synthesis of natural product-based sphingosine derivatives
TRANSCRIPT
University of Mississippi University of Mississippi
eGrove eGrove
Electronic Theses and Dissertations Graduate School
2013
Synthesis Of Natural Product-Based Sphingosine Derivatives Synthesis Of Natural Product-Based Sphingosine Derivatives
Zarana Daksh Chauhan University of Mississippi
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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
Copyright © 2013 by Zarana Daksh Chauhan
All rights reserved
ii
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.
iii
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.
iv
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
v
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
vi
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
vii
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.
viii
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.
ix
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
x
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
xi
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
xii
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
xiii
Scheme 20. Proposed synthetic route for penazetidine A .............................................................49
xiv
LIST OF TABLE
Table 1. Sphingosine kinase inhibitors and their characteristics ...................................................21
1
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
2
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
3
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
4
Figure 1. Schematic illustration of signaling pathways for sustained PKC
activation. Adapted from ref.8 (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,
5
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
protein-coupled receptors (GPCRs).23
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
6
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,
7
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
8
Figure 3. Biosynthesis of ceramide, adapted from ref.27, 29
9
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
is found in liver and heart.30
Three different splice isoforms of SK1 have been detected in
10
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
additional amino acids than SK2a.29
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
of substrates.31
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
localization signal and an SH3 binding motif.31
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
S1P signaling depends on SK1 subcellular localization.32
In animal model, targeting SK1 was
shown to decrease the occurrence and complexity of disease, while targeting SK2 was shown to
11
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
12
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
S1P1 receptors. However, the function of S1P1 in Purkinje cell is poorly understood.22,
35
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
13
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
involved in mast cell migration, whereas S1P2 receptors are engaged in degranulation.37
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
14
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
15
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
oncogene.42
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
16
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
17
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
specific S1P receptors, and targeting SK1 and/or SK2 enzymes.41
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
18
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
19
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
20
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
acute myelogenous leukemia xenograft tumors.54
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
substrate for SK2.32
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
21
Table 1. Sphingosine kinase inhibitors and their characteristics
SK
Inhibitors
Mechanism
of action
Type of
Inhibition Comments
SKi Inhibit SK1
and SK2
Non-
Competitive
Stimulates cathepsin-B-mediated
degradation of SK1, no effects on PKC,
ERK and PI3K.32,
41
SKi-178 Inhibit SK1 Competitive
Addition of methyl and methoxy groups
to the parent compound led to reduce
cytotoxicity and increase selectivity for
SK1.32
BML-258 Inhibit SK1 Competitive
Suppressed tumor growth, decreased
proliferation, and induced apoptosis in
the tumor.54
ABC294640 Inhibit SK2 Competitive
Showed good pharmacological profiles,
anticancer activity, anti-inflammatory
activity and low toxicity in in vivo & in
vitro studies.55
D,L-threo-
DHS Inhibit SK1 Competitive Inhibits other kinases.
32
DMS Inhibit SK1
and SK2
Competitive for
SK1/Non-
Competitive for
SK2
Lack of specificity, inhibit various other
cellular kinases.56
PF543 Inhibit SK1 Competitive Inhibits S1P formation in whole blood.57
F-12509A
(Natural
product)
Inhibit SK1
and SK2 Competitive
Isolated from a dicomycete
Trichopezizella barbata , induce
apoptosis.41
B-5354c
(Natural
product)
Inhibit SK1
and SK2
Non-
Competitive
Isolated from a marine bacterium,
decreased S1P production in human
platelets.41, 52
S-15183a and
S-15183b
(Natural
product)
SK
(Specificity
not
determined)
Not determined
Isolated from a fungus Zopfiella
intermis, decreased S1P production in in
vitro studies.41
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
22
types of S1P receptors except S1P2. FTY720 has low affinity for S1P2. There are five different
types of S1P receptors including S1P1, S1P2, S1P3, S1P4, and S1P5.58, 59, 60
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
inhibits ERK1, ERK2, and Akt phosphorylation, and activates caspases, JNK, and PKCδ.41
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
23
phosphorylated by SK2 and acts as an agonist or partial agonist for S1P1, S1P4 and S1P5 receptors
and antagonist for S1P3 receptor.60
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
24
Figure 10. Structures of S1P receptor agonists and antagonists, adapted from ref.60
25
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-
26
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
27
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
(PKC) inhibitor.68
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.
