Hsp 90 are ubiquitous chaperone proteins that are expressed in all living cells (except

Archaea)11 upon exposure to elevated temperatures/stress and are believed to play a critical role in

acquiring thermotolerance.36,37 Heat shock proteins function in the regulation of cell fate and

intracellular cell survival, facilitation of protein folding and avoiding miss-folding and protein

11

aggregation.11 Hsp 90 is the target of known bioactive natural products such as geldanamycin (GA)

and radicicol (RD),38,39 shown in Figure 1.7, and it is a well-known target for the development of

small molecule inhibitors in cancer drug discovery.40 Thus, Hsp 90 or its related orthologs may

serve as viable target in antiprotozoal drug discovery, owing to their expression in these cells.

Figure 1.8: Radicicol (RD) and Geldanamycin (GA)

1.5.1 Heat shock proteins 90 (Hsp 90) as antiparasitic drug target

The biological significance of Hsp 9041 in a number of protozoan parasites such as

Plasmodium spp,42 Toxoplasma gondii,43 and trypanosomatids (Trypanosoma and Leishmania

spp.)44,45 and as a drug target has been established.30,46,47 Both geldanamycin and radicicol are not

stable in vivo and exhibit liver toxicity, hence they are not promising candidates for use in humans.

However, analogs of geldanamycin, IPI-504, 17-AAG and 17-DMAG,48 shown in Figure 1.8,

have been investigated as less toxic candidates. IPI-504 in particular contains a reduced

hydroquinoline derivative, water (H2O) soluble and can be formulated for parenteral

administration.48 The compound 17-AAG (tanespimycin), an analog of geldanamycin and active

against L. infantum, L. panamensis49 and L. amazonensis,50 has undergone clinical trials as an anti-

parasitic drug.

12

Figure 1.9: Analogs of geldanamycin

Other analogs of radicicol such as VER-52296, shown in Figure 1.9, and geldanamycin (19-

methyl- and 19-phenylgeldanamycin) may find use as antiparasitic agents.38 Similarly, the non-

radicicol drug, paclitaxel (taxol), has also proven effective on Leishmania Hsp 90.45

1.5.2 Heat Shock Proteins 90 (Hsp 90) in Leishmania

Heat shock genes of Leishmania species have been cloned and sequenced and the results

indicate that genes encoding the 90 KDa (83 KDa) form are organised in a multi-copy tandem

clusters having up to fourteen (14) copies per haploid tandem.51 It is one of the most abundant

proteins in the Leishmania parasite52,53 accounting for almost 3% of all cellular proteins. Their

constitutive expression upon transmission to their mammalian hosts due to the rise in temperature45

13

suggests they are crucial for the survival of the parasite.51 Indeed Hsp 90 (Hsp 83) homeostasis is

critical for proliferation – differentiation of the parasite from promastigote (insect stage) to

amastigote (pathogenic stage).45,53,54 Thus, the rise in ambient temperature encountered upon

transmission to a mammal can be viewed as a signal for cellular differentiation and not as stress.

Predictably, the pharmacological inhibition of Leishmania donovani Hsp 90,45 as shown in Figure

1.10, or the related parasite Trypanosoma cruzi55 using known inhibitors such as geldanamycin or

radicicol leads to the arrest of proliferating promastigotes of the cell cycle, induction of Hsp 90

protein synthesis and conversion towards amastigote-like morphology.

Figure 1.11: L. donovani promastigotes following 24h incubation (a) at 25 oC, pH 7;

(b) at 37 oC, pH 5.5; (c) at 25 oC, pH 7 with 200 ng/mL GA.45

It was therefore hypothesized that compounds that can inhibit the protozoan ortholog of Hsp 90

(Hsp 83) could prove useful in disrupting amastigote growth. Thus, optimization of the initial hit

compound, 1.001 (described in Chapter 2) was initiated, in our search for potential anti-

leishmanial agents. To this end, several analogs were synthesized in order to improve upon the

pharmacokinetic properties of the intial compound. Critical to the drug discovery efforts was the

design of compounds that would not only be selective for the protozoan ortholog of Hsp 90 (Hsp

83), but also meet the following suggested criteria: orally bioavailable, safe, universally effective

14

over a short course (preferably no more than a 10 day regimen), affordable and capable of

withstanding harsh environmental conditions.56

1.6 Biological Assays

The anti-leishmanial activity of the synthesized compounds were evaluated in multiple

assays including, the axenic amastigote assay (AA, initial screening assay), Figure 1.11, an

infected macrophage assay (IM, high content screen assay) and a J774 cytotoxicity assay. Thus

each of the analogs was evaluated in the initial screening assay (AA) as well in a high content

screening (HCS) infected macrophage assay (with J774 murine macrophages), in order to

investigate their anti-leishmanial activity inside of cells. The latter not only allowed the evaluation

of amastigote growth inside these cells, but also evaluated the cytotoxicity of the compounds to

ensure that the observed activity is not the result of killing the host cells.57 Detailed procedures for

these assays are provided in the appendices.

Figure 1.12: 96 Well plate with Leishmania ready to be read in a spectrophotometer

15

1.7 References

(1) IDSA : Bad Bugs, No Drugs: As antibiotic discovery stagnates, a public health crisis brews.

http://www.idsociety.org/Policy___Advocacy/Antimicrobial_Resistance/Bad_Bugs,_No_Drugs_

_As_Antibiotic_Discovery_Stagnates,_a_Public_Health_Crisis_Brews/ (accessed Nov 29, 2017).

(2) Loedige, M. Design and synthesis of novel antileishmanial compounds. International Journal

of Medicinal Chemistry 2015, 2015, 1-21.

(3) Murray, C.; Lopez, A. Global mortality, disability, and the contribution of risk factors: Global

Burden of Disease Study. The Lancet 1997, 349, 1436-1442.

(4) Pelgrift, R.; Friedman, A. Nanotechnology as a therapeutic tool to combat microbial resistance.

Advanced Drug Delivery Reviews 2013, 65, 1803-1815.

(5) Jones, K.; Patel, N.; Levy, M.; Storeygard, A.; Balk, D.; Gittleman, J.; Daszak, P. Global trends

in emerging infectious diseases. Nature 2008, 451, 990-993.

(6) Kahn, L. The growing number of immunocompromised http://thebulletin.org/growing-

number-immunocompromised. (accessed Nov 29, 2017).

(7) World Health Organization (WHO) document on Global Health and Aging

http://www.who.int/ageing/publications/global_health.pdf (accessed Nov 29, 2017).

(8) Alanis, A. Resistance to antibiotics: are we in the post-antibiotic Era? Archives of Medical

Research 2005, 36, 697-705.

(9) Neglected Tropical Diseases http://www.who.int/neglected_diseases/diseases/en/ (accessed

Nov 29, 2017).

(10) den Boer, M.; Argaw, D.; Jannin, J.; Alvar, J. Leishmaniasis impact and treatment access.

Clinical Microbiology and Infection 2011, 17, 1471-1477.

16

(11) de Menezes, J.; Guedes, C.; Petersen, A.; Fraga, D.; Veras, P. Advances in development of

new treatment for leishmaniasis. BioMed Research International 2015, 2015, 1-11.

(12) Hotez, P.J; Molyneux, D.H.; Fenwick, A.; Kumaresan, J.; Sachs, S.E.; Sachs, J.D and Savioli,

L. Control of neglected tropical diseases. New England Journal of Medicine 2007, 357, 1018-

1027.

(13) Laursen, L. Tropical disease: A neglected cause. Nature 2016, 533, S68-S69.

(14) Pockley, A.; Calderwood, S.; Santoro, M. Prokaryotic and eukaryotic heat shock proteins in

infectious disease; Springer Netherlands: Dordrecht, 2010.

(15) Hombach, A.; Clos, J. No stress – Hsp 90 and signal transduction in Leishmania. Parasitology

2014, 141, 1156-1166.

(16) Desjeux, P. Leishmaniasis: current situation and new perspectives. Comparative Immunology,

Microbiology and Infectious Diseases 2004, 27, 305-318.

(17) Technical Report Series: Report of a meeting of the WHO Expert Committee on the Control

of Leishmaniases, Geneva, 22–26 March 2010

http://www.who.int/leishmaniasis/resources/technical_report_series/en/ (accessed Nov 29,

2017).

(18) Werbovetz, K.; Sackett, D.; Salem, M. Antileishmanial dinitroaniline sulfonamides with

activity against parasite tubulin. US 2005/0227982 A1, PCT/US03/12587, 2005.

(19) Global plan to combat neglected tropical diseases, 2008–2015

http://whqlibdoc.who.int/hq/2007/WHO_CDS_NTD_2007.3_eng.pdf (accessed Nov 29, 2017).

(20) CDC - Leishmaniasis - Biology https://www.cdc.gov/parasites/leishmaniasis/biology.html

(accessed Mar 9, 2018).

17

(21) Bogdan, C.; Röllinghoff, M. The immune response to Leishmania: mechanisms of parasite

control and evasion. International Journal for Parasitology 1998, 28, 121-134.

(22) Maltezou, H. Drug resistance in visceral leishmaniasis. Journal of Biomedicine and

Biotechnology 2010, 2010, 1-8.

(23) Nagle, A.; Khare, S.; Kumar, A.; Supek, F.; Buchynskyy, A.; Mathison, C.; Chennamaneni,

N.; Pendem, N.; Buckner, F.; Gelb, M. et al. Recent developments in drug discovery for

leishmaniasis and human African trypanosomiasis. Chemical Reviews 2014, 114, 11305-11347.

(24) Field, M.; Horn, D.; Fairlamb, A.; Ferguson, M.; Gray, D.; Read, K.; De Rycker, M.; Torrie,

L.; Wyatt, P.; Wyllie, S. et al. Anti-trypanosomatid drug discovery: an ongoing challenge and a

continuing need. Nature Reviews Microbiology 2017, 15, 217-231.

(25) Don, R.; Ioset, J. Screening strategies to identify new chemical diversity for drug development

to treat kinetoplastid infections. Parasitology 2013, 141, 140-146.

(26) Liévin-Le Moal, V.; Loiseau, P. Leishmania hijacking of the macrophage intracellular

compartments. FEBS Journal 2015, 283, 598-607.

(27) Loiseau, P.; Cojean, S.; Schrével, J. Sitamaquine as a putative antileishmanial drug candidate:

from the mechanism of action to the risk of drug resistance. Parasite 2011, 18, 115-119.

(28) Sundar, S.; Sinha, P.; Mohamed, K.; Al-Banna, M.; Miller, A.; Buckley, R.; Dixon, S.

Pharmacokinetics of oral Sitamaquine taken with or without food and safety and efficacy for

treatment of visceral leishmaniais: A randomized study in Bihar, India. The American Journal of

Tropical Medicine and Hygiene 2011, 84, 892-900.

(29) Fexinidazole/Miltefosine combination (VL) – DNDi https://www.dndi.org/diseases-

projects/portfolio/completed-projects/fexinidazole-vl/ (accessed Nov 29, 2017).

18

(30) Angel, S.; Matrajt, M.; C. Echeverria, P. A Review of recent patents on the protozoan parasite

Hsp 90 as a drug target. Recent Patents on Biotechnology 2013, 7, 2-8.

(31) Wang, M.; Zhu, X.; Srivastava, A.; Liu, Q.; Sweat, J.; Pandharkar, T.; Stephens, C.; Riccio,

E.; Parman, T.; Munde, M. et al. Novel arylimidamides for treatment of visceral leishmaniasis.

Antimicrobial Agents and Chemotherapy 2010, 54, 2507-2516.

(32) Chandarlapaty, S.; Sawai, A.; Ye, Q.; Scott, A.; Silinski, M.; Huang, K.; Fadden, P.; Partdrige,

J.; Hall, S.; Steed, P. et al. SNX2112, a synthetic heat shock protein 90 inhibitor, has potent

antitumor activity against HER kinase dependent cancers. Clinical Cancer Research 2008, 14,

240-248.

(33) Compound 1 is commercially available from Ark Pharm Inc. in >98% purity and was used

without further purification.

(34) Reddy, N.; Voorhees, P.; Houk, B.; Brega, N.; Hinson, J.; Jillela, A. Phase I Trial of the Hsp

90 inhibitor PF-04929113 (SNX5422) in adult patients with recurrent, refractory hematologic

malignancies. Clinical Lymphoma Myeloma and Leukemia 2013, 13, 385-391.

(35) Infante, J.; Weiss, G.; Jones, S.; Tibes, R.; Bauer, T.; Bendell, J.; Hinson, J.; Von Hoff, D.;

Burris, H.; Orlemans, E. et al. Phase I dose-escalation studies of SNX-5422, an orally bioavailable

heat shock protein 90 inhibitor, in patients with refractory solid tumours. European Journal of

Cancer 2014, 50, 2897-2904.

(36) Carper, S.W.; Duffy, J.J.; Gerner, E.W. Heat shock proteins in thermotolerance and other

cellular processes. Cancer Research 1987, 47, 5249-5255.

(37) Nussenzweig, A.; Burgman, P.; Li, G. The role of heat shock proteins in thermotolerance.

Thermobiology 1997, 261-285.

19

(38) Kitson, R.; Moody, C. Learning from Nature: Advances in Geldanamycin- and Radicicol-

based inhibitors of Hsp 90. The Journal of Organic Chemistry 2013, 78, 5117-5141.

(39) Sharma, S.; Agatsuma, T.; Nakano, H. Targeting of the protein chaperone, Hsp 90, by the

transformation suppressing agent, radicicol. Oncogene 1998, 16, 2639-2645.

(40) Trepel, J.; Mollapour, M.; Giaccone, G.; Neckers, L. Targeting the dynamic Hsp 90 complex

in cancer. Nature Reviews Cancer 2010, 10, 537-549.

(41) Figueras, M.J.; Echeverria, P.C.; O. Angel, S. Protozoan Hsp 90-heterocomplex: molecular

interaction network and biological significance. Current Protein & Peptide Science 2014, 15, 245-

255.

(42) Shahinas, D.; Folefoc, A.; Pillai, D. Targeting plasmodium falciparum Hsp 90: towards

reversing antimalarial resistance. Pathogens 2013, 2, 33-54.

(43) Angel, S.; Figueras, M.; Alomar, M.; Echeverria, P.; Deng, B. Toxoplasma gondii Hsp 90:

potential roles in essential cellular processes of the parasite. Parasitology 2014, 141, 1138-1147.

(44) Meyer, K.; Shapiro, T. Potent antitrypanosomal activities of heat shock protein 90 Inhibitors

in vitro and in vivo. The Journal of Infectious Diseases 2013, 208, 489-499.

(45) Wiesgigl, M.; Clos, J. Heat shock protein 90 homeostasis controls stage differentiation in

leishmania donovani. Molecular Biology of the Cell 2001, 12, 3307-3316.

(46) Pallavi, R.; Roy, N.; Nageshan, R.; Talukdar, P.; Pavithra, S.; Reddy, R.; Venketesh, S.;

Kumar, R.; Gupta, A.; Singh, R. et al. Heat shock protein 90 as a drug target against protozoan

infections. Journal of Biological Chemistry 2010, 285, 37964-37975.

(47) Giannini, G.; Battistuzzi, G. Exploring in vitro and in vivo Hsp 90 inhibitors activity against

human protozoan parasites. Bioorganic & Medicinal Chemistry Letters 2015, 25, 462-465.

20

(48) Huang, K.; Veal, J.; Fadden, R.; Rice, J.; Eaves, J.; Strachan, J.; Barabasz, A.; Foley, B.;

Barta, T.; Ma, W. et al. Discovery of novel 2-aminobenzamide inhibitors of heat shock protein 90

as potent, selective and orally active antitumor agents. Journal of Medicinal Chemistry 2009, 52,

4288-4305.

(49) Varela-M, R.; Mollinedo-Gajate, C.; Muro, A.; Mollinedo, F. The Hsp 90 inhibitor 17-AAG

potentiates the antileishmanial activity of the ether lipid edelfosine. Acta Tropica 2014, 131, 32-

36.

(50) Petersen, A.; Guedes, C.; Versoza, C.; Lima, J.; de Freitas, L.; Borges, V.; Veras, P. 17-AAG

kills intracellular leishmania amazonensis while reducing inflammatory responses in infected

macrophages. PLoS ONE 2012, 7, e49496.

(51) Shapira, M.; Pinelli, E. Heat-shock protein 83 of Leishmania mexicana amazonensis is an

abundant cytoplasmic protein with a tandemly repeated genomic arrangement. European Journal

of Biochemistry 1989, 185, 231-236.

(52) Brandau, S.; Dresel, A.; Clos, J. High constitutive levels of heat-shock proteins in human-

pathogenic parasites of the genus Leishmania. Biochemical Journal 1995, 310, 225-232.

(53) Wiesgigl, M.; Clos, J. The heat shock protein 90 of Leishmania donovani. Medical

Microbiology and Immunology 2001, 190, 27-31.

(54) Hombach, A.; Ommen, G.; Chrobak, M.; Clos, J. The Hsp 90-Sti1 interaction is critical for

Leishmania donovaniproliferation in both life cycle stages. Cellular Microbiology 2012, 15, 585-

600.

(55) Graefe, S.; Wiesgigl, M.; Gaworski, I.; Macdonald, A.; Clos, J. Inhibition of Hsp 90 in

Trypanosoma cruzi induces a stress response but no stage differentiation. Eukaryotic Cell 2002, 1,

936-943.

21

(56) Freitas-Junior, L.; Chatelain, E.; Kim, H.; Siqueira-Neto, J. Visceral leishmaniasis treatment:

What do we have, what do we need and how to deliver it? International Journal for Parasitology:

Drugs and Drug Resistance 2012, 2, 11-19.

(57) Siqueira-Neto, J.; Moon, S.; Jang, J.; Yang, G.; Lee, C.; Moon, H.; Chatelain, E.; Genovesio,

A.; Cechetto, J.; Freitas-Junior, L. An image-based high-content screening assay for compounds

targeting intracellular Leishmania donovani amastigotes in human macrophages. PLoS Neglected

Tropical Diseases 2012, 6, e1671.

22

Chapter 2

Indazole analogs

2.1 Drug design and Medicinal Chemistry Strategies

The identification of a novel target1 is primarily the initial step in designing and developing

a new series of anti-leishmanial agents. The initial hit/lead compound, 1.001 (SNX-2112),2 shown

in Figure 2.1, was identified via high throughput screen (HTS). This compounds was not only

active in inhibiting axenic amastigote growth of Leishmania donovani, but was also found to be

an active human Hsp 90 inhibitor, the primary biochemical target.3,4

This compound possesses an indazole heterocyclic core and an anthranilamide portion. Molecules

possessing the indazole heterocyclic core exhibit a variety of potent biological activities such as

serotonin receptor antagonist,5 anti-cancer activity6,7 and anti-inflammatory activity.6 Others have

been shown to be an active HIV protease inhibitor,8 or exhibit anti-hypertensive properties.9

Examples of indazole-based biologically activite molecules are shown in Figure 2.2.

23

Figure 2.2: Indazole-based biologically active molecules

Docking2 and X-ray crystallographic10 studies on analogs of 1.001 depicted 3 critical hydrogen

bonds upon binding to human Hsp 90, two of which are contributed by the anthranilamide portion,

as shown in Figure 2.3. The protozoan ortholog of Hsp 90 has also been crystallized by Silva and

coworkers11 and has been shown to exhibit similar binding characteristics.

24

Figure 2.3: (A) Docking of an analog of 1.001 into Hsp 90. (B) 2D Schematic of the binding site.

(C) Binding of another analog to the protein structure (Hsp 90). (D) Close-up view of active site region

In the initial discovery of 1.001, SNX-2112, and its prodrug analog, 1.002, SNX-5422, as discussed

in Chapter 1, Huang and coworkers2 focused mainly on the anthranilamide portion in their attempt

to optimize for pharmacokinetic properties. Thus, little attention was paid to the indazole

heterocyclic core as well as the two (2) methyl substituents, but they noted that their presence was

required for activity. The overall synthetic and structure and activity relationship (SAR) strategies

adopted in our search for anti-leishmanial agents, fully incorporated the binding properties from

the aforementioned models (docking) as well as modifications to the anthranilamide and indazole

portions of the molecule. The necessity/importance of the two (2) methyl groups was also

evaluated for activity.

25

Thus anti-leishmanial compounds based on the Hsp 90 inhibitor, SNX-2112, were synthesized,

with the primary goal of designing compounds that would be selective for the protozoan ortholog

of Hsp 90 (Hsp 83). In an attempt to confirm the initial hit, a simplified version of 1.001 was

synthesized for a number of carefully considered reasons; not only was it feasible and synthetically

less challenging to make compound 2.008, as shown in Scheme 2.1, as the first test compound,

but a new data set to compare the anti-leishmanial activity would also prove useful. Furthermore,

to optimize for pharmacokinetics and hopefully restrict the cytotoxicity that may arise when the

compound binds to human Hsp 90, the -CF3 group was deleted from the indazole heterocyclic core

and a methyl (-CH3) group was installed instead.12

2.2 Synthesis of initial analog, 2.008

A variety of synthetic methods has been developed for indazole synthesis;13,14 however, the

synthetic approach adopted has the advantage of being flexible enough to make a broad range of

different modifications via robust synthetic chemistry, as shown in Scheme 2.1. This would enable

the rapid preparation of several novel anti-leishmanial agents for SAR and structural optimization

studies. Indazole heterocyclic rings can be achieved through condensation reaction involving a

diketone and a hydrazinyl derivative.15

2.2.1 Synthesis of initial test compound, 2.008: 2-((3-methoxypropyl)amino)-4-(3,6,6-

trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzamide, 2.008.

Synthesis of the initial indazole analog 2.008, shown in Scheme 2.1, required a triketone

2.003 and an arylhydrazinyl derivative 2.005 for the key step. The triketone 2.003 was

conveniently prepared from dimedone (2.001) and acetic anhydride (2.002) in the presence of N,N-

diisopropylethylamine (DIPEA, Hunig’s base) and a catalytic amount of 4-dimethylamino-

26

pyridine (DMAP).16,17 Substitution of the fluorine of 2-bromo-4-fluorobenzonitrile, 2.004, with

hydrazine in tetrahydrofuran (THF) under room temperature conditions afforded the known

hydrazinyl compound 2.005.18 The fluoride substitution was feasible due to the presence of the

cyano-group on the benzene ring which acts an activator in the reaction process, while itself being

a convenient synthetic precursor for the carboxamide later in the synthesis.

Scheme 2.1: Synthesis of initial analog 2.008

Reagents and conditions: (a) iPrNEt, DMAP, DCM, rt, 89%; (b) H2N-NH2, rt, THF, 94%; (c) 2.003, EtOH,

AcOH, rt, 59%; (d) H2N(CH2)3OMe, Pd(OAc)2, DPPF, NaOtBu, Toluene, mW, 120 oC, 64%; (e) H2O2,

aqueous NaOH, EtOH, DMSO, mW, 100 oC, 72%.

27

The advanced intermediate condensation product, 2.006, was obtained by treating the triketone, 2-

acetyl-5,5-dimethylcyclohexane-1,3-dione, 2.003, with 2-bromo-4-hydrazinylbenzonitrile, 2.005,

in a mixture of ethanol (EtOH) and acetic acid (AcOH). This product was able to undergo the

traditional Hartwig-Buchwald amination19 reaction using conditions involving palladium acetate

[Pd(OAc)2], 1,1’-bis(dimethylphosphino)ferrocene (DPPF), and sodium tert-butoxide (NaOtBu)

in toluene at 120 oC under microwave irradiation (mW) to install the methoxy propylamine side

chain in compound 2.007. This procedure was followed by hydrogen peroxide (H2O2) catalysed

hydration of the benzonitrile to the corresponding benzamide20 in a mixture of EtOH and dimethyl

sulfoxide (DMSO) in the presence of aqueous sodium hydroxide (NaOH) to afford the target

compound 2.008.

The addition of DMSO to the reaction system is known to both improve yields and increase the

rate of the hydration process.2 Gratifyingly, the synthesized compound 2.008 retained its activity

in the axenic amastigote assay with an IC50 of 0.65µM. As expected, the reaction conditions of the

final hydration step also yielded the acid by-product compound, 2.009, Figure 2.4, which also

showed good activity in the assays. The excellent in vitro activity of the initial analog led to the

explororation of closely related analogs based on Hsp 90 inhibitors, guided by computational

modelling.

Figure 2.4: Acid by-product of initial analog

28

2.2.2 Computational modelling strategies

In order to optimize for activity and improve upon the pharmacokinetic properties and/or

selectivity of the anti-leishmanial agents, a computational model for binding into the active site of

human Hsp 90 was developed to guide the design, synthesis and development of further analogs.

Publicly available coordinates (3D0B)10 were used to dock a related quinazoline analog 2.010,

Figure 2.5, with the active site of Hsp 90 in Autodock 1.5.6.21

The observations from the docking studies included a depiction of the methyl groups orienting

towards a hydrophobic pocked,12 as shown in Figure 2.6. The choice of the quinazoline analog

2.010 was based not only on its good activity in the assays, but it also restricts most of the rotatable

bonds to facilitate computational analysis. Moreover, and perhaps most crucial, is the fact that it

maintains the amide-like nature of the initial analog as well as all of the hydrogen-bonds that is

expected to exist.

29

Figure 2.6: Docking studies: Minimized poses of 2.010 showing methyl groups accessing hydrophobic pockets.

Ball and stick (A) and Surface (B) models showing 2.010 accessing the active site. (C) Showing a channel

exposing methyl groups which should accommodate modifications at this site

Based on the docking studies, the necessity of the dimethyl substituents in the initial lead

compound was investigated, given the fact that their presence was the result of using 5,5-

dimethylcyclohexane-1,3-dione (dimedone) as the starting material. Thus, compound 2.013 was

prepared using 2-acetylcyclohexane-1,3-dione as the starting material, Scheme 2.2, and following

similar synthetic steps as outlined above.

Scheme 2.2: Synthesis of desmethyl analog of 1.001

Reagents and conditions: (a) DIPEA, DMAP, DCM, rt, 67%; (b) i. 2.005, EtOH, AcOH, rt, 52%; (ii)

H2N(CH2)3OMe, Pd(OAc)2, DPPF, NaOtBu, Toluene, mW, 120 oC, 74%; (iii) H2O2, aqueous NaOH, EtOH,

DMSO, mW, 100 oC, 81%.

30

Predictably, preliminary SAR studies of this analog proved that, the geminal dimethyl groups are

critical for antiprotozoal activity, since compound 2.013 showed no inhibitory activity in the axenic

amastigote assay (IC50 25 µM), suggesting that the hydrophobic pocket where they were oriented

could be exploited to gain additional activity and/or selectivity. Based upon this finding, a plan

was put in place to maintain the methyl groups or diversify the entire indazoles bicyclic core while

maintaining the anthranilamide portion of the molecule. In all of the synthetic efforts it was ensured

that the compounds conformed to the traditional physicochemical parameters such as Lipinski’s

Rule of 5,22 in order to maximize the likelihood of identifying compounds with optimal

pharmacokinetic properties and acceptable oral bioavailability.

2.2.3 Synthesis of methyl substituted analog, 2.017: 2-((3-methoxypropyl)amino)-5-

methyl-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzamide

A methyl substituted version, 2.017, of 1.001, Figure 2.7, was also prepared using similar

synthetic routes as outlined previously. However, the condensation reaction between the methyl

substituted arylhydrazinyl derivative, 2014, and the triketone, 2.003, was not possible under room

temperature conditions, Scheme 2.3.

31

The rationale for the synthesis of 2.017 was based on the striking similarities between the

anthranilamide portion of 1.001 and the known active Hsp 90 inhibitor, XL888,23 which is at

various stages in clinical trials for different cancers.24 The methyl substituent on the

anthranilamide of XL888 was designed to twist the phenyl ring out of planarity. Thus a methyl

group was introduced into the first test compound in order to elicit a similar twisting effect and to

evaluate its effect on anti-leishmanial activity as shown in Scheme 2.3.

Scheme 2.3: Synthesis of methyl substituted analog

Reagents and conditions: (a) i. 2.003, MeOH, AcOH, rt; ii. Reflux, 73%; (b) (i) H2N(CH2)3OMe, Pd(OAc)2,

DPPF, NaOtBu, Toluene, mW, 120 oC, 66%; (ii) H2O2, aqueous NaOH, EtOH, DMSO, mW, 100 oC, 70%.

The methylsubstituted arylhydrazinyl derivative 2.014 was obtained in 70% yield by treating 2-

bromo-4-fluoro-5-methylbenzonitrile with hydrazine in THF. This compound was coupled to

2.003 under refluxing conditions to obtain the key intermediate 2.016. Ordinarily, this reaction

32

would go smoothly under room temperature conditions, however owing to the presence of sterics,

the intermediate 2.015 (confirmed by LCMS) could not proceed to 2.016 until after being subjected

to refluxing conditions. This was followed by the Hartwig-Buchwald amination and cyanide

hydration to arrive at the target compound in 70% yield.

33

2.3 Results and discussion

The anti-leishmanial activity of the synthesized analogs were evaluated in the laboratory

of Prof. Dennis Kyle by Dr. Brian Vesely, (formerly in the USF Department of Public Health), in

the initial high throughput axenic amastigote assay as well as in a high content screening (HCS)

infected-macrophage amastigote assay. The latter employed transformed J774 human macrophage

cell24 lines, which allowed the evaluation of amastigote growth inside the cells as well as

determined the cytotoxicity of the analogs to ensure that the observed activity was not a result of

killing the host cells. Figure 2.8 depicts the test compounds and Table 2.1 displays the results of

their corresponding biological evaluations. Miltefosine – a currently marketed anti-leishmanial

drug was used as the standard molecule.

Figure 2.8: Test compounds

34

Compound

IC50 (µM)

Selectivity

(Cytotox.

IC50/IM

IC50

ID

R

R1

R2 Axenic

Amastigote J774

Cytotoxicity Infected

Macrophage

Miltefosine 3.26 > 50 1.43 > 35

1.001 0.42 0.06 0.61 <0.1

2.008 NH2 H CH

3 0.65 3.52 0.88 4.0

2.009 OH H CH3 5.19 18.96 0.88 21.55

2.013 NH2 H H >25 >50 10.00 >5.0

2.017 NH2 CH

3 CH

3 2.65 12.55 0.65 19.31

Table 2.1: Biological data for indazole series

Each of these analogs was active in inhibiting L. donovani amastigote growth, with 2.009 and

2.017 showing good selectivity at approximately 22 and 20 respectively. The initial synthetic

analog 2.008 retained its anti-leishmanial activity in the sub-micromolar region with a four (4) fold

selectivity over J774 cell lines, which compared very well to the cytotoxicity of the initial hit

compound, 1.001. Removal/deletion of the two (2) geminal dimethyl groups in compound 2.013

led to a complete loss of activity, signifying that both methyl groups are required for activity.

Juxtaposition of this data with the docking studies suggests that this position can be exploited to

gain additional selectivity, activity and/or improved pharmacokinetics.

Compound 2.009 (acid-by-product) and 2.017 (methyl-substituted analog) exhibited considerably

more activity in the infected macrophage assay with IC50’s of 0.88 and 0.65 µM respectively, over

35

the initial compound 2.008 as well as the clinically used drug, miltefosine. More profound was

that compound 2.017, the most potent analog thus far, had selectivity of almost 20.

36

2.4 Experimental section

All reagents and solvents, unless specifically stated, were used as obtained from their

commercial sources without further purification. Air and moisture sensitive reactions were

performed under an inert atmosphere using either argon or nitrogen in a previously oven-dried or

flame-dried reaction flask, and addition of reagents were done using a syringe. Dry tetrahydrofuran

(THF) was obtained via distillation from sodium benzophenone ketyl. All microwave (mW)

reactions were carried out with an Anton Paar monowave 300 instrument. Thin layer

chromatography (TLC) analyses were performed using 200 µM pre-coated Sorbtech fluorescent

TLC plates and spots were visualized using UV light and/or by staining with different stains

including iodine vapor, ceric ammonium nitrate, acidic vanillin or acidic anisaldehyde. Analytical

LCMS data was obtained using an Agilent 1100 HPLC/MSD system equipped with a diode array

detector running a MeOH/H2O gradient. Purification of compounds was carried out using Sorbtech

silica gel (60Å porosity, 40-63µM particle size) in fritted medium pressure liquid chromatography

(MPLC) cartridges and eluted with Thomson Instrument SINGLE StEP pumps. Reverse phase

preparative HPLC (prepHPLC) purification was achieved on an Agilent preparative 1200

LC/6120B equipped with a single quadrupole mass spectrometer detector.

High resolution mass spectral (HRMS) data was performed on an Agilent 6540 Ultra-High-

Definition (UHD) Liquid chromatography quadrupole time-of-flight mass spectrometry

(LC/QToF-MS) with electrospray ionization (ESI) detection. Nuclear magnetic resonance (NMR)

spectrometry was run on a Varian Inova 500 MHz, Varian Inova 400 MHz or Varian Mercury 400

MHz spectrometer and data was processed using the ACD/NMR Processor program. Chemical

shifts are reported in ppm with residual solvent peaks referenced as internal standard. All

37

compounds were determined to be >95% pure by HPLC analysis and further confirmed by NMR

analysis.

General synthetic procedures:

2-Acetyl-5,5-dimethylcyclohexane-1,3-dione: To a solution of 5,5-dimethylcyclohexane-1,3-

dione (10.0 g, 71.3 mmol), Hunig's base (13.0 mL, 74.4 mmol) and DMAP (0.435 g, 3.560 mmol)

in DCM (200 mL) was added acetic anhydride (7.0 mL, 74 mmol) and the reaction mixture was

stirred overnight at room temperature (rt). The solution was then concentrated on a rotary

evaporator, and the resultant yellow oil was partitioned between hexanes and 1 N HCl. The organic

phase was then washed with brine, dried over MgSO4, filtered and concentrated on a rotary

evaporator to give the product as a brown oil which was purified by flash column chromatography

(hexanes/ethyl acetate 9:1 to 2:1) to afford 2.003 as a light yellow oil (11.6 g, 89%). 1H NMR (500

MHz, CDCl3) 18.01 (s, 1H), 2.49 (s, 3H), 2.45 (s, 2H), 2.26 (s, 2H), 0.98 (s, 6H) ppm. 13C NMR

(126 MHz, CDCl3) 202.3, 197.8, 195.0, 112.3, 52.4, 46.8, 30.5, 28.4, 28.1 ppm. LRMS (ESI)

m/z calc’d for C10H14O3 [M+H]+: 183.10, found 183.10.

2-Acetylcyclohexane-1,3-dione: This compound was synthesized in the same manner as

compound 2.003 (67%). 1H NMR (500 MHz, CDCl3) 18.04 (s, 1H), 2.59 (t, J = 6.5 Hz, 2H),

38

2.51 (s, 3H), 2.38 - 2.44 (m, 2H), 1.91 (td, J = 13.1, 6.53 Hz, 2H) ppm. 13C NMR (126 MHz,

CDCl3) 203.0, 198.6, 195.3, 113.4, 38.5, 33.2, 28.7, 18.9 ppm. LRMS (ESI) m/z calc’d for

C8H10O3 [M+H]+: 155.06, found 155.

2-Bromo-4-hydrazinylbenzonitrile: To a solution of 2-bromo-4-fluorobenzonitrile (2.51 g, 12.5

mmol) in dry THF (15 mL) was added anhydrous hydrazine (5.00 mL, 159 mmol) and the reaction

mixture was stirred overnight at rt leading to the formation of an off-white solid precipitate.

Additional THF (50 mL) was added to dissolve the solid which was then washed with saturated

sodium bicarbonate solution. The isolated organic phase was concentrated under reduced pressure

and the white solid was washed with H2O, followed by diethyl ether (30 mL) and dried under

reduced pressure to afford the title compound (2.5 g, 94%). 1H NMR (500 MHz, DMSO) 8.04

(s, 1H), 7.47 (d, J = 8.8 Hz, 1H), 7.06 (d, J = 1.5 Hz, 1H), 6.69 - 6.77 (m, 1H), 4.38 (s, 2H) ppm.

13C NMR (126 MHz, DMSO) 156.7, 135.4, 125.9, 119.6, 113.6, 110.4, 98.5 ppm. LRMS (ESI)

m/z calc’d for C7H6BrN3 [M + H]+: 211.97/213.97, found 212.08/214.08.

2-Bromo-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzonitrile: To a

solution of 2-bromo-4-hydrazinylbenzonitrile, 2.005, (2.156 g, 10.17 mmol) and 2-acetyl-5,5-

39

dimethylcyclohexane-1,3-dione, 2.003, (1.86 g, 10.2 mmol) in EtOH (40 mL) was added acetic

acid (1 mL), and the reaction mixture was stirred at room temperature for 72 hours. After formation

of the product, monitored by TLC/LCMS, the mixture was concentrated on a rotary evaporator

and purified by flash column chromatography eluting with hexanes and ethyl acetate (9:1 to 2:1 to

1:1) to obtain the target compound as an orange solid (2.2 g, 59%). 1H NMR (500 MHz, DMSO)

8.08 - 8.13 (m, 2H), 7.80 (dd, J = 8.6, 2.20 Hz, 1H), 3.00 (s, 2H), 2.39 (s, 3H), 2.33 (s, 2H), 1.01

(s, 6H) ppm. 13C NMR (126 MHz, DMSO) 193.3, 151.0, 150.0, 142.9, 136.4, 127.1, 125.9,

122.6, 117.7, 117.3, 113.3, 52.1, 36.6, 35.9, 28.2, 13.6 ppm. LRMS (ESI) m/z calc’d for

C17H16N3OBr [M + H]+: 358.0/360.0, found 357.8/359.8.

2-bromo-4-(3-methyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzonitrile: This compound

was synthesized in the same manner as compound 2.006 (52%). 1H NMR (500 MHz, DMSO)

8.08 - 8.12 (m, 2H), 7.78 - 7.83 (m, 1H), 3.07 (t, J = 6.1 Hz, 2H), 2.37 - 2.44 (m, 5H), 2.05 (quin,

J = 6.4 Hz, 2H) ppm. 13C NMR (126 MHz, DMSO) 194.0, 152.2, 150.3, 143.0, 136.3, 127.0,

125.8, 122.6, 118.6, 117.3, 113.3, 38.2, 23.5, 23.4, 13.6 ppm. LRMS (ESI) m/z calc’d for

C15H12BrN3O [M + H]+: 330.0/332.0, found 330.2/332.2.

40

Synthesis of 2-((3-methoxypropyl)amino)-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-

indazol-1-yl)benzonitrile: A mixture of 2-bromo-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-

1H-indazol-1-yl)benzonitrile, 2.006, (400.0 mg, 1.117 mmol), 1,1"

bis(diphenylphosphino)ferrocene (61.50 mg, 0.741 mmol), sodium 2-methylpropan-2-olate (2150

mg, 2.233 mmol), Pd(OAc)2 (15.04 mg, 0.067 mmol) and 3-methoxypropan-1-amine (199 mg,

2.233 mmol) in dry toluene was added to 30 mL microwave vial and the content was heated to

120 oC for 18 minutes in a microwave reactor. Upon cooling, DCM was added, transferred to a

round bottomed flask, concentrated on a rotary evaporator and purified by flash column

chromatography (eluting with 50% ethyl acetate in hexanes). The product was isolated as an off-

white solid (524 mg, 64%). 1H NMR (500 MHz, DMSO) 7.62 (d, J = 8.3 Hz, 1H), 6.87 (s, 1H),

6.82 (d, J = 8.3 Hz, 1H), 6.45 - 6.53 (m, 1H), 3.41 (t, J = 5.6 Hz, 2H), 3.19 - 3.29 (m, 5H), 2.93 (s,

2H), 2.25 - 2.43 (m, 5H), 1.75 - 1.86 (m, 2H), 1.00 (s, 6H) ppm. 13C NMR (126 MHz, DMSO) δ

193.7, 151.7, 150.4, 149.2, 143.5, 135.1, 117.9, 117.1, 110.7, 105.4, 93.7, 70.4, 58.4, 52.1, 40.7,

36.9, 35.8, 28.6, 28.1, 13.5 ppm. LRMS (ESI) m/z calc’d for C21H26N4O2 [M + H]+: 367.2, found

367.2.

41

2-((3-methoxypropyl)amino)-4-(3-methyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-

yl)benzonitrile: This compound was synthesized in the same manner as compound 2.007 (74%).

1H NMR (500 MHz, CD3OD) 7.54 (d, J = 8.3 Hz, 1H), 6.92 (d, J = 2.0 Hz, 1H), 6.82 (dd, J =

8.3, 2.0 Hz, 1H), 3.54 (t, J = 5.6 Hz, 2H), 3.36 - 3.39 (m, 2H), 3.35 (s, 3H), 3.04 (t, J = 6.4 Hz,

2H), 2.49 - 2.53 (m, 2H), 2.46 (s, 3H), 2.11 - 2.17 (m, 2H), 1.88 - 1.95 (m, 2H) ppm. 13C NMR

(126 MHz, CD3OD) 195.4, 151.6, 150.1, 143.2, 133.9, 117.6, 116.7, 110.4, 105.1, 94.3, 70.7,

57.6, 40.9, 37.6, 28.3, 23.4, 23.2, 12.0 ppm. LRMS (ESI) m/z calc’d for C19H22N4O2 [M + H]+:

339.2, found 339.4.

2-((3-methoxypropyl)amino)-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-

yl)benzamide: A mixture of 2-((3-methoxypropyl)amino)-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-

tetrahydro-1H-indazol-1-yl)benzonitrile, 2.007, (88.00 mg, 0.246 mmol), 50% w/w NaOH (9.000

µL, 0.246 mmol), and 30% H2O2 (143 µL, 4.67 mmol) in EtOH (800 µL) and DMSO (200 µL)

was added to a 2 mL microwave vial. The reaction mixture was heated to 100°C for 40 minutes in

a microwave reactor. Upon cooling, the mixture was transferred into a separatory funnel followed

42

by the addition of H2O and extracted with ethyl acetate and the organic solvent was removed in

vacuo. The residue was purified by preparative HPLC using an acetonitrile (ACN)/H2O gradient

and lyophilized to give the product as a white fluffy solid (68 mg, 72%). 1H NMR (500 MHz,

CDCl3) 8.03 - 8.16 (m, 1H), 7.47 (d, J = 8.3 Hz, 1H), 6.80 (d, J = 1.5 Hz, 1H), 6.62 (dd, J = 8.3,

1.5 Hz, 1H), 5.78 - 5.93 (m, 1H), 3.50 (t, J = 6.0 Hz, 2H), 3.27 - 3.36 (m, 5H), 2.81 (s, 2H), 2.53

(s, 3H), 2.38 (s, 2H), 1.93 (quin, J = 6.4 Hz, 2H), 1.77 - 1.87 (m, 1H), 1.09 (s, 6H) ppm. 13C NMR

(101 MHz, CDCl3) 193.5, 171.3, 150.6, 150.0, 149.1, 142.7, 129.6, 117.2, 112.6, 109.5, 106.8,

70.2, 58.7, 52.4, 40.7, 37.6, 35.8, 29.1, 28.4, 13.4 ppm. HRMS m/z: [M + H]+ calc’d for

C21H28N4O3 385.2240; Found 385.2248.

2-((3-methoxypropyl)amino)-4-(3-methyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-

yl)benzamide: This compound was synthesized in the same manner as compound 2.008 and was

obtained as a white fluffy solid (81%). 1H NMR (500 MHz, CD3OD) 7.70 (d, J = 8.3 Hz, 1H),

6.84 (d, J = 2.0 Hz, 1H), 6.70 (dd, J = 8.3, 2.0 Hz, 1H), 3.52 (t, J = 6.0 Hz, 2H), 3.27 - 3.34 (m,

8H), 3.03 (t, J = 6.4 Hz, 2H), 2.45 - 2.53 (m, 5H), 2.11 - 2.17 (m, 2H), 1.89 - 1.94 (m, 2H) ppm.

13C NMR (126 MHz, CD3OD) δ 195.5, 172.5, 151.4, 150.7, 149.7, 142.0, 130.0, 117.2, 113.3,

108.8, 105.7, 69.9, 57.5, 39.5, 37.7, 28.7, 23.4, 23.1, 12.0 ppm. HRMS m/z: [M + H]+ calc’d for

C19H24N4O3 357.1927; Found 357.1926.

43

2-((3-methoxypropyl)amino)-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-

yl)benzoic acid: This compound was obtained as a side product during the final hydration step of

2.008 as an off-white solid. 1H NMR (500 MHz, CDCl3) 8.11 (d, J = 8.8 Hz, 1H), 7.10 (br. s.,

1H), 6.87 (d, J = 7.8 Hz, 1H), 3.58 (t, J = 5.6 Hz, 2H), 3.32 - 3.47 (m, 5H), 2.86 - 2.94 (m, 2H),

2.56 (s, 3H), 2.40 - 2.46 (m, 2H), 2.00 - 2.10 (m, 2H), 1.22 - 1.31 (m, 1H), 1.09 (s, 6H) ppm. 13C

NMR (101 MHz, CDCl3) 193.5, 171.3, 150.6, 150.3, 149.3, 144.1, 133.9, 117.5, 110.9, 109.6,

107.5, 70.3, 58.7, 52.3, 41.9, 37.7, 35.9, 28.6, 28.4, 13.4 ppm. HRMS m/z: [M+H]+ calc’d for

C21H27N3O4 386.2080.

2-bromo-4-hydrazinyl-5-methylbenzonitrile: Using conditions identical to those described

above, 2.014 was obtained as white crystalline solid (70%). 1H NMR (500 MHz, DMSO) 7.53

(s, 1H), 7.33 (s, 1H), 7.28 (s, 1H), 4.36 (s, 2H), 1.99 (s, 3H) ppm. 13C NMR (126 MHz, DMSO-

d6) 154.5, 134.7, 123.8, 120.2, 119.6, 112.7, 98.5, 17.0 ppm. LRMS (ESI) m/z calc’d for

C8H8BrN3 [M+H]+: 226.08/228.08, found 225.90/227.90.

44

2-bromo-5-methyl-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzonitrile:

Using conditions identical to those described above, 2.016, was obtained as an orange solid

(73%). 1H NMR (500 MHz, DMSO) 8.11 (s, 1H), 7.97 (s, 1H), 2.57 (s, 2H), 2.38 (s, 3H), 2.31

(s, 2H), 2.07 (s, 3H), 1.00 (s, 6H) ppm. 13C NMR (126 MHz, DMSO) 193.1, 151.9, 148.9, 142.0,

137.7, 136.8, 132.0, 122.3, 117.1, 115.9, 115.8, 52.4, 35.9, 35.0, 28.1, 17.2, 13.5 ppm. LRMS (ESI)

m/z calc’d for C18H18BrN30 [M+H]+: 372.27/374.27, found 372.2/374.2

2-((3-methoxypropyl)amino)-5-methyl-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-

indazol-1-yl)benzonitrile: Using conditions identical to those above, 2.016 (b) i was obtained as

a brownish solid (66%). 1H NMR (400 MHz, DMSO) δ 7.53 (s, 1H), 6.65 (s, 1H), 6.24-6.31 (m,

1H), 3.36 (d, J = 1.0 Hz, 2H), 3.13-3.22 (m, 5H), 2.34 (s, 3H), 2.28 (s, 2H), 1.85 (s, 3H), 1.74

(quin, J = 1.0 Hz, 2H), 0.97 (s, 6H) ppm. 13C NMR (101 MHz, DMSO) δ 192.9, 151.2, 150.0,

148.2, 142.5, 135.8, 122.1, 117.7, 115.5, 110.3, 95.8, 70.4, 58.3, 52.4, 40.7, 35.8, 35.3, 28.7, 28.1,

16.1, 13.5 ppm. LRMS (ESI) m/z calc’d for C22H28N4O2 [M+H]+: 381.49, found 381.40.

45

Synthesis of 2-((3-methoxypropyl)amino)-5-methyl-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-

tetrahydro-1H-indazol-1-yl)benzamide: Using conditions identical to those described above,

2.017 was obtained as a pale yellow solid (70%). 1H NMR (500 MHz, DMSO) δ 8.05 (t, J = 5.5

Hz, 1H), 7.84-8.00 (m, 1H), 7.64 (s, 1H), 7.30 (br s, 1H), 6.53 (s, 1H), 3.39 (t, J = 6.1 Hz, 2H),

3.21 (s, 3H), 3.07 – 3.16 (m, 2H), 2.52 (s, 2H), 2.38 (s, 3H), 2.30 (s, 2H), 1.89 (s, 3H), 1.75 (quin,

J = 6.5 Hz, 2H), 1.00 (s, 6H) ppm. 13C NMR (126 MHz, DMSO) 193.1, 171.2, 151.1, 148.9,

147.9, 140.8, 132.1, 119.4, 115.5, 115.3, 109.9, 70.0, 58.4, 52.4, 39.9, 35.8, 35.4, 29.1, 28.2, 16.5,

13.6 ppm. HRMS m/z: [M+H]+ calc’d for C22H30N4O3 399.2396; Found 399.2407.

46

2.5 References

(1) Eckstein, J. ISOA/ARF Drug Development Tutorial

http://www.alzforum.org/sites/default/files/legacy/drg/tut/ISOATutorial.pdf (accessed Dec 2,

2017).

(2) Huang, K.; Veal, J.; Fadden, R.; Rice, J.; Eaves, J.; Strachan, J.; Barabasz, A.; Foley, B.;

Barta, T.; Ma, W. et al. Discovery of novel 2-aminobenzamide inhibitors of heat shock protein 90

as potent, selective and orally active antitumor agents. Journal of Medicinal Chemistry 2009, 52,

4288-4305.

(3) Chandarlapaty, S.; Sawai, A.; Ye, Q.; Scott, A.; Silinski, M.; Huang, K.; Fadden, P.; Partdrige,

J.; Hall, S.; Steed, P. et al. SNX2112, a synthetic heat shock protein 90 inhibitor, has potent

antitumor activity against HER kinase dependent cancers. Clinical Cancer Research 2008, 14,

240-248.

(4) Okawa, Y.; Hideshima, T.; Steed, P.; Vallet, S.; Hall, S.; Huang, K.; Rice, J.; Barabasz, A.;

Foley, B.; Ikeda, H. et al. SNX-2112, a selective Hsp 90 inhibitor, potently inhibits tumor cell

growth, angiogenesis, and osteoclastogenesis in multiple myeloma and other hematologic tumors

by abrogating signaling via Akt and ERK. Blood 2008, 113, 846-855.

(5) Lummis, S.; Thompson, A. Agonists and antagonists induce different palonosetron

dissociation rates in 5-HT3A and 5-HT3AB receptors. Neuropharmacology 2013, 73, 241-246.

(6) Bräse, S.; Gil, C.; Knepper, K. The recent impact of solid-phase synthesis on medicinally

relevant benzoannelated nitrogen heterocycles. Bioorganic & Medicinal Chemistry 2002, 10,

2415-2437.

(7) Harris, P.; Boloor, A.; Cheung, M.; Kumar, R.; Crosby, R.; Davis-Ward, R.; Epperly, A.;

Hinkle, K.; Hunter, R.; Johnson, J. et al. Discovery of 5-[[4-[(2,3-Dimethyl-2H-indazol-6-

yl)methylamino]-2-pyrimidinyl]amino]-2-methyl-benzenesulfonamide (Pazopanib), a novel and

potent vascular endothelial growth factor receptor inhibitor. Journal of Medicinal Chemistry

2008, 51, 4632-4640.

47

(8) Patel, M.; Rodgers, J.; McHugh, R.; Johnson, B.; Cordova, B.; Klabe, R.; Bacheler, L.;

Erickson-Viitanen, S.; Ko, S. Unsymmetrical cyclic ureas as HIV-1 protease inhibitors: Novel

biaryl indazoles as P2/P2′ substituents. Bioorganic & Medicinal Chemistry Letters 1999, 9,

3217-3220.

(9) Goodman, K.; Cui, H.; Dowdell, S.; Gaitanopoulos, D.; Ivy, R.; Sehon, C.; Stavenger, R.;

Wang, G.; Viet, A.; Xu, W. et al. Development of dihydropyridone indazole amides as selective

Rho-kinase inhibitors. Journal of Medicinal Chemistry 2007, 50, 6-9.

(10) Barta, T.; Veal, J.; Rice, J.; Partridge, J.; Fadden, R.; Ma, W.; Jenks, M.; Geng, L.; Hanson,

G.; Huang, K. et al. Discovery of benzamide tetrahydro-4H-carbazol-4-ones as novel small

molecule inhibitors of Hsp 90. Bioorganic & Medicinal Chemistry Letters 2008, 18, 3517-3521.

(11) Silva, K.; Seraphim, T.; Borges, J. Structural and functional studies of Leishmania

braziliensis Hsp 90. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 2013, 1834,

351-361.

(12) Kanwar, A.; Eduful, B.; Barbeto, L.; Carletti Bonomo, P.; Lemus, A.; Vesely, B.; Mutka, T.;

Azhari, A.; Kyle, D.; Leahy, J. Synthesis and activity of a new series of antileishmanial agents.

ACS Medicinal Chemistry Letters 2017, 8, 797-801.

(13) Sather, A.; Berryman, O.; Rebek, J. Synthesis of fused indazole ring systems and application

to nigeglanine hydrobromide. Organic Letters 2012, 14, 1600-1603.

(14) Lukin, K.; Hsu, M.; Fernando, D.; Leanna, M. New practical synthesis of indazoles via

condensation ofo-fluorobenzaldehydes and their O-methyloximes with hydrazine. The Journal of

Organic Chemistry 2006, 71, 8166-8172.

(15) Rahmatzadeh, S.; Karami, B.; Khodabakhshi, S. A Modified and practical synthetic route to

indazoles and pyrazoles using tungstate sulfuric acid. Journal of the Chinese Chemical Society

2014, 62, 17-20.

48

(16) Akkrem, A.; Lakhvich, F.; Budai, S.; Khlebnicova, T.; Petrusevich, I. A new simple

synthesis of 2-acylcyclohexane-1,3-diones. Synthesis 1978, 1978, 925-927.

(17) De Buyck, L.; De Pooter, H.; Verhé, R.; De Kimpe, N.; Schamp, N. Synthesis of 5-alkyl-

3,3-dimethyl-1,2,4-cyclopentanetriones, involving hydrogenation of 2-acyldimedones and

polychlorination. Bulletin des Sociétés Chimiques Belges 2010, 90, 825-835.

(18) Voss, G.; Eichner, S. Simplified synthesis of the precursor for the azo dye chlorindazone ds.

Journal für praktische Chemie 2000, 342, 201-204.

(19) Yang, B.; Buchwald, S. Palladium-catalyzed amination of aryl halides and sulfonates.

Journal of Organometallic Chemistry 1999, 576, 125-146.

(20) Sawaki, Y.; Ogata, Y. Mechanism of the reaction of nitriles with alkaline hydrogen peroxide.

reactivity of peroxycarboximidic acid and application to superoxide ion reaction. Bulletin of the

Chemical Society of Japan 1981, 54, 793-799.

(21) Trott, O.; Olson, A. AutoDock Vina: Improving the speed and accuracy of docking with a

new scoring function, efficient optimization, and multithreading. Journal of Computational

Chemistry 2009, NA-NA.

(22) Lipinski, C.; Lombardo, F.; Dominy, B.; Feeney, P. Experimental and computational

approaches to estimate solubility and permeability in drug discovery and development settings.

Advanced Drug Delivery Reviews 1997, 23, 3-25.

(23) Bussenius, J.; Blazey, C.; Aay, N.; Anand, N.; Arcalas, A.; Baik, T.; Bowles, O.; Buhr, C.;

Costanzo, S.; Curtis, J. et al. Discovery of XL888: A novel tropane-derived small molecule

inhibitor of Hsp 90. Bioorganic & Medicinal Chemistry Letters 2012, 22, 5396-5404.

(24) XL888 Clinical Trials

https://clinicaltrials.gov/ct2/results?cond=XL888&term=&cntry=&state=&city=&dist=

(accessed Mar 21, 2018).

49

(25) Siqueira-Neto, J.; Moon, S.; Jang, J.; Yang, G.; Lee, C.; Moon, H.; Chatelain, E.;

Genovesio, A.; Cechetto, J.; Freitas-Junior, L. An image-based high-content screening assay for

compounds targeting intracellular leishmania donovani amastigotes in human macrophages.

PLoS Neglected Tropical Diseases 2012, 6, e1671.

50

Chapter 3

Tetrahydroquinoline Analog

3.1 Tetrahydroquinoline scaffold in biologically active molecules

The privileged scaffold, tetrahydroquinoline, is an important substructure of various

natural products and pharmaceutical agents with a broad range of biological activities.1 For

example, the incorporation of macrocyclic rings onto a tetrahydroquinoline scaffold led to the

discovery of angiogenesis agents and inhibitors of an early embryonic development.2 Substituted

tetrahydroquinolines are the core substructure of a number of key pharmacological agents and drug

molecules including potent NF-kB inhibitors and cytotoxic agents,3 anti-tubercular agents,4

neurotropic agents,5 HIV protease inhibitors,6 and anti-tumor agents.7 The rest include,8 anti-

bacterial, anti-malarial, antagonist of neuronal Na+ channels, inhibitors of Na+/H+ exchange,

agonist of serotonin 5-HT3 receptor, antagonist of histamine H3 receptor, agonist of dopaminergic

D2 receptors, agonist of the large conductance calcium-activated potassium channel, agonist of β3

adrenergic receptors and positive allosteric modulator of the α7 nicotinic acetyl receptor agents.8

This subject has been thoroughly reviewed.8 Some selected tetrahydroquinoline compounds and

their biological activities are shown in Figure 3.1. However, to our knowledge, this scaffold has

not been previously explored as an anti-leishmanial agents.

51

Figure 3.1: Selected tetrahydroquinoline compounds and their biological activities

3.2 Rationale for designing tetrahydroquinoline analogs

The strategy to optimize for activity of the initial hit/lead compound was focused on the

SAR studies in two main areas: The anthranilamide portion and the indazole bicyclic core, guided

by docking studies. This led to the search for a novel scaffold that would be able to mimic the

exact same binding orientation as 1.001, as shown in Figure 3.2, hence the choice for the

privileged scaffold – tetrahydroquinoline.

52

Figure 3.2: Binding interactions of 1.001 and 3.013

It was rationalized that the nitrogen of tetrahydroquinoline analog 3.013 would serve as a viable

mimic for the carbonyl in 1.001. Thus, synthesis which incorporated the tetrahydroquinoline

substructure was initiated. Owing to the broad range of biological activities of the privileged

scaffold, there has been considerable interest in the design and development of new and efficient

synthetic methodologies aimed at obtaining tetrahydroquinoline derivatives in high yields and

good purity.1,5,9,10,11 The approach to the synthesis of the tetrahydroquinoline scaffold in analog

3.013 followed a palladium-catalysed oxidation of a γ-hydroxyenaminone, as described in Scheme

3.1.

3.3 Synthesis of the first tetrahydroquiniline analog: 4-((7,7-dimethyl-5,6,7,8-

tetrahydroquinolin-5-yl)amino)-2-((3-methoxypropyl)amino)benzamide.

Synthesis of the tetrahydroquinoline scaffold, Scheme 3.1, was initiated by a condensation

reaction between 5,5-dimethylcyclohexane-1,3-dione, 3.001, and the corresponding 3-

aminopropanol in the presence of molecular sieves to yield γ-hydroxyenaminone, 3.002.12

53

Scheme 3.1: Synthesis of 7,7-dimethyl-5,6,7,8-tetrahydroquinolin-5-amine

Reagents and conditions: (a) H2N(CH2)3OH, PhMe, reflux, 95%; (b) Pd(OAc)2, PPh3, K2CO3, mesityl bromide,

DMF, 150 oC, 50%; (c) H2NOMe, HCl, DIPEA, 81%; Pd/C, H2, TFA, Parr shaker.

Palladium-catalysed oxidation of 3.002 was followed by cyclization and spontaneous

aromatization to provide the corresponding quinolin-5-one, 3.003, in 67% yield. The subsequent

oxime intermediate 3.004 was obtained in decent yield (81%) by treating 3.003 with

methoxyamine hydrochloride in the presence of Hunig’s base.13,14 An attempt at hydrogenating

the oxime in trifluoroacetic acid (TFA) to provide the free amine 3.005 yielded no product.15 An

alternative method was then sought for the synthesis of the tetrahydroquinolinamine, 3.005,

Scheme 3.2.

54

Scheme 3.2: Preparation of advanced intermediate 3.008 via azide formation

Reagents and conditions: (a) NaBH4, MeOH, 98%; (b) DPPA, DBU, Toluene/THF, 72%, (c) Ph3P, pyridine,

H2O, THF, 72%; (d) 2.004, NaH, DMSO, 7%.

Tetrahydroquinolinone 3.003 was subjected to sodium borohydride reduction in MeOH which

provided the corresponding alcohol 3.006. The alcohol was converted to azide 3.007 using

diphenylphosphoryl azide (DPPA) in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene

(DBU).16 The azide thus formed was able to undergo the Staudinger reduction with

triphenylphosphine and H2O16,17 to yield the free amine, tetrahydroquinolinamine, 3.005.

Nucleophilic aromatic substitution of 3.005 with 2.004 yielded the key advanced intermediate

3.008, however the yield was very low. In an attempt to optimize the reaction and increase yield,

the alcohol 3.006 was converted to a tosyl group,18 3.009, in 80% yield. Functional group

interconversion19 was also effected on 2.004 with a resulting 55% yield to the corresponding

aminobromobenzonitrile 3.010, Scheme 3.3 - with the aim of coupling 3.009 and 3.010 together

via SN2 reaction, but this failed to provide the desired product.

55

Scheme 3.3: Preparation of advanced intermediate 3.008 via tosylation

Reagents and conditions: (a) TsCl, Et3N, 80%; (b) 3,4,5-trimethoxybenzylamine (TMBA), TFA, DCM, 140 oC, 55%; (c) 3.009, pTSA

At this stage, the synthesis of quinazolin-5-one 3.003 as well as aminobromobenzonitrile 3.010

had been achieved – the perfect couple for a condensation reaction. Thus, condensation reaction

involving 3.003 and 3.010 was initiated using the Dean-Stark trap apparatus leading to the

formation of imine 3.011, and subsequent sodium borohydride reduction in MeOH afforded the

advanced intermediate 3.008, as depicted in Scheme 3.4.

56

Scheme 3.4: Synthesis of tetrahydroquinoline analog

Reagents and conditions: (a) pTSA, PhMe, Dean-Stark, 175 oC, 59%; (b) NaBH4, MeOH, 37%; (c)

H2N(CH2)3OCH3, Pd(OAc)2, DPPF, NaOtBu, mW, 120 oC, 52%; (d) aqueous NaOH, H2O2, EtOH, DMSO, mW,

100 oC, 72%.

With 3.008 bearing bromine as a synthetic handle, the nucleophilic replacement was achieved

using the traditional Hartwig-Buchwald amination20 conditions which allowed the installation of

the methoxypropylamine side chain. This was followed by H2O2 catalysed hydration of the

benzonitrile21 to afford the final product, 3.013, as the first tetrahydroquinoline analog.

57

3.4 Synthesis of N7-(7,7-dimethyl-5,6,7,8-tetrahydroquinolin-5-yl)quinazoline-4,7-

diamine

Inspired by the activity of the quinazoline analog 2.010 used in the docking studies,

preparation of a tetrahydroquinoline-quinazoline analog was initiated. Key to this synthesis was

the avoidance of late stage structural modifications as shown in Scheme 3.5.

Scheme 3.5: Tetrahydroquinoline-Quinazoline analog

Reagents and conditions: (a) Formamidine acetate, NaH, DMF, rt; (b) NaH, Dioxane

However, reaction of the advanced intermediate 3.008 with formamidine acetate,22 and a

nucleophilic aromatic substitution reaction between 3.005 and 3.015 in the presence of sodium

hydride (NaH) or Hunig’s base23 yielded no product. Failure of the above reactions led to the

preparation of quinazoline-4,7-diamine 3.022, Scheme 3.6, targeted at coupling to 3.003. In the

58

course of this synthesis, an attempt was made to couple the various quinazoline-intermediates to

different derivatives of the tetrahydroquinoline scaffold as shown in Scheme 3.6.

Scheme 3.6: Synthesis of quinazoline-4,7-diamine for

tetrahydroquinoline-quinazoline analog

Reagents and conditions: CuI, NaN3, CH3NH(CH2)2NHCH3, CsCO3, EtOH, 90 oC, 62%; (b) HCONH2, 150 oC,

mW, 70%; (c) Pd/C, H2, MeOH, 82%; (d) 3.003, NaBH3CN, MeOH Or 3.021, DIPEA, MeOH; (e) i. SOCl2,

DMF. ii. 7N NH3/MeOH, rt, 50%; (f) (Boc)2O, TEA, MeOH; (g) Pd/C, H2, THF, MeOH; (h) Pd/C, H2, DMF,

THF/MeOH, rt, 93%; (i) 3.003, pTSA, rt, reflux.

59

In the above Scheme 3.6, copper-catalysed direct amination24 of 2-chloro-4-nitrobenzoic acid

3.015 using sodium azide (NaN3) as the amino source under mild conditions provided 2-amino-4-

nitrobenzoic acid 3.016. This compound was then treated with an excess amount of formamide25

and irradiated in a microwave reactor at 150 oC to obtain the quinazoline derivative 3.017. The

nitro group in compound 3.017 was reduced to the amino derivative 3.018, and treated with 3.003

in the presence of sodium cyanoborohydride (NaBH3CN) but yielded none of the desired product.

The amino-derivative 3.018 underwent SN2 displacement reaction with 3.020, but the reaction did

not produce the desired product.

The nitro-quinazoline derivative 3.017 was treated with SOCl2 in the presence of DMF (catalytic)

followed by ammonia to provide aminonitro-quinazoline derivative 3.021. It was envisioned that,

protecting the amine at the 4-position with an appropriate protecting group such as Boc26 and

reducing the nitro-group to the corresponding amine in compound 3.014 could serve as a viable

precursor to coupling to either 3.003 or 3.020. However, the Boc-protection reaction did not yield

the desired product. The diaminoquinazoline compound 3.022 was also obtained, but again,

coupling reactions failed. In the latter reaction, there was no anticipation of the amine at the 4-

position interfering in the reaction process.

A lot of effort and labour went into designing a second tetrahydroquinoline analog, however data

for 3.013 showed that the compound was not sufficiently active and also considerably cytotoxic

(towards J774 cell lines). This did not warrant the pursuance of another analog of this series. Thus,

attention was shifted from this scaffold to focus on other series that showed more promise as anti-

leishmanial agents which will be discussed in Chapter 4.

60

3.5 Results and Discussion

Table 3.1 shows the biological evaluation of tetrahydroquinoline analog 3.013 in the axenic

amastigote, J774 cytotoxicity and infected macrophage assays. This was performed in the

laboratory of Prof. Dennis Kyle by Dr. Brian Vesely, formerly in the USF Department of Public

Health.

Compound

ID

IC50 (µM)

Selectivity

(Cytotox.

