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Design and synthesis of novel anti-inflammatory lipid mediators and anticancer small molecules

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Design and synthesis of novel anti-inflammatory lipid mediators and anticancer small molecules

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DESIGN AND SYNTHESIS OF NOVEL ANTI-INFLAMMATORY
LIPID MEDIATORS AND ANTICANCER SMALL MOLECULES

by

Jasim Uddin

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMISTRY)

May 2008

Copyright 2008 Jasim Uddin
ii

Dedication

TO
My parents for their unconditional love and care
My wife Srabon for her endless love, support, devotion and sacrifice
My daughter Shanaya for being such joy to our life
My brothers and sisters, and my nephew Rony for their love
iii
Acknowledgements

First and foremost, I would like to thank my advisor Professor Nicos Petasis for
giving me the opportunity to work under his tutelage, and for all the education,
guidance, and encouragement I have received during my time as a student in his group.
Professor Petasis is a brilliant organic chemist, great teacher and a nice person whom I
have learned a great deal, and for all that I will be forever grateful.
I would specially like to thank Professor G. K. Surya Prakash for being so wonderful
and supportive of me from the very first day of my life at USC. I will forever
remember him not only as a great scientist, but also as a wonderful human being who
taught me many aspect of life, which I will carry in the rest of my life.
I would like to thank my dissertation committee members, Professor Axel H.
Schönthal, Professor Robert Bau, and Professor Roy A. Periana for their helpful
suggestions and discussions, especially to Professor Schönthal for his absolute patience
and compassion to teach me cancer cell biology. Special thanks to Professor Golam
Rasul for his constant support, encouragement and help for the last five years.
I would now specially like to thank my wife Srabon. No person has meant more to
me during my stay at USC than my wife. I became father of a daughter—Shanaya, both
my wife and Shanaya have been such a joy and wonderful part of my life, and every
moment with them makes me a better person. I will forever remember her unwavering
love, suffering, constant sacrifice and support for me even at the hardest period of her
life.
iv
I must thank the past and present members of Petasis group for being a second
family. I have learned a lot from them. I personally thank Rong, Jeff, Brad, Raquel,
Fotini, Petros, Wei, Kalyan, Gosia, Jeremy, Kevin, Charles-1 and Charles-2 for their
help and friendship during the last five years.
I gratefully acknowledge and specially thank Panja, Thomas and Sujit for all their
help, support and friendship. Anytime I needed help with anything, they were right
there for me, I will be forever grateful to them. I would like to thank Adel Kardosh from
Dr. Schönthal group for his thoughtful discussion and friendship.
I would specially like to thank Carole for being so nice, and helpful for the entire
period of my time here at Loker Institute. I would also like to thank Jessy, David,
Michele and Heather for their kind support. Jim and Allan Kershaw are also
acknowledged for the technical help with NMR and making useful glass-ware.
My life in the chemistry department was fun. I don’t think I would have been as
happy at USC if it wasn’t for the friends outside the lab. Special thanks to Shamol,
Mong, Greg, Jessie, Tissa, Rehana, CJ, Kaitlin, Mark, Brian, Larryn, Somesh, Kenney,
Bill, Steve, Ying, Mirandah, Sayantan and many more to mention. I will always
treasure their friendship, and all the great times we had together, especially watching
the USC football games at Coliseum.
Last but not the least—I convey my deep appreciation to my parents, my brothers
and sisters, my niece and nephews back home in Bangladesh for their unconditional
love, strong support, and encouragement. Being thousands of miles away from them, it
is hard, but I feel their love and support in every single moment.
v
To everyone else that I haven’t mention, thanks for making my life at USC so
wonderful and enjoyable. I wish everyone nothing but the best.

vi
Table of Contents

Dedication ii
Acknowledgements iii
List of Figures x
List of Schemes xiii
Abstract xv
Chapter 1: Endogenous Lipid Mediators Derived from EPA and DHA:
Identification, Biosynthesis and Biological Activities 1
1.1 Introduction 1
1.2 Structures of Lipid Mediators Derived from EPA 7
1.3 Biosynthesis of EPA-derived Lipid Mediators 8
1.4 Structures of DHA-derived Resolvins, Aspirin-Triggered Resolvins and
Neuroprotectins 10
1.5 Biosynthesis of DHA-derived Resolvins and Neuroprotectins 11
1.5.1 Biosynthesis of 17R-series Resolvins (AT-RvD1 - AT-RvD4) 12
1.5.2 Biosynthesis of 17S-series Resolvins (RvD1 - RvD4) and Neuroprotectin
(NPD1)
17,
13
1.6 Biological Activities of Resolvins and Neuroprotectin D1 in Animal Disease
Models and their Formation in Cells, Tissues and Organs 15
Table 1. Lipid Mediators, their Bioactions and sites of Formation 17
1.7 Conclusion 18
Chapter 2. First Total Synthesis of Resolvin E1 (RvE1) 19
2.1 Introduction 19
2.2 Results and Discussion 20
2.2.1 Synthetic Strategy for RvE1 20
2.2.2 Synthesis of Terminal Alkyne Intermediate (2.2) 22
2.2.3 Synthesis of Middle Vinyl Bromide (2.3) 23
2.2.4 Synthesis of Bottom Vinyl Iodide Intermediate (2.4). 25
2.2.5 Synthesis of top bis-alkyne intermediate (2.21) 27
2.2.6 Final Assembly of the Resolvin E1 (RvE1) Methyl Ester (2.1) 28
2.3 Conclusion. 31
2.4 Experimental 33

vii
Chapter 3. Resolvin D1 and Aspirin-Triggered Resolvin D1: Total Synthesis,
Stereochemical Assignments and Biological Actions 69
3.1 Introduction 69
3.2 Results and Discussion 70
3.2.1 First Total Synthesis of RvD1 70
3.2.1.1 Retrosynthetic Analysis of RvD1 (3.1) 70
3.2.1.2 Acetonation of 2-deoxy-D-ribose (3.3) 72
3.2.1.4 Synthesis of (2E, 5-trimethylsilyl-2-penten-4-ynyl-)
triphenylphosphonium bromide (3.4) 73
3.2.1.5 Synthesis of 3S, 1E, 5Z, 3-hydroxy-1-iodoocta-1,5-diene (3.5) 74
3.2.1.6 Assembly of three modules (3.2, 3.3 and 3.4) by Wittig reaction
for the synthesis of top alkyne intermediate (3.20) 76
3.2.1.7 Final assembly of RvD1 methyl ester (3.1) 80
3.2.2 First Total Synthesis of Aspirin-Triggered RvD1 (AT-RvD1) 83
3.3 Stereochemical Assignments of RvD1 and AT-RvD1 86
3.4 Biological Activities of RvD1 and AT-RvD1 91
3.5 Conclusion 92
3.6 Experimental for RvD1 and AT-RvD1 94
Chapter 4. Total Synthesis of Resolvin D2 (7S-RvD2) and its 7R-epimer
(7R-RvD2) 139
4.1 Introduction 139
4.2 Results and Discussion 140
4.2.1 Total synthesis of 7R-resolvin D2 (7R-RvD2) and 7S-resolvin
D2 (7S-RvD2) 140
4.2.1.1 Retrosynthetic analysis 141
4.2.1.2 Synthesis of common terminal alkyne (4.8) for both 7R-RvD2
and 7S-RvD2 143
4.2.1.3 Synthesis of 7R-vinyl iodide (4.3) for 7R-RvD2 145
4.2.1.4 Synthesis of 7S-vinyl iodide (4.3') for 7S-RvD2 147
4.2.1.5 Final assembly of 7R-RvD2 and 7S-RvD2 methyl ester 148
4.3 Conclusion 150
4.4 Experimental 152
Chapter 5. Synthetic Efforts towards the First Total Synthesis of Resolvin D3
(RvD3) and Aspirin-Triggered Resolvin D3 (AT-RvD3) 184
5.1 Introduction 184
5.2 Results and Discussion 186
5.2.1 First Total Synthesis of RvD3 and AT-RvD3 186
5.3 Conclusion 193
5.4 Experimental 194

viii
Chapter 6. First Total Synthesis of Resolvin D4 (RvD4) and Aspirin-Triggered
Resolvin D4 (AT-RvD4) 208
6.1 Introduction 208
6.2 Results and Discussion 209
6.2.1 Retrosynthetic Analysis of RvD4 and AT-RvD4 209
6.2.2 Synthesis of aldehyde 6.5 211
6.2.3 Wittig reaction between the aldehyde and the Wittig reagent 214
6.2.4 Coupling of propargyl bromide and terminal alkyne 215
6.2.5 Final assembly of the RvD4 and AT-RvD4 by Sonogashira coupling 216
6.2.6 Revised synthetic strategy for RvD4 and AT-RvD4 218
6.3 Conclusion 220
6.4 Experimental 221
Chapter 7. Metabolic Inactivation of Lipoxins and Resolvins and Design &
Synthesis of their Biostable Analogs 241
7.1 Introduction 241
7.2 Metabolic inactivation lipoxins and design and synthesis of biostable lipoxin
analogs 242
7.2.1 Metabolic inactivation pathways of lipoxins 242
7.2.2 Design of Lipoxin analogs 244
7.2.3 Synthesis of p-F-phenoxy-15-epi-Lipoxin (ATLa) 246
7.3 Metabolic Inactivation of Resolvin E1 (RvE1) and Synthesis of Biostable
p-F-phenoxy-RvE1 Analog 251
7.3.1 Metabolic Inactivation Pathways of RvE1 251
7.3.2 Synthesis of p-F-phenoxy-RvE1 analog to prevent metabolic
inactivation 256
7.3.3 Biological Activities of p-F-phenoxy-RvE1 Analog 258
7.4 Metabolic Inactivation of Resolvin D1 (RvD1) and AT-RvD1 and Synthesis
of their Biostable Analogs 260
7.4.1 Metabolic inactivation pathways of RvD1 and AT-RvD1 260
7.4.2 Bioactions of RvD1 metabolites 264
7.4.3 Design and synthesis of biostable AT-RvD1 analogs 266
7.5 Conclusion 270
7.6 Experimental 272

ix
Chapter 8. Design and Synthesis of Anticancer Small Molecules 309
8 Introduction 309
8.1 Apoptosis 309
8.2 Regulations of Cell Cycle 311
8.3 Cycloxygenase 2 (COX-2) and Cancer 314
8.4 COX-2 Inhibitors as Anticancer Agents 315
8.5 Design and Synthesis of Celebrex Analogs 316
8.6 Design and Synthesis of Celebrex, DMC and Vioxx Analogs 319
8.7 Anticancer Activities of Celecoxib and DMC 325
8.7.1 COX-2 independent anti-tumor activity of celecoxib on Burkitt’s
lymphoma 325
8.7.2 DMC as a promising drug for multiple myeloma 329
10.8 Conclusion 329
8.9 Experimental 331
Bibliography 341

x
List of Figures

Figure 1. Structures of EPA derived lipid mediators. ...................................................... 8
Figure 2. Biosynthetic pathways of E-series of resolvin and epi-LXA
5
. ......................... 9
Figure 3. The main group of lipid mediators derived from DHA and their aspirin-
triggered epimers. ............................................................................................ 11

Figure 4. Proposed biosynthesis of aspirin-triggered (AT-Rv) D series of resolvins. ... 13
Figure 5. Biosynthetic pathways of 17S-D series of resolvins....................................... 14
Figure 6. Biosynthesis of neuroprotectin D1 and related compounds............................ 15
Figure 7. Retrosynthetic analysis of RvE1 methyl ester (2.1)........................................ 21
Figure 8. Retrosynthetic Analysis of Resolvin D1 (RvD1)............................................ 71
Figure 9. Decoupled
1
H-NMR spectrum (expanded) of 3.16 (DMSO-d
6
, 400 MHz).... 77
Figure 10. Retrosynthetic analysis of AT-RvD1 methyl ester (3.1') ............................. 83
Figure 11. Structures of RvD1 and AT-RvD1 methyl esters. ........................................ 86
Figure 12.
1
H-NMR assignments of RvD1 and AT-RvD1 methyl ester based on the
analysis of COSY spectra. The NMR spectrum of olefinic region of
RvD1 methyl ester...................................................................................... 88

Figure 13. HPLC chromatogram at UV absorbance 301 nm, and MS/MS analysis of
synthetic RvD1, AT-RvD1 and biogenic RvD1............................................................. 89
Figure 14. Comparison of enzymatic RvD1 obtained via LOX-catalyzed synthesis.... 90
Figure 15. Anti-inflammatory actions of RvD1 and AT-RvD1. .................................... 91
Figure 16. Structures of 7R-RvD2 (4.1) and 7S-RvD2 (4.1')....................................... 140
Figure 17. Retrosynthetic analysis of 7R-RvD2 (4.1) methyl ester. ............................ 141
Figure 18. Structures of RvE1, RvD3 and AT-RvD3. ................................................. 186
xi
Figure 19. Retrosynthetic analysis of RvD3 and AT-RvD3......................................... 188
Figure 20. Structures of RvD4 (6.1) and AT-RvD4 (6.1'). .......................................... 209
Figure 21. First retrosynthetic analysis of RvD4 and AT-RvD4.................................. 211
Figure 22. Revised retrosynthetic strategy for RvD4 and AT-RvD4........................... 218
Figure 23. Enzymatic oxido/reductase mediated inactivation of lipoxins and related
lipid mediators............................................................................................ 242

Figure 24. Metabolic inactivation of LXA
4
and LTB
4
................................................. 243
Figure 25. Structures of lipoxins analogs with the modifications in the omega-end. .. 245
Figure 26. Design of Lipoxin analogs .......................................................................... 245
Figure 27. Retrosynthetic analysis of p-F-Phenoxy-epi-LXA
4
. ................................... 246
Figure 28. Reaction progress monitored by UV at 340 nm.......................................... 252
Figure 29. LC-ESI-MS/MS chromatograms and spectra of RvE1............................... 253
Figure 30. GC-MS spectrum of the derivatized methoximation product of 18-oxo-
RvE1........................................................................................................... 254

Figure 31. RvE1 metabolism in cells and tissues......................................................... 255
Figure 32. Biological activities of major RvE1 metabolite, 18-oxo-RvE1. ................. 256
Figure 33.
1
H-NMR spectrum (panel A), and bioconversion rate of RvE1 analog...... 259
Figure 34. Conversion of RvD1, AT-RvD1 and LXA4 by 15-PGDH......................... 261
Figure 35. Analysis of RvD1 metabolites. ................................................................... 262
Figure 36. RvD1 metabolism in murine lung............................................................... 264
Figure 37. Bioactions of RvD1 and its metabolites 17-oxo-RvD1 .............................. 265
Figure 38. Retrosynthetic analysis of AT-RvD1 analogs............................................. 267
Figure 39. Regulation of cell cycle............................................................................... 313
xii
Figure 40. Structure of celecoxib and DMC. ............................................................... 317
Figure 41. Structures of Modified Drugs. .................................................................... 320
Figure 42. Cell proliferation in the presence of celecoxib and DMC on Raji and
Ramos cell lines.......................................................................................... 326

Figure 43. Tumor formation in animals treated with celecoxib or DMC..................... 327
Figure 44. Cyclin A and cyclin B expression in tumor tissue in vivo. ......................... 328

xiii
List of Schemes

Scheme 1. Synthesis of top terminal alkyne (2.2) for RvE1. ......................................... 23
Scheme 2. Synthesis of middle vinyl bromide (2.3) for RvE1....................................... 24
Scheme 3. Synthesis of bottom vinyl iodide (2.4) for RvE1.......................................... 26
Scheme 4. Synthesis of bis-alkyne intermediate (2.21) for RvE1.................................. 28
Scheme 5. Final assembly of resolvin E1 (RvE1) methyl ester (2.1)............................. 29
Scheme 6. Synthesized RvE1 methyl ester analogs. ...................................................... 31
Scheme 7. Acetonation of 2-deoxy-D-ribose. ................................................................ 73
Scheme 8. Synthesis of Wittig salt 3.2........................................................................... 73
Scheme 9. Synthesis of Wittig salt 3.4........................................................................... 74
Scheme 10. Synthesis of vinyl iodide (3.5) of RvD1. .................................................... 75
Scheme 11. Synthesis of the top terminal alkyne intermediate 3.20.............................. 79
Scheme 12. Final assembly of resolvin D1 (RvD1) methyl ester (3.1).......................... 81
Scheme 13. Synthesis of vinyl iodide 3.5' for AT-RvD1............................................... 84
Scheme 14. Final coupling reaction for AT-RvD1 methyl ester (3.1').......................... 85
Scheme 15. Synthesis of bottom terminal alkyne 4.8 for both 7R-RvD2 and
7S-RvD2. .................................................................................................... 143

Scheme 16. Synthesis of 7R-vinyl iodide (4.3) for 7R-RvD2. ..................................... 146
Scheme 17. Synthesis of 7S-vinyl iodide (4.3') for 7S-RvD2...................................... 148
Scheme 18. Final assembly of 7R-RvD2 (4.1) and 7S-RvD2 (4.1')............................. 149
Scheme 19. Synthesis of top terminal alkyne (5.2). ..................................................... 190
Scheme 20. Synthesis of bis-alkyne intermediate (5.11) for RvD3 and AT-RvD3. .... 191
xiv
Scheme 21. Synthetic steps for RvD3 and AT-RvD3 are yet to be done..................... 193
Scheme 22. Synthesis of aldehyde 6.5 for RvD4 and AT-RvD4. ................................ 212
Scheme 23. Synthesis of terminal alkyne 6.3 for RvD4 and AT-RvD4....................... 214
Scheme 24. Synthesis of bis-alkyne intermediate 6.2. ................................................. 215
Scheme 25. Final Sonogashira coupling of vinyl iodides and terminal alkyne............ 217
Scheme 26. Synthesis of propargyl bromide for RvD4................................................ 219
Scheme 27. Final assembly of RvD4 and AT-RvD4 which are yet to be done. .......... 220
Scheme 28. Synthesis of p-F-phenoxy-vinyl iodide (7.3)............................................ 247
Scheme 29. Final assembly of p-F-phenoxy-LXA
4
methyl ester (7.1). ....................... 250
Scheme 30. Final assembly of p-F-Phenoxy-RvE1 (7.14) analog. .............................. 257
Scheme 31. Synthesis of racemic vinyl bromide for 17R/S-RvD1. ............................. 268
Scheme 32. Final assembly of AT-RvD1 analogs........................................................ 270
Scheme 33. Synthesis of 2,5-dimethyl celecoxib (DMC). ........................................... 319
Scheme 34. Synthesis of modified celecoxib (8.8). ..................................................... 322
Scheme 35. Synthesis of modified DMC. .................................................................... 323
Scheme 37. Synthesis of modified Vioxx (8.16).......................................................... 324

xv
Abstract

This dissertation deals with two distinct projects. First—Design and synthesis of novel
anti-inflammatory lipid mediators—It is well documented that omega-3
polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA) display beneficial actions in many human diseases. The
underlying molecular mechanism for these actions remains of tremendous interest, but
yet to be established until recently Serhan and colleagues identified a novel class of
lipid mediators generated from EPA and DHA during the resolution phase of
inflammation via cell-cell interactions that displayed potent anti-inflammatory, pro-
resolving activities. The lipid mediators derived from EPA are designated as E-series of
resolvins such as RvE1, and the lipid mediators generated from DHA are denoted as D-
series of resolvins such as RvD1, RvD2, RvD3 and RvD4. In addition, aspirin triggers
the endogenous formation of epimeric series of D-series of resolvins named aspirin-
triggered resolvins such as AT-RvD1, AT-RvD2, AT-RvD3 and AT-RvD4. These lipid
mediators are generated in very minute quantities in stereochemically pure form and
possess potent anti-inflammatory, pro-resolving bioactions. The first part of this
dissertation deals with the first asymmetric total syntheses of these lipid mediators for
their complete stereochemical assignments, and for further biological studies. The first
asymmetric total syntheses of RvE1, RvD1, AT-RvD1, 7S-RvD2, 7R-RvD2 were
accomplished, and efforts towards the total syntheses of RvD3, AT-RvD3, RvD4 and
AT-RvD4 described. A highly convergent and practical approach heavily relied on
Pd
0
/Cu
I
medicated cross-coupling, Wittig, and modified Wittig reactions were
xvi
employed to embark these molecules in their stereochemically pure form. The absolute
stereochemical assignments of RvE1, RvD1 and AT-RvD1 were established by
matching the physical and biological properties of stereochemically pure synthetic one
with their biogenic counterparts. The metabolic inactivation pathways of RvE1, RvD1
and AT-RvD1 were also investigated, and described here in this dissertation. The
elucidation of the biochemical inactivation pathways of these lipid mediators including
their arachidonic acid derived cousins lipoxins provided the rationale for the design of
their biostable analogs. A number of analogs have been designed, and synthesized, and
the designed biostable analogs were found to be superior to their natural counterparts.
Our efforts provided the basis for the development of potential new therapeutics for the
treatment of inflammation and inflammation associated diseases.
Second—Design and synthesis of anticancer small molecules—Celecoxib (Celebrex®)
is a non-steroidal anti-inflammatory drug (NSAID), which is widely used for the
treatment of patients with arthritis and osteoarthritis. It is unique among other COX-2
inhibitors because of its ability to induce cancer cell death. Despite promising results,
the underlying molecular mechanisms for its anticancer properties are poorly
understood, and somewhat controversial. To evaluate any apparent involvement of
COX-2 for its anticancer activity, we have designed a close structural analog of
celecoxib, named 2,5-dimethyl celecoxib (DMC), which is not a COX-2 inhibitor. The
synthesis of DMC and its structural analogs were discussed in this dissertation. Our
collaborator performed a series of experiments both in vivo and in vitro, and found that
DMC potently mimics the all anti-tumor activities of celecoxib and finally came to a
xvii
conclusion that celecoxib does its anti-proliferative and pro-apoptotic effects without
any apparent involvement of COX-2.
1
Chapter 1: Endogenous Lipid Mediators Derived from EPA
and DHA: Identification, Biosynthesis and Biological
Activities

1.1 Introduction

Beneficial effects of omega-3 polyunsaturated fatty acids (PUFAs), such as
docosahexanoic acid (DHA) and eicosapentaenoic acid (EPA), in human health and
diseases have been suggested for over a half of a century ago. In 1929-1930, a
systematic study by George O. Burr and Mildred M. Burr
1
first demonstrated in
laboratory rats that exclusion of fat-free diets resulted the failure of multiple organs, and
ultimately caused the premature death. Although the importance of PUFAs were first
recognized by Burr and Burr, the major interest in omega-3 PUFAs followed the
brilliant work done by Bang and Dyerberg. In 1971, they reported
2
that Greenland
Eskimos had an exceptionally low incidence of heart disease and arthritis despite the
fact that they consumed a high-fat diet. Their extensive follow-up studies soon
uncovered
3
that the low rates of heart disease of Greenland Eskimos were associated
with their higher consumption of whale, seal and fish, which are rich in omega-3
PUFAs such as DHA and EPA. Since then, recognizing the unique benefits of DHA

1
(i) Burr, G. O.; Burr, M. M. J. Biol. Chem. 1929, 82, 345. (ii) Burr G.O.; Burr, M. M. J. Biol. Chem.
1930, 86, 587.
2
Bang, H. O.; Dyerberg, J.; Nielsew, A. B. Lancet 1971, 1, 1143.
3
(i) Bang, H. O.; Dyerberg, J. Acta. Med. Scand. 1972, 192, 85; (ii) Dyerberg, J.; Bang, H. O.; Hjorne, N.
Am. J. Clin. Nutr .1975, 28, 958; (iii) Bang, H. O.; Dyerberg, J.; Hjorne, N. Acta. Med. Scand. 1976, 200,
69; (iv) Dyerberg, J.; Bang, H. O.; Stofferson, E. Lancet 1978, 2, 117; (v) Bang, H. O.; Dyerberg, J.;
Sinclair, H. M. J. Am. Clin. Nutr. 1980, 33, 2657.
2
and EPA, extensive studies have been done for several decades implicates DHA and
EPA as beneficial in many human diseases including cardiovascular disease,
arthrosclerosis, asthma and cancer.
4
Several other studies on human demonstrated that
omega-3 dietary supplementation considerably reduced the sudden cardiac death,
5

coronary heart disease and Crohn’s disease.
6
Realizing the wide range of unique of
benefits of EPA and DHA and serious consequences of their deficiencies, the National
Heart Association (see http://www.american-heart.org) and US National Institutes of
Health (see http://www.nih.gov) recently published recommended daily intakes of
omega-3 polyunsaturated fatty acids. Since the early studies on lipids and lipids derived
mediators, it became conclusive that the local bioactive lipid mediators play a crucial
role for the regulation of various intracellular and intercellular functions, including
inflammation.
7
Inflammation and the inflammatory response are the part of the body’s
normal, innate immune response to infection and injury. In normal physiological
conditions, inflammation functions to begin the immunological process of the clearance
of invading toxins and pathogens, and to resolve the damaged tissue to the non-
inflammed state to maintain the tissue homeostasis.
8
Complete resolution of an acute
inflammatory response and its return to homeostasis are essential for healthy tissues.