28
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
29
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
30
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
As shown in Scheme 3, cyclization of 6 was carried out by treatment with K2CO3 to afford 7.75
The synthetic protocol described in Scheme 3 afforded azetidine 7 in excellent yield.
Scheme 3. Synthetic pathway for the formation of azetidine
31
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
32
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
33
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).
34
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
35
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
36
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
37
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
38
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
39
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,
21 was treated with PPh3 and diisopropyl azodicarboxylate (DIAD) to afford 22.76
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
40
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
41
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
42
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
43
phosphine reagent such as PPh3 and an oxidizing azo reagent such as DIAD or DEAD.100
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
PPh3 and DIAD in the presence of THF.76
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).
44
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
experiments. The 13
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.
45
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
46
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
47
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.
48
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
49
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
50
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.
51
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
173.085 [M+Na]+,
1H 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); 13
C 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.
Using an adapted method of Venkatesan, et al.,110
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
52
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
Hz, 1H), 3.82 – 3.78 (m, 1H), 3.68 – 3.60 (m, 2H), 3.05 (s, 2H); 13
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); 13
C 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.
53
Using an adapted method of Badorrey et al.,111
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.
ESI-MS: m/z 444.139 [M+Na]+,
1H 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); 13
C 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
54
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
(d, J = 17.1 Hz, 6H), 0.05 – 0.04 (d, J = 3.1 Hz, 6H); 13
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.
Using an adapted method of Pastó M et al.,85
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); 13
C NMR (101
55
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); 13
C 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.
56
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:
m/z 600.689 [M+Na]+,
1H 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
(s, 9H), 0.85 (s, 9H), 0.04 – 0.02 (d, J = 9.0 Hz, 9H), -0.03 (s, 3H); 13
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-
57
MS: m/z 486.381[M+Na]+,
1H 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
(s, 3H), -0.09 (s, 3H); 13
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
white solid in 65% yield. ESI-MS: m/z 468.433 [M+Na]+,
1H 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),
-0.09 – -0.11 (d, J = 7.0 Hz, 6H); 13
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,
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%
yield. ESI-MS: m/z 484.388 [M+Na]+, Melting Point: 104-106 °C;
1H 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,
6H), 2.21 (s, 3H), 0.86 (s, 9H), 0.02 (s, 3H), -0.04 (s, 3H); 13
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
59
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
compound 25 (total yield 81%). ESI-MS: m/z 512.46 [M+Na]+;
1H 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,
3H), -0.22 (s, 3H); 13
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
60
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.
Compound (±)-26A: 15% yield. ESI-MS: m/z 504.402 [M+H]+,
1H 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
(s, 3H); 13
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.
Compound (±)-26B: 55% yield. ESI-MS: m/z 504.462 [M+H]+,
1H 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); 13
C 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.
61
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-
74 °C; 1H 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,
6H), 2.19 (s, 3H), 0.82 (s, 9H), -0.11 (s, 3H), -0.14 (s, 3H); 13
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-
96 °C; 1H 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,
6H), 2.16 (s, 3H), 0.81 (s, 9H), -0.12 (s, 3H), -0.14 (s, 3H); 13
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.
62
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
[M+Na]+,
1H 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); 13
C 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.
63
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-
NMR and 13
C-NMR evaluation. ESI-MS: m/z 330.26 [M+H]+,
1H 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.
64
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); 13
C 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.
65
BIBLIOGRAPHY
66
1. Newman, D.J. and G.M. Cragg, Natural Products as Sources of New Drugs over the Last
25 Years. Journal of Natural Products, 2007. 70(3): p. 461-477.
2. Skropeta, D., N. Pastro, and A. Zivanovic, Kinase inhibitors from marine sponges. Mar
Drugs, 2011. 9(10): p. 2131-54.
