A Dissertation

entitled

Design, Synthesis, and Process Chemistry Studies of

Agents Having Anti-Cancer Properties

by

Amarjit Luniwal

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

Doctor of Philosophy Degree in Medicinal Chemistry

______Paul W. Erhardt, Ph.D., Committee Chair

______Ronald E. Viola, Ph.D., Committee Member

______L. M. V. Tillekeratne, Ph.D., Committee Member

______Steven M. Peseckis, Ph.D., Committee Member

______Jeffrey G. Sarver, Ph.D., Committee Member

______Patricia R. Komuniecki, Ph.D., Dean, College of Graduate Studies

The University of Toledo

May 2011

Copyright 2011, Amarjit Luniwal

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

An abstract of

Design, Synthesis and Process Chemistry Studies of Agents Having Anti-Cancer Properties

by

Amarjit Luniwal

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

The University of Toledo May 2011

Breast cancer is the second leading cause of cancer deaths in American women. The use of receptor modulators is the most common treatment for early stage breast cancer and for prevention of its recurrence after surgery. However, among the available agents in this class, none display an ideal therapeutic profile. Therefore, it was aimed to design and synthesize selective modulators (SERMs) which can overcome problems associated with the currently available agents. Toward that end, process chemistry studies were undertaken to enhance a synthetic route to natural glyecollin I (GLY I) which is a novel SERM that has promising anti-estrogenic and anti- cancer properties. Reaction yields across several steps were improved by optimizing reaction conditions and a few steps were improved by adopting alternative synthetic protocols. In doing so, not only were the total number of steps reduced from 15 to 13, but the overall yield was also tripled, i.e. from 3% to 9%. Molecular modeling and receptor docking studies were carried out during the design of GLY I related analogs obtained from intermediates produced during the scale-up syntheses. While performing such studies it was deduced that the active sites of the two subtypes of estrogen

iii receptors(ERs), namely ERα and ERβ, are very similar except for two key aspects. First, the active site His residue in ERα lays slightly closer to the active site Arg and Glu as compared to the one found in ERβ. Second, the imidazole ring of the active site His residue in the two ERs is oriented quite differently in three-dimensional space. The new designed and synthesized GLY I related analogs possess pharmacophores that may be able to exploit these differences in the active sites. This, in turn, could lead to more selective estrogen receptor modulation. These analogs also possess varying degrees of flexibility while displaying their key pharmacophores important for receptor binding and selectivity.

The pursuit of improved SERMs that can be derived directly from the GLY I chemistry turned next toward total synthesis of the second most prevalent member of the family, namely GLY II. Unlike GLY I, for a specific synthesis of GLY II the

2,2-dimethylbenzopyran ring needs to be constructed first. Various chemical trials were made to accomplish this before linking two key synthons, namely 2,2- dimethylchromanone carboxylic methyl ester and an α-iodo ketone through an SN2 displacement reaction followed by an intramolecular Wittig olefination. The olefin has been instilled with the two dihydroxyl groups required for construction of a fused- benzopyran-benzofuran ring system. A robust, 12-step chemical route has been developed to the synthetic point of a key, late-stage intermediate which is just a few, additional steps away from the final target molecule.

Model compounds were synthesized to develop and understand the chemistry associated with the key reactions utilized in the specific GLY II synthetic route. These compounds also led to the synthesis of three additional natural products,

iv namely 6a-hydroxymedicarpin, vestitol and bolusanthin III. Importantly, all of them possess key structural features required for binding to ERs. Therefore, they are additionally interesting for development of structure-activity relationships (SAR) that eventually can be translated into improved SERMs.

Finally, analogs were similarly designed to explore SERM SAR after a new and highly practical synthetic route to a benzofuran scaffold was discovered during the specific synthesis of GLY II. The benzofuran type system represents an interesting and novel scaffold to pursue ER modulation because it is able to display polar hydroxyl groups at almost equivalent distances to those found in . These analogs were synthesized to experimentally test the two thereotical concepts pertaining to differential spacing and altered orientation of the His-imidazole ring within the ERs active site.

As a second, major part of this dissertation work, the research has contributed toward the Center for Drug Design and Development‟s (CD3‟s) pursuit of chemotherapeutic treatments for advanced, hormone-independent cancer of the prostate (CaP). Similar to breast cancer in women, CaP is the second leading cause of cancer deaths in American men. Presently, there are no agents available that effectively address late-stage, advanced

CaP. In this same regard, however, the CD3 previously has identified a very promising small, peptidomimetic molecule that may be able to provide some benefit in this setting.

This lead compound has been designated as „CD3-246.‟ Moving forward with this technology, these thesis-related efforts involved scale-up synthetic studies and a new epimerization-free convergent route to CD3-246. In addition, an exploration of the reaction mechanism for pentamethylbenzene-mediated O-debenzylation of phenolic ethers was investigated because of its key role in the CD3-246 process chemistry.

v Experimental evidence was gathered to support that this reaction predominantly follows an SN1 pathway.

vi

Acknowledgments

Today, when I thankfully reflect upon my experiences over the last five years; I feel privileged for getting help, guidance and well wishes from so many people. I am truly gratified and owe thanks to all of them.

First and foremost, it gives me immense joy and pleasure to thank my mentor, advisor and local guardian Dr. Paul Erhardt. Without his guidance, encouragement and constant support, I could never have accomplished this. His impressive enthusiasm and dedication to his work, has made a profound impact upon me. Without a doubt he is a great scientist with great accomplishments, but what makes him so special and wonderful is his caring and loving attitude toward his students. He has been and will always remain a source of my inspiration and motivation. Vocabulary fails to describe how privileged and indebted

I feel to have him as my mentor and guide.

I would also like to thank all my research committee members, Drs. Tillekeratne,

Viola, Sarver, and Peseckis. I am truly honored to have them on my committee and I greatly appreciate their time, help and valuable suggestions.

I feel joyous in thanking all former and current CD3 group and synthetic lab members,

Jill, Nicole, Mike Reese, Pam, Dr. Nagy, Crystal, Lei, Jidong, Mohammed, Rahul,

Dhana, Renuka, Rachael, Neha and Mike for all their support and companionship.

vii I sincerely thank Dr. Y. W. Kim, Mr. Steven Moder, Tony and all other chemistry stockroom staff members for all their help during this entire time.

I owe special thanks to all MBC and College of Pharmacy colleagues, staff and faculty for their support and friendship. I am very thankful to all former and current members of organic chemistry journal club for providing a very useful platform for organic chemistry discussions and learning.

I am grateful to all my friends, especially Shan and Ritesh, for their support, encouragement and companionship during entire course of my PhD studies. I owe special thanks to my „buddy‟ Shan. He has been very helpful, encouraging and cooperative thoughout this entire duration.

I am so pleased to thank my parents, brothers, sisters, sisters-in-law, brothers-in-law, nieces and nephews for their blessing, wishes and love. I owe special thanks to my elder brother, Naresh, for his unwavering support, affection, and encouragement.

Finally, my heartfelt thanks and appreciations go to my beloved wife, Rekha, for all her love, care, and for the compromises and adjustments she has made so that I can realize my dreams. I greatly appreciate her support and encouragement during this entire time, especially when the „going gets tough.‟

Last but not least, I thank the almighty for providing strength to endure and for all His blessings.

viii

Contents

Abstract iii

Acknowledgments vii

Contents ix

List of Tables xiii

List of Figures xiv

List of Schemes xvi

List of Abbreviations xix

List of Spectra and Chromatograms xxii

1 Introduction

1.1 Overview 1

1.2 and Estrogen Receptors 2

1.3 Selective Estrogen Receptor Modulators (SERMs) 5

1.4 Isoflavonoids 6

1.4.1 Phytoalexins and 7

1.5 Initial Biological Testing of SERM Analogs 9

1.51 Binding Assays 9

1.5.2 Cell Lines 9

1.5.3 Cell Culture 10

1.5.4 Growth Inhibition Assays 10

1.6 Peptidylglycine-α-Amidating Monooxygenase (PAM) Enzyme and

ix CD3-246 11

1.6.1 Early Synthetic Studies for CD3-246 Synthesis 14

1.6.2 Mechanistic Investigations for O-Debenzylation of Phenolic

Ethers Using PMB and Trifluoroacetic Acid 17

1.7 Advanced Biological Testing of CD3-246 18

2 Results and Discussion 20

2.1 Scale-up of and Synthesis of Related Analogs 20

2.1.1 Scale-up Studies 20

2.1.2 GLY I Analogs 28

2.1.2.1 Spatial Analysis of ERα and ERβ Active Sites and Their

Natural Ligand Estradiol 29

2.1.2.2 Molecular Docking Studies of Analogs 32

2.1.2.3 Analysis of Docking Results for Gly I Related Analogs 36

2.1.2.4 Synthesis of Analogs 39

2.2 Synthesis of Glyceollin II (GLY II) 42

2.2.1 Early Synthetic Efforts and Initial Retro-Analyses 42

2.2.2 Later Retro-Analysis and Synthesis 50

2.2.3 Recommendations for Future Synthetic Efforts 57

2.3 Synthesis of Vestitol, Bolusanthin III and 6a-Hydroxymedicarpin 58

2.4 Design and Synthesis of New SERM Analogs 61

2.4.1 Design of Benzofuran and Furo[2,3-f]-2H-1-benzopyran Analogs

to Study SERM SAR 62

2.4.2 Design of Final Targets for SERM SAR 64

x 2.4.3 Molecular Docking Studies 64

2.4.4 Analysis of Molecular Docking Studies 65

2.4.5 Synthesis of Benzofuran Based Targets 67

2.4.6 Synthesis of Furo[2,3-f]-2H-1-benzopyran Based Analogs 69

2.5 Summary of Studies Aimed At Preparing Compounds Having Improved

SERM Properties 71

2.6 Scale-up and Process Chemistry Enhancement for Production of CD3-246 72

2.6.1 Scale-up Synthesis of CD3-246 Using the „Boc/Bzl‟ Strategy 73

2.6.2 Development of a Convergent Synthetic Route 75

2.6.2.1 Retro-Analysis 76

2.6.2.2 Synthesis 76

2.6.3 Mechanistic Investigations for O-Debenzylation of Phenolic Ethers

Using Pentamethylbenzene and Trifluoroacetic Acid 81

2.6.4 Summary of Studies Related to Peptide Chemistry and Mechanistic

Invetigations 85

3 Experimental 86

3.1 General Description 86

3.2 Scale-up Synthesis of Glyceollin I 87

3.2.1 GLY I Analogs 96

3.3 Synthesis of Glyceollin II (GLY II) 105

3.4 Syntheses of Vestitol, Bolusanthin III and 6a-Hydroxymedicarpin 122

3.5 Synthesis of New SERM Analogs 129

3.6 Scale-up and Process Chemistry Enhancement for Production of CD3-246 139

xi 3.7 Molecular Modeling and Docking Studies 149

References 152

Appendices

A List of Spectra and Chromatograms 167

xii

List of Tables

2.1 Stability data determined by 1H NMR for 3 in different solvents

at different temperatures…………………………………………………………...24

2.2 Yield and scale comparison for synthesis of GLY I………………………………28

2.3 Internal validation deviations…………………………………………………….34

2.4 External validation of ERα and ERβ docking model using training set………….35

2.5 Surflexdock scores for GLY I analogs using ERα and ERβ..…………………….39

2.6 Variables examined during reaction condition „b‟ in Scheme 2.5.10 (A)………..49

2.7 Formation of cylic ketals and thioketals………………………………………….52

2.8 Wittig salt formation reaction condition optimization……………………………53

2.9 Reaction conditions for elimination reaction……………………………………..54

2.10 Reaction optimization of de-benzylation in presence of tertiary alcohol…………56

2.11 Predicted binding affinities of target analogs……………………………………..67

2.12 Reaction conditions for „bomb‟ reaction………………………………………….75

2.13 Details for different batches of CD3-246 prepared by the optimized

„Boc/Bzl‟ route…………...………………………………………………………..75

2.14 Screening of various bases to improve on zwitterion solubility………………….77

2.15 Acylation reaction condition optimization results……………………………….77

xiii

List of Figures

1.1 Natural estrogens and synthetic ER ligands………………………………………2

1.2 Some interesting isoflavonoids and phytoalexins……………………………...... 7

1.3 Structures of major soy isoflavonoids and pterocarpanoids……………………..8

1.4 Lead compound CD3-246 and an earlier analog from the NCI‟s former

lung cancer research program……….……………………………………………13

2.1 GLY I analogs and structures of E2, GLO and GLY I used for external

validation of docking models…………………………………………………….29

2.2 A) THC bound ERα active site; B) THC bound ERβ active site; C) E2

bound ERα (1ERE x-ray structure)……………………………..……………….30

2.3 H-bond interactions between estradiol and active site residue of ERα………….31

2.4 A) THC bound ERα and ERβ active sites overlapping; B) Glyceollin I

(GLY I), Glyceollin II (GLY II) and Glyceollin III (GLY II)……………………32

2.5 Internal validation for ERα…………………………………………………...... 33

2.6 Internal validation for ERβ………………….……………………………...... 34

2.7 Correlation plot between experimental binding affinity and docking

model predicted binding affinity………………………………...………………35

2.8 External validation highest scoring surflexdock poses of training set

(A) with ERα, (B) with ERβ…………………………………………………….35

xiv 2.9 Highest scoring poses of ligands with active sites: (A) ERα; (B) ERβ…………..36

2.10 Binding interactions of CD3-654 and CD3-714-R…………………….………..37

2.11 Comparative views of the most flexible analogs CD3-649, CD3-650

and CD3-658 interactions with: (A) ERα active site; (B) ERβ active site……….37

2.12 Distance map of three analogs having mostly H-bonding capability……………38

2.13 Pharmacophore mapping of estradiol and energy-minimized T2………….…….63

2.14 List of benzofuran based SERM targets………………………………………….64

2.15 List of furo[2,3-f]-2H-1-benzopyran based SERM targets………………………64

2.16 Surflexdock poses of test set data points for ERα..………………………………66

2.17 Surflexdock poses of test set data points for ERβ (A) data points with

one or more benzyl group; (B) without any benzyl group, (C) Highest

scoring data point with a phenolic hydroxyl pharmacophore

is showing π-π interaction…………………………………….…………………66

2.18 Epimerization at α-carbon of a carboxy terminal activated peptide…………….79

2.19 600 MHz NMR spectrum recorded in deuterated DMSO for key

intermediate CD3-404 obtained via the convergent synthesis strategy.

Spectrum depicts epimerization …………………………………….…………...80

2.20 600 MHz NMR spectrum recoded in deuterated DMSO for key

intermediate CD3-404 obtained via the convergent synthesis strategy.

Spectrum suggests that no significant epimerization …………………………...80

2.21 600 MHz NMR spectrum recorded in deuterated DMSO for final

product CD3-246 obtained via the convergent synthesis strategy.

Spectrum suggests that no significant epimerization ………………….……….81

xv

List of Schemes

1.1 Key steps in biosynthesis of estrogens ….……………………………………….3

1.2 PAM enzymatic action mechanism………………………………….………….13

1.3 Synthesis of CD3-246 using „Boc/Bzl‟ strategy………………………………..15

1.4 Synthesis of CD3-246 using „Boc/Bzl‟ strategy (continued)…………………..15

1.5 Synthesis of CD3-246 using Fmoc chemistry…………………………………..16

2.1 Scale-up Synthesis of GLY I…………………………………………………….22

2.2 Proposed mechanism for in situ generation of MOM-Cl……………………….22

2.3 Proposed mechanism for Selectfuor-assisted selective α-iodination…………...23

2.4 Synthesis of chiral ligand……………………………………………………….27

2.5 Synthesis of completely open GLY I scaffold analogs…………………………39

2.6 Synthesis of partially open GLY I scaffold analogs……………………………40

2.7 Synthesis of additional GLY I analogs…………………………………………41

2.8 Synthesis of additional GLY I analogs………………………………………….41

2.9 Initial retro-synthetic analysis……….………………………………………….43

2.10 Toward synthesis of 2,2-dimethyl-7-hydroxy-2H-[1]-benzopyran-6-car-

boxaldehyde (A)……………………………………………………………………44

2.11 Toward synthesis of dimethylchromanone……………………………………...44

2.12 Toward synthesis of dimethylchromanone using alternate route……………….45

xvi 2.13 Synthesis of 4-chromanone ester……………………………………………...... 46

2.14 Alternative synthesis of „4-chromanone‟ ester…………………………………..46

2.15 Proposed mechanism for alternate synthesis of 4-chromanone ester……………46

2.16 Synthesis of 4-chromanone Weinreb amide……………………………………..47

2.17 Ring closing metathesis based retro-analysis for GLY II synthesis……………...48

2.18 Synthesis of intermediate B……………………………………………………...48

2.19 Stille coupling……………………………………………………………………49

2.20 Retro-synthetic analysis of GLY II from common building blocks……………..50

2.21 Synthesis of GLY II………………………………………………………………51

2.22 Synthesis of GLY II (continued)…………………………………………………52

2.23 Synthesis of GLY II (continued)…………………………………………...... 54

2.24 Synthesis of GLY II (continued)………………………………………..……….55

2.25 Reaction optimization for selective debenzylation………………………………56

2.26 Replacement of benzyl with TBDMS and subsequent reduction……………….57

2.27 Proposed route for final GLY II synthesis………………………………………58

2.28 Synthesis of vestitol and bolusanthin III…………………………………...... 60

2.29 Proposed degradation mechanism for 23……………………...... 61

2.30 Synthesis of 6a-hydroxymedicarpin………………………………………...... 61

2.31 Fortuitous formation of benzofuran scaffold……………………………………62

2.32 Synthesis of benzofuran-based targets T1 and T2………………………………68

2.33 Synthesis of benzofuran-based targets T3, T4, T5 and T6………………………69

2.34 Synthesis of targets T7 and T8...... 70

2.35 Synthesis of targets T9 and T10…………………………………………………71

xvii 2.36 Scale-up Synthesis of CD3-246 using „Boc/Bzl‟ strategy………………………74

2.37 Retro-analysis of convergent synthesis for CD3-246………………………….76

2.38 Synthesis of N-acyl-(D)-tyrosine……………………………………………….76

2.39 Synthesis of „dipeptide‟ synthon 35 & 35a…………………………………….78

2.40 Convergent coupling step……………………………………………………….78

2.41 Synthesis of model compounds…………………………………………...... 82

2.42 Role of „PMB‟ in ether cleavage………………………………………………..83

2.43 Effect of substitution on reaction time………………………………………….84

2.44 Concerted (SN2) type of displacement………………………………………….84

2.45 Stepwise (SN1) type of displacement……………………………………...... 85

xviii

List of Abbreviations

Non-Chemical Substances and Groups

(g)………………..Gaseous 3β-HSD………….3β-Hydroxysteroid dehydrogenase ANOVA………...Analysis of variance ArgATCC……….American Type Culture Collection Av………………Average BA………………Binding affinity CC………………Column chromatography CD3……………..Center for Drug Design and Development CI……………….Confidence interval COSY…………..Correlation spectroscopy (in context of NMR) D………………..Dextro Dev……………..Deviation DOD…………….Department of Defense E1……………….. E2……………….Estradiol E3………………. ER………………Estrogen receptor ERE……………..Estrogen response element ERT……………..Estrogen replacement therapy FBS……………..Fetal bovine serum GI50……………..50% Growth inhibition GST……………..Glutathione-S-transferase HEK…..………...Human Embryonic Kidney HRT…………….Hormone replacement therapy ID……………….Identification IP………………..Intraperitoneal IV……………….Intravenous J…………………Coupling constant LBD……………..Ligand binding domain LC………………Liquid chromatography M……………….Molar MCF……………Michigan Cancer Foundation MS……………...Mass spectroscopy Molec S………...Molecular sieve NB……………...Non-bonding

xix NCI……………..National Cancer Institute NS………………NuSerum OD………………Optical density PAL……………..α-Hydroxypeptidylglycine-α-amidating Lyase PAM…………….Peptidylglycine-α-amidating Monooxygenase PC……………….Prostate cancer PDB……………...Protein data bank PHM……………..Peptidylglycine-α-hydroxylating Monooxygenase PK……………….Pharmacokinetic prep-TLC………...Preparative-thin layer chromatography psi………………..Pressure per square inch QC………………Quality control RCM…………….Ring closing metathesis Rf………………...Retention factor RMSD…………..Root mean square deviation RPMI………….....Roswell Park Memorial Institute RT……………….Retention time rt…………………Room temperature SAR……………..Structure-activity relationship SC……………….Subcutaneous SERM…………...Selective estrogen receptor modulator Std………………Standard TR-FRET……….Terbium–based time-resolved fluorescence energy transfer

Chemical Substrates and Groups

(DHQD)2PHAL…1,4-Bis(9-O-dihydroquinidinyl)phthalazine BnBr……………..Benzyl bromide Boc………………tert-Butyloxy carbonyl BuLi……………..Butyl lithium Bzl……………….Benzyl CDI………………1,1-Carbonyldiimidazole DBU……………..1,8-Diazobicyclo[5.4.0]undecane DCC……………...N,N'-Dicyclohexylcarbodiimide DCM………...... Dichlormethane Dehydro………….6a,11a-Dehydro DIBAL-H…………Diisobutylaluminum hydride DIPEA……………Diisopropylethyl amine DMF………………Dimethyl formamide DMSO…………….Dimethylsulfoxide DPPA……………..Diphenylphosphoryl azide Et2O………………Diethyl ether Et3N………………Triethyl amine EtOAc…………….Ethyl acetate EtOH……………..Ethanol Fmoc……………..9-Fluorenylmethyloxycarbonyl

xx GLO………………… GLY …………...…….Glyceollin HATU………………..Hexafluorophospho-azabenzotriazoloxy-tetramethyluronium HOAt ………………..Hydroxyazabenzotriazole HOBt……………..….Hydroxybenzotriazole LDA………….………Lithium diisopropylamide LiHMDS……….…….Lithium bis(trimethylsilyl)amide MEM………….……..Methoxyethoxymethyl MeOH………………..Methanol MOM…………….…..Methoxymethyl MPM………………….4-Methoxyphenyl methyl Ms2O…………………Methanesulfonic anhydride NMO…………………N-Methylmorpholine N-oxide OTs…………………...Tosyloxy PBA………….………4-Phenylbut-3-enoic acid PMB…………….……Pentamethylbenzene PPTS…………………Pyridinium p-toluenesulfonate PyBOP……………….Hexafluorophosphate-benzotriazoloxy-tripyrrolidinophosphonium Tba………………..…4-(2-Thienyl)butyric acid TBDMS………….…..tert-Butyldimethylsilyl tBu………………...…tert-Butyl TFA………………….Trifluoroacetic acid THC………………….Tetrahydrochrysene-2,8-diol THF…………………..Tetrahydrofuran TMS……………...... Tetramethylsilane

xxi

List of Spectra and Chromatograms

1H NMR spectrum of compound 3……………………….………………………….168

1H NMR spectrum of compound 4……………………….………………………….168

13C NMR spectrum of compound 4………………………………….………………168

1H NMR spectrum of compound 5..………………………………….……………....169

13C NMR spectrum of compound 5………………………………….………………169

1H NMR spectrum of compound 6..………………………………….……………....169

13C NMR spectrum of compound 6………………………………….………………170

1 H NMR spectrum of compound (DHQD)2PHAL………………….……………....170

1H NMR spectrum of compound CD3-656………………………….……………....170

13C NMR spectrum of compound CD3-656…...…………………….………………171

1H NMR spectrum of compound CD3-699………………….……………………....171

1H NMR spectrum of compound 7………………………….………………….…....171

13C NMR spectrum of compound 7…...…………………….…………………….…172

1H NMR spectrum of compound GLY-I………………….………………………....172

HPLC Chromatogram compound GLY-I………………..….………………….…....172

1H NMR spectrum of compound CD3-649………………………….……………....173

13C NMR spectrum of compound CD3-649…...…………………….………………173

1H NMR spectrum of compound CD3-650………………………….……………....174

xxii 13C NMR spectrum of compound CD3-650…...…………………….………………174

1H NMR spectrum of compound CD3-653………………………….……………....175

13C NMR spectrum of compound CD3-653…...…………………….………………175

1H NMR spectrum of compound CD3-654………………………….……………....175

13C NMR spectrum of compound CD3-654…...…………………….………………176

1H NMR spectrum of compound CD3-714………………………….……………....176

13C NMR spectrum of compound CD3-714…...…………………….………………176

1H NMR spectrum of compound CD3-667………………………….……………....177

13C NMR spectrum of compound CD3-667…...…………………….………………177

1H NMR spectrum of compound CD3-666………………………….……………....177

13C NMR spectrum of compound CD3-666…...…………………….………………178

1H NMR spectrum of compound CD3-523 (-)-glycinol…………….…………….....178

13C NMR spectrum of compound CD3-523 (-)-glycinol…………….…………….....178

HPLC Chromatogram for racemic (±)-glycinol…………..….………………….…....179

HPLC Chromatogram of compound CD3-523 (-)-glycinol….………………….…....179

1H NMR spectrum of compound CD3-640………………………….…………….....180

13C NMR spectrum of compound CD3-640…...…………………….……………….180

1H NMR spectrum of compound CD3-639………………………….…………….....180

13C NMR spectrum of compound CD3-639…...…………………….………………181

1H NMR spectrum of compound CD3-698………………………….……………....181

13C NMR spectrum of compound CD3-698…...…………………….………………181

1H NMR spectrum of compound GLY-II………………………….……………...... 182

13C NMR spectrum of compound GLY-II …...…………………….………………..182

xxiii 13C NMR spectrum of compound Dehydro-GLY-II ……………….……………...... 182

1H NMR spectrum of compound 7a..………………………………….…………….183

13C NMR spectrum of compound 7a………………………………….……………..183

1H NMR spectrum of compound 8a..………………………………….…………….183

13C NMR spectrum of compound 8a ……………….……………...... 184

1H NMR spectrum of compound 8..………………………………….………………184

13C NMR spectrum of compound 8………………………………….……………….184

1H NMR spectrum of compound A’..………………………………….…………….185

13C NMR spectrum of compound A’ ……………….……………...... 185

1H NMR spectrum of compound 9..………………………………….……………...185

13C NMR spectrum of compound 9 ……………….……………...... 186

1H NMR spectrum of compound 10..………………………………….…………….186

13C NMR spectrum of compound 10……………….……………...... 187

1H NMR spectrum of compound 10a..………………………………….…………...187

13C NMR spectrum of compound 10a……………….……………...... 188

1H NMR spectrum of compound 11a..………………………………….…………...188

1H NMR spectrum of compound 11b..………………………………….…………...188

1H NMR spectrum of compound 11..………………………………….……………..189

13C NMR spectrum of compound 11 ……………….……………...... 189

1H NMR spectrum of compound 12..………………………………….……………..189

13C NMR spectrum of compound 12……………….……………...... 190

1H NMR spectrum of compound 13..………………………………….…………...... 190

1H NMR spectrum of compound 13..………………………………….…………….190

xxiv 1H NMR spectrum of compound 13a..………………………………….…………...191

1H NMR spectrum of compound 13b..………………………………….……………..191

13C NMR spectrum of compound 13b ……………….……………...... 191

1H NMR spectrum of compound 15..………………………………….………………192

13C NMR spectrum of compound 15……………….……………...... 192

1H NMR spectrum of compound 17..………………………………….…………...... 192

1H NMR spectrum of compound 18..………………………………….………………193

13C NMR spectrum of compound 18……………….……………...... 193

1H NMR spectrum of compound 18a.………………………………….…………...... 193

COSY spectrum of compound 18a……………….……………...... 194

1H NMR spectrum of compound 18b..………………………………….…………...... 194

1H NMR spectrum of compound 18c..………………………………….………………194

1H NMR spectrum of compound 19..………………………………….………………195

13C NMR spectrum of compound 19……………….……………...... 195

COSY spectrum of compound 19……………….……………...... 195

1H NMR spectrum of compound 21..………………………………….………………196

13C NMR spectrum of compound 21……………….……………...... 196

1H NMR spectrum of compound 21b.………………………………….…………...... 196

13C NMR spectrum of compound 21b ……………….……………...... 197

1H NMR spectrum of compound 22..………………………………….………………197

13C NMR spectrum of compound 22……………….……………...... 197

1H NMR spectrum of compound 23.………………………………….…………...... 198

13C NMR spectrum of compound 23 ……………….……………...... 198

xxv 1H NMR spectrum of compound 23a.………………………………….………………198

COSY spectrum of compound 23a…………….……………...... 199

13C NMR spectrum of compound 23a…………….……………...... 199

1H NMR spectrum of compound 23b.………………………………….…………...... 199

13C NMR spectrum of compound 23b ……………….……………...... 200

1H NMR spectrum of compound 16..………………………………….………………200

13C NMR spectrum of compound 16……………….……………...... 200

1H NMR spectrum of compound 24..………………………………….………………201

1H NMR spectrum of compound 6a-Hydroxymedicarpin…………….…………...... 201

13C NMR spectrum of compound 6a-Hydroxymedicarpin ……………….……...... 201

1H NMR spectrum of compound T1..………………………………….………………202

13C NMR spectrum of compound T1……………….……………...... 202

1H NMR spectrum of compound T2..………………………………….………………202

13C NMR spectrum of compound T2 ……………….……...... 203

1H NMR spectrum of compound T3..………………………………….………………203

13C NMR spectrum of compound T3……………….……………...... 203

1H NMR spectrum of compound T4..………………………………….………………204

13C NMR spectrum of compound T4 ……………….……...... 204

1H NMR spectrum of compound T5..………………………………….………………204

13C NMR spectrum of compound T5……………….……………...... 205

1H NMR spectrum of compound T6..………………………………….………………205

13C NMR spectrum of compound T6 ……………….……...... 205

1H NMR spectrum of compound 25..………………………………….………………206

xxvi 13C NMR spectrum of compound 25……………….……………...... 206

1H NMR spectrum of compound T7..………………………………….………………206

13C NMR spectrum of compound T7……………….……………...... 207

13C NMR spectrum of compound T8 ……………….……...... 207

1H NMR spectrum of compound 26..………………………………….………………207

1H NMR spectrum of compound 26..………………………………….………………208

13C NMR spectrum of compound 27……………….……………...... 208

1H NMR spectrum of compound 27..………………………………….………………208

1H NMR spectrum of compound T9..………………………………….………………209

13C NMR spectrum of compound T9……………….……………...... 209

1H NMR spectrum of compound T10.………………………………….………………210

13C NMR spectrum of compound T10…………….……………...... 210

1H NMR spectrum of compound 29..………………………………….………………210

13C NMR spectrum of compound 29……………….……………...... 211

1H NMR spectrum of compound 30..………………………………….………………211

1H NMR spectrum of compound CD3-404…………………………….………………211

1H NMR spectrum of compound CD3-246…………………………….………………212

1H NMR spectrum of compound Fmoc-Met-Gly-tbu ester….……….………………212

13C NMR spectrum of compound Fmoc-Met-Gly-tbu ester …...... 212

1H NMR spectrum of compound 36a.………………………………….………………213

1H NMR spectrum of compound 37..………………………………….………………213

13C NMR spectrum of compound 37……………….……………...... 213

1H NMR spectrum of compound 38..………………………………….………………214

xxvii 13C NMR spectrum of compound 38……………….……………...... 214

1H NMR spectrum of compound 39..………………………………….………………214

13C NMR spectrum of compound 39……………….……………...... 215

1H NMR spectrum of compound 40..………………………………….………………215

13C NMR spectrum of compound 40……………….……………...... 215

xxviii

Chapter 1

Introduction

1.1 Overview

This thesis describes synthetic medicinal chemistry studies directed toward human hormone-related cancers. The first several sections pertain to breast cancer and the last two sections pertain to prostate cancer. Breast cancer is the second leading cause of cancer deaths in American women.1 During their early stages, the growth of these types of tumors is generally dependent upon stimulation by the estrogen hormone pathway.

Thus, the use of estrogen receptor modulators is the most common strategy to initially treat early stage breast cancer and to prevent its reoccurrence after surgery. However, among the available agents in this class, none display an ideal therapeutic profile. My thesis research in this area has focused upon the pursuit of improved selective estrogen receptor modulator (SERM) compounds. Several of such target molecules along with many of their synthetic intermediates have been entered into the Center for Drug Design and Development‟s (CD3‟s) compound library so that they can be screened by the CD3‟s

Biological Testing Core Laboratory in the future. These types of compounds have been assigned “CD3-#” codes which have also been incorporated into my thesis for cross- reference purposes.

Similar to breast cancer in women, prostate cancer is the most frequent type and second leading cause of cancer deaths in American men.1 During its early stages, growth is generally dependent upon stimulation by the androgen hormone pathway. While

1 ablation of the latter by chemotherapeutic agents and surgical procedures are reasonably effective during the early stages of disease, prostate cancer often re-emerges in an aggressive growth and metastases phase that is no longer dependent upon hormone control. Since there are no treatment options that are clearly effective for the latter, this situation represents a very significant unmet medical need. My thesis research in this area has focused upon the chemical development and reaction mechanism details associated with a novel compound called „CD3-246.‟ This was previously identified within the CD3 by prior investigators during their search for new molecules that may be able to provide therapy in the setting of advanced, hormone-independent prostate cancer.

1.2 Estrogens and Estrogen Receptors

Estrogens are part of the nuclear hormone family of steroidal compounds. They play a pivotal role in reproductive endocrinology and are important for maintaining homeostasis in a woman‟s body.2 Estrone (E1), estradiol (E2) and estriol (E3) are the three main estrogens found in humans. The structures for these hormones are shown in Fig. 1.1 along with two synthetic estrogen receptor (ER) ligands (discussed later).

O OH OH OH H H H

HO HO HO E1 E2 E3

N O O O N N

CYP2D6 O

HO OH HO S 4-Hydroxytamoxifen (Active form) (Prodrug)

Fig. 1.1. Natural estrogens and synthetic ER ligands.

2 Ovary and testis are the two main sources for production of the estrogens,3 for which the biochemical pathway is depicted in Scheme 1.1. Alternatively, other tissues such as bone, cardiovascular and the brain can also synthesize estrogens via aromatization of androgens by aromatase.3, 4

Cholestrol side-chain O OH cleavage enzyme 3 -HSD

Androstenedione HO O HO Cholesterol Aromatase Aromatase O OH OH

OH

HO HO HO E1 E3 E2

Scheme 1.1. Key steps in biosynthesis of estrogens (3β-HSD: 3β-Hydroxysteroid dehydrogenase).5

Estrogens mediate their effects through ERs, also known as ligand-activated transcription factors.2 ERα6 and ERβ7 are two subtypes of estrogen receptors,8 structurally belonging to the same nuclear receptor superfamily. Because ERs usually reside in cytoplasm and move into the nucleus upon ligand binding, they are classified as „type I nuclear receptors.‟9 The DNA binding domain of both ERs has ca. 90% structural homology in comparison to ca. 53% amino acid identity in their ligand binding domains. This suggests that they recognize and bind to similar estrogen recognition element (EREs) on DNA while each may have a distinct spectrum of preferred ligands.10 It has been shown experimentally that both receptors show similar affinities toward classical EREs and display similar binding affinity (0.2 nM for ERα and 0.6 nM for ERβ) to the natural

3 ligand estradiol.7, 10, 11 However, many other ligands such as those present as natural dietary components like and , preferably bind to ERβ.11 Both receptors have differing tissue distributions. ERα is predominantly found in the ovary, uterus, endomertium and breast.12 ERβ is expressed in several tissues such as mammary gland, uterus, ovary, prostate, epididymus, testis, pituitary, kidney, thymus, bone and the central nervous system.12, 13 Because of alternative splicing, multiple ERβ isoforms have been observed.14 However, among all known isoforms (ERβ1, -β2, -β4 and –β5), only

ERβ1 is a fully functional isoform. All the others lack innate activities in their homodimers. Alternatively, when the latter heterodimerise with ERβ1, the ERβ1 induced transactivation can be enhanced in a ligand-dependent manner.9

Understanding the biological effects of sex hormones, especially estrogens and progestins, has helped in both the development of oral contraceptives15 and in the use of estrogen replacement therapy (ERT) to treat postmenopausal symptoms.16 Hormone replacement therapy (HRT), a combination of estrogen and progestin, is used for the treatment and prevention of which lowers the risk of fractures in elderly women by elevating bone mineral density.2 Such treatments have proven to be very beneficial for many women, particularly throughout the menopausal years.17 Though the benefits of estrogen replacement are well known, the Collaborative Group on Hormonal

Factors in Breast Cancer in a re-analysis of data from 51 published studies (up to 1997), concluded that ERT actually increased the risk of breast cancer.18 Several other studies have likewise found a direct correlation between the duration of ERT with development of estrogen receptor-positive (ER+) breast cancer.19, 20 These early findings provided rationale for the development of and eventually for selective estrogen

4 receptor modulators (SERMs) as alternative treatments for breast cancer and osteoporosis2 that can be deployed with less side-effect toxicity.

1.3 Selective Estrogen Receptor Modulators (SERMs)

The discovery of tamoxifen as a that blocks the estrogen- stimulated growth of breast cancer,21 provided a chemical structural basis for the further pursuit of selective estrogen receptor modulators.2 SERMs can act as estrogen antagonists in some tissues and as agonists in others.17, 22, 23 Ideally, a SERM would provide postmenopausal women the benefits of estrogen therapy (such as reduced risk of bone fracture, urologic and vaginal atrophy, and hot flashes) without any of its side-effects (i.e. an increased risk of breast cancer, endometrial cancer, coronary heart disease, stroke, and venous thromboembolic events).24 Jordan et al. in their seminal work,25 first observed that the anti-estrogens, tamoxifen and keoxifene (now known as raloxifene), helped maintain bone density both in intact as well as overiectomized female rats. This was in contrast to the then contemporary understanding where in animal models estrogen was known to block bone loss such that a loss of bone density in overiectomized animals, rather than a gain, would have been the expected outcome from an anti-estrogen.17 This observation eventually led to testing of tamoxifen as a chemopreventive agent in women with high risk for breast cancer.26 The need for development of tamoxifen analogues, which not only treat and prevent osteoporosis and artheroclerosis (the two main side effects of reduced body estrogens in postmenopausal women) while also preventing breast cancer,27 led to the further development of raloxifene.2 Currently, tamoxifen and raloxifene are two SERMS being marketed for postmenopausal women in the United

5 States. Each offers benefits as well as drawbacks.24 Tamoxifen reduces the risk of estrogen receptor positive (ER+) breast cancer but is found to increase the risk of endometrial cancer.24, 28 Alternatively, raloxifene is associated with a reduced risk of osteoporosis29 and breast cancer30 and has negligible impact on the risk of endometrial cancer. It does not lower the risk of coronary heart disease or stroke.24, 31 Both agents are linked with higher risks of venous thromboembolic events and hot flashes, and neither ameliorates urogenital atrophy.24 Furthermore, tamoxifen‟s benefits in treating breast cancer are typically dose-duration limited, with resistance generally being observed after three-years of treatment and, even worse, certain studies suggesting that an increased cancer risk can eventually occur.32, 33 Because of these shortcomings, the quest for an ideal SERM is still a very active area of medicinal chemistry research.

1.4 Isoflavonoids

Flavonoids and isoflavonoids show a wide range of physiological activities. Because of their antioxidant properties, isoflavonoids remain of interest for the treatment of free radical mediated disorders such as cancer, Alzheimer‟s, Parkinson‟s and certain cardiovascular diseases.34 As a result, these polyphenolic compounds have recently gained popularity in the fields of nutrition, health and medicine.34 Several reports have attributed biological activities such as antimicrobial, antioxidant and antiviral etc. to phenolic constituents in Glycyrrhiza species.35 Vestitol and 2‟,7-dihydroxy-4‟- methoxyisoflav-3-ene/bolusanthin III36 (Fig. 1.2) are isolated from Glycyrrhiza pallidiflora.35, 37, 38 Vestitol and related isoflavonoids are considered to be useful chemopreventive agents for peptic ulcer and gastric cancer in Helicobacter Pylori

6 infected individuals where they have shown good activity against antibiotic resistant as well as nonresistant H. pylori.39 A study conducted in a large group of Japanese patients with duodenal ulcer, gastric hyperplasia or non-ulcer dyspepsia, has found an association between H. pylori and gastric cancer development.40 Because their display of hydroxyl- groups is similar to that found among some of the SERMs, these types of isoflavonoids also became of interest to us as possible selective anti-estrogenic agents for the potential treatment of breast cancer.

HO O HO O HO O HO O H OH

O O HO OCH3 HO OCH3 Vestitol Bolusanthin III OCH3 6a-Hydroxymedicarpin OCH3

Fig. 1.2. Some interesting isoflavonoids and phytoalexins.

1.4.1 Phytoalexins and Glyceollins

Phytoalexins are low molecular weight antimicrobial molecules that are synthesized de novo by plants in response to stress from various insults.41, 42 It is thought that they help protect plants against infections. , cis-fused benzofuran-benzopyran tetracyclic ring systems,43 are an important class of phytoalexins. Medicarpin and 6a- hydroxymedicarpin (Fig. 1.2) are two important pterocarpans found in nature.44, 45

Medicarpin is a potent antifungal agent that can also induce apoptosis in human leukemia cells.46 6a-Hydroxymedicarpin is a 6a-hydroxy derivative of medicarpin that constitutes the aglycone moiety of licoagroside E found in G. pallidiflora.45

Many reports have associated high dietary intake of products to lower prevalence of prostate and breast cancer in Asian populations.47, 48 Within the context of

7 beneficial natural products that can reside within edible plants, the phytoalexins represent a very special and interesting class. The glyceollins (GLYs) I, II and III (Fig. 1.3), are 6a- hydroxypterocarpan phytoalexins produced by soy plants in response to attack on seeds by various fungi41 or attack on roots by soybean-cyst root nematodes.49, 50

The GLYs exhibit marked anti-estrogenic activity in some tissues which is contrary to the estrogenic activity displayed by the well-known isoflavonoids such as genistein and diadzein that are normally present in soy when it is not stressed.41, 42 In addition to their established anti-fungal51 and anti-bacterial52 activities, the CD3‟s collaborators have found that the GLYs possess good anticancer properties [IC50 3.2 μM (ERα) and 6.4 μM

(ERβ)] by using in vitro assays with MCF7 and HEK297 cell lines.41, 53, 54 This promising activity has also been observed in in vivo models using human tumor xenografts implanted into mice.53, 55 Mechanistically, GLY I has been found to have good anti- estrogenic activity.42, 56 Because of these interesting biological activities, the GLYs hold considerable promise to be developed as chemo-preventive agents in women with high risk of breast cancer.

O O O O O O OH OH OH

O O O GLY I GLY II OH OH GLY III OH HO O HO O

OH O O Genestein OH Diadzein OH

Fig. 1.3. Structures of major soy isoflavonoids and pterocarpanoids.

8 1.5 Initial Biological Testing of SERM Analogs

In order to develop SAR, all of the target compounds and selected intermediates will be examined by the CD3‟s Bioanalytical Testing Laboratory. In vitro functional receptor binding assays will be performed first. Active compounds will be further tested in vitro by using cell growth inhibition assays.

1.5.1 Binding Assays

Invitrogen‟s LanthaScreen™ TR-FRET Estrogen Receptor α and β Coactivator Assay kits will be used for determination of ERα and ERβ responses upon exposure to the various test compounds. These assays provide a sensitive and robust method for high- throughput screening of potential ERα and ERβ ligands as agonists or antagonists of ligand-dependent coactivator recruitment. The kits use a terbium-labeled anti- glutathione-S-transferase (GST) antibody, a fluorescein-labeled coactivator peptide, and an ER α and β ligand-binding domain (ER α-LBD and ER β-LBD) that is tagged with

GST in a homogenous „mix-and-read‟ assay format.

1.5.2 Cell Lines

The estrogen receptor positive (ER+) human breast cancer cell line MCF7, the estrogen receptor negative (ER-) human ovarian cancer cell line NCI-ADR-RES, and the androgen receptor negative (AR-) human prostate cancer (PC) cell lines DU145 and PC3, were generously provided by the National Cancer Institute, Division of Cancer Treatment and

Diagnosis Tumor Repository. The androgen receptor positive (AR+) human PC cell line

9 LNCaP along with an ER- immortalized “normal” human breast cell line MCF12A, were obtained from the American type culture collection (ATCC).

1.5.3 Cell Culture

All cell lines except MCF12A are routinely maintained in RPMI-1640 media containing

2.0 mM L-glutamine supplemented with 20 mM HEPES, 0.2% sodium bicarbonate, 5% fetal bovine serum (FBS), 5% NuSerum (NS) IV® (BD Biosciences, Bedford, MA), and

50 µg/mL gentamicin. MCF12A cells were maintained in a 1:1 mixture of DMEM and F-

12 Ham media containing 2.5 mM L-glutamine and 15 mM HEPES, supplemented with

0.2% sodium bicarbonate, 10 mg/mL insulin, 500 µg/L hydrocortisone, 20 µg/L

Epidermal Growth Factor, 100 µg/L cholera toxin, 5% fetal bovine serum, and 50 µg/mL gentamicin. All cells are cultured at 37°C in a 5% CO2/100% humidity environment.

1.5.4 Growth Inhibition Assays

Cell growth inhibition assays will be performed by a sulforhodamine B staining method described previously.57-59 Cell loading densities yielding exponential growth over the 48- hour exposure period with final optical density (OD) values less than 2.0 will be determined in preliminary tests. Three media conditions will be employed during testing.

For intermediate E2 level tests (0.6 nM E2, 0.7 nM testosterone), all cell lines will be loaded in maintenance media and test agents will be added in 5% FBS/5% NS RPMI maintenance media, using the following cell loading densities: DU145 2,300 cells per well (cpw), PC3 1,300 cpw, LNCaP 3,600 cpw, MCF12A 1,300 cpw, MCF7 2,000 cpw, and NCI-ADR-RES 3,500 cpw. For tests with MCF7, NCI-ADR-RES, and MCF12A cell

10 lines in high E2 conditions, cell loading will be carried out as described for the intermediate E2 conditions, but agents will be loaded in 5% FBS/5% NS media with added E2 to give a final media concentration during the exposure period of 100 nM E2.

For low E2 tests (0.003 nM E2, 0.01 nM testosterone) with MCF7, NCI-ADR-RES, and

MCF12A cell lines, MCF7 and NCI-ADR-RES cells will be precultured for at least 4 days in RPMI with 5% FBS (same as RPMI maintenance media but without added NS), after which these two cell lines will be loaded in the 5% FBS media while MCF12A cells will be loaded in their normal maintenance media at cell densities of MCF12A 1,600 cpw, MCF7 3,500 cpw, and NCI-ADR-RES 6,000 cpw, and test agents will be loaded in the 5% FBS RPMI media for all three cell lines. For each E2 condition to be tested, the percent of control cell growth will be determined for each cell line at each agent concentration by comparing OD readings to those of matched vehicle-only control cells

(0.25% DMSO). Agent concentrations causing 50% growth inhibition (GI50) versus vehicle-only control cells will be estimated by a Hill-equation fit of the data. A log- normal distribution provided the best approximation for the GI50 measurements, so statistical analyses will be performed using log10(GI50) values. All results will be listed as mean ±95% confidence interval (CI). Statistically significant differences will be determined by analysis of variance (ANOVA) using a Bonferroni post-hoc analysis for pair-wise comparisons, with p <0.05 used as the criteria for statistical significance.

1.6 Peptidylglycine-α-Amidating Monooxygenase (PAM) Enzyme and CD3-246

As indicated in section 1.1, male hormones typically promote tumor growth during early stages of prostate cancer such that their ablation is generally considered an effective

11 initial therapy. However, with disease progression the tumors can begin to develop in the absence of these hormones. It is believed that advanced cancer acquires this ability through overpopulation of the tumor with cells that can generate their own hormone signaling pathways and local growth factors. Among these types of factors, there is evidence that the advanced cancer cells over-express an enzyme called “PAM.”

Therefore, PAM inhibition could be an alternative new way to treat advanced stages of prostate cancer.60 A brief description of PAM is provided in the next paragraph.

For many mammalian and insect hormones that are secreted as C-terminal glycine- extended peptides, post-translational α-amidation is a critical step for them to become biologically active.61, 62 The enzyme Peptidylglycine-α-amidating Monooxygenase

(PAM) catalyzes such amidation reactions through oxidative cleavage of the glycine N-

61, 63 Cα bond. This enzyme consists of two domains, namely Peptidylglycine-α- hydroxylating monooxygenase (PHM) and α-Hydroxypeptidylglycine-α-amidating lyase

61, 64 (PAL). The first domain, PHM, requires a copper, oxygen (O2) and ascorbate to mediate a stereoselective hydroxylation where only the pro-(S)-hydrogen atom is selectively removed63 from the α-carbon of the terminal glycine residue. This is the rate limiting step65 because the product, a α-hydroxylglycine-extended peptide, is then rapidly

64 cleaved at the N-Cα bond by PAL. The latter reaction involves a zinc-dependent dealkylation of the α-hydroxy amide intermediate to generate the α-amidated peptide and glyoxalate as side-product.61 This two-step bioactivation pathway is shown in Scheme

1.2.

12 2 Ascorbate (AA)n NH2 + O2 -amidated peptide H HR OH R H S + (AA) (AA)n n PHM PAL OOC N OOC N OCH COO H H Glyoxalate Peptidylglycine 2 Semidehydroascorbate Peptidyl -hydroglycine + H2O Scheme 1.2. PAM enzymatic action mechanism (adopted from S. T. Prigge et al.).63

In previous research at the CD3 funded by an Idea Development Award from the

DOD Prostate Cancer Research Program, a directed library of approximately fifty N- acylated novel tri-peptides were evaluated for PAM inhibition. Compared to a previous

NCI lung cancer PAM research program which included substrate-like inhibitors such as phenylbutenoic acid (PBA, Fig. 1.4),66 as well as the nonspecific copper chelating inhibitors disulfiram and diethyldithiocarbamate (DDC), the CD3‟s approach was novel because it utilized much more sophisticated, hybrid molecules that combined the best of substrate binding and PAM-specific copper chelating properties. Based upon structure- activity relationship (SAR) results from this initial library, CD3-246 became the lead compound.60 The chemical structure of CD3-246 is depicted in Fig. 1.4.