4
(i) Calder, P. C. Am. J. Clin. Nutr. 2006, 83, 1505. (ii) Calder, P. C. Lipids 2001, 36, 1007.
5
Albert, C. M.; Campos, H.; Stampfer, M. J.; Ridker, P. M.; Manson, J. E.; Willet, W. C.; Ma, J. N. Engl.
J. Med. 2002, 346, 1113.
6
Belluzi, A.; Brignola, C.; Capieri, M.; Pera, A.; Boschi, S.; Miglioli, M. N. Engl. J. Med. 1996, 334,
1557.
7
Gallin, J. I.; Snyderman, R.; Fearon, D. T.; Haynes, B. F.; and Nathan, C. (eds), 1999, Inflammation:
Basic Principles and Clinical Correlates, 3
rd
Ed., Pippincott Williams & Wilkins, Philadelphia.
8
Majno, G.; Joris, I. 2004, Cells, Tissues, and Disease: Principles of General Pathology, 2
nd
Ed., Oxford
University Press, New York.
3
Unresolved inflammation is heavily associated in many chronic diseases not previously
known to involve inflammation including Alzheimer’s disease, cardiovascular disease,
9

and cancer,
10
in addition to those well known to be associated with inflammation such
as arthritis and periodontal disease.
11
In 1999, GISSI-Prevenzione investigation on
dietary supplementation on omega-3 PUFAs showed a remarkable ~45% reduction in
sudden death, 3940 in >11000 patients with cardiovascular disease, when taking almost
a gram of omega-3 fatty acids per day.
12
It was noteworthy in the GISSI trail that
patients with both groups of the study were taking aspirin daily that was unaccounted
for in their analysis. The mechanisms by which omega-3 fatty acids exert their actions
have been described as the inhibition of the production of pro-inflammatory
prostaglandins (PGs) and leukotrienes (LTs) synthesis, which is because of the
replacement of the substrate arachidonic acid (AA) by DHA or EPA for the 5-LOX in
order to produce less potent 5-series LTs or COX mediated production to anti-
thrombotic prostanoids.
13
but surprisingly the molecular basis, and the underlying

9
Helgadottir, H.; Manolescu, A.; Thorleifsson, G.; Gretarsdottir, S.; Jonsdottir, H.; Thorsteinsdottir, U.;
Samani, J. J.; Godmundsson, G.; Grant, S. F. A.; Thorgeirsson, G.; Sveinbjornsdottir, S.; Valdimarsson, E.
M.; Matthiasson, S. E.; Johannsson, H.; Gudmundsdottir, O.; Gurney, M. E.; Sainz, J.; Thorhallsdottir, M.;
Anderesdottir, A.; Frigge, M. L.; Topol, E. J.; Kong, A.; Gudnason, V.; Hakonarson, H.; Gulcher, J. R.;
Stefansson, K. Nat. Genetics 2004, 36, 233.
10
(i) Erlinger, T. P.; Platz, E. A.; Rifai, N.; Helzlsouer, K. J. JAMA 2004, 291, 585. (ii) Pasche, B.;
Serhan, C. N. JAMA 2004, 291, 623.
11
(i) Gallin, J. I.; Snyderman, R.; Fearon, D. T.; Haynes, B. F.; Nathan, C. (eds) Inflammation: Basic
Principles and Clinical Correlations 1999, Lippincott Williams & Wilkins, Philadelphia, 1360 pp. (ii)
Van Dyke, T. E.; Serhan, C. N. J. Dent. Res. 2003, 82, 82.
12
GISSI-Prevenzione Investigators Lancet 1999, 354, 447.
13
De Caterina, R.; Enders, S.; Kristensen, S. D.; Schmidt, E. B. n-3 fatty acids and vascular disease 1993,
Springer-Verlag, UK, 1-116.
4
biochemical mechanisms, and their direct connection to human disease and treatment
remained to be discovered.

Several questions to be asked to uncover the molecular mechanisms for wide range of
beneficial effects of omega-3 fatty acids in many human diseases.
(i) Are omega-3 fatty acids, such as DHA and EPA, converted to
endogenous potent lipid mediators relevant to human disease and health
as in the case with omega-6 arachidonic acid? If so, what are their
structures? What are the biosynthetic pathways? And what are their
biological or pharmacological effects in human or in animals?

(ii) What is the role of aspirin for the omega-3 fatty acid’s protective action?
Is aspirin act differently or there are overlaps in their actions?

To address those questions in laboratory experimental settings, Serhan and colleagues
first reported in 2000 that EPA is transformed into anti-inflammatory, pro-resolving
lipid mediators, such as resolvin E1 and other bioactive molecules in murine exudates
treated with aspirin and EPA, providing a potential mechanism for omega-3 beneficial
actions in many human diseases.
14
The identification and structure elucidation were
performed using LC-tandem MS/MS and GC/MS based lipidomic analyses of bioactive
fractions obtained from the resolution of phase of inflammation following solid phase
extraction and reversed-phase HPLC (RP-HPLC). The basic structures of these lipid
mediators were determined as follows.
15

14
(i) Serhan, C. N.; Clish, C. B.; Brannon, J.; Colgan, S. P.; Chiang, N.; Gronert, K. J. Exp. Med. 2000,
192, 1197.
15
Lu, Y.; Hong, S.; Tjonahen, E.; Serhan, C. N. J. Lipid Res. 2005, 46, 790.
5
(i) Isolation of lipid mediators from the resolution phase of exudates and
tissues.

(ii) LC-UV-MS/MS data acquisition on the isolated compounds and then
constructed lipid mediators databases with physical properties such as
MS spectra, MS/MS fragmentation patterns, HPLC retention time, and
UV spectra.

(iii) These databases were systematically researched in step-wise fashion as
described below using UV chromophores and MS/MS fragmentation
patterns, and then HPLC retention time for matching and to identify
whether known or potential novel lipid mediators were present within
the resolution phase of exudates.

(iv) If new, novel lipid mediators were encountered, their basic structures
were elucidated from MS/MS fragmentation pattern.

The criteria for identification of a known bioactive lipid mediator for LC-UV-MS/MS-
based lipidomic analyses are: (i) HPLC retention time should match by co-elution with
standard authentic lipid mediators; (ii) UV λ
max
and band-shape should match with the
standard authentic samples; (iii) MS/MS spectra should have the fragment ions of [M-
H], [M-H-CO
2
], [M-H-nH
2
O] (n is the number of hydroxyl groups in the lipid
mediators); and (iv) The ions generated from at least one or two cleavages on the bonds
directly linked to the carbon of the functional group.

Since DHA is associated with significantly lower risk of heart disease, coronary heart
disease mortality and sudden death, and risk of a second heart-attack in many, and
6
abundant in brain and retina, and displays an impact in many physiological process,
16

Serhan and colleagues have undertaken a series of experiments on murine in vivo and
on human PMNs in vitro with DHA, with and without aspirin. The authors
17
used
murine dorsal skin pouches that spontaneously resolve in rats and adapted them for
mice in order to include both genetics and to set-up a lipidomic analysis using LC-UV-
MS/MS for identification and characterization of potential novel lipid mediators that are
indeed generated during the resolution phase of inflammation.
16
After 4 hours of this
experimental contained inflammation in mice called a pouch (that allows for an accurate
measurement of the neutrophil infiltration, and most importantly the lipid mediators
generation during the resolution phase of inflammation), PMN numbers begin to drop
within exudates, which were taken at a regular timed intervals, focusing on the period of
“spontaneous resolution” and lipid mediators profile were determined using tandem LC-
UV-MS/MS. The authors identified and characterized the planar structures of three
distinct series of DHA-derived polyoxygenated lipid mediators, which were
endogenously produced through a series of enzymatic oxygenations during the cell-cell
interactions in the resolution phase of inflammation. They termed this family of
compounds as “Resolvins (Rv)” (resolution phase interactions products), because they
generated in the resolution phase, and they are involved in chemically redundant cell
signaling that play protective roles in dampening inflammation to promote resolution.
Lipid mediators generated when treated with aspirin termed as aspirin-triggered

16
Bazan, N. G.; Rodriquez, E. B.; Gordon, W. C. Can. J. Physiol. Pharmacol. 1993, 71, 690.
17
(i) Serhan, C. Clish, C. B.; Brannon, J.; Colgan, S. P.; Gronet, K. J. Exp. Med. 2000, 192, 1197. (ii)
Serhan, C. N.; Hong, S.; Gornert, K.; Colagan, S. P.; Devchand, P. R.; Mirick, G.; Moussignac, R. L. J.
Exp. Med. 2002, 196, 1025.
7
resolvins (AT-Rv). The lipid mediators derived from EPA are designated as resolvin E
series, such as RvE1 while those formed from DHA are denoted as either resolvin D
series, such as RvD1, AT-RvD1, RvD2, AT-RvD2, RvD3, AT-RvD3, RvD4 and AT-
RvD4 or protectins such as neuroprotectins D1 (NPD1).
16

1.2 Structures of Lipid Mediators Derived from EPA

The family of resolvins derived from EPA and aspirin is 18R E series of resolvins,
specifically resolvin E1 (RvE1)
18
and more recently resolvin E2 (RvE2).
19
Microscale
structural elucidation gave the basic structures of the potent bioactive compounds
generated in the resolution of phase of inflammation from EPA and aspirin. The
resolvin E1 was determined to be 5, 12, 18R-trihydroxyeicosapentaenoic acid
18
and
Lipoxin A
5
(epi-LXA
5
)
17
was determined to be 5, 6, 18R-trihydroxy-eicosanpentaneoic
acid and recently resolvin E2 was determined to be 5S, 18-dihydroxy-eicosapentaenoic
acid,
19
as depicted in Figure 1.

18
Serhan, C. Clish, C. B.; Brannon, J.; Colgan, S. P.; Gronet, K. J. Exp. Med. 2000, 192, 1197.
19
Tjonahen, F.; Oh, S. F.; Siegelman, J.; Elangovan, S.; Percarpio, K, B.; Hong, S.; Arita, M.; Serhan, C.
N. Chemistry & Biology 2006, 13, 1193.
8
OH OH
OH
COOH
OH HO
OH
COOH
RvE1
15-epi-LXA
5
R
R
OH
OH
COOH
RvE2
R

Figure 1. Structures of EPA derived lipid mediators.

1.3 Biosynthesis of EPA-derived Lipid Mediators

In vascular endothelial cells derived from blood vessels, the recombinant COX-2 treated
with aspirin generated 18R-HEPE as well as 15R-HEPE from EPA, which are blocked
by selective COX-2 inhibitors.
20
Cytochrome p450, which can be released from
vascular endothelial cells and mucosal epithelial cells as well as from microbial origins,
can also convert EPA to 18R-HEPE and 15R-HEPE. These substrates are taken up the
by the activated human polymorphonuclear neutrophils (PMNs), and 5-LOX act on
them to generate 5S-hydroperoxy-18R-HEPE, 5S-hydroperoxy-15R-HEPE, which can
be converted to 5S, 6-epoxy-18R-HEPE and 5R, 6-epoxy-15R-HEPE. The reduction of
5S-hydroperoxy-18R-hydroxy eicosapentaenoic acid generates resolvin E2 (RvE2) as
shown in Figure 2. The enzymatic hydrolysis of 5S, 6-epoxy-18R-HEPE generated

20
Serhan, C. Clish, C. B.; Brannon, J.; Colgan, S. P.; Gronet, K. J. Exp. Med. 2000, 192, 1197.
9
RvE1 (Figure 2). Similarly, an enzymatic hydrolysis of 5R,6-epoxy-15R-HEPE
produced 15-epi-LXA
5
as shown in Figure 2. The biological results demonstrate that
RvE2, together with RvE1, may contribute to the beneficial actions of omega-3 fatty
acids in human diseases. Moreover, they indicate that the 5-lipoxygenase in human
leukocytes is a pivotal enzyme that can produce both pro- and anti-inflammatory
chemical mediators.
18

COOH
Eicosapentaenoic Acid (EPA)
COOH COOH
OH
18
OH
15
COX-2
+ Aspirin
COX-2
+ Aspirin
Vascular
Endothelial Cells
Vascular
Endothelial Cells
OH
18R-HEPE 15R-HEPE
OOH
COOH
5-LO
COOH
O
OH
PMN 5-LO + Epoxidation
OH
OH
COOH
OH
COOH
O
Enzymatic
epoxidation
OH
COOH
OH HO
OH
OH
COOH
OH
Reduction
Enzymatic
hydrolysis
Enzymatic
hydrolysis
15-epi-LXA
5
RvE1
RvE2

Figure 2. Biosynthetic pathways of E-series of resolvin and epi-LXA
5
.
10
1.4 Structures of DHA-derived Resolvins, Aspirin-Triggered Resolvins
and Neuroprotectins

Serhan and colleagues
21
were determined the planar and partial stereostructures of lipid
mediators derived from DHA by extensive analyses of data obtained from the advanced
analytical techniques, together with biosynthetic considerations. Inflammatory exudates
obtained from within the resolution phase formed within dorsal skin air pouches after
injection of TNF-α, DHA and aspirin. The polyoxygenated compounds were extracted
by solid phase extraction, and then the combined extracts were analyzed by both liquid
chromatography-UV-diode array detector-tandem mass spectrometry (LC-UV-MS/MS)
and GC/MS (with derivatized products). The analysis of HPLC retention time, UV and
MS/MS data confirmed a series of novel lipid mediators derived from both DHA and
EPA. The bioactive resolvins derived from DHA include three distinct series, which are
depicted in Figure 3.

(i) 17S series of resolvins: RvD1, RvD2, RvD3, RvD4
(ii) 17R series (AT) of resolvins: AT-RvD1, AT-RvD2, AT-RvD3, AT-RvD4
(iii) Docosatrienes: Neuroprotectin D1 (NPD1)

21
(i) Serhan, C. Clish, C. B.; Brannon, J.; Colgan, S. P.; Gronet, K. J. Exp. Med. 2000, 192, 1197. (ii)
Serhan, C. N.; Hong, S.; Gornert, K.; Colagan, S. P.; Devchand, P. R.; Mirick, G.; Moussignac, R. L. J.
Exp. Med. 2002, 196, 1025.(iii) Hong, S.; Gronert, K.; Devchand, P. R.; Moussignac, R. L. J. Biol. Chem.
2003, 278, 14677. (iv) Serhan, C. N.; Arita, M.; Hong, S.; Gotlinger, K. Lipids 2004, 39, 1125.
11
OH HO
COOH
RvD1
OH
OH HO
COOH
AT-RvD1
OH
HO OH
OH
COOH
HO OH
OH
COOH
OH
COOH
OH
OH
OH
COOH
OH
OH
OH
COOH
OH
OH
OH
COOH
OH
OH
R
S
S
R
S
R
S
R
RvD2
AT-RvD2
RvD3
AT-RvD3
RvD4
AT-RvD4
OH OH
COOH
NPD1
R

Figure 3. The main group of lipid mediators derived from DHA and their aspirin-
triggered epimers.

1.5 Biosynthesis of DHA-derived Resolvins and Neuroprotectins

The above mentioned DHA-derived or other lipid mediators are biosynthesized in
specific cell-types via epoxide-containing intermediates in well-regulated enzymatic
processes in very small quantities with precise geometric and entiomeric forms. It is
very important to know their biosynthetic pathways to determine their absolute
stereochemistries. Three major lypoxygenases, such as 5-LOX, 12-LOX, 15-LOX and
acetylated COX-2, are involved in the biosynthesis processes, which are described
below.

12
1.5.1 Biosynthesis of 17R-series Resolvins (AT-RvD1 - AT-RvD4)
17,19

Resolving exudates from mice given aspirin and DHA converted DHA to a novel 17R-
hydroxy-docosahexanoic acid (17R-HDHA), and several related of bioactive
compounds. Earlier literatures indicated that DHA is not a substrate for human
recombinant COX-2.
22
But surprisingly human endothelial cells expressing COX-2
treated with aspirin (aspirin acetylated the COX-2, but the enzyme is still active for
DHA) transform DHA to 17R-HDHA. Human PMN convert 17R-HDHA to two
compounds via 5-LOX, each of the compounds are rapidly transformed in to two
epoxide intermediates, 7S, 8-epoxy-17R-HDHA and 4S, 5-epoxy-17R-HDHA.
Enzymatic ring opening of these two novel epoxide intermediates give the 17R series
resolvins designated as AT-RvD1, AT-RvD2, AT-RvD3 and AT-RvD4. The
stereochemistries of the hydroxyl groups are depicted in likely configuration based on
results with recombinant enzymes. For complete biosynthetic pathways, please see
Figure 4.

22
(i) Corey, E. J.; Shih, C.; Cashman, J. R. Proc. Natl. Acad. Sci. USA 1983, 80, 3581. (ii) Serhan, C. N.;
Oliw, E. J. Clin. Invest. 2001, 107, 1481.
13
DHA
COX-2
+ Aspirin
COOH
HOO
17R-H(p)DHA
17
COOH
HO
COOH
HO
HOO
OOH
COOH
COOH
HO O
O
HO
COOH
HO
HO
OH
HO
OH
OH
COOH
OH OH
COOH
OH
HO
COOH
OH
HO
AT-RvD1 AT-RvD2 AT-RvD3 AT-RvD4
5-LOX
PMN
5-LOX
PMN
7S-hydroperoxy-17R-HDHA 4S-hydroperoxy-17R-HDHA
7S, 8-epoxy-17R-HDHA 4-epoxy-17R-HDHA
Enzymatic epoxidation Enzymatic epoxidation
Enzymatic
Hydrolysis
Enzymatic
Hydrolysis

Figure 4. Proposed biosynthesis of aspirin-triggered (AT-Rv) D series of resolvins.

1.5.2 Biosynthesis of 17S-series Resolvins (RvD1 - RvD4) and
Neuroprotectin (NPD1)
17,23

Mice without aspirin treatment and added DHA, the endogenous DHA was converted in
vivo to a 17S series of resolvins (RvD1 – RvD4) in similar fashion as it did with aspirin.
In the experimental model added DHA was given to quantify the amount of lipid
mediators to confirm biosynthesis. Tissues contain DHA that is available upon

23
Marcheselli, V. L.; Hong, S.; Lukiw, W. J.; Hua Tian, X.; Gronert, K.; Musto, A.; Hardy, M.; Gimenez,
J. M.; Chiang, N.; Serhan, C. N.; Bazan, N. G. J. Biol. Chem. 2003, 278, 43807.
14
activation to produce 17S-HDHA by LOX pathways, which then rapidly converted to
RvD1-RvD4 through a series of enzymatic processes as shown in the Figure 5.

DHA
COOH
HOO
17S-H(p)DHA
17
COOH
HO
COOH
HO
HOO
OOH
COOH
COOH
HO O
O
HO
COOH
HO
HO
OH
HO
OH
OH
COOH
OH OH
COOH
OH
HO
COOH
OH
HO
RvD1 RvD2 RvD3 RvD4
5-LOX
PMN
5-LOX
PMN
7S-hydroperoxy-17S-HDHA 4S-hydroperoxy-17S-HDHA
7S, 8-epoxy-17S-HDHA 4-epoxy-17S-HDHA
Enzymatic epoxidation Enzymatic epoxidation
Enzymatic
Hydrolysis
Enzymatic
Hydrolysis
15-LOX

Figure 5. Biosynthetic pathways of 17S-D series of resolvins.

Of the docosatriene-derived family, 10, 17S-DT, the neuroprotectin D1 (NPD1)
pathway as shown in Figure 6, proved a potent regulator of PMN influx in exudates at
sites where it is formed from endogenous precursors,
17
and limits stroke brain injury.
19

15
DHA
COOH
HOO
17S-H(p)DHA
17
15-LOX
Blood, leukocytes, brain, glial cells
kindney, lung and retina
COOH
HO
COOH
COOH
17S-HDHA
Reduction
Second
Oxygenation
OH
OH
Enzymatic
epoxidation
O
Enzymatic hydrolysis
Neuroprotectin D1 (NPD1)
COOH
OH
OH

Figure 6. Biosynthesis of neuroprotectin D1 and related compounds.

1.6 Biological Activities of Resolvins and Neuroprotectin D1 in Animal
Disease Models and their Formation in Cells, Tissues and Organs

Resolvins and neuroprotectins are endogenously and stereospecifically produced lipid
mediators from EPA and DHA that play a critical and broad role in human health and
diseases, especially those related to inflammation and resolution. It is now proved that
the presence of aspirin uniquely facilitates the resolution of inflammation. Regarding
their anti-inflammatory properties, the D series of resolvins block TNF-α induced IL-1β
transcripts and are potent regulators of PMN transmigration and inflammation in brain,
16
skin, and peritonitis in vivo.
24
It was also demonstrated that both D and E series of
resolvins down-regulated neutrophil infiltration.
25
The neuroprotectin D1 proved to be
a potent regulator of PMN influx in exudates at sites where it is formed from
endogenous DHA, limiting stroke brain injury
23
and retinal pigmented cellular
damage.
26
Their unique biological activities and the site of formation are summarized
in Table 1.

24
(i) Hong, S.; Gronert, K.; Devchand, P.; Moussignac, R. L.; Serhan, C. N. J. Biol. Chem. 2003, 278,
14677. (ii) Marcheselli, V. L.; Hong, S.; Lukiw, W. J.; Hua Tian, X.; Gronert, K.; Musto, A.; Hardy, M.;
Gimenez, J. M.; Chiang, N.; Serhan, C. N.; Bazan, N. G. J. Biol. Chem. 2003, 278, 43807.
25
Serhan, C. N. Pharmacology & Therapy 2005, 105, 7.
26
Mukerjee, P. K.; Marcheselli, V. L.; Serhan, C. N.; Bazan, N. G. Proc. Natl. Acad. Sci. USA 2004, 101,
8491.
17
Table 1. Lipid Mediators, their Bioactions and sites of Formation.
23-25

Lipid
Mediator
Bioactions Formation Sites
RvD1 Reduces PMN infiltration in murine skin air
pouch. Reduces peritonitis, and cytokine
expression in microglial cells.

Human PMN,
Ischemic-injury kidney.
AT-RvD1 Same as RvD1 Murine peritonitis
exudates and murine
brain stroke.
RvD2 Same as other D-series of resolvins Ischemic-injury kidney
and trout brain.
AT-D2 to AT-
D4
Same as other resolvins Murine dorsal air pouch
exudates.
RvE1 Reduces PMN infiltration, murine skin air
pouch inflammation and peritonitis.
Gastrointestinal protection
27
in TNBS colitis.
Murine dorsal air pouch
exudates, peritonitis
exudates, human
plasma, and trout brain.
RvE2 Stopped zymosan-induced olymorphonuclear
(PMN) leukocyte infiltration and displayed
potent anti-inflammatory properties in murine
peritonitis.
28

Murine dorsal air pouch
exudates and human
PMN.

NPD1 Reduces PMN infiltration, protects from
retinal injury. Diminished production in
human Alzheimer’s disease and promotes
neural cell survival.
29

Promotes corneal epithelial cell wound
healing.
30

Murine brain stroke,
human retinal pigment
epithelium, human
whole blood, ischemic-
injury kidney, trout
brain.

27
Arita, M.; Yoshida, M.; Hong, S.; Tjonahen, E.; Glickman, J. N.; Petasis, N. A.; Blumberg, R. S.;
Serhan. C. N. Proc. Natl. Acad. Sci. USA 2005, 102, 7671.
28
Tjonahen, F.; Oh, S. F.; Siegelman, J.; Elangovan, S.; Percarpio, K, B.; Hong, S.; Arita, M.; Serhan, C.
N. Chemistry & Biology 2006, 13, 1193.
29
Lukiw, W. J.; Cui, J. G.; Marcheselli, V. L.; Bodker, M.; Botkjaer, A.; Gotlinger, K.; Serhan, C. N.;
Bazan, N. G. J. Clin. Invest. 2005, 115, 2774.
30
Gronert, K.; Maheshwari, N.; Khan, N.; Hassan, I. R.; Dunn, M.; Schwartzman, M. L. J. Biol. Chem.
2005, 280, 15267.
18
1.7 Conclusion

Omega-3 polyunsaturated fatty acids have long been known to be important in human
health and diseases. The underlying molecular biochemical mechanism just began to
uncover. After taking EPA and DHA, they converted to potent polyoxygenated lipid
mediators in very minute quantities with stereochemically pure forms via a series of
well-defined enzymatic processes with unique structures and properties. Both resolvins
and neuroprotectins are generated in their epimeric forms when aspirin is given in the
mammalian systems. These aspirin triggered epimers also exhibit the same
characteristics of biological and structural features that resolve inflammation and PMN-
mediated injury. The studies on PUFAs derived pro-resolving, anti-inflammatory
polyoxygenated lipid mediators are uncovering surprising new avenues in anti-
inflammation and inflammation associated diseases, putting PUFA metabolites right at
the forefront of potential drug therapy. To determine their absolute stereostructures,
and to establish a structure-activity relationship, these novel lipid mediators have be to
asymmetrically synthesized in enantiomerically and geometrically pure forms, and then
design and synthesize their analogs or mimetics for the discovery of enzymatically
stable and biologically superior drugs.