3. Hu, G.-P., et al., Statistical Research on Marine Natural Products Based on Data
Obtained between 1985 and 2008. Mar Drugs, 2011. 9(4): p. 514-525.
4. Belarbi el, H., et al., Producing drugs from marine sponges. Biotechnol Adv, 2003.
21(7): p. 585-98.
5. Sipkema, D., et al., The life and death of sponge cells. Biotechnology and
Bioengineering, 2004. 85(3): p. 239-247.
6. Montaser, R. and H. Luesch, Marine natural products: a new wave of drugs? Future Med
Chem, 2011. 3(12): p. 1475-89.
7. Henkel, T., et al., Statistical Investigation into the Structural Complementarity of Natural
Products and Synthetic Compounds. Angewandte Chemie International Edition, 1999.
38(5): p. 643-647.
8. Nishizuka, Y., Protein kinase C and lipid signaling for sustained cellular responses. The
FASEB Journal, 1995. 9(7): p. 484-96.
9. Dhanasekaran, N. and E. Premkumar Reddy, Signaling by dual specificity kinases.
Oncogene, 1998. 17(11 Reviews): p. 1447-55.
10. Manning, G., et al., The Protein Kinase Complement of the Human Genome. Science,
2002. 298(5600): p. 1912-1934.
11. Goldstein, D.M., N.S. Gray, and P.P. Zarrinkar, High-throughput kinase profiling as a
platform for drug discovery. Nat Rev Drug Discov, 2008. 7(5): p. 391-397.
12. Kobayashi, J., Sphingosine-related Marine Alkaloids: Cyclic Amino Alcohols.
Heterocycles, 1996. 42(2): p. 943-970.
13. Meyer, M. and M. Guyot, New Sphingosines from the Marine Sponge Grayella
cyatophora. Journal of Natural Products, 2002. 65(11): p. 1722-1723.
14. Mellor, H. and P.J. Parker, The extended protein kinase C superfamily. Biochem. J.,
1998. 332(2): p. 281-292.
15. Murakami, A., et al., Absence of Down-Regulation and Translocation of The [eegr]
Isoform of Protein Kinase C in Normal Human Keratinocytes. J Investig Dermatol, 1996.
106(4): p. 790-794.
67
16. Shier, W.T., Sphingosine Analogs: an Emerging New Class of Toxins that Includes the
Fumonisins. 1992. 11(3): p. 241-257.
17. Thudichum, J.L.W., A treatise on the chemical constitution of the brain1962, Hamden,
Conn.,: Archon Books. xliii p., facsim.,.
18. Carter, H.E., W.J. Haines, and et al., Biochemistry of the sphingolipides; preparation of
sphingolipides from beef brain and spinal cord. J Biol Chem, 1947. 169(1): p. 77-82.
19. Takabe, K., et al., "Inside-out" signaling of sphingosine-1-phosphate: therapeutic targets.
Pharmacol Rev, 2008. 60(2): p. 181-95.
20. Spiegel, S. and S. Milstien, Sphingosine 1-phosphate, a key cell signaling molecule. J
Biol Chem, 2002. 277(29): p. 25851-4.
21. Spiegel, S. and S. Milstien, Sphingosine-1-phosphate: an enigmatic signalling lipid. Nat
Rev Mol Cell Biol, 2003. 4(5): p. 397-407.
22. Hla, T., Physiological and pathological actions of sphingosine 1-phosphate. Semin Cell
Dev Biol, 2004. 15(5): p. 513-20.
23. Z. Qu, Differential Expression of Sphingosine-1-Phosphate Receptors in Abdominal
Aortic Aneurysms. Mediators of Inflammation, 2012.
24. Lee, M.-J., et al., Vascular Endothelial Cell Adherens Junction Assembly and
Morphogenesis Induced by Sphingosine-1-Phosphate. Cell, 1999. 99(3): p. 301-312.
25. Huang, Y.L., W.P. Huang, and H. Lee, Roles of sphingosine 1-phosphate on
tumorigenesis. World J Biol Chem, 2011. 2(2): p. 25-34.
