Synthesis of Natural Product-Based Sphingosine Derivatives

University of Mississippi eGrove

Electronic Theses and Dissertations Graduate School

2013

Synthesis Of Natural Product-Based Sphingosine Derivatives

Zarana Daksh Chauhan University of Mississippi

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Recommended Citation Chauhan, Zarana Daksh, "Synthesis Of Natural Product-Based Sphingosine Derivatives" (2013). Electronic Theses and Dissertations. 690. https://egrove.olemiss.edu/etd/690

This Thesis is brought to you for free and open access by the Graduate School at eGrove. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of eGrove. For more information, please contact [email protected]. SYNTHESIS OF NATURAL PRODUCT-BASED

SPHINGOSINE DERIVATIVES

A Thesis

Presented for the

Master of Science

Degree

The University of Mississippi

Zarana Daksh Chauhan

August 2013

ABSTRACT

Marine sponges represent an excellent source of sphingolipids and their metabolites, especially sphingosine-1-phosphate (S1P). S1P plays an important role in cell differentiation, survival, proliferation and the immune response. The enzyme sphingosine kinase (SK) is critical for the phosphorylation of sphingosine to S1P and has been examined as a druggable target for the treatment of hyper-proliferative diseases, inflammation, cancer and other pathological conditions. Natural products that consist of an azetidine ring represents an interesting class of conformationally constrained scaffolds that may serve as effective lead compounds for SK inhibitor development. Hence, the goal of this thesis research was to synthesize natural-product based sphingosine analogs from easily accessible precursors.

Our initial approach was to synthesize azetidine from the commercially available cinnamyl alcohol. The described synthetic methodology is proficient and scalable for the synthesis of fully substituted azetidines. We were successful at preparing two azetidine analogs,

(±)-30 and (±)-31, via an intramolecular Mitsunobu reaction as a key step. Another crucial technique is the Grubbs’ cross-metathesis reaction to the side-chain extension. This reaction provides an opportunity to develop various analogs through side-chain modification. Compound

(±)-30 was synthesized in 11 steps and compound (±)-31 was synthesized in 10 steps with affordable overall yield. The described synthetic methodology represents a promising tool for the invention of various analogs of the natural products and the synthesized compounds will serve as lead compounds for the future optimizations.

DEDICATION

This work is dedicated to my beloved husband, Daksh Chauhan

and to my parents, Ramanbhai and Induben Chauhan,

for their continuous support and love.

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LIST OF ABBREVIATIONS AND SYMBOLS

13C NMR Carbon Nuclear Magnetic Resonance

1H NMR Hydrogen Nuclear Magnetic Resonance

ATP Adenosine triphosphate

BOC t-Butoxycarbonyl

CNS Central nervous system

COX Cyclooxygenase

DAG Diacylglycerol

DCM Dichloromethane (CH2Cl2)

DEAD Diethyl azodicarboxylate

DIAD Diisopropyl azodicarboxylate

DIEA Diisoproylethylamine

DME Dimethoxyethane

DMF N,N-Dimethylformamide

DMSO Dimethylsulfoxide

EDG Endothelial differentiation gene

ERK Extracellular signal-regulated kinase

ESI Electrospray ionization

Et2O Diethyl ether

EtOAc Ethyl acetate

FTY720 Fingolimod

GPCR G protein-coupled receptor

HADC Histone deacetylase

HMBC Heteronuclear multiple-bond correlation

HMQC Heteronuclear multiple-quantum correlation

IBX 2-iodoxybenzoic acid

IP3 Inositol trisphosphate

JNK c-Jun N-terminal protein kinase

K2CO3 Potassium carbonate m-CPBA meta-Chloroperoxybenzoic acid

MgSO4 Magnesium sulphate

MS Mass spectrometry

Na2S2O3 Sodium thiosulfate

Na2SO4 Sodium sulfate

NaH Sodium hydride

NaHCO3 Sodium bicarbonate

NaN3 Sodium azide n-Bu2SnO Dibutyltin oxide

NF-ĸB Nuclear factor kappa light chain enhancer of activated B cells

NH4Cl Ammonium chloride

NH4OH Ammonium hydroxide

NO Nitric oxide

NOE Nuclear Overhauser effect

Pd/C Palladium on carbon

PI3K Phosphoinositide 3-kinase

PKC Protein kinase C

PLC Phospholipase C

PNS Peripheral nervous system

PPh3 Triphenylphosphine

Rac1 Ras-related C3 botulinum toxin substrate 1

ROK Rho-associated protein kinase

RT Room temperature

S1P Sphingosine-1-phosphate

SAR Structure-activity relationship sGC soluble isoform of Guanylate cyclase

SK Sphingosine kinase

SPT Serine palmitoyltransferease

TBAF Tetra-N-butylammonium fluoride

TBS tert-Butyldimethylsilyl

TBSCl tert-Butyldimethylsilyl chloride

TBS-OTf tert-butyldimethylsilyl triflate

TFA Trifluoroacetic acid

THF Tetrahydrofuran

TLC Thin layer chromatography

TRAF2 TNF receptor-associated factor 2

TsCl p-toluenesulfonyl chloride

ACKNOWLEDGMENTS

I would like to thank Dr. John M. Rimoldi for his continuous support, guidance, and encouragement throughout my academic career. Dr. Rimoldi has the attitude to continually express a spirit of quest and enthusiasm in regard to research and teaching. I thank Dr. Rimoldi for giving me an opportunity to serve as a teaching assistant in his Pharmacogenetics and

Pharmacoimmunology class. Without his constant help this work would have been impossible.

I am thankful to Dr. Rama Sharma Gadepalli for giving me constructive comments and suggestions in my research project. I am also grateful to all the past and present Rimoldi labmates, Dr. Rama Gadepalli, Dr. Robert Smith, Dr. Sarah Scarry, Brian Morgan, Kimberly

Foster and Eric Bow, who made my research project interesting by sharing their remarkable ideas & thoughts. Thank you guys so much!

I would like to thank my committee members, Dr. Stephen Cutler and Dr. Christopher

McCurdy, for their support and guidance. I am deeply grateful to Dr. Cutler for providing me financial support throughout my graduate studies. I would also like to thank Dr. John

Williamson, Dr. Robert Doerksen, Dr. Ronald Borne and Dr. Mitchell Avery for their input in my graduate career. I owe much gratitude for the financial support provided by to the National

Institutes of Health - National Institute of General Medical Sciences (NIH-NIGMS) Grant no.

P20 GM 104932.

vii

I owe huge thanks to my beloved husband whose outstanding love & support, enthused me and encouraged me to complete my thesis project. I own special thanks to my parents for their undivided interest and support throughout my academic career.

Lastly, I thank to my friends and all the students of Department of Medicinal Chemistry who helped me in my work and to God who made this work possible.

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TABLE OF CONTENTS

ABSTRACT ...... ii

DEDICATION ...... iii

LIST OF ABBREVIATIONS AND SYMBOLS ...... iv

ACKNOWLEDGMENTS ...... vii

LIST OF FIGURES ...... xi

LIST OF SCHEMES...... xii

LIST OF TABLES ...... xiv

I. INTRODUCTION ...... 1

1.1. PATHWAY FOR BIOSYNTHESIS AND METABOLISM OF SPHINGOSINE-1-PHOSPHATE ...... 5

1.2. SPHINGOSINE KINASE (SK) ...... 9

1.3. FUNCTION OF SPHINGOSINE-1-PHOSPHATE ...... 11

1.3.1. Effect of S1P on the vascular system ...... 11

1.3.2. Effect of S1P on the central and peripheral nervous system ...... 12

1.3.3. Effect of S1P in inflammation ...... 13

1.3.4. Effect of S1P in cancer ...... 14

1.3.5. Effect of S1P on the reproductive system ...... 16

1.4. MEDICINAL APPROACHES TO TARGET S1P PATHWAYS ...... 17

1.4.1. S1P-specific antibodies ...... 17

1.4.2. SK inhibitors ...... 18

1.4.3. S1P receptor modulators...... 21

1.5. CRYSTAL STRUCTURE OF THE GPCR S1P1 RECEPTOR ...... 25

II. SYNTHETIC METHODOLOGIES TOWARDS SPHINGOSINE DERIVATIVES ...... 27

2.1 INTRODUCTION AND THE PURPOSE OF REASERCH ...... 27

2.2. EXPLORATORY STUDIES ...... 29

2.3. RESULT AND DISCUSSION ...... 31

2.4. BIOLOGICAL INVESTIGATION ...... 47

2.5. CLOSING THOUGHTS ...... 48

2.6. FUTURE DIRECTIONS ...... 48

III. EXPERIMENTAL ...... 50

BIBLIOGRAPHY ...... 65

APPENDIX : SPECTRAL DATA...... 75

VITA ...... 138

LIST OF FIGURES

Figure 1. Schematic illustration of signaling pathways for sustained PKC activation ...... 4

Figure 2. Structure of sphingosine and sphingosine-1-phosphate ...... 5

Figure 3. Biosynthesis of ceramide ...... 8

Figure 4. Biosynthesis and metabolism of sphingosine and S1P ...... 9

Figure 5. Schematic representation of human sphingosine kinase isoforms ...... 10

Figure 6. Schematic representation of role of S1P in vascular system ...... 12

Figure 7. Schematic representation of role of sphingolipids in inflammation ...... 14

Figure 8. Schematic representation of role of S1P in cancer ...... 16

Figure 9. Structures of SK inhibitors ...... 19

Figure 10. Structures of S1P receptor agonists and antagonists ...... 24

Figure 11. Structural features related to S1P1 receptor ...... 25

Figure 12. Structures of azetidine alkaloids ...... 28

Figure 13. 1H-NMR spectrum of (±)-3 ...... 34

Figure 14. Proposed mechanism for the formation of (±)-5 ...... 35

Figure 15. 1H-NMR spectrum of (±)-23 ...... 41

Figure 16. Mechanism of Mitsunobu reaction ...... 43

Figure 17. J- Values of compound (±)-27A and (±)-27B ...... 45

Figure 18. Commonly used catalysts for olefin cross metathesis ...... 45

Figure 19. Mechanism of olefin cross metathesis ...... 46

LIST OF SCHEMES

Scheme 1. Schematic representation of the formation of azetidine ...... 29

Scheme 2. Initial synthetic approaches for the formation of azetidine ...... 30

Scheme 3. Synthetic pathway for the formation of azetidine ...... 30

Scheme 4. Retrosynthetic analysis of (±)-30 ...... 31

Scheme 5. Epoxidation of cinnamyl alcohol ...... 32

Scheme 6. Ring opening of epoxy alcohol using sodium azide ...... 32

Scheme 7. Protection of primary and secondary alcohol ...... 33

Scheme 8. Cyclization of 3 & MS spectrum of (±)-4 and (±)-5 ...... 35

Scheme 9. Ring opening of epoxy alcohol using ammonia ...... 36

Scheme 10. Sulfonylation of amine salt (±)-14 & MS spectrum of (±)-15 and (±)-16 ...... 37

Scheme 11. Oxidation of alcohol & MS spectrum of (±)-17, (±)-18 and (±)-19...... 38

Scheme 12. TBS protection of diol ...... 39

Scheme 13. Deprotection of primary TBS-protected alcohol & ring formation ...... 39

Scheme 14. Oxidation of alcohol & MS spectrum of (±)-23 and (±)-24 ...... 40

Scheme 15. Oxidation of alcohol using IBX ...... 41

Scheme 16. Vinylation and allylation of aldehyde ...... 42

Scheme 17. Ring closure reaction of (±)-25 ...... 44

Scheme 18. Olefin cross-metathesis reaction of (±)-27A ...... 46

Scheme 19. Deprotection of (±)-28 ...... 47

xii

Scheme 20. Proposed synthetic route for penazetidine A ...... 49

xiii

LIST OF TABLE

Table 1. Sphingosine kinase inhibitors and their characteristics ...... 21

xiv

I. INTRODUCTION

The diversity of plant and animal species in marine environments represent unique sources in the discovery and development of novel drugs.1, 2, 3 Marine natural products began to receive attention in 1951 when the unknown nucleosides, spongouridin and spongothymidin, were isolated from the sponge Cryptotethya crypta.3 More than 20,000 compounds were isolated from marine sources since the 1960s.3 In addition, marine sponges are excellent sources of lipids, terpenes and alkaloids. To prevent predation and for security from infection, sponges generate cytotoxic compounds. More than 10% marine sponge species have engendered cytotoxic activity which implies that they are an efficient source for the production of effective medicines.2, 4 The pharmaceutically active compounds found in sponges include immunomodulators, anticancer, anti-viral, antifouling, and antimalarial agents. Unfortunately, only a few of these compounds have resulted in marketed drugs due to the limited production of sponge biomass and trace amounts of the desired bioactive compounds in sponges.4, 5 In order to understand the biological activity of marine sponges and further develop a bioactive compounds into a commercialized product, constant supply is a prerequisite. The supply issue is the major problem in the field of marine natural products. However, advanced technologies such as improved sampling techniques, fermentation, total synthesis and biotechnology have resolved this critical issue. A feasible tactic for the total synthesis of bioactive natural products has provided the large-scale production and allowed to develop SAR and lead optimization studies. However, marine natural

1 products often possess complex chemical structures with well-defined stereochemistry.