OH

O O O NH OH S NH NH OH O O CD3-246 S PBA Fig. 1.4. Lead compound CD3-246 and an earlier analog from the NCI‟s former lung cancer research program.

13 This initial library was prepared by manual, solid-phase synthesis which served well to produce up to 100 mg quantities needed to examine SAR in conjunction with the CD3‟s front-line biological screens. Alternatively, a more scalable method of synthesis was required to produce larger, multi-gram quantities so that the CD3 could further examine

CD3-246 in more advanced, secondary testing protocols. Toward that end, a solution phase synthetic route was sought that could take advantage of readily available and inexpensive starting materials while requiring only a few steps.

1.6.1 Early Synthetic Studies for CD3-246 Synthesis

A small scale solution phase synthesis of the lead compound CD3-246 was developed using a „Boc/Bzl‟ strategy67 as depicted in Schemes 1.3 and 1.4. Because of the presence of sulfur, the benzyl group from the (D)-tyrosine side-chain could not be removed even when excess catalyst (200% by weight), high hydrogen pressure (100 psi) and high temperature (100 ºC) were simultaneously deployed on either the di-benzyl-protected intermediate CD3-404 or the hydrolyzed ester, mono-benzyl-protected intermediate 1b.

Alternatively, this benzyl group could be removed by using a method first described and later scaled-up by H. Yoshino et al.68, 69 which deploys pentamethylbenzene (PMB) in trifluoroacetic acid (TFA). Scheme 1.3 illustrates a practical and inexpensive production of key intermediate CD3-404. Scheme 1.4 depicts two paths, A & B, used to complete the synthesis. Path „A‟, wherein step „f‟ is followed by step „g‟, proved to provide a more readily isolated product, as well as being more efficient overall. The overall yield for the entire route was about 20%.

14 OBn

O O O O Cl H N OBn BocHN BocHN OBn 3 * NH NH OBn OH a NH b c BocHN * NH O O O O

S S S S

d

OBn OBn

O O O e * NH OBn NH OBn NH NH ClH3N * NH O O O O S CD3-404 S S

Scheme 1.3. Synthesis of CD3-246 using a „Boc/Bzl‟ strategy: a) CDI, CH2Cl2, 1 h then H-Gly-OBzl·p-tosylate, NEt3, CH2Cl2, rt, 18 h; b) HCl(g), EtOAc, 1.5 h; c) Boc-D- Tyr(Bzl)-OH, CDI, CH2Cl2, 0.5 h then H-Met-Gly-OBzl·HCl, NEt3, CH2Cl2, rt, 24 h; d) HCl(g), EtOAc, 1 h; e) Thienylbutyric acid, CDI, CH2Cl2, rt, 4 h, then H-D-Tyr(Bzl)- Met-Gly-OBzl·HCl, CH2Cl2, NEt3, rt, 18 h.

OH

O O NH OBn NH * NH f g OH O O OBn 65% S

A 75%

1a S O O

h t

a NH OH O O P NH * NH NH OBn O O NH * NH S O O CD3-246 S

S OBn

B

CD3-404 h

S t f a

P O O 24% g NH OH NH * NH 78% O O S 1b S Scheme 1.4. Synthesis of CD3-246 using a „Boc/Bzl‟ strategy (continued): f) PMB, TFA, o rt, 2 h; g) K2CO3, THF, rt, 24 h, then 60 C, 1 h.

In parallel to the aforementioned efforts, investigations of alternative protecting groups were also undertaken, with most attention being given to an „Fmoc/tBu‟ strategy.67 As shown in Scheme 1.5, for this approach the N-terminus of the amino acid building blocks are protected by a 9-fluorenylmethoxycarboxy (Fmoc) group which is sensitive to basic reaction conditions but is stable to acidic conditions. The carboxyl group of the C-terminus Gly residue was protected as its t-butyl ester. By taking this

15 approach it was envisioned that protection of the tripeptide‟s Tyr side-chain could be accomplished with the acid sensitive t-butyl group so as to completely eliminate the need for using catalytic hydrogenolysis. Thus, this leads to a process wherein removal of all protecting groups can be accomplished as a single step at the end of the synthesis, namely the t-butyl group from both the Tyr side-chain and the C-terminus ester.

t-BuO O O O H H N N Ot-Bu Ox • H2N Ot-Bu Fmoc OH a Fmoc N b N c O H H H O O N Ot-Bu FmocH2N N H S S S O O

S

d OH Ot-Bu t-BuO

O O O H O O H N OH H S N N f N Ot-Bu e N Ot-Bu (CH2)3 S N N Ox • H2N N H H (CH2)3 H H H O O O O O O S CD3-246 S S

Scheme 1.5. Synthesis of CD3-246 using an Fmoc chemistry: a) AcO·NH3-Gly-COOt- Bu, CDI, DIPEA, CH2Cl2, rt, 3 h; b) Octanethiol, DBU, THF, rt, then oxalic acid/EtOAc, 6 h; c) CDI, DIPEA, CH2Cl2, rt, 6 h; d) Octanethiol, DBU, THF, rt, then oxalic acid/EtOAc, 6 h; e) 4-(2-Thienyl)butyric acid, CDI, DIPEA, CH2Cl2, rt, 6 h; (f) 1:1 TFA:CH2Cl2, rt, 2 h.

While this approach was shown to work on small scale and seemingly might have provided slightly higher yields, some of the starting materials were more expensive and it required using a large excess of 1-octanethiol during removal of the Fmoc groups. In addition, separation of the liberated free amines as their oxalate salts was not straightforward. In the end, the „Boc/Bzl‟ route was deemed to be the more practical once the methodology for the final deprotection step with PMB had become adopted.

Therefore, this route through path „A‟ was selected for scale-up synthesis of CD3-246.

The PMB reaction is itself of interest from a chemical mechanism point of view and we

16 have additionally undertaken separate studies in that regard. A brief background for the latter is provided in the next Section.

1.6.2 Mechanistic Investigations for O-Debenzylation of Phenolic Ethers Using PMB and Trifluoroacetic Acid

Careful manipulation of protecting groups is a key aspect for the synthesis of multifunctional molecules in modern chemistry. The benzyl group has found frequent use for the protection of alcohols and .70, 71 Equally common is the use of acidic conditions for final removal of acid labile protecting groups on peptide chains prepared via a Boc/Bzl strategy during solid phase peptide synthesis.67, 72 A combination of thioanisole and trifluoroacetic acid (TFA) has been used successfully to overcome the common problem of formation of 3-benzylated tyrosine73 during deprotection of O- benzyltyrosine (Tyr(Bzl)).68, 74 Alternatively, palladium catalyzed hydrogenolysis is a commonly used procedure for removal of benzyl protecting groups.75 71 However, the presence of a divalent sulfur often renders the latter method ineffective.76 Use of PMB along with TFA has been found to be very effective for deprotection of a Tyr(Bzl) residue.68 The Yoshino group found that PMB is a better reagent than anisole for such manipulations.68 Later, they demonstrated the utility of this reagent for large-scale peptide synthesis while using PMB-TFA for removal of a benzyl-type side-chain protecting group in their synthesis of an octapeptide.69 Recently, this interesting reagent has been used in combination with a Lewis acid for the cleavage of aryl benzyl ethers.70

However, to date no one has demonstrated the utility of PMB-TFA in the presence of sulfur. Likewise, the selectivity for phenolic O-debenzylation over hydrogenolysis of

17 benzyl esters does not appear to have received significant attention. Finally, a detailed description for its reaction mechanism does not appear to have been published.

1.7 Advanced Biological Testing of CD3-246

In vivo studies will be performed in castrated male Ncr nude mice (purchased from

Taconic, Inc., Hudson, NY) by the CD3‟s Bioanalytical Testing Laboratory. A total of 60 animals are planned for this study. The minimum effective dosing rate, 32 mg/kg delivery three times per week or 100 mg/kg/wk in total, will be employed for each of the delivery routes in this study. Delivery routes will include 3x32 mg/kg intraperitoneal (IP) injections per week to duplicate previous conditions as a positive-treatment control, 5 x

20 mg/kg intermittent intravenous (IV) delivery per week, 100 mg/kg/wk continuous IV delivery via an implantable pump (Alzet, Inc., Cupertino, CA; model 2006 implantable pump) and 100 mg/kg/wk continuous subcutaneous (SC) delivery to the flank via the same pump model. The agent formulations used in these tests will be the most successful options for each route that are to be determined in preceding experiments. Matched vehicle-only control groups will be included for each type of delivery. Tests for toxicological effects will include monitoring of body weight and behavioral observations77 three times per week, as well as hematology and blood chemistry panels determined on five vehicle-only control animals and five treated animals at the beginning and end of the study. Based on previous experience in the CD3‟s labs, the use of five animals for control and treated toxicology measurements can be expected to yield a statistical power of 0.812 to detect differences of at least 3% between all treated and untreated groups. In addition to the toxicology results, terminal (one per animal)

18 pharmacokinetic (PK) plasma samples for LC-MS/MS analysis of CD3-246 and major metabolites will also be collected from treated animals at the end of the study. For continuous IV and SC agent delivery, plasma samples will be collected from each of the five treated animals to estimate steady plasma concentrations. For IP and intermittent IV administration, ten additional treated animals (making a total of 15) will be included for each route, and terminal plasma samples will be collected at five different time points after the final dose with three samples collected for each time point, including immediately after the final dose and at 30, 60, 90, and 360 min after the final dose. These samples will supply limited but useful PK information on the plasma concentrations and bioavailability of each route (with direct comparison to IV delivery) and elimination half- life of the test agent. Full autopsies will be performed on all animals prematurely removed from the study due to toxic effects and at the end of the study on any animals exhibiting toxicological problems, while post-mortem inspection of the agent/vehicle delivery site will be performed on all animals to check for signs of localized irritation.

19

Chapter 2

Results and Discussion

2.1 Scale-up of Glyceollin I and Synthesis of Related Analogs

My pursuit of improved SERM compounds began with a scale-up synthesis of glyceollin

I (GLY I) and development of a synthetic route to GLY II, along with several of their intermediates and related analogs that can be derived directly from this composite of flavonoid-related chemistry. In addition, analog design took advantage of computational studies of the estrogen receptors (ERs) followed by molecular modeling studies of selected ligand docking paradigms.

2.1.1 Scale-up Studies

Efforts were made to improve reaction yields and to reduce the number of steps for synthesis of GLY I using the original 14-step synthesis as shown in Scheme 2.1.42 Scale- up was carried out as a team effort. Although I performed every step in this synthesis at several levels of increasing scale, only those steps that I personally optimized will be discussed in detail. The latter include steps c, d, and f to j.

The synthesis began with orthogonal protection of the two phenolic hydroxyl groups on commercially available acetophenone (2a) to synthesize intermediate 2 by sequential protection. The p-hydroxyl of 2a was first protected by treatment with methoxymethyl chloride (MOM-Cl). This highly toxic and carcinogenic reagent was generated in situ

20 from „methylal‟ using Zn+2 as catalyst. The zinc ion catalyzes formation of a reactive

„oxonium‟ ion through activation of acetyl chloride. The „oxonium‟ ion, in turn, is captured by the chloride counter ion to form MOM-Cl (this mechanism is shown in

Scheme 2.2).78 The o-hydroxyl was protected next by treatment with benzyl bromide.

The diprotected ketone 2 was then converted to the α-iodo ketone 3. Reaction conditions for selective iodination of 2 to 3 were extensively investigated. An experimental survey of alternative reagents79 to replace the expensive catalyst SelectfluorTM were unsuccessful. Reaction conditions were then optimized on a gram scale of the starting material (ca. 3.5 mmol). It was found that use of CH2Cl2 and MeOH (1:5) in 0.2 M concentration was important for obtaining high yields of regioselectively iodinated product. Use of freshly prepared saturated aqueous solutions of sodium thiosulfate pentahydrate (Na2S2O3·5H2O) to neutralize traces of unused iodine was also found to be important. The scale of the reaction was increased in two- to five-fold increments starting from 1 g (ca. 3.5 mmol) to 113 g (ca. 0.4 mol). Beyond this level of reaction, apparatus size (larger than 2 L round bottom flask) and proper stirring of the reaction mixture became limiting factors.

21 HO OH BnO OH a H O OBn BnO O 1c 1 O OH BnO O d e, f OR HO OH RO OBn RO OBn BnO OR' b c OH 4 5 I 2a O 2 O 3 O R = MOM g R' = TBDMS

O O HO O BnO O OH i OH h OH OBn

O O OH CD3-656 6 OR' 7 OR' OR' i

O O O O OH j OH

O O GLY I OH CD3-699 OR'

Scheme 2.1. Scale-up Synthesis of GLY I: a) (i) BnBr, KHCO3, CH3CN, reflux, 15 h; (ii) NaBH4, MeOH, 0 °C then rt, 6 h, 61%, over two steps; b) (i) DIPEA, CH3COCl, CH3OCH2OCH3, cat. Zn(OAc)2, EtOAc, 0 °C then rt, 18 h; (ii) BnBr, K2CO3, Me2CO, reflux, 18 h, 74% over two steps; c) I2, cat. Selectfluor, CH2Cl2:MeOH (1:5), rt, 20 h, 79%; d) K2CO3, Me2CO, reflux, 20 h, 72%; e) (i)PPh3 · HBr, CH3CN, rt, 1 h; (ii) t- BuOK, MeOH, reflux, 24 h, 70% over two steps; f) PPh3 · HBr, CH2Cl2, rt, 2 h, then Et3N, TBDMS-Cl, rt, 12 h, 70%; g) OsO4, (DHQD)2PHAL, CH2Cl2, -20 °C, 20 h, 86%; h) Cat. Pd/C, H2, EtOH, rt, 2 h, 93%; i) Diethoxy-3-methylbut-2-enal, 3-picoline, p- xylene, 110 °C, 18 h, 60%; j) Et3N · 3HF, pyridine, CH2Cl2, rt, 5 h, 90%.

Zn++ O Zn++ O O O H C + Cl + CH3COOCH3 H C CH + 3 3 3 Cl

O Cl H3C

Scheme 2.2. Proposed mechanism for in situ generation of MOM-Cl.78

After observing periodic losses in yield attributed to variable quality of the

SelectfluorTM from commercial sources, a standardized small-scale reaction (5 g, 17.5 mmol) was always performed as a process quality control (QC) measure before using reagent from a new bottle at large levels, even when a new bottle had an identical batch number. The progress of this reaction was followed by both TLC and 1H NMR. The

22 appearance of a peak at ca. δ 4.4 for the protons on the carbon bearing the iodo accompanied by disappearance of the methylketone peak at ca. δ 2.5 was diagnostic. The postulated reaction mechanism for SelectfluorTM meditated selective α-iodination is shown in Scheme 2.3. Beyond knowing that this transformation proceeds via a reactive iodinium species,80 the mechanism for this interesting reaction appears to remain unreported. Regioselective iodination at the α-carbon in the presence of a benzene ring is very delicate, especially when the latter‟s electron density is increased by substitution with electron-donating benzyloxy and methoxy groups. Perhaps SelectfluorTM helps to overcome this difficulty by activating elemental iodine toward an electrophilic attack

(Scheme 2.3). In addition, its positive charges may alleviate the overall electron density on the benzene ring if the two species were to hover in close proximity somewhat similar to a „stacking‟ arrangement within the transition state.

Cl N RO OR' RO OR' I I 2BF4 N F O OH

RO OR' I F

OH RO OR' NHF + N I O Cl

Scheme 2.3. Proposed mechanism for Selectfluor-assisted selective α-iodination.

Salicyl alcohol 1 was obtained uneventfully in two steps from commercially available aldehyde 1c after regio-selective protection of the p-hydroxyl group with a benzyl moiety followed by reduction of the aldehyde functionality to a primary alcohol. The ether

23 linkage between α-iodo ketone 3 and salicyl alcohol 1 was then established under basic conditions after refluxing the reaction mixture for 20-24 h in Me2CO. Because various solutions of 3 were observed to undergo decomposition at elevated temperature, stability studies were undertaken. These results are summarized in Table 2.1. Different solvents were examined and this eventually led to the selection of Me2CO as the preferred solvent for both stability and reaction time. Initial optimization of the reaction conditions was carried out at a 0.8 g (ca. 2 mmol) scale. This was increased to 115 g (ca. 140 mmol) after all variables had been examined. Reaction progress was followed by TLC and 1H NMR.

The reaction was stopped after disappearance of a diagnostic peak in 3 that appeared at ca. δ 4.4 for the protons on the carbon bearing the iodo. Purification of both 3 and 4 was achieved by crystallization.

Table 2.1. Stability data determined by 1H NMR for 3 in different solvents at different temperatures; t is time after reaction set up.

Solvent Approx. % degradation t = 0 h t = 6 h t = 17 h t = 24 h a CH2Cl2 reflux 0 0 0 - a CH3CN 70 °C 0 60 70 -

Me2CO rt 0 0 2.0 6 reflux 0 0 60 95 DMF 70 °C 0 -b 95 -a a No reaction was performed upto this time point; b No observation was made until t is 17 h time point.

Cyclization of 4 was carried out using an intramolecular Wittig olefination reaction.

Substantial amounts of degradation of 4 were observed during synthesis of the Wittig salt, an intermediate needed for olefination. After careful analysis of data collected for characterization of the side-product, it was concluded that the methoxymethyl (MOM)

24 protecting group was gradually being cleaved due to the presence of a small excess of hydrobromic acid in the reagent. Careful monitoring of reaction progress by TLC revealed that the reaction could be completed after just 1 h of stirring at room temperature. Furthermore, it was determined that portion-wise addition of the reagent was preferred to adding the entire reagent in one lot. This led to complete conversion of 4 to the desired triphenylphosphine bromide salt with negligible formation of the side- product. The salt was directly used in the next step without further purification to obtain the intramolecular Wittig product.

The next conversion involved a two-step de-protection/re-protection maneuver in order to obtain intermediate 5 where R‟ is TBDMS rather than MOM (Scheme 2.1).

Feasibility studies were performed to avoid this two-step maneuver using model chemistry to see if MOM could be carried through the entire synthesis and eventually removed at the final step. Thus, an attempt was made to find a selectivity window so that

MOM group could be removed while keeping in mind that GLY I is sensitive toward mildly acidic and basic conditions. Magnesium bromide, known to be very selective toward de-protection of MOM groups,81 was tried on the racemic penultimate intermediate. This gave a very complex mixture of side-products. In another approach, model chemistry was performed to check the stability of the t-butyldimethylsilyl

(TBDMS) protecting group during intramolecular Wittig olefination. This would allow the MOM group to be replaced by TBDMS at the beginning of the synthesis. However, under the Wittig conditions, the TBDMS group was found to cleave. Finally, the intermediate obtained after removal of the MOM group was found to be very susceptible to oxidation and polymerization, likely exacerbated by the extended conjugation that

25 occurs when an additional double-bond is present in the benzopyran ring. The ready formation of this energetically favored species and the outcomes from our feasibility studies suggested that a one-pot, two-step maneuver for exchange of protecting groups would still be the most practical. In the end, a one-pot strategy was achieved to provide 5 in a manner that not only improved the overall reaction yield, but also reduced total reaction time and purification procedures.

Asymmetric synthesis of 6 was achieved using Sharpless dihydroxylation where bis- dihydroquinidine phthalazine [(DHQD)2PHAL] was used as a chiral ligand to introduce chiral selectivity. Examination of hydroquinidine 4-chlorobenzoate (a less expensive chiral ligand) even after using high excess, did not provide enantiomeric excess at 97% or above which we had set as a process specification for quality control (QC) purposes. A shortage in the supply of good quality (DHQD)2PHAL from commercial sources then made in-house synthesis of the chiral ligand imperative. The ligand was synthesized following a two-step literature protocol in large quantities as shown in Scheme 2.4.82 QC measures were adopted to establish the utility of the synthesized ligand. Melting point and 1H NMR data were compared with literature data82 and then the chemical reaction efficiency of each batch of synthesized catalyst was tested in a 50 mg standard reaction using QC-approved OsO4. Assessment of the product by chiral HPLC had to meet a specification of >97% ee. In the end, not only were the supply issues overcome by preparing this catalyst ourselves, a cost comparison also indicated that after our synthesis of ca. 200 g, we were able to effect a saving of nearly $ 4000.00.

26 Et Et HN NH N N N N N N O O a Cl Cl b O O MeO OMe

N N Phthalhydrazide 7b (DHQD)2PHAL

Scheme 2.4. Synthesis of chiral ligand: a) PCl5, Cat. DMF, 145 °C, 8 h, 81%; b) K2CO3, Toluene, KOH, 135 °C, 14 h, 59%.

With QC approved (mp, TLC, 1H NMR, CHN and >97% ee) 6 in hand, the stage was set for the next four steps in quick succession. To our delight, intermediate 6 could be coaxed into cyclizing to the benzofuran ring system during the course of the hydrolytic cleavage of the benzyl protecting groups.83, 84 This provided a high-quality and previously difficult to make intermediate, readily available in excellent yield (ca. 93%). This also eliminated the use of column chromatography up to this entire point in the overall synthetic pathway. Furthermore, these modifications not only improved scalability of the route, but also improved overall yield as well. As before, isoprenylation and cyclization of the 2,2-dimethylbenzopyran ring gave a mixture of two regio-isomers. The desired isomer was purified using gravity column chromatography. In the final step, removal of the silyl group yielded GLY I (ca. 2 g, ca. 6 mmol) in good yield (ca. 9% over 11 linear steps). Buffering of the reaction mixture with excess pyridine significantly reduced formation of the otherwise prominent dehydro-GLY I side-product (Fig. 2.1).

Table 2.2 provides a side-by-side comparison of the yields and reaction scale-up levels for each of the steps in Scheme 2.1 before and after process optimization. The overall yields were ca. 3% versus ca. 9%. In addition, after optimization only one column purification became required such that this procedure should be highly amenable to even further scale-up.

27 Table 2.2. Yield and scale comparison for synthesis of GLY I.

Before optimization After optimization Reac. Av. % Largest Scalea Av. % Largest Scale step Yielda Yield a 61b 1 mmol (0.14 g) 61b 0.72 mol (100 g) b 56 1 mmol (0.15 g) 74 1.3 mol (200 g) c 70 1 mmol (0.29 g) 79 0.43 mol (124 g) d 72 1 mmol (0.41 g) 72 0.28 mol (116 g) e 78 1 mmol (0.51 g) 70 58 mmol (30 g) f 78 1 mmol (0.48 g) 70 41 mmol (20 g) g 70 1 mmol (0.54 g) 86 18 mmol (10 g) h 64 0.1 mmol (39 mg) 93 8.5 mmol (5 g) i 50 0.1 mmol (40 mg) 60 6.6 mmol (3 g) j 69 0.1 mmol (43 mg) 90 2.2 mmol (1 g) Overall 2.7 ca. 25 mg after 13 stepsc 9.1 ca. 5 g after 11 stepsc a Data are taken from Rahul Khupse‟s PhD dissertation and lit.42; b Across two steps; c Counting longest linear flow of steps.

2.1.2 GLY I Analogs

GLY I related analogs obtained from intermediates produced during scale-up synthesis are shown in Fig. 2.1. Analogs with „R‟ suffix (CD3-653-R, CD3-698-R, CD3-700-R and CD3-714-R) were synthesized as racemic molecules. However, only the (R)- enantiomers were used for docking purposes because of their structural similarity to the

6a-chiral center in (S)-glycinol (GLO) and (S)-glyceollin I (GLY I).

28 Direct Analogs O OH O OBn HO HO O BnO O

OMOM OH OH OH OH CD3-649 CD3-650 THC

HO O BnO O HO O

HO OTBDMS BnO OH HO OH CD3-653-R CD3-654 CD3-714-R HO O HO O BnO O OH OH OH OH OBn O OH HO OTBDMS OTBDMS CD3-666 CD3-667 OH CD3-656

Methoxy Analogs O OBn HO O HO O BnO O

OMe OH CD3-658 CD3-700-R HO OCH3 CD3-174 HO OMe

Natural Compounds and Their Derivatives O O O OH O OH 6a O O OH HO HO O HO OH O OH O CD3-698-R GLO Dehydro-GLO Dehydro-GLY II Dihydroglabridin

O OH O OH O 6a 6a O 17 OH 3 OH O O O HO H O OH O OH E2 GLY I Dehydro-GLY I GLY II

Fig. 2.1. GLY I analogs and structures of E2, GLO and GLY I used for external validation of docking models.

2.1.2.1 Spatial Analysis of ERα and ERβ Active Sites and Their Natural Ligand Estradiol

X-ray data with THC (tetrahydrochrysene-2,8-diol, Fig. 2.1) bound ERs (PDB ID 1L2I and 1L2J for ERα and ERβ, respectively)85 were used because of lack of available x-ray crystal structure data for both ERα and ERβ bound with the natural ligand estradiol (E2) from humans. As a model, THC similarly displays two key alcohol groups somewhat analogous to those present within the pharmacophore of the natural ligand E2 (Fig. 2.2).

29 While the x-ray structure for rat ERβ bound with E2 (PDB ID 2J7X, unpublished results) is available, the overall sequence of the active site in humans is different as evidenced by the fact that the active site „triad‟ Glu 305, Arg 346 and His 475 in human ERβ is represented by Glu 260, Arg 346 and His 430 (from PDB ID 2J7X and 1QKN86) in rat protein.

Fig. 2.2. A) THC bound ERα active site; B) THC bound ERβ active site; C) E2 bound ERα (1ERE x-ray structure87).

As shown in Fig. 2.2, the ERα active site (Fig. 2.2A, 16.15±0.03 Å from His 524 NE2 to Arg 394 N20; 15.08±0.03 Å from His 524 NE2 to Glu 353 OE1) is slightly shorter than the ERβ active site (Fig. 2.2B, 16.49±0.07 Å from His 475 NE2 to Arg 346 N20;

15.74±0.07 Å from His 475 NE2 to Glu 305 OE1) when THC is bound. However, the ligand‟s conformation and length remains the same in both receptor types. This length is only slightly longer than that observed for E2: 11.9 Å for THC compared to estradiol‟s 3-

OH - 17β-OH of 10.8 Å.87 Moreover, as shown in Fig. 2.3, E2 forms three hydrogen bonds with active site residues within ERs by using its terminal hydroxyl groups. The phenolic hydroxyl interacts with an Arg and a Glu residue whereas the 17-β hydroxyl

30 forms a hydrogen bond with a His residue.87 These hydrogen bond interactions, combined with hydrophobic interactions between the lipophilic core of E2 and complementary receptor residues, jointly contribute to the high binding affinity that E2 displays for the

ER.

Fig. 2.3. H-bond interactions between estradiol and active site residue of ERα (Adapted by permission from Macmillan Publishers Ltd: Nature, A. M. Brzozowski et al., Nature, 1997, 389, 753-8, copyright 1997).87

It is important to observe that there is a significant overlap among Glu and Arg residues of the ERα and ERβ active sites (Fig 2.4 A). Alternatively, the imidazole rings of His residues in the two active sites are oriented differently. This altered orientation of the imidazole ring of the His residue in ER active sites could a play very significant role in achieving selectivity between the two ERs. Upon close examination of Fig. 2.2 and

Fig. 2.4, it can be imagined that for ligands aligned so as to bind with the active sites‟

Arg and Glu residues, the imidazole ring in ERβ will be more accessible to an analog‟s π system (for potential π-π stacking interactions) than the one in ERα.

31 O B OH O

O OH GLY I

O O O O OH OH

O O GLY II GLY III OH OH

Fig. 2.4. A) THC bound ERα and ERβ active sites overlapping; B) Glyceollin I (GLY I), Glyceollin II (GLY II) and Glyceollin III (GLY II)

Finally it is important to note that, mechanistically, GLY I has been found to have pronounced anti-estrogenic properties in these models.42, 56 GLY I has only a single phenolic hydroxyl located at one terminus. Taken together, this suggests that there is a different mode of interaction with the active site residues that can prompt GLY I‟s unique antagonistic properties. Our molecular dynamic studies suggest that in some binding modes, GLY I interacts with the active site His residue‟s imadazole-ring through a π-π stacking event that involves its 2,2-dimethylbenzopyran ring. Interestingly, a review of x- ray data from the RCSB Protein Data Bank for several other structures bound to either

ERα or ERβ did not show any clear evidence for π-π stacking types of interactions between the ligands and active site residues per se. But then, none of these examined structures also contain GLY I‟s unique 2,2-dimethylbenzopyran ring.

2.1.2.2 Molecular Docking Studies of Analogs

Docking studies were carried out to examine our hypothesis that the benefit of π-π stacking interactions and differences in size and shape of the two active sites might provide for selectivity and potential development of new SERMs. Docking models were

32 generated for both ERα and ERβ using Sybyl‟s SurflexdockTM suite. The models were validated internally, as well as externally, using a training set to confirm their reliability before using them for binding affinity prediction of test set/targets. For the internal validation ligand, THC was docked using the standard dock models and the top scoring conformation of THC was aligned with the bound crystal structure of the ligand. As shown in Fig. 2.5, not only did the two conformations (dock model conformation shown in gray and crystal conformation shown in green) aligned one-over-the-other very tightly, but also all ten of the top scoring conformations generated by the dock model are oriented and overlap with the crystal structure in a very similar fashion (Fig. 2.5B). Likewise, the dock models for the ERβ selected top scoring conformations are also in proper alignment with the ligand crystal structure (Fig. 2.6). As shown in Table 2.3, almost all of the atoms for the Surflexdock-generated structure of ligand are within 2 Å distances from the atoms of the reference molecule (crystal structure of ligand). Root mean square deviation

(RMSD)88 values are 0.33 and 0.55 for ERα and ERβ, respectively.

Fig. 2.5. Internal validation for ERα: (A) Docking model generated conformation (in grey) of THC overlapped over x-ray conformation (in green); (B) All ten top scoring poses of THC, generated by docking model, oriented in a similar mode into receptor active site.

33

Fig. 2.6. Internal validation for ERβ: (A) Docking model generated conformation (in grey) of THC overlapped over x-ray conformation (in green); (B) All ten top scoring poses of THC, generated by docking model, oriented in a similar mode, into receptor active site.

Table 2.3. Internal validation deviations.

ER type Mean Std. RMS # Atoms with Dev Dev Dev >2 Å Std. Dev ERα 0.20 0.26 0.33 2 ERβ 0.32 0.46 0.55 1

As mentioned above, the internally validated docking models were cross-validated using a test data set of structurally similar compounds with known binding affinities for the

ERs (Fig. 2.1, Table 2.4). Results of the external validation are shown in Table 2.4. As summarized in Fig. 2.7, the strong predictive power of these models is demonstrated by excellent correlation found between the experimental and predicted binding affinities of the training set molecules (with a >0.9 cross-validation correlation co-efficient (r2) value for both receptors). Moreover, as shown in Fig. 2.8, all the test set data points have similar modes of binding with the active site residues.

34 Table 2.4. External validation of ERα and ERβ docking model using test set (E2, GLO, GLY I, for their chemical structure see Fig. 2.1).

S. No. Test ERα ERβ Dataset BAa pKdb pKdc BAa pKdb pKdc (nM) (nM) 1 E2 0.289 9.70 7.09 0.67 9.22 5.34 2 GLO90 13.8 7.86 5.25 9.1 8.04 5.32 3 GLY I41 3.2 x 103 5.49 1.19 6.4 x 103 5.19 3.42 a Experimental receptor binding affinity (BA); b Calculated from exp. BA; c Highest Surflexdock score.

Fig. 2.7. Correlation plot between experimental binding affinity (calculated pKd values on the x-axis) and docking model predicted binding affinity (highest surflexdock scores, predicted pKd values, on the y-axis).

Fig. 2.8. External validation highest scoring surflexdock poses of test set (A) with ERα, (B) with ERβ.

35 2.1.2.3 Analysis of Docking Results for Gly I Related Analogs

Docking studies were performed using the docking models validated in section 2.1.2.2.

As shown in Figs. 2.9 and 2.10, the orientation of the ERβ active site His 475 residue‟s imidazole ring causes the π-π stacking interaction to become enhanced. This allows most of the analogs having a flexible benzyl group to show better binding scores in the ERβ docking model when compared to the one for ERα (see Table 2.5 and Fig. 2.1).

Fig. 2.9. Highest scoring poses of ligands with active sites: (A) ERα; (B) ERβ.

36

Fig. 2.10. Binding interactions of CD3-654 (in grey and red; highest pKd score with ERβ) and CD3-714-R (in magenta, highest pKd score with ERα): (A) depicts interaction with ERα as mainly H-bonding while (B) depicts with ERβ as both H-bonding and π-π stacking (CD3-654).

Similarly, Fig. 2.11 shows that even the highly flexible molecules, such as CD3-650 and

658, show better scores in the ERβ model because the His 475‟s imidazole is positioned in a manner that it is highly amenable to stacking with a π electron cloud.

Fig. 2.11. Comparative views of the most flexible analogs CD3-649 (in green), CD3-650 (in orange) and CD3-658 (in grey and red) interactions with: (A) ERα active site; (B) ERβ active site.

37 As delineated in Table 2.5, this stacking effect appears to have played a significant role by contributing to more than a one-hundred fold difference in binding scores between the highest scoring analogs CD3-714-R and CD3-654 for ERα and ERβ, respectively. The reason that CD3-714-R (ERα pKd 7.41, Table 2.5, entry 1) has a lower score may be because it mainly interacts with active site residues through H-bonding interactions (as shown in Fig. 2.10). The higher dock score of CD3-654 (ERβ pKd 9.66, Table 2.5, entry

9) is perhaps due to the fact that it not only interacts through H-bonds involving Glu 305 and Arg 346, but its benzyl group also has strong π-π transfer interactions with the imidazole ring of the active site His 475 (Fig. 2.10, notice how the benzyl ring is pointing away from the imidazole ring in ERα whereas it is stacked very nicely in ERβ). Overall, analogs with an ability for π-π transfer show better affinity toward ERβ. For instance,

CD3-650 (Fig 2.1 and Table 2.5, entry 8) is a highly flexible molecule wherein the latter has improved its ability to strategically position the benzyl and phenolic groups so that its affinity is significantly improved (Fig. 2.11). Moreover, as shown in Fig. 2.12, the three analogs CD3-714-R, dehydro-GLO, and GLO have varying distances between their two key pharmacophores (O11 and O20). Interestingly, their docking scores show a high degree of correlation with these distances, especially in the ERα model (Table 2.5).

Fig. 2.12. Distance map of three analogs having mostly H-bonding capability.

38 Table 2.5. Surflexdock scores for GLY I analogs using ERα and ERβ.

S.No. Ligand ERα ERβ pKda Kd (M) b Kd (nM)c pKda Kd (M) Kd (nM) c b 1 CD3-714-R 7.41 4E-08 38.90451 6.26 5.5E-07 549.541 2 CD3-649 7.07 9E-08 85.1138 7.2 6.3E-08 63.0957 3 Dehydro-GLO 6.67 2E-07 213.7962 6.47 3.4E-07 338.844 4 CD3-174 4.79 2E-05 16218.1 5.05 8.9E-06 8912.51 5 GLO 4.6 3E-05 25118.86 4.99 1E-05 10232.9 6 CD3-700-R 4.49 3E-05 32359.37 5.73 1.9E-06 1862.09 7 CD3-658 1.84 0.0145 14454398 6.48 3.3E-07 331.131 8 CD3-650 -0.78 6.0256 6.03E+09 6.61 2.5E-07 245.471 9 CD3-654 -1.22 16.596 1.66E+10 9.66 2.2E-10 0.21878 10 GLY II -1.83 67.608 6.76E+10 3.93 0.00012 117490 11 Dehydro-GLY II -1.97 93.325 9.33E+10 2.15 0.00708 7079458 12 CD3-667 -3.17 1479.1 1.48E+09 8.56 2.8E-09 2.7523 13 Dehydro-GLY I -3.75 5623.4 5.62E+12 2.67 0.00214 2137962 14 CD3-698-R -4.51 32359 3.24E+13 0.67 0.2138 2.1E+08 15 CD3-653-R -6.23 2E+06 1.7E+15 1.33 0.04677 4.7E+07 16 CD3-666 -6.87 7E+06 7.41E+15 1.56 0.02754 2.8E+07 17 CD3-656 -10.2 2E+10 1.58E+19 1.63 0.02344 2.3E+07 a Surflexdock scores; b Calculated using pKd = -logKd; c Calculated.

2.1.2.4 Synthesis of Analogs

Target compounds CD3-649 and CD3-650 (Fig. 2.1) represent completely open or highly flexible scaffold versions of GLY I. Both targets were readily accessible from intermediate 4 as shown in Scheme 2.5.

O O HO O BnO O a b HO OMOM 4 BnO OH OH OH CD3-649 CD3-650

Scheme 2.5. Synthesis of completely open GLY I scaffold analogs: a) Cat. 10% Pd/C, H2, EtOAc 15 psi, rt 10 h, 90%; b) MgBr2 Et2O, EtSH, CH2Cl2, rt, 18 h, 53%.

39 Catalytic hydrogenolysis of both benzyl protecting groups converted 4 to CD3-649.

Careful selection of reaction conditions was found to be critical for selective removal of these benzyl groups without affecting the aromatic ketone. For removal of the MOM group, a solution of 4 and ethanethiol was treated with MgBr2·Et2O overnight to obtain

CD3-650 as a white precipitate (Scheme 2.5).

Target compounds CD3-653, CD3-654 and CD3-714 (Fig. 2.1) represent partially opened or semi-rigid scaffold versions of GLY I. These were readily accessible from intermediate 5 as shown in Scheme 2.6. Removal of the benzyl groups and t- butyldimethylsilyl group from 5 yielded CD3-653 and CD3-654, respectively (Scheme

2.6). Target compound CD3-714 was obtained by treating the benzyl deprotected intermediate from 5 with conditions similar to the synthesis of CD3-654. Significant degradation was observed when CD3-654 was subjected to catalytic hydrogenolysis, as well as when using the PMB/TFA protocol for removal of benzyl groups to obtain CD3-

714.

HO O HO O BnO O a b c 5

HO OH HO OTBDMS BnO OH CD3-714 CD3-653 CD3-654

Scheme 2.6. Synthesis of partially open GLY I scaffold analogs: a) cat. 10% Pd/C, H2, EtOAc, 35 psi, rt, 15 h, 74%; b) Et3N · 3HF, CH2Cl2, rt, 6 h, 63%; c) Et3N · 3HF, CH2Cl2, rt, 8 h, 73%.

As shown in Scheme 2.7 and 2.8 several additional analogs having various levels of rigidity relative to GLY I‟s central scaffold became available from intermediate 6.

Interestingly, CD3-639 (dehydro-GLY I) and 6b (dehydro-GLY II) are more rigid than

GLY I which has higher rigidity than CD3-698 (Fig. 2.1 and Scheme 2.8).

40 HO O BnO O HO O OH OH OH c a b 6 OH OH OH HO OH BnO OH HO OR 6a CD3-667 CD3-666

HO O HO O HO O OH OH d e R = TBDMS O O O (CD3-523) CD3-640 OH CD3-656 OR (-)-Glycinol OH

Scheme 2.7. Synthesis of additional GLY I analogs: a) Et3N·3HF, CH2Cl2, rt, 6 h, 74%; b) cat. Pd/C, H2, Me2CO, 30 psi, rt, 12 h, 72%; c) Cat. Pd/C, H2, Me2CO, 30 psi, rt, 6 h, ca. 40%; d) HF, CH2Cl2, rt, 4 h, 60%; e) Et3N·3HF, pyridine, CH2Cl2, MeOH, rt, 10 h, 65%.

O O O O O O OH a b

O O HO OH CD3-699 OTBDMS CD3-639 OH CD3-698

O O O O O O OH d OH c

O O O GLY II OH 7 OTBDMS Dehydo-GLYII (6b) OH

Scheme 2.8. Synthesis of additional GLY I analogs (continued): a) HF, CH2Cl2, rt, 4 h, 76%; b) Cat. Pd/C, H2, MeOH, 35 psi, rt, 12 h, 74%; c) HF, CH2Cl2, rt, 6-8 h, 55%; d) Et3N·3HF, pyridine, CH2Cl2, rt, 5-6 h, 74%.

The common intermediate 6 was converted to CD3-666 and CD3-667 by treatment with Pd/C under a H2 atmosphere, and with Et3N·3HF, respectively. Target 6a was achieved with sequential removal of the silyl group, TBDMS, and the benzyl using hydrofluoric acid and catalytic hydrogenation, respectively. Alternately, as described above (Section 2.1.1, Scheme 2.1), after careful optimization of reaction conditions, simultaneous debenzylation and cyclization of the furan ring occurred so as to provide

CD3-656 when 6 was treated with Pd/C under a H2 atmosphere. The first total synthesis

41 of natural glycinol (GLO) was achieved from CD3-656 upon treatment with Et3N·3HF at ca. pH 5.5 (Scheme 2.7).83 Alternately, CD3-656 and CD3-699 were exposed to hydrofluoric acid to obtain CD3-640 and CD3-639, respectively. Also, treating 7 with similar conditions provided 6b. Removal of the silyl group and elimination of a H2O molecule occurred in the same reaction pot, likely driven by enhanced conjugation between the two phenyl ring systems as previously discussed. Under catalytic hydrogenation conditions CD3-639 yielded CD3-698, an interesting analog which will shed additional light on the importance of π-π stacking type interactions for biological activity of GLY I (see section 2.1.2.1 to 2.1.2.3).

2.2 Synthesis of Glyceollin II (GLY II)

My continued pursuit of improved SERM compounds that can be derived directly from the GLY chemistry, turned next toward the total synthesis of the second most prevalent family member, namely GLY II (Fig. 2.1). The obtainment of synthetic intermediates and their readily accessible analogs was again regarded as being important for assembly of the CD3‟s overall „directed library‟ of SERM-like compounds.

2.2.1 Early Synthetic Efforts and Initial Retro-Analyses

Our initial retro-analysis for the synthesis of GLY II is shown in Scheme 2.9. Contrary to the GLY I synthesis (Section 2.1), it was our intent to build the 2,2-dimethyl-2H-1- benzopyran ring system first so as to achieve a specific synthesis of GLY II. It was envisioned that after construction, the olefin could be masked by various methods (“x”) during several intermediate steps and then revealed again at the end of the synthesis.

42 O O O O O O OH OH OH

X X O HO OH O OR RO OR RO GLY II E D X =LG

O OR HO OH O OH O OH O OR O O + X X X OR O O OR O O A B C

Scheme 2.9. Initial retro-synthetic analysis.

Toward that end we started with the synthesis of intermediate „A‟. As illustrated in

Scheme 2.10, when commercially available 2,4-dihyroxybenzaldehyde (1c) was treated with 3,3-dimethyl acrylic diethoxy diacetal, instead of getting the desired 2,2-dimethyl-7- hydroxy-2H-[1]-benzopyran-6-carboxaldehyde (A), an alternate product was formed, namely 2,2-dimethyl-5-hydroxy-2H-[1]-benzopyran-6-carboxaldehyde (7a). To our surprise, this reaction exclusively formed intermediate 7a in excellent yield rather than the expected mixture of the two possible regio-isomers, the latter being observed repeatedly during late-stage „northern ring‟ closure to form the penultimate intermediate in our GLY I syntheses. We think that this interesting selectivity comes from the presence of the additional free hydroxyl group located ortho to the site of the pyran ring closure, perhaps through some type of „neighboring group effect.‟ Alternatively, perhaps this hydroxyl is more electron dense in comparison to the other91 such that it might then influence the reaction more predominantly. Therefore, 2,2-dimethyl-3,4-dihydro-7- hydroxy-4-oxo-2H-[1]-chro-man-one-6-car-box-aldehyde or „4-chromanone‟ (Scheme

2.11) was instead attempted in a coupling reaction with a ketone such as intermediate „B.‟

It was envisioned that after coupling, the generated ketoaldehyde could undergo a

43 carbonyl umpolung92 cyclization via a 1,3-dithiane mediated93, 94or triazolium salt mediated benzoin cyclization.95

HO OH O OH OEt a O OH + H H + OEt H 3,3-Dimethylacrylic 1c O A O diethoxy acetal 7a O >95%

Scheme 2.10. Toward synthesis of 2,2-dimethyl-7-hydroxy-2H-[1]-benzopyran-6- carboxaldehyde (A): a) 3-Picoline , p-xylene, 120 oC, 20 h, 95%.

This type of reaction sequence was reported to work very well in a literature procedure96 that utilized 2,4-resorcinol. However, reaction yields were extremely low when this method was tried on our substrate, 2,4-dihydroxybenzaldehyde so as to potentially obtain „4-chromanone‟. One possibility for this difference could be that under acidic conditions, protonation of the aldehyde becomes feasible because it can form an

„o-quinone methide‟ species. If this is the case, we thought it might be better to first convert the aldehyde functionality into an olefin so that the chromanone moiety could be assembled in a subsequent step.

(i) (ii) O OH HO OH O OH HO OH a a H H

O O O O '4-Chromanone' Scheme 2.11. Toward synthesis of dimethylchromanone: a) Dimethylacrylic acid, CH3SO3H, P2O5, 60% (for 7-hydroxychromanone), 5-10% (for „4-chromanone‟).

44 As illustrated in Scheme 2.12, olefination was first attempted without having any protecting group on the phenolic hydroxyl groups so as to keep the total number of steps at a minimum.97 However, successful olefination occurred only when we protected both hydroxyls, such as with methoxyethoxymethyl (MEM). Attempts to continue to build the

4-chromanone scaffold on the olefin after de-protection of the MEM groups, however, turned-out to be a futile exercise (Scheme 2.12)

O OH HO OH a HO OH x H O O CH2 b

MEMO OMEM MEMO OMEM a HO OH O OH c d H x CH O 2 CH2 O CH2

Scheme 2.12. Toward synthesis of dimethylchromanone using alternate route: a) CH3PPh3I, n-BuLi, THF; then add dihydroxy aldehyde in THF slowly; b) MEM-Cl, DIPEA, DMF, 0 °C 30 min, rt, 3-4 h, 70%; c) CH3PPh3Br, THF, 0 °C to rt, nBuLi, 6-8 h, 60%; d) Dimethylacrylic acid, CH3SO3H, P2O5.

After these set-backs, we turned our attention toward synthesis of masked aldehyde groups such as an ester or a Weinreb amide (Scheme 2.13 and 2.16), so that the aldehyde could be revealed after reduction with mild reducing agents like diisobutylaluminum hydride (DIBAL-H).98 When we first tried to make 4-chromanone methyl ester, the „5- hydroxyester‟ was formed as the major component with only very small percentages of the desired ester (Scheme 2.13). Alternatively and to our pleasant surprise, when the two reactants (the acrylic acid and 2,4-dihyroxymethylbenzoate) were pulverized simultaneously in a „mortar & pestle‟ for 5-10 min before addition99 to the reaction mixture, the product profile shifted in favor of the desired material. „7-Hydroxyester‟ was

45 crystallized in ca. 52% yield. As shown in Scheme 2.14, the desired ester was also synthesized by a three-step procedure involving an initial Friedel-Crafts acylation. The acetylated intermediate was then subjected to Aldol reaction conditions where a base like

LDA assisted in generation of an enolate that can undergo a nucleophilic attack on

Me2CO. The Aldol product was then cyclized into the desired „4-chromanone‟ ester via a carbocation transitory species as depicted in Scheme 2.15.

HO OH O OH HO OH O a b OMe OMe OMe O OH O O O O OMe O (8a) (8) '5-Hydroxyester' '7-Hydroxyester'

Scheme 2.13. Synthesis of 4-chromanone ester: a) 3,3-Dimethylacrylic acid, P2O5, CH3SO3H, 70 °C, 3-6 h, 60%; b) 3,3-Dimethylacrylic acid (pulverized first with benzoate), P2O5, CH3SO3H, 70 °C, 3-6 h, 54%.

HO OH HO OH O OH a b OMe OMe OMe O O O O O 8

Scheme 2.14. Alternative synthesis of „4-chromanone‟ ester: a) Amberlyst 15 resin, o o o CH3COCl, 70 C, 5-6 h, 96 C, 4-5 h, 40%; b) (i) LDA, CH3COCH3, THF, -78 C, 5-6 h, 65%; (ii) HCl(g), MeOH, 0 oC, then reflux, 3-4 h, 70%.

HO OH HO OH O H-Base H OMe MeO Base O O O OLi

O OH HO OH HO OH

OMe OMe OMe OH O O O O O O

Scheme 2.15. Proposed mechanism for alternate synthesis of 4-chromanone ester.

46 The „7-hydroxy ester‟(8) (Scheme 2.13) was hydrolyzed using lithium hydroxide to liberate the free carboxylic acid which was then converted into the Weinreb amide as shown in Scheme 2.16.100 Attempts to synthesize „4-chromanone‟ acid using a protocol similar to the synthesis of „7-hydroxy ester‟ resulted in low yielding reactions (in the range of 10-20%). Although the amide was synthesized successfully, yields were in the range of only 20-30%. Furthermore, this procedure was judged to be rather tedious when critically assessed relative to the overall length and complexity of the total synthesis needed for the final target compound.

HO OH O OH O OH a b OMe OH NOMe

O O O O O 'Weinreb amide'

Scheme 2.16. Synthesis of 4-chromanone Weinreb amide: a) (i) 3,3-Dimethylacrylic acid, P2O5, CH3SO3H, 70 °C, 3 h, 54%; (ii) LiOH, H2O, THF, reflux, 2-4 h, 60%; b) Weinreb salt (CH3NH(OMe) · HCl), DMAP, DCC, DMF, rt, 12 h, 30%.