19
Chapter 2. First Total Synthesis of Resolvin E1 (RvE1)

2.1 Introduction
Resolvin E1 (RvE1) was first discovered as a potent anti-inflammatory, pro-resolving
local mediator from aspirin treated eicosapentaenoic acid (EPA) that is generated in the
mouse exudates during the spontaneous resolution phase of inflammation, and act
locally at the sites of inflammation.
31
The biosynthetic pathways of RvE1, and its
biological activities were described in the chapter 2. Although the basic structure of
RvE1 was determined by lipidomic analyses and biosynthetic considerations, however,
the complete stereochemical assignments including the precise geometrical
configuration were incomplete. Therefore, because RvE1 is produced in subnanogram
quantities in vivo, and to assign the complete stereochemistry of RvE1, to study its
further biological activities, to establish a structure-activity relationship (SAR), and
finally to design and synthesis of metabolically stable & biologically superior analogs,
it was necessary to accomplish its asymmetric total synthesis, and then determine the
absolute stereostructure by matching its physical and most importantly biological
properties with the biogenic RvE1. In our laboratory we have undertaken a project for
over a decade to synthesize those lipid mediators and then systematically study their
biological properties in collaboration of Serhan group at Harvard Medical School.
Describe below is the first asymmetric total synthesis of RvE1 in its enantiomerically
and geometrically pure form.

31
Serhan, C. N.; Clish, C. B.; Brannon, J.; Colgan, S. P.; Gronet, K. J. Exp. Med. 2000, 192, 1197.
20
2.2 Results and Discussion
2.2.1 Synthetic Strategy for RvE1

Since a large number of naturally occurring lipid mediators and their analogs need to be
synthesized for the determination of their absolute stereochemistry and for the
systematic investigation of their biological properties, and functions, a carefully
constructed flexible, but a convergent synthetic plan was employed that addressed the
several key issues, such as chemical and thermal stability of these conjugated polyene
molecules to get the correct double-bond geometries, precise control of stereochemistry
at the hydroxyl bearing stereocenters, structural complexity, and protecting group
strategy. The naturally occurring RvE1 (2.1) poses similar challenges, as described
above, for its total synthesis. The retrosynthetic analysis of RvE1 (2.1) is presented in
the Figure 7.

21
Asymmetric Reduction
OH OH
OH
Chiral Glycidol
Chiral Glycidol
Selective Reduction
Selective Reduction
HWE
Pd
0
/Cu
I
Coupling
Pd
0
/Cu
I
Coupling
OTBS
TMS OTBS
Br OTBS
I
2.1
2.2 2.3 2.4
COOMe
COOMe

Figure 7. Retrosynthetic analysis of RvE1 methyl ester (2.1).
In our carefully constructed synthetic plan, we decided to employ 6Z and 14Z-double
bonds from the bis-acetylenic precursor of RvE1 in the last step by selective
hydrogenation using Zn (Cu/Ag) amalgam to avoid Z/E-isomerization and losses from
over-hydrogenation. The key retrosynthetic disconnection of our approach to RvE1
involved the formation of two carbon—carbon bonds using Pd
0
/Cu
I
mediated cross-
coupling of terminal alkynes and vinyl halides (iodide and bromide), called Sonogashira
coupling as depicted in Figure 7. The stereochemistry of the hydroxyl group at C-5 was
employed by an asymmetric reduction from the corresponding ketone using S-Alpine
borane. The stereochemistries of the rest of the chiral centers were introduced from
chiral starting material such (S)-glycidol as shown in the Figure 7. The major
22
advantages of our approach are: (a) it is highly convergent with less number of linear
steps, involved only the connections of three major building blocks (2.2, 2.3 and 2.4),
which allowed us to make a number of RvE1 analogs for the absolute stereostructure
determination, and also for the study of their structure-activity relationship (SAR), (b)
simultaneous generation of the two Z-double bonds by a very mild selective reduction
of triple bonds, by which we have prepared isotopically labeled RvE1 analogs that can
be used as probes for biological target identifications, (c) high degree of control of the
geometry of three E-double bonds, (d) flexibility to construct novel acetylenic and other
RvE1 analogs. The first asymmetric total synthesis of RvE1 by employing the above-
mentioned strategy is described below.
2.2.2 Synthesis of Terminal Alkyne Intermediate (2.2)
There are several ways of making this terminal alkyne (2.2) with the S-stereochemistry
at C-5. We have followed a literature procedure employed by others for the synthesis of
leukotriene B
4
.
32
The synthetic steps with reaction conditions are shown in the Scheme
1.

32
(i) for review please see: Nicolaou, K. C.; Ramphal, J. Y.; Petasis, N. A.; Serhan, C. N. Angew. Chem.
Int. Ed. Engl. 1991, 30, 1100. (ii) Corey, E. J.; Marfat, A.; Goto, G.; Brion, F. J. Am. Chem. Soc. 1980,
102, 7984. (iii) Nicolaou, K. C.; Chung, Y. S.; Hernandez, P. E.; Taffer, I. M.; Zipkin, R. E. Tetrahedron
Lett. 1986, 27, 1881. (iv) Avignon-Tropis, A.; Berjeaud, J. M.; Pougny, J. R.; Frechard-Ortuno, I.;
Guillerm, D.; Linstrumelle, G. J. Org. Chem. 1992, 57, 6521. (v) Chemin, D.; Linstrumelle, G.
Tetrahedron 1992, 48, 1943. (vi) Kerdesky, F. A.; Schimdt, S. P.; Brooks, D. W. J. Org. Chem. 1993, 58,
3516.
23
Cl
O O
OMe
O O
OMe
TMS
OH O
OMe
TMS
2.5
2.6
OTBS O
OMe
2.2 2.7
Reagents and conditions: (a) bis-trimethylsilyl acetylene, AlCl
3
,CH
2
Cl
2
,
-10
o
C, 4h, 93%; (b) S-Alpine borane, THF, -10
o
C to rt by overnight, 73%,
90% ee; (c) (i) TBS-Cl, imidazole, DMAP, CH
2
Cl
2
, rt, overnight, 95%; (ii)
K
2
CO
3
, MeOH, rt, overnight, 76%.
a
b
c

Scheme 1. Synthesis of top terminal alkyne (2.2) for RvE1.
The Lewis acid such as AlCl
3
mediated addition of bis(trimethylsilyl)acetylene to
methyl-4-chloroformyl-butanoate (2.5)
33
in CH
2
Cl
2
at -10
o
C gave a ketone intermediate
(2.6) with excellent yield (93%), which was then enantioselectively reduced to a
secondary alcohol with a S-stereochemistry at C-5 using (S)-Alpine Borane at -10
o
C in
THF. The enantiomeric excess (ee) was determined to be 90% by preparing the Mosher
ester and its
1
H-NMR analysis.
34
Protection of the hydroxyl group by TBS group and
the subsequent desilylation of TMS group by K
2
CO
3
in MeOH furnished the desired
terminal alkyne (2.2) with excellent overall yield (49%).
2.2.3 Synthesis of Middle Vinyl Bromide (2.3)

The synthesis of the vinyl bromide (2.3) was started with the ring opening of the
appropriate TBS-protected chiral glycidol with TMS-acetylene using n-BuLi in

33
Walton, D. R. M.; Waugh, F. J. Organomet. Chem. 1972, 37, 45.
34
Dale, J. A.; Dull, D. L.; Mosher, H. S. J. Org. Chem. 1969, 34, 2543.
24
presence of BF
3
.OEt
2
.
35
The stereochemistry at the hydroxyl group bearing chiral
center was achieved from the chiral glycidol used, which allowed us to assemble at least
a pair of diastereomers (one from R-glycidol and another one from S-glycidol) of RvE1.
For our synthetic efforts to embark the natural RvE1, we have started with S-glycidol,
as shown in Scheme 2, to give the secondary alcohol 2.9 with the appropriate
stereochemistry. The BF
3
.OEt
2
complex facilitates the ring opening at the least
substituted site of the glycidol to give a single enantiomer 2.9 exclusively with excellent
yield (90%).

O
OTBS
OTBS
TMS OH
2.10 2.11
2.12 2.3
TMS OTBS
Br
(i) TMS-acetylene, n-BuLi,
BF
3
.OEt
2
, THF, -78
o
C, 2h
90%
2.9
OTBS
TMS OTBS
(i) TBS-Cl, imidazole,
DMAP, CH
2
Cl
2
, rt
99%
OH
TMS OTBS
(i) CSA, MeOH-CH
2
Cl
2

(1:1), 0
o
C, 30 min
65%
(i) Swern oxidation,
-78
o
C, 4h
92%
O
TMS OTBS
(i) 2.14, LDA, THF, -78
o
C, 5h
55%
Br
Br P
Br
O
EtO
OEt
P
OEt
EtO OEt
2.14
2.13
90
o
C, overnight
88%
S-Glycidol (2.8)

Scheme 2. Synthesis of middle vinyl bromide (2.3) for RvE1.

35
Mohr, P.; Tamm, C. Tetrahedron Lett. 1987, 28, 391.
25
The newly generated secondary hydroxyl group in 2.9 was silylated with TBS-Cl,
imidazole and DMAP to furnish 2.10 quantitatively, which was then selectively de-
silylated at the primary position by the mild action of camphorsulfonic acid (CSA) in
MeOH-CH
2
Cl
2
at 0
o
C for 30 min leading to the primary alcohol 2.11 with a modest
65% yield.
36
If not careful with the reaction condition and temperature, this type of
selective deprotection can lead to the de-silylation of both TBS groups to give a mess in
the reaction mixture. The primary alcohol 2.11 was then oxidized by the Swern
oxidation
37
at -78
o
C to give an aldehyde 2.12 with an excellent yield (92%). A vinyl
bromo-phosphonate 2.14 was prepared
38
by heating 1,3-dibromo-propene (cis/trans-
mixture) with ethylphosphite 2.13 as neat. The LDA mediated Horner-Wadsworth-
Emmons (HWE)
39
reaction between the aldehyde 2.12 and the vinyl bromo
phosphonate 2.14 at -78
o
C in THF yielded the desired building block 2.3 with E-
geometry in moderate yield (55%).

2.2.4 Synthesis of Bottom Vinyl Iodide Intermediate (2.4).
Synthesis of the bottom vinyl iodide, as shown in Scheme 3, was started with the
protection of the enantiomerically pure glycidol, either S-glycidol or R-glycidol, which
allowed us to accomplish both epimers of RvE1, 18R-RvE1 and 18S-RvE1, respectively.

36
Nicolaou, K. C.; Ninkovic, S.; Sarabia, F.; Vourloumis, D.; He, Y.; Vallberg, H.; Finlay, M. R. V.;
Yang, Z. J. Am. Chem. Soc. 1997, 119, 7974.
37
Mancuso, A. J.; Huang, S. L.; Swern, D. J. Org. Chem. 1978, 43, 2480.
38
Arbuzov, B. A. Pure Appl. Chem. 1964, 9, 307.
39
Wadsworth, W. S.; Emmons, W. D. J. Am. Chem. Soc. 1961, 83, 1733.
26
The CuI mediated opening of the TBS-protected epoxide by CH
3
Li in THF at -78
o
C
gave the diol 2.15 with excellent yield (93%).
40
The newly generated secondary alcohol
was protected by a TBDPS-group followed the subsequent deprotection of the TBS-
group with the mild action of camphorsulfonic acid in MeOH-CH
2
Cl
2
(1:1) at room
temperature afforded the primary alcohol 2.17 in excellent yield (92%).
3
The oxidation
of the primary alcohol (2.17) was done by Swern oxidation
7
at -78
o
C in CH
2
Cl
2
to give
the aldehyde 2.18 in excellent yield (91%), followed by the subsequent Takai
olefination
41
with CrCl
2
and CHI
3
to give the desired vinyl iodide 2.4 with excellent
overall yield (39%).
O
OTBS OTBS
OH
OTBS
OTBDPS
OH
OTBDPS
O
OTBDPS
OTBS
I
2.15
ab
c
d e
Reagenst and conditions: (a) CH
3
Li,CuI,THF,-78
o
C,
4h and then rt for overnight, 93%; (b) TBDPS-Cl, imidazole,
DMAP, CH
2
Cl
2
, rt, overnight, 95%; (c) CSA, Et
3
N, MeOH-
CH
2
Cl
2
(1:1), 0
o
C, 45 min, 92%; (d) Swern oxidation, CH
2
Cl
2
,-78
o
C,
4h, 91%; (e) (i) CrCl
2
, CHI
3
, THF, 0
o
C, 3h; (ii) TBAF, THF, rt, overnight,
56% in two steps; (iii) TBS-OTf, lutidine, CH
2
Cl
2
, rt, overnight, 95%.
2.16
2.17 2.18 2.4
S-Glycidol (2.8)

Scheme 3. Synthesis of bottom vinyl iodide (2.4) for RvE1.

40
Mohr, P.; Tamm, C. Tetrahedron Lett. 1987, 28, 391.
41
Takai, K.; Nitta, K.; Utimoto, K. J. Am. Chem. Soc. 1986, 108, 7408.
27
2.2.5 Synthesis of top bis-alkyne intermediate (2.21)

With the two key intermediates 2.2 and 2.3 in hands, the next step was to run a
carbon—carbon bond forming reaction to couple these two intermediates. The standard
Pd
0
/Cu
I
mediated Sonogashira coupling
42
between the terminal alkyne 2.2, and vinyl
bromide 2.3 afforded the 8E/8Z-diasteromers of bis-alkyne 2.19 with excellent yield
(90%) as shown in Scheme 4. The 8Z-isomer was, however, easily isomerized to the
corresponding 8E-isomer 2.20 with the catalytic amount of sublimed iodine in dry
CH
2
Cl
2
. The Na
2
CO
3
mediated desilylation of TMS-group furnished the terminal 8E,
10E-bis-alkyne 2.21 in excellent overall yield (73%).

42
Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett. 1975, 16, 4467.
28
OTBS
TMS OTBS
2.2
2.19
2.20
2.21
OTBS
a
b
c
2.3
TMS OTBS
Br
TMS OTBS OTBS
OTBS OTBS
COOMe
COOMe
COOMe
COOMe
Reagents and conditions: (a) Pd(Ph
3
)
4
,CuI, C
6
H
6
,Et
3
N, rt,
overnight, 90%; (b) I
2
(catalytic), CH
2
Cl
2
, rt, 3 days, 85%; (c)
K
2
CO
3
, MeOH, rt, overnight, 95%.

Scheme 4. Synthesis of bis-alkyne intermediate (2.21) for RvE1.

2.2.6 Final Assembly of the Resolvin E1 (RvE1) Methyl Ester (2.1)

The final coupling of terminal bis-alkyne (2.21) and vinyl iodide (2.4) under the
standard Pd
0
/Cu
I
conditions with catalytic amount of palladium[tetrakis-
(triphenylphosphine)] and cuprous iodide in presence of triethylamine in benzene at
room temperature afforded the tri-protected 8E, 10E, 16E bis-alkyne precursor of
resolvin E1 (2.22) with excellent yield (80%) as shown in Scheme 5. The coupling
reaction mixture has to be oxygen free to avoid homo-coupling of terminal alkyne,
which gives an undesired side product. The product obtained from Sonogashira
coupling was desilylated using tetrabutylammonium fluoride (TBAF) in THF. The
29
extreme basicity of the fluoride anion, especially in anhydrous condition produced a
significant amount of free acid, which was further esterified using freshly prepared
diazomethane as shown in the Scheme 5 to furnish the bis-acetylenic-5S, 12R, 18R, 8E,
10E, 16E-RvE1 methyl ester precursor (2.23) in excellent yield (95% in two steps). It is
worth to mention that in deprotection step, addition of small amount of water (5-10%)
usually reduces the amount of free acid formed.
2.21
OTBS OTBS
OTBS
I
2.4
OTBS OTBS
OTBS
OH OH
OH
OH OH
OH
2.22
2.23
RvE1 Methyl Ester (2.1)
a
b
c
COOMe
COOMe
COOMe
COOMe
Reagenst and conditions: (a) Pd(Ph
3
)
4
, CuI, NEt
3
, C
6
H
6
, rt, overnight,
80%; (b) TBAF, THF, 0
o
C to rt, overnight followed by CH
2
N
2
, ether, 2h,
95%; (c) Zn (Cu/Ag), H
2
O-MeOH (1:1), 40
o
C, overnight, 80%.
R

Scheme 5. Final assembly of resolvin E1 (RvE1) methyl ester (2.1).
30
The selective reduction of the triple bonds in conjugation with extended double bonds
was a challenging task. Over the years, Scientists were using Lindlar catalyst and its
modified method for Z-selective partial hydrogenation, which sometimes produced
thermodynamically stable all E-isomer and/or over hydrogenated products.
43
Moreover,
selective hydrogenation by Lindlar catalyst requires the reaction to be monitored
continuously by HPLC in order to prevent losses due to the over-hydrogenation of the
conjugated polyene system, which is a very tedious and time consuming task. However,
the method introduced by Boland
44
using Zn(Cu/Ag) in aqueous-MeOH (1:1) at 30-40
o
C gave excellent results in our experimental conditions. The stereospecific Z-reduction
of the two triple bonds to RvE1 methyl ester (2.1) was carried-out with the pre-activated
freshly prepared Zn(Cu/Ag) in CH
3
OH-H
2
O (1:1) at 40
o
C for overnight. The reaction
preceded smoothly without the formation any side-product and gave excellent yield
(80%). The final product RvE1 methyl ester (2.1) was purified by a reversed-phase
HPLC on ODS column using MeOH-H
2
O mixture as the eluting solvent. The final
product RvE1 methyl ester was characterized by the extensive analyses of 1-& 2D-
NMR, UV and mass-spectral data.

43
(i) Nicolaou, K. C.; Veale, C. A.; Webber, S. E.; Katerinopoulos, H. J. Am. Soc. 1985, 107, 7515. (ii)
Choi, J.; Yoon, N. M. Tetrahedron Lett. 1996, 37, 1057. (iii) Ho, T. L.; Liu, S. H. Synth. Commun. 1987,
17, 969.
44
Boland, W.; Schroer, N.; Sieler, C.; Feigel, M. Helv. Chim. Acta. 1987, 70, 1025.
31
2.3 Conclusion.

We have accomplished the first total synthesis of EPA-derived endogenously produced
pro-resolving, anti-inflammatory lipid mediator RvE1 methyl ester. Our approach is
highly convergent and very practical which allowed us
45
to make several other
diastereomers and analogs as shown in the Scheme 6 by employing either same or
similar synthetic strategy.
OH OH
OH
COOMe
18S-RvE1 Methyl Ester
OH OH
CD
3
OH
COOMe
18R-RvE1-D
3
Methyl Ester
OH OH
COOMe
18-dehydro-RvE1 Methyl Ester
OH OH
COOMe
HO
6E, 8Z, 10E, 14E, 16E, 18R-RvE1
OH
HO
OH
COOMe
6E, 8E, 10E, 14E, 16E, 18R-RvE1

Scheme 6. Synthesized RvE1 methyl ester analogs.

45
Yang, R. Synthesis of Study of Novel Lipid Mediators 2006, PhD thesis.
32
Our strategy is potentially useful for the large scale production from easily available
starting materials without using any biologically and environmentally toxic metals or
reagents. These synthesized molecules sent to our collaborator at Harvard Medical
School for biological and pharmacological studies, which will be discussed in the later
chapter of this dissertation.
33
2.4 Experimental

All reactions, unless otherwise noted, were carried in flame dried flasks under argon
atmosphere. “Dried and concentrated” refers to removal of residual water with
anhydrous MgSO
4
, followed by evaporation of the solvent on the rotary evaporator.
THF was freshly distilled from sodium-benzophenone, benzene and dichloromethane
from CaH
2
and anhydrous DMF, EtOH, and MeOH were purchased from commercial
sources.
1
H and
13
C NMR spectra were recorded on a Varian Mercury 400 and a
Bruker AC-250 using residual
1
H or
13
C signals of deuterated solvents as internal
standards. UV spectra were recorded on a Hewlett-Packard 8350 instrument. HPLC
analyses were performed on a Rainin dual pump HPLC system equipped with a
Phenomenex ODS column and an UV-VIS detector.

O O
OMe
TMS
2.6

2.4.1 Methyl 5-oxo-7-trimethylsilyl-hept-6-ynoate (2.6). To a stirred suspension of
AlCl
3
(8.0 g, 60.0 mmol) in dry CH
2
Cl
2
(40 mL) at -5
o
C, a solution of methyl-4-
chloroformyl butanoate 2.5 (5.0 g, 4.2 mL, 30.4 mmol) in dry CH
2
Cl
2
(5.0 mL) was
added by a cannula. The suspension was stirred for 30 min at -5
o
C. The reaction
mixture was then cooled at -78
o
C and then added the bis-(trimethylsilyl)-acetylene
(5.23 g, 6.9 mL, 30.7 mmol) in dry CH
2
Cl
2
(15 mL) by a cannula. The temperature was
34
then raised to -10
o
C and stirred for 3 h. After stirring at -10
o
C for 3 h, the reaction was
the quenched with an ice-cold HCl (0.1 M, 15 mL) to get two clear phases. The organic
phase was first separated, and then the aqueous layer was extracted with ether (30 mL x
3). The combined organic extract was washed with brine, and dried over MgSO
4
. The
crude product was purified on a silica column using 15 % EtOAc/hexanes as the mobile
phase to furnish the pure title compound, 2.6 (6.4 g, 93%).
1
H-NMR (250 MHz, CDCl
3
)
δ
H
3.65 (s, 3H), 2.62 (t, J = 7.0 Hz, 2H), 2.34 (t, J = 7.7 Hz, 2H), 1.94 (quintet, J = 7.0
Hz, 2H), 0.21 (s, 9H);
13
C-NMR (62 MHz, CDCl
3
) δ
C
186.4, 173.9, 101.5, 97.6, 51.3,
43.9, 32.3, 18.6, -1.3.

OH O
OMe
TMS
2.7

2.4.2 Methyl 5S, 7-trimethylsilyl-5-hydroxy-hept-6-ynoate (2.7). To a solution of a
keto-ester, 2.6 (6.4 g, 28.0 mmol) in dry THF (20 mL) at -10
o
C was added dropwise a
solution of S-Alpine borane (84.0 mL of 0.5M solution, 42.0 mmol) in THF. The
reaction mixture was then warmed to room temperature by overnight, and stirred for
almost 3 days. The excess S-Alpine borane was quenched by adding acetaldehyde (6.5
mL) to the reaction mixture at 0
o
C. After stirring at 0
o
C for 30 min, a solution of
ethanolamine (3.0 mL) was added dropwise to the reaction mixture, and stirred for 10
min. The product was extracted with ether, washed with brine, and concentrated in
vacuo. The crude product was then purified by a silica column chromatography using
30 % ether/hexanes as the solvent system to give the pure alcohol 2.7 (4.61 g, 73%)
35
with 90% ee, which was determined by the Mosher Ester analysis.
1
H-NMR (250 MHz,
CDCl
3
) δ
H
4.32 (m, 1H), 3.63 (s, 3H), 2.33 (t, J = 7.0 Hz, 3H), 1.71 (m, 4H), 0.12 (s,
9H);
13
C-NMR (62.5 MHz, CDCl
3
) δ
C
174.1, 106.4, 89.5, 62.2, 51.4, 37.0, 33.7, 20.5, -
0.4.

OTBS O
OMe
TMS
2.24

2.4.3 Methyl 5S, 5-(t-butyldimethylsilyloxy)-7-trimethylsilyl-hept-6-ynoate (2.24).
To a mixture of imidazole (1.5 g, 21.0 mmol), TBS-Cl (3.3 g, 21.0 mmol), and DMAP
(0.1 g, 0.8 mmol) in dry CH
2
Cl
2
(30 mL) at 0
o
C was added a solution of hydroxyl ester
2.7 (4.6 g, 20.0 mmol). The reaction mixture was warmed to room temperature, and
stirred for overnight. The reaction mixture was quenched with a saturated aqueous
solution of NH
4
Cl, extracted with ether (30 mL x 3), washed with brine, dried over
MgSO
4
, and concentrated under reduced pressure to give a crude product. The crude
product was purified on silica column using 3% EtOAc/hexanes as the eluant to afford
the pure product 2.24 (6.5 g, 95%) as a colorless oil.
1
H-NMR (250 MHz, CDCl
3
) δ
H

4.31 (t, J = 6.2 Hz, 1H), 3.64 (s, 3H), 2.32 (t, J = 7.0 Hz, 2H), 1.68 (m, 4H), 0.86 (s,
9H), 0.12 (s, 9H), 0.10 (s, 3H), 0.07 (s, 3H);
13
C-NMR (62.5 MHz, CDCl
3
) δ
C
174.0,
107.4, 88.7, 62.9, 51.5, 37.7, 33.5, 25.8, 20.8, -0.4, -4.5, -5.1.