26. Merrill, A.H., Jr., De novo sphingolipid biosynthesis: a necessary, but dangerous,
pathway. J Biol Chem, 2002. 277(29): p. 25843-6.
27. Hanada, K., et al., CERT-mediated trafficking of ceramide. Biochimica et Biophysica
Acta (BBA) - Molecular and Cell Biology of Lipids, 2009. 1791(7): p. 684-691.
28. Hanada, K., Serine palmitoyltransferase, a key enzyme of sphingolipid metabolism.
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2003.
1632(1–3): p. 16-30.
29. Pitson, S.M., Regulation of sphingosine kinase and sphingolipid signaling. Trends
Biochem Sci, 2011. 36(2): p. 97-107.
30. Chen, L.Y., et al., Cytosolic phospholipase A2alpha activation induced by S1P is
mediated by the S1P3 receptor in lung epithelial cells. Am J Physiol Lung Cell Mol
Physiol, 2008. 295(2): p. L326-35.
68
31. Yokota, S., et al., Asp177 in C4 domain of mouse sphingosine kinase 1a is important for
the sphingosine recognition. FEBS Letters, 2004. 578(1–2): p. 106-110.
32. Orr Gandy, K.A. and L.M. Obeid, Targeting the sphingosine kinase/sphingosine 1-
phosphate pathway in disease: Review of sphingosine kinase inhibitors. Biochim Biophys
Acta, 2013. 1831(1): p. 157-66.
33. Igarashi, J. and T. Michel, Sphingosine-1-phosphate and modulation of vascular tone.
Cardiovasc Res, 2009. 82(2): p. 212-20.
34. Chae, S.S., R.L. Proia, and T. Hla, Constitutive expression of the S1P1 receptor in adult
tissues. Prostaglandins Other Lipid Mediat, 2004. 73(1-2): p. 141-50.
35. Liu, C.H. and T. Hla, The Mouse Gene for the Inducible G-Protein-Coupled
Receptoredg-1. Genomics, 1997. 43(1): p. 15-24.
36. Van Brocklyn, J.R., et al., Sphingosine 1-Phosphate-induced Cell Rounding and Neurite
Retraction Are Mediated by the G Protein-coupled Receptor H218. Journal of Biological
Chemistry, 1999. 274(8): p. 4626-4632.
37. Nixon, G.F., Sphingolipids in inflammation: pathological implications and potential
therapeutic targets. Br J Pharmacol, 2009. 158(4): p. 982-93.
38. Pettus, B.J., et al., The sphingosine kinase 1/sphingosine-1-phosphate pathway mediates
COX-2 induction and PGE2 production in response to TNF-alpha. FASEB J, 2003.
17(11): p. 1411-21.
39. French, K.J., et al., Discovery and evaluation of inhibitors of human sphingosine kinase.
Cancer Res, 2003. 63(18): p. 5962-9.
40. Herzinger, T., et al., Sphingosine-1-phosphate signaling and the skin. Am J Clin
Dermatol, 2007. 8(6): p. 329-36.
41. Pyne, N.J. and S. Pyne, Sphingosine 1-phosphate and cancer. Nature Reviews Cancer,
2010. 10(7): p. 489-503.
42. Sabbadini, R.A., Targeting sphingosine-1-phosphate for cancer therapy. Br J Cancer,
2006. 95(9): p. 1131-5.
43. Maceyka, M., et al., Sphingosine-1-phosphate signaling and its role in disease. Trends
Cell Biol, 2012. 22(1): p. 50-60.
44. Anelli, V., et al., Role of sphingosine kinase-1 in paracrine/transcellular angiogenesis
and lymphangiogenesis in vitro. FASEB J, 2010. 24(8): p. 2727-38.
69
45. Suomalainen, L., et al., Sphingosine-1-Phosphate in Inhibition of Male Germ Cell
Apoptosis in the Human Testis. Journal of Clinical Endocrinology & Metabolism, 2003.
88(11): p. 5572-5579.