Noting that reduction of the ester intermediate using DIBAL-H invariably produced the primary alcohol rather than stopping at the aldehyde stage, led us to next consider a slight change in the overall synthetic strategy. Instead of building a ketoaldehyde substrate (C) (Scheme 2.9) so as to cyclize through a benzoin type strategy, we decided to construct two olefins and then join them through a ring closing metathesis (RCM) protocol using Grubb‟s catalyst (Scheme 2.17). Toward that end, we began a synthesis of intermediate B’ using commercially available 2,4-dihyroxyacetophenone. As before, the two phenolic hydroxyl groups were first protected with benzyl moieties and the resulting ketone was then selectively converted into the α-iodo compound (9). The iodinated

47 ketone was converted into an acetoxy intermediate (10) which led to synthesis of an allylic alcohol when it was subjected to Wittig olefination conditions. This alcohol could then be replaced with a good leaving group such as p-tosylate or triflate (Scheme 2.17) so as to make it amenable toward an SN2 displacement reaction.

O O O O O O OH OH

O O HO OH O RO OR RO OR GLY II E' D'

X = I/Br/LG RCM

OR HO OH O OH O OH OR O O + X Br Br OR O O OR O A' B' C'

Scheme 2.17. Ring closing metathesis based retro-analysis for GLY II synthesis.

O OH OBn O OBn O OBn OR X a I b OAc c OH

OH BnO BnO BnO OR 9 10 B'

Scheme 2.18. Synthesis of intermediate B: a) (i) BnBr, K2CO3, CH3CN, reflux, 24 h, o 90%; (ii) I2, Selectfluor, CH2Cl2:MeOH (1:5), rt, 18 h, 94%; b) AcOH, DBU, THT, 0 C to rt, 4-5 h; c) CH3PPh3Br, nBuLi, THF, 3-4 h, 50%.

In parallel, efforts were also made to synthesize vinyl-4-chromanone (derivable from intermediate A’, Scheme 2.17). To that end, 4-bromoresorcinol was converted to its 4- chromanone derivative A’ through sequential Friedel-Crafts and Michael addition reactions. Because of the commercial availability of the corresponding vinyltin reagent, a

Stille coupling reaction was attempted to displace the bromo with a vinyl group.

Numerous reaction conditions having different catalyst, additives, solvents and

48 temperature/duration differences were tried (Table 2.6). While such attempts typically worked fine on a simple model compound, displacement of the bromo in our substrate never became realized in any practical amounts (Scheme 2.19). There is some literature to suggest that the low reactivity of chloride/bromide derivatives toward Stille coupling is confounded by the presence of electron-donating substituents and by substitution at the ortho-position.101 While our substrate has an acyl-group which could potentially activate it toward oxidative addition, because of its meta-position relative to the bromide, it turned-out to not be useful for such manipulations.

(A)

HO OH O OH O OH O OH O OH a b + + + Br Br SnBu3 Br O O O O A' i ii iii

O (B) O Model reaction b + SnBu3 I 'Vinyltin reagent' Scheme 2.19. Stille coupling: a) 3,3-Dimethylacrylic acid, polyphosphoric acid, 90 °C, 1- 2 h; b) see Table 2.6.

Table 2.6. Variables examined during reaction condition „b‟ in Scheme 2.5.10 (A).

Exp # Catalysta Additive Temp.(oC) Time (h) % i % iic % iiic b 1. Pd(PPh3)4 110 6-12 ca. 40 20-30 b 1. Pd(PPh3)4 170 6-12 ca. 40 20-30 102 b 3. PdCl2 tBu3P CsF CuI 45 6-12 60-70 10-20 102 b 4. Pd(PPh3)4 CsF CuI 45 6-12 ca. 40 10-20 103 d b 5. Pd(PPh3)4 BHT 110 4-8 Ca. 30 10-20 a About 10-20 %mol of catalyst was used; b Undetectable quantities; c Rough estimates based on pre-TLC isolation; d Butylated Hydroxy Toluene.

49 2.2.2 Later Retro-Analysis and Synthesis

After being met by these dead-ends, we finally decided to pursue the synthetic route shown in Scheme 2.20. In this strategy, the two synthons (8 and 9) are to be coupled through an ether linkage by an SN2 type displacement of a leaving group with under basic conditions. Similar to the previous synthesis of GLY I, intramolecular Wittig olefination followed by a Sharpless dihydroxylation will cyclize the benzopyran ring leaving two hydroxyl groups in the requisite orientation. Finally, a selective elimination and second SN2 displacement by phenol can then lead to the synthesis of GLY II.

Intramolecular Wittig olefination & Sharpless asymmetric dihydroxylation O OBn O O O O O O OH OH OBn O O HO O OH OH BnO OBn SN2 diplacement 12 11 SN2 displacement

O HO OH O OH O OBn + OMe I OH OMe + O O O OBn 8 9

Scheme 2.20. Retro-synthetic analysis of GLY II from common building blocks.

The ongoing synthesis of GLY II is shown below in Schemes 2.18-2.23. Intermediate 8

(Scheme 2.13) was coupled with 9 (Scheme 2.18) via an ether linkage to obtain 10a.

Protection of both ketones through cyclic ketals or thioketals was attempted next

(Scheme 2.21).

50 O OBn O OH BnO OBn O O OMe + a OMe I OBn O 8 O 9 O O 10a O b

O OBn R R OBn R R OBn O O O O O O d c OMe OBn OBn OBn O OH R R OH R R O 11 11b 11a

Scheme 2.21. Synthesis of GLY II: a) K2CO3, Me2CO, reflux, 20 h, 72%; b) EtOH, cat. H2SO4, triethylorthoformate, Molec S 3Å, rt, 20 h; c) LiAlH4, THF, 0 °C, 5 h; d) PPTS, H2O, Me2CO, rt, 12 h, 74% after 3 steps.

Many unsuccessful attempts were made to form 1,3-dioxanes and 1,3- using

1,3-propanediol and 1,2-ethanediol, respectively (Table 2.7). Similarly, formation of 1,3- dithiolane using 1,2-ethanedithiol proved difficult. Finally, a three-step dual-protection, reduction and dual-deprotection scheme for the two ketones and methyl ester provided 11 in excellent yield. It was observed that the use of triethyl-ortho-formate alone was not able to drive this reaction to completion. Addition of activated molecular sieves (Molec.

S) was found to be important for the ketalization at both positions and for completion of the reaction. Also, the timing of the addition of the molecular sieves was found to be critical. Molecular sieves added at the beginning were never able to drive the reaction to completion, the TLC profile looking quite similar to reaction runs without molecular sieves. For optimal reaction conditions, molecular sieves were added after 4-6 h of stirring at room temp, a point when all starting material had completely dissolved and the initial suspension had become a clear, brownish-colored solution.

51 Table 2.7. Formation of cylic ketals and thioketals.

Exp.# Reagent Catalyst Solvent Temp Yield 1. 1,2-ethanediol104 p-TsOH Toluene reflux a 2. 1,3-propanediol104 p-TsOH Toluene reflux a 105 b 3. 1,2-ethanedithiol BF3 · Et2O MeOH rt a All starting material decomposed, no isolable product was formed; b No isolable product was formed.

A typical protection reaction was then found to be complete after stirring for ca. 18 h.

The crude ketals could be utilized directly in the next reduction step without further purification. The ester was reduced to its primary alcohol using lithium aluminum hyride

(LiAlH4), this reaction usually being complete within 4 h. EtOAc was used to quench the excess reducing reagent and the pH of the reaction was adjusted to ca. 2 using 0.1M HCl during work-up. At higher pH, insoluble aluminum hydrides were formed and interfered with isolation of reduced product. The crude product was subjected to deprotection without further purification. Pyridinium-p-toluensulfonate (PPTS) was selected as the reagent for de-protection of the ketals using a H2O/Me2CO mixture as solvent. Upon completion, the product was isolated as a white precipitate which could be re-crystallized from Me2CO/MeOH. This sequential maneuver has been optimized to a multi-gram level and conveniently provides intermediate 11 in crystalline form.

O OBn O O O O OBn b 11 a OBn O PPh Br O 3 12 OBn

Scheme 2.22. Synthesis of GLY II (continued): a) PPh3·HBr, CH3CN, 60 °C, 1 h; b) t- BuOK, MeOH, reflux, 36 h, 94% after two steps.

52 Compound 11 was then converted to the Wittig salt by reaction with triphenylphosphine hydrobromide. Reaction temperature and duration were optimized

(Table 2.8) after observing that, unlike in the GLY I synthesis, the reaction remained incomplete even after stirring for 12 h at room temperature. The crude Wittig salt was used in the next step without further purification. This cyclization provided intermediate

12, the reaction behaving well after only a quick optimization. Fortuitously, the product from this transformation precipitates out of the reaction mixture upon cooling to room temperature (Scheme 2.22).

Table 2.8. Wittig salt formation reaction condition optimization.

Exp.# Reagent Equiv. Temp Time TLC (oC) (h) a 1 PPh3 · HBr 1 rt 12 b c 2. PPh3 · HBr 2 rt 6 d 3. PPh3 · HBr 2 60 3-4 a b Starting material spot with additional lower Rf spot; Additional one equiv. was added; c TLC profile did not change much; d Starting material disappeared with single product spot.

Intermediate 12 was reduced to the secondary alcohol 13 using LiAlH4 (Scheme

2.23) so that it can be converted into a group which remains stable during dihydroxylation and is also amenable toward a subsequent elimination reaction leading to intermediate 13b. A delicate balance was sought in this case because after dihydroxylation, intermediate 13a will have a tertiary alcohol that could be very labile toward high-temperature elimination protocols.106 Attempts to convert the secondary alcohol into an alkyl aryl selenide using 2-nitrophenylselenocyanate so that, after oxidation, it could yield intermediate 13b remained unsuccessful.107-109 In addition to that various reagents such as sulfonates and acetate were screened for synthesis of 13b

53 (summarized in Table 2.9). All but acetate (Table 2.9, entry 7) gave elimination intermediate 14. However, the formed acetate does not eliminate when we then tried to optimize reaction conditions to form olefin 14 under basic conditions. Various bases such as DBU, DIPEA, potassium tert-butoxide (K-OtBu), and the bulky base lithium bis(trimethylsilyl)amide (LiHMDS), left starting material unchanged or decomposed without any sign for product formation. We did not try other strong and less sterically hindered bases, such nBuLi, because we were fearful about elimination of tertiary- alcohol in the dihydroxylated intermediate 13a.

O O O O a OBn b 12 OBn

OH OR OBn 13 13a OBn

b

O O O O OH OBn OBn

HO 14 OBn 13b OBn

Scheme 2.23. Synthesis of GLY II (continued): a) LiAlH4, THF, 0 °C, 4 h, 64%; b) see Table 2.9.

Table 2.9. Reaction conditions for elimination reaction.

Exp.# Reagent Base % % of 14 yield (%) d 13a a 1. Tf2O DMAP ca. 90 ca. 40 a 2. Tf2O NEt3 ca. 90 ca. 40 3. TsCl DMAP a b b a b b 4. Ms2O DIPEA 5. MsCl DIPEA a b b 6. MsCl Imidazole a b b c 7. Ac2O DMAP 100 80 110 a 8. I2/PPh3 Imidazole ca. 80 60 a No product was observed; b Similar results as above experiments; c 14 was not observed; d Typically these reactions were run for 1-3 h at 0 oC.

54 O O O O OBn a OH b OH OBn 12

O OH OH HO OBn 15 15a OBn

b' c

O O O O OH OBn OH OBn

OH HO HO 15a 13b OBn OBn

Scheme 2.24. Synthesis of GLY II (continued): a) OsO4, NMO 60% aq, CH3SO2NH2, H2O/Me2CO (1:10), rt, 15 h, 60%; b) LiAlH4, THF, 0 °C, 4 h, 70%; b‟) LiAlH4, THF, 0 o °C, 4 h, then EtOAc, pH 1-2, 25%; c) Tf2O, NEt3, 0 C to rt, 12 h, 30%.

As shown in Scheme 2.24, instead of attempting to reduce the ketone first, dihydroxylation across the olefin using Upjohn‟s dihydroxylation protocol was achieved to obtain intermediate 15. The „dihyroxyl ketone‟ 15 was subjected to LiAlH4 mediated reduction. During acidic work-up of the reduced product, a very small amount of

„dihydroxyl olefin‟ 13b was observed. This protocol has been optimized at a „mg‟ scale for 13b. Simultenously, 13b has been synthesized from intermediate 15a as well. In this route the secondary alcohol is first converted to a triflate and because its such a good leaving group, under basic conditions it simultenously converts to the eliminated product,

13b.

Many reaction conditions were attempted on the methoxy-containing model compound (Scheme 2.25) to devise a way to selectively remove two benzyl groups from the intermediate 13b. Our biggest concern in this case was not so much about de- protection of benzyl in the presence of olefin, but to do so in the presence of a tertiary- alcohol which could be very susceptible toward dehydration because of increased

55 conjugation in the resulting intermediate. In this regard, intermediate 16 served well for such explorations (summarized in Table 2.10).

O O O O OH OBn OH OH

HO HO

13b OBn OH Model Chemistry

HO O BnO O HO O OH OH OBn OH OH a a HO HO HO 16a OMe 16 OMe 16b OMe

Scheme 2.25. Reaction optimization for selective debenzylation: a) see Table 2.10 for details.

Table 2.10. Reaction optimization of de-benzylation in presence of tertiary alcohol.

Exp.# Reagent Solvent Temp.(oC) Time (h) % 16 % 16b % 16a 70 a b c 1. BCl3/PMB CH2Cl2 -78 1-4 a b c 2. BBr3 CH2Cl2 -78 1-2 h 111 a b d 3. DDQ CH3CN rt 12-16 h 112 a b d. 4. TMS-I CH2Cl2 0 to rt 6-8 h 5. Li[naphthalenide]113 THF rt 12-18 h e b b a Almost all starting material was consumed; b Could not see detectable quantities; c immediately on addition of reagent, it starts forming, ca. 25% yield; d Not observed; e Starting material remain un-reacted.

Ultimately, in the absence of a reliable method for selective removal of the two benzyl groups from intermediate 13b, it was decided to remove the benzyls before generation of the olefin (i.e. after elimination of the alcohol obtained from reduction of the ketone) and replace them with another orthogonal protecting group such as a silyl group so that the newly formed olefin does not interfere during the subsequent de- protection step. As shown in scheme 2.26 intermediate 17 was obtained from 15 after hydrogenolysis. Its re-protection with t-butyldimethylsilyl chloride (TBDMS-Cl) to make

56 18 occurred smoothly. For reduction of TBDMS protected ketone 17, both sodium borohydride and LiAlH4 mediated reduction were attempted. It was observed that reduction using sodiumborohydride (NaBH4) is very slow, the reaction taking a very long time to reach completion even when very large excesses of reagent were deployed. A

LiAlH4 mediated reduction was also attempted. Because LiAlH4 can chelate with the tertiary alcohol through „neighboring group‟ participation, the TBDMS group at the ortho position most likely, based on reaction mechanism,114 collapses and thus the reaction ultimately provides mixture of intermediate 18a and 18b as diastereomeric mixture. Also, formation of intermediate 18c was observed during this reaction. Interestingly, the latter can act as a precursor for an important keto-analog of GLY II upon closure of the benzofuran ring (Scheme 2.26). This type of keto-analog will be analogous to estrone which has ketone functionality instead of the 17β-hydroxyl as found in estradiol)

O O O O OH a OH b OH OTBDMS 15

O OH O HO OH 17 18 OTBDMS

O O O O O O OH OTBDMS OH c OH OH OH 18 + + OH HO OH HO O HO 18a OTBDMS 18b OTBDMS 18c OTBDMS

Scheme 2.26. Replacement of benzyl with TBDMS and subsequent reduction: a) Cat. o Pd/C, EtOAc, 35 psi, rt, 10 h, 80%; b) TBDMS-Cl, DIPEA, CH2Cl2, 0 C to rt, 12 h, o 70%; c) LiAlH4, 0 C to rt, 4-6 h, 30% (18a), 40% (18b), 10% (18c) ; when c) NaBH4, MeOH, 24-30 h, 60% 18a.

2.2.3 Recommendations for Future Synthetic Efforts

As shown in Scheme 2.27, completion of the synthesis of GLY II can be now attempted following either of two pathways. At a very small scale, formation of intermediate 19 has

57 already been observed. However, reproducibility is yet to be demonstrated at large scale.

Selective removal of the silyl group from the ortho-hydroxyl group will take advantage of chemistry we have already learned during reduction of ketone 18 where LiAlH4 was found to be very effective for such manipulations due to neighbouring group participation. There is literature precedent for mesylate mediated benzofuran ring closure84 and that might lead to intermediate 7 (Scheme 2.1). This penultimate intermediate would then lead to a final synthesis of GLY II after silyl deprotection.

O O O O O O OH OTBDMS OH OTBDMS OH OH

OH OH HO HO OTBDMS 20 18a 19 OTBDMS OTBDMS

O O O O O O OH OH OH OH OH

OH HO HO O 7 18b OTBDMS 19a OTBDMS OTBDMS

O O O O O O OH OH OH O

O O OH GLY II OH 7 OTBDMS GLY II

Scheme 2.27. Proposed route for final GLY II synthesis.

2.3 Synthesis of Vestitol, Bolusanthin III and 6a-Hydroxymedicarpin

All of these natural products became accessible from our desire to have good models for understanding the chemistry associated with key reactions we deployed during our GLY

II synthetic efforts. For example, near the end of our final synthetic strategy (see Section

2.2.2), we envisioned removal of benzyl groups from a substrate having an olefin and an allylic system. Also, the final step involves construction of a benzofuran ring as part of a

58 fused, benzofuran-benzopyran tetracyclic pterocarpan ring system by using an SN2 displacement type of protocol.84 Thus, 23 served as a good model for deprotection of the benzyl group in the presence of an olefin while also providing access to the synthesis of vestitol and bolusanthin III (Scheme 2.28).

The synthesis started with protection of the free phenolic group on 4-methoxy-2- hydroxyacetophenone (21a) with a benzyl group under basic conditions followed by selective α-iodination of intermediate 21b to provide intermediate α-iodo ketone 21 using either Selectfluor42 or copper oxide.79 Alternatively, selective benzyl protection of 2,4- dihydroxybenzaldehyde (1c) at the para position under very mild basic conditions produced aldehyde 1b which was converted to salicyl alcohol 1 using sodium borohydride. Coupling of the alcohol 1 and the ketone 21 through a nucleophilic substitution reaction formed ether 22 as shown in Scheme 2.28. The latter was converted into a Wittig salt by treatment with triphenyphosphine hydrobromide and then directly subjected to an intramolecular Wittig olefination so as to obtain 23. The total synthesis of vestitol was then achieved in eight steps (29% overall yield)115 when intermediate 23 was subjected to catalytic hydrogenolysis wherein not only did the two benzyls cleave, but the olefin was reduced as well.

59 MeO OH MeO OBn MeO OBn a b O OBn I BnO O 21a O 21b O 21 O e + OMe OH 22 HO OH c BnO OH d BnO OH H H f 1c O 1b O 1 OH

HO O BnO O HO O OH g OBn h OH

(23a) OMe 23 OMe (23b) OMe Vestitol Bolusanthin III

Scheme 2.28. Synthesis of vestitol and bolusanthin III: a) BnBr, K2CO3, CH3CN, reflux, 24 h, 96%; b) Selectfluor, I2, CH2Cl2/MeOH(1:5), rt, 20 h, 84%; c) BnBr, KHCO3, o CH3CN, reflux, 15 h, 85%; d) NaBH4, EtOH, 0 C to rt, 10-12 h, 75%; e) K2CO3, Me2CO, reflux, 16 h, 78%; f) (i) PPh3. HBr, CH3CN, rt, 1 h; (ii) t-BuOK, MeOH, reflux, 24 h, 70% over two steps; g) Cat. Pd/C, EtOAc, 35 psi, 12-14 h, 84%; h) BCl3, CH2Cl2, 15 min, -78 oC, 61%.

Alternatively, for synthesis of its 3-ene relative (bolusanthin III) we first tried to remove the benzyl groups with PMB/TFA, as well as with boron tribromide.

Unexpectedly, both reagents proved to be harsh enough to degrade starting material 23 without providing any desired product. One plausible mechanism for such ready decomposition is depicted in Scheme 2.29. We think that because of resonance stabilization, the allylic ether preferably becomes protonated or chelated with the Lewis acid in the case of boron tribromide. After protonation and subsequent flow of electrons from the conjugated benzyl ether, the allylic ether linkage collapses and the transitory carbocation resonates across the molecule before being converted into various side- products. Ultimately, this synthesis was accomplished by treating 23 with BCl3 in the presence of PMB at -78 oC.70 This eight-step procedure resulted in the first total synthesis of bolusanthin III (21% overall yield).115

60 H

BnO O BnO OH

BnO OCH3 BnO OCH3

BnO OH BnO OH Degradation

BnO OCH3 BnO OCH3

Scheme 2.29. Proposed degradation mechanism for 23.

BnO O HO O HO O OH a OH OH 23 a b OCH3 OCH HO HO 3 O BnO HO OCH3 16 24 6a- Hydroxymedicarpin

Scheme 2.30. Synthesis of 6a-hydroxymedicarpin: a) Cat. OsO4, NMO, CH3SO2NH2, Me2CO/H2O (10:1), rt, 10 h, 75%; b) Cat. Pd/C, H2, 35 psi, rt, 10 h, 80 %; c) Ms2O, o pyridine, CH2Cl2, 0 C to rt, 16-20 h, 35%.

For the synthesis of 6a-hydroxymedicarpin, intermediate 23 was subjected to Upjohn dihydroxylation conditions to instill two hydroxyl groups. Intermediate 16 produced intermediate 24 in ca. 80% yield under catalytic hydrogenolysis conditions. This penultimate intermediate was cyclized into a pterocarpan through nucleophilc substitution displacement of the activated secondary alcohol by the phenol.84 This first total synthesis of another natural target molecule, 6a-hydroxymedicarpin, was thus achieved in 10 steps (13% overall yield) (Scheme 2.30).116

2.4 Design and Synthesis of New SERM Analogs

This section discusses the further design and synthesis of new target molecules that can additionally contribute to the development of SERM SAR. The fortuitous synthesis of

61 intermediate 22b instead of 22a (Scheme 2.31) during part of our GLY II efforts, along with our familiarity about estrogen receptor SAR, came together in a manner that allowed us to recognize the possible utility of this scaffold to generate new analogs for estrogen modulation. The benzofuran type system represents an interesting and novel scaffold to pursue ER modulation. Additional new targets were designed based upon our earlier spatial analysis of the estrogen receptor (ER) active sites and their natural ligand, estradiol (E2), and upon molecular docking studies associated with these new scaffold systems in particular.

O OBn BnO OH MeO OBn a BnO O + H x I OMe 1b O 21 O O 22a a

BnO O O OBn

22b OMe

Scheme 2.31. Fortuituos formation of benzofuran scaffold: a) K2CO3, Me2CO, reflux, 4 h, 75%.

2.4.1 Design of Benzofuran and Furo[2,3-f]-2H-1-benzopyran Analogs to Study SERM SAR

Preliminary pharmacophore mapping as shown in Fig. 2.13 suggested that the benzofuran scaffold is able to display polar hydroxyl groups at almost equivalent distance to those found in estradiol. This finding makes a strong case to use this interesting scaffold for new estrogen modulators when coupled with its ease of synthesis as shown in Scheme

2.29.

62 HO HO HO O O H O O OH OH OH

~12 Å ~11Å ~ 10 Å OMe OMe T2 Etradiol T2

Fig. 2.13. Pharmacophore mapping of estradiol and energy-minimized T2 (Fig. 2.14).

To experimentally test the two key concepts pertaining to differential spacing and altered orientation of the His imidazole ring within the active site as described in section 2.1.2.1, the benzofuran-based analogs shown in Figs 2.14 and 2.15 were designed. Docking studies were performed using the validated docking models described in section 2.1.2.2.

These type of studies can further clarify the potential utility of possible π-π interactions to achieve selective receptor binding. After their synthesis and testing, it was envisioned that the SAR could correlate with a π-π type of interaction. The rotable bonds through which these rings are connected with the core of these molecules, may actually enhance the possibility for π-π interactions. This is because the flexible nature of the linkage may allow them to better orient in a manner even more suitable for stacking with the imidazole ring of the active site His residue in ERβ. Even if the latter is not found, it should be noted that all of the analogs shown in Fig. 2.14 also possess possible hydrogen bonding features in very similar orientations as those displayed by estradiol (Fig. 2.13).

The targets T2, T6, T4, T5 can additionally examine possible binding selectivity between the two ER subtypes because of differential spacing between two ends of their active sites. Alternatively, analogs T1 and T3 will serve as negative controls for the analogs that have hydrogen-bond donating capability.

63

T1. - R = R1 = Bn, R2 = Me RO O O T2. - R = R1 = H, R2 = Me OR1 T3. - R = R1 = Bn, R2 = MOM T4. - R = R1 = Bn, R2 = H T5. - R = Bn, R1 = R2 = H OR2 T6. - R = R1 = R2 = H

Fig. 2.14. List of benzofuran based SERM targets.

2.4.2 Design of Final Targets for SERM SAR

T7. - R = Me, R1 = Bn O O O OR1 T8. - R = Me, R1 = H T9. - R = H, R1 = Bn T10. - R = R1 = H OR

Fig. 2.15. List of furo[2,3-f]-2H-1-benzopyran based SERM targets.

Additional targets were designed (Fig. 2.15) to incorporate structural features similar to

GLY I so as to test whether the latter‟s structural complexity is important for its activity.

Also, exposing one phenolic hydroxyl at a time (ortho or para, T8 and T10) can examine if there is any change in binding affinities because of their different distances from the dimethylpyran ring.

2.4.3 Molecular Docking Studies

Docking studies were carried out to support our hypothesis that ease of π-π stacking interactions and differences in size and shape of the two active sites can provide desired selectivity for development of new SERMs. The validated docking models generated for

64 both ERα and ERβ using Sybyl‟s SurflexdockTM suite, as previously described in section

2.1.2.2, were used for this study as well.

2.4.4 Analysis of Molecular Docking Results

A comparative examination of the docking results shown in Fig 2.16A and Fig 2.17A helped explain the influence of the imidazole ring orientation on possible π-π interactions with the targets. It appears that targets with a benzyl group on the benzofuran ring (T1,

T3, T4, T5) could efficiently participate in π-π stacking interactions with the imidazole ring of the His residue of ERβ. They are ranked among pools of the highest scoring analogs (Table 2.11). On the other hand, the ERα docking model (Fig. 2.16A) ranking penalized these analogs because of the lack of an efficient stacking possibility and because these extra groups are likely having negative steric interactions with the imidazole ring. Thus, they become ranked among the most poorly scoring analogs (Table

2.11). However, the altered orientation of the imidazole ring does not seem to impact its

H-bond interactions as much as π-π stacking. By comparison of Fig. 2.16B and Fig.

2.17B, ligands with appropriately displayed H-bonding pharmacophores seem to be able to bind similarly and also have similar binding affinities.

65

Fig. 2.16 Surflexdock poses of test set data points for ERα: (A) Data points with one or more benzyl group, (B) without any benzyl group (T2 in magenta, T6 in orange, T8 in red T10 in gray, THC crystal structure in green), (C) highest scoring data point.

Fig. 2.17 Surflexdock poses of test set data points for ERβ: (A) data points with one or more benzyl group, (B) without any benzyl group (T2 in magenta, T6 in orange, T8 in red T10 in gray, THC crystal structure in green), (C) Highest scoring data point with a phenolic hydroxyl pharmacophore is showing π-π interaction.

The difference in distances between the two ends of the active site (i.e. between His, and

Args or Glus or both) within the two receptor subtypes seemed to have an important influence on the overall score for any given analog. A slightly longer active site, as found in ERβ, could have significant effects on the strength of H-bond interactions since the electronic interactions are inversely proportional to the square of distance. That could be one plausible explanation for the almost one-hundred-fold selectivity shown by analog

T6 between the two receptor subtypes (Table 2.11 entry 6). Moreover, the significance of

66 distance on H-bonding interactions is clearly portrayed when one compares Fig. 2.16B and Fig. 2.17B while also considering the dock scores given in Table 2.11. It seems that analogs T2 and T8, containing a free hydroxyl group at the ortho position, have a significantly different orientation in ERβ (Fig. 2.17B) in comparison to ERα (Fig. 2.16B).

This may be because the shorter distance between the two binding pharmacophores (ca.

10 Å, Fig. 2.13) within these analogs becomes exaggerated during their interaction with

ERβ due to the longer spatial display of active site residues in ERβ. Because their interactions with the imidazole ring of the His residue are not as strong, they bind with alternative binding sites and ultimately become oriented differently.

Table 2.11. Predicted binding affinities of target analogs

S. No. Target pKda ERα pKda ERβ 1. T1 -1.05 6.87 2. T2 3.24 5.68 3. T3 -6.39 5.56 4. T4 -4.66 8.40 5. T5 -2.66 7.36 6. T6 6.77 5.04 7. T7 - 6.12 2.04 8. T8 -3.25 2.66 9. T9 1.26 5.34 T10 0.18 3.42 a Highest surflexdock scores, b Calculated binding affinity.

2.4.5 Synthesis Benzofuran Based Targets

Syntheses of targets T1 and T2 are shown in Scheme 2.32. Initially, aldehyde 1b

(Scheme 2.28) was refluxed with α-iodo intermediate 21 (Scheme 2.28) using K2CO3 in

Me2CO in an attempt to obtain intermediate 22a as shown in Scheme 2.31. After 3-4 h of stirring under reflux, the reaction was complete and there was only one spot on TLC

67 subsequent to a quick acidic work up. Upon characterization, however, this product turned-out to be the benzofuran derivative, which will serve as a target in itself, T1.

Perhaps after displacement of α-iodo by the phenoxide, the in situ generated enolate makes an intramolecular nucleophilic attack on the aldehyde functionality of the intermediate 22a. The resulting Aldol product could then lose a H2O molecule under reflux conditions so as to form the condensation product. This type of transformation is known as a Rap-Stoermer condensation.117 The two benzyl groups on intermediate T1 were removed using pentamethylbenzene (PMB) and trifluoroacetic acid (TFA) to obtain

T2. Targets T4, T5, and T6 were obtained uneventfully from T3 which was obtained by coupling of α-iodo ketone 3 with salicyaldehyde 1b followed by an intramolecular Aldol condensation as described in Scheme 2.33.

O OBn BnO OH H3CO OBn BnO O a + H I OMe 1b O 21 O O

Intramolecular Aldol condensation

HO O O BnO O O OH b OBn

T2 T1 OMe OMe

Scheme 2.32. Synthesis of benzofuran-based targets T1 and T2: a) K2CO3, Me2CO, reflux, 4 h, 75%; b) PMB, TFA/CH2Cl2 (2:1), rt, 10 h, 88%.

68 BnO O O OH

T5 OH b BnO O O BnO OH MOMO OBn a OBn + I 1b O 3 O T3 OMOM b

HO O O BnO O O OH c OBn

T6 T4 OH OH

Scheme 2.33. Synthesis of benzofuran-based targets T3, T4, T5 and T6: a) K2CO3, Me2CO, reflux, 4 h, 75%; b) HCl(g), EtOAc, rt, 2 h, 27 % (T5) and 70% (T4); c) PMB, TFA: CH2Cl2 (2:1), rt, 12 h, 90%.

2.4.6 Synthesis of Furo[2,3-f]-2H-1-benzopyran Based Analogs

Synthesis of this series of compounds (Scheme 2.34) followed a similar route as in the benzofuran-based analogs except for the fact that these analogs needed a stronger base

(lithium diisopropylamide, LDA) to complete the intramolecular Aldol condensation.

Intermediate 7a (Scheme 2.10) was then reacted with α-iodo ketone 21 (Scheme 2.28) under basic conditions in order to obtain target T7, but intermediate 25 was instead isolated. Perhaps the additional substitution with an electron-donating group on the benzene bearing an aldehyde functionality makes the aldehyde less reactive toward nucleophilic attack. Therefore, as mentioned above, a stronger base such as LDA was used to generate the enolate. Though we did not isolate the Aldol reaction intermediate,

TLC and 1H NMR confirmed its formation which eventually condensed into target T7 in situ on elevation of temperature. The sterically bulky Lewis acid BCl3, instead of TFA, was used for acidolysis of the benzyl group on T7 to obtaint target T8. This was mainly

69 because of apprehensions we held about potential decomposition of starting material and the product as they have a tertiary and allylic ether linkage which could be sensitive toward a protic acid such as TFA.

O OBn O O OH a O O O O b OBn H 21 OMe OH 7a O O 25 OMe

O O O O O O OH c OBn

T8 T7 OMe OMe

Scheme 2.34. Synthesis of targets T7 and T8: a) 3-Picoline , p-xylene, 120 oC, 20 h, o o o 95%; b) LDA, THF, -78 C, 3-4 h then 50 C, 2-3 h, 90%; c) BCl3, PMB, CH2Cl2, -78 C, 5-10 min, 78%.

Additional furo[2,3-f]-2H-1-benzopyran based targets T9 and T10 were synthesized by careful selection of protecting groups (Scheme 2.35). Toward that end, intermediate 7a was coupled with iodo intermediate 3 (Scheme 2.1) to obtain intermediate 26 which was then treated with LDA to form intermediate 27 as described previously. Treatment with triphenylphosphine hydrobromide allowed acidolysis of the MOM protecting group to convert intermediate 27 into target T9. Again, BCl3 with PMB was used to take both benzyl and MOM groups off the phenols for direct conversion of 27 into target T10.

70 O OBn O OH O O O O a b O OBn H 3 OMOM 7a O O 26 OH OMOM

Condensation

O O O O O O O O O OBn c OBn d OH

T9 27 T10 OH OMOM OH

Scheme 2.35. Synthesis of targets T9 and T10: a) K2CO3, Me2CO, reflux, 12-14 h, 83%; o o o c) LDA, THF, -78 C, 4-5 h then 50 C, 2-3 h, 83%; c) PPh3·HBr, CH2Cl2, 0 C to rt, 6-8 o h, 63%.; d) BCl3, PMB, CH2Cl2, -78 C, 30 min, 70%.

2.5 Summary of Studies Aimed At Preparing Compounds Having Improved SERM Properties

During our pursuit of better SERM compounds, I was able to accomplish the total syntheses of four natural compounds: namely (S)-glycinol, 6a-hydroxymedicarpin, bolusanthin III and vestitol. The first total synthesis of (S)-glycinol was achieved after

10-steps in ca. 10% overall yield. We recently published these methods.118 The racemic syntheses of 6a-hydroxymedicarpin (10 steps, 13% overall yield), bolusanthin III (8 steps, 21% yield) and vestitol (8 steps, 29% yield) have been presented at national meetings and their manuscripts are nearing completion for submission.115, 119

Additionally, good progress has been made toward the specific synthesis of GLY II.

After numerous experiments, chemistry has been developed for the practical synthesis of

TBDMS protected tricyclic ketone 18 which can serve as a key intermediate (11 linear steps in 15% overall yield). Part of the efficiency of this synthetic route lies in the fact that it is devoid of any column chromatography purification steps.

During scale-up and process chemistry enhancement studies, the synthesis of GLY I was improved by reducing the total number of synthetic steps from 15 to 13 and this, in

71 turn, was accompanied by a 3-fold improvement in overall yield (3% to 9%). Moreover, this synthetic procedure has been made highly amenable for further scale-up by eliminating several column chromatography purification steps; only one column purification step remains present in the new 13-step route. Importantly, more than 28 final target compounds (including direct GLY I analogs and new SERM analogs) have been added to the CD3‟s „SERM-directed‟ compound library for future biological and

SAR studies.

Finally, molecular modeling and docking studies were performed to enhance our understanding about the binding interactions for these analogs with ERs, as well as for several standard agents. During these studies we deduced a key finding wherein the two different estrogen receptor active sites have very differently oriented His residue imidazole rings. To our knowledge, this important difference between the two active sites has never been exploited to enhance selective binding. We perceive that this important insight about the active sites could serve as a novel starting-point for the further development of better SERMs. Several of the compounds that were added to the CD3‟s directed library are intended to examine this possibility. Their biological testing data may thus lend experimental support to our theoretical perceptions in the near future.

Additional publications for these potentially exciting findings will then follow as appropriate.

2.6 Scale-up and Process Chemistry Enhancement for Production of CD3-246

This final section of my dissertation describes studies that contribute toward the CD3‟s pursuit of chemotherapeutic treatments for advanced, hormone-independent prostate

72 cancer. Specifically, CD3-246 is a lead compound that was previously identified by the

CD3 to merit progression into early drug development activities because of its promising profile for the possible treatment of this life-threatening clinical indication (see Section

1.5). An important first step during drug development activities is to optimize and undertake process chemistry experiments so that they become suitable for the scale-up production of initial multi-gram and eventual kilogram supplies of the lead compound.

2.6.1 Scale-up Synthesis of CD3-246 Using the ‘Boc/Bzl’ Strategy

The scale-up synthesis of CD3-246 started with activation of the carboxyl terminal of

Boc-Met-OH using 1,1‟-carbonyl diimidazole (CDI) as a coupling agent (Scheme 2.36).

Activation of the acid functionality for an amino acid can be performed either as a separate pre-activation step, or in situ as part of the reaction process.67 The activated Boc-

Met-OH was coupled with commercially available H-Gly-OBzl·p-tosylate (28) to generate dipeptide intermediate 29. A solution of intermediate 29 in EtOAc was treated with HCl gas at 0 oC to remove the „Boc‟ protecting group. The free amine was, in turn, reacted with activated Boc-D-Tyr(Bzl)-OH to produce „tripeptide‟ intermediate 30.

Acylation of this intermediate took place by reacting it with activated 4-(2-thienyl)butyric acid after removal of the „Boc‟ protecting group from the tyrosine N-terminus under acidic conditions that were similar those used on intermediate 29. This sequence produced acylated tripeptide CD3-404.

At this point various chemical investigations were again undertaken to simultaneously remove both of the benzyl groups on intermediate CD3-404, primarily by focusing upon palladium catalyzed hydrogenolysis. Some of the results from these studies are

73 summarized in Table 2.12. A bench-top hydrogenator from ThalesNano, Inc., Hatboro,

PA was used for the „bomb‟ reaction conditions. Even with this well-controlled system, all of such efforts were again unsuccessful. Therefore, the two benzyl groups present on intermediate CD3-404 were removed sequentially. First, the ether-linked benzyl was removed by treating with a combination of PMB and TFA under ambient conditions. The crude, penultimate intermediate 31 was then utilized in the next step without further purification. The benzyl ester was hydrolyzed under basic conditions to produce the lead compound CD3-246. A batch-wise record of synthesized CD3-246 was delineated (Table

2.13) for quality control (QC) purposes. The batches which met the desired „QC‟ standard of 95% or higher purity by HPLC and tight melting point, were combined so as to provide ca. 15 g of CD3-246 in ca. 19% overall yield.

OBn OBn O OBn BocHN OBn O O O OTs H3N a NH b c NH OBn NH OBn O O BocHN * NH NH * NH O O O O S S 28 29 30 S CD3-404 S

d

OH OH

O O O O NH OH NH OBn NH * NH NH * NH O O O O S S c CD3-246 S 31 S Scheme 2.36. Scale-up Synthesis of CD3-246 using „Boc/Bzl‟ strategy: a) Boc-Met-OH, CDI, CH2Cl2, 1 h then H-Gly-OBzl·p-tosylate, NEt3, CH2Cl2, rt, 18 h, 88%; b) (i) HCl(g), o EtOAc, 0 C, 1.5 h; (ii) Boc-D-Tyr(Bzl)-OH, CDI, CH2Cl2, 0.5 h then H-Met-Gly- OBzl·HCl, NEt3, CH2Cl2, rt, 24 h, 73% over two steps; c) (i) HCl(g), EtOAc, 1 h; (ii) Thienylbutyric acid, CDI, CH2Cl2, rt, 4 h, then H-D-Tyr(Bzl)-Met-Gly-OBzl·HCl, CH2Cl2, NEt3, rt, 18 h, 70% over two steps; d) (i) PMB, TFA, rt, 2 h; (ii) 31, K2CO3, THF, rt, 24 h, then 60 oC for 1 h, 42% over two steps.

74 Table 2.12. Reaction conditions for „bomb‟ reaction.

S.No. Scale Solvents Flow rate 10% Pd/Ca Pressure Temp TLC (mmol) (mL/min) (mg) (psi) (oC) b 1. 0.7 CH2Cl2/MeOH 0.5 140 mg 15 100 (1:1, 7 mL) b 2. 0.7 CH2Cl2/MeOH 0.5 140 mg 600 50 (1:1, 7mL) b 3. 0.7 CH2Cl2/MeOH 0.5 140 mg 1500 100 (1:1, 7mL) a Pre-packed cartirage (H-30) was used with 140 mg Pd/C; b No sign of product formation, s.m. remained intact.

Table 2.13. Details for different batches of CD3-246 prepared by the optimized „Boc/Bzl‟ route.

Batch CHN data Melting Point HPLC Batch Size (°C) (% purity) (g) (mole of H2O) 1 0 140-142 99.4 0.98 2 - 145-150 96.95 0.65 3 0.25 148-154 95.83 2.91 4 - 131-142 95.2 2.2 5 0.25 146-152 97.57 3.34 6 - 130-134 95.57 1.21 7 - 143-150 97.64 1.31

2.6.2 Development of a Convergent Synthetic Route

Although low-gram quantities of CD3-246 could be readily produced by the aforementioned strategy, further optimization was additionally undertaken with an eye toward producing 100 gram to kilogram quantities. Noting that our prior routes were linear in their assembly of the peptide chain, we decided to explore a convergent synthetic route. Retro-analysis of the latter is shown below as Scheme 2.37.

75 2.6.2.1 Retro-Analysis

Synthesis of CD3-246 was envisioned to be achieved by coupling two synthons, namely

N-acyl-tyrosine (36) and „dipeptide‟ synthon 35. Synthon 36 could be synthesized through coupling of 4-(2-thienyl)butyric acid (Tba) with side-chain protected H-(D)-Tyr-

OH (32). On the other hand, the „dipeptide‟ synthon could be realized through a peptide coupling reaction between N-terminus protected H-Met-OH (33) and C-terminus protected H-Gly-OH (34).

OH OR S

O O O O H H N OH N S N N S N OH + H N OR H H H 2 O O O O 36 35 CD3-246 S

OR S

O O + OH OH + H2N S OH H2N R1-HN OR O O Tba 32 33 34 Scheme 2.37. Retro-analysis of convergent synthesis for CD3-246.

2.6.2.2 Synthesis

OR OR

O O + a OH OH S OH H2N S N H O O Tba 32 36

R = Bzl R = Bzl R = tBu (32a) R = tBu (36a)

Scheme 2. 38. Synthesis of N-acyl-(D)-tyrosine: a) CDI, DBU, CH2Cl2, rt, 14-18 h, 81%.

In order to keep the total number of steps at a minimum, side-chain-protected tyrosine with both terminals free was chosen for the synthesis of synthon 36. But with both terminals free, the (D)-tyrosine formed an internal salt (zwitterion) and therefore was

76 highly insoluble in the reaction solvent. To improve the solubility of zwitterionic 32, various organic bases were screened as shown in Table 2.14. Among all the bases, 1,8- diazobicyclo[5.4.0]undecane (DBU) best solved the solubility issue.

Table 2.14. Screening of various bases to improve on zwitterion solubility.

S. No. Organic Base pKa 1. Triethylamine ~ 10.8 2. Hunig‟s Base/DIPEA ~ 11.4 3. Collidine ~ 6.7 4. 1,8-Diazabicyclo[5.4.0]undecane (DBU) ~ 12

Various reaction condition optimizations for the coupling between Tba and 32 are summarized in Table 2.15. In the case for acylation of D-tyrosine, the reaction went well with less than one equivalent of coupling agent CDI when a benzyl moiety served as the protecting group for the side-chain. Although reaction yields are similar, the overall work-up of the reaction was more efficient when less equivalents of CDI were used

(Table 2.15).

Table 2.15. Acylation reaction condition optimization results.

S. # equivalents Scale Reac. time Yield (%) No. Tba CDI H-D-Tyr(tBu)-OH (32a) DBU (mmol) (h) 1. 1.0 1.2 1.0 1 1 12 -18 73a 2. 1.0 1.2 1.0 1.0 5 ” 75a 3 1.0 1.2 1.0 1.0 10 ” 70 S. # equivalents Scale Reac. time Yield (%) No. Tba CDI H-D-Tyr(Bzl)-OH (32) DBU (mmol) (h) 1. 1.0 1.2 1.0 1.0 1.0 Ca. 20 70 2. 1.2 1.0 1.1 1.1 1 Ca. 20 65b 3. 1.2 1.0 1.1 1.1 10 Ca. 20 81 4. 1.2 1.0 1.1 1.1 60 Ca. 24 62 a Average of n = 2 reactions; b Work-up was easy and less emulsification was built during work-up

77 Syntheses of „dipeptide‟ synthons 35 and 35a (Scheme 2.39) were achieved by coupling correspondingly N-protected methionine (Fmoc/Boc) with benzyl (34) or tert-butyl (34a) esters of the amino acid glycine. The same procedure as described in Scheme 2.36 was followed for acidolysis of the „Boc‟ from Boc-Met-Gly-OBzl to obtain intermediate 35.

For preparation of 35a, intermediate Fmoc-Met-Gly-OtBu was treated with 1-octanethiol in presence of catalytic DBU. 1-Octanethiol acts as a scavenging agent to trap the highly reactive dibenzofulvene.67 Both the free amine or salt forms of the „dipeptide‟ could be used in the next coupling step.

S S

O O a H OH + H2N N R1-HN OR H2N OR O O 33 34 35 R = Bzl R = Bzl R1 = Boc/ Fmoc R = t-Bu (34a) R = t-Bu (35a) Scheme 2.39. Synthesis of „dipeptide‟ synthon 35 & 35a: a) for synthesis of 35, as HCl salt, procedure as described in scheme 2.36 was used; (i) CDI, CH2Cl2, DIPEA, rt, 18h, 40%; (ii) 1-octanethiol, cat. DBU, 3-4 h, 40% (35a)

OBn OBn S

O O O O H a H N + OH N OBn ClH3N OBn S N S N N H H H O O O O 35 36 CD3-404 S Scheme 2.40. Convergent coupling step: a) DPPA, DIPEA, DMF, 0°C, 15 h, 61%.

Many different reaction conditions were then examined on small scale to couple these two fragments (35 and 36) so as to optimize the yield while avoiding epimerization120 at the α-carbon of the activated amino acid functionality (Fig 2.18). The problematic epimerization is readily discernable by NMR (Fig. 2.19)

78

OBn

Base BH O H O O H O HN R1 X R1 R1 O O O N N O N Aromatizes R R R Oxazolone Epimerized S

Fig. 2.18 Epimerization at α-carbon of a carboxy terminal activated peptide.

We tried several different coupling reagents with varying racemization suppressive abilities such as hydroxybenzotriazole (HOBt), hydroxyazabenzotriazole (HOAt), and various uranium and phosphonium salts (HATU, PyBOP etc.) alone or in combination with copper salts such as copper chloride.120 After numerous experiments, we eventually determined that diphenylphosphine azide (DPPA) is a particularly well-behaved reagent that does not cause epimerization in this reaction (Scheme 2.40).121 The latter has now been scaled-up to the ca. 1 gram level. Completion of this route again takes advantage of the „Boc/Bzl‟ studies wherein PMB is conveniently deployed to de-protect the side-chain of the (D)-Tyr residue, and then the C-terminus ester is cleaved by basic hydrolysis

(Scheme 2.36). The overall yield is again around 20%.

79

OBn

O O H 20 N 9 O S N 8 N H H O OBn S

Fig. 2.19. 600 MHz NMR spectrum recorded in deuterated DMSO for key intermediate CD3-404 obtained via the convergent synthesis strategy. Spectrum depicts epimerization that can occur during synthesis. While assignments are tentative, they are supported by

COSY spectra.

2.50 1.97

OBn

O O H N O S N N H H O OBn

S

CD3-404

7.36

7.35

5.11

7.38 3.33

7.41

5.01

5.01

6.89

7.14

2.12

6.87

7.15

3.88

7.28

7.28

2.20

6.78

7.42

2.11

2.64

2.63

6.78

6.91 3.89

3.87

8.42

8.16

8.31 8.18

8.32

6.91

1.72

1.72

2.21

4.46

4.48

2.20

1.70

1.73

1.71

2.72

2.84 2.65

4.30

4.30

8.41

2.86 2.87

0.96 0.91 9.59 1.80 2.61 1.83 0.98 1.00 1.91 1.10 0.98 1.86 1.88 2.73 2.84

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 ppm Fig. 2.20. 600 MHz NMR spectrum recorded in deuterated DMSO for key intermediate CD3-404 obtained via the convergent synthesis strategy. Spectrum suggests that no significant epimerization has occurred during synthesis. While assignments are tentative, they are supported by COSY spectra.

80

2.50 2.50 1.98 OH

O O H N O S N N H H O OH

S 6.63

6.61 CD3-246

7.00 7.02

2.67

7.29

3.71

3.72 2.12

7.30

2.65

6.79

8.27 6.92

8.27

2.68

8.13 8.14

8.29

1.73

3.35

2.26

1.72

9.17

4.42

4.43

1.74

4.31

4.30

2.79

1.71

1.70

2.27

2.81

2.64

2.82 4.28

0.94 1.89 1.03 1.94 1.96 1.02 1.96 1.16 2.85 1.95 2.89 2.98 0.49

9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 ppm Fig. 2.21. 600 MHz NMR spectrum recorded in deuterated DMSO for final product CD3- 246 obtained via the convergent synthesis strategy. Spectrum suggests that no significant epimerization has occurred during synthesis. While assignments are tentative, they are supported by COSY spectra.