IC50/

IM IC50) Axenic

Amastigote

J774 Cytotoxicity

Infected

Macrophage

Miltefosine 3.26 > 50 1.43 > 35

3.013 2.55 6.75 5.38 1.3

Table 3.1: Biological data for tetrahydroquinoline analog

The tetrahydroquinoline analog 3.013 showed activity in inhibiting amastigote growth in the

assays, however there was no great improvement over the activities of the indazole series, as

discussed in Chapter 2. Moreover, the level of selectivity depicted by this analog indicates poor

selectivity in the infected macrophage assay compared to cytotoxicity (with a selectivity factor of

1.3).

61

3.6 Experimental

General synthetic procedures:

7,7-dimethyl-7,8-dihydroquinolin-5(6H)-one: A solution of 5,5-dimethylcyclohexane-1,3-dione

(10.0 g, 71.3 mmol) and 3-aminopropan-1-ol (6.00 mL, 71.3 mmol) in toluene (250 mL) was

heated to in a Dean-Stark trap until the separation of H2O had finished. The solvent was removed

in vacuo and the residual viscous oil was triturated with ethyl acetate to give a yellow crystalline

solid, 3-((3-hydroxypropyl)amino)-5,5-dimethylcyclohex-2-enone (13.3 g, 95%). LRMS (ESI)

m/z calculated for C11H19NO2 [M+H]+: 198.28, found 198.0. A portion of this material was used

directly in the next step without further purification.

To a solution of 3-((3-hydroxypropyl)amino)-5,5-dimethylcyclohex-2-enone (10 g, 50.7 mmol) in

DMF (60 mL) was added mesityl bromide (11.12 g, 55.90 mmol), triphenylphosphine (0.799 g,

3.040 mmol), palladium (II) acetate (0.342 g, 1.522 mmol) and potassium carbonate (8.52 g, 61.7

mmol). The resulting mixture was heated to 150°C for 2 hours. Upon cooling to room temperature,

the mixture was filtered through a celite pad and evaporated in vacuo to give an oily residue, which

was purified by column chromatography to yield 7,7-dimethyl-7,8-dihydroquinolin-5(6H)-one

(4.4 g, 50%). 1H NMR (400 MHz, CDCl3) 8.68 (dd, J = 4.7, 1.6 Hz, 1H), 8.24 (dd, J = 7.8, 1.6

Hz, 1H), 7.27 (dd, J = 4.7, 7.8 Hz, 1H), 3.03 (s, 2H), 2.53 (s, 2H), 1.10 (s, 6H) ppm. 13C NMR

(101 MHz, CDCl3) 198.0, 162.2, 153.8, 134.6, 126.5, 122.1, 52.0, 46.4, 33.0, 28.2 ppm. LRMS

(ESI) m/z calc’d for C11H13NO [M+H]+: 176.23, found 176.20.

62

4-amino-2-bromobenzonitrile: A mixture of 2-bromo-4-fluorobenzonitrile (3.00 g, 15.0 mmol)

and (3,4,5-trimethoxyphenyl)methanamine (3.16 g, 16.0 mmol) was added to a 48 mL reaction

vial, sealed and then stirred at 140 oC for 1 hour. Upon cooling to room temperature, DCM (7.5

mL) and trifluoroacetic acetic (7.5 mL) were sequentially added, sealed and allowed to stir at room

temperature for 24 hours. The resulting mixture was concentrated, diluted with ethyl acetate (100

mL), washed with saturated aqueous sodium bicarbonate (2 x 50 mL) and dried over anhydrous

sodium sulfate, filtered, concentrated and purified by gradient flash chromatography, eluting with

0% to 40% ethyl acetate in hexanes to yield the target compound as a pale yellow crystalline solid

(1.734 g, 55%). 1H NMR (400 MHz, CDCl3) δ 7.36 (d, J = 8.2 Hz, 1H), 6.87 (d, J = 2.3 Hz, 1H),

6.56 (dd, J = 8.4, 2.2 Hz, 1H), 4.19 (br s, 2H) ppm. 13C NMR (126 MHz, CDCl3)

ppm. LRMS (ESI) m/z calc’d for C7H5BrN2

[M+H]+: 198.04/200.04, found 198.08/200.08.

2-bromo-4-((7,7-dimethyl-5,6,7,8-tetrahydroquinolin-5-yl)amino)benzonitrile: Step 1: A

mixture of 7,7-dimethyl-7,8-dihydroquinolin-5(6H)-one (0.50 g, 2.85 mmol) and 4-amino-2-

63

bromobenzonitrile (0.562 g, 2.850 mmol) in toluene (15.0 mL) was heated to reflux in a Dean

Stark trap until the separation of H2O had finished. The reaction was monitored by LC/MS for 3 -

4 days until no starting material was seen. The solution was then concentrated on a rotary

evaporator to give the product as a residual viscous oil which was purified by column

chromatography using hexanes and ethyl acetate (1:1) as eluent, to give (E)-2-bromo-4-((7,7-

dimethyl-7,8-dihydroquinolin-5(6H)-ylidene)amino)benzonitrile as a brownish yellow solid (0.6

g, 59%). LRMS (ESI) m/z calc’d for C18H16BrN3 [M+H]+: 355.25/357.25, found 355.20/357.20.

This material was used in the next step.

Step 2: To a stirred solution of (E)-2-bromo-4-((7,7-dimethyl-7,8-dihydroquinolin-5(6H)-

ylidene)amino)benzonitrile (900 mg, 2.54 mmol) in MeOH (11 mL) was added sodium

borohydride (192 mg, 5.08 mmol) portionwise at 0°C. The reaction mixture was stirred at rt for

> 5 h and monitored by LCMS. Upon completion of the reaction, the mixture was concentrated on

a rotary evaporator and purified with flash column chromatography using hexanes and ethyl

acetate (2:1) as the eluent to give the product as a pale white solid (675 mg, 37%). 1H NMR (400

MHz, CDCl3) 8.47 (br. s., 1H), 7.68 (d, J = 7.4 Hz, 1H), 7.40 (d, J = 8.6 Hz, 1H), 7.12 - 7.18 (m,

1H), 6.88 (s, 1H), 6.57 (d, J = 8.2 Hz, 1H), 4.74 (d, J = 7.4 Hz, 1H), 4.41 (d, J = 8.6 Hz, 1H), 2.81

(s, 2H), 2.01 - 2.09 (m, 1H), 1.52 - 1.58 (m, 1H), 1.13 (s, 3H), 1.07 (s, 3H) ppm. 13C NMR (101

MHz, CDCl3) δ 156.6, 151.5, 148.6, 135.3, 135.2, 131.3, 126.8, 121.7, 118.5, 115.7, 111.2, 102.0,

77.0, 49.5, 46.0, 42.7, 31.1, 30.6, 25.9 ppm. LRMS (ESI) m/z calc’d for C18H18BrN3 [M+H]+:

357.27/359.27, found 357.20/359.20.

64

4-((7,7-dimethyl-5,6,7,8-tetrahydroquinolin-5-yl)amino)-2-((3

methoxypropyl)amino)benzonitrile: To a mixture of 2-bromo-4-((7,7-dimethyl-5,6,7,8-

tetrahydroquinolin-5-yl)amino)benzonitrile (100.0 mg, 0.281 mmol), palladium (II) acetate (3.200

mg, 0.014 mmol), 1,1'-bis(diphenylphosphino)ferrocene (15.50 mg, 0.187 mmol) and sodium tert-

butoxide (54.00 mg, 0.562 mmol) in toluene (0.5 mL) was added 3-methoxypropan-1-amine

(50.10 mg, 0.562 mmol) in a 10 mL microwave vial. The reaction mixture was heated to 120 oC

in a microwave reactor for 16 minutes. Upon cooling, methylene chloride was added to the reaction

mixture and concentrated on a rotary evaporator. The resulting solid was adsorbed onto silica gel

using MeOH (3X) and purified by flash chromatography eluting with 100% ethyl acetate. Product

was recovered as a brown oil (105.5 mg, 52%). 1H NMR (500 MHz, DMSO) δ 8.33-8.40 (m, 1H),

7.62 (d, J = 7.6 Hz, 1H), 7.19 (dd, J = 7.8, 4.7 Hz, 1H), 7.08-7.15 (m, 1H), 6.68 (d, J = 8.8 Hz,

1H), 6.04 (dd, J = 1.7, 8.6 Hz, 1H), 5.87 (s, 1H), 5.61 (t, J = 5.5 Hz, 1H), 4.65-4.78 (m, 1H), 3.29-

3.40 (m, 4H), 3.06-3.20 (m, 3H), 2.61-2.73 (m, 2H), 1.83-1.92 (m, 1H), 1.68-1.79 (m, 2H), 1.54

(dd, J = 11.3, 12.4 Hz, 1H), 1.04-1.07 (s, 3H), 0.99 (s, 3H) ppm. 13C NMR (126 MHz, DMSO) δ

156.5, 153.6, 152.3, 148.3, 135.3, 134.4, 132.9, 121.9, 120.2, 102.8, 92.6, 82.4, 70.7, 58.4, 48.6,

46.4, 42.4, 40.7, 31.5, 30.7, 28.7, 26.1 ppm. LCMS (ESI) m/z calc’d for C22H28N4O [M+H]+:

365.49, found 365.33.

65

4-((7,7-dimethyl-5,6,7,8-tetrahydroquinolin-5-yl)amino)-2-((3-

methoxypropyl)amino)benzamide: A mixture of 4-((7,7-dimethyl-5,6,7,8-tetrahydroquinolin-5-

yl)amino)-2-((3-methoxypropyl)amino)benzonitrile (95.00 mg, 0.261 mmol), 50% w/w NaOH

(0.100 mL, 0.261 mmol), 30% H2O2 (0.15 mL, 4.89 mmol) in EtOH (1 mL) and DMSO (0.45 mL)

was added to a 10 mL microwave vial. The reaction mixture was heated to 100°C for 45 minutes.

The product was extracted with H2O and ethyl acetate and following removal of the solvent on

rotary evaporator, the product was purified by flash column chromatography to give the product

as a pale brown solid (72 mg, 72%). 1H NMR (500 MHz, CDCl3) 8.48 (d, J = 3.9 Hz, 1H), 8.28

(br s, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.25-7.29 (m, 1H), 7.15 (dd, J = 7.8, 4.9 Hz, 1H), 5.89-5.97

(m, 2H), 5.49 (br s, 2H), 4.75-4.85 (m, 1H), 4.16 (br d, J = 8.3 Hz, 1H), 3.51 (t, J = 6.1 Hz, 2H),

3.35 (s, 3H), 3.23 (br t, J = 6.6 Hz, 2H), 2.83 (d, J = 3.4 Hz, 2H), 2.07-2.14 (m, 1H), 1.87-1.99 (m,

2H), 1.54-1.63 (m, 1H), 1.16 (s, 3H), 1.10 (s, 3H) ppm. 13C NMR (126 MHz, CDCl3) 171.9,

156.6, 152.6, 152.1, 148.3, 135.5, 132.8, 130.5, 121.6, 103.1, 100.4, 93.2, 70.4, 58.7, 49.3, 46.2,

43.3, 39.8, 31.3, 30.6, 29.3, 26.0 ppm. HRMS m/z calc’d for C22H30N4O2 [M+H]+: 383.2447;

Found 383.2450.

66

3.7 References

(1) Sheikhhosseini, E.; Farrokhi, E.; Bigdeli, M. Synthesis of novel tetrahydroquinoline

derivatives from α,α′-bis(substituted-benzylidene)cycloalkanones. Journal of Saudi Chemical

Society 2016, 20, S227-S230.

(2) Reddy Guduru, S.; Chamakuri, S.; Chandrasekar, G.; Kitambi, S.; Arya, P.

Tetrahydroquinoline-derived macrocyclic toolbox: the discovery of antiangiogenesis agents in

zebrafish assay. ACS Medicinal Chemistry Letters 2013, 4, 666-670.

(3) Jo, H.; Choi, M.; Kumar, A.; Jung, Y.; Kim, S.; Yun, J.; Kang, J.; Kim, Y.; Han, S.; Jung, J. et

al. Development of novel 1,2,3,4-tetrahydroquinoline scaffolds as potent nf-κb inhibitors and

cytotoxic agents. ACS Medicinal Chemistry Letters 2016, 7, 385-390.

(4) Kumar, A.; Srivastava, S.; Gupta, G.; Chaturvedi, V.; Sinha, S.; Srivastava, R. Natural

product inspired diversity oriented synthesis of tetrahydroquinoline scaffolds as antitubercular

agent. ACS Combinatorial Science 2011, 13, 65-71.

(5) Goli, N.; Mainkar, P.; Kotapalli, S.; K, T.; Ummanni, R.; Chandrasekhar, S. Expanding the

tetrahydroquinoline pharmacophore. Bioorganic & Medicinal Chemistry Letters 2017, 27, 1714-

1720.

(6) Carlson, H.; Damm, K.; Meagher, K. Compositions and methods relating to HIV protease

inhibitors. 2008, PCT Int. Appl. WO 2009036341.

(7) Avendaño, C.; Menéndez, J. Alkylating and non-alkylating compounds interacting with the

dna minor groove. Medicinal Chemistry of Anticancer Drugs 2008, 177-198.

(8) Sridharan, V.; Suryavanshi, P.A.; Menendez, J.C. Advances in the chemistry of

tetrahydroquinolines. Chemical reviews 2011, 111 (11), 7157-7259.

(9) Yadav, J.; Subba Reddy, B.; Sunitha, V.; Srinivasa Reddy, K.; Ramakrishna, K.

Montmorillonite KSF-catalyzed one-pot synthesis of hexahydro-1H-pyrrolo[3,2-c]quinoline

derivatives. Tetrahedron Letters 2004, 45, 7947-7950.

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(10) Spanedda, M.; Hoang, V.; Crousse, B.; Bonnet-Delpon, D.; Bégué, J. Aza-Diels–Alder

reaction in fluorinated alcohols. A one-pot synthesis of tetrahydroquinolines. Tetrahedron Letters

2003, 44, 217-219.

(11) Powell, D.; Batey, R. Lanthanide(III)-catalyzed multi-component aza-Diels–Alder reaction

of aliphatic N-arylaldimines with cyclopentadiene. Tetrahedron Letters 2003, 44, 7569-7573.

(12) Pita, B.; Masaguer, C.; Ravina, E. New synthetic approaches to CNS drugs. A

straightforward, efficient synthesis of tetrahydroindol-4-ones and tetrahydroquinolin-5-ones via

palladium-catalyzed oxidation of hydroxyenaminones. Tetrahedron Letters, 2002, 43 (44), 7927-

7932.

(13) Zhao, H.; Vandenbossche, C.; Koenig, S.; Singh, S.; Bakale, R. An efficient synthesis of

enamides from ketones. Organic Letters 2008, 10, 505-507.

(14) Holmes, A.; Smith, A.; Williams, S.; Hughes, L.; Lidert, Z.; Swithenbank, C. Stereoselective

synthesis of (.+-.)-indolizidines 167B, 205A, and 207A. Enantioselective synthesis of (-)-

indolizidine 209B. The Journal of Organic Chemistry 1991, 56, 1393-1405.

(15) Harada, K.; Shiono, S. Stereoselective Catalytic Hydrogenation of α-Hydroxy Ketoximes.

Bulletin of the Chemical Society of Japan 1984, 57, 1040-1045.

(16) Hopkins, B.; Ma, M.; Chan, T.; Sun, L.; Zhang, L.; Kumaravel, G.; Lyssikatos, J.; Koch, K.;

Miao, H. Biaryl inhibitors of Bruton's tyrosine kinase. Cross reference to related applications.

2015, WO 2015/089337 A1, PCT/US2014/069853.

(17) Staudinger, H.; Meyer, J. Über neue organische phosphorverbindungen iii.

phosphinmethylenderivate und phosphinimine. Helvetica Chimica Acta 1919, 2, 635-646.

(18) Marcotullio, M.; Campagna, V.; Sternativo, S.; Costantino, F.; Curini, M. A new, simple

synthesis of n-tosyl pyrrolidines and piperidines. Synthesis 2006, 2006, 2760-2766.

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(19) Huang, K.; Hughes, P.; Ma, W.; Ommen, A.; Woodward, A.; Veal, J.; Barta, T. Benzene,

pyridine, and pyridazine derivatives as Hsp-90 inhibitors and their preparation, pharmaceutical

compositions and use in the treatment of proliferative diseases. 2008, WO 2008024970 A2, PCT

Int. Appl US 2006 60823457.

(20) Yang, B.; Buchwald, S. Palladium-catalyzed amination of aryl halides and sulfonates.

Journal of Organometallic Chemistry 1999, 576, 125-146.

(21) Sawaki, Y.; Ogata, Y. Mechanism of the reaction of nitriles with alkaline hydrogen peroxide.

reactivity of peroxycarboximidic acid and application to superoxide ion reaction. Bulletin of the

Chemical Society of Japan 1981, 54, 793-799.

(22) Tomazic, A.; Hynes, J. Synthesis of some novel halogenated 2,4-diaminoquinazolines.

Journal of Heterocyclic Chemistry 1992, 29, 915-920.

(23) Jia, Z.; Song, Y.; Q, X.; Kane, B.; Bauer, S.; Pandey, A. Preparation of benzamides and

nicotinamides as spleen tyrosine kinase inhibitors. 2012, WO 20120614180A2.

(24) Zhao, H.; Fu, H.; Qiao, R. copper-catalyzed direct amination of ortho-functionalized

haloarenes with sodium azide as the amino source. The Journal of Organic Chemistry 2010, 75,

3311-3316.

(25) Kulkarni, S.; Singh, S.; Shah, J.; Low, W.; Talele, T. Synthesis and SAR optimization of

quinazolin-4(3H)-ones as poly(ADP-ribose)polymerase-1 inhibitors. European Journal of

Medicinal Chemistry 2012, 50, 264-273.

(26) Nishida, H.; Saitoh, F.; Hirabayashi, T.; Chackalamannil, S.; Chan, T.; Chelliah, M.V.,

Clasby, M.C., Dwyer, M.P.; Greenlee, W.J.; Xia, Y. Morpholine compounds as factor ixa

inhibitors. 2010, WO 2010065717 A1, PCT/US2009/066548.

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Chapter 4

Tropane Analogs

4.1 Tropanes

Tropanes are bicyclic amines possessing both pyrrolidine and piperidine rings that share a

common nitrogen and two (2) carbon atoms. Thus, the typical structural element of all tropanes is

the azabicyclo[3.2.1]octane skeleton.1 Tropanes occur naturally as part of esters in plant species,

which are generally secondary metabolites of these plants, as shown in Figure 4.1.

Examples of plant species containing tropane alkaloids include Datura species (thorn apple,

angel’s trumpet and jimson weed), Hyoscyamus niger, Atropa belladonna (deadly nightshade),

Mandragora officinarum, Brugmansia, Atropanthe, Duboisia, Scopolia, Anisodus and

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Anthrotroche.2 Tropane alkaloids (ornithine-derived compounds) are extracted from the world’s

oldest plant medicines and have a variety of pharmacological applications, including analgesics,

hallucinations and poisons.3 Owing to their central nervous system (CNS) activity, tropane

alkaloids are often abused – one such commonly abused tropane is cocaine.

Tropane derivatives are one of the most economically important pharmaceuticals, Figure 4.2,4 and

over twenty (20) active pharmaceutical ingredients (API) possessing the tropane scaffold have

found use as anti-emetics, anesthetics, bronchodilators, anti-spasmodics and mydriatics.5

Figure 4.2: Major applications of tropane moiety and annual global revenue 4

Many different synthetic protocols6,7,8,9 have been developed for the synthesis of tropane

derivatives, due to their many useful applications. However, Robinson’s synthesis of tropinone

71

developed in 1917 continues to be the ideal choice for the synthesis of tropanes and its

derivatives.10

4.2 XL-888 – potent Hsp 90 inhibitor: A novel tropane derivative

Pursuant to our laboratory’s ongoing efforts to identify novel anti-leishmanial compounds

based on Hsp 90 inhibitors and prior docking studies, the intriguing striking similarities shared by

the anthranilamide portion of 1.001 with the potent Hsp 90 inhibitor, XL-88811 and its analogs –

(tropane derivatives used for the treatment of cancer), Figure 4.3, led to the exploration of a

hybridized version of a compound that contained our anthraniliamide. Aminopiperidine analogs

(non-bridged) were also prepared simultaneously to allow us evaluate the necessity/importance of

the bicyclic core.

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4.3 Synthesis of piperidine analogs

Three (3) piperidine analogs were synthesized, Figure 4.4: benzylamide 4.014, amide

4.018, and an ester 4.021. A convergent synthetic approach was used, wherein the

aminopiperidinyl nicotinamide/nicotinate and the anthranilamide portions were each synthesized

separately, and then coupled together using an appropriate peptide coupling reagent.

Figure 4.4: Piperidine analogs

4.3.1 Synthesis of piperidine benzylamide analog: N1-(1-(5-(benzylcarbamoyl)pyridin-2-

yl)piperidin-4-yl)-3-((3-methoxypropyl)amino)terephthalamide:

Scheme 4.1: Synthesis of Boc-protected piperidine

Reagents and conditions: (a) Boc2O, TEA, DCM, rt, 85%; (b) Pd/C, H2, MeOH, rt, 90%.

The primary amine functionality of commercially available benzyl-protected piperidine 4.001,

Scheme 4.1, was reacted with di-tert-butyl-dicarbonate in DCM at room temperature to provide

the respective Boc-protected intermediate, 4.002,12 which subsequently underwent

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hydrogenation13 to afford the target compound – Boc-protected piperidine, 4.003. This compound

would subsequently undergo nucleophilic aromatic substitution (SNAr) with benzyl-6-

chloronicotinamide, 4.008, as described in Scheme 4.2.

Scheme 4.2: Synthesis of 6-(4-aminopiperidin-1-yl)-N-benzylnicotinamide

Reagents and conditions: (a) i. SOCl2, DMF, Toluene, 80 oC. ii. BnNH2, DCM, 0 oC - rt, 68%; (b) 4.003,

DIPEA, ACN, reflux, 79%; (c) 1M HCl/Et2O, 0 oC - rt, 82%

Reaction of 6-chloronicotinic acid (4.004) with thionyl chloride and then benzylamine yielded N-

benzyl-6-chloronicotinamide14 (4.005) in an overall yield of 68%. This compound was able to

undergo aromatic nucleophilic substitution reaction with 4.003 to furnish compound 4.006,15,16

which was subsequently deprotected under acidic conditions15 to arrive at the free amine in

compound 4.007. This compound would subsequently be coupled to 4.013 via peptide coupling.

The synthesis of 4.013 is provided in Scheme 4.3.

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Scheme 4.3: Synthesis of 4-carbamoyl-3-((3-methoxypropyl)amino)benzoic acid

Reagents and conditions: (a) NBS, DCM, 89%; (b) i. tBuONO, BF3/Etherate, ii. CuCN, NaCN, Toluene, 0 oC

- rt, 83%; (c) CH3O(CH2)3NH2, XANTPHOS, Pd2(dba)3, CsCO3, Dioxane, 81%; (d.i) Aqueous NaOH, H2O2,

DMSO, EtOH (d.ii) Excess aqueous NaOH, 78%

Ethyl 4-aminobenzoate was monobrominated17 with N-bromosuccinimide (NBS) to give

compound 4.009 which was subsequently converted to ethyl 3-bromo-4-cyanobenzoate, 4.010,

through cyanidation via diazonium salt formation. This functional group interconversion was

initially attempted using sodium nitrite (NaNO2) as described,18 however, what worked best was

the use of tert-butyl nitrite and boron trifluoride diethyl etherate followed by the addition of copper

(I) cyanide and sodium cyanide.19 An attempt at installing the methoxypropylamine side chain in

compound 4.013 was not possible via the regular Hartwig-Buchwald amination conditions,20 thus

a related reaction condition was employed that uses 4,5-bis(diphenylphosphino)-9,9-

dimethylxanthene (XANTPHOS), cesium carbonate and tris(dibenzylideneacetone)-dipalladium

(Pd2(dba)3),21 with a resulting yield of 81%. With compound 4.013 in hand, the nitrile group

underwent H2O2-catalysed hydration22,23 and the carboxylate was hydrolysed under basic

conditions (saponification) to yield the carboxylic acid 4.013. However, the presence of aqueous

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NaOH in the reaction media resulted in a mixture of both the hydrated derivative and partly

saponified product (confirmed by LC/MS). Thus, excess aqueous NaOH was added to force the

reaction to the overall target compound 4.013 with a yield of 78%. This action also led to the

formation of a diacid side product (verified via LCMS and NMR) as shown in Figure 4.4, which

was difficult to separate via prepHPLC.

Compound 4.007 was added to 4.013 under appropriate reaction conditions using HATU21 as the

coupling reagent, which condensed and formed the amide bond as shown in compound 4.014,

Scheme 4.4.

Scheme 4.4: Synthesis of piperidine-benzyl analog, 4.014

Reagents and conditions: (a) HATU, DIPEA, DMF, 48%

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Following the successful synthesis of the benzylamide analog, 4.014, synthesis of amide and

ester analogs we initiated.

4.3.2 Synthesis of piperidine amide analog: N1-(1-(5-carbamoylpyridin-2-yl)piperidin-4-

yl)-3-((3-methoxypropyl)amino)terephthalamide

Scheme 4.5: Synthesis of piperidine-amide analog 4.018

Reagents and conditions: (a) SOCl2, NH4OH, Toluene, 73%; (b) i. 4.003, DIPEA, ACN, reflux, 78%. ii. 1M

HCl/Etherate, 87%; (c) 4.013, HATU, DIPEA, DMF, 48%

Reaction of chloronicotinic acid, 4.015, with thionyl chloride (SOCl2) and aqueous ammonia

(NH4OH)24 yielded 2-chloro-5-pyridinecarboxamide 4.016 in overall yield of 73%. The use of

ammonia gas for this purpose has been reported,25 however NH4OH was found to be more

convenient for this purpose. Following procedures described above, compound 4.017 was

achieved over two (2) steps in 68% overall yield. To compound 4.017 was added 4.013 under

appropriate reaction conditions using HATU21 as the coupling reagent which condensed and

formed the amide bond as shown in compound 4.018.

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4.3.3 Synthesis of piperidin-ester analog: ethyl 6-(4-(4-carbamoyl-3-((3-

methoxypropyl)amino)benzamido)piperidin-1-yl)nicotinate

Scheme 4.6: Synthesis of piperidine-ester analog 4.021

Reagents and condtions: (a) Triethylorthoacetate, toluene, 95%; (b) i. 4.003, DIPEA, CAN, reflux, 78%. ii, 1M

HCl/Etherate, 84%; (c) 4.013, HATU, DIPEA, 55%

6-chloronicotinic acid was treated with triethylorthoacetate to yield the corresponding ethyl-6-

chloronicotinate.26,27 Using a ratio of 1:3 (halonicotinic acid: triethyl orthoacetate) led to an almost

quantitative yield. Following procedures identical to the ones described above, 4.020 was achieved

over two (2) steps in 66% overall yield. To compound 4.020 was added 4.013 under appropriate

reaction conditions using HATU21 as the coupling reagent, which condensed and formed the amide

bond as shown in compound 4.021.

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4.4 Synthesis of tropane/bridged analogs

Scheme 4.7 describes the synthesis of all three (3) bridged/tropane analogs 4.030, 4.031

and 4.032 (benzylamide, amide and ester) wherein R has been defined as -NHBn, -OEt or –NH2.

Scheme 4.7: Synthesis of tropane analogs 4.030, 4.031 and 4.032

Reagents and conditions: (a) NaBH(AcO)3, BnNH2, AcOH, DCM, 45%; (b) Pd(OH)2, (NH4)HCO2, EtOH,

92%; (c) DIPEA, CbzCl, DCM, rt, 69%; (d) HCl/Et2O, rt, 84%; (e) R = -OEt: TEA, Dioxane, 51%, R = -

NHBn: TEA, ACN, 48%, R = -NH2: TEA, ACN, 61%; (f) Pd(OH)2, H2, MeOH, 72-78%; (g) 4.013, HATU,

DIPEA, DMF, 48-55%.

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In the above synthetic scheme, Scheme 4.7, the primary amine functionality in the known

compound 4.02428 was introduced stereoselectively under reductive amination conditions by

treating Boc-protected tropinone, 4.022, with sodium triacetoxyborohydride in the presence of

benzylamine to yield N-benzyl protective derivative 4.023. This was followed by debenzylation

with Pearlman’s catalyst29 using ammonium formate as the hydrogen source to obtain 4.024.

Reaction between tropane 4.024 and benzyl chloroformate (CbzCl) provided the N-Cbz protected

derivative, 4.025.30,31 Deprotection of the Boc-group under acidic conditions, followed by aromatic

nucleophilic substitution reaction with 4.027 furnished compound 4.028.21 This compound

underwent deprotection using the Pearlman’s catalyst29 in the presence of hydrogen to arrive at the

free amine in compound 4.029. To this compound was added 4.013 under appropriate reaction

conditions using HATU21 as the coupling reagent which condensed to form the amide bond as

shown in compounds 4.030, 4.031 and 4.032, Scheme 7.

The acid by-product, compound 4.033, shown in Figure 4.5, was isolated using preparative HPLC.

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4.4.1 Alternative synthesis to tropane/bridged analogs

In order to optimize the reaction conditions with increased yields, alternative synthetic

route to the tropane/bridged analogs was simultaneously pursued as shown below in Scheme 4.8.

However, yields recorded were not superior to what was found in Scheme 4.7.

Scheme 4.8: Alternative synthesis to tropane/bridged analogs

Reagents and conditions: (a) NaBH(AcO)3, BnNH2, AcOH, DCM, 45%; (b) Pd(OH)2, (NH4)HCO2, EtOH, 92%;

(c) 4.013, HATU, TEA, DMF, 52%; (d) 1M HCl/Et2O, 18-39%; (e) 4.027, TEA, ACN or Dioxane, 47-58%.

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4.5 Results and Discussions

Results of the biological evaluations (performed in the laboratory of Prof. Dennis Kyle by

Dr. Brian Vesely, formerly in the USF Department of Public Health) are displayed in Table 4.1.

Evidently the apparent hydrophobic pocket can accommodate larger groups. Each of the

synthesized bridged/non-bridged analogs showed inhibitory activity against amastigote growth,

however compounds were less selective in the infected macrophage assay. In general terms, the

compounds did not demonstrate significant cytotoxicity on the J774 cell lines, implying that the

observed activity is specific to Leishmania amastigotes.

Compound IC50 (µM) Selectivity

(Cytotox.

IC50/IM

IC50

ID Axenic

Amastigote J774

Cytotoxicity Infected

Macrophage

miltefosine 3.26 >50 1.43 35.0

4.014 8.19 24.19 2.62 9.2

4.018 20.00 >50.00 >10.00 <5.0

4.021 20.00 5.17 0.64 8.1

4.030 8.80 7.77 0.89 8.7

4.031 10.52 2.88 3.56 0.81

4.032 1.80 >50.00 5. 07 >9.9

4.033 20.00 50.00 >10.00 >5.0

Table 4.1: Biological data for piperidine (non-bridged) and tropane (bridged) analogs

With the exception of compound 4.032, each compound showed considerably more activity in the

HCS infected macrophage assay than the axenic amastigote assay, suggesting that these

compounds may be exerting their anti-leishmanial activity via multiple targets, a phenomenon

previously observed with opioid receptor antagonists.32 Compounds 4.021 (non-bridged) and

4.030 (bridged) were identified as being more potent in the infected macrophage assay than

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miltefosine, which is currently being marketed as treatment for leishmaniasis. However, these

compounds were less selective in the assays. The amide analogs, 4.018 and 4.031, which were not

particularly soluble, compared to the ester or benzamide analogs, provided unusual results, which

could be attributed to their solubility issues.

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4.6 Experimental

General experimental procedures:

Tert-butyl piperidin-4-ylcarbamate: To a solution of 1-benzylpiperidin-4-amine (1.00 mL, 4.90

mmol) in DCM (20 mL) was added TEA (1.00 mL, 7.17 mmol), followed by di-tert-butyl

dicarbonate (1.26 g, 5.78 mmol). The reaction mixture was stirred overnight at room temperature

and then diluted with DCM (20 mL). The organic phase was washed with aqueous NaHCO3

solution (15 mL), brine (15 mL), dried over anhydrous MgSO4 and concentrated on a rotary

evaporator. The resulting solid was dried in a lyophilizer to give tert-butyl (1-benzylpiperidin-4-

yl)carbamate as a pale white solid (1.419 g, 85%). LRMS (ESI) m/z calc’d for C17H26N2O2

[M+H]+: 291.41, found 291.1. This material was used in the next step without further purification.

To a solution of tert-butyl (1-benzylpiperidin-4-yl)carbamate (1.40 g, 4.82 mmol) in MeOH (80

mL) was added 20% palladium on carbon (0.300 g, 0.282 mmol) at room temperature. After

stirring overnight under H2 atmosphere, the reaction mixture was filtered through a celite pad and

evaporated to provide tert-butyl piperidin-4-ylcarbamate as a white powder (870 mg, 90%).1H

NMR (400 MHz, DMSO) δ ppm 6.69 - 6.77 (m, 1 H) 2.81 - 2.91 (m, 3 H) 2.35 - 2.46 (m, 3 H)

1.58 - 1.65 (m, 2 H) 1.35 - 1.37 (s, 9 H) 1.16 - 1.25 (m, 2 H) ppm. 13C NMR (101 MHz, DMSO-

d6) δ 155.2, 77.8, 48.4, 45.6, 33.6, 28.7 ppm. LRMS (ESI) m/z calc’d for C10H20N2O2 [M+H]+:

201.28, found 201.30.

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N-benzyl-6-chloronicotinamide: Thionyl chloride (5.18 mL, 71.4 mmol) and DMF (77.00 µL,

0.999 mmol) were added to a stirred suspension of 6-chloronicotinic acid (7.50 g, 47.6 mmol) in

toluene (75 mL) and heated to 80 °C for 2 hours. Upon cooling, the reaction mixture was

concentrated under reduced pressure and the residue was dissolved in DCM (75 mL). Benzylamine

(15.28 mL, 143.0 mmol) was then added at 0 °C over 20 minutes and allowed to stir overnight at

room temperature. Saturated aqueous NaHCO3 (100 mL) and DCM (50 mL) were added under

vigorous stirring, the phases were separated and the aqueous phase was extracted with DCM (2 x

50 mL). The combined organic extracts were washed with saturated aqueous NaHCO3, dried

(Na2SO4) and concentrated on a rotary evaporator. The residue was dissolved in ethyl acetate (10

mL) at reflux, n-hexane (5.0 mL) was added and the solution was slowly cooled to room

temperature and then over ice. Precipitation began on scratching with a glass rod. The precipitate

was filtered off, washed with cold n-hexane/ethyl acetate 1:2 and dried in a lyophilizer to yield N-

benzyl-6-chloronicotinamide as light brown crystals (11.92 g, 68%). 1H NMR (500 MHz, DMSO)

δ 9.30 (br t, J = 5.7 Hz, 1H), 8.89 (d, J = 2.3 Hz, 1H), 8.28 (dd, J = 8.3, 2.5 Hz, 1H), 7.65 (d, J =

8.3 Hz, 1H), 7.33 (d, J = 4.6 Hz, 4H), 7.22-7.36 (m, 1H), 4.50 (d, J = 6.0 Hz, 2H) ppm. 13C NMR

(126 MHz, DMSO) δ 164.2, 153.0, 149.5, 139.5, 139.1, 129.7, 128.8, 127.8, 127.4, 124.6, 43.2

ppm. LRMS (ESI) m/z calc’d for C13H11ClN2O [M+H]+ = 247.49, found 247.40.

85

Tert-butyl (1-(5-(benzylcarbamoyl)pyridin-2-yl)piperidin-4-yl)carbamate: To a stirred

solution of N-benzyl-6-chloronicotinamide (162.0 mg, 0.405 mmol) in ACN was added tert-butyl

piperidin-4-ylcarbamate (158.0 mg, 0.809 mmol) and DIPEA (339 mg, 2.63 mmol). The reaction

mixture was stirred at reflux for 48 h. Upon cooling to room temperature, the mixture was diluted

with EtOAc and then washed with H2O. The organic layer was washed with citric acid, brine and

dried over anhydrous sodium sulphate. Filteration, concentration and purification by flash column

chromatography (hexanes: EtOAc, 1:1) afforded tert-butyl (1-(5-(benzylcarbamoyl)pyridin-2-

yl)piperidin-4-yl)carbamate as an off-white crystalline solid (213 mg, 79%). 1H NMR (500 MHz,

DMSO) δ 8.78 (t, J = 6.0 Hz, 1H), 8.63 (d, J = 2.4 Hz, 1H), 7.96 (dd, J = 9.0, 2.5 Hz, 1H), 7.27-

7.34 (m, 4H), 7.19-7.27 (m, 1H), 6.85 (d, J = 9.1 Hz, 2H), 4.45 (d, J = 6.0 Hz, 2H), 4.31 (br d, J =

13.3 Hz, 2H), 3.40-3.59 (m, 1H), 2.91-3.04 (m, 2H), 1.77 (br d, J = 10.6 Hz, 2H), 1.38 (s, 9H),

1.28-1.33 (m, 2H) ppm. 13C NMR (126 MHz, DMSO) δ 165.4, 159.9, 155.2, 148.5, 140.4, 137.0,

128.7, 127.6, 127.1, 118.3, 105.9, 78.0, 48.0, 43.9, 42.8, 31.8, 28.7 ppm. LRMS (ESI) m/z calc’d

for C23H30N4O3 [M+H]+: 411.52, found 411.32.

6-(4-Aminopiperidin-1-yl)-N-benzylnicotinamide: To a solution of tert-butyl (1-(5-

(benzylcarbamoyl)pyridin-2-yl)piperidin-4-yl)carbamate (1.32 g, 3.22 mmol) in DCM was added

86

a 1.0M solution of HCl in diethyl ether (150 mL) at 0°C. After stirring for 20 minutes at 0 °C, the

mixture was allowed to warm to room temperature and stirred for 2 days. The reaction mixture

was concentrated to give 6-(4-aminopiperidin-1-yl)-N-benzylnicotinamide as a pale white powder

(820 mg, 82%). 1H NMR (500 MHz, DMSO) δ 9.29 (br s, 1H), 8.61 (s, 1H), 8.23-8.44 (m, 4H),

7.25-7.39 (m, 5H), 4.45 (s, 2H), 3.38 (q, J = 7.0 Hz, 2H), 3.26-3.31 (m, 1H), 2.06 (br d, J = 10.8

Hz, 2H), 1.53-1.71 (m, 2H), 1.03-1.37 (m, 2H) ppm. 13C NMR (126 MHz, DMSO) δ 163.4, 154.7,

149.5, 140.3, 139.9, 128.8, 127.8, 127.3, 119.1, 110.6, 47.32, 44.8, 43.0, 29.4 ppm. LRMS (ESI)

m/z calc’d for C18H22N4O [M+H]+: 311.40, found 311.20.

Ethyl 4-amino-3-bromobenzoate: To a solution of ethyl 4-aminobenzoate (1.850 g, 11.20 mmol)

and DCM (10 mL) was added NBS (2.207 g, 12.40 mmol) and the mixture was allowed to stir at

room temperature overnight. The resulting mixture was then extracted with H2O and brine, dried

over anhydrous MgSO4 and concentrated. The residue was purified by column chromatography

(n-hexane/ethyl acetate, 9:1) to afford ethyl 4-amino-3-bromobenzoate as a yellow solid (2.433 g,

89%). 1H NMR (500 MHz, DMSO) δ 7.88 (d, J = 1.9 Hz, 1H), 7.64 (dd, J = 1.9, 8.5 Hz, 1H), 6.80

(d, J = 8.5 Hz, 1H), 6.18 (s, 2H), 4.21 (q, J = 7.1 Hz, 2H), 1.27 (t, J = 7.1 Hz, 3H) ppm. 13C NMR

(126 MHz, DMSO) 165.2, 150.7, 134.1, 130.3, 118.3, 114.6, 106.3, 60.5, 14.7 ppm. LRMS (ESI)

m/z calc’d for C9H10BrNO2 [M+H]+: 245.09/247.09, found 245.09/247.09.

87

Ethyl 3-bromo-4-cyanobenzoate: A solution of ethyl 4-amino-3-bromobenzoate (5.000 g, 20.48

mmol) in DCM (50 mL) was cooled to -10°C and treated dropwise with 90% tert-butyl nitrite (3.34

mL, 28.1 mmol) followed by boron trifluoride diethyl etherate (4.08 mL, 33.1 mmol) and the

suspension was allowed to warm to room temperature and stirred for 4h.

Diethyl ether (20 mL) was then added, and the off-white solid was collected by filtration, washed

with diethyl ether and dried briefly under high vacuum. The solid was then suspended in toluene

(40 mL) and cooled to 0°C. A solution of copper (I) cyanide (2.6 g, 29 mmol) and sodium cyanide

(3.55 g, 72.4 mmol) in H2O (25 mL) was added dropwise over 10 min. The mixture was allowed

to cool to room temperature, and ethyl acetate (50 mL) and H2O (50 mL) were added. The aqueous

phase was separated and extracted with ethyl acetate (2 x 50 mL). The organic phases were

combined, washed with H2O (2 x 30 mL) and brine, dried over Na2SO4, filtered and concentrated

at reduced pressure. The solid residue obtained was adsorbed onto silica gel and chromatographed

(hexanes/ethyl acetate 95:5 to 85:15) to afford ethyl 3-bromo-4-cyanobenzoate (4.3 g, 83%). 1H

NMR (500 MHz, DMSO) δ 8.26 (d, J = 1.4 Hz, 1H), 8.03-8.13 (m, 2H), 4.35 (q, J = 7.1 Hz, 2H),

1.33 (t, J = 7.1 Hz, 3H) ppm. 13C NMR (126 MHz, DMSO) 163.8, 135.9, 135.7, 133.4, 129.0,

125.4, 118.8, 117.1, 62.4, 14.4 ppm. LRMS (ESI) m/z calc’d for C10H8BrNO2 [M+H]+:

255.08/257.08, found 255.20/257.20.

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Ethyl 4-cyano-3-((3-methoxypropyl)amino)benzoate: A mixture of ethyl 3-bromo-4-

cyanobenzoate (500.0 mg, 1.968 mmol), XANTPHOS (228.0 mg, 0.394 mmol), cesium carbonate

(1.28 g, 3.93 mmol),3-methoxypropan-1-amine (233 mg, 2.61 mmol), and Pd2(dba)3 (180.0 mg,

0.197 mmol) in dioxane (5 mL) was stirred at 100°C for 17 h. The cooled reaction mixture was

partitioned between ethyl acetate (100 mL) and H2O (50 mL), the organic phase was washed with

brine (50 mL), dried over sodium sulfate, filtered and concentrated. Column chromatography on

silica gel (hexanes:ethyl acetate 9:1) afforded ethyl 4-cyano-3-((3-methoxypropyl)amino)benzoate

as a brown oil (418 mg, 81%). 1H NMR (400 MHz, CDCl3) δ 7.41 (d, J = 7.8 Hz, 1H), 7.31 (d, J

= 1.2 Hz, 1H), 7.23 - 7.29 (m, 1H), 4.36 (q, J = 7.0 Hz, 2H), 3.50 - 3.60 (m, 2H), 3.32 - 3.44 (m,

5H), 1.88 - 2.00 (m, 2H), 1.37 (t, J = 7.0 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3) 165.8,

150.5, 135.5, 132.7, 117.2, 116.6, 111.2, 99.1, 71.2, 61.5, 58.8, 41.9, 28.7, 14.3 ppm. LRMS (ESI)

m/z calc’d for C14H18N2O3 [M+H]+: 263.31, found 263.21.

4-Carbamoyl-3-((3-methoxypropyl)amino)benzoic acid: A mixture of ethyl 4-cyano-3-((3-

methoxypropyl)amino)benzoate (1.00 g, 3.81 mmol), 50% w/w NaOH (0.20 mL, 3.81 mmol), and

H2O2 (2.22 mL, 72.4 mmol), in EtOH (15 mL) and DMSO (6.58 mL) was added to a 30 mL

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microwave vial. The reaction was heated to 100°C in a microwave reactor for 45 minutes. Upon

cooling, the mixture was extracted with H2O and ethyl acetate, and the organic phase was

concentrated on a rotary evaporator to give a solid substance which was purified with flash column

chromatography to afford a mixture of ethyl 4-carbamoyl-3-((3-methoxypropyl)amino)benzoate

and 4-cyano-3-((3-methoxypropyl)amino)benzoic acid as a yellow solid.

The mixture was then subjected to saponification conditions with excess aqueous NaOH in EtOH

to yield a mixture of 4-carbamoyl-3-((3-methoxypropyl)amino)benzoic acid and 2-((3-

methoxypropyl)amino)terephthalic acid (diacid, by product) as a light yellow solid (78%).

4.013: LRMS (ESI) m/z calcd for C12H16N2O4 [M+H]+: 252.27, found 253.20.

4.013a: LRMS (ESI) m/z calc’d for C12H15NO5 [M+H]+: 254.25, found 254.20.

N1-(1-(5-(benzylcarbamoyl)pyridin-2-yl)piperidin-4-yl)-3-((3-

methoxypropyl)amino)terephthalamide: A 15 mL vial was charged with a magnetic spin bar, 6-

(4-aminopiperidin-1-yl)-N-benzylnicotinamide (320.0 mg, 1.031 mmol), 4-carbamoyl-3-((3-

methoxypropyl)amino)benzoic acid (295.0 mg, 1.169 mmol), DMF (5.0 mL) and DIPEA (1.00

mL, 5.73 mmol). With stirring, HATU (730 mg, 1.92 mmol) was added and the reaction was

warmed to 50 °C for 3 h. The reaction was then diluted with H2O and extracted with ethyl acetate

(3x). The combined organic extracts were dried with MgSO4, filtered and concentrated in vacuo

to yield the product which was purified via silica gel chromatography using 100% ethyl acetate as

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eluent to provide the title compound. The title compound was further purified by preparative HPLC

to give N1-(1-(5-(benzylcarbamoyl)pyridin-2-yl)piperidin-4-yl)-3-((3-

methoxypropyl)amino)terephthalamide as a pale white solid (270 mg, 48%). 1H NMR (500 MHz,

DMSO) δ 8.80 (t, J = 6.0 Hz, 1H), 8.66 (d, J = 2.4 Hz, 1H), 8.22 (d, J = 7.9 Hz, 1H), 8.16 (t, J =

5.3 Hz, 1H), 7.86-8.02 (m, 2H), 7.63 (d, J = 8.2 Hz, 1H), 7.27-7.48 (m, 4H), 7.02-7.27 (m, 3H),

6.88-6.98 (m, 2H), 4.40-4.49 (m, 4H), 3.99-4.23 (m, 1H), 3.42 (t, J = 6.1 Hz, 2H), 3.09-3.29 (m,

5H), 3.03 (br t, J = 11.9 Hz, 2H), 1.73-1.88 (m, 4H), 1.44-1.59 (m, 2H) ppm. 13C NMR (126 MHz,

DMSO) 171.5, 166.0, 165.4, 160.0, 150.1, 148.6, 140.3, 138.5, 137.0, 129.4, 128.7, 127.6, 127.1,

118.3, 116.0, 112.9, 110.2, 106.0, 70.0, 58.4, 47.4, 44.3, 42.8, 40.1, 31.3, 29.2 ppm. HRMS m/z:

[M + H]+ calc’d for C30H36N6O4 545.2876; Found 545.2885.

A mixture of 6-chloronicotinic acid (8.31 g, 52.7 mmol) and thionyl chloride (5.78 mL, 79.0 mmol)

in toluene (30 mL) was heated to reflux for 15 hrs and cooled to room temperature. The solvent

was evaporated to dryness in vacuo and the residue thus obtained was dissolved in toluene (30

mL) and added to aqueous ammonia (50 mL). The reaction mixture was then stirred at room

temperature for 8 hrs and filtered to give a brown solid which was recrystallized from distilled

water (300 mL) to obtain 4.016 as a brownish crystalline solid (6.02 g 73%). LRMS (ESI) m/z

calc’d for C5H5ClN2O [M+H]+: 157.57, found 157.60.

91

To a stirred solution of 6-chloronicotinamide (420 mg, 2.68 mmol) in ACN (10 mL) was added

tert-butyl piperidin-4-ylcarbamate (540 mg, 2.70 mmol) and DIPEA (1.4 mL, 8.2 mmol). The

reaction mixture was stirred at reflux for 2 days. The resulting mixture was cooled to rt and

concentrated in vacuo to obtain a solid residue which was purified using column chromatography

using DCM/MeOH as the eluent to obtain tert-butyl (1-(5-carbamoylpyridin-2-yl)piperidin-4-

yl)carbamate as the title compound (421 mg, 49%). This compound underwent acidic deprotection

in 1.0 M HCl in ether to obtain the title compound, 4.017 (190 mg, 69%). LRMS (ESI) m/z calc’d

for C11H16N4O [M+H]+: 221.28, found 221.20.

N1-(1-(5-carbamoylpyridin-2-yl)piperidin-4-yl)-3-((3-

methoxypropyl)amino)terephthalamide: Using conditions identical to those described above,

the amide was obtained as an off white solid (48%). 1H NMR (500 MHz, DMSO) δ 8.60 (d, J =

2.5 Hz, 1H), 8.20 (d, J = 7.8 Hz, 1H), 8.15 (t, J = 5.4 Hz, 1H), 7.94 (dd, J = 2.5, 8.8 Hz, 1H), 7.85

- 7.90 (m, 1H), 7.68 - 7.75 (m, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.19 - 7.26 (m, 1H), 7.06 - 7.12 (m,

1H), 7.04 (d, J = 1.0 Hz, 1H), 6.93 (dd, J = 8.3, 1.5 Hz, 1H), 6.87 (d, J = 8.8 Hz, 1H), 4.41 (d, J =

13.2 Hz, 2H), 4.04 - 4.14 (m, 1H), 3.40 (t, J = 6.4 Hz, 2H), 3.22 (s, 3H), 3.18 (q, J = 6.4 Hz, 2H),

92

3.01 (t, J = 11.7 Hz, 2H), 1.74 - 1.87 (m, 4H), 1.50 (dd, J = 12.0, 3.67 Hz, 2H) ppm. 13C NMR

(126 MHz, DMSO) 171.5, 167.1, 165.9, 160.0, 150.0, 148.8, 138.6, 137.2, 129.3, 118.3, 116.1,

113.0, 110.2, 105.9, 70.0, 58.4, 47.4, 44.3, 31.2, 29.2 ppm. HRMS m/z: [M+H]+ calc’d for

C23H30N6O4 455.2407; Found 455.2403.

To a slurry of 6-chloronicotinic acid (5.00 g, 31.7 mmol) in anhydrous toluene (35 mL) was added

dropwise with stirring triethyl orthoacetate (17.6 mL, 95.0 mmol). The mixture was heated to

reflux overnight and allowed to cool to room temperature and the resultant solution washed with

sat NaHCO3 (50 mL). The organic phase was dried with MgSO4 and the solvent removed in vacuo

to afford the title compound, ethyl 6-chloronicotinate, 4.019 (5.6 g 95%) as a brownish oil. LRMS

(ESI) m/z calc’d for C8H8ClNO2 [M+H]+: 186.61, found 186.54.

To a solution of ethyl 6-chloronicotinate (200.0 mg, 1.078 mmol) in EtOH (2 mL) was added tert-

butyl piperidin-4-ylcarbamate (256.0 mg, 1.278 mmol) and DIPEA (0.223 mL, 1.278 mmol). The

mixture was heated to reflux overnight. The resulting mixture was cooled to rt and concentrated in

vacuo to obtain a solid residue which was purified using column chromatography using

hexane/EtOAc as the eluent to obtain ethyl 6-(4-((tert-butoxycarbonyl)amino)piperidin-1-

yl)nicotinate (339 mg, 76%). This compound underwent acidic deprotection in 1.0 M HCl in ether

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to obtain the title compound, 4.020 (176 mg, 82%). LRMS (ESI) m/z calc’d for C13H19N3O2

[M+H]+: 250.31, found 250.30.

Ethyl 6-(4-(4-carbamoyl-3-((3-methoxypropyl)amino)benzamido)piperidin-1-yl)nicotinate:

Using conditions identical to those described above, the ester was obtained as a white fluffy solid

(55%). 1H NMR (500 MHz, DMSO) δ 8.63 (d, J = 2.3 Hz, 1H), 8.21 (d, J = 7.9 Hz, 1H), 8.15 (t,

J = 5.3 Hz, 1H), 7.84-7.96 (m, 2H), 7.62 (d, J = 8.2 Hz, 1H), 7.23 (br s, 1H), 7.04 (s, 1H), 6.92 (t,

J = 9.6 Hz, 2H), 4.45 (br d, J = 13.3 Hz, 2H), 4.23 (q, J = 7.1 Hz, 2H), 4.06-4.15 (m, 1H), 3.34-

3.43 (m, 2H), 3.16-3.23 (m, 1H), 3.22 (s, 4H), 3.06 (br t, J = 11.8 Hz, 2H), 1.75-1.89 (m, 4H),

1.40-1.55 (m, 2H), 1.27 (t, J = 7.1 Hz, 3H) ppm. 13C NMR (126 MHz, DMSO) 171.5, 165.9,

165.5, 160.5, 150.8, 150.1, 138.5, 138.6, 129.4, 116.1, 113.9, 112.8, 110.2, 106.1, 70.0, 60.3, 58.4,

47.2, 44.1, 40.1, 31.4, 29.2, 14.7 ppm. HRMS m/z: [M+H]+ calc’d for C25H33N5O5 484.2560;

Found 484.2560.

Tert-butyl 3-amino-8-azabicyclo[3.2.1]octane-8-carboxylate: A solution of tert-butyl 3-oxo-8-

azabicyclo[3.2.1]octane-8-carboxylate (2.500 g, 11.10 mmol), benzylamine (1.400 mL, 12.43

mmol) and sodium triacetoxyborohydride (3.530 g, 16.65 mmol) in a mixture of glacial acetic acid

(7.5 mL) and methylene chloride (66 mL) was allowed to stir for 16 hours at room temperature.

94

The solvents were evaporated under reduced pressure and the residue was dissolved in ethyl acetate

(50 mL) and washed with saturated aqueous sodium bicarbonate solution (13 mL) and H2O (13

mL). The organic solution was dried (MgSO4), filtered, and evaporated under reduced pressure.

The residue was purified by column chromatography on silica gel (98:2:0.25 DCM: MeOH: conc.

aq. NH3) to afford tert-butyl 3-(benzylamino)-8-azabicyclo[3.2.1]octane-8-carboxylate as a white

solid (1.77 g, 45%). LRMS (ESI) m/z calc’d for C19H28N2O2 [M+H]+: 317.48, found 317.40.

To a solution of tert-butyl 3-(benzylamino)-8-azabicyclo[3.2.1]octane-8-carboxylate (2.40 g, 7.58

mmol) in EtOH (15 mL) was added ammonium formate (2.39 g, 37.9 mmol) and 20% palladium

hydroxide on carbon (0.320 g, 2.275 mmol). The resulting suspension was stirred at 50° until gas

evolution had ceased. The mixture was then filtered through celite, concentrated and

chromatographed on silica gel using MeOH and DCM to afford the target compound, tert-butyl 3-

amino-8-azabicyclo[3.2.1]octane-8-carboxylate (1.58 g, 92%). 1H NMR (500 MHz, CD3OD) δ

4.13-4.20 (m, 2H), 3.09-3.18 (m, 1H), 2.16-2.29 (m, 2H), 1.94-2.03 (m, 4H), 1.40-1.59 (m, 13H).

13C NMR (126 MHz, CD3OD-d4) δ 153.8, 79.5, 52.5, 42.7, 37.1, 28.5, 27.3. LRMS (ESI) m/z

calc’d for C12H22N2O2 [M+H]+: 227.32, found 227.40.

Benzyl (8-(5-carbamoylpyridin-2-yl)-8-azabicyclo[3.2.1]octan-3-yl)carbamate: To a solution

of tert-butyl 3-amino-8-azabicyclo[3.2.1]octane-8-carboxylate (500.0 mg, 2.209 mmol) in DCM

(5 mL) was added benzyl chloroformate (0.41 mL, 2.87 mmol) and DIPEA (0.60 mL, 3.31 mmol).

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The solution was stirred vigorously overnight at room temperature, concentrated and purified on

silica gel to provide tert-butyl 3-(((benzyloxy)carbonyl)amino)-8-azabicyclo[3.2.1]octane-8-

carboxylate as a solid (549 mg, 69%). LRMS (ESI) m/z calc’d for C20H28N2O4 [M+H]+: 361.45,

found 361.31. A portion of this material was used in the next step.

A suspension of tert-butyl 3-(((benzyloxy)carbonyl)amino)-8-azabicyclo[3.2.1]octane-8-

carboxylate (200.0 mg, 0.555 mmol) in a 1M solution of HCl in diethyl ether was allowed to stir

overnight. After completion of the reaction, monitored by LC/MS, the solution was concentrated

on a rotary evaporator to give benzyl 8-azabicyclo[3.2.1]octan-3-ylcarbamate (121 mg, 84%).

LRMS (ESI) m/z calc’d for C15H20N2O2 [M+H]+: 261.34, found 261.20. This material was used

in the next step without further purification.

To a stirred solution of 6-chloronicotinamide (144.0 mg, 0.922 mmol) in ACN (5.0 mL) was added

benzyl 8-azabicyclo[3.2.1]octan-3-ylcarbamate (200.0 mg, 0.768 mmol) and DIPEA (0.47 mL,

2.69 mmol). The reaction mixture was stirred at reflux for 48 h. The solid was collected by

filtration and washed with ACN. After drying, the title compound, benzyl (8-(5-carbamoylpyridin-

2-yl)-8-azabicyclo[3.2.1]octan-3-yl)carbamate was obtained as brownish solid (178 mg, 61%). 1H

NMR (500 MHz, DMSO) δ 8.59 (d, J = 2.4 Hz, 1H), 7.91 (dd, J = 2.5, 8.8 Hz, 1H), 7.71 (br.s.,

1H), 7.24-7.40 (m, 6H), 7.08 (br. S., 1H), 6.65-6.73 (m, 1H), 5.02 (s, 2H), 4.49 (br. S., 2H), 3.50

(d, J = 4.4 Hz, 1H), 2.07-2.14 (m, 2H), 1.96-2.04 (m, 2H), 1.87-1.94 (m, 2H), 1.73-1.77 (m, 2H)

ppm. 13C NMR (126 MHz, DMSO) d 171.9, 167.3, 157.9, 156.3, 149.4, 137.7, 137.1, 128.8, 128.2,

118.0, 107.4, 65.6, 52.2, 43.6, 32.8, 27.6 ppm. LRMS (ESI) m/z calc’d for C21H24N4O3 [M+H]+:

381.45, found 381.4.

96

6-(3-amino-8-azabicyclo[3.2.1]octan-8-yl)nicotinamide: To a solution of benzyl (8-(5-

carbamoylpyridin-2-yl)-8-azabicyclo[3.2.1]octan-3-yl)carbamate (400.0 mg, 1.051 mmol) in

MeOH (10 mL) was added Pd(OH)2/C (43.10 mg, 0.307 mmol). The resulting mixture was stirred

under a hydrogen-filled balloon overnight, filtered through a celite pad, concentrated and purified

by flash column chromatography (DCM:MeOH:NH4OH) to afford the title compound (197 mg,

76%). 1H NMR (500 MHz, CD3OD) 8.59 (d, J = 2.5 Hz, 1H), 7.95 (dd, J = 9.1, 2.48 Hz, 1H),

6.69 (d, J = 9.0 Hz, 1H), 4.56 (br s, 2H), 3.34 (m, 2H), 2.91-3.10 (m, 1H), 2.26 (td, J = 13.9, 6.0

Hz, 2H), 2.15-2.21 (m, 2H), 2.06-2.12 (m, 2H), 1.93-1.94 (m, 2H), 1.53-1.58 (m, 2H) ppm. 13C

NMR (126 MHz, CD3OD) 169.6, 158.0, 148.8, 136.8, 116.6, 107.1, 52.6, 42.9, 36.2, 28.1 ppm.

LRMS (ESI) m/z calc’d for C13H18N4O [M+H]+: 247.31, found 247.30.

N1-(8-(5-(benzylcarbamoyl)pyridin-2-yl)-8-azabicyclo[3.2.1]octan-3-yl)-3-((3-

methoxypropyl)amino)terephthalamide: Using conditions identical to those described above,

compound 4.030 was obtained as an off white solid (48%). 1H NMR (500 MHz, DMSO) 8.77 (t,

J = 6.1 Hz, 1H), 8.64 (d, J = 2.0 Hz, 1H), 8.20 (t, J = 5.4 Hz, 1H), 8.14 (d, J = 4.4 Hz, 1H), 7.97

(dd, J = 8.8, 2.5 Hz, 1H), 7.86 - 7.93 (m, 1H), 7.65 (d, J = 8.3 Hz, 1H), 7.18 - 7.34 (m, 6H), 6.97

97

(s, 1H), 6.86 (d, J = 7.8 Hz, 1H), 6.75 (d, J = 9.3 Hz, 1H), 4.56 (br. s., 2H), 4.44 (d, J = 5.9 Hz,

2H), 3.82 (br. s., 1H), 3.40 (t, J = 6.1 Hz, 2H), 3.17 - 3.25 (m, 5H), 2.19 (d, J = 7.3 Hz, 2H), 2.03

- 2.10 (m, 2H), 1.96 - 2.02 (m, 2H), 1.90 (d, J = 14.2 Hz, 2H), 1.80 (t, J = 6.4 Hz, 2H) ppm. 13C

NMR (126 MHz, DMSO) 171.5, 167.2, 165.5, 158.0, 149.9, 149.2, 140.4, 139.2, 136.8, 129.5,

128.7, 127.6, 127.1, 118.2, 116.1, 112.9, 110.3, 107.5, 70.1, 58.4, 52.4, 43.0, 42.8, 40.6, 32.4, 29.1,

27.9 ppm. HRMS m/z: [M+H]+ calc’d for C32H38N6O4 571.3033; Found 571.3042.

N1-(8-(5-carbamoylpyridin-2-yl)-8-azabicyclo[3.2.1]octan-3-yl)-3-((3-

methoxypropyl)amino)terephthalamide: Using conditions identical to those described above,

compound 4.031 was obtained as a light yellow solid (55%). 1H NMR (500 MHz, DMSO) 8.62

(d, J = 2.5 Hz, 1H), 8.21 (t, J = 5.4 Hz, 1H), 8.16 (d, J = 3.9 Hz, 1H), 7.95 (dd, J = 8.8, 2.0 Hz,

1H), 7.87 - 7.93 (m, 1H), 7.73 (br. s., 1H), 7.67 (d, J = 8.3 Hz, 1H), 7.25 - 7.30 (m, 1H), 7.09 (br.

s., 1H), 6.99 (s, 1H), 6.86 - 6.90 (m, 1H), 6.75 (d, J = 8.8 Hz, 1H), 4.57 (br. s., 2H), 3.80 - 3.86 (m,

1H), 3.42 (t, J = 6.4 Hz, 3H), 3.25 (s, 3H), 3.19 - 3.24 (m, 2H), 2.17 - 2.23 (m, 2H), 2.08 (td, J =

14.2, 5.1 Hz, 3H), 1.98 - 2.04 (m, 2H), 1.91 (d, J = 14.2 Hz, 3H), 1.82 (quin, J = 6.5 Hz, 2H) ppm.

13C NMR (126 MHz, DMSO) 171.5, 167.3, 167.2, 158.1, 150.0, 149.5, 139.2, 137.2, 129.5,

118.1, 116.1, 112.9, 110.3, 107.5, 70.1, 58.4, 52.4, 43.1, 39.8, 32.4, 29.1, 27.9 ppm. HRMS m/z:

[M+H]+ calc’d for C25H32N6O4 481.2563; found 481.2571.

98

Ethyl 6-(3-(4-carbamoyl-3-((3-methoxypropyl)amino)benzamido)-8-azabicyclo[3.2.1]octan-

8-yl)nicotinate: Using conditions identical to those described above, compound 4.032 was

obtained as a white solid (55%). 1H NMR (500 MHz, DMSO) 8.79 (d, J = 2.5 Hz, 1H), 8.35 (t,

J = 5.4 Hz, 1H), 8.31 (d, J = 4.0 Hz, 1H), 8.06 (dd, J = 9.1, 2.2 Hz, 2H), 7.80 (d, J = 8.3 Hz, 1H),

7.36 - 7.47 (m, 1H), 7.12 (s, 1H), 7.02 (d, J = 7.8 Hz, 1H), 6.91 (d, J = 8.8 Hz, 1H), 4.66 - 4.82

(m, 2H), 4.39 (q, J = 7.3 Hz, 2H), 3.98 (br. s., 1H), 3.55 (t, J = 6.1 Hz, 2H), 3.32 - 3.40 (m, 5H),

2.35 (d, J = 7.3 Hz, 2H), 2.13 - 2.25 (m, 4H), 2.08 (d, J = 14.7 Hz, 2H), 1.91 - 2.00 (m, 2H), 1.43

(t, J = 7.1 Hz, 3H) ppm. 13C NMR (126 MHz, DMSO) 171.5, 167.2, 165.6, 158.5, 151.5, 149.9,

139.2, 138.3, 129.4, 116.1, 113.8, 112.9, 110.3, 107.6, 70.1, 60.3, 58.4, 52.5, 43.0, 39.5, 32.8, 29.1,

27.7, 14.8. HRMS m/z: [M+H]+ calc’d for C27H35N5O5 510.2716; Found 510.2727 ppm.

4-(((1R,3r,5S)-8-(5-(ethoxycarbonyl)pyridin-2-yl)-8-azabicyclo[3.2.1]octan-3-yl)carbamoyl)-

2-((3-methoxypropyl)amino)benzoic acid: Compound 4.033 was obtained in the manner

described above. 1H NMR (500 MHz, CDCl3) 8.84 (d, J = 2.0 Hz, 1H), 8.09 (d, J = 8.3 Hz, 1H),

8.01 (d, J = 8.3 Hz, 1H), 7.18 (s, 1H), 6.76 (d, J = 8.3 Hz, 1H), 6.67 (d, J = 6.4 Hz, 1H), 6.59 (d,

99

J = 7.3 Hz, 1H), 4.74 (br. s., 2H), 4.36 (q, J = 6.9 Hz, 2H), 4.26 (q, J = 6.0 Hz, 1H), 3.48 - 3.57

(m, 3H), 3.31 - 3.43 (m, 5H), 2.26 - 2.42 (m, 4H), 2.09 - 2.20 (m, 2H), 1.88 - 2.03 (m, 4H), 1.38

(t, J = 7.1 Hz, 3H). HRMS m/z [M+H] calc’d for C27H34N4O6 511.2478.

100

4.7 References

(1) Lounasmaa, M.; Tamminen, T. Chapter 1 The Tropane Alkaloids. The Alkaloids: Chemistry

and Pharmacology 1993, 1-114.

(2) Griffin, W.; Lin, G. Chemotaxonomy and geographical distribution of tropane alkaloids.

Phytochemistry 2000, 53, 623-637.

(3) Kukula-Koch, W.; Widelski, J. Alkaloids. Pharmacognosy 2017, 163-198.