36
OTBS O
OMe
2.2

2.4.4 Methyl 5S, 5-(t-butyldimethylsilyloxy)hept-6-ynoate (2.2). To a solution of
TMS protected alkyne 2.24 (4.6 g, 34.2 mmol) in MeOH (50 mL) was added one scoop
of Na
2
CO
3
, and stirred for overnight. The cloudy mixture was concentrated in vacuuo
to remove MeOH, and then added H
2
O (25 mL) to quench the reaction, extracted with
ether (30 mL x 3), washed with brine, dried over MgSO
4
. The crude was purified over
silica column using 5% EtOAc/hexanes as the eluant to afford the alkyne 2.2 (2.6 g,
76%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
) δ
H
4.33 (td, J = 6.8, 2.0 Hz, 1H),
3.64 (s, 3H), 2.35 (d, J = 2.0 Hz, 1H), 2.34 (t, J = 7.6 Hz, 2H), 1.75 (m, 2H), 1.68 (m,
2H), 0.87 (s, 9H), 0.10 (s, 3H), 0.08 (s, 3H);
13
C-NMR (62.5 MHz, CDCl
3
) δ
C
173.9,
85.2, 72.4, 62.3, 51.6, 37.7, 33.6, 25.6, 20.7, 18.2, -4.7, -5.3.

O
OTBS
2.8

2.4.5 TBS-protected S-glycidol (2.8). To a mixture of imidazole (5.5 g, 81.0 mmol),
TBS-Cl (12.2 g, 81.0 mmol), and DMAP (0.4 g, 3.3 mmol) in dry CH
2
Cl
2
(100 mL) at 0
o
C was added S-glycidol 2.25 (5.0 g, 67.5 mmol). The reaction mixture was warmed to
room temperature, and stirred for overnight. The reaction mixture was quenched with a
saturated aqueous solution of NH
4
Cl, extracted with ether (100 mL x 3), washed with
brine, dried over MgSO
4
, and concentrated under reduced pressure to give a crude
product. The crude product was purified on silica column using 5% EtOAc/hexane as
37
the eluant to give the TBS-protected glycidol 2.8 (12.5 g, 98%) as a colorless oil.
1
H-
NMR (400 MHz, CDCl
3
) δ
H
3.84 (1H, dd, J = 11.5, 3.1 Hz), 3.65 (1H, dd, J = 11.5, 4.8
Hz), 3.06 (1H, m), 2.76 (1H, dd, J = 5.3, 4.5 Hz), 2.62 (1H, dd, J = 5.3, 2.6 Hz).
13
C-
NMR (100 MHz, CDCl
3
) δ
C
64.1, 52.0, 44.2, 25.9, 18.0, -5.0.

OTBS
TMS OH
2.9

2.4.6 2R, 1-(t-butyldimethylsilyloxy)-5-trimethylsilyl-pent-4-yn-2-ol (2.9). To a
solution of TMS-acetylene (11.30 mL, 80.0 mmol) in dry THF (90 mL) at -78
o
C was
added n-BuLi (50 mL of 1.6 M solution in hexane, 80.0 mmol) dropwise. The mixture
was stirred for 15 min at -78
o
C. A solution of BF
3
.OEt
2
complex (10.13 mL, 80.0
mmol) was then added at the same temperature and stirred for an additional 15 min. A
solution of protected S-glycidol 2.8 (10.0 g, 53.0 mmol) in dry THF (10 mL) was added
to the reaction mixture at -78
o
C by a cannula. The reaction mixture was stirred at -78
o
C for 2 h, and warmed to room temperature by removing the dry-ice bath, and stirred
for additional 30 min. The reaction was then quenched with a saturated aqueous
solution of NH
4
Cl, extracted with ether (100 mL x 3), washed with brine, dried over
MgSO
4
. The organic extracts were combined and concentrated in vacuo. The crude
product was then purified on a silica column using 10% EtOAc/hexanes as the solvent
system to afford pure title compound 2.9 (14.2 g, 95%) as a colorless oil.
1
H-NMR
(400 MHz, CDCl
3
) δ
H
3.78 (m, 1H), 3.71 (dd, J = 10.4, 4.4 Hz, 1H), 3.62 (dd, J = 9.6,
5.2 Hz, 1H), 2.53 (d, J = 5.2 Hz, 1H, -OH), 2.45 (t, J = 6.0 Hz, 2H), 0.90 (s, 9H), 0.14 (s,
38
9H), 0.08 (s, 6H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
102.8, 86.9, 70.1, 65.4, 25.8, 24.5,
18.3, -0.03, -5.4, -5.5.

OTBS
TMS OTBS
2.10

2.4.7 2R, 1, 2-di-(t-butyldimethylsilyloxy)-5-trimethylsilyl-pent-4-yn (2.10). To a
mixture of imidazole (2.57 g, 37.8 mmol), TBS-Cl (5.7 g, 37.8 mmol), and DMAP (0.2
g, 1.6 mmol) in dry CH
2
Cl
2
(60 mL) at 0
o
C was added the alcohol 2.9 (9.04 g, 31.5
mmol). The reaction mixture was warmed to room temperature, and stirred for
overnight. The reaction mixture was quenched with a saturated aqueous solution of
NH
4
Cl, extracted with ether (60 mL x 3), washed with brine, dried over MgSO
4
, and
concentrated under reduced pressure to give a crude product. The crude product was
purified on silica column using 3% EtOAc/hexanes as the eluant to furnish the pure
product 2.10 (12.5 g, 99%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
) δ
H
3.75
(quintet, J = 6.5 Hz, 1H), 3.48 (dd, J = 6.5, 4.5 Hz, 2H), 2.45 (dd, J = 17.2, 5.2 Hz, 1H),
2.25 (dd, J = 16.8, 6.4 Hz, 1H), 0.84 (s, 18H), 0.08 (s, 9H), 0.06 (s, 3H), 0.03 (s, 3H),
0.00 (s, 3H), -0.01 (s, 3H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
104.7, 85.8, 72.1, 66.7,
26.0, 25.9, 25.7, 18.4, 18.1, 0.1, -4.5, -4.6, -5.3, -5.4.

OH
TMS OTBS
2.11

39
2.4.8 2R, 2-(t-butyldimethylsilyloxy)-5-trimethylsilyl-pent-4-yn-1-ol (2.11). To a
solution of protected diol 2.10 (13.9 g, 34.67 mmol) in a 1:1 mixture of CH
2
Cl
2
:MeOH
(100 mL) was added camphorsulfonic acid (5.6 g, 24.27 mmol) at 0
o
C. The progress of
the reaction was monitored by TLC. The reaction was over by an hour, it was then
quenched with Et
3
N (4.8 mL, 34.67 mmol). The solvent was evaporated to dryness to
give a crude mixture, which was then purified on a silica gel column using 7%
EtOAc/hexane to afford the title primary alcohol 2.11 (6.8 g, 74%).
1
H-NMR (400
MHz, CDCl
3
) δ
H
3.84 (m, 1H), 3.60 (ddd, J = 9.6, 6.4, 4.0 Hz, 1H), 3.52 (ddd, J = 10.0,
7.2, 4.0 Hz, 1H), 2.41 (dd, J = 16.8, 6.8 Hz, 1H), 2.34 (dd, J = 17.2, 6.4 Hz, 1H), 2.01 (t,
J = 4.0 Hz, 1H, -OH), 0.86 (s, 9H), 0.09 (s, 9H), 0.08 (s, 3H), 0.06 (s, 3H);
13
C-NMR
(62.5 MHz, CDCl
3
) δ
C
103.2, 86.7, 71.5, 66.1, 25.8, 25.2, 18.0, -0.1, -4.8.

O
TMS OTBS
2.12

2.4.9 2R, 2-(t-butyldimethylsilyloxy)-5-trimethylsilyl-pent-4-ynal (2.12). To a
solution of DMSO (4.06 mL, 52.5 mmol) in dry CH
2
Cl
2
(100 mL) at -78
o
C was slowly
added oxalyl chloride (3.05 mL, 35.0 mmol) and stirred for 15 min, and then added the
primary alcohol 2.11 (5.0 g, 17.5 mmol) in CH
2
Cl
2
(10 mL) through a cannula, and
stirred for 50 min. Et
3
N (12.2 mL, 87.5 mmol) was then added to the reaction mixture,
and stirred for 3 h at -78
o
C. The reaction mixture was then brought to room
temperature by removing the dry-ice bath and stirred for 30 min. The white reaction
mixture was quenched with a saturated aqueous solution of NH
4
Cl, extracted with ether
40
(100 mL x 3), washed with brine, dried over anhydrous MgSO
4
, and concentrated under
reduced pressure. The crude product was purified on a silica column using 5%
EtOAc/hexanes as the eluant to give the aldehyde 2.12 (4.6 g, 92%) as a colorless oil.
1
H-NMR (360 MHz, CDCl
3
) δ
H
9.60 (s, 1H), 4.09 (m, 1H), 2.60 (dd, J = 15.2, 5.6 Hz,
1H), 2.43 (dd, J = 15.3, 10.1 Hz, 1H), 0.97 (s, 9H), 0.27 (s, 9H), 0.18 (s, 3H), 0.12 (s,
3H);
13
C-NMR (90 MHz, CDCl
3
) δ
C
204.2, 103.4, 86.5, 70.6, 25.7, 25.3, 18.6, -0.1, -4.2.

P
Br
O
EtO
OEt
2.14

2.4.10 Ethyl phosphonate 2.14. A mixture of ethyl phosphite 2.13 (8.3 g, 50 mmol)
and cis/trans-1,3 dibromo-1-propene (5.0g, 25 mmol) was refluxed as neat at 120
o
C for
overnight. The excess phosphite and ethyl bromide by-product were removed by a
using high vacuum and a rotatory evaporator heated the water-bath at 60
o
C until all
removed. The product purity was confirmed by taking a
1
H-NMR (400 MHz, CDCl
3
)
δ
H
6.35 (m, 1H), 6.15 (m, 1H), 4.11-4.04 (m, 4H), 2.78 (ddd, J = 21.6, 7.2, 0.8 Hz, 1H),
2.55 (ddd, J = 21.6, 7.2, 0.8 Hz, 1H), 1.30 (m, 6H).

TMS OTBS
Br
2.3

2.4.11 1(E/Z), 5R, 5-(t-butyldimethylsilyloxy)-8-trimethylsilyl-octa-1,3-dien-7-yne
(2.3). To a solution of ethyl phosphonate 2.14 (5.42 g, 21.08 mmol) in dry THF (25 mL)
at -78
o
C was added dropwise the freshly prepared LDA (14.75 mmol) in THF (5.0
41
mL). The mixture was then stirred for 15 min at -78
o
C. A solution of aldehyde 2.12
(3.0 g, 10.54 mmol) in dry THF (5.0 mL) was slowly added to the reaction mixture at -
78
o
C. The reaction mixture was stirred for 4 h at -78
o
C, and then brought to room
temperature and stirred for an additional 50 min until no aldehyde was detected by TLC.
The reaction was quenched with a saturated aqueous solution of NH
4
Cl, extracted with
ether (30 mL x 3), washed with brine, dried over MgSO4. The crude product was
purified on a silica column using 1% EtOAc/pentane to give the pure vinyl bromide 2.3
(2.2 g, 55%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.59 (m, 2H), 6.14 (m,
1H), 5.94 (m, 1H), 4.32 (q, J = 6.8 Hz, 1H), 2.44 (dd, J = 16.0, 7.2 Hz, 1H), 2.35 (dd, J
= 16.0, 7.2 Hz, 1H), 0.90 (s, 9H), 0.12 (s, 9H), 0.08 (s, 3H), 0.06 (s, 3H);
13
C-NMR
(125 MHz, CDCl
3
) δ
C
135.3, 129.3, 104.1, 86.0, 72.0, 31.6, 30.0, 25.8, 18.3, 14.1, 0.00,
-4.5, -4.7.

OTBS
OH
2.15

2.4.12 2R-1-(t-butyldimethylsilyloxy)-2-hyroxy-butane (2.15). To a suspension of
CuI (0.4 g, 2.03 mmol) in dry THF (40 mL) was added dropwise a 1.6M solution of
CH
3
Li (20 mL, 24.0 mmol) at -10
o
C and stirred for 10 min. The resulting mixture was
then cooled at -78
o
C and a solution of TBS-protected S-glycidol 2.8 (4.0 g, 21.2 mmol)
in THF (5 mL) was added by a cannula. The resulting mixture was stirred at -78
o
C for
4 h, and then slowly warmed to room temperature by overnight. The reaction was
quenched with a saturated aqueous solution of NH
4
Cl, extracted with ether (30 mL x 3),
42
washed with brine, and dried over MgSO
4
, and concentrated in vacuo to give a crude oil.
The crude product was then purified on silica column using 10% EtOAc/hexanes as the
solvent system to give the pure product 2.15 (4.6 g, 93%) as a colorless oil.
1
H-NMR
(400 MHz, CDCl
3
) δ
H
3.61 (dd, J = 9.6, 3.2 Hz, 1H), 3.37 (dd, J = 9.6, 8.0 Hz, 1H),
3.53 (m, 1H), 2.39 (d, J = 4.0 Hz, 1H, -OH), 1.43 (m, 2H), 0.93 (t, J = 7.6 Hz, 3H), 0.90
(s, 9H), 0.05 (s, 6H).

OTBS
OTBDPS
2.16

2.4.13 2R-1-(t-butyldimethylsilyloxy)-2-(t-butyldiphenylsilyloxy)-butane (2.16). To
a solution of TBDPS-Cl (6.85 mL, 26.4 mmol), imidazole (1.8 g, 26.4 mmol) and
DMAP (0.12 g, 0.98 mmol) in dry CH
2
Cl
2
(30 mL) at 0
o
C was added the alcohol 2.15
(4.5 g, 22.0 mmol) in CH
2
Cl
2
(5.0 mL) through a cannula. The mixture was warmed to
room temperature and stirred for overnight. The reaction was quenched with a saturated
aqueous solution of NH
4
Cl, extracted with ether (40 mL x 3), washed with brine, dried
over MgSO
4
, and concentrated under reduced pressure. The crude was purified on a
silica gel column using 1% EtOAc/hexane as the eluant to afford 2.16 (9.3 g, 95%) as a
colorless oil.
1
H-NMR (400 MHz, CDCl
3
) δ
H
7.68 (m, 4H), 7.38 (m, 6H), 3.70 (quintet,
J = 5.0 Hz, 1H), 3.44 (t, J = 5.0 Hz, 2H), 1.58-1.46 (m, 2H), 1.05 (s, 9H), 0.90 (t, J =
7.6 Hz, 3H), 0.81 (s, 9H), -0.08 (s, 3H), -0.11 (s, 3H).

43
OH
OTBDPS
2.17

2.4.14 2R-2-(t-butyldiphenylsilyloxy)-but-1-ol (2.17). To a solution of protected diol
2.16 (4.8 g, 10.84 mmol) in a 1:1 mixture of CH
2
Cl
2
:MeOH (40 mL) was added
camphorsulfonic acid (2.26 g, 9.70 mmol) at room temperature. The progress of the
reaction was monitored by TLC. The reaction was over by 30 min, it was then
quenched with Et
3
N (1.40 mL, 10.84 mmol). The solvent was evaporated to dryness,
and then added a saturated aqueous solution of NH
4
Cl, extracted with ether (50 mL x 3),
washed with brine, and dried over MgSO
4
. The crude product was purified on a silica
gel column using 10 %EtOAc/hexanes to afford the primary alcohol 2.17 (3.3 g, 92%).

O
OTBDPS
2.18

2.4.15 2R-2-(t-butyldiphenylsilyloxy)-butanal (2.18). To a solution of DMSO (3.17
mL, 41.0 mmol) in dry CH
2
Cl
2
(50 mL) at -78
o
C was slowly added oxalyl chloride
(2.26 mL, 26.0 mmol) and stirred for 15 min, and then added the alcohol 33 (4.5 g,
13.69 mmol) in CH
2
Cl
2
(5 mL) through a cannula, and stirred for 50 min. Et
3
N (9.05
mL, 65.0 mmol) was then added to the reaction mixture, and stirred for 3 h at -78
o
C,
and allowed it to come to the room temperature. The reaction was quenched with a
saturated aqueous solution of NH
4
Cl, extracted with ether (40 mL x 3), washed with
brine, dried over anhydrous MgSO
4
, and concentrated under reduced pressure. The
crude product was purified on a silica column using 10 %EtOAc/hexanes as the eluant
44
to give the aldehyde 34 (4.01 g, 91%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
)
δ
H
9.56 (d, J = 1.6 Hz, 1H), 7.62 (m, 4H), 7.40-7.31 (m, 6H), 3.96 (td, J = 6.0, 1.6 Hz,
1H), 1.70-1.58 (m, 2H), 1.10 (s, 9H), 0.87 (t, J = 7.6 Hz, 3H).

OTBS
I
2.4

2.4.16 3R, 3-(t-butyldimethylsilyloxy)-1-iodo-pent-1-en (2.4). To a stirring
suspension of CrCl
2
(7.62 g, 62.0 mmol) in dry THF (50 mL) at 0
o
C was added a
solution of CHI
3
(9.04 g, 23.0 mmol) and aldehyde 2.18 (2.5 g, 7.6 mmol) in dry THF
(5 mL). The reaction mixture was stirred at 0
o
C for 3 h, then warmed upto room
temperature and stirred for an additional 1 h. The reaction was quenched with H
2
O (30
mL), extracted with pentane (50 mL x 3), washed with brine, dried over MgSO
4
, and
evaporated to dryness to give a crude product. The crude was then dissolved in dry
THF (10 mL) and was added 1.0 M solution of TBAF (9.2 mL, 9.2 mmol) at 0
o
C and
stirred for overnight. The reaction was quenched with a saturated aqueous solution of
NH
4
Cl (15 mL), extracted with ether (15 mL x 3), washed with brine, dried over
MgSO
4
, and concentrated under reduced pressure. The crude product was purified on a
silica column using 7% EtOAc/hexane to give a pure vinyl iodide (0.9 g, 56% for two
steps).
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.55 (dd, J = 14.4, 6.4 Hz, 1H), 6.33 (dd, J =
14.4, 0.8 Hz, 1H), 4.01 (qd, J = 6.4, 0.8 Hz, 1H), 1.54 (quintet, J = 7.6 Hz, 2H), 0.91 (t,
J = 7.6 Hz, 3H). The OH group of the vinyl iodide (0.9 g, 4.24 mmol) was protected
again using TBS-OTf (1.26 mL, 5.50 mmol) and 2,6-lutidine (1.48 mL, 12.70 mmol) in
45
dry CH
2
Cl
2
(10 mL). The reaction mixture was stirred at room temperature for
overnight. The reaction was quenched with a saturated aqueous solution of NH
4
Cl,
extracted with pentane (15 mL x 3), washed with brine, and dried over MgSO
4
. The
crude product was purified on a silica column using 1% EtOAc/pentane as the eluant to
give pure vinyl iodide 2.4 (1.3 g, 95%) as a colorless oil.
1
H-NMR (250 MHz, CDCl
3
)
δ
H
6.49 (dd, J = 14.4, 5.8 Hz, 1H), 6.17 (dd, J = 14.5, 1.5 Hz, 1H), 4.00 (qd, J = 6.0, 1.5
Hz, 1H), 1.47 (quintet, J = 7.6 Hz, 2H), 0.87 (s, 9H), 0.85 (t, J = 7.6 Hz, 3H), 0.02 (s,
3H), 0.00 (s, 3H).

TMS OTBS O
OMe
2.19
OTBS

2.4.17 Methyl 8(E/Z), 10E, 5S, 12R-bis(t-butyldimethylsilyloxy)-15-trimethylsilyl-
pentadeca-8, 10-diene-6, 14-diynoate (2.19). To a solution of vinyl bromide 2.3 (1.0 g,
2.58 mmol) in benzene (4 mL) was added Et
3
N (3.2 mL, 23.0 mmol) and the terminal
alkyne 2.2 (0.62 g, 2.30 mmol) in benzene (1.0 mL) by a cannula. The mixture was
freeze-thaw several times at -78
o
C to remove oxygen. The reaction mixture was
warmed to room temperature followed by the addition of Pd(Ph
3
)
4
(265 mg, 0.23 mmol)
and CuI (87.5 mg, 0.46 mmol). The reaction mixture was protected from the light by
warping the flask with the aluminum foil. The reaction mixture was then stirred at
room temperature for overnight. The reaction was quenched with saturated aqueous
solution of NH
4
Cl, extracted with ether (25 mL x 3), washed with brine. The organic
layers were combined, dried over MgSO
4
, and concentrated in vacuuo to give the crude
46
product, which was then purified on a silica column using 1% EtOAc/hexane to give the
protected bis-alkyne 2.19 (1.0 g, 90%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.63 (dd, J =
15.2, 10.8 Hz, 1H), 6.27 (t, J = 11.2 Hz, 1H), 5.80 (dd, J = 15.6, 6.4 Hz, 1H), 5.38 (d, J
= 11.2 Hz, 1H), 4.46 (t, J = 6.4 Hz, 1H), 4.28 (q, J = 6.4 Hz, 1H), 3.60 (s, 3H), 2.29 (m,
4H), 1.67 (m, 4H), 0.84 (s, 18H), 0.07 (s, 9H), 0.06 (s, 3H), 0.05 (s, 3H), 0.03 (s, 3H),
0.00 (s, 3H).

TMS OTBS O
OMe
2.20
OTBS

2.4.18 Methyl 8E, 10E, 5S, 12R-bis(t-butyldimethylsilyloxy)-15-trimethylsilyl-
pentadeca-8, 10-diene-6, 14-diynoate (2.20). The mixture of 8Z/8E isomers of 2.19
(0.9 g, 1.78 mmol) was dissolved in dry CH
2
Cl
2
(100 mL), then a small crystal of
sublimed I
2
(30 mg) was added to the mixture and stirred at room temperature for three
days under the sunlight. The violet color solution was then quenched with a saturated
aqueous solution of Na
2
S
2
O
5
, extracted with ether (40 mL x 3), and washed with brine.
The organic layers were combined, dried over anhydrous MgSO
4
, filtered, and
concentrated under reduced pressure. The residue was purified on a silica gel column
using 3% EtOAc/hexane as the eluant to give the desired 8E, 10E isomer, 2.20 (0.75 g,
85%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.84 (dd, J = 14.0, 10.4 Hz, 1H), 6.46 (d, J =
14.0 Hz, 1H), 6.40 (dd, J = 15.2, 10.8 Hz, 1H), 5.95 (dd, J = 14.8, 6.8 Hz, 1H), 4.42 (t, J
= 5.2 Hz, 1H), 4.32 (q, J = 6.4 Hz, 1H), 3.65 (s, 3H), 2.44-2.32 (m, 4H), 1.75 (m, 1H),
47
1.60-1.54 (m, 3H), 0.89 (s, 18H), 0.13 (s, 9H), 0.09 (s, 3H), 0.08 (s, 9H), 0.05 (s, 3H),
0.04 (s, 3H).

OTBS O
OMe
2.21
OTBS

2.4.19 Methyl 8E, 10E, 5S, 12R-bis(t-butyldimethylsilyloxy)pentadeca-8, 10-diene-6,
14-diynoate (2.21). To a solution of TMS protected alkyne 2.20 (0.60 g, 1.48 mmol) in
MeOH (70 mL) was added one scoop of Na
2
CO
3
, and stirred for overnight. The cloudy
mixture was concentrated in vacuo to remove MeOH, and then added H
2
O (25 mL) to
quench the reaction, extracted with ether (35 mL x 3), washed with brine, dried over
MgSO
4
. The crude was purified over silica column using 2% EtOAc/hexanes as the
eluant to afford the alkyne 2.21 (0.50 g, 95%) as a colorless oil.
1
H-NMR (400 MHz,
CDCl
3
) δ
H
6.86 (dd, J = 13.6, 10.8 Hz, 1H), 6.47 (d, J = 14.0 Hz, 1H), 6.43 (dd, J =
15.6, 10.4 Hz, 1H), 6.00 (dd, J = 15.2, 6.0 Hz, 1H), 4.42 (t, J = 5.6 Hz, 1H), 4.34 (q, J =
6.8 Hz, 1H), 3.65 (s, 3H), 2.34 (m, 4H), 2.00 (t, J = 1.8 Hz, 1H), 1.75 (m, 1H), 1.58 (m,
3H), 0.89 (s, 18H), 0.10 (s, 3H), 0.08 (s, 3H), 0.06 (s, 3H), 0.05 (s, 3H);
13
C-NMR (125
MHz, C
6
D
6
) δ
C
173.9, 140.7, 137.4, 129.2, 111.1, 93.4, 83.4, 80.9, 71.3, 70.2, 63.0, 51.5,
37.9, 33.5, 28.3, 25.8, 20.6, 18.1, -4.4, -4.6, -4.9, -5.0.