2.6.3 Mechanistic Investigations for O-Debenzylation of Phenolic Ethers Using Pentamethylbenzene and Trifluoroacetic Acid

As described in preceding Sections 1.5 and 2.6.2, during development of solution phase syntheses of our sulfur-containing peptidomimetic lead compound CD3-246, all efforts toward O-debenzylation of the so-protected tyrosine side-chain were futile. Alternatively, we found the PMB-TFA strategy to be very effective for selective cleavage of an O- benzyl group in the presence of a sulfur and, furthermore, that it had little effect upon a neighboring benzyl ester (all shown in Scheme 1.4).122 Other than knowing that 1-benzyl-

2,3,4,5,6-pentamethylbenzene is formed as a side-product,123 to our knowledge the detailed mechanism of this simple but highly practical and rather interesting reaction remains unknown. Therefore, a study was designed to investigate the reaction mechanism for this transformation. First, model compounds 2-benzyloxynaphthol (37) and 2-(4‟-

81 methoxybenzyloxy) naphthol (38) (Scheme 2.41) were synthesized to investigate how

PMB facilitates O-debenzylation of phenolic ethers. These compounds along with a para- nitrobenzyl derivative (Scheme 2.42) were judiciously chosen because they confer varying degrees of reactivity toward acidolysis of the ether linkage. For instance, compound 38 is very susceptible toward benzylic carbocation generation whereas the nitro-derivative, because of its electron-withdrawing nature, substantially decreases the stability of the corresponding benzylic carbocation and hence suppresses the latter‟s formation.

As shown in Scheme 2.41, model compounds (37) and (38) were synthesized from commercially available 2-naphthol by treatment with benzyl bromide and para- methoxybenzyl bromide under basic reactions conditions, respectively.71, 124, 125

a

HO BnO 37

b

HO PMBO 38

Scheme 2.41. Synthesis of model compounds: a) K2CO3, BnBr, Me2CO, reflux, 6 h, 80%; b) K2CO3, MPM-Br, Me2CO, reflux, 10 h, 75%.

Scheme 2.42 illustrates the role played by pentamethylbenzene (PMB) in ether cleavage.

When 37 was treated with TFA in the presence of PMB, the reaction exclusively follows the desired path (Line I). This was not the case when it was treated under the same solvent and temp conditions with hexamethylbenzene which thus serves as a negative control (Line II ). In addition, as shown in reaction Line III, deuterium exchange

82 experiment was carried out to see if the proton on „PMB‟ is exchangeable under these same reaction conditions.126, 127

TFA:CH2Cl2 (2:1) + 37 + (I) 4 h, rt HO Bn 39

TFA:CH2Cl2 (2:1) 37 + Side Products + 4 h, rt (II)

H H D

d-TFA:CH2Cl2 (2:1) + (III) 2 h, rt

<10% >90% Scheme 2.42. Role of „PMB‟ in ether cleavage.

Further experiments were then carried out to demonstrate whether deprotection follows an SN1 or SN2 type of displacement (Scheme 2.43). As delineated in Line I (Scheme

2.42), a typical cleavage reaction takes about 4 h to complete. When the naphthol is substituted with a para-methoxybenzyl group (a good electron-donating group) the reaction was complete within 5 min (Line IV, Scheme 2.43). Alternatively, there was no cleavage or decomposition of the 2-para-nitrobenzylnaphthyl ether (which is substituted by a strong electron-withdrawing group) under similar reaction conditions even when the reaction was run for a very prolonged time (Line V, Scheme 2.43).

83 TFA:DCM (2:1) + 38 + (IV) 5 min, rt HO MPM 40

TFA:DCM (2:1) + 18 h, rt + (V) O O

NO2 NO2 '2-Nitrobenzyloxynaphthalene' 'PMB'

Scheme 2.43. Effect of substitution on reaction time.

As evident in Scheme 2.42 and 2.43, there is a large increase in reaction rate upon para-substitution on the benzyl group with an electron-donating group (OMe). In contrast, there is a significant decrease in the rate of cleavage when the benzyl group is substituted with an electron-withdrawing group (NO2). Based on these observations, we propose that it is likely that the reaction predominantly follows a nucleophilic substitution type-I (SN1) pathway. The SN2 and SN1 possibilities are depicted in Schemes 2.42 and

2.43, respectively. Essentially, PMB then plays a key role in quickly trapping the benzyl carbocation so that, overall, the reaction can proceed quite cleanly.

H O O + CF3CO2 + CF3CO2H

H O CF CO H + + CF3CO2 H + 3 2 H HO Bn

Scheme 2.44. Concerted (SN2) type of displacement.

84 O H O + + CF3CO2H + HO CF3CO2 CH2 Benzylic carbocation

H CF CO + 3 2 H + CF3CO2H

CH 2 Bn 21

Scheme 2.45. Stepwise (SN1) type of displacement.

2.6.4 Summary of Studies Related to Peptide Chemistry and Mechanistic Investigations

During the scale-up and process chemistry enhancement studies for preparation of the lead compound CD3-246, I was able to optimize reaction conditions for a multigram scale synthesis using a „Boc/Bzl‟ strategy. Application of the PMB/TFA procedure during the synthesis has clearly demonstrated the utility of this simple and effective protocol for O-debenzylation of sulfur-containing substrates in general, as well as for the specific case of our peptide synthesis. Additionally, an epimerization-free convergent synthesis of the lead compound has also been achieved. This methodology could prove very useful in further „gram to kilogram‟ scale production of the lead compound. Finally, the reaction mechanism for PMB mediated O-debenzylation of phenolic ethers was clarified. Sufficient experimental evidence has been gatherd to support our hypothesis that PMB-mediated debenzylation of phenols predominantly occurs via a nucleophilic substitution type-1 (SN1) pathway.

85

Chapter 3

Experimental

3.1 General Description

Chemical reactions were conducted under nitrogen in anhydrous solvents unless stated otherwise. Anhydrous solvents were purchased from commercial sources and were used without additional purification except for: (i) Acetone (Me2CO) which was further dried over 3 Å molecular sieves; and (ii) Tetrahydrofuran (THF) which was further distilled under nitrogen over sodium-benzophenone. All other reagents obtained from commercial suppliers were used without further purification. Thin-layer chromatography (TLC) was done on 250 μm fluorescent TLC plates (Baker-flex, Silica Gel IB-F from VWR

International, LLC) and visualized by using UV light or iodine vapor. Normal-phase flash column chromatography (CC) was performed using silica gel (200-425 mesh 60 Å pore size) and ACS grade solvents. Chiral high performance liquid chromatography (HPLC) assays were performed on a Waters HPLC system using a Chiralcel OD 25 cm x 0.46 cm column with UV detection at 230 nm. Chiral HPLC retention time is indicated as RT.

Optical rotations were determined on a Rudolph Research model AUTOPOL III automatic polarimeter. Molecular modeling and docking studies were performed by deploying SYBYL 8.0 (from Tripos, Inc.) on a Linux workstation. Melting points (mps) were determined on an Electrothermal digital melting point apparatus and are uncorrected. NMR spectra were recorded on either a Varian Inova-600 spectrometer at

86 600 MHz, or a Unity-400 spectrometer at 400 MHz. Peak locations were referenced using either tetramethylsilane (TMS) or residual nondeuterated solvent as an internal standard. Proton coupling constants are expressed in Hertz. 13C NMR chemical shifts are reported to the first decimal place unless peaks are very close wherein for such instances values are reported to a second decimal place. Spin multiplicity for 1H NMR are reported using the following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, quin = quintet, br = broadened, dd = doublet doublet, dt = doublet triplet, dq = doublet quartet and other combinations derived from those listed. Elemental analyses were performed by Atlantic Microlab, Inc., Norcross, GA, and are regarded as acceptable when experimental values are within ±0.4% of the calculated values.

3.2 Scale-up Synthesis of Glyceollin I

2-Benzyloxy-4-methoxymethylenoxyacetophenone (2)

To a solution of dimethoxymethanal (150.5 g, 1.97 mol) and zinc acetate (43.88 mg, 0.24 mmol) in ethyl acetate (350 ml), acetyl chloride (154.7 g, 1.97 mol) was added over 2-3 h. The reaction mixture was stirred for an additional 2-3 h and then cooled to 0 oC. A solution of 2a (200.0 g, 1.31 mol) in EtOAc (615 mL) was added then slowly, followed by dropwise addition of diisopropylethylamine (211.6 g, 1.63 mol). The reaction mixture was stirred for 18 h while being monitored by TLC. After completion, H2O (300 mL) was added and the mixture further stirred for 1 h. The organic phase was washed with 1M

NaOH, brine and dried over anhyd Na2SO4. Solvents were evaporated under vacuum to obtain a yellowish oily residue (250 g) which was utilized directly in the next step.

87 To a solution of the above product (128.3 g) in Me2CO (1.3 L), benzl bromide (168.6 g, 0.98 mol) and K2CO3 (96.74 g) were added under flow of N2. The reaction mixture was stirred under reflux while progress was monitored by TLC. After completion, the mixture was filtered and solvents were evaporated under vacuum. The product was recrystallized from MeOH to obtain 2 (249.0 g, 0.97 mol, 74% in 2 steps) as off-white

o 42 o 1 crystals: mp 71-73 C (lit. mp 70-71 C); TLC Rf = 0.63 [EtOAc/hexanes (1:2)]; H

NMR (CDCl3, 600 MHz) δ 7.82 (1H, d, J = 8.4 Hz), 7.40 (5H, m), 6.68 (2H, m), 5.19

(2H, s), 5.14 (2H, s), 3.47 (3H, s), 2.55 (3H, s).

1-(2’-Benzyloxy-4’-methoxymethylenoxy)phenyl-2-iodoethanone (3)

To a solution of acetophenone 2 (124.26 g, 0.43 mol) in anhyd CH2Cl2 (280 mL) and anhyd MeOH (1.7 L), SelectfluorTM (100 g, 0.26 mol) was added followed by elemental iodine (49.88 g, 0.22 mol) under a flow of N2. The reaction mixture was stirred for 24 h.

The reaction progress was monitored by TLC and 1H NMR. After completion, the mixture was filtered and and the ppt was washed with CH2Cl2 extensively. The combined organic phase was evaporated under vacuum at 25-30 oC. The residue was again dissolved in CH2Cl2. The organic layer was washed with freshly prepared sodium thiosulfate solution (10% w/v, 3x125 mL), dried over anhyd Na2SO4 and evaporated under vacuum. The residue was recrystallized from MeOH to obtain 3 (108.1g, 0.26 mol,

42 o 61%) as yellowish crystals: mp 76–78 °C (lit. mp 66-68 C); TLC Rf = 0.65

1 [EtOAc/hexanes (1:2)]; H NMR (CDCl3, 600 MHz) δ 7.88 (1H, d, J = 9.6 Hz), 7.49 (2H, m), 7.41 (3H, m), 6.71 (2H, m), 5.20 (2H, s), 5.17 (2H, s), 4.40 (2H, s), 3.48 (3H, s).

88 2-(5’-Benzyloxy-2’-hydroxymethyl)phenoxy-1-(2’-benzyloxy-4’-methoxy-methy- lenoxy)phenylethanone (4)

To a solution of α-iodo ketone 3 (115.7 g, 0.28 mol) and salicyl alcohol 1 (107.5 g, 0.46 mol) in Me2CO (1.4 L), K2CO3 (46.32 gm, 0.34 mol) was added. The reaction mixture was stirred at reflux for 18-20 h. Reaction progress was followed by TLC and 1H NMR.

After completion, solvents were evaporated under vacuum and residue was dissolved in

EtOAc/H2O (1:1) mixture. The organic layer was washed with 0.1 M HCl, saturated

NaHCO3, H2O, brine and dried over anhyd Na2SO4 and evaporated under vacuum. The solid residue was recrystallized from EtOAc/hexane (700 mL, 1:1) to obtain 4 (94.0 g,

42 o 0.18 mol, 65%) as white solid: mp 122.3-124.2 °C [lit. mp 115-118 C]; TLC Rf = 0.3

1 [EtOAc/Hexane (1:2)]; H NMR ((CD3)2CO, 400 MHz) δ 7.87 (1H, d, J = 8.8 Hz), 7.64

(2H, d, J = 7.6 Hz), 7.41 (6H, m), 7.33 (1H, m), 7.24 (2H, m), 6.93 (1H, d, J = 2 Hz),

6.77 (1H, dd, J = 2, 8.8 Hz), 6.56 (1H, dd, J = 2.4, 8.4 Hz), 6.26 (1H, m), 5.32 (2H, s),

5.31 (2H, s), 5.22 (2H, s), 4.57 (2H, d, J = 6.4 Hz), 4.11 (1H, t, J = 6.4 Hz), 3.45 (3H, s).

2’,7-Dibenzyloxy-4’-(t-butyldimethylsilyloxy)-isoflav-3-ene (5)

To a solution of 2‟,7-dibenzyloxy-4‟-(methoxymethalenoxy)-isoflav-3-ene (1.0 g, 2.0 mmol) in anhyd CH2Cl2 (10 mL), triphenylphosphine hydrobromide salt (0.74 g, 2.2 mmol) was added. The reaction mixture was stirred at rt for 1-2 h while followed by TLC

(EtOAc/hexanes 1:2). After completion, Et3N (0.37 g, 3.6 mmol) and TBDMS-Cl (0.35 g, 2.3 mmol) were added. The reaction was stirred at rt for 12-15 h. After completion, solvents were evaporated under vacuum at 30 oC. The solid residue was dissolved in

76 o CH2Cl2 (100 mL) and 15 g of silica reagent (NaHSO4/SiO2, pre-activated at 120 C for

89 48 h) was added to it. The mixture was stirred vigorously for ca. 1 h. The filtrates were passed through a pad of silica. The solvents were evaporated under vacuum and the residue was recrystallized from CH2Cl2/MeOH (1:5) to obtain 5 (0.77 g, 1.4 mmol, 70%)

o 42 o as white crystals: mp 104-106 C [lit. mp 106-107 C]; TLC Rf = 0.42 [EtOAc/hexanes

1 (1:2)]; H NMR ((CD3)2CO, 600 MHz) δ 7.40 (10H, m), 7.25 (1H, d, J = 8.4 Hz), 7.02

(1H, d, J = 8.4 Hz), 6.62 (2H, m), 6.57 (1H, dd, J = 2.4, 8.4 Hz), 6.51 (1H, dd, J = 2.4,

8.4 Hz), 6.47 (1H, d, J = 2.4 Hz), 5.15 (2H, s), 5.10 (2H, s), 4.94 (2H, d, J = 1.2 Hz), 0.97

13 (9H, s), 0.19 (6H, s); C NMR ((CD3)2CO, 150 MHz) δ 160.4, 158.1, 157.5, 155.6, 138.2,

137.9, 130.0, 129.8, 129.3, 129.2, 128.7, 128.57, 128.52, 128.3, 128.29, 128.28, 121.8,

118.1, 113.1, 108.9, 105.9, 102.9, 90.1, 70.9, 70.4, 68.9, 25.9, -4.3, -11.4; Anal. (%) calcd for C17H18O7, C 76.30, H 6.95, found, C 76.02, H 7.09.

(+)-4′-t-Butyldimethylsilyloxy-2′,7-(dibenzyloxy)isoflavan-3,4-diol (6)

To a solution chiral ligand (DHQD)2PHAL (0.86 g, 1.1 mmol) in CH2Cl2 (5 mL), OsO4

(0.25 g, 0.98 mmol) was added. After stirring at -20 °C for 1 h, a solution of 5 (0.48 g,

0.87 mmol) in CH2Cl2 (10 mL) was slowly added over 10-15 min and the mixture was stirred at -20 °C for 18-20 h. The reaction progress was monitored by TLC. After completion, the reaction was allowed to warm to rt, and 10% sodium sulfite (15 mL, pH

∼9.0) and 10% sodium bisulfite (15 mL, pH ∼4) solution was added. After stirring at rt for 2 h, a mixture of THF/EtOAc (1:4, 50 mL) was added to the reaction mixture and further stirred at 55 °C (external oil bath temp) for additional 3-4 h. The reaction mixture was cooled to rt and filtered. The aq phase was extracted with EtOAc (3x25 mL). The combined organic phase was washed wit 0.1 M HCl, brine and dried over anhyd Na2SO4

90 and evaporated under vacuum. The product was recrystallized from EtOAc/hexanse to

25 obtain 6 (0.44 g, 0.75 mmol, 86%, >98% ee) as a white solid: mp 75-77 °C; [α] D +6.7 (c

1.6, MeOH); TLC Rf = 0.28 [EtOAc/hexanes (1:3)]; Chiral HPLC RT = 10.35 min

[Standard racemate RT = 10.38 and 15.31 min], mobile phase was 2-propanol/hexanes

1 (25:75) at 1.0 mL/min; H NMR ((CD3)2CO, 600 MHz) δ 7.59 (1H, dd, J = 2.4. 8.4 Hz,

Ar-H5), 7.39 (11H, m), 6.58 (2H, m), 6.49 (1H, d, J = 8.4 Hz, 3J = 2.4Hz), 6.38 (1H, d, J

= 2.4 Hz), 5.52 (1H, d, J = 6.6 Hz, H4), 5.20 (2H, s), 5.07 (2H, s), 4.73 (1H, d, J = 11.4

Hz), 4.26 (1H, m), 4.21 (1H, m), 4.02 (1H, d, J = 11.4 Hz), 0.96 (9H, s), 0.17 (6H, s); 13C

NMR ((CD3)2CO, 100 MHz) δ 159.9, 157.4, 157.2, 155.7, 138.4, 137.8, 130.7, 130.1,

129.3, 129.2, 128.6, 128.6, 128.4, 128.2, 128.1, 123.5, 118.3, 112.4, 108.7, 106.1, 102.1,

72.0, 70.8, 7.2, 67.6, 67.4, 25.9, 18.6, -4.3; Anal. (%) calcd for C35H40O6Si, C 71.89, H

6.89, found, C 71.83, H 6.92.

1,4-Dichlorophthalazine (7b)

A flame dried (under strong flow of N2) 500 mL three-neck round bottom flask, equipped with a condenser and a mechanical stirrer, was loaded with commercially available phthalhydrazide (8.1 g, 50 mmol), phosphorous pentachloride (21.8 g, 105 mmol), and catalytic DMF (ca. 12 μL). The condenser was fitted with a calcium chloride drying tube.

Temperature of the solid mixture was gradually increased to 145 °C (external oil bath temp.) with gentle heating. The mixture started melting at around 130 °C and turned orange. The liquefied mixture was heated with constant mechanical stirring for ca 7 h.

After cessation of HCl gas evolution, the condenser was replaced with a distillation apparatus to distill off the phosphorus oxychloride. After cooling to rt the residual off-

91 white solid was reduced to fine powder and was dissolved in CH2Cl2 (ca. 120 mL) and was stirred at rt. After 1 h the solution was filtered and the filtrate was added to 25 g neutral alumina (activity 1, fisher scientific). The combined organic phase was dried over anhyd Na2SO4 and evaporated under vacuum to obtain an off-white solid residue and was recrystalized from THF to obtain 7b (8.0 g, 40.2 mmol, 81%) as white crystals: mp

128 o 165.7–166.5 °C (Lit. mp 162-163.5 C); TLC Rf = 0.22 [CH2Cl2]; Anal. (%) calcd for

C8H4Cl2N2, C, 48.28, H, 2.03, N, 14.07, found C, 48.39, H, 1.89, N, 14.08.

1,4-Bis(9-O-dihydroquinidinyl)phthalazine ((DHQD)2PHAL)

To a solution of dihydroquinidine (5.0 g, 15.3 mmol) and 1,4-dichlorophalazine 7b (1.5 g, 7.8 mmol) in anhyd toluene (50 mL), K2CO3 (3.2 g, 22.9 mmol) was added under a flow of N2. The reaction vessel was fitted with a Dean-Stark-condenser and the reaction mixture was refluxed for 2 h at 135 °C (external oil bath temp). Then, KOH pellets (1.5 g, 22.9 mmol) were added to the reaction mixture and the reaction mixture was refluxed

(with azeotropic removal of water) for additional 12-16 h. The reaction was followed by

TLC. After completion, the orange solution was cooled to rt and 15-20 mL of H2O was added. The aq phase was extracted with EtOAc (3x20 mL). The combined organic phase was washed with water, brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum. The yellowish solid residue was dissolved in absolute EtOH (250 mL) and to this a solution of sulfuric acid (ca. 5 g) in absolute EtOH (50 mL) was added slowly with vigorous stirring. After 30 min at rt, white ppt was formed which was filtered and washed with a small amount of cold absolute EtOH (5 mL) and with Et2O (25-30 mL). The precipitate was dissolved in water (ca. 25 mL) and was basified with saturated sodium

92 bicarbonate (pH 9-10). After extracting with EtOAc (3x25 mL), the organic phase was dried over anhyd Na2SO4. The solvents were removed under vacuum to obtain

128 DHQD)2PHAL (3.5 g, 4.5 mmol, 59%) as off-white solid: mp 114-133 °C [Lit. mp

o 1 133-135 C]; TLC Rf = 0.21 [MeOH/CH2Cl2 (1:151) and one drop of NEt3]; H NMR

(400 MHz, CDCl3) δ 8.64 (2H, d, J = 4.2 Hz), 8.31 (2H, m,), 7.97 (2H, d, J = 9 Hz), 7.91

(2H, m), 7.55 (2H, d, J = 3.0 Hz), 7.43 (2H, d, J = 4.8 Hz), 7.35 (2H, dd, J = 2.4, 9.0 Hz),

6.95 (2H, d, J = 6.6 Hz), 3.89 (6H, s), 3.40 (2H, q, J = 7.8 Hz), 2.72 (8H, m), 1.94 (2H, m), 1.68 (2H, s), 1.53 (4H, m), 1.42 (8H, m), 0.79 (6H, t, J = 7.2 Hz); Anal. (%) calcd for

C48H54N6O4·0.5H2O·0.5 C4H8O2, C 72.19, H 7.15, N 10.10, found C, 71.86 H 6.97, N

10.02.

(-)-9-(-t-Butyl dimethylsilyloxy)glycinol (CD3-656)

To a solution of 6 (0.5 g, 0.85 mmol) in anhydrous EtOH (10 mL) at 0 oC, 10% Pd/C (0.1 g) was added. The mixture was stirred at rt for 4 h under hydrogen atmosphere (35 psi) and was followed by TLC. Prolonged reaction times can cause reductions in overall yield. After completion, the reaction mixture was passed through a pad of Celite and was washed with EtOH (3x10 mL). The solvents were evaporated under vacuum to obtain

25 CD3-656 (0.28 g, 0.72 mmol, 85%) as an off-white powder: mp 196-198 °C; [α] D -

1 209.5 (c 0.3, MeOH); TLC Rf = 0.41 [MeOH/CH2Cl2/hexanes (1:10:10)]; H NMR

((CD3)2CO, 600 MHz) δ 8.57 (1H, s), 7.31 (1H, d, J = 8.4 Hz), 7.26 (1H, d, J = 7.8 Hz),

6.56 (1H, dd, J = 8.4 Hz, J = 2.4 Hz), 6.46 (1H, dd, J = 2.4, 8.4 Hz), 6.33 (1H, d, J = 2.4

Hz), 6.27 (1H, d, J = 1.8 Hz), 5.28 (1H, s), 5.03 (1H, s), 4.13 (1H, d, J = 11.4 Hz ), 4.01

13 (1H, d, J = 11.4 Hz), 0.97 (9H, s), 0.20 (6H, s); C NMR ((CD3)2CO, 150 MHz) δ 161.6,

93 159.6, 158.6, 157.0, 133.1, 125.0, 123.6, 113.3, 113.0, 110.7, 103.7, 103.2, 90.1, 85.8,

76.6, 70.5, 25.9, -4.39, -4.40, -11.45, -11.46; Anal. (%) calcd for C21H26O5Si, C 65.26, H

6.78, found, C 65.75, H 6.76.

9-(t-Butyldimethylsillyloxy)glyceollin I (CD3-699)

To a mixture of 6a-hydroxypterocarpan CD3-656 (1.37g, 3.5 mmol) in anhyd p-xylene

(ca. 20 mL), 3,3-dimethylacryl diethoxy diacetal (1.4 mL, 7.0 mmol) and 3-picoline (90

μL, 0.9 mmol) were added successively under a flow of N2. The reaction flask was assembled with distillation assembly and was stirred at 125 oC (internal temp 120 oC).

Progress of the reaction was followed by TLC. After completion, the reaction mixture was directly applied to a column and was purified by flash column chromatography using a step gradient [first hexanes/CH2Cl2 (2:1), 450 mL then was changed to hexanes/CH2Cl2/EtOAc (20: 10:1) to obtain CD3-699 (1.05g, 2.3 mmol, 65% yield) as

1 off-white solid: mp 69-75 °C; TLC Rf 0.57 [EtOAc/hexanes (1:2)]; H NMR ((CD3)2CO,

600 MHz) δ 7.28 (1H, d, J = 8.4 Hz), 7.24 (1H, d, J = 8.4 Hz), 6.57 (1H, d, J = 10.2 Hz),

6.46 (1H, m), 6.28 (1H, d, J = 2.4 Hz), 5.66 (1H, d, J = 10.2 Hz), 5.28 (1H, s), 5.09 (1H, s, ), 4.20 (1H, d, J = 11.4 Hz), 4.08 (1H, d, J = 11.4 Hz), 1.39 (3H, s), 1.35 (3H, s), 0.97

(9H, s), 0.20 (6H, s); Anal. (%) calcd for C21H26O5Si, C 68.99, H 7.13, found, C 68.91, H

7.27.

9-(t-Butyldimethylsilyloxy)glyceollin II (7)

Intermediate 7 is obtained as regioisomer of CD3-699 during latter‟s synthesis as

1 yellowish to off-white semi-solid: TLC Rf = 0.44 [hexanes/EtOAc (1:1]; H NMR

94 (CD3COCD3, 600 MHz) δ 7.27 (1H, d, J = 7.8 Hz), 7.15 (1H, s), 6.46 (1H, dd, J = 1.8,

7.8 Hz), 6.41 (1H, d, J = 9.6 Hz), 6.28 (1H, d, J = 1.8 Hz), 6.22 (1H, s), 5.66 (1H, d, J =

9.6 Hz), 5.26 (1H, s), 5.07 (1H, s), 4.15 (1H, d, J = 11.4 Hz), 4.03(1H, d, J = 11.4 Hz),

13 1.39 (3H, s), 1.36 (3H, s), 0.97 (9H, s), 0.20 (6H, s); C ((CD3)2CO, 150 MHz) δ 161.0,

158.0, 156.1, 154.6, 129.3, 129.1, 124.4, 121.5, 116.5, 113.5, 112.8, 104.8, 102.6, 100.3,

89.4, 85.0, 84.9, 76.5, 76.0, 69.9, 28.2 25.3, 25.2, 18.0, -5.0; Anal. (%) calcd for

C20H18O5·0.75 H2O, C 68.27, H 5.59, found C 68.36, H 5.59.

(-)-Glyceollin I (GLY I)

To a solution of CD3-699 (0.75 g, 1.7 mmol) in CH2Cl2 (30 mL), Et3N.3HF (30 mmol) and excess pyridine (45 mmol) were added. The reaction mixture was stirred at rt for 6 h and was followed by TLC. After completion, the mixture was concentrated to half of its original volume under vacuum at 20 oC and then was directly applied to the column gravity column chromatography (silica ca. 20 g, EtOAc/hexanes (1:1)). The eluting fractions were collected and solvents were removed under vacuum and resulting residue was lyophilized after dissolving in minimal amount of MeOH to obtain GLY I (0.47 g,

o 25 1.4 mmol, 83%) as pinkish brown solid: mp 95-101 C; [α] D -202.6 (c 0.15, EtOAc)

TLC Rf = 0.33 (MeOH /CH2Cl2/hexanes (1:10:10)); Chiral HPLC RT = 11.75 min

[Standard racemate RT = 11.74 and 13.51 min], mobile phase was 2-propanol/hexanes

1 (10:90) at 1.5 mL/min; H NMR (CD3OD, 600 MHz) δ 7.21 (1H, d, J = 8.4 Hz), 7.16

(1H, d, J = 8.0 Hz), 6.60 (1H, d, J =10 Hz), 6.46 (1H, d, J = 8.4 Hz), 6.40 (1H, dd, J =

2.0, 8.4 Hz), 6.22 (1H, d, J = 2 Hz), 5.62 (1H, d, J = 10 Hz), 5.16 (1H, s), 4.16 (1H, d, J =

95 11.6 Hz), 3.93 (1H, d, J = 11.2 Hz), 1.38 (3H, s), 1.35 (3H, s); Anal. (%) calcd for

C20H18O5·0.1H2O, C 70.62, H 5.39, found, C 70.35, H 5.65.

3.2.1 GLY I Analogs

2-(5’-hydroxy,2’-hydroxymethyl)phenoxy-1-(2’-hydroxy-4’-methyoxy-methylen- oxy)-phenylethanone (CD3-649)

To a solution of 4 (0.4 g, 0.77 mmol) in EtOAc (50 mL) at 0 oC, 10% Pd/C (100 mg) was added. The mixture was stirred at rt for 8-10 h under hydrogen atmosphere (35 psi) and was followed by TLC. After completion, the reaction mixture was passed through a pad of Celite which was washed extensively with EtOAc. The solvents were evaporated under vacuum to obtain CD3-649 (0.23 g, 0.69 mmol, 90%) as off-white solid: mp 140-

o 1 150 C; TLC = 0.59 [MeOH/CH2Cl2 (1:9)]; H NMR ((CD3)2CO, 600 MHz) δ 8.02 (1H, d, J = 9.0 Hz), 7.17 (1H, d, J = 7.8 Hz), 6.65 (1H, dd, J = 2.4, 9.0 Hz), 6.59 (1H, d, J =

2.4), 6.46 (1H, d, J = 1.8 Hz), 6.42 (1H, dd, J = 1.8, 7.8 Hz), 5.48 (2H, s), 5.30 (2H, s),

13 4.61 (2H, s), 3.83 (1H, br), 3.45 (3H, s); C NMR ((CD3)2CO, 150 MHz) δ 199.8, 164.9,

158.5, 157.5, 132.4, 129.8, 122.7, 113.2, 109.3, 108.1, 104.1, 100.7, 94.7, 90.1, 70.2,

60.4, 56.47; Anal. (%) calcd for C17H18O7, C 61.07, H 5.43, found C 60.76, H 5.43.

2-(5’-Benzyloxy-2’-hydroxymethyl)phenoxy-1-(2’-benzyloxy-4’-hydroxy)-phenyl- ethanone (CD3-650)

To solution of 4 (0.51 g, 1 mmol) in CH2Cl2 (7 mL) and Et2O (30 mL), MgBr2·etharate

(1.3 g, 5 mmol) was added. To the mixture ethanthiol (0.12 mL, 2.6 mmol) was added.

The reaction mixture was stirred at rt and was followed by TLC. After completion, the

96 reaction mixture was filtered and the white precipitates were washed with Et2O (2-3 times) and dried. The traces of ethanethiol were removed by a quick pad filtration using

EtOAc/hexane (1:3). The solvents were removed under vacuum to obtain CD3-650 (0.25

o g, 0.53 mmol, 53%) as white solid: mp 161-165 C; TLC Rf = 0.63 [MeOH/CH2Cl2

1 (1:10)]; H NMR (d6-DMSO, 600 MHz) δ 10.53 (1H, s), 7.70 (1H, d, J = 9 Hz), 7.55

(2H, m), 7.36 (7H, m), 7.23 (2H, m), 6.62 (1H, d, J = 1.8 Hz), 6.56 (1H, dd, J = 2.4, 8.4

Hz), 6.50 (1H, dd, J = 1.8, 8.4 Hz), 6.15 (1H, d, J = 1.8 Hz), 5.20 (2H, s), 5.12 (2H, s),

13 4.96 (2H, s), 4.85 (1H, t, J = 6.0 Hz), 4.45 (2H, d, J = 6.0 Hz); C NMR (d6-DMSO, 150

MHz) δ 192.3, 163.9, 160.7, 158.2, 155.8, 137.0, 136.1, 132.2, 128.6, 128.3, 128.23,

128.21, 127.9, 127.8, 123.1, 116.4, 108.6, 104.9, 100.0, 99.7, 73.2, 70.2, 69.2, 57.8, -

12.2; Anal. (%) calcd for C29H26O6, C 74.03, H 5.57, found, C 74.17, H 5.76.

2’,7-Dihydroxy-4’-t-butyldimethylsilyloxyisoflavane (CD3-653)

To a chilled solution of 5 (0.1 g, 0.18 mmol) in ethyl acetate (10 mL) 10% w/w Pd/C (ca.

20 mg, 20 % w/w) was added. The mixture was stirred at rt for 12-14 h under hydrogen atmosphere (35 psi) and was followed by TLC. After completion, the reaction mixture was passed through a pad of Celite which was washed extensively with EtOAc. The solvents were dried over anhyd Na2SO4 and evaporated under vacuum. The residue was purified by flash chromatography to obtain CD3-653 (50 mg, 0.13 mmol, 74%) as off-

o 1 white solid: mp 148-157 C; TLC Rf = 0.44 [MeOH/CH2Cl2 (1:15)]; H NMR ((CD3)2CO,

600 MHz) δ 8.43 (1H, s), 8.08 (1H, s), 7.01 (1H, d, J = 8.4 Hz), 6.89 (1H, d, J = 7.8 Hz),

6.49 (1H, d, J = 2.4 Hz), 6.36 (2H, m), 6.27 (1H, d, J = 2.4 Hz), 4.24 (1H, m), 3.97 (1H, t, J = 10.2 Hz), 3.47 (1H, m), 2.96 (1H, m), 2.81 (1H, m), 0.97 (9H, s), 0.20 (6H, s); 13C

97 NMR ((CD3)2CO, 150 MHz) δ 157.4, 156.5, 156.0, 155.9, 130.9, 128.6, 121.6, 114.2,

112.1, 108.7, 108.0, 103.6, 70.3, 32.6, 30.9, 25.9, 18.7, -4.3; Anal. (%) calcd for

C21H28O4Si, C 67.71, H 7.58 found, C 67.76, H 7.77.

2’,7-Dibenzyloxy-4’-hydroxyisoflavan-3-ene (CD3-654)

To a solution of 5 (0.1 g, 0.18 mmol) in CH2Cl2 (5 mL), Et3N·3HF (1.8 mmol) was added. The reaction mixture was stirred at rt for 4-6 h and was followed by TLC. After completion, the mixture was passed through a pad of silica (15 g, 15 mm dia) and was washed with mixture of EtOAc/hexanes (1:1, 3x20 mL). The solvents were evaporated under vacuum to obtain, CD3-654 (50 mg, 0.14 mmol, 63%) as off-white solid: mp 57-62 o 1 C; TLC Rf = 0.65 [EtOAc/hexanes (1:1)]; H NMR ((CD3)2CO, 600 MHz) δ 8.58 (1H, s), 7.40 (10H, m), 77.19 (1H, d, J = 7.8 Hz), 7.00 (1H, d, J = 8.4 Hz), 6.60 (1H, d, J =

2.4), 6.5 6 (2H, m), 6..47 (2H, m), 5.11 (2H, s), 5.09 (2H, s), 4.92 (2H, d, J = 1.8 Hz); 13C

NMR (CD3OD, 150 MHz) δ 159.4, 158.5, 157.6, 154.7, 137.3, 137.1, 129.6, 129.2,

128.3, 128.2, 127.7, 127.6, 127.5, 127.4, 127.2, 120.3, 119.7, 117.6, 107.9, 107.7, 102.1,

100.4, 70.2, 69.8, 68.4; Anal. (%) calcd for C29H24O4·0.2H2O, C 79.14, H 5.59, found, C

78.77, H 5.52.

2’,4’,7-Trihydroxyisoflavane (CD3-714)

To a solution of CD3-653 (0.2 g, 0.53 mmol) in MeOH (1 mL) and CH2Cl2 (6 mL),

Et3N.3HF (5 mmol) in CH2Cl2 was added. The reaction mixture was stirred at rt for 8 h and was followed by TLC. After completion, the mixture was reduced to half of its original volume under vacuum at 20 oC and then was directly applied to the column for a

98 quick pad filtration (silica ca. 10 g, eluted first with hexane then with EtOAc). The combined organic phase was evaporated under vacuum. the semisolid residue was lyophilized to obtain CD3-714 (0.110 g, 0.38 mmol, 73%) as an off-white solid powder:

o 1 mp > 170 C; TLC Rf = 0.23 [MeOH/CH2Cl2 (1:11)]; H NMR (CD3OD, 600 MHz) δ

6.86 (2H, m), 6.30 (2H, m), 6.24 (1H, m, dd, J = 2.4, 7.6 Hz), 6.21 (1H, d, J = 2.4 Hz),

4.21 (1H, m), 3.91 (1H, t, J = 10.2 Hz), 3.40 (1H, m), 2.92 (1H, m), 2.76 (1H, m); 13C

NMR (CD3OD, 100 MHz) δ 157.8, 157.4, 157.1, 156.3, 131.2, 128.7, 120.1, 115.0,

108.9, 107.6, 103.7, 103.5, 71.2, 33.0, 31.4; Anal. (%) calcd for

C15H14O4·0.2H2·0.2CH4O·0.01C4H8O2, C 69.61, H 5.86, found C 69.27, H 6.26.

2’,7-Dibenzyloxy-3,4,4’-trihydroxyisoflavane (CD3-667)

To a solution of 6 (0.12 g, 0.2 mmol) in CH2Cl2 (6 mL), Et3N.3HF (5 mmol) was added.

The reaction mixture was stirred at rt for 12 h. and was followed by TLC. After completion, the mixture was reduced to half of its original volume under vacuum at 20 oC and then was directly applied to the column for column chromatography (silica ca. 20 g,

(MeOH/CH2Cl2 (1:20)). The solvents were removed under vacuum to obtain CD3-667

o (75 mg, 0.15 mmol, 74%) as an off white solid: mp 83-95 C; TLC Rf = 0.33

1 [MeOH/CH2Cl2 (1:20)]; H NMR ((CD3)2CO, 600 MHz) δ 8.80 (1H, br),7.50 (1H, d, J =

9.0 Hz), 7.46 (4H, m), 7.3 (11H, m), 6.57 (1H, d, J = 2.4 Hz), 6.55 (1H, dd, J = 2.4, 8.4

Hz), 6.43 (1H, dd, J = 2.4, 9.0 Hz), 6.35 (1H, d, J = 2.4 Hz), 5.48 (1H, d, J = 6 Hz), 5.13

(2H, s), 5.05 (2H, s), 4.68 (1H, d, J = 11.4 Hz), 4.37 (1H, d, J = 6.6 Hz), 4.20 (1H, s),

13 4.00 (1H, d, J = 11.4 Hz); C (CD3OD, 150 MHz) δ 160.5, 159.6, 158.1, 156.1, 138.7,

138.2, 130.9, 130.2, 129.6, 129.4, 128.9, 128.7, 128.5, 128.4, 121.3, 118.5, 109.3, 108.3,

99 102.7, 101.8, 72.6, 71.2, 70.8, 70.7, 68.4; Anal. (%) calcd for C29H26O6·1.2H2O·0.1

CH2Cl2, C 69.82, H 5.76, found C 69.50, H 5.36.

2’,3,4,4’,7-Pentahdyroxyisoflavane (6a)

To a solution of CD3-667 (50 mg, 0.13 mmol) in anhyd EtOH (5 mL) at 0 oC, 10% Pd/C

(15 mg) was added. The mixture was stirred at rt for 6 h under hydrogen atmosphere (35 psi) and was followed by TLC. After completion, the reaction mixture was passed through a pad of Celite which was washed extensively with EtOAc. The solvents were evaporated under vacuum. The residue was purified by flash column chromatography

[MeOH/ CH2Cl2 (1:9)] to obtain 6a (25 mg, 90 μmol, 66%) as brownish semisolid: TLC

1 Rf = 0.15 [MeOH/CH2Cl2 (1:9)]; H NMR (CD3OD, 600 MHz) δ 7.16 (2H, m), 6.38 (1H, dd, J = 2.4, 8.4 Hz), 6.27 (1H, d, J = 1.8 Hz), 6.22 (2H, m), 5.13 (1H, s), 4.47 (1H, d, J =

13 11.4 Hz), 4.09 (1H, d, J = 11.4 Hz); C NMR (CD3OD, 150 MHz) δ 159.3, 159.2, 157.8,

155.9, 131.5, 128.9, 118.8, 116.5, 109.7, 107.6, 104.5, 103.2, 73.3, 69.9, 69.1.

4’-t-Butyldimethylsilyloxy-2’,3,4,7-tetrahydroxyisoflavane (CD3-666)

To a chilled solution of 6 (60 mg, 0.1mmol) in Me2CO, 10% w/w Pd/C (ca. 15 mg) was added. The mixture was stirred at rt for 10-12 h under hydrogen atmosphere (35 psi) and was followed by TLC. After completion, the reaction mixture was passed through a pad of Celite which washed extensively with EtOAc. The solvents were evaporated under vacuum to obtainThe reaction mixture was set up on Parr hydrogenator under H2 (30 psi) atmosphere at rt. The reaction was complete after 10-12 h. After completion, the mixture was passed through a pad of celite and the pad was washed with MeOH (2x5 mL). The

100 combined organic solvents were evaporated under vacuum. The crude was purified by column chromatography (silica gel, [MeOH/ CH2Cl2/hexanes (1:10:10)] to obtain CD3-

666 (30 mg, 73 μmol, 72%) as off-white solid: mp 92 oC turns glassy and gets darker

o 1 then melts at 190-195 C; TLC Rf = 0.14 [MeOH/CH2Cl2/hexanes (1:10:10)]; H NMR

(CD3OD, 600 MHz) δ 7.24 (1H, d, J = 8.4 Hz), 7.18 (1H, d, J =8.4 Hz ), 6.39 (1H, dd, J

= 2.4, 8.4 Hz), 6.32 (1H, d, J = 2.4 Hz), 6.29 (1H, dd, J = 2.4, 8.4 Hz), 6.23 (1H, d, J =

2.4 Hz), 5.17 (1H, s), 4.52 (1H,d, J = 11.4 Hz), 4.07 (1H, d, J = 11.4 Hz), 0.97 (9H, s),

13 0.18 (6H, s); C NMR (CD3OD, 100 MHz) δ 159.0, 157.6, 155.9, 131.3, 129.0, 125.0,

121.1, 116.7, 112.2, 109.74, 109.24, 103.1, 73.2, 70.1, 69.0, 50.5, 26.1, 19.0, -4.3; Anal.

(%) calcd for C21H28O6Si, C 62.35, H 6.98, found C 62.10, H 6.74.

(-)-Glycinol (CD3-523)

To a solution of CD3-656 (25 mg, 65 μmol) in CH2Cl2/MeOH (1.2 mL, 5:1), Et3N·3HF

(33 μL, 195 μmol) buffered to pH 5-6 with excess pyridine was added. The reaction mixture was stirred at rt for 10 h. The reaction was followed by TLC. After completion, the mixture was directly applied to column chromatography [ca. 10 g silica gel;

MeOH/CH2Cl2 (1:10)]. The eluting solvents were evaporated under vacuum, and the residue was lyophilized to obtain CD3-523 (14 mg, 51 μmol, 78%) as a yellow solid: mp

25 108-112 °C; [α] D -221.0 (c 0.3, MeOH); TLC Rf = 0.49 [EtOAc/hexanes (7:3)]; Chiral

HPLC RT = 17.92 min [Standard racemate RT = 15.60 and 18.30 min], mobile phase was

2-propanol/hexanes (15:85) at 1.5 mL/min 1H NMR ((CD3)2CO, 600 MHz) δ 8.55 (1H, s), 8.47 (1H, s), 7.30 (1H, d, J= 9 Hz), 7.20 (1H, d, J = 7.8 Hz), 6.55 (1H, dd, J = 2.4, 8.4

Hz), 6.42 (1H, dd, J = 2.4, 8.4 Hz), 6.31 (1H, d, J = 2.4 Hz), 6.24 (1H, d, J = 2.4 Hz),

101 5.26 (1H, s), 4.95 (1H, s), 4.11 (1H, d, J = 11.4 Hz), 4.02 (1H, d, J = 11.4 Hz); 13C NMR

(CD3OD, 150 MHz) δ 162.1, 161.1, 160.0, 157.3, 133.2, 125.1, 121.2, 113.0, 111.0,

109.2, 104.0, 98.9, 85.9, 77.2, 70.2; HREIMS m/z calcd for C15H12O5 272.0685, found

272.0678; Anal. (%) calcd for C5H12O5·0.5H2O·0.4CH4O, C 62.90, H 5.00, found C

63.16, H 5.38.

6a,11a-Dehydroglycinol (CD3-640)

To a solution of CD3-640 (50 mg, 0.13 mmol) in MeOH/CH2Cl2 (1:5, 6 mL), excess HF

(0.1 mL) and Et3N 3HF (0.1 mL) were added. The reaction mixture was stirred for 4-5 h at rt. Reaction progress was monitored by TLC. After completion the mixture was directly passed through a pad of silica (3 cm thick, 35 mm dia) and washed with

EtOAc/hexanes (1:1, 50 mL). The filtrate was dried over anhyd Na2SO4 and evaporated under vacuum. The residue was purified by flash column chromatography

[EtOAc/hexanes (7:3)] to obtain CD3-640 (20mg, 80 μmol, 60%) as an off-white solid:

o 1 mp 195-200 C; TLC Rf = 0.68 [EtOAc/hexanes (7:3)]; H NMR ((CD3OD, 600 MHz) δ

7.25 (1H, d, J = 7.8 Hz), 7.20 (1H, d, J = 8.4 Hz), 6.92 (1H, d, J = 1.8 Hz), 6.74 (1H, dd,

J = 8.4 Hz, J = 1.8 Hz), 6.42 (1H, d, J = 2.4, 7.8 Hz), 6.35 (1H, d, J = 2.4 Hz), 5.49 (2H,

13 s); C NMR (CD4OD, 150 MHz) δ 160.0, 157.7, 156.5, 156.4, 148.1, 121.8, 119.6,

119.5, 113.1, 110.0, 109.0, 106.6, 104.6, 99.1, 66.3; HREIMS predicted 254.0579 for

C15H10O4, found, 254.0575; Anal. (%) calcd for C15H10O4·0.2 H2O·0.2CH3OH, C 69.09,

H 4.27; found, C 68.77, H 4.26.

102 6a,11a-Dehydroglyceollin I (CD3-639)

To a solution of CD3-699 (50 mg, 0.11 mmol) in CH2Cl2 (5 mL), excess HF (0.1 mL) and Et3N·3HF (0.1 mL) were added. The reaction mixture was stirred at rt for 4-5 h.

Reaction progress was followed by TLC. After completion the mixture was directly passed through a pad of silica (3 cm thick, 35mm dia) and washed with EtOAc/hexanes

(1:1, 50 mL). After evaporation of solvents under vacuum, the residue was purified using column chromatography [EtOAc/hexanes (1:1)] to obtain CD3-639 (27mg, 80 μmol,

o 1 76%) as off-white solid: mp >170 C; TLC Rf = 0.67 [EtOAc/hexanes (1:1)]; H NMR

((CD3OD, 600 MHz) δ 7.18 (1H, d, J = 8.4 Hz), 7.15 (1H, d, J = 7.8 Hz), 6.90 (1H, d, J =

1.8 Hz), 6.74 (1H, dd, J = 1.8, 8.4 Hz), 6.62 (1H, d, J = 10.2 Hz), 6.36 (1H, d, J = 8.4

13 Hz), 5.56 (1H, d, J = 10.2 Hz), 5.51 (2H, s), 1.39 (6H, s); C NMR (CD4OD, 100 MHz)

δ 157.7, 156.6, 155.1, 150.4, 147.8, 130.9, 120.7, 119.6, 119.4, 117.4, 113.1, 111.5,

110.9, 110.2, 106.9, 99.1, 77.1, 66.6, 28.0; Anal. (%) calcd for C20H16O4, C 74.99, H

5.03, found C 74.98, H 59.

3,4-Dihydroxyglabridin (CD3-698)

To a chilled solution of CD3-639 (20 mg, 62 μmol) in MeOH (3-5 mL), 10% w/w Pd/C

(5 mg) was added. The mixture was stirred at rt for 10-12 h under hydrogen atmosphere

(35 psi) and was followed by TLC. After completion, the reaction mixture was passed through a pad of Celite which washed extensively with MeOH. The solvents were dried over anhyd Na2SO4 and evaporated under vacuum. The residue was purified by prep-

TLC [EtOAc/hexanes (1:2)] to obtain CD3-698 (15 mg, 46 μmmol, 74%) as off white

o 1 solid: mp 112-115 C; TLC Rf = 0.23 [EtOAc/hexanes (2:1)]; H NMR (CD3OD, 600

103 MHz) δ 6.86 (1H, d, J = 8.4 Hz), 6.76 (1H, d, J = 8.4 Hz), 6.30 (1H, d, J = 2.4 Hz), 6.24

(2H, m, H4), 4.29 (1H, m), 3.92 (1H, t, J = 10.2 Hz), 3.39 (1H, m), 2.94 (1H, m), 2.74

13 (1H, m), 2.60 (2H, t, J = 6.6 Hz), 1.74 (1H, m), 1.27 (6H, s); C NMR (CD3OD, 150

MHz) δ 157.9, 157.2, 153.8, 153.3, 128.6, 128.4, 120.0, 114.4, 110.11, 110.05, 107.5,

103.4, 74.5, 71.3, 33.4, 33.0, 31.7, 26.9, 26.7, 18.1; Anal. (%) calcd for

C20H22O4·0.3H2O·0.3CH4O, C 71.42, H 7.03, found C 71.80, H 7.42.