(4) Hassan, Q. H. Tropanes. https://www.chm.bris.ac.uk/motm/tropane/tropane.pdf (accessed

December 15, 2017).

(5) Grynkiewicz, G.; Gadzikowska, M. Tropane alkaloids as medicinally useful natural products

and their synthetic derivatives as new drugs. Pharmacological Reports 2008, 60, 439-463.

(6) Majewski, M.; Lazny, R. Synthesis of tropane alkaloids via enantioselective deprotonation of

tropinone. Journal of Organic Chemistry 1995, 60, 5825-5830.

(7) Noyori, R.; Baba, Y.; Hayakawa, Y. New, general synthesis of tropane alkaloids. Journal of

the American Chemical Society 1974, 96, 3336-3338.

(8) Hayakawa, Y.; Baba, Y.; Makino, S.; Noyori, R. Carbon-carbon bond formation promoted by

transition metal carbonyls. 19. General synthesis of tropane alkaloids via the polybromo ketone-

iron carbonyl reaction. Journal of the American Chemical Society 1978, 100, 1786-1791.

(9) Davies, H.; Matasi, J.; Hodges, L.; Huby, N.; Thornley, C.; Kong, N.; Houser, J.

Enantioselective synthesis of functionalized tropanes by rhodium(ii) carboxylate-catalyzed

decomposition of vinyldiazomethanes in the presence of pyrroles. The Journal of Organic

Chemistry 1997, 62, 1095-1105.

(10) Robinson, R. A synthesis of tropinone. Journal of Chemical Society Transactions 1917, 111,

762-768.

101

(11) Bussenius, J.; Blazey, C.; Aay, N.; Anand, N.; Arcalas, A.; Baik, T.; Bowles, O.; Buhr, C.;

Costanzo, S.; Curtis, J. et al. Discovery of XL888: A novel tropane-derived small molecule

inhibitor of Hsp 90. Bioorganic & Medicinal Chemistry Letters 2012, 22, 5396-5404.

(12) Um, S.; Park, M.; Park, S.; Han, H.; Kwon, Y.; Sin, H. Synthesis of new glycyrrhetinic acid

(GA) derivatives and their effects on tyrosinase activity. Bioorganic & Medicinal Chemistry

2003, 11, 5345-5352.

(13) Zhou, H.; Liao, X.; Cook, J. Regiospecific, enantiospecific total synthesis of the 12-alkoxy-

substituted indole alkaloids, (+)-12-methoxy-Na-methylvellosimine, (+)-12-Methoxyaffinisine,

and (−)-Fuchsiaefoline. Organic Letters 2004, 6, 249-252.

(14) Hilpert, H.; Hoffmann-Emery, F.; Rimmler, G.; Rogers-Evans, M.; Stahr, H.; Waldmeier, P.

Process for the preparation of pyridine derivatives 2009, CA 232681 C.

(15) Tielin, W.; Xuechun, Z. Carbamates/urea derivatives containing piperidine and piperazine

rings as H3 receptor inhibitors 2012, PCT/1B2012/055424.; WO 2013/050987 A1.

(16) Hoffmann-Emery, F.; Hilpert, H.; Scalone, M.; Waldmeier, P. Efficient synthesis of novel

nk1receptor antagonists: selective 1,4-addition of grignard reagents to 6-chloronicotinic acid

derivatives. Journal of Organic Chemistry 2006, 71, 2000-2008.

(17) Aoyama, A.; Endo-Umeda, K.; Kishida, K.; Ohgane, K.; Noguchi-Yachide, T.; Aoyama, H.;

Ishikawa, M.; Miyachi, H.; Makishima, M.; Hashimoto, Y. Design, synthesis, and biological

evaluation of novel transrepression-selective liver X receptor (LXR) ligands with 5,11-dihydro-

5-methyl-11-methylene-6H-dibenz[b,e]azepin-6-one skeleton. Journal of Medicinal Chemistry

2012, 55, 7360-7377.

(18) Hussain, M.; Ahmed, V.; Hill, B.; Ahmed, Z.; Taylor, S. A re-examination of the

difluoromethylenesulfonic acid group as a phosphotyrosine mimic for PTP1B inhibition.

Bioorganic & Medicinal Chemistry 2008, 16, 6764-6777.

102

(19) Wang, G.; Wang, X.; Wang, W.; Hasvold, L.; Sullivan, G.; Hutchins, C.; O’Conner, S.;

Gentiles, R.; Sowin, T.; Cohen, J. et al. Design and synthesis of o-trifluoromethylbiphenyl

substituted 2-amino-nicotinonitriles as inhibitors of farnesyltransferase. Bioorganic & Medicinal

Chemistry Letters 2005, 15, 153-158.

(20) Yang, B.; Buchwald, S. Palladium-catalyzed amination of aryl halides and sulfonates.

Journal of Organometallic Chemistry 1999, 576, 125-146.

(21) Rice, K.; Aay, N.; Arcalas, A.; Baik, T.; Blazey, C.; Bowles, O.; Buhr, C.; Bussenius, J.;

Costanzo, S.; Curtis, J. et al. Tropane compounds 2011, US 8,012,956 B2.

(22) Sawaki, Y.; Ogata, Y. Mechanism of the reaction of nitriles with alkaline hydrogen peroxide.

reactivity of peroxycarboximidic acid and application to superoxide ion reaction. Bulletin of the

Chemical Society of Japan 1981, 54, 793-799.

(23) Huang, K.; Veal, J.; Fadden, R.; Rice, J.; Eaves, J.; Strachan, J.; Barabasz, A.; Foley, B.;

Barta, T.; Ma, W. et al. Discovery of novel 2-aminobenzamide inhibitors of heat shock protein 90

as potent, selective and orally active antitumor agents. Journal of Medicinal Chemistry 2009, 52,

4288-4305.

(24) Wang, Q.; Sun, H.; Huang, R. Synthesis and herbicidal activity of (Z)-ethoxyethyl 2-cyano-

3-(2-methylthio-5-pyridylmethylamino)acrylates. Heteroatom Chemistry 2004, 15, 67-70.

(25) Hekmatshoar, R.; Sadjadi, S.; Sadjadi, S.; Heravi, M.; Beheshtiha, Y.; Bamoharram, F. New

and general nitrogen heterocycle synthesis: use of heteropoly acids as a heterogeneous recyclable

catalyst. Synthetic Communications 2010, 40, 1708-1716.

(26) Gillett, S.; Garst, M.; Johnson, A. Methods for preparing esters of halonicotinic acids 1996,

PCT/US95/13153; WO 96/12701.

(27) Gibson, S.; Romero, D.; Jacobs, H.; Gopalan, A. Concurrent esterification and N-acetylation

of amino acids with orthoesters: a useful reaction with interesting mechanistic implications.

Tetrahedron Letters 2010, 51, 6737-6740.

103

(28) Stupple, P.; Batchelor, D.; Corless, M.; Dorr, P.; Ellis, D.; Fenwick, D.; Galan, S.; Jones, R.;

Mason, H.; Middleton, D. et al. An imidazopiperidine Series of CCR5 antagonists for the

treatment of HIV: the discovery ofN-{(1S)-1-(3-fluorophenyl)-3-[(3-endo)-3-(5-isobutyryl-2-

methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-1-yl)-8-azabicyclo[3.2.1]oct-8-

yl]propyl}acetamide (PF-232798). Journal of Medicinal Chemistry 2011, 54, 67-77.

(29) Bernotas, R.; Cube, R. The use of pearlman's catalyst for selective N-debenzylation in the

presence of benzyl ethers. Synthetic Communications 1990, 20, 1209-1212.

(30) Layton, M.; Rodzinak, K.; Kelly, M.; Sanderson, P. N-alkyl-azacycloalkyl nmda/nr2b

antagonists 2006, WO2006113471 A2, PCT/US2006/014139.

(31) Xie, C.; Runnegar, M.; Snider, B. Total synthesis of (±)-cylindrospermopsin. Journal of the

American Chemical Society 2000, 122, 5017-5024.

(32) De Muylder, G.; Ang, K.; Chen, S.; Arkin, M.; Engel, J.; McKerrow, J. A screen against

leishmania intracellular amastigotes: comparison to a promastigote screen and identification of a

host cell-specific hit. PLoS Neglected Tropical Diseases 2011, 5, e1253.

104

Chapter 5

Conclusions

5.1 Summary and conclusions

A novel series of compounds based on the known Hsp 90 inhibitor agents SNX-2112 and

XL-888 that potentially inhibit Leishmania donovani amastigote growth has been discovered.

These compounds have been tested in multiple assays for their anti-leishmanial activity, and the

overall synthetic schemes developed allowed the preparation of several intermediates and different

analogs.

Docking studies of a selected member compound clearly defined the probable binding interactions

and available hydrophobic pocket that could be exploited, which assisted in the design of further

analogs for evaluation. The synthesized analogs included indazole, tetrahydroquinoline, piperidine

and tropane derivatives. Based on previous docking and x-ray crystallographic studies of analogs

of 1.001, it was envisioned that the primary benzamide, indazole carbonyl, tetrahydroquinoline

nitrogen or the piperidine/tropane nitrogen groups represented key structural features of these

series of compounds that confer binding into the Hsp 90 active site.

The initial strategy provided a means of evaluating the necessity of the dimethyl groups at the 6-

position on the indazole scaffold for activity. The impact a twisting effect would have on the overall

Hsp 90 binding affinity was also anticipated with the introduction of a methyl group at the 5-

105

position on the benzene ring (anthranilamide portion). Additionally, the indazole scaffold was

replaced with the privileged scaffold, tetrahydroquinoline, or piperidine/tropane scaffold. It was

anticipated that such SAR strategies would enhance the activity of these molecules since docking

studies revealed that the active site can accommodate considerably larger groups.

Clearly, the results (pages 34, 60 and 81) indicate that these changes resulted in a significant effect

on the anti-leishmanial activities, suggesting significant binding affinities. Deletion of the geminal

dimethyl groups led to a complete loss of activity in the assays. This result clearly defined the

importance of the geminal dimethyl groups on the indazole bicyclic core and represents a crucial

SAR discovery in the search for potent anti-leishmanial agents. For instance, the 6-position of the

indazole bicyclic core can further be exploited to gain additional selectivity and/or improved

pharmacokinetics.

Several of these compounds displayed more potent activity than the clinically used miltefosine in

both assays (axenic amastigote and infected macrophage) and were found to have an excellent

toxicity profile as well. Compound 2.017 exhibited superior inhibitory activity to all of the analogs

including miltefosine. In fact, compound 2.017 was found to be more than twice as active as

miltefosine in the infected macrophage assay and with selectivity of approximately 20-fold over

human macrophage cell lines.

Given that some compounds displayed considerably more activity in the amastigote-infected

macrophage assay than the initial axenic amastigote assay, it was investigated whether the

observed activity is indeed the result of protozoan Hsp 90 inhibition. Thus, most of these

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compounds were assayed using an Hsp 90 fluorescence polarization assay,1 (Figure 5.1) run by

Hardik Patel in the laboratory of Prof. Gabriela Chiosis.

As expected, compound 1.001 (shown in diagram as 1) exhibited inhibitory activity against human

Hsp 90, but none of the other synthesized compounds displayed any significant inhibitory activity

against the same. Remarkably, the desmethylated analog 2.013 (shown in Figure 5.1 as 10) which

showed no inhibitory activity in the assays was more potent against human Hsp 90 than the rest of

the compounds that displayed activity in the anti-leishmanial assays.

Based on these findings, it is proposed that these compounds may be exerting their anti-leishmanial

activity through a different mechanism (such as having an allosteric effect) or that these

compounds are selective for the protozoan ortholog of Hsp 90 (Hsp 83). The biochemical pathway

is currently being investigated and it is targeted at undertsnading the observed activity.

Figure 5.1: Hsp 90 fluorescence polarization assay. *Numberings are as received

and not as used in manuscript

107

In conclusion, SAR studies of Hsp 90 inhibitors can lead to the discovery of potent anti-leishmanial

agents with an excellent toxicity profile and great selectivity over human macrophage cells.

References

(1) Kim, J.; Felts, S.; Llauger, L.; He, H.; Huezo, H.; Rosen, N.; Chiosis, G. Development of

fluorescence polarization assay for the molecular chaperone Hsp 90. J. Biomol. Screening 2004,

9, 375-381.

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Chapter 6

Introduction to Alzheimer’s diseases

6.1 Alzheimer’s disease

Alzheimer’s disease (AD) is a progressive and irreversible neurodegenerative disease and

a complicated multi-factorial disorder characterized by the accumulation of neuritic plaques (senile

plaques) consisting of the amyloid-β peptides (Aβ) in the brain, associated with

hyperphosphorylated tau-based neurofibrillary tangles (NF), synapse loss, mitochondrial damage

and ultimately cognitive decline,1,2,3 Figure 6.1. It is the most common form of dementia among

elderly people and one of the major health problems in western societies,4 associated with

tremendous cost to patients and their families - in both human and financial terms.

Figure 6.1: Cross sections of the brain show atrophy, or shrinking,

of brain tissue caused by Alzheimer’s disease

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Common symptoms of AD include memory loss with deficit in attention and language,

disorientation, confusion and difficulty carrying on conversations or responding to their

environment. The risk of developing AD increases with age and people who are aged 65 years and

older are particularly at high risk. About thirty five (35) million people are afflicted with the disease

worldwide and this number is projected to upsurge due to aging population – with current

healthcare cost standing at many billions of dollars annually.5 In 2010, the global economic impact

stood at an estimated $605 billion, but the social and emotional cost is incalculable.6

In the United States alone nearly 5.5 million people are afflicted with the disease and the number

is projected to rise to between 11 and 16 million by 20507 and this could potentially get worse if

no effective intervention is found in time. It is currently the 6th leading cause of death in the United

States. In 2012, $200 billion was spent for caring for AD patients and related dementia cases and

this figure is projected to rise to $1.1 trillion by 2050.8 AD disease is indeed one of the most

important economic, health and social crises of the current century.9

6.2 Etiology and pathology of Alzheimer’s disease

Notwithstanding the extensive study on the etiology and pathology of the disease, the exact

underlying mechanism is yet to be fully understood, due to its complex pathological

characteristics.10 However, the two (2) main pathological hallmarks of AD are the accumulation

of Aβ peptide (Aβ - plaques) within the extracellular space of the brain and tau accumulation

(neurofibrillary tangles), which are intraneural inclusion of hyperphosphorylated tau.2 These have

been recognized as being at the root of the disease pathogenesis. Albeit, designing effective new

medicaments has been hampered by the lack of knowledge about the precise cause of the disease.

110

A number of hypotheses have been proposed in an attempt to explain the etiology and pathology

of the disease, however no one hypothesis fully explains the precise mechanism of the disease

progression.6 The three main hypotheses that currently lend credence to the molecular basis of the

disease are the cholinergic hypothesis, the amyloid hypothesis and the tau hypotheses. These

hypotheses currently serve as the basis for therapeutic development.

6.2.1 The cholinergic hypothesis

This first hypothesis postulates that the loss of cholinergic neurons that subsequently leads

to dysfunction of cholinergic activity may play a role in the loss of memory and related cognitive

impairment – (often observed in the brain of AD patients).6,9 Thus, restoring cholinergic function

through the use of acetylcholinesterase inhibitors (AChEIs) or by modulating other cholinergic

receptors (muscarinic and nicotinic ACh), can lead to a reduction of the serious lack of cognitive

function.10 Acetylcholinesterase (AChE) plays a major role in nerve signal transmission, but

AChEIs prevent the breakdown of acetylcholine, leading to an increase in the concentration of

acetylcholine in the brain with improved cholinergic transmission, and ultimately improved

cognitive function. This hypothesis led to the development of the only drugs currently approved

by the FDA to treat AD (tacrine, donepezil, rivastigmine and galanthamine).10

6.2.2 Amyloid hypothesis

This hypothesis postulates that intramembrane proteolysis of the transmembrane protein,

β-amyloid precursor protein (APP), generates insoluble Aβ fragments (synaptotoxic/neurotoxic

peptide), that leads to plaque aggregation and subsequent development of neurofibrillary tangles.11

This occurs due to imbalanced production of Aβ, clearance and aggregation, resulting in a series

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of biochemical events that ultimately lead to AD. Aβ, typically a 42 amino acid peptide, is derived

from the sequential proteolytic cleavage of APP, by three aspartic acid proteases called β-secretase

(BACE-1), γ-secretase (presenilin) and α-secretase,5,12,13 as shown in Figure 6.2.

Figure 6.2: Formation of Aβ and Aβ fibrils from APP by sequential

cleavage by β- and γ-secretase12

The Aβ pathology has been the target of current drug discovery – the approach aims at modulating

or inhibiting the proteolysis of APP to reduce the synaptotoxic Aβ formation, increase Aβ

clearance, neutralize Aβ toxicity and/or remove existing Aβ aggregates.14,15 Inhibition of the

proteolytic enzymes in preclinical studies have indeed demonstrated reduction in Aβ peptides in

the brain, although BACE-1 inhibition is associated with a myriad of challenges. Aside from

cleaving APP, BACE-1 also cleaves many substrates that are involved with important

physiological roles, thus resulting in undesired side effects. Additionally, due to the elongated

active site, developed inhibitors are usually bulky and do not readily cross the blood brain barrier

(BBB).14,16 Conversely, in BACE-1 knockout mice, Aβ production was significantly reduced and

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no apparent AD pathology was observed, but most crucial was the fact that there was no consistent

phenotypic differences between the control.17,18,19 This establishes BACE-1 enzyme inhibition as

a potential therapeutic target, however, no BACE-1 inhibitor has ever been approved by the FDA.

In spite of the current challenges, a number of BACE-1 inhibitors (such as AZD3293, CTS-21166,

E2609, LYS2811376, LY2886721, MK-8931, HPP854, PF-05297909, TAK-070 and VTP-37948)

are currently in clinical trials,10,20 as a potential treatment for AD.

Additionally, inhibition of γ-secretase is also associated with challenges, due to the large numbers

of transmembrane proteins cleaved by it, including Notch, which could lead to serious mechanism

based side effects due to deficiency in selectivity.5 Notch inhibition has been the cause of

premature termination of clinical trials owing to toxicity and deleterious effects on cognition and

functionality of test subjects.21 Notwithstanding these challenges, there are a number of γ-secretase

inhibitors and modulators currently being studied in clinical trials including Avagacestat (BMS-

708163), Begacestat (GSI-953), NIC5-15, CHF5074 and E2012.10

6.2.3 Tau hypothesis

This hypothesis postulates that the accumulation of neurotoxic neurofibrillary tangles in

the brain is the ultimate pathway to AD, the result of atypical tau hyperphosphorylation.6 The

current understanding is that the accumulation of Aβ in the pathogenic progression of the disease

triggers a cascade of events, leading to tauopathy (tau hyperphosphorylation), mitochondrial

dysfuction, synaptic and neuronal degeneration that eventually manifest in the symptoms of

dementia,4 Figure 6.3.

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Figure 6.3: Signalling from Aβ via Tau drives AD progression22

There is a strong correlation between the formation of neurofibrillary tangles and the severity of

AD.23,24 Under normal physiological conditions soluble tau peptides function to stabilize axonal

microtubules, however, during early stages of AD (pathological conditions) they become

hyperphosphorylated (which weakens their association to microtubules) and causes them to

aggregate due to imbalanced activity of tau kinases and phosphatases.25,26,27

6.3 Current treatment

Current treatment options for AD are all symptomatic and they do not alter the underlying

biology of the disease process, but disease-modifying drugs are currently in development.28

Currently there are only five different drugs, Figure 6.4, approved by the United States Food and

Drugs Administration (US FDA) for treating AD: One (1) glutamate N-methyl-D-aspartate

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(NMDA) receptor antagonist, Memantine (Eli Lilly) and four (4) AChEIs, Tacrine (Warner-

Lambert Co.), Donepezil (Pfizer), rivastigmine (Novartis) and galantamine (Janssen).10,16,29

Figure 6.4. Current drugs used to treat AD

Tacrine, Donepezil and memantine are synthetic drugs whereas galanthamine and rivastigmine are

AChEIs derived from plant alkaloids.30

Many clinical trials have been conducted with the aim of reducing the accumulation of Aβ plaques

between neurons and tau-based neurofibrillary tangles within neurons but these have failed to

provide the desired result – cognitive improvement.6 One such compound to fail is semagacestat,

an Elli Lilly drug. Notwithstanding the numerous failures in clinical trials, Figure 6.5 summarizes

the biological and small molecule drug candidates currently being studied in clinical trials as of

January, 2017.31

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Figure 6.5: Agents in clinical trials for the treatment of AD in 201731

Alternative approaches to combating AD include immunotherapy32,33 and tau-targeted

therapy.16,26,34 The rest are stem cells, drugs modulating cholesterol and vascular-related risk

factors, antioxidants, enhances of mitochondrial function, neurotropins and anti-inflammatory

medicaments.14,16,32 Immunotherapy has been regarded as a promising approach to combating AD,

although there have been reports of adverse side effects.35,36,37 Immunotherapy is administered as

either active immunization (vaccination) or passive immunization (monoclonal antibodies), and

several antibodies are currently being investigated in both preclinical and clinical trials.14,16,28,38 In

spite of the enormous advances made so far, there are currently no new drugs that address the

116

underlying mechanisms of AD - either to slow the disease progression or stop it entirely. The lack

of effective treatment for AD continues to be a major unmet medical need.

6.4 Virtual screening of Compounds

In an effort to identify new compounds as potential treatments for AD, our laboratory in

collaboration with the laboratories of Professors David Kang (USF Byrd’s Alzheimer’s Institute)

and Yu Chen (USF Morsani College of Medicine), conducted a virtual screen of >4 million

compounds that could potentially be docked onto the catalytic pocket of the phosphatase slingshot

homology-2 (SSH2), using the ZINC molecule library and the program (DOCK 3.5.5.4).39 This

program was developed at the University of California, San Francisco, in the Shoichet laboratory.

Among the compound candidates from the screen results, two (2) promising lead compounds were

identified as shown in Figure 6.6, as slingshot homology-1 (SSH1) inhibitors, based on testing in

cell-based assays.

Figure 6.6: Lead compounds from virtual screen

The lead compounds contained either a pyridone or a thiazole moiety with a carboxylic acid

functionality, of which the catalytic residues of contact on SSH1 protein were obtained. These

residues were 100% conserved between SSH1, SSH2 and SSH3 (homologs) and were found to

117

reduce Aβ-induced cofilin dephosphorylation as well as significantly reduce Aβ secretion. Among

the several studies conducted with these compounds, results indicated that Aβ oligomers led to

rapid activation of SSH1 and Cofilin, both of which promote Aβ aggregation.2

6.5 Cofilin-Slingshot pathway in AD

The biochemical processes leading to the production of Aβ closely involve the actin

cytoskeleton, which plays a crucial role both in the maturation and maintenance of synapses,1 by

regulating synaptic plasticity, receptor anchoring and spine morphology. Of the many actin binding

proteins, the Cofilin family and actin-depolymerizing factor (ADF) function to regulate actin

filament dynamics and reorganization, which destabilizes F-Actin via severing and

depolymerization of actin filaments.1 There is considerable evidence that support the role of

Cofilin in Aβ induced dendritic spine changes, aggregation of Cofilin-Actin rods and increased

Cofilin activity in the brains of AD patients.2 Cofilin is inactivated by phosphorylation on serine

3 (Ser3) by LIM kinase isoform 1 (LIMK1) and the same is reactivated by SSH1-mediated

dephosphorylation (SSH1 activation),40,41,42 by the activity of calcineurin. SSH1 is inactivated via

Ca2+ dependent protein kinase II and phosphoinositide-dependent protein kinase.43 LIMK1 and

SSH1 show the highest substrate specificity among the many kinases and phosphatases that can

affect Cofilin. LIMK1 is also a substrate for SSH1 activity and can control Cofilin activation via

an upstream regulation of LIMK1, either directly or indirectly. Thus the SSH1-Cofilin activation

molecular pathway is essential for both Aβ production and tau pathologies, which ultimately leads

to mitochondrial and synaptic dysfunction,2 Figure 6.7.

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Figure 6.7: Progressive pathogenic cycle of AD

Thus, compounds that can inhibit the SSH1 enzyme/pathway (leading to the inhibition of excessive

Cofilin activation) represents a potentially viable therapeutic option in the fight against AD

pathogenesis.2 It was therefore hypothesized that small molecules that can inhibit the SSH1

pathway can break the AD pathogenic pathway via inhibition of Aβ production and Aβ induced

neurotoxicity.

6.6 In vitro phosphatase enzyme inhibitory assay

Phosphatase inhibitory activity of the synthesized compounds was evaluated using the

para-nitrophenyl phosphate (pNPP) phosphatase assay. Phosphatases control the removal of

phosphate groups (PO4-3) from peptide molecules and this leads to the regulation of many

important cellular processes, including differentiation, cell attachement, proliferation and

apoptosis. pNPP is a chromogenic substrate for many phosphatases (alkaline, acid, protein

tyrosine, and serine/threonine phosphatases), and the pNPP phosphatase assay has been optimized

to detect phosphatase activity via the hydrolysis of pNPP to para-nitrophenol (pNP).

119

The pNP produced during the reaction of a phosphatase with pNPP has an intense absorption at

405 nm and can therefore be conveniently measured on a spectrophotometer. Compounds that

inhibit a particular phosphatase should therefore be able to prevent the hydrolysis of pNPP to pNP

– the chromogenic species. Each of the compounds was treated with SSH1-N461 enzyme followed

by the addition of pNPP and incubated at 37 oC and the absorbances measured at intervals of 1

hour. Lower absorbances is an indication of phosphatase inhibitory activity.

120

6.7 References

(1) Barone, E.; Mosser, S.; Fraering, P. Inactivation of brain Cofilin-1 by age, Alzheimer's

disease and γ-secretase. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease

2014, 1842, 2500-2509.

(2) Woo, J.; Zhao, X.; Khan, H.; Penn, C.; Wang, X.; Joly-Amado, A.; Weeber, E.; Morgan, D.;

Kang, D. Slingshot-Cofilin activation mediates mitochondrial and synaptic dysfunction via Aβ

ligation to β1-integrin conformers. Cell Death & Differentiation 2015, 22, 921-934.

(3) Walsh, D.; Klyubin, I.; Fadeeva, J.; Cullen, W.; Anwyl, R.; Wolfe, M.; Rowan, M.; Selkoe, D.

Naturally secreted oligomers of amyloid β protein potently inhibit hippocampal long-term

potentiation in vivo. Nature 2002, 416, 535-539.

(4) Muñoz-Torrero, D.; Camps, P. Huprines for alzheimer's disease drug development. Expert

Opinion on Drug Discovery 2007, 3, 65-81.

(5) Truong, A.; Tóth, G.; Probst, G.; Sealy, J.; Bowers, S.; Wone, D.; Dressen, D.; Hom, R.;

Konradi, A.; Sham, H. et al. Design of an orally efficacious hydroxyethylamine (HEA) BACE-1

inhibitor in a preclinical animal model. Bioorganic & Medicinal Chemistry Letters 2010, 20,

6231-6236.

(6) Rafii, M.; Aisen, P. Advances in Alzheimer ’s disease drug development. BMC Medicine

2015, 13.

(7) MacMillan, K.; Naidoo, J.; Liang, J.; Melito, L.; Williams, N.; Morlock, L.; Huntington, P.;

Estill, S.; Longgood, J.; Becker, G. et al. Development of proneurogenic, neuroprotective small

molecules. Journal of the American Chemical Society 2011, 133, 1428-1437.

(8) 2012 Alzheimer's disease facts and figures

https://www.alz.org/downloads/facts_figures_2012.pdf (accessed Mar 15, 2018).

121

(9) Almansour, A.; Suresh Kumar, R.; Arumugam, N.; Basiri, A.; Kia, Y.; Ashraf Ali, M. An

expedient synthesis, acetylcholinesterase inhibitory activity, and molecular modeling study of

highly functionalized hexahydro-1,6-naphthyridines. BioMed Research International 2015, 2015,

1-9.

(10) Liu, Z.; Zhang, A.; Sun, H.; Han, Y.; Kong, L.; Wang, X. Two decades of new drug

discovery and development for Alzheimer's disease. RSC Advances 2017, 7, 6046-6058.

(11) Hardy, J. The amyloid hypothesis of alzheimer's disease: progress and problems on the road

to therapeutics. Science 2002, 297, 353-356.

(12) Singh, D.; Gupta, M.; Kesharwani, R.; Sagar, M.; Dwivedi, S.; Misra, K. Molecular drug

targets and therapies for Alzheimer’s disease. Translational Neuroscience 2014, 5, 203-217

(13) Zhang, H.; Ma, Q.; Zhang, Y.; Xu, H. Proteolytic processing of Alzheimer’s β-amyloid

precursor protein. Journal of Neurochemistry 2011, 120, 9-21.

(14) Mangialasche, F.; Solomon, A.; Winblad, B.; Mecocci, P.; Kivipelto, M. Alzheimer's

disease: clinical trials and drug development. The Lancet Neurology 2010, 9, 702-716.

(15) Castellani, R.; Smith, M. Compounding artefacts with uncertainty, and an amyloid cascade

hypothesis that is ‘too big to fail’. The Journal of Pathology 2011, 224, 147-152.

(16) Yiannopoulou, K.; Papageorgiou, S. Current and future treatments for Alzheimer’s disease.

Therapeutic Advances in Neurological Disorders 2012, 6, 19-33.

(17) Roberds, S. BACE knockout mice are healthy despite lacking the primary beta-secretase

activity in brain: implications for Alzheimer's disease therapeutics. Human Molecular Genetics

2001, 10, 1317-1324.

(18) Luo, Y.; Bolon, B.; Kahn, S.; Bennett, B.; Babu-Khan, S.; Denis, P.; Fan, W.; Kha, H.;

Zhang, J.; Gong, Y. et al. Mice deficient in BACE1, the Alzheimer's β-secretase, have normal

phenotype and abolished β-amyloid generation. Nature Neuroscience 2001, 4, 231-232.

122

(19) Cai, H.; Wang, Y.; McCarthy, D.; Wen, H.; Borchelt, D.; Price, D.; Wong, P. BACE1 is the

major β-secretase for generation of Aβ peptides by neurons. Nature Neuroscience 2001, 4, 233-

234.

(20) Vassar, R. BACE1 inhibitor drugs in clinical trials for Alzheimer’s disease. Alzheimer's

Research & Therapy 2014, 6, 1-14.

(21) Imbimbo, B.P; Giardina, G.A.M. Gamma-secretase inhibitors and modulators for the

treatment of alzheimer's disease: disappointments and hopes. Current Topics in Medicinal

Chemistry 2011, 11, 1555-1570.

(22) Bloom, G. Amyloid-β and tau. Journal of American Medical Association Neurology 2014,

71, 505.

(23) Arriagada, P.; Growdon, J.; Hedley-Whyte, E.; Hyman, B. Neurofibrillary tangles but not

senile plaques parallel duration and severity of Alzheimer's disease. Neurology 1992, 42, 631-

631.

(24) Bierer, L.; Hof, P.; Purohit, D.; Carlin, L.; Schmeidler, J.; Davis, K.; Perl, D. Neocortical

neurofibrillary tangles correlate with dementia severity in Alzheimer's disease. Archives of

Neurology 1995, 52, 81-88.

(25) Gendron, T.; Petrucelli, L. The role of tau in neurodegeneration. Molecular

Neurodegeneration 2009, 4, 13.

(26) Iqbal, K.; del C. Alonso, A.; Chen, S.; Chohan, M.; El-Akkad, E.; Gong, C.; Khatoon, S.; Li,

B.; Liu, F.; Rahman, A. et al. Tau pathology in Alzheimer disease and other tauopathies.

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 2005, 1739, 198-210.

(27) Mandelkow, E. Tau in Alzheimer's disease. Trends in Cell Biology 1998, 8, 425-427.

(28) Galimberti, D.; Scarpini, E. Disease-modifying treatments for Alzheimer’s disease.

Therapeutic Advances in Neurological Disorders 2011, 4, 203-216.

123

(29) Shidore, M.; Machhi, J.; Shingala, K.; Murumkar, P.; Sharma, M.; Agrawal, N.; Tripathi, A.;

Parikh, Z.; Pillai, P.; Yadav, M. Benzylpiperidine-linked diarylthiazoles as potential anti-

Alzheimer’s agents: synthesis and biological evaluation. Journal of Medicinal Chemistry 2016,

59, 5823-5846.

(30) Howes, M. Alkaloids and drug discovery for neurodegenerative diseases. Natural Products

2013, 1331-1365.

(31) Cummings, J.; Lee, G.; Mortsdorf, T.; Ritter, A.; Zhong, K. Alzheimer's disease drug

development pipeline: 2017 (accessed Jan 29, 2018).

(32) Salomone, S.; Caraci, F.; Leggio, G.; Fedotova, J.; Drago, F. New pharmacological

strategies for treatment of Alzheimer's disease: focus on disease modifying drugs. British Journal

of Clinical Pharmacology 2012, 73, 504-517.

(33) Town, T. Alternative abeta immunotherapy approaches for Alzheimers disease. CNS &

Neurological Disorders - Drug Targets 2009, 8, 114-127.

(34) Mudher, A.; Lovestone, S. Alzheimer's disease – do tauists and baptists finally shake

hands?. Trends in Neurosciences 2002, 25, 22-26.

(35) Check, E. Nerve inflammation halts trial for Alzheimer's drug. Nature 2002, 415, 462-462.

(36) Morgan, D. Immunotherapy for Alzheimer’s disease. Journal of Internal Medicine 2010,

269, 54-63.

(37) Gilman, S.; Koller, M.; Black, R.; Jenkins, L.; Griffith, S.; Fox, N.; Eisner, L.; Kirby, L.;

Rovira, M.; Forette, F. et al. Clinical effects of A immunization (AN1792) in patients with AD in

an interrupted trial. Neurology 2005, 64, 1553-1562.

(38) Brody, D.; Holtzman, D. Active and passive immunotherapy for neurodegenerative

disorders. Annual Review of Neuroscience 2008, 31, 175-193.

124

(39) Irwin, J.; Shoichet, B. ZINC − A free database of commercially available compounds for

virtual screening. Journal of Chemical Information and Modeling 2005, 45, 177-182.

(40) Bernstein, B.; Chen, H.; Boyle, J.; Bamburg, J. Formation of actin-ADF/cofilin rods

transiently retards decline of mitochondrial potential and ATP in stressed neurons. American

Journal of Physiology-Cell Physiology 2006, 291, C828-C839.

(41) Kurita, S.; Watanabe, Y.; Gunji, E.; Ohashi, K.; Mizuno, K. Molecular dissection of the

mechanisms of substrate recognition and f-actin-mediated activation of cofilin-phosphatase

slingshot-1. Journal of Biological Chemistry 2008, 283, 32542-32552.

(42) Niwa, R.; Nagata-Ohashi, K.; Takeichi, M.; Mizuno, K.; Uemura, T. Control of actin

reorganization by slingshot, a family of phosphatases that dephosphorylate ADF/Cofilin. Cell

2002, 108, 233-246.

(43) Bravo-Cordero, J.; Magalhaes, M.; Eddy, R.; Hodgson, L.; Condeelis, J. Functions of cofilin

in cell locomotion and invasion. Nature Reviews Molecular Cell Biology 2013, 14, 405-415.

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Chapter 7

Pyridones Analogs

7.1 Pyridones

Nitrogen-containing heterocyclic molecules are of vital importance due to their abundance

in various biologically active synthetic organic molecules and natural products. Among the

nitrogen-containing heterocyclic molecules, 2-pyridones are recognized as an important structural

motif1 and exhibit a broad range of biological properties. Synthetic 2-pyridone molecules have

found many uses including as an anti-epileptic drug (Perampanel) and a phosphodiesterase 3

inhibitor (Amrinone), Figure 7.1. Other uses of pyridone containing compounds include anti-

cancer, anti-fungal, anti-hepatitis B, anti-HIV, anti-Mycobacterium tuberculosis agents, human

rinovirus 3C protease activities and MEK-1 inhibition.1

Figure 7.1: Biologically active compounds containing 2-pyridone

Important to our laboratory’s drug discovery effort is the development of pyridones and related

analogs as a potential treatment for Alzheimer’s disease. Synthesized anti-AD molecules

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possessing the pyridone core that exhibit potent important biological activities include γ-secretase

modulators2 and acetylcholinesterase inhibitor,3 Figure 7.2. Due to the complexity in etiology of

AD, Sheng and colleagues4 have prepared a multifactorial 4-pyridone agent that integrates H3

receptor antagonist, metal chelation, anti-β-amyloid peptide aggregation and radical scavenging

into a single molecule, Figure 7.2.

Figure 7.2: Pyridones as treatment for AD

A number of pyridone-related analogs such as pyradazinones (ABT-288, CEP-26401) and

pyrimidinone (MK0249) are currently being evaluated at various stages in clinical trials targeting

antagonism of the H3 receptor as therapeutic candidates for AD,4 Figure 7.3.

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Figure 7.3: H3 receptor antagonists in clinical trials

7.2 Pyridone alkaloids

Pyridone alkaloids have a pyridone nucleus and may contain either a tetracyclic or tricyclic

ring.5 Many of these alkaloids exhibit profound biological neuroprotective and neurological

properties. For instance, cytisine, shown in Figure 7.4, is a naturally occurring alkaloid possessing

a pyridone nucleus with a variety of uses including smoke cessation.6 It acts as a partial agonist by

binding to the nicotinic acetylcholine receptor and has been shown to have an affinity for the α4β2

receptor subtype to exert its activity.

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Figure 7.4: Naturally occurring pyridone alkaloids

Huperzine A, also a naturally occurring sesquiterpene alkaloid isolated from the Chinese herb

Huperzia serrata by Liu and colleagues7 in the early 1980’s, is currently in various stages of

clinical trials as a treatment for dementia, schizophrenia, traumatic brain injury, epilepsy, cognitive

functioning, cocaine dependence and Alzheimer’s disease.8 As a natural selective inhibitor of

acetylcholinesterase,9 huperzine A acts in a similar fashion to donepezil – an oral drug used to treat

dementia and AD. Owing to its antioxidant and neuroprotective properties, huperzine A is

currently available as a neutraceutical. The prodrug analog of huperzine A, ZT-1, also derived

from natural sources9 is a potent selective inhibitor of acetylcholinesterase inhibitor. Data from

phase I clinical trials indicates that ZT-1 has good pharmacokinetics properties. Militarinone A,

produced by the entomogenous deuteromycete Paecilomyces militaris is also reported as having

distinct neuritogenic properties in PC12 cells at 10 µM concentrations.10,11

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7.3 The Huprines

A class of compounds recognized as being perhaps the most promising drugs as AChEIs

for the treatment of AD are the huprines, a (-)-huperzine A/tacrine hybrid.12,13 These are designed

to span as much as possible the active site of AChE, Figure 7.5. These 4-aminoquinoline-based

compounds share close resemblance to the cytisine alkaloid and are proven to inhibit brain

acetylcholinesterase enzyme – both ex vivo and in vivo and have been shown to improve learning

and memory in middle aged mice.14 This potent class of compounds is also known to interact with

other cholinergic targets including nicotinic and muscarinic receptors.

Figure 7.5: The huprines as AChE inhibitors

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7.4 Synthesis of pyridone analogs as SSH1 inhibitors

Owing to the profound biological importance of 2-pyridones and related analogs, several

synthetic schemes have been devised for their synthesis.15,16,17 The synthetic approach adopted

makes use of an α,β-unsaturated chromone to provide the target intermediate bearing the pyridone

core,18 as shown in Scheme 7.1. This route allows for the introduction of a wide variety of different

amines (less nucleophilic or sterically hindered) at the penultimate step of the synthesis, whilst

ensuring quick determination of the most active analog for further development.

Scheme 7.1: Synthesis of 2-Pyridone analogs

Reagents and conditions: (a) DMF, POCl3, H2O, 0 oC – rt, 62-75%; (b) CH2(COOCH3)2, TEA, EtOH, mW,

83%; (c) R-NH2, AcOH or CsF, EtOH, 75 oC, 55 - 75%; (d) Aqueous NaOH, TBAI, DMSO, mW, 30min, 55 -

88%.

Starting with commercially available hydroxyacetophenone, 7.001, the key starting compound, 3-

formylchromone derivative, 7.002, was prepared in yields of up to 75% using the Vilsmeier-Haack

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cyclization reaction19,20,21 employing dry DMF and POCl3. The compound thus obtained, 7.002, is

the key intermediate for the synthesis of many important heterocyclic systems owing to its three

(3) electron-deficient sites, Figure 7.6, vis-à-vis C2 carbon for Michael addition; aldehyde carbon

for Knoevenagel condensation and the C4 carbon of the carbonyl group.22

Depending upon the type of substrate and reaction conditions, a number of different heterocyclic

systems can be obtained from the chromone derivative, due to its ability to serve as a Michael

acceptor. This compound, 7.002, was subjected to Knoevenagel reaction conditions23,24 using

diethyl malonate in the presence of TEA to obtain the α,β-unsaturated derivative 7.003. Treatment

of 7.003 with different primary amines (A-D) provided the corresponding pyridones in decent

yields via a cascade reaction process, that is, aza-Michael addition, chromone ring opening and

ring closure.18 Thus compounds 7.004 (A-D) were obtained in good yields, however, depending

upon the type of amine used (thiazole-2-amine, benzylamine, aniline, or 4-(thiophen-2-yl)thiazol-

2-amine), the catalyst (CsF or AcOH) and/or solvents (EtOH or DCM) needed to be varied for

optimal yields.18 Compounds 7.004 (A-D) were then subjected to saponification conditions to

afford the final compounds 7.005 (A-D) using aqueous NaOH in EtOH. The saponification

procedure proved to be a much more difficult task than anticipated. The following reaction

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conditions were attempted with no success: KOH/MeOH;25 FeCl3-promoted O-alkylation cleavage

of the esters;26 TMS-I/CCl4/H2O;27 TMS-Cl, NaI/CH3CN28 and LiOH/CHCl3. Recognizing that

the lack of apparent reactivity was largely due to the poor solubility of this series of compounds,

the saponification process was achieved by using DMSO as solvent, tetrabutylammonium iodide

(TBAI) as a phase transfer reagent and an excess amount of aqueous NaOH under microwave

irradiation conditions. Compounds 7.005 (A-D) were achieved in yields ranging from 55 – 88%.

7.5 Masking phenol: Intended mechanistic studies

Computational modelling studies, as shown in Figure 7.7, suggested that the most crucial

features involved in binding to the SSH1 active site are the thiazole nitrogen, which forms a

hydrogen bond with SSH1 Arg 399, the carboxylate and the pyridone carbonyl. Neither the

phenolic hydrogen nor the ketone functionality appeared to play any role in binding. Thus, in order

to assess whether the phenolic functionality is involved in binding, the phenolic site was blocked

via alkylation with a methyl group29 in order to assess this property in compound 7.007, Scheme

7.2.

Figure 7.7: Compound 7.005A and its binding orientation in SSH2 active site

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Scheme 7.2: O-Alkylation of the phenolic functionality

Reagents and conditions: (a) K2CO3, MeI, TBAI, ACN, H2O, DMSO, 42%; (b) Aqueous NaOH, TBAI,

DMSO, mW, 30 min, 51%.

Compound 7.004A was able to undergo O-alkylation with methyl iodide (MeI) in the presence of

K2CO3 to yield the ether derivative 7.006, which was subsequently treated with aqueous NaOH to

provide the target compound 7.007 as the O-methylated ether analog of the initial compound.

It was envisioned that if indeed the phenolic functionality is not involved in binding, it could

represent an opportunity to be used as a handle for molecular probes30 for elucidating the

mechanism of action of these compound, Figure 7.8. That is, addition of fluorescein would

potentially allow visualization in living systems;31 addition of biotin would help pull down

biological receptors32,33 and the attachment of an azide would likely allow the formation of a

covalent bond with a biomolecule under copper catalysis.34

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Figure 7.8: Compound 7.005A alkylated with proposed linkers for mechanistic studies.

7.6 Derivatization at the 5-position of compound 7.005A

In order to ensure that the compounds were not only active but have acceptable

pharmacokinetic properties, derivatization at the 5-position via either carbon-carbon bond

formation (Suzuki coupling)35,36 or carbon-nitrogen bond formation (Hartwig-Buchwald)37 was

initiated. Given that these compounds have solubility issues, it was envisioned that this would also

be a suitable site for the incorporation of solubilizing groups. A synthesis of the proposed analogs

was initiated by first incorporating a synthetic handle, bromine, at the 5-position, which would be

exploited using Suzuki methodology, where different boronic acids could be used for the rapid

exploration of this portion of the molecule, Scheme 7.3. Additionally, functionality might also be

achieved using the well-established Hartwig-Buchwald amination for the installation of amines at

this position. The goal was to make additional changes to the molecule at the very end of the

synthesis.

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Scheme 7.3: Synthesis of bromo-substituted analog

Reagents and conditions: (a) i. DMF, POCl3, H2O, 0 oC – rt, 69%; ii. CH2(COOCH3)2, TEA, EtOH, mW, 82%;

iii. Thiazol-2-amine, AcOH, EtOH, 75 oC, 71%; (b). Aqueous NaOH, TBAI, DMSO, mW, 30min, 64%.

Following similar synthetic routes outlined previously, the bromo-analog, 7.010, was achieved in

good yields. This compound and its precursor, 7.009, was subjected to Suzuki crosss coupling36,38

or the Hartwig-Buchwald37 reaction conditions to introduce diversity at the 5-position. In order to

test the viability of the coupling reactions on this substrate, phenylboronic acid was initially

selected for Suzuki-coupling and benzylamine for the Hartwig-Buchwald amination reaction.

The Suzuki coupling on both 7.009 and 7.010 using potassium phosphate or potassium carbonate,

bis(triphenylphosphine)palladium chloride [Pd2(PPh3)2Cl2], H2O and DMF38 did not produce the

desired product. The Hartwig-Buchwald reaction also failed on the acid, 7.010, however an

expected side-product, benzylamide, 7.011, was obtained in 11% yield as shown in Scheme 7.4,

route a. In order to test the side product in the assays, this reaction was optimized and a yield of

89% - detailed procedure is provided under experimental. Thus, an alternative approach was

sought to effect both the C-C coupling and C-N coupling on the compound.

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Scheme 7.4: Hartwig-Buchwald reaction

Reagents and conditions: (a) BnNH2, DPPF, NaOtBu, Pd(OAc)2, 11%; (b) BnNH2, CsCO3, XANTPHOS,

Pd2(dba)3.

One such alternative approach was the use of XANTPHOS and Pd2(dba)339 in the Hartwig-

Buchwald amination reaction. The combination of XANTPHOS and Pd2(dba)3 had proven to give

excellent yields on substrates with an existing ester group. Indeed, this methodology has previously

been used on a similar substrate with yield in excess of 80%, Figure 7.4, Chapter 4. However, in

this case, the reaction with 4.009 failed to provide the desired product. The solubility issues

associated with this series of analogs may account for the lack of reactivity.

Figure 7.9: Hartwig-Buchwald amination with XANTPHOS and Pd2(dba)3

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The C-N coupling was then attempted via nitration40,41 and anticipated reducing the nitro

group,41,42,43,44 which would then be followed by SN2 reaction with benzyl bromide to achieve the

target analog, but this was accompanied by its own set of synthetic and purification challenges,

Scheme 7.6. Neither 7.01 nor 7.015 could be reduced with SnCl2,41,42,43 and iron (Fe) reduction44

of 7.014 resulted in a product that could not be isolated. Compounds 7.017 and 7.018 also

decomposed upon standing.

Scheme 7.5: Synthesis of nitro analog

Reagents and conditions: (a) Fuming HNO3, AcOH, 25%; (b) DMF, POCl3, H2O, 0 oC – rt, 88%;

(c) CH2(COOCH3)2, TEA, EtOH, mW, 61%; (b) Thiazol-2-amine, AcOH, EtOH, 75 oC, 51%; (e) Aqueous

NaOH, TBAI, DMSO, mW, 30min, 59%.

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7.6.1 Synthesis of a the phenyl analog via Suzuki coupling of starting material

Although undesirable, the phenyl group was installed at the beginning of the synthesis,

using Suzuki methodlogy,36,38 as shown in Scheme 7.6. Compound 7.020 was obtained in the

crude form and following similar synthetic routes outlined previously, compound 7.021 was

obtained in good yields.

Scheme 7.6: Synthesis of phenyl analog

Reagents and conditions: (a) Phenylboronic acid, PdCl2(PPh3)2, CH3CHOHCH3, H2O; (b) i. DMF, POCl3, H2O,

0 oC – rt, 69%; ii. CH2(COOCH3)2, TEA, EtOH, mW, 82%; iii. Thiazol-2-amine, AcOH, EtOH, 75 oC, 71%; iv.

Aqueous NaOH, TBAI, DMSO, mW, 30min, 64%.

7.7 Modifications to the bottom portion of the molecule

Although computational modelling did not predict changes to the bottom portion of the

molecule would result in significant impact on SSH1 activity, modern drug discovery hinges on

the concept of parallel lead optimization - which ensures that most active drug candidates also

possesses acceptable pharmacokinetic properties. Thus, modifications in this portion may lead to

the identification of novel compounds with the greatest chance of success as a potential drug. The

initial choice of an ideal candidate for replacing the bottom portion was a quinazoline scaffold

(7.022), Figure 7.10, since the nitrogens in the quinazoline perfectly overlap the ketone and phenol

oxygens in the original molecule, and may ultimately mimic any apparent binding interactions

thereof.

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Figure 7.10: Modification to bottom portion of molecule

Given that thiazol-2-ylboronic acid is expensive (1 g/$1435.00) for the proposed synthetic route

for the top portion of the molecule, Scheme 7.6, a related isostere (phenylboronic acid) was

pursued instead, Figure 7.11. To this end, it was proposed that a species such as 7.023 could be

conveniently coupled to 7.024 via Suzuki cross coupling reaction.

Figure 7.11: Retrosynthetic scheme for the synthesis of 7.039

7.7.1 Synthesis of iodo and boronic ester derivatives: top portion of quinazoline analog

The top portion of 7.039 was synthesized according to the synthetic scheme outlined below,

starting with commercially available 2-hydroxynicotinic acid, 7.025, Scheme 7.7. It was

envisioned that either the iodo-derivative, 7.028, or the boronic ester, 7.029, could serve as an ideal

140

candidate for coupling to an appropriate boronic acid/ester or halo-derivative of quinazoline45 via

Suzuki methodology.36,38

Scheme 7.7: Synthesis of iodo and boronic ester derivatives

Reagents and conditions: (a) SOCl2, DCM, THF, MeOH, 100%; (b) Phenylboronic acid, Cu(OAc)2, Pyr, DCM,

56%; (c) NIS, DCM, 89%; (d) Cs2CO3, Pd(OAc)2, BISPIN, MeOH, 41%

The commercially available 2-hydroxynicotinic acid, 7.025, was treated with thionyl chloride,

followed by MeOH to obtain methyl 2-hydroxynicotinate, 7.026, in quantitative yield.46 This

compound underwent N-arylation (copper-catalyzed C-N coupling) in the presence of the

corresponding phenylboronic acid, Cu(OAc)2 and pyridine in DCM to yield 7.029.46 Iodination

of 7.029 with N-iodosuccinimide (NIS) in DCM47 provided the iodo derivative 7.028, which was

subsequently converted to the corresponding boronic ester, 7.029, using bis(pinacolato)diboron

(BISPIN), Cs2CO3 and Pd(OAc)2 in MeOH.48 With compounds 7.028 and 7.029 in hand,

compound 7.031 was synthesized to test the viability of the Suzuki reaction, Scheme 7.8

(substituting the quinazoline for a phenyl group). Phenylboronic acid was added to 7.028 in the

presence of Pd(PPh3)Cl2 and K2CO3 in dioxane and H2O to provide 7.030 in 72%. This compound

141

was able to undergo saponification using aqueous NaOH in dioxane and H2O to provide the acid

product, 7.031.

Scheme 7.8: Synthesis of phenyl analog: Modification to bottom portion

Reagents and conditions: (a) Phenylboronic acid, Pd(PPh3)Cl2, K2CO3, Dioxane, H2O, 73%; (b) Aqueous

NaOH, Dioxane, H2O, 50 oC, 97%.

7.7.2 Synthesis of quinazoline analog bottom portion

Following the successful synthesis of 7.031, synthesis of a series of quinazolines, 7.033,

7.034, 7.035 or 7.036 was initiated which would potentially be coupled to either 7.028 or 7.029

via Suzuki methodology.38,45

Scheme 7.9: Synthesis of quinazoline derivatives

Reagents and conditions: (a) HCONH2, 81%; (b) SOCl2, DMF, 50%

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The anthranilic acid, 7.031, was treated with an excess amount of formamide49 and heated to 120

oC under microwave irradiation to obtain compound 7.032, which was subsequently treated with

thionyl chloride and a catalytic amount of DMF50 to obtain compound 7.033. However, because

chlorides are not particularly great candidates for Suzuki cross coupling reactions, compound

7.032 was initially treated with CBr4 in the presence of PPh3 in toluene51 but could not achieve the

desired product. A similar conversion was also attempted with TBABr, P2O5 in toluene;52 PBr3,

DMF; Appel type reaction on 7.033 with TMSBr in CH3CN53 or with HBr-AcOH,54 however all

these failed to yield the desired product – any formed bromo-quinazoline derivative quickly

hydrolysed back to the keto form, 7.032. The O-triflate quinazoline derivative, 7.03555 was

synthesized but this also hydrolysed to the keto form upon standing. An attempt to convert 7.033

to the corresponding boronic ester with BISPIN also resulted in 7.032, even in the presence of

molecular sieves and the use of dry solvents. Notwithstanding these challenges, the Suzuki cross

coupling reaction was attempted using 7.033 and 7.29 as shown in Scheme 7.10, however, a trace

amount of the desired product, 7.037, was observed along with a significant amount of 7.032.

Scheme 7.10: Synthesis of quinazoline analog

Reagents and conditions:; (b) 7.033, Pd(OAc)2, Cs2CO3, DMF, mW (c) Aqueous NaOH, Dioxane, H2O, 60 oC.

143

7.8 Summary of synthesized pyridone analogs

A summary of the synthesized pyridones are provided in Figure 7.12, and their

phosphatase inhibitory activities are discussed jointly with the thiazole series in Chapter 8.

Figure 7.12: Summary of pyridone test compounds

144

7.9 Experimental

General Experimental procedures:

4-Oxo-4H-chromene-3-carbaldehyde: A solution of 1-(2-hydroxyphenyl)ethanone (17.7 mL,

147 mmol) in DMF (68.0 mL, 881 mmol) was cooled over ice for 1 hour. POCl3 (28.8 mL, 308

mmol) was slowly added to the solution and the mixture was heated to 45°C for 1 hour. While

stirring, a mixture of ice and H2O was added and the mixture was stirred for 4 hours. The resulting

crystalline solid/precipitate was filtered off, washed thoroughly with H2O and dried under vacuum.

The acidic crude product was stirred in ACN for 10 minutes, filtered and washed with MeOH and

dried under vacuum. The pure product, 4-oxo-4H-chromene-3-carbaldehyde, 7.002, was isolated

as a light yellow solid (16 g, 63% yield). 1H NMR (500 MHz, DMSO) 10.11 (s, 1H), 8.91 (s,

1H), 8.13 (dd, J = 1.5, 7.8 Hz, 1H), 7.87 (ddd, J = 8.6, 7.1, 2.0 Hz, 1H), 7.74 (dd, J = 8.3, 1.0 Hz,

1H), 7.53 - 7.61 (m, 1H) ppm. 13C NMR (126 MHz, DMSO) 188.8, 175.3, 163.9, 156.1, 135.7,

127.2, 125.7, 125.1, 120.4, 119.4 ppm. LRMS (ESI) m/z calc’d for C10H6O3 [M+H]+: 175.16,

found 175.06.

Diethyl 2-((4-oxo-4H-chromen-3-yl)methylene)malonate: A mixture of 7.002 (2.000 g, 11.48

mmol), diethyl malonate (2.023 g, 12.63 mmol) and TEA (0.160 mL, 1.148 mmol) in EtOH (20.0

mL) in a 30 mL microwave vial was heated to 100 °C for 80 mins. The resulting mixture was

added to H2O and neutralized with HCl and extracted with EtOAc, washed with brine and dried

145

over anhydrous sodium sulfate, and concentrated in vacuum. The crude product could either be

purified by column chromatography (EtOAc:hexane 1:5) or recrystallized from hexane and EtOAc

to obtain diethyl 2-((4-oxo-4H-chromen-3-yl)methylene)malonate, 7.003, as a light brown solid

(3.01 g, 9.53 mmol, 83% yield). 1H NMR (500 MHz, DMSO) 8.71 (d, J = 1.0 Hz, 1H), 8.08

(dd, J = 8.1, 1.7 Hz, 1H), 7.85 (ddd, J = 8.6, 7.1, 1.5 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.50 - 7.57

(m, 2H), 4.15 - 4.26 (m, 4H), 1.24 (t, J = 7.1 Hz, 3H), 1.14 (t, J = 7.1 Hz, 3H) ppm. 13C NMR (126

MHz, DMSO) 174.4, 164.9, 164.3, 159.8, 155.7, 135.3, 134.3, 128.1, 126.8, 125.9, 123.5, 119.0,

118.6, 61.8, 61.3, 14.5, 14.2 ppm. LRMS (ESI) m/z calc’d for C17H16O6 [M+H]+: 317.31, found

317.20.

Ethyl 1-benzyl-5-(2-hydroxybenzoyl)-2-oxo-1,2-dihydropyridine-3-carboxylate: To a 50 mL

round bottomed flask was added 7.003 (500.0 mg, 1.581 mmol), benzylamine (129.0 µL, 1.179

mmol) and acetic acid (45.00 µL, 0.786 mmol) in EtOH (5 mL). The reaction was stirred at 75 oC

for 24 - 36 hours and monitored by TLC. After completion of the reaction, the mixture was

evaporated under vacuum and purified by flash column chromatography (hexane and EtOAc) to

afford the desired product, 7.004B, (453 mg, 75%). 1H NMR (500 MHz, DMSO) 10.25 (s, 1H),

8.69 (d, J = 2.8 Hz, 1H), 8.30 (d, J = 2.8 Hz, 1H), 7.24 - 7.44 (m, 7H), 6.96 (d, J = 7.9 Hz, 1H),

6.92 (t, J = 7.5 Hz, 1H), 5.23 (s, 2H), 4.20 (q, J = 7.2 Hz, 2H), 1.22 (t, J = 7.0 Hz, 3H) ppm. 13C

NMR (126 MHz, DMSO) 191.4, 164.4, 158.3, 156.2, 149.5, 143.4, 136.6, 133.4, 130.5, 129.1,

146

128.3, 128.2, 125.1, 119.7, 119.3, 117.1, 116.2, 61.2, 53.2, 14.5 ppm. LRMS (ESI) m/z calc’d for

C22H19NO5 [M+H]+: 378.40, found 378.38.

1-benzyl-5-(2-hydroxybenzoyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid: A mixture of

7.004B (200.0 mg, 0.530 mmol), 50% w/w NaOH (70.00 µL, 1.325 mmol) and

tetrabutylammonium iodide (9.790 mg, 0.026 mmol) in DMSO (5 mL) in a 30 mL microwave vial

was heated to 75°C in a microwave reactor for 30 minutes. The resulting mixture was allowed to

cool to room temperature, acidified with aqueous HCl to a pH of 3 and the solid was collected

using vacuum filtration, rinsed with ethyl acetate and ACN and identified as 1-benzyl-5-(2-

hydroxybenzoyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid, 7.005B, (163 mg, 0.466 mmol,

88% yield). 1H NMR (500 MHz, DMSO) 13.57 (br. s., 1H), 10.33 (s, 1H), 8.89 (d, J = 2.9 Hz,

1H), 8.47 (d, J = 2.9 Hz, 1H), 7.41 - 7.47 (m, 1H), 7.34 - 7.40 (m, 5H), 7.29 - 7.34 (m, 1H), 6.93 -

7.01 (m, 2H), 5.37 (s, 2H) ppm. 13C NMR (126 MHz, DMSO) 191.4, 164.8, 162.9, 156.2, 149.0,

144.7, 135.9, 133.9, 130.7, 130.7, 129.2, 128.5, 128.5, 124.8, 119.9, 118.8, 117.1, 53.6 ppm.

HRMS m/z: [M + H]+ calc’d for C20H15NO5 350.1028; Found 350.1026.

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Ethyl 5-(2-hydroxybenzoyl)-2-oxo-1-(thiazol-2-yl)-1,2-dihydropyridine-3-carboxylate:, 7.

Using conditions identical to those described above 7.004A was obtained as a yellow powder

(55%). 1H NMR (500 MHz, DMSO) 10.34 (s, 1H), 9.35 (d, J = 2.9 Hz, 1H), 8.49 (d, J = 2.5 Hz,

1H), 7.80 (d, J = 3.4 Hz, 1H), 7.75 (d, J = 3.4 Hz, 1H), 7.40 - 7.49 (m, 2H), 6.95 - 7.04 (m, 2H),

4.30 (q, J = 6.9 Hz, 2H), 1.30 (t, J = 7.1 Hz, 3H) ppm. 13C NMR (126 MHz, DMSO) 191.4,

163.6, 156.9, 156.2, 155.1, 142.5, 141.8, 138.6, 133.9, 130.6, 124.7, 121.7, 121.4, 119.9, 117.7,

117.1, 61.7, 14.5 ppm. LRMS (ESI) m/z calc’d for C18H14N2O5S [M+H]+: 371.38, found 371.20.

5-(2-Hydroxybenzoyl)-2-oxo-1-(thiazol-2-yl)-1,2-dihydropyridine-3-carboxylic acid: Using

conditions identical to those described above, 7.005A was obtained as a light brown solid (68%).

1H NMR (500 MHz, DMSO) 13.23 (br. s., 1H), 10.35 (s, 1H), 9.37 (d, J = 2.5 Hz, 1H), 8.49 (d,

J = 2.5 Hz, 1H), 7.81 (d, J = 3.4 Hz, 1H), 7.77 (d, J = 3.4 Hz, 1H), 7.41 - 7.48 (m, 2H), 6.95 - 7.03

(m, 2H) ppm. 13C NMR (126 MHz, DMSO) 191.5, 164.8, 157.9, 156.2, 155.1, 142.8, 141.4,

138.6, 133.9, 130.6, 124.7, 121.8, 121.5, 119.9, 118.1, 117.1 ppm. HRMS m/z: [M + H]+ calc’d

for C16H10N2O5S 343.0389; Found 343.0379.

148

Ethyl 5-(2-hydroxybenzoyl)-2-oxo-1-phenyl-1,2-dihydropyridine-3-carboxylate: Using

conditions identical to those described above, 7.004C was obtained as a yellow solid (61%). 1H

NMR (400 MHz, CDCl3) 11.36 (s, 1H), 8.59 (d, J = 1.2 Hz, 1H), 8.18 (br. s., 1H), 7.58 (d, J =

8.2 Hz, 1H), 7.45 - 7.55 (m, 4H), 7.39 (d, J = 7.4 Hz, 2H), 7.07 (d, J = 8.6 Hz, 1H), 6.94 (t, J = 7.6

Hz, 1H), 4.38 (q, J = 7.0 Hz, 2H), 1.36 (t, J = 7.0 Hz, 3H) ppm. 13C NMR (101 MHz, CDCl3) δ

194.2, 164.1, 162.2, 158.4, 147.0, 144.1, 139.6, 136.4, 131.4, 129.5, 129.4, 126.4, 126.3, 120.9,

119.2, 118.7, 115.9, 61.6, 14.2 ppm. LRMS (ESI) m/z calc’d for C21H17NO5 [M+H]+: 364.37,

found 364.27.

5-(2-Hydroxybenzoyl)-2-oxo-1-phenyl-1,2-dihydropyridine-3-carboxylic acid: Using

conditions identical to those above, 7.005C was obtained as a brownish solid (77%). 1H NMR (500

MHz, DMSO) 13.47 (br. s., 1H), 10.41 (s, 1H), 8.57 (d, J = 2.9 Hz, 1H), 8.37 (d, J = 2.5 Hz,

1H), 7.49 - 7.61 (m, 5H), 7.37 - 7.45 (m, 2H), 6.91 - 7.01 (m, 2H) ppm. 13C NMR (126 MHz,

DMSO) 191.1, 164.8, 162.6, 156.3, 149.0, 144.7, 139.7, 134.0, 130.8, 130.0, 129.8, 127.2, 124.7,

120.0, 118.2, 118.1, 117.2 ppm. HRMS m/z: [M + H]+ calc’d for C19H13NO5 336.0872; Found

336.0868.

149

Ethyl 5-(2-hydroxybenzoyl)-2-oxo-1-(4-(thiophen-2-yl)thiazol-2-yl)-1,2-dihydropyridine-3-

carboxylate: Using conditions identical to those described above 7.005D was obtained as a solid

(66%). 1H NMR (500 MHz, DMSO) 10.37 (s, 1H), 9.42 (d, J = 2.5 Hz, 1H), 8.48 (d, J = 2.5 Hz,

1H), 7.96 (s, 1H), 7.56 (d, J = 3.9 Hz, 2H), 7.47 - 7.52 (m, 1H), 7.45 (dd, J = 7.8, 1.5 Hz, 1H),

7.12 (t, J = 4.2 Hz, 1H), 7.05 (d, J = 8.3 Hz, 1H), 6.98 - 7.02 (m, 1H), 4.31 (q, J = 7.3 Hz, 2H),

1.30 (t, J = 7.09 Hz, 3H) ppm. 13C NMR (126 MHz, DMSO) 191.4, 163.6, 157.0, 156.3, 154.8,

144.4, 142.6, 141.8, 137.7, 133.8, 130.5, 128.7, 127.0, 125.2, 124.6, 121.2, 119.8, 117.7, 117.1,

114.1, 61.7, 14.5 ppm. LRMS (ESI) m/z calc’d for C22H16N2O5S2 [M + H]+: 453.50, found 453.42.

5-(2-hydroxybenzoyl)-2-oxo-1-(4-(thiophen-2-yl)thiazol-2-yl)-1,2-dihydropyridine-3-

carboxylic acid: Using conditions identical to those described above, 7.005D was obtained as a

solid (76%). 1H NMR (500 MHz, DMSO) 13.25 (br. s., 1H), 10.38 (s, 1H), 9.43 (d, J = 2.5 Hz,

1H), 8.48 (d, J = 2.5 Hz, 1H), 7.97 (s, 1H), 7.55 - 7.59 (m, 2H), 7.47 - 7.52 (m, 1H), 7.45 (dd, J =

7.6, 1.7 Hz, 1H), 7.12 (dd, J = 5.1, 3.7 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 6.98 - 7.02 (m, 1H) ppm.

150

13C NMR (126 MHz, DMSO) 191.5, 164.8, 157.9, 156.3, 154.8, 144.5, 142.9, 141.4, 137.7,

133.9, 130.5, 128.7, 127.0, 125.2, 124.7, 121.4, 119.9, 118.0, 117.1, 114.2 ppm. HRMS m/z: [M

+ H]+ calc’d for C20H12N2O5S2 425.0266; Found 425.0248.