48
OTBS O
OMe
OTBS
OTBS
2.22

2.4.20 Methyl 5S, 12R, 18R, 8E, 10E, 16E, tris(t-butyldimethylsilyloxy)-eicosa-
8,10,16-triene-6,14-diynoate (2.22). To a solution of vinyl iodide 2.4 (264 mg, 0.81
mmol) in benzene (3 mL) was added Et
3
N (0.80 mL, 6.30 mmol) and the alkyne 2.21
(315 mg, 0.63 mmol) in benzene (1.0 mL) by a cannula. The mixture was freeze-thaw
several times at -78
o
C to remove oxygen. The reaction mixture was warmed to room
temperature followed by the addition of Pd(Ph
3
)
4
(73 mg, 0.063 mmol) and CuI (25 mg,
0.13 mmol). The reaction mixture was protected from the light by warping the flask
with the aluminum foil. The reaction mixture was then stirred at room temperature for
overnight. The reaction was quenched with saturated aqueous solution of NH
4
Cl,
extracted with ether (25 mL x 3), washed with brine. The organic layers were
combined, dried over MgSO
4
, and concentrated in vacuuo to give the crude product,
which was then purified on a silica column using 2% EtOAc/hexane to give the TBS-
protected alkyne precursor of resolvin E1, 2.22 (354 mg, 80%).
1
H-NMR (400 MHz,
CDCl
3
) 6.52 (dd, J = 15.2, 11.2 Hz, 1H), 6.24 (dd, J = 16.0, 11.2 Hz, 1H), 6.01 (dd, J =
16.0, 5.6 Hz, 1H), 5.85 (dd, J = 14.8, 5.6 Hz, 1H), 5.58 (d, J = 15.6 Hz, 1H), 5.60 (d, J
= 15.6 Hz, 1H), 4.49 (t, J = 8.0 Hz, 1H), 4.32 (q, J = 6.0 Hz, 1H), 4.06 (q, J = 6.0 Hz,
1H), 3.66 (s, 3H), 2.50-2.30 (m, 4H), 1.78-1.69 (m, 4H), 1.48 (quintet, J = 7.6 Hz, 2H),
0.88 (s, 27H), 0.85 (t, J = 7.6 Hz, 3H), 0.12 (s, 3H), 0.10 (s, 3H), 0.07 (s, 3H), 0.04 (s,
3H), 0.03 (s, 3H), 0.02 (s, 3H);
13
C-NMR (125 MHz, C
6
D
6
) δ
C
171.1, 145.7, 141.2,
49
138.4, 129.4, 111.5, 109.9, 94.0, 87.5, 84.1, 81.1, 74.0, 72.0, 63.6, 50.9, 38.3, 33.6, 31.3,
29.2, 26.0, 21.2, 18.4, 9.2, -4.2, -4.4, -4.6, -4.7, -4.8, -5.1.

OH O
OMe
OH
OH
2.23

2.4.21 Methyl 5S, 12R, 18R, 8E, 10E, 16E, eicosa-5, 12, 18-trihydroxy-8, 10, 16-
triene-6, 14-diynoate (2.23). To a solution of tri-protected precursor of RvE1, 2.22
(160 mg, 0.23 mmol) in THF (5 mL) at 0
o
C was added TBAF (1.10 mL of 1M solution
in THF, 1.10 mmol). The reaction mixture was stirred for overnight at room
temperature, and then quenched with saturated aqueous solution of NH
4
Cl, extracted
with ether (15 mL x 3), washed with brine, and dried over MgSO
4
. The combined ether
extract was then treated with freshly prepared diazomethane to convert the free acid to
the methyl ester. The solution was then bubbled with nitrogen to remove excess
diazomethane. The crude product was purified over a silica column using 3%
MeOH/CH
2
Cl
2
to afford the bis-acetylenic precursor of RvE1 methyl ester 2.23 (81 mg
measured by UV, 99%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.55 (dd, J = 15.6, 11.2 Hz,
1H), 6.32 (dd, J = 14.8, 10.4 Hz, 1H), 6.07 (dd, J = 16.4, 6.0 Hz, 1H), 5.84 (dd, J = 15.2,
6.0 Hz, 1H), 5.67 (d, J = 16.4 Hz, 1H), 5.62 (d, J = 16.4 Hz, 1H), 4.51 (m, 1H), 4.34
(quintet, J = 5.0 Hz, 1H), 4.06 (quintet, J = 6.0 Hz, 1H), 3.66 (s, 3H), 2.57 (m, 2H), 2.36
(t, J = 7.2 Hz, 2H), 2.05 (d, J = 4.8 Hz, 1H, -OH), 1.95 (d, J = 4.8 Hz, 1H, -OH), 1.51 (d,
J = 4.8 Hz, 1H, -OH), 1.76 (m. 4H), 1.53 (m, 2H), 0.91 (t, J = 7.6 Hz, 3H);
13
C-NMR
50
(125 MHz, C
6
D
6
) δ
C
173.8, 145.2, 141.1, 136.7, 130.0, 111.3, 109.9, 92.7, 85.9, 84.1,
81.3, 73.5, 70.2, 62.5, 51.5, 37.1, 33.6, 29.9, 28.7, 20.6, 9.5.

OH O
OMe
OH
OH
RvE1 Methyl Ester (2.1)

2.4.22 Methyl 5S, 12R, 18R, 6Z, 8E, 10E, 14Z, 16E, eicosa-5, 12, 18-trihydroxy-6, 8,
10, 14, 16-pentaoate (1).
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.49 (dd, J = 15.6, 8.4 Hz, 1H), 6.46 (dd, J = 15.6, 10.8
Hz, 1H), 6.30 (dd, J = 14.4, 10.8 Hz, 1H), 6.22 (dd, J = 14.4, 10.8 Hz, 1H), 6.14 (t, J =
10.8 Hz, 1H), 6.07 (t, J = 11.2 Hz, 1H), 5.77 (dd, J = 14.8, 6.4 Hz, 1H), 5.71 (dd, J =
14.8, 6.8 Hz, 1H), 5.43 (quintet, J = 9.6 Hz, 2H), 4.58 (m, 1H), 4.25 (m, 1H), 4.08 (m,
1H), 3.65 (s, 3H), 2.46 (q, J = 6.8 Hz, 2H), 2.34 (t, J = 6.8 Hz, 2H), 1.65 (m, 6H), 0.91
(t, J = 7.6 Hz, 3H);
13
C-NMR (125 MHz, CDCl
3
) δ
C
177.3, 137.9, 137.1, 134.9, 134.1,
131.1, 130.3, 129.6, 128.4, 126.9, 125.3, 73.6, 71.8, 67.6, 37.1, 36.2, 33.8, 30.6, 21.2,
9.9.

51

3.00
2.29 2.29
7.87
2.17
3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 PPM

1
H-NMR (250 MHz, CDCl
3
) of 2.6

TMS
OMe
O O
2.6
52

1.00
3.00 2.97
4.30
9.43
5.56
5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 2.2

OTBS
OMe
O
2.2
53
160 140 120 100 80 60 40 20 0 PPM

13
C-NMR (100 MHz, CDCl
3
) of 2.2

54
0.87
1.00
1.10
1.55
9.68
7.86
5.24
0.84
4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 2.9
100 80 60 40 20 0 PPM

13
C-NMR (100 MHz, CDCl
3
) of 2.9
TMS OH
OTBS
2.9
55
0.99
18.33 18.39
2.07
0.99 0.98
4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 2.10

100 80 60 40 20 0 PPM

13
C-NMR (100 MHz, CDCl
3
) of 2.10
TMS OTBS
OTBS
2.10
56
0.94 1.00 0.97
1.96
0.90
8.85
12.64
4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 2.11

TMS OTBS
OH
2.11
57
0.45
1.05
4.00
0.99
0.70
6.15
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of 2.14

Br P
O
EtO
EtO
2.14
58
1.86
0.92 0.93 0.95
2.40
11.40
16.94
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 2.3

TMS OTBS
Br
2.3
59
0.89 0.87 0.88 0.90 1.00 0.94
3.00
4.13 4.05
20.06
19.89
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 2.19
OTBS TMS
OTBS
COOMe
2.19
60
0.95
1.20
0.70
0.97 0.96 0.95
3.00
3.99
2.54
0.97
23.23
19.57
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 2.20
OTBS TMS OTBS
COOMe
2.20
61
0.96
1.93
0.96 0.95 0.96
3.00
4.13
0.81
0.94
3.41
19.02
11.27
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 2.21

OTBS OTBS
COOMe
2.21
62

0.96 0.97 1.00
0.84
1.93
3.04
9.21
5.11
3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 2.15

OH
OTBS
2.15
63
4.00
5.95
0.97
2.05
2.48
9.36
2.54 2.52
9.58
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 2.16

OTBDPS
OTBS
2.16
64
0.68
4.00
5.90
0.94
2.37
9.57
5.65
9 8 7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of 2.18

OTBDPS
O
2.18
65
0.79
0.83 0.85
3.00
3.12
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of 2.24

0.60 0.61
0.74
2.00
12.50
4.59
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 2.4

OTBS
I
2.4
OH
I
2.24
66

1.06 1.10 1.17 1.10
2.05
1.03 0.98 1.01
3.00
3.91 4.10
2.25
33.27
17.78
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 2.22

OTBS OTBS
COOMe
OTBS
2.22
67
0.91 0.96 0.99 0.96
1.93
1.02 0.98 1.03
3.00
2.28 2.23
0.94 0.90
4.59
5.03
3.48
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of 2.23

OH OH
COOMe
OH
2.23
68
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of 2.1

OH OH
OMe
O
OH
2.1
6.6 6.4 6.2 6.0 5.8 5.6 5.4 PPM
69
Chapter 3. Resolvin D1 and Aspirin-Triggered Resolvin D1:
Total Synthesis, Stereochemical Assignments and Biological
Actions

3.1 Introduction

The two major omega-3 polyunsaturated fatty acids (PUFAs) in fish oil are
eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA). Experiments in animals,
as well as in human suggest that omega-3 polyunsaturated fatty acids rich fish
consumptions and dietary omega-3 supplementation reduces the incidence of many
human diseases including atherosclerosis, cardiovascular disease, cardiac sudden death,
stroke, asthma, and among others.
46
DHA is highly enriched in both brain and retina,
and is a critical component of cognitive decline and neural development
47
and shows
promise for slowing the progression of neurodegenerative diseases including
Alzheimer’s disease. The molecular mechanism of the beneficial effects of DHA or
other omega-3 fatty acids is of significant importance, yet poorly understood, until
recently Serhan and colleagues discovered
3
a new class of lipid mediators derived from
both DHA and EPA. These newly discovered lipid mediators were identified in the
resolving inflammatory exudates and in tissues enriched with DHA, and displayed

46
For very recent reviews, please see the following articles and references therein (i) Barnham, K. J.;
Masters, C. L.; Bush, A. I. Nat. Rev. Drug. Discov. 2004, 3, 205. (ii) Calder, P. J. Am. Clin. Nutr. 2006,
83, 1505. (iii) Yokoyama, M.; Origasa, H.; Matsuzaki, M.; Matsuzawa, Y.; Saito, Y.; Ishikawa, Y.;
Oikawa, S.; Sasaki, J.; Hishida, H.; Itakura, H.; Kita, T.; Kitabatake, A.; Nakaya, N.; Sakata, T.; Shimada,
K.; Shirato, K. The Lancet 2007, 369, 1090.
47
Lukiw, W. J.; Cui, J. G.; Marcheselli, V. L.; Bodker, M.; Botkjaer, A.; Gotlinger, K.; Serhan, C. N.;
Bazan, N. G. J. Clin. Invest. 2005, 115, 2774, and references therein.
70
potent bioactions.
48
These novel families of compounds were termed as resolvins
(resolution phase interaction products), and protectins. During the resolution phase of
inflammation, DHA is converted through a series of enzymatic processes to resolvin D
series and their aspirin-triggered epimers, and protectins, including RvD1 and AT-
RvD1.
3
Both RvD1 and AT-RvD1 exhibit potent anti-inflammatory activities in vivo.
Due to their physiological importance, and very minute natural abundance, and in order
to determine their absolute stereochemistry, and to expedite their further biological and
medicinal studies, and structure-activity relationship, it is extremely important to
accomplish their asymmetric total synthesis. In the light of the importance of the
determination of absolute stereochemistry, and unraveling the unknown medicinal and
biological activities, we have embarked the first total syntheses of the naturally
occurring resolvin D1 (RvD1) and its aspirin-triggered epimer, AT-resolvin D1 (AT-
RvD1) described herein.
3.2 Results and Discussion
3.2.1 First Total Synthesis of RvD1
3.2.1.1 Retrosynthetic Analysis of RvD1 (3.1)

In developing an efficient strategy for a highly convergent route to the asymmetric total
synthesis of resolvin D1 (3.1), there are several challenges and key issues that had to be
considered. The polyene system in RvD1 contains six double bonds in which three of
them are E-double bonds in conjugation with a Z-double bond, which is prone to easy

48
(i) Serhan, C. N.; Clish, C. B.; Brannon, J.; Colgan, S. P.; Chiang, N.; Gronert, K. J. Exp. Med. 2000,
192, 1197. (ii) Serhan, C. N.; Hong, S.; Gronert, K.; Colgan, S. P.; Devchand, P. R.; Mirick, G.;
Moussignac, R. L. J. Exp. Med. 2002, 196, 1025. (iii) Hong, S.; Gronert, K.; Devchand, P.; Moussignac,
R. L.; Serhan, C. N. J. Biol. Chem. 2003, 278, 14677.
71
isomerization mediated by acid, light, heat or transition metals. In our retrosynthetic
analysis, as shown in the Figure 8, we carefully envisaged to introduce the conjugated
Z-double bond in the last step by a mild selective reduction of a triple bond after
assembling the entire carbon framework of RvD1. The isolated 4Z double bond was
introduced from a modified Wittig reaction, and another isolated 19Z-double bond was
incorporated from the partial hydrogenation of a triple bond using Lindlar catalyst.
Since the absolute stereo-configuration of resolvin D1 (3.1) is not fully known, we
planned to introduce the stereochemistry of the hydroxyl groups at C-7, C-8 and C-17
from readily accessible known chiral building blocks from chiral pool such as 2-deoxy-
D-ribose (3.3) and chiral glycidol, as shown in Figure 8.

OH HO
COOMe
OH
Wittig
Chiral pool
Wittig
Pd
0
/Cu
I
coupling Lindlar reduction
Selective
hydrogenation
BrPh
3
P COOMe
3.1
O OH
O
O
TMS
PPh
3
I
OTBS
3.2 3.3 3.4 3.5

Figure 8. Retrosynthetic Analysis of Resolvin D1 (RvD1).
72
Finally, to maximize the synthetic convergency and the synthesis of number of RvD1
analogs including AT-RvD1 and its analogs, the target was divided into four main
modules (3.2, 3.3, 3.4 and 3.5) by carefully examining their connections by highly
efficient carbon—carbon bond forming reactions such as the powerful Pd
0
/Cu
I
mediated
cross coupling reaction, and Wittig reaction.
3.2.1.2 Acetonation of 2-deoxy-D-ribose (3.3)

According to the retrosynthetic strategy described above, the total synthesis of the target
molecule RvD1 (3.1) is began with the acetonation of the 2-deoxy-D-ribose (3.6) using
2-methoxypropene in presence of p-TSA in DMF at 0
o
C to give the 2-deoxy-3, 4-
isopropylidene-D-erythro-pentopyranose (3.3),
49
as shown in Scheme 7. 2-Deoxy-D-
ribose exits mainly as pyranose form at 0
o
C, although the proportion of furanose form
in water increases substantially at higher temperature.
50
Exploiting this behavior of 2-
deoxy-D-ribose, acetonation with 2-methoxypropene at 0
o
C in DMF proceeded
smoothly to give the desired six membered product (3.3) as a mixture of α- and β-
anomers (17:83, determined by
1
H-NMR) in high yield (78%).
51

49
Barbat, J.; Gelas, J.; Horton, D. Carbohydrate Research 1983, 116, 312.
50
Lemieux, R. U.; Anderson, L.; Conner, A. H. Carbohydrate Research 1971, 20, 59.
51
(i) Geng, X.; Danishefsky, S. J. Org. Lett. 2004, 6, 413. (ii) Rodriguez, A. R.; Spur, B. W. Tetrahedron
Lett. 2001, 42, 6057.
73
2-deoxy-D-ribose (3.6)
O
HO OH
OH
O
OH
O O
3.3
2-methoxypropene, p-TSA
DMF, 0
o
C, 3h, 78%;

Scheme 7. Acetonation of 2-deoxy-D-ribose.

3.2.1.3 Synthesis of 3-(carbmethoxypropyl)triphenylphosphonium bromide (3.2)
52

The title compound 3.2 was prepared in very high yield (93%) by heating an equimolar
mixture of methyl-4-bromo-butyrate (3.7) and triphenylphosphine in toluene under
reflux condition,
7
as shown in Scheme 8.

MeO
O
Br
MeO
O
PPh
3
Br
3.7 3.2
PPh
3
Toluene
100
o
C, reflux

Scheme 8. Synthesis of Wittig salt 3.2.

3.2.1.4 Synthesis of (2E, 5-trimethylsilyl-2-penten-4-ynyl-) triphenylphosphonium
bromide (3.4)

The title Wittig reagent (3.4) was prepared from a commercially available starting 1-
hydroxy-2E-penten-4-yne (3.8) by following a procedure
53
as shown in Scheme 9.
After the introduction of TMS-group at the terminal alkyne carbon, and the vinyl

52
Narayanan, K.; Berlin, D. K. J. Org. Chem. 1980, 45, 2240.
53
Nicolaou, K. C.; Veal, C. A.; Webber, S. E.; Katerinopoulos, H. J. Am. Chem. Soc. 1985, 107, 7515.
74
hydroxyl group was converted to a vinyl bromide (3.9) by NBS in presence of PPh
3
.
The vinyl bromide (3.9) was then converted to a phosphonium salt according to a
standard procedure as shown in Scheme 9. All the reactions were highly efficient.

OH
OH
TMS
Br
TMS
3.8 3.9
PPh
3
Br
TMS
3.4 3.10
a
b
c
Reagents and conditions: (a) n-BuLi, TMS-Cl, -78
o
C to rt, 4h, then
acetic acid, 30 min, 90%; (b) PPh
3
, NBS, CH
2
Cl
2
, 0
o
C to rt, 30 min, 95%;
(c) PPh
3
, C
6
H
6
, rt, 2 days, 95%.

Scheme 9. Synthesis of Wittig salt 3.4.

3.2.1.5 Synthesis of 3S, 1E, 5Z, 3-hydroxy-1-iodoocta-1,5-diene (3.5)
The synthesis of bottom vinyl iodide (3.5) with S-stereochemistry at C-3 is shown in
Scheme 10. For the construction of 17S-stereochemistry in RvD1, a commercially
available R-glycidol (3.11) was envisaged as the suitable starting material. As
described in Scheme 10, our approach gave us two advantages; first—the precise
control over the stereochemistry at the hydroxyl bearing chiral center at C-17, second—
an easy access to the synthesis of vinyl iodide intermediate for AT-RvD1 by simply
75
replacing the chiral glycidol, such as the introduction of 17R-stereochemistry in AT-
RvD1 from S-glycidol, which will be discussed later in this chapter.

As shown in Scheme 10, the R-glycidol (3.11) was first silylated with a TBS-group.
The epoxide ring of the protected glycidol was opened by 1-butyne gas with n-BuLi in
presence of BF
3
.OEt
2
at -78
o
C in dry THF. The opening occurs exclusively at the least
substituted site of the protected glycidol gave the secondary alcohol (3.12) in excellent
yield (85%).
O
HO
TBSO
OH
R-Glycidol (3.11)
HO
OTBDPS
HO
OTBDPS
3.12
3.13
O
OTBDPS OTBS
I
a
Reagents and conditions: (a) (i) TBS-Cl, imidazole, DMAP, CH
2
Cl
2
, rt, overnight, 98%; (ii)
1-butyne, n-BuLi, BF
3
.Et
2
O, THF, -78
o
C, 2h, 85%;(b) (i) TBDPS-Cl,imidazole,DMAP,
CH
2
Cl
2
, overnight, 95%; (ii) CSA, MeOH:CH
2
Cl
2
(1:1), NEt
3
,rt, 1h,99%;(c) Lindlar
catalyst, H
2
gas, quinoline, EtOAc, rt, 2h, 99%; (d) Swern oxidation, -78
o
C, 4h, 95%; (e) (i)
CrCl
2
, CHI
3
, THF, 0
o
C, 3h; (ii) TBAF, THF, rt, 2h, 70% for two steps; (iii) TBS-OTf, lutidine,
CH
2
Cl
2
, rt, overnight, 98%.
b
c
d
3.14 3.15 3.5
e S

Scheme 10. Synthesis of vinyl iodide (3.5) of RvD1.

The newly generated secondary alcohol group was silylated with a TBDPS-group,
followed by the desilylation of TBS-group by camphorsulfonic acid in MeOH-CH
2
Cl
2

(1:1) at room temperature to give a primary alcohol (3.13) with excellent yield (94% in
76
two steps). The selective desilylation of primary hydroxyl group was done by the mild
action of camphorsulfonic acid at room temperature as described in the literature.
54
The
Z-double was introduced exclusively by the partial hydrogenation of the triple bond in
3.13 using Lindlar catalyst, quinoline and EtOAc as solvent. It is worth mentioning that
when the MeOH was used as solvent, a mixture of Z/E-isomers was produced, but in
EtOAc the reaction yielded Z-isomer exclusively, and quantitatively. Swern oxidation
55

of the primary alcohol in dry CH
2
Cl
2
at -78
o
C gave the corresponding aldehyde (3.15)
in excellent yield (98%). Finally the aldehyde (3.15) was converted to the
corresponding vinyl iodide (3.5) by Takai olefination
56
using CrCl
2
and CHI
3
in dry
THF.
3.2.1.6 Assembly of three modules (3.2, 3.3 and 3.4) by Wittig reaction for the
synthesis of top alkyne intermediate (3.20)

Having achieved the synthesis four key building blocks (3.2, 3.3, 3.4 and 3.5) for the
construction of entire RvD1, we now focused on the formation of two carbon—carbon
double bond formations by Wittig reaction to synthesize the top terminal alkyne (3.20)
as shown in Scheme 11. At first, the Z-selective Wittig olefination was carried out by
exploiting the masked aldehyde character of C1 of isopropylidene protected D-ribose
(3.3) with the anion generated from the 3-(carbmethoxypropyl)triphenylphosphonium

54
Nicolaou, K. C.; Ninkovic, S.; Sarabia, F.; Vourloumis, D.; He, Y.; Vallberg, H.; Finlay, M, R. V.;
Yang, Z. J. Am. Chem. Soc. 1997, 119, 7974.
55
Mancuso, A. J.; Huang, S. L.; Swern, D. J. Org. Chem. 1978, 43, 2480.
56
Takai, K.; Nitta, K.; Utimoto, K. J. Am. Chem. Soc. 1986, 108, 7408.
77
bromide (3.4) by KN(TMS)
2
at -78
o
C in THF.
57
The reaction proceeded smoothly with
high stereoselectivity to afford Z-isomer (3.16) exclusively (98% Z-isomer detected by
1
H-NMR analysis) in high yield (75%) as shown in Scheme 11. The setting up this
reaction initially at low temperature (-78
o
C) was important since the use of higher
temperature at the beginning resulted the production of chromatographically separable
undesired 4E-isomer. The 4Z-double geometry of the double bond in 3.16 was
confirmed from the
1
H-NMR coupling constants (J = 10.8 Hz), which was further
confirmed by a series of decoupled
1
H-NMR (DMSO-d
6
, 400 MHz) experiments as
shown in Figure 9.
5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 PPM

Figure 9. Decoupled
1
H-NMR spectrum (expanded) of 3.16 (DMSO-d
6
, 400 MHz).

57
(i) Bennet III, R. B.; Choi, J. R.; Montgomery, W. D.; Cha, J. K. J. Am. Chem. Soc. 1989, 111, 2580. (ii)
Baldwin, J. E.; Calridge, T. D. W.; Culshaw, A. J.; Heupel, F. A.; Lee, V.; Spring, D. R.; Whitehead, R.
C.; Boughtflower, R. J.; Mutton, I. M.; Upton, R. J. Angew. Chem. Int. Ed. 1998, 37, 2661. (iii) Baldwin,
J. E.; Calridge, T. D. W.; Culshaw, A. J.; Heupel, F. A.; Lee, V.; Spring, D. R.; Whitehead, R. C. Chem.
Eur. J. 1999, 5, 3154.
d (J = 10.8 Hz)d (J =10.8Hz)
Decoupled
1
H-NMR spectrum
78
Dess-Martin oxidation
58
of the primary alcohol (3.16) gave the corresponding aldehyde
(3.17) in high yield (85%). The second set of Wittig reaction was carried out between
the triphenylphosphonium salt (3.4) and the aldehyde (3.17) using n-BuLi at -78
o
C
afforded the 9Z,11E/9E,11E (2:1) mixture in high yield (85%) as shown in Scheme 11.