(-)-Glyceollin II (GLY II) (Obtained from scale-up synthesis of GLY I)

To a solution of 7 (90 mg, 0.2 mmol) in CH2Cl2 (6 mL), Et3N·3HF (33 μL, 195μmol) buffered to pH 5-6 with excess pyridine was added. The reaction mixture was stirred at rt for 6 h. The reaction was followed by TLC. After completion the mixture was concentrated to half of its original volume under vacuum at 20 oC and then was directly applied to the column for column chromatography (silica ca. 10 g, EtOAc/hexanes:

(1:1)). The solvents were evaporated under vacuum to obtain GLY II (50 mg, 0.15

o 1 mmol, 74%) as off white solid: mp 95-101 C; TLC Rf : 0.44 [EtOAc/hexanes (1:1)]; H

NMR (CD3OD, 600 MHz) δ 7.15 (1H, d, J = 8.4 Hz), 7.09 (1H, s), 6.39 (1H, dd, J = 1.8,

8.4 Hz), 6.36 (1H, d, J = 9.6 Hz), 6.23 (1H, s), 6.22 (1H, d, J = 1.8 Hz), 5.60 (1H, d, J =

10.2 Hz), 5.15 (1H, s), 4.10 (1H, dd, J = 0.6, 11.4 Hz), 3.90 (1H, d, J = 11.4 Hz), 1.38

13 (3H, s), 1.37 (3H, s); C NMR (CD3OD, 150 MHz) δ 162.1, 161.2, 157.1, 155.7, 130.3,

129.8, 125.1, 122.6, 121.1, 117.8, 114.3, 109.4, 105.2, 98.9, 85.7, 77.6, 77.1, 70.9, 28.3,

28.2; Anal. (%) calcd for C20H18O5·0.75H2O, C 68.27, H 5.59, found C 68.36, H 5.59.

104 6a,11a-Dehydroglyceollin II (Dehydro-GLY II)

To a solution of 7 (50 mg, 0.11 mmol) in CH2Cl2 (5 mL), excess HF (0.1 mL) and

Et3N·3HF (0.1 mL) were added. The reaction mixture was stirred for 8-10 h at room temp. Reaction progress was followed by TLC. After completion the mixture was directly passed through a pad of silica (3 cm thick, 35 mm dia) and washed with EtOAc/hexanes

(1:1, 50 mL). After evaporation of solvents under vacuum, the residue was purified using column chromatography [EtOAc/hexanes (1:2)] to obtain Dehydro-GLY II (20 mg, 62

1 μmol, 57%) as yellowish semi-solid: TLC Rf = 0.36 [EtOAc/hexanes (1:2)]; H NMR

((CD3)2CO, 600 MHz) δ 8.61 (1H, br), 7.31 (1H, d, J = 8.4 Hz), 7.13 (1H, s), 7.01 (1H, d,

J = 1.8 Hz), 6.83 (1H, dd, J = 1.8, 8.4 Hz), 6.30 (1H, d, J = 9.6 Hz), 6.31 (1H, s), 5.65

13 (1H, d, J = 9.6 Hz), 5.56 (2H, s), 1.40 (6H, s); C NMR ((CD3)2CO, 150 MHz) δ 157.2,

156.4, 155.6, 155.1, 146.9, 129.5, 122.2, 119.9, 119.1, 118.2, 116.0, 113.1, 110.4, 107.4,

105.2, 99.0, 77.3, 66.0, 28.1.

3.3 Synthesis of Glyeollin II (GLY II)

2,2-Dimethyl-5-hydroxy-2H-1-benzopyran-6-carboxaldehyde (7a)

To a solution of 2,4-dihdroxybenzaldehyde (1c) (1.0 g, 7.24 mmol) in anhyd p-xylene

(ca. 20 mL), 3,3-dimethylacrylic diethoxy diacetal (1.67 mL, 8.83 mmol) and 3-picoline

(176 μL, 1.81 mmol) were added successively under a flow of N2. The reaction mixture was stirred at 125 oC (internal temp 120 oC) for 24 h. The reaction was monitored by

TLC. After completion, the reaction mixture was cooled to rt and was diluted with EtOAc

(30 mL). The reaction mixture was washed with 1 M aq HCl, H2O, brine, dried over anhyd Na2SO4 and dried over anhyd Na2SO4 and evaporated under vacuum to obtain 5

105 (1.4 gm, 6.8 mmol, 95%) as brownish semisolid: TLC Rf = 0.74 [EtOAc/hexanes (1:2)];

1 H NMR (CDCl3, 600 MHz) δ 11.64 (1H, s), 9.65 (1H, s), 7.28 (1H, d, J =8. 4 Hz), 6.67

(1H, d, J = 10.2 Hz), 6.42 (1H, d, J = 8.4 Hz), 5.60 (1H, d, J = 10.2 Hz), 1.46 (6H, s); 13C

NMR (CDCl3, 150 MHz) δ 194.5, 160.5, 158.6, 134.6, 128.5, 115.1, 115.0, 109.3, 108.7,

78.1, 28.6, 28.3; Anal. (%) calcd for C12H12O3·0.075H2O, C 70.11, H 5.96, found, C

70.43, H 6.35.

2,2-Dimethyl-5-hydroxy-4-oxo-6-chromancarboxylic acid, methyl ester (8a)

o To a mixture of methane sulfonic acid (10 mL) and P2O5 (1.5 g) stirred at 70 C for 1 h, methyl-2,4-dihydroxybenzoate (0.6 g, 3.5 mmol) and methyl 3,3-dimethylacrylate(0.42 g,

3.5 mmol) were added sequentially. The reaction mixture was stirred at 70 oC for 4-6 h.

After completion, the mixture was cooled and poured into ice/H2O and was extracted with EtOAc (3x30 mL). The combined organic phase was washed with H2O and brine, dried and evaporated under vacuum. The residue was purified by flash column chromatography to obtain 8a (0.5 g, 2 mmol, 60%) as white solid: mp 101-103 oC [lit.129

o 1 mp 104 C]; TLC Rf = 0.69 [EtOAc/hexanes (1:3)]; H NMR (CDCl3, 600 MHz) δ 11.63

(1H, s), 7.76 (1H, d, J = 8.4 Hz), 6.58 (1H, d, J = 8.4 Hz), 3.94 (3H, s), 2.63 (2H, s), 1.50

13 (6H, s); C NMR (CDCl3, 100 MHz) δ 170.6, 167.2, 161.3, 156.0, 129.7, 118.2, 108.8,

108.6, 52.4, 44.7, 34.2, 27.1.

2,2-Dimethyl-7-hydroxy-4-oxo-6-chromancarboxylic acid, methyl ester (8)

o To a mixture of methane sulfonic acid (240 mL) and P2O5 (15 g) stirred at 70 C for 1h, a powder (mixed and pulverized together for 5-10 min in a „mortar & pastle‟) of methyl-

106 2,4-dihydroxybenzoate (25.2 g, 150 mmol) and methyl 3,3-dimethylacrylate(15 g, 150 mmol) was added in portions. The reaction mixture was stirred at 70 oC for 4-6 h. After completion, the mixture was cooled and poured into ice/H2O and was extracted with

EtOAc (3x150 mL). The combined organic phase was washed with H2O and brine, dried and evaporated under vacuum. The residue was purified by extraction. It was dissolved in

EtOAc then extracted back in aq 2 M NaOH (4x150 mL). The combined aq phase was washed with Et2O and then was neutralized with 1 M HCl to pH 3 and was extracted in

CH2Cl2 (4x50 mL). The combined organic layer was washed with H2O, brine, dried over anhyd Na2SO4 and evaporated under vacuum to obtain 8 (19.6 g, 78.4 mmol, 52%) as

o 130 o off-white solid: mp 133-134 C [lit. mp 132-133 C]; TLC Rf = 0.34 [EtOAc/hexanes

1 (1:6)]; H NMR (CDCl3, 600 MHz) δ 11.29 (1H, s), 8.47 (1H, s), 6.44 (1H, s), 3.94 (3H,

13 s), 2.70 (2H, s), 1.46 (6H, s); C NMR (CDCl3, 150 MHz) δ 190.5, 170.0, 167.3, 165.1,

131.0, 113.7, 107.2, 104.73, 104.70, 80.3, 52.3, 48.5, 26.7; Anal. (%) calcd for C13H14O5,

C 62.39, H 5.64, found C 62.58, H 5.78.

6-Bromo-2,2-dimethyl-7-hydroxy-4-chromanone (A’)

A properly mixed and powdered mixture of 4-bromoresorcinol (2.0 g, 10.6 mmol) and

3,3-dimethylacrylic acid (1.1 g, 10.6 mmol) was added to polyphosphoric acid (10 g) at

90 oC in portions. The reaction mixture was stirred at 90 oC for 1-2 h. After completion,

H2O (50 mL) was added to the mixture afer cooling to rt. The aq phase was extracted with Et2O (4x30 mL). The combined organic phase was washed with 10% NaHCO3, brine and dried over anhyd Na2SO4. The solvents were evaporated under vacuum to obtain A’ (1.2 g, 4.4 mmol, 42%) as off-white solid: TLC Rf = 0.5 [EtOAc/hexanes

107 1 (1:2)]; H NMR (CDCl3, 400 MHz) δ 8.00 (1H, s), 6.56 (1H, s), 6.01 (1H, br), 2.66 (2H,

13 s), 1.44 (6H, s); C NMR ((CD3)2CO, 150 MHz) δ 189.7, 161.5, 161.2, 131.3, 115.6,

105.1, 103.5, 80.6, 48.4, 26.58, 26.57.

1-(2’,4’-dibenzyloxy)phenyl-2-iodoethanone (9)

To solution of 2,4-dihydroxyacetophenone 2a (20.0 g, 131 mmol) in CH3CN (350 mL),

K2CO3 (40 g, 295 mmol) benzyl bromide (35 mL, 295 mmol) were added. The reaction mixture was stirred at reflux for 20-24 h. The reaction progress was followed by TLC and

1H NMR. After completion, the solvents were evaporated under vacuum the residue was dissolved in EtOAc/H2O. The organic phase was washed with 0.1 M HCl, H2O, brine , dried over anhyd Na2SO4 and evaporated under vacuum. The residue was recrystallized from EtOAc/hexanes to obtain dibenzylacetophenone (40 g, 120.3 mmol, 92%) as white

1 solid: mp 80-81 °C; TLC Rf = 0.59 (EtOAc/hexanes, 1:2); H NMR (CDCl3, 400 MHz) δ

7.84 (1H, m), 7.33 (10H, m), 6.62 (2H, m), 5.11 (2H, s), 5.08 (2H, s), 2.55 (3H, s).

To a stirred clear solution of dibenzylacetophenone (34.8 g, 104.6 mmol) in anhyd

TM CH2Cl2 (90 mL) and anhyd MeOH (450 mL, first dissolved in CH2Cl2), Selectfluor

(22.32 g, 62.8 mmol) followed by elemental iodine (13.32 g, 52.3 mmol) was added. The reaction mixture was stirred at rt for 20-24 h. The progress of reaction was monitored by

TLC and 1H NMR. After completion, the reaction mixture was filtered and ppt was washed with CH2Cl2 extensively. The combined filtrate was applied to rotavap and the resulting solid residue was dissolved in CH2Cl2. The organic phase was washed with freshly prepared 10% w/v sodium thiosulfate solution, H2O, brine, dried over anhyd

Na2SO4. The organic solvents were evaporated under vacuum at 25 degree. The residue

108 was recrystallized from Me2CO/MeOH (110 mL, 1:10) to obtain 9. (45.0 g, 98.2 mmol,

o 1 94%) as yellowish crystals: mp 105-110 C; TLC Rf = 0.53 [EtOAc/hexanes (1:2)]; H

NMR (CDCl3, 600 MHz) δ 7.91 (1H, d, J = 8.4 Hz), 7.42 (10H, m), 6.65 (1H, dd, J = 1.8,

8.4 Hz), 6.61 (1H, d, J = 1.8 Hz), 5.14 (2H, s), 5.09 (2H, s), 4.40 (2H, s); 13C NMR

(CDCl3, 150 MHz) δ 192.1, 164.2, 159.6, 135.8, 135.4, 134.1, 128.8, 128.7, 128.5,

128.3, 127.9, 127.5, 106.8, 100.0, 71.0, 70.3, 9.9; Anal. (%) calcd for C20H19IO3·0.2H2O,

C 57.21, H 4.23, found C 57.02, H 3.85.

2-Acetoxy-1-(2’,4’-dibenzyloxy)phenylethanone (10)

To a solution of glacial acetic acid (0.87 mL) in THF (30 mL), DBU (2.3 mL) was added and the reaction mixture was vigorously stirred. The reaction mixture was cooled to 0 oC and a solution of 9 (4.5 g, 9.8 mmol) in THF (10 mL) was added dropwise. The reaction mixture was stirred at rt for 3-4 h and was followed by TLC. After completion, the solvents were evaporated under vacuum at 35 oC. The oily residue was dissolved in

CH2Cl2 (15 mL). The organic phase was washed with 0.1 M HCl, 10% NaHCO3, brine, dried over anhyd Na2SO4 and evaporated under vacuum. The residue was purified by flash column chromatography to obtain 10 (3.52 g, 9 mmol, 92%) white solid: mp 65-85 o 1 C; TLC Rf = 0.24 [EtOAc/hexanes (1:4)]; H NMR (CDCl3, 600 MHz) δ 8.99 (1H, d, J

= 9.0 Hz), 7.39 (10H, m), 6.66 (1H, dd, J = 2.4, 9.0 Hz), 6.60 (1H, d, J = 2.4 Hz), 5.13

13 (2H, s), 5.12 (2H, s), 5.10 (2H, s), 2.17 (3H, s); C NMR (CDCl3, 100 MHz) δ 191.0,

170.5, 164.3, 160.4, 135.8, 135.3, 133.2, 128.8, 128.73, 128.70, 128.5, 128.3, 127.8,

127.5, 118.0, 106.9, 99.9, 70.9, 70.3, 70.0, 20.6; Anal. (%) calcd for C24H22O5·0.6H2O, C

71.84, H 5.83, found, C 71.55, H 5.62.

109 Methyl-7-(1’-(2’,4’-dibenzyloxy)phenyl-1’-oxo))ethoxy-2,2-dimethyl-4-chromanone-

6-carboxylate (10a)

To a solution of 8 (4.6 g, 18.4 mmol) and 9 (7.67 g, 16.7 mmol) in acetone (170 mL),

K2CO3 (3.0 g) were added under a flow of N2. The reaction mixture was stirred at reflux and the reaction was monitored by TLC. After completion, brine (20 mL) was added to the reaction mixture. The organic solvents were evaporated under vacuum. The yellowish solid residue was dissolved in a mixture of water and ethyl acetate (ca. 500 mL). The aqueous phase was washed with ethyl acetate (2x80 mL) and the combined organic phase was washed with 2 M NaOH, 1 M HCl, brine, dried over anhyd Na2SO4 and evaporated under vacuum. The solid residue was recrystallized from EtOAc/hexanes to obtain 10a (8 g, 13.8 mmol, 83%) as yellow crystals: mp 155-159 °C; TLC Rf = 0.45 [EtOAc/hexanes

1 (1:1); H NMR (CDCl3, 600 MHz) δ 8.43 (1H, s), 8.01 (1H, d, J = 9.0 Hz), 7.40 (11H, m), 6.68 (1H, m), 6.66 (1H, m), 5.99 (1H, s), 5.21 (2H, s), 5.12 (4H, s), 3.83 (3H, s), 2.66

13 (2H, s), 1.44 (6H, s); C NMR (CDCl3, 100 MHz) δ 190.9, 190.2, 165.0, 164.5, 164.3,

163.8, 160.4, 135.8, 135.2, 133.4, 132.1, 129.0, 128.8, 128.7, 128.4, 128.1, 127.5, 118.1,

114.0, 113 .3, 107.1, 101.2, 99.9, 80.3, 74.5, 71.1, 70.4, 51.8, 48.3, 26.7; Anal. (%) calcd for C35H32O5, C 72.40, H 5.56, found C 72.06, H 5.28.

7-(2’,4’-dibenzyloxy-1’-oxo)ethoxy-6-hydroxymethyl-2,2-dimethyl-4-chromanone

(11)

To a mixture of 10a (4.2 g, 7.2 mmol) in anhyd EtOH (80 mL), triethyl-ortho-formate

(120 mL), conc. H2SO4 (1.8 mL) was added dropwise under a flow of N2. The mixture was stirred for 2-3 h until it turn into a brownish solution then 3 Å molecular sieves (42

110 g) were added to the reaction solution and was stirred are rt using mechanical stirrer.

Reaction progress was followed by TLC. After completion, the volatiles were evaporated under vacuum. The solid/semi-solid residue was dissolved in Et2O (500 mL) and H2O

(150 mL) mixture. The aqueous phase was extracted with Et2O (2x100 mL). The combined organic phase was washed with 10% NaHCO3, brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum to obtain 11a (ca. 5.8 g) as semi-solid residue which was directly used in the next step without any further purification: 1H NMR

((CD3)2CO, 600 MHz) δ 7.78 (1H, d, J = 9.0 Hz), 7.74 (1H, s), 7.56 (2H, m), 7.48 (2H, m), 7.39 (4H, m), 7.33 (2H, s), 6.72 (1H, d, J = 2.4 Hz), 6.65 (1H, dd, J = 2.4, 9.0 Hz),

6.28 (1H, s), 5.16 (2H, s), 5.09 (2H, s), 4.72 (1H, s), 4.50 (2H, s), 3.68 (3H, s) 3.54 (8H, m), 1.39 (6H, s), 1.14 (12H, m).

To a solution of the above diketal in anhyd THF (70-95 mL) at 0 °C, lithium aluminu hydride (LiAlH4) (12 mmol) was added dropwise. The reaction mixture was stirred at rt for 3-4 h and the reaction progression was followed by TLC. After completion, the reaction was quenched with addition of EtOAc (80 mL). The pH of reaction was adjusted to 3-4 with addition of 0.1 M HCl. The aqueous phase was extracted with EtOAc (3x50 mL). The combined organic phase was washed with 10% NaHCO3, brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum to obtain off white solid/semi-

1 solid alcohol 11b which was directly used in the next step: H NMR (CDCl3, 600 MHz) δ

7.79 (1H, d, J = 9.0 Hz), 7.37 (10H, m), 7.16 (1H, s), 6.61 (1H, dd, J = 2.4, 9.0 Hz), 6.57

(1H, d, J = 2.4 Hz), 6.22 (1H, s), 5.04 (2H, s), 5.01 (2H, s), 4.42 (2H, s), 4.35 (2H, d, J =

7.2 Hz), 4.10 (8H, q, J = 7.2 Hz), 1.24 (12H, 7.2 Hz).

111 To a solution of the above alcohol in Me2CO (160 mL) and H2O (30 mL), pyridinium p-toluene sulfonate (6 g) was added. The reaction mixture was stirred at rt for 20-24 h and the reaction progress was followed by TLC. After completion, organic solvents were evaporated under vacuum. The resulting mixture was filtered and the ppt was washed with small amount of Et2O and dried under vacuum the residue was dissolved in CH2Cl2.

The organic phase was washed with 10% NaHCO3, brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum to obtain 11 (3.15 g, 5.7 mmol, 79%) as off white to

o 1 pinkish solid: mp 176-182 C; TLC Rf = 0.37 [EtOAc/hexanes (3:2)]; H NMR (CDCl3,

600 MHz) δ 8.05 (1H, d, J = 9.0 Hz), 7.72 (1H, s), 7.51 (4H, d, J = 5.4 Hz), 7.42 (6H, m),

6.71 (1H, dd, J = 2.4, 9.0 Hz), 6.67 (1H, d, J = 2.4 Hz), 5.88 (1H, s), 5.148 (2H, s), 5.142

(2H, s), 5.10 (2H, s), 4.65 (2H, d, J = 6.6 Hz), 3.76 (1H, t, J = 6.6 Hz), 2.65 (2H, s), 1.46

13 (6H, s); C NMR (CDCl3, 150 MHz) δ 191.7, 190.9, 164.8, 163.1, 161.6, 160.7, 135.7,

135.1, 133.4, 129.1, 129.0, 128.7, 128.4, 128.3, 127.5, 127.2, 123.8, 117.6, 113.5, 107.2,

100.6, 99.8, 79.6, 74.1, 71.3, 70.4, 62.0, 48.3, 26.7; Anal. (%) calcd for C34H32O7, C

73.90, H 5.84, found C 73.79, H 5.87.

2,2-Dimethyl-7-(2’4’-dibenzyloxy)phenyl-2H-pyro-[2’,3’-g]-4-chromanone (12)

To a solution of 11 (1.2 g, 2.1 mmol) in anhyd CH3CN (20 mL), triphenyl phosphine hydrobromide (PPh3·HBr) (1.48 g, 2.1 mmol) was added under a flow of N2. The reaction mixture was stirred at 60 °C for 1-2 h and the reaction progress was followed by TLC.

After completion, solvents were evaporated under vacuum and the obtained off-white

Wittig salt was directly used in the next step without further purification.

112 To a solution of the above salt in anhyd MeOH (210 mL), K-tOBu (0.51 g, 4.2 mmol) was added. The reaction mixture was stirred at reflux for 24-30 h and was followed by

TLC. After completion, the reaction volume was reduced to one fourth under vacuum.

The mixture was then allowed to stand at 0 °C for 2 h. The yellow ppt was filtered and washed with cold MeOH (-78 °C, 2x10 mL) and were dried. The solid residues were dissolved in CH2Cl2 and washed with 0.1 M HCl, brine, dried over anhyd Na2SO4 and evaporated under vacuum to obtain 12 (1.05 g, 2.03 mmol, 94%) as yellowish solid: mp

1 157-161 °C; TLC Rf 0.42 (EtOAc/hexanes (1:2)); H NMR (CDCl3, 600 MHz) δ 7.54

(1H, s), 7.37 (10H, m), 7.22 (1H, d, J = 8.4 Hz), 6.37 (2H, m), 6.54 (1H, s), 6.32 (1H, s),

13 5.05 (2H, s), 5.04 (2H, s), 5.02 (2H, s), 2.66 (2H, s), 1.44 (6H, s); C NMR (CDCl3, 150

MHz) δ 191.1, 161.3, 160.6, 159.9, 157.3, 136.5, 136.2, 130.4, 129.4, 128.65, 128.63,

128.11, 128.10, 127.5, 127.4, 124.5, 120.7, 120.3, 117.6, 114.6, 106.0, 104.0, 100.7, 79.5,

77.2, 76.9, 76.7, 70.4, 70.1, 68.7, 48.5, 26.6; Anal. (%) calcd for C34H30O5·0.3H2O, C

77.93, H 5.89, found, C 77.56, H 5.88.

2,2-Dimethyl-7-(2’4’-dibenzyloxy)phenyl-2H-pyro-[2’,3’-g]-4-hydroxychromane (13)

o To a solution of 12 (100 mg, 0.18 mmol) in anhyd THF (1 mL) at 0 C, LiAlH4 (0.2 mmol) was added under a flow of N2. The reaction mixture was stirred at rt for 8-10 h and was followed by TLC. After completion, EtOAc (2 mL) was added followed by 0.1

M HCl. The aq phase was extracted with EtOAc (3x5 mL). The combined organic phase was washed with 0.1 M HCl, 10% NaHCO3, brine and dried over anhyd Na2SO4. The organic solvents were evaporated under vacuum. The residue was recrystallized from

CH2Cl2/hexanes to obtain 13 (60 mg, 0.12 mmol, 64%) as semi-solid residue: TLC Rf =

113 1 0.28 [EtOAc/hexanes (1:2)]; H NMR (CDCl3, 600 MHz) δ 7.37 (11H, m), 7.24 (1H, d, J

= 8.4 Hz), 7.13 (1H, s), 6.61 (1H, d, J = 2.4 Hz), 6.59 (1H, dd, J = 2.4, 8.4 Hz), 6.54 (1H, s), 6.29 (1H, s), 5.05 (2H, s), 5.02 (2H, s), 4.95 (2H, s), 4.78 (1H, q, J = 6.6 Hz), 2.15

(1H, dd, J = 6.6, 8.4 Hz), 1.84 (1H, dd, J = 6.6, 8.4 Hz), 1.433 (3H, s), 1.31 (3H, s); Anal.

(%) calcd for C34H32O5, C 78.44, H 6.20, found C 78.21, H 6.28.

2,2-Dimethyl-7-(2’4’-dibenzyloxy)phenyl-2H-pyro-[2’,3’-g]-chromene (14)

To a solution of p-tosyl chloride (57 mg, 0.3 mmol) and dimethylaminopyridine (DMAP)

o (37 mg, 0.3 mmol) in CH2Cl2 (1 mL) stirred at 0 C for 15-20 min, a solution of 13 (52 mg, 0 1 mmol) in anhyd CH2Cl2 (1 mL) was added dropwise. The reaction mixture was stirred at 0 oC for 4-6 h and was followed The reaction progress was monitored by TLC.

After completion, reaction was diluted with CH2Cl2 and quenched with NH4Cl solution.

The aq phase was extracted with CH2Cl2 (2x2 mL) and the combined organic phase was washed with 0.1 M HCl, 10% sodium bicarbonoate, brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum. The residue was purified by flash column chromatography to obtain 14 (25 mg, 50 μmol, 50%) as semisolid residue: TLC Rf = 0.51

1 [EtOAc/hexanes (1:2)]; H NMR (CDCl3, 600 MHz) δ 7.38 (10H, m), 7.24 (1H, d, J =

8.4 Hz), 6.66 (1H, s), 6.61 (1H, d, J = 2.4 Hz), 6.58 (1H, dd, J = 2.4, 8.4 Hz), 6.50 (1H, s), 6.30 (1H, s), 6.25 (1H, d, J = 10.2 Hz), 5.48 (1H, d, J = 9.6 Hz), 5.05 (2H, s), 5.02

(2H, s), 4.95 (2H, s), 1.41 (6H, s).

4-Acetoxy-2,2-dimethyl-7-(2’4’-dibenzyloxy)phenyl-2H-pyro-[2’,3’-g]chromane

(13a)

114 o To a solution of DMAP (19 mg, 0.15 mmol) in CH2Cl2 (0.5 mL) at 0 C, acetic anhydride

(16 mg, 0.15 mmol) was added under N2 atmosphere. After stirring for 15-20 min, 13 (26 mg, 50 μmol) was added. The reaction mixture was stirred at 0 oC for 1-2 h and was followed by. After completion, reaction was diluted with CH2Cl2 and quenched with

NH4Cl solution. The aq phase was extracted with CH2Cl2 (2x2 mL) and the combined organic phase was washed with 0.1 M HCl, 10% NaHCO3, brine, dried over anhyd

Na2SO4 and evaporated to dryness under vacuum. The residue was purified by flash column chromatography to obtain 13a (20 mg, 36 μmol, 72%) as semisolid residue: TLC

1 Rf = 0.5 [EtOAc/hexanes (1:2)]; H NMR (CDCl3, 600 MHz) δ 7.37 (10H, m), 7.23 (1H, d, J = 8.4 Hz), 6.91 (1H, s), 6.60 (1H, d, J = 2.4 Hz), 6.58 (1H, dd, J = 2.4, 8.4 Hz), 6.52

(1H, s), 6.30 (1H, s), 5.92 (1H, t, J = 6.0 Hz), 5.05 (2H, s), 5.02 (2H, s), 4.95 (2H, m),

2.20 (1H, m), 2.10 (3H, s) 1.97 (1H, m), 1.41 (3H, s), 1.37 (3H, s).

2,2-Dimethyl-7-(2’4’-dibenzyloxy)phenyl-2H-(4’,5’-dihydro-4’,5’-dihydroxy)-pyro-

[2’,3’-g]-chromene (13b)

Procedure A: To a solution of 15 (100 mg, 0.18 mmol) in anhyd. THF (5 mL) at 0 oC,

LiAlH4 (0.48 mmol) was added under a flow of N2. The reaction mixture was stirred at rt for about 6-8 h and was followed by TLC. After completion, EtOAc (8-10 mL) was added to reaction mixture at 0 oC. The reaction mixture was stirred at rt for ca. 3-4 h.

Then 1 M HCl (3-4 mL) was added at 0 oC. The reaction mixture was stirred at rt and was followed by TLC. After ca. 3-4 h TLC spots for the intermediate diastereomers vanished completely with dominant product spot. After completion, the aqueous layer was extracted with EtOAc (3x5 mL). The combined organic phase was washed with 10%

115 NaHCO3, brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum. The residue was purified by prep-TLC (EtOAc/hexanes (1:2)) to obtain 13b (40 mg, 74 μmol,

o o 41% over two steps): mp turns glassy at 68 C and melts at ca. 140 C; TLC Rf = 0.84

1 (EtOAc/hexanes (1:1)); H NMR ((CD3)2CO, 600 MHz) δ 7.63 (1H, d, J = 9.0 Hz), 7.39

(10H, m), 7.05 (1H, s), 6.76 (1H, d, J = 2.4 Hz), 6.64 (1H, dd, J = 2.4, 9.0 Hz), 6.33 (1H, d, J = 9.6 Hz), 6.10 (1H, s), 5.55 (1H, d, J = 10.2 Hz), 5.46 (1H, m), 5.20 (2H, m), 5.10

(2H, s), 4.70 (1H, d, J = 11.4 Hz), 4.27 (1H, m), 4.21 (1H, m), 4.01 (1H, d, J = 11.4 Hz),

13 1.365 (3H, s), 1.361 (3H, s); C NMR ((CD3)2CO, 150 MHz) δ 160.6, 157.6, 155.6,

155.5, 154.1, 138.2, 137.8, 130.2, 129.3, 129.2, 128.75, 128.72, 128.6, 128.4, 128.3,

127.8, 122.9, 122.7, 118.2, 115.7, 106.5, 103.6, 101.8, 76.6, 72.0, 70.9, 70.4, 70.15,

70.11, 67.58, 67.57, 67.4, 28.2, 28.1; Anal. (%) calcd for C34H32O6·0.25H2O, C 75.47, H

6.05, found C 75.07, H 6.44.

Procedure B: To a solution of 15 (10 mg, 18 μmol) and Et3N (110 mg, 1 mmol) in anhyd

THF (1 mL) at 0 oC, triflic anhydride (40 μmol) was added. The reaction mixture was stirred at rt for 8-10 h and was followed by TLC. After completion, the reaction was

o quenched with addition of MeOH followed by 5% NaHCO3 at 0 C. The reaction mixture was diluted with EtOAc/H2O mixture. The aq phase was extracted with EtOAc. The combined organic phase was washed with 0.1 M HCl, 10% NaHCO3, brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum. The residue was purified by prep-TLC to obtain 13b (5 mg, 9 μmol, 50%) as semi-solid residue.

2,2-Dimethyl-7-(2’4’-dibenzyloxy)phenyl-2H-(4’,5’-dihydro-4’,5’-dihydroxy)-pyro-

[2’,3’-g]-4-chromanone (15)

116 To a mixture of 12 (0.45 g, 0.87 mmol) in a mixture of Me2CO (25 mL) and H2O (2.5 mL), N-Methyl morphonline N-oxide (216 μL, 1.3 mmol) and methanesulfonamide (0.27 g, 1.05 mmol) were added. After stirring for 5 min OsO4 (0.87 μmol) was added. The mixture was stirred at rt for about 18 h and was monitored by TLC. After completion,

10% sodium bisulfite (20 mL) was added and after stirring for additional 1 h the solvents were evaporated under vacuum. The residue was dissolved in EtOAc/H2O mixture. The aq phase was extracted with EtOAc (3x10 mL). The combined organic phase was washed with 0.1M HCl, 5% NaHCO3, brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum. The solid residue was recrystallized from EtOAc/hexanes to obtain 15

o (0.4 g, 0.73 mmol, 83%) as off-white solid: mp 196 -198 C; TLC Rf = 0.54

1 [EtOAc/hexanes (1:2)]; H NMR (CDCl3, 600 MHz) δ 8.00 (1H, s), 7.38 (11H, m, H8),

6.67 (1H, d, J = 3.0 Hz), 6.65 (1H, dd, J = 2.4, 8.4 Hz); 6.34 (1H, s); 5.22 (1H, m), 5.10

(2H, s), 5.02 (2H, s), 4.45 (1H, d, J = 12.0 Hz), 4.34 (1H, d, J = 11.4 Hz); 4.17 (1H, s),

13 2.80 (1H, d, J = 5.4 Hz), 2.64 (2H, m), 1.44 (3H, s), 1.41 (3H, s); C NMR (CDCl3, 150

MHz) δ 191.1, 161.0, 160.2, 160.0, 157.0, 136.4, 135.3, 129.0, 128.9, 128.7, 128.6,

128.6, 128.1, 127.6, 127.5, 119.7, 117.5, 114.9, 106.0, 104.0, 101.3, 79.4, 71.2, 70.7,

70.1, 69.0, 67.5, 48.6, 27.0, 26.3; Anal. (%) calcd for C34H32O7, C 73.90, H 5.84, found C

74.25, H 5.78.

2,2-Dimethyl-7-(2’4’-dibenzyloxy)phenyl-2H-(4’,5’-dihydro-4’,5’-dihydroxy)-pyro-

[2’,3’-g]-4-hydroxychromane (15a)

o To a solution of 15 (15 mg, 27 μmol) in anhyd THF (1 mL) at 0 C, LiAlH4 (60 μmol) was added under a flow of N2. The reaction mixture was stirred at rt for 4-5 h and was

117 followed by TLC. After completion, EtOAc (2 mL) was added followed by addition of small amount of 0.1 M HCl. The aq phase was extracted with EtOAc (3x5 mL). The combined organic phase was washed with 0.1 M HCl, 10% NaHCO3, brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum. The residue was purified by prep-TLC to obtain 15a (12 mg, 21 μmol, 64%) as colorless semi-solid: TLC Rf = 0.28

1 [EtOAc/hexanes (1:2)]; H NMR (CDCl3, 600 MHz) δ 7.51 (1H, s), 7.45 (1H, 9.0 Hz),

7.36 (10H, m), 6.66 (1H, d, J = 8.4 Hz), 6.55 (1H, dd, J = 2.4, 8.4 Hz), 6.27 (1H, s), 5.17

(1H, d, J = 5.4 Hz), 5.10 (2H, s), 5.01 (2H, s), 4.81 (1H, m), 4.41 (1H, d, J = 11.4 Hz),

4.25 (1H, d, J = 11.4 Hz), 4.21 (1H, s), 2.28 (1H, d, J = 5.4 Hz), 2.13 (1H, m), 1.82 (1H, m), 1.42 (3H, s), 1.28 (3H, s).

2,2-Dimethyl-7-(2’4’-dihydroxy)phenyl-2H-(4’,5’-dihydro-4’,5’-dihydroxy)-pyro-

[2’,3’-g]-4-chromanone (17)

To a solution of 15 (60 mg, 0.11 mmol) in ethyl acetate (15 mL) at 0 oC, 10% Pd/C (30 mg) was added. The reactor was applied to Parr hydrogenator and hydrogenolysis was carried out under hydrogen atmosphere at 15 psi. The reaction mixture was stirred at rt for 8-10 h. After completion the reaction mixture was passed through a celite pad and pad was washed with EtOAc (2x15 mL). The combined organic phase was evaporated under vacuum to obtain 17 (40 mg, quantitative yield) as semi-solid residue: TLC Rf = 0.27

1 [MeOH/CH2Cl2 (1:15)]; H NMR ((CD3)2CO, 600 MHz) δ 9.11 (1H, br), 8.35 (1H, br),

7.94 (1H, s), 7.21 (1H, d, J = 8.4 Hz), 6.35 (1H, d, J = 1.8 Hz), 6.31 (1H, dd, J = 2.4, 8.4

Hz), 6.27 (1H, s), 5.36 (1H, s), 4.96 (1H, br), 4.52 (1H, d, J = 12.0 Hz), 4.21 (1H, d, J =

12.0 Hz); 3.30 (1H, s), 2.66 (2H, s), 1.41 (6H, s).

118 2,2-Dimethyl-7-(2’4’-di-t-butyldimethylsilyloxy)phenyl-2H-(4’,5’-dihydro-4’,5’- dihydroxy)-pyro-[2’,3’-g]-4-chromanone (18)

To a solution of 17 (60 mg, 0.16 mmol) and Et3N (174 mg, 1.7 mmol) in anhyd CH2Cl2

o (2-3 mL) at 0 C, TBDMS-Cl (260 mg, 1.7 mmol) was added under a flow of N2. The reaction mixture was stirred for 10-12 h and was followed by TLC. After completion, saturated NH4Cl solution (5 mL) followed by MeOH (2 mL) was added. The aq phase was extracted with CH2Cl2 (3x5 mL). The combined organic phase was washed with 0.1

M HCl, 5% NaHCO3, brine, dried over anhyd Na2SO4 and was evaporated under vacuum. The residue was purified by flash column chromatography to obtain 18 (70 mg,

o 0.12 mmol, 72%) as white solid: mp 87.2-99.1 C; TLC Rf = 0.39 [EtOAc/hexanes (1:3)];

1 H NMR (CDCl3, 600 MHz) δ 7.98 (1H, s), 7.30 (1H, d, J = 8.4 Hz), 6.42 (1H, dd, J =

2.4, 8.4 Hz), 6.37 (1H, d, J = 2.4 Hz), 6.35 (1H, s), 5.14 (1H, d, J = 6.0 Hz), 4.43 (1H, s),

4.36 (1H, d, J = 11.4 Hz), 4.33 (1H, d, J = 11.4 Hz), 2.88 (1H, d, J = 6.0 Hz), 2.64 (2H, m), 1.44 (3H, s), 1.41 (3H, s), 1.01 (9H, s), 0.95 (9H, s), 0.37 (3H, s), 0.36 (3H, s), 0.18

13 (6H, s); C NMR (CDCl3, 100 MHz) δ 191.1, 161.0, 160.3, 156.4, 154.0, 128.9, 128.5,

121.4, 117.4, 114.9, 113.2, 110.6, 104.0, 79.3, 71.3, 69.0, 67.5, 48.6, 27.0, 26.4, 25.8,

25.5, 18.2, 18.1, -3.7, -3.8, -4.4; Anal. (%) calcd for C32H48O7Si2·0.05H2O, C 63.87, H

8.06, found, C 63.48, H 7.97.

2,2-Dimethyl-7-(2’4’-di-t-butyldimethylsilyloxy)phenyl-2H-(4’,5’-dihydro-4’,5’- dihydroxy)-pyro-[2’,3’-g]-4-hydroxychromane (18a)

Procedure A: To a solution of 18 (10 mg, 16 μmol) in MeOH (1 mL) at 0 oC, sodium borohydride (4 mg, 0.1 mmol) was added. The reaction mixture was stirred at rt for 30-36

119 h. Reaction progress was monitored by TLC. After completion, small amount of NH4Cl solution was added and pH of the reaction mixture was adjusted to pH ~3-4 with addition of 0.1 M HCl. The aq phase was extracted with EtOAc (3x5 mL). The combined organic phase was washed with 0.1 M HCl, 5% NaHCO3, brine, dried over anhyd Na2SO4 and was evaporated under vacuum. The residue was purified using prep-TLC

[EtOAc/hexanes (1:2)] to obtain 18a (6 mg, 10 μmol, 60%) as semi-solid residue: TLC Rf

1 = 0.56 [MeOH/dcm (1:15)]; H NMR (CDCl3, 600 MHz) δ 7.49 (1H, s), 7.33 (1H, d, J =

8.4 Hz), 6.42 (1H, dd, J = 2.4, 8.4 Hz), 6.37 (1H, d, J = 2.4 Hz), 6.28 (1H, s), 5.11 (1H, d,

J = 6.0 Hz), 4.90 (1H, br), 4.47 (1H, s), 4.29 (1H, d, J = 11.4 Hz), 4.26 (1H, d, J = 11.4

Hz), 2.91 (1H, d, J = 6.0 Hz), 2.12 (1H, m), 1.82 (1H, m), 1.41 (3H, s), 1.31 (3H, s), 1.02

(9H, s), 0.96 (1H, s), 0.37 (3H, s), 0.36 (3H, s), 0.18 (6H, s).

o Procedure B: To a solution of 18 (20 mg, 33 μmol) in anhyd THF (1 mL) at 0 C, LiAlH4

(0.1 mmol) was added under N2 atmosphere. The reaction mixture was stirred at rt for 3-4 h and was followed by TLC. After completion, EtOAc (2 mL) was added followed by 0.1

M HCl. The aq phase was extracted with EtOAc (3x5 mL). The combined organic phase was washed with 0.1 M HCl, 10% NaHCO3, brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum. The residue was further purified using prep-TLC to obtain 18a (6 mg, 10 μmol, 30%) and two side products: 18b (2,2-Dimethyl-7-(2’,- hydroxy-4’-t-butyldimethylsilyloxy)phenyl-2H-(4’,5’-dihydro-4’,5’-dihydroxy)-pyro-

[2’,3’-g]-4-hydroxychromane) (7 mg, 14 μmol, 40%) as semi-solid residue [TLC Rf =

1 0.33 [EtOAc/hexanes (1:1)]; H NMR (CDCl3, 600 MHz) δ 8.78 (1H, s), 7.54 (1H, s),

6.78 (1H, d, J = 9.0 Hz), 6.36 (1H, d, J = 1.8 Hz), 6.32 (2H, m), 4.80 (1H, d, J = 6.0 Hz),

4.65 (1H, s), 4.22 (1H, d, J = 12.0 Hz), 4.00 (1H, d, J = 12.0 Hz), 3.87 (1H, s), 3.40 (1H,

120 br), 2.50 (1H, br), 2.07 (1H, m), 1.77 (1H, m), 1.41 (3H, s), 1.23 (3H, s), 0.95 (9H, s),

0.18 (6H, s)] and 18c (2,2-Dimethyl-7-(2’-hydroxy-4’-t-butyldimethylsilyloxy)phenyl-

2H-(4’,5’-dihydro-4’,5’-dihydroxy)-pyro-[2’,3’-g]-chromanone) (3 mg, 6 μmol, 18%)

1 as semi-solid residue [TLC Rf = 0.52 [EtOAc/hexanes (1:1)]; H NMR (CDCl3, 600

MHz) δ 8.64 (1H, s), 8.02 (1H, s), 6.94 (1H, d, J = 8.4 Hz), 6.42 (1H, d, J = 2.4 Hz), 6.41

(1H, s), 6.34 (1H, dd, J = 2.4, 8.4 Hz), 5.22 (1H, d, J = 4.8 Hz), 4.39 (1H, d, J = 12.0 Hz),

4.20 (1H, d, J = 12.6 Hz), 2.71 (1H, m), 2.67 (1H, d, J = 4.2 Hz), 1.45 (3H, s), 1.44 (3H, s), 0.96 (9H, s), 0.19 (6H, s)].

2,2-Dimethyl-7-(2’4’-di-t-butyldimethylsilyloxy)phenyl-2H-(4’,5’-dihydro-4’,5’- dihydroxy)-pyro-[2’,3’-g]-chromene (19)

Isolated from a dried up NMR tube containing 18a in CDCl3. The residue was dissolved in Me2CO and was applied to pre-TLC [EtOAc/hexanes (1:1)]. The band with higher Rf than the that of 18 was scrapped and extracted using MeOH and CH2Cl2 mixture (5%) to

1 obtain 19 (ca. 1 mg, 2 μmol): TLC Rf = 0.85 [EtOAc/hexanes (1:1)]; H NMR

((CD3)2CO, 600 MHz) δ 7.53 (1H, d, J = 8.4 Hz), 7.06 (1H, s), 6.49 (1H, dd, J = 2.4, 8.4

Hz), 6.39 (1H, d, J = 2.4 Hz), 6.34 (1H, d, J = 9.6 Hz), 6.13 (1H, s), 5.56 (1H, d, J = 9.6

Hz), 5.43 (1H, d, J = 6.6 Hz), 4.66 (1H, d, J = 11.4 Hz), 4.27 (1H, d, 6.6 Hz), 4.16 (1H, s), 4.00 (1H, d, J = 11.4 Hz); 1.37 (3H, s), 1.36 (3H, s), 0.98 (9H, s), 0.96 (9H, s), 0.338

13 (3H, s), 0.335 (3H, s), 0.22 (6H, s); C NMR ((CD3)2CO, 150 MHz) δ 156.8, 155.5,

154.2, 130.4, 128.8, 127.8, 122.7, 115.8, 113.3, 111.4, 103.7, 76.7, 71.8, 70.0, 67.3, 28.2,

26.3, 25.9, 18.9, -3.6, -4.2.

121 3.4 Syntheses of Vestitol, Bolusanthin III and 6a-Hydroxymedicarpin

4-Benzyloxy-2-hydroxybenzaldehyde (1b)

To a solution of 2,4-dihydroxybenzaldehyde (1c) (100 g, 0.72 mol) in CH3CN (1.5 L),

NaHCO3 (72.0 g, 0.86 mol) and benzyl bromide (145.4 g, 0.85 mol) were added and the reaction mixture was stirred under reflux. The reaction progression was followed by TLC and on completion the reaction mixture was cooled to room temperature and filtered. The organic solvents were evaporated under vacuum. The off white residue was recrystallized from MeOH to obtain 1b (140 g, 0.61 mol) in 85% yield: mp 77–79 oC [lit.42 mp 78-80 o 1 C); TLC Rf 0.73 [EtOAc/hexanes (1:2)]; H NMR ((CD3)2CO, 600 MHz) δ 11.50 (1H, s), 9.88 (1H, br), 7.67 (1H, d, J = 5.4 Hz), 7.50 (2H, d, J = 7.2), 7.41 (2H, m), 7.36 (1H, m), 6.71 (1H, m), 6.57 (1H, s), 5.24 (2H, s).

4-Benzyloxysalicyl alcohol (1)

To a mixture of 1b (6 g, 28.2 mmol) in anhyd EtOH (120 mL), sodium borohydride (850 mg) was added portion-wise at 0 oC. On addition of sodium borohydride the reaction mixture turned clear with evolution of gases. The reaction mixture was allowed to stir at

0 oC for 1 h and then for 8-10 h at rt. The reaction progression was followed by TLC.

After completion, the reaction mixture was reduced to one-fourth of its original volume and then was neutralized with 0.1 M sulfuric acid. The precipitates were formed on addition of H2O (ca. 500 mL). The ppt was filtered and recrystallized from toluene to obtain 1 (3.8 g, 16.5 mmol, 58%) as off white crystals: mp 89.0-92.0 [lit.42 mp 88.0-90.0 o 1 C]; TLC Rf = 0.33 [EtOAc/hexanes (1:2)]; H NMR (DMSO-d6, 400 MHz) δ 9.36 (1H,

122 br), 7.36 (5H, m), 7.12 (1H, d, J = 8.0 Hz), 6.42 (2H, m), 5.01 (2H, s), 4.80 (1H, br), 4.38

(2H, s).

2-Benzyloxy-4-methoxyacetophenone (21b)

To a solution of 4-methoxy-2-hydroxyacetophenone (21a) (5.0 g, 30 mmol) in CH3CN

(80 mL), K2CO3 (5.0 g, 36 mmol) benzyl bromide (5.0 g, 29 mmol) were added. The reaction mixture was allowed to stir under refluxing conditions. The reaction progress was followed by TLC and 1H NMR. After 24 h the solvents were evaporated under vacuum and the off white residue was dissolved in EtOAc and washed with 2 M sodium hydroxide, 0.1 M HCl, H2O, and brine. After drying over anhyd Na2SO4 and then filtration, the volatiles were evaporated under vacuum to obtain 21b (7.4 g, 28.8 mmol,

o 1 96%) as white solid: mp 84-88 C; TLC Rf = 0.40 [EtOAc/hexanes (1:3)]; H NMR

(CDCl3, 600 MHz) δ 7.85 (1H, d, J = 8.4 Hz), 7.40 (5H, m), 6.53 (2H, m), 5.13 (2H, s),

13 3.83 (3H, s), 2.56 (3H, s); C NMR (CDCl3, 150 MHz) δ 197.8, 164.3, 160.1, 135.9,

132.7, 128.7, 128.2, 127.6, 121.4, 105.3, 99.4, 70.6, 55.5, 32.2.

1-(2’-Benzyloxy-4’-methoxy)phenyl-2-iodoethanone (21)

To a solution of 21b (22.7 g, 88.6 mmol) in anhyd CH2Cl2 (100mL) and anhyd MeOH

(500 mL), SelectfluorTM (18.9 g, 53.3 mmol) was added followed by adition of elemental iodine (11.25g, 44.3 mmol). The reaction mixture was stirred for 20 h. The progress of reaction was monitored by TLC and 1H NMR. After completion, reaction mixture was filtered and the ppt was washed with CH2Cl2. The combined filtrate was evaporated under vacuum and the solid residue was dissolved in CH2Cl2 (200 mL) and washed with

123 freshly prepared 10% sodium thiosulfate solution (3x125 mL). The organic layer was dried over anhyd Na2SO4, filtered and evaporated to dryness. The solid residues were purified by recrystalisation from Me2CO/MeOH (1:10, 100 mL) to obtain 21. (28.4 g,

o 74.3 mmol, 84%) as yellowish crystals: mp 96-100 C; TLC Rf = 0.54 [EtOAc/hexanes

1 (1:3)]; H NMR (CDCl3, 600 MHz) δ 7.91 (1H, d, J = 9.0 Hz), 7.44 (5H, m), 6.57 (1H, dd, J = 9.0 Hz), 6.53 (1H, d, J = 2.4 Hz), 5.17 (2H, s), 4.40 (2H, s), 3.84 (3H, s); 13C

NMR (CDCl3, 150 MHz) δ 165.1, 159.7, 135.5, 134.1, 128.84, 128.83, 128.5, 127.9,

117.4, 106.0, 99.3, 71.0, 55.6, 9.9; Anal. (%) calcd for C16H15IO3·0.5 H2O, C 49.13, H

4.12, found C 48.82, H 3.78.