Ethyl 5-(2-methoxybenzoyl)-2-oxo-1-(thiazol-2-yl)-1,2-dihydropyridine-3-carboxylate: To a

mixture of 7.004A (100.0 mg, 0.270 mmol), iodomethane (57.50 mg, 0.405 mmol), K2CO3 (56.00

mg, 0.405 mmol) and tetrabutylammonium iodide (9.970 mg, 0.027 mmol) in ACN (1.5 mL) was

added a few drops of DMSO and H2O and heated to 70°C overnight. The resulting mixture was

concentrated and purified using DCM/MeOH as the eluent to afford ethyl 5-(2-methoxybenzoyl)-

2-oxo-1-(thiazol-2-yl)-1,2-dihydropyridine-3-carboxylate, 7.006, (43.6 mg, 0.113 mmol, 42%

yield). 1H NMR (500 MHz, DMSO) 9.27 (d, J = 2.5 Hz, 1H), 8.46 (d, J = 2.9 Hz, 1H), 7.79 (d,

J = 3.4 Hz, 1H), 7.76 (d, J = 3.4 Hz, 1H), 7.59 - 7.65 (m, 1H), 7.44 (dd, J = 7.3, 2.0 Hz, 1H), 7.26

(d, J = 8.3 Hz, 1H), 7.11 - 7.16 (m, 1H), 4.30 (q, J = 6.9 Hz, 2H), 3.75 (s, 3H), 1.30 (t, J = 7.1 Hz,

3H) ppm. 13C NMR (101 MHz, DMSO) δ 190.6, 163.5, 157.1, 156.8, 155.0, 142.0, 141.8, 138.5,

133.5, 129.9, 126.7, 121.6, 121.5, 121.4, 117.6, 112.8, 61.6, 56.3, 14.5 ppm. LRMS (ESI) m/z

calc’d for C19H16N2O5S [M + H]+: 385.41, found 385.36.

151

5-(2-Methoxybenzoyl)-2-oxo-1-(thiazol-2-yl)-1,2-dihydropyridine-3-carboxylic acid: Using

conditions identical to those above, 7.007 was obtained as a solid (51%). 1H NMR (500 MHz,

DMSO) 13.24 (br. s., 1H), 9.29 (d, J = 2.5 Hz, 1H), 8.46 (d, J = 2.5 Hz, 1H), 7.80 (d, J = 3.4 Hz,

1H), 7.76 (d, J = 3.4 Hz, 1H), 7.61 (dt, J = 8.0, 1.7 Hz, 1H), 7.45 (dd, J = 7.3, 1.5 Hz, 1H), 7.26

(d, J = 8.3 Hz, 1H), 7.13 (t, J = 7.3 Hz, 1H), 3.74 (s, 3H) ppm. 13C NMR (101 MHz, DMSO) δ

190.7, 164.7, 158.9, 157.9, 157.2, 155.0, 142.2, 141.5, 138.6, 133.6, 129.9, 126.7, 121.8, 121.3,

117.9, 112.7, 56.1 ppm. HRMS m/z [M + H]+ calc’d for C17H12N2O5S: 357.0545; Found 357.0545.

6-bromo-4-oxo-4H-chromene-3-carbaldehyde: Using conditions identical to those described

above, 7.008 (a) i was obtained as a tan solid (69%). 1H NMR (500 MHz, DMSO) 10.08 (s, 1H),

8.94 - 8.95 (m, 1H), 8.19 (d, J = 2.5 Hz, 1H), 8.04 (dd, J = 8.8, 2.5 Hz, 1H), 7.76 (d, J = 8.8 Hz,

1H) ppm. 13C NMR (126 MHz, DMSO) 188.5, 174.2, 164.2, 155.0, 138.2, 127.8, 126.7, 122.0,

120.5, 119.7 ppm. LRMS (ESI) m/z calc’d for C10H5BrO3 [M + H]+: 254.05/256.05, found

254.10/256.10.

152

Diethyl 2-((6-bromo-4-oxo-4H-chromen-3-yl)methylene)malonate: Using conditions identical

to those described above, 7.008 (a) ii was obtained as a brown solid (82%). 1H NMR (500 MHz,

DMSO) 8.74 (s, 1H), 8.13 (d, J = 2.5 Hz, 1H), 8.02 (dd, J = 8.8, 2.5 Hz, 1H), 7.72 (d, J = 8.8

Hz, 1H), 7.49 (d, J = 1.0 Hz, 1H), 4.15 - 4.26 (m, 4H), 1.24 (t, J = 7.1 Hz, 3H), 1.11 - 1.19 (m,

3H) ppm. 13C NMR (126 MHz, DMSO) 173.2, 164.8, 164.2, 160.1, 154.7, 137.9, 133.8, 128.5,

127.9, 125.0, 121.8, 119.2, 118.6, 61.9, 61.4, 14.4, 14.2 ppm. LRMS (ESI) m/z calc’d for

C17H15BrO6 [M + H]+: 395.20/397.20, found 395.28/397.28.

Ethyl 5-(5-bromo-2-hydroxybenzoyl)-2-oxo-1-(thiazol-2-yl)-1,2-dihydropyridine-3-

carboxylate: Using conditions identical to those described above, 7.009 was obtained as a solid

(71%). 1H NMR (500 MHz, DMSO) 10.63 (s, 1H), 9.33 (d, J = 2.5 Hz, 1H), 8.47 (d, J = 2.5 Hz,

1H), 7.81 (d, J = 3.4 Hz, 1H), 7.77 (d, J = 3.4 Hz, 1H), 7.60 (dd, J = 8.6, 2.7 Hz, 1H), 7.54 (d, J =

2.5 Hz, 1H), 6.98 (d, J = 8.8 Hz, 1H), 4.30 (q, J = 6.9 Hz, 2H), 1.30 (t, J = 7.1 Hz, 3H) ppm. 13C

NMR (126 MHz, DMSO) 189.7, 163.5, 156.8, 155.1, 155.0, 142.2, 142.0, 138.6, 136.0, 132.4,

127.0, 121.7, 121.5, 119.4, 117.4, 110.9, 61.7, 14.5 ppm. LRMS (ESI) m/z calc’d for

C18H13BrN2O5S [M + H]+: 448.28/450.28, found 448.17/450.17.

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5-(5-Bromo-2-hydroxybenzoyl)-2-oxo-1-(thiazol-2-yl)-1,2-dihydropyridine-3-carboxylic

acid: Using conditions identical to those described above, 7.010 was obtained as a solid (64%).

1H NMR (500 MHz, DMSO) 13.26 (br. s., 1H), 10.64 (s, 1H), 9.35 (d, J = 2.9 Hz, 1H), 8.47 (d,

J = 2.9 Hz, 1H), 7.83 (d, J = 3.4 Hz, 1H), 7.78 (d, J = 3.4 Hz, 1H), 7.60 (dd, J = 8.6, 2.7 Hz, 1H),

7.54 (d, J = 2.9 Hz, 1H), 6.98 (d, J = 8.8 Hz, 1H) ppm. 13C NMR (126 MHz, DMSO) 189.8,

164.8, 157.8, 155.1, 155.0, 142.5, 141.7, 138.6, 136.0, 132.4, 127.8, 121.7, 121.1, 119.4, 117.7,

110.9 ppm. HRMS m/z calculated for C16H9BrN2O5S [M + H]+ 420.9494; Found 422.9459.

N-benzyl-5-(5-bromo-2-hydroxybenzoyl)-2-oxo-1-(thiazol-2-yl)-1,2-dihydropyridine-3-

carboxamide: A mixture of ethyl 5-(5-bromo-2-hydroxybenzoyl)-2-oxo-1-(thiazol-2-yl)-1,2-

dihydropyridine-3-carboxylate (200.0 mg, 0.445 mmol), phenylmethanamine (57.20 mg, 0.534

mmol) and potassium 2-methylpropan-2-olate (100 mg, 0.89 mmol) in toluene (1.0 mL) in a

microwave vial was heated to 65°C for 1 hour. After completion of the reaction, the crude was

purified in hexane/ethyl acetate to afford N-benzyl-5-(5-bromo-2-hydroxybenzoyl)-2-oxo-1-

(thiazol-2-yl)-1,2-dihydropyridine-3-carboxamide (202 mg, 89%). 1H NMR (500 MHz, DMSO)

12.77 (s, 1H), 10.59 (s, 1H), 8.97 (d, J = 2.5 Hz, 1H), 8.68 (d, J = 2.5 Hz, 1H), 7.59 (dd, J = 8.8,

154

2.5 Hz, 1H), 7.53 (d, J = 3.4 Hz, 1H), 7.50 (d, J = 2.5 Hz, 1H), 7.35 - 7.41 (m, 4H), 7.30 - 7.34 (m,

2H), 6.97 (d, J = 8.8 Hz, 1H), 5.41 (s, 2H) ppm. 13C NMR (126 MHz, DMSO) 183.8, 172.4,

162.0, 160.8, 157.3, 155.3, 149.5, 144.0, 138.1, 136.0, 135.8, 132.5, 129.2, 128.5, 127.3, 119.5,

118.3, 117.3, 115.2, 110.9, 53.8 ppm. HRMS m/z [M + H]+ calc’d for C23H16BrN3O4S 510.0123;

found 512.0114.

1-(4-Hydroxy-[1,1'-biphenyl]-3-yl)ethan-1-one: To a stirred solution of 1-(5-bromo-2-

hydroxyphenyl)ethanone (1.00 g, 4.65 mmol), phenylboronic acid (1.14 g, 9.30 mmol), K2CO3

(1.29 g, 9.30 mmol) in 2-propanol and H2O (14 mL: 3.5 mL) under argon atmosphere was added

Pd(PPh3)2Cl2 (0.07 g, 0.10 mmol). The contents were stirred at room temperature for 20 minutes

and slowly heated to reflux for 12 hours. The solvents were removed under reduced pressure, the

reaction mixture was extracted with ethyl acetate (2X) and washed with H2O, dried over sodium

sulfate and concentrated under pressure to obtain the crude product which was used directly in the

next step without further purification.

4-oxo-6-phenyl-4H-chromene-3-carbaldehyde: Using conditions identical to those described

above, 7.020 (b) i, was obtained as a solid (58%). 1H NMR (500 MHz, DMSO) 10.12 - 10.15

(m, 1H), 8.94 - 8.96 (m, 1H), 8.30 (d, J = 2.0 Hz, 1H), 8.16 - 8.20 (m, 1H), 7.83 - 7.87 (m, 1H),

7.76 (d, J = 7.3 Hz, 2H), 7.52 (t, J = 7.6 Hz, 2H), 7.40 - 7.46 (m, 1H) ppm. 13C NMR (101 MHz,

155

DMSO) δ 188.9, 175.3, 163.9, 155.5, 139.0, 138.5, 134.0, 129.7, 128.7, 127.4, 125.4, 122.9, 120.4,

120.2 ppm. LRMS (ESI) m/z calc’d for C16H10O3 [M + H]+: 251.25, found 251.20.

Dimethyl 2-((4-oxo-6-phenyl-4H-chromen-3-yl)methylene)malonate: Using conditions

identical to those above, 7.020 (b) ii was obtained as a solid and used in the next step without

further purification (63%). LRMS (ESI) m/z calc’d for C21H16O6 [M + H]+: 365.35, found 365.25.

Methyl 5-(4-hydroxy-[1,1'-biphenyl]-3-carbonyl)-2-oxo-1-(thiazol-2-yl)-1,2-

dihydropyridine-3-carboxylate:

Using conditions identical to those described above, 7.020 (b) iii was obtained as a solid (70%).

1H NMR (500 MHz, DMSO) 10.50 (s, 1H), 9.43 (d, J = 2.5 Hz, 1H), 8.54 (d, J = 2.5 Hz, 1H),

7.76 - 7.81 (m, 3H), 7.70 (d, J = 2.5 Hz, 1H), 7.62 - 7.65 (m, 2H), 7.39 - 7.44 (m, 2H), 7.28 - 7.33

(m, 1H), 7.11 (d, J = 8.8 Hz, 1H), 3.84 (s, 3H) ppm. 13C NMR (101 MHz, DMSO) δ 191.1, 164.1,

156.8, 155.8, 155.1, 142.9, 142.0, 139.5, 138.6, 132.0, 131.9, 129.3, 128.5, 127.4, 126.6, 125.1,

121.7, 121.0, 117.8, 52.9 ppm. LRMS (ESI) m/z calc’d for C23H16N2O5S [M + H]+: 433.45, found

433.32.

156

5-(4-hydroxy-[1,1'-biphenyl]-3-carbonyl)-2-oxo-1-(thiazol-2-yl)-1,2-dihydropyridine-3-

carboxylic acid: Using conditions identical to those described above, 7.021 was obtained as a

solid (89%). 1H NMR (500 MHz, DMSO) 13.26 (br. s., 1H), 10.51 (s, 1H), 9.43 (d, J = 2.5 Hz,

1H), 8.54 (d, J = 2.5 Hz, 1H), 7.76 - 7.82 (m, 3H), 7.70 (d, J = 2.5 Hz, 1H), 7.62 - 7.65 (m, 2H),

7.40 - 7.44 (m, 2H), 7.28 - 7.33 (m, 1H), 7.11 (d, J = 8.3 Hz, 1H) ppm. 13C NMR (101 MHz,

DMSO) δ 191.2, 164.8, 157.9, 155.8, 155.1,142.8, 141.6, 193.5, 138.6, 132.0, 131.9, 129.4, 128.5,

127.4, 126.6, 125.2, 121.8, 121.5, 118.1, 117.8 ppm. HRMS m/z [M + H]+ calc’d for C22H14N2O5S

419.0702; Found 419.0690.

Methyl 2-hydroxynicotinate: To a suspension of 2-hydroxynicotinic acid (10.0 g, 71.9 mmol) in

anhydrous DCM (150 mL) was added thionyl chloride (26.0 mL, 356 mmol) followed by

anhydrous THF (150 mL) . The reaction mixture was stirred at room temperature for 1 hr., and

then excess MeOH (300 mL) was added to the suspension until a homogenous solution was

obtained. The reaction mixture was filtered and the filtrate was concentrated under reduced

pressure to give a pale yellow oil which solidified to an off white solid ( 11 g, 100%) under vacuum.

A portion of the crude material was used directly in subsequent step without further purification.

LRMS (ESI) m/z calculated for C7H7NO3 [M + H]+: 154.14, found 154.10.

157

Methyl 5-iodo-2-oxo-1-phenyl-1,2-dihydropyridine-3-carboxylate.

Step 1: To a mixture of methyl 2-hydroxynicotinate (6.00 g, 39.2 mmol) , phenylboronic acid (14.3

g, 118 mmol), copper (II) acetate (14.23 g, 78.00 mmol) and pyridine (12.68 mL, 157.0 mmol) in

DCM was added 5 g of 4 Å molecular sieves. The reaction mixture was stirred at room temperature

overnight and open to the atmosphere. The solvent was removed in vacuo and extracted 3 times

with aqueous ammonium chloride solution and DCM and the organic phase was purified using

column chromatography (hexane:ethyl acetate=1:2) to obtain methyl 2-oxo-1-phenyl-1,2-

dihydropyridine-3-carboxylate (5 g, 21.81 mmol, 56% yield), which was used in the next step.

Step 2: To a solution of methyl 2-oxo-1-phenyl-1,2-dihydropyridine-3-carboxylate (3.000 g, 13.09

mmol) in DCM (19.45 mL) and TFA (16.21 mL) was added N-iodosuccinimide (2.940 g, 13.09

mmol) under an argon atmosphere. After stirring for 19 h at ambient temperature the solvent was

concentrated in vacuo. To the residue were added ethyl acetate (200 mL) and saturated aq. sodium

hydrogen carbonate (150 mL) and the organic phase was collected, washed with H2O and brine,

dried over sodium sulfate, and concentrated in vacuo. The residue was the subtitled compound,

methyl 5-iodo-2-oxo-1-phenyl-1,2-dihydropyridine-3-carboxylate (4.12 g, 89% yield) as a pale

yellow solid. 1H NMR (500 MHz, DMSO) 8.20 (d, J = 2.9 Hz, 1H), 8.16 (d, J = 2.9 Hz, 1H),

7.43 - 7.53 (m, 3H), 7.38 - 7.43 (m, 2H), 3.73 (s, 3H) ppm. 13C NMR (126 MHz, DMSO) 164.3,

157.3, 151.4, 148.5, 140.1, 129.5, 129.2, 127.4, 123.0, 63.9, 52.5 ppm. LRMS (ESI) m/z calc’d

for C13H10INO3 [M + H]+: 356.13, found 356.0.

158

A flame dried roud bottomed flask was charged with cesium carbonate (1.84 g, 5.63 mmol),

BISPIN (2.860 g, 11.26 mmol), palladium(II) acetate (0.063 g, 0.282 mmol) and methyl 5-iodo-2-

oxo-1-phenyl-1,2-dihydropyridine-3-carboxylate (1.00 g, 2.82 mmol) and MeOH (20 mL) under

argon atmosphere. The reaction mixture was heated to reflux for 42 h. After completion of the

reaction, the mixture was passed over a short column of neutral Al2O3 with diethyl ether. The

solvent was evaporated under reduced pressure to obtain the crude material, methyl 2-oxo-1-

phenyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2-dihydropyridine-3-carboxylate

(0.550 g, 55%). LRMS (ESI) m/z calc’d for C19H22BNO5 [M + H]+: 356.20, found 356.01.

2-oxo-1,5-diphenyl-1,2-dihydropyridine-3-carboxylic acid: Methyl 5-iodo-2-oxo-1-phenyl-

1,2-dihydropyridine-3-carboxylate (300.0 mg, 0.845 mmol), potassium carbonate (292.0 mg,

2.112 mmol), phenylboronic acid (155.0 mg, 1.267 mmol) and

bis(triphenylphosphine)palladium(II) chloride (29.60 mg, 0.042 mmol) in a round bottom flask

was added dioxane (6.80 mL) and H2O (1.70 mL). The resulting mixture was stirred under argon

159

at 80 °C for 24 hours and allowed to cool to rt. The cooled reaction mixture was filtered through

alumina and concentrated on a rotary evaporator to obtain methyl 2-oxo-1,5-diphenyl-1,2-

dihydropyridine-3-carboxylate (187 mg, 73%).

Step 2: To a solution of methyl 2-oxo-1,5-diphenyl-1,2-dihydropyridine-3-carboxylate (300.0 mg,

0.983 mmol) in dioxane (2 mL) and H2O (1 mL) was added 50% w/w NaOH (104 µL, 1.965

mmol) and the mixture was heated to 50 oC for 3 hr. The resulting solution was concentrated on a

rotary evaporator and purified using column chromatography (hexanes and EtOAc, 4:1, as eluent)

to obtain compound 7.031 as a brown solid, 97%. 1H NMR (400 MHz, DMSO) 14.28 (s, 1H),

8.70 - 8.73 (s, 1H), 8.49 - 8.53 (s, 1H), 7.35 - 7.76 (m, 10H) ppm. 13C NMR (126 MHz, DMSO)

165.1, 163.2, 144.6, 142.5, 139.7, 134.7, 129.8, 129.7, 129.5, 128.4, 127.4, 126.6, 121.2, 117.9

ppm. HRMS m/z: [M + H]+ calc’d for C18H13NO3 [M + H]+ 292.0974; Found 292.0972.

A mixture of 2-aminobenzoic acid (10.0 g, 72.9 mmol) and formamide (21.3 mL, 535 mmol) in a

microwave vessel was heated in a microwave reactor to 150 °C for 1 hour. The slurry was cooled

to rt and stirred in NaHCO3 and the resulting solid was filtered and washed with water followed

by with diethyl ether and dried on a lyophilizer to provide quinazolin-4(3H)-one, 7.032 (7.51 g,

71%).1H NMR (500 MHz, DMSO) δ 12.22 (br. s., 1H), 8.11 (dd, J = 8.1, 1.2 Hz, 1H), 8.08 (s, 1H),

7.77 - 7.82 (m, 1H), 7.65 (d, J = 8.3 Hz, 1H), 7.48 - 7.53 (m, 1H) ppm. 13C NMR (126 MHz,

DMSO) δ 161.2, 149.2, 145.9, 134.7, 127.6, 127.2, 126.3, 123.1 ppm. LRMS (ESI) m/z calc’d for

C8H6N2O [M + H]+: 147.15, found 147.1.

160

To a solution of quinazolin-4(3H)-one (2.000 g, 13.68 mmol) in thionyl chloride (52.3 mL, 717

mmol) was added DMF (0.8 mL) and the mixture was heated to reflux overnight. The reaction

mixture was cooled to rt and evaporated to dryness to provide crude compound which was

purification by column chromatography to provide 4-chloroquinazoline, 7.033 (0.650 g 29%). 1H

NMR (500 MHz, DMSO) δ 8.65 (s, 1H), 8.14 (dd J = 8.1, 1.2 Hz, 1H), 7.87 - 7.91 (m, 1H), 7.77

(d, J = 7.8 Hz, 1H), 7.58 - 7.62 (m, 1H) ppm. 13C NMR (126 MHz, DMSO) δ 160.3, 147.6, 144.3,

135.6, 128.2, 126.8, 124.4, 122.3 ppm. LRMS (ESI) m/z calc’d for C8H5ClN2 [M + H]+: 164.59,

found 164.48.

161

7.10 References

(1) Poomathi, N.; Perumal, P.; Ramakrishna, S. An efficient and eco-friendly synthesis of 2-

pyridones and functionalized azaxanthone frameworks via indium triflate catalyzed domino

reaction. Green Chemistry 2017, 19, 2524-2529.

(2) Sheng, R.; Tang, L.; Jiang, L.; Hong, L.; Shi, Y.; Zhou, N.; Hu, Y. Novel 1-Phenyl-3-

hydroxy-4-pyridinone derivatives as multifunctional agents for the therapy of alzheimer’s

disease. ACS Chemical Neuroscience 2015, 7, 69-81.

(3) Huang, X.; Aslanian, R.; Zhou, W.; Zhu, X.; Qin, J.; Greenlee, W.; Zhu, Z.; Zhang, L.; Hyde,

L.; Chu, I. et al. The discovery of pyridone and pyridazone heterocycles as γ-secretase

modulators. ACS Medicinal Chemistry Letters 2010, 1, 184-187.

(4) Almansour, A.; Suresh Kumar, R.; Arumugam, N.; Basiri, A.; Kia, Y.; Ashraf Ali, M. An

expedient synthesis, acetylcholinesterase inhibitory activity, and molecular modeling study of

highly functionalized hexahydro-1,6-naphthyridines. BioMed Research International 2015,

2015, Article ID 965987, 9 pages.

(5) Aniszewski, T. Alkaloids - secrets of life; Elsevier: Amsterdam, 2007.

(6) Walker, N.; Howe, C.; Glover, M.; McRobbie, H.; Barnes, J.; Nosa, V.; Parag, V.; Bassett,

B.; Bullen, C. Cytisine versus nicotine for smoking cessation. New England Journal of Medicine

2014, 371, 2353-2362.

(7) Liu, J.; Zhu, Y.; Yu, C.; Zhou, Y.; Han, Y.; Wu, F.; Qi, B. The structures of huperzine A and

B, two new alkaloids exhibiting marked anticholinesterase activity. Canadian Journal of

Chemistry 1986, 64, 837-839.

(8) Huperzine A - List Results - ClinicalTrials.gov

https://clinicaltrials.gov/ct2/results?cond=&term=huperzine+A&cntry1=&state1=&recrs=#table

Top (accessed Jan 31, 2018).

162

(9) Liu, Z.; Zhang, A.; Sun, H.; Han, Y.; Kong, L.; Wang, X. Two decades of new drug

discovery and development for Alzheimer's disease. RSC Advances 2017, 7, 6046-6058.

(10) Cheng, Y.; Schneider, B.; Riese, U.; Schubert, B.; Li, Z.; Hamburger, M. (+)-N-

Deoxymilitarinone A, a neuritogenic pyridone alkaloid from the insect pathogenic fungus

paecilomycesfarinosus. Journal of Natural Products 2006, 69, 436-438.

(11) Schmidt, K.; Günther, W.; Stoyanova, S.; Schubert, B.; Li, Z.; Hamburger, M. Militarinone

A, a neurotrophic pyridone alkaloid from paecilomycesmilitaris1. Organic Letters 2002, 4, 197-

199.

(12) Ziemianin, A.; Ronco, C.; Dolé, R.; Jean, L.; Renard, P.; Lange, C. Screening of new

huprines—Inhibitors of acetylcholinesterases by electrospray ionization ion trap mass

spectrometry. Journal of Pharmaceutical and Biomedical Analysis 2012, 70, 1-5.

(13) Howes, M. Alkaloids and drug discovery for neurodegenerative diseases. Natural Products

2013, 1331-1365.

(14) Defaux, J.; Sala, M.; Formosa, X.; Galdeano, C.; Taylor, M.; Alobaid, W.; Kelly, J.; Wright,

C.; Camps, P.; Muñoz-Torrero, D. Huprines as a new family of dual acting trypanocidal–

antiplasmodial agents. Bioorganic & Medicinal Chemistry 2011, 19, 1702-1707.

(15) Chun, Y.; Ryu, K.; Ko, Y.; Hong, J.; Hong, J.; Shin, H.; Lee, S. One-pot synthesis of 2-

pyridones via chemo- and regioselective tandem blaise reaction of nitriles with propiolates.

Journal of Organic Chemistry 2009, 74, 7556-7558.

(16) Imase, H.; Noguchi, K.; Hirano, M.; Tanaka, K. Convergent and rapid assembly of

substituted 2-pyridones through formation of N-alkenyl alkynylamides followed by gold-

catalyzed cycloisomerization. Organic Letters 2008, 10, 3563-3566.

(17) Patel, B.; Mason, A.; Barrett, A. Synthesis of 6-substituted-4-hydroxy-2-pyridinones via

intramolecular ketene trapping of functionalized enamine-dioxinones. Organic Letters 2011, 13,

5156-5159.

163

(18) Pintiala, C.; Lawson, A.; Comesse, S.; Daïch, A. A versatile domino process for the

synthesis of substituted 3-aminomethylene-chromanones and 2-pyridones catalyzed by CsF.

Tetrahedron Letters 2013, 54, 2853-2857.

(19) Nohara, A.; Umetani, T.; Sanno, Y. A facile synthesis of chromone-3-carboxaldehyde,

chromone-3-carboxylic acid and 3-hydroxymethylchromone. Tetrahedron Letters 1973, 14,

1995-1998.

(20) Tu, Q.; Li, D.; Sun, Y.; Han, X.; Yi, F.; Sha, Y.; Ren, Y.; Ding, M.; Feng, L.; Wan, J.

Design and syntheses of novel N′-((4-oxo-4H-chromen-3-yl)methylene)benzohydrazide as

inhibitors of cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase. Bioorganic &

Medicinal Chemistry 2013, 21, 2826-2831.

(21) Awadallah, F.; El-Waei, T.; Hanna, M.; Abbas, S.; Ceruso, M.; Oz, B.; Guler, O.; Supuran,

C. Synthesis, carbonic anhydrase inhibition and cytotoxic activity of novel chromone-based

sulfonamide derivatives. European Journal of Medicinal Chemistry 2015, 96, 425-435.

(22) Kumar, V.; Chatterjee, A.; Banerjee, M. A mild and efficient route to 3-vinylchromones in

aqueous micellar media. Synthetic Communications 2015, 45, 2364-2377.

(23) Knoevenagel, E. Condensation von malonsäure mit aromatischen aldehyden durch

ammoniak und amine. Berichte der deutschen chemischen Gesellschaft 1898, 31, 2596-2619.

(24) Lepitre, T.; Denhez, C.; Sanselme, M.; Othman, M.; Lawson, A.; Daïch, A. Original

Mitsunobu-triggered sequence involved in a one-pot domino process toward tetracyclic systems

bearing a bis-N,O-acetal junction. The Journal of Organic Chemistry 2016, 81, 8837-8849.

(25) Khurana, J.; Chauhan, S.; Bansal, G. Facile hydrolysis of esters with koh-methanol at

ambient temperature. Monatshefte fur Chemie / Chemical Monthly 2004, 135, 83-87.

(26) Lian, X.; Fu, S.; Ma, T.; Li, S.; Zeng, W. An efficient FeCl3-promoted O-alkyl cleavage of

esters to carboxylic acids. Applied Organometallic Chemistry 2011, 25, 443-447.

164

(27) Jung, M.; Lyster, M. Quantitative dealkylation of alkyl esters via treatment with

trimethylsilyl iodide. A new method for ester hydrolysis. Journal of the American Chemical

Society 1977, 99, 968-969.

(28) Olah, G.; Narang, S.; Gupta, B.; Malhotra, R. Synthetic methods and reactions. 62.

Transformations with chlorotrimethylsilane/sodium iodide, a convenient in situ

iodotrimethylsilane reagent. The Journal of Organic Chemistry 1979, 44, 1247-1251.

(29) Stoochnoff, B.; Benoiton, N. The methylation of some phenols and alcohols with sodium

hydride / methyl iodide in tetrahydrofuran at room temperature. Tetrahedron Letters 1973, 14,

21-24.

(30) Schenone, M.; Dancík, V.; Wagner, B.; Clemons, P. Target identification and mechanism of

action in chemical biology and drug discovery. Nature Chemical Biology 2013, 9, 232-240.

(31) Nasir, M.; Jolley, M. Fluorescence polarization: An analytical tool for immunoassay and

drug discovery. Combinatorial Chemistry and High Throughput Screening 1999, 2, 177-190.

(32) Xie, Z.; Jia, Y.; Li, H. Studying protein–protein interactions by biotin AP-tagged pulldown

and LTQ-orbitrap mass spectrometry. Methods in Molecular Biology 2017, 129-138.

(33) Wysocka, J. Identifying novel proteins recognizing histone modifications using peptide

pull-down assay. Methods 2006, 40, 339-343.

(34) Kolb, H.; Sharpless, K. The growing impact of click chemistry on drug discovery. Drug

Discovery Today 2003, 8, 1128-1137.

(35) Suzuki, A. Synthetic studies via the cross-coupling reaction of organoboron derivatives with

organic halides. Pure and Applied Chemistry 1991, 63.

(36) Malacea, R.; Chahdoura, F.; Devillard, M.; Saffon, N.; Gómez, M.; Bourissou, D. ortho-

(Dimesitylboryl)phenylphosphines: positive boryl effect in the palladium-catalyzed Suzuki-

Miyaura coupling of 2-chloropyridines. Advanced Synthesis & Catalysis 2013, 355, 2274-2284.

165

(37) Yang, B.; Buchwald, S. Palladium-catalyzed amination of aryl halides and sulfonates.

Journal of Organometallic Chemistry 1999, 576, 125-146.

(38) Biediger, R.; Bui, H.; Henry, K.; Thrash, T. Modulators of C3A receptor and methods of use

thereof. WO/2008/079371; PCT/US2007/026237, 2008.

(39) Rice, K.; Aay, N.; Arcalas, A.; Baik, T.; Blazey, C.; Bowles, O.; Buhr, C.; Bussenius, J.;

Costanzo, S.; Curtis, J. Tropane compounds. US 8,012,956 B2, 2011, 2011.

(40) Verner, E.; Katz, B.; Spencer, J.; Allen, D.; Hataye, J.; Hruzewicz, W.; Hui, H.; Kolesnikov,

A.; Li, Y.; Luong, C. et al. Development of serine protease inhibitors displaying a multicentered

short (<2.3 Å) hydrogen bond binding mode: inhibitors of urokinase-type plasminogen activator

and factor Xa. Journal of Medicinal Chemistry 2001, 44, 2753-2771.

(41) Panteleon, V.; Marakos, P.; Pouli, N.; Mikros, E.; Andreadou, I. Synthesis, conformational

analysis and free radical scavenging activity of some new spiropyranoquinolinones. Chemical &

Pharmaceutical Bulletin 2003, 51, 522-529.

(42) Isaad, J.; Achari, A. Biosourced 3-formyl chromenyl-azo dye as Michael acceptor type of

chemodosimeter for cyanide in aqueous environment. Tetrahedron 2011, 67, 5678-5685.

(43) Hammond, M.; Gurnani, P. Reduction of nitroarene to aniline using tin chloride and acetic

acid. ChemSpider Synthetic Pages 2013.

(44) D Patil, R. Chemoselective reduction of nitroarenes to aromatic amines with commercial

metallic iron powder in water under mild reaction conditions. Organic Chemistry: Current

Research 2015, 04.

(45) Shim, Y.; Kim, K.; Chi, D.; Lee, K.; Cho, H. Formylchromone derivatives as a novel class

of protein tyrosine phosphatase 1B inhibitors. Bioorganic & Medicinal Chemistry Letters 2003,

13, 2561-2563.

166

(46) Kim, T.; Bauer, D.; Bellon, S.; Boezio, A.; Booker, S.; Choquette, D.; D'amico, D.;

D'angelo, N.; Dominguez, C.; Fellows, I. Substituted amide derivatives as protein kinase

inhibitors. WO2006116713 A1; PCT/US2006/016344, 2006.

(47) Hansen, P.; Lawitz, K.; LÖNN, H.; Nikitidis, A. 2-pyridine derivatives as inhibitors of

neutrophile elastase. WO2006098683 A1; PCT/SE2006/000327, 2006.

(48) Zhang, J.; Wu, H.; Zhang, J. Cesium carbonate mediated borylation of aryl iodides with

diboron in methanol. European Journal of Organic Chemistry 2013, 2013, 6263-6266.

(49) Kulkarni, S.; Singh, S.; Shah, J.; Low, W.; Talele, T. Synthesis and SAR optimization of

quinazolin-4(3H)-ones as poly(ADP-ribose)polymerase-1 inhibitors. European Journal of

Medicinal Chemistry 2012, 50, 264-273.

(50) Zhang, Y.; Cockerill, S.; Guntrip, S.; Rusnak, D.; Smith, K.; Vanderwall, D.; Wood, E.;

Lackey, K. Synthesis and SAR of potent EGFR/erbB2 dual inhibitors. Bioorganic & Medicinal

Chemistry Letters 2004, 14, 111-114.

(51) Kijrungphaiboon, W.; Chantarasriwong, O.; Chavasiri, W. Cl3CCN/PPh3 and CBr4/PPh3:

two efficient reagent systems for the preparation of N-heteroaromatic halides. Tetrahedron

Letters 2012, 53, 674-677.

(52) Kieffer, C.; Verhaeghe, P.; Primas, N.; Castera-Ducros, C.; Gellis, A.; Rosas, R.; Rault, S.;

Rathelot, P.; Vanelle, P. Sonogashira cross-coupling reaction in 4-chloro-2-

trichloromethylquinazoline series is possible despite a side dimerization reaction. Tetrahedron

2013, 69, 2987-2995.

(53) Serrano, C.; Looper, R. Synthesis of cytimidine through a one-pot copper-mediated

amidation cascade. Organic Letters 2011, 13, 5000-5003.

(54) Coats, S.; Bian, H.; Connolly, P.; Bignan, G.; Cai, C.; Dax, S.; De, C.; Lin, S.; Liu, L.;

Macielag, M. Pyrimidine compounds as delta opioid receptor modulators 2011, WO2011053705

A1; PCT/US2010/054489.

167

(55) Bernotas, R.; Ullrich, J.; Travins, J.; Wrobel, J.; Unwalla, R. Quinazoline compounds 2009,

WO2009020683 A2; PCT/US2008/063685.

168

Chapter 8

Thiazole Analogs

8.1 Thiazoles

Thiazoles are aromatic heterocyclic organic compounds with a five membered molecular

ring structure (C3H3NS), having nitrogen and sulfur heteroatoms as part of the ring, Figure 8.1.

The aromaticity of the thiazole ring is based on the delocalization of a lone pair electrons of the

sulfur heteroatom which completes the 6π electrons that satisfy the Huckel’s rule.1 Thiazole was

discovered in 1887 by Hantzsch and Wader and its structure was confirmed by Popp two (2) years

later.2

Figure 8.1: Structure of thiazole

8.1.1 Thiazole based Therapeutics

The thiazole ring is present in many different natural products and medicinally important

compounds, Figure 8.2, including vitamin B1 (thiamine),3 the β-lactam antibiotic, penicillin G;4

the potent anti-fungal, ruvaconazole;5 the anti-retroviral drug, ritonavir;6 the respiratory stimulant

and antidote for barbiturate/opiate overdose, amiphenazole;7 the anti-microbial agent,

sulfathiazole;8 broad spectrum anti-fungal agent, abafungin;9 the fungicide, angiogenesis inhibitor,

chelating agent for lead, mercury and antimony poisoning, thiabendazole;10 the nonsteroidal anti-

inflammatory drug (NSAID) for joint and muscular pain, fentiazac11 and the schistosomicide and

169

periodontitis, niridazole.12,13 Among others are the monobactam antibiotic, aztreonam;14,15

xanthine oxidase inhibitor, feboxostat;16 the dopamine D3 receptor agonist used to treat

Parkinson’s disease, pramipexazole;17 treatment for typeII diabetes mellitus, teneligliptin18 and the

antipsychotic agent used for the treatment of schizophrenia, lurasidone.19 All of the above

mentioned drugs are currently in use in the clinic.

Figure 8.2: Some thiazole based drugs in clinical use.

170

There are a number of thiazole derivatives currently under clinical investigation and they include

anticancer agents20 CNS modulators,21 anticonvulsants,22 acetylcholine receptor ligands23 opioid

receptor antagonists,24 anti-tubercular agents,25 anti-diabetic agents,26 anti-obesity agents,27 anti-

inflammatory agents,28 anti-viral agents,29 antimicrobial agents,30 and anti-Alzheimer’s agents.

Development of thiazole-based anti-Alzheimer’s agents, Figure 8.3, include compounds that

inhibit the dual specific tyrosine phosphorylation regulated kinase 1A phosphorylation on Thr 212,

8.001,31 AD related glycogen synthase kinase 3β inhibitor, 8.002,32 A2AR antagonists, 8.003,33

strong inhibitor of plaque deposition, 8.004,34 potent A3R antagonist, 8.005,35 (112) and dopamine

d4 receptor antagonists, 8.006.36

Figure 8.3: Some thiazole based compounds as anti-AD agents in development

Thiazoles have established themselves as an important scaffold in medicinal chemistry through

their profound therapeutic effects. Owing to their therapeutic profile in organic and biological

171

chemistry, various synthetic methods have been developed for the construction of the thiazole ring.

These include the Hantzch, Tchernic, Gabriel and Cook-Heilborn thiazole synthesis.37 The

Hantzsch synthesis, which involves the reaction between thioamides and haloketones, was used

for the synthesis of all the thiazole analogs.

8.2 Synthesis of thiazole analogs

Thiazoles can be synthesised via the Hantszch thiazole synthesis37,38 involving the reaction

between an α-haloketone species and a thiourea derivative. The retrosynthetic scheme, Figure 8.4,

for the Hantszch thiazole synthesis is shown below.

Figure 8.4 Retrosynthetic scheme of thiazole synthesis

The required α-haloketone derivative (α-bromothiophene), 8.008, Scheme 8.1, was initially

prepared by treating commercially available 2-acetylthiophene (2-AT) with two equivalents of

copper (II) bromide in DCM,39 but the best results came from treating 2-AT with an equimolar

quantity of bromine in MeOH and H2O at 0 oC to rt40 or by using DCM as the solvent at rt41 or

with AcOH as the solvent38 to provide a mixture of the desired bromide, 8.008, and the

corresponding dibromide, which was easily separable via column chromatography.

172

Scheme 8.1: Synthesis of thiazole analogs

Reagents and conditions: (a) Br2, MeOH, H2O, 72%; (b) HCl/Ether, rt, 82%; (c) NH3SCN, NaHSO3, 52%; (d)

EtOH, 79 - 87%; (e) Aqueous NaOH, EtOH, 69 - 86%.

The thiourea derivative, 8.011, for the Hantzch thiazole synthesis was prepared from the

corresponding HCl-salt of ethyl aminobenzoate (EAB),42 8.009, in H2O. Alternatively, the

thiourea, 8.011, can be synthesized in aqueous HCl (2N) using the free amine form, 8.009. Thus,

the addition of 2.1 equivalents of ammonium thiocyanate to the amine solution of ethyl

aminobenzoate, 8.010, in H2O in the presence of catalytic amounts of sodium bisulfite42 readily

afforded the thiourea, 8.011, which was isolated as a solid. The ester thiazoles, 8.012 (A-E), were

173

consistently obtained in yields greater than 78% when the respective α-bromothiophenes were

coupled together with the thiourea, 8.011, in the Hanszch thiazole synthesis.40 The products thus

obtained were able to undergo saponification with aqueous NaOH in DMSO to obtain compounds

8.013 (A – E). A methyl substituent was introduced in compound 8.013 B for a twisting effect and

to observe how this might affect the overall activity. Compound 8.014 was synthesized in the same

manner starting with 3-acetylthiophene.

8.2.1 Phenyl analogs of thiazoles

In the pursuit of active SSH1 inhibitor that could potentially be used as a treatment for AD,

a systematic exploration of the thiazole series was carried out by first substituting the thiophene

for benzene (phenyl) or methoxy-substituted phenyl derivatives, and employing similar synthetic

routes as outlined previously. Benzene (phenyl group) is a known thiophene isostere43 and could

offer superior binding orientation. Thus, compounds 8.015 – 8.019 were prepared (Figure 6.2),

and subsequently tested in the biochemical assay. The para-methoxy analog, 8.019, was initially

obtained from commercial sources, which showed good SSH1 inhibitory activity in the assay and

was synthesized to confirm its activity. The meta- and ortho-methoxy substituted analogs (8.017,

8.018) were also synthesized in an attempt to improve upon the observed activity.

174

Figure 8.5: Phenyl and substituted phenyl analogs of thiazole

8.2.2 Pyridine analogs of thiazoles

Three pyridine analogs were prepared according to the synthetic procedures described

previously. However, α-bromination of the pyridinylethanones were carried out using HBr and Br2

in acetic acid,44,45 which gave the corresponding α-bromoketones with yields consistently greater

than 84%. Since pyridine derivatives are known to exhibit excellent activities against a wide range

of biological targets,46 it was envisioned that pyridine analog could improve upon the activity and

exhibit improved pharmacokinetics. The synthesized pyridine analogs are provided in Figure 8.6.

175

Figure 8.6: Pyridine analogs of thiazoles

8.2.3 tert-butyl analog of thiazoles

A tert-butyl analog, 8.023, was also prepared to investigate the effect removal of

aromaticity would have on the SSH1 inhibitory activity, Figure 8.7.

Figure 8.7: tert-butyl analog

8.2.4 Phosphonic Acid analog

A recently obtained crystal structure of the active site of SSH1 indicated the presence of a

number of inorganic phosphate groups. Thus, it was envisioned that compounds that could

compete with the inorganic phosphate groups might offer superior binding and may lead to the

development of potent compounds as SSH1 inhibitors. To this end a phosphonate version of the

thiazole analog was prepared to evaluate its activty. The synthetic scheme for the phophonic acid

analog is provided below, Scheme 8.2.

176

Scheme 8.2: Synthesis of phosphonic acid analog 8.029

Reagents and conditions: (a) HCl/Ether, 94%; (b) NH3SCN, NaHSO3, 89%; (c) 8.008A, EtOH, 76%; (d) Diethyl

phosphonate, TEA, Pd(PPh3)4, 62%; (e) Conc. HCl, 32%.

The bromo-thiazole derivative, 8.027, was synthesized according to the previously described

procedure, and was able to undergo palladium-catalyzed cross coupling reaction with diethyl

phosphonate47 to give the aryl phosphonate product, 8.028, in 62% yield. This product was then

hydrolyzed in concentrated HCl48 to give the corresponding acid, 8.029.

177

8.3 Summary of synthesized thiazole compounds

Figure 8.8: Summary of thiazole test compounds

178

Figure 8.9: Pyridone analogs

179

8.4 Results and discussion

The SSH1 inhibitory activity of the synthesized compounds was evaluated in the pNPP

phosphatase assay by Dr. Zao Xingyu in the laboratory of Prof. David Kang, USF Byrd’s

Alzheimer’s Institute. Graphs 1, shows the SAR results observed with analogs 7.005 (A-D) and

8.013A.

Graph 1: SSH1 inhibitory activity of analogs 8.013A, 7.005A-D

The activity of the thiazole lead compound, 8.013A, was confirmed, but the initial pyridone

hit/lead compound, 7.005A, did not show activity. Among the compounds tested, only the

thiophene-thiazole analog, 7.005D, showed some activity. It is worth noting that this compound

shares striking similarities with 8.013A.

180

With the activity of 8.013A confirmed, the activity of the remaining pyridone analogs were

evaluated, using 8.013A as the standard compound. Graph 2 shows the SAR studies results

observed with analogs 7.007, 7.010, 7.011 and 7.021.

Graph 2: SSH1 inhibitory activity of analogs 8.013A, 7.007, 7.010, 7.011, 7.021.

8.013A maintained its SSH1 inhibitory activity, however, there was no inhibitory activity observed

from the remaining pyridone analogs. Strikingly, the amide analog, 7.011, obtained as a by-

product, appeared to show increased activity and enhanced the enzyme’s ability to catalyze the

dephosphorylation from para-nitrophenyl phosphate to para-nitrophenol, the chromogenic species.

Following the successful confirmation of SSH1 inhibitory activity of 8.013A, a series of

compounds were acquired from commercial sources (Chembridge) and their activities compared

with 8.013A. Graphs 3,4 and 5 show the results of this SSH1 inhibitory comparison studies.

181

Graph 3: Comparison of 8.013A to E1-series of analogs.

[Chembridge IDs: E1B 5976776; E1E 6748322; E1C 7425371; E1D 7369557]

Clearly, 8.013A is more potent than the set of E-series of compounds shown above. E1B, E1C and

E1E exhibit comparable activity, which can be distinguished by running multiple concentrations.

Graph 4: Comparison of 8.013A to C2-series of analogs

[Chembridge IDs: C2B 5935324; C2C 7905874; C2D 5663386; C2E 7984560; C2F 5807736; C2G 7412943]

182

C2B is a paramethoxyphenyl thiazole analog and appears to be the only C2 analog with good

SSH1 inhibitory activity. Other related analogs were synthesized in an attempt to improve upon

the activity of the methoxyphenyl thiazole analogs.

Graph 5: Comparison of 8.013A to E-series analogs

[IDs: E1 7965015; E2 7992372; E3 7958228; E4 7114024;E5 7970657]

E1, E2 and E3 are structurally similar and exhibit nearly identical activity. E1 – E4 showed superior

activity to 8.013A. In an attempt to improve upon the observed activity, SAR studies of 8.013A

was explored and a range of diverse analogs was prepared and screened in the pNPP assay for their

inhibitory effects, Graph 6.

183

Graph 6: SAR studies of thiazole series

Although several (10) of these synthesized compounds (10) showed good activity as SSH1

inhibitor in the pNPP assay, eight (8) showed superior inhibitory activity at 8 hours (Graph 6),

over the initial hit/lead compound, 8.013A. Analogs that showed better inhibitory activity over

8.013A include 8.013B (methyl thiazole), 8.013C (5-methyl thiophene), 8.013D (4-methyl

thiophene), 8.013E (3-methyl thiophene), 8.014 (3-thiophene analog), 8.019 (para-methoxyphenyl

analog), 8.021 (3-pyridine analog) and 8.022 (4-pyridine analog).

184

8.5 Experimental section

General synthetic procedures:

2-bromo-1-(thiophen-2-yl)ethan-1-one: To a solution of 2-acetylthiophene (5.00 g, 39.6 mmol)

in MeOH (20 mL) at 0°C was added bromine (2.2 mL, 42 mmol) dropwise. The mixture was

allowed to stir for 45 min and H2O (20 mL) was added to the resulting mixture and stirred overnight

at room temperature. At the completion of the reaction H2O (20 mL) was added and the resulting

mixture was extracted twice with EtOAc. The organic layer was washed with sodium bicarbonate

and H2O, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain

2-bromo-1-(thiophen-2-yl)ethanone, 8.007A, (5.85 g, 72%) as a brown semi-solid. LRMS (ESI)

m/z calc’d for C6H5BrOS [M+H]+: 204.07/206.07, found 205.17/207.17. A portion of the crude

material was used in a subsequent step without further purification.

Ethyl 4-thioureidobenzoate: Ethyl 4-aminobenzoate (20.0 g, 121 mmol) was stirred in 1N HCl

in diethyl ether (200 mL) overnight and the resulting off-white precipitate was filtered off, dried

and identified as ethyl 4-aminobenzoate hydrochloride (23.9 g, 98%). This compound (23.0 g, 114

mmol) was dissolved in H2O (160 mL), followed by the addition of ammonium thiocyanate (18.0

g, 240 mmol) and sodium bisulfite (0.60 g, 5.70 mmol). The reaction mixture was heated at 85 °C

for 15 h and then refluxed for 24 hours. The solution was cooled to room temperature and the

precipitate filtered off and washed with H2O and 2N HCl. The product was dried under vacuum

185

and identified as mixture of the thiourea, 8.011, and ethyl amnobenzoate, 8.009, as a light yellow

solid (6.50 g, 25.4%). LRMS (ESI) m/z calc’d for C10H12N2O2S [M+H]+: 225.28, found 225.08.

Ethyl 4-((4-(thiophen-2-yl)thiazol-2-yl)amino)benzoate: To a solution of 2-bromo-1-(thiophen-

2-yl)ethanone (3.60 g, 17.6 mmol) in EtOH (37 mL) was added ethyl 4-thioureidobenzoate (4.330

g, 19.31 mmol). The mixture was allowed to stir at 60 °C for 3.5 hours and cooled to room

temperature. Aqueous sodium bicarbonate was added to the resulting mixture, which was extracted

twice with ethyl acetate. The combined organic phase was washed with H2O and brine, dried over

anhydrous magnesium sulfate and concentrated under reduced pressure. The resulting residue thus

obtained was purified by column chromatography (EtOAc/hexane = 1/2) to obtain the title

compound, 8.012A, (4.58 g, 79%). 1H NMR (500 MHz, DMSO) δ 10.74 (s, 1H), 7.92 (d, J = 8.7

Hz, 2H), 7.78 (d, J = 8.8 Hz, 2H), 7.56 - 7.44 (m, 2H), 7.25 (s, 1H), 7.09 (dd, J = 5.0, 3.7 Hz, 1H),

4.26 (q, J = 7.1 Hz, 2H), 1.30 (t, J = 7.1 Hz, 3H) ppm. 13C NMR (126 MHz, DMSO) 165.8,

162.7, 145.5, 145.3, 138.8, 131.1, 128.4, 125.9, 124.1, 122.5, 116.5, 103.0, 60.7, 14.7 ppm. LRMS

(ESI) m/z calc’d for C16H14N2O2S2 [M+H]+: 331.42, found 331.40.

186

4-((4-(Thiophen-2-yl)thiazol-2-yl)amino)benzoic acid: To 8.012A (4.000 g, 12.11 mmol) in

EtOH (15 mL) was added 50% w/w sodium hydroxide (2.00 g, 24.2 mmol). The mixture was

heated to 75 °C in a microwave reactor for 35 minutes. The resulting mixture was acidified with

2N HCl and the solid collected via suction filtration, washed with EtOH and EtOAc and dried

under vacuum to obtain the title compound as a solid, 8.013A, (3.15 g, 86%). 1H NMR (500 MHz,

DMSO) 12.56 (br. s., 1H), 10.71 (s, 1H), 7.91 (d, J = 8.8 Hz, 2H), 7.75 (d, J = 8.8 Hz, 2H), 7.50

(m, 2H), 7.25 (s, 1H), 7.09 (dd, J = 5.0, 3.6 Hz, 1H) ppm. 13C NMR (126 MHz, DMSO) 167.4,

162.7, 145.3, 145.2, 138.9, 131.2, 128.4, 125.9, 124.1, 123.4, 116.4, 102.9 ppm. HRMS m/z:

[M+H]+ calc’d for C14H10N2O2S2 303.0262; Found 303.0242.

2-bromo-1-(thiophen-2-yl)propan-1-one: To a stirred solution of 1-(thiophen-2-yl)propan-1-one

(5.00 g, 35.7 mmol) in DCM (95 mL) was added bromine (1.80 mL, 35.7 mmol) dropwise. The

solution was stirred for 1 hour, then neutralized by the addition of aqueous sodium carbonate. The

aqueous phase was extracted three times with DCM, and the combined organic extracts were

concentrated under reduced pressure to afford 2-bromo-1-(thiophen-2-yl)propan-1-one, 8.007B,

in nearly quantitative yield (7.5 g, 96%). LRMS (ESI) m/z calc’d for C7H7BrOS [M+H]+:

187

218.10/220.10, found 218.23/220.23. A portion of this material was used in the next step without

further purification.

Ethyl 4-((5-methyl-4-(thiophen-2-yl)thiazol-2-yl)amino)benzoate: Using conditions identical

to those described above, the coupling reaction between 8.007B and 8.011 in EtOH yielded the

target compound, 8.012B, as a solid (87%). 1H NMR (400 MHz, DMSO) 10.57 (s, 1H), 7.86 (d,

J = 8.8 Hz, 2H), 7.73 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 4.6 Hz, 1H), 7.34 (d, J = 3.2 Hz, 1H), 7.10

(dd, J = 5.1, 3.5 Hz, 1H), 4.23 (q, J = 7.0 Hz, 2H), 2.45 (s, 3H), 1.27 (t, J = 7.0 Hz, 3H) ppm. 13C

NMR (101 MHz, DMSO) 165.8, 158.8, 158.6, 145.6, 140.8, 140.0, 138.8, 130.9, 128.2, 122.2,

116.7, 116.3, 60.6, 14.7, 12.2 ppm. LRMS (ESI) m/z calc’d for C17H16N2O2S2 [M+H]+: 345.45,

found 345.40.

4-((5-Methyl-4-(thiophen-2-yl)thiazol-2-yl)amino)benzoic acid: Using conditions identical to

those described above, 8.012B was converted into 8.013B and obtained as a solid (77%). 1H NMR

(400 MHz, DMSO) 10.67 (s, 1H), 7.85 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 8.8 Hz, 2H), 7.49 (d, J

= 4.6 Hz, 1H), 7.33 (d, J = 3.1 Hz, 1H), 7.10 (dd, J = 5.1, 3.5 Hz, 1H), 2.44 (s, 3H) ppm. 13C NMR

(101 MHz, DMSO) δ 167.5, 158.7, 145.4, 140.0, 138.9, 131.1, 128.3, 125.7, 124.9, 123.1, 116.6,

116.3, 12.3 ppm. HRMS m/z: [M-H]- calc’d for C15H12N2O2S2 315.0262; Found 315.0261.

188

4-((4-(5-Methylthiophen-2-yl)thiazol-2-yl)amino)benzoic acid: Using conditions identical to

those described above, 8.012C was converted into 8.013C and obtained as a solid (82%). 1H NMR

(400 MHz, DMSO) 10.95 (s, 1H), 7.86 (d, J = 8.8 Hz, 2H), 7.77 (d, J = 8.8 Hz, 2H), 7.27 (d, J

= 3.5 Hz, 1H), 7.09 (s, 1H), 6.73 - 6.76 (m, 1H), 2.50 (s, 1H), 2.42 (s, 3H) ppm. 13C NMR

(101MHz, DMSO) δ 167.5, 162.7, 145.4, 145.3, 139.1, 136.6, 131.1, 126.7, 123.9, 123.2, 116.4,

102.0, 15.5 ppm. HRMS m/z: [M-H]- calc’d for C15H12N2O2S2 315.0262; Found 315.0257.

4-((4-(4-Methylthiophen-2-yl)thiazol-2-yl)amino)benzoic acid: Using conditions identical to

those described above, 8.012D was converted into 8.013D and obtained as a solid (69%). 1H NMR

(400 MHz, DMSO) 11.02 (s, 1H), 7.86 (d, J = 8.8 Hz, 2H), 7.75 (d, J = 8.8 Hz, 2H), 7.31 (s,

1H), 7.15 (s, 1H), 7.02 (s, 1H), 2.50 (s, 1H), 2.19 (s, 3H) ppm. 13C NMR (101MHz, DMSO) δ

167.5, 162.7, 145.4, 145.2, 138.6, 138.3, 131.1, 126.1, 123.2, 121.0, 116.4, 102.7, 16.0 ppm.

HRMS m/z: [M-H]- calc’d for C15H12N2O2S2 315.0262; Found 315.0260.

189

4-((4-(3-methylthiophen-2-yl)thiazol-2-yl)amino)benzoic acid. Using conditions identical to

those described above, 8.012E was converted into 8.013E and obtained as a solid (73%). 1H NMR

(400 MHz, DMSO) 10.91 (s, 1H), 7.86 (d, J = 8.8 Hz, 2H), 7.77 (d, J = 8.8 Hz, 2H), 7.35 (d, J

= 5.1 Hz, 1H), 6.96 (s, 1H), 6.92 (d, J = 5.1 Hz, 1H), 2.50 (s, 1H), 2.38 (s, 3H) ppm. 13C NMR

(101 MHz, DMSO-d6) δ 167.4, 162.0, 145.3, 145.0, 133.9, 132.8, 132.1, 131.1, 124.4, 123.3,

116.4, 103.9, 16.2 ppm. HRMS m/z: [M-H]- calc’d for C15H12N2O2S2 317.0262; Found 315.0260.

4-((4-(Thiophen-3-yl)thiazol-2-yl)amino)benzoic acid: Using conditions identical to those

described above, 8.014 was obtained as a solid (69%). 1H NMR (400 MHz, DMSO) 10.81 (br.

s., 1H), 7.84 - 7.90 (m, 3H), 7.79 - 7.84 (m, 2H), 7.54 - 7.57 (m, 2H), 7.22 (s, 1H). 13C NMR (101

MHz, DMSO) δ 167.5, 162.7, 146.9, 145.5, 139.1, 131.2, 127.3, 126.4, 123.2, 122.3, 116.5, 104.0

ppm. HRMS m/z: [M-H]- calc’d for C14H10N2O2S2 301.0105; Found 301.0105.

190

4-((4-Phenylthiazol-2-yl)amino)benzoic acid: Using conditions identical to those described

above, and starting with acetophenone, 8.015 was obtained as a solid (68%). 1H NMR (400 MHz,

DMSO) 11.03 (br. s., 1H), 7.83 - 7.93 (m, 7H), 7.37 - 7.43 (m, 3H), 7.25 - 7.31 (m, 1H) ppm.

13C NMR (101 MHz, DMSO) δ 167.5, 162.8, 150.4, 145.5, 134.8, 131.2, 129.1, 128.1, 126.2,

123.2, 116.4, 104.7 ppm. HRMS m/z: [M-H]- calc’d for C16H12N2O2S 295.0541; Found 295.0543.

4-((5-Methyl-4-phenylthiazol-2-yl)amino)benzoic acid: Using conditions identical to those

described above, and starting with propiophenone, 8.016 was obtained as a solid (71%). 1H NMR

(400 MHz, DMSO) 10.62 (br. s., 1H), 7.85 (d, J = 8.7 Hz, 2H), 7.71 (d, J = 8.8 Hz, 2H), 7.64 (d,

J = 8.7 Hz, 2H), 7.40 - 7.45 (m, 2H), 7.29 - 7.34 (m, 1H), 2.40 (s, 3H) ppm. 13C NMR (101 MHz,

DMSO) 167.4, 159.4, 145.3, 144.8, 134.8, 131.2, 128.8, 128.5, 127.8, 123.3, 118.0, 116.6, 12.3

ppm. HRMS m/z: [M-H]- calc’d for C17H14N2O2S 309.0698; Found 309.0698.

191

4-((4-(2-Methoxyphenyl)thiazol-2-yl)amino)benzoic acid: Using conditions identical to those

described above, and starting with 2-methoxy-substituted-acetophenone, 8.017 was obtained as a

solid (88%). 1H NMR (400 MHz, DMSO) 10.75 (br. s., 1H), 8.12 (dd, J = 7.8, 1.56 Hz, 1H),

7.88 (d, J = 8.7 Hz, 2H), 7.79 (d, J = 8.7 Hz, 2H), 7.45 (s, 1H), 7.28 (ddd, J = 8.5, 7.1, 2.0 Hz,

1H), 7.00 - 7.11 (m, 2H), 3.89 (s, 3H) ppm. 13C NMR (101MHz, DMSO) δ 167.5, 161.2, 157.0,

146.2, 145.4, 131.2, 129.8, 129.2, 123.2, 122.8, 121.0, 116.4, 112.0, 108.5, 55.9 ppm. HRMS m/z:

[M+H]+ calc’d for C17H14N2O3S 327.0803; Found 327.0804.

4-((4-(3-Methoxyphenyl)thiazol-2-yl)amino)benzoic acid: Using conditions identical to those

described above, and starting with 3-methoxy-substituted-acetophenone, 8.018 was obtained as a

solid (89%). 1H NMR (400 MHz, DMSO) 10.82 (br. s., 1H), 7.89 (d, J = 8.7 Hz, 2H), 7.78 - 7.82

(d, J = 8.8 Hz, 2H), 7.43 - 7.50 (m, 3H), 7.31 (t, J = 8.0 Hz, 1H), 6.87 (td, J = 1.4, 8.2 Hz, 1H),

3.79 (s, 3H), 2.50 (s, 1H) ppm. 13C NMR (101 MHz, DMSO) δ 167.5, 162.6, 160.0, 150.4, 145.5,

136.1, 131.2, 130.2, 123.2, 118.6, 116.4, 113.7, 111.6, 105.0, 55.5 ppm. HRMS m/z: [M-H]- calc’d

for C17H14N2O3S 325.0647; Found 325.0646.

192

4-((4-(4-Methoxyphenyl)thiazol-2-yl)amino)benzoic acid: Using conditions identical to those

described above, and starting with 4-methoxy-substituted-acetophenone, 8.019 was obtained as a

solid (89%). 1H NMR (400 MHz, DMSO-d6) δ 10.91 (s, 1H), 7.89 - 7.81 (m, 7H), 7.22 (s, 1H),

6.96 (br d, J = 8.2 Hz, 2H), 3.75 (s, 3H). 13C NMR (101MHz, DMSO-d6) δ 167.5, 162.7, 159.3,

150.2, 145.6, 131.2, 127.7, 127.5, 123.1, 116.4, 114.5, 102.5, 55.6. HRMS m/z: [M-H]- calc’d for

C17H14N2O3S 325.0647; Found 325.0640.

2-Bromo-1-(pyridin-2-yl)ethan-1-one: To a solution of 2-acetylpyridine (5.00 g, 41.3 mmol) and

conc. HBr (48% in acetic acid; 13.9 g, 83.0 mmol) was added bromine (6.60 g, 41.3 mmol)

dropwise at room temperature with stirring. The reaction mixture was heated to 60 oC for 5 hr. The

solvents were then evaporated to afford a yellowish solid, which was washed with diethyl ether

via filtration (89%). The bromoketone was used in the next step without further purification.

LRMS (ESI) m/z calc’d for C7H6BrNO [M + H]+: 199.04/201.04, found 199.21/201.21.

193

Ethyl 4-((4-(pyridin-2-yl)thiazol-2-yl)amino)benzoate: Using conditions identical to those

described above, 8.020 i was obtained as a solid (74%). 1H NMR (400 MHz, DMSO) 11.02 (s,

1H), 8.75 (d, J = 5.5 Hz, 1H), 8.38 - 8.49 (m, 2H), 8.19 (s, 1H), 7.88 - 7.98 (m, 4H), 7.74 - 7.81

(m, 1H), 4.25 (q, J = 7.0 Hz, 2H), 1.28 (t, J = 7.0 Hz, 3H) ppm. 13C NMR (101 MHz, DMSO)

165.8, 163.9, 146.0, 145.6, 144.9, 143.3, 143.2, 131.0, 125.3, 124.0, 123.0, 117.2, 115.9, 60.7, 14.7

ppm. LRMS (ESI) m/z calc’d for C17H15N3O2S [M + H]+: 326.39, found 326.40.

4-((4-(Pyridin-2-yl)thiazol-2-yl)amino)benzoic acid: Using conditions identical to those

described above, 8.020 was obtained as a solid (91%). 1H NMR (400 MHz, DMSO) δ 11.25 (s,

1H), 8.74 (br d, J = 5.4 Hz, 1H), 8.47 - 8.38 (m, 2H), 8.31 (s, 1H), 7.95 - 7.88 (m, 5H), 7.79 - 7.73

(m, 1H) ppm. 13C NMR (101 MHz, DMSO) δ 167.4, 163.8, 145.0, 144.4, 141.6, 140.7, 139.5,

133.5, 131.2, 127.7, 123.6, 116.8, 110.5 ppm. HRMS m/z: [M-H]- calc’d for C15H11N3O2S

296.0494; Found 296.0495.

194

4-((4-(Pyridin-3-yl)thiazol-2-yl)amino)benzoic acid: Using conditions identical to those

described above, and starting with 3-acetylpyridine, 8.021 was obtained as a solid (87%). 1H NMR

(400 MHz, DMSO) δ 11.13 (s, 1H), 9.36 (d, J = 1.6 Hz, 1H), 8.95 (br d, J = 8.2 Hz, 1H), 8.77 (br

d, J = 5.3 Hz, 1H), 8.02 (dd, J = 8.1, 5.6 Hz, 1H), 7.96 (s, 1H), 7.90 (dd, J = 8.1, 5.6 Hz, 2H), 7.85

(m, J = 5.6, 8.1 Hz, 2H), 2.50 (s, 1H) ppm. 13C NMR (101 MHz, DMSO) δ 167.44, 163.77, 145.02,

144.42, 141.62, 140.73, 139.51, 133.49, 131.18, 127.69, 123.61, 116.76, 110.45 ppm. HRMS m/z:

[M-H]- calc’d for C15H11N3O2S 296.0494; Found 296.0497.

4-((4-(Pyridin-4-yl)thiazol-2-yl)amino)benzoic acid: Using conditions identical to those

described above, and starting with 4-acetylpyridine, 8.022 was obtained as a solid (85%). 1H NMR

(400 MHz, DMSO-d6) 11.28 (s, 1H), 8.86 (d, J = 6.6 Hz, 2H), 8.45 (d, J = 6.7 Hz, 2H), 8.36 (s,

1H), 7.92 (d, J = 8.8 Hz, 2H), 7.86 (d, J = 8.8 Hz, 2H), 2.50 (s, 1H). 13C NMR (101 MHz, DMSO-

d6) δ = 167.42, 163.68, 148.90, 145.78, 144.90, 142.36, 131.18, 123.78, 122.84, 116.96, 116.84.

HRMS m/z: [M-H]- calc’d for C15H11N3O2S 296.0494; Found 296.0504.

195

Ethyl 4-((4-(tert-butyl)thiazol-2-yl)amino)benzoate: Using conditions identical to those

described above, and starting with commercially available 1-bromo-3,3-dimethylbutan-2-one,

8.023 i was obtained as a solid (83%). 1H NMR (400 MHz, DMSO) 10.49 (s, 1H), 7.87 (d, J =

8.7 Hz, 2H), 7.70 (d, J = 8.7 Hz, 2H), 6.50 (s, 1H), 4.23 (q, J = 7.03 Hz, 2H), 1.23 - 1.28 (m, 12H)

ppm. 13C NMR (101 MHz, DMSO) 165.8, 162.2, 162.0, 145.9, 131.0, 121.9, 116.1, 101.2, 60.5,

34.9, 30.0, 14.7 ppm. LRMS (ESI) m/z calc’d for C16H20N2O2S [M+H]+: 305.41, found 305.39.