Therefore, it is difficult to synthesize compounds with extraordinary spatial orientation.6, 7

Secondary metabolites from marine sponges are well-known for their protein kinase inhibitory activity, with more than 70 compounds identified with promising activity and selectivity. For instance, lasonolide A from Forecpia sp., hymenialdisine from Axinella verrucosa, variolin B from Kirkpatrickia varialosa, hymenialdisines from Stylissa massa and liphagal from Aka coralliphaga produce significant activities against various kinases.2 Protein kinases are a superfamily of enzymes that are ubiquitous among animal, plant and bacteria kingdoms, and their altered production or mutations are involved in diverse diseases, such as cancer, Alzheimer’s disease and atherosclerosis.2 The human genome contains over 500 protein kinases, representing approximately 1.7% of human genes.8, 9, 10 Protein kinases catalyze the addition of phosphate group from adenosine triphosphate (ATP) to a specific amino acid substrate. The covalent bond formed between phosphate group and amino acid substrates are provided by the hydroxyl group of serine/threonine or tyrosine. Hence, protein kinases are recognized as serine/threonine kinases or tyrosine kinases depending on the specific amino acid substrates.2, 9 Furthermore, protein kinases play critical roles in regulating cellular processes such as cell proliferation, metabolism, transcription, differentiation, cell movement, cell survival and apoptosis. Deregulation and mutation of protein kinases are linked to diseases such as, cancer, immune system disorders, osteoporosis, central nervous system diseases, and metabolic disorders. More than 130 protein kinase inhibitors are either in Phase I or Phase II clinical trials and the majority are being evaluated against different types of cancer.2, 10, 11

Sphingolipids are components of the cell walls or cell membranes and are engaged in a variety of biological processes including cell differentiation, growth, proliferation, apoptosis and

2 the immune response.12, 13 Sphingosine is recognized as an effective and reversible inhibitor of protein kinase C (PKC). PKC is activated by the presence of Ca2+ or diacylglycerol as shown in

Figure 1. It is also triggered by tumor-promoting phorbol esters. The activation of PKC is related to its intracellular translocation from the cytoplasm, and consequently down regulation via proteolytic cleavage of PKC. PKC is involved in the phosphorylation of membrane-related proteins. Sphingosine hinders the translocation and down regulation of PKC. Inhibition of PKC by sphingosine is competitive with diacylglycerol and phorbol esters and non-competitive with

Ca2+.8, 14, 15, 16

Figure 1. Schematic illustration of signaling pathways for sustained PKC 8 activation. Adapted from ref. (PIP2-phosphatidylinositol-4,5-bisphosphate; PC-phosphatidylcholine; PLC-phospholipase C; PLD-phospholipase D; PLA2-phospholipase A2; PA-phosphatidic acid; DAG-diacylglycerol; PKC- protein kinase C; IP3-Inositol trisphosphate)

Sphingosine was discovered by the German physician and neurochemist Johann Ludwig

Wilhelm Thudichum.17, 18 Sphingosine is an unsaturated monoaminodihydroxy alcohol and was origionally obtained from the hydrolysis of phrenosin (Figure 2).18 It is metabolized to sphingosine-1-phosphate (S1P) which is a potent signaling molecule.19 S1P has been identified in many organisms such as plants, yeast, worms, flies and mammals.20, 21 In mammals, it is produced and secreted from the platelets.22 S1P is a major component of serum and its approximate concentration is 400 nM. It is also secreted from specific cells, like mast cells,

4 monocytic cells and red blood cells. It is bound to serum albumin in plasma and has been detected in high density lipoproteins and oxidized low density lipoproteins. However, the mechanisms of secretion at cellular levels are still an enigma. It has also been detected in numerous organ systems such as, gastrointestinal, reproductive, pulmonary, and endocrine.

Studies indicate that S1P is engaged in physiological regulation of many organ systems.22 It stimulates signaling pathways by binding to five different receptor subtypes expressed as S1P1-5 which are the constituents of the endothelial differentiation gene (EDG). EDG is a family of G

23 protein-coupled receptors (GPCRs). S1P1 and S1P5 activate mainly Gi, S1P2 activates all G proteins, S1P3 activates Gi, Gq, and G12/13, and S1P4 couples to Gi and G12 in response to S1P. As a result, S1P involves in various biological functions depending on S1P receptor subtypes and G proteins.20, 24 S1P regulates cell migration, proliferation, and angiogenesis via activating extracellular signal-regulated kinase (ERK)1/2, phosphoinositide 3-kinase (PI3K), phospholipase

C (PLC), Ras and Rho-dependent pathways.25 However, the biological activities of S1P receptors still remain elusive.

Figure 2. Structure of sphingosine and sphingosine-1-phosphate, adapted from ref.26

1.1 PATHWAY FOR BIOSYNTHESIS AND METABOLISM

OF SPHINGOSINE-1-PHOSPHATE

Understanding the intracellular regulation of S1P demands a description of the various enzymes responsible for its synthesis and metabolism. Sphingosine and ceramide are required for the production of S1P; sphingosine kinases (SKs) are responsible for the metabolism of

5 sphingosine to S1P. Sphingosine is produced from the metabolism of sphingolipids and the deacylation of ceramide. There are two routes that engage in the production of ceramide: the first is the de novo biosynthesis pathway of sphingolipids; the second is the endocytic recycling pathway of sphingolipids.19, 20

The de novo sphingolipid biosynthesis pathway is prevalent among cells and tissues; loss of function by mutation of the enzyme serine palmitoyltransferease (SPT) can affect cell growth and development. The de novo pathway initially involves the condensation of L-serine and palmitoyl CoA to afford 3-ketodihydrosphingosine (KDS) as shown in Figure 3. This reaction takes place at the cytosolic leaflet of the endoplasmic reticulum. It is the rate-limiting reaction and is catalyzed by SPT, essential for the management of sphingolipid levels in cells. KDS, produced from SPT, is quickly converted to dihydrosphingosine by 3-ketodihydrosphingosine reductase. Dihydrosphingosine is N-acylated to produce dihydroceramide catalyzed by the enzyme ceramide synthase. Dihydroceramide is then desaturated to form ceramide by the enzyme dihydroceramide desaturatase. Ceramide is subsequently converted to sphingosine by the enzyme ceramidase, which is then phosphorylated to S1P by SK (Figure 4). 19, 20, 27, 28, 26 The decomposition of S1P is facilitated by two different routes: the reversible dephosphorylation of

S1P to sphingosine by the enzyme S1P phosphatases and the irreversible inactivation of S1P to hexadecenal and phosphoethanolamine by a pyridoxal phosphate-dependent S1P lyase.

Hexadecenal and phosphoethanolamine are the precursors for phosphatidylethanolamine biosynthesis.21

In the endocytic sphingolipid recycling pathway, the enzymes sphingomyelinases are responsible for hydrolyzation of sphingomyelin to ceramide and phosphorylcholine. There are three different types of sphingomyelinases, characterized based on their pH optima into acidic,

6 neutral, and alkaline forms. Acidic sphingomyelinases are expressed in the lysosomal compartment, while neutral sphingomyelinases are expressed in the plasma membrane, endoplasmic reticulum, Golgi, and nucleus. Alkaline sphingomyelinases are mainly expressed in the intestinal tract and the bile. Several studies have suggested that stress factors and proinflammatory cytokines can increase sphingomyelinase activity and ceramide levels which lead to cell death. Like sphingomyelinases, ceramidases also have three different forms such as acidic, neutral, and alkaline forms, that deacylate ceramide to sphingosine.19

Figure 3. Biosynthesis of ceramide, adapted from ref.27, 29

Figure 4. Biosynthesis and metabolism of sphingosine and S1P, adapted from ref.21, 29

1.2 SPHINGOSINE KINASE (SK)

SK is the key enzyme in the transformation of sphingosine to S1P. There are two isoforms of SK, SK1 and SK2. The high level of SK1 is detected in lung and spleen, whereas SK2

30 is found in liver and heart. Three different splice isoforms of SK1 have been detected in

9 humans, SK1a, SK1b, and SK1c. These isoforms vary only at their N-terminal regions. As shown in figure 5, SK1b contains 14 additional amino acids whereas SK1c contains 86 additional amino acids compared to SK1a. There are two isoforms of SK2, SK2a and SK2b, with SK2b having 36

29 additional amino acids than SK2a. Surprisingly, the structural features of SK are not fully understood.

Figure 5. Schematic representation of human sphingosine kinase isoforms. Reprinted with permission from Elsevier (Trends Biochem Sci, 2011. 36(2): p. 97- 107).29

All identified SKs contain five highly conserved regions, denoted as C1–C5. These conserved regions are believed to be involved in protein structure management and the binding

31 of substrates. For instance, GLY82 in conserved regions of SK1 is essential for catalysis whereas ASP278 is essential for substrate binding. The other domains of SK1 contain

Ca2+/Calmodulin, TRAF2 and the conserved ATP binding site.32 Moreover, a nuclear export signal is found in SK1 while the C-terminus in SK2 contains a proline rich domain, nuclear

31 localization signal and an SH3 binding motif. SK1 have residues that attach to acidic phospholipids and phosphatidylserine. SK1 is found mostly in the cytosol, where it is translocated to the plasma membrane and subsequently phosphorylated by ERK. The outcome of

32 S1P signaling depends on SK1 subcellular localization. In animal model, targeting SK1 was shown to decrease the occurrence and complexity of disease, while targeting SK2 was shown to

10 increase the progression of disease. This implies that SK1 and SK2 play different roles under normal or disease conditions.29

1.3 FUNCTION OF SPHINGOSINE-1-PHOSPHATE

1.3.1 Effect of S1P on the vascular system.

High concentrations of S1P have been detected in plasma and serum. S1P participates in signal transduction using the GPCRs found on vascular endothelial and smooth muscle cells. S1P is involved in the activation of endothelial cells that lead to production of an imperative messenger, nitric oxide (NO) (Figure 6).22 NO stimulates the soluble isoform of guanylate cyclase (sGC) in the vascular smooth muscle cells, which in turn generates the messenger molecule cyclic guanosine monophosphate (cGMP). cGMP production promotes the vasorelaxation responses induced by S1P actions. In contrast, S1P also stimulates S1P2 and S1P3 receptors on vascular smooth muscle cells which in turn activate the small G-protein

RhoA, Rho-associated protein kinase (ROK) and PKC. Stimulation of the RhoA/ROK/PKC route promotes the vasoconstriction responses induced by S1P receptors.33 This suggests that

S1P can act as a vasoconstrictor or a vasorelaxor depending on the expression patterns of S1P receptor subtypes in endothelial cells and vascular smooth muscle cells. A number of studies revealed that S1P1 receptor on endothelial cell regulates vascular maturation and induces endothelial cell proliferation. This suggests that S1P is a powerful controller of angiogenesis.22, 33, 34

Figure 6. Schematic representation of role of S1P in vascular system, adapted from ref.33 (Rac1-Ras-related C3 botulinum toxin substrate 1; NO - nitric oxide; eNOS - endothelial isoform of NO synthase; RhoA - Ras homolog gene family, member A; ROK - Rho-associated protein kinases; GC - soluble isoform of guanylate cyclase; cGMP - cyclic guanosine monophosphate)

1.3.2 Effect of S1P on the central and peripheral nervous system.

S1P receptors have been identified in the central and peripheral nervous system. For example, the white matter, hippocampus and the cerebellum express high levels of S1P1 receptor.

Astrocytes (specialized glial cells) and Purkinje cells (cerebellum) also express high level of

22, 35 S1P1 receptors. However, the function of S1P1 in Purkinje cell is poorly understood. S1P is involved in neurite rounding, growth cone collapse and axonal collapse processes that are imperative for the development of neuronal pathways.22, 36 This indicates that S1P may play a role in the development and remodeling of the CNS. In the peripheral nervous system, S1P

12 receptors have been detected in glial cells. In vitro studies imply that S1P prevents apoptosis of

Schwann cells (which prevents apoptosis of glial cells) by the PI-3-kinase/Akt signaling pathway. This suggests that S1P may have a crucial role in the nervous system; however its function in the CNS and PNS is poorly understood.22

1.3.3 Effect of S1P in inflammation.

S1P is abundant in platelets and has been detected in other cell types including neutrophils, erythrocytes, and mononuclear cells.23 These cells can release S1P via activation of growth factor receptors, G-protein-coupled receptors, and cytokine receptors. The released S1P produces various physiological actions, such as, inflammatory responses (Figure 7). Mounting evidence reveals that S1P plays an essential role in inflammatory process since S1P1 and S1P2 receptors are detected in mast cell and may regulate mast cell activation. S1P1 receptors also are

37 involved in mast cell migration, whereas S1P2 receptors are engaged in degranulation.