1-(2’-Benzyloxy-4’-methoxy)phenyl-2-(6’-benzyloxy-2’-hydroxymethyl)phenyl-etha- none (22)

To a solution of salicyl alcohol 1 (0.19 g, 0.82 mmol) and α-iodo ketone 21 (0.28 g, 0.74 mmol) in Me2CO (15 mL), K2CO3 (0.14 g, 0.98 mmol) was added under a flow of N2.

The reaction mixture was refluxed for 16-18 h. Reaction progress was followed The reaction progress was monitored by TLC. After completion, the solvents were evaporated and the solid residue was dissolved in EtOAc (120 mL) and was washed with 1 M NaOH,

H2O, brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum to obtain.

The solid residue was purified using flash column chromatography to obtain 22 (0.28 g,

o 0.57 mmol, 78%) as off-white solid: mp 150-153 C; TLC Rf = 0.17 [EtOAc/hexanes

1 (1:2)]; H NMR ((CD3)2CO, 600 MHz) δ 7.89 (1H, d, J = 8.4 Hz), 7.45 (10H, m), 7.22

(1H, d, J = 9.0 Hz), 6.83 (1H, d, J = 2.4 Hz), 6.68 (1H, dd, J = 2.4, 9.0 Hz), 6.56 (1H, dd,

J = 2.4, 8.4 Hz), 6.26 (1H, d, J = 2.4 Hz), 5.33 (2H, s), 5.21 (2H, s), 4.99 (2H, s), 4.98

124 13 (2H, d, J = 6.6 Hz), 4.15 (1H, t, J = 6.6 Hz), 3.90 (3H, s); C NMR ((CD3)2CO, 100

MHz) δ 193.7, 166.3, 161.7, 160.1, 158.0, 138.2, 137.0, 133.2, 129.6, 129.5, 129.3,

129.2, 129.1, 128.6, 124.4, 118.9, 107.5, 106.0, 101.0, 99.8, 74.6, 71.7, 70.5, 60.9, 56.1;

Anal. (%) calcd for C30H28O6·0.25H2O, C 73.68, H 5.87, found C 73.33, H 5.71.

2’,7-Dibenzyloxy-4’-methoxyisoflav-3-ene (23)

To a suspension of 22 (97 mg, 0.2 mmol) in anhyd CH3CN (4 mL), PPh3·HBr (70 mg, 0.2 mmol) was added under a flow of N2. The reaction mixture was stirred at rt and was followed The reaction progress was monitored by TLC. After completion, solvents were evaporated under vacuum to obtain an off-white residue which was directly used in the next step without further purification.

To a solution the above phosphonium salt in anhyd MeOH (15 mL), t-BuOK (45 mg,

0.4 mmol) was added under a flow of N2. The reaction mixture was refluxed for 16-20 h.

The reaction progress was monitored by TLC. After completion, the mixture was reduced to one-third of original volume under vacuum and was filtered. The precipitates were dissolved in CH2Cl2 (30 mL). The organic phase was washed with H2O, brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum to obtain 23 (50 mg, 0.11 mmol,

o 70% over 2 steps) as off-white solid: mp 119-122 C; TLC Rf = 0.65 [EtOAc/hexanes

(1:2)]; 1H NMR ((CD3)2CO, 600 MHz) δ 7.41 (10H, m), 7.28 (1H, d, J = 8.4 Hz), 7.01

(1H, d, J = 7.8 Hz), 6.70 (1H, d, J = 2.4 Hz), 6.60 (1H, s), 6.57 (2H, m, H6), 6.46 (1H, d,

13 J = 1.8 Hz), 5.16 (2H, s), 5.10 (2H, s), 4.93 (2H, s), 3.8 (3H, s); C NMR ((CD3)2CO,

150 MHz) δ 161.7, 160.3, 158.2, 155.5, 138.2, 137.8, 130.0, 129.9, 129.3, 129.4, 129.23,

128.7, 128.6, 128.5, 128.3, 128.2, 121.5, 121.4, 118.1, 108.9, 106.3, 102.8, 100.5, 71.0,

125 70.4, 68.9, 55.6, 3.4; Anal. (%) calcd for C30H26O4·0.5H2O, C 78.41, H 5.92, found C

78.30, H 5.69.

(±)-Vestitol (23a)

To a solution of 23 (50 mg, 0.11 mmol) in EtOAc (15 ml) at 0 oC, 10 % w/w Pd/C (15-20 mg) was added. The mixture was stirred at rt under hydrogen atmosphere (35 psi) and was followed by TLC. After completion, the reaction mixture was passed through a pad of Celite and was washed with EtOAc (3x10 mL). The solvents were dried over anhyd

Na2SO4 and evaporated under vacuum and the residue was further purified using flash column chromatography [EtOAc/hexanes (1:1)] to obtain 23a (25 mg, 90 μmol, 84%) as

1 off-white powder: mp 172-179 °C; TLC Rf = 0.44 [EtOAc/hexanes (1:1)]; H NMR

((CD3)2CO, 600 MHz) δ 8.6 (2H, br), 7.05 (1H, d, J = 8.4 Hz), 6.88 (1H, d, J = 8.4 Hz),

6.50 (1H, d, J = 2.4 Hz), 6.42 (1H, dd, J = 2.4, 8.4 Hz), 6.35 (1H, dd, J = 2.4, 8.4 Hz),

6.27 (1H, d, J = 2.4 Hz), 4.23 (1H, m), 3.97 (1H, t, J = 10.2 Hz), 3.71 (3H, s), 3.47 (1H,

13 m), 2.96 (1H, m), 2.79 (1H, m); C NMR ((CD3)2CO, 100 MHz) δ 160.3, 157.4, 156.6,

156.0, 130.9, 128.6, 120.8, 114.2, 108.6, 105.5, 103.5, 102.4, 70.4, 55.2, 32.5, 30.9; Anal.

(%) calcd for C16H16O4, C 70.57, H 5.92, found C 70.22, H 5.98.

Bolusanthin III (23b)

To a solution of 23 (0.45 g, 0.1 mmol) and pentamethylbenzene (1.48 g, 10 mmol) in

o anhyd CH2Cl2 (30 mL) at -78 C, BCl3 (0.2 mmol) was dropwise added under N2. The reaction mixture was stirred at -78 oC and after 15-20 min the reaction was quenched with 20 mL of CHCl3/MeOH (10:1) mixture. The resulting mixture was warmed to rt.

126 The organic solvents were evaporated under vacuum. The residues were purified by column chromatography [Silica gel 35 mm dia, 8 inch thick, EtOAc/hexanes (1:2)] to

o obtain 23b (0.17 g, 0.61 mmol, 61%) as brownish solid: mp 150-154 C; TLC Rf = 0.48

1 [EtOAc/hexanes (1:2)]; H NMR (CD3OD, 600 MHz) δ 7.14 (1H, d, J = 8.4 Hz), 6.88

(1H, d, J = 8.4 Hz), 6.53 (1H, s), 6.42 (1H, dd, J = 2.4, 8.4 Hz), 6.37 (1H, d, J = 2.4 Hz),

6.33 (1H, dd, J = 2.4, 8.4 Hz), 6.24 (1H, d, J = 1.8 Hz), 4.95 (2H, s), 3.75 (3H, s); 13C

NMR (CD3OD, 150 MHz) δ 161.8, 159.1, 157.3, 155.9, 130.1, 130.0, 128.4, 121.4,

119.9, 117.6, 109.4, 106.1, 103.4, 102.4, 69.2, 55.6; Anal. (%) calcd for C16H14O4·0.1

H2O, C 70.63, H 5.26, found C 70.42, H 5.20.

2’,7-Dibenzyloxy-4’-methoxyisoflavan-3,4-diol (16)

To a solution of 23 (1.42 g, 3.16 mmol) in Me2CO (100 mL) and H2O (10 mL), methanesulfonamide (0.9 g, 3.79 mmol), N-morpholine oxide (NMO) (60% aq sol, 1.0 mL, 4.74 mmol) and OsO4 (0.12 mmol) were added. The reaction mixture was stirred at rt for 12-15 h. Reaction progress was followed by TLC. After completion, 2.5 g of sodium sulfite was added and was stirred for 30-40 min. The volatile solvents were evaporated under vacuum. The residue was dissolved in EtOAc and H2O (1:1, 100-150 mL). The aqueous phase was extracted with EtOAc (2x50 mL), then the combined organic phase was washed with 1 M HCl, H2O, brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum and the residue was recrystallized from

EtOAc/hexanes to obtain 16 (1.12 g, 2.4 mmol, 75%) as off white solid: mp 141-146 oC;

1 TLC Rf 0.5 [EtOAc/hexanes (1:1)]; H NMR ((CD3)2CO, 600 MHz) δ 7.63 (1H, d, J =

8.4 Hz), 7.38 (11H, m), 6.66 (1H, d, J = 2.4 Hz), 6.55 (2H, m), 6.37 (1H, d, J = 2.4 Hz),

127 5.50 (1H, d, J = 6.0 Hz), 5.20 (2H, m, ), 5.07 (2H, s), 4.71 (1H, d, J = 11.4 Hz), 4.49 (1H, m), 4.18 (1H, d, J = 3.0 Hz), 4.01 (1H, d, J =11.4 Hz), 3.77 (3H, s); 13C NMR

((CD3)2CO, 150 MHz) δ 161.4, 159.9, 157.6, 155.7, 138.4, 137.8, 130.8, 130.1, 129.3,

129.2, 128.7, 128.5, 128.32, 128.30, 122.7, 118.3, 108.6, 105.5, 102.1, 101.0, 90.1, 71.9,

70.9, 70.2, 70.1, 67.6, 55.5; Anal. (%) calcd for C30H28O6·0.2 H2O, C 73.82, H 5.86, found C73.47, H 5.57

4’-Methoxy-2’,3’,4’,7-tetrahydroxyisoflavan (24)

To a solution of 16 (50 mg, 0.1 mmol) in EtOAc (5 mL) at 0 oC, 10% w/w Pd/C (10 mg) was added. The mixture was stirred at rt for 18-20 h under hydrogen atmosphere (35 psi) and was followed by TLC. After completion, the reaction mixture was passed through a pad of Celite which was washed extensively with EtOAc. The solvents were dried over anhyd Na2SO4 and evaporated under vacuum. The residue was purified by flash column chromatography (silica gel, MeOH/CH2Cl2 (1:15) to obtain 24 (25 mg, 83 μmol, 82%) as

1 semi-solid residue: TLC Rf = 0.48 [MeOH/CH2Cl2 (1:9)]; H NMR ((CD3)2CO, 400

MHz) δ 7.21 (2H, dd, J = 2.4, 8.4 Hz), 6.42 (1H, dd, J = 2.4, 8.4 Hz), 6.37 (1H, d, J = 2.4

Hz), 6.33 (1H, dd, J = 2.4, 8.4 Hz), 6.26 (1H, d, J = 2.4 Hz), 5.07 (1H, s), 4.34 (1H, d, J =

13 11.4 Hz), 4.15 (1H, d, J = 11.4 Hz), 3.71 (3H, s); C NMR ((CD3)2CO, 100 MHz) δ

161.4, 159.0, 158.4, 155.3, 131.6, 128.4, 118.9, 116.1, 109.4, 105.7, 103.3, 102.9, 73.4,

69.5, 68.6, 55.3.

128 6a-Hydroxymedicarpin

To solution of 24 (20 mg, 65 μmol) in anhyd CH2Cl2 (1 mL), methanesulfonic anhydride

(12 mg, 70 μmol) and pyridine (0.35 mmol) were added under a flow of N2. The reaction mixture was stirred at rt for 16-18 h and reaction progress was monitored by TLC. Moist

Et2O (6 mL) was added to the mixture and the organic phase was washed with H2O, dried over anhyd Na2SO4, evaporated and the residue was purified by prep-TLC to obtain the target molecule (8 mg, 28 μmol, 41%) as semi-solid residue: TLC Rf = 0.46

1 [EtOAc/hexanes (1:1)]; H NMR ((CD3)2CO, 600 MHz) δ 8.60 (1H, br), 7.31 (1H, d, J =

8.4 Hz), 7.28 (1H, d, J = 8.4 Hz), 6.55 (1H, dd, J = 2.4, 8.4 Hz), 6.49 (1H, dd, J = 2.4, 8.4

Hz), 6.34 (1H, d, J = 2.4 Hz), 6.31 (1H, d, J = 2.4 Hz), 5.29 (1H, s), 5.01 (1H, br), 4.12

13 (1H, d, J = 11.4 Hz), 4.06 (1H, d, J = 11.4 Hz), 3.74 (3H, s); C NMR (CDCl3, 150 MHz)

δ 163.0, 161.9, 157.0, 133.1, 125.0, 113.2, 110.7, 107.7, 103.7, 97.0, 86.0, 70.4, 55.6; MS m/z: [M + Na] calcd 309.27; found, 309.30; Anal. (%) calcd for C16H14O5·0.3H2O

·0.2C4H8O2·0.2CH4O, C 64.67, H 5.43; found, C 65.05, H 5.83.

3.5 Synthesis of New SERM Analogs

6-Benzyloxy-2-(2’-benzyloxy-4’-methoxy)benzoyl-1-benzofuran (T1)

To a solution of benzylaldehyde 1b (0.685 g, 3.0 mmol) and the α-iodo ketone 21 (1 g,

2.6 mmol) in Me2CO (20 mL), K2CO3 (0.432 g, 3.12 mmol) was added under a flow of

N2. The reaction mixture was stirred at reflux for overnight. Reaction progress was monitored using TLC. After completion, the solvents were evaporated under vacuum and residue was dissolved in EtOAc/water [100 mL (1:1)]. The organic phase was washed with 1 M sodium hydride, 0.1 M HCl, brine, dried over anhyd Na2SO4 and evaporated to

129 dryness under vacuum. The residue was recrystallized from MeOH to obtain T1 (0.9 g,

o 1.9 mmol, 75%) as white solid: mp 134-137 C; TLC Rf = 0.42 [EtOAc/hexanes (1:3)];

1 H NMR ((CD3)2CO, 600 MHz) δ 7.66 (1H, d, J = 8.4 Hz), 7.52 (2H, m), 7.48 (1H, d, J =

9.0 Hz), 7.40 (3H, m), 7.34 (1H, m), 7.25 (1H, d, J = 1.8 Hz), 7.23 (2H, m), 7.17 (3H, m),

7.05 (1H, dd, J = 2.4, 8.4 Hz), 6.81 (1H, d, J = 2.4 Hz), 6.68 (1H, dd, J = 1.8, 8.4 Hz),

13 5.24 (2H, s), 5.16 (2H, s), 3.89 (3H, s); C NMR ((CD3)2CO, 150 MHz) δ 183.5, 164.2,

160.6, 159.2, 157.7, 154.2, 137.6, 137.4, 131.8, 129.1, 128.8, 128.6, 128.4, 128.2, 127.8,

124.4, 122.3, 121.5, 115.7, 115.2, 106.0, 100.6, 97.2, 70.8, 70.7, 55.7, -11.5; Anal. (%) calcd for C30H24O5·0.1 H2O, C 77.27, H, 5.23, found C 76.93, H5.14.

6-Hydroxy-2-(2’-hydroxy-4’-methoxy)benzoyl-1-benzofuran (T2)

To a solution of T1 (150 mg, 0.32 mmol) in anhyd CH2Cl2 (10 mL), trifluoroacetic acid

(TFA, 20 mL) saturated with pentamethylbenzene (PMB) (4 g, 26.9 mmol) was added under a flow of N2. The reaction mixture was stirred at rt for overnight and was followed by TLC. After completion, solvents were removed under vacuum. The residue was purified by flash column chromatography (silica gel, 10 inch, 35 mm dia, EtOAc/hexanes

(1:2)). The combined solvents were evaporated under vacuum to obtain T2 (80 mg, 0.28

o 1 mmol, 88%) as yellow solid: mp 187-192 C; TLC Rf = 0.16 [EtOAc/hexanes (1:3)]; H

NMR ((CD3)2CO, 600 MHz) δ 9.17 (1H, s), 8.48 (1H, d, J = 9.0 Hz), 7.77 (1H, s), 7.68

(1H, d, J = 8.4 Hz), 7.11 (1H, d, J = 1.2 Hz), 6.97 (1H, dd, J = 1.8, 8.4 Hz), 6.60 (1H, dd,

13 J = 2.4, 9.0 Hz), 6.51 (1H, d, J = 2.4 Hz), 3.91 (3H, s); C NMR ((CD3)2CO, 150 MHz)

δ 185.0, 167.6, 167.2, 158.4, 152.4, 134.4, 124.7, 120.4, 117.5, 115.3, 113.4, 108.5,

130 103.7, 101.7, 98.5, 56.1; Anal. (%) calcd for C16H12O5, C 67.60, H 4.25, found C 67.26,

H 4.44.

6-Benzyloxy-2-(2’-benzyloxy-4’-methoxymethylenoxy)benzoyl-1-benzofuran (T3)

To a solution of aldehyde 2b (3.22 g, 14 mmol) and the α-iodo ketone 3 (5.0 g, 12 mmol) in Me2CO (100 mL), K2CO3 (2.5 g, 18 mmol) was added under a flow of N2. The reaction mixture was stirred under reflux for 12-15 h and was followed by TLC. After reaction completion, the solvents were evaporated under vacuum and the residue was dissolved in EtOAc/H2O (1:1). The organic phase was washed with 1 M NaOH, 0.1 M

HCl, brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum. The residue was recrystallized from MeOH to obtain T3 (4.5 g, 9 mmol, 75%) as off white

o 1 power: mp: 97.5-100 C; TLC Rf = 0.22 [EtOAc/hexanes (1:3)]; H NMR ((CDCl3, 600

MHz) δ 7.50 (2H, m), 7.45 (2H, d, J = 7.8 Hz), 7.397 (2H, t, J = 7.8 Hz), 7.33 (1H, m),

7.24 (3H, m), 7.20 (3H, m), 7.09 (1H, m), 7.00 (1H, m), 6.74 (1H, m), 6.71 (1H, m), 5.21

13 (2H, d, J = 0.6 Hz), 5.12 (2H, s), 5.08 (2H, s), 3.49 (3H, s); C NMR (CDCl3, 100 MHz)

δ 183.3, 160.9, 159.9, 158.3, 157.2, 153.0, 136.2, 136.1, 131.4, 128.6, 128.3, 128.1,

127.7, 127.5, 126.9, 123.5, 122.2, 120.7, 116.3, 114.7, 107.5, 101.9, 96.8, 94.3, 70.4,

70.3, 56.2; Anal. (%) calcd for C31H26O6, C 75.29, H 5.30, found C 75.16, H 5.41.

6-Benzyloxy-2-(2’-benzyloxy-4’-hydroxy)benzoyl-1-benzofuran (T4)

To a solution of T3 (1.0 g, 2 mmol) in anhyd EtOAc (50 mL) at 0 oC, HCl(g) was bubbled through for 15-20 min (pH 1). The reaction mixture was stirred for 2 h at rt and was followed by TLC. After completion, the mixture was washed with H2O and NaHCO3

131 and with brine. The organic phase was dried over anhyd Na2SO4 and was evaporated under vacuum. The residue was purified by flash column chromatography

[EtOAc/hexanes (1:2)] to obtain T4 (0.63 g, 1.4 mmol, 70%) as yellow solid: mp 136–

o 1 140 C; TLC Rf = 0.1 [EtOAc/hexanes (1:3)]; H NMR (CDCl3, 600 MHz) δ 7.51 (1H, d,

J = 9.0 Hz), 7.40 (6H, m), 7.28 (1H, s), 7.21 (5H, m), 7.09 (1H, d, J = 1.8 Hz), 7.00 (1H, dd, J = 1.8, 8.4 Hz), 6.55 (1H, d, J = 1.8 Hz), 6.49 (1H, dd, J = 1.8, 8.4 Hz), 5.39 (1H, s),

13 5.12 (2H, s), 5.00 (2H, s); C NMR (CDCl3, 100 MHz) δ 183.4, 159.9, 159.5, 158.8,

157.2, 153.0, 136.2, 136.0, 131.9, 128.6, 128.3, 128.1, 127.7, 127.5, 126.7, 125.2, 123.5,

121.3, 120.7, 116.2, 114.7, 107.4, 100.9, 96.8, 70.47, 70.40; Anal. (%) calcd for

C29H22O5·0.05 H2O, C 77.17, H 4.93 found C 76.77, H 5.07.

6-Benzyloxy-2-(2’, 4’-dihydroxy)benzoyl-1-benzofuran (T5)

To a solution of T3 (1.0 g, 2 mmol) in anhyd EtOAc (50 mL) at 0 oC, HCl (g) was bubbled through for 15-20 min (pH ca. 1). The reaction mixture was stirred for 2 h at rt and was monitored by TLC. After reaction completion the mixture was washed with H2O and NaHCO3 and finally with brine. The organic phase was dried over anhyd Na2SO4 and dried over anhyd Na2SO4 and dried over anhyd Na2SO4 and evaporated under vacuum.

The residue was purified by flash column chromatography [EtOAc/hexanes (1:2)] to

o obtain T5 (0.2 g, 0.55 mmol, 27%) as yellow solid: mp 173-176 C; TLC Rf = 0.19

1 [EtOAc/Hexanes (1:3)]; H NMR (CDCl3, 600 MHz) δ 8.36 (1H, d, J = 8.4 Hz), 7.61

(2H, m), 7.47 (2H, m), 7.42 (2H, m), 7.36 (1H, m), 7.17 (1H, d, J = 1.8 Hz), 7.06 (2H, dd, J = 1.8, 8.4 Hz), 6.48 (2H, m), 5.50 (1H, br), 5.16 (2H, s), -0.56 (1H, s); 13C NMR

(CDCl3 , 150 MHz) δ 184.3, 166.4, 162.4, 160.0, 157.3, 151.9, 136.1, 134.1, 128.7,

132 128.2, 127.4, 123.4, 120.3, 116.4, 115.1, 107.9, 103.7, 96.7, 70.5; Anal. (%) calcd for

C22H16O5, C 73.33, H 4.48, found C 73.40, 4.48.

6-Hydroxy-2-(2,’4’-dihydroxy)benzoyl-1-benzofuran (T6)

To solution of T4 (0.15 g, 0.33 mmol) in anhyd CH2Cl2 (2 mL), a solution of trifluoroacetic (4 mL) saturated with PMB (1 g, 6.7 mmol) was added under flow of N2.

The reaction mixture was stirred at rt for 3-4 h. The reaction was monitored by TLC.

After completion, reaction mixture was diluted with EtOAc (25 mL) and then was poured into H2O (50 mL). The mixture was neutralized with saturated NaHCO3 to the neutral pH. The aqueous phase was extracted with EtOAc (3x25 mL). The combined organic phase was washed with 10% NaHCO3, 1 M aq HCl, H2O, brine, dried over anhyd

Na2SO4 and evaporated to dryness under vacuum. The residue was further purified by flash column chromatography (ca. 400 mL, EtOAc/hexanes (1:3)) to obtained T6 (80 mg,

o 0.3 mmol, 90%) as yellow solid: mp 308-313 C; TLC Rf = 0.29 [EtOAc/hexanes (1:1)];

1 H NMR (DMSO-d6, 600 MHz) δ 10.73 (1H, br), 10.22 (1H, br), 8.09 (1H, d, J = 8.4

Hz), 7.72 (1H, s), 7.62 (1H, d, J = 8.4 Hz), 7.03 (1H, s), 6.88 (1H, dd, J = 1.8, 8.4 Hz),

6.46 (1H, dd, J = 1.8, 8.4 Hz), 6.34 (1H, d, J = 1.8 Hz), -1.08 (1H, s); 13C NMR (DMSO- d6 , 100 MHz) δ 183.3, 164.5, 164.4, 159.1, 156.9, 150.7, 133.5, 124.0, 118.9, 116.9,

114.5, 112.5, 108.4, 102.8, 97.5; Anal. (%) calcd for C15H10O5·0.11 H2O, C 66.18, H

3.78, found, C 65.80, H 4.11.

133 1-(2’-Benzyloxy-4’-methoxy)phenyl-2-(2’,2’-dimethyl-6’-carboaldehyde)-2H-1-benz- opyranoxyethanone (25)

To a solution of the aldehyde 7a (0.41 g, 2.0 mmol) and the α-iodo ketone 21 (0.69 g, 1.8 mmol) in Me2CO (15 mL), K2CO3 (0.33 g, 2.4 mmol) was added under a flow of N2. The reaction mixture was stirred under reflux for overnight. The progress of reaction was monitored using TLC. After reaction completion, the solvents were evaporated under vacuum and the residue was dissolved in EtOAc/water [100 mL (1:1)]. The organic phase was washed with 1 M NaOH, 0.5 M HCl, brine and was dried over anhyd Na2SO4 and was evaporated to dryness under vacuum and the residue was recrystallized from

EtOAc/hexanes to obtain 25 (0.30 g, 0.68 mmol, 40%) as off white power: mp: 143-146 o 1 C; TLC Rf = 0.27 [EtOAc/hexanes (1:2)]; H NMR ((CD3)2CO, 600 MHz) δ 10.34 (1H, s), 7.92 (1H, d, J = 8.4 Hz), 7.59 (1H, d, J = 9.0 Hz), 7.53 (1H, m), 7.37 (3H, m), 6.77

(1H, d, J = 2.4 Hz), 6.68 (1H, dd, J = 2.4, 8.4 Hz), 6.64 (1H, d, J = 8.4 Hz), 6.55 (1H, d, J

10.2 Hz), 5.72 (1H, d, J = 9.6 Hz), 5.26 (2H, s), 5.25 (2H, s), 3.88 (3H, s), 1.40 (6H, s);

13 C NMR ((CD3)2CO, 100 MHz) δ 192.8, 189.0, 166.2, 161.5, 160.1, 159.7, 136.9, 133.0,

131.3, 129.7, 129.4, 129.1, 129.1, 123.8, 118.8, 116.8, 114.9, 113.5, 107.5, 99.9, 82.3,

77.9, 71.6, 56.0, 28.0; Anal. (%) calcd for C28H26O6·0.5 H2O·0.04 CH2Cl2, C 71.52, H

5.80, found, C 71.23, H 5.40.

2,2-Dimethyl-6-(2’-benzyloxy-4’-methoxy)benzoyl-2H-furo[2,3-f]-1-benzopyran (T7)

To a solution of 25 (25 mg, 54 μmol) in anhyd THF (5 mL) at -78 oC, lithium diisopropyl amide (LDA) (50 μL, 2.0 M stock solution, 108 μmol) was added dropwise. The reaction was stirred at -78 oC for 30-40 min and then at rt for 1 h followed by stirring at 50 oC for

134 2-4 h. Progression of the reaction was followed by TLC. After completion, it was quenched with saturated NH4Cl solution. The aq phase was extracted with EtOAc (2x10 mL). The combined organic phase was washed with 0.1 M HCl, H2O, brine, dried over anhyd Na2SO4 and was evaporated to dryness under vacuum. The residue was further purified by flash column chromatography [EtOAc/hexanes (1:2)] to obtain T7 (20 mg, 45

o μmol, 90%) as yellowish powder: mp 132-137 C; TLC Rf = 0.59 [EtOAc/hexanes (1:2)];

1 H NMR (CDCl3, 600 MHz) δ 7.52 (1H, d, J = 8.4 Hz), 7.34 (1H, d, J = 8.4 Hz), 7.23

(6H, m), 6.86 (1H, d, J = 9.6 Hz), 6.78 (1H, d, J = 8.4 Hz), 6.57 (2H, m), 5.68 (1H, d, J =

13 9.6 Hz), 5.08 (2H, s), 3.85 (3H, s), 1.47 (6H, s); C NMR (CDCl3, 100 MHz) δ 183.4,

163.2, 158.6, 153.5, 153.1, 152.3, 136.2, 131.7, 130.2, 128.3, 127.7, 126.8, 122.5, 121.6,

120.8, 116.7, 115.7, 114.5, 106.5, 104.8, 100.4, 77.2, 70.4, 55.5, 28.1, 27.8; Anal. (%) calcd for C28H24O5·0.4 H2O, C 75.12, H 5.58, found C 75.01, H 5.72.

2,2-Dimethyl-6-(2’-hydroxy-4’-methoxy)benzoyl-2H-furo[2,3-f]-1-benzopyran (T8)

To a solution of T7 (0.11 g, 0.22 mmol) in anhyd CH2Cl2 (12 mL), PMB (1 g, 6.7 mmol) was added under argon. The resulting solution was stirred at -78 oC for 10-15 min followed by slow addition of BCl3 (0.22 mmol). The reaction mixture was stirred at -78 oC for 2-4 h and was monitored by TLC. After completion reaction was quenched with a

o mixture of CHCl3 and MeOH (10:1, ca 15 mL) at -78 C and was stirred for additional 30 min and then was warmed rt. The volatiles were evaporated under vacuum and the residue was purified by flash column chromatography [EtOAc/hexanes (1:2)] to obtain

o T8 (ca. 60 mg, 0.17 mmol, 78%) as yellow powder: mp 117.0-122.0 C; TLC Rf = 0.81

1 [EtOAc/hexanes (1:2)]; H NMR (CDCl3, 600 MHz) δ 8.35 (1H, d, J = 9.0 Hz), 7.58 (1H,

135 s), 7.44 (1H, d, J = 8.4 Hz), 7.87 (1H, d, J = 10.2 Hz), 7.84 (1H, d, J = 8.4 Hz), 6.55 (1H, dd, J = 2.4, 9.0 Hz), 6.51 (1H, d, J = 2.4 Hz), 5.75 (1H, d, J = 10.2 Hz), 3.89 (3H, s), 1.50

13 (6H, s), -0.41 (1H, s); C NMR (CDCl3, 150 MHz) δ 166.8, 166.1, 153.6, 152.4, 133.2,

130.5, 122.4, 120.4, 116.7, 115.5, 114.8, 112.8, 108.0, 106.3, 101.0, 55.6, 29.6, 27.8;

Anal. (%) calcd for C21H18O5·0.1 C4H8O2, C 71.56, H 5.28, found C 71.88, H 5.65.

1-(2’-Benzyloxy-4’-methoxymethylenoxy)phenyl-2-(2’,2’-dimethyl-6-carbo-aldehyd- e)-2H-1-benzopyranoxyethanone (26)

To a solution of aldehyde 7a (0.25 g, 1.2 mmol) and α-iodo ketone 3 (0.62 g, 1.5 mmol) in Me2CO (15 mL), K2CO3 (0.25 g, 1.8 mmol) was added under N2. The reaction mixture was stirred under reflux for 12-15 h and reaction progress was monitored using TLC.

After completion, the solvents were removed and residue was dissolved in EtOAc/water

[100 mL (1:1)]. The organic phase was washed with 1 M NaOH, 0.5 M HCl, brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum. The residue was further purified by flash column chromatography [EtOAc/hexanes (1:3)] to obtain 26 (0.5 g, 1.02

o mmol, 85%) as off white power: mp 96-102 C; TLC Rf = 0.23 [EtOAc/hexanes (1:2)];

1 H NMR CDCl3, 600 MHz) δ 10.15 (1H, s), 8.04 (1H, d, J = 8.4 Hz), 7.62 (1H, d, J = 8.4

Hz), 7.29 (5H, m), 6.74 (1H , dd, J = 2.4, 8.4 Hz), 6.68 (1H, d, J = 1.8 Hz), 6.63 (1H, d, J

= 8.4 Hz), 6.56 (1H, d, J = 9.6 Hz), 5.57 (1H, d, J = 10.2 Hz), 5.21 (2H, s), 5.09 (2H, s),

13 5.08 (2H, s), 3.47 (3H, s), 1.41 (6H, s); C NMR (CDCl3, 100 MHz) δ 192.1, 188.5,

162.8, 160.2, 159.5, 158.5, 135.0, 132.9, 130.3, 129.7, 128.6, 128.5, 127.8, 122.5, 118.6,

116.0, 114.2, 113.2, 108.8, 100.5, 94.1, 82.0, 71.0, 56.3, 28.1; Anal. (%) calcd for

C29H28O7, C 71.30, H 5.78, found C 71.15, H 5.86.

136 2,2-Dimethyl-6-(2’-benzyloxy-4’-methoxymethylenoxy)benzoyl-2H-furo[2,3-f]-1- benzopyran (27)

To a solution of ketoaldehyde 26 (25 mg, 51 μmol) in anhyd THF (5 mL) at -78 oC, lithium diisopropyl amide (LDA) (51 μL of 2.0 M solution, 102 μmol) was added dropwise. The reaction was stirred at -78 oC for 30-40 min and then at rt for 1 h then at

50 oC for 2-4 h. The reaction progression was followed by TLC. After completion the reaction was quenched with saturated NH4Cl solution. The aqueous phase was extracted with EtOAc (2x10 mL). The combined organic phase was washed with 0.1 M HCl, H2O, brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum to obtain 27 (20 mg, 42 μmol, 83%) as yellowish semi-solid residue: TLC Rf = 0.57 [EtOAc/hexanes

1 (1:2)]; H NMR (CDCl3, 600 MHz) δ 7.49 (1H, d, J = 8.4 Hz), 7.34 (1H, d, J = 8.4 Hz),

7.23 (6H, m), 6.86 (1H, d, J = 9.6 Hz), 6.79 (1H, d, J = 9.0 Hz), 6.74 (1H, d, J = 1.8 Hz),

6.72 (1H, dd, J = 1.8, 8.4 Hz), 5.69 (1H,, d, J = 9.6 Hz), 5.21 (2H, s), 5.08 (2H,s), 3.50

13 (3H, s), 1.47 (6H, s); C NMR (CDCl3, 150 MHz) delta 183.3, 163.2, 158.3, 153.6,

153.0, 152.4, 136.1, 131.4, 130.2, 128.3, 127.7, 126.9, 126.9, 122.5, 122.4, 120.7, 117.0,

115.7, 114.5, 106.5, 104.8, 100.4, 70.4, 55.5, 29.7, 27.8, 27.7; Anal. (%) calcd for

C29H26O6, C 74.03, H 5.57, found C 74.17, H 6.31.

2,2-Dimethyl-6-(2’-benxyloxy-4’-hydroxy)benzoyl-2H-furo[2,3-f]-1-benzopyran (T9)

o To a solution of intermediate 27 (70 mg, 0.15 mmol) in anhyd CH2Cl2 (5 mL) at 0 C, triphenylphosphine hydrobromide (PPh3·HBr) (0.14 g, 0.4 mmol) was added under argon.

The resulting solution was stirred at 0 oC for 30-40 min and then at rt for 2-4 h and the reaction progress was monitored by TLC. After completion, the reaction was quenched

137 with addition of 5% aq NaHCO3 (5 mL). After stirring for 20-30 min, the aq phase was extracted with CH2Cl2 (3x10 mL). The combined organic phase was washed with 0.1 M aq HCl, brine and was dried over anhyd Na2SO4. After filtration the volatiles were evaporated to dryness under vacuum. The residue was further purified by flash column chromatography [EtOAc/hexanes (1:3)] to obtain T9 (40 mg, 0.09 mmol, 63%) as yellow

o 1 solid: mp 183-189 C; TLC Rf = 0.25 [EtOAc/hexanes (1:2)]; H NMR (CDCl3, 600

MHz) δ 7.44 (1H, d, J = 8.4 Hz), 7.34 (1H, d, J = 8.4 Hz), 7.25 (1H, s), 7.20 (5H, m),

6.83 (1H, d, J = 9.6 Hz), 6.79 (1H, d, J = 9.0 Hz), 6.55 (1H, d, J = 1.8 Hz), 6.50 (1H, dd,

J = 1.8, 8.4 Hz), 6.32 (1H, br), 5.67 (1H, d, J = 9.6 Hz), 5.01 (2H, s), 1.46 (6H, s); 13C

NMR ((CD3)2CO, 150 MHz) δ 183.5, 162.4, 159.7, 154.5, 154.1, 152.7, 137.6, 132.3,

131.7, 128.9, 128.3, 127.8, 123.6, 121.8, 121.4, 116.0, 115.8, 115.0, 108.3, 107.0, 101.4,

77.67, 70.7, 28.1, 27.8; Anal. (%) calcd for C27H22O5·0.1 H2O, C 75.72, H 5.22, found C

75.36, H 5.62.

2,2-Dimethyl-6-(2’,4’-dihydroxy)benzoyl-2H-furo[2,3-f]-1-benzopyran (T10)

To a solution of intermediate 27 (80 mg, 0.17 mmol) in anhyd CH2Cl2 (10 mL), pentamethylbenzene (1 g, 6.7 mmol) was added under argon. The resulting solution was

o stirred at -78 C for 10-15 min followed by slow addition of BCl3 (0.65 mmol). The reaction was monitored by TLC. After completion reaction was quenched with CHCl3 and MeOH (10:1, ca 15 mL) mixture and the reaction mixture was allowed to stir for 30-

40 min and then was warmed up to rt. The volatiles were evaporated under vacuum and the crude mixture was purified by flash chromatography [EtOAc/hexanes (1:3)] to obtain

o T10 (40 mg, 0.12 mmol, 70%) as yellow powder: mp 132.0-137.0 C; TLC Rf = 0.46

138 1 [EtOAc/hexanes (1:2)]; H NMR (CDCl3, 400 MHz) δ 12.78 (1H, s), 8.33 (1H, d, J = 8.8

Hz), 7.57 (1H, s,), 7.43 (1H, d, J = 8.8 Hz), 6.84 (2H, m), 6.49 (2H, m), 6.19 (1H, br),

13 5.73 (1H, d, J = 10.0 Hz), 1.49 (6H, s); C NMR (CDCl3, 100 MHz) δ 184.4, 166.4,

162.8, 153.7, 151.8, 133.9, 130.5, 122.4, 120.4, 116.9, 115.4, 114.9, 113.2, 108.1, 106.3,

103.7, 103.6, 77.2, 28.0, 27.8; Anal. (%) calcd for C20H16O5·0.2 H2O, C 70.66, H 4.86, found C 70.47, H 5.09.

3.6 Scale-up and Process Chemistry Enhancement for Production of CD3-246

Boc-Met-Gly-OBzl (29)

To solution of N-Boc-Met-OH 28 (25.5 g, 53.4 mmol) in anhyd CH2Cl2 (110 mL) at rt,

N,N-carbonyldiimidazole (CDI) (9.02 g, 55.63 mmol) was added under a flow of N2. The reaction mixture was stirred at rt for 1 h. Then to this activated acid, a solution of H-Gly-

OBzl·p-tosylate (15.0 g, 44.4 mmol) and Et3N (6.2 mL, 44.5 mmol) in anhyd CH2Cl2

(105 mL) was cannulated under N2. The reaction mixture was stirred at rt overnight and was followed by TLC. After completion, saturated NH4Cl solution was added to quench the reaction. The organic phase was washed with 10% NaHCO3, 0.1 M HCl, brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum. The solid residue was initially precipitated from CH2Cl2/hexanes and then the ppt was recrystallized from Et2O

o to obtain 29 (15.6 g, 39.4 mmol, 88%) as a white solid: mp 73-76 C; TLC Rf = 0.49

1 [EtOAc/hexanes (1:1)]; H NMR (CDCl3, 600 MHz) δ 7.36 (5H, m), 6.74 (1H, br), 5.21

(1H, m), 5.18 (2H, s), 4.34 (1H, m), 4.13 (1H, m), 4.05 (1H, m), 2.59 (2H, t, J = 7.2 Hz),

13 2.10 (3H, s), 2.10 (1H, m), 1.94 (1H, m), 1.44 (9H, s); C NMR (CDCl3, 150 MHz) δ

139 171.7, 169.3, 155.5, 135.0, 128.6, 128.5, 128.4, 80.2, 67.2, 53.2, 41.3, 31.4, 30.0, 28.2,

15.2.

Boc-D-Tyr(Bzl)-Met-Gly-OBzl (30)

A solution of the dipeptide 29 (27.67 g, 70 mmol) in anhyd EtOAc (260 mL) at 0 oC, was bubbled (2-3 bubbles per sec) through with HCl(g) for 1-2 h. The reaction mixture was stirred at rt for 12-14 h and was followed by TLC. After completion, the organic solvents were evaporated under vacuum to obtain a white solid residue (23.3 g) which was directly used in the next step without further purification.

A solution of the above white solid residue (23.3 g, 70 mmol) and Et3N (9.76 mL, 70 mmol) in anhyd CH2Cl2 (175 mL), was cannulated into a solution of N-Boc-D-Tyr(Bzl)-

OH (31.20 g, 84 mmol) and CDI (14.2 g, 87.5 mmol) in anhyd CH2Cl2 (180 mL) under a flow of N2. The reaction mixture was stirred overnight at rt and was followed by TLC.

After completion, saturated NH4Cl solution was added to quench the reaction. The organic phase was washed with 10% NaHCO3, 0.1 M HCl, brine, dried over anhyd

Na2SO4 and evaporated to dryness under vacuum. The solid residue was precipitated from CH2Cl2/hexanes to obtain 30 (33.3 g, 51.2 mmol, 73% over two steps) as white

1 solid: mp 131-137 °C; TLC Rf = 0.75 [MeOH/CH2Cl2 (1:12)]; H NMR [CD3)2SO, 600

MHz] δ 8.33 (1H, t, J = 6.0 Hz), 8.16 (1H, d, J = 8.4 Hz), 7.34 (10H, m), 7.10 (2H, d, J =

8.4 Hz), 7.05 (1H, d, J = 7.2 Hz), 6.86 (1H, d, J = 8.4 Hz), 5.08 (2H, s), 5.01 (2H, s), 4.27

(1H, m), 4.07 (1H, m), 3.86 (2H, d, J = 5.4 Hz), 2.77 (1H, m), 2.67 (1H, m), 2.16 (2H, m), 1.93 (3H, s), 1.84 (1H, m), 1.72 (1H, m), 1.27 (9H, s).

140 N-4-(2-Thienyl)butyroyl-D-Tyr(Bzl)-Met-Gly-OBzl (CD3-404)

A solution of the tripeptide 30 (20.1 g, 30.9 mmol) in anhyd EtOAc (200 mL) at 0 oC, was bubbled (2-3 bubbles per sec) through with HCl(g) for 1-2 h. The reaction mixture was stirred at rt for 12-14 h and was followed by TLC. After completion, the organic solvents were evaporated under vacuum to obtain a white solid residue (18.1 g) which was directly used in the next step without further purification.

A solution of the above white solid residue (18.1g, 30.9 mmol) and Et3N (4.32 mL,

30.9 mmol) in anhyd CH2Cl2 (110 mL), was cannulated into a solution of 4-(2- thienyl)butyric acid (5.41 mL, 37.2 mmol) and CDI (6.28 g, 38.71 mmol) in anhyd

CH2Cl2 (70 mL) under a flow of N2. The reaction mixture was stirred overnight at rt and was followed by TLC. After completion, saturated NH4Cl solution was added to quench the reaction. The organic phase was washed with 10% NaHCO3, 0.1 M HCl, brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum. The solid residue was precipitated from CH2Cl2/Et2O to obtain CD3-404 (15.2 g, 21.6 mmol, 70% over two

1 steps): mp 168-175 °C; TLC Rf = 0.54 [MeOH/CH2Cl2 (1:12)]; H NMR ((CD3)2SO, 600

MHz) δ 8.31 (1H, t, J = 6.0 Hz), 8.12 (1H, d, J = 7.8 Hz), 8.05 (1H, d, J = 8.4 Hz), 7.36

(10H, m, H13), 7.28 (1H, d, J = 5.4 Hz), 7.16 (2H, m), 7.01 (3H, m), 6.76 (1H, d, J = 3.6

Hz), 5.12 (2H, s), 4.99 (2H, s), 4.50 (1H, m), 4.38 (1H, m), 3.92 (1H, dd, J = 6.0, 17.4

Hz), 3.85 (1H, dd, J = 6.0, 17.4 Hz), 2.85 (1H, dd, J = 6.6, 13.2 Hz), 2.71 (1H, dd, J =

8.4, 13.2 Hz), 2.59 (2H, m), 2.44 (2H, m), 2.08 (2H, m), 2.00 (3H, s), 1.92 (1H, m), 1.80

(1H, m), 1.70 (2H, m).

141 N-4-(2-Thienyl)butyroyl-D-Tyr-Met-Gly-OH (CD3-246).

To a two neck round bottom flask loaded with acylated tripeptide intermediate CD3-404

(5.6 g, 8 mmol), trifluoroacetic acid (67 mL) saturated with pentamethylbenzene (12 g,

80 mmol) was added under a flow of N2. The reaction mixture was stirred at rt for 3 h and was followed by TLC. After completion, reaction mixture was diluted with CH2Cl2 and the volatiles were evaporated to dryness under vacuum at 35 oC. To the residue,

H2O/Et2O (1:1, 120 mL) mixture was added and was stirred vigorously. A white ppt was formed which was collected after filtration. After drying under vacuum the ppt was directly used in the next step without further purification.

The above solid was dissolved in THF (80 mL) and the pH of the solution was adjusted to about 13-14 with 0.5% aq NaOH solution (200 mL). The reaction mixture was stirred for 6 h at rt and was followed by TLC. After completion, the reaction mixture was filtered and the pH of the filtrate was adjusted to 4-5 using 2 M aq HCl. The mixture was reduced to one-fourth of its original volume under vacuum at 35 oC. The resulting semisolid residue was dissolved in a H2O/Et2O (1:1) mixture. The aqueous phase was further acidified to pH 1-2 and was extracted with Et2O. The combined organic phase was washed with brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum. A solution of the solid residue in Me2CO was decolorized using activated charcoal and organic solvents were dried over anhyd Na2SO4 and evaporated under vacuum. The residue obtained was precipitated from Me2CO/Et2O to obtain CD3-246

(1.73 g, 3.3 mmol, 41.5% over two steps) as a white solid: mp 147-150 °C; TLC Rf =

1 0.31 [CH2Cl2/Me2CO/AcOH (20:10:1)]; H NMR [(CD3)2SO] δ 9.15 (1H, s), 8.25 (1H, m), 8.11 (1H, d, J = 7.2 Hz), 7.29 (1H, d, J = 4.8 Hz), 7.01 (2H, d, J = 8.4 Hz), 6.92 (1H,

142 m), 6.78 (1H, d, J = 3.0 Hz), 6.62 (2H, d, J = 8.4 Hz), 4.42 (1H, dd, J = 7.8 , 15.0 Hz),

4.30 (1H, m), 3.71 (2H, m), 2.80 (1H, dd, J = 6.6, 13.8 Hz), 2.66 (3H, m), 2.25 (2H, m),

2.12 (2H, m), 1.98 (3H, s), 1.87 (1H, m), 1.71 (3H, m); Anal. (%) calcd for

C24H31N3O6S2·0.3 H2O, C 54.69, H, 6.04, N, 7.97, found C 54.38, H 5.91, N 7.75.

NH2-Met-Gly-OBzl Hydrogen Chloride (35)

To a solution of Boc-Met-OH (11.07 g, 44.4 mmol) in CH2Cl2 (75 mL), N,N- carbonyldiimidazole (CDI) (6.64 g, 41 mmol) was added under a flow of N2. The reaction mixture was stirred at rt for 1 h. To the activated acid, a solution of H-Gly-

OBzl·p-tosylate (12.5 g, 37 mmol) and Et3N (5.13 mL, 37 mmol) in anhyd CH2Cl2 was cannulated under a flow of N2. The reaction mixture was stirred at room temp for 12-14 h and was followed by TLC. After completion, the reaction mixture was washed with 5% aq NaHCO3, 0.1 M HCl, brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum. The solid residue was precipitated from CH2Cl2/hexanes to obtain 29 (11.4 g,

o 28.8 mmol, 78%) as a white solid: mp 73-76 C; TLC Rf = 0.49 [EtOAc/hexanes (1:1)];

1 H NMR (CDCl3, 600 MHz) δ 7.36 (5H, m), 6.74 (1H, br), 5.21 (1H, m), 5.18 (2H, s),

4.34 (1H, m), 4.13 ( 1H, m), 4.05 (1H, m), 2.59 (2H, t, J = 7.2 Hz), 2.109 (3H, s), 2.100

13 (1H, m), 1.94 (1H, m), 1.44 (9H, s); C NMR (CDCl3, 150 MHz) δ 171.7, 169.3, 155.5,

135.0, 128.6, 128.5, 128.4, 80.2, 67.2, 53.2, 41.3, 31.4, 30.0, 28.2, 15.2.

To a solution of 29 (10.0 g, 30 mmol) in anhyd EtOAc (120 mL) at 0 oC, was bubbled

(2-3 bubble per sec) through HCl(g) for 40-50 min. The reaction mixture was stirred at rt and was followed by TLC. After completion, solvents and volatiles were evaporated under vacuum. The obtained white solid residue representing HCl salt of de-protected

143 dipeptide 35 (9.0 g) was directly used in the eventual coupling step without further purification.

NH2-Met-Gly-OtBu·oxalate (35a)

To a solution of N-Fmoc-methionine 33 (0.64g, 1.72 mmol) in CH2Cl2 (10 mL), CDI

(0.33g, 2.07 mmol) was added under a flow of N2. The reaction mixture was stirred at rt for 20 min. To this, a solution of H-Gly-OBzl·acetate (0.3 g, 1.56 mmol) and DIPEA (0.4 g, 3.12 mmol) in CH2Cl2 (15 mL), was cannulated under a flow of N2. The combined reaction mixture was stirred at rt for 2 h and was follwed by TLC. After completion, reaction mixture was diluted with Et2O (70 mL) and filtered. The filtrate was washed with 0.1M HCl, 10% NaHCO3, brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum. The residue was purified by flash column chromatography using

[CH2Cl2/MeOH (50:1)] to obtain N-Fmoc-Met-Gly-OtBu (0.49, 0.65 mmol, 65%) a

o 1 white solid: mp 108-110 C; TLC Rf = 0.22 [MeOH/CH2Cl2.(1:50)]; H NMR (CDCl3,

400 MHz) δ 7.76 (2H, d, J = 7.6 Hz), 7.58 (2H, d, J = 7.2 Hz), 7.39 (2H, t, J = 7.2 Hz),

7.31 (2H, t, J = 7.2 Hz), 6.54 (1H, s), 5.63 (1H, d, J = 7.6 Hz), 4.41 (3H, m), 4.21 (1H, t,

J = 6.8 Hz), 3.91 (2H, m), 2.58 (2H, br), 2.11 (4H, br), 1.93 (1H, m), 1.46 (9H, s); 13C

NMR (CDCl3, 100 MHz ) δ 171.1, 168.4, 156.0, 143.7, 143.6, 141.2, 127.7, 127.0, 125.0,

119.9, 82.4, 67.0, 53.6, 47.0, 42.0, 31.5, 29.9, 27.9, 15.1.