4-((4-(tert-butyl)thiazol-2-yl)amino)benzoic acid: Using conditions identical to those described

above, 8.023 was obtained as a solid (92%). 1H NMR (400 MHz, DMSO) 10.51 - 10.70 (m, 1H),

7.84 (d, J = 8.7 Hz, 2H), 7.69 (d, J = 8.7 Hz, 2H), 6.48 (s, 1H), 1.24 (s, 9H) ppm. 13C NMR (101

MHz, DMSO) δ 167.4, 162.6, 160.8, 145.4, 131.2, 123.3, 116.5, 101.0, 34.7, 29.9 ppm. HRMS

m/z: [M-H]- calc’d for C14H16N2O2S 275.0854; Found 275.0855.

196

N-(4-bromophenyl)-4-(thiophen-2-yl)thiazol-2-amine: Using conditions identical to those

described above, 8.027 was obtained as a light brown solid (76%). LRMS (ESI) m/z calc’d for

C13H9BrN2S2 [M+H]+: 336.25/338.25, found 336.40/338.40.

(4-((4-(Thiophen-2-yl)thiazol-2-yl)amino)phenyl)phosphonic acid: A mixture of 8.029 (1.000

g, 2.97 mmol), diethyl phosphonate (1.640 g, 11.86 mmol), TEA (1.080 g, 10.67 mmol) and

tetrakis(triphenylphosphine)palladium(0) [Pd(PPh3)4] (0.514 g, 0.445 mmol) was irradiated under

microwave conditions for 4 hours to obtain the phosphonate ester which was then treated with

concentrated HCl to give 8.029 as a dark brown solid. 1H NMR (400 MHz, DMSO) δ 10.53 (s,

1H), 7.66 (dd, J = 8.5, 2.8 Hz, 2H), 7.55 - 7.52 (m, 3H), 7.44 (d, J = 3.2 Hz, 1H), 7.40 (d, J = 5.0

Hz, 1H), 7.14 (s, 1H), 7.02 (d, J = 3.9 Hz, 1H) ppm. 13C NMR (101 MHz, DMSO) δ 163.0, 145.1,

143.7, 139.0, 132.2, 128.4, 125.8, 124.0, 116.5, 116.3, 102.5 ppm. HRMS m/z: [M-H]- calc’d for

C13H11N2O3PS2 336.9870; Found 336.9880.

197

8.6 References

(1) Siddiqui, N.; Arshad, M.; Ahsan, W.; Alam, M. Thiazole: A valuable insight into recent

advances, synthesis and biological activities. International Journal of Pharmaceutical Sciences

and Drug Research 2009, 1, 136-143.

(2) Yadav, P.; Devprakash, D.; Senthilkumar, G. Benzothiazole: different methods of synthesis

and diverse biological activities. International Journal of Pharmaceutical Sciences and Drug

Research 2011, 3, 01-07.

(3) Patt, W.; Hamilton, H.; Taylor, M.; Ryan, M.; Taylor, D.; Connolly, C.; Doherty, A.;

Klutchko, S.; Sircar, I. Structure-activity relationships of a series of 2-amino-4-thiazole-

containing renin inhibitors. Journal of Medicinal Chemistry 1992, 35, 2562-2572.

(4) Garrod, L. Relative antibacterial activity of three penicillins. British Medical Journal 1960, 1,

527-529.

(5) Pasqualotto, A.; Thiele, K.; Goldani, L. Novel triazole antifungal drugs: focus on

isavuconazole, ravuconazole and albaconazole. Current Opinion in Investigational Drugs 2000,

11, 165-174.

(6) Henry, J.; Hill, I. Fatal interaction between ritonavir and MDMA. Lancet 1998, 352, 1751-

1752.

(7) Dotevall, G.; Herner, B. Treatment of acute primidone poisoning with bemegride and

amiphenazole. British Medical Journal 1957, 2, 451-452.

(8) Figueroa - Valverde, L.; Díaz - Cedillo, F.; Camacho - Luis, A.; García - Cervera, E.; Pool -

Gómez, E.; López - Ramos, M.; Sarabia - Alcocer, B.; May - Gil, I.; Sarao - Álvarez, A.; Ancona

- Leon, G. Antimicrobial activity induced by a sulfathiazole derivative on Staphylococcus

aureus and Vibrio cholerae . International Journal of PharmTech Research 2013, 5, 1247 - 1253.

198

(9) Borelli, C.; Schaller, M.; Niewerth, M.; Nocker, K.; Baasner, B.; Berg, D.; Tiemann, R.;

Tietjen, K.; Fugmann, B.; Lang-Fugmann, S. et al. Modes of action of the new arylguanidine

abafungin beyond interference with ergosterol biosynthesis and in vitro activity against

medically important fungi. Chemotherapy 2008, 54, 245-259.

(10) Upadhyay, M.; West, E.; Sharma, A. Keratitis due to Aspergillus flavus successfully treated

with thiabendazole. British Journal of Ophthalmology 1980, 64, 30-32.

(11) Brown, K.; Cater, D.; Cavalla, J.; Green, D.; Newberry, R.; Wilson, A. Nonsteroidal

antiinflammatory agents. 1. 2,4-Diphenylthiazole-5-acetic acid and related compounds. Journal

of Medicinal Chemistry 1974, 17, 1177-1181.

(12) Barat, R.; Srinatha, A.; Pandit, J.; Mittal, N.; Anupurba, S. Ethylcellulose inserts of an

orphan drug for periodontitis: preparation, in vitro, and clinical studies. Drug Delivery 2007, 14,

531-538.

(13) Barat, R.; Srinatha, A.; Pandit, J.; Ridhurkar, D.; Balasubramaniam, J.; Mittal, N.; Mishra,

D. Niridazole biodegradable inserts for local long-term treatment of periodontitis: possible new

life for an orphan drug. Drug Delivery 2006, 13, 365-373.

(14) Brogden, R.; Heel, R. Aztreonam. Drugs 1986, 31, 96-130.

(15) Cunha, B. Aztreonam. Urology 1993, 41, 249-258.

(16) Pacher, P. Therapeutic effects of xanthine oxidase inhibitors: renaissance half a century after

the discovery of allopurinol. Pharmacological Reviews 2006, 58, 87-114.

(17) Ghosh, B.; Antonio, T.; Zhen, J.; Kharkar, P.; Reith, M.; Dutta, A. Development of (S)-N6-

(2-(4-(isoquinolin-1-yl)piperazin-1-yl)ethyl)-N6-propyl-4,5,6,7-tetrahydrobenzo[d]-thiazole-2,6-

diamine and its analogue as a d3 receptor preferring agonist: potent in vivo activity in

parkinson’s disease animal models. Journal of Medicinal Chemistry 2010, 53, 1023-1037.

199

(18) Scheen, A. A review of gliptins in 2011. Expert Opinion on Pharmacotherapy 2011, 13, 81-

99.

(19) Citrome, L. Lurasidone for schizophrenia: a review of the efficacy and safety profile for this

newly approved second-generation antipsychotic. International Journal of Clinical Practice

2010, 65, 189-210.

(20) Pang, T.; Zhang, Z.; Gu, M.; Qiu, B.; Yu, L.; Cao, P.; Shao, W.; Su, M.; Li, J.; Nan, F. et al.

Small molecule antagonizes autoinhibition and activates amp-activated protein kinase in cells.

Journal of Biological Chemistry 2008, 283, 16051-16060.

(21) Di Fabio, R.; Pellacani, A.; Faedo, S.; Roth, A.; Piccoli, L.; Gerrard, P.; Porter, R.; Johnson,

C.; Thewlis, K.; Donati, D. et al. Discovery process and pharmacological characterization of a

novel dual orexin 1 and orexin 2 receptor antagonist useful for treatment of sleep disorders.

Bioorganic & Medicinal Chemistry Letters 2011, 21, 5562-5567.

(22) Malik, S.; Bahare, R.; Khan, S. Design, synthesis and anticonvulsant evaluation of N-

(benzo[d]thiazol-2-ylcarbamoyl)-2-methyl-4-oxoquinazoline-3(4H)-carbothioamide derivatives:

A hybrid pharmacophore approach. European Journal of Medicinal Chemistry 2013, 67, 1-13.

(23) Dinklo, T.; Shaban, H.; Thuring, J.; Lavreysen, H.; Stevens, K.; Zheng, L.; Mackie, C.;

Grantham, C.; Vandenberk, I.; Meulders, G. et al. Characterization of 2-[[4-fluoro-3-

(trifluoromethyl)phenyl]amino]-4-(4-pyridinyl)-5-thiazolemethanol (JNJ-1930942), a novel

positive allosteric modulator of the {alpha}7 nicotinic acetylcholine receptor. Journal of

Pharmacology and Experimental Therapeutics 2010, 336, 560-574.

(24) Sadashiva, C.; Narendra Sharath Chandra, J.; Kavitha, C.; Thimmegowda, A.; Subhash, M.;

Rangappa, K. Synthesis and pharmacological evaluation of novel N-alkyl/aryl substituted

thiazolidinone arecoline analogues as muscarinic receptor 1 agonist in Alzheimer's dementia

models. European Journal of Medicinal Chemistry 2009, 44, 4848-4854.

200

(25) Andreani, A.; Granaiola, M.; Leoni, A.; Locatelli, A.; Morigi, R.; Rambaldi, M. Synthesis

and antitubercular activity of imidazo[2,1-b]thiazoles. European Journal of Medicinal Chemistry

2001, 36, 743-746.

(26) Yang, S.; Je Lee, W.; Kim, E.; Dal Nam, K.; Hahn, H.; Young Choi, S.; Cho, S. Effects of N-

adamantyl-4-methylthiazol-2-amine on hyperglycemia, hyperlipidemia and oxidative stress in

streptozotocin-induced diabetic rats. European Journal of Pharmacology 2014, 736, 26-34.

(27) Ramtohul, Y.; Black, C.; Chan, C.; Crane, S.; Guay, J.; Guiral, S.; Huang, Z.; Oballa, R.;

Xu, L.; Zhang, L. et al. SAR and optimization of thiazole analogs as potent stearoyl-CoA

desaturase inhibitors. Bioorganic & Medicinal Chemistry Letters 2010, 20, 1593-1597.

(28) Kalkhambkar, R.; Kulkarni, G.; Shivkumar, H.; Rao, R. Synthesis of novel triheterocyclic

thiazoles as anti-inflammatory and analgesic agents. European Journal of Medicinal Chemistry

2007, 42, 1272-1276.

(29) Tiew, K.; Dou, D.; Teramoto, T.; Lai, H.; Alliston, K.; Lushington, G.; Padmanabhan, R.;

Groutas, W. Inhibition of Dengue virus and West Nile virus proteases by click chemistry-derived

benz[d]isothiazol-3(2H)-one derivatives. Bioorganic & Medicinal Chemistry 2012, 20, 1213-

1221.

(30) Sadek, B.; Al-Tabakha, M.; Fahelelbom, K. Antimicrobial Prospect of Newly Synthesized

1,3-Thiazole Derivatives. Molecules 2011, 16, 9386-9396.

(31) Ogawa, Y.; Nonaka, Y.; Goto, T.; Ohnishi, E.; Hiramatsu, T.; Kii, I.; Yoshida, M.; Ikura, T.;

Onogi, H.; Shibuya, H. et al. Development of a novel selective inhibitor of the Down syndrome-

related kinase Dyrk1A. Nature Communications 2010, 1, 1-9.

(32) Ahn, J.; Radhakrishnan, M.; Mapelli, M.; Choi, S.; Tidor, B.; Cuny, G.; Musacchio, A.; Yeh,

L.; Kosik, K. Defining Cdk5 ligand chemical space with small molecule inhibitors of tau

phosphorylation. Chemistry & Biology 2005, 12, 811-823.

201

(33) Slee, D.; Chen, Y.; Zhang, X.; Moorjani, M.; Lanier, M.; Lin, E.; Rueter, J.; Williams, J.;

Lechner, S.; Markison, S. et al. 2-Amino-N-pyrimidin-4-ylacetamides as A2A receptor

antagonists: 1. Structure−activity relationships and optimization of heterocyclic substituents.

Journal of Medicinal Chemistry 2008, 51, 1719-1729.

(34) Kounnas, M.; Danks, A.; Cheng, S.; Tyree, C.; Ackerman, E.; Zhang, X.; Ahn, K.; Nguyen,

P.; Comer, D.; Mao, L. et al. Modulation of γ-secretase reduces β-amyloid deposition in a

transgenic mouse model of Alzheimer's disease. Neuron 2010, 67, 769-780.

(35) Miwatashi, S.; Arikawa, Y.; Matsumoto, T.; Uga, K.; Kanzaki, N.; Imai, Y.; Ohkawa, S.

Synthesis and biological activities of 4-phenyl-5-pyridyl-1,3-thiazole derivatives as selective

adenosine A3 antagonists. Chemical & Pharmaceutical Bulletin 2008, 56, 1126-1137.

(36) Sampson, D.; Zhu, X.; Eyunni, S.; Etukala, J.; Ofori, E.; Bricker, B.; Lamango, N.; Setola,

V.; Roth, B.; Ablordeppey, S. Identification of a new selective dopamine D4 receptor ligand.

Bioorganic & Medicinal Chemistry 2014, 22, 3105-3114.

(37) Potewar, T.; Ingale, S.; Srinivasan, K. Efficient synthesis of 2,4-disubstituted thiazoles using

ionic liquid under ambient conditions: a practical approach towards the synthesis of Fanetizole.

Tetrahedron 2007, 63, 11066-11069.

(38) Ikemoto, N.; Liu, J.; Brands, K.; McNamara, J.; Reider, P. Practical routes to the

triarylsulfonyl chloride intermediate of a β3 adrenergic receptor agonist. Tetrahedron 2003, 59,

1317-1325.

(39) Gambacorti-Passerini, C.; Mologni, L.; Scapozza, L.; Bisson, W.; Goekjian, P.; Benoit, J. 2-

acylaminothiazoles for the treatment of cancer. WO2015001024 A1; PCT/EP2014/064197, 2015.

(40) Chang, Y.; Jung, Y.; Kim, H.; Kim, J.; Kim, N.; Son, J. New bicyclic compound for

modulating g protein-coupled receptors. WO2012093809 A3; PCT/KR2011/010304, 2012.

(41) Carroll, F.; Blough, B.; Abraham, P.; Mills, A.; Holleman, J.; Wolckenhauer, S.; Decker, A.;

Landavazo, A.; McElroy, K.; Navarro, H. et al. Synthesis and biological evaluation of bupropion

202

analogues as potential pharmacotherapies for cocaine addiction. Journal of Medicinal Chemistry

2009, 52, 6768-6781.

(42) Zafar, A.; Melendez, R.; Geib, S.; Hamilton, A. Hydrogen bond controlled aggregation of

guanidinium-carboxylate derivatives in the solid state. Tetrahedron 2002, 58, 683-690.

(43) Burger, A. Isosterism and bioisosterism in drug design. Progress in Drug Research 1995,

37, 288-371.

(44) Bhuniya, D.; Mukkavilli, R.; Shivahare, R.; Launay, D.; Dere, R.; Deshpande, A.; Verma,

A.; Vishwakarma, P.; Moger, M.; Pradhan, A. et al. Aminothiazoles: Hit to lead development to

identify antileishmanial agents. European Journal of Medicinal Chemistry 2015, 102, 582-593.

(45) Bellale, E.; Naik, M.; VB, V.; Ambady, A.; Narayan, A.; Ravishankar, S.; Ramachandran, V.;

Kaur, P.; McLaughlin, R.; Whiteaker, J. et al. Diarylthiazole: An Antimycobacterial Scaffold

Potentially Targeting PrrB-PrrA Two-Component System. Journal of Medicinal Chemistry 2014,

57, 6572-6582.

(46) Altaf, A.; Shahzad, A.; Gul, Z.; Rasool, N.; Badshah, A.; Lal, A.; Khan, E. A review on the

medicinal importance of pyridine derivatives. Journal of Drug Design and Medicinal Chemistry

2015, 1, 1-11.

(47) Wang, P.; Lu, J.; Zhang, Z. An improved procedure for the synthesis of aryl phosphonates

by palladium-catalysed cross-coupling of aryl halides and diethyl phosphite in polyethylene

glycol. Journal of Chemical Research 2013, 37, 359-361.

(48) Jansa, P.; Baszczyňski, O.; Procházková, E.; Dracínský, M.; Janeba, Z. Microwave-assisted

hydrolysis of phosphonate diesters: an efficient protocol for the preparation of phosphonic acids.

Green Chemistry, 2012, 14, 2282-2288.

203

Chapter 9

Conclusions

9.1 Summary and conclusions

Current treatment for AD only temporarily relieves symptoms, but does not alter the

underlying biology of the disease. Patients who receive the current standard of care ultimately

progress to the advanced form of the disease. In spite of the tenacious efforts to identify compounds

for treating AD, the lack of effective treatment for the disease continues to be a major unmet

medical need.

Novel pyridones and thiazoles that potentially inhibit the SSH1 phosphatase enzyme pathway,

which could lead to inhibition of excessive Cofilin activation have been successfully synthesized.

The pyridone analogs were obtained via a aza-bicyclo-Michael type reaction, and the thiazoles

were prepared through a Hantzsch thiazole synthesis. The synthetic approaches allowed for the

quick preparation of several different analogs for evaluation. Each synthesized compound was

evaluated for in vitro SSH1 inhibitory activity employing the pNPP phosphatase assay. As a non-

proteinaceous and non-specific substrate, pNPP was employed to evaluate protein phosphatases

using a spectrophotometric assay. This assay is based on the ability of the enzymes (phosphatases)

to catalyze the hydrolysis of pNPP to pNP (para-nitrophenol). The pNP is a chromogenic species

with an absorbance at 405 nm. The majority of the synthesized compounds showed moderate to

high SSH1 inhibitory activity.

204

The initial plan was to confirm the activity of the lead compounds by first synthesizing compounds

7.005A and 8.013A, together with other pyridone analogs. The SAR studies were initially centered

on the pyridone nitrogen where varying choices of amines were used. This allowed the quick

synthesis of different analogs and effortlessly determine the most active candidate for further

development. Modelling suggested that the crucial features of the pyridone series were the

carboxylic acid functionality, pyridone carbonyl and the thiazole nitrogen. As a result, compound

7.007, was prepared by masking the phenolic hydrogen to confirm the findings. Moreover, the

thiazole nitrogen was believed to bind to Arg 399, thus it was envisioned that decreasing or

increasing this binding interaction may have a clear impact on the activity. To this end, compound

7.005C, of which the thiazole ring was substituted for a phenyl group was prepared. Hence the

sulfur was replaced with a carbon-carbon double bond and the nitrogen with a carbon atom.

Compound 7.005D - a thiazole-thiophene N-substituted analog was also prepared aimed, at

evaluating the importance of the thiophene ring. This compound proved to be the only analog

among the pyridone series that showed activity in the assay.

Surprisingly, data from the initial screening results showed that the pyridones series were not

particularly active as SSH1 inhibitors, but the activity of the thiazole analog, 8.013A, was

confirmed. Among the pyridone series, compound 7.011 exhibited an increased activity, that is, it

appeared to enhance the phosphatase ability at catalyzing the hydrolysis of pNPP to pNP. Attention

was thus shifted to focus on the thiazole series for SAR studies, since compound 8.013A exhibited

superior and comparable SSH1 phosphatase inhibitory activity over a number of compounds

presumed to be SSH1 inhibitors (from commercial sources).

205

A lead optimization campaign was initiated with 8.013A to identify compounds that would not

only be more potent and selective, but also possess acceptable pharmacokinetic properties. Firstly,

a methyl substituted thiazole analog, 8.013B, was prepared in order to investigate the twisting

effect of the methyl substituent on the activity of this analog. Additionally, to investigate the effect

of substituents on this series, compounds 8.013C-D was prepared via a ‘methyl walk’ on the

thiophene ring. Subsequently the thiophene ring was substituted for the known thiophene-isostere,

phenyl group, as well phenyl-substituted analogs in compounds 8.015, 8.016, 8.017, 8.018 and

8.019. The thiophene ring was replaced with 2-,3- and 4-substituted pyridine rings (8.020, 8.021,

8.022). Finally, one drastic change to the lead molecule was made by replacing the thiphene ring

with a tert-butyl group. This was intended to remove aromaticity from the molecule and to observe

the effect this will have on the SSH1 inhibity activity.

Following the revelation of the presence of inorganic phosphate groups within the active site of

SSH1 via crystal structure, it was envisioned that compounds bearing the phosphonic acid

functionality may achieve superior binding and consequently better activity by competing with the

existing inorganic phosphate groups. To this end, a phosphonic acid analog 8.029 was prepared.

Compound 8.013C possessing a methyl group on the thiazole ring was one of the most potent

compounds in this series. Similarly, compounds which differ from 8.013C by having the methyl

substituent on the thiophene rings (8.013E, 8.013D and 8.013B) demonstrated potent SSH1

inhibitory activity. These findings suggest that methyl substitutions on either the thiazole or

thiophene rings are important for anti-SSH1 activity. Compound 8.021, the 3-pyridine analog,

exhibited unusual activity – its profound inhibition was only seen at 6 – 7 hours after incubation.

206

Prior to those stated hours, compound 8.021 showed no inhibitory effect. The 4-pyridine analog,

8.022, was equipotent to 8.013A, however the 2-pyridone analog, 8.020, showed no activity. This

suggests that the position of the nitrogen on the pyridine ring is important. Moving the sulfur in

the thiophene ring was also shown to enhance activity and compound 8.014 was observed to be

slightly better than 8.013A. Multiple concentrations have to be tested in order to identify the best

compound.

The exchange of the thiophene ring for a phenyl group was shown to be deleterious for SSH1

inhibitory activity, however, the methoxy-substituted phenyl analogs exhibited SSH1 inhibitory

activity, with the best compound being the para-methoxy analog, 8.019. No SSH1 inhibition was

observed for the tert-butyl, 8.023, and phosphonic acid analogs, 8.029.

Data obtained from this series further establishes the importance of the thiazole scaffold in

medicinal chemistry. The introduction of a methyl group increased the SSH1 inhibitory activity of

the series, giving rise to the most potent compound seen to date. The methyl groups probably twists

the compound in the right orientation to facilitate binding. Further investigations into the

molecular target and mechanistic studies of these compounds are currently underway.

207

Appendices

(Assays, NMR Spectra and Copyright permission)

208

Appendix – I

Biological Assays

1. Leishmaniasis

All data were obtained using 96-well plates. Control compounds were used in order to ensure

proper assay behaviour. All reported assay data are the average of three consecutive readings

(N=3) and variability were typically in the range of less than +/- 10%. Miltefosine was obtained

from commercial sources (Sigma-Aldrich) and was used as acquired.

1.a. L. donovani axenic amastigote assay

Cell Line

L. donovani axenic amastigotes were cultured in RPMI 1640 at a pH of 5.5 with 7.5 g/L

Hepes (Invitrogen Corp., Carlsbad, CA), 5.86 g/L MES (Sigma-Aldrich, St Louis, MO), 2 g/L

sodium bicarbonate (Fisher Scientific, Pittsburgh, PA), 10 mg/L Hemin (Sigma-Aldrich, St Louis,

MO), 100 μM Xanthine (Sigma-Aldrich, St Louis, MO), 40 mg/L Tween-80 (Sigma-Aldrich, St

Louis, MO), 1% Penicillin-Streptomycin, 5 g/L Trypton-Peptone (BD Bioscience, Sparks, MD),

and 20% 16 hours heat inactivated FBS. L. donovani was incubated at 37 °C. All culturing was

done using non-vented 25 cm2 tissue culture flasks (Coning, Lowell, MA)

Cell viability assay

The viability of amastigotes was determined by the Cell Titer 96 Aqueous Assay (Promega,

Madison, WI) that employs a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5(3-

carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium; MTS] and electron-coupling

reagent, phenazine methosulphate (PMS). Test compounds were serially diluted in 100 μL of PBS

209

in 96 cell plates using a Biomeck 3000 (Beckman Coulter, Miami, FL). Ten μl of each well was

transferred to another 96 well plate and then receives 90 μl or parasites in media. The parasites

were in a concentration to have 6.600 parasites per well. After 72 hours 20 μl of MTS solution is

added to each well in the 96 well plates. The plates were then incubated 37 °C for four hours to

achieve optimal color development. After 4 h of incubation, the OD values were determined at 490

nm using a Spectra Max M2 (Molecular Devices, Sunnyvale, CA). The results were presented as

the percentage of survivors (OD value with test compound divided by that of untreated control).

Curve fitting using non-linear regression was done using trifox to obtain IC50 values.

1.b. Cytotoxicity assay

Cell Line

J774A.1 (ATCC, Manassas, VA) macrophages were cultured in RPMI 1640 (Invitrogen

Corp., Carlsbad, CA) at a pH of 7.2 containing 10% fetal bovine serum (Invitrogen Corp.,

Carlsbad, CA) and 1% penicillin-streptomycin (Invitrogen Corp., Carlsbad, CA) The cells were

incubated at 37 °C in a humidified atmosphere containing 5% CO2 and serially passaged biweekly.

Cell viability assay

The viability of the macrophages was determined by the Cell Titer 96 Aqueous Assay

(Promega, Madison, WI) that employs a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5(3-

carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium; MTS] and electron-coupling

reagent, phenazine methosulphate (PMS). Test compounds were serially diluted in 100 μl of PBS

in 96 cell plates using a Biomeck 3000 (Beckman Coulter, Miami, FL). Ten μl of each well was

transferred to another 96 well plate and then receives 90 μl or parasites in media. The parasites

210

were in a concentration to have 50,000 parasites per well. After 72 hours 20 μl of MTS solution is

added to each well in the 96 well plates. The plates were then incubated 37 °C for four hours to

achieve optimal color development. After 4 h of incubation, the OD values were determined at 490

nm using a Spectra Max M2 (Molecular Devices, Sunnyvale, CA). The results were presented as

the percentage of survivors (OD value with test compound divided by that of untreated control).

Curve fitting using non-linear regression was done using trifox to obtain IC50 values.

2. Hsp 90 Assay

The Hsp 90 fluorescence polarization assay was run by Hardik Patel in the laboratory of

Prof. Gabriela Chiosis using the protocol described in their manuscript (Kim, J.; Felts, S.; Llauger,

L.; He, H.; Huezo, H.; Rosen, N.; Chiosis, G. Development of a fluorescence polarization ssay

for the molecular chaperone Hsp 90. J. Biomol. Screening 2004, 9, 375-381) and using their

standard compound PU-H71 as a control compound.

3. p-Nitrophenyl phosphate phosphatase assay

SSH1-N461 Purification

SSH1-N461 (truncated SSH1 with catalytic function) was subcloned into a pFastBac1

vector and transferred into a DH10Bac E. coli to generate a Bacmid, which was transfected with

Sf9 insect cells and cultured in Sf900-SFM medium for 72 hours, to generate P1 virus. The P1

virus generated was further transfected with new Sf9 cells and cultured for 72 hours and the

harvested cells were added in lysis buffer (Tris 20mM, NaCl 150 mM, Triton-X100 1%, 10 mM

imidazole, with protease inhibitors) and centrifuged at 12,000 g for 15 minutes, following which

the supernatant was collected. To the supernatant was added Ni sepharose high performance beads

211

and shaken for 1 hour at 4 oC, washed 3 times with lysis buffer and centrifuged for 2 minutes at

700 g. The SSH1-N461 was then eluted with the elution buffer – Tris 20 mM, NaCl 150 mM, 200

mM imidazole), and dialysated with dialysis buffer – Tris 20 mM, NaCl 150 mM, DTT 1 mM).

pNPP Assay Procedure

Each compound 10 mM 1µL (final 100 µM) was incubated with 10X reaction buffer 10

µL (Tris 500 mM pH 7.4, NaCl 1M, MgCl2 20 mM, DTT 10 mM) with SSH1-N461 (final 1 µM)

at room temperature for 2 hours. pNPP 500 mM 20 µL (final 100 mM), was then added to the

mixture (total volume 100 µL) and incubated at 37 oC and absorbances read at intervals of 1 hour.

212

Appendix II

Chapter 2 – NMR Spectra

JWL-BE1-002_PROTON_01

18 16 14 12 10 8 6 4 2 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Nor

mal

ized

Inte

nsity

6.002.072.092.751.04

M03(s)

M02(s)

M04(s)

M01(s)

M05(s)

0.95

0.98

1.03

2.262.

452.49

18.0

1

JWL-BE1-002_CARBON_01

200 180 160 140 120 100 80 60 40 20 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

Nor

mal

ized

Inte

nsity

M09(s)

M07(s)

M06(s)

M08(s)

M05(s)

M01(s)

M02(s)

M03(s)

M04(s)

28.1

028

.43

30.5

5

46.7

7

52.3

5

112.

25

195.

0119

7.78

202.

30

213

JWL-BE1-002_W_O_METHYL_PROTON_01.esp

18 16 14 12 10 8 6 4 2 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

2.002.012.901.831.03

M04(m)

M02(t)

M03(s)

M05(dt)

M01(s)

1.88

1.90

1.91

1.94

2.402.41

2.51

2.59

2.6118

.04

JWL-BE1-002_W_O_METHYL_CARBON_01

200 180 160 140 120 100 80 60 40 20 0

Chemical Shif t (ppm)

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

M07(s)

M06(s)

M02(s)

M08(s)

M05(s)

M04(s)

M03(s)

M01(s)

18.9

4

28.7

3

33.1

8

38.5

2

113.

37

195.

3419

8.64

203.

01

214

001 PROTON.esp

11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.05

0.10

0.15

0.20

Nor

mal

ized

Inte

nsity

2.001.000.940.931.04

M02(d)

M03(d)M01(s)

M05(s)

M04(m)

4.38

6.72

6.73

6.74

7.06

7.46

7.48

8.04

001 CARBON

180 160 140 120 100 80 60 40 20 0

Chemical Shif t (ppm)

0

0.05

0.10

0.15

0.20

0.25

0.30

No

rma

lize

d I

nte

nsi

ty

M05(s)

M01(s)

M02(s)

M04(s)

M07(s)

M03(m)

M06(s)

98

.52

11

0.3

71

13.6

0

11

9.5

6

13

5.3

7

15

6.6

5

215

003 PROTON

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

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

6.002.062.982.041.101.97

M05(s)

M04(s)

M03(s)

M06(s)

M02(m)

M01(dd)

1.01

2.33

2.39

3.00

7.79

7.797.80

7.81

8.098.

108.

11

003 CARBON

200 180 160 140 120 100 80 60 40 20 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

M03(m)

M09(s)

M06(s)

M15(s)

M10(s)

M12(s)

M13(s)

M16(s)M02(s)

M11(s)

M14(s)

M01(s)

M04(s)

M05(s)

M08(s)

13.5

6

28.1

935.9

036

.56

52.1

2

113.

3411

7.32

117.

72122.

6112

5.89

127.

14

136.

35

142.

87

151.

03

193.

35

216

003 W Me PROTON

11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

2.185.242.141.092.00

M05(m)

M04(m)

M03(t)

M02(m)

M01(quin)

2.02

2.042.

052.

062.

072.

392.

41

3.06

3.07

3.097.79

7.807.817.81

8.09

8.11

003 WO Me CARBON

200 180 160 140 120 100 80 60 40 20 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

M02(s)

M01(s)

M10(s)

M04(s)

M05(s)

M13(s)M06(s)

M11(s)

M07(s)M12(s)

M08(s)

M14(s)

M15(s)

M03(s)

13.6

4

23.4

323

.53

38.1

6

113.

3011

7.3311

8.62

122.

6112

5.84

127.

05

136.

33

142.

96

150.

2515

2.20

193.

98

217

004 Br dsp PROTON 2

12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

6.002.215.281.935.021.941.041.050.970.98

M02(s)

M07(s)

M03(d)

M01(d)

M05(t)

M04(m)

M10(s)

M06(m)

M09(m)

M08(m)

1.00

1.801.

811.

822.

322.

39

2.93

3.27

3.31

3.41

3.42

6.486.

496.

816.

836.

87

7.61

7.63

004 Br dsp CARBON

200 180 160 140 120 100 80 60 40 20 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

M20(s) M17(s)M10(s)

M15(s)

M16(s)

M19(s)

M14(s)

M13(s)

M18(s)

M07(s)

M08(s)

M06(s)M09(s)

M12(s)

M11(s) M01(s)

M02(s)

M03(s)

M05(s)

M04(s)

13.5

128.1

028

.62

35.8

1

40.7

2

52.0

9

58.3

9

70.4

2

93.7

4

105.

35

110.

68

117.

1411

7.92

135.

07

143.

4614

9.25

150.

4115

1.71

193.

75

218

004 Br dsp WO PROTON

11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

2.322.222.632.142.052.981.611.920.980.881.00

M10(d)

M03(s)M06(s)

M11(d)

M07(m)

M08(t)

M09(dd)

M04(m)

M05(t)

M02(m)

M01(m)

1.911.92

1.94

2.132.14

2.16

2.46

2.51

2.523.

043.

35

3.54

3.55

6.81

6.81

6.83

6.836.

926.

92

7.53

7.55

004 Br dsp WO CARBON

200 180 160 140 120 100 80 60 40 20 0

Chemical Shif t (ppm)

0

0.05

0.10

0.15

0.20

0.25

0.30

Nor

mal

ized

Inte

nsity

M02(s)M12(m)

M03(s)

M01(s)

M11(s)

M10(s)M15(m)

M17(s)

M04(s)

M08(s)

M14(s)

M06(m)

M16(s)

M09(m)

M05(s)

M07(s)M18(s)

M13(m)

12.0

1

23.1

523

.36

28.3

1

37.6

3

57.5

6

70.7

0

105.

10

110.

42

133.

94

150.

0515

1.57

195.

42

219

004 final

11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

6.000.942.242.043.102.084.962.051.361.121.031.100.93

M04(d)

M10(s)

M13(s)

M06(t)

M05(dd)

M08(s)

M03(d)

M09(s)

M11(quin)

M07(m)

M12(m)

M02(m)

M01(m)

7.48 7.46

6.80

6.63

6.61

3.51

3.50

3.33

3.30

3.29

2.81

2.53

2.38

1.95 1.

931.

921.

91

1.09

JWL-BE1-004_ACTUAL_MASS_CARBON_01

200 180 160 140 120 100 80 60 40 20 0

Chemical Shif t (ppm)

0.05

0.10

0.15

0.20

0.25

0.30

No

rma

lize

d I

nte

nsity

M19(s)

M17(s)

M07(s)

M01(s)

M04(s)

M08(s)

M05(s)

M20(s)

M02(s)

M06(s)

M13(s)

M16(s)

M10(s)

M03(s)

M18(s)

M11(br. s.)

M15(s)

M12(s)

M09(s)

M14(s)

19

3.47

17

1.29

15

0.59

14

9.95

14

9.13

14

2.66

12

9.58

11

7.18

11

2.56

10

9.51

10

6.76

70

.21

58

.70

52

.37

40

.68

37

.59

35

.79

29

.07

28

.38

13

.39

220

004 WO Me PROTON

11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

2.002.064.731.738.361.930.820.770.92

M02(d)

M03(dd)

M01(d)

M06(t)

M04(t)

M09(m)

M08(m)

M05(m)

M07(m)

7.71

7.69 6.

846.

716.

716.

696.

69

3.53

3.52

3.33

3.30

3.30

3.29

3.04 3.

032.

472.

16 2.15 2.14

1.92 1.

911.

89

004 WO Me CARBON13CMETHANOL-d419 C's

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

0

0.05

0.10

0.15

0.2011

.99

11.9

911

.99

11.9

911

.99

11.9

911

.99

11.9

911

.99

11.9

911

.99

11.9

911

.99

11.9

911

.99

11.9

911

.99

11.9

911

.99

11.9

911

.99

11.9

9

23.0

723

.07

23.0

723

.07

23.0

723

.07

23.0

723

.07

23.0

723

.07

23.0

723

.07

23.0

723

.07

23.0

723

.07

23.0

723

.07

23.0

723

.07

23.0

723

.07

23.3

823

.38

23.3

823

.38

23.3

823

.38

23.3

823

.38

23.3

823

.38

23.3

823

.38

23.3

823

.38

23.3

823

.38

23.3

823

.38

23.3

823

.38

23.3

823

.38

28.7

528

.75

28.7

528

.75

28.7

528

.75

28.7

528

.75

28.7

528

.75

28.7

528

.75

28.7

528

.75

28.7

528

.75

28.7

528

.75

28.7

528

.75

28.7

528

.75

37.6

737

.67

37.6

737

.67

37.6

737

.67

37.6

737

.67

37.6

737

.67

37.6

737

.67

37.6

737

.67

37.6

737

.67

37.6

737

.67

37.6

737

.67

37.6

737

.67

39.5

239

.52

39.5

239

.52

39.5

239

.52

39.5

239

.52

39.5

239

.52

39.5

239

.52

39.5

239

.52

39.5

239

.52

39.5

239

.52

39.5

239

.52

39.5

239

.52

57.5

157

.51

57.5

157

.51

57.5

157

.51

57.5

157

.51

57.5

157

.51

57.5

157

.51

57.5

157

.51

57.5

157

.51

57.5

157

.51

57.5

157

.51

57.5

157

.51

69.9

269

.92

69.9

269

.92

69.9

269

.92

69.9

269

.92

69.9

269

.92

69.9

269

.92

69.9

269

.92

69.9

269

.92

69.9

269

.92

69.9

269

.92

69.9

269

.92

105.

7210

5.72

105.

7210

5.72

105.

7210

5.72

105.

7210

5.72

105.

7210

5.72

105.

7210

5.72

105.

7210

5.72

105.

7210

5.72

105.

7210

5.72

105.

7210

5.72

105.

7210

5.72

108.

8110

8.81

108.

8110

8.81

108.

8110

8.81

108.

8110

8.81

108.

8110

8.81

108.

8110

8.81

108.

8110

8.81

108.

8110

8.81

108.

8110

8.81

108.

8110

8.81

108.

8110

8.81

113.

3611

3.36

113.

3611

3.36

113.

3611

3.36

113.

3611

3.36

113.

3611

3.36

113.

3611

3.36

113.

3611

3.36

113.

3611

3.36

113.

3611

3.36

113.

3611

3.36

113.

3611

3.36

117.

2311

7.23

117.

2311

7.23

117.

2311

7.23

117.

2311

7.23

117.

2311

7.23

117.

2311

7.23

117.

2311

7.23

117.

2311

7.23

117.

2311

7.23

117.

2311

7.23

117.

2311

7.23

130.

0013

0.00

130.

0013

0.00

130.

0013

0.00

130.

0013

0.00

130.

0013

0.00

130.

0013

0.00

130.

0013

0.00

130.

0013

0.00

130.

0013

0.00

130.

0013

0.00

130.

0013

0.00

141.

9714

1.97

141.

9714

1.97

141.

9714

1.97

141.

9714

1.97

141.

9714

1.97

141.

9714

1.97

141.

9714

1.97

141.

9714

1.97

141.

9714

1.97

141.

9714

1.97

141.

9714

1.97

149.

7114

9.71

149.

7114

9.71

149.

7114

9.71

149.

7114

9.71

149.

7114

9.71

149.

7114

9.71

149.

7114

9.71

149.

7114

9.71

149.

7114

9.71

149.

7114

9.71

149.

7114

9.71

150.

7415

0.74

150.

7415

0.74

150.

7415

0.74

150.

7415

0.74

150.

7415

0.74

150.

7415

0.74

150.

7415

0.74

150.

7415

0.74

150.

7415

0.74

150.

7415

0.74

150.

7415

0.74

151.

3615

1.36

151.

3615

1.36

151.

3615

1.36

151.

3615

1.36

151.

3615

1.36

151.

3615

1.36

151.

3615

1.36

151.

3615

1.36

151.

3615

1.36

151.

3615

1.36

151.

3615

1.36

172.

4717

2.47

172.

4717

2.47

172.

4717

2.47

172.

4717

2.47

172.

4717

2.47

172.

4717

2.47

172.

4717

2.47

172.

4717

2.47

172.

4717

2.47

172.

4717

2.47

172.

4717

2.47

195.

5019

5.50

195.

5019

5.50

195.

5019

5.50

195.

5019

5.50

195.

5019

5.50

195.

5019

5.50

195.

5019

5.50

195.

5019

5.50

195.

5019

5.50

195.

5019

5.50

195.

5019

5.50

221

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

0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

13.3

713

.37

13.3

713

.37

13.3

713

.37

13.3

713

.37

13.3

713

.37

13.3

713

.37

13.3

713

.37

13.3

713

.37

13.3

713

.37

13.3

713

.37

13.3

713

.37

28.3

728

.37

28.3

728

.37

28.3

728

.37

28.3

728

.37

28.3

728

.37

28.3

728

.37

28.3

728

.37

28.3

728

.37

28.3

728

.37

28.3

728

.37

28.3

728

.37

28.6

228

.62

28.6

228

.62

28.6

228

.62

28.6

228

.62

28.6

228

.62

28.6

228

.62

28.6

228

.62

28.6

228

.62

28.6

228

.62

28.6

228

.62

28.6

228

.62

35.8

635

.86

35.8

635

.86

35.8

635

.86

35.8

635

.86

35.8

635

.86

35.8

635

.86

35.8

635

.86

35.8

635

.86

35.8

635

.86

35.8

635

.86

35.8

635

.86

37.7

137

.71

37.7

137

.71

37.7

137

.71

37.7

137

.71

37.7

137

.71

37.7

137

.71

37.7

137

.71

37.7

137

.71

37.7

137

.71

37.7

137

.71

37.7

137

.71

41.9

341

.93

41.9

341

.93

41.9

341

.93

41.9

341

.93

41.9

341

.93

41.9

341

.93

41.9

341

.93

41.9

341

.93

41.9

341

.93

41.9

341

.93

41.9

341

.9352

.34

52.3

452

.34

52.3

452

.34

52.3

452

.34

52.3

452

.34

52.3

452

.34

52.3

452

.34

52.3

452

.34

52.3

452

.34

52.3

452

.34

52.3

452

.34

52.3

4

58.6

658

.66

58.6

658

.66

58.6

658

.66

58.6

658

.66

58.6

658

.66

58.6

658

.66

58.6

658

.66

58.6

658

.66

58.6

658

.66

58.6

658

.66

58.6

658

.66

70.2

670

.26

70.2

670

.26

70.2

670

.26

70.2

670

.26

70.2

670

.26

70.2

670

.26

70.2

670

.26

70.2

670

.26

70.2

670

.26

70.2

670

.26

70.2

670

.26

107.

4910

7.49

107.

4910

7.49

107.

4910

7.49

107.

4910

7.49

107.

4910

7.49

107.

4910

7.49

107.

4910

7.49

107.

4910

7.49

107.

4910

7.49

107.

4910

7.49

107.

4910

7.49

109.

6310

9.63

109.

6310

9.63

109.

6310

9.63

109.

6310

9.63

109.

6310

9.63

109.

6310

9.63

109.

6310

9.63

109.

6310

9.63

109.

6310

9.63

109.

6310

9.63

109.

6310

9.63

110.

9211

0.92

110.

9211

0.92

110.

9211

0.92

110.

9211

0.92

110.

9211

0.92

110.

9211

0.92

110.

9211

0.92

110.

9211

0.92

110.

9211

0.92

110.

9211

0.92

110.

9211

0.92

117.

5111

7.51

117.

5111

7.51

117.

5111

7.51

117.

5111

7.51

117.

5111

7.51

117.

5111

7.51

117.

5111

7.51

117.

5111

7.51

117.

5111

7.51

117.

5111

7.51

117.

5111

7.51

133.

8913

3.89

133.

8913

3.89

133.

8913

3.89

133.

8913

3.89

133.

8913

3.89

133.

8913

3.89

133.

8913

3.89

133.

8913

3.89

133.

8913

3.89

133.

8913

3.89

133.

8913

3.89

144.

0814

4.08

144.

0814

4.08

144.

0814

4.08

144.

0814

4.08

144.

0814

4.08

144.

0814

4.08

144.

0814

4.08

144.

0814

4.08

144.

0814

4.08

144.

0814

4.08

144.

0814

4.08

149.

3814

9.38

149.

3814

9.38

149.

3814

9.38

149.

3814

9.38

149.

3814

9.38

149.

3814

9.38

149.

3814

9.38

149.

3814

9.38

149.

3814

9.38

149.

3814

9.38

149.

3814

9.38

150.

3315

0.33

150.

3315

0.33

150.

3315

0.33

150.

3315

0.33

150.

3315

0.33

150.

3315

0.33

150.

3315

0.33

150.

3315

0.33

150.

3315

0.33

150.

3315

0.33

150.

3315

0.33

150.

6315

0.63

150.

6315

0.63

150.

6315

0.63

150.

6315

0.63

150.

6315

0.63

150.

6315

0.63

150.

6315

0.63

150.

6315

0.63

150.

6315

0.63

150.

6315

0.63

150.

6315

0.63

171.

3317

1.33

171.

3317

1.33

171.

3317

1.33

171.

3317

1.33

171.

3317

1.33

171.

3317

1.33

171.

3317

1.33

171.

3317

1.33

171.

3317

1.33

171.

3317

1.33

171.

3317

1.33

193.

5519

3.55

193.

5519

3.55

193.

5519

3.55

193.

5519

3.55

193.

5519

3.55

193.

5519

3.55

193.

5519

3.55

193.

5519

3.55

193.

5519

3.55

193.

5519

3.55

193.

5519

3.55

JWL-BE1-004_ACID_PROTON_01

11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

6.001.092.272.083.031.894.941.851.010.811.00

M02(br. s.)

M08(m)

M01(d)

M07(s)

M04(t)

M11(m)

M06(m)

M10(m)

M03(d)

M09(m)

M05(m) 1.13

1.14

1.26

2.04

2.05

2.07

2.42

2.56

2.90

3.34

3.39

3.58

3.59

6.86

6.877.108.

108.

12

222

001B PROTON1HDMSO-d68 H's

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

3.01

2.01

0.94

0.85

1.00

M03(s)

M02(s)

DMSO-d6

M01(s)

M04(s)

M05(s)

Water

001B CARBON13CDMSO-d68 C's

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

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

M02(s)

M03(s)

M04(s)

M05(s)

M06(s)

M01(s)

M08(s)

M07(s)

DMSO-d6

16.9

816

.98

16.9

816

.98

16.9

816

.98

16.9

816

.98

16.9

816

.98

16.9

816

.98

16.9

816

.98

16.9

816

.98

16.9

816

.98

16.9

816

.98

16.9

816

.98

98.4

698

.46

98.4

698

.46

98.4

698

.46

98.4

698

.46

98.4

698

.46

98.4

698

.46

98.4

698

.46

98.4

698

.46

98.4

698

.46

98.4

698

.46

98.4

698

.46

112.

7111

2.71

112.

7111

2.71

112.

7111

2.71

112.

7111

2.71

112.

7111

2.71

112.

7111

2.71

112.

7111

2.71

112.

7111

2.71

112.

7111

2.71

112.

7111

2.71

112.

7111

2.71

120.

2012

0.20

120.

2012

0.20

120.

2012

0.20

120.

2012

0.20

120.

2012

0.20

120.

2012

0.20

120.

2012

0.20

120.

2012

0.20

120.

2012

0.20

120.

2012

0.20

120.

2012

0.20

123.

8212

3.82

123.

8212

3.82

123.

8212

3.82

123.

8212

3.82

123.

8212

3.82

123.

8212

3.82

123.

8212

3.82

123.

8212

3.82

123.

8212

3.82

123.

8212

3.82

123.

8212

3.82

134.

7213

4.72

134.

7213

4.72

134.

7213

4.72

134.

7213

4.72

134.

7213

4.72

134.

7213

4.72

134.

7213

4.72

134.

7213

4.72

134.

7213

4.72

134.

7213

4.72

134.

7213

4.72

154.

4915

4.49

154.

4915

4.49

154.

4915

4.49

154.

4915

4.49

154.

4915

4.49

154.

4915

4.49

154.

4915

4.49

154.

4915

4.49

154.

4915

4.49

154.

4915

4.49

154.

4915

4.49

223

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

5.85

2.97

2.01

3.03

2.11

0.97

0.99

DMSO-d6

Water

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.31

2.31

2.31

2.31

2.31

2.31

2.31

2.31

2.31

2.31

2.31

2.31

2.31

2.31

2.31

2.31

2.31

2.31

2.31

2.31

2.31

2.31

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.57

2.57

2.57

2.57

2.57

2.57

2.57

2.57

2.57

2.57

2.57

2.57

2.57

2.57

2.57

2.57

2.57

2.57

2.57

2.57

2.57

2.57

7.97

7.97

7.97

7.97

7.97

7.97

7.97

7.97

7.97

7.97

7.97

7.97

7.97

7.97

7.97

7.97

7.97

7.97

7.97

7.97

7.97

7.97

8.11

8.11

8.11

8.11

8.11

8.11

8.11

8.11

8.11

8.11

8.11

8.11

8.11

8.11

8.11

8.11

8.11

8.11

8.11

8.11

8.11

8.11

003B CARBON13CDMSO-d617 C's

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

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

M01(s)

M02(s)

M03(s)

M07(s)

M08(s)

M09(s)

M10(s)

M04(s)

M05(s)

M06(s)M11(s)

M12(s)

M13(s)

M14(s)

M15(s)

M16(s)

M17(s)

DMSO-d6

13.5

313

.53

13.5

313

.53

13.5

313

.53

13.5

313

.53

13.5

313

.53

13.5

313

.53

13.5

313

.53

13.5

313

.53

13.5

313

.53

13.5

313

.53

13.5

313

.53

17.1

917

.19

17.1

917

.19

17.1

917

.19

17.1

917

.19

17.1

917

.19

17.1

917

.19

17.1

917

.19

17.1

917

.19

17.1

917

.19

17.1

917

.19

17.1

917

.19

28.1

028

.10

28.1

028

.10

28.1

028

.10

28.1

028

.10

28.1

028

.10

28.1

028

.10

28.1

028

.10

28.1

028

.10

28.1

028

.10

28.1

028

.10

28.1

028

.10

34.9

634

.96

34.9

634

.96

34.9

634

.96

34.9

634

.96

34.9

634

.96

34.9

634

.96

34.9

634

.96

34.9

634

.96

34.9

634

.96

34.9

634

.96

34.9

634

.96

35.9

135

.91

35.9

135

.91

35.9

135

.91

35.9

135

.91

35.9

135

.91

35.9

135

.91

35.9

135

.91

35.9

135

.91

35.9

135

.91

35.9

135

.91

35.9

135

.91

52.4

152

.41

52.4

152

.41

52.4

152

.41

52.4

152

.41

52.4

152

.41

52.4

152

.41

52.4

152

.41

52.4

152

.41

52.4

152

.41

52.4

152

.41

52.4

152

.41

115.

7711

5.77

115.

7711

5.77

115.

7711

5.77

115.

7711

5.77

115.

7711

5.77

115.

7711

5.77

115.

7711

5.77

115.

7711

5.77

115.

7711

5.77

115.

7711

5.77

115.

7711

5.77

115.

9011

5.90

115.

9011

5.90

115.

9011

5.90

115.

9011

5.90

115.

9011

5.90

115.

9011

5.90

115.

9011

5.90

115.

9011

5.90

115.

9011

5.90

115.

9011

5.90

115.

9011

5.90

117.

1311

7.13

117.

1311

7.13

117.

1311

7.13

117.

1311

7.13

117.

1311

7.13

117.

1311

7.13

117.

1311

7.13

117.

1311

7.13

117.

1311

7.13

117.

1311

7.13

117.

1311

7.13

122.

2912

2.29

122.

2912

2.29

122.

2912

2.29

122.

2912

2.29

122.

2912

2.29

122.

2912

2.29

122.

2912

2.29

122.

2912

2.29

122.

2912

2.29

122.

2912

2.29

122.

2912

2.29

131.

9813

1.98

131.

9813

1.98

131.

9813

1.98

131.

9813

1.98

131.

9813

1.98

131.

9813

1.98

131.

9813

1.98

131.

9813

1.98

131.

9813

1.98

131.

9813

1.98

131.

9813

1.98

136.

7913

6.79

136.

7913

6.79

136.

7913

6.79

136.

7913

6.79

136.

7913

6.79

136.

7913

6.79

136.

7913

6.79

136.

7913

6.79

136.

7913

6.79

136.

7913

6.79

136.

7913

6.7913

7.72

137.

7213

7.72

137.

7213

7.72

137.

7213

7.72

137.

7213

7.72

137.

7213

7.72

137.

7213

7.72

137.

7213

7.72

137.

7213

7.72

137.

7213

7.72

137.

7213

7.72

137.

72

142.

0214

2.02

142.

0214

2.02

142.

0214

2.02

142.

0214

2.02

142.

0214

2.02

142.

0214

2.02

142.

0214

2.02

142.

0214

2.02

142.

0214

2.02

142.

0214

2.02

142.

0214

2.02

148.

9214

8.92

148.

9214

8.92

148.

9214

8.92

148.

9214

8.92

148.

9214

8.92

148.

9214

8.92

148.

9214

8.92

148.

9214

8.92

148.

9214

8.92

148.

9214

8.92

148.

9214

8.92

151.

9415

1.94

151.

9415

1.94

151.

9415

1.94

151.

9415

1.94

151.

9415

1.94

151.

9415

1.94

151.

9415

1.94

151.

9415

1.94

151.

9415

1.94

151.

9415

1.94

151.

9415

1.94

193.

0919

3.09

193.

0919

3.09

193.

0919

3.09

193.

0919

3.09

193.

0919

3.09

193.

0919

3.09

193.

0919

3.09

193.

0919

3.09

193.

0919

3.09

193.

0919

3.09

193.

0919

3.09

224

JWL-BE2-004B_PROTON_01

Chemical Shift (ppm)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

No

rm

alize

d I

nte

nsity

0

0.1

0.2

0.3

0.4

0.5

0.6

6.0

0

1.9

8

2.9

7

2.0

3

2.9

7

1.9

3

5.1

4

1.9

6

0.8

7

0.9

3

0.9

0

M07(s)

M06(s)

M05(s)

M08(d)

M03(s)

M10(m)

M02(s)

Water

M09(m)

M01(s)M04(quin)

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

0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

13.

48

13.

48

13.

48

13.

48

13.

48

13.

48

13.

48

13.

48

13.

48

13.

48

13.

48

13.

48

13.

48

13.

48

13.

48

13.

48

13.

48

13.

48

13.

48

13.

48

13.

48

13.

481

6.1

41

6.1

41

6.1

41

6.1

41

6.1

41

6.1

41

6.1

41

6.1

41

6.1

41

6.1

41

6.1

41

6.1

41

6.1

41

6.1

41

6.1

41

6.1

41

6.1

41

6.1

41

6.1

41

6.1

41

6.1

41

6.1

4

28.

09

28.

09

28.

09

28.

09

28.

09

28.

09

28.

09

28.

09

28.

09

28.

09

28.

09

28.

09

28.

09

28.

09

28.

09

28.

09

28.

09

28.

09

28.

09

28.

09

28.

09

28.

09

28.

71

28.

71

28.

71

28.

71

28.

71

28.

71

28.

71

28.

71

28.

71

28.

71

28.

71

28.

71

28.

71

28.

71

28.

71

28.

71

28.

71

28.

71

28.

71

28.

71

28.

71

28.

71

35.

29

35.

29

35.

29

35.

29

35.

29

35.

29

35.

29

35.

29

35.

29

35.

29

35.

29

35.

29

35.

29

35.

29

35.

29

35.

29

35.

29

35.

29

35.

29

35.

29

35.

29

35.

29

35.

79

35.

79

35.

79

35.

79

35.

79

35.

79

35.

79

35.

79

35.

79

35.

79

35.

79

35.

79

35.

79

35.

79

35.

79

35.

79

35.

79

35.

79

35.

79

35.

79

35.

79

35.

79

40.

75

40.

75

40.

75

40.

75

40.

75

40.

75

40.

75

40.

75

40.

75

40.

75

40.

75

40.

75

40.

75

40.

75

40.

75

40.

75

40.

75

40.

75

40.

75

40.

75

40.

75

40.

75

52.

38

52.

38

52.

38

52.

38

52.

38

52.

38

52.

38

52.

38

52.

38

52.

38

52.

38

52.

38

52.

38

52.

38

52.

38

52.

38

52.

38

52.

38

52.

38

52.

38

52.

38

52.

38

58.

33

58.

33

58.

33

58.

33

58.

33

58.

33

58.

33

58.

33

58.

33

58.

33

58.

33

58.

33

58.

33

58.

33

58.

33

58.

33

58.

33

58.

33

58.

33

58.

33

58.

33

58.

33

70.

42

70.

42

70.

42

70.

42

70.

42

70.

42

70.

42

70.

42

70.

42

70.

42

70.

42

70.

42

70.

42

70.

42

70.

42

70.

42

70.

42

70.

42

70.

42

70.

42

70.

42

70.

42

95.

78

95.

78

95.

78

95.

78

95.

78

95.

78

95.

78

95.

78

95.

78

95.

78

95.

78

95.

78

95.

78

95.

78

95.

78

95.

78

95.

78

95.

78

95.

78

95.

78

95.

78

95.

78

110

.27

110

.27

110

.27

110

.27

110

.27

110

.27

110

.27

110

.27

110

.27

110

.27

110

.27

110

.27

110

.27

110

.27

110

.27

110

.27

110

.27

110

.27

110

.27

110

.27

110

.27

110

.27

115

.51

115

.51

115

.51

115

.51

115

.51

115

.51

115

.51

115

.51

115

.51

115

.51

115

.51

115

.51

115

.51

115

.51

115

.51

115

.51

115

.51

115

.51

115

.51

115

.51

115

.51

115

.51

117

.70

117

.70

117

.70

117

.70

117

.70

117

.70

117

.70

117

.70

117

.70

117

.70

117

.70

117

.70

117

.70

117

.70

117

.70

117

.70

117

.70

117

.70

117

.70

117

.70

117

.70

117

.70

122

.05

122

.05

122

.05

122

.05

122

.05

122

.05

122

.05

122

.05

122

.05

122

.05

122

.05

122

.05

122

.05

122

.05

122

.05

122

.05

122

.05

122

.05

122

.05

122

.05

122

.05

122

.05

135

.77

135

.77

135

.77

135

.77

135

.77

135

.77

135

.77

135

.77

135

.77

135

.77

135

.77

135

.77

135

.77

135

.77

135

.77

135

.77

135

.77

135

.77

135

.77

135

.77

135

.77

135

.77

142

.56

142

.56

142

.56

142

.56

142

.56

142

.56

142

.56

142

.56

142

.56

142

.56

142

.56

142

.56

142

.56

142

.56

142

.56

142

.56

142

.56

142

.56

142

.56

142

.56

142

.56

142

.56

148

.17

148

.17

148

.17

148

.17

148

.17

148

.17

148

.17

148

.17

148

.17

148

.17

148

.17

148

.17

148

.17

148

.17

148

.17

148

.17

148

.17

148

.17

148

.17

148

.17

148

.17

148

.17

149

.98

149

.98

149

.98

149

.98

149

.98

149

.98

149

.98

149

.98

149

.98

149

.98

149

.98

149

.98

149

.98

149

.98

149

.98

149

.98

149

.98

149

.98

149

.98

149

.98

149

.98

149

.98

151

.18

151

.18

151

.18

151

.18

151

.18

151

.18

151

.18

151

.18

151

.18

151

.18

151

.18

151

.18

151

.18

151

.18

151

.18

151

.18

151

.18

151

.18

151

.18

151

.18

151

.18

151

.18

192

.94

192

.94

192

.94

192

.94

192

.94

192

.94

192

.94

192

.94

192

.94

192

.94

192

.94

192

.94

192

.94

192

.94

192

.94

192

.94

192

.94

192

.94

192

.94

192

.94

192

.94

192

.94

225

004BB PROTON1HDMSO-d630 H's

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

5.66

1.93

2.83

1.93

2.78

1.74

1.98

2.81

1.93

0.93

0.93

0.95

0.92

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.73

1.73

1.73

1.73

1.73

1.73

1.73

1.73

1.73

1.73

1.73

1.73

1.73

1.73

1.73

1.73

1.73

1.73

1.73

1.73

1.73

1.73

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.89

1.89

1.89

1.89

1.89

1.89

1.89

1.89

1.89

1.89

1.89

1.89

1.89

1.89

1.89

1.89

1.89

1.89

1.89

1.89

1.89

1.89

2.30

2.30

2.30

2.30

2.30

2.30

2.30

2.30

2.30

2.30

2.30

2.30

2.30

2.30

2.30

2.30

2.30

2.30

2.30

2.30

2.30

2.30

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

2.38

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.12

3.12

3.12

3.12

3.12

3.12

3.12

3.12

3.12

3.12

3.12

3.12

3.12

3.12

3.12

3.12

3.12

3.12

3.12

3.12

3.12

3.12

3.13

3.13

3.13

3.13

3.13

3.13

3.13

3.13

3.13

3.13

3.13

3.13

3.13

3.13

3.13

3.13

3.13

3.13

3.13

3.13

3.13

3.13

3.21

3.21

3.21

3.21

3.21

3.21

3.21

3.21

3.21

3.21

3.21

3.21

3.21

3.21

3.21

3.21

3.21

3.21

3.21

3.21

3.21

3.21

6.53

6.53

6.53

6.53

6.53

6.53

6.53

6.53

6.53

6.53

6.53

6.53

6.53

6.53

6.53

6.53

6.53

6.53

6.53

6.53

6.53

6.53

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.64

7.64

7.64

7.64

7.64

7.64

7.64

7.64

7.64

7.64

7.64

7.64

7.64

7.64

7.64

7.64

7.64

7.64

7.64

7.64

7.64

7.64

8.04

8.04

8.04

8.04

8.04

8.04

8.04

8.04

8.04

8.04

8.04

8.04

8.04

8.04

8.04

8.04

8.04

8.04

8.04

8.04

8.04

8.04

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.06

8.06

8.06

8.06

8.06

8.06

8.06

8.06

8.06

8.06

8.06

8.06

8.06

8.06

8.06

8.06

8.06

8.06

8.06

8.06

8.06

8.06

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

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

DMSO-d6

13.5

613

.56

13.5

613

.56

13.5

613

.56

13.5

613

.56

13.5

613

.56

13.5

613

.56

13.5

613

.56

13.5

613

.56

13.5

613

.56

13.5

613

.56

13.5

613

.56

16.5

016

.50

16.5

016

.50

16.5

016

.50

16.5

016

.50

16.5

016

.50

16.5

016

.50

16.5

016

.50

16.5

016

.50

16.5

016

.50

16.5

016

.50

16.5

016

.50

28.1

628

.16

28.1

628

.16

28.1

628

.16

28.1

628

.16

28.1

628

.16

28.1

628

.16

28.1

628

.16

28.1

628

.16

28.1

628

.16

28.1

628

.16

28.1

628

.16

29.1

129

.11

29.1

129

.11

29.1

129

.11

29.1

129

.11

29.1

129

.11

29.1

129

.11

29.1

129

.11

29.1

129

.11

29.1

129

.11

29.1

129

.11

29.1

129

.11

35.4

135

.41

35.4

135

.41

35.4

135

.41

35.4

135

.41

35.4

135

.41

35.4

135

.41

35.4

135

.41

35.4

135

.41

35.4

135

.41

35.4

135

.41

35.4

135

.41

35.8

235

.82

35.8

235

.82

35.8

235

.82

35.8

235

.82

35.8

235

.82

35.8

235

.82

35.8

235

.82

35.8

235

.82

35.8

235

.82

35.8

235

.82

35.8

235

.82

39.8

939

.89

39.8

939

.89

39.8

939

.89

39.8

939

.89

39.8

939

.89

39.8

939

.89

39.8

939

.89

39.8

939

.89

39.8

939

.89

39.8

939

.89

39.8

939

.89

52.4

252

.42

52.4

252

.42

52.4

252

.42

52.4

252

.42

52.4

252

.42

52.4

252

.42

52.4

252

.42

52.4

252

.42

52.4

252

.42

52.4

252

.42

52.4

252

.42

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

70.0

370

.03

70.0

370

.03

70.0

370

.03

70.0

370

.03

70.0

370

.03

70.0

370

.03

70.0

370

.03

70.0

370

.03

70.0

370

.03

70.0

370

.03

70.0

370

.03

109.

9210

9.92

109.

9210

9.92

109.

9210

9.92

109.

9210

9.92

109.

9210

9.92

109.

9210

9.92

109.

9210

9.92

109.

9210

9.92

109.

9210

9.92

109.

9210

9.92

109.

9210

9.92

115.

3311

5.33

115.

3311

5.33

115.

3311

5.33

115.

3311

5.33

115.

3311

5.33

115.

3311

5.33

115.

3311

5.33

115.

3311

5.33

115.

3311

5.33

115.

3311

5.33

115.

3311

5.33

115.

4811

5.48

115.

4811

5.48

115.

4811

5.48

115.

4811

5.48

115.

4811

5.48

115.

4811

5.48

115.

4811

5.48

115.

4811

5.48

115.

4811

5.48

115.

4811

5.48

115.

4811

5.48

119.

4511

9.45

119.

4511

9.45

119.

4511

9.45

119.

4511

9.45

119.

4511

9.45

119.

4511

9.45

119.

4511

9.45

119.

4511

9.45

119.

4511

9.45

119.

4511

9.45

119.

4511

9.4513

2.05

132.

0513

2.05

132.

0513

2.05

132.

0513

2.05

132.

0513

2.05

132.

0513

2.05

132.

0513

2.05

132.

0513

2.05

132.

0513

2.05

132.

0513

2.05

132.

0513

2.05

132.

05

140.

8314

0.83

140.

8314

0.83

140.

8314

0.83

140.

8314

0.83

140.

8314

0.83

140.

8314

0.83

140.

8314

0.83

140.

8314

0.83

140.

8314

0.83

140.

8314

0.83

140.

8314

0.83

147.

8614

7.86

147.

8614

7.86

147.

8614

7.86

147.

8614

7.86

147.

8614

7.86

147.

8614

7.86

147.

8614

7.86

147.

8614

7.86

147.

8614

7.86

147.

8614

7.86

147.

8614

7.86

148.

9414

8.94

148.

9414

8.94

148.

9414

8.94

148.

9414

8.94

148.

9414

8.94

148.

9414

8.94

148.

9414

8.94

148.

9414

8.94

148.

9414

8.94

148.

9414

8.94

148.

9414

8.94

151.

0715

1.07

151.

0715

1.07

151.

0715

1.07

151.

0715

1.07

151.

0715

1.07

151.

0715

1.07

151.

0715

1.07

151.

0715

1.07

151.

0715

1.07

151.

0715

1.07

151.

0715

1.07

171.

2017

1.20

171.

2017

1.20

171.

2017

1.20

171.

2017

1.20

171.

2017

1.20

171.

2017

1.20

171.

2017

1.20

171.

2017

1.20

171.

2017

1.20

171.

2017

1.20

171.

2017

1.20

193.