The cyclooxygenases, COX-1 and COX-2, are oxidative enzymes involved in prostaglandin synthesis. COX-2 facilitates inflammatory events in response to the cytokines interleukin-1β and tumor necrosis factor-α (TNF-α). In certain cell types, TNF elevates intracellular levels of ceramide via stimulating the endocytic recycling pathway of sphingolipid

(sphingomyelin hydroxylation) and/or triggering the de novo biosynthesis pathway. In many cells, TNF activates S1P production by stimulating the enzyme SK.38 S1P stimulates cytosolic phospholipase A2 (cPLA2) and releases arachidonic acid through S1P3 receptor-mediated increase of intracellular calcium and Rho kinase activation. Arachidonic acid is oxygenated to prostaglandins which are involved in a number of physiological and pathological processes.30

This suggests that S1P may have a role in pathological inflammatory diseases.38

Figure 7. Schematic representation of role of sphingolipids in inflammation, Adapted from ref.37 (C1P - ceramide 1-phosphate; c/EBP - CCAAT/enhancer binding proteins; COX-2 - cyclooxygenase-2; cPLA2 - cytosolic phospholipase A2; LPS - lipopolysaccharide; NF-ĸB - nuclear factor-ĸB; S1P - sphingosine 1- phosphate; SK - sphingosine kinase; SMase - sphingomyelinase; TNF - tumor necrosis factor-α.)

1.3.4 Effect of S1P in cancer.

Several reports have documented the finding that S1P plays a significant role in the development of tumor growth. In addition, elevated amount of SK mRNA has been observed in tumors of uterus, ovary, breast, colon, stomach, and lung.39, 40 The equilibrium between ceramide, sphingosine and S1P stipulates a rheostat model. According to this mechanism, a cell is directed to apoptosis through ceramide and sphingosine signaling or is survives by S1P.41, 42

SK is critical checkpoint enzyme in the rheostat model since it transfers death-supporter sphingolipids such as ceramide and sphingosine, into the growth-supporter S1P. Cancer cells make good use of rheostat by fostering circumstances that encourage the formation of S1P.

Various cancer cells attain this through the enzyme SK1 suggesting that SK1 could be a possible

42 oncogene. Furthermore, studies suggest that inhibition of SK1 decreases tumor angiogenesis, growth, and chemoresistance in various xenograft models, whereas overexpression of SK1 increases these events.43

In the tumor microenvironment, S1P is released by blood platelets, fibroblasts, and mast cell. Tumor cells enhance expression of SK, which in turn augment the level of S1P in the tumor microenvironment (Figure 8). The produced S1P from tumor cells act in an autocrine fashion to enhance growth, cell migration, survival and metastatic potential of tumor cells or in a paracrine fashion to encourage endothelial cell based angiogenesis and regulate lymphangiogenesis and immune cells.42, 43, 44 In addition, S1P enhances cancer development through intracellular targets including HDAC1/2 and NF-ĸB and also supports cancer cells to resist therapy. Recent studies revealed that S1P1 receptor activates signal transducer and activator of transcription-3 (STAT3) in cancer cells and in the tumor microenvironment which is crucial for cancer development and metastasis. S1P production is also able to enhance proangiogenic growth factors such as vascular endothelial growth factor (VEGF), IL-6 and IL-8 from tumor cells. Taken together, S1P modulators represent a central target in the treatment of cancer.42, 43

Figure 8. Schematic representation of role of S1P in cancer. Reprinted by permission from Macmillan Publishers Ltd: Nature Publishing Group. (British Journal of Cancer, 2006. 95(9): p. 1131-35).42 (SPH-Sphingosine; SPHK- Sphingosine kinase; VEGF-vascular endothelial growth factor; IL-6- Interleukin 6; IL-8- Interleukin 8)

1.3.5 Effect of S1P on the reproductive system.

S1P receptors are detected in some cells of the testes and the ovaries. In the male, S1P1 is detected in the seminiferous tubule of the testicle and this receptor has been expressed by mature spermatid during the preparation of spermatogenesis. Moreover, S1P is involved in the inhibition of the male germ cells apoptosis. S1P has an ability to protect the initial phases of spermatogenesis suggesting that S1P inhibits the sperm cell loss induced by radiation.22, 45, 46 In the female, the oocyte apoptosis and female infertility is induced by cytotoxic chemotherapy treatment. S1P inhibits the oocyte apoptosis. Although, S1P may engage in the male and female

16 germ cells, but the further studies are required to investigate the mechanism of S1P in reproductive system.22

1.4 MEDICINAL APPROACHES TO TARGET S1P PATHWAYS

Cancer cells release S1P that are proficient to bind S1P receptors on endothelial cells and foster cell proliferation that leads to tumor neovascularization. In tumors, S1P can also work on fibroblasts through growth factors that may nurture cancer development. Hence, S1P receptors antagonists might be proficient to impede fibrosis, neovascularization and proliferation of cancer cells. Furthermore, a compound with SK1 inhibitory activity might bolster apoptosis of cancer cells and fibroblasts. Thus, a compound with dual activity would be tremendously beneficial to prevent cancer expansion.47 Several therapeutic approaches have been developed to control S1P signaling in cancer such as production of S1P-specific antibodies, agonizing/antagonizing

41 specific S1P receptors, and targeting SK1 and/or SK2 enzymes.

1.4.1 S1P-specific antibodies.

Prevention of cancer using S1P-specific antibodies is a nascent approach in the S1P research arena. The anti-S1P antibodies work as “molecular sponges” to reduce S1P from blood and other compartments. They nullify extracellular tumor supporting growth factors which lead to lower their bioavailability.48 A recently established murine monoclonal antibody, LT1002, known as Sfingomab, has high binding affinity and specificity for S1P. This anti-S1P monoclonal antibody has shown anti-neovascularization effects, minimized tumor progression as well as lowered tumor vascularization by inhibiting vascular endothelial growth factor in several in vivo and in vitro assays.49 LT1009, the humanized variant of LT1002, known as

Sonepcizumab, also has high binding affinity and specificity for S1P and it is as effective as

LT1002. S1P plays an imperative role in the lymphocyte trafficking between blood and lymphoid tissue. The intravenous administration of LT1002 and LT1009 was shown to decrease numbers of circulating lymphocytes in in vivo study. Currently LT1009 is in Phase 1 clinical trial for the treatment of cancer and age-related macular degeneration (AMD). It was formulated as ASONEPTM for cancer and iSONEPTM for AMD. In Phase 1 clinical trial, LT1009 was tested against patients with different types of solid tumors such as prostate, renal, ovarian, colorectal, and breast. It has been revealed that LT1009 decrease the tumor progression for around 8-12 months in majority of patients. Based on the successful results in cancer patients, LT1009 is being selected for Phase II clinical trials. As such, this anti-S1P monoclonal antibody could be a marketable drug that would be very beneficial for the patients who are battling against life- threatening disease such as cancer.41, 48, 50, 51

1.4.2 SK inhibitors.

In cancer cells, it is imperative to reduce S1P signaling. SK is an essential enzyme that catalyzes the production of S1P. Drug discovery efforts have been focused on identifying new and selective inhibitors of SK1 and SK2. The structures of several SK inhibitors are shown in

Figure 9 and their properties are shown in Table 1. The first non-lipid small molecule SK inhibitor reported was 2-(p-hydroxyanilino)-4-(pchlorophenyl) thiazole, also recognized as SKi or SKI-II, a non-specific inhibitor. SKi was shown to reduce the production of S1P, inhibit proliferation and generate apoptosis in many tumor cell lines. It also showed good bioavailability and minimization of tumor progression in in vivo studies.32, 47 Structural editing of SKi led to discovery of SKi-178 which is selective inhibitor of SK1. SKi-178 is more potent and less cytotoxic than the parent molecule.32

Figure 9. Structures of SK inhibitors, adapted from ref.32, 51, 52, 53

The first selective SK1 inhibitor discovered was (2R,3S,4E)-N-methyl-5-(4’- pentylphenyl)-2-aminopent-4-ene-1,3-diol, also known as BML-258; a water soluble sphingosine analogue. It did not demonstrate any inhibitory effects on SK2, PKC, or various other cellular

19 kinases. BML-258 reduced tumor growth and survival and induced apoptosis in human leukemia cell lines. It also inhibited the phosphorylation of Akt and ERK1/2 and decreased the growth of

54 acute myelogenous leukemia xenograft tumors. A selective non-lipid SK2 inhibitor developed is 3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide, also known as

ABC294640. ABC294640 was shown to prevent cell proliferation and migration in tumor cell lines. It also showed excellent oral bioavailability and promoted tumor cell autophagy that generated non-apoptotic cell death and reduced tumor growth in several in vivo studies.55, 56

D,L-threo-dihydrosphingosine (D,L-threo-DHS) is the synthesized form of natural D- erythro-dihydrosphingosine. D,L-threo-DHS functions as an inhibitor of SK1 and functions as a

32 substrate for SK2. L-threo-DHS, also known as safingol, is the first SK1 inhibitor that has been used in clinical trials for the treatment of cancer. It showed enhanced cytotoxic effects when used in combination with other anticancer agents.51 N,N-dimethylsphingosine (DMS) is another non- specific inhibitor of SK activity. It also interferes with PKC, 3-phosphoinositide-dependent kinase, and casein kinase II. It was shown to reduce tumor growth and induce apoptosis in several studies.41, 56

There are reports of natural products such as F-12509A, B-5354c, S-15183a, and S-

15183b that have shown good SK inhibitory activity in vitro, but their specificity for SK isoforms is vague and their large-scale production is dubious.41, 56

Table 1. Sphingosine kinase inhibitors and their characteristics SK Mechanism Type of Comments Inhibitors of action Inhibition Stimulates cathepsin-B-mediated Inhibit SK Non- SKi 1 degradation of SK , no effects on PKC, and SK Competitive 1 2 ERK and PI3K.32, 41 Addition of methyl and methoxy groups Inhibit SK Competitive to the parent compound led to reduce SKi-178 1 cytotoxicity and increase selectivity for 32 SK1. Suppressed tumor growth, decreased Inhibit SK Competitive BML-258 1 proliferation, and induced apoptosis in the tumor.54 Showed good pharmacological profiles, anticancer activity, anti-inflammatory ABC294640 Inhibit SK Competitive 2 activity and low toxicity in in vivo & in vitro studies.55 D,L-threo- Inhibit SK Competitive Inhibits other kinases.32 DHS 1 Competitive for Inhibit SK1 SK1/Non- Lack of specificity, inhibit various other DMS 56 and SK2 Competitive for cellular kinases. SK2 57 PF543 Inhibit SK1 Competitive Inhibits S1P formation in whole blood. F-12509A Isolated from a dicomycete Inhibit SK (Natural 1 Competitive Trichopezizella barbata , induce and SK product) 2 apoptosis.41 B-5354c Isolated from a marine bacterium, Inhibit SK Non- (Natural 1 decreased S1P production in human and SK Competitive product) 2 platelets.41, 52 S-15183a and SK Isolated from a fungus Zopfiella S-15183b (Specificity Not determined intermis, decreased S1P production in in (Natural not vitro studies.41 product) determined)

1.4.3 S1P receptor modulators.

Recently, the discovery for S1P therapeutics has emphasized compounds which have the ability to target specific S1P receptors. In particular, the unique immunosuppressant indicated for the treatment of multiple sclerosis, fingolimod (GilenyaTM) or FTY720, is phosphorylated through SK2 and the resulting phosphorylated FTY720 acts as an agonist or partial agonist for all

21 types of S1P receptors except S1P2. FTY720 has low affinity for S1P2. There are five different

58, 59, 60 types of S1P receptors including S1P1, S1P2, S1P3, S1P4, and S1P5. FTY720 is the first oral drug for the treatment of multiple sclerosis, and it was approved by FDA in 2010.51 FTY720 easily crosses the blood–brain barrier and produces several responses in the CNS. Multiple sclerosis is an inflammatory disease in which lymphocytes move out of lymph nodes and circulate throughout the body and damage myelin sheath of neurons that lead to inflammation, demyelination, neuroaxonal injury, and neurodegeneration. S1P is detected in the lymph and serum. The high concentration of S1P (found in circulation) is responsible for lymphocytes migration into the circulation. Fingolimod is structurally similar to S1P and down-regulates S1P receptors on T cells. As a result, lymphocytes remain sequestered in the lymph node.61

Moreover, FTY720 was shown to induce apoptosis in various cancer cell lines such as prostate, liver, lung, breast and bladder.62, 63 It also reduced tumor growth and metastasis without any noticeable toxic side effect in a mouse melanoma model. In ovarian cancer cells, FTY720 was shown to promote autophagy and cell death via caspase 3-independent necrotic pathways.62, 63 It

41 inhibits ERK1, ERK2, and Akt phosphorylation, and activates caspases, JNK, and PKCδ.