46. Otala, M., et al., Protection from Radiation-Induced Male Germ Cell Loss by
Sphingosine-1-Phosphate. Biology of Reproduction, 2004. 70(3): p. 759-767.
47. Pyne, S., R. Bittman, and N.J. Pyne, Sphingosine kinase inhibitors and cancer: seeking
the golden sword of Hercules. Cancer Res, 2011. 71(21): p. 6576-82.
48. O'Brien, N., et al., Production and characterization of monoclonal anti-sphingosine-1-
phosphate antibodies. J Lipid Res, 2009. 50(11): p. 2245-2257.
49. Caballero, S., et al., Anti-sphingosine-1-phosphate monoclonal antibodies inhibit
angiogenesis and sub-retinal fibrosis in a murine model of laser-induced choroidal
neovascularization. Exp Eye Res, 2009. 88(3): p. 367-377.
50. Sabbadini, R.A., Sphingosine-1-phosphate antibodies as potential agents in the treatment
of cancer and age-related macular degeneration. Br J Pharmacol, 2011. 162(6): p. 1225-
1238.
51. Adan-Gokbulut, A., et al., Novel agents targeting bioactive sphingolipids for the
treatment of cancer. Curr Med Chem, 2013. 20(1): p. 108-22.
52. Kono, K., et al., Characterization of B-5354c, a new sphingosine kinase inhibitor,
produced by a marine bacterium. J Antibiot (Tokyo), 2000. 53(8): p. 759-64.
53. Kono, K., et al., S-15183a and b, new sphingosine kinase inhibitors, produced by a
fungus. J Antibiot (Tokyo), 2001. 54(5): p. 415-20.
54. Paugh, S.W., et al., A selective sphingosine kinase 1 inhibitor integrates multiple
molecular therapeutic targets in human leukemia. Blood, 2008. 112(4): p. 1382-1391.
55. French, K.J., et al., Pharmacology and Antitumor Activity of ABC294640, a Selective
Inhibitor of Sphingosine Kinase-2. Journal of Pharmacology and Experimental
Therapeutics, 2010. 333(1): p. 129-139.
56. Beljanski, V., C. Knaak, and C.D. Smith, A novel sphingosine kinase inhibitor induces
autophagy in tumor cells. J Pharmacol Exp Ther, 2010. 333(2): p. 454-64.
57. Schnute, M.E., et al., Modulation of cellular S1P levels with a novel, potent and specific
inhibitor of sphingosine kinase-1. Biochem J, 2012. 444(1): p. 79-88.
58. GRÄLER, M.H. and E.J. GOETZL, The immunosuppressant FTY720 down-regulates
sphingosine 1-phosphate G-protein-coupled receptors. The FASEB Journal, 2004. 18(3):
p. 551-553.
70
59. Noda, H., et al., Fingolimod phosphate promotes the neuroprotective effects of microglia.
J Neuroimmunol, 2013. 256(1–2): p. 13-18.
60. Welch, S.P., L.J. Sim-Selley, and D.E. Selley, Sphingosine-1-phosphate receptors as
emerging targets for treatment of pain. Biochem Pharmacol, 2012. 84(12): p. 1551-1562.
61. Pelletier, D. and D.A. Hafler, Fingolimod for multiple sclerosis. N Engl J Med, 2012.
366(4): p. 339-47.
62. Pchejetski, D., et al., FTY720 (Fingolimod) Sensitizes Prostate Cancer Cells to
Radiotherapy by Inhibition of Sphingosine Kinase-1. Cancer Res, 2010. 70(21): p. 8651-
8661.
63. Zhang, N., et al., FTY720 induces necrotic cell death and autophagy in ovarian cancer
cells: a protective role of autophagy. Autophagy, 2010. 6(8): p. 1157-67.
64. Parrill, A.L., S. Lima, and S. Spiegel, Structure of the first sphingosine 1-phosphate
receptor. Sci Signal, 2012. 5(225): p. pe23.
65. Hanson, M.A., et al., Crystal structure of a lipid G protein-coupled receptor. Science,
2012. 335(6070): p. 851-5.