0319

3.03

193.

0319

3.03

193.

0319

3.03

193.

0319

3.03

193.

0319

3.03

193.

0319

3.03

193.

0319

3.03

193.

0319

3.03

193.

0319

3.03

193.

0319

3.03

193.

0319

3.03

226

Appendix III

Chapter 3 – NMR Spectra

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.86

1.08

1.03

1.00

CHLOROFORM-d

Water

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

6.54

6.54

6.54

6.54

6.54

6.54

6.54

6.54

6.54

6.54

6.54

6.54

6.54

6.54

6.54

6.54

6.54

6.54

6.54

6.54

6.54

6.54

6.55

6.55

6.55

6.55

6.55

6.55

6.55

6.55

6.55

6.55

6.55

6.55

6.55

6.55

6.55

6.55

6.55

6.55

6.55

6.55

6.55

6.55

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.57

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.87

6.87

6.87

6.87

6.87

6.87

6.87

6.87

6.87

6.87

6.87

6.87

6.87

6.87

6.87

6.87

6.87

6.87

6.87

6.87

6.87

6.87

7.35

7.35

7.35

7.35

7.35

7.35

7.35

7.35

7.35

7.35

7.35

7.35

7.35

7.35

7.35

7.35

7.35

7.35

7.35

7.35

7.35

7.35

7.37

7.37

7.37

7.37

7.37

7.37

7.37

7.37

7.37

7.37

7.37

7.37

7.37

7.37

7.37

7.37

7.37

7.37

7.37

7.37

7.37

7.37

227

JWL-BE1-501_PROTON_01

11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.05

0.10

0.15

Nor

mal

ized

Inte

nsity

6.002.012.000.950.890.91

M04(s) M06(s)

M05(s)

M02(dd)

M01(dd)

M03(dd)

1.10

2.53

3.03

7.25

7.267.27

7.288.

238.23

8.258.25

8.678.68

8.68

8.69

JWL-BE1-005_CARBON_01

220 200 180 160 140 120 100 80 60 40 20 0 -20

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

M02(s)

M04(s)

M05(s)

M07(s)

M01(s)

M03(s)

M09(s)

M06(s)

M08(s)

28.2

432

.95

46.3

752.0

1

122.

14

134.

55

153.

78

162.

22

198.

01

228

JWL-BE2-013_P_PROTON_01

Chemical Shift (ppm)11 10 9 8 7 6 5 4 3 2 1 0

Nor

mal

ized

Inte

nsity

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

3.053.000.841.401.970.910.961.000.990.950.970.960.90

M13(s)

M12(s)

M02(s)

M09(s)

M04(d)

M05(d)

M11(m)M01(d)

M03(m)

M06(br. s.)M08(d)

M10(m)M07(d)

8.47

8.47

8.47

8.47

8.47

8.47

8.47

8.47

8.47

8.47

8.47

8.47

8.47

8.47

8.47

8.47

8.47

8.47

8.47

8.47

8.47

8.47

7.69

7.69

7.69

7.69

7.69

7.69

7.69

7.69

7.69

7.69

7.69

7.69

7.69

7.69

7.69

7.69

7.69

7.69

7.69

7.69

7.69

7.69 7.67

7.67

7.67

7.67

7.67

7.67

7.67

7.67

7.67

7.67

7.67

7.67

7.67

7.67

7.67

7.67

7.67

7.67

7.67

7.67

7.67

7.67 7.41

7.41

7.41

7.41

7.41

7.41

7.41

7.41

7.41

7.41

7.41

7.41

7.41

7.41

7.41

7.41

7.41

7.41

7.41

7.41

7.41

7.41 7.39

7.39

7.39

7.39

7.39

7.39

7.39

7.39

7.39

7.39

7.39

7.39

7.39

7.39

7.39

7.39

7.39

7.39

7.39

7.39

7.39

7.39

7.15

7.15

7.15

7.15

7.15

7.15

7.15

7.15

7.15

7.15

7.15

7.15

7.15

7.15

7.15

7.15

7.15

7.15

7.15

7.15

7.15

7.15

6.88

6.88

6.88

6.88

6.88

6.88

6.88

6.88

6.88

6.88

6.88

6.88

6.88

6.88

6.88

6.88

6.88

6.88

6.88

6.88

6.88

6.88

6.58

6.58

6.58

6.58

6.58

6.58

6.58

6.58

6.58

6.58

6.58

6.58

6.58

6.58

6.58

6.58

6.58

6.58

6.58

6.58

6.58

6.58

6.56

6.56

6.56

6.56

6.56

6.56

6.56

6.56

6.56

6.56

6.56

6.56

6.56

6.56

6.56

6.56

6.56

6.56

6.56

6.56

6.56

6.56

4.75

4.75

4.75

4.75

4.75

4.75

4.75

4.75

4.75

4.75

4.75

4.75

4.75

4.75

4.75

4.75

4.75

4.75

4.75

4.75

4.75

4.75

4.73

4.73

4.73

4.73

4.73

4.73

4.73

4.73

4.73

4.73

4.73

4.73

4.73

4.73

4.73

4.73

4.73

4.73

4.73

4.73

4.73

4.73 4.

424.

424.

424.

424.

424.

424.

424.

424.

424.

424.

424.

424.

424.

424.

424.

424.

424.

424.

424.

424.

424.

424.

404.

404.

404.

404.

404.

404.

404.

404.

404.

404.

404.

404.

404.

404.

404.

404.

404.

404.

404.

404.

404.

40

2.81

2.81

2.81

2.81

2.81

2.81

2.81

2.81

2.81

2.81

2.81

2.81

2.81

2.81

2.81

2.81

2.81

2.81

2.81

2.81

2.81

2.81

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.06

2.06

2.06

2.06

2.06

2.06

2.06

2.06

2.06

2.06

2.06

2.06

2.06

2.06

2.06

2.06

2.06

2.06

2.06

2.06

2.06

2.06 2.04

2.04

2.04

2.04

2.04

2.04

2.04

2.04

2.04

2.04

2.04

2.04

2.04

2.04

2.04

2.04

2.04

2.04

2.04

2.04

2.04

2.04

2.03

2.03

2.03

2.03

2.03

2.03

2.03

2.03

2.03

2.03

2.03

2.03

2.03

2.03

2.03

2.03

2.03

2.03

2.03

2.03

2.03

2.03

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.55

1.55

1.55

1.55

1.55

1.55

1.55

1.55

1.55

1.55

1.55

1.55

1.55

1.55

1.55

1.55

1.55

1.55

1.55

1.55

1.55

1.55

1.52

1.52

1.52

1.52

1.52

1.52

1.52

1.52

1.52

1.52

1.52

1.52

1.52

1.52

1.52

1.52

1.52

1.52

1.52

1.52

1.52

1.52

1.13

1.13

1.13

1.13

1.13

1.13

1.13

1.13

1.13

1.13

1.13

1.13

1.13

1.13

1.13

1.13

1.13

1.13

1.13

1.13

1.13

1.13

1.07

1.07

1.07

1.07

1.07

1.07

1.07

1.07

1.07

1.07

1.07

1.07

1.07

1.07

1.07

1.07

1.07

1.07

1.07

1.07

1.07

1.07

013 C

Chemical Shift (ppm)192 184 176 168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24

No

rma

lize

d I

nte

nsity

0

0.1

0.2

0.3

0.4

0.5

0.6

M19(s)

M04(m)

M05(m)

M09(m)M07(m)

M08(m)

M06(m)

M02(m)

M01(m)

M11(m)

M10(m)

M03(m)

M13(m)M17(m)

M16(m)

M14(m) M18(m)

M15(m)

M12(m)

77

.00

77

.00

77

.00

77

.00

77

.00

77

.00

77

.00

77

.00

77

.00

77

.00

77

.00

77

.00

77

.00

77

.00

77

.00

77

.00

77

.00

77

.00

77

.00

77

.00

77

.00

77

.00

229

Chemical Shift (ppm)11 10 9 8 7 6 5 4 3 2

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

2.98

2.89

1.16

2.14

1.19

2.15

3.48

3.77

1.07

1.06

1.04

1.07

1.05

1.00

1.07

1.06

1.00

Water

DMSO-d6

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

1.54

1.54

1.54

1.54

1.54

1.54

1.54

1.54

1.54

1.54

1.54

1.54

1.54

1.54

1.54

1.54

1.54

1.54

1.54

1.54

1.54

1.541.

721.

721.

721.

721.

721.

721.

721.

721.

721.

721.

721.

721.

721.

721.

721.

721.

721.

721.

721.

721.

721.

72

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.74

1.75

1.75

1.75

1.75

1.75

1.75

1.75

1.75

1.75

1.75

1.75

1.75

1.75

1.75

1.75

1.75

1.75

1.75

1.75

1.75

1.75

1.75

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.87

1.88

1.88

1.88

1.88

1.88

1.88

1.88

1.88

1.88

1.88

1.88

1.88

1.88

1.88

1.88

1.88

1.88

1.88

1.88

1.88

1.88

1.88

2.67

2.67

2.67

2.67

2.67

2.67

2.67

2.67

2.67

2.67

2.67

2.67

2.67

2.67

2.67

2.67

2.67

2.67

2.67

2.67

2.67

2.672.

692.

692.

692.

692.

692.

692.

692.

692.

692.

692.

692.

692.

692.

692.

692.

692.

692.

692.

692.

692.

692.

69

3.06

3.06

3.06

3.06

3.06

3.06

3.06

3.06

3.06

3.06

3.06

3.06

3.06

3.06

3.06

3.06

3.06

3.06

3.06

3.06

3.06

3.06

3.08

3.08

3.08

3.08

3.08

3.08

3.08

3.08

3.08

3.08

3.08

3.08

3.08

3.08

3.08

3.08

3.08

3.08

3.08

3.08

3.08

3.08

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.10

3.11

3.11

3.11

3.11

3.11

3.11

3.11

3.11

3.11

3.11

3.11

3.11

3.11

3.11

3.11

3.11

3.11

3.11

3.11

3.11

3.11

3.11

3.16

3.16

3.16

3.16

3.16

3.16

3.16

3.16

3.16

3.16

3.16

3.16

3.16

3.16

3.16

3.16

3.16

3.16

3.16

3.16

3.16

3.163.17

3.17

3.17

3.17

3.17

3.17

3.17

3.17

3.17

3.17

3.17

3.17

3.17

3.17

3.17

3.17

3.17

3.17

3.17

3.17

3.17

3.17

3.31

3.31

3.31

3.31

3.31

3.31

3.31

3.31

3.31

3.31

3.31

3.31

3.31

3.31

3.31

3.31

3.31

3.31

3.31

3.31

3.31

3.31

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.38

3.38

3.38

3.38

3.38

3.38

3.38

3.38

3.38

3.38

3.38

3.38

3.38

3.38

3.38

3.38

3.38

3.38

3.38

3.38

3.38

3.38

4.69

4.69

4.69

4.69

4.69

4.69

4.69

4.69

4.69

4.69

4.69

4.69

4.69

4.69

4.69

4.69

4.69

4.69

4.69

4.69

4.69

4.694.

714.

714.

714.

714.

714.

714.

714.

714.

714.

714.

714.

714.

714.

714.

714.

714.

714.

714.

714.

714.

714.

71

4.72

4.72

4.72

4.72

4.72

4.72

4.72

4.72

4.72

4.72

4.72

4.72

4.72

4.72

4.72

4.72

4.72

4.72

4.72

4.72

4.72

4.72

4.74

4.74

4.74

4.74

4.74

4.74

4.74

4.74

4.74

4.74

4.74

4.74

4.74

4.74

4.74

4.74

4.74

4.74

4.74

4.74

4.74

4.745.

605.

605.

605.

605.

605.

605.

605.

605.

605.

605.

605.

605.

605.

605.

605.

605.

605.

605.

605.

605.

605.

60

5.61

5.61

5.61

5.61

5.61

5.61

5.61

5.61

5.61

5.61

5.61

5.61

5.61

5.61

5.61

5.61

5.61

5.61

5.61

5.61

5.61

5.615.

875.

875.

875.

875.

875.

875.

875.

875.

875.

875.

875.

875.

875.

875.

875.

875.

875.

875.

875.

875.

875.

87

6.05

6.05

6.05

6.05

6.05

6.05

6.05

6.05

6.05

6.05

6.05

6.05

6.05

6.05

6.05

6.05

6.05

6.05

6.05

6.05

6.05

6.056.67

6.67

6.67

6.67

6.67

6.67

6.67

6.67

6.67

6.67

6.67

6.67

6.67

6.67

6.67

6.67

6.67

6.67

6.67

6.67

6.67

6.67

6.68

6.68

6.68

6.68

6.68

6.68

6.68

6.68

6.68

6.68

6.68

6.68

6.68

6.68

6.68

6.68

6.68

6.68

6.68

6.68

6.68

6.68

7.10

7.10

7.10

7.10

7.10

7.10

7.10

7.10

7.10

7.10

7.10

7.10

7.10

7.10

7.10

7.10

7.10

7.10

7.10

7.10

7.10

7.10

7.12

7.12

7.12

7.12

7.12

7.12

7.12

7.12

7.12

7.12

7.12

7.12

7.12

7.12

7.12

7.12

7.12

7.12

7.12

7.12

7.12

7.12

7.19

7.19

7.19

7.19

7.19

7.19

7.19

7.19

7.19

7.19

7.19

7.19

7.19

7.19

7.19

7.19

7.19

7.19

7.19

7.19

7.19

7.19

7.20

7.20

7.20

7.20

7.20

7.20

7.20

7.20

7.20

7.20

7.20

7.20

7.20

7.20

7.20

7.20

7.20

7.20

7.20

7.20

7.20

7.207.

627.

627.

627.

627.

627.

627.

627.

627.

627.

627.

627.

627.

627.

627.

627.

627.

627.

627.

627.

627.

627.

62

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.638.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.36

8.37

8.37

8.37

8.37

8.37

8.37

8.37

8.37

8.37

8.37

8.37

8.37

8.37

8.37

8.37

8.37

8.37

8.37

8.37

8.37

8.37

8.37

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

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

26.1

026

.10

26.1

026

.10

26.1

026

.10

26.1

026

.10

26.1

026

.10

26.1

026

.10

26.1

026

.10

26.1

026

.10

26.1

026

.10

26.1

026

.10

26.1

026

.10

28.7

428

.74

28.7

428

.74

28.7

428

.74

28.7

428

.74

28.7

428

.74

28.7

428

.74

28.7

428

.74

28.7

428

.74

28.7

428

.74

28.7

428

.74

28.7

428

.74

30.6

830

.68

30.6

830

.68

30.6

830

.68

30.6

830

.68

30.6

830

.68

30.6

830

.68

30.6

830

.68

30.6

830

.68

30.6

830

.68

30.6

830

.68

30.6

830

.68

31.5

431

.54

31.5

431

.54

31.5

431

.54

31.5

431

.54

31.5

431

.54

31.5

431

.54

31.5

431

.54

31.5

431

.54

31.5

431

.54

31.5

431

.54

31.5

431

.54

40.7

440

.74

40.7

440

.74

40.7

440

.74

40.7

440

.74

40.7

440

.74

40.7

440

.74

40.7

440

.74

40.7

440

.74

40.7

440

.74

40.7

440

.74

40.7

440

.74

42.4

542

.45

42.4

542

.45

42.4

542

.45

42.4

542

.45

42.4

542

.45

42.4

542

.45

42.4

542

.45

42.4

542

.45

42.4

542

.45

42.4

542

.45

42.4

542

.45

46.3

646

.36

46.3

646

.36

46.3

646

.36

46.3

646

.36

46.3

646

.36

46.3

646

.36

46.3

646

.36

46.3

646

.36

46.3

646

.36

46.3

646

.36

46.3

646

.36

48.5

648

.56

48.5

648

.56

48.5

648

.56

48.5

648

.56

48.5

648

.56

48.5

648

.56

48.5

648

.56

48.5

648

.56

48.5

648

.56

48.5

648

.56

48.5

648

.5658

.40

58.4

058

.40

58.4

058

.40

58.4

058

.40

58.4

058

.40

58.4

058

.40

58.4

058

.40

58.4

058

.40

58.4

058

.40

58.4

058

.40

58.4

058

.40

58.4

0

70.7

170

.71

70.7

170

.71

70.7

170

.71

70.7

170

.71

70.7

170

.71

70.7

170

.71

70.7

170

.71

70.7

170

.71

70.7

170

.71

70.7

170

.71

70.7

170

.71

82.4

182

.41

82.4

182

.41

82.4

182

.41

82.4

182

.41

82.4

182

.41

82.4

182

.41

82.4

182

.41

82.4

182

.41

82.4

182

.41

82.4

182

.41

82.4

182

.41

92.6

092

.60

92.6

092

.60

92.6

092

.60

92.6

092

.60

92.6

092

.60

92.6

092

.60

92.6

092

.60

92.6

092

.60

92.6

092

.60

92.6

092

.60

92.6

092

.60

102.

8010

2.80

102.

8010

2.80

102.

8010

2.80

102.

8010

2.80

102.

8010

2.80

102.

8010

2.80

102.

8010

2.80

102.

8010

2.80

102.

8010

2.80

102.

8010

2.80

102.

8010

2.8012

0.21

120.

2112

0.21

120.

2112

0.21

120.

2112

0.21

120.

2112

0.21

120.

2112

0.21

120.

2112

0.21

120.

2112

0.21

120.

2112

0.21

120.

2112

0.21

120.

2112

0.21

120.

21

121.

9312

1.93

121.

9312

1.93

121.

9312

1.93

121.

9312

1.93

121.

9312

1.93

121.

9312

1.93

121.

9312

1.93

121.

9312

1.93

121.

9312

1.93

121.

9312

1.93

121.

9312

1.93

132.

9513

2.95

132.

9513

2.95

132.

9513

2.95

132.

9513

2.95

132.

9513

2.95

132.

9513

2.95

132.

9513

2.95

132.

9513

2.95

132.

9513

2.95

132.

9513

2.95

132.

9513

2.95

134.

4213

4.42

134.

4213

4.42

134.

4213

4.42

134.

4213

4.42

134.

4213

4.42

134.

4213

4.42

134.

4213

4.42

134.

4213

4.42

134.

4213

4.42

134.

4213

4.42

134.

4213

4.42

135.

3213

5.32

135.

3213

5.32

135.

3213

5.32

135.

3213

5.32

135.

3213

5.32

135.

3213

5.32

135.

3213

5.32

135.

3213

5.32

135.

3213

5.32

135.

3213

5.32

135.

3213

5.32

148.

3114

8.31

148.

3114

8.31

148.

3114

8.31

148.

3114

8.31

148.

3114

8.31

148.

3114

8.31

148.

3114

8.31

148.

3114

8.31

148.

3114

8.31

148.

3114

8.31

148.

3114

8.31

152.

2715

2.27

152.

2715

2.27

152.

2715

2.27

152.

2715

2.27

152.

2715

2.27

152.

2715

2.27

152.

2715

2.27

152.

2715

2.27

152.

2715

2.27

152.

2715

2.27

152.

2715

2.27

153.

6415

3.64

153.

6415

3.64

153.

6415

3.64

153.

6415

3.64

153.

6415

3.64

153.

6415

3.64

153.

6415

3.64

153.

6415

3.64

153.

6415

3.64

153.

6415

3.64

153.

6415

3.64

156.

5315

6.53

156.

5315

6.53

156.

5315

6.53

156.

5315

6.53

156.

5315

6.53

156.

5315

6.53

156.

5315

6.53

156.

5315

6.53

156.

5315

6.53

156.

5315

6.53

156.

5315

6.53

230

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

3.08

3.12

1.07

2.15

1.32

2.12

2.07

3.01

2.11

1.01

1.06

1.85

2.04

0.99

1.00

0.98

0.98

1.00

Water

CHLOROFORM-d

1.10

1.10

1.10

1.10

1.10

1.10

1.10

1.10

1.10

1.10

1.10

1.10

1.10

1.10

1.10

1.10

1.10

1.10

1.10

1.10

1.10

1.101.

161.

161.

161.

161.

161.

161.

161.

161.

161.

161.

161.

161.

161.

161.

161.

161.

161.

161.

161.

161.

161.

161.

581.

581.

581.

581.

581.

581.

581.

581.

581.

581.

581.

581.

581.

581.

581.

581.

581.

581.

581.

581.

581.

58

1.61

1.61

1.61

1.61

1.61

1.61

1.61

1.61

1.61

1.61

1.61

1.61

1.61

1.61

1.61

1.61

1.61

1.61

1.61

1.61

1.61

1.61

1.90

1.90

1.90

1.90

1.90

1.90

1.90

1.90

1.90

1.90

1.90

1.90

1.90

1.90

1.90

1.90

1.90

1.90

1.90

1.90

1.90

1.90

1.92

1.92

1.92

1.92

1.92

1.92

1.92

1.92

1.92

1.92

1.92

1.92

1.92

1.92

1.92

1.92

1.92

1.92

1.92

1.92

1.92

1.92

1.93

1.93

1.93

1.93

1.93

1.93

1.93

1.93

1.93

1.93

1.93

1.93

1.93

1.93

1.93

1.93

1.93

1.93

1.93

1.93

1.93

1.93

1.94

1.94

1.94

1.94

1.94

1.94

1.94

1.94

1.94

1.94

1.94

1.94

1.94

1.94

1.94

1.94

1.94

1.94

1.94

1.94

1.94

1.94

2.11

2.11

2.11

2.11

2.11

2.11

2.11

2.11

2.11

2.11

2.11

2.11

2.11

2.11

2.11

2.11

2.11

2.11

2.11

2.11

2.11

2.11

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.832.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

2.83

3.22

3.22

3.22

3.22

3.22

3.22

3.22

3.22

3.22

3.22

3.22

3.22

3.22

3.22

3.22

3.22

3.22

3.22

3.22

3.22

3.22

3.22

3.23

3.23

3.23

3.23

3.23

3.23

3.23

3.23

3.23

3.23

3.23

3.23

3.23

3.23

3.23

3.23

3.23

3.23

3.23

3.23

3.23

3.23

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.51

3.51

3.51

3.51

3.51

3.51

3.51

3.51

3.51

3.51

3.51

3.51

3.51

3.51

3.51

3.51

3.51

3.51

3.51

3.51

3.51

3.51

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

4.15

4.15

4.15

4.15

4.15

4.15

4.15

4.15

4.15

4.15

4.15

4.15

4.15

4.15

4.15

4.15

4.15

4.15

4.15

4.15

4.15

4.15

4.17

4.17

4.17

4.17

4.17

4.17

4.17

4.17

4.17

4.17

4.17

4.17

4.17

4.17

4.17

4.17

4.17

4.17

4.17

4.17

4.17

4.17

4.77

4.77

4.77

4.77

4.77

4.77

4.77

4.77

4.77

4.77

4.77

4.77

4.77

4.77

4.77

4.77

4.77

4.77

4.77

4.77

4.77

4.774.79

4.79

4.79

4.79

4.79

4.79

4.79

4.79

4.79

4.79

4.79

4.79

4.79

4.79

4.79

4.79

4.79

4.79

4.79

4.79

4.79

4.79

4.80

4.80

4.80

4.80

4.80

4.80

4.80

4.80

4.80

4.80

4.80

4.80

4.80

4.80

4.80

4.80

4.80

4.80

4.80

4.80

4.80

4.80

4.82

4.82

4.82

4.82

4.82

4.82

4.82

4.82

4.82

4.82

4.82

4.82

4.82

4.82

4.82

4.82

4.82

4.82

4.82

4.82

4.82

4.825.

495.

495.

495.

495.

495.

495.

495.

495.

495.

495.

495.

495.

495.

495.

495.

495.

495.

495.

495.

495.

495.

49

5.92

5.92

5.92

5.92

5.92

5.92

5.92

5.92

5.92

5.92

5.92

5.92

5.92

5.92

5.92

5.92

5.92

5.92

5.92

5.92

5.92

5.92

5.93

5.93

5.93

5.93

5.93

5.93

5.93

5.93

5.93

5.93

5.93

5.93

5.93

5.93

5.93

5.93

5.93

5.93

5.93

5.93

5.93

5.93

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

5.95

7.16

7.16

7.16

7.16

7.16

7.16

7.16

7.16

7.16

7.16

7.16

7.16

7.16

7.16

7.16

7.16

7.16

7.16

7.16

7.16

7.16

7.16

7.17

7.17

7.17

7.17

7.17

7.17

7.17

7.17

7.17

7.17

7.17

7.17

7.17

7.17

7.17

7.17

7.17

7.17

7.17

7.17

7.17

7.17

7.26

7.26

7.26

7.26

7.26

7.26

7.26

7.26

7.26

7.26

7.26

7.26

7.26

7.26

7.26

7.26

7.26

7.26

7.26

7.26

7.26

7.267.28

7.28

7.28

7.28

7.28

7.28

7.28

7.28

7.28

7.28

7.28

7.28

7.28

7.28

7.28

7.28

7.28

7.28

7.28

7.28

7.28

7.28

7.83

7.83

7.83

7.83

7.83

7.83

7.83

7.83

7.83

7.83

7.83

7.83

7.83

7.83

7.83

7.83

7.83

7.83

7.83

7.83

7.83

7.83

7.84

7.84

7.84

7.84

7.84

7.84

7.84

7.84

7.84

7.84

7.84

7.84

7.84

7.84

7.84

7.84

7.84

7.84

7.84

7.84

7.84

7.84

8.28

8.28

8.28

8.28

8.28

8.28

8.28

8.28

8.28

8.28

8.28

8.28

8.28

8.28

8.28

8.28

8.28

8.28

8.28

8.28

8.28

8.288.

478.

478.

478.

478.

478.

478.

478.

478.

478.

478.

478.

478.

478.

478.

478.

478.

478.

478.

478.

478.

478.

47

8.48

8.48

8.48

8.48

8.48

8.48

8.48

8.48

8.48

8.48

8.48

8.48

8.48

8.48

8.48

8.48

8.48

8.48

8.48

8.48

8.48

8.48

Chemical Shift (ppm)170 160 150 140 130 120 110 100 90 80 70 60 50 40

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

25.9

525

.95

25.9

525

.95

25.9

525

.95

25.9

525

.95

25.9

525

.95

25.9

525

.95

25.9

525

.95

25.9

525

.95

25.9

525

.95

25.9

525

.95

25.9

525

.95

29.2

729

.27

29.2

729

.27

29.2

729

.27

29.2

729

.27

29.2

729

.27

29.2

729

.27

29.2

729

.27

29.2

729

.27

29.2

729

.27

29.2

729

.27

29.2

729

.27

30.6

430

.64

30.6

430

.64

30.6

430

.64

30.6

430

.64

30.6

430

.64

30.6

430

.64

30.6

430

.64

30.6

430

.64

30.6

430

.64

30.6

430

.64

30.6

430

.64

31.3

031

.30

31.3

031

.30

31.3

031

.30

31.3

031

.30

31.3

031

.30

31.3

031

.30

31.3

031

.30

31.3

031

.30

31.3

031

.30

31.3

031

.30

31.3

031

.30

39.7

639

.76

39.7

639

.76

39.7

639

.76

39.7

639

.76

39.7

639

.76

39.7

639

.76

39.7

639

.76

39.7

639

.76

39.7

639

.76

39.7

639

.76

39.7

639

.76

43.2

843

.28

43.2

843

.28

43.2

843

.28

43.2

843

.28

43.2

843

.28

43.2

843

.28

43.2

843

.28

43.2

843

.28

43.2

843

.28

43.2

843

.28

43.2

843

.28

46.1

846

.18

46.1

846

.18

46.1

846

.18

46.1

846

.18

46.1

846

.18

46.1

846

.18

46.1

846

.18

46.1

846

.18

46.1

846

.18

46.1

846

.18

46.1

846

.18

49.2

849

.28

49.2

849

.28

49.2

849

.28

49.2

849

.28

49.2

849

.28

49.2

849

.28

49.2

849

.28

49.2

849

.28

49.2

849

.28

49.2

849

.28

49.2

849

.2858

.72

58.7

258

.72

58.7

258

.72

58.7

258

.72

58.7

258

.72

58.7

258

.72

58.7

258

.72

58.7

258

.72

58.7

258

.72

58.7

258

.72

58.7

258

.72

58.7

2

70.3

570

.35

70.3

570

.35

70.3

570

.35

70.3

570

.35

70.3

570

.35

70.3

570

.35

70.3

570

.35

70.3

570

.35

70.3

570

.35

70.3

570

.35

70.3

570

.35

76.8

076

.80

76.8

076

.80

76.8

076

.80

76.8

076

.80

76.8

076

.80

76.8

076

.80

76.8

076

.80

76.8

076

.80

76.8

076

.80

76.8

076

.80

76.8

076

.80

77.3

177

.31

77.3

177

.31

77.3

177

.31

77.3

177

.31

77.3

177

.31

77.3

177

.31

77.3

177

.31

77.3

177

.31

77.3

177

.31

77.3

177

.31

77.3

177

.31

93.2

093

.20

93.2

093

.20

93.2

093

.20

93.2

093

.20

93.2

093

.20

93.2

093

.20

93.2

093

.20

93.2

093

.20

93.2

093

.20

93.2

093

.20

93.2

093

.20

100.

4310

0.43

100.

4310

0.43

100.

4310

0.43

100.

4310

0.43

100.

4310

0.43

100.

4310

0.43

100.

4310

0.43

100.

4310

0.43

100.

4310

0.43

100.

4310

0.43

100.

4310

0.43

103.

0910

3.09

103.

0910

3.09

103.

0910

3.09

103.

0910

3.09

103.

0910

3.09

103.

0910

3.09

103.

0910

3.09

103.

0910

3.09

103.

0910

3.09

103.

0910

3.09

103.

0910

3.0912

1.64

121.

6412

1.64

121.

6412

1.64

121.

6412

1.64

121.

6412

1.64

121.

6412

1.64

121.

6412

1.64

121.

6412

1.64

121.

6412

1.64

121.

6412

1.64

121.

6412

1.64

121.

64130.

5113

0.51

130.

5113

0.51

130.

5113

0.51

130.

5113

0.51

130.

5113

0.51

130.

5113

0.51

130.

5113

0.51

130.

5113

0.51

130.

5113

0.51

130.

5113

0.51

130.

5113

0.51

132.

8113

2.81

132.

8113

2.81

132.

8113

2.81

132.

8113

2.81

132.

8113

2.81

132.

8113

2.81

132.

8113

2.81

132.

8113

2.81

132.

8113

2.81

132.

8113

2.81

132.

8113

2.8113

5.53

135.

5313

5.53

135.

5313

5.53

135.

5313

5.53

135.

5313

5.53

135.

5313

5.53

135.

5313

5.53

135.

5313

5.53

135.

5313

5.53

135.

5313

5.53

135.

5313

5.53

135.

53

148.

3014

8.30

148.

3014

8.30

148.

3014

8.30

148.

3014

8.30

148.

3014

8.30

148.

3014

8.30

148.

3014

8.30

148.

3014

8.30

148.

3014

8.30

148.

3014

8.30

148.

3014

8.30

152.

0715

2.07

152.

0715

2.07

152.

0715

2.07

152.

0715

2.07

152.

0715

2.07

152.

0715

2.07

152.

0715

2.07

152.

0715

2.07

152.

0715

2.07

152.

0715

2.07

152.

0715

2.0715

2.65

152.

6515

2.65

152.

6515

2.65

152.

6515

2.65

152.

6515

2.65

152.

6515

2.65

152.

6515

2.65

152.

6515

2.65

152.

6515

2.65

152.

6515

2.65

152.

6515

2.65

152.

65

156.

6115

6.61

156.

6115

6.61

156.

6115

6.61

156.

6115

6.61

156.

6115

6.61

156.

6115

6.61

156.

6115

6.61

156.

6115

6.61

156.

6115

6.61

156.

6115

6.61

156.

6115

6.6117

1.91

171.

9117

1.91

171.

9117

1.91

171.

9117

1.91

171.

9117

1.91

171.

9117

1.91

171.

9117

1.91

171.

9117

1.91

171.

9117

1.91

171.

9117

1.91

171.

9117

1.91

171.

91

231

Appendix IV

Chapter 4 – NMR Characterization

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

2.39

9.25

2.40

2.78

3.27

0.98

H 6(s)

H 4(m)H 1(m)

H 5(m)

H 2(m)

H 3(m)

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

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

28.7

228

.72

28.7

228

.72

28.7

228

.72

28.7

228

.72

28.7

228

.72

28.7

228

.72

28.7

228

.72

28.7

228

.72

28.7

228

.72

28.7

228

.72

28.7

228

.72

33.5

533

.55

33.5

533

.55

33.5

533

.55

33.5

533

.55

33.5

533

.55

33.5

533

.55

33.5

533

.55

33.5

533

.55

33.5

533

.55

33.5

533

.55

33.5

533

.55

45.4

645

.46

45.4

645

.46

45.4

645

.46

45.4

645

.46

45.4

645

.46

45.4

645

.46

45.4

645

.46

45.4

645

.46

45.4

645

.46

45.4

645

.46

45.4

645

.46

48.3

948

.39

48.3

948

.39

48.3

948

.39

48.3

948

.39

48.3

948

.39

48.3

948

.39

48.3

948

.39

48.3

948

.39

48.3

948

.39

48.3

948

.39

48.3

948

.39

77.7

477

.74

77.7

477

.74

77.7

477

.74

77.7

477

.74

77.7

477

.74

77.7

477

.74

77.7

477

.74

77.7

477

.74

77.7

477

.74

77.7

477

.74

77.7

477

.74

155.

1715

5.17

155.

1715

5.17

155.

1715

5.17

155.

1715

5.17

155.

1715

5.17

155.

1715

5.17

155.

1715

5.17

155.

1715

5.17

155.

1715

5.17

155.

1715

5.17

155.

1715

5.17

232

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

2.11

4.92

0.95

1.02

0.93

1.06

Water

DMSO-d6

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

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

DMSO-d6

43.1

843

.18

43.1

843

.18

43.1

843

.18

43.1

843

.18

43.1

843

.18

43.1

843

.18

43.1

843

.18

43.1

843

.18

43.1

843

.18

43.1

843

.18

43.1

843

.18

124.

5912

4.59

124.

5912

4.59

124.

5912

4.59

124.

5912

4.59

124.

5912

4.59

124.

5912

4.59

124.

5912

4.59

124.

5912

4.59

124.

5912

4.59

124.

5912

4.59

124.

5912

4.59

127.

3612

7.36

127.

3612

7.36

127.

3612

7.36

127.

3612

7.36

127.

3612

7.36

127.

3612

7.36

127.

3612

7.36

127.

3612

7.36

127.

3612

7.36

127.

3612

7.36

127.

3612

7.36

127.

7812

7.78

127.

7812

7.78

127.

7812

7.78

127.

7812

7.78

127.

7812

7.78

127.

7812

7.78

127.

7812

7.78

127.

7812

7.78

127.

7812

7.78

127.

7812

7.78

127.

7812

7.78

128.

8012

8.80

128.

8012

8.80

128.

8012

8.80

128.

8012

8.80

128.

8012

8.80

128.

8012

8.80

128.

8012

8.80

128.

8012

8.80

128.

8012

8.80

128.

8012

8.80

128.

8012

8.80

129.

6512

9.65

129.

6512

9.65

129.

6512

9.65

129.

6512

9.65

129.

6512

9.65

129.

6512

9.65

129.

6512

9.65

129.

6512

9.65

129.

6512

9.65

129.

6512

9.65

129.

6512

9.65

139.

0913

9.09

139.

0913

9.09

139.

0913

9.09

139.

0913

9.09

139.

0913

9.09

139.

0913

9.09

139.

0913

9.09

139.

0913

9.09

139.

0913

9.09

139.

0913

9.09

139.

0913

9.09

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

149.

4614

9.46

149.

4614

9.46

149.

4614

9.46

149.

4614

9.46

149.

4614

9.46

149.

4614

9.46

149.

4614

9.46

149.

4614

9.46

149.

4614

9.46

149.

4614

9.46

149.

4614

9.46

153.

0515

3.05

153.

0515

3.05

153.

0515

3.05

153.

0515

3.05

153.

0515

3.05

153.

0515

3.05

153.

0515

3.05

153.

0515

3.05

153.

0515

3.05

153.

0515

3.05

153.

0515

3.05

164.

1616

4.16

164.

1616

4.16

164.

1616

4.16

164.

1616

4.16

164.

1616

4.16

164.

1616

4.16

164.

1616

4.16

164.

1616

4.16

164.

1616

4.16

164.

1616

4.16

164.

1616

4.16

233

JWL-BE1-037A_PROTON_011HDMSO-d630 H's

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

2.16

9.07

2.17

2.02

1.04

1.99

2.01

1.89

0.96

3.86

0.97

0.93

1.00

Water

DMSO-d6

1.38

1.38

1.38

1.38

1.38

1.38

1.38

1.38

1.38

1.38

1.38

1.38

1.38

1.38

1.38

1.38

1.38

1.38

1.38

1.38

1.38

1.38

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.76

1.78

1.78

1.78

1.78

1.78

1.78

1.78

1.78

1.78

1.78

1.78

1.78

1.78

1.78

1.78

1.78

1.78

1.78

1.78

1.78

1.78

1.78

2.95

2.95

2.95

2.95

2.95

2.95

2.95

2.95

2.95

2.95

2.95

2.95

2.95

2.95

2.95

2.95

2.95

2.95

2.95

2.95

2.95

2.952.

972.

972.

972.

972.

972.

972.

972.

972.

972.

972.

972.

972.

972.

972.

972.

972.

972.

972.

972.

972.

972.

97

2.99

2.99

2.99

2.99

2.99

2.99

2.99

2.99

2.99

2.99

2.99

2.99

2.99

2.99

2.99

2.99

2.99

2.99

2.99

2.99

2.99

2.99

3.52

3.52

3.52

3.52

3.52

3.52

3.52

3.52

3.52

3.52

3.52

3.52

3.52

3.52

3.52

3.52

3.52

3.52

3.52

3.52

3.52

3.52

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.54

3.54

3.54

3.54

3.54

3.54

3.54

3.54

3.54

3.54

3.54

3.54

3.54

3.54

3.54

3.54

3.54

3.54

3.54

3.54

3.54

3.544.

294.

294.

294.

294.

294.

294.

294.

294.

294.

294.

294.

294.

294.

294.

294.

294.

294.

294.

294.

294.

294.

294.

324.

324.

324.

324.

324.

324.

324.

324.

324.

324.

324.

324.

324.

324.

324.

324.

324.

324.

324.

324.

324.

324.

444.

444.

444.

444.

444.

444.

444.

444.

444.

444.

444.

444.

444.

444.

444.

444.

444.

444.

444.

444.

444.

44

4.45

4.45

4.45

4.45

4.45

4.45

4.45

4.45

4.45

4.45

4.45

4.45

4.45

4.45

4.45

4.45

4.45

4.45

4.45

4.45

4.45

4.45

6.84

6.84

6.84

6.84

6.84

6.84

6.84

6.84

6.84

6.84

6.84

6.84

6.84

6.84

6.84

6.84

6.84

6.84

6.84

6.84

6.84

6.84

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

6.86

7.23

7.23

7.23

7.23

7.23

7.23

7.23

7.23

7.23

7.23

7.23

7.23

7.23

7.23

7.23

7.23

7.23

7.23

7.23

7.23

7.23

7.23

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.30

7.32

7.32

7.32

7.32

7.32

7.32

7.32

7.32

7.32

7.32

7.32

7.32

7.32

7.32

7.32

7.32

7.32

7.32

7.32

7.32

7.32

7.32

7.33

7.33

7.33

7.33

7.33

7.33

7.33

7.33

7.33

7.33

7.33

7.33

7.33

7.33

7.33

7.33

7.33

7.33

7.33

7.33

7.33

7.337.95

7.95

7.95

7.95

7.95

7.95

7.95

7.95

7.95

7.95

7.95

7.95

7.95

7.95

7.95

7.95

7.95

7.95

7.95

7.95

7.95

7.95

7.96

7.96

7.96

7.96

7.96

7.96

7.96

7.96

7.96

7.96

7.96

7.96

7.96

7.96

7.96

7.96

7.96

7.96

7.96

7.96

7.96

7.96

7.98

7.98

7.98

7.98

7.98

7.98

7.98

7.98

7.98

7.98

7.98

7.98

7.98

7.98

7.98

7.98

7.98

7.98

7.98

7.98

7.98

7.988.

628.

628.

628.

628.

628.

628.

628.

628.

628.

628.

628.

628.

628.

628.

628.

628.

628.

628.

628.

628.

628.

62

8.63

8.63

8.63

8.63

8.63

8.63

8.63

8.63

8.63

8.63

8.63

8.63

8.63

8.63

8.63

8.63

8.63

8.63

8.63

8.63

8.63

8.63

8.77

8.77

8.77

8.77

8.77

8.77

8.77

8.77

8.77

8.77

8.77

8.77

8.77

8.77

8.77

8.77

8.77

8.77

8.77

8.77

8.77

8.778.

788.

788.

788.

788.

788.

788.

788.

788.

788.

788.

788.

788.

788.

788.

788.

788.

788.

788.

788.

788.

788.

78

8.80

8.80

8.80

8.80

8.80

8.80

8.80

8.80

8.80

8.80

8.80

8.80

8.80

8.80

8.80

8.80

8.80

8.80

8.80

8.80

8.80

8.80

JWL-BE1-037A_CARBON_0113CDMSO-d64 C's

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

0

0.05

0.10

0.15

28.7

228

.72

28.7

228

.72

28.7

228

.72

28.7

228

.72

28.7

228

.72

28.7

228

.72

28.7

228

.72

28.7

228

.72

28.7

228

.72

28.7

228

.72

28.7

228

.72

31.7

531

.75

31.7

531

.75

31.7

531

.75

31.7

531

.75

31.7

531

.75

31.7

531

.75

31.7

531

.75

31.7

531

.75

31.7

531

.75

31.7

531

.75

31.7

531

.75

42.7

642

.76

42.7

642

.76

42.7

642

.76

42.7

642

.76

42.7

642

.76

42.7

642

.76

42.7

642

.76

42.7

642

.76

42.7

642

.76

42.7

642

.76

42.7

642

.76

43.8

543

.85

43.8

543

.85

43.8

543

.85

43.8

543

.85

43.8

543

.85

43.8

543

.85

43.8

543

.85

43.8

543

.85

43.8

543

.85

43.8

543

.85

43.8

543

.85

48.0

048

.00

48.0

048

.00

48.0

048

.00

48.0

048

.00

48.0

048

.00

48.0

048

.00

48.0

048

.00

48.0

048

.00

48.0

048

.00

48.0

048

.00

48.0

048

.00

78.0

278

.02

78.0

278

.02

78.0

278

.02

78.0

278

.02

78.0

278

.02

78.0

278

.02

78.0

278

.02

78.0

278

.02

78.0

278

.02

78.0

278

.02

78.0

278

.02

105.

8510

5.85

105.

8510

5.85

105.

8510

5.85

105.

8510

5.85

105.

8510

5.85

105.

8510

5.85

105.

8510

5.85

105.

8510

5.85

105.

8510

5.85

105.

8510

5.85

105.

8510

5.85

118.

2811

8.28

118.

2811

8.28

118.

2811

8.28

118.

2811

8.28

118.

2811

8.28

118.

2811

8.28

118.

2811

8.28

118.

2811

8.28

118.

2811

8.28

118.

2811

8.28

118.

2811

8.28

127.

1112

7.11

127.

1112

7.11

127.

1112

7.11

127.

1112

7.11

127.

1112

7.11

127.

1112

7.11

127.

1112

7.11

127.

1112

7.11

127.

1112

7.11

127.

1112

7.11

127.

1112

7.11

127.

6012

7.60

127.

6012

7.60

127.

6012

7.60

127.

6012

7.60

127.

6012

7.60

127.

6012

7.60

127.

6012

7.60

127.

6012

7.60

127.

6012

7.60

127.

6012

7.60

127.

6012

7.60

128.

7012

8.70

128.

7012

8.70

128.

7012

8.70

128.

7012

8.70

128.

7012

8.70

128.

7012

8.70

128.

7012

8.70

128.

7012

8.70

128.

7012

8.70

128.

7012

8.70

128.

7012

8.70

136.

9813

6.98

136.

9813

6.98

136.

9813

6.98

136.

9813

6.98

136.

9813

6.98

136.

9813

6.98

136.

9813

6.98

136.

9813

6.98

136.

9813

6.98

136.

9813

6.98

136.

9813

6.98

140.

3814

0.38

140.

3814

0.38

140.

3814

0.38

140.

3814

0.38

140.

3814

0.38

140.

3814

0.38

140.

3814

0.38

140.

3814

0.38

140.

3814

0.38

140.

3814

0.38

140.

3814

0.3814

8.52

148.

5214

8.52

148.

5214

8.52

148.

5214

8.52

148.

5214

8.52

148.

5214

8.52

148.

5214

8.52

148.

5214

8.52

148.

5214

8.52

148.

5214

8.52

148.

5214

8.52

148.

52

155.

3015

5.30

155.

3015

5.30

155.

3015

5.30

155.

3015

5.30

155.

3015

5.30

155.

3015

5.30

155.

3015

5.30

155.

3015

5.30

155.

3015

5.30

155.

3015

5.30

155.

3015

5.30

159.

8915

9.89

159.

8915

9.89

159.

8915

9.89

159.

8915

9.89

159.

8915

9.89

159.

8915

9.89

159.

8915

9.89

159.

8915

9.89

159.

8915

9.89

159.

8915

9.89

159.

8915

9.89

165.

3916

5.39

165.

3916

5.39

165.

3916

5.39

165.

3916

5.39

165.

3916

5.39

165.

3916

5.39

165.

3916

5.39

165.

3916

5.39

165.

3916

5.39

165.

3916

5.39

165.

3916

5.39

234

JWL-BE1-037_B_PROTON_011HDMSO-d622 H's

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

2.00

2.33

2.07

0.60

1.61

1.61

5.19

3.86

0.96

1.00

Water

DMSO-d6

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.58

1.59

1.59

1.59

1.59

1.59

1.59

1.59

1.59

1.59

1.59

1.59

1.59

1.59

1.59

1.59

1.59

1.59

1.59

1.59

1.59

1.59

1.59

1.62

1.62

1.62

1.62

1.62

1.62

1.62

1.62

1.62

1.62

1.62

1.62

1.62

1.62

1.62

1.62

1.62

1.62

1.62

1.62

1.62

1.622.

052.

052.

052.

052.

052.

052.

052.

052.

052.

052.

052.

052.

052.

052.

052.

052.

052.

052.

052.

052.

052.

05

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

2.07

3.27

3.27

3.27

3.27

3.27

3.27

3.27

3.27

3.27

3.27

3.27

3.27

3.27

3.27

3.27

3.27

3.27

3.27

3.27

3.27

3.27

3.27

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.35

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.37

3.373.

383.

383.

383.

383.

383.

383.

383.

383.

383.

383.

383.

383.

383.

383.

383.

383.

383.

383.

383.

383.

383.

383.

403.

403.

403.

403.

403.

403.

403.

403.

403.

403.

403.

403.

403.

403.

403.

403.

403.

403.

403.

403.

403.

40

8.60

8.60

8.60

8.60

8.60

8.60

8.60

8.60

8.60

8.60

8.60

8.60

8.60

8.60

8.60

8.60

8.60

8.60

8.60

8.60

8.60

8.608.

608.

608.

608.

608.

608.

608.

608.

608.

608.

608.

608.

608.

608.

608.

608.

608.

608.

608.

608.

608.

608.

60

9.29

9.29

9.29

9.29

9.29

9.29

9.29

9.29

9.29

9.29

9.29

9.29

9.29

9.29

9.29

9.29

9.29

9.29

9.29

9.29

9.29

9.29

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

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.829

.42

29.4

229

.42

29.4

229

.42

29.4

229

.42

29.4

229

.42

29.4

229

.42

29.4

229

.42

29.4

229

.42

29.4

229

.42

29.4

229

.42

29.4

229

.42

29.4

2

42.9

942

.99

42.9

942

.99

42.9

942

.99

42.9

942

.99

42.9

942

.99

42.9

942

.99

42.9

942

.99

42.9

942

.99

42.9

942

.99

42.9

942

.99

42.9

942

.99

44.7

744

.77

44.7

744

.77

44.7

744

.77

44.7

744

.77

44.7

744

.77

44.7

744

.77

44.7

744

.77

44.7

744

.77

44.7

744

.77

44.7

744

.77

44.7

744

.77

47.3

247

.32

47.3

247

.32

47.3

247

.32

47.3

247

.32

47.3

247

.32

47.3

247

.32

47.3

247

.32

47.3

247

.32

47.3

247

.32

47.3

247

.32

47.3

247

.32

110.

5611

0.56

110.

5611

0.56

110.

5611

0.56

110.

5611

0.56

110.

5611

0.56

110.

5611

0.56

110.

5611

0.56

110.

5611

0.56

110.

5611

0.56

110.

5611

0.56

110.

5611

0.56

119.

0611

9.06

119.

0611

9.06

119.

0611

9.06

119.

0611

9.06

119.

0611

9.06

119.

0611

9.06

119.

0611

9.06

119.

0611

9.06

119.

0611

9.06

119.

0611

9.06

119.

0611

9.06

127.

2712

7.27

127.

2712

7.27

127.

2712

7.27

127.

2712

7.27

127.

2712

7.27

127.

2712

7.27

127.

2712

7.27

127.

2712

7.27

127.

2712

7.27

127.

2712

7.27

127.

2712

7.2712

7.77

127.

7712

7.77

127.

7712

7.77

127.

7712

7.77

127.

7712

7.77

127.

7712

7.77

127.

7712

7.77

127.

7712

7.77

127.

7712

7.77

127.

7712

7.77

127.

7712

7.77

127.

77

128.

7512

8.75

128.

7512

8.75

128.

7512

8.75

128.

7512

8.75

128.

7512

8.75

128.

7512

8.75

128.

7512

8.75

128.

7512

8.75

128.

7512

8.75

128.

7512

8.75

128.

7512

8.75

139.

8613

9.86

139.

8613

9.86

139.

8613

9.86

139.

8613

9.86

139.

8613

9.86

139.

8613

9.86

139.

8613

9.86

139.

8613

9.86

139.

8613

9.86

139.

8613

9.86

139.

8613

9.86

140.

2914

0.29

140.

2914

0.29

140.

2914

0.29

140.

2914

0.29

140.

2914

0.29

140.

2914

0.29

140.

2914

0.29

140.

2914

0.29

140.

2914

0.29

140.

2914

0.29

140.

2914

0.29

149.

5114

9.51

149.

5114

9.51

149.

5114

9.51

149.

5114

9.51

149.

5114

9.51

149.

5114

9.51

149.

5114

9.51

149.

5114

9.51

149.

5114

9.51

149.

5114

9.51

149.

5114

9.51

154.

7415

4.74

154.

7415

4.74

154.

7415

4.74

154.

7415

4.74

154.

7415

4.74

154.

7415

4.74

154.

7415

4.74

154.

7415

4.74

154.

7415

4.74

154.

7415

4.74

154.

7415

4.74

163.

3916

3.39

163.

3916

3.39

163.

3916

3.39

163.

3916

3.39

163.

3916

3.39

163.

3916

3.39

163.

3916

3.39

163.

3916

3.39

163.

3916

3.39

163.

3916

3.39

163.

3916

3.39

235

JWL-BE1-038_MONO_SUBS_BROMO_PROTON_011HDMSO-d610 H's

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

3.00

2.04

2.03

1.01

0.95

0.84

DMSO-d6*

DMSO-d6*

DMSO-d6

Water

1.25

1.25

1.25

1.25

1.25

1.25

1.25

1.25

1.25

1.25

1.25

1.25

1.25

1.25

1.25

1.25

1.25

1.25

1.25

1.25

1.25

1.25

1.27

1.27

1.27

1.27

1.27

1.27

1.27

1.27

1.27

1.27

1.27

1.27

1.27

1.27

1.27

1.27

1.27

1.27

1.27

1.27

1.27

1.27

1.28

1.28

1.28

1.28

1.28

1.28

1.28

1.28

1.28

1.28

1.28

1.28

1.28

1.28

1.28

1.28

1.28

1.28

1.28

1.28

1.28

1.28

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.19

4.20

4.20

4.20

4.20

4.20

4.20

4.20

4.20

4.20

4.20

4.20

4.20

4.20

4.20

4.20

4.20

4.20

4.20

4.20

4.20

4.20

4.20

4.22

4.22

4.22

4.22

4.22

4.22

4.22

4.22

4.22

4.22

4.22

4.22

4.22

4.22

4.22

4.22

4.22

4.22

4.22

4.22

4.22

4.22

4.23

4.23

4.23

4.23

4.23

4.23

4.23

4.23

4.23

4.23

4.23

4.23

4.23

4.23

4.23

4.23

4.23

4.23

4.23

4.23

4.23

4.23

6.18

6.18

6.18

6.18

6.18

6.18

6.18

6.18

6.18

6.18

6.18

6.18

6.18

6.18

6.18

6.18

6.18

6.18

6.18

6.18

6.18

6.18

6.79

6.79

6.79

6.79

6.79

6.79

6.79

6.79

6.79

6.79

6.79

6.79

6.79

6.79

6.79

6.79

6.79

6.79

6.79

6.79

6.79

6.796.81

6.81

6.81

6.81

6.81

6.81

6.81

6.81

6.81

6.81

6.81

6.81

6.81

6.81

6.81

6.81

6.81

6.81

6.81

6.81

6.81

6.81

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.63

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.65

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

7.88

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

0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.5514

.72

14.7

214

.72

14.7

214

.72

14.7

214

.72

14.7

214

.72

14.7

214

.72

14.7

214

.72

14.7

214

.72

14.7

214

.72

14.7

214

.72

14.7

214

.72

14.7

2

39.4

839

.48

39.4

839

.48

39.4

839

.48

39.4

839

.48

39.4

839

.48

39.4

839

.48

39.4

839

.48

39.4

839

.48

39.4

839

.48

39.4

839

.48

39.4

839

.48

39.6

539

.65

39.6

539

.65

39.6

539

.65

39.6

539

.65

39.6

539

.65

39.6

539

.65

39.6

539

.65

39.6

539

.65

39.6

539

.65

39.6

539

.65

39.6

539

.65

39.8

139

.81

39.8

139

.81

39.8

139

.81

39.8

139

.81

39.8

139

.81

39.8

139

.81

39.8

139

.81

39.8

139

.81

39.8

139

.81

39.8

139

.81

39.8

139

.81

39.9

839

.98

39.9

839

.98

39.9

839

.98

39.9

839

.98

39.9

839

.98

39.9

839

.98

39.9

839

.98

39.9

839

.98

39.9

839

.98

39.9

839

.98

39.9

839

.98

40.1

540

.15

40.1

540

.15

40.1

540

.15

40.1

540

.15

40.1

540

.15

40.1

540

.15

40.1

540

.15

40.1

540

.15

40.1

540

.15

40.1

540

.15

40.1

540

.15

40.3

140

.31

40.3

140

.31

40.3

140

.31

40.3

140

.31

40.3

140

.31

40.3

140

.31

40.3

140

.31

40.3

140

.31

40.3

140

.31

40.3

140

.31

40.3

140

.31

40.4

840

.48

40.4

840

.48

40.4

840

.48

40.4

840

.48

40.4

840

.48

40.4

840

.48

40.4

840

.48

40.4

840

.48

40.4

840

.48

40.4

840

.48

40.4

840

.48

60.5

160

.51

60.5

160

.51

60.5

160

.51

60.5

160

.51

60.5

160

.51

60.5

160

.51

60.5

160

.51

60.5

160

.51

60.5

160

.51

60.5

160

.51

60.5

160

.51

106.

3410

6.34

106.

3410

6.34

106.

3410

6.34

106.

3410

6.34

106.

3410

6.34

106.

3410

6.34

106.

3410

6.34

106.

3410

6.34

106.

3410

6.34

106.

3410

6.34

106.

3410

6.34

114.

5511

4.55

114.

5511

4.55

114.

5511

4.55

114.

5511

4.55

114.

5511

4.55

114.

5511

4.55

114.

5511

4.55

114.

5511

4.55

114.

5511

4.55

114.

5511

4.55

114.

5511

4.55

118.

2811

8.28

118.

2811

8.28

118.

2811

8.28

118.

2811

8.28

118.

2811

8.28

118.

2811

8.28

118.

2811

8.28

118.

2811

8.28

118.

2811

8.28

118.

2811

8.28

118.

2811

8.28

130.

2913

0.29

130.

2913

0.29

130.

2913

0.29

130.

2913

0.29

130.

2913

0.29

130.

2913

0.29

130.

2913

0.29

130.

2913

0.29

130.

2913

0.29

130.

2913

0.29

130.

2913

0.29

134.

1313

4.13

134.

1313

4.13

134.

1313

4.13

134.

1313

4.13

134.

1313

4.13

134.

1313

4.13

134.

1313

4.13

134.

1313

4.13

134.

1313

4.13

134.

1313

4.13

134.

1313

4.13

150.

7015

0.70

150.

7015

0.70

150.

7015

0.70

150.

7015

0.70

150.

7015

0.70

150.

7015

0.70

150.

7015

0.70

150.

7015

0.70

150.

7015

0.70

150.

7015

0.70

150.

7015

0.70

165.

1716

5.17

165.

1716

5.17

165.

1716

5.17

165.

1716

5.17

165.

1716

5.17

165.

1716

5.17

165.

1716

5.17

165.

1716

5.17

165.

1716

5.17

165.

1716

5.17

165.

1716

5.17

236

JWL-BE1-039_PROTON_011HDMSO-d68 H's

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

3.00

2.03

1.84

0.75

DMSO-d6*

DMSO-d6*

Water

1.32

1.32

1.32

1.32

1.32

1.32

1.32

1.32

1.32

1.32

1.32

1.32

1.32

1.32

1.32

1.32

1.32

1.32

1.32

1.32

1.32

1.32

1.33

1.33

1.33

1.33

1.33

1.33

1.33

1.33

1.33

1.33

1.33

1.33

1.33

1.33

1.33

1.33

1.33

1.33

1.33

1.33

1.33

1.33

1.34

1.34

1.34

1.34

1.34

1.34

1.34

1.34

1.34

1.34

1.34

1.34

1.34

1.34

1.34

1.34

1.34

1.34

1.34

1.34

1.34

1.34

4.33

4.33

4.33

4.33

4.33

4.33

4.33

4.33

4.33

4.33

4.33

4.33

4.33

4.33

4.33

4.33

4.33

4.33

4.33

4.33

4.33

4.33

4.34

4.34

4.34

4.34

4.34

4.34

4.34

4.34

4.34

4.34

4.34

4.34

4.34

4.34

4.34

4.34

4.34

4.34

4.34

4.34

4.34

4.34

4.36

4.36

4.36

4.36

4.36

4.36

4.36

4.36

4.36

4.36

4.36

4.36

4.36

4.36

4.36

4.36

4.36

4.36

4.36

4.36

4.36

4.36

4.37

4.37

4.37

4.37

4.37

4.37

4.37

4.37

4.37

4.37

4.37

4.37

4.37

4.37

4.37

4.37

4.37

4.37

4.37

4.37

4.37

4.37

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.05

8.07

8.07

8.07

8.07

8.07

8.07

8.07

8.07

8.07

8.07

8.07

8.07

8.07

8.07

8.07

8.07

8.07

8.07

8.07

8.07

8.07

8.07

8.09

8.09

8.09

8.09

8.09

8.09

8.09

8.09

8.09

8.09

8.09

8.09

8.09

8.09

8.09

8.09

8.09

8.09

8.09

8.09

8.09

8.09

8.26

8.26

8.26

8.26

8.26

8.26

8.26

8.26

8.26

8.26

8.26

8.26

8.26

8.26

8.26

8.26

8.26

8.26

8.26

8.26

8.26

8.26

8.27

8.27

8.27

8.27

8.27

8.27

8.27

8.27

8.27

8.27

8.27

8.27

8.27

8.27

8.27

8.27

8.27

8.27

8.27

8.27

8.27

8.27

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

0

0.05

0.10

0.15

0.20

0.25

0.30

14.4

314

.43

14.4

314

.43

14.4

314

.43

14.4

314

.43

14.4

314

.43

14.4

314

.43

14.4

314

.43

14.4

314

.43

14.4

314

.43

14.4

314

.43

14.4

314

.43

39.4

839

.48

39.4

839

.48

39.4

839

.48

39.4

839

.48

39.4

839

.48

39.4

839

.48

39.4

839

.48

39.4

839

.48

39.4

839

.48

39.4

839

.48

39.4

839

.48

39.6

439

.64

39.6

439

.64

39.6

439

.64

39.6

439

.64

39.6

439

.64

39.6

439

.64

39.6

439

.64

39.6

439

.64

39.6

439

.64

39.6

439

.64

39.6

439

.64

39.8

139

.81

39.8

139

.81

39.8

139

.81

39.8

139

.81

39.8

139

.81

39.8

139

.81

39.8

139

.81

39.8

139

.81

39.8

139

.81

39.8

139

.81

39.8

139

.81

39.9

839

.98

39.9

839

.98

39.9

839

.98

39.9

839

.98

39.9

839

.98

39.9

839

.98

39.9

839

.98

39.9

839

.98

39.9

839

.98

39.9

839

.98

39.9

839

.98

40.1

540

.15

40.1

540

.15

40.1

540

.15

40.1

540

.15

40.1

540

.15

40.1

540

.15

40.1

540

.15

40.1

540

.15

40.1

540

.15

40.1

540

.15

40.1

540

.15

40.3

140

.31

40.3

140

.31

40.3

140

.31

40.3

140

.31

40.3

140

.31

40.3

140

.31

40.3

140

.31

40.3

140

.31

40.3

140

.31

40.3

140

.31

40.3

140

.31

40.4

840

.48

40.4

840

.48

40.4

840

.48

40.4

840

.48

40.4

840

.48

40.4

840

.48

40.4

840

.48

40.4

840

.48

40.4

840

.48

40.4

840

.48

40.4

840

.48

62.4

062

.40

62.4

062

.40

62.4

062

.40

62.4

062

.40

62.4

062

.40

62.4

062

.40

62.4

062

.40

62.4

062

.40

62.4

062

.40

62.4

062

.40

62.4

062

.40

117.

1211

7.12

117.

1211

7.12

117.

1211

7.12

117.

1211

7.12

117.

1211

7.12

117.

1211

7.12

117.

1211

7.12

117.

1211

7.12

117.

1211

7.12

117.

1211

7.12

117.

1211

7.12

118.

8411

8.84

118.

8411

8.84

118.

8411

8.84

118.

8411

8.84

118.

8411

8.84

118.

8411

8.84

118.

8411

8.84

118.

8411

8.84

118.

8411

8.84

118.

8411

8.84

118.

8411

8.84

125.

3512

5.35

125.

3512

5.35

125.

3512

5.35

125.

3512

5.35

125.

3512

5.35

125.

3512

5.35

125.

3512

5.35

125.

3512

5.35

125.

3512

5.35

125.

3512

5.35

125.

3512

5.35

129.

0412

9.04

129.

0412

9.04

129.

0412

9.04

129.

0412

9.04

129.

0412

9.04

129.

0412

9.04

129.

0412

9.04

129.

0412

9.04

129.

0412

9.04

129.

0412

9.04

129.

0412

9.04

133.

3613

3.36

133.

3613

3.36

133.

3613

3.36

133.

3613

3.36

133.

3613

3.36

133.

3613

3.36

133.

3613

3.36

133.

3613

3.36

133.

3613

3.36

133.

3613

3.36

133.

3613

3.36

135.

9113

5.91

135.

9113

5.91

135.

9113

5.91

135.

9113

5.91

135.

9113

5.91

135.

9113

5.91

135.

9113

5.91

135.

9113

5.91

135.

9113

5.91

135.

9113

5.91

135.

9113

5.91

163.

8516

3.85

163.

8516

3.85

163.

8516

3.85

163.

8516

3.85

163.

8516

3.85

163.

8516

3.85

163.

8516

3.85

163.

8516

3.85

163.

8516

3.85

163.

8516

3.85

163.

8516

3.85

237

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

2.92

2.02

4.95

2.06

1.97

1.00

1.00

0.98

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

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

14.2

714

.27

14.2

714

.27

14.2

714

.27

14.2

714

.27

14.2

714

.27

14.2

714

.27

14.2

714

.27

14.2

714

.27

14.2

714

.27

14.2

714

.27

14.2

714

.2728

.66

28.6

628

.66

28.6

628

.66

28.6

628

.66

28.6

628

.66

28.6

628

.66

28.6

628

.66

28.6

628

.66

28.6

628

.66

28.6

628

.66

28.6

628

.66

28.6

6

41.8

941

.89

41.8

941

.89

41.8

941

.89

41.8

941

.89

41.8

941

.89

41.8

941

.89

41.8

941

.89

41.8

941

.89

41.8

941

.89

41.8

941

.89

41.8

941

.89

58.8

358

.83

58.8

358

.83

58.8

358

.83

58.8

358

.83

58.8

358

.83

58.8

358

.83

58.8

358

.83

58.8

358

.83

58.8

358

.83

58.8

358

.83

58.8

358

.8361

.51

61.5

161

.51

61.5

161

.51

61.5

161

.51

61.5

161

.51

61.5

161

.51

61.5

161

.51

61.5

161

.51

61.5

161

.51

61.5

161

.51

61.5

161

.51

61.5

171.2

371

.23

71.2

371

.23

71.2

371

.23

71.2

371

.23

71.2

371

.23

71.2

371

.23

71.2

371

.23

71.2

371

.23

71.2

371

.23

71.2

371

.23

71.2

371

.23

99.1

499

.14

99.1

499

.14

99.1

499

.14

99.1

499

.14

99.1

499

.14

99.1

499

.14

99.1

499

.14

99.1

499

.14

99.1

499

.14

99.1

499

.14

99.1

499

.14

111.

2011

1.20

111.

2011

1.20

111.

2011

1.20

111.

2011

1.20

111.

2011

1.20

111.

2011

1.20

111.

2011

1.20

111.

2011

1.20

111.

2011

1.20

111.

2011

1.20

111.

2011

1.20

116.

5911

6.59

116.

5911

6.59

116.

5911

6.59

116.

5911

6.59

116.

5911

6.59

116.

5911

6.59

116.

5911

6.59

116.

5911

6.59

116.

5911

6.59

116.

5911

6.59

116.

5911

6.59

117.

2211

7.22

117.

2211

7.22

117.

2211

7.22

117.

2211

7.22

117.

2211

7.22

117.

2211

7.22

117.

2211

7.22

117.

2211

7.22

117.

2211

7.22

117.

2211

7.22

117.

2211

7.22

132.

7013

2.70

132.

7013

2.70

132.

7013

2.70

132.

7013

2.70

132.

7013

2.70

132.

7013

2.70

132.

7013

2.70

132.

7013

2.70

132.

7013

2.70

132.

7013

2.70

132.

7013

2.70

135.

5013

5.50

135.

5013

5.50

135.

5013

5.50

135.

5013

5.50

135.

5013

5.50

135.

5013

5.50

135.

5013

5.50

135.

5013

5.50

135.

5013

5.50

135.

5013

5.50

135.

5013

5.50

150.

4715

0.47

150.

4715

0.47

150.