To a solution of N-Fmoc-Met-Gly-OtBu (1 g, 2.01 mmol) in THF (15 mL), and 1- octanethiol (3.0 g, 20.6 mmol) and a solution of 1,8-diazabicyclo[5.4.0]undecane (DBU)

(1-2 drops) in THF (5 ml) was added. The reaction mixture was stirred at rt for 3 h. and was followed by TLC. After completion, the volatiles were evaporated under vacuum.

144 The residue was treated with saturated oxalic acid/EtOAc solution (50 mL) and stirred at rt for 3 h. The desired NH2-Met-Gly-OtBu·oxalate 35a was obtained as a white ppt.

N-4-(2-Thienyl)butyroyl-D-Tyr(Bzl)-OH (36)

To a solution of 4-(2-thienyl)butyric acid (1.74 mL, 12 mmol) in CH2Cl2 (20 mL), N,N- carbonyldiimidazole (CDI) (1.62 g, 10 mmol) was added at rt under a flow of N2 abd was stirred at rt for 2 h. After that a solution of H-(D)-Tyr(Bzl)-OH (2.98 g, 11 mmol) and

DBU (1.64 mL, 11 mmol) in anhyd CH2Cl2 was cannulated into it under a flow of N2.

The reaction mixture was stirred at rt overnight and was followed by TLC. After completion, the reaction mixture was filtered and the filtrate was washed with 0.1 M aq

HCl, H2O, brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum. The solid residue was precipitated from EtOAc/hexanes to obtain 36 (3.5 g, 8.1 mmol, 81%)

1 as off-white solid: mp 161-165 °C; TLC Rf = 0.44 [MeOH/CH2Cl2 (1:12)]; H NMR

[(CD3)2CO, 600 MHz] δ 7.45 (2H, m), 7.38 (2H, m), 7.31 (1H, m), 7.19 (3H, m) 6.90

(3H, m), 5.05 (1H, s), 4.67 (1H, m), 3.14 (1H, m), 2.92 (1H, m), 2.76 (2H, m), 2.22 (2H,

13 m), 1.87 (2H, m); C NMR [(CD3)2CO, 150 MHz] δ 173.4, 172.4, 158.5, 145.4, 138.4,

131.1, 130.5, 129.1, 128.5, 128.3, 127.4, 125.2, 123.8, 115.3, 70.2, 54.3, 37.2, 35.3, 28.4;

Anal. (%) calcd for C24H25NO4S, C 68.06, H 5.95, N 3.31, found C 68.04, H 5.91, N

3.32.

N-4-(2-Thienyl)butyroyl-D-Tyr(tBu)-OH (36a)

To a solution of 4-(2-thienyl)butyric acid (1.52 mL, 10 mmol) in CH2Cl2 (20 mL), N,N- carbonyldiimidazole (CDI) (2.02 g, 12 mmol) was added under a flow of N2. The

145 resulting solution was stirred for 2 h at rt. After that a solution of H-(D)-Tyr(tBu)-OH

(2.61 g, 10 mmol) and 1,8-diazabicyclo[5.4.0]undecane (DBU) (1.5 mL, 10 mmol) in anhyd CH2Cl2 (20 mL) was cannulated into it under a flow of N2. The reaction mixture was stirred at rt overnight and was followed by TLC. After completion, the reaction mixture was filtered and the filtrate was washed with 0.1 M HCl, H2O, brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum. The solid residue was precipitated from EtOAc/hexanes to obtain 36a (2.73 g, 7.0 mmol, 70%) as off-white

1 solid: TLC Rf = 0.52 [MeOH/CH2Cl2 (1:12)]; H NMR (DMSO-d6, 600 MHz] δ 12.66

(1H, br), 8.17 (1H, d, J = 8.0 Hz), 7.29 (1H, m), 7.12 (2H, d, J = 8.4 Hz), 6.91 (1H, m),

6.84 (2H, 8.0 Hz), 6.77 (1H, d, J = 2.8 Hz), 4.40 (1H, m), 3.01 (1H, dd, J = 4.8, 14 Hz),

2.78 (1H, m), 2.66 (2H, t, J = 7.6 Hz), 2.01 (2H, m), 1.72 (2H, m), 1.22 (9H, s); 13C

NMR (DMSO-d6, 100 MHz) δ 173.9, 172.3, 154.1, 144.9, 133.0, 130.2, 127.5, 125.1,

124.1, 124.0, 78.2, 54.0, 36.6, 34.9, 29.1, 28.1.

N-4-(2-Thienyl)butyroyl-D-Tyr(Bzl)-Met-Gly-OBzl (CD3-404) (by convergent route)

To a solution of acyl-Tyr(Bzl)-OH 36 (100 mg, 0.24 mmol) and NH2-Met-Gly-OBzl·HCl

35 (88 mg, 0.26 mmol) in anhyd DMF at 0 oC, a solution of diphenylphosphoryl azide

(DPPA) (60 μL, 0.27 mmol) in DMF was cannulated under a flow of N2, followed by the immediate addition of diisopropylethylamine (DIPEA) (90 μL, 0.24 mmol). The reaction mixture was stirred at 0 oC for 4 h, and then at rt for overnight. The reaction progress was followed by TLC. After completion, the reaction mixture was poured into a mixture of

H2O (100 mL) and Et2O (40 mL) was added with vigorous shaking. A white ppt was formed at the interface of the two layers. The ppt was collected after filtration and was washed with Et2O. The ppt was dried under vacuum to obtain epimerization free

146 (distinguished by 1H NMR as discussed in chapter 2, section 2.6) intermediate CD3-404

o (103 mg, 0.15 mmol, 61%) as a white solid: mp 168.175 C; TLC Rf = 0.54

1 [MeOH/CH2Cl2 (1:12)]; H NMR [(CD3)2SO, 600 MHz] δ 8.31 (1H, t, J = 6.0 Hz), 8.12

(1H, d, J = 7.8 Hz), 8.05 (1H, d, J = 8.4 Hz), 7.36 (10 H, m), 7.28 (1H, d, J = 5.4 Hz),

7.16 (2H, m), 7.01 (3H, m), 6.76 (1H, d, J = 3.6 Hz), 5.12 (2H, s), 4.99 (2H, s), 4.50 (1H, m), 4.38 (1H, m), 3.92 (1H, dd, J = 6.0, 17.4 Hz), 3.85 (1H, dd, J = 6.0, 17.4 Hz), 2.85

(1H, dd, J = 6.6, 13.2 Hz), 2.71 (1H, dd, J = 8.4, 13.2 Hz), 2.59 (2H, m), 2.44 (2H, m),

2.08 (2H, m), 2.00 (3H, s), 1.92 (1H, m), 1.80 (1H, m), 1.70 (2H, m).

2-Benzyloxynaphthalene (37)

To a solution of 2-naphathol (0.91 g, 6.3 mmol) in Me2CO (25 mL), K2CO3 (1.74 g, 12.6 mmol) and benzyl bromide (0.50 mL, 4.2 mmol) were added under a flow of N2. The reaction mixture was stirred at reflux for 24 h and was followed by by TLC. After completion, the organic solvents were evaporated under vacuum and the residue was dissolved in a mixture of H2O/ Et2O (1:1). The aq phase was extracted with Et2O and the combined organic phase was washed with brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum. The residue was recryllized from Et2O to obtain 37

o 131 o (0.69 g, 2.94 mmol, 70%) as white solid: mp 102-104 C [lit. mp 97-99 C]; TLC Rf =

1 0.72 [EtOAc/hexanes (1:2)]; H NMR (CDCl3, 600 MHz) δ 7.76 (2H, m,), 7.72 (1H, d, J

= 7.8 Hz), 7.49 (2H, d, J = 7.2 Hz), 7.42 (3H, m), 7.34 (2H, m), 7.23 (2H, m); 13C NMR

(CDCl3, 150 MHz) δ 156.6, 136.8, 134.4, 129.4, 129.0, 128.6, 128.0, 127.6, 127.5, 126.7,

126.3, 123.6, 119.0, 107.0, 69.9.

147 2-(4’-Methoxy)benzyloxynaphthalene (38)

To a solution of 2-naphathol (0.91 g, 6.3 mmol) in Me2CO (25 mL), K2CO3 (1.74 g, 12.6 mmol) and 4-methoxy benzyl chloride (0.57 mL, 4.2 mmol) were added under a flow of

N2. The reaction mixture was stirred under reflux for 24 h and was followed by TLC.

After completion, the organic solvents were evaporated under vacuum and the residue dissolved in a mixture of H2O/Et2O (1:1). The aq phase was extracted with Et2O and the combined organic phase was washed with brine, dried over anhyd Na2SO4 and evaporated to dryness under vacuum. The residue was recrystallized from Et2O to obtain

o 38 (0.78 g, 2.94 mmol, 70%) off-white crystals: mp 150.3-153.6 C; TLC Rf = 0.46

1 [EtOAc/hexanes (1:8)]; H NMR (CDCl3, 600 MHz) δ 7.76 (3H, m), 7.44 (3H, m), 7.35

13 (1H, m), 7.22 (2H, m), 6.95 (2H, m), 5.11 (2H, s), 3.84 (3H, s); C NMR (CDCl3, 600

MHz) δ 159.73, 157.03, 134.74, 129.65, 129.61, 129.25, 129.12, 127.87, 127.02, 126.59,

123.89, 119.37, 114.27, 107.28, 70.05, 55.56. Anal. (%) calcd for C18H16O2·0.1H2O, C

81.24, H 6.14, found C 81.21, H 6.04.

General De-Protection Procedure

To a solution of 37 (20 mg, 86 μmol) in anhyd CH2Cl2 (1mL), pentamethylbenzene

(127.5 mg, 860 μmol) dissolved in trifluoroacetic acid (2 mL) was added under a flow of nitrogen. The reaction mixture was stirred at rt for 4 h and was followed by TLC. After completion, the solvents were evaporated under vacuum to dryness and the residue was applied to prep-TLC. The prep-TLC was developed using EtOAc/hexanes (1:8) as eluting system. The bands were removed and analyzed to obtain 39 (15 mg, 55 μmol), for characterization see below.

148 1-Benzyl-2,3,4,5,6-pentamethylbenzene (39)

Isolated from de-protection reaction of 2-benzyloxynaphthalene using penta-methyl- benzene (10 equiv) with TFA/CH2Cl2 (2:1) as described in general deprotection

o 68 o procedure: mp 115.6-117.0 C [lit. mp 111-112 C]; TLC Rf = 0.36 [EtOAc/hexanes

1 (1:32)]; H NMR (CDCl3, 600 MHz) δ 7.24 (2H, m), 7.16 (1H, m), 7.04 (2H, d, J = 6.0

13 Hz), 4.12 (2H, s), 2.28 (3H, s), 2.25 (6H, s), 2.18 (6H, s); C NMR (CDCl3, 100 MHz) δ

140.5, 133.7, 133.1, 132.7, 132.4, 128.2, 127.9, 125.5, 36.1, 16.9, 16.85, 16.83; Anal. (%) calcd for C18H22, C 90.70, H 9.30, found, C 90.54, H 9.38.

1-(4’-Methoxy)benzyl-2,3,4,5,6-pentamethylbenzene (40)

Isolated from de-protection reaction of 2-(4‟-methoxy)benzyloxynaphthalene using pentamethylbenzene (10 equiv.) with TFA/CH2Cl2 (2:1) as described in general de-

o 1 protection procedure: mp 94.4-97.2 C, TLC Rf = 0.86 [EtOAc/hexanes (1:6)]; H NMR

(CDCl3, 600 MHz) δ 6.95 (2H, d, J = 8.4), 6.83 (2H, d, J = 8.4 Hz), 4.05 (2H, s), 3.77

13 (3H, s), 2.28 (3H, s), 2.25 (6H, s), 2.18 (6H, s); C NMR (CDCl3, 150 MHz,) δ 157.8,

134.3, 133.2, 132.9, 132.77, 132.71, 129.0, 113.9, 77.4, 77.2, 77.0, 55.4, 35.4, 17.2, 17.1,

17.0; Anal. (%) calcd for C19H24O·0.15 H2O·0.1 CH2Cl2, C 82.05, H 8.83, found, C

81.84, H 8.75.

3.7 Molecular Modeling and Docking Studies

All molecular modeling and docking studies were performed using SYBYL 8.0 from

Tripos, Inc., on a Linux workstation. The SKETCH option in SYBYL was used to generate three-dimensional structures for each compound using default settings.

149 Gasteiger-Huckel charges were applied to the molecules after adding all hydrogen atoms before energy calculations were performed using the Tripos force-field with a distance- dependent dielectric constant (4.0) and non-bonding cut-off (NB) of 8.0. The Powell conjugate-gradient algorithm with a termination criterion of 0.05 kcal/mol was used for energy minimizations. For pharmacophore mapping, distances were measured using

SYBYL‟s standard „compute options.‟

Docking studies were performed using the Surflexdock docking package on SYBYL

8.0. The crystal structure with THC (tetrahydrochrysene-2,8-diol) bound human ERs ligand binding domain (PDB ID 1L2I and 1L2J) were used for docking and active site spatial analysis studies. Only the chain „A‟ of the protein structure was used for all the analyses. The protein was prepared for docking with extraction of the ligand from active sites, followed by removal of all other substructures including water, and then addition of hydrogens to the protein. The binding pocket in ERα was searched via „protomol generation‟ from co-crystallized ligand (THC) where the threshold was 0.5 and where the

„bloat value‟ was zero. In the case of ERβ, the „protomol‟ was generated using an automatic mode where the threshold was 0.3 and the „bloat value‟ was one. These specific conditions were chosen as the most appropriate for each case based upon the performances of the generated models during internal and external validation.

For internal validation, the docking model-generated conformation of the ligand was aligned with that for the ligand‟s co-crystalized structure as a reference molecule using both the „Match Atoms‟ and „Fit Atoms‟ alignment methods. These methods provide

RMSD values (0.33 for ERα and 0.55 for ERβ, as mentioned in Section 2.1.2.2) reflect

150 the least squares fit between two molecules when they are undergoing such superimposing.

151

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94. Corey, E. J.; Seebach, D. Synthesis of 1,n-dicarbonyl derivatives using carbanions from 1,3-dithianes. Angew. Chem. Int. Ed. 1965, 4, 1077-8.

95. Takikawa, H.; Hachisu, Y.; Bode, J. W.; Suzuki, K. Catalytic enantioselective crossed aldehyde-ketone benzoin cyclization. Angew. Chem. Int. Ed. 2006, 45, 3492-4.

96. Camps, F.; Coll, J.; Messeguer, A.; Pericas, M. A.; Ricart, S.; Bowers, W. S.; Soderlund, D. M. An improved procedure for the preparation of 2,2-dimethyl-4- chromanones. Synthesis 1980, 725-7.

97. Lay, L. Simple synthesis of versatile coumarin scaffolds. Synth. Commun. 2006, 36, 2203-2209.

98. Nahm, S.; Weinreb, S. M. N-Methoxy-N-methylamides as effective acylating agents. Tetrahedron Lett. 1981, 22, 3815-18.

99. Beard, R. L.; Duong, T. T.; Takeuchi, J. A.; Li, L.; Tsang, K. Y.; Liu, X.; Vasudevan, J.; Wang, L.; Sinha, S. C.; Yuan, H.; Chandraratna, R. A. Preparation of 7-[(7-alkoxy)chrom-3-en-6-yl]heptatrienoates and 7-[(3-alkoxy)-5,6- dihydronaphthalen-2-yl]heptatrienoates having serum glucose reducing activity. US 6887896, 2005.

162 100. Silva, F.; Reiter, M.; Mills-Webb, R.; Sawicki, M.; Klaer, D.; Bensel, N.; Wagner, A.; Gouverneur, V. Pd(II)-Catalyzed Cascade Wacker-Heck Reaction: Chemoselective Coupling of Two Electron-Deficient Reactants. J. Org. Chem. 2006, 71, 8390-8394.

101. Littke, A. F.; Schwarz, L.; Fu, G. C. Pd/P(t-Bu)3: A Mild and General Catalyst for Stille Reactions of Aryl Chlorides and Aryl Bromides. J. Am. Chem. Soc. 2002, 124, 6343-6348.

102. Mee, S. P. H.; Lee, V.; Baldwin, J. E. Stille coupling made easier - the synergic effect of copper(I) salts and the fluoride ion. Angew. Chem. Int. Ed. 2004, 43, 1132-1136.

103. McKean, D. R.; Parrinello, G.; Renaldo, A. F.; Stille, J. K. Synthesis of functionalized styrenes via palladium-catalyzed coupling of aryl bromides with vinyl tin reagents. J. Org. Chem. 1987, 52, 422-4.

104. Li, Z.; Zard, S. Z. A Flexible Radical-Based Approach to TMS-Substituted Propargyl Alcohols and to 2,3-Allenols. Org. Lett. 2009, 11, 2868-2871.

105. Angeles, A. R.; Waters, S. P.; Danishefsky, S. J. Total Syntheses of (+)- and (-)- Peribysin E. J. Am. Chem. Soc. 2008, 130, 13765-13770.

106. Cope, A. C.; LeBel, N. A. Amine oxides. VII. Thermal decomposition of the N- oxides of N-methylazacycloalkanes. J. Am. Chem. Soc. 1960, 82, 4656-62.

107. Grieco, P. A.; Gilman, S.; Nishizawa, M. Organoselenium chemistry. A facile one-step synthesis of alkyl aryl selenides from alcohols. J. Org. Chem. 1976, 41, 1485-6.

108. Kadota, I.; Abe, T.; Uni, M.; Takamura, H.; Yamamoto, Y. A cross-metathesis approach to the stereocontrolled synthesis of the AB ring segment of ciguatoxin. Tetrahedron Lett. 2008, 49, 3643-3647.

109. Liu, D.; Acharya, H. P.; Yu, M.; Wang, J.; Yeh, V. S. C.; Kang, S.; Chiruta, C.; Jachak, S. M.; Clive, D. L. J. Total Synthesis of the Marine Alkaloid Halichlorine: Development and Use of a General Route to Chiral Piperidines. J. Org. Chem. 2009, 74, 7417-7428.

163 110. Filali, H.; Ballereau, S.; Chahdi, F. O.; Baltas, M. Synthesis of functionalized bicyclic precursors of heptulosonic acid analogues. Synthesis 2009, 251-256.

111. Rahim, M. A.; Matsumura, S.; Toshima, K. Deprotection of benzyl ethers using 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) under photoirradiation. Tetrahedron Lett. 2005, 46, 7307-7309.

112. Ballester, P.; Capo, M.; Costa, A.; Deya, P. M.; Gomila, R.; Decken, A.; Deslongchamps, G. Dual Binding Mode of Methylmethanetriacetic Acid to Tripodal Amidopyridine Receptors. J. Org. Chem. 2002, 67, 8832-8841.

113. Liu, H.-J.; Yip, J.; Shia, K.-S. Reductive cleavage of benzyl ethers with lithium naphthalenide. A convenient method for debenzylation. Tetrahedron Lett. 1997, 38, 2253-2256.

114. de Vries, E. F. J.; Brussee, J.; van der Gen, A. Intramolecular Reductive Cleavage of tert-Butyldimethylsilyl Ethers. Selective Mono-Deprotection of Bis-Silyl- Protected Diols. J. Org. Chem. 1994, 59, 7133-7.

115. Luniwal, A.; Erhardt, P. W. In Total synthesis of vestitol and its 3-ene relative, American Chemical Society: pp ORGN-941.

116. Luniwal, A.; Erhardt, P. W. Total synthesis of 3-hydroxymedicarpin. Abstracts of Papers, 239th ACS National Meeting, San Francisco, CA, United States, March 21-25, 2010, AGFD-211.

117. Rao, M. L. N.; Awasthi, D. L.; Banerjee, D. Microwave-mediated solvent free Rap-Stoermer reaction for efficient synthesis of benzofurans. Tetrahedron Lett. 2007, 48, 4.

118. Luniwal, A.; Khupse, R. S.; Reese, M.; Fang, L.; Erhardt, P. W. Total Syntheses of Racemic and Natural Glycinol. J. Nat. Prod. 2009, 72, 2072-2075.

119. Luniwal, A.; Erhardt, P. W. In Total synthesis of 3-hydroxymedicarpin, American Chemical Society: pp AGFD-211.

120. Han, S.-Y.; Kim, Y.-A. Recent development of peptide coupling reagents in organic synthesis. Tetrahedron 2004, 60, 2447-2467.

164

121. Shioiri, T.; Ninomiya, K.; Yamada, S. Diphenylphosphoryl azide. New convenient reagent for a modified Curtius reaction and for peptide synthesis. J. Am. Chem. Soc. 1972, 94, 6203-5.

122. Luniwal, A.; Mittal, R.; Erhardt, P. W. Mechanistic Studies for O-debenzylation of Sulfur-Containing Substrates Having Therapeutic Importance by Using Pentamethylbenzene/TFA. 31st Natl. Med. Chem. Symp. Pittsburgh, PA 2008, Poster # 123.

123. Yoshino, H.; Tsuchiya, Y.; Saito, I.; Tsujii, M. Promoting effect of pentamethylbenzene on the deprotection of O-benzyltyrosine and Nε- benzyloxycarbonyllysine with trifluoroacetic acid. Chem. Pharm. Bull. 1987, 35, 3438-41.

124. Nagaoka, H.; Schmid, G.; Iio, H.; Kishi, Y. A synthesis of the aromatic segment of rifamycin S. Tetrahedron Lett. 1981, 22, 899-902.

125. Venuti, M. C.; Loe, B. E.; Jones, G. H.; Young, J. M. Topical nonsteroidal antipsoriatic agents. 2. 2,3-(Alkylidenedioxy)naphthalene analogues of lonapalene. J. Med. Chem. 1988, 31, 2132-6.

126. Werstiuk, N. H.; Timmins, G. Protium-deuterium exchange of alkylated benzenes in dilute acid at elevated temperatures. Can. J. Chem. 1989, 67, 1744-7.

127. Lau, W.; Kochi, J. K. Arene activation with mercury(II) and thallium(III) electrophiles. Mechanistic relevance of charge-transfer transitions in π- complexes as intermediates. J. Am. Chem. Soc. 1986, 108, 6720-32.

128. Willi Amberg, Y. L. B., Raj K. Chadha, Gerard A. Crispino, William D. Davis, Jens Hartung, Kyu-Sung Jeong, Yasukazu Ogino, Tomoyuki Shibata, and K. Barry Sharpless. Syntheses and Crystal Structures of the Cinchona Alkaloid Derivatives Used as Ligands in the Osmium-Catalyzed Asymmetric Dihydroxylation of Olefins. J. Org. Chem. 1993, 58.

129. Huls, R. Friedel-Crafts reaction between methyl β-resorcylate and 3,3- dimethylacryloyl chloride. Bull. Soc. R. Sci. Liege 1954, 23, 31-40.

165 130. Nickl, J. The Friedel-Crafts reaction of 4-carbomethoxyresorcinol with 3,3- dimethylacrylyl chloride. Chem. Ber. 1959, 92, 1989-98.

131. Banerjee, S. K.; Gupta, B. D.; Singh, K. A simple one-step synthesis of phenyl ethers from phenyl acetates. J. Chem. Soc., Chem. Commun. 1982, 815-16.

166

Appendix A

List of Spectra and Chromatograms

167 23 Jan 2011 Acquisition Time (sec) 1.5000 Comment iodination 124.26 gms first ppt Date Sep 25 2008 File Name Jupiter\data1\amar\inova600\PhD\iodo_acetophenone Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

TMS 0.00

3 3.48

4.41

5.21

7.26

5.18

7.26

6.70

1.56

7.49

7.43

7.88 -0.01

7.90

7.50

6.72

7.40 6.72

1.00 2.95 1.95 1.96 2.00 2.93

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

Acquisition Time (sec) 1.3654 Comment STANDARD 1H OBSERVE Date Mar 29 2011 File Name Jupiter\data1\amar\vxrs400\PhD\gly_synt_#5_4 Frequency (MHz) 399.95 Nucleus 1H Number of Transients 32 Original Points Count 16384 Points Count 16384 Pulse Sequence s2pul

Solvent ACETONE-D6 Sweep Width (Hz) 5999.70 Temperature (degree C) 29.000

5.21 4.98 3.45 4 5.30

Acetone-d6

7.40

4.57

7.38

2.04

7.43

7.86

7.88

6.92

7.63

6.26

6.25

6.93

2.03

7.65

7.20

7.22

7.37

7.45

6.78

6.76

6.77

6.56

6.75

2.05

2.03 1.95

0.94 1.90 9.11 1.01 1.01 1.00 4.04 2.05 2.01 0.64 2.91

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

Acquisition Time (sec) 0.6025 Comment 13C OBSERVE Date Mar 29 2011 File Name Jupiter\data1\amar\vxrs400\PhD\c13_gly_synt_#5_4 Frequency (MHz) 100.58 Nucleus 13C Number of Transients 10000 Original Points Count 32768 Points Count 32768 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 27192.39 Temperature (degree C) 29.000

Acetone-d6

129.62 129.31 128.60 30.19 30.00 29.80 29.61

206.11 4

94.92

101.10

133.05

109.37

74.70

101.81

71.82

106.06

70.53

30.38

29.22

56.40

136.99

60.92

119.76

124.49

163.84

138.31

160.12

193.94

157.98 161.57

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

168 23 Jan 2011 Acquisition Time (sec) 1.5000 Comment #8 Date Jul 9 2009 File Name Jupiter\data1\amar\inova600\PhD\gly-interm-8 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

Acetone-d6

2.04 0.97 0.20

2.04

2.85 5.09

5.15 5

4.93

2.03

6.61

0.98

7.39

7.38

7.23

7.25

6.62

7.01

7.45

7.49

7.02

0.20

6.47

2.81

0.97

7.33

6.52

6.57

6.58 6.56

8.423.79 2.00 4.09 4.33 3.90 18.49 10.99

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

23 Jan 2011 Acquisition Time (sec) 0.5000 Comment STANDARD CARBON PARAMETERS Date Jul 9 2009 File Name Jupiter\data1\amar\inova600\PhD\c13-gly-interm-8 Frequency (MHz) 150.85 Nucleus 13C Number of Transients 20000 Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

Acetone-d6

29.80 25.97

5

129.23

128.52

129.35

-4.36

128.34

102.90

70.41

129.86

70.96

121.82

113.16

68.95

108.94

105.99

122.28

-11.45

158.15

118.13

160.40

137.90

157.52

155.60

130.05 138.25

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

23 Jan 2011 Acquisition Time (sec) 1.5000 Comment #9 Date Jul 10 2009 File Name Jupiter\data1\amar\inova600\PhD\gly-compd9 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

Acetone-d6

2.04 0.95 0.16

6

5.07

7.45

2.03

2.05

7.46

5.18

5.19

7.38

7.33

7.32

2.85

6.58

7.30

6.58

6.37

0.95

7.58

7.60

5.50

4.20 4.02

4.00

6.57

7.28

0.99

7.29

4.72

4.74

4.26

0.15

5.51

2.81

4.19 2.07

0.86 7.07 1.95 1.05 0.96 2.00 1.00 0.70 1.00 8.49 5.13

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

169 23 Jan 2011 Acquisition Time (sec) 0.6025 Comment #9 Date Jul 10 2009 File Name Jupiter\data1\amar\vxrs400\PhD\c13-gly-compd9 Frequency (MHz) 100.58 Nucleus 13C Original Points Count 32768 Points Count 32768 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 27192.39 Temperature (degree C) 29.000

Acetone-d6

206.07 30.19 29.99 29.80 29.60 29.41

25.96

128.12 129.37

129.19 6

30.38

70.25

70.86

106.18

102.11

108.70

130.76

112.48

67.60

159.90

-4.36

157.18

72.03

155.69

18.69

138.43

118.36

123.52

137.89

67.48 205.86

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

23 Jan 2011 Acquisition Time (sec) 1.3654 Comment diland (DHQD)2PHAL after dried in oven at 60 c over nite and then over 6 hrs at 55-60c under high vaccum Date Jan 7 2009 File Name Jupiter\data1\amar\vxrs400\PhD\DHQD2PHAL-synth Frequency (MHz) 399.95 Nucleus 1H Number of Transients 32 Original Points Count 16384 Points Count 16384

Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 5999.70 Temperature (degree C) 29.000 3.89

Chloroform-d

7.25 (DHQD)2PHAL

0.79

7.96

8.63

8.64 7.98

7.43

7.42

1.38

7.54

7.55

0.81

0.77

1.39

8.29

7.92

8.32

7.33 1.53

6.95

6.94

8.31 7.90

8.30

2.04

1.68

2.66

2.71

1.55

1.54 1.24

2.78

3.39

3.40 2.76 2.74

2.68

2.69

1.93 2.64

2.00 2.01 2.05 2.05 2.07 2.03 5.96 2.02 7.86 1.91 2.41 7.92 5.46

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

23 Jan 2011 Acquisition Time (sec) 1.5000 Comment gly-compd11 Date Jul 10 2009 File Name Jupiter\data1\amar\inova600\PhD\gly-compd11 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent ACETONE-D6

Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

0.96 0.19

CD3-656

Acetone-d6

2.04

5.03

2.04

5.27

7.25

4.02

6.27

7.26

0.97

6.32

6.31

4.00

6.45 4.12

6.44

7.30

7.31

6.44

6.56

2.89

6.56

6.54

2.03

4.14

8.57 0.19

0.79 0.96 0.95 0.90 0.85 1.00 8.38 5.17

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

170 Acquisition Time (sec) 0.5000 Comment STANDARD CARBON PARAMETERS Date Jul 10 2009 File Name Jupiter\data1\amar\inova600\PhD\c13-gly-compd11 Frequency (MHz) 150.85 Nucleus 13C Number of Transients 10000 Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul

Acetone-d6

206.14 30.06 29.80 29.67 29.54 25.92

CD3-656

113.39

125.00

85.86

70.55

133.19

103.24

-4.41

76.69

103.77

158.64

110.71

157.03

-11.49

123.59

90.10 161.69

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

Acquisition Time (sec) 1.5000 Comment #12a (NB5P2C2-12A) Date Mar 31 2009 File Name Jupiter\data1\amar\inova600\PhD\NB5P2C2-12A Frequency (MHz) 599.88 Nucleus 1H Number of Transients 64 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

Acetone-d6

2.04 2.04 0.96 0.19 1.38

CD3-699 1.35

2.03

2.85

5.27

6.46

5.66

6.28

5.65

7.27

0.96

7.28

5.09

7.23

4.09

6.48

7.25 4.19

6.55

2.81

6.57 6.45

0.88

4.07

4.21 0.19

1.88 1.88 0.90 0.94 0.95 0.66 1.06 2.77 7.83 4.82

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

Acquisition Time (sec) 1.5000 Comment 12b_3 Date Jul 19 2009 File Name Jupiter\data1\amar\inova600\PhD\12b-3 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent ACETONE-D6

Acetone-d6

2.04 0.96 0.19

7

2.04

1.38

1.36

2.03

7.15

6.21

5.26

0.96

7.25 6.27

7.27 5.66

5.64

6.46

4.03

4.13

6.47 6.45 6.39

2.85

5.07

4.01 4.15

1.04 1.05 0.89 1.02 0.96 0.58 1.01 2.87 8.36 5.24

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

171 25 Oct 2010 Acquisition Time (sec) 0.5000 Comment 12b-3 Date Jul 19 2009 File Name Jupiter\data1\amar\inova600\c13-12b-3 Frequency (MHz) 150.85 Nucleus 13C Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

Acetone-d6

206.12 29.80 25.92

7

28.25

104.78

122.20

129.75

113.48

103.27

-4.39

18.69

117.15

77.17

125.04

85.64 70.52

156.72

155.22

161.65

90.11 158.69

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

Acquisition Time (sec) 1.5000 Comment NB5P5 DMSO Date Jun 9 2009 File Name Jupiter\data1\amar\inova600\PhD\nb5p5 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 256 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent DMSO-D6 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

DMSO-d6

3.33 2.49 -0.01 -0.02

GLY-I

1.32

1.35

9.53

5.97

0.08

-0.11

5.18

6.17

5.68

7.15 5.66

7.17 4.01

4.04

7.19 6.46

7.20

6.47

6.45

6.49

6.34

3.99 4.06

0.92 1.98 1.96 0.96 0.96 0.95 1.00 3.06

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

0.12 12.122 0.10 GLY-I (-)-Glyceollin

0.08 AU 0.06

0.04

0.02 13.595 0.00

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 Minutes

Name Retention Area % Area Height Int Type Amount Units Peak Type Peak Codes Time 1 12.122 4658497 99.19 132714 bv Unknown 2 13.595 38047 0.81 942 vb Unknown

172

File Name Jupiter\data1\amar\inova600\PhD\NB5P8-2 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

Acetone-d6

5.47 5.30 3.44 2.04 4.60

CD3-649 2.04

6.58

6.45

6.58

2.05

8.01

8.02

7.16

7.18

6.43

6.62

6.64

6.41 6.41

1.00 0.94 1.93 2.06 1.872.06 2.03 0.78 3.11 0.83

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

Acquisition Time (sec) 0.5000 Comment NB5P8-2(pd/c) Date May 13 2009 File Name Jupiter\data1\amar\inova600\PhD\c13-NB5P8-2-pd-c Frequency (MHz) 150.85 Nucleus 13C Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

Acetone-d6

206.13 30.19 29.93 29.80 29.54 29.41

CD3-649

94.77

70.23

132.48

109.39

129.86

104.15

56.47

100.73

108.16

60.41

164.92

113.27

157.58 122.75

158.53 199.82

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

173 Acquisition Time (sec) 1.5000 Comment NB5P8C Date Apr 29 2009 File Name Jupiter\data1\amar\inova600\PhD\NB5P8C Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent DMSO-D6 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

DMSO-d6

2.49

4.95

5.12 5.19

CD3-650 3.33

10.52

7.38

7.36

4.44

4.45

7.54

7.69

7.70

7.55

6.61

6.15

7.22 6.15

4.84

7.20

2.51

6.51

6.49 6.49 7.39

0.90 1.00 6.29 1.91 1.00 1.97 1.92 1.88 1.20

10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

File Name Jupiter\data1\amar\inova600\PhD\c13-NB5P8C Frequency (MHz) 150.85 Nucleus 13C Number of Transients 10000 Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent DMSO-D6 Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

TMS

127.71 39.51 39.37 39.09 0.00

128.07

128.26 128.47

CD3-650

38.95

39.79

57.76

69.16

70.14

99.96

108.55

73.15

99.62

132.12

160.63

104.82

158.07

155.70 136.03

136.93

163.78

123.02

116.29

192.18 -12.36

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

174 File Name Jupiter\data1\amar\inova600\PhD\NB5P11C Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent ACETONE-D6

Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

0.97 0.19

CD3-653

Acetone-d6

2.04

2.04

6.49

3.97

0.96

6.49

6.27

7.00

7.01

6.36

6.88

6.89 2.03

8.43

3.98

3.95

8.07

3.30

0.18

3.31

2.95

2.93

4.22

4.24 4.23

4.24

2.97

2.96

2.81 0.98

2.81 2.80

3.46

2.79 3.47

0.78 0.82 0.99 1.94 1.00 1.02 1.02 0.31 1.04 8.87 5.33

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

File Name Jupiter\data1\amar\inova600\PhD\C13-NB5P11C Frequency (MHz) 150.85 Nucleus 13C Number of Transients 10000 Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

Acetone-d6

206.18 128.62 70.39 32.61 30.99 29.93 29.67 29.54 25.98

112.19

108.70

103.61

108.00 130.97

CD3-653 -4.31

29.41

156.03

157.45

114.24

121.68 18.70

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

Acquisition Time (sec) 1.5000 Comment NB5P12C-2 Date May 15 2009 File Name Jupiter\data1\amar\inova600\PhD\NB5P12C-2 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

Acetone-d6

2.04 2.04 5.09

CD3-654 5.10

4.91

7.38

7.39

6.57

7.18

7.45

7.19

6.99

6.59

7.49

7.00

6.60

6.46

7.33

6.45

7.50

2.85 8.55

0.69 11.77 1.00 2.10 2.01

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

175 Acquisition Time (sec) 0.5000 Comment c13-NB5P12C Date May 8 2009 File Name Jupiter\data1\amar\inova600\PhD\c13-NB5P12C Frequency (MHz) 150.85 Nucleus 13C Number of Transients 10000 Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul

Solvent METHANOL-D4 Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

128.37

47.85

127.40 47.99

CD3-654

68.45

69.82

107.93

102.10

47.57

70.23

120.39

129.21

100.39

137.10

154.69

158.55

129.65

159.47

117.63

137.39

157.63 48.28

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

Acquisition Time (sec) 1.5000 Comment NB5P13-2 Date May 18 2009 File Name Jupiter\data1\amar\inova600\PhD\NB5P13-2 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent METHANOL-D4 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

Methanol-d4

4.91 3.30 3.30

EtOAc 2.00

CD3-714

EtOAc

1.23 6.30

6.30 EtOAc

6.85

3.91

6.21

6.87

6.20

1.24

1.22

4.09

4.08

3.89

2.92

2.90

4.19

4.21

2.94

2.92

2.77 2.76

3.39

4.07 2.76

4.21

2.74

3.40 3.41

1.97 1.94 1.01 1.00 1.04 1.021.03

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

Acquisition Time (sec) 0.6025 Comment NB5P13 Date Nov 26 2009 File Name Jupiter\data1\amar\vxrs400\PhD\c13-NB5P13 Frequency (MHz) 100.58 Nucleus 13C Number of Transients 16960 Original Points Count 48202 Points Count 65536 Pulse Sequence s2pul Solvent METHANOL-D4 Sweep Width (Hz) 40000.00 Temperature (degree C) 29.000

Methanol-d4

131.19 128.75 71.21 49.42 49.21 49.00 48.78 33.06 31.41

103.76 108.96

CD3-714 107.60

103.52

48.36

115.05

49.64

156.33

120.11

157.18 157.88

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

176 Acquisition Time (sec) 1.5000 Comment STANDARD PROTON PARAMETERS Date Apr 27 2009 File Name Jupiter\data1\amar\inova600\PhD\NB4P47-100 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul

Solvent METHANOL-D4 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000 4.92

CD3-667 3.31

5.02

7.40

6.39

5.09

7.35

7.26

5.45

7.31

7.41

7.48 6.40

6.53

7.50

4.00

7.25

3.98

4.71

4.73

6.41

6.58 6.58

1.01 6.99 1.011.96 0.97 1.984.94 0.98 1.01 4.45

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

Acquisition Time (sec) 0.5000 Comment NB4P47-100 Date Apr 27 2009 File Name Jupiter\data1\amar\inova600\PhD\C13-NB4P47-100 Frequency (MHz) 150.85 Nucleus 13C Number of Transients 10000 Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent METHANOL-D4 Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

Methanol-d4

129.65 129.46 128.54 128.43 49.15 49.00 48.86

CD3-667 48.71

130.97

109.34

108.31 102.74

101.86

48.57

49.28

70.89

71.30

70.80

68.47

156.16

159.65

118.51

138.75

138.23

160.57

158.16

121.35 72.65

160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm

File Name Jupiter\data1\amar\inova600\PhD\NB5P9 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent METHANOL-D4 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

Methanol-d4

4.90 3.30 0.96 0.17

CD3-666

3.31

3.29

6.31

5.17

0.97

6.22

7.23

6.32

7.24

4.08

4.06

0.96

7.17

7.18

4.51

4.52

6.39

6.37 0.17

0.97 1.92 0.97 1.00 0.93 8.25 5.09

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

177 File Name Jupiter\data1\amar\vxrs400\PhD\c13-NB5P9 Frequency (MHz) 100.58 Nucleus 13C Original Points Count 32768 Points Count 32768 Pulse Sequence s2pul Solvent METHANOL-D4 Sweep Width (Hz) 27192.39 Temperature (degree C) 29.000

Methanol-d4 49.00

CD3-666

26.13

129.05 112.25

131.35

109.23

103.19

109.73

70.12

69.00

-4.30

157.59

116.70

121.14 50.51

159.08

73.27

19.04

155.89 125.08

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

Frequency (MHz) 599.88 Nucleus 1H Number of Transients 64 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

Acetone-d6 2.04

CD3-523 (-)-Glycinol

2.85

2.03

5.25

6.23

7.20 7.19

4.94

2.82

6.31 4.02

6.30

4.10

7.29

7.30

4.00

6.54 4.11

6.55 6.42

6.40

2.06

8.46 8.55

1.47 1.04 2.18 1.00 1.07

12 11 10 9 8 7 6 5 4 3 2 1 0 -1 ppm

Frequency (MHz) 150.85 Nucleus 13C Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent METHANOL-D4 Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

CD3-523 (-)-Glycinol Methanol-d4

49.28 49.14 49.00 48.86

133.25

111.05

125.12

109.28

98.92

104.01

70.92

49.42

85.90

48.57

157.33

160.08

161.12

77.18

162.14

113.03 121.26

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

178

0.025

0.020 15.544

(±)-Glycinol 18.214

0.015 AU 0.010

0.005

0.000

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 Minutes

Name Retention Area % Area Height Int Type Amount Units Peak Type Peak Time Codes 1 15.544 1297067 49.38 24828 bv Unknown 2 18.214 1329407 50.62 21331 vb Unknown

0.020 17.921

0.015 (-)-Glycinol

AU 0.010

0.005

0.000 15.247

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 Minutes

Name Retention Area % Area Height Int Type Amount Units Peak Type Peak Time Codes 1 15.247 16532 1.16 254 bb Unknown 2 17.921 1414067 98.84 23412 bb Unknown

179 Acquisition Time (sec) 1.5000 Comment NB5P20-lypho Date Jun 30 2009 File Name Jupiter\data1\amar\inova600\PhD\NB5P20-lypho Frequency (MHz) 599.88 Nucleus 1H Number of Transients 256 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent METHANOL-D4 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

Methanol-d4

4.91 3.30

CD3-640

5.48

7.24

6.35

7.25

7.19

6.91

7.21

6.91

6.41

6.74

6.73 6.41

0.90 1.01 0.89 1.00 2.44

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

File Name Jupiter\data1\amar\inova600\PhD\C13-NB5P20-LYPHO Frequency (MHz) 150.85 Nucleus 13C Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent METHANOL-D4 Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

Methanol-d4 49.00

CD3-640

121.84

104.60

66.30

113.13

119.58

99.15

109.50

156.48

106.64

157.75

160.06

110.05 148.14

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

File Name Jupiter\data1\amar\inova600\PhD\NB5P7-2 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent METHANOL-D4

Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000 1.38

CD3-639

5.51 4.91

Methanol-d4

3.30

5.66

7.14

6.90

5.65

7.17

7.15

7.19 3.30

6.36

6.60 6.35

6.62

6.74

6.73 6.72

1.98 0.97 1.00 0.99 1.00 1.97 5.96

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm

180 File Name Jupiter\data1\amar\vxrs400\PhD\C13-NB5P7-2 Frequency (MHz) 100.58 Nucleus 13C Original Points Count 32768 Points Count 32768 Pulse Sequence s2pul Solvent METHANOL-D4 Sweep Width (Hz) 27192.39 Temperature (degree C) 29.000

Methanol-d4

130.94 120.75 119.68 117.40 113.20 99.15 66.62 49.43 49.21 49.00 48.79 28.08 110.20

CD3-639

48.36

106.98

49.64

77.15

157.76

156.64

110.97

155.12

147.81 150.41

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

File Name Jupiter\data1\amar\inova600\PhD\NB5P6C-B1 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent METHANOL-D4 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

Methanol-d4

4.90 3.30 3.30 1.27

CD3-698

6.30

2.60

6.25

3.92

6.23

6.86

6.87

1.74

-0.01

6.75

2.59

6.77

2.61

3.91

3.94

1.76

1.73

1.73

2.94

2.92

4.28

4.28

4.30

2.96

4.29

3.39 2.76

2.76

3.40

3.41 1.25

1.04 1.93 1.01 1.03 1.02 1.01 2.01 2.07 7.27

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

Acquisition Time (sec) 0.5000 Comment NB5P6C-2 Date Apr 25 2009 File Name Jupiter\data1\amar\inova600\PhD\C13-NB5P6C-2 Frequency (MHz) 150.85 Nucleus 13C Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent METHANOL-D4 Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

CD3-698

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

181 File Name Jupiter\data1\amar\inova600\PhD\NB5P22-glyII Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent METHANOL-D4 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

H2O Methanol-d4 TMS

4.91 3.30 3.30 0.00

GLY-II

1.37

1.38

7.09

6.23

5.15

7.14

6.22

3.91

7.15

5.62

5.60

3.89

6.35

6.37

4.09

6.39

6.39

4.11

-0.01 0.00

1.00 1.93 1.00 0.92 1.05 1.04 5.67

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

File Name Jupiter\data1\amar\inova600\PhD\c13-NB5P22-glyII Frequency (MHz) 150.85 Nucleus 13C Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent METHANOL-D4 Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

Methanol-d4 49.00

GLY-II

105.22 28.24

122.64

130.26 125.15 98.95

129.85

85.71

70.90

109.37

77.62

-0.03

117.84

157.06 114.35

121.13 77.11

161.18

162.14 155.70

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

Acquisition Time (sec) 0.5000 Comment STANDARD CARBON PARAMETERS Date Oct 18 2010 File Name Jupiter\data1\amar\inova600\PhD\c13-NB5P22-dehydroGLYII Frequency (MHz) 150.85 Nucleus 13C Number of Transients 9000 Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

Acetone-d6

30.06 29.80 29.54 28.17

206.16 Dehydro-GLY-II

66.09

105.26

119.90

129.57

122.20

30.19

118.21

113.13

99.00

77.32

107.18

155.66

110.45

155.17

146.93

156.43

116.09 157.19

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

182 Acquisition Time (sec) 1.5000 Comment NB5P27 Date Jan 29 2010 File Name Jupiter\data1\amar\inova600\PhD\NB5P27 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 35998 Points Count 65536 Pulse Sequence s2pul Solvent CHLOROFORM-D

Sweep Width (Hz) 11999.40 Temperature (degree C) 29.000 1.46

7a TMS

0.00

9.66

11.65

5.62

7.29

7.30

5.60

6.43

6.69 6.68

6.42

7.26 1.49

0.93 1.08 1.37 1.00 1.10 7.13

12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

Acquisition Time (sec) 0.5000 Comment STANDARD CARBON PARAMETERS Date Jan 29 2010 File Name Jupiter\data1\amar\inova600\PhD\c13-NB5P27 Frequency (MHz) 150.85 Nucleus 13C Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent CHLOROFORM-D

Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

134.68 28.40

77.01

76.80

109.40

77.22 128.57

7a 115.21

194.54

78.17

115.08

158.64

160.52

108.79 28.62

TMS

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

File Name Jupiter\data1\amar\inova600\PhD\NB6P11C-isomer Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul

Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000 1.50

8a TMS

0.00

3.95

2.63

7.26

11.64

6.59

7.76

7.77

6.58 1.56

0.69 0.99 1.00 2.93 2.04 5.86

12 11 10 9 8 7 6 5 4 3 2 1 0 -1 ppm

183 Acquisition Time (sec) 0.6025 Comment NB6P11C-isomer Date Jan 9 2010 File Name Jupiter\data1\amar\vxrs400\PhD\c13-NB6P11C-isomer Frequency (MHz) 100.58 Nucleus 13C Original Points Count 32768 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 27192.39 Temperature (degree C) 29.000

Chloroform-d

77.31 77.00 76.69 44.73 27.21 129.80

8a 108.68

52.45

34.24

118.22

0.02

161.35

108.86

167.24

156.00 170.64

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

File Name Jupiter\data1\amar\inova600\PhD\NB6P11C-crude-ppt Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

TMS 0.00

8

1.47

3.94

2.71

8.48

6.44

11.30 7.27

0.76 0.98 1.00 3.10 2.07 6.12

12 11 10 9 8 7 6 5 4 3 2 1 0 -1 ppm

Acquisition Time (sec) 0.5000 Comment STANDARD CARBON PARAMETERS Date Oct 23 2010 File Name Jupiter\data1\amar\inova600\PhD\c13-NB6P11C Frequency (MHz) 150.85 Nucleus 13C Number of Transients 1000 Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul

Solvent CHLOROFORM-D Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000 26.77 Chloroform-d

8

76.79

77.21

48.51

80.33

131.06

104.73

167.30

52.38

113.76

165.13

107.26

190.51 170.07

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

184 Acquisition Time (sec) 1.3654 Comment NB5P32C Date Nov 18 2009 File Name Jupiter\data1\amar\vxrs400\PhD\NB5P32C Frequency (MHz) 399.95 Nucleus 1H Number of Transients 32 Original Points Count 16384 Points Count 16384 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 5999.70 Temperature (degree C) 29.000

Acetone-d6

2.67 2.05 2.03 1.42

6.52 A’

7.85

2.05 2.03

0.86 1.00 2.13 6.35

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

Acquisition Time (sec) 0.6025 Comment 13C OBSERVE Date Nov 18 2009 File Name Jupiter\data1\amar\vxrs400\PhD\c13-NB5P32C Frequency (MHz) 100.58 Nucleus 13C Number of Transients 10000 Original Points Count 32768 Points Count 32768 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 27192.39 Temperature (degree C) 29.000