58
(i) Dess, D. B.; Marin, J. C. J. Org. Chem. 1983, 48, 4155. (ii) Dess, D. B.; Martin, J. C. J. Am. Chem.
Soc. 1991, 113, 7277.
79
HO
O O
COOMe
O
OH
O O
O O
COOMe
TMS
Reagents and conditions: (a) KHMDS, THF, -78
o
C, then 3.2 at -78
o
C, brought to rt
by overnight, 75% (b) Dess-Martin, CH
2
Cl
2
,rt, 2h,85%; (c) 3.4, n-BuLi, -78
o
Cto
0
o
C, then 3.17 at -78
o
C, 3h, 85%; (d) I
2
,CH
2
Cl
2
,overnight,85%; (e) (i)1M HCl,
MeOH:H
2
O (1:1), 40 min, 82%; (ii) TBS-OTf, 2,6-lutidine, CH
2
Cl
2
, rt, overnight,
95%; (iii) Na
2
CO
3
, MeOH, rt, overnight, 88%.
9
MeO
O
PPh
3
Br
O
O O
COOMe
O O
COOMe
TMS
OTBS TBSO
COOMe
PPh
3
Br
TMS
3.2 3.3 3.16
3.4 3.17
a
b
c
3.18
3.19
3.20
d
e

Scheme 11. Synthesis of the top terminal alkyne intermediate 3.20.

80
The undesired 9Z-isomer was easily isomerized to 9E, however, a possible problem was
the stability of the 4Z-double bond, which might isomerizes to 4E-double bond even
though it is a quite stable, an isolated Z-double bond. In our experimental conditions
with catalytic amount of sublimed I
2
in CH
2
Cl
2
at room temperature resulted the
formation of desired 4Z, 9E, 11E (3.19) isomer in very high yield (85%). The iodine
induced isomerization also gave a trace amount of undesired 4E, 9E, 11E isomer (~5%
confirmed by
1
H-NMR analysis), which was separable by flash column chromatography.

In order to avoid acid catalyzed deprotection in the final stage of the molecule, which
could make a mess in the last step, we decided to deprotect the isopropylidene group at
this stage by 1N HCl in MeOH:H
2
O (1:1) , and the protect the free hydroxyl groups
again by TBS-OTf, and 2,6-lutidine, followed by the Na
2
CO
3
mediated desilylation of
TMS-group afforded the terminal alkyne (3.20) in excellent yield (88%) as shown in
Scheme 11.

3.2.1.7 Final assembly of RvD1 methyl ester (3.1)

With terminal alkyne (3.20) and the fourth module, vinyl iodide (3.5) in hands, we now
focused on the Pd
0
/Cu
I
mediated cross coupling reaction, called Sonogashira coupling,
59

59
(i) Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett. 1975, 16, 4467. (ii) Nicolaou, K. C.;
Ramphal, J. Y.; Petasis, N. A.; Serhan, C. N. Angew. Chem. Int. Ed. 1991, 30, 1100, and references
therein. (iii) Petasis, N. A.; Aritopoulou-Zanze, I.; Fokin, V. V.; Bernasconi, G.; Keledjian, R.; Yang, R.;
Uddin, J.; Nagulapalli, K. C.; Serhan, C. N. Prostaglandins, Leukotrienes and Essential Fatty Acids 2005,
73, 301, and references therein.
81
of these two advantaged stage intermediates to construct the entire carbon-framework of
RvD1 methyl ester (3.1). The reaction sequences are depicted in Scheme 12.
OTBS TBSO
COOMe
3.20
Reagents and conditions: (a) Pd(Ph
3
)
4
, CuI, C
6
H
6
, Et
3
N, rt, overnight,
95%; (b) TBAF, THF, rt, overnight followed by CH
2
N
2
, ether, rt, 2 h,
90%; (c) Zn (Cu/Ag), H
2
O:MeOH (1:1), 40
o
C, overnight, 85%.
OTBS
I
3.5
3.21
3.22
b
OH HO
COOMe
OH
RvD1 Methyl ester (3.1)
OTBS TBSO
COOMe
OTBS
OH HO
COOMe
OH
c
a
S

Scheme 12. Final assembly of resolvin D1 (RvD1) methyl ester (3.1).

Under standard Sonogashira reaction conditions with catalytic amount of
palladium[tetrakis-(triphenylphosphine)] and CuI in presence of triethylamine in
benzene at room temperature, the coupling of terminal alkyne (3.20) with the vinyl
iodide (3.5) proceeded smoothly in excellent yield (95%) furnishing the complete
82
carbon framework of resolvin D1 (3.21). Subsequent desilylation of TBS groups in
3.21 with fluoride anion in THF gave the trihydroxy compound 3.22 along with the
formation of free carboxylic acid. However, the free acid was conveniently converted
to the corresponding methyl ester 3.22 by treating it with the freshly prepared CH
2
N
2
in
ether at room temperature. Finally, the selective hydrogenation of the acetylenic
precursor of RvD1 methyl ester (3.22) by Zn(Ag/Cu) amalgam
60
in MeOH:H
2
O (1:1) at
40
o
C resulted the final product of 7S, 8R, 17S, 4Z, 9E, 11E, 13Z, 15E, 19Z-resolvin D1
(RvD1) methyl ester (3.1) in excellent yield (85%) without the formation any side-
product or over-hydrogenated product. The final product was purified by a RP-HPLC
on ODS column (Phenomenex C18, 10.2 x 250 mm) using MeOH-H
2
O (2:1) as the
solvent system. The structure of the final RvD1 methyl ester was characterized by the
extensive analyses of 1-and 2D NMR, UV, and Mass spectral data.

60
Boland, W.; Schroer, N.; Sieler, C.; Feigel, M. Helv. Chim. Acta 1987, 70, 1025.
83
3.2.2 First Total Synthesis of Aspirin-Triggered RvD1 (AT-RvD1)
The aspirin-triggered resolvin D1 (AT-RvD1) and resolvin D1 (RvD1) are
diastereomers, differ the stereoconfiguration at C-17. In RvD1, the stereochemistry is
17S and in AT-RvD1 it is 17R. In our carefully designed synthetic plan, we envisaged
setting the stereochemistry at C-17 from the chiral glycidol, for example 17S
stereochemistry in RvD1 was derived from the R-glycidol as described above in
Schemes 5 and 7. The 17R stereochemistry in AT-RvD1 could be introduced in the
same way from the commercially available S-glycidol as shown in retrosynthetic
analysis of AT-RvD1 (3.1') in Figure 10.

OH HO
COOMe
OH
Wittig
Chiral pool
Wittig
Pd
0
/Cu
I
coupling Lindlar reduction
Selective
hydrogenation
BrPh
3
P COOMe
3.1'
O OH
O
O
TMS
PPh
3
I
OTBS
3.2 3.3 3.4 3.5'
R
R
O
OH
(S) glycidol

Figure 10. Retrosynthetic analysis of AT-RvD1 methyl ester (3.1')

84
The synthesis of vinyl iodide intermediate (3.5') was described in Scheme 13, and rests
of the intermediates (3.2, 3.3 and 3.4) were synthesized by the same reaction sequences
as described for the RvD1. The carbon—carbon bond forming reactions of 3.2, 3.3 and
3.4 to give to the top terminal alkyne 3.20 were accomplished as described for RvD1 in
this chapter as shown in Scheme 11.

O
HO
TBSO
OH
S-Glycidol (3.11')
HO
OTBDPS
HO
OTBDPS
3.12'
3.13'
O
OTBDPS OTBS
I
a
Reagents and conditions: (a) (i) TBS-Cl, imidazole, DMAP, CH
2
Cl
2
, rt, overnight, 98%; (ii)
1-butyne, n-BuLi, BF
3
.Et
2
O, THF, -78
o
C, 2 h, 82%; (b) (i) TBDPS-Cl, imidazole, DMAP,
CH
2
Cl
2
, overnight, 85%; (ii) CSA, MeOH:CH
2
Cl
2
(1:1), NEt
3
,rt, 1h,99%;(c) Lindlar
catalyst, H
2
gas, quinoline, EtOAc, rt, 2h, 90%; (d) Swern oxidation, -78
o
C, 4h, 99%; (e) (i)
CrCl
2
, CHI
3
,THF, 0
o
C, 3h; (ii) TBAF, THF, rt, 2h; (iii) TBS-OTf, lutidine, CH
2
Cl
2
,rt,
overnight, (52% in three steps).
b
c
d
3.14' 3.15' 3.5'
e R

Scheme 13. Synthesis of vinyl iodide 3.5' for AT-RvD1.

The entire carbon skeleton of AT-RvD1 was accomplished from the Pd
0
/Cu
I
mediated
coupling reaction from the common terminal alkyne (3.20) and the vinyl iodide (3.5')
using the standard conditions as shown in Scheme 14. Desilylation TBS group by
TBAF, followed by the Z-selective partial hydrogenation of alkyne precursor 3.22'
afforded the AT-RvD1 methyl ester (3.1') with desired stereochemistries in excellent
85
overall yield. The final product was purified by running a preparative reversed-phase
HPLC on ODS column (Phenomenex C18, 10.2 x 250 mm) using MeOH-H
2
O (2:1) as
the solvent system. The structure of the final AT-RvD1 methyl ester was elucidated
from the analyses of 1- and 2D-NMR (CDCl
3
, 400 MHz), UV and MS spectra data.

OTBS TBSO
COOMe
3.20
Reagents and conditions: (a) Pd(Ph
3
)
4
, CuI, C
6
H
6
, Et
3
N, rt, overnight,
95%; (b) TBAF, THF, rt, overnight followed by CH
2
N
2
, ether, rt, 2 h,
91%; (c) Zn (Cu/Ag), H
2
O:MeOH (1:1), 40
o
C, overnight, 86%.
OTBS
I
3.5'
3.21'
3.22'
b
OH HO
COOMe
OH
AT-RvD1 Methyl ester (3.1')
OTBS TBSO
COOMe
OTBS
OH HO
COOMe
OH
c
a
R

Scheme 14. Final coupling reaction for AT-RvD1 methyl ester (3.1')

86
3.3 Stereochemical Assignments of RvD1 and AT-RvD1

The stereochemistry of the conjugated tetraene-moiety and R/S-configurations of
hydroxyl group bearing carbon centers in RvD1 and AT-RvD1 were determined in
collaboration with Serhan’s group and published.
61
The complete stereochemical
assignments were established by matching the physical and biological properties of
RvD1 and AT-RvD1 obtained from asymmetric total organic synthesis and
enzymatically produced biogenic compounds as follows.

OH HO
COOMe
OH
Resolvin D1 (RvD1)
OH HO
COOMe
OH
Aspirin-Triggered Resolvin D1 (AT-RvD1)
S R

Figure 11. Structures of RvD1 and AT-RvD1 methyl esters.

The structure and stereochemistry of synthetic RvD1 and AT-RvD1 were
unambiguously established from their stereocontrolled organic total syntheses using
known chiral starting materials as described in the synthetic section of this chapter.
According to the reaction sequences and chiral starting materials used, the
stereochemistry of resolvin D1 (RvD1) methyl ester was set to be
4Z,7S,8R,9E,11E,13Z,15E,17S,19Z-docosahexaenoate, and AT-RvD1 methyl ester was
planned to be 4Z,7S,8R,9E,11E,13Z,15E,17R,19Z-docosahexaenoate. The Z/E
configurations of the double bonds in RvD1 methyl ester and AT-RvD1 methyl ester as

61
Sun, Y. P.; Oh, S.; Uddin, J.; Yang, R.; Gotlinger, K.; Campbell, E.; Colgan, S. P.; Petasis, N. A.;
Serhan, C. N. J. Biol. Chem. 2007, 282, 9323.
87
well as the proton assignments were determined from the
1
H-NMR (400 MHz, CDCl
3
)
coupling constants and COSY analysis (Figure 12). Both RvD1 and AT-RvD1 methyl
ester gave a similar UV absorbance at λ
max
301 nm with shoulders at 289 nm and 315
nm, which are the characteristic peaks for a conjugated tetraene moiety.

88

Figure 12.
1
H-NMR assignments of RvD1 and AT-RvD1 methyl ester based on the analysis of
COSY spectra. The NMR spectrum of olefinic region of RvD1 methyl ester is shown. J. Biol.
Chem. 2007, 282, 9323.
89
RP-HPLC analysis of RvD1 and AT-RvD1 displayed different behavior as, expected
since they are diastereomers, when co-injected. AT-RvD1 eluted first with retention
time 13.9 min, and then RvD1 came out at 15.1 min. Under the experimental setting
with column and mobile phase that allowed the separation of these two diastereomers
by ~1.2 min as shown in the Figure 13. Even though they eluted with different
retention time, however, MS/MS spectrum obtained from both RvD1 and AT-RvD1 and
biogenic RvD1 gave essentially the same fragmentation patterns as expected (Figure
13).

Figure 13. HPLC chromatogram at UV absorbance 301 nm, and MS/MS analysis of
synthetic RvD1, AT-RvD1 and biogenic RvD1. J. Biol. Chem. 2007, 282, 9323.
90
Having established the physical properties of synthetic RvD1, and its 17R-epimer, next
is to compare whether it was identical to the biogenic RvD1 to assign the complete
stereochemistry. The biological activities, UV absorbance, RP-HPLC chromatograms
(Figure 13), GC/MS and MS/MS fragmentation patterns (Figure 13) of biogenic RvD1
and AT-RvD1 were identical with the synthetically produced stereochemically pure
RvD1 and AT-RvD1 thus confirming their absolute stereostructures as
4Z,7S,8R,9E,11E,13Z,15E,17S,19Z for RvD1 and 4Z,7S,8R,9E,11E,13Z, 15E,17R, 19Z
for AT-RvD1.

Figure 14. Comparison of enzymatic RvD1 obtained via LOX-catalyzed synthesis
and total organic synthesis. J. Biol. Chem. 2007, 282, 9323.
91
3.4 Biological Activities of RvD1 and AT-RvD1

RvD1 and AT-RvD1 are generated locally in response to inflammatory stimuli, and
each proved to potent regulators of both human and murine PMN.
62
When freshly
isolated human PMNs were incubated with RvD1 and AT-RvD1, they both stopped
transendothelial migration of human neutrophils (EC
50
~30 nm).
63
In murine peritonitis
in vivo, RvD1 and AT-RvD1 found to be equipotent at nanogram dosages, limiting
PMN infiltration in dose-dependent fashion (Figure 15).
18

Figure 15. Anti-inflammatory actions of RvD1 and AT-RvD1.
J. Biol. Chem. 2007, 282, 9323.

62
(i) Serhan, C. N.; Hong, S.; Gronert, K.; Colgan, S. P.; Devchand, P. R.; Mirick, G.; Moussignac, R. L.
J. Exp. Med. 2002, 196, 1025. (ii) Hong, S.; Gronert, K.; Devchand, P.; Moussignac, R. L.; Serhan, C. N.
J. Biol. Chem. 2003, 278, 14677.
63
Sun, Y. P.; Oh, S.; Uddin, J.; Yang, R.; Gotlinger, K.; Campbell, E.; Colgan, S. P.; Petasis, N. A.;
Serhan, C. N. J. Biol. Chem. 2007, 282, 9323.
92

The relative potencies of RvD1 and AT-RvD1 were established using the synthetically
produced pure compounds because biogenically produced materials were given a
mixture of RvD1 and AT-RvD1 as well as other additional resolvins.
17
Dose-dependent
comparison of the reduction of PMN infiltration using synthetic RvD1 and AT-RVD1
in peritonitis and air pouch at equal doses of 100 ng/mouse demonstrated that RvD1 and
AT-RvD1 exhibited similar potency and efficacy with maximal inhibition of ~35%
(Figure 15), although at the dose of 10 ng/mouse, AT-RvD1 was more active than
RvD1 (~23% reduction of leukocytic infiltration compared to ~10%) (Figure 15).
Serhan and colleagues recently reported
19
that RvD1 was generated in response to
bilateral ischemia/reperfusion injury in mouse kidneys. Administration of RvD1 along
with other resolvins before or after ischemia resulted a considerable reduction in
functional and morphological kidney injury.
64

3.5 Conclusion

The endogenous DHA in brain, synapses and retina was converted to resolvin D1
(RvD1) without aspirin, and the exogenously given DHA and aspirin in mice converted
DHA to AT-RvD1 in resolving exudates. Both RvD1 and AT-RvD1 are highly potent,
anti-inflammatory, pro-resolving lipid mediators. The first total syntheses RvD1 methyl
ester and AT-RvD1 methyl ester were accomplished and described herein. Our
approach was not only very practical to synthesize the molecules and their analogs in

64
Duffied, J. S.; Hong, S.; Vaidya, V.; Lu, Y.; Fredman, G.; Serhan, C. N. J. Immunol. 2006, 177, 5902.
93
ample quantities for further chemical and biological studies, but also commenced in
highly convergent way from easily available stating materials and reagents. We heavily
relied on very efficient Wittig and modified-Wittig reactions, and Pd
0
/Cu
I
mediated
cross-coupling reaction to construct the entire carbon frame-works of RvD1 and AT-
RvD1, and the absolute stereochemistries were unambiguously incorporated from
natural chiral pool and from known chiral glycidols. The geometrically and
enantiomerically pure synthetic RvD1 and AT-RvD1 matched with the physical and
biological properties of those enzymatically generated for the complete stereochemical
assignments. RvD1 was established to be 7S,8R,17S-trihydroxy-
4Z,9E,11E,13Z,15E,19Z-docosahexaneoic acid, and AT-RvD1 was proved to be
7S,8R,17R-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaneoic acid. Both RvD1 and
AT-RvD1 are potent regulators of PMN infiltration in vivo and in vitro. AT-RvD1 was
found to be superior drug at lower doses compared to RvD1, but both compounds were
bioactive at the lowest dose administrated (1 ng/mouse), demonstrating the extreme
potencies of these mediators.

94
3.6 Experimental for RvD1 and AT-RvD1

All reactions, unless otherwise noted, were carried in flame dried flasks under argon
atmosphere. “Dried and concentrated” refers to removal of residual water with
anhydrous MgSO4, followed by evaporation of the solvent on the rotary evaporator.
THF was freshly distilled from sodium-benzophenone, benzene and dichloromethane
from CaH2 and anhydrous DMF, EtOH, and MeOH were purchased from commercial
sources. 1H and 13C NMR spectra were recorded on a Varian Mercury 400 and a
Bruker AC-250 using residual 1H or 13C signals of deuterated solvents as internal
standards. UV spectra were recorded on a Hewlett-Packard 8350 instrument. HPLC
analyses were performed on a Rainin dual pump HPLC system equipped with a
Phenomenex ODS column and an UV-VIS detector.

OH
TMS
3.9

3.6.1 5-Trimethylsilyl-2E-penten-4-yn-1-ol (3.9). To a solution of 2-pentene-4-yn-1-
ol 3.8 (5.0 g, 60.90 mmol) in dry THF (100 mL) at -78
o
C was added drop-wise a 1.6M
solution of n-BuLi (48.72 mL, 121.80 mmol). The resulting brown slurry was stirred
vigorously for 1 h, and then TMS-Cl (15.5 mL, 121.80 mmol) was slowly added. The
reaction mixture was brought to room temperature and stirred for 4 h. The resulting
slurry was treated with acetic acid (15.0 mL), and stirred for 30 min, and then
neutralized with a saturated aqueous solution of NaHCO
3
. The product was extracted
with ether (100 mL x 3), washed with brine. The combined ether extracts was dried
95
over MgSO
4
, filtered, and concentrated in vacuo. The crude product was purified on
silica column chromatography using 12% EtOAc/hexane to give pure 3.9 (8.4 g, 90%).
1
H-NMR (250 MHz, CDCl
3
) δ
H
6.24 (1H, dt, J = 16.0, 5.3 Hz), 5.57 (1H, dt, J = 16.0,
1.3 Hz), 4.13 (2H, dd, J = 5.3, 2.0 Hz), 0.14 (9H, s).

Br
TMS
3.10

3.6.2 1-Bromo-5-trimethylsilyl-2E-penten-4-yne (3.10). To a solution of 5-
trimethylsilyl-2-penten-4-yn-1-ol 3.9 (4.10 g, 26.5 mmol) in dry CH
2
Cl
2
(50 mL) was
added triphenylphosphine (7.6 g, 29.2 mmol) and N-bromosuccinimde (5.2 g, 29.2
mmol) at 0
o
C. The reaction mixture was warmed to room temperature and stirred for
30 min, and then quenched with a saturated solution of NaHCO
3
. The crude product
was extracted with ether (50 mL x 3), washed with brine, dried over MgSO
4
, and
concentrated under reduced pressure. A flash column chromatography on silica gel
using 1% EtOAc/hexane as the eluant to afford the bromide 3.10 (5.1 g, 90%) as a
colorless oil.
1
H- NMR (400 MHz, CDCl
3
) δ
H
6.28 (1H, dt, J = 15.5, 8.0 Hz), 5.73 (1H,
dt, J = 15.5, 1.8 Hz), 3.94 (2H, d, J = 8.0 Hz), 0.17 (9H, s).

PPh
3
Br
TMS
3.4

3.6.3 Phosphonium bromide salt (3.4). The bromide 3.10 (3.68 g, 16.93 mmol) and
triphenylphosphine (5.3 g, 20.31 mmol) were stirred in the dark in dry benzene (5 mL)
96
for 24 h. The off-white solid product was filtered, washed with ether (100 mL x 3) and
dried in high vacuum next to P
2
O
5
for overnight to give the salt 3.4 (7.5 g, 95%).
1
H-
NMR (400 MHz, CDCl
3
), δ
H
7.80 (9H, m), 7.65 (6H, m), 6.18 (1H, dd, J = 16.0, 7.6
Hz), 5.88 (1H, dt, J = 15.6, 7.6 Hz), 5.0 (2H, q, J = 7.6 Hz), 0.09 (9H, s);
13
C-NMR
(100 MHz, CDCl
3
) δ
C
134.8, 133.5, 130.1, 127.5, 120.1, 117.6, 116.7, 101.6, 97.3, 27.9,
-0.6.

O
TBSO
3.23

3.6.4 TBS-protected R-glycidol (3.23). To a mixture of imidazole (5.5 g, 81.0 mmol),
TBS-Cl (12.2 g, 81.0 mmol), and DMAP (0.4 g, 3.3 mmol) in dry CH
2
Cl
2
(100 mL) at 0
o
C was added R-glycidol 3.11 (5.0 g, 67.5 mmol). The reaction mixture was warmed to
room temperature, and stirred for overnight. The reaction mixture was quenched with a
saturated aqueous solution of NH
4
Cl, extracted with ether (100 mL x 3), washed with
brine, dried over MgSO
4
, and concentrated under reduced pressure to give a crude
product. The crude product was purified on silica column using 5% EtOAc/hexane as
the eluant to give the protected glycidol 3.23 (12.5 g, 98%) as a colorless oil.
1
H-NMR
(400 MHz, CDCl
3
) δ
H
3.84 (1H, dd, J = 11.5, 3.1 Hz), 3.65 (1H, dd, J = 11.5, 4.8 Hz),
3.06 (1H, m), 2.76 (1H, dd, J = 5.3, 4.5 Hz), 2.62 (1H, dd, J = 5.3, 2.6 Hz).
13
C-NMR
(100 MHz, CDCl
3
) δ
C
64.1, 52.0, 44.2, 25.9, 18.0, -5.0.

97
TBSO
OH
3.12

3.6.5 2S, 1-(t-butyldimethylsilyloxy)hept-4-yn-2-ol (3.12). To a solution of 1-propyne
(6.9 g, 127.4 mmol) in dry THF (100 mL) at -78
o
C was slowly added n-BuLi (79.6 mL
of 1.6 M solution in hexane, 127.4 mmol). The mixture was stirred for 15 min, then
added BF
3
.Et
2
O (16.14 mL, 127.4 mmol) solution at -78
o
C and stirred for additional 15
min. A solution of 3.23 (12.0 g, 63.7 mmol) in THF (10 mL) was slowly added to the
flask through a cannula. The reaction mixture was stirred for 2 h at -78
o
C, and then
warmed to room temperature and stirred for an additional 45 min at rt. The reaction
was quenched with a saturated solution of NH
4
Cl, extracted with ether (100 mL x 3),
washed with brine. The organic layers were combined, dried over MgSO
4
, and
concentrated under reduced pressure. The crude product was purified on silica gel
column using 7% EtOAc/hexane to give 3.12 (13.1 g, 85%).
1
H-NMR (400 MHz,
CDCl
3
) δ
H
3.71 (1H, m), 3.67 (1H, dd, J = 9.6, 4.4 Hz), 3.56 (1H, dd, J = 9.6, 6.0 Hz),
2.35 (2H, m), 2.13 (2H, m), 1.08 (3H, t, J = 7.6 Hz), 0.87 (9H, s), 0.05 (6H, s).
13
C-
NMR (100 MHz, CDCl
3
) δ
C
83.8, 75.0, 70.4, 65.5, 25.9, 23.3, 18.3, 14.2, 12.4, -6.0.