4715

0.47

150.

4715

0.47

150.

4715

0.47

150.

4715

0.47

150.

4715

0.47

150.

4715

0.47

150.

4715

0.47

150.

4715

0.47

150.

4715

0.47

165.

8416

5.84

165.

8416

5.84

165.

8416

5.84

165.

8416

5.84

165.

8416

5.84

165.

8416

5.84

165.

8416

5.84

165.

8416

5.84

165.

8416

5.84

165.

8416

5.84

165.

8416

5.84

238

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

2.08

3.95

1.95

5.42

2.10

1.03

3.92

1.99

2.71

3.98

0.99

1.85

2.01

0.93

1.01

DMSO-d6*

DMSO-d6*

DMSO-d6

Water

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

0

0.005

0.010

0.015

0.020

DMSO-d6

29.2

029

.20

29.2

029

.20

29.2

029

.20

29.2

029

.20

29.2

029

.20

29.2

029

.20

29.2

029

.20

29.2

029

.20

29.2

029

.20

29.2

029

.20

29.2

029

.20

31.2

931

.29

31.2

931

.29

31.2

931

.29

31.2

931

.29

31.2

931

.29

31.2

931

.29

31.2

931

.29

31.2

931

.29

31.2

931

.29

31.2

931

.29

31.2

931

.29

40.9

840

.98

40.9

840

.98

40.9

840

.98

40.9

840

.98

40.9

840

.98

40.9

840

.98

40.9

840

.98

40.9

840

.98

40.9

840

.98

40.9

840

.98

40.9

840

.98

42.7

742

.77

42.7

742

.77

42.7

742

.77

42.7

742

.77

42.7

742

.77

42.7

742

.77

42.7

742

.77

42.7

742

.77

42.7

742

.77

42.7

742

.77

42.7

742

.77

44.2

944

.29

44.2

944

.29

44.2

944

.29

44.2

944

.29

44.2

944

.29

44.2

944

.29

44.2

944

.29

44.2

944

.29

44.2

944

.29

44.2

944

.29

44.2

944

.29

47.3

747

.37

47.3

747

.37

47.3

747

.37

47.3

747

.37

47.3

747

.37

47.3

747

.37

47.3

747

.37

47.3

747

.37

47.3

747

.37

47.3

747

.37

47.3

747

.37

58.4

058

.40

58.4

058

.40

58.4

058

.40

58.4

058

.40

58.4

058

.40

58.4

058

.40

58.4

058

.40

58.4

058

.40

58.4

058

.40

58.4

058

.40

58.4

058

.40

70.0

470

.04

70.0

470

.04

70.0

470

.04

70.0

470

.04

70.0

470

.04

70.0

470

.04

70.0

470

.04

70.0

470

.04

70.0

470

.04

70.0

470

.04

70.0

470

.04

106.

0410

6.04

106.

0410

6.04

106.

0410

6.04

106.

0410

6.04

106.

0410

6.04

106.

0410

6.04

106.

0410

6.04

106.

0410

6.04

106.

0410

6.04

106.

0410

6.04

106.

0410

6.04

110.

2211

0.22

110.

2211

0.22

110.

2211

0.22

110.

2211

0.22

110.

2211

0.22

110.

2211

0.22

110.

2211

0.22

110.

2211

0.22

110.

2211

0.22

110.

2211

0.22

110.

2211

0.22

112.

9511

2.95

112.

9511

2.95

112.

9511

2.95

112.

9511

2.95

112.

9511

2.95

112.

9511

2.95

112.

9511

2.95

112.

9511

2.95

112.

9511

2.95

112.

9511

2.95

112.

9511

2.95

118.

3911

8.39

118.

3911

8.39

118.

3911

8.39

118.

3911

8.39

118.

3911

8.39

118.

3911

8.39

118.

3911

8.39

118.

3911

8.39

118.

3911

8.39

118.

3911

8.39

118.

3911

8.39

127.

1212

7.12

127.

1212

7.12

127.

1212

7.12

127.

1212

7.12

127.

1212

7.12

127.

1212

7.12

127.

1212

7.12

127.

1212

7.12

127.

1212

7.12

127.

1212

7.12

127.

1212

7.12

127.

6012

7.60

127.

6012

7.60

127.

6012

7.60

127.

6012

7.60

127.

6012

7.60

127.

6012

7.60

127.

6012

7.60

127.

6012

7.60

127.

6012

7.60

127.

6012

7.60

127.

6012

7.60

128.

7112

8.71

128.

7112

8.71

128.

7112

8.71

128.

7112

8.71

128.

7112

8.71

128.

7112

8.71

128.

7112

8.71

128.

7112

8.71

128.

7112

8.71

128.

7112

8.71

128.

7112

8.71

129.

3612

9.36

129.

3612

9.36

129.

3612

9.36

129.

3612

9.36

129.

3612

9.36

129.

3612

9.36

129.

3612

9.36

129.

3612

9.36

129.

3612

9.36

129.

3612

9.36

129.

3612

9.36

137.

0113

7.01

137.

0113

7.01

137.

0113

7.01

137.

0113

7.01

137.

0113

7.01

137.

0113

7.01

137.

0113

7.01

137.

0113

7.01

137.

0113

7.01

137.

0113

7.01

137.

0113

7.01

140.

3714

0.37

140.

3714

0.37

140.

3714

0.37

140.

3714

0.37

140.

3714

0.37

140.

3714

0.37

140.

3714

0.37

140.

3714

0.37

140.

3714

0.37

140.

3714

0.37

140.

3714

0.37

148.

5614

8.56

148.

5614

8.56

148.

5614

8.56

148.

5614

8.56

148.

5614

8.56

148.

5614

8.56

148.

5614

8.56

148.

5614

8.56

148.

5614

8.56

148.

5614

8.56

148.

5614

8.56

150.

0615

0.06

150.

0615

0.06

150.

0615

0.06

150.

0615

0.06

150.

0615

0.06

150.

0615

0.06

150.

0615

0.06

150.

0615

0.06

150.

0615

0.06

150.

0615

0.06

150.

0615

0.06

159.

9915

9.99

159.

9915

9.99

159.

9915

9.99

159.

9915

9.99

159.

9915

9.99

159.

9915

9.99

159.

9915

9.99

159.

9915

9.99

159.

9915

9.99

159.

9915

9.99

159.

9915

9.99

165.

3816

5.38

165.

3816

5.38

165.

3816

5.38

165.

3816

5.38

165.

3816

5.38

165.

3816

5.38

165.

3816

5.38

165.

3816

5.38

165.

3816

5.38

165.

3816

5.38

165.

3816

5.38

165.

8816

5.88

165.

8816

5.88

165.

8816

5.88

165.

8816

5.88

165.

8816

5.88

165.

8816

5.88

165.

8816

5.88

165.

8816

5.88

165.

8816

5.88

165.

8816

5.88

165.

8816

5.88

171.

5017

1.50

171.

5017

1.50

171.

5017

1.50

171.

5017

1.50

171.

5017

1.50

171.

5017

1.50

171.

5017

1.50

171.

5017

1.50

171.

5017

1.50

171.

5017

1.50

171.

5017

1.50

239

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

2.00

3.68

1.87

1.86

2.54

1.86

1.07

1.82

0.94

0.94

0.92

0.84

0.85

0.98

0.84

0.71

0.99

0.94

0.94

0.88

DMSO-d6

Water

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

0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.4029

.19

29.1

929

.19

29.1

929

.19

29.1

929

.19

29.1

929

.19

29.1

929

.19

29.1

929

.19

29.1

929

.19

29.1

929

.19

29.1

929

.19

29.1

929

.19

29.1

9

31.2

731

.27

31.2

731

.27

31.2

731

.27

31.2

731

.27

31.2

731

.27

31.2

731

.27

31.2

731

.27

31.2

731

.27

31.2

731

.27

31.2

731

.27

31.2

731

.27

44.2

744

.27

44.2

744

.27

44.2

744

.27

44.2

744

.27

44.2

744

.27

44.2

744

.27

44.2

744

.27

44.2

744

.27

44.2

744

.27

44.2

744

.27

44.2

744

.27

47.3

647

.36

47.3

647

.36

47.3

647

.36

47.3

647

.36

47.3

647

.36

47.3

647

.36

47.3

647

.36

47.3

647

.36

47.3

647

.36

47.3

647

.36

47.3

647

.36

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

70.0

370

.03

70.0

370

.03

70.0

370

.03

70.0

370

.03

70.0

370

.03

70.0

370

.03

70.0

370

.03

70.0

370

.03

70.0

370

.03

70.0

370

.03

70.0

370

.03

105.

9310

5.93

105.

9310

5.93

105.

9310

5.93

105.

9310

5.93

105.

9310

5.93

105.

9310

5.93

105.

9310

5.93

105.

9310

5.93

105.

9310

5.93

105.

9310

5.93

105.

9310

5.93

110.

2111

0.21

110.

2111

0.21

110.

2111

0.21

110.

2111

0.21

110.

2111

0.21

110.

2111

0.21

110.

2111

0.21

110.

2111

0.21

110.

2111

0.21

110.

2111

0.21

110.

2111

0.21

112.

9511

2.95

112.

9511

2.95

112.

9511

2.95

112.

9511

2.95

112.

9511

2.95

112.

9511

2.95

112.

9511

2.95

112.

9511

2.95

112.

9511

2.95

112.

9511

2.95

112.

9511

2.95116.

0711

6.07

116.

0711

6.07

116.

0711

6.07

116.

0711

6.07

116.

0711

6.07

116.

0711

6.07

116.

0711

6.07

116.

0711

6.07

116.

0711

6.07

116.

0711

6.07

116.

0711

6.07

118.

3311

8.33

118.

3311

8.33

118.

3311

8.33

118.

3311

8.33

118.

3311

8.33

118.

3311

8.33

118.

3311

8.33

118.

3311

8.33

118.

3311

8.33

118.

3311

8.33

118.

3311

8.33

129.

3512

9.35

129.

3512

9.35

129.

3512

9.35

129.

3512

9.35

129.

3512

9.35

129.

3512

9.35

129.

3512

9.35

129.

3512

9.35

129.

3512

9.35

129.

3512

9.35

129.

3512

9.35

137.

3013

7.30

137.

3013

7.30

137.

3013

7.30

137.

3013

7.30

137.

3013

7.30

137.

3013

7.30

137.

3013

7.30

137.

3013

7.30

137.

3013

7.30

137.

3013

7.30

137.

3013

7.30

138.

6113

8.61

138.

6113

8.61

138.

6113

8.61

138.

6113

8.61

138.

6113

8.61

138.

6113

8.61

138.

6113

8.61

138.

6113

8.61

138.

6113

8.61

138.

6113

8.61

138.

6113

8.61

148.

8514

8.85

148.

8514

8.85

148.

8514

8.85

148.

8514

8.85

148.

8514

8.85

148.

8514

8.85

148.

8514

8.85

148.

8514

8.85

148.

8514

8.85

148.

8514

8.85

148.

8514

8.85

150.

0615

0.06

150.

0615

0.06

150.

0615

0.06

150.

0615

0.06

150.

0615

0.06

150.

0615

0.06

150.

0615

0.06

150.

0615

0.06

150.

0615

0.06

150.

0615

0.06

150.

0615

0.06

159.

9715

9.97

159.

9715

9.97

159.

9715

9.97

159.

9715

9.97

159.

9715

9.97

159.

9715

9.97

159.

9715

9.97

159.

9715

9.97

159.

9715

9.97

159.

9715

9.97

159.

9715

9.97

165.

8616

5.86

165.

8616

5.86

165.

8616

5.86

165.

8616

5.86

165.

8616

5.86

165.

8616

5.86

165.

8616

5.86

165.

8616

5.86

165.

8616

5.86

165.

8616

5.86

165.

8616

5.86

167.

1316

7.13

167.

1316

7.13

167.

1316

7.13

167.

1316

7.13

167.

1316

7.13

167.

1316

7.13

167.

1316

7.13

167.

1316

7.13

167.

1316

7.13

167.

1316

7.13

167.

1316

7.13

171.

5017

1.50

171.

5017

1.50

171.

5017

1.50

171.

5017

1.50

171.

5017

1.50

171.

5017

1.50

171.

5017

1.50

171.

5017

1.50

171.

5017

1.50

171.

5017

1.50

171.

5017

1.50

240

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

2.95

2.07

4.11

2.05

4.82

2.16

1.03

2.01

2.01

1.99

0.99

0.95

0.99

1.82

2.03

0.82

DMSO-d6*

DMSO-d6*

Water

DMSO-d6

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

0

0.05

0.10

DMSO-d6

14.7

314

.73

14.7

314

.73

14.7

314

.73

14.7

314

.73

14.7

314

.73

14.7

314

.73

14.7

314

.73

14.7

314

.73

14.7

314

.73

14.7

314

.73

14.7

314

.73

29.1

929

.19

29.1

929

.19

29.1

929

.19

29.1

929

.19

29.1

929

.19

29.1

929

.19

29.1

929

.19

29.1

929

.19

29.1

929

.19

29.1

929

.19

29.1

929

.19

31.3

731

.37

31.3

731

.37

31.3

731

.37

31.3

731

.37

31.3

731

.37

31.3

731

.37

31.3

731

.37

31.3

731

.37

31.3

731

.37

31.3

731

.37

31.3

731

.37

40.1

440

.14

40.1

440

.14

40.1

440

.14

40.1

440

.14

40.1

440

.14

40.1

440

.14

40.1

440

.14

40.1

440

.14

40.1

440

.14

40.1

440

.14

40.1

440

.14

44.1

244

.12

44.1

244

.12

44.1

244

.12

44.1

244

.12

44.1

244

.12

44.1

244

.12

44.1

244

.12

44.1

244

.12

44.1

244

.12

44.1

244

.12

44.1

244

.12

47.2

247

.22

47.2

247

.22

47.2

247

.22

47.2

247

.22

47.2

247

.22

47.2

247

.22

47.2

247

.22

47.2

247

.22

47.2

247

.22

47.2

247

.22

47.2

247

.2258

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

858

.38

58.3

8

60.4

060

.40

60.4

060

.40

60.4

060

.40

60.4

060

.40

60.4

060

.40

60.4

060

.40

60.4

060

.40

60.4

060

.40

60.4

060

.40

60.4

060

.40

60.4

060

.4070

.02

70.0

270

.02

70.0

270

.02

70.0

270

.02

70.0

270

.02

70.0

270

.02

70.0

270

.02

70.0

270

.02

70.0

270

.02

70.0

270

.02

70.0

270

.02

70.0

2

106.

1310

6.13

106.

1310

6.13

106.

1310

6.13

106.

1310

6.13

106.

1310

6.13

106.

1310

6.13

106.

1310

6.13

106.

1310

6.13

106.

1310

6.13

106.

1310

6.13

106.

1310

6.13

110.

2111

0.21

110.

2111

0.21

110.

2111

0.21

110.

2111

0.21

110.

2111

0.21

110.

2111

0.21

110.

2111

0.21

110.

2111

0.21

110.

2111

0.21

110.

2111

0.21

110.

2111

0.21

112.

9411

2.94

112.

9411

2.94

112.

9411

2.94

112.

9411

2.94

112.

9411

2.94

112.

9411

2.94

112.

9411

2.94

112.

9411

2.94

112.

9411

2.94

112.

9411

2.94

112.

9411

2.94

113.

9411

3.94

113.

9411

3.94

113.

9411

3.94

113.

9411

3.94

113.

9411

3.94

113.

9411

3.94

113.

9411

3.94

113.

9411

3.94

113.

9411

3.94

113.

9411

3.94

113.

9411

3.94

116.

0811

6.08

116.

0811

6.08

116.

0811

6.08

116.

0811

6.08

116.

0811

6.08

116.

0811

6.08

116.

0811

6.08

116.

0811

6.08

116.

0811

6.08

116.

0811

6.08

116.

0811

6.08

129.

3612

9.36

129.

3612

9.36

129.

3612

9.36

129.

3612

9.36

129.

3612

9.36

129.

3612

9.36

129.

3612

9.36

129.

3612

9.36

129.

3612

9.36

129.

3612

9.36

129.

3612

9.36

138.

4713

8.47

138.

4713

8.47

138.

4713

8.47

138.

4713

8.47

138.

4713

8.47

138.

4713

8.47

138.

4713

8.47

138.

4713

8.47

138.

4713

8.47

138.

4713

8.47

138.

4713

8.47

138.

6013

8.60

138.

6013

8.60

138.

6013

8.60

138.

6013

8.60

138.

6013

8.60

138.

6013

8.60

138.

6013

8.60

138.

6013

8.60

138.

6013

8.60

138.

6013

8.60

138.

6013

8.60

150.

0515

0.05

150.

0515

0.05

150.

0515

0.05

150.

0515

0.05

150.

0515

0.05

150.

0515

0.05

150.

0515

0.05

150.

0515

0.05

150.

0515

0.05

150.

0515

0.05

150.

0515

0.05

150.

8515

0.85

150.

8515

0.85

150.

8515

0.85

150.

8515

0.85

150.

8515

0.85

150.

8515

0.85

150.

8515

0.85

150.

8515

0.85

150.

8515

0.85

150.

8515

0.85

150.

8515

0.85

160.

5416

0.54

160.

5416

0.54

160.

5416

0.54

160.

5416

0.54

160.

5416

0.54

160.

5416

0.54

160.

5416

0.54

160.

5416

0.54

160.

5416

0.54

160.

5416

0.54

160.

5416

0.54

165.

4916

5.49

165.

4916

5.49

165.

4916

5.49

165.

4916

5.49

165.

4916

5.49

165.

4916

5.49

165.

4916

5.49

165.

4916

5.49

165.

4916

5.49

165.

4916

5.49

165.

4916

5.49

165.

9116

5.91

165.

9116

5.91

165.

9116

5.91

165.

9116

5.91

165.

9116

5.91

165.

9116

5.91

165.

9116

5.91

165.

9116

5.91

165.

9116

5.91

165.

9116

5.91

165.

9116

5.91

171.

4917

1.49

171.

4917

1.49

171.

4917

1.49

171.

4917

1.49

171.

4917

1.49

171.

4917

1.49

171.

4917

1.49

171.

4917

1.49

171.

4917

1.49

171.

4917

1.49

171.

4917

1.49

241

JWL-BE1-053_TROP_DeBeNZ_PROTON_011HMETHANOL-d422 H's

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

13.0

0

4.40

2.21

1.03

2.08

METHANOL-d4

METHANOL-d4

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

0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

METHANOL-d4

27.2

927

.29

27.2

927

.29

27.2

927

.29

27.2

927

.29

27.2

927

.29

27.2

927

.29

27.2

927

.29

27.2

927

.29

27.2

927

.29

27.2

927

.29

27.2

927

.29

27.9

527

.95

27.9

527

.95

27.9

527

.95

27.9

527

.95

27.9

527

.95

27.9

527

.95

27.9

527

.95

27.9

527

.95

27.9

527

.95

27.9

527

.95

27.9

527

.95

28.5

028

.50

28.5

028

.50

28.5

028

.50

28.5

028

.50

28.5

028

.50

28.5

028

.50

28.5

028

.50

28.5

028

.50

28.5

028

.50

28.5

028

.50

28.5

028

.50

37.1

437

.14

37.1

437

.14

37.1

437

.14

37.1

437

.14

37.1

437

.14

37.1

437

.14

37.1

437

.14

37.1

437

.14

37.1

437

.14

37.1

437

.14

37.1

437

.14

42.7

142

.71

42.7

142

.71

42.7

142

.71

42.7

142

.71

42.7

142

.71

42.7

142

.71

42.7

142

.71

42.7

142

.71

42.7

142

.71

42.7

142

.71

42.7

142

.71

51.6

551

.65

51.6

551

.65

51.6

551

.65

51.6

551

.65

51.6

551

.65

51.6

551

.65

51.6

551

.65

51.6

551

.65

51.6

551

.65

51.6

551

.65

51.6

551

.65

52.5

552

.55

52.5

552

.55

52.5

552

.55

52.5

552

.55

52.5

552

.55

52.5

552

.55

52.5

552

.55

52.5

552

.55

52.5

552

.55

52.5

552

.55

52.5

552

.55

79.4

579

.45

79.4

579

.45

79.4

579

.45

79.4

579

.45

79.4

579

.45

79.4

579

.45

79.4

579

.45

79.4

579

.45

79.4

579

.45

79.4

579

.45

79.4

579

.45

153.

7515

3.75

153.

7515

3.75

153.

7515

3.75

153.

7515

3.75

153.

7515

3.75

153.

7515

3.75

153.

7515

3.75

153.

7515

3.75

153.

7515

3.75

153.

7515

3.75

153.

7515

3.75

242

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

2.39

2.10

2.20

1.99

1.02

1.82

2.08

1.36

1.02

6.23

0.97

1.05

1.00

DMSO-d6*

DMSO-d6*

Water

DMSO-d6

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

0

0.05

0.10

0.15

Impurity

27.5

527

.55

27.5

527

.55

27.5

527

.55

27.5

527

.55

27.5

527

.55

27.5

527

.55

27.5

527

.55

27.5

527

.55

27.5

527

.55

27.5

527

.55

27.5

527

.55

32.8

132

.81

32.8

132

.81

32.8

132

.81

32.8

132

.81

32.8

132

.81

32.8

132

.81

32.8

132

.81

32.8

132

.81

32.8

132

.81

32.8

132

.81

32.8

132

.81

43.6

243

.62

43.6

243

.62

43.6

243

.62

43.6

243

.62

43.6

243

.62

43.6

243

.62

43.6

243

.62

43.6

243

.62

43.6

243

.62

43.6

243

.62

43.6

243

.62

52.2

352

.23

52.2

352

.23

52.2

352

.23

52.2

352

.23

52.2

352

.23

52.2

352

.23

52.2

352

.23

52.2

352

.23

52.2

352

.23

52.2

352

.23

52.2

352

.23

65.5

365

.53

65.5

365

.53

65.5

365

.53

65.5

365

.53

65.5

365

.53

65.5

365

.53

65.5

365

.53

65.5

365

.53

65.5

365

.53

65.5

365

.53

65.5

365

.53

107.

4110

7.41

107.

4110

7.41

107.

4110

7.41

107.

4110

7.41

107.

4110

7.41

107.

4110

7.41

107.

4110

7.41

107.

4110

7.41

107.

4110

7.41

107.

4110

7.41

107.

4110

7.41

118.

0411

8.04

118.

0411

8.04

118.

0411

8.04

118.

0411

8.04

118.

0411

8.04

118.

0411

8.04

118.

0411

8.04

118.

0411

8.04

118.

0411

8.04

118.

0411

8.04

118.

0411

8.04

128.

2212

8.22

128.

2212

8.22

128.

2212

8.22

128.

2212

8.22

128.

2212

8.22

128.

2212

8.22

128.

2212

8.22

128.

2212

8.22

128.

2212

8.22

128.

2212

8.22

128.

2212

8.22

128.

8112

8.81

128.

8112

8.81

128.

8112

8.81

128.

8112

8.81

128.

8112

8.81

128.

8112

8.81

128.

8112

8.81

128.

8112

8.81

128.

8112

8.81

128.

8112

8.81

128.

8112

8.81

137.

0813

7.08

137.

0813

7.08

137.

0813

7.08

137.

0813

7.08

137.

0813

7.08

137.

0813

7.08

137.

0813

7.08

137.

0813

7.08

137.

0813

7.08

137.

0813

7.08

137.

0813

7.08

137.

6613

7.66

137.

6613

7.66

137.

6613

7.66

137.

6613

7.66

137.

6613

7.66

137.

6613

7.66

137.

6613

7.66

137.

6613

7.66

137.

6613

7.66

137.

6613

7.66

137.

6613

7.66

149.

4314

9.43

149.

4314

9.43

149.

4314

9.43

149.

4314

9.43

149.

4314

9.43

149.

4314

9.43

149.

4314

9.43

149.

4314

9.43

149.

4314

9.43

149.

4314

9.43

149.

4314

9.43

156.

2515

6.25

156.

2515

6.25

156.

2515

6.25

156.

2515

6.25

156.

2515

6.25

156.

2515

6.25

156.

2515

6.25

156.

2515

6.25

156.

2515

6.25

156.

2515

6.25

156.

2515

6.25

157.

8915

7.89

157.

8915

7.89

157.

8915

7.89

157.

8915

7.89

157.

8915

7.89

157.

8915

7.89

157.

8915

7.89

157.

8915

7.89

157.

8915

7.89

157.

8915

7.89

157.

8915

7.89

167.

2616

7.26

167.

2616

7.26

167.

2616

7.26

167.

2616

7.26

167.

2616

7.26

167.

2616

7.26

167.

2616

7.26

167.

2616

7.26

167.

2616

7.26

167.

2616

7.26

167.

2616

7.26

171.

8717

1.87

171.

8717

1.87

171.

8717

1.87

171.

8717

1.87

171.

8717

1.87

171.

8717

1.87

171.

8717

1.87

171.

8717

1.87

171.

8717

1.87

171.

8717

1.87

171.

8717

1.87

243

JWL-BE1-061_1_PROTON_011HMETHANOL-d418 H's

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.93

2.32

2.16

2.25

2.02

1.31

1.60

2.49

1.41

1.22

1.11

METHANOL-d4

METHANOL-d4

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

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Impurity

28.1

328

.13

28.1

328

.13

28.1

328

.13

28.1

328

.13

28.1

328

.13

28.1

328

.13

28.1

328

.13

28.1

328

.13

28.1

328

.13

28.1

328

.13

28.1

328

.13

36.2

436

.24

36.2

436

.24

36.2

436

.24

36.2

436

.24

36.2

436

.24

36.2

436

.24

36.2

436

.24

36.2

436

.24

36.2

436

.24

36.2

436

.24

36.2

436

.24

42.9

442

.94

42.9

442

.94

42.9

442

.94

42.9

442

.94

42.9

442

.94

42.9

442

.94

42.9

442

.94

42.9

442

.94

42.9

442

.94

42.9

442

.94

42.9

442

.94

52.5

652

.56

52.5

652

.56

52.5

652

.56

52.5

652

.56

52.5

652

.56

52.5

652

.56

52.5

652

.56

52.5

652

.56

52.5

652

.56

52.5

652

.56

52.5

652

.56

107.

1110

7.11

107.

1110

7.11

107.

1110

7.11

107.

1110

7.11

107.

1110

7.11

107.

1110

7.11

107.

1110

7.11

107.

1110

7.11

107.

1110

7.11

107.

1110

7.11

107.

1110

7.11

116.

6311

6.63

116.

6311

6.63

116.

6311

6.63

116.

6311

6.63

116.

6311

6.63

116.

6311

6.63

116.

6311

6.63

116.

6311

6.63

116.

6311

6.63

116.

6311

6.63

116.

6311

6.63

136.

8113

6.81

136.

8113

6.81

136.

8113

6.81

136.

8113

6.81

136.

8113

6.81

136.

8113

6.81

136.

8113

6.81

136.

8113

6.81

136.

8113

6.81

136.

8113

6.81

136.

8113

6.81

148.

8314

8.83

148.

8314

8.83

148.

8314

8.83

148.

8314

8.83

148.

8314

8.83

148.

8314

8.83

148.

8314

8.83

148.

8314

8.83

148.

8314

8.83

148.

8314

8.83

148.

8314

8.83

158.

0415

8.04

158.

0415

8.04

158.

0415

8.04

158.

0415

8.04

158.

0415

8.04

158.

0415

8.04

158.

0415

8.04

158.

0415

8.04

158.

0415

8.04

158.

0415

8.04

158.

0415

8.04

169.

5416

9.54

169.

5416

9.54

169.

5416

9.54

169.

5416

9.54

169.

5416

9.54

169.

5416

9.54

169.

5416

9.54

169.

5416

9.54

169.

5416

9.54

169.

5416

9.54

169.

5416

9.54

244

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

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0.11

0.12

0.13

27.8

627

.86

27.8

627

.86

27.8

627

.86

27.8

627

.86

27.8

627

.86

27.8

627

.86

27.8

627

.86

27.8

627

.86

27.8

627

.86

27.8

627

.86

27.8

627

.8629

.12

29.1

229

.12

29.1

229

.12

29.1

229

.12

29.1

229

.12

29.1

229

.12

29.1

229

.12

29.1

229

.12

29.1

229

.12

29.1

229

.12

29.1

229

.12

29.1

2

32.4

532

.45

32.4

532

.45

32.4

532

.45

32.4

532

.45

32.4

532

.45

32.4

532

.45

32.4

532

.45

32.4

532

.45

32.4

532

.45

32.4

532

.45

32.4

532

.45

43.0

543

.05

43.0

543

.05

43.0

543

.05

43.0

543

.05

43.0

543

.05

43.0

543

.05

43.0

543

.05

43.0

543

.05

43.0

543

.05

43.0

543

.05

43.0

543

.05

52.3

652

.36

52.3

652

.36

52.3

652

.36

52.3

652

.36

52.3

652

.36

52.3

652

.36

52.3

652

.36

52.3

652

.36

52.3

652

.36

52.3

652

.36

52.3

652

.36

58.4

558

.45

58.4

558

.45

58.4

558

.45

58.4

558

.45

58.4

558

.45

58.4

558

.45

58.4

558

.45

58.4

558

.45

58.4

558

.45

58.4

558

.45

58.4

558

.45

70.0

870

.08

70.0

870

.08

70.0

870

.08

70.0

870

.08

70.0

870

.08

70.0

870

.08

70.0

870

.08

70.0

870

.08

70.0

870

.08

70.0

870

.08

70.0

870

.08

107.

4610

7.46

107.

4610

7.46

107.

4610

7.46

107.

4610

7.46

107.

4610

7.46

107.

4610

7.46

107.

4610

7.46

107.

4610

7.46

107.

4610

7.46

107.

4610

7.46

107.

4610

7.46

110.

3211

0.32

110.

3211

0.32

110.

3211

0.32

110.

3211

0.32

110.

3211

0.32

110.

3211

0.32

110.

3211

0.32

110.

3211

0.32

110.

3211

0.32

110.

3211

0.32

110.

3211

0.32

112.

9211

2.92

112.

9211

2.92

112.

9211

2.92

112.

9211

2.92

112.

9211

2.92

112.

9211

2.92

112.

9211

2.92

112.

9211

2.92

112.

9211

2.92

112.

9211

2.92

112.

9211

2.92

116.

0911

6.09

116.

0911

6.09

116.

0911

6.09

116.

0911

6.09

116.

0911

6.09

116.

0911

6.09

116.

0911

6.09

116.

0911

6.09

116.

0911

6.09

116.

0911

6.09

116.

0911

6.09

118.

1211

8.12

118.

1211

8.12

118.

1211

8.12

118.

1211

8.12

118.

1211

8.12

118.

1211

8.12

118.

1211

8.12

118.

1211

8.12

118.

1211

8.12

118.

1211

8.12

118.

1211

8.12

129.

4712

9.47

129.

4712

9.47

129.

4712

9.47

129.

4712

9.47

129.

4712

9.47

129.

4712

9.47

129.

4712

9.47

129.

4712

9.47

129.

4712

9.47

129.

4712

9.47

129.

4712

9.47

137.

1513

7.15

137.

1513

7.15

137.

1513

7.15

137.

1513

7.15

137.

1513

7.15

137.

1513

7.15

137.

1513

7.15

137.

1513

7.15

137.

1513

7.15

137.

1513

7.15

137.

1513

7.15

139.

2213

9.22

139.

2213

9.22

139.

2213

9.22

139.

2213

9.22

139.

2213

9.22

139.

2213

9.22

139.

2213

9.22

139.

2213

9.22

139.

2213

9.22

139.

2213

9.22

139.

2213

9.22

149.

4614

9.46

149.

4614

9.46

149.

4614

9.46

149.

4614

9.46

149.

4614

9.46

149.

4614

9.46

149.

4614

9.46

149.

4614

9.46

149.

4614

9.46

149.

4614

9.46

149.

4614

9.46

149.

9614

9.96

149.

9614

9.96

149.

9614

9.96

149.

9614

9.96

149.

9614

9.96

149.

9614

9.96

149.

9614

9.96

149.

9614

9.96

149.

9614

9.96

149.

9614

9.96

149.

9614

9.96

158.

0315

8.03

158.

0315

8.03

158.

0315

8.03

158.

0315

8.03

158.

0315

8.03

158.

0315

8.03

158.

0315

8.03

158.

0315

8.03

158.

0315

8.03

158.

0315

8.03

158.

0315

8.0316

7.20

167.

2016

7.20

167.

2016

7.20

167.

2016

7.20

167.

2016

7.20

167.

2016

7.20

167.

2016

7.20

167.

2016

7.20

167.

2016

7.20

167.

2016

7.20

167.

2016

7.20

167.

20

167.

2916

7.29

167.

2916

7.29

167.

2916

7.29

167.

2916

7.29

167.

2916

7.29

167.

2916

7.29

167.

2916

7.29

167.

2916

7.29

167.

2916

7.29

167.

2916

7.29

167.

2916

7.29

171.

5117

1.51

171.

5117

1.51

171.

5117

1.51

171.

5117

1.51

171.

5117

1.51

171.

5117

1.51

171.

5117

1.51

171.

5117

1.51

171.

5117

1.51

171.

5117

1.51

171.

5117

1.51

JWL-BE1-061_2-RECRYS_PROTON_01

11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

Nor

mal

ized

Inte

nsity

2.001.931.842.071.871.762.801.951.071.830.940.960.920.870.790.930.860.781.080.930.930.93

245

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

-0.01

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

27.8

327

.83

27.8

327

.83

27.8

327

.83

27.8

327

.83

27.8

327

.83

27.8

327

.83

27.8

327

.83

27.8

327

.83

27.8

327

.83

27.8

327

.83

27.8

327

.8329

.09

29.0

929

.09

29.0

929

.09

29.0

929

.09

29.0

929

.09

29.0

929

.09

29.0

929

.09

29.0

929

.09

29.0

929

.09

29.0

929

.09

29.0

929

.09

29.0

9

32.4

232

.42

32.4

232

.42

32.4

232

.42

32.4

232

.42

32.4

232

.42

32.4

232

.42

32.4

232

.42

32.4

232

.42

32.4

232

.42

32.4

232

.42

32.4

232

.42

40.5

540

.55

40.5

540

.55

40.5

540

.55

40.5

540

.55

40.5

540

.55

40.5

540

.55

40.5

540

.55

40.5

540

.55

40.5

540

.55

40.5

540

.55

40.5

540

.55

42.7

642

.76

42.7

642

.76

42.7

642

.76

42.7

642

.76

42.7

642

.76

42.7

642

.76

42.7

642

.76

42.7

642

.76

42.7

642

.76

42.7

642

.76

42.7

642

.76

43.0

343

.03

43.0

343

.03

43.0

343

.03

43.0

343

.03

43.0

343

.03

43.0

343

.03

43.0

343

.03

43.0

343

.03

43.0

343

.03

43.0

343

.03

43.0

343

.03

52.3

752

.37

52.3

752

.37

52.3

752

.37

52.3

752

.37

52.3

752

.37

52.3

752

.37

52.3

752

.37

52.3

752

.37

52.3

752

.37

52.3

752

.37

52.3

752

.37

58.4

258

.42

58.4

258

.42

58.4

258

.42

58.4

258

.42

58.4

258

.42

58.4

258

.42

58.4

258

.42

58.4

258

.42

58.4

258

.42

58.4

258

.42

58.4

258

.42

70.0

570

.05

70.0

570

.05

70.0

570

.05

70.0

570

.05

70.0

570

.05

70.0

570

.05

70.0

570

.05

70.0

570

.05

70.0

570

.05

70.0

570

.05

70.0

570

.05

107.

5410

7.54

107.

5410

7.54

107.

5410

7.54

107.

5410

7.54

107.

5410

7.54

107.

5410

7.54

107.

5410

7.54

107.

5410

7.54

107.

5410

7.54

107.

5410

7.54

107.

5410

7.54

110.

2911

0.29

110.

2911

0.29

110.

2911

0.29

110.

2911

0.29

110.

2911

0.29

110.

2911

0.29

110.

2911

0.29

110.

2911

0.29

110.

2911

0.29

110.

2911

0.29

110.

2911

0.29

112.

9011

2.90

112.

9011

2.90

112.

9011

2.90

112.

9011

2.90

112.

9011

2.90

112.

9011

2.90

112.

9011

2.90

112.

9011

2.90

112.

9011

2.90

112.

9011

2.90

112.

9011

2.90

116.

0711

6.07

116.

0711

6.07

116.

0711

6.07

116.

0711

6.07

116.

0711

6.07

116.

0711

6.07

116.

0711

6.07

116.

0711

6.07

116.

0711

6.07

116.

0711

6.07

116.

0711

6.07

118.

1611

8.16

118.

1611

8.16

118.

1611

8.16

118.

1611

8.16

118.

1611

8.16

118.

1611

8.16

118.

1611

8.16

118.

1611

8.16

118.

1611

8.16

118.

1611

8.16

118.

1611

8.16

127.

0912

7.09

127.

0912

7.09

127.

0912

7.09

127.

0912

7.09

127.

0912

7.09

127.

0912

7.09

127.

0912

7.09

127.

0912

7.09

127.

0912

7.09

127.

0912

7.09

127.

0912

7.09

127.

5812

7.58

127.

5812

7.58

127.

5812

7.58

127.

5812

7.58

127.

5812

7.58

127.

5812

7.58

127.

5812

7.58

127.

5812

7.58

127.

5812

7.58

127.

5812

7.58

127.

5812

7.58

128.

6812

8.68

128.

6812

8.68

128.

6812

8.68

128.

6812

8.68

128.

6812

8.68

128.

6812

8.68

128.

6812

8.68

128.

6812

8.68

128.

6812

8.68

128.

6812

8.68

128.

6812

8.68

129.

4412

9.44

129.

4412

9.44

129.

4412

9.44

129.

4412

9.44

129.

4412

9.44

129.

4412

9.44

129.

4412

9.44

129.

4412

9.44

129.

4412

9.44

129.

4412

9.44

129.

4412

9.44

136.

8213

6.82

136.

8213

6.82

136.

8213

6.82

136.

8213

6.82

136.

8213

6.82

136.

8213

6.82

136.

8213

6.82

136.

8213

6.82

136.

8213

6.82

136.

8213

6.82

136.

8213

6.82

140.

4014

0.40

140.

4014

0.40

140.

4014

0.40

140.

4014

0.40

140.

4014

0.40

140.

4014

0.40

140.

4014

0.40

140.

4014

0.40

140.

4014

0.40

140.

4014

0.40

140.

4014

0.40

149.

1514

9.15

149.

1514

9.15

149.

1514

9.15

149.

1514

9.15

149.

1514

9.15

149.

1514

9.15

149.

1514

9.15

149.

1514

9.15

149.

1514

9.15

149.

1514

9.15

149.

1514

9.15

149.

9414

9.94

149.

9414

9.94

149.

9414

9.94

149.

9414

9.94

149.

9414

9.94

149.

9414

9.94

149.

9414

9.94

149.

9414

9.94

149.

9414

9.94

149.

9414

9.94

149.

9414

9.94

158.

0215

8.02

158.

0215

8.02

158.

0215

8.02

158.

0215

8.02

158.

0215

8.02

158.

0215

8.02

158.

0215

8.02

158.

0215

8.02

158.

0215

8.02

158.

0215

8.02

158.

0215

8.02

165.

5116

5.51

165.

5116

5.51

165.

5116

5.51

165.

5116

5.51

165.

5116

5.51

165.

5116

5.51

165.

5116

5.51

165.

5116

5.51

165.

5116

5.51

165.

5116

5.51

165.

5116

5.51

167.

1816

7.18

167.

1816

7.18

167.

1816

7.18

167.

1816

7.18

167.

1816

7.18

167.

1816

7.18

167.

1816

7.18

167.

1816

7.18

167.

1816

7.18

167.

1816

7.18

167.

1816

7.18

171.

4817

1.48

171.

4817

1.48

171.

4817

1.48

171.

4817

1.48

171.

4817

1.48

171.

4817

1.48

171.

4817

1.48

171.

4817

1.48

171.

4817

1.48

171.

4817

1.48

171.

4817

1.48

jwl-be1-060_2_P2_PROTON_01.esp

11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

2.001.961.952.151.924.782.091.151.951.931.081.021.015.701.030.951.101.041.040.971.12

246

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

0

0.05

0.10

0.15

DMSO-d6

14.7

514

.75

14.7

514

.75

14.7

514

.75

14.7

514

.75

14.7

514

.75

14.7

514

.75

14.7

514

.75

14.7

514

.75

14.7

514

.75

14.7

514

.75

14.7

514

.75

27.7

027

.70

27.7

027

.70

27.7

027

.70

27.7

027

.70

27.7

027

.70

27.7

027

.70

27.7

027

.70

27.7

027

.70

27.7

027

.70

27.7

027

.70

27.7

027

.70

29.0

929

.09

29.0

929

.09

29.0

929

.09

29.0

929

.09

29.0

929

.09

29.0

929

.09

29.0

929

.09

29.0

929

.09

29.0

929

.09

29.0

929

.09

29.0

929

.09

32.8

332

.83

32.8

332

.83

32.8

332

.83

32.8

332

.83

32.8

332

.83

32.8

332

.83

32.8

332

.83

32.8

332

.83

32.8

332

.83

32.8

332

.83

32.8

332

.83

39.4

639

.46

39.4

639

.46

39.4

639

.46

39.4

639

.46

39.4

639

.46

39.4

639

.46

39.4

639

.46

39.4

639

.46

39.4

639

.46

39.4

639

.46

39.4

639

.46

43.0

143

.01

43.0

143

.01

43.0

143

.01

43.0

143

.01

43.0

143

.01

43.0

143

.01

43.0

143

.01

43.0

143

.01

43.0

143

.01

43.0

143

.01

43.0

143

.0152

.50

52.5

052

.50

52.5

052

.50

52.5

052

.50

52.5

052

.50

52.5

052

.50

52.5

052

.50

52.5

052

.50

52.5

052

.50

52.5

052

.50

52.5

052

.50

52.5

0

58.4

258

.42

58.4

258

.42

58.4

258

.42

58.4

258

.42

58.4

258

.42

58.4

258

.42

58.4

258

.42

58.4

258

.42

58.4

258

.42

58.4

258

.42

58.4

258

.42

60.3

560

.35

60.3

560

.35

60.3

560

.35

60.3

560

.35

60.3

560

.35

60.3

560

.35

60.3

560

.35

60.3

560

.35

60.3

560

.35

60.3

560

.35

60.3

560

.3570

.05

70.0

570

.05

70.0

570

.05

70.0

570

.05

70.0

570

.05

70.0

570

.05

70.0

570

.05

70.0

570

.05

70.0

570

.05

70.0

570

.05

70.0

570

.05

70.0

5

107.

6110

7.61

107.

6110

7.61

107.

6110

7.61

107.

6110

7.61

107.

6110

7.61

107.

6110

7.61

107.

6110

7.61

107.

6110

7.61

107.

6110

7.61

107.

6110

7.61

107.

6110

7.61

110.

2911

0.29

110.

2911

0.29

110.

2911

0.29

110.

2911

0.29

110.

2911

0.29

110.

2911

0.29

110.

2911

0.29

110.

2911

0.29

110.

2911

0.29

110.

2911

0.29

110.

2911

0.29

112.

9011

2.90

112.

9011

2.90

112.

9011

2.90

112.

9011

2.90

112.

9011

2.90

112.

9011

2.90

112.

9011

2.90

112.

9011

2.90

112.

9011

2.90

112.

9011

2.90

112.

9011

2.90

113.

7511

3.75

113.

7511

3.75

113.

7511

3.75

113.

7511

3.75

113.

7511

3.75

113.

7511

3.75

113.

7511

3.75

113.

7511

3.75

113.

7511

3.75

113.

7511

3.75

113.

7511

3.75

116.

0811

6.08

116.

0811

6.08

116.

0811

6.08

116.

0811

6.08

116.

0811

6.08

116.

0811

6.08

116.

0811

6.08

116.

0811

6.08

116.

0811

6.08

116.

0811

6.08

116.

0811

6.08

129.

4512

9.45

129.

4512

9.45

129.

4512

9.45

129.

4512

9.45

129.

4512

9.45

129.

4512

9.45

129.

4512

9.45

129.

4512

9.45

129.

4512

9.45

129.

4512

9.45

129.

4512

9.45

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

139.

1513

9.15

139.

1513

9.15

139.

1513

9.15

139.

1513

9.15

139.

1513

9.15

139.

1513

9.15

139.

1513

9.15

139.

1513

9.15

139.

1513

9.15

139.

1513

9.15

139.

1513

9.15

149.

9414

9.94

149.

9414

9.94

149.

9414

9.94

149.

9414

9.94

149.

9414

9.94

149.

9414

9.94

149.

9414

9.94

149.

9414

9.94

149.

9414

9.94

149.

9414

9.94

149.

9414

9.94

151.

4715

1.47

151.

4715

1.47

151.

4715

1.47

151.

4715

1.47

151.

4715

1.47

151.

4715

1.47

151.

4715

1.47

151.

4715

1.47

151.

4715

1.47

151.

4715

1.47

151.

4715

1.47

158.

4715

8.47

158.

4715

8.47

158.

4715

8.47

158.

4715

8.47

158.

4715

8.47

158.

4715

8.47

158.

4715

8.47

158.

4715

8.47

158.

4715

8.47

158.

4715

8.47

158.

4715

8.47

165.

5816

5.58

165.

5816

5.58

165.

5816

5.58

165.

5816

5.58

165.

5816

5.58

165.

5816

5.58

165.

5816

5.58

165.

5816

5.58

165.

5816

5.58

165.

5816

5.58

165.

5816

5.58

167.

2216

7.22

167.

2216

7.22

167.

2216

7.22

167.

2216

7.22

167.

2216

7.22

167.

2216

7.22

167.

2216

7.22

167.

2216

7.22

167.

2216

7.22

167.

2216

7.22

167.

2216

7.22

171.

4817

1.48

171.

4817

1.48

171.

4817

1.48

171.

4817

1.48

171.

4817

1.48

171.

4817

1.48

171.

4817

1.48

171.

4817

1.48

171.

4817

1.48

171.

4817

1.48

171.

4817

1.48

JWL-BE1-062_2_PROTON

11 10 9 8 7 6 5 4 3 2 1

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

3.002.152.044.162.004.672.011.132.062.030.990.940.890.780.871.400.871.020.88

247

JWL-BE1-062_2_ACID_PROTON_01

11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

3.003.712.204.134.512.421.142.102.101.070.880.950.840.891.070.99

248

Appendix V

Chapter 7 – NMR Spectra

JWL-BE2-071_PROTON_01

12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shift (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

1.000.890.960.910.840.86

JWL-BE2-071_CARBON_01

200 180 160 140 120 100 80 60 40 20 0

Chemical Shift (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

119.

3612

0.43

125.

1012

5.74

127.

18

135.

66

156.

06

163.

91

175.

34

188.

84

249

JWL-BE2-072_PROTON_01

12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shift (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

3.003.004.061.990.971.040.990.93

JWL-BE1-072_CARBON_01

200 180 160 140 120 100 80 60 40 20 0

Chemical Shift (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Norm

alize

d In

tensi

ty

14.

2314.

45

61.

3261.

84

118

.58

119

.02

123

.53

125

.91

128

.15

134

.25

135

.34

155

.69

159

.78

164

.31

164

.90

174

.38

250

JWL-BE2-074_P2_MAY16_PROTON_01

12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shift (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

3.002.112.102.090.970.970.940.951.04

JWL-BE2-074_MAY2016_CARBON_01

200 180 160 140 120 100 80 60 40 20 0

Chemical Shift (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

14.5

5

61.6

7

117.

1511

7.75

119.

9012

1.42

121.

65124.

69

130.

5713

3.85

138.

5614

1.81

142.

49

155.

0615

6.21

156.

8516

3.60

191.

42

251

JWL-BE1-074_1_P3_19MAY16_PROTON_01

13 12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

2.112.070.880.890.830.871.001.01

JWL-BE1-074_1_P3_19MAY16_CARBON_01

200 180 160 140 120 100 80 60 40 20 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsi

ty

19

1.4

7

16

4.7

9

15

7.9

0 15

6.2

11

55.0

5

14

2.7

91

41.4

51

38.6

01

33.8

91

30.5

91

24.7

21

21.7

91

21.5

01

19.9

31

18.1

31

17.1

4

252

JWL-BE1-073_PROTON_01

12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

3.002.041.951.051.037.161.001.101.13

JWL-BE1-073_CARBON_01

200 180 160 140 120 100 80 60 40 20 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

19

1.43

16

4.43 15

8.32

15

6.21

14

9.48

14

3.37

13

6.59

13

0.52

12

9.07

12

8.31

12

8.22

11

9.32 1

16.

23

61

.18

53

.18

14

.53

253

JWL-BE2-073_1_PPT_PROTON_01

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2

Chemical Shif t (ppm)

0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

No

rma

lize

d I

nte

nsity

2.002.070.945.011.100.941.041.020.96

JWL-BE2-073_1_PPT_CARBON_01

200 180 160 140 120 100 80 60 40 20 0

Chemical Shif t (ppm)

0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

Nor

mal

ized

Inte

nsity

191.

39

164.

7916

2.91 15

6.25

149.

05

144.

73

135.

93

130.

7012

9.18

128.

4612

4.84

119.

9211

8.78

117.

14

53.6

0

254

BE2-077_PROTON_01

12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsi

ty

3.001.951.000.941.993.900.960.910.890.89

BE2-077_CARBON_01

200 180 160 140 120 100 80 60 40 20 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

194.

18

164.

0616

2.17

158.

41

146.

9714

4.10

139.

6313

6.39

129.

49 126.

43

119.

2411

8.69

115.

91

61.6

1

14.2

0

255

JWL-BE2-077_1_May16_PROTON_01

13 12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

2.192.115.230.970.871.001.11

JWL-BE2-077_1_May16_CARBON_01

220 200 180 160 140 120 100 80 60 40 20 0 -20

Chemical Shif t (ppm)

0

0.05

0.10

0.15

0.20

0.25

0.30

Nor

mal

ized

Inte

nsity

164.

83

156.

27

139.

68

130.

8012

9.81

127.

2012

4.65

119.

9811

7.17

256

JWL-BE2-075_AB_PROTON_01

12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

3.002.081.081.070.991.011.111.840.920.880.961.03

JWL-BE2-075_AB_CARBON_01

220 200 180 160 140 120 100 80 60 40 20 0 -20

Chemical Shif t (ppm)

0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

No

rma

lize

d I

nte

nsi

ty

19

1.4

3

16

3.5

71

57.0

01

56.2

81

54.8

31

44.4

3

13

0.5

11

28.6

81

25.2

01

24.6

31

21.2

11

17.6

71

17.0

51

14.0

7

61

.70 14

.55

257

JWL-BE2-075_1_PROTON_01

13 12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

1.000.960.910.941.041.710.850.820.850.940.85

JWL-BE2-075_1_CARBON_01

200 180 160 140 120 100 80 60 40 20 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

19

1.50

16

4.78

15

7.93

15

6.28

15

4.84

14

4.47

14

2.92

14

1.42 1

37.

661

30.

541

28.

691

25.

231

24.

681

21.

371

18.

021

17.

061

14.

18

258

JWL-BE2-076_PROTON_01

11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

3.002.892.121.121.051.011.110.880.890.811.15

JWL-BE2-076_CARBON_01

Chemical Shift (ppm)192 184 176 168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24

No

rm

alize

d In

ten

sity

-0.01

0

0.01

0.02

0.03

0.04

0.05

0.06

M07(m)

M11(m)

M10(m)

M08(m)

M14(m)M17(m)

M13(m)M15(m)

M16(m)

M19(m)

M09(m)

M18(m)

M06(m)

M12(m) M05(m)

M04(m)

M02(m)

M03(m)M01(m)

259

JWL-BE2-076_1_PROTON_01

13 12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

3.001.061.041.011.060.880.870.800.851.06

JWL-BE2-076_1_CARBON_01

Chemical Shift (ppm)192 184 176 168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24

No

rma

lize

d In

ten

sity

-0.01

0

0.01

0.02

0.03

0.04

0.05

0.06

M03(m)M07(m)

M16(m)

M05(m)

M06(m)

M08(m)

M14(m)

M13(m)

M09(m)

M10(m)

M11(m)

M15(m)

M17(m)

M12(m)

M02(m)

M01(m)

260

JWL-BE2-078_PROTON_01

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

1.171.190.960.961.00

JWL-BE2-078_CARBON_01

220 200 180 160 140 120 100 80 60 40 20 0 -20

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsi

ty

18

8.5

2

17

4.1

6

16

4.2

3

15

5.0

5

13

8.1

8

12

7.7

91

26.6

81

22.0

11

20.4

81

19.6

5

261

JWL-BE2-079_PROTON_01

11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

3.002.623.620.840.860.920.700.83

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

Abs

olut

e In

tens

ity

0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

14.2

514

.25

14.2

514

.25

14.2

514

.25

14.2

514

.25

14.2

514

.25

14.2

514

.25

14.2

514

.25

14.2

514

.25

14.2

514

.25

14.2

514

.25

14.2

514

.25

14.4

414

.44

14.4

414

.44

14.4

414

.44

14.4

414

.44

14.4

414

.44

14.4

414

.44

14.4

414

.44

14.4

414

.44

14.4

414

.44

14.4

414

.44

14.4

414

.44

61.3

961

.39

61.3

961

.39

61.3

961

.39

61.3

961

.39

61.3

961

.39

61.3

961

.39

61.3

961

.39

61.3

961

.39

61.3

961

.39

61.3

961

.39

61.3

961

.39

61.8

961

.89

61.8

961

.89

61.8

961

.89

61.8

961

.89

61.8

961

.89

61.8

961

.89

61.8

961

.89

61.8

961

.89

61.8

961

.89

61.8

961

.89

61.8

961

.89

118.

6511

8.65

118.

6511

8.65

118.

6511

8.65

118.

6511

8.65

118.

6511

8.65

118.

6511

8.65

118.

6511

8.65

118.

6511

8.65

118.

6511

8.65

118.

6511

8.65

118.

6511

8.65

119.

1911

9.19

119.

1911

9.19

119.

1911

9.19

119.

1911

9.19

119.

1911

9.19

119.

1911

9.19

119.

1911

9.19

119.

1911

9.19

119.

1911

9.19

119.

1911

9.19

119.

1911

9.1912

1.76

121.

7612

1.76

121.

7612

1.76

121.

7612

1.76

121.

7612

1.76

121.

7612

1.76

121.

7612

1.76

121.

7612

1.76

121.

7612

1.76

121.

7612

1.76

121.

7612

1.76

121.

76

125.

0112

5.01

125.

0112

5.01

125.

0112

5.01

125.

0112

5.01

125.

0112

5.01

125.

0112

5.01

125.

0112

5.01

125.

0112

5.01

125.

0112

5.01

125.

0112

5.01

125.

0112

5.01

128.

4812

8.48

128.

4812

8.48

128.

4812

8.48

128.

4812

8.48

128.

4812

8.48

128.

4812

8.48

128.

4812

8.48

128.

4812

8.48

128.

4812

8.48

128.

4812

8.48

128.

4812

8.48

133.

8313

3.83

133.

8313

3.83

133.

8313

3.83

133.

8313

3.83

133.

8313

3.83

133.

8313

3.83

133.

8313

3.83

133.

8313

3.83

133.

8313

3.83

133.

8313

3.83

133.

8313

3.83

137.

9113

7.91

137.

9113

7.91

137.

9113

7.91

137.

9113

7.91

137.

9113

7.91

137.

9113

7.91

137.

9113

7.91

137.

9113

7.91

137.

9113

7.91

137.

9113

7.91

137.

9113

7.91

154.

6815

4.68

154.

6815

4.68

154.

6815

4.68

154.

6815

4.68

154.

6815

4.68

154.

6815

4.68

154.

6815

4.68

154.

6815

4.68

154.

6815

4.68

154.

6815

4.68

154.

6815

4.6816

0.08

160.

0816

0.08

160.

0816

0.08

160.

0816

0.08

160.

0816

0.08

160.

0816

0.08

160.

0816

0.08

160.

0816

0.08

160.

0816

0.08

160.

0816

0.08

160.

0816

0.08

160.

08

164.

2416

4.24

164.

2416

4.24

164.

2416

4.24

164.

2416

4.24

164.

2416

4.24

164.

2416

4.24

164.

2416

4.24

164.

2416

4.24

164.

2416

4.24

164.

2416

4.24

164.

2416

4.24

164.

8116

4.81

164.

8116

4.81

164.

8116

4.81

164.

8116

4.81

164.

8116

4.81

164.

8116

4.81

164.

8116

4.81

164.

8116

4.81

164.

8116

4.81

164.

8116

4.81

164.

8116

4.81

173.

2217

3.22

173.

2217

3.22

173.

2217

3.22

173.

2217

3.22

173.

2217

3.22

173.

2217

3.22

173.

2217

3.22

173.

2217

3.22

173.

2217

3.22

173.

2217

3.22

173.

2217

3.22

262

JWL-BE2-080_PROTON_01

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

3.002.111.060.891.000.920.930.840.861.03

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

Abs

olut

e In

tens

ity

0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

14.5

414

.54

14.5

414

.54

14.5

414

.54

14.5

414

.54

14.5

414

.54

14.5

414

.54

14.5

414

.54

14.5

414

.54

14.5

414

.54

14.5

414

.54

14.5

414

.54

61.7

161

.71

61.7

161

.71

61.7

161

.71

61.7

161

.71

61.7

161

.71

61.7

161

.71

61.7

161

.71

61.7

161

.71

61.7

161

.71

61.7

161

.71

61.7

161

.71

110.

8911

0.89

110.

8911

0.89

110.

8911

0.89

110.

8911

0.89

110.

8911

0.89

110.

8911

0.89

110.

8911

0.89

110.

8911

0.89

110.

8911

0.89

110.

8911

0.89

110.

8911

0.89

117.

3611

7.36

117.

3611

7.36

117.

3611

7.36

117.

3611

7.36

117.

3611

7.36

117.

3611

7.36

117.

3611

7.36

117.

3611

7.36

117.

3611

7.36

117.

3611

7.36

117.

3611

7.36

119.

3711

9.37

119.

3711

9.37

119.

3711

9.37

119.

3711

9.37

119.

3711

9.37

119.

3711

9.37

119.

3711

9.37

119.

3711

9.37

119.

3711

9.37

119.

3711

9.37

119.

3711

9.37

121.

7412

1.74

121.

7412

1.74

121.

7412

1.74

121.

7412

1.74

121.

7412

1.74

121.

7412

1.74

121.

7412

1.74

121.

7412

1.74

121.

7412

1.74

121.

7412

1.74

121.

7412

1.74

127.

0412

7.04

127.

0412

7.04

127.

0412

7.04

127.

0412

7.04

127.

0412

7.04

127.

0412

7.04

127.

0412

7.04

127.

0412

7.04

127.

0412

7.04

127.

0412

7.04

127.

0412

7.04

132.

3913

2.39

132.

3913

2.39

132.

3913

2.39

132.

3913

2.39

132.

3913

2.39

132.

3913

2.39

132.

3913

2.39

132.

3913

2.39

132.

3913

2.39

132.

3913

2.39

132.

3913

2.39

135.

9713

5.97

135.

9713

5.97

135.

9713

5.97

135.

9713

5.97

135.

9713

5.97

135.

9713

5.97

135.

9713

5.97

135.

9713

5.97

135.

9713

5.97

135.

9713

5.97

135.

9713

5.97

138.

5913

8.59

138.

5913

8.59

138.

5913

8.59

138.

5913

8.59

138.

5913

8.59

138.

5913

8.59

138.

5913

8.59

138.

5913

8.59

138.

5913

8.59

138.

5913

8.59

138.

5913

8.59

142.

0214

2.02

142.

0214

2.02

142.

0214

2.02

142.

0214

2.02

142.

0214

2.02

142.

0214

2.02

142.

0214

2.02

142.

0214

2.02

142.

0214

2.02

142.

0214

2.02

142.

0214

2.02

142.

2214

2.22

142.

2214

2.22

142.

2214

2.22

142.

2214

2.22

142.

2214

2.22

142.

2214

2.22

142.

2214

2.22

142.

2214

2.22

142.

2214

2.22

142.

2214

2.22

142.

2214

2.22

155.

0015

5.00

155.

0015

5.00

155.

0015

5.00

155.

0015

5.00

155.

0015

5.00

155.

0015

5.00

155.

0015

5.00

155.

0015

5.00

155.

0015

5.00

155.

0015

5.00

155.

0015

5.00

155.

1415

5.14

155.

1415

5.14

155.

1415

5.14

155.

1415

5.14

155.

1415

5.14

155.

1415

5.14

155.

1415

5.14

155.

1415

5.14

155.

1415

5.14

155.

1415

5.14

155.

1415

5.14

156.

8115

6.81

156.

8115

6.81

156.

8115

6.81

156.

8115

6.81

156.

8115

6.81

156.

8115

6.81

156.

8115

6.81

156.

8115

6.81

156.

8115

6.81

156.

8115

6.81

156.

8115

6.81

163.

5316

3.53

163.

5316

3.53

163.

5316

3.53

163.

5316

3.53

163.

5316

3.53

163.

5316

3.53

163.

5316

3.53

163.

5316

3.53

163.

5316

3.53

163.

5316

3.53

163.

5316

3.53

189.

7418

9.74

189.

7418

9.74

189.

7418

9.74

189.

7418

9.74

189.

7418

9.74

189.

7418

9.74

189.

7418

9.74

189.

7418

9.74

189.

7418

9.74

189.

7418

9.74

189.

7418

9.74

263

JWL-BE2-080_1_PROTON_01

13 12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

1.000.830.900.810.800.830.870.980.87

Chemical Shift (ppm)192 184 176 168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24

No

rm

alize

d I

nte

nsity

0

0.05

0.10

0.15

0.20

0.25

11

0.9

01

10.9

01

10.9

01

10.9

01

10.9

01

10.9

01

10.9

01

10.9

01

10.9

01

10.9

01

10.9

01

10.9

01

10.9

01

10.9

01

10.9

01

10.9

01

10.9

01

10.9

01

10.9

01

10.9

01

10.9

01

10.9

0

11

7.7

11

17.7

11

17.7

11

17.7

11

17.7

11

17.7

11

17.7

11

17.7

11

17.7

11

17.7

11

17.7

11

17.7

11

17.7

11

17.7

11

17.7

11

17.7

11

17.7

11

17.7

11

17.7

11

17.7

11

17.7

11

17.7

1

11

9.3

71

19.3

71

19.3

71

19.3

71

19.3

71

19.3

71

19.3

71

19.3

71

19.3

71

19.3

71

19.3

71

19.3

71

19.3

71

19.3

71

19.3

71

19.3

71

19.3

71

19.3

71

19.3

71

19.3

71

19.3

71

19.3

7

12

1.6

51

21.6

51

21.6

51

21.6

51

21.6

51

21.6

51

21.6

51

21.6

51

21.6

51

21.6

51

21.6

51

21.6

51

21.6

51

21.6

51

21.6

51

21.6

51

21.6

51

21.6

51

21.6

51

21.6

51

21.6

51

21.6

5

12

1.8

41

21.8

41

21.8

41

21.8

41

21.8

41

21.8

41

21.8

41

21.8

41

21.8

41

21.8

41

21.8

41

21.8

41

21.8

41

21.8

41

21.8

41

21.8

41

21.8

41

21.8

41

21.8

41

21.8

41

21.8

41

21.8

4

12

7.0

71

27.0

71

27.0

71

27.0

71

27.0

71

27.0

71

27.0

71

27.0

71

27.0

71

27.0

71

27.0

71

27.0

71

27.0

71

27.0

71

27.0

71

27.0

71

27.0

71

27.0

71

27.0

71

27.0

71

27.0

71

27.0

7

13

2.4

11

32.4

11

32.4

11

32.4

11

32.4

11

32.4

11

32.4

11

32.4

11

32.4

11

32.4

11

32.4

11

32.4

11

32.4

11

32.4

11

32.4

11

32.4

11

32.4

11

32.4

11

32.4

11

32.4

11

32.4

11

32.4

1

13

5.9

91

35.9

91

35.9

91

35.9

91

35.9

91

35.9

91

35.9

91

35.9

91

35.9

91

35.9

91

35.9

91

35.9

91

35.9

91

35.9

91

35.9

91

35.9

91

35.9

91

35.9

91

35.9

91

35.9

91

35.9

91

35.9

9

13

8.6

21

38.6

21

38.6

21

38.6

21

38.6

21

38.6

21

38.6

21

38.6

21

38.6

21

38.6

21

38.6

21

38.6

21

38.6

21

38.6

21

38.6

21

38.6

21

38.6

21

38.6

21

38.6

21

38.6

21

38.6

21

38.6

2

14

1.6

71

41.6

71

41.6

71

41.6

71

41.6

71

41.6

71

41.6

71

41.6

71

41.6

71

41.6

71

41.6

71

41.6

71

41.6

71

41.6

71

41.6

71

41.6

71

41.6

71

41.6

71

41.6

71

41.6

71

41.6

71

41.6

7

14

2.4

51

42.4

51

42.4

51

42.4

51

42.4

51

42.4

51

42.4

51

42.4

51

42.4

51

42.4

51

42.4

51

42.4

51

42.4

51

42.4

51

42.4

51

42.4

51

42.4

51

42.4

51

42.4

51

42.4

51

42.4

51

42.4

5

15

5.0

11

55.0

11

55.0

11

55.0

11

55.0

11

55.0

11

55.0

11

55.0

11

55.0

11

55.0

11

55.0

11

55.0

11

55.0

11

55.0

11

55.0

11

55.0

11

55.0

11

55.0

11

55.0

11

55.0

11

55.0

11

55.0

1

15

5.1

41

55.1

41

55.1

41

55.1

41

55.1

41

55.1

41

55.1

41

55.1

41

55.1

41

55.1

41

55.1

41

55.1

41

55.1

41

55.1

41

55.1

41

55.1

41

55.1

41

55.1

41

55.1

41

55.1

41

55.1

41

55.1

4

15

7.7

81

57.7

81

57.7

81

57.7

81

57.7

81

57.7

81

57.7

81

57.7

81

57.7

81

57.7

81

57.7

81

57.7

81

57.7

81

57.7

81

57.7

81

57.7

81

57.7

81

57.7

81

57.7

81

57.7

81

57.7

81

57.7

8

16

4.7

51

64.7

51

64.7

51

64.7

51

64.7

51

64.7

51

64.7

51

64.7

51

64.7

51

64.7

51

64.7

51

64.7

51

64.7

51

64.7

51

64.7

51

64.7

51

64.7

51

64.7

51

64.7

51

64.7

51

64.7

51

64.7

5

18

9.8

11

89.8

11

89.8

11

89.8

11

89.8

11

89.8

11

89.8

11

89.8

11

89.8

11

89.8

11

89.8

11

89.8

11

89.8

11

89.8

11

89.8

11

89.8

11

89.8

11

89.8

11

89.8

11

89.8

11

89.8

11

89.8

1

264

265

JWL-BE2-084_PROTON_01

12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

1.001.961.960.910.970.890.740.76

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

Ab

solu

te I

nte

nsity

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

120

.17

120

.17

120

.17

120

.17

120

.17

120

.17

120

.17

120

.17

120

.17

120

.17

120

.17

120

.17

120

.17

120

.17

120

.17

120

.17

120

.17

120

.17

120

.17

120

.17

120

.17

120

.17

120

.44

120

.44

120

.44

120

.44

120

.44

120

.44

120

.44

120

.44

120

.44

120

.44

120

.44

120

.44

120

.44

120

.44

120

.44

120

.44

120

.44

120

.44

120

.44

120

.44

120

.44

120

.44

122

.85

122

.85

122

.85

122

.85

122

.85

122

.85

122

.85

122

.85

122

.85

122

.85

122

.85

122

.85

122

.85

122

.85

122

.85

122

.85

122

.85

122

.85

122

.85

122

.85

122

.85

122

.85

125

.39

125

.39

125

.39

125

.39

125

.39

125

.39

125

.39

125

.39

125

.39

125

.39

125

.39

125

.39

125

.39

125

.39

125

.39

125

.39

125

.39

125

.39

125

.39

125

.39

125

.39

125

.39

127

.41

127

.41

127

.41

127

.41

127

.41

127

.41

127

.41

127

.41

127

.41

127

.41

127

.41

127

.41

127

.41

127

.41

127

.41

127

.41

127

.41

127

.41

127

.41

127

.41

127

.41

127

.41

129

.68

129

.68

129

.68

129

.68

129

.68

129

.68

129

.68

129

.68

129

.68

129

.68

129

.68

129

.68

129

.68

129

.68

129

.68

129

.68

129

.68

129

.68

129

.68

129

.68

129

.68

129

.68

134

.00

134

.00

134

.00

134

.00

134

.00

134

.00

134

.00

134

.00

134

.00

134

.00

134

.00

134

.00

134

.00

134

.00

134

.00

134

.00

134

.00

134

.00

134

.00

134

.00

134

.00

134

.00

138

.54

138

.54

138

.54

138

.54

138

.54

138

.54

138

.54

138

.54

138

.54

138

.54

138

.54

138

.54

138

.54

138

.54

138

.54

138

.54

138

.54

138

.54

138

.54

138

.54

138

.54

138

.54

138

.98

138

.98

138

.98

138

.98

138

.98

138

.98

138

.98

138

.98

138

.98

138

.98

138

.98

138

.98

138

.98

138

.98

138

.98

138

.98

138

.98

138

.98

138

.98

138

.98

138

.98

138

.98

155

.53

155

.53

155

.53

155

.53

155

.53

155

.53

155

.53

155

.53

155

.53

155

.53

155

.53

155

.53

155

.53

155

.53

155

.53

155

.53

155

.53

155

.53

155

.53

155

.53

155

.53

155

.53

163

.99

163

.99

163

.99

163

.99

163

.99

163

.99

163

.99

163

.99

163

.99

163

.99

163

.99

163

.99

163

.99

163

.99

163

.99

163

.99

163

.99

163

.99

163

.99

163

.99

163

.99

163

.99

175

.34

175

.34

175

.34

175

.34

175

.34

175

.34

175

.34

175

.34

175

.34

175

.34

175

.34

175

.34

175

.34

175

.34

175

.34

175

.34

175

.34

175

.34

175

.34

175

.34

175

.34

175

.34

188

.85

188

.85

188

.85

188

.85

188

.85

188

.85

188

.85

188

.85

188

.85

188

.85

188

.85

188

.85

188

.85

188

.85

188

.85

188

.85

188

.85

188

.85

188

.85

188

.85

188

.85

188

.85

266

088

12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

3.001.131.122.222.171.033.090.870.921.03

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

0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

DMSO-d652

.87

52.8

752

.87

52.8

752

.87

52.8

752

.87

52.8

752

.87

52.8

752

.87

52.8

752

.87

52.8

752

.87

52.8

752

.87

52.8

752

.87

52.8

752

.87

52.8

7

117.