The success of FTY720 has provided proof-of-principal evidence that selective S1P receptor modulators have clinical value in disease management. The structures of the S1P agonists and antagonists are illustrated in Figure 10. SEW2871 was the first selective S1P receptor agonist discovered. Structurally, it differs from S1P; hence there is no need of phosphorylation for receptor binding. In animal studies, this compound was shown to produce lymphopenia. CYM-5442 is another selective S1P1 receptor agonist. Like SEW2871, there is no need of phosphorylation for receptor binding. KRP-203 is an S1P1 receptor agonist, but also has the ability to bind and activate other receptors such as S1P1, S1P4 and S1P5. VPC01091 is

22 phosphorylated by SK2 and acts as an agonist or partial agonist for S1P1, S1P4 and S1P5 receptors

60 and antagonist for S1P3 receptor.

There are several selective S1P receptor antagonists that have been discovered in recent years. VPC44116 is a competitive S1P1 receptor antagonist and is less potent for the S1P3 receptor, and has the ability to act as a partial agonist at S1P4 and S1P5 receptors. The prodrug

VPC03090 is a S1P1/3 receptor antagonist. It is phosphorylated by SK2 and the resulting phosphorylated VPC03090 acts as an antagonist for S1P1 and S1P3 receptors, and an agonist or partial agonist for S1P4 and S1P5 receptors. JTE-013 was developed as an S1P2 receptor antagonist whereas CAY10444 is selective S1P3 receptor antagonist. W146 is an S1P1 receptor antagonist whereas its enantiomer, W140, has less affinity for the S1P1 receptor. However, none of these compounds have superior profiles and further studies are required to reveal the potential of these antagonists.60

Figure 10. Structures of S1P receptor agonists and antagonists, adapted from ref.60

1.5 CRYSTAL STRUCTURE OF THE GPCR S1P1 RECEPTOR

Recently, the crystal structure of the S1P1 receptor has been solved. The crystal structure of the S1P1 receptor will serve as a valuable tool for medicinal chemists to construct more selective small molecule modulators of the S1P1 receptor, and this will be advantageous for designing and understanding the agonistic and antagonistic functions of other S1P receptors.64

The crystallization was carried out by fusing the S1P1 receptor to a T4-lysozyme in complex with the selective antagonist (R)-3-amino-(3-hexylphenylamino) - 4-oxobutylphosphonic acid

(ML056). The resultant crystalized receptor has seven trans-membrane helices organized in a conserved bundle.60, 65 There are three extracellular loops (ECL) including ECL1, ECL2 and

ECL3 as shown in Figure 11.

Figure 11. Structural features related to S1P1 receptor. Reprinted with permission from the American Association for the Advancement of Science (Science, 2012. 335(6070): p. 851-55).65 (ECL1, ECL2 and ECL3 are shown in gold, orange and yellow ribbon respectively. ML056 is illustrated in green ball and stick model.)

There are two important features of the crystal structure of the S1P1 receptor. First, the N- terminus folds over the ECL1and ECL2 to form a helical cap that confines the extracellular access to the amphipathic binding pocket. The ECL1 and ECL2 are tightly packed against the N-

25 terminus helix. S1P obtains access to the binding pocket from within the membrane bilayer, probably via a gap between transmembrane helices I and VII. Second, the development of a cluster of positively charged and polar residues created by helices III and VII, ECL 2 and the N- terminal cap, furnish a high affinity interaction to the phosphate group of the sphingolipid. The information obtained from the crystal structure of S1P1 receptor may help to construe and determine the crystal structure of other S1P receptors.60, 65

II. SYNTHETIC METHODOLOGIES TOWARDS

SPHINGOSINE DERIVATIVES

2.1 INTRODUCTION AND THE PURPOSE OF REASERCH

Heterocyclic sphingoid secondary metabolites have been isolated from marine sources,

For instance, in 1991 Kobayashi et al. isolated the sphingosine relatives, penaresidin A and B, from the marine sponge penares sp. that were characterized as actomyosin ATPase-activators.66,

67 Crews and coworkers isolated penazetidine A from the marine sponge Penares sollasi in 1994, structurally related to the penaresidins and was identified as a relatively potent protein kinase C

68 (PKC) inhibitor. The crude extract of this marine sponge showed an IC50 = 0.3 μg/mL against

PKCβ1 while it was inactive against protein tyrosine kinases. Furthermore, penazetidine A exhibited substantial activity against PKC (IC50 = l μM), and displayed considerable in vitro cytotoxicity against murine and human cancer cell lines.2, 69 The structures of penaresidin A / B and penazetidine A are shown in Figure 12. These compounds have received attention due to their unique structure and interesting biological activity. There are several total synthesis described in the literature; however, the protocols suffer from poor stereo control and lengthy reaction sequences.70 Therefore, the goal of this thesis research is to develop new methodologies for the construction of the azetidine ring with emphasis on preparing sphingosine analogs from readily available precursors. Natural products that consist of an azetidine ring represents an intriguing class of conformationally constrained scaffolds that may serve as valuable lead compounds for SK inhibitor development.

Figure 12. Structures of azetidine alkaloids, adapted from ref. 70

The quest for biologically active compounds from marine origins has revealed that sponges represent a rich resource for novel protein kinase inhibitors.2 The azetidine ring, a four membered ring comprising a nitrogen atom, is found as a substructural element in a number of natural products. Azetidine ring-containing compounds exhibit various biological activities, such as antihypertensive, anticonvulsant, analgesic, antiepileptic, and anticancer. For instance, ABT-

594 demonstrates more potent analgesic activity compared to that of morphine.71 Although azetidines are important substructures in drug discovery and lead optimization, they are synthetically challenging to construct, particularly with substitutions on the azetidine ring system. Huszthy et al., reported, “azetidines are one of the most difficult amines to synthesize because of the unfavorable enthalpy of activation in four-membered ring formation and the high susceptibility to cleavage of the strained hetero ring”.72 Since the parent azetidine was first synthesized in 1888 in impure form, it has been challenging for synthetic chemists to discover new and efficient methods for their construction. There are several conventional methods

28 reported and usually involve an intramolecular displacement of a leaving group from the γ-amino nucleophile. The most reliable and often used chemistry protocols are described in Scheme 1.

These cyclizations can also be achieved by the reduction of an azido group using Raney-nickel, palladium on carbon (Pd/C) or using other reducing agents. It has also been reported that the

Mitsunobu reaction offers a robust method to form azetidines from protected NH precursors.71

Scheme 1. Schematic representation of the formation of azetidine

2.2 EXPLORATORY STUDIES

The inspiration for synthesis of azetidine derivatives as sphingosine analogs originated from the identification of the PKC inhibitor, penazetidine A. We hypothesized that azetidines could be derived from commercially available cinnamyl alcohol. The Prilezhaev’s epoxidation protocols73 would produce enantiomeric epoxides that could be elaborated to the aminodiol with subsequent cyclization to azetidines. As shown in Scheme 2, the cyclization of 1 was carried out by treatment with various reducing agents (10% Pd/C, lithium aluminum hydride and triphenylphosphine (PPh3)) to afford 2. We also attempted form the azetidine ring by treatment of 3 with NaH to afford 4 and 5. However, the procedures used for the formation of azetidine

29 suffered from low yield and undesired side products. Moreover, it was problematic to deal with purification process during the synthesis and was also difficult to introduce key alkyl group on the C-4 carbon of the azetidine ring system. Hence, efforts towards these initial reaction protocols were discontinued.

Scheme 2. Initial synthetic approaches for the formation of azetidine

It was realized that modification of the amine protecting group, particularly with N- sulfonyl group, may offer an advantage with the ring cyclization chemistry. The resultant sulfonamide is the strong electron-withdrawing agent due to the sulfonyl group and it is weak acid which upon reaction with weak base eliminates the leaving group and undergo cyclization.74

75 As shown in Scheme 3, cyclization of 6 was carried out by treatment with K2CO3 to afford 7.

The synthetic protocol described in Scheme 3 afforded azetidine 7 in excellent yield.

Scheme 3. Synthetic pathway for the formation of azetidine

2.3 RESULT AND DISCUSSION

We believed that a proficient technique could be developed toward the synthesis of target compound 30 using an intramolecular cyclization approach. The retrosynthetic analysis of 30 is depicted in Scheme 4. Compound 30 was envisioned to be derived from a Grubbs’ cross- metathesis reaction73 of a long chain alkene with the vinyl substituted 27A. The azetidine ring would be formed from a Mitsunobu reaction76 of 25 using a suitable amine protecting group, namely, a sulfonamide. Compound 25 would be prepared from the precursor alcohol 21 via oxidation and subsequent Grignard reaction.77 The protected amino diol 21 would be synthesized from cinnamyl alcohol using epoxidation and amine ring opening reactions.

Scheme 4. Retrosynthetic analysis of (±)-30

The synthesis began with the formation of known epoxy alcohol 8 from cinnamyl alcohol using the Prilezhaev reaction.73 Typically, meta-chloroperoxybenzoic acid (m-CPBA) is the preferred peroxyacid for the formation of epoxides from alkene precursors. This reaction is reported to be stereospecific, and the product obtained was isolated in 89% yield in racemic

31 form, which was used without purification.73, 78 Scheme 5 summarizes the epoxidation of cinnamyl alcohol.

Scheme 5. Epoxidation of cinnamyl alcohol

Initially, we performed a regiochemical ring opening reaction of epoxide 8 using sodium azide to afford azido alcohol 9. The reduction of azide 9 with triphenhylphosphine and water

(Staudinger reaction) and subsequent N-protection of the resulting amine with tert- butoxycarbonyl anhydride to yield 10 was carried out in a single step (Scheme 6).79

Scheme 6. Ring opening of epoxy alcohol using sodium azide.

The next step was the selective protection of the primary over the secondary alcohol to activate the primary alcohol as a leaving group (tosyloxy) for the formation of azetidine ring system. The reaction was carried out by treatment of 1,2-diol 10 with catalytic dibutyltin oxide (n-Bu2SnO) with removal of water to produce tin acetal 11. Compound 11 undergoes sulfonylation in the presence of p-toluenesulfonyl chloride (TsCl) at the primary position to afford selective monotosylate 13 in 71% yield. As shown in Scheme 7, the tin acetal 11 activates the primary

32 alcohol and temporary protects secondary alcohol to produce selective monotosylate 13 with high regioselectivity in a single operation.80, 81

Scheme 7. Protection of primary and secondary alcohol

With 13 in hand, the protection of secondary alcohol was conducted by treatment of 13 with tert-butyldimethylsilyl triflate (TBS-OTf) and 2,6-lutidine to afford TBS-protected alcohol

3. Surprisingly, we observed that TBS-OTf is not only protecting the secondary alcohol but also replacing the N-Boc group to N-tert-butyldimethylsilyloxycarbonyl group (silyl carbamate,

(Scheme 7)). The N-Boc group can be activated by fluoride ion that would react with an electrophile to produce silyl carbamate type compound.82 The structure of compound 3 was confirmed by MS, 1H-NMR and 13C-NMR analysis. Diagnostic signals in the proton NMR spectrum of compound 3 (δ 0.91 – 0.88 (d, J = 9.7 Hz, 18H), 0.27 – 0.23 (d, J = 17.1 Hz, 6H),

0.05 – 0.04 (d, J = 3.1 Hz, 6H)) clearly indicated that the N-Boc group of 13 was converted to silyl carbamate group (Figure 13).

Figure 13. 1H-NMR spectrum of (±)-3

After protection, the cyclization reaction of 3 was performed in the presence of sodium hydride (NaH) to afford azetidine 4. Interestingly, another product was formed from this reaction, namely, the five-membered cyclic carbamate 5 (Scheme 8). It is known that silyl carbamates can serve as nucleophiles and participate in substitution reactions with electrophiles.83 Activation of the N-carbamate of 4 with sodium hydride afforded 4 and 5 in an

SN2 manner; this would result in inversion of stereochemistry if the leaving group was a functionalized secondary alcohol.84 The proposed mechanism for the conversion of cyclic

34 carbamate 5 is shown in Figure 14. Products were confirmed based on TLC and MS analysis.