TBSO
OTBDPS
3.24

3.6.6 2S, 1-(t-butyldimethylsilyloxy)-2-(t-butyldiphenylsilyloxy)hept-4-yne (3.24).
To a solution of TBDPS-Cl (7.72 mL, 29.6 mmol), imidazole (2.01 g, 29.6 mmol) and
DMAP (0.15 g, 1.23 mmol) in dry CH
2
Cl
2
(50 mL) at 0
o
C was added the alcohol 3.12
(6.0 g, 24.75 mmol) in CH
2
Cl
2
(5.0 mL) through a cannula. The mixture was warmed
98
to room temperature and stirred for overnight. The reaction was quenched with a
saturated aqueous solution of NH
4
Cl, extracted with ether (50 mL x 3), washed with
brine, dried over MgSO
4
, and concentrated under reduced pressure. The crude was
purified on a silica gel column using 1%EtOAc/hexane as the eluant to afford 3.24
(11.29 g, 95%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
) δ
H
7.69 (4H, m), 7.40-
7.32 (6H, m), 3.80 (1H, m), 3.54 (1H, dd, J = 10.0, 5.6 Hz), 3.49 (1H, dd, J = 10.0, 6.0
Hz), 2.36 (1H, ddt, J = 16.8, 6.0, 2.4 Hz), 2.25 (1H, ddt, J = 16.8, 5.6, 2.4 Hz), 2.09 (2H,
qt, J = 5.6, 2.4 Hz), 1.07 (3H, t, J = 7.5 Hz), 1.03 (9H, s), 0.81 (9H, s), -0.06 (3H, s), -
0.09 (3H, s);
13
C-NMR (100 MHz, CDCl
3
) δ
C
135.9, 135.8, 134.3, 134.1, 129.6, 129.5,
127.5, 83.1, 76.3, 72.6, 65.6, 23.9, 14.2, 12.5, -5.5.

HO
OTBDPS
3.13

3.6.7 2S, 2-(t-butyldiphenylsilyloxy)hept-4-yn-1-ol (3.13). To a solution of protected
diol 3.24 (9.3 g, 19.34 mmol) in a 1:1 mixture of CH
2
Cl
2
:MeOH (100 mL) was added
camphorsulfonic acid (2.69 g, 11.60 mmol) at room temperature. The progress of the
reaction was monitored by TLC. The reaction was over by 30 min, it was then
quenched with Et
3
N (1.62 mL, 11.60 mmol). The solvent was evaporated to dryness,
and then added a saturated aqueous solution of NH
4
Cl, extracted with ether (50 mL x 3),
washed with brine, and dried over MgSO
4
. The crude product was purified on a silica
gel column using 9%EtOAc/hexane to afford the primary alcohol 3.13 (7.0 g, 99%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
7.65 (4H, m), 7.43-7.34 (6H, m), 3.87 (1H, m), 3.61
(2H, d, J = 4.8 Hz), 2.37 (1H, ddt, J = 16.8, 8.0, 2.4 Hz), 2.25 (1H, ddt, J = 16.8, 7.2,
99
2.4 Hz), 2.05 (2H, qt, J = 7.2, 2.4 Hz), 1.05 (9H, s), 1.01 (3H, t, J = 7.2).
13
C-NMR
(100 MHz, CDCl
3
) δ
C
135.8, 135.6, 133.5, 133.4, 129.9, 129.8, 127.8, 127.6, 83.8, 75.2,
72.5, 65.6, 34.1, 26.9, 14.0, 12.3.

HO
OTBDPS
3.14

3.6.8 2S, 4Z, 2-(t-butyldiphenylsilyloxy)hept-4-en-1-ol (3.14). To a solution of 3.13
(6.0 g, 16.38 mmol) in EtOAc (300 mL) was added 210 mg of Lindlar catalyst and 4
drops quinoline. The reaction mixture stirred at room temperature under the static
atmosphere of hydrogen, and its progress was monitored by TLC. The reaction was
over by 2 h. The reaction mixture was then filtered through celite and solvent was
evaporated under reduced pressure. The crude product was purified on a silica column
using 12%EtOAc/hexane as the eluant to furnish 3.14 (6.0 g, 99%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
) δ
H
7.61-7.57 (4H, m), 7.36-7.27 (6H, m), 5.34 (1H, dtt, J =
10.8, 7.2, 1.2 Hz), 5.14 (1H, dtt, J = 10.8, 7.6, 1.6 Hz), 3.75 (1H, m), 3.55-3.42 (2H, m),
2.27 (1H, m), 2.12 (1H, m), 1.86-1.74 (2H, m), 1.05 (9H, s), 0.81 (3H, t, J = 7.6 Hz);
13
C-NMR (100 MHz, CDCl
3
) δ
C
137.7, 135.7, 134.4, 133.7, 133.6, 129.8, 129.7, 127.7,
127.6, 73.8, 65.5, 31.5, 19.3, 14.1.

O
OTBDPS
3.15

3.6.9 2S, 4Z, 2-(t-butyldiphenylsilyloxy)hept-4-enal (3.15). To a solution of DMSO
(0.40 mL, 5.19 mmol) in dry CH
2
Cl
2
(10 mL) at -78
o
C was slowly added oxalyl
100
chloride (0.30 mL, 3.46 mmol) and stirred for 15 min, and then added the alcohol 3.14
(0.64 g, 1.73 mmol) in CH
2
Cl
2
(5 mL) through a cannula, and stirred for 50 min. Et
3
N
(1.2 mL, 8.65 mmol) was then added to the reaction mixture, and stirred for 3 h at -78
o
C. The white reaction mixture was quenched with a saturated aqueous solution of
NH
4
Cl, extracted with ether (20 mL x 3), washed with brine, dried over anhydrous
MgSO
4
, and concentrated under reduced pressure. The crude product was purified on a
silica column using 9%EtOAc/hexane as the eluant to give the aldehyde 3.15 (0.61 g,
95%) as a colorless oil.

OTBS
I
3.5

3.6.10 3S, 1E, 5Z, 3-(t-butyldimethylsilyloxy)-1-iodoocta-1,5-diene (3.5). To a
stirring suspension of CrCl
2
(2.0 g, 16.5 mmol) in THF (20 mL) at 0
o
C was added a
solution of CHI
3
(2.6 g, 6.60 mmol) and aldehyde 3.15 (0.60 g, 1.65 mmol) in THF (10
mL). The reaction mixture was stirred at 0
o
C for 3 h, then warmed to room
temperature and stirred for an additional 1 h. The reaction was quenched with H
2
O (20
mL), extracted with pentane (30 mL x 3), washed with brine, dried over MgSO
4
, and
evaporated to dryness to give a crude product. The crude was then dissolved in THF (5
mL), was added 1.0 M solution of TBAF (1.65 mL, 1.65 mmol) to the flask at 0
o
C and
stirred for 2 h. The reaction was quenched with a saturated aqueous solution of NH
4
Cl
(15 mL), extracted with ether (15 mL x 3), washed with brine, dried over MgSO
4
, and
concentrated under reduced pressure. The crude product was purified on a silica
column using 10% EtOAc/hexane to give a pure vinyl iodide (290 mg, 70% for 2 steps).
101
The OH group of the vinyl iodide (290 mg, 1.15 mmol) was protected again using TBS-
OTf (0.52 mL, 2.30 mmol) and 2,6-lutidine (0.40 mL, 3.45 mmol) in dry CH
2
Cl
2
(15
mL). The reaction mixture was stirred at room temperature for overnight. The reaction
was quenched with a saturated aqueous solution of NH
4
Cl, extracted with pentane (15
mL x 3), washed with brine, and dried over MgSO
4
. The crude product was purified on
a silica column using 1% EtOAc/pentane as the eluant to give pure vinyl iodide 3.5 (412
mg, 98%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.50 (1H, dd, J = 14.4, 6.0
Hz), 6.18 (1H, dd, J = 14.4, 1.2 Hz), 5.45 (1H, dtt, J = 10.8, 7.6, 1.2 Hz), 5.27 (1H, dtt,
J = 10.8, 7.2, 1.2 Hz), 4.06 (1H, m), 2.20 (2H, m), 1.99 (2H, m), 0.93 (3H, t, J = 7.6 Hz),
0.02 (3H, s), 0.00 (3H, s);
13
C-NMR (100 MHz, CDCl
3
) δ
C
148.7, 135.3, 134.2, 123.5,
75.0, 35.5, 26.9, 25.9, 25.7, 20.7, 18.2, 18.5, -4.6, -4.8.

MeO
O
PPh
3
Br
3.2

3.6.11 (4-Methoxy-4-oxobutyl)triphenylphosphonium bromide (3.2). To a solution
of methyl-bromo-butyrate 3.7 (5.0 g, 27.62 mmol) in dry toluene (75 mL) was added
PPh
3
(8.69 g, 33.14 mmol). The mixture was refluxed under argon atmosphere for a
period of overnight. After the flask was cooled to room temperature, 20 mL of ether
was added to the flask, and triturated with dry ether, and filtered off the solvent to give
a white solid. The white solid was washed with ether (50 mL x 3), and dried over P
2
O
5

for overnight to give pure salt 3.2 (11.5 g, 93%).
1
H-NMR (400 MHz, CD
3
OD) δ
H

7.90-7.76 (15H, m), 3.68 (3H, s), 3.51 (2H, m), 2.63 (2H, t, J = 6.8 Hz), 1.92 (2H, m);
102
13
C-NMR (100 MHz, CDCl
3
) δ
C
172.5, 134.4, 133.0, 132.9, 129.9, 129.7, 117.6, 116.7,
51.0, 32.5, 21.2, 19.2.

O
OH
O O
3.3

3.6.12 2-Deoxy-3,4-isopropylidene-D-erythro-pentapyranose (3.3). To a solution of
2-deoxy-D-ribose 3.6 (4.0 g, 30.00 mmol) in anhydrous DMF (50 mL) at 0
o
C was
added a desiccant (Drierite, 1.0 g), and 2-methoxypropene (4.32 g, 60 mmol), followed
by p-toluenesulfonic acid (20 mg). The reaction mixture was stirred for 3 h at 0
o
C.
Sodium carbonate (~5.0 g) was added to the reaction mixture, and stirred for an
additional 1 h at rt. The solids were filtered off, and the filtrate was evaporated to give
a syrup, which was purified on a silica gel column using EtOAc/Hexane (2:3) as the
eluant to afford the acetonide, 3.3 (4.0 g, 78%) as a colorless liquid.
1
H-NMR (400
MHz, DMSO-d
6
) δ
H
6.45 (d, J = 6.4 Hz), 6.28 (d, J = 5.2 Hz) (OH-1 anomers in 4:1
ratio), 4.93 (m), 4.55 – 3.38 (m), 3.65-3.77 (m), 3.44 (m), 1.59-1.97 (m), 1.35 (s), 1.22
(s).
HO
O O
COOMe
3.16

3.6.13 Synthesis of 3.16. To a stirring suspension of (3-
carbomethoxy)triphenylphosphonium bromide, 3.2 (8.55 g, 19.30 mmol) in anhydrous
THF (30 mL) was added drop-wise KHMDS (36.18 mL, 18.09 mmol) at -78
o
C over 10
103
min. The mixture was allowed to warm to 0
o
C over 30 min to give an orange-red
solution. The mixture was cooled again at -78
o
C, and a solution of 3.3 (1.40 g, 8.04
mmol) in THF was added drop-wise through cannula. The yellow-colored mixture was
stirred at -78
o
C to room temperature over the course of overnight. The reaction
mixture was quenched with saturated aqueous NH
4
Cl solution (30 mL), extracted with
ether (50 mL x 3), washed with brine, dried over MgSO
4
, and concentrated in vacuo to
give a yellow oil. Flash column chromatography on silica gel using 40%
EtOAc/Hexane afforded 3.16 (1.5 g, 75%, Z:E ~98:2 by
1
H-NMR) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
) δ
Η
5.43 (2H, m), 4.15 (2H, m), 3.63 (3H, s), 3.61 (2H, m),
2.35 (5H, m), 2.27 (1H, m), 1.44 (3H, s), 1.33 (3H, s).
13
C-NMR (100 MHz, CD
3
OD)
δ
C
173.4, 129.6, 127.8, 107.4, 78.1, 76.7, 60.2, 51.7, 33.6, 28.5, 27.6, 25.9, 22.99.

O
O O
COOMe
3.17

3.6.14 Synthesis of aldehyde 3.17. A solution of primary alcohol 3.16 (0.40 g, 1.55
mmol) in dry CH
2
Cl
2
(5 mL) was added drop-wise to a solution of Dess-Martin
periodinane (1.3 g, 3.09 mmol) in CH
2
Cl
2
(15 mL) at room temperature. The reaction
mixture was stirred for 2 h. The reaction mixture was then quenched with a 1:1 solution
of saturated aqueous Na
2
S
2
O
3
(10 mL) and saturated aqueous NaHCO
3
(10 mL). The
aqueous phase was extracted with ether (20 mL x 3), washed with brine, dried over
MgSO
4
and concentrated under reduced pressure. A flash column chromatography on
silica gel using 35% EtOAc/Hexane as eluant to yield the aldehyde 3.17 (0.34 g, 85%)
104
as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
) δ
H
9.65 (1H, d, J = 2.0 Hz), 5.45 (2H,
m), 4.36 (1H, q, J = 6.8 Hz), 4.29 (1H, d, J = 7.2, 3.2 Hz), 2.33 (6H, m), 1.59 (3H, s),
1.38 (3H, s).
O O
COOMe
TMS
3.18

3.6.15 Synthesis of 3.18. To a suspension of phosphonium salt 3.4 (1.12 g, 2.34 mmol)
in dry THF (10 mL) was added n-BuLi (1.2 mL of 1.6 M solution in hexane, 1.87 mmol)
at -78
o
C. The dark red solution was allowed to warm to 0
o
C and stirred for 30 min
before re-cooled to -78
o
C. The aldehyde 3.17 (0.30 g, 1.17 mmol) solution in 5 mL
THF was added to the reaction mixture drop-wise via a cannula. The dark red mixture
was then brought to room temperature and stirred for 3.5 h before quenched with
saturated aqueous NH
4
Cl solution, extracted with ether (20 mL x 3) and washed with
brine. The organic layers were combined and dried over anhydrous MgSO
4
, filtered,
and concentrated under reduced pressure. The residue was purified over a silica column
using 8% EtOAc/Hexane as the eluant to afford 3.18 (0.34 g, 87%) as the mixture
9Z/9E (2:1 by
1
H-NMR) isomers.
1
H-NMR (400 MHz, CDCl
3
) of 9Z isomer δ
H
6.86
(1H, dd, J = 15.2, 11.2 Hz), 6.17 (1H, t, J = 15.6 Hz), 5.65 (1H, d, J = 15.6 Hz), 5.53
(1H, t , J = 10.0 Hz), 5.40 (2H, m), 4.99 (1H, dd, J = 8.4, 6.4 Hz), 4.16 (1H, m), 3.64
(3H, s), 2.32 (4H, m), 2.24 (1H, m), 2.12 (1H, m), 1.45 (3H, s), 1.34 (3H, s), 0.17 (9H,
s).
1
H-NMR (400 MHz, CDCl
3
) of 9E isomer is described below.

105
O O
COOMe
TMS
3.19

3.6.16 Isomerization of 3.18. The mixture of 9Z/9E isomers of 3.18 (0.35 g, 0.93
mmol) was dissolved in dry CH
2
Cl
2
(80 mL), then a small crystal of sublimed I
2
(25 mg)
was added to the mixture and stirred at room temperature for overnight (18 h). The
violet color solution was then quenched with a saturated aqueous solution of Na
2
S
2
O
5
,
extracted with ether (25 mL x 3), and washed with brine. The organic layers were
combined, dried over anhydrous MgSO
4
, filtered, and concentrated under reduced
pressure. The residue was purified on a silica gel column using 7% EtOAc/hexane as
the eluant to give the a trace amount of chromatographically separable 4E, 9E, 11E
isomer (0.04 g, 5%) and the desired 4Z, 9E, 11E isomer 3.19 (0.30 g, 85%).
1
H-NMR
(400 MHz, CDCl
3
) δ
H
6.62 (1H, dd, J = 15.2, 10.4 Hz), 6.26 (1H, dd, J = 15.2, 10.4 Hz),
5.74 (1H, dd, J = 15.2, 7.6 Hz), 5.61 (1H, d, J = 15.6 Hz), 5.40 (2H, m), 4.56 (1H, t, J =
6.8 Hz), 4.15 (1H, m), 3.65 (3H, s), 2.33 (4H, m), 2.21 (1H, m), 2.11 (1H, m), 1.14 (3H,
s), 1.13 (3H, s), 0.16 (9H, s).

OTBS TBSO
COOMe
3.20

3.6.17 Methyl (7S, 8R, 4Z, 9E, 11E)-7,8-bis(t-butyldimethylsilyloxy)-tetradeca-
4,9,11-trien-13-ynoate (3.20). To a solution of 3.19 (0.25 g, 0.66 mmol) was added
1M HCl (1.5 mL) and 1.5 mL of MeOH, and stirred for 40 min. The reaction was
quenched with saturated aqueous NaHCO
3
solution and extracted with ether (15 mL x
106
3). The organic layers were combined, dried over MgSO
4
, filtered and concentrated
under reduced pressure. The crude product was purified on a silica column using 5%
MeOH/CH
2
Cl
2
to give the diol (0.18 g, 82%), which was further protected using TBS-
OTf and 2,6-lutidine in dry CH
2
Cl
2
(20 mL) at room temperature for overnight. The
reaction was quenched with saturated aqueous NH
4
Cl solution, extracted with ether (20
mL x 3), washed with brine. The organic layers were combined and dried over MgSO
4
,
and concentrated in vacuo. The crude was purified on silica column using 4%
EtOAc/hexane to give the protected diol (0.29 g, 95%).
1
H-NMR (400 MHz, CDCl
3
) δ
H

6.61 (1H, dd, J = 15.6, 10.8 Hz), 6.12 (1H, dd, J = 15.6, 10.8 Hz), 5.76 (1H, dd, J =
15.6, 6.8 Hz), 5.55 (1H, d, J = 15.6 Hz), 5.44 (1H, dt, J = 10.8, 7.2 Hz), 5.39 (1H, dt, J
= 10.8, 7.2 Hz), 3.97 (1H, dd, J = 7.2, 4.4 Hz), 3.64 (3H, s), 3.59 (1H, dd, J = 6.0, 4.0
Hz), 2.31 (2H, m), 2.20 (2H, m), 0.85 (3H, s), 0.83 (3H, s), 0.17 (9H, s), 0.00 (3H, s), -
0.01 (3H, s), -0.04 (3H, s);
13
C-NMR (100 MHz, CDCl
3
) δ
C
173.6, 142.3, 137.5, 130.5,
128.9, 127.4, 110.6, 104.3, 97.0, 76.4, 76.2, 51.5, 36.8, 33.8, 31.5, 29.7, 25.8, 23.0, 18.2,
18.1, -0.1, -4.2, -4.4, -4.7. The TMS group from the alkyne was deprotected as follows:
to a solution of TMS protected alkyne (0.28 g, 0.50 mmol) in MeOH was added a scoop
of Na
2
CO
3
, and stirred for overnight. The cloudy mixture was concentrated in vacuuo
to remove MeOH, and then added H
2
O to quench the reaction, extracted with ether (15
mL x 3), washed with brine, dried over MgSO
4
. The crude was purified over silica
column using 2% EtOAc/hexane as the eluant to afford the alkyne 3.20 (0.22 g, 88%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.63 (1H, dd, J = 15.6, 10.8 Hz), 6.13 (1H, dd, J = 15.6,
10.8 Hz), 5.77 (1H, dd, J = 15.6, 6.8 Hz), 5.51 (1H, dd, J = 15.6, 2.0 Hz), 5.43 (1H, m),
107
5.37 (1H, m), 3.40 (1H, dd, J = 6.8, 5.2 Hz), 3.63 (3H, s), 3.58 (1H, dd, J = 10.8, 6.0
Hz), 2.99 (1H, d, J = 2.8 Hz), 2.30 (4H, m), 2.22-2.17 (2H, m), 0.85 (3H, s), 0.82 (3H,
s), 0.00 (3H, s), -0.01 (3H, s), -0.02 (3H, s), -0.04 (3H, s).

OTBS TBSO
COOMe
OTBS
3.21
S
S
R

3.6.18 Methyl (7S, 8R, 17S, 4Z, 9E, 11E, 15E, 19Z)-tris(t-butyldimethylsilyloxy)-
docosa-4,9,11,15,19-pentaen-13-ynoate (3.21). To a solution of vinyl iodide 3.5 (106
mg, 0.29 mmol) in benzene (2 mL) was added Et
3
N (0.16 mL, 1.12 mmol) and the
alkyne 3.20 (110 mg, 0.223 mmol). The mixture was freeze-thaw at -78
o
C to remove
oxygen. The reaction mixture was warmed to room temperature followed by the
addition of Pd(Ph
3
)
4
and CuI, and protected from light. It was then stirred at room
temperature for overnight. The reaction was quenched with saturated aqueous solution
of NH
4
Cl, extracted with ether (15 mL x 3), washed with brine. The organic layers
were combined, dried over MgSO
4
, and concentrated in vacuuo to give a crude product.
The crude was purified on a silica column using 3% EtOAc/hexane to give the protected
alkyne precursor of 17S-RvD1 methyl ester (3.21, 155 mg, 95%).
1
H-NMR (400 MHz,
CDCl
3
) δ
H
6.54 (1H, dd, J = 15.6, 10.8 Hz), 6.13 (1H, dd, J = 15.6, 10.8 Hz), 6.10 (1H,
dd, J = 15.6, 5.2 Hz), 5.81-5.63 (3H, m), 5.46-5.43 (2H, m), 5.38 (1H, m), 5.33-5.26
(1H, m), 4.17 (1H, m), 3.97 (1H, dd, J = 6.4, 3.6 Hz), 3.65 (3H, s), 3.58 (1H, m), 2.32
(4H, m), 2.26-2.19 (4H, m), 2.00 (2H, m), 0.93 (3H, t, J = 7.6 Hz), 0.87 (9H, s), 0.86
(9H, s), 0.83 (9H, s), 0.03 (3H, s), 0.01 (3H, s), 0.00 (3H, s), -0.01 (6H, s), -0.03 (3H, s).
108
OH HO
COOMe
OH
3.22
S

3.6.19 Methyl (7S, 8R, 17S, 4Z, 9E, 11E, 15E, 19Z)-7, 8, 17-trihydroxy-docosa-4, 9,
11, 15, 19-pentaen-13-ynoate (3.22). To a solution of TBS-protected triol 3.21 (145
mg, 0.20 mmol) in THF (5 mL) at 0
o
C was added TBAF (1.2 mL of 1M solution in
THF, 1.20 mmol). The reaction mixture was stirred for overnight at room temperature,
and then quenched with saturated aqueous solution of NH
4
Cl, extracted with ether (15
mL x 3), washed with brine, and dried over MgSO
4
. The combined ether extract was
then treated with freshly prepared diazomethane to convert the free acid to the methyl
ester. The solution was then bubbled with nitrogen to remove excess diazomethane.
The crude product was purified over a silica column using 3% MeOH/CH
2
Cl
2
to afford
3.22 (70 mg, 92%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.53 (1H, dd, J = 15.6, 10.8 Hz),
6.30 (1H, dd, J = 15.6, 10.8 Hz), 6.10 (1H, dd, J = 16.4, 6.0 Hz), 5.80 (2H, d, J = 15.6
Hz), 5.70 (1H, d, J = 15.6 Hz), 5.52 (1H, m), 5.41 (2H, m), 5.27 (1H, m), 4.14 (2H, m),
3.65 (1H, m), 3.61 (3H, s), 2.35-2.32 (3H, m), 2.28-2.25 (4H, m), 2.11 (1H, m), 2.0 (2H,
m), 0.91 (3H, t, J = 7.6 Hz);
13
C-NMR (100 MHz, CDCl
3
) δ
C
174.0, 144.8, 140.6, 135.5,
133.6, 131.6, 130.6, 126.7, 123.1, 111.9, 109.9, 90.4, 89.4, 74.6, 73.8, 71.5, 51.7, 34.8,
33.4, 29.9, 22.5, 20.6, 14.1.