66. Delgado, A., et al., Chapter Eight - Natural Products as Platforms for the Design of
Sphingolipid-Related Anticancer Agents, in Advances in Cancer Research, S.N. James,
Editor 2013, Academic Press. p. 237-281.
67. Yoda, H., T. Oguchi, and K. Takabe, Novel asymmetric synthesis of penaresidin B as a
potent actomyosin ATPase activator. Tetrahedron Letters, 1997. 38(18): p. 3283-3284.
68. Yajima, A., H. Takikawa, and K. Mori, Synthesis of Sphingosine Relatives, XVIII!.
Synthesis of Penazetidine A, an Alkaloid Inhibitor of Protein Kinase C Isolated from the
Marine Sponge Penares sollasi. Liebigs Annalen, 1996. 1996(7): p. 1083-1089.
69. Alvi, K.A., et al., Penazetidine A, an alkaloid inhibitor of protein kinase C. Bioorg Med
Chem Lett, 1994. 4(20): p. 2447-2450.
70. Raghavan, S. and V. Krishnaiah, An Efficient Stereoselective Synthesis of Penaresidin A
from (E)-2-Protected Amino-3,4-unsaturated Sulfoxide. J Org Chem, 2009. 75(3): p. 748-
761.
71. Alvarez-Builla, J. Vol. Weinheim Germany. 2011: Wiley-VCH Verlag & Co.
72. Huszthy, P., et al., Efficient synthesis of azetidine through N-trityl- or N-
dimethoxytritylazetidines starting from 3-amino-l-propanol or 3-halopropylamine
hydrohalides. Journal of Heterocyclic Chemistry, 1993. 30(5): p. 1197-1207.
71
73. Kürti, L. and B. Czakó, Strategic applications of named reactions in organic synthesis :
background and detailed mechanisms2005, Amsterdam ; Boston: Elsevier Academic
Press. lii, 758 p.
74. Edder, C., et al., Unusual Electronic Effects of Electron-Withdrawing Sulfonamide
Groups in Optically and Magnetically Active Self-Assembled Noncovalent
Heterodimetallic d−f Podates. Inorg Chem, 2000. 39(22): p. 5059-5073.
75. Feng, X., et al., Efficient synthesis of chiral β-and γ-N-tosylaminoalcohols from 1-aryl-2-
aminopropane-1,3-diols. Russian Journal of Organic Chemistry, 2006. 42(4): p. 496-500.
76. Ghorai, M.K., A. Kumar, and K. Das, Lewis Acid-Mediated Unprecedented Ring-
Opening Rearrangement of 2-Aryl-N-tosylazetidines to Enantiopure (E)-Allylamines. Org
Lett, 2007. 9(26): p. 5441-5444.
77. Hirose, T., et al., Total Synthesis and Determination of the Absolute Configuration of
Guadinomines B and C2. Chemistry – A European Journal, 2008. 14(27): p. 8220-8238.
78. Bach, R.D. and O. Dmitrenko, Spiro versus Planar Transition Structures in the
Epoxidation of Simple Alkenes. A Reassessment of the Level of Theory Required. The
Journal of Physical Chemistry A, 2003. 107(21): p. 4300-4306.
79. Kimura, T., et al., Preparation of morpholine type cinnamides as amyloid-β production
inhibitors, 2007, Eisai R&D Management Co., Ltd., Japan . p. 87pp.
80. Martinelli, M.J., et al., Dibutyltin Oxide Catalyzed Selective Sulfonylation of α-
Chelatable Primary Alcohols. Org Lett, 1999. 1(3): p. 447-450.
81. Martinelli, M.J., R. Vaidyanathan *, and V. Van Khau, Selective monosulfonylation of
internal 1,2-diols catalyzed by di-n-butyltin oxide. Tetrahedron Letters, 2000. 41(20): p.
3773-3776.
82. Sakaitani, M. and Y. Ohfune, Syntheses and reactions of silyl carbamates. 1.
Chemoselective transformation of amino protecting groups via tert-butyldimethylsilyl
carbamates. J Org Chem, 1990. 55(3): p. 870-876.