Due to the instability of silyl carbamate 4, it was problematic to purify using silica gel column chromatography.

Scheme 8. Cyclization of (±)-3 & MS spectrum of (±)-4 and (±)-5

Figure 14. Proposed mechanism for the formation of (±)-5

Although this protocol provided good yields and stereocontrol, the N-Boc group had no significant impact on the formation of azetidine. Moreover, problems occurred during synthesis and prevented the expansion of further chemistry. In order to understand the role of different N- protecting groups on the formation of azetidine, it became necessary to investigate alternative methods earlier in the synthesis.

Pastó M. et al. reported an outstanding method for the efficient synthesis of vicinal amino alcohols from precursor epoxides. The vicinal amino alcohol motif is a structural pharmacophore in a variety of natural products and therapeutics many of which comprise various biological activities.85, 86 Furthermore, a free amino group renders the opportunity of utilizing diverse N- protecting group tactics (e.g. N-Ts or N-Ns) in the synthesis of amino acids and biomolecules.

With 8 in hand, the ring-opening of epoxy alcohol was investigated by treatment of 8 with ammonia in the presence of isopropyl alcohol to afford amino alcohols in excellent yield

(Scheme 9). This reaction is safe, inexpensive, stereospecific and regioselective, hence, furnishes a greener alternative to the typical two- or three-step reaction sequences involving either the use of poor atom-economic conditions such as benzhydrylamine or the use of hazardous reagent such as azide.85, 87 The obtained product was converted to the hydrochloride salt by treatment with an

HCl saturated solution of methanol to yield 14 in excellent yield. The product was confirmed based on LC-MS, 1H-NMR and 13C-NMR analysis.

Scheme 9. Ring opening of epoxy alcohol using ammonia

An alternate protecting group strategy was employed, to overcome the obstacles using an

N-carbonate protecting group in earlier synthetic attempts. We selected to employ an N-sulfonyl protection of the amine as a reasonable group, due to its stability and lack of O-alkylation or group transfer reactions.88 We attempted several N-sulfonylation methods but all the procedures involved the formation of desired product with undesired N,O-disulfonylated compound as a side product (Scheme 10). In a typical procedure, the amine salt 14 was treated with 2- mesitylenesulfonyl chloride in the presence of diisoproylethylamine (DIEA) to afford sulfonamide 15 in good yield. The low yield was attributed to competing sulfonylation of the hydroxyl groups, which were partially suppressed with conducting the reaction at 0 oC.

Scheme 10. Sulfonylation of amine salt (±)-14 & MS spectrum of (±)-15 and (±)-16

The next step was to selectively oxidize the primary alcohol in order to produce a method to install various R-groups in advance of ring cyclization strategies. We attempted several oxidation methods, but none of them proved useful in selective oxidation of the primary over the secondary alcohol. The procedures used for formation of the aldehyde suffered from over- oxidation and was problematic to deal with product purification. The products described in

Scheme 11 were confirmed based on TLC and MS analysis.

Scheme 11. Oxidation of alcohol & MS spectrum of (±)-17, (±)-18 and (±)-19

For these reasons, we chose an alternate strategy to generate the desired aldehyde product, which invoked a dual protection and selective mono-deprotection strategy. Silylation

38 was achieved by reaction of 15 with tert-butyldimethylsilyl chloride in DMF and imidazole to afford bis-silylated 20 in excellent yield (Scheme 12).89

Scheme 12. TBS protection of diol

The next step involved the selective deprotection of the primary TBS-protected alcohol.

Accordingly, 20 was treated with THF/TFA/H2O (4:1:1) at room temperature to yield 21 in good yield. To enhance yield while preserving the secondary TBS-protected alcohol, the reaction mixture was constantly monitored. The reaction was stopped when the production of bis-silyl deprotected product was observed and the remaining starting material was consumed.90, 91 In order to evaluate the impact of the sulfonamide group, initially we directed our efforts towards the formation of the azetidine using Mitsunobu reaction conditions (Scheme 13). In this attempt,

76 21 was treated with PPh3 and diisopropyl azodicarboxylate (DIAD) to afford 22.

Scheme 13. Deprotection of primary TBS-protected alcohol & ring formation.

The success of the Mitsunobu reaction paved the way for introduction of alkyl side chains via Grignard reactions using aldehyde precursors.92 We attempted to oxidize 21 to 23 using a variety of oxidation methods, including Swern oxidation93 and Parikh-Doering oxidation94 methods. However, the low yields coupled with the formation of side products were observed

39 during the synthesis (Scheme 14). Furthermore, the resulting product aldehyde was prone to decomposition upon silica gel purification.

Scheme 14. Oxidation of alcohol & MS spectrum of (±)-23 and (±)-24

Saito et al. reported an efficient and selective method for oxidation of primary achohols to aldehydes using 2-iodoxybenzoic acid (IBX).95 Therefore, compound 21 was treated with IBX in the presence of DMSO at RT to afford 23 in excellent yield (Scheme 15). The obtained product was taken to the next step without silica gel purification, in order to circumvent decomposition chemistry. The 1H-NMR spectral profile clearly indicated that compound 23 is an aldehyde not a ketone or carboxylic acid (Figure 15). IBX was prepared by treatment of 2-iodobenzoic acid with

Oxone in presence of deionized water in 85% yield. This method is convenient, non-hazardous and environmentally safe.96

Scheme 15. Oxidation of alcohol using IBX

Figure 15. 1H-NMR spectrum of (±)-23

After successfully synthesizing the key aldehyde 23, vinylation was carried out using

Grignard reaction conditions. The reaction was performed by treatment of 23 with

41 vinylmagnesium bromide to afford 25 in 81% yield and diastereomeric ratio of 5:1 (Scheme 16).

Since the addition of Grignard reagents occurs on both faces of the carbonyl group of aldehyde, the prochiral aldehyde induces diastereomeric mixtures of the resultant alcohols.73, 97 The resulting isomers were purified and taken to the next step as a mixture.

Stereoselective addition of various organometallics such as In, Sn, and Zn to prochiral carbonyls in the presence of aqueous medium has been scrutinized from synthetic and mechanistic perspectives.98, 99 Therefore, 23 was treated with Zn dust & allyl bromide in the presence of aqueous NH4Cl to afford 26 in excellent yield (Scheme 16). This Zn-mediated

Barbier-type reaction98 is faster and the isolation of the product is straightforward. The diastereomeric products were separated using silica gel chromatography, and characterized using spectroscopic techniques.

Scheme 16. Vinylation and allylation of aldehyde

The Mitsunobu reaction is an adaptable and frequently used approach for the dehydrative coupling of an acid or nucleophile (or pro-nucleophile) with an alcohol by using reducing

100 phosphine reagent such as PPh3 and an oxidizing azo reagent such as DIAD or DEAD. The nucleophilic addition of PPh3 to DIAD produces a phosphonium intermediate that binds to the alcohol and activates it as a leaving group. The deprotonated sulfonamide displaces the leaving group (phosphine oxide (Ph3PO)) and completes the ring formation. The resultant azetidine has inverted stereochemistry due to the presence of secondary alcohol in starting material.100, 101, 102

The diagrammatic representation of the mechanism of the Mitsunobu reaction is outlined in

Figure 16. The intramolecular ring closure reaction of 25 was investigated by treatment with

76 PPh3 and DIAD in the presence of THF. The reaction produced two major products, 27A and

27B.

Figure 16. Mechanism of Mitsunobu reaction, adapted from ref. 73, 102

Aryl azetidine (27) represents the key intermediate towards the synthesis of final compounds. Diastereomers were separated by column chromatography (Scheme 17).

Scheme 17. Ring closure reaction of (±)-25

The structure elucidation of 27A and 27B was investigated by MS and NMR spectroscopic techniques. The molecular weights of 27A and 27B are identical and were confirmed on the basis of their MS spectra, with a [M+Na]+ m/z 494. The 1H-1H 2D COSY NMR spectrum disclosed correlations between protons at δ 4.75 – 4.73 (H2), 4.50 – 4.46 (H4), 3.95 –

3.92 (H3) for 27A and δ 5.13 – 5.12 (H2), 4.88 – 4.85 (H4), 4.37 – 4.35 (H3) for 27B of azetidine ring. The correlation between carbons and protons was determined by 2D HMQC and HMBC

13 experiments. The C-NMR data of 27A and 27B are δ 76.77(C2), 71.71(C4), 69.72(C3) and

74.41(C2), 73.41(C4), 72.52 (C3), respectively. The NMR spectral profile clearly indicated that

27A and 27B are fully substituted azetidines. The difficulty of determining the configuration of azetidine alcohol by 1D or 2D NOE spectral analysis has been reported due to the vicinity of the

H2 and H4 proton resonances. Hence, the configurational assignment has been determined by their coupling constants values. The cis isomers usually show higher coupling constants values than the corresponding trans isomers.103, 104 It was confirmed based on J-values that 27A is a trans-isomer and 27B is cis-isomer. The J-values are shown in Figure 17.

Figure 17. J- Values of compound (±)-27A and (±)-27B

The next step was to perform an olefin cross-metathesis reaction on the azetidine precursors in order to extend the scope of the chemistry in the production of analogs. Olefin cross-metathesis reactions are efficient techniques to introduce carbon-carbon double bonds with homologation. Ruthenium carbenoids (Grubbs’ Catalyst) are favorably active catalysts for different types of olefin-metathesis reactions. These catalysts work very well for electron- deficient and sterically demanding olefins.105 Commonly used catalysts are depicted in Figure

18.

Figure 18. Commonly used catalysts for olefin cross metathesis, adapted from ref.106

A Grubbs’ 2nd generation catalyst was chosen as preferred catalyst for our cross metathesis reactions because of its tremendous functional-group tolerance, simplicity to control

45 under ambient condition, a satisfactorily high rate of reaction propagation, and commercial availability.106 The general mechanism of cross metathesis is illustrated in Figure 19.

Figure 19. Mechanism of olefin cross metathesis, adapted from ref.105

With 27A in hand, the cross metathesis reaction was conducted by treatment of 27A with

Grubbs’ 2nd generation catalyst and tridecane providing 28 in modest yield. The important aspect of the cross metathesis reaction is the cis:trans (E/Z) selectivity. It is well-stated in the literature that longer reaction times usually lead to higher quantities of trans-olefin products (Scheme

18).106 The resultant product was identified by MS and NMR spectroscopic methods.

Scheme 18. Olefin cross-metathesis reaction of (±)-27A

The final step in the synthesis included the deprotection of N-sulfonyl group by reaction with sodium naphthalenide and subsequently deptrotection of TBS group by tetrabutylammonium fluoride (TBAF), affording 30. The product was converted to the hydrochloride salt by treatment with methanolic HCl (Scheme 19).

In order to determine the influence of N-sulfonyl group on biological activity, the deprotection of silyl group on 28 was achieved by treatment with TBAF to afford 31. The final products were confirmed by MS, 1H-NMR and 13C-NMR evaluation. The synthesized azetidine analogs (30 and 31) may act as SK inhibitors and/or S1P receptor modulators. This synthetic strategy we employed is very efficient and scalable with the flexibility to introduce a variety of functionality and route to analog synthesis.

Scheme 19. Deprotection of (±)-28

2.4 BIOLOGICAL INVESTIGATION

Among the synthesized compounds, (±)-30 and (±)-31 were selected for biological evaluation as potential SK inhibitors. Dr. Daniel Baker and his colleagues at the University of Memphis are currently testing these derivatives as SK1 inhibitors.

2.5 CLOSING THOUGHTS

The research described documents an efficient and practical route for the synthesis of

2,3,4-trisubstituted azetidines using the intramolecular Mitsunobu reaction as key method for creating the azetidine ring. We were successful at preparing and structurally verifying two azetidine analogs, (±)-30 and (±)-31, each of which display structural resemblance to other natural sphingosine analogs.

The key to this synthetic pathway invoked is the use of the weak acid N-sulfonyl group as a nucleophile in the intramolecular Mitsunobu reaction. The nucleophilic sulfonamide displaces the activated alcohol as phosphine oxide and completes the azetidine ring formation. Another imperative approach in this synthesis is the Grubbs’ cross-metathesis reaction for side-chain elongation. This reaction provides an opportunity to develop various analogs through side-chain modification by using simple alkenes.

Although the total synthesis of penazetidine A was not completed, the key intermediate

((±)-27) represents a promising compound for constructing this natural product and its analogs.

Several problems were encountered and overcome during this thesis research. The first and foremost was the selection of a proper N-protecting group. The use of an N-sulfonyl group overcomes the problem associated with azetidine ring formation. The described synthetic methodology will stimulate opportunities to develop various azetidine analogs as a lead compounds for not only for sphingosine kinase, but also for other pertinent biological targets.