7711

7.77

117.

7711

7.77

117.

7711

7.77

117.

7711

7.77

117.

7711

7.77

117.

7711

7.77

117.

7711

7.77

117.

7711

7.77

117.

7711

7.77

117.

7711

7.77

117.

7711

7.77

121.

6712

1.67

121.

6712

1.67

121.

6712

1.67

121.

6712

1.67

121.

6712

1.67

121.

6712

1.67

121.

6712

1.67

121.

6712

1.67

121.

6712

1.67

121.

6712

1.67

121.

6712

1.67

125.

1012

5.10

125.

1012

5.10

125.

1012

5.10

125.

1012

5.10

125.

1012

5.10

125.

1012

5.10

125.

1012

5.10

125.

1012

5.10

125.

1012

5.10

125.

1012

5.10

125.

1012

5.10

126.

6012

6.60

126.

6012

6.60

126.

6012

6.60

126.

6012

6.60

126.

6012

6.60

126.

6012

6.60

126.

6012

6.60

126.

6012

6.60

126.

6012

6.60

126.

6012

6.60

126.

6012

6.60

129.

3512

9.35

129.

3512

9.35

129.

3512

9.35

129.

3512

9.35

129.

3512

9.35

129.

3512

9.35

129.

3512

9.35

129.

3512

9.35

129.

3512

9.35

129.

3512

9.35

129.

3512

9.35

132.

0113

2.01

132.

0113

2.01

132.

0113

2.01

132.

0113

2.01

132.

0113

2.01

132.

0113

2.01

132.

0113

2.01

132.

0113

2.01

132.

0113

2.01

132.

0113

2.01

132.

0113

2.01

138.

5513

8.55

138.

5513

8.55

138.

5513

8.55

138.

5513

8.55

138.

5513

8.55

138.

5513

8.55

138.

5513

8.55

138.

5513

8.55

138.

5513

8.55

138.

5513

8.55

138.

5513

8.55

141.

9614

1.96

141.

9614

1.96

141.

9614

1.96

141.

9614

1.96

141.

9614

1.96

141.

9614

1.96

141.

9614

1.96

141.

9614

1.96

141.

9614

1.96

141.

9614

1.96

141.

9614

1.96

142.

8914

2.89

142.

8914

2.89

142.

8914

2.89

142.

8914

2.89

142.

8914

2.89

142.

8914

2.89

142.

8914

2.89

142.

8914

2.89

142.

8914

2.89

142.

8914

2.89

142.

8914

2.89

155.

0515

5.05

155.

0515

5.05

155.

0515

5.05

155.

0515

5.05

155.

0515

5.05

155.

0515

5.05

155.

0515

5.05

155.

0515

5.05

155.

0515

5.05

155.

0515

5.05

155.

0515

5.05

155.

7915

5.79

155.

7915

5.79

155.

7915

5.79

155.

7915

5.79

155.

7915

5.79

155.

7915

5.79

155.

7915

5.79

155.

7915

5.79

155.

7915

5.79

155.

7915

5.79

155.

7915

5.79

156.

8115

6.81

156.

8115

6.81

156.

8115

6.81

156.

8115

6.81

156.

8115

6.81

156.

8115

6.81

156.

8115

6.81

156.

8115

6.81

156.

8115

6.81

156.

8115

6.81

156.

8115

6.81

164.

1016

4.10

164.

1016

4.10

164.

1016

4.10

164.

1016

4.10

164.

1016

4.10

164.

1016

4.10

164.

1016

4.10

164.

1016

4.10

164.

1016

4.10

164.

1016

4.10

164.

1016

4.10

191.

1219

1.12

191.

1219

1.12

191.

1219

1.12

191.

1219

1.12

191.

1219

1.12

191.

1219

1.12

191.

1219

1.12

191.

1219

1.12

191.

1219

1.12

191.

1219

1.12

191.

1219

1.12

267

088

13 12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

1.001.022.032.000.962.860.890.920.990.99

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

0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

117

.78

117

.78

117

.78

117

.78

117

.78

117

.78

117

.78

117

.78

117

.78

117

.78

117

.78

117

.78

117

.78

117

.78

117

.78

117

.78

117

.78

117

.78

117

.78

117

.78

117

.78

117

.78

118

.14

118

.14

118

.14

118

.14

118

.14

118

.14

118

.14

118

.14

118

.14

118

.14

118

.14

118

.14

118

.14

118

.14

118

.14

118

.14

118

.14

118

.14

118

.14

118

.14

118

.14

118

.14

121

.50

121

.50

121

.50

121

.50

121

.50

121

.50

121

.50

121

.50

121

.50

121

.50

121

.50

121

.50

121

.50

121

.50

121

.50

121

.50

121

.50

121

.50

121

.50

121

.50

121

.50

121

.50

121

.79

121

.79

121

.79

121

.79

121

.79

121

.79

121

.79

121

.79

121

.79

121

.79

121

.79

121

.79

121

.79

121

.79

121

.79

121

.79

121

.79

121

.79

121

.79

121

.79

121

.79

121

.79

125

.16

125

.16

125

.16

125

.16

125

.16

125

.16

125

.16

125

.16

125

.16

125

.16

125

.16

125

.16

125

.16

125

.16

125

.16

125

.16

125

.16

125

.16

125

.16

125

.16

125

.16

125

.16

126

.62

126

.62

126

.62

126

.62

126

.62

126

.62

126

.62

126

.62

126

.62

126

.62

126

.62

126

.62

126

.62

126

.62

126

.62

126

.62

126

.62

126

.62

126

.62

126

.62

126

.62

126

.62

129

.36

129

.36

129

.36

129

.36

129

.36

129

.36

129

.36

129

.36

129

.36

129

.36

129

.36

129

.36

129

.36

129

.36

129

.36

129

.36

129

.36

129

.36

129

.36

129

.36

129

.36

129

.36

132

.02

132

.02

132

.02

132

.02

132

.02

132

.02

132

.02

132

.02

132

.02

132

.02

132

.02

132

.02

132

.02

132

.02

132

.02

132

.02

132

.02

132

.02

132

.02

132

.02

132

.02

132

.02

138

.60

138

.60

138

.60

138

.60

138

.60

138

.60

138

.60

138

.60

138

.60

138

.60

138

.60

138

.60

138

.60

138

.60

138

.60

138

.60

138

.60

138

.60

138

.60

138

.60

138

.60

138

.60

139

.48

139

.48

139

.48

139

.48

139

.48

139

.48

139

.48

139

.48

139

.48

139

.48

139

.48

139

.48

139

.48

139

.48

139

.48

139

.48

139

.48

139

.48

139

.48

139

.48

139

.48

139

.48

142

.83

142

.83

142

.83

142

.83

142

.83

142

.83

142

.83

142

.83

142

.83

142

.83

142

.83

142

.83

142

.83

142

.83

142

.83

142

.83

142

.83

142

.83

142

.83

142

.83

142

.83

142

.83

155

.07

155

.07

155

.07

155

.07

155

.07

155

.07

155

.07

155

.07

155

.07

155

.07

155

.07

155

.07

155

.07

155

.07

155

.07

155

.07

155

.07

155

.07

155

.07

155

.07

155

.07

155

.07

155

.79

155

.79

155

.79

155

.79

155

.79

155

.79

155

.79

155

.79

155

.79

155

.79

155

.79

155

.79

155

.79

155

.79

155

.79

155

.79

155

.79

155

.79

155

.79

155

.79

155

.79

155

.79

157

.93

157

.93

157

.93

157

.93

157

.93

157

.93

157

.93

157

.93

157

.93

157

.93

157

.93

157

.93

157

.93

157

.93

157

.93

157

.93

157

.93

157

.93

157

.93

157

.93

157

.93

157

.93

164

.80

164

.80

164

.80

164

.80

164

.80

164

.80

164

.80

164

.80

164

.80

164

.80

164

.80

164

.80

164

.80

164

.80

164

.80

164

.80

164

.80

164

.80

164

.80

164

.80

164

.80

164

.80

191

.23

191

.23

191

.23

191

.23

191

.23

191

.23

191

.23

191

.23

191

.23

191

.23

191

.23

191

.23

191

.23

191

.23

191

.23

191

.23

191

.23

191

.23

191

.23

191

.23

191

.23

191

.23

268

JWL-BE2-118_1_PROTON_01

15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsi

ty

10.010.740.700.67

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

Abso

lute

Inte

nsity

0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

TMS

DMSO-d6

117.

8711

7.87

117.

8711

7.87

117.

8711

7.87

117.

8711

7.87

117.

8711

7.87

117.

8711

7.87

117.

8711

7.87

117.

8711

7.87

117.

8711

7.87

117.

8711

7.87

117.

8711

7.87

121.

2312

1.23

121.

2312

1.23

121.

2312

1.23

121.

2312

1.23

121.

2312

1.23

121.

2312

1.23

121.

2312

1.23

121.

2312

1.23

121.

2312

1.23

121.

2312

1.23

121.

2312

1.23

126.

6112

6.61

126.

6112

6.61

126.

6112

6.61

126.

6112

6.61

126.

6112

6.61

126.

6112

6.61

126.

6112

6.61

126.

6112

6.61

126.

6112

6.61

126.

6112

6.61

126.

6112

6.61

127.

4012

7.40

127.

4012

7.40

127.

4012

7.40

127.

4012

7.40

127.

4012

7.40

127.

4012

7.40

127.

4012

7.40

127.

4012

7.40

127.

4012

7.40

127.

4012

7.40

127.

4012

7.40

129.

5312

9.53

129.

5312

9.53

129.

5312

9.53

129.

5312

9.53

129.

5312

9.53

129.

5312

9.53

129.

5312

9.53

129.

5312

9.53

129.

5312

9.53

129.

5312

9.53

129.

5312

9.53129.

6812

9.68

129.

6812

9.68

129.

6812

9.68

129.

6812

9.68

129.

6812

9.68

129.

6812

9.68

129.

6812

9.68

129.

6812

9.68

129.

6812

9.68

129.

6812

9.68

129.

6812

9.68

129.

7912

9.79

129.

7912

9.79

129.

7912

9.79

129.

7912

9.79

129.

7912

9.79

129.

7912

9.79

129.

7912

9.79

129.

7912

9.79

129.

7912

9.79

129.

7912

9.79

129.

7912

9.79

139.

7113

9.71

139.

7113

9.71

139.

7113

9.71

139.

7113

9.71

139.

7113

9.71

139.

7113

9.71

139.

7113

9.71

139.

7113

9.71

139.

7113

9.71

139.

7113

9.71

139.

7113

9.7114

2.45

142.

4514

2.45

142.

4514

2.45

142.

4514

2.45

142.

4514

2.45

142.

4514

2.45

142.

4514

2.45

142.

4514

2.45

142.

4514

2.45

142.

4514

2.45

142.

4514

2.45

142.

45

144.

5914

4.59

144.

5914

4.59

144.

5914

4.59

144.

5914

4.59

144.

5914

4.59

144.

5914

4.59

144.

5914

4.59

144.

5914

4.59

144.

5914

4.59

144.

5914

4.59

144.

5914

4.59

163.

2116

3.21

163.

2116

3.21

163.

2116

3.21

163.

2116

3.21

163.

2116

3.21

163.

2116

3.21

163.

2116

3.21

163.

2116

3.21

163.

2116

3.21

163.

2116

3.21

163.

2116

3.21

165.

0716

5.07

165.

0716

5.07

165.

0716

5.07

165.

0716

5.07

165.

0716

5.07

165.

0716

5.07

165.

0716

5.07

165.

0716

5.07

165.

0716

5.07

165.

0716

5.07

165.

0716

5.07

269

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

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

123

.06

123

.06

123

.06

123

.06

123

.06

123

.06

123

.06

123

.06

123

.06

123

.06

123

.06

123

.06

123

.06

123

.06

123

.06

123

.06

123

.06

123

.06

123

.06

123

.06

123

.06

123

.06

126

.27

126

.27

126

.27

126

.27

126

.27

126

.27

126

.27

126

.27

126

.27

126

.27

126

.27

126

.27

126

.27

126

.27

126

.27

126

.27

126

.27

126

.27

126

.27

126

.27

126

.27

126

.27

127

.17

127

.17

127

.17

127

.17

127

.17

127

.17

127

.17

127

.17

127

.17

127

.17

127

.17

127

.17

127

.17

127

.17

127

.17

127

.17

127

.17

127

.17

127

.17

127

.17

127

.17

127

.17

127

.62

127

.62

127

.62

127

.62

127

.62

127

.62

127

.62

127

.62

127

.62

127

.62

127

.62

127

.62

127

.62

127

.62

127

.62

127

.62

127

.62

127

.62

127

.62

127

.62

127

.62

127

.62

134

.74

134

.74

134

.74

134

.74

134

.74

134

.74

134

.74

134

.74

134

.74

134

.74

134

.74

134

.74

134

.74

134

.74

134

.74

134

.74

134

.74

134

.74

134

.74

134

.74

134

.74

134

.74

145

.87

145

.87

145

.87

145

.87

145

.87

145

.87

145

.87

145

.87

145

.87

145

.87

145

.87

145

.87

145

.87

145

.87

145

.87

145

.87

145

.87

145

.87

145

.87

145

.87

145

.87

145

.87

149

.16

149

.16

149

.16

149

.16

149

.16

149

.16

149

.16

149

.16

149

.16

149

.16

149

.16

149

.16

149

.16

149

.16

149

.16

149

.16

149

.16

149

.16

149

.16

149

.16

149

.16

149

.16

161

.20

161

.20

161

.20

161

.20

161

.20

161

.20

161

.20

161

.20

161

.20

161

.20

161

.20

161

.20

161

.20

161

.20

161

.20

161

.20

161

.20

161

.20

161

.20

161

.20

161

.20

161

.20

12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

1.000.900.990.890.960.96

270

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

0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

122.

2812

2.28

122.

2812

2.28

122.

2812

2.28

122.

2812

2.28

122.

2812

2.28

122.

2812

2.28

122.

2812

2.28

122.

2812

2.28

122.

2812

2.28

122.

2812

2.28

122.

2812

2.2812

4.39

124.

3912

4.39

124.

3912

4.39

124.

3912

4.39

124.

3912

4.39

124.

3912

4.39

124.

3912

4.39

124.

3912

4.39

124.

3912

4.39

124.

3912

4.39

124.

3912

4.39

124.

39126.

7512

6.75

126.

7512

6.75

126.

7512

6.75

126.

7512

6.75

126.

7512

6.75

126.

7512

6.75

126.

7512

6.75

126.

7512

6.75

126.

7512

6.75

126.

7512

6.75

126.

7512

6.75

128.

2412

8.24

128.

2412

8.24

128.

2412

8.24

128.

2412

8.24

128.

2412

8.24

128.

2412

8.24

128.

2412

8.24

128.

2412

8.24

128.

2412

8.24

128.

2412

8.24

128.

2412

8.24

135.

6213

5.62

135.

6213

5.62

135.

6213

5.62

135.

6213

5.62

135.

6213

5.62

135.

6213

5.62

135.

6213

5.62

135.

6213

5.62

135.

6213

5.62

135.

6213

5.62

135.

6213

5.62

144.

3414

4.34

144.

3414

4.34

144.

3414

4.34

144.

3414

4.34

144.

3414

4.34

144.

3414

4.34

144.

3414

4.34

144.

3414

4.34

144.

3414

4.34

144.

3414

4.34

144.

3414

4.34

147.

6214

7.62

147.

6214

7.62

147.

6214

7.62

147.

6214

7.62

147.

6214

7.62

147.

6214

7.62

147.

6214

7.62

147.

6214

7.62

147.

6214

7.62

147.

6214

7.62

147.

6214

7.62

160.

3416

0.34

160.

3416

0.34

160.

3416

0.34

160.

3416

0.34

160.

3416

0.34

160.

3416

0.34

160.

3416

0.34

160.

3416

0.34

160.

3416

0.34

160.

3416

0.34

160.

3416

0.34

12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

1.000.970.950.940.90

271

Appendix VI

Chapter 6 – NMR Spectra

H11HDMSO-d618 H's

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

Abs

olut

e In

tens

ity

0

0.05

0.10

0.15

0.20

0.25

0.30

2.691.950.940.881.691.911.831.00

C1313CDMSO-d64 C's

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

Abs

olut

e In

tens

ity

0

0.05

0.10

0.15

0.20

14.7

014

.70

14.7

014

.70

14.7

014

.70

14.7

014

.70

14.7

014

.70

14.7

014

.70

14.7

014

.70

14.7

014

.70

14.7

014

.70

14.7

014

.70

14.7

014

.70

60.6

960

.69

60.6

960

.69

60.6

960

.69

60.6

960

.69

60.6

960

.69

60.6

960

.69

60.6

960

.69

60.6

960

.69

60.6

960

.69

60.6

960

.69

60.6

960

.69

102.

9810

2.98

102.

9810

2.98

102.

9810

2.98

102.

9810

2.98

102.

9810

2.98

102.

9810

2.98

102.

9810

2.98

102.

9810

2.98

102.

9810

2.98

102.

9810

2.98

102.

9810

2.98

116.

4911

6.49

116.

4911

6.49

116.

4911

6.49

116.

4911

6.49

116.

4911

6.49

116.

4911

6.49

116.

4911

6.49

116.

4911

6.49

116.

4911

6.49

116.

4911

6.49

116.

4911

6.49

124.

1412

4.14

124.

1412

4.14

124.

1412

4.14

124.

1412

4.14

124.

1412

4.14

124.

1412

4.14

124.

1412

4.14

124.

1412

4.14

124.

1412

4.14

124.

1412

4.14

124.

1412

4.14

125.

8812

5.88

125.

8812

5.88

125.

8812

5.88

125.

8812

5.88

125.

8812

5.88

125.

8812

5.88

125.

8812

5.88

125.

8812

5.88

125.

8812

5.88

125.

8812

5.88

125.

8812

5.88

128.

4412

8.44

128.

4412

8.44

128.

4412

8.44

128.

4412

8.44

128.

4412

8.44

128.

4412

8.44

128.

4412

8.44

128.

4412

8.44

128.

4412

8.44

128.

4412

8.44

128.

4412

8.44

131.

0513

1.05

131.

0513

1.05

131.

0513

1.05

131.

0513

1.05

131.

0513

1.05

131.

0513

1.05

131.

0513

1.05

131.

0513

1.05

131.

0513

1.05

131.

0513

1.05

131.

0513

1.05

138.

8413

8.84

138.

8413

8.84

138.

8413

8.84

138.

8413

8.84

138.

8413

8.84

138.

8413

8.84

138.

8413

8.84

138.

8413

8.84

138.

8413

8.84

138.

8413

8.84

138.

8413

8.84

145.

2614

5.26

145.

2614

5.26

145.

2614

5.26

145.

2614

5.26

145.

2614

5.26

145.

2614

5.26

145.

2614

5.26

145.

2614

5.26

145.

2614

5.26

145.

2614

5.26

145.

2614

5.26

145.

4714

5.47

145.

4714

5.47

145.

4714

5.47

145.

4714

5.47

145.

4714

5.47

145.

4714

5.47

145.

4714

5.47

145.

4714

5.47

145.

4714

5.47

145.

4714

5.47

145.

4714

5.47

162.

6516

2.65

162.

6516

2.65

162.

6516

2.65

162.

6516

2.65

162.

6516

2.65

162.

6516

2.65

162.

6516

2.65

162.

6516

2.65

162.

6516

2.65

162.

6516

2.65

162.

6516

2.65

165.

8416

5.84

165.

8416

5.84

165.

8416

5.84

165.

8416

5.84

165.

8416

5.84

165.

8416

5.84

165.

8416

5.84

165.

8416

5.84

165.

8416

5.84

165.

8416

5.84

165.

8416

5.84

272

JWL-BE2-068_1_PROTON_01

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

1.000.921.892.092.101.051.08

JWL-BE2-068_1_CARBON_01

200 180 160 140 120 100 80 60 40 20 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsi

ty

10

2.8

5

11

6.4

0

12

4.1

11

25.8

71

28.4

41

31.2

3

13

8.8

81

45.2

014

5.2

6

16

2.7

4

16

7.4

4

273

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

Abso

lute

Inte

nsity

0

0.05

0.10

0.15

0.20

0.25

DMSO-d6

12.2

612

.26

12.2

612

.26

12.2

612

.26

12.2

612

.26

12.2

612

.26

12.2

612

.26

12.2

612

.26

12.2

612

.26

12.2

612

.26

12.2

612

.26

12.2

612

.26

116.

2611

6.26

116.

2611

6.26

116.

2611

6.26

116.

2611

6.26

116.

2611

6.26

116.

2611

6.26

116.

2611

6.26

116.

2611

6.26

116.

2611

6.26

116.

2611

6.26

116.

2611

6.26

124.

9312

4.93

124.

9312

4.93

124.

9312

4.93

124.

9312

4.93

124.

9312

4.93

124.

9312

4.93

124.

9312

4.93

124.

9312

4.93

124.

9312

4.93

124.

9312

4.93

124.

9312

4.93

125.

6912

5.69

125.

6912

5.69

125.

6912

5.69

125.

6912

5.69

125.

6912

5.69

125.

6912

5.69

125.

6912

5.69

125.

6912

5.69

125.

6912

5.69

125.

6912

5.69

125.

6912

5.69

128.

3112

8.31

128.

3112

8.31

128.

3112

8.31

128.

3112

8.31

128.

3112

8.31

128.

3112

8.31

128.

3112

8.31

128.

3112

8.31

128.

3112

8.31

128.

3112

8.31

128.

3112

8.31

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

138.

8613

8.86

138.

8613

8.86

138.

8613

8.86

138.

8613

8.86

138.

8613

8.86

138.

8613

8.86

138.

8613

8.86

138.

8613

8.86

138.

8613

8.86

138.

8613

8.86

138.

8613

8.86

140.

0414

0.04

140.

0414

0.04

140.

0414

0.04

140.

0414

0.04

140.

0414

0.04

140.

0414

0.04

140.

0414

0.04

140.

0414

0.04

140.

0414

0.04

140.

0414

0.04

140.

0414

0.0414

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

39

158.

7415

8.74

158.

7415

8.74

158.

7415

8.74

158.

7415

8.74

158.

7415

8.74

158.

7415

8.74

158.

7415

8.74

158.

7415

8.74

158.

7415

8.74

158.

7415

8.74

158.

7415

8.74

167.

4516

7.45

167.

4516

7.45

167.

4516

7.45

167.

4516

7.45

167.

4516

7.45

167.

4516

7.45

167.

4516

7.45

167.

4516

7.45

167.

4516

7.45

167.

4516

7.45

167.

4516

7.45

JWL-BE2-130_2_PROTON_01

12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

3.041.041.030.992.192.141.00

274

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

Abso

lute

Inte

nsity

0

0.05

0.10

0.15

0.20

0.25

DMSO-d6

15.4

615

.46

15.4

615

.46

15.4

615

.46

15.4

615

.46

15.4

615

.46

15.4

615

.46

15.4

615

.46

15.4

615

.46

15.4

615

.46

15.4

615

.46

15.4

615

.46

101.

9710

1.97

101.

9710

1.97

101.

9710

1.97

101.

9710

1.97

101.

9710

1.97

101.

9710

1.97

101.

9710

1.97

101.

9710

1.97

101.

9710

1.97

101.

9710

1.97

101.

9710

1.97

116.

3911

6.39

116.

3911

6.39

116.

3911

6.39

116.

3911

6.39

116.

3911

6.39

116.

3911

6.39

116.

3911

6.39

116.

3911

6.39

116.

3911

6.39

116.

3911

6.39

116.

3911

6.39

123.

9212

3.92

123.

9212

3.92

123.

9212

3.92

123.

9212

3.92

123.

9212

3.92

123.

9212

3.92

123.

9212

3.92

123.

9212

3.92

123.

9212

3.92

123.

9212

3.92

123.

9212

3.92

126.

6612

6.66

126.

6612

6.66

126.

6612

6.66

126.

6612

6.66

126.

6612

6.66

126.

6612

6.66

126.

6612

6.66

126.

6612

6.66

126.

6612

6.66

126.

6612

6.66

126.

6612

6.66

131.

1113

1.11

131.

1113

1.11

131.

1113

1.11

131.

1113

1.11

131.

1113

1.11

131.

1113

1.11

131.

1113

1.11

131.

1113

1.11

131.

1113

1.11

131.

1113

1.11

131.

1113

1.11

136.

6213

6.62

136.

6213

6.62

136.

6213

6.62

136.

6213

6.62

136.

6213

6.62

136.

6213

6.62

136.

6213

6.62

136.

6213

6.62

136.

6213

6.62

136.

6213

6.62

136.

6213

6.62

139.

1413

9.14

139.

1413

9.14

139.

1413

9.14

139.

1413

9.14

139.

1413

9.14

139.

1413

9.14

139.

1413

9.14

139.

1413

9.14

139.

1413

9.14

139.

1413

9.14

139.

1413

9.14

145.

2614

5.26

145.

2614

5.26

145.

2614

5.26

145.

2614

5.26

145.

2614

5.26

145.

2614

5.26

145.

2614

5.26

145.

2614

5.26

145.

2614

5.26

145.

2614

5.26

145.

2614

5.26

145.

4014

5.40

145.

4014

5.40

145.

4014

5.40

145.

4014

5.40

145.

4014

5.40

145.

4014

5.40

145.

4014

5.40

145.

4014

5.40

145.

4014

5.40

145.

4014

5.40

145.

4014

5.40

162.

6516

2.65

162.

6516

2.65

162.

6516

2.65

162.

6516

2.65

162.

6516

2.65

162.

6516

2.65

162.

6516

2.65

162.

6516

2.65

162.

6516

2.65

162.

6516

2.65

162.

6516

2.65

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

JWL-BE2-131_2_PROTON_01

12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

3.201.001.061.031.062.272.290.93

275

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

Abso

lute

Inte

nsity

0

0.05

0.10

0.15

15.9

815

.98

15.9

815

.98

15.9

815

.98

15.9

815

.98

15.9

815

.98

15.9

815

.98

15.9

815

.98

15.9

815

.98

15.9

815

.98

15.9

815

.98

15.9

815

.98

102.

6710

2.67

102.

6710

2.67

102.

6710

2.67

102.

6710

2.67

102.

6710

2.67

102.

6710

2.67

102.

6710

2.67

102.

6710

2.67

102.

6710

2.67

102.

6710

2.67

102.

6710

2.67

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

121.

0112

1.01

121.

0112

1.01

121.

0112

1.01

121.

0112

1.01

121.

0112

1.01

121.

0112

1.01

121.

0112

1.01

121.

0112

1.01

121.

0112

1.01

121.

0112

1.01

121.

0112

1.01

123.

2412

3.24

123.

2412

3.24

123.

2412

3.24

123.

2412

3.24

123.

2412

3.24

123.

2412

3.24

123.

2412

3.24

123.

2412

3.24

123.

2412

3.24

123.

2412

3.24

123.

2412

3.2412

6.10

126.

1012

6.10

126.

1012

6.10

126.

1012

6.10

126.

1012

6.10

126.

1012

6.10

126.

1012

6.10

126.

1012

6.10

126.

1012

6.10

126.

1012

6.10

126.

1012

6.10

126.

1013

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

27

138.

6313

8.63

138.

6313

8.63

138.

6313

8.63

138.

6313

8.63

138.

6313

8.63

138.

6313

8.63

138.

6313

8.63

138.

6313

8.63

138.

6313

8.63

138.

6313

8.63

138.

6313

8.63

145.

2414

5.24

145.

2414

5.24

145.

2414

5.24

145.

2414

5.24

145.

2414

5.24

145.

2414

5.24

145.

2414

5.24

145.

2414

5.24

145.

2414

5.24

145.

2414

5.24

145.

2414

5.2414

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

39

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.6816

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

46

JWL-BE2-133_2_PROTON_01

12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsi

ty

3.141.000.950.951.022.142.260.92

276

JWL-BE2-132_2_PROTON_01

12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

3.390.911.091.091.102.332.391.00

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

Abso

lute

Inte

nsity

0

0.05

0.10

0.15

15.9

815

.98

15.9

815

.98

15.9

815

.98

15.9

815

.98

15.9

815

.98

15.9

815

.98

15.9

815

.98

15.9

815

.98

15.9

815

.98

15.9

815

.98

15.9

815

.98

102.

6710

2.67

102.

6710

2.67

102.

6710

2.67

102.

6710

2.67

102.

6710

2.67

102.

6710

2.67

102.

6710

2.67

102.

6710

2.67

102.

6710

2.67

102.

6710

2.67

102.

6710

2.67

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

121.

0112

1.01

121.

0112

1.01

121.

0112

1.01

121.

0112

1.01

121.

0112

1.01

121.

0112

1.01

121.

0112

1.01

121.

0112

1.01

121.

0112

1.01

121.

0112

1.01

121.

0112

1.01

123.

2412

3.24

123.

2412

3.24

123.

2412

3.24

123.

2412

3.24

123.

2412

3.24

123.

2412

3.24

123.

2412

3.24

123.

2412

3.24

123.

2412

3.24

123.

2412

3.24

123.

2412

3.2412

6.10

126.

1012

6.10

126.

1012

6.10

126.

1012

6.10

126.

1012

6.10

126.

1012

6.10

126.

1012

6.10

126.

1012

6.10

126.

1012

6.10

126.

1012

6.10

126.

1012

6.10

126.

1013

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

1.12

131.

1213

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

2713

8.27

138.

27

138.

6313

8.63

138.

6313

8.63

138.

6313

8.63

138.

6313

8.63

138.

6313

8.63

138.

6313

8.63

138.

6313

8.63

138.

6313

8.63

138.

6313

8.63

138.

6313

8.63

138.

6313

8.63

145.

2414

5.24

145.

2414

5.24

145.

2414

5.24

145.

2414

5.24

145.

2414

5.24

145.

2414

5.24

145.

2414

5.24

145.

2414

5.24

145.

2414

5.24

145.

2414

5.24

145.

2414

5.2414

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

3914

5.39

145.

39

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.6816

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

4616

7.46

167.

46

277

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

Abso

lute

Inte

nsity

0

0.05

0.10

0.15

0.20

0.25

0.30

104.

0410

4.04

104.

0410

4.04

104.

0410

4.04

104.

0410

4.04

104.

0410

4.04

104.

0410

4.04

104.

0410

4.04

104.

0410

4.04

104.

0410

4.04

104.

0410

4.04

104.

0410

4.04

116.

4911

6.49

116.

4911

6.49

116.

4911

6.49

116.

4911

6.49

116.

4911

6.49

116.

4911

6.49

116.

4911

6.49

116.

4911

6.49

116.

4911

6.49

116.

4911

6.49

116.

4911

6.49

122.

3312

2.33

122.

3312

2.33

122.

3312

2.33

122.

3312

2.33

122.

3312

2.33

122.

3312

2.33

122.

3312

2.33

122.

3312

2.33

122.

3312

2.33

122.

3312

2.33

122.

3312

2.33

126.

4012

6.40

126.

4012

6.40

126.

4012

6.40

126.

4012

6.40

126.

4012

6.40

126.

4012

6.40

126.

4012

6.40

126.

4012

6.40

126.

4012

6.40

126.

4012

6.40

126.

4012

6.40

127.

2912

7.29

127.

2912

7.29

127.

2912

7.29

127.

2912

7.29

127.

2912

7.29

127.

2912

7.29

127.

2912

7.29

127.

2912

7.29

127.

2912

7.29

127.

2912

7.29

127.

2912

7.29

131.

2113

1.21

131.

2113

1.21

131.

2113

1.21

131.

2113

1.21

131.

2113

1.21

131.

2113

1.21

131.

2113

1.21

131.

2113

1.21

131.

2113

1.21

131.

2113

1.21

131.

2113

1.21

137.

1213

7.12

137.

1213

7.12

137.

1213

7.12

137.

1213

7.12

137.

1213

7.12

137.

1213

7.12

137.

1213

7.12

137.

1213

7.12

137.

1213

7.12

137.

1213

7.12

137.

1213

7.12

145.

4514

5.45

145.

4514

5.45

145.

4514

5.45

145.

4514

5.45

145.

4514

5.45

145.

4514

5.45

145.

4514

5.45

145.

4514

5.45

145.

4514

5.45

145.

4514

5.45

145.

4514

5.45

146.

8614

6.86

146.

8614

6.86

146.

8614

6.86

146.

8614

6.86

146.

8614

6.86

146.

8614

6.86

146.

8614

6.86

146.

8614

6.86

146.

8614

6.86

146.

8614

6.86

146.

8614

6.86

162.

7416

2.74

162.

7416

2.74

162.

7416

2.74

162.

7416

2.74

162.

7416

2.74

162.

7416

2.74

162.

7416

2.74

162.

7416

2.74

162.

7416

2.74

162.

7416

2.74

162.

7416

2.74167.

5016

7.50

167.

5016

7.50

167.

5016

7.50

167.

5016

7.50

167.

5016

7.50

167.

5016

7.50

167.

5016

7.50

167.

5016

7.50

167.

5016

7.50

167.

5016

7.50

167.

5016

7.50

JWL-BE2-129_2_PROTON_01

12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsi

ty

1.001.812.203.240.79

278

JWL-BE2-070_1_PROTON_01

12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

1.183.447.000.93

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

0

0.05

0.10

0.15

0.20

0.25

0.30

DMSO-d6

104.

6710

4.67

104.

6710

4.67

104.

6710

4.67

104.

6710

4.67

104.

6710

4.67

104.

6710

4.67

104.

6710

4.67

104.

6710

4.67

104.

6710

4.67

104.

6710

4.67

104.

6710

4.67

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

116.

4011

6.40

123.

1312

3.13

123.

1312

3.13

123.

1312

3.13

123.

1312

3.13

123.

1312

3.13

123.

1312

3.13

123.

1312

3.13

123.

1312

3.13

123.

1312

3.13

123.

1312

3.13

123.

1312

3.13

126.

1712

6.17

126.

1712

6.17

126.

1712

6.17

126.

1712

6.17

126.

1712

6.17

126.

1712

6.17

126.

1712

6.17

126.

1712

6.17

126.

1712

6.17

126.

1712

6.17

126.

1712

6.17

129.

1212

9.12

129.

1212

9.12

129.

1212

9.12

129.

1212

9.12

129.

1212

9.12

129.

1212

9.12

129.

1212

9.12

129.

1212

9.12

129.

1212

9.12

129.

1212

9.12

129.

1212

9.12

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

134.

7913

4.79

134.

7913

4.79

134.

7913

4.79

134.

7913

4.79

134.

7913

4.79

134.

7913

4.79

134.

7913

4.79

134.

7913

4.79

134.

7913

4.79

134.

7913

4.79

134.

7913

4.79

145.

5314

5.53

145.

5314

5.53

145.

5314

5.53

145.

5314

5.53

145.

5314

5.53

145.

5314

5.53

145.

5314

5.53

145.

5314

5.53

145.

5314

5.53

145.

5314

5.53

145.

5314

5.53

150.

4415

0.44

150.

4415

0.44

150.

4415

0.44

150.

4415

0.44

150.

4415

0.44

150.

4415

0.44

150.

4415

0.44

150.

4415

0.44

150.

4415

0.44

150.

4415

0.44

150.

4415

0.44

162.

7716

2.77

162.

7716

2.77

162.

7716

2.77

162.

7716

2.77

162.

7716

2.77

162.

7716

2.77

162.

7716

2.77

162.

7716

2.77

162.

7716

2.77

162.

7716

2.77

162.

7716

2.77

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

279

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

Abso

lute

Inte

nsity

0

0.05

0.10

0.15

0.20

0.25

12.3

412

.34

12.3

412

.34

12.3

412

.34

12.3

412

.34

12.3

412

.34

12.3

412

.34

12.3

412

.34

12.3

412

.34

12.3

412

.34

12.3

412

.34

12.3

412

.34

116.

5711

6.57

116.

5711

6.57

116.

5711

6.57

116.

5711

6.57

116.

5711

6.57

116.

5711

6.57

116.

5711

6.57

116.

5711

6.57

116.

5711

6.57

116.

5711

6.57

116.

5711

6.57

118.

0311

8.03

118.

0311

8.03

118.

0311

8.03

118.

0311

8.03

118.

0311

8.03

118.

0311

8.03

118.

0311

8.03

118.

0311

8.03

118.

0311

8.03

118.

0311

8.03

118.

0311

8.03

123.

2912

3.29

123.

2912

3.29

123.

2912

3.29

123.

2912

3.29

123.

2912

3.29

123.

2912

3.29

123.

2912

3.29

123.

2912

3.29

123.

2912

3.29

123.

2912

3.29

123.

2912

3.29

128.

5112

8.51

128.

5112

8.51

128.

5112

8.51

128.

5112

8.51

128.

5112

8.51

128.

5112

8.51

128.

5112

8.51

128.

5112

8.51

128.

5112

8.51

128.

5112

8.51

128.

5112

8.51

128.

8112

8.81

128.

8112

8.81

128.

8112

8.81

128.

8112

8.81

128.

8112

8.81

128.

8112

8.81

128.

8112

8.81

128.

8112

8.81

128.

8112

8.81

128.

8112

8.81

128.

8112

8.81

131.

1613

1.16

131.

1613

1.16

131.

1613

1.16

131.

1613

1.16

131.

1613

1.16

131.

1613

1.16

131.

1613

1.16

131.

1613

1.16

131.

1613

1.16

131.

1613

1.16

131.

1613

1.16

134.

7913

4.79

134.

7913

4.79

134.

7913

4.79

134.

7913

4.79

134.

7913

4.79

134.

7913

4.79

134.

7913

4.79

134.

7913

4.79

134.

7913

4.79

134.

7913

4.79

134.

7913

4.79

144.

8414

4.84

144.

8414

4.84

144.

8414

4.84

144.

8414

4.84

144.

8414

4.84

144.

8414

4.84

144.

8414

4.84

144.

8414

4.84

144.

8414

4.84

144.

8414

4.84

144.

8414

4.84145.

2514

5.25

145.

2514

5.25

145.

2514

5.25

145.

2514

5.25

145.

2514

5.25

145.

2514

5.25

145.

2514

5.25

145.

2514

5.25

145.

2514

5.25

145.

2514

5.25

145.

2514

5.25

159.

3615

9.36

159.

3615

9.36

159.

3615

9.36

159.

3615

9.36

159.

3615

9.36

159.

3615

9.36

159.

3615

9.36

159.

3615

9.36

159.

3615

9.36

159.

3615

9.36

159.

3615

9.36

167.

4316

7.43

167.

4316

7.43

167.

4316

7.43

167.

4316

7.43

167.

4316

7.43

167.

4316

7.43

167.

4316

7.43

167.

4316

7.43

167.

4316

7.43

167.

4316

7.43

167.

4316

7.43

JWL-BE2-127_1_PROTON_01

12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

2.781.001.991.911.751.860.74

280

JWL-BE2-139_1_PROTON_01

12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Nor

mal

ized

Inte

nsity

3.002.341.150.912.082.061.040.84

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

Abso

lute

Inte

nsity

0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

55.9

255

.92

55.9

255

.92

55.9

255

.92

55.9

255

.92

55.9

255

.92

55.9

255

.92

55.9

255

.92

55.9

255

.92

55.9

255

.92

55.9

255

.92

55.9

255

.92

108.

5010

8.50

108.

5010

8.50

108.

5010

8.50

108.

5010

8.50

108.

5010

8.50

108.

5010

8.50

108.

5010

8.50

108.

5010

8.50

108.

5010

8.50

108.

5010

8.50

108.

5010

8.50

112.

0211

2.02

112.

0211

2.02

112.

0211

2.02

112.

0211

2.02

112.

0211

2.02

112.

0211

2.02

112.

0211

2.02

112.

0211

2.02

112.

0211

2.02

112.

0211

2.02

112.

0211

2.02

116.

4211

6.42

116.

4211

6.42

116.

4211

6.42

116.

4211

6.42

116.

4211

6.42

116.

4211

6.42

116.

4211

6.42

116.

4211

6.42

116.

4211

6.42

116.

4211

6.42

116.

4211

6.42

121.

0412

1.04

121.

0412

1.04

121.

0412

1.04

121.

0412

1.04

121.

0412

1.04

121.

0412

1.04

121.

0412

1.04

121.

0412

1.04

121.

0412

1.04

121.

0412

1.04

121.

0412

1.04

129.

1612

9.16

129.

1612

9.16

129.

1612

9.16

129.

1612

9.16

129.

1612

9.16

129.

1612

9.16

129.

1612

9.16

129.

1612

9.16

129.

1612

9.16

129.

1612

9.16

129.

1612

9.16

129.

7612

9.76

129.

7612

9.76

129.

7612

9.76

129.

7612

9.76

129.

7612

9.76

129.

7612

9.76

129.

7612

9.76

129.

7612

9.76

129.

7612

9.76

129.

7612

9.76

129.

7612

9.76

131.

2413

1.24

131.

2413

1.24

131.

2413

1.24

131.

2413

1.24

131.

2413

1.24

131.

2413

1.24

131.

2413

1.24

131.

2413

1.24

131.

2413

1.24

131.

2413

1.24

131.

2413

1.24

145.

4414

5.44

145.

4414

5.44

145.

4414

5.44

145.

4414

5.44

145.

4414

5.44

145.

4414

5.44

145.

4414

5.44

145.

4414

5.44

145.

4414

5.44

145.

4414

5.44

145.

4414

5.44

146.

2314

6.23

146.

2314

6.23

146.

2314

6.23

146.

2314

6.23

146.

2314

6.23

146.

2314

6.23

146.

2314

6.23

146.

2314

6.23

146.

2314

6.23

146.

2314

6.23

146.

2314

6.2315

7.04

157.

0415

7.04

157.

0415

7.04

157.

0415

7.04

157.

0415

7.04

157.

0415

7.04

157.

0415

7.04

157.

0415

7.04

157.

0415

7.04

157.

0415

7.04

157.

0415

7.04

157.

04

161.

1616

1.16

161.

1616

1.16

161.

1616

1.16

161.

1616

1.16

161.

1616

1.16

161.

1616

1.16

161.

1616

1.16

161.

1616

1.16

161.

1616

1.16

161.

1616

1.16

161.

1616

1.16

167.

4816

7.48

167.

4816

7.48

167.

4816

7.48

167.

4816

7.48

167.

4816

7.48

167.

4816

7.48

167.

4816

7.48

167.

4816

7.48

167.

4816

7.48

167.

4816

7.48

167.

4816

7.48

281

Chemical Shift (ppm)11 10 9 8 7 6 5 4 3 2

Abs

olut

e In

tens

ity

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

1.103.001.101.142.452.282.310.93

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

Abso

lute

Inte

nsity

0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

55.5

455

.54

55.5

455

.54

55.5

455

.54

55.5

455

.54

55.5

455

.54

55.5

455

.54

55.5

455

.54

55.5

455

.54

55.5

455

.54

55.5

455

.54

55.5

455

.54

105.

0310

5.03

105.

0310

5.03

105.

0310

5.03

105.

0310

5.03

105.

0310

5.03

105.

0310

5.03

105.

0310

5.03

105.

0310

5.03

105.

0310

5.03

105.

0310

5.03

105.

0310

5.03111.

6111

1.61

111.

6111

1.61

111.

6111

1.61

111.

6111

1.61

111.

6111

1.61

111.

6111

1.61

111.

6111

1.61

111.

6111

1.61

111.

6111

1.61

111.

6111

1.61

111.

6111

1.61

113.

7111

3.71

113.

7111

3.71

113.

7111

3.71

113.

7111

3.71

113.

7111

3.71

113.

7111

3.71

113.

7111

3.71

113.

7111

3.71

113.

7111

3.71

113.

7111

3.71

113.

7111

3.71

116.

3911

6.39

116.

3911

6.39

116.

3911

6.39

116.

3911

6.39

116.

3911

6.39

116.

3911

6.39

116.

3911

6.39

116.

3911

6.39

116.

3911

6.39

116.

3911

6.39

116.

3911

6.39

118.

6411

8.64

118.

6411

8.64

118.

6411

8.64

118.

6411

8.64

118.

6411

8.64

118.

6411

8.64

118.

6411

8.64

118.

6411

8.64

118.

6411

8.64

118.

6411

8.64

118.

6411

8.64

123.

2012

3.20

123.

2012

3.20

123.

2012

3.20

123.

2012

3.20

123.

2012

3.20

123.

2012

3.20

123.

2012

3.20

123.

2012

3.20

123.

2012

3.20

123.

2012

3.20

123.

2012

3.20130.

2113

0.21

130.

2113

0.21

130.

2113

0.21

130.

2113

0.21

130.

2113

0.21

130.

2113

0.21

130.

2113

0.21

130.

2113

0.21

130.

2113

0.21

130.

2113

0.21

130.

2113

0.21

131.

2213

1.22

131.

2213

1.22

131.

2213

1.22

131.

2213

1.22

131.

2213

1.22

131.

2213

1.22

131.

2213

1.22

131.

2213

1.22

131.

2213

1.22

131.

2213

1.22

131.

2213

1.22

136.

1413

6.14

136.

1413

6.14

136.

1413

6.14

136.

1413

6.14

136.

1413

6.14

136.

1413

6.14

136.

1413

6.14

136.

1413

6.14

136.

1413

6.14

136.

1413

6.14

136.

1413

6.14

145.

4514

5.45

145.

4514

5.45

145.

4514

5.45

145.

4514

5.45

145.

4514

5.45

145.

4514

5.45

145.

4514

5.45

145.

4514

5.45

145.

4514

5.45

145.

4514

5.45

145.

4514

5.45

150.

3615

0.36

150.

3615

0.36

150.

3615

0.36

150.

3615

0.36

150.

3615

0.36

150.

3615

0.36

150.

3615

0.36

150.

3615

0.36

150.

3615

0.36

150.

3615

0.36

150.

3615

0.36

159.

9715

9.97

159.

9715

9.97

159.

9715

9.97

159.

9715

9.97

159.

9715

9.97

159.

9715

9.97

159.

9715

9.97

159.

9715

9.97

159.

9715

9.97

159.

9715

9.97

159.

9715

9.97

162.

6316

2.63

162.

6316

2.63

162.

6316

2.63

162.

6316

2.63

162.

6316

2.63

162.

6316

2.63

162.

6316

2.63

162.

6316

2.63

162.

6316

2.63

162.

6316

2.63

162.

6316

2.63

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

282

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

Abso

lute

Inte

nsity

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

3.001.870.707.111.00

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

Abso

lute

Inte

nsity

0

0.05

0.10

0.15

0.20

0.25

0.30

55.6

155

.61

55.6

155

.61

55.6

155

.61

55.6

155

.61

55.6

155

.61

55.6

155

.61

55.6

155

.61

55.6

155

.61

55.6

155

.61

55.6

155

.61

55.6

155

.61

102.

4810

2.48

102.

4810

2.48

102.

4810

2.48

102.

4810

2.48

102.

4810

2.48

102.

4810

2.48

102.

4810

2.48

102.

4810

2.48

102.

4810

2.48

102.

4810

2.48

102.

4810

2.48

114.

4711

4.47

114.

4711

4.47

114.

4711

4.47

114.

4711

4.47

114.

4711

4.47

114.

4711

4.47

114.

4711

4.47

114.

4711

4.47

114.

4711

4.47

114.

4711

4.47

114.

4711

4.4711

6.37

116.

3711

6.37

116.

3711

6.37

116.

3711

6.37

116.

3711

6.37

116.

3711

6.37

116.

3711

6.37

116.

3711

6.37

116.

3711

6.37

116.

3711

6.37

116.

3711

6.37

116.

37

123.

0812

3.08

123.

0812

3.08

123.

0812

3.08

123.

0812

3.08

123.

0812

3.08

123.

0812

3.08

123.

0812

3.08

123.

0812

3.08

123.

0812

3.08

123.

0812

3.08

123.

0812

3.08

127.

5212

7.52

127.

5212

7.52

127.

5212

7.52

127.

5212

7.52

127.

5212

7.52

127.

5212

7.52

127.

5212

7.52

127.

5212

7.52

127.

5212

7.52

127.

5212

7.52

127.

5212

7.52

131.

1613

1.16

131.

1613

1.16

131.

1613

1.16

131.

1613

1.16

131.

1613

1.16

131.

1613

1.16

131.

1613

1.16

131.

1613

1.16

131.

1613

1.16

131.

1613

1.16

131.

1613

1.16

145.

5514

5.55

145.

5514

5.55

145.

5514

5.55

145.

5514

5.55

145.

5514

5.55

145.

5514

5.55

145.

5514

5.55

145.

5514

5.55

145.

5514

5.55

145.

5514

5.55

145.

5514

5.55

150.

2415

0.24

150.

2415

0.24

150.

2415

0.24

150.

2415

0.24

150.

2415

0.24

150.

2415

0.24

150.

2415

0.24

150.

2415

0.24

150.

2415

0.24

150.

2415

0.24

150.

2415

0.24

159.

3215

9.32

159.

3215

9.32

159.

3215

9.32

159.

3215

9.32

159.

3215

9.32

159.

3215

9.32

159.

3215

9.32

159.

3215

9.32

159.

3215

9.32

159.

3215

9.32

159.

3215

9.32

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

162.

6816

2.68

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

167.

4916

7.49

283

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

Abso

lute

Inte

nsity

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

1.164.711.062.291.161.00

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

Abso

lute

Inte

nsity

0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

DMSO-d6

110.

4511

0.45

110.

4511

0.45

110.

4511

0.45

110.

4511

0.45

110.

4511

0.45

110.

4511

0.45

110.

4511

0.45

110.

4511

0.45

110.

4511

0.45

110.

4511

0.45

110.

4511

0.45

116.

7611

6.76

116.

7611

6.76

116.

7611

6.76

116.

7611

6.76

116.

7611

6.76

116.

7611

6.76

116.

7611

6.76

116.

7611

6.76

116.

7611

6.76

116.

7611

6.76

116.

7611

6.76

123.

6112

3.61

123.

6112

3.61

123.

6112

3.61

123.

6112

3.61

123.

6112

3.61

123.

6112

3.61

123.

6112

3.61

123.

6112

3.61

123.

6112

3.61

123.

6112

3.61

123.

6112

3.61

127.

6912

7.69

127.

6912

7.69

127.

6912

7.69

127.

6912

7.69

127.

6912

7.69

127.

6912

7.69

127.

6912

7.69

127.

6912

7.69

127.

6912

7.69

127.

6912

7.69

127.

6912

7.69

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

140.

7314

0.73

140.

7314

0.73

140.

7314

0.73

140.

7314

0.73

140.

7314

0.73

140.

7314

0.73

140.

7314

0.73

140.

7314

0.73

140.

7314

0.73

140.

7314

0.73

140.

7314

0.73

144.

4214

4.42

144.

4214

4.42

144.

4214

4.42

144.

4214

4.42

144.

4214

4.42

144.

4214

4.42

144.

4214

4.42

144.

4214

4.42

144.

4214

4.42

144.

4214

4.42

144.

4214

4.42

145.

0214

5.02

145.

0214

5.02

145.

0214

5.02

145.

0214

5.02

145.

0214

5.02

145.

0214

5.02

145.

0214

5.02

145.

0214

5.02

145.

0214

5.02

145.

0214

5.02

145.

0214

5.02

163.

7716

3.77

163.

7716

3.77

163.

7716

3.77

163.

7716

3.77

163.

7716

3.77

163.

7716

3.77

163.

7716

3.77

163.

7716

3.77

163.

7716

3.77

163.

7716

3.77

163.

7716

3.77

167.

4416

7.44

167.

4416

7.44

167.

4416

7.44

167.

4416

7.44

167.

4416

7.44

167.

4416

7.44

167.

4416

7.44

167.

4416

7.44

167.

4416

7.44

167.

4416

7.44

167.

4416

7.44

284

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

Abso

lute

Inte

nsity

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.582.072.131.031.081.061.031.031.00

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

Abso

lute

Inte

nsity

0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

DMSO-d6

110.

4511

0.45

110.

4511

0.45

110.

4511

0.45

110.

4511

0.45

110.

4511

0.45

110.

4511

0.45

110.

4511

0.45

110.

4511

0.45

110.

4511

0.45

110.

4511

0.45

110.

4511

0.45

116.

7611

6.76

116.

7611

6.76

116.

7611

6.76

116.

7611

6.76

116.

7611

6.76

116.

7611

6.76

116.

7611

6.76

116.

7611

6.76

116.

7611

6.76

116.

7611

6.76

116.

7611

6.76

123.

6112

3.61

123.

6112

3.61

123.

6112

3.61

123.

6112

3.61

123.

6112

3.61

123.

6112

3.61

123.

6112

3.61

123.

6112

3.61

123.

6112

3.61

123.

6112

3.61

123.

6112

3.61

127.

6912

7.69

127.

6912

7.69

127.

6912

7.69

127.

6912

7.69

127.

6912

7.69

127.

6912

7.69

127.

6912

7.69

127.

6912

7.69

127.

6912

7.69

127.

6912

7.69

127.

6912

7.69

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

139.

5113

9.51

140.

7314

0.73

140.

7314

0.73

140.

7314

0.73

140.

7314

0.73

140.

7314

0.73

140.

7314

0.73

140.

7314

0.73

140.

7314

0.73

140.

7314

0.73

140.

7314

0.73

140.

7314

0.73

144.

4214

4.42

144.

4214

4.42

144.

4214

4.42

144.

4214

4.42

144.

4214

4.42

144.

4214

4.42

144.

4214

4.42

144.

4214

4.42

144.

4214

4.42

144.

4214

4.42

144.

4214

4.42

145.

0214

5.02

145.

0214

5.02

145.

0214

5.02

145.

0214

5.02

145.

0214

5.02

145.

0214

5.02

145.

0214

5.02

145.

0214

5.02

145.

0214

5.02

145.

0214

5.02

145.

0214

5.02

163.

7716

3.77

163.

7716

3.77

163.

7716

3.77

163.

7716

3.77

163.

7716

3.77

163.

7716

3.77

163.

7716

3.77

163.

7716

3.77

163.

7716

3.77

163.

7716

3.77

163.

7716

3.77

167.

4416

7.44

167.

4416

7.44

167.

4416

7.44

167.

4416

7.44

167.

4416

7.44

167.

4416

7.44

167.

4416

7.44

167.

4416

7.44

167.

4416

7.44

167.

4416

7.44

167.

4416

7.44

285

Chemical Shift (ppm)

12 11 10 9 8 7 6 5 4 3 2 1 0

Norm

alize

d In

tens

ity

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.001.561.610.761.591.660.78

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

Abso

lute I

ntens

ity

0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

DMSO-d6

116.

8411

6.84

116.

8411

6.84

116.

8411

6.84

116.

8411

6.84

116.

8411

6.84

116.

8411

6.84

116.

8411

6.84

116.

8411

6.84

116.

8411

6.84

116.

8411

6.84

116.

8411

6.84

116.

9611

6.96

116.

9611

6.96

116.

9611

6.96

116.

9611

6.96

116.

9611

6.96

116.

9611

6.96

116.

9611

6.96

116.

9611

6.96

116.

9611

6.96

116.

9611

6.96

116.

9611

6.96

122.

8412

2.84

122.

8412

2.84

122.

8412

2.84

122.

8412

2.84

122.

8412

2.84

122.

8412

2.84

122.

8412

2.84

122.

8412

2.84

122.

8412

2.84

122.

8412

2.84

122.

8412

2.84

123.

7812

3.78

123.

7812

3.78

123.

7812

3.78

123.

7812

3.78

123.

7812

3.78

123.

7812

3.78

123.

7812

3.78

123.

7812

3.78

123.

7812

3.78

123.

7812

3.78

123.

7812

3.78

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

131.

1813

1.18

142.

3614

2.36

142.

3614

2.36

142.

3614

2.36

142.

3614

2.36

142.

3614

2.36

142.

3614

2.36

142.

3614

2.36

142.

3614

2.36

142.

3614

2.36

142.

3614

2.36

142.

3614

2.36

144.

9014

4.90

144.

9014

4.90

144.

9014

4.90

144.

9014

4.90

144.

9014

4.90

144.

9014

4.90

144.

9014

4.90

144.

9014

4.90

144.

9014

4.90

144.

9014

4.90

144.

9014

4.90

145.

7814

5.78

145.

7814

5.78

145.

7814

5.78

145.

7814

5.78

145.

7814

5.78

145.

7814

5.78

145.

7814

5.78

145.

7814

5.78

145.

7814

5.78

145.

7814

5.78

145.

7814

5.78

148.

9014

8.90

148.

9014

8.90

148.

9014

8.90

148.

9014

8.90

148.

9014

8.90

148.

9014

8.90

148.

9014

8.90

148.

9014

8.90

148.

9014

8.90

148.

9014

8.90

148.

9014

8.90

163.

6816

3.68

163.

6816

3.68

163.

6816

3.68

163.

6816

3.68

163.

6816

3.68

163.

6816

3.68

163.

6816

3.68

163.

6816

3.68

163.

6816

3.68

163.

6816

3.68

163.

6816

3.68

167.

4216

7.42

167.

4216

7.42

167.

4216

7.42

167.

4216

7.42

167.

4216

7.42

167.

4216

7.42

167.

4216

7.42

167.

4216

7.42

167.

4216

7.42

167.

4216

7.42

167.

4216

7.42

286

BE2-128_1_CARBON_01

Chemical Shift (ppm)192 184 176 168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24

No

rm

alize

d In

ten

sity

0

0.05

0.10

0.15

0.20

M01(s)

M03(s)

M04(s)

M05(s)

M06(s)

M07(s)M08(s)

M09(s)

M10(s)

29.9

32

9.9

32

9.9

32

9.9

32

9.9

32

9.9

32

9.9

32

9.9

32

9.9

32

9.9

32

9.9

32

9.9

32

9.9

32

9.9

32

9.9

32

9.9

32

9.9

32

9.9

32

9.9

32

9.9

32

9.9

32

9.9

3

34.7

03

4.7

03

4.7

03

4.7

03

4.7

03

4.7

03

4.7

03

4.7

03

4.7

03

4.7

03

4.7

03

4.7

03

4.7

03

4.7

03

4.7

03

4.7

03

4.7

03

4.7

03

4.7

03

4.7

03

4.7

03

4.7

0

101

.05

101

.05

101

.05

101

.05

101

.05

101

.05

101

.05

101

.05

101

.05

101

.05

101

.05

101

.05

101

.05

101

.05

101

.05

101

.05

101

.05

101

.05

101

.05

101

.05

101

.05

101

.05

116

.45

116

.45

116

.45

116

.45

116

.45

116

.45

116

.45

116

.45

116

.45

116

.45

116

.45

116

.45

116

.45

116

.45

116

.45

116

.45

116

.45

116

.45

116

.45

116

.45

116

.45

116

.45

123

.28

123

.28

123

.28

123

.28

123

.28

123

.28

123

.28

123

.28

123

.28

123

.28

123

.28

123

.28

123

.28

123

.28

123

.28

123

.28

123

.28

123

.28

123

.28

123

.28

123

.28

123

.28

131

.15

131

.15

131

.15

131

.15

131

.15

131

.15

131

.15

131

.15

131

.15

131

.15

131

.15

131

.15

131

.15

131

.15

131

.15

131

.15

131

.15

131

.15

131

.15

131

.15

131

.15

131

.15

145

.38

145

.38

145

.38

145

.38

145

.38

145

.38

145

.38

145

.38

145

.38

145

.38

145

.38

145

.38

145

.38

145

.38

145

.38

145

.38

145

.38

145

.38

145

.38

145

.38

145

.38

145

.38

160

.84

160

.84

160

.84

160

.84

160

.84

160

.84

160

.84

160

.84

160

.84

160

.84

160

.84

160

.84

160

.84

160

.84

160

.84

160

.84

160

.84

160

.84

160

.84

160

.84

160

.84

160

.84

162

.64

162

.64

162

.64

162

.64

162

.64

162

.64

162

.64

162

.64

162

.64

162

.64

162

.64

162

.64

162

.64

162

.64

162

.64

162

.64

162

.64

162

.64

162

.64

162

.64

162

.64

162

.64

167

.42

167

.42

167

.42

167

.42

167

.42

167

.42

167

.42

167

.42

167

.42

167

.42

167

.42

167

.42

167

.42

167

.42

167

.42

167

.42

167

.42

167

.42

167

.42

167

.42

167

.42

167

.42

JWL-BE2-128_1_PROTON_01

12 11 10 9 8 7 6 5 4 3 2 1 0

Chemical Shif t (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

9.000.951.972.020.80

287

Chemical Shift (ppm)12 11 10 9 8 7 6 5 4 3 2

Abs

olut

e In

tens

ity

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.610.750.870.902.902.131.00

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

0

0.05

0.10

0.15

DMSO-d6

102.

4710

2.47

102.

4710

2.47

102.

4710

2.47

102.

4710

2.47

102.

4710

2.47

102.

4710

2.47

102.

4710

2.47

102.

4710

2.47

102.

4710

2.47

102.

4710

2.47

102.

4710

2.47

116.

3111

6.31

116.

3111

6.31

116.

3111

6.31

116.

3111

6.31

116.

3111

6.31

116.

3111

6.31

116.

3111

6.31

116.

3111

6.31

116.

3111

6.31

116.

3111

6.31

116.

3111

6.31

116.

4511

6.45

116.

4511

6.45

116.

4511

6.45

116.

4511

6.45

116.

4511

6.45

116.

4511

6.45

116.

4511

6.45

116.

4511

6.45

116.

4511

6.45

116.

4511

6.45

116.

4511

6.45

123.

9812

3.98

123.

9812

3.98

123.

9812

3.98

123.

9812

3.98

123.

9812

3.98

123.

9812

3.98

123.

9812

3.98

123.

9812

3.98

123.

9812

3.98

123.

9812

3.98

123.

9812

3.98

125.

7712

5.77

125.

7712

5.77

125.

7712

5.77

125.

7712

5.77

125.

7712

5.77

125.

7712

5.77

125.

7712

5.77

125.

7712

5.77

125.

7712

5.77

125.

7712

5.77

125.

7712

5.77

128.

4312

8.43

128.

4312

8.43

128.

4312

8.43

128.

4312

8.43

128.

4312

8.43

128.

4312

8.43

128.

4312

8.43

128.

4312

8.43

128.

4312

8.43

128.

4312

8.43

128.

4312

8.4313

2.23

132.

2313

2.23

132.

2313

2.23

132.

2313

2.23

132.

2313

2.23

132.

2313

2.23

132.

2313

2.23

132.

2313

2.23

132.

2313

2.23

132.

2313

2.23

132.

2313

2.23

132.

23

139.

0313

9.03

139.

0313

9.03

139.

0313

9.03

139.

0313

9.03

139.

0313

9.03

139.

0313

9.03

139.

0313

9.03

139.

0313

9.03

139.

0313

9.03

139.

0313

9.03

139.

0313

9.03

143.

6614

3.66

143.

6614

3.66

143.

6614

3.66

143.

6614

3.66

143.

6614

3.66

143.

6614

3.66

143.

6614

3.66

143.

6614

3.66

143.

6614

3.66

143.

6614

3.66

143.

6614

3.66

145.

1314

5.13

145.

1314

5.13

145.

1314

5.13

145.

1314

5.13

145.

1314

5.13

145.

1314

5.13

145.

1314

5.13

145.

1314

5.13

145.

1314

5.13

145.

1314

5.13

145.

1314

5.13

163.

0316

3.03

163.

0316

3.03

163.

0316

3.03

163.

0316

3.03

163.

0316

3.03

163.

0316

3.03

163.

0316

3.03

163.

0316

3.03

163.

0316

3.03

163.

0316

3.03

163.

0316

3.03

288

Appendix VII

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