Acetone-d6

206.08 131.31 48.50 30.19 29.99 29.80 29.61 26.60

105.15 A’

29.22

30.38

80.63

189.69

103.49

161.17

161.56 115.63

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

Acquisition Time (sec) 1.5000 Comment NB5P36-2 Date Dec 24 2009 File Name Jupiter\data1\amar\inova600\PhD\NB5P36-2 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000 TMS

9 0.00

4.41

7.26

7.41

5.09

5.15

7.41

0.01

7.42

1.56

7.47

6.62

7.91

7.93

7.48

-0.10

7.40

6.66 6.65

0.10 -0.03 6.66

1.00 8.50 1.04 2.12 1.97

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

185 File Name Jupiter\data1\amar\inova600\PhD\c13-NB5P36 Frequency (MHz) 150.85 Nucleus 13C Number of Transients 1000 Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul

Solvent CHLOROFORM-D Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

128.72 128.80

127.92 Chloroform-d 9

127.56

77.00

76.79

77.21

70.32

71.06

100.09

134.12

106.80

9.96

135.45

159.69

164.22

135.88

192.15 117.60

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

Acquisition Time (sec) 1.5000 Comment NB6P14-2 Date Jan 11 2010 File Name Jupiter\data1\amar\inova600\PhD\NB6P14-2 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

TMS

7.43 7.41 5.14 5.13 5.10 2.18 0.00 7.26

7.41 10

6.61

7.99

8.01

6.61

1.58

6.67

7.39 6.67

1.00 10.15 2.12 6.25 3.06

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

186 File Name Jupiter\data1\amar\vxrs400\PhD\c13-NB6P14 Frequency (MHz) 100.58 Nucleus 13C Number of Transients 20000 Original Points Count 32768 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 27192.39 Temperature (degree C) 29.000

Chloroform-d

128.70 128.54 128.32 127.84 70.93 70.31 70.02

99.92

133.20

106.93

76.69

77.00

77.32 20.70

10

135.33

135.84

164.30

191.07

118.07

160.45

170.56 128.95

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

Acquisition Time (sec) 1.5000 Comment NB6P19C-5 Date Feb 14 2010 File Name Jupiter\data1\amar\inova600\PhD\NB6P19C-5 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

TMS

7.26 5.13 3.84 1.45 0.00

10a

7.43

5.22

2.67

7.44

1.57

8.43

5.99

7.42

6.66

8.01

8.03

6.69

6.70 7.40

0.90 0.96 9.02 1.98 1.00 3.72 2.74 1.88 5.28

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

187 Acquisition Time (sec) 0.6025 Comment NB6P19C Date Dec 25 2009 File Name Jupiter\data1\amar\vxrs400\PhD\c13-NB6P19C Frequency (MHz) 100.58 Nucleus 13C Original Points Count 48202 Points Count 65536 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 40000.00 Temperature (degree C) 29.000

TMS

77.28 76.97 76.65 0.00

10a

26.76

127.49

128.97 128.05

128.71

70.38

99.89

71.12

107.02

133.38

48.37

132.13

101.22

51.87

74.48

80.33

190.85

164.41 135.76

190.14

113.29

113.95

160.36

163.77

118.10

164.23

-0.25

164.98 0.25

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

Acquisition Time (sec) 1.5000 Comment NB6P23-5(lowerband) Date Feb 4 2010 File Name Jupiter\data1\amar\inova600\PhD\NB6P23-5-monoprotectd Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

TMS

2.05 1.41

11a 0.00

3.70

1.20

1.26

7.26

2.64

5.01

1.43

1.27

4.50 1.19

5.07

7.38

4.12

4.13

8.28

1.44

6.29

7.40

1.59

7.46

6.56

4.35 4.34

3.51

7.47

7.77

3.50

7.79

3.46

6.57

4.10

3.47

3.45 3.53

0.77 0.92 11.16 2.02 1.00 2.15 1.82 2.623.90 1.92 9.96

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

Acquisition Time (sec) 1.5000 Date Jan 7 2010 File Name Jupiter\data1\amar\inova600\PhD\NB6P23C-1 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 64 Original Points Count 24000 Points Count 32768

Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

4.11 4.09 2.03 1.25 1.24 1.23

11b

1.37 4.12

Chloroform-d 4.08

1.17

7.25

1.34

4.42

5.00

1.68

1.16

5.03

7.36

7.35 3.46 3.44

6.22

7.15

0.91

4.35 4.34

0.86

7.40 3.80

3.78

7.78

7.42 2.02

6.57 2.86

7.79 4.04

0.93

7.44

1.38 0.90

0.90 9.74 1.07 1.99 1.00 3.98 2.76 26.58 44.91

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

188 File Name Jupiter\data1\amar\inova600\PhD\NB6P24-5 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

TMS

7.26 1.56 1.46 0.00

11

7.51

7.51

5.11

5.14

2.65

5.15

7.44

5.88

7.72

4.65

7.42

4.66

8.04

0.01

8.06

6.68

-0.01

6.68

3.76

6.70

6.72

5.30

7.42 3.77 3.75

0.98 0.95 3.20 8.87 0.99 1.00 3.43 1.91 1.09 1.90 5.58

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

Acquisition Time (sec) 0.5000 Comment STANDARD CARBON PARAMETERS Date Oct 23 2010 File Name Jupiter\data1\amar\inova600\PhD\c13-NB6P24 Frequency (MHz) 150.85 Nucleus 13C Number of Transients 10000 Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

Chloroform-d

76.79

77.00

77.21 26.70

11

129.17

128.35

127.54

128.77

70.45

71.30

62.00

48.38

99.84

133.39

74.13

100.62

107.21

127.24

79.64

135.12

160.73

161.68 123.85

135.73

163.17

191.72

164.80

113.58

117.68 190.95

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

Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768

Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

7.37 1.44

5.02

2.66

7.38 5.06

12

7.55

6.33 7.42

6.61 TMS

6.54

7.22

7.24

0.00

7.34

5.29 7.43

0.949.76 1.99 0.90 3.97 2.00 5.93

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

189 File Name Jupiter\data1\amar\inova600\PhD\c13-NB6P24C-5 Frequency (MHz) 150.85 Nucleus 13C Number of Transients 10000 Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul

Solvent CHLOROFORM-D Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

26.69

128.63 127.49

Chloroform-d

12

70.46

76.79

77.00

124.53

77.21

70.19

104.03

68.79

120.33

48.60

106.09

100.71

129.42

79.56

130.41

136.23

157.30

160.62

136.58

159.99

117.66

161.36

191.14

120.71 114.65

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

Acquisition Time (sec) 1.5000 Comment NB6P31 Date Feb 16 2010 File Name Jupiter\data1\amar\inova600\PhD\NB6P31-2 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

TMS

7.26 1.56 0.00

13

1.43

1.31

7.38

5.02

2.18

5.06

4.95

6.29

7.13

7.40

6.61

7.23

6.55

7.41

7.43

2.15

2.14

2.17 1.57

1.84

-0.01

4.79

4.78

4.81 4.77

10.58 1.10 3.31 1.00 4.24 1.25 1.99 1.24 3.31

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

Acquisition Time (sec) 1.5000 Comment NB6P32-top Date Feb 26 2010 File Name Jupiter\data1\amar\inova600\PhD\NBNB6P32-top Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D

Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

7.26 1.56 1.41 0.00

14

7.38

5.02

5.06

4.95

6.67

7.37

6.31

7.40

7.42

6.61

7.23

6.50 5.50

7.25

5.48

6.24

6.26

6.60 -0.01

8.58 1.80 1.16 0.94 1.52 4.76

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

190

13a Acquisition Time (sec) 1.5000 Comment mv(1 Date Feb 27 2010 File Name Jupiter\data1\amar\inova600\PhD\NB6P39C-OAc Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D

Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

7.37 7.26 5.06 5.02 2.11 1.57 1.41 1.37 TMS

13a 0.00

2.05

2.18

4.95

6.91

6.31

7.40

6.61

7.22

7.24

6.53

1.26

7.41

6.61

5.93

7.43

1.99 0.92

1.25

1.98

2.19

7.44

1.96

4.13

4.12

1.95

2.20

2.21

5.94

5.92

0.91

0.90

0.93

2.28

1.42

1.88

0.88

4.14 4.10

4.93

4.23 0.88

11.49 1.09 2.36 1.00 1.11 4.60 0.51 3.44 3.83 1.59

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

Acquisition Time (sec) 1.5000 Comment NB7P6C-1 Date Jul 24 2010 File Name Jupiter\data1\amar\inova600\PhD\NB6P38-5 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent ACETONE-D6

Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

1.37 1.36

13b

2.87

2.05

5.11

1.96

2.05

7.48

7.35

6.11

7.06

7.40

7.46 5.21

1.20

7.33

5.56

2.06

5.55

6.76 2.84

7.49

6.32

6.34

7.63

4.02

7.64

5.47

4.00

6.65

4.22

6.64 1.21

1.18 4.70

0.95 6.03 0.83 1.00 0.84 0.79 0.88 2.00 1.00 0.61 1.59 1.59 5.43

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 ppm

File Name Jupiter\data1\amar\inova600\PhD\c13-NB7P10 Frequency (MHz) 150.85 Nucleus 13C Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

Acetone-d6

206.14 30.06 29.93 29.67 29.54

13b

129.25

128.48

129.39

128.31

29.41

70.42

28.18

70.93

122.72 103.68

101.80

106.53

130.20

127.80

70.15

67.58

76.65

160.60

115.70

154.12

137.83

157.64

67.46

72.02

138.24

155.58

122.93 118.21

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

191 Acquisition Time (sec) 1.5000 Comment NB67P4C Date Jul 13 2010 File Name Jupiter\data1\amar\inova600\PhD\NB7P4C Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D

Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

7.40 7.39 5.10 5.02 1.44 1.41

7.26

0.00

6.34

1.59

7.38 8.00

4.17 15

2.63

2.65

4.35

7.40 6.67

7.42

7.43

4.45

2.81

4.34

5.23

5.23

4.47

6.57

6.56

2.61 2.68

1.01 11.28 1.08 1.00 1.07 2.14 1.10 2.16 6.19

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

Acquisition Time (sec) 0.5000 Comment NB7P4C Date Aug 18 2010 File Name Jupiter\data1\amar\inova600\PhD\c13-NB7P4C Frequency (MHz) 150.85 Nucleus 13C Number of Transients 29500 Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

Chloroform-d

129.02 128.66 127.60 127.51 77.21 77.00 76.79 26.38

15 27.08

70.75

70.17

104.04

101.36

67.57

48.64

106.01

71.29

-0.01

79.41

114.96

135.39

160.25

160.00

119.75

117.54

161.04

191.11

157.04 136.43

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

File Name Jupiter\data1\amar\inova600\PhD\NB7P5C Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

Acetone-d6

2.04 2.04 1.41

17 2.03

1.95

2.86

2.65

2.83

6.26

1.18

6.34

7.94

4.22

7.20

4.20

7.21

1.20

1.17

4.51

4.03

4.04

5.35

4.52

8.31

4.95 9.06

0.38 0.49 1.00 1.09 3.07 1.13 0.91 1.10 1.14 2.10 6.08

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

192 File Name Jupiter\data1\amar\inova600\PhD\NB7P6-3-diol-TBDMS Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul

Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

1.61 1.02 0.96 0.19

0.38 0.37

18

1.45

1.42

7.27

6.36

6.38

7.99 4.35

2.64

2.66

4.36

7.30

7.32

0.00

0.96

4.45

2.86 2.85

6.42

6.43

5.15 5.14

0.18

4.33

2.61

5.30 0.94

1.00 1.05 1.95 1.06 2.05 1.04 2.08 6.04 8.91 5.625.41

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

Acquisition Time (sec) 0.6025 Comment 13C OBSERVE Date Jul 26 2010 File Name Jupiter\data1\amar\vxrs400\PhD\c13-NB7P5-4 Frequency (MHz) 100.58 Nucleus 13C Number of Transients 30000 Original Points Count 32768 Points Count 32768 Pulse Sequence s2pul

Solvent CHLOROFORM-D Sweep Width (Hz) 27192.39 Temperature (degree C) 29.000

25.89 25.56

18 Chloroform-d

67.56

77.00

128.98

-4.41

71.31

27.08

128.56

76.68

48.65

77.31

113.25

110.68

121.46 104.05

69.08

-3.87

156.47

114.94

161.06

154.04

191.13

79.38

160.30

-3.77

18.14

117.48 18.28

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

File Name Jupiter\data1\amar\inova600\PhD\NB7P16-1-LB Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D

Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

7.26 5.30 1.03 0.96 0.19 0.00

18a

1.58

0.36

0.38

1.42

1.31

6.29

7.49 6.38

4.27

4.47

7.33

4.29

0.01

6.43 6.42

-0.01

6.43

4.25

5.11 5.11

2.92

2.91 2.12

2.14 2.11

2.15 1.84 1.83

4.31 1.82 4.79

1.00 1.98 1.02 1.18 0.96 2.02 0.96 1.07 1.13 2.89 6.38 4.153.98

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

193 Date Stamp Aug 16 2010 Frequency (MHz) (599.88, 599.88) Nucleus (1H, 1H) Number of Transients 1 Original Points Count (512, 1024) Points Count (1024, 1024) Pulse Sequence gcosy Solvent CDCl3 Sweep Width (Hz) (5256.24, 5256.24) Temperature (degree C) 0.000

1.5 18a 2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5 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

Acquisition Time (sec) 1.5000 Comment NB7P8C-3-MB Date Aug 14 2010 File Name Jupiter\data1\amar\inova600\PhD\NB7P8C-3-MB Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D

Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

0.96 0.19 1.61

7.26 18b

1.42 1.24

TMS

6.33

0.00

8.79

7.55

6.37

1.25

3.88

4.22

4.24

0.98

6.79

6.80

2.05

4.02

6.32 4.00

0.92 0.94 1.00 1.98 1.05 1.08 1.00 0.94 0.95 1.05 1.09 3.73 9.10 5.32

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

Acquisition Time (sec) 1.5000 Comment NB7P8C-2 LB Date Jul 30 2010 File Name Jupiter\data1\amar\inova600\PhD\NB7P8C-2LB Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

TMS

7.26 1.58 0.96 0.19

18c 0.00

1.44

1.46

6.41

8.02

2.67

2.68

8.65

6.42

0.96

3.85

4.38

6.94

6.95

4.40

4.22

6.36 4.20

6.34

5.22 5.22

0.19

6.34

2.72 2.71

1.00 0.92 1.15 2.94 1.03 1.10 1.02 2.55 4.83 6.39 4.38

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

194 Acquisition Time (sec) 1.5000 Comment STANDARD PROTON PARAMETERS Date Oct 17 2010 File Name Jupiter\data1\amar\inova600\PhD\NB7P16_1_LB_preTLCtopband Frequency (MHz) 599.88 Nucleus 1H Number of Transients 1000 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

Acetone-d6

2.04 0.98 0.96

19 0.21

0.33

1.35

1.37

6.13

7.06

6.39

5.56

7.55

7.57

4.16

4.00

4.37 3.98

4.36

6.32

6.34

6.48

6.49

5.42

4.64

5.43

4.66

5.54 5.41

1.27 1.11 1.21 1.04 1.00 1.20 0.89 1.35 4.29 10.44 3.69

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

19 Oct 2010 Acquisition Time (sec) 0.5000 Comment STANDARD CARBON PARAMETERS Date Oct 18 2010 File Name Jupiter\data1\amar\inova600\c13-NB7P16-1-LB_preTLCsecondband Frequency (MHz) 150.85 Nucleus 13C Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

Acetone-d6

29.80 26.30

19

25.93

28.27

113.30

18.97

128.85

130.43

103.77

122.72

70.01

111.42

127.88

-4.28

76.72

-3.63

71.81

156.80

115.87

67.36

155.59 154.26

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

Acquisition Time (sec) (0.0415, 0.1855) Comment STANDARD PROTON PARAMETERS Date 17 Oct 2010 19:50:50 Date Stamp Oct 17 2010 Frequency (MHz) (599.88, 599.88) Nucleus (1H, 1H) Number of Transients 1 Original Points Count (229, 1024) Points Count (1024, 1024) Pulse Sequence gcosy Solvent Acetone Sweep Width (Hz) (5519.14, 5519.14) Temperature (degree C) 0.000

3.5

4.0 19 4.5

5.0

5.5

6.0

6.5

7.0

7.5

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5

195 Acquisition Time (sec) 1.5000 Comment NB5P41C Date Sep 25 2009 File Name Jupiter\data1\amar\inova600\PhD\NB5P41C Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D

Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

3.83 4.40

21

-0.01

7.25

5.16

7.90

7.92

7.41

6.52

6.56

6.57

7.48

7.49

1.56

0.02

7.49 0.09

Chloroform-d

0.93 3.04 1.01 2.01 2.00 3.02

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

Acquisition Time (sec) 0.5000 Comment NB5P41C Date Sep 25 2009 File Name Jupiter\data1\amar\inova600\PhD\c13-NB5P41C Frequency (MHz) 150.85 Nucleus 13C Number of Transients 1000 Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent CHLOROFORM-D

Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

128.81 -0.01

127.95 Chloroform-d

77.00 55.60

21 71.06

134.15

99.30

9.98

105.98

135.51

159.71

165.11 117.43

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

File Name Jupiter\data1\amar\inova600\PhD\NB5P40C Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

TMS

3.82 2.55 -0.01

21b 5.13

Chloroform-d

7.40

7.43

6.52

7.84

7.86

6.52

7.44

6.54

7.38

7.35

7.25

1.67

6.54 7.36

0.95 1.96 1.97 2.00 3.05 3.03

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

196 File Name Jupiter\data1\amar\inova600\PhD\c13-NB5P40C Frequency (MHz) 150.85 Nucleus 13C Number of Transients 10000 Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

Chloroform-d TMS

128.68 128.26 127.61 99.42 70.64 -0.03

105.29

132.71

55.50

32.19 77.00

21b

164.33

135.96

121.42

197.77 160.07

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

File Name Jupiter\data1\amar\inova600\PhD\NB5P45C Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent ACETONE-D6

Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000 3.90

Acetone-d6 2.04

22

5.20

5.33

4.98

7.88

7.89

7.39

7.40

6.83 6.82

6.25

7.44

6.25

7.20 6.69

7.63

6.68

7.64

7.21

4.56

7.42

6.67

7.45

7.38 6.55

7.25 6.56

6.54

7.32

4.57 4.15

1.00 1.92 3.951.03 0.99 0.99 1.97 1.98 1.92 0.61 2.89

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 ppm

Acquisition Time (sec) 0.6025 Comment NB5P45C Date Oct 10 2009 File Name Jupiter\data1\amar\vxrs400\PhD\c13-NB5P45C Frequency (MHz) 100.58 Nucleus 13C Original Points Count 32768 Points Count 32768 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 27192.39 Temperature (degree C) 29.000

Acetone-d6

206.09 129.61 129.18 128.59 99.88 29.80

74.68

133.19

107.56

106.00 56.12

22

70.50

101.09

71.79

193.75

138.27

157.99

60.95

137.04

160.09 161.77

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm

197 Acquisition Time (sec) 1.5000 Comment STANDARD PROTON PARAMETERS Date Oct 8 2009 File Name Jupiter\data1\amar\inova600\PhD\NB5P46 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul

Solvent ACETONE-D6 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

3.80 2.04 2.04

2.03

5.16 5.09

23 4.92

4.92

7.38

7.40

7.28

7.45

7.50

7.29

6.59

7.00

6.69

7.01

6.46

7.51

6.46 6.57

3.90 1.00 2.02 2.01 1.95 3.01

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 ppm

File Name Jupiter\data1\amar\inova600\PhD\c13-NB5P46 Frequency (MHz) 150.85 Nucleus 13C Number of Transients 20000 Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

Acetone-d6

206.12 29.80

128.67 129.33

23

68.98

70.41

128.33

108.92

102.89

55.68

71.00

121.58

129.96

100.55

106.30

158.27

118.15

161.73

130.08

138.25

160.36 155.56

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

File Name Jupiter\data1\amar\inova600\PhD\NB5P49-acetone Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

3.71 Acetone-d6 6.48

23a 2.04

6.49

3.97

6.27

6.27

7.04

7.05

2.04

6.36

6.88

3.98

6.89

3.95

6.43

2.05

2.94

4.21

4.22

2.95

4.23

4.24

4.23

2.98

2.96

2.81 2.80

3.46

2.80

2.78

3.46

3.47

3.45

3.48 3.45

3.72

8.10 8.52

0.60 0.62 1.000.99 1.97 1.02 1.01 2.97 0.98 1.24 1.02

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 ppm

198 Acquisition Time (sec) (0.0768, 0.1536) Comment NB5P49-acetone Date 30 Nov 2009 13:40:04 Date Stamp Nov 30 2009 Frequency (MHz) (599.88, 599.88) Nucleus (1H, 1H) Number of Transients 1 Original Points Count (256, 512) Points Count (256, 2048) Pulse Sequence gcosy Solvent Acetone Sweep Width (Hz) (3332.36, 3332.36) Temperature (degree C) 0.000

2.7

2.8

2.9 3.0 23a 3.1

3.2

3.3

3.4

3.5

3.6

3.7

3.8

3.9

4.0

4.1

4.2

4.3

4.4

4.5

4.6 4.0 3.5 3.0 Jupiter\data1\amar\inova600\PhD\cosy-NB5P49-acetone.fid\cosy-NB5P49-acetone.fid.

File Name Jupiter\data1\amar\vxrs400\PhD\c13-NB5P49-vestitol Frequency (MHz) 100.58 Nucleus 13C Original Points Count 60253 Points Count 65536 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 50000.00 Temperature (degree C) 29.000

Acetone-d6

206.16 30.19 29.99 29.80 29.60

23a 29.41

70.43

30.98

102.39

108.67 103.58

105.55

128.67

29.22

130.96

32.53

55.29

114.23

157.44

120.83

156.66

160.31

205.96

156.03 206.35

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

Acquisition Time (sec) 1.5000 Comment NB5P49-4 Date Oct 25 2009 File Name Jupiter\data1\amar\inova600\PhD\NB5P49-4 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent METHANOL-D4 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

Methanol-d4

4.91 3.74 3.30

23b

6.37

6.53

7.13

6.86

7.15

6.36

6.88

6.24 6.40

0.93 1.00 0.98 2.94 4.65

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

199 Acquisition Time (sec) 0.5000 Comment NB5P49-4 Date Oct 25 2009 File Name Jupiter\data1\amar\inova600\PhD\c13-NB5P49-4 Frequency (MHz) 150.85 Nucleus 13C Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent METHANOL-D4 Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

Methanol-d4

49.43 49.29 49.14 49.00 48.86 48.72 48.58

128.44 23b

69.26

55.62

130.02

103.49

109.49

121.41

106.18

102.42

117.64

161.81

157.38

130.10

155.98

159.11 119.98

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 ppm

Acquisition Time (sec) 1.5000 Comment STANDARD PROTON PARAMETERS Date Oct 29 2009 File Name Jupiter\data1\amar\inova600\PhD\NB5P46C Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul

Solvent ACETONE-D6 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000 3.77

16

2.04

5.06

2.04

7.47

7.38

7.33

5.20

5.19

7.30 6.37

6.36

7.45

6.66

6.66

7.62

2.05

7.63

7.31

7.48

4.02

4.00

4.18

6.57

6.55

6.54

5.50

4.70

4.72

5.51 4.28

4.27 2.85

1.04 7.03 2.04 1.00 1.01 2.05 1.03 0.78 1.05 3.05

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 ppm

File Name Jupiter\data1\amar\inova600\PhD\c13-NB5P46C Frequency (MHz) 150.85 Nucleus 13C Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000 Acetone-d6

16

206.13 30.06 29.93 29.67 29.54

129.38

129.21

128.30

70.25

70.91

130.83

102.12

101.06

108.68 105.52

55.53

70.17

67.63

138.45

137.86

161.48

159.95

157.63

71.97

118.31

155.71

122.72 90.09

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

200 Acquisition Time (sec) 1.3654 Comment STANDARD 1H OBSERVE Date Nov 4 2009 File Name Jupiter\data1\amar\vxrs400\PhD\NB5P50-2 Frequency (MHz) 399.95 Nucleus 1H Number of Transients 32 Original Points Count 16384 Points Count 16384 Pulse Sequence s2pul

Solvent ACETONE-D6 Sweep Width (Hz) 5999.70 Temperature (degree C) 29.000 3.71

24 7.21

7.18 Acetone-d6

5.06

6.36

2.04

4.16

6.26

6.25

6.37

4.32

4.13

2.05

4.35

5.62

1.11

6.42

6.43

3.55

2.05

3.57

2.03 1.12

2.07 1.00 1.00 1.011.02 2.92 1.18

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

Acquisition Time (sec) 1.5000 Comment STANDARD PROTON PARAMETERS Date Oct 14 2010 File Name Jupiter\data1\amar\inova600\PhD\NB6P3_secondband Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul

Solvent ACETONE-D6 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000 3.74 6a-Hydroxymedicarpin

Acetone-d6

2.04

2.04

5.29

4.06

7.27 6.34

4.11

7.29 2.05

6.33 6.31

7.30

7.31

4.04

2.88

4.13

6.55

6.49

6.54

6.54

5.01 8.59

0.92 1.93 0.99 0.93 0.87 1.03 2.96

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

Acquisition Time (sec) 0.5000 Comment STANDARD CARBON PARAMETERS Date Oct 14 2010 File Name Jupiter\data1\amar\inova600\PhD\c13-NB6P3c13-NB6P3-secband Frequency (MHz) 150.85 Nucleus 13C Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul

Solvent ACETONE-D6 Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

30.19 30.06 29.80 29.54

206.15 6a-Hydroxymedicarpin

55.68

97.07

107.71

133.15

125.04

70.42

86.06

163.06

157.04

161.93

110.69

103.72

206.01 113.22

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

201 Acquisition Time (sec) 1.5000 Comment NB5P43 Date Sep 26 2009 File Name Jupiter\data1\amar\inova600\PhD\NB5P43 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent ACETONE-D6

Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

3.89 2.06 2.05 2.05 5.24

T1 5.17

7.40

7.18

7.41

7.48

7.50

7.66

6.81

2.85

6.81

7.17

7.54

7.23

2.82

7.18

7.26

7.07

6.69

6.68

7.36

2.08 2.07 1.96

1.01 2.912.92 1.01 1.00 2.00 2.85

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

Acquisition Time (sec) 0.5000 Comment NB5P43 Date Sep 26 2009 File Name Jupiter\data1\amar\inova600\PhD\c13-NB5P43 Frequency (MHz) 150.85 Nucleus 13C Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

Acetone-d6

30.06 29.80 29.67 29.54

206.12 T1

129.31

127.96

128.99

70.94

70.83

55.93

124.56 97.40

100.77

106.21

115.41

132.01

115.83

137.57

159.38

137.81

164.33

122.51 121.64

160.80

-11.39

157.87

183.64 154.33

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

Acquisition Time (sec) 1.5000 Comment NB5P43-2 Date Oct 3 2009 File Name Jupiter\data1\amar\inova600\PhD\NB5P43-2 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent ACETONE-D6

Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000 3.92

T2 Acetone-d6

2.04

2.04

7.67

2.05

7.77

7.68

6.52

6.52

7.10

8.47

8.48

6.98

6.97

6.61 6.60

2.87

5.62

-0.40 9.13

0.72 1.00 1.00 1.01 0.98 2.94 0.37

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

202 Frequency (MHz) 150.85 Nucleus 13C Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

Acetone-d6 29.80

T2

56.13

124.79

134.41

98.49

101.77

115.38

117.58

108.57

120.43

113.44

185.05

158.42

167.24

152.45

167.62 159.94

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

File Name Jupiter\data1\amar\inova600\PhD\NB6P27 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000 EtOAc

EtOAc

T3

5.21 5.11 5.08 3.49 2.04 1.25

EtOAc

7.27

7.20

1.26

1.24

7.44

4.11

4.12

7.39

6.75

7.50

7.50

7.09

7.51

6.75

0.00

6.73

7.00

6.71

6.99

4.13 4.10

1.97 5.00 0.97 1.96 3.96 2.95

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

Acquisition Time (sec) 0.6025 Comment NB6P27 Date May 4 2010 File Name Jupiter\data1\amar\vxrs400\PhD\c13-NB6P27-2 Frequency (MHz) 100.58 Nucleus 13C Number of Transients 10000 Original Points Count 32768 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 27192.39 Temperature (degree C) 29.000

Chloroform-d

128.33 127.49 126.91

T3 128.66

77.00 76.68

77.31

96.86

70.45

94.29

123.52

107.53

114.73

131.48

101.91

70.38

116.32

56.22

136.27

122.26

160.90

120.70

153.00

158.38

159.90

157.28 183.36

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

203 Acquisition Time (sec) 1.5000 Comment STANDARD PROTON PARAMETERS Date Apr 26 2010 File Name Jupiter\data1\amar\inova600\PhD\NB6P27C Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

TMS

7.26 1.58 0.00

T4

5.07

5.12

5.39

7.46

7.28

7.21

7.21

6.55

7.47

7.51

7.52

7.20

7.09

7.09

2.05

7.41

6.51

7.01

6.49 6.49

2.85 4.75 1.01 1.06 0.96 1.97

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

Acquisition Time (sec) 0.6025 Comment 13C OBSERVE Date Apr 25 2010 File Name Jupiter\data1\amar\vxrs400\PhD\C13-NB6P27C Frequency (MHz) 100.58 Nucleus 13C Number of Transients 24500 Original Points Count 32768 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 27192.39 Temperature (degree C) 29.000

Chloroform-d

128.67 128.38 127.50 126.78 77.00

T4

114.76

123.53

107.42 100.96

131.91

70.47

96.85

116.27

159.56

136.07

136.26

159.90

120.71

157.28

183.39 158.86

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

Acquisition Time (sec) 1.5000 Comment STANDARD PROTON PARAMETERS Date Apr 26 2010 File Name Jupiter\data1\amar\inova600\PhD\NB6P27C Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul

Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

7.26 1.58 0.00

T5

5.07

5.12

5.39

7.46

7.28

7.21

7.40

7.21

6.55

7.47

7.51

7.52

7.20

6.56

7.09

2.05

7.39

6.51

7.01

6.49

7.00 6.49

2.92 4.85 1.07 1.03 0.91 1.86

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

204 File Name Jupiter\data1\amar\inova600\PhD\c13-NB6P27C-diol Frequency (MHz) 150.85 Nucleus 13C Number of Transients 2000 Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

Acetone-d6

206.14 129.32 128.53 30.06 29.80 29.54

71.01

128.79

115.89

97.40

124.54 109.23

T5 135.02

117.01

29.41

103.81

30.19

137.74

113.00

161.07

121.22

158.10

184.88

165.85 152.95

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

Acquisition Time (sec) 1.5000 Comment NB7P20 NB6P27C PMB Date Sep 14 2010 File Name Jupiter\data1\amar\inova600\PhD\NB7P20 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent DMSO-D6 Sweep Width (Hz) 8000.00

Temperature (degree C) 29.000

7.73 3.38 3.36 2.50

T6

6.35

7.04

7.62

-1.09

7.64

8.09

8.11

6.89

6.88

6.47

3.34

10.23 10.73

0.72 0.82 1.01 1.98 1.04 1.02 0.56

10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 -1.0 ppm

File Name Jupiter\data1\amar\vxrs400\PhD\c13-NB7P20 Frequency (MHz) 100.58 Nucleus 13C Number of Transients 31000 Original Points Count 32768 Points Count 32768 Pulse Sequence s2pul Solvent DMSO-D6 Sweep Width (Hz) 27192.39 Temperature (degree C) 29.000

DMSO-d6

39.91 39.71 39.50 39.08

T6 40.13

97.52

124.05

116.98

114.57

108.44

102.80

133.58

159.10

183.31

150.70

164.39

118.92

112.52 156.98

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

205 Acquisition Time (sec) 1.5000 Comment NB6P2 Date Nov 11 2009 File Name Jupiter\data1\amar\inova600\PhD\NB6P2 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

Acetone-d6

3.88 2.04 1.39

25

2.04

5.24

2.87

5.26

10.33

7.36

7.57

7.91 5.73

7.92

7.59

5.71

7.37

6.76

2.83

6.53

6.55

6.64

6.63

6.68 6.67

0.80 1.00 3.06 3.05 1.00 3.80 2.88 5.99

10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

Acquisition Time (sec) 0.6025 Comment NB6P2 Date Jan 29 2010 File Name Jupiter\data1\amar\vxrs400\PhD\c13-NB6P2 Frequency (MHz) 100.58 Nucleus 13C Number of Transients 10000 Original Points Count 32768 Points Count 32768 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 27192.39 Temperature (degree C) 29.000

Acetone-d6

206.07 30.19 29.99 29.80 29.61 28.09 129.12

25

129.43

82.32

99.90

107.59

113.55

133.06

71.66

116.88

56.10

189.04 30.38

77.93

166.21

192.88

118.79

123.85

114.94

136.96

159.75

161.50 160.16

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

Acquisition Time (sec) 1.5000 Comment NB7P21C-2 Date Sep 17 2010 File Name Jupiter\data1\amar\inova600\PhD\NB7P21C-2 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D

Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000 1.47

T7

3.50

5.22

7.26

5.09

7.24

7.48

7.34

7.50 5.70

5.68

6.80

6.78

6.86 6.88 6.71 TMS

1.00 6.56 2.06 0.96 2.08 2.76 5.53

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

206 Acquisition Time (sec) 0.6025 Comment NB7P21C Date Sep 15 2010 File Name Jupiter\data1\amar\vxrs400\PhD\c13-NB7P21C Frequency (MHz) 100.58 Nucleus 13C Number of Transients 25000 Original Points Count 32768 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 27192.39 Temperature (degree C) 29.000

Chloroform-d

128.38 126.86 77.31 77.00 76.68 27.81

T7

122.50

114.52

100.44

55.53

130.21

70.47

104.85

115.76

116.72

131.72

136.22

120.81

163.28

153.55

153.13

158.59

106.51

183.39

29.70 152.45

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

Acquisition Time (sec) 0.5000 Comment Nb7P22C Date Sep 17 2010 File Name Jupiter\data1\amar\inova600\PhD\c13-NB7P22C Frequency (MHz) 150.85 Nucleus 13C Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

Chloroform-d

122.45 115.51 108.03 101.03 77.00 27.84

T8 114.88

133.21

55.62

130.58

116.70

29.69

106.39

120.43

112.89

153.67

166.82

166.13 152.42

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

Acquisition Time (sec) 1.5000 Comment NB6P1C-2 Date Sep 15 2010 File Name Jupiter\data1\amar\inova600\PhD\NB6P1C-2 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D

Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

3.48 1.41 5.21

26 5.09

5.08

7.29

10.15

7.31

7.62

8.04

8.06 7.63 5.58

6.69

5.57

6.55

6.57

6.65

6.63

7.27 1.70

7.28 TMS

6.75

7.32 0.00

0.85 1.00 1.01 5.25 1.04 1.00 3.98 3.09 5.80

10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

207 Acquisition Time (sec) 0.6025 Comment 13C OBSERVE Date Sep 15 2010 File Name Jupiter\data1\amar\vxrs400\PhD\c13-NB6P1C-2 Frequency (MHz) 100.58 Nucleus 13C Number of Transients 10000 Original Points Count 32768 Points Count 32768 Pulse Sequence s2pul

Solvent CHLOROFORM-D Sweep Width (Hz) 27192.39 Temperature (degree C) 29.000

28.10 128.67

26 127.85

100.51

94.15

108.79

132.90

188.56

82.04

129.76

116.09

71.03

113.24

56.31

77.00

77.32

76.67

135.07

122.54

192.17

162.86

160.23

118.60

159.59 158.58

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

Acquisition Time (sec) 1.5000 Comment NB7P21C-2 Date Sep 17 2010 File Name Jupiter\data1\amar\inova600\PhD\NB7P21C-2 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

27 1.47

3.50

5.22

7.26

5.09

6.75

7.23

7.48

7.34

7.50

5.70

5.68

6.80

6.78

6.86

6.88 6.73 6.71 TMS

1.08 6.80 3.05 1.00 2.17 3.17 5.91

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

Acquisition Time (sec) 0.6025 Comment NB7P21C Date Sep 15 2010 File Name Jupiter\data1\amar\vxrs400\PhD\c13-NB7P21C Frequency (MHz) 100.58 Nucleus 13C Number of Transients 25000 Original Points Count 32768 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D

Sweep Width (Hz) 27192.39 Temperature (degree C) 29.000

77.00

128.38 126.86

27 27.81

122.50 114.52

100.44

55.53

130.21

70.47

104.85

115.76

116.72

131.72

136.22

120.81

163.28

153.55

153.13

158.59

106.51

183.39

29.70 152.45

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

208 Acquisition Time (sec) 1.5000 Comment NB7P23 Date Sep 18 2010 File Name Jupiter\data1\amar\inova600\PhD\NB7P23 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D

Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

7.26 1.47 T9

EtOAc H2O

5.02

2.05

1.65 7.26

EtOAc

1.26 7.20

7.19 EtOAc TMS

7.21

7.44

7.34

6.56 5.67

5.69 CH2Cl2

7.45

1.25

7.19

6.80

6.56

6.78

6.82

6.84

1.28

0.00

4.12

6.52 4.13

6.50

6.32 5.30

1.00 5.34 1.91 1.10 0.96 1.17 2.28 6.41

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

14 Oct 2010 Acquisition Time (sec) 0.5000 Comment STANDARD CARBON PARAMETERS Date Oct 14 2010 File Name Jupiter\data1\amar\inova600\c13-NB7P23 Frequency (MHz) 150.85 Nucleus 13C Number of Transients 10000 Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent ACETONE-D6 Sweep Width (Hz) 37735.85 Temperature (degree C) 22.000

Acetone-d6

206.13 30.06 29.80

T9 29.54

27.86

127.86

128.97

30.18

115.08

115.86

131.73

132.36

70.72

77.67

123.66

116.05

101.47

108.31

121.86

137.63

152.71

107.02

121.41

183.57

154.11

159.79

154.50

206.06 162.45

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

209 Acquisition Time (sec) 1.3654 Comment NB7P23C Date Sep 19 2010 File Name Jupiter\data1\amar\vxrs400\PhD\NB7P23C Frequency (MHz) 399.95 Nucleus 1H Number of Transients 32 Original Points Count 16384 Points Count 16384 Pulse Sequence s2pul Solvent CHLOROFORM-D

Sweep Width (Hz) 5999.70 Temperature (degree C) 29.000 1.50

T10

7.26

7.58

12.79

5.72

5.75

7.42

8.32

8.34

6.83

6.85

6.51 6.52

6.49 TMS 6.16

0.71 1.00 0.88 1.76 2.05 0.96 4.93

13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 ppm

T10

27.84

122.49

114.94

133.98

130.58

116.94

103.72

108.12

166.49

77.23

153.73

113.24

162.81

151.84

184.45

120.42

28.05 106.39

250 200 150 100 50 0 ppm

17 Jan 2011 Acquisition Time (sec) 1.5000 Comment Boc-d-met-gly-obzl Date Jan 17 2010 File Name Jupiter\data1\amar\inova600\PhD\Boc-d-met-gly-obzl Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

TMS

2.11 1.44 0.00 5.18

29

7.36

7.27

7.37

2.59

7.38

2.58

2.60

4.13

4.12

2.10

4.07

2.12

4.06

4.16 2.09

7.39

0.01

6.75 5.20

1.95

5.22

1.94

4.36 4.35

1.96

4.04 -0.01

4.45 0.90 2.66 0.84 0.98 1.87 3.661.00 8.36

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

210 17 Jan 2011 Acquisition Time (sec) 0.6025 Comment 13C OBSERVE Date Jan 17 2010 File Name Jupiter\data1\amar\vxrs400\PhD\c13-boc-d-met-gly-obzl Frequency (MHz) 100.58 Nucleus 13C Original Points Count 32768 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 27192.39 Temperature (degree C) 29.000

Chloroform-d

77.32 77.00 76.69 28.29

29 67.27

128.41

128.63

15.22

30.06

41.31

169.36

31.45

135.00

171.73

53.21 0.01

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

17 Jan 2011 Acquisition Time (sec) 1.5000 Comment boc_D_Tyr_MG_obzl Date Apr 9 2007 File Name Jupiter\data1\amar\inova600\PhD\boc_D_tmg_obzl_batch_3_DCMwash Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent DMSO-D6 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

DMSO-d6

7.35 5.75 2.49 1.96 1.29

5.11

5.04 3.32

7.36 30

7.37

7.41

6.89

7.12 6.88

3.88

3.89

7.14

7.42

7.31

7.32

2.18

7.07

7.08

1.20

8.34

8.18

8.20

1.99

2.19

2.17

4.09 2.79

4.10

2.78

2.70

4.29 4.29

2.71

7.30

8.33

2.82

1.87

1.67

1.89

4.28

4.08 4.30

0.93 8.38 1.94 2.10 1.74 2.13 1.00 1.16 2.14 0.83 1.05 1.86 3.09 1.17 7.91

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 ppm

Acquisition Time (sec) 1.5000 Comment STANDARD PROTON PARAMETERS Date Apr 12 2007 File Name Jupiter\data1\amar\inova600\PhD\Bzlfinal_5gmsscale Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul

DMSO-d6

2.49 1.96

CD3-404

7.35

3.31

7.34

5.10

7.36

7.37

7.40

5.01

6.88

7.13

6.86

2.11

7.14

3.87

7.27

7.27

6.90 2.19

7.41

6.77

6.77 2.10

3.86

2.63

3.88

2.62

8.41

8.15

8.29 8.16

8.30

1.71

1.71

4.47 4.45 2.20

2.19

1.70 1.69

1.72

2.84 2.71

4.29 2.64

4.29

8.42 8.40

2.85

2.86

4.30 4.28 4.27

1.00 8.99 1.88 2.77 1.95 1.00 1.05 2.02 1.11 1.01 1.97 1.952.61 3.07

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 ppm

211 17 Jan 2011 Acquisition Time (sec) 1.5000 Comment menu('main') Date Sep 24 2010 File Name Jupiter\data1\amar\inova600\PhD\Dr_sarver_sample_CD3-246 Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent DMSO-D6 Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

DMSO-d6

2.49 2.49 1.97 6.62

6.60 CD3-246

6.99

7.01

2.66

7.28

3.70

2.11

3.71

7.29

2.64

6.78

8.26 6.91

8.26

2.67

8.12

8.13

8.28

1.72

3.34

2.25

1.71

9.16

4.41

4.42

1.71

1.73

4.30

2.78

1.70

2.26

2.80

2.63

2.81

4.28

1.87

4.27

4.43

1.67 2.28

0.95 1.92 0.99 1.97 2.00 1.06 1.99 1.20 2.88 1.99 2.97 3.03 0.53

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

17 Jan 2011 Acquisition Time (sec) 1.5000 Comment fmoc-met-gly-o-tBu Date May 31 2008 File Name Jupiter\data1\amar\inova600\PhD\fmoc-met-gly-o-tBu Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul

Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000 1.45

Fmoc-Met_Gly-tbu ester 2.11

Chloroform-d

7.25

7.30

7.39

7.75

7.76

1.65

4.41

4.40

7.40

7.58

0.87

7.59

2.57

4.21 2.58

3.90

3.91 0.86

3.96

6.59 4.22 3.96

5.56

5.57

0.88 1.99

2.00 2.06 2.05 0.89 0.88 2.71 1.04 2.03 1.88 3.64 1.01 9.10 0.93 0.83

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 ppm

Acquisition Time (sec) 0.6025 Comment 13C OBSERVE Date Oct 12 2010 File Name Jupiter\data1\amar\vxrs400\PhD\c13-Fmoc-met-gly-OtBu Frequency (MHz) 100.58 Nucleus 13C Number of Transients 1000 Original Points Count 32768 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 27192.39 Temperature (degree C) 29.000

Chloroform-d

127.69 127.05 125.03 119.96 77.31 77.00 76.68 47.09 31.57 29.95 28.00

Fmoc-Met_Gly-tbu ester

42.01

15.14

67.02

168.46

141.25

53.64 171.13

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

212 17 Jan 2011 Acquisition Time (sec) 1.3654 Comment STANDARD 1H OBSERVE Date Oct 12 2010 File Name Jupiter\data1\amar\vxrs400\PhD\Thba-D-Tyr_tBu_OH Frequency (MHz) 399.95 Nucleus 1H Number of Transients 32 Original Points Count 16384 Points Count 16384 Pulse Sequence s2pul

Solvent DMSO-D6 Sweep Width (Hz) 5999.70 Temperature (degree C) 29.000 1.22

36a

6.85 7.11

6.83 DMSO-d6

7.13

2.07

2.09

2.65

6.77

6.91

1.71

7.28 2.63

7.29

6.76

8.17 8.15

2.67

1.73

2.11

1.69

12.65

2.99

2.77

2.49

4.40

3.02

4.39

3.03

4.38

4.41

3.34

4.37

1.75

4.43 1.67

0.70 1.02 2.112.08 1.00 0.35 0.96 2.00 2.11 1.90 8.51

12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

Acquisition Time (sec) 1.5000 Comment benzyl-2-naphthol Date Aug 29 2010 File Name Jupiter\data1\amar\inova600\PhD\benzyl-2-naphthol Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul

Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000 5.18

d-chloroform

7.23 7.25

37

7.41

7.77 7.49

7.42

7.50

7.40

7.34

7.44 7.35

7.72

7.75 7.73

7.78

7.35

7.33

7.45 7.33

2.95 1.95 2.99 2.00 2.59 2.00

7.5 7.0 6.5 6.0 5.5 5.0 ppm

Acquisition Time (sec) 0.5000 Comment menu('main') Date Aug 29 2010 File Name Jupiter\data1\amar\inova600\PhD\C13-benzyl-2-naphthol Frequency (MHz) 150.85 Nucleus 13C Number of Transients 28000 Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 37735.85

Temperature (degree C) 22.000 128.61

Chloroform-d 123.68

128.02 37

107.04

129.44 76.79

77.00

127.58

77.21

119.04

126.77

69.98

126.35

134.45

136.83 156.69

155 150 145 140 135 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 ppm

213 25 Oct 2010 Acquisition Time (sec) 1.5000 Comment STANDARD PROTON PARAMETERS Date Oct 16 2007 File Name Jupiter\data1\amar\inova600\4-methoxy-2-naphathalEther Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul

Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

5.10 3.83 38

Chloroform-d

7.43

6.95 6.94

7.41 No. (ppm) Value 1 [3.78 .. 3.88]2.997

2 [5.04 .. 5.15]2.000 7.23

7.25 3 [6.89 .. 6.99]1.960 7.22

7.74 4 [7.18 .. 7.28]2.317

7.76 5 [7.28 .. 7.39]1.032

7.34 7.21

7.44

7.74

7.76 7.72

7.78 6 [7.39 .. 7.49]3.002

7.20 7 [7.69 .. 7.82]3.023

7.45 7.33

3.02 3.00 2.32 1.96 2.00 3.00

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 ppm

Acquisition Time (sec) 0.5000 Comment 4-methoxy-2-naphathyl-Ether Date Oct 16 2007 File Name Jupiter\data1\amar\inova600\PhD\C13-4-methoxy-2-2naphathyl-Ether Frequency (MHz) 150.85 Nucleus 13C Number of Transients 1000 Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 37735.85

Temperature (degree C) 22.000

129.38 129.34 127.62 126.75 123.62 119.10 114.00

107.01 38 126.32

Chloroform-d 69.78

134.47

77.00

76.79

77.21

128.98

159.46

55.30 156.76

160 155 150 145 140 135 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 55 50 45 ppm

Acquisition Time (sec) 1.5000 Comment benxylPMB Date Aug 16 2006 File Name Jupiter\data1\amar\inova600\PhD\benzylPMB Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D

TMS 0.00

39

2.17

2.24

2.27

4.11

7.23

1.54

7.04

7.03

7.22 7.15

2.171.93 2.00 5.98

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

214 Acquisition Time (sec) 0.6025 Comment bzl-pmb Date Aug 30 2010 File Name Jupiter\data1\amar\vxrs400\PhD\c13-bzl-pmb Frequency (MHz) 100.58 Nucleus 13C Number of Transients 30000

Original Points Count 32768 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D

128.29 127.92

125.54 39

16.84

36.11 132.78

132.47 Chloroform-d

77.00

76.68

16.97

77.31

133.76 140.58

140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

Acquisition Time (sec) 1.5000 Comment 4-methoxy benzyl PMB Date Aug 16 2006 File Name Jupiter\data1\amar\inova600\4-meoxybenzylPMB Frequency (MHz) 599.88 Nucleus 1H Number of Transients 16 Original Points Count 24000 Points Count 32768 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 8000.00 Temperature (degree C) 29.000

TMS 0.00

40 2.17 2.24

No. (ppm) Value

1 [2.14 .. 2.21] 6.202 3.76 2 [2.21 .. 2.26] 6.251

3 [2.27 .. 2.31] 3.061 2.27

4 [3.73 .. 3.81] 3.084 1.57 5 [3.99 .. 4.10] 2.142 6 [6.74 .. 6.84] 2.073

7 [6.91 .. 6.97] 2.000 4.04

7.26 8 [7.23 .. 7.29] 0.922

6.79

6.94 6.78 6.95

0.92 2.07 2.14 3.08 6.25

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

Acquisition Time (sec) 0.5000 Comment 4-methoxy-benzyl-PMB Date Oct 17 2007 File Name Jupiter\data1\amar\inova600\PhD\C-13-4-methoxy-benzyl-PMB Frequency (MHz) 150.85 Nucleus 13C Number of Transients 4000 Original Points Count 37736 Points Count 65536 Pulse Sequence s2pul Solvent CHLOROFORM-D Sweep Width (Hz) 37735.85

Temperature (degree C) 22.000 128.77

113.68 Chloroform-d

16.86

77.00

76.79 77.21

40

35.18

132.46

132.72

16.96

55.20

133.01

157.54 134.10

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

215