2.6 FUTURE DIRECTIONS

Since the synthesis was performed in racemic fashion, it was difficult to ascertain the impact of stereochemistry on biological activity. To resolve stereochemistry issues it will be

48 necessary to repeat the synthetic pathways using Sharpless asymmetric epoxidation (SAE)73 as a first step to install chirality. The attractive feature of this reaction is the use of (+) or (-)-diethyl tartrate to afford enantioenriched epoxides with more than 90% enantiomeric excess.73, 107, 108

Penazetidine A can be synthesized using this Sharpless epoxide product as a starting material.

The possible synthetic pathway for penazetidine A is outlined in Scheme 20. Yajima and coworkers synthesized penazetidine A in 6% overall yield in eighteen steps.68 Our proposed synthetic methodology will lead to the production of penazetidine A in eleven steps from commercially available cinnamyl alcohol.

Scheme 20. Proposed synthetic route for penazetidine A

III. EXPERIMENTAL

General methods:

Cinnamyl alcohol was purchased from Sigma Aldrich. All chemicals, catalysts, reagents and solvents used for reaction and chromatography were purchased at the best commercial grade.

Moisture-free reactions were carried out in oven or flame dried glassware in the presence of argon using dry solvents. Tetrahydrofuran (THF) was purified and dried by sodium metal and benzophenone (indicator) using distillation technique. Dichloromethane (CH2Cl2) was purified and dried by distillation process in the presence of calcium hydride. Anhydrous dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF) and dimethoxyethane (DME) were purchased from Sigma Aldrich and used without further purification. Thin layer chromatography

(TLC) was carried out on aluminum backed silica gel 60 F254 coated TLC sheets with UV 254 from EMD Chemicals Inc. Flash column chromatography was obtained using a SiliaFlash®

P60 40-63µm 60Å Silica Gels from SiliCycle Inc. 1D and 2D NMR spectra were recorded on either Bruker Advance DRX 400 (400 MHz) or Bruker Advance DRX 500 (500 MHz) spectrometers. 1H NMR coupling patterns are designated as singlet (s), doublet (d), triplet (t),

(quartet (q), double doublet (dd), double double doublet (ddd), double triplet (dt), multiplet (m).

Mass spectrometry (MS) data were recorded using electrospray ionization (ESI) on a Waters

Micromass ZQ mass spectrometer. Elemental Analysis was determined using a PerkinElmer

2400 Series II CHNS/O Analyzer. Melting points were detected on a Mel-Temp Apparatus.

General procedure for Epoxidation:

Using an adapted method of Balamurugan, et al.,109 to a solution of cinnamyl alcohol (74.52 mmol, 1 equiv.) in dry CH2Cl2 (300 ml), m-CPBA (111.78 mmol, 1.5 equiv.) was added. After 2 h of continuous stirring at RT, 50 ml of saturated aqueous Na2SO3 solution was added to the reaction mixture and stirred for another 15 min. Then the resultant mixture was washed twice with saturated aqueous NaHCO3 and brine. The organic layer was separated, dried over anhydrous Na2SO4, filtered and concentrated in vacuo to afford epoxide 8 as a white solid in

89% yield. The desired product was used in the next step without purification. ESI-MS: m/z

+ 1 173.085 [M+Na] , H NMR (400 MHz, CDCl3) δ 7.32 – 7.25 (m, 5H), 4.02 – 3.98 (dd, 1H), 3.90

(s, 1H), 3.77 –3.73 (dd, J = 12.7, 4.1 Hz, 1H), 3.22 – 3.20 (m, 1H), 2.96 (s, 1H); 13C NMR (101

MHz, CDCl3) δ 136.69, 128.53, 128.34, 125.79, 62.66, 61.40, 55.75; DEPT-135 (101 MHz,

CDCl3) δ 128.53, 128.34, 125.79, 62.66, 61.40, 55.75.

110 Using an adapted method of Venkatesan, et al., epoxide 8 (26.63 mmol, 1 equiv.), NaN3

(53.26 mmol, 2 equiv.) and NH4Cl (56.26 mmol, 2 equiv.) were added to a solvent mixture of methanol (29 ml) and water (5 ml) and warmed at 65 °C for 8 h. The resultant mixture was cooled to RT and filtered. The filtrate was concentrated in vacuo and the residue was diluted with EtOAc, extracted two times with water and brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was purified by column chromatography (70% petroleum ether / EtOAc; stains orange with p-anisaldehyde) to

51 afford azido diol 9 as a yellow oily liquid in 80% yield. ESI-MS: m/z 216.201 [M+Na]+, 1H

NMR (400 MHz, CDCl3) δ 7.41 – 7.32 (ddd, J = 19.6, 13.2, 7.2 Hz, 5H), 4.57 – 4.55 (d, J = 7.1

13 Hz, 1H), 3.82 – 3.78 (m, 1H), 3.68 – 3.60 (m, 2H), 3.05 (s, 2H); C NMR (101 MHz, CDCl3) δ

136.10, 128.94, 128.78, 127.81, 74.03, 67.00, 62.88.

Using an adapted method of Kimura et al.,79 to a solution of azido diol 9 (1.4518 mmol, 1 equiv.) in THF (4.8 ml) was added PPh3 (1.4518 mmol, 1 equiv.) under an atmosphere of argon.

The reaction mixture was stirred at RT for 10 min. and water (0.32 ml) was added. After 5 h of continuous stirring at 60 °C, it was cooled to RT. Thereafter, di-tert-butyl dicarbonate (1.5969 mmol, 1.1 equiv.) was added and stirred at RT for 2 h. The resultant mixture was concentrated in vacuo and the crude product was purified by column chromatography (80% EtOAc/ether; stains purple with ninhydrin) to afford N-Boc protected diol 10 as a white solid in 90% yield. ESI-MS: m/z 290.15 [M+Na]+, 1H NMR (400 MHz, MeOD) δ 7.36 – 7.23 (m, 5H), 4.70 – 4.68 (d, 1H),

3.86 – 3.82 (q, J = 5.8 Hz, 1H), 3.54 – 3.50 (dd, J = 11.8, 4.3 Hz, 1H), 3.46 – 3.42 (dd, J = 11.3,

5.8 Hz, 1H), 1.42 (s, 9H), 1.28 (s, 2H); 13C NMR (101 MHz, MeOD) δ 156.35, 139.91, 127.74,

127.43, 126.80, 78.97, 73.65, 62.95, 56.92, 27.37; DEPT-135 (101 MHz, MeOD) δ 127.74,

127.44, 126.80, 73.66, 62.94, 56.91, 27.37.

111 Using an adapted method of Badorrey et al., n-Bu2SnO (0.032 mmol, 0.029 equiv.) was added to a solution of N-Boc protected diol 10 (0.864 mmol, 1 equiv.) in dry CH2Cl2 (3.35 ml) at

RT under an atmosphere of argon. Subsequently, a solution of Et3N (1.039 mmol, 1.2 equiv.) in dry CH2Cl2 (3.35 ml) and TsCl (1.039 mmol, 1.2 equiv.) were added. After 5 h of continuous stirring at RT, the reaction mixture was treated with saturated aqueous NaCl solution. The organic layer was separated and the aqueous layer was washed three times with CH2Cl2. The combined organic layers were washed with brine, and dried over anhydrous MgSO4, filtered and concentrated in vacuo. The crude product was purified by column chromatography (40% EtOAc

/ hexane; stains purple with ninhydrin) to afford monotosylate 13 as a white solid in 71% yield.

+ 1 ESI-MS: m/z 444.139 [M+Na] , H NMR (400 MHz, CDCl3) δ 7.75 – 7.73 (d, J = 8.1 Hz, 2H),

7.33 – 7.22 (m, 7H), 5.44 (s, 1H), 4.68 (s, 1H), 4.18 – 4.14 (dd, J = 10.2, 3.5 Hz, 1H), 3.96 (s,

1H), 3.83 – 3.79 (dd, J = 10.2, 7.1 Hz, 1H), 2.44 (s, 3H), 1.39 (s, 9H); 13C NMR (101 MHz,

CDCl3) δ 155.40, 145.07, 137.57, 132.47, 129.91, 128.66, 127.96, 127.52, 71.06, 64.38, 56.37,

28.26, 21.63.

Using an adapted method of M.D. Lebar et al.,112 a solution of monotosylate 13 (0.593 mmol, 1 equiv.) in dry CH2Cl2 (5.6 ml) at 0 °C was added 2, 6-lutidine (1.7793 mmol, 3 equiv.) and TBS-

OTf (1.4827 mmol, 2.5 equiv.) under an argon atmosphere. After 2 h of continuous stirring at the

53 same temperature, the reaction mixture was treated with water. The organic layer was separated and the aqueous layer was washed two times with CH2Cl2. The combined organic layers were washed with water and brine, dried over anhydrous MgSO4, filtered and concentrated in vacuo.

The crude product was purified by column chromatography (30% EtOAc / hexane; stains purple with ninhydrin) to afford silyl carbamate 3 as a white solid in 59% yield. ESI-MS: m/z 616.320

+ [M+Na] , 1H NMR (400 MHz, CDCl3) δ 7.74 – 7.72 (d, J = 8.1 Hz, 2H), 7.34 – 7.27 (m, 7H),

5.29 – 5.27 (d, J = 8.0 Hz, 1H), 4.68 – 4.65 (dd, J = 8.1, 4.2 Hz, 1H), 4.26 – 4.22 (q, J = 5.2 Hz,

1H), 3.70 – 3.69 (d, J = 5.7 Hz, 2H), 2.46 (s, 3H), 0.91 – 0.88 (d, J = 9.7 Hz, 18H), 0.27 – 0.23

13 (d, J = 17.1 Hz, 6H), 0.05 – 0.04 (d, J = 3.1 Hz, 6H); C NMR (101 MHz, CDCl3) δ 154.08,

144.91, 137.18, 132.68, 129.85, 128.49, 128.11, 128.00, 127.94, 71.89, 70.47, 63.59, 56.93,

25.75, 25.53, 21.63, 18.06, 17.54, -4.61, -4.69, -5.15; DEPT-135 (101 MHz, CDCl3) δ 129.85,

128.49, 128.12, 128.01, 127.94, 71.89, 70.47, 56.92, 25.75, 25.52, 21.63, -4.61, -4.69, -5.16.

85 Using an adapted method of Pastó M et al., epoxide 8 (1.331mmol, 1eqiv.), NH4OH (2.1 ml, 40 equiv. of a 30% solution in water), in 4.2 ml of isopropanol were stirred in a sealed tube at 70 °C for 48 h. The resulting mixture was concentrated in vacuo to afford amino diol (40% EtOAc / hexane; stains purple with ninhydrin) as an oil. The obtained product was converted to the hydrochloride salt by treatment with HCl saturated solution of methanol to yield 14 which was taken to the next step without purification. 90% yield. ESI-MS: m/z 168.147 [M+H]+, 1H NMR

(400 MHz, MeOD) δ 7.55 – 7.43 (m, 5H), 4.49 – 4.48 (d, J = 3.5 Hz, 1H), 4.14 – 4.10 (m, 1H),

3.43 – 3.39 (dd, J = 11.1, 5.6 Hz, 1H), 3.35 – 3.30 (dd, J = 11.2, 4.8 Hz, 1H); 13C NMR (101

MHz, MeOD) δ 133.36, 133.31, 128.75, 128.41, 128.22, 70.85, 62.59, 56.94; DEPT-135 (101

MHz, MeOD) δ 128.75, 128.41, 128.21, 70.85, 62.59, 56.94.

Using an adapted method of Kamioka et al.,113 to a stirred solution of amine salt 14 (4.861 mmol, 1 equiv.) in anhydrous DMF (12 ml) at 0 °C was added DIEA (13.6108 mmol, 2.8 equiv.) and 2,4,6-trimethylbenzene-1-sulfonyl chloride (4.6179 mmol, 0.95 equiv.) under an argon atmosphere. The reaction mixture was stirred for 4 h at the same temperature, then treated with cold 1M HCl solution and extracted two times with EtOAc. The combined EtOAc layers were washed with saturated aqueous NaHCO3 and brine, dried over anhydrous MgSO4, filtered and concentrated in vacuo. The crude product was purified by column chromatography (50% EtOAc

/ hexane; stains orange with p-anisaldehyde) to afford sulfonamide 15 as a white solid in 58% yield. ESI-MS: m/z 372.227 [M+Na]+. Melting Point: 116-118 °C. 1H NMR (400 MHz, MeOD)

δ 7.09 – 7.04 (m, 5H), 6.78 (s, 2H), 4.58 (s, 1H), 4.25 – 4.23 (d, J = 6.4 Hz, 1H), 3.87 – 3.83 (q, J

= 6.0 Hz, 1H), 3.56 – 3.46 (ddt, J = 17.4, 11.4, 5.5 Hz, 2H), 2.47 (s, 6H), 2.19 (s, 3H); 13C NMR

(101 MHz, MeOD) δ 141.85, 138.53, 137.76, 134.48, 131.30, 127.53, 127.37, 126.79, 73.88,

63.06, 59.23, 21.77, 19.48; DEPT-135 (101 MHz, MeOD) δ 131.30, 127.53, 127.37, 126.79,

73.88, 63.06, 59.23, 21.77, 19.48. Elemental Analysis Calcd for C18H23NO4S: C, 61.87; H, 6.63;

N, 4.01; Found: C, 61.89; H, 6.46; N, 3.96.