83. Sakaitani, M. and Y. Ohfune, A new mode of cyclic carbamate formation via tert-
butyldimethylsilyl carbamate. Stereoselective syntheses of statine and its analogue.
Tetrahedron Letters, 1987. 28(34): p. 3987-3990.
84. Sakaitani, M. and Y. Ohfune, Syntheses and reactions of silyl carbamates. 2. A new mode
of cyclic carbamate formation from tert-butyldimethylsilyl carbamate. J Am Chem Soc,
1990. 112(3): p. 1150-1158.
72
85. Pastó, M., et al., Synthesis of enantiopure amino alcohols by ring-opening of
epoxyalcohols and epoxyethers with ammonia. Tetrahedron Letters, 2003. 44(46): p.
8369-8372.
86. Bergmeier, S.C., The Synthesis of Vicinal Amino Alcohols. Tetrahedron, 2000. 56(17): p.
2561-2576.
87. Medina, E., et al., Enantioselective synthesis of N-Boc-1-naphthylglycine. Tetrahedron:
Asymmetry, 1997. 8(10): p. 1581-1586.
88. Kamal, A., et al., Base-free monosulfonylation of amines using tosyl or mesyl chloride in
water. Tetrahedron Letters, 2008. 49(2): p. 348-353.
89. Kiren, S. and L.J. Williams, Configuration of the psymberin amide side chain. Org Lett,
2005. 7(14): p. 2905-8.
90. David Crouch, R., Selective monodeprotection of bis-silyl ethers. Tetrahedron, 2004.
60(28): p. 5833-5871.
91. Frank, S.A. and W.R. Roush, Studies on the Synthesis of (−)-Spinosyn A: Application of
the Steric Directing Group Strategy to Transannular Diels−Alder Reactions. J Org
Chem, 2002. 67(12): p. 4316-4324.
92. Pandey, S.K., A. Bisai, and V.K. Singh, Activation of DMSO by Phosphonitrilic
Chloride: An Efficient Method for Oxidation of Alcohols. Synthetic Communications,
2007. 37(23): p. 4099-4103.
93. Mancuso, A.J., S.-L. Huang, and D. Swern, Oxidation of long-chain and related alcohols
to carbonyls by dimethyl sulfoxide "activated" by oxalyl chloride. J Org Chem, 1978.
43(12): p. 2480-2482.
94. Parikh, J.R. and W.v.E. Doering, Sulfur trioxide in the oxidation of alcohols by dimethyl
sulfoxide. J Am Chem Soc, 1967. 89(21): p. 5505-5507.
95. Saito, T., H. Fuwa, and M. Sasaki, Synthetic studies on goniodomin A: convergent
assembly of the C15–C36 segment via palladium-catalyzed organostannane–thioester
coupling. Tetrahedron, 2011. 67(2): p. 429-445.
96. Frigerio, M., M. Santagostino, and S. Sputore, A User-Friendly Entry to 2-Iodoxybenzoic
Acid (IBX). J Org Chem, 1999. 64(12): p. 4537-4538.
97. Felkin, H. and G. Swierczewski, Activation of Grignard reagents by transition metal
compounds. Tetrahedron, 1975. 31(22): p. 2735-2748.
73
98. Chattopadhyay, A., (R)-2,3-O-Cyclohexylideneglyceraldehyde, a Versatile Intermediate
for Asymmetric Synthesis of Homoallyl and Homopropargyl Alcohols in Aqueous
Medium. J Org Chem, 1996. 61(18): p. 6104-6107.
99. Li, C.J., Organic reactions in aqueous media with a focus on carbon-carbon bond
formations: a decade update. Chem Rev, 2005. 105(8): p. 3095-165.
100. But, T.Y.S. and P.H. Toy, The Mitsunobu Reaction: Origin, Mechanism, Improvements,
and Applications. Chemistry – An Asian Journal, 2007. 2(11): p. 1340-1355.
101. Swamy, K.C.K., et al., Mitsunobu and Related Reactions: Advances and Applications.
Chem Rev, 2009. 109(6): p. 2551-2651.