Using an adapted method of Kiren et al.,89 to a stirred solution of sulfonamide 15 (5.40 mmol, 1 equiv.) in anhydrous DMF (13 ml) at 0 °C was added imidazole (32.4 mmol, 6 equiv.) and

TBSCl (24.3 mmol, 4.5 equiv.) under an argon atmosphere. The reaction mixture was stirred for

48 h at RT, then treated with H2O and extracted three times with Et2O. The combined Et2O layers were washed with H2O and brine, dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was purified by column chromatography (15% EtOAc / hexane; stains brownish yellow with p-anisaldehyde) to afford bis-silylated 20 in 80% yield. ESI-MS:

+ 1 m/z 600.689 [M+Na] , H NMR (400 MHz, CDCl3) δ 7.09 – 7.06 (m, 5H), 6.74 (s, 2H), 5.95 –

5.93 (d, J = 6.4 Hz, 1H), 4.45 – 4.43 (m, 1H), 3.91 – 3.87 (dt, J = 8.3, 4.2 Hz, 1H), 3.50 – 3.46

(dd, J = 10.3, 4.0 Hz, 1H), 3.25 – 3.21 (dd, J = 10.2, 7.6 Hz, 1H), 2.48 (s, 6H), 2.21 (s, 3H), 0.91

13 (s, 9H), 0.85 (s, 9H), 0.04 – 0.02 (d, J = 9.0 Hz, 9H), -0.03 (s, 3H); C NMR (101 MHz, CDCl3)

δ 141.50, 138.72, 137.43, 134.58, 131.51, 127.97, 127.60, 127.26, 75.03, 64.65, 60.95, 25.87,

25.80, 22.86, 22.65, 20.76, 18.19, 18.04, -4.64, -5.03, -5.55, -5.58. Elemental Analysis Calcd for

C30H51NO4SSi2: C, 62.34; H, 8.89; N, 2.42; Found: C, 61.93; H, 8.75; N, 2.39.

Using an adapted method of Liu et al.,114 to a stirred solution of bis-silylated 20 (0.64 mmol, 1 equiv.) in THF (8 ml) at 0 °C was added H2O (2 ml) and TFA (2 ml). The reaction mixture was stirred for 75 min at RT and constantly monitored by TLC. Thereafter, it was treated with the saturated aqueous NaHCO3 and extracted three times with DCM. The combined DCM layers were washed with H2O and brine, dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was purified by column chromatography (15% EtOAc / hexane; stains brownish yellow with p-anisaldehyde) to afford compound 21 as a white solid in 60% yield. ESI-

+ 1 MS: m/z 486.381[M+Na] , H NMR (500 MHz, CDCl3) δ 7.10 (dt, J = 14.0, 6.9 Hz, 3H), 6.99

(d, J = 7.1 Hz, 2H), 6.76 (s, 2H), 5.84 (d, J = 7.0 Hz, 1H), 4.44 – 4.42 (t, 1H), 3.94 – 3.92 (t, 1H),

3.75 – 3.73 (d, J = 9.8 Hz, 1H), 3.49 – 3.48 (d, 1H), 2.47 (s, 6H), 2.23 (s, 3H), 0.82 (s, 9H), 0.05

13 (s, 3H), -0.09 (s, 3H); C NMR (126 MHz, CDCl3) δ 141.86, 138.71, 137.74, 134.33, 131.62,

127.98, 127.46, 127.43, 74.99, 63.44, 60.68, 25.74, 22.81, 20.78, 18.02, -4.71, -5.08; DEPT-135

(126 MHz, CDCl3) δ 131.62, 127.98, 127.47, 127.43, 74.98, 63.44, 60.68, 25.75, 22.81, 20.79, -

4.71, -5.08. Elemental Analysis Calcd for C24H37NO4SSi: C, 62.16; H, 8.04; N, 3.02; Found: C,

62.38; H, 7.95; N, 3.18.

Using an adapted method of Ghorai et al.,76 to a stirred solution of compound 21 (0.25 mmol, 1 equiv.) and PPh3 (0.375 mmol, 1.5 equiv.) in dry THF (1 ml) was added a solution of DIAD

(0.375 mmol, 1.5 equiv.) in dry THF (0.5 ml) dropwise at 0 °C over a period of 10 min under an argon atmosphere. The resulting mixture was stirred for 2 h at RT. Thereafter, solvent was removed under reduced pressure and the crude residue was purified by column chromatography

(10% EtOAc / hexane; stains brownish orange with p-anisaldehyde) to afford azetidine 22 as a

+ 1 white solid in 65% yield. ESI-MS: m/z 468.433 [M+Na] , H NMR (400 MHz, CDCl3) δ 7.15 –

7.12 (m, 5H), 6.74 (s, 2H), 4.87 – 4.85 (d, J = 5.7 Hz, 1H), 4.29 – 4.24 (q, J = 6.1 Hz, 1H), 3.92

– 3.89 (t, J = 6.7 Hz, 1H), 3.85 – 3.82 (t, J = 6.6 Hz, 1H), 2.52 (s, 6H), 2.18 (s, 3H), 0.83 (s, 9H),

13 -0.09 – -0.11 (d, J = 7.0 Hz, 6H); C NMR (101 MHz, CDCl3) δ 142.60, 140.15, 137.63, 131.71,

131.51, 128.05, 127.90, 126.56, 74.81, 69.61, 55.62, 25.58, 22.91, 20.83, 17.82, -4.82, -5.08;

DEPT-135 (101 MHz, CDCl3) δ 131.51, 128.05, 127.90, 126.56, 74.81, 69.61, 55.62, 25.58,

22.91, 20.83, -4.82, -5.09. Elemental Analysis Calcd for C24H35NO3SSi: C, 64.68; H, 7.92; N,

3.14; Found: C, 65.38; H, 8.03; N, 3.04.

Using an adapted method of Saito et al.,95 to a stirred solution of mono-alcohol 21 (0.711 mmol,

1 equiv.) in anhydrous DMSO (3.1 ml) at RT was added IBX (2.844 mmol, 4 equiv.) under an argon atmosphere. The resulting mixture was stirred overnight at the same temperature, then it was treated with a 1/1 mixture of saturated aqueous NaHCO3 and Na2S2O3 solution and extracted three times with Et2O. The combined Et2O layers were washed with brine, dried over anhydrous

Na2SO4, filtered and concentrated in vacuo to afford pure aldehyde 23 as a white solid in 90%

+ 1 yield. ESI-MS: m/z 484.388 [M+Na] , Melting Point: 104-106 °C; H NMR (500 MHz, CDCl3)

δ 9.42 (s, 1H), 7.13 – 7.08 (dt, J = 14.1, 6.8 Hz, 3H), 7.02 – 7.01 (d, J = 7.0 Hz, 2H), 6.76 (s,

2H), 5.62 –5.60 (d, J = 7.7 Hz, 1H), 4.52 – 4.49 (t, 1H), 4.35 – 4.34 (d, J = 4.7 Hz, 1H), 2.48 (s,

13 6H), 2.21 (s, 3H), 0.86 (s, 9H), 0.02 (s, 3H), -0.04 (s, 3H); C NMR (126 MHz, CDCl3) δ

201.22, 142.17, 138.81, 135.98, 131.74, 128.14, 128.03, 127.75, 79.95, 58.97, 25.73, 25.66,

22.80, 20.76, 18.07, -4.82, -5.25; DEPT-135 (126 MHz, CDCl3) δ 201.24, 131.74, 128.13,

128.03, 127.75, 79.95, 58.96, 25.66, 22.80, 20.76, -4.83, -5.25.

Using an adapted method of Hirose et al.,77 to a stirred solution of aldehyde 23 (0.368 mmol, 1 equiv.) in anhydrous Et2O (3.68 ml) at -78 °C was added 1.0M solution of vinylmagnesium bromide in THF (1.104 mmol, 3 equiv.) dropwise under an argon atmosphere. The resulting

58 mixture was stirred for 7 h at the same temperature, then it was treated with a 1/1 mixture of saturated aqueous NaHCO3 and Et2O. The organic layer was separated and washed with NH4Cl,

H2O, and brine, dried over anhydrous Na2SO4, filtered and concentrated in vacuo to furnish

+ 1 compound 25 (total yield 81%). ESI-MS: m/z 512.46 [M+Na] ; H NMR (500 MHz, CDCl3) δ

7.07 – 6.97 (m, 10H), 6.73 – 6.70 (d, J = 15.0 Hz, 4H), 6.63 – 6.62 (d, J = 5.0 Hz, 1H), 6.00 –

5.93 (ddd, J = 17.0, 10.4, 6.2 Hz, 1H), 5.87 – 5.81 (ddd, J = 16.2, 10.6, 4.9 Hz, 1H), 5.58 – 5.56

(d, J = 8.1 Hz, 1H), 5.31 – 5.25 (dd, J = 17.0, 12.1 Hz, 2H), 5.22 – 5.19 (dd, J = 10.3, 5.1 Hz,

2H), 4.55 – 4.53 (t, J = 5.3 Hz, 1H), 4.45 – 4.42 (t, 1H), 4.26 (s, 1H), 4.12 – 4.05 (dt, J = 21.2,

5.2 Hz, 2H), 3.82 – 3.81 (d, J = 5.1 Hz, 1H), 2.46 –2.45 (d, J = 5.9 Hz, 12H), 2.20 – 2.19 (d, J =

8.2 Hz, 6H), 1.45 (s, 1H), 0.84 – 0.83 (d, J = 3.1 Hz, 18H), 0.10 (s, 3H), 0.02 (s, 3H), -0.07 (s,

13 3H), -0.22 (s, 3H); C NMR (126 MHz, CDCl3) δ 141.78, 141.46, 138.59, 138.50, 138.30,

137.70, 137.43, 137.08, 135.23, 134.36, 131.76, 131.59, 131.50, 127.92, 127.85, 127.79, 127.74,

127.59, 127.55, 127.35, 127.30, 117.40, 116.46, 78.50, 76.32, 74.13, 72.49, 61.65, 59.81, 30.36,

25.97, 25.88, 25.76, 25.72, 25.67, 25.57, 22.79, 20.73, 18.16, 18.06, -4.09, -4.53, -4.62, -5.00;

DEPT-135 (126 MHz, CDCl3) δ 137.42, 137.07, 131.59, 131.50, 127.92, 127.85, 127.79, 127.60,

127.35, 127.30, 117.41, 116.46, 78.49, 76.30, 74.13, 72.50, 61.65, 59.81, 25.98, 25.88, 22.79,

20.73, -4.53, -4.62.

Using an adapted method of Chattopadhyay et al.,98 to a cold (10 °C ) and stirred solution of compound 23 (0.547 mmol, 1 equiv.), Zn dust (1.367 mmol, 2.5 equiv.) and allyl bromide (1.367 mmol, 2.5 equiv.) in THF (0.5 mL) was added a saturated aqueous solution of NH4Cl (0.3 mL) dropwise via syringe over a period of 30 min. under argon atmosphere. The mixture was stirred

59 for 3 h at RT, filtered, and the precipitate was washed with DCM. The aqueous layer was separated and treated with 5% HCl and extracted three times with DCM. The combined DCM layers were washed with 10% NaHCO3, H2O and brine, dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was purified by column chromatography (15-20%

EtOAc / hexane; stains dark purple with p-anisaldehyde) to afford compound 26A and 26B.

+ 1 Compound (±)-26A: 15% yield. ESI-MS: m/z 504.402 [M+H] , H NMR (400 MHz, CDCl3) δ

7.10 – 7.06 (d, J = 7.2 Hz, 3H), 7.04 – 7.01 (m, 2H), 6.74 (s, 2H), 6.63 – 6.63 (d, J = 5.1 Hz, 1H),

5.63 – 5.53 (m, 1H), 5.06 – 4.98 (m, 2H), 4.66 –4.63 (t, J = 5.0 Hz, 1H), 3.73 – 3.71 (m, 1H),

2.45 (s, 6H), 2.21 (s, 3H), 2.16 – 2.10 (dt, J = 14.0, 5.8 Hz, 2H), 0.90 (s, 9H), 0.08 (s, 3H), -0.05

13 (s, 3H); C NMR (101 MHz, CDCl3) δ 141.38, 138.36, 138.20, 135.51, 134.01, 131.50, 128.02,

127.33, 127.23, 118.04, 74.97, 70.09, 62.16, 38.55, 25.87, 22.82, 20.76, 18.07, -4.56, -4.81;

DEPT-135 (101 MHz, CDCl3) δ 134.01, 131.50, 128.02, 127.33, 127.23, 118.04, 74.97, 70.09,

62.16, 38.55, 25.87, 22.82, 20.76, -4.56, -4.81.

+ 1 Compound (±)-26B: 55% yield. ESI-MS: m/z 504.462 [M+H] , H NMR (500 MHz, CDCl3) δ

7.07 – 7.05 (t, J = 7.3 Hz, 3H), 6.99 – 6.98 (d, J = 7.0 Hz, 2H), 6.74 (s, 2H), 5.79 – 5.71 (td, J =

16.6, 8.3 Hz, 1H), 5.40 – 5.38 (d, J = 8.3 Hz, 1H), 5.14 – 5.11 (t, 2H), 4.45 – 4.42 (dd, J = 8.1,

5.4 Hz, 1H), 4.07 – 4.05 (t, J = 5.2 Hz, 1H), 3.54 – 3.49 (dq, J = 11.9, 6.7, 5.8 Hz, 1H), 2.46 (s,

6H), 2.22 (s, 3H), 1.29 (s, 1H), 0.87 (s, 9H), 0.83 (s, 2H), 0.14 (s, 3H), 0.04 (s, 3H); 13C NMR

(126 MHz, CDCl3) δ 141.87, 138.65, 137.74, 134.80, 134.23, 131.64, 127.98, 127.93, 127.80,

127.42, 118.63, 78.40, 71.51, 59.64, 37.29, 26.01, 22.80, 20.77, 18.25, -4.15, -4.24; DEPT-135

(126 MHz, CDCl3) δ 134.80, 131.65, 127.93, 127.80, 127.42, 118.64, 78.39, 71.50, 59.64, 37.29,

26.01, 22.80, 20.77, -4.15, -4.24.

The same procedure was used for the synthesis of 22 was followed using compound 25 (0.247 mmol, 1 equiv.), PPh3 (0.3705 mmol, 1.5 equiv.), dry THF (1 ml), DIAD (0.3705 mmol, 1.5 equiv.), and dry THF ((0.5 ml) for DIAD). Chromatographic purification (10%-15% EtOAc / hexane; stains brownish orange with p-anisaldehyde) furnished fully substituted azetidine 27 as a white solid (total yield 60%).

Compound (±)-27A: White solid. 35% yield. ESI-MS: m/z 494.458 [M+Na]+, Melting Point: 71-

1 74 °C; H NMR (400 MHz, CDCl3) δ 7.20 – 7.18 (d, J = 7.8 Hz, 5H), 6.74 (s, 2H), 5.97 – 5.89

(ddd, J = 17.5, 10.3, 7.6 Hz, 1H), 5.35 – 5.31 (d, J = 17.2 Hz, 1H), 5.18 – 5.16 (d, J = 10.3 Hz,

1H), 4.75 – 4.73 (d, J = 5.9 Hz, 1H), 4.50 – 4.46 (t, 1H), 3.95 – 3.92 (t, J = 5.9 Hz, 1H), 2.54 (s,

13 6H), 2.19 (s, 3H), 0.82 (s, 9H), -0.11 (s, 3H), -0.14 (s, 3H); C NMR (101 MHz, CDCl3) δ

142.75, 140.35, 137.86, 135.35, 131.64, 131.50, 128.08, 127.88, 126.85, 118.46, 71.71, 69.72,

25.55, 22.99, 20.85, 17.81, -4.77; DEPT-135 (101 MHz, CDCl3) δ 135.35, 131.54, 131.50,

128.08, 127.88, 126.85, 118.46, 76.77, 71.71, 69.72, 25.55, 22.99, 20.85, -4.77.

Compound (±)-27B: White solid. 23% yield. ESI-MS: m/z 494.32 [M+Na]+, Melting Point: 94-

1 96 °C; H NMR (500 MHz, CDCl3) δ 7.08 – 7.00 (dt, J = 22.2, 9.0 Hz, 5H), 6.65 (s, 2H), 6.62 –

6.57 (m, 1H), 5.48 – 5.46 (d, J = 10.1 Hz, 1H), 5.44 – 5.41 (d, J = 17.0 Hz, 1H), 5.13 – 5.12 (d,

J = 6.0 Hz, 1H), 4.88 – 4.85 (dd, J = 9.7, 6.9 Hz, 1H), 4.37 – 4.35 (t, J = 6.4 Hz, 1H), 2.50 (s,

13 6H), 2.16 (s, 3H), 0.81 (s, 9H), -0.12 (s, 3H), -0.14 (s, 3H); C NMR (126 MHz, CDCl3) δ

142.13, 139.71, 137.14, 134.02, 132.63, 131.26, 127.86, 127.76, 126.44, 120.72, 74.41, 73.41,

72.52, 25.53, 22.79, 20.74, 17.96, -4.92, -5.07; DEPT-135 (126 MHz, CDCl3) δ 132.62, 131.25,

127.85, 127.75, 126.44, 120.73, 74.41, 73.41, 72.52, 25.53, 22.79, 20.74, -4.92.

Using an adapted method of Sheddan et al.,115 a stirred solution of azetidine 27A (0.212 mmol, 1 equiv.) and Tridecane (0.848 mmol, 4 equiv.) in degassed DCM (11 ml) was added a solution of

Grubbs’ II generation catalyst (7 mol %, 0.0148 mmol) in degassed DCM (3 ml). Degassing of

DCM was carried out by 3 cycles of the freeze and pump technique. The reaction mixture was refluxed at 40 °C for 48 h. Thereafter, the solvent was evaporated under reduced pressure and the crude product was purified by column chromatography (5%-10% EtOAc / hexane; stains bluish grey with p-anisaldehyde) to afford compound 28 as oil in 70% yield. ESI-MS: m/z 648.63

+ 1 [M+Na] , H NMR (500 MHz, CDCl3) δ 7.23 – 7.20 (m, 5H), 6.76 (s, 2H), 5.74 – 5.68 (dt, J =

13.6, 6.4 Hz, 1H), 5.50 – 5.45 (dd, J = 14.9, 8.1 Hz, 1H), 4.79 – 4.78 (d, J = 5.7 Hz, 1H), 4.41 –

4.39 (t, 1H), 3.93 – 3.90 (t, J = 5.7 Hz, 1H), 2.56 (s, 6H), 2.20 (s, 3H), 1.95 (s, 2H), 1.30 (s,

18H), 0.93 – 0.90 (t, J = 6.8 Hz, 3H), 0.83 (s, 9H), -0.11 – -0.12 (d, J = 6.5 Hz, 6H); 13C NMR

(126 MHz, CDCl3) δ 142.57, 140.33, 138.25, 135.92, 131.99, 131.48, 128.11, 127.85, 127.15,

126.84, 77.22, 71.17, 69.88, 32.25, 31.98, 29.74, 29.70, 29.67, 29.57, 29.41, 29.16, 28.96, 25.68,

25.60, 25.53, 23.02, 22.74, 20.87, 17.86, 14.18, -4.75; DEPT-135 (126 MHz, CDCl3) δ 135.93,

131.48, 128.11, 127.85, 127.15, 126.84, 77.22, 71.16, 69.89, 32.26, 31.98, 29.74, 29.71, 29.67,

29.57, 29.42, 29.17, 28.96, 25.60, 23.03, 22.99, 22.75, 20.88, 14.20, -4.75.

Using an adapted method of Yajima et al.,68 sodium (0.76 mmol, 8 equiv.) was added to the solution of naphthalene (0.95 mmol, 10 equiv.) in dry DME (1 ml) under an argon atmosphere.

The mixture was stirred for 90 min. at RT. The resulting mixture was added dropwise via syringe to a mixture of compound 28 (0.095 mmol, 1 equiv.) in dry DME (0.5 ml) at -78 °C under an argon. The reaction mixture was stirred for 40 min. at the same temperature and then treated with water. After stirring for 30 min, the reaction mixture was extracted thrice with DCM. The combined DCM layers were washed with brine, dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was purified by column chromatography (20%-40%

EtOAc / hexane; greyish blue with p-anisaldehyde) to afford compound 29 in 42% yield. ESI-

MS: m/z 444.44 [M+H]+.

Using an adapted method of Wang et al. and Burtoloso et al.,116, 117 to a stirred solution of compound 29 (0.022 mmol, 1 equiv.) in dry THF (0.5 ml) at 0 °C was added 1.0 M solution of

TBAF in THF (0.033 mmol, 1.5 equiv.) and the resulting mixture was stirred for overnight at

RT. Then it was treated with the saturated aqueous NaHCO3 and extracted thrice with DCM. The combined DCM layers were washed with brine, dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was purified by column chromatography (100%

EtOAc; stains greyish blue with p-anisaldehyde) to afford azetidine alcohol. The obtained product was converted to the hydrochloride salt by treatment with HCl saturated solution of methanol to furnish 30 as a solid in 40% yield. The product was confirmed based on MS, 1H-

13 + 1 NMR and C-NMR evaluation. ESI-MS: m/z 330.26 [M+H] , H NMR (400 MHz, CDCl3) δ

7.43 – 7.33 (m, 5H), 5.91 – 5.78 (qd, J = 16.2, 15.8, 6.2 Hz, 2H), 4.72 – 4.70 (d, J = 5.5 Hz, 1H),

4.38 – 4.35 (t, 1H), 4.31 – 4.28 (t, 1H), 2.19 – 2.14 (q, 2H), 1.99 (s, 1H), 1.27 (s, 18H), 0.90 –

0.86 (t, 3H); DEPT-135 (101 MHz, CDCl3) δ 136.46, 128.43, 127.35, 126.60, 125.94, 74.59,

70.02, 63.69, 32.68, 31.92, 29.68, 29.64, 29.61, 29.50, 29.35, 29.29, 29.24, 22.69, 14.11.

The above procedure used in the synthesis of compound 30 was followed using compound 28

(0.0592 mmol, 1 equiv.) dry THF (1.42 ml) and 1.0 M solution of TBAF in THF (0.0592 mmol,

1 equiv.). Chromatographic purification (100% EtOAc; stains greyish blue with p-anisaldehyde) furnished compound 31 as a white solid in 56% yield. ESI-MS: m/z 534.38 [M+Na]+. 1H NMR

(400 MHz, CDCl3) δ 7.23 – 7.18 (dq, J = 6.9, 3.9 Hz, 5H), 6.75 (s, 2H), 5.71 – 5.64 (dt, J = 13.6,

6.6 Hz, 1H), 5.47 – 5.42 (dd, J = 15.4, 7.8 Hz, 1H), 4.80 – 4.78 (d, J = 6.0 Hz, 1H), 4.41 – 4.38

(t, 1H), 3.94 – 3.93 (d, J = 5.7 Hz, 1H), 2.98 – 2.96 (d, J = 6.7 Hz, 1H), 2.52 (s, 6H), 2.18 (s,

3H), 1.91 – 1.90 (d, 2H), 1.27 (s, 18H), 0.90 – 0.87 (t, J = 6.5 Hz, 3H); 13C NMR (101 MHz,

CDCl3) δ 142.81, 140.40, 138.11, 136.03, 131.59, 131.56, 128.22, 127.98, 126.89, 126.62, 76.55,

71.00, 69.72, 32.23, 31.94, 29.72, 29.67, 29.64, 29.53, 29.38, 29.20, 28.83, 23.00, 22.70, 20.88,

14.13; DEPT-135 (101 MHz, CDCl3) δ 136.03, 131.56, 128.22, 127.98, 126.89, 126.63, 76.55,

71.00, 69.72, 32.22, 31.94, 29.71, 29.67, 29.64, 29.53, 29.37, 29.20, 28.83, 23.00, 22.70, 20.88,

14.13.

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APPENDIX SPECTRAL DATA

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

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VITA

Zarana Daksh Chauhan was born in Surat, Gujarat, India on May 9th, 1983. She received her Bachelor of Pharmacy degree from Veer Narmad South Gujarat University in May 2004. She worked as a Lab Technician at Maliba Pharmacy College from August 2005 to February 2008 in

India. She joined the Master’s program in the Department of Medicinal Chemistry at the

University of Mississippi in the fall of 2010 and successfully fulfilled all the requirements for a

Master’s degree in the summer of 2013.

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