102. Mitsunobu, O. and M. Yamada, Preparation of Esters of Carboxylic and Phosphoric
Acid <I>via</I> Quaternary Phosphonium Salts. Bulletin of the Chemical Society of
Japan, 1967. 40(10): p. 2380-2382.
103. Pinto, D.C.G.A., Chapter eight - Advanced NMR techniques for structural
characterization of heterocyclic structures,. Recent Research Developments in
Heterocyclic Chemistry, 2007.
104. Salgado, A., et al., Synthesis of 4-alkyl-1-benzhydryl-2-(methoxymethyl)azetidin-3-ols by
regio- and stereoselective alkylation of 1-benzhydryl-3-(N-alkyl)imino-2-
(methoxymethyl)azetidine. Tetrahedron, 2002. 58(14): p. 2763-2775.
105. Love, J.A., et al., A Practical and Highly Active Ruthenium-Based Catalyst that Effects
the Cross Metathesis of Acrylonitrile. Angewandte Chemie International Edition, 2002.
41(21): p. 4035-4037.
106. Xue, Z. and M.F. Mayer, Entropy-driven ring-opening olefin metathesis polymerizations
of macrocycles. Soft Matter, 2009. 5(23): p. 4600-4611.
107. Katsuki, T. and K.B. Sharpless, The first practical method for asymmetric epoxidation. J
Am Chem Soc, 1980. 102(18): p. 5974-5976.
108. Martin, V.S., et al., Kinetic resolution of racemic allylic alcohols by enantioselective
epoxidation. A route to substances of absolute enantiomeric purity? J Am Chem Soc,
1981. 103(20): p. 6237-6240.
109. Balamurugan, R., R.B. Kothapalli, and G.K. Thota, Gold-Catalysed Activation of
Epoxides: Application in the Synthesis of Bicyclic Ketals. European Journal of Organic
Chemistry, 2011. 2011(8): p. 1557-1569.
110. Venkatesan, K., Srinivasan K. , A stereoselective synthesis of (S)-dapoxetine starting
from trans-cinnamyl alcohol. ARKIVOC, 2008. (xvi): p. 302-310.
74
111. Badorrey, R., M.D. Díaz-de-Villegas, and J.A. Gálvez, Expedient asymmetric synthesis of
(2S,3S)-Boc-phenylalanine epoxide, a key intermediate for the synthesis of biologically
active compounds. Tetrahedron: Asymmetry, 2009. 20(19): p. 2226-2229.
112. Lebar, M.D. and B.J. Baker, Synthesis of the C3–14 fragment of palmerolide A using a
chiral pool based strategy. Tetrahedron, 2010. 66(8): p. 1557-1562.
113. Kamioka, S., et al., Chiral Tetraazamacrocycles Having Four Pendant-Arms. Org Lett,
2009. 11(11): p. 2289-2292.
114. Liu, W.-B., et al., Asymmetric N-Allylation of Indoles Through the Iridium-Catalyzed
Allylic Alkylation/Oxidation of Indolines. Angewandte Chemie International Edition,
2012. 51(21): p. 5183-5187.
115. Sheddan, N.A. and J. Mulzer, Cross Metathesis as a General Strategy for the Synthesis of
Prostacyclin and Prostaglandin Analogues. Org Lett, 2006. 8(14): p. 3101-3104.
116. Wang, Y. and W.-M. Dai, Total synthesis of diastereomeric marine butenolides
possessing a syn-aldol subunit at C10 and C11 and the related C11-ketone. Tetrahedron,
2010. 66(1): p. 187-196.
117. Burtoloso, A.C.B. and C.R.D. Correia, Asymmetric synthesis of cis-2,4-disubstituted
azetidin-3-ones from metal carbene chemistry. Journal of Organometallic Chemistry,
2005. 690(24–25): p. 5636-5646.
75
APPENDIX
SPECTRAL DATA
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81
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83
84
85
86
87
88
89
90
91
92
93
94
95
96
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105
106
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108
109
110
111
112
113
114
115
116
117
118
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126
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138
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.