109
OH HO
COOMe
OH
3.1

3.6.20 Methyl (7S, 8R, 17S, 4Z, 9E, 11E, 13Z, 15E, 19Z)-trihydroxy-
docosahexaenoate (3.1). (i) preparation of activated Zn. Argon was passed through a
suspension of 0.50 g of Zn dust (98+%, <10m, Aldrich) in 3.0 mL of H
2
O for 15 min,
and then 0.05 g of Cu(OAc)
2
.H
2
O was added, stirring was continued for 20 min before
0.05 g of AgNO
3
was introduced slowly to the mixture. The suspension was stirred for
further 30 min under argon. The grey colored Zn-amalgam was carefully washed with
H
2
O (3.0 mL x 2), MeOH (3.0 mL x 2), acetone (3.0 mL x 2) and ether (3.0 mL x 2),
respectively. The ether moist amalgam was added 1:1 (v/v) H
2
O (2.0 mL) and MeOH
(2.0 mL), and was ready to use.
(ii) Stereospecific partial reduction of the triple bond. To the above mentioned
activated Zn-amalgam suspension in H
2
O-MeOH (1:1) was added a solution of alkyne
3.22 (5.0 mg) in MeOH (1.0 mL). The reaction mixture was stirred for overnight in the
dark at 40
o
C. The reaction progress was monitored by reversed-phase HPLC (21%
MeOH/H
2
O), and found that the reduction was completed by overnight. The metal was
then filtered off, and H
2
O-MeOH solution was concentrated in vacuo, dissolved again in
MeOH, filtered through HPLC filters. The MeOH was dried, and then added 1.20 mL
of MeOH and 0.60 mL of H
2
O to the solid. The product was then purified by a
reversed-phase HPLC on ODS using 45% H
2
O/MeOH as mobile phase to give the 17S-
RvD1 methyl ester, 3.1 (4.2 mg, 85%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.76 (2H, ddd,
J = 16.8, 10.0, 7.0 Hz), 6.44 (1H, dd, J = 14.8, 10.8 Hz), 6.30 (1H, dd, J = 14.8, 10.8
110
Hz), 6.01 (2H, dd, J = 10.4, 6.8 Hz), 5.84 (2H, ddd, J = 14.8, 10.8, 6.8 Hz), 5.63 (1H, dt,
J = 10.8, 8.0 Hz), 5.52 (2H, m), 5.39 (1H, dt, J = 10.8, 8.0 Hz), 4.43 (2H, m), 3.75 (1H,
m), 3.70 (3H, s), 2.45 (2H, m), 2.42 (2H, m), 2.37 (2H, m), 2.34 (1H, m), 2.23 (1H, m),
2.11 (2H, m), 1.0 (3H, t, J = 7.2 Hz);
13
C-NMR (100 MHz, CDCl
3
) δ
C
174.0, 136.9,
135.5, 133.0, 132.8, 131.6, 130.9, 129.5, 129.4, 128.7, 126.8, 125.5, 123.5, 75.0, 73.8,
71.9, 51.7, 35.3, 33.4, 30.0, 22.6, 20.7, 14.2.

111
0.83
0.17
0.88
0.16
0.85
0.17
0.92
0.39
0.91
0.18
0.87
1.19
0.87
2.66
3.35
6 5 4 3 2 PPM

1
H-NMR (400 MHz, DMSO-d
6
) of acetonide 3.3.

O
OH
O O
Mixture of α and β-anomers of 3.3 (17:83))
112
15.15
3.00
2.44
2.23 2.26
8 7 6 5 4 3 2 PPM

1
H-NMR (400 MHz, CDCl
3
) of Wittig Salt 3.2.
180 160 140 120 100 80 60 40 20 PPM

13
C-NMR (100 MHz, CDCl
3
) of Wittig Salt 3.2.

MeO
O
PPh
3
Br
3.2
113
0.65 0.58
2.00
9.30
7 6 5 4 3 2 1 0 PPM

1
H-NMR (250 MHz, CDCl
3
) of TMS-protected vinyl alcohol (3.9).
0.93 0.94
2.13
9.00
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of TMS-protected vinyl bromide (3.10).

OH
TMS
3.9
Br
TMS
3.10
114
1.09 1.10
2.30
8.63
6.52
9.86
8 7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of Wittig salt (3.4).

140 120 100 80 60 40 20 0 PPM

13
C-NMR (100 MHz, CDCl
3
) of Wittig salt (3.4).
PPh
3
Br
TMS
3.4
115
0.96 1.00
0.82
0.92 0.93
10.18
5.77
4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 PPM

1
H-NMR (400 MHz, CDCl
3
) of protected R-glycidol (3.23).

80 70 60 50 40 30 20 10 0 PPM

13
C-NMR (100 MHz, CDCl
3
) of protected R-glycidol (3.23).

O
TBSO
3.23
116
1.26
1.08 1.05
2.22
1.91
3.00
12.21
6.24
4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 PPM

1
H-NMR (400 MHz, CDCl
3
) of secondary alcohol 3.12.

TBSO
OH
3.12
117
3.95
6.00
0.94
2.05
0.96 0.94
1.88
12.35
9.19
5.10
8 7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of protected diol from R-glycidol 3.24.

4.14
6.00
0.98
2.14
0.93 0.92
1.93
12.53
8 7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of primary alcohol from R-glycidol (3.13).

TBSO
OTBDPS
3.24
HO
OTBDPS
3.13
118

4.06
6.00
0.95 0.98 1.01
2.17
0.99
0.89
2.86
9.64
2.72
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of Z-alkene from R-glycidol (3.14).

HO
OTBDPS
3.14
119
1.00 1.02 1.08 0.99 0.92
2.38 2.23
3.02
17.58
9.29
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) spectrum of vinyl iodide 3.5 from R-glycidol.

OTBS
I
3.5
120

1.92
0.93
1.95 1.96
2.69
1.94
3.11
2.92 3.00
5 4 3 2 1 PPM

1
H-NMR (400 MHz, DMSO-d
6
) spectrum of Z-alkene 3.16.
1.84 1.78
2.52
1.87
4.76
1.14
2.90
3.00
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) spectrum of Z-alkene 3.16.
HO
O O
COOMe
3.16
121
5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 PPM

Decoupled
1
H-NMR (400 MHz, DMSO-d
6
) spectrum of Z-alkene 3.16.

Decoupled
1
H-NMR spectrum
d (J = 10.8 Hz)d (J =10.8Hz)
122
0.71
2.05
1.91
3.00
6.30
3.34 3.29
8 6 4 2 0 PPM

1
H-NMR (400 MHz, CDCl
3
) spectrum of aldehyde 3.17.

O
O O
COOMe
3.17
123

7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) spectrum of 3.18.

O O
COOMe
TMS
9
3.18
124
0.99 1.06 1.05 1.03
2.11
1.04 1.04
3.00
4.37
1.02 1.15
3.30 3.27
7.90
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) spectrum of 3.19.

O O
COOMe
TMS
3.19
125
1.02 1.03 0.99 0.95
2.15
0.96
3.00
1.16
4.22
2.20
19.33
7.52
11.65
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) spectrum of TMS-protected alkyne 3.25.

OTBS TBSO
COOMe
TMS
3.25
1.02 1.03 0.99 0.95
2.15
6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 PPM
126
1.07 1.14 1.08 1.07
2.17
0.99
3.00
0.98
0.74
3.91
2.07
19.01
11.23
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) spectrum of terminal alkyne 3.20.

180 160 140 120 100 80 60 40 20 0 PPM

13
C-NMR (100 MHz, CDCl
3
) spectrum of terminal alkyne 3.20.

OTBS TBSO
COOMe
3.20
1.07
1.14 1.08 1.07
2.17
6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 PPM
127

1.07
2.11
3.19
4.34
1.01 0.94
3.00
1.20
4.07 4.10
1.86
3.30
28.16
16.57
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) spectrum of protected RvD1 methyl ester 3.21.

OTBS TBSO
COOMe
OTBS
3.21
1.07
2.11
3.19
4.34
6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 PPM
128
1.14 1.19 1.14
2.21
1.14 1.13
2.20
1.05
1.99
4.16
3.52
4.48
1.01
1.95
3.00
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) spectrum of alkyne precursor of RvD1 methyl ester 3.22.

180 160 140 120 100 80 60 40 20 PPM

13
C-NMR (100 MHz, CDCl
3
) spectrum of alkyne precursor of RvD1 methyl ester 3.22.
OH HO
COOMe
OH
3.22
1.14 1.19 1.14
2.21
1.14 1.13
2.20
1.05
6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 PPM
150 140 130 120 110 100 90 PPM
129

1.64
0.94
1.10
1.61
1.69
0.92
2.10
0.95
1.71
0.91
3.00
0.91
7.96
1.25
2.02
2.80
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) spectrum of RvD1 methyl ester 3.1.

160 140 120 100 80 60 40 20 PPM

13
C-NMR (100 MHz, CDCl
3
) spectrum of RvD1 methyl ester (3.1).

OH HO
COOMe
OH
3.1
1.64
0.94
1.10
1.61
1.69
0.92
2.10
0.95
6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 PPM
150 140 130 120 110 PPM
130
0.98 1.00
0.86 0.96 0.95
10.04
5.69
3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 PPM

1
H-NMR (400 MHz, CDCl
3
) spectrum of protected S-glycidol 3.26.

O
TBSO
3.26
131
0.84
1.33 1.20
0.80
2.10 2.05
3.00
11.30
6.22
4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 PPM

1
H-NMR (400 MHz, CDCl
3
) spectrum of secondary alcohol 3.12'.
TBSO
OH
3.12'
132
3.96
6.00
1.00
2.10
0.78 0.80
1.67
12.05
9.54
5.09
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) spectrum of protected diol from S-glycidol (3.27).

TBSO
OTBDPS
3.27
133
4.08
6.00
0.97
2.14
0.92 0.97
1.99
12.66
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) spectrum of primary alcohol 3.13' from S-glycidol.

HO
OTBDPS
3.13'
134
4.07
6.00
0.97 1.00 0.99
2.13
0.94
1.57
2.79
9.75
2.69
8 7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) spectrum of Z-alkene (3.14') from S-glycidol.

HO
OTBDPS
3.14'
135
0.73 0.77
0.90 0.91
0.85
1.89
2.01
0.75
3.00
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) spectrum of vinyl iodide (3.28) from S-glycidol.

0.99 1.00 1.11 1.04 0.98
2.10 1.95
2.75
12.27
5.94
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) spectrum of vinyl iodide (3.5') from S-glycidol.

OH
I
3.28
OTBS
I
3.5'
136
1.17
2.30
3.42
4.47
1.06 1.01
3.00
1.03
4.10
4.53
2.00
3.56
3.25
32.03
17.52
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) spectrum of TBS-protected AT-RvD1 methyl ester 3.21'.

OTBS TBSO
COOMe
OTBS
3.21'
1.17
2.30
3.42
4.47
6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 PPM
137
1.18 1.19 1.12
2.23
1.12 1.16
2.26
1.02
2.20
1.22
3.33
9.47
1.62
2.55
3.00
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) spectrum of alkyne precursor of AT-RvD1 methyl ester
3.22'.

180 160 140 120 100 80 60 40 20 PPM

13
C-NMR (100 MHz, CDCl
3
) spectrum of alkyne precursor of AT-RvD1 methyl ester
3.22'.
1.18 1.19 1.12
2.23
1.12 1.16
2.26
1.02
6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 PPM
170 160 150 140 130 120 110 100 90 PPM
OH HO
COOMe
OH
3.22'
138
2.13
1.01 1.04
2.08 2.07
0.98
2.10
0.99
2.15
1.16
3.10
11.17
2.21
1.02
5.31
3.00
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) spectrum of AT-RvD1 methyl ester 3.1'.

180 160 140 120 100 80 60 40 20 PPM

13
C-NMR (100 MHz, CDCl
3
) spectrum of AT-RvD1 methyl ester 3.1'.

OH HO
COOMe
OH
3.1'
2.13
1.01 1.04
2.08 2.07
0.98
2.10
0.99
6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 PPM
170 160 150 140 130 120 110 PPM
139
Chapter 4. Total Synthesis of Resolvin D2 (7S-RvD2) and its
7R-epimer (7R-RvD2)

4.1 Introduction

The beneficial effects of omega-3 fatty acids such as DHA and EPA in human health
and diseases are well-documented, and subject of interests since early 1929.
65
Despite
many decades of research on omega-3 polyunsaturated fatty acids, the cellular and
molecular mechanisms accounting for their bioactions remained to be established, and
their direct connection to human diseases is still an important biomedical challenge.
Recently, Serhan and colleagues discovered new families of lipid mediators derived
from both EPA and DHA.
66
The DHA-derived lipid mediators termed as D-series of
resolvins (resolution phase interaction products) such as resolvin D1 (RvD1), resolvin
D2 (RvD2) etc. and protectins such as neuroprotectins (NPD1).
2
Their detailed
biosynthetic pathways were described in Chapter 1 of this dissertation. In absence of
aspirin, and added DHA, 15-lipoxygenase (15-LOX) generates 17S-HDHA as the initial
product in tissues rich in DHA such as in brain, synapses, and retina, which was then
converted to 7(S)-hydroperoxy-17S-HDHA by the action of 5-LOX in PMN.
Enzymatic epoxidation of 7(S)-hydroperoxy-17S-HDHA followed by the enzymatic
hydrolysis gave resolvin D2 (7S-RvD2) in sub-nanomolar quantities. Although the

65
Burr, G. O.; Burr, M. M. J. Biol. Chem. 1929, 82, 345.
66
(i) Serhan, C. N.; Clish, C. B.; Brannon, J.; Colgan, S. P.; Chiang, N.; Gronert, K. J. Exp. Med. 2000,
192, 1197. (ii) Serhan, C. N.; Hong, S.; Gronert, K.; Colgan, S. P.; Devchand, P. R.; Mirick, G.;
Moussignac, R. L. J. Exp. Med. 2002, 196, 1025. (iii) Hong, S.; Gronert, K.; Devchand, P.; Moussignac,
R. L.; Serhan, C. N. J. Biol. Chem. 2003, 278, 14677.

140
basic structure of RvD2 was established from the LC-UV-MS/MS based lipidomic
analysis, but the absolute stereochemistry is yet to be fully determined. Because of their
extreme minute natural abundance, it has to be synthesized in its stereochemically pure
form for comparison to assign the complete stereochemistry. In this Chapter, we
describe the asymmetric total synthesis of resolvin D2 and its 7R-epimer in order to
determine absolute stereochemistry, and to further expedite their biological and
pharmacological studies.
4.2 Results and Discussion
4.2.1 Total synthesis of 7R-resolvin D2 (7R-RvD2) and 7S-resolvin D2 (7S-
RvD2)

HO OH
OH
COOH
7R-RvD2 (4.1)
HO OH
OH
COOH
R S
7S-RvD2 (4.1')

Figure 16. Structures of 7R-RvD2 (4.1) and 7S-RvD2 (4.1').

The 7R-RvD2 and 7S-RvD2 are diastereomers, differ only the stereochemistry at C-7 as
shown in Figure 16. Both compounds have three Z-double bonds, and one of them is in
conjugation with three E-double bonds, which is prone to easy isomerization by heat,
light and transition metals. We have devised a simple strategy for the asymmetric total
141
synthesis of these two diastereomers (4.1 and 4.1'), and their analogs using common
intermediates as described herein.
4.2.1.1 Retrosynthetic analysis
The highly convergent retrosynthetic disassembly for 7R-reolvin D2 (4.1) and 7S-RvD2
(4.1') is outlined in Figure 17.

HO OH
OH
COOMe
Wittig Wittig
Pd
0
/Cu
I
Coupling
Lindlar Hydrogenation
Chiral Glycidol
Natural Chiral Pool
Selective Reduction
O OH
O
O TMS
PPh
3
Br
3.3 3.4
7R-RvD2 (4.1)
BrPh
3
P
4.2
7
OTBS
COOMe
7
I
4.3
O
OH
S-Glycidol

Figure 17. Retrosynthetic analysis of 7R-RvD2 (4.1) methyl ester.

142
The total synthesis of 7S-RvD2 is published by Spur,
67
and we used a similar strategy
but entirely different sequence of reactions for both 7R-RvD2 and 7S-RvD2. In order to
achieve the highest optical purity, we envisaged to incorporate the chirality of the
hydroxyl group bearing carbon centers from known natural chiral pool and
commercially available known chiral glycidols. The 7R-configuration in 7R-RvD2 (4.1)
was planned to derive from S-glycidol, and 7S-configuration in 7S-RvD2 (4.1') was
from R-glycidol, whereas the 16R and 17S stereoconfigurations could arise from 2-
deoxy-D-ribose (3.3). The labile Z-double bond, which is in conjugation with E-double
bonds in the tetraene moiety, was planned to introduce in the last step using the mild
stereoselective reduction of a triple bond in order to avoid light, heat or transition
metals mediated isomerization. The 4Z-double bond was designed to construct by
partial hydrogenation from a triple bond using Lindlar catalyst, and the 19Z-double
bond was envisaged to incorporate from a Z-selective Wittig reaction. The key
advantages of our synthetic strategy are: first—our approach, which is depicted in
Figure 17, is a convergent one involving four intermediates (3.3, 3.4, 4.2, 4.3), three of
them (3.3, 3.4 and 4.2 ) are already in hands synthesized for other resolvins; second—
the high degree of stereoselectivity originating from the optically pure commercially
available starting materials; third—using highly efficient carbon—carbon bond-forming
reactions such as Pd
0
/Cu
I
mediated Sonogashira coupling reaction and Wittig reaction
for the connection of the advantaged stage intermediates to construct the entire carbon
frame-work of these two lipid mediators.

67
Robriguez, A. R.; Spur, B. W. Tetrahedron Lett. 2004, 45, 8717.
143
4.2.1.2 Synthesis of common terminal alkyne (4.8) for both 7R-RvD2 and 7S-RvD2
Three advanced stage intermediates such as (1-propyl)triphenylphosphonium bromide
(4.2), 3,4-O-isopropylidene-2-deoxy-D-ribose (3.3) and Wittig salt (3.4) were
synthesized for the synthesis of other resolvins (please see Chapter 3). These common
intermediates were used here for the synthesis of both 7R-RvD2 (4.1) and 7S-RvD2
(4.1')

Reagents and conditions: (a) NaHMDS, Et
2
O, -78
o
C for 15 min, then 3.3 at -
78
o
C & brought to rt by overnight, 61%; (b) Dess-Martin periodinane, NaHCO
3
,
CH
2
Cl
2
,rt, 2h, 70%; (c) n-BuLi, -78
o
Cto0
o
C, then 3.4 at -78
o
C, 3 h, 80%; (d)
I
2
, CH
2
Cl
2
, overnight, 100%; (e) (i) 1M HCl, MeOH-H
2
O (1:1), rt, 40 min, 90%; (ii)
TBS-OTf, 2,6-lutidine, CH
2
Cl
2
, rt, overnight, 88%; (iii) Na
2
CO
3
,MeOH, rt,
overnight, 100%.
HO
OO
OO
TMS
BrPh
3
P
4.2
O OH
O
O
3.3
O
OO
TMS
PPh
3
Br
3.4
4.4
4.5
4.6
OO
TMS
4.7
4.8
TBSO OTBS
a
b
c
d
e

Scheme 15. Synthesis of bottom terminal alkyne 4.8 for both 7R-RvD2 and 7S-RvD2.
144

Having these three crucial pieces in hands, we now focused on their connections by
carbon—carbon bond forming reactions as shown in Scheme 15. Exploiting the
masked aldehyde behavior of C1 in 3,4-O-isopropyliedene-2-deoxy-D-ribose (3.3), a Z-
selective Wittig olefination was carried out with the anion generated from the (1-
propyl)triphenylphosphonium bromide (4.2) by NaHMDS at -78
o
C in dry ether. The
reaction proceeded smoothly with high stereoselectivity to afford Z-isomer (4.4)
exclusively in moderate yield (61%) as shown in Scheme 15. When the reaction was
carried-out in dry THF under same experimental conditions, a mixture of E/Z-isomers
was obtained. The setting up this reaction initially at low temperature (-78
o
C) was
important since the use of higher temperature at the beginning also resulted the
production of chromatographically separable undesired E-isomer. The Z-double
geometry of the double bond in 4.4 was confirmed from the
1
H-NMR coupling
constants (J = 10.0 Hz), which was further confirmed by a series of decoupled
1
H-NMR
(CDCl
3
, 400 MHz) experiments. Dess-Martin oxidation
68
of the primary alcohol (4.4)
in CH
2
Cl
2
at room temperature afforded the corresponding aldehyde (4.5) in moderate
yield (71%). The second set of Wittig reaction was carried out between the
triphenylphosphonium salt (3.4) and the aldehyde (4.5) using n-BuLi
69
at -78
o
C to 0
o
C
in dry THF furnished a mixture of 8Z/8E in high yield (80%) as shown in Scheme 15.
However, the 8Z-double bond was easily isomerized to 8E-double bond quantitatively

68
(i) Dess, D. B.; Marin, J. C. J. Org. Chem. 1983, 48, 4155. (ii) Dess, D. B.; Martin, J. C. J. Am. Chem.
Soc. 1991, 113, 7277.
69
(i) Nicolaou, K. C.; Veal, C. A.; Webber, S. E.; Katerinopolos, H. J. Am. Chem. Soc. 1985, 107, 7515.
(ii) Nicolaou, K. C.; Webber, S. E. Synthesis 1986, 453.
145
with the catalytic amount of sublimed I
2
in dry CH
2
Cl
2
without interfering the 3Z-
double bond. The isopropylidene group was deprotected at this stage with 1M HCl in
MeOH:H
2
O (1:1) with few drops of CH
2
Cl
2
to dissolve the compound. The
deprotection was done at this stage by thinking that the acid induced deprotection might
cause some problems at the final stage of the molecule. The free diol was then
immediately protected again with TBS-OTf and 2, 6-lutidine in CH
2
Cl
2
afforded the
TMS-protected 3Z,8E,10E-enyn in excellent yield (88%). The Na
2
CO
3
mediated
desilylation of terminal TMS-group afforded the terminal alkyne 4.8 quantitatively.

4.2.1.3 Synthesis of 7R-vinyl iodide (4.3) for 7R-RvD2
The synthesis of 7R-vinyl iodide (4.3) commenced with the masking of commercially
available pent-4-ynoic acid (4.9) as an ortho-ester called OBO, which was first
introduced by E. J. Corey for the synthesis of prostaglandins and related molecules.
70
A
standard procedure for introduction of OBO
71
was followed as shown in Scheme 16.
The carboxylic acid group was first coupled with 3-hydroxymehtyl-3-methyloxetane
(4.10) by DCC in presence of catalytic amount of DMAP in CH
2
Cl
2
to give the ester
4.11 quantitatively. Treatment of the oxetane ester (4.11) with the catalytic amount of
BF
3
.OEt
2
in CH
2
Cl
2
re-arranged the ester to an OBO ester (4.12) in moderate yield
(78%). The OBO protecting group is very stable in bases including n-BuLi, but very
labile in acidic media including silica column.

70
For a review please see, Corey, E. J. Angew. Chem. Int. Ed. Engl. 1991, 30, 455, and references therein.
71
Corey, E. J., Raju, N. Tetrahedron Lett. 1983, 24, 5571.
146
O
OTBS
OH
O
O
HO O
O
O
O
O
O
OH
TBSO
O
O
HO
OH
HO
OTBDPS
COOMe
O
OTBDPS
COOMe
OTBDPS
COOMe I
4.11
4.12
4.9 4.10
a
b
Reagents and conditions: (a) DCC, DMAP, CH
2
Cl
2
, rt, overnight, 100%; (b) BF
3
.OEt
2
,
CH
2
Cl
2
,Et
3
N, rt, 1 h, 78%; (c) (i) n-BuLi, BF
3
.OEt
2
,THF,-78
o
C for 15 min, then 5,3 h; (ii) 1N
HCl, THF-H
2
O(1:1), 0
o
C, 1h, 81% in twosteps;(d) (i)1M LiOH, THF-H
2
O (1:1), rt, 3 h; (ii)
CH
2
N
2
, Et
2
O, 1 h, 87% in two steps; (iii) TBDPS-Cl, imidazole, DMAP, CH
2
Cl
2
, overnight, 93%;
(iv) CSA, MeOH:CH
2
Cl
2
(1:1), NEt
3
,rt, 1 h,90%;(iv) Lindlar catalyst,H
2
gas, quinoline,
EtOAc, rt, 2h, 96%; (e) Swern Oxidation, -78
o
C, 4 h, 95%; (f) CrCl
2
, CHI
3
, THF, 0
o
C, 3 h, 55%.
4.13
4.14 4.15
4.16 4.3
e
c
d
f
R

Scheme 16. Synthesis of 7R-vinyl iodide (4.3) for 7R-RvD2.

The ring opening the TBS-protected S-glycidol (4.13) by OBO-protected terminal
alkyne (4.12) using n-BuLi in presence of BF
3
.OEt
2
in dry THF at -78
o
C gave a
secondary alcohol.
72
Since OBO-masking group is very prone even to silica column,
we treated the newly produced crude secondary alcohol with 1M HCl in THF-H
2
O (1:1)
at 0
o
C for 1 h to give the ester 4.14, which was then hydrolyzed by LiOH in THF-H
2
O
(1:1) at room temperature to free carboxylic acid followed by the esterification using

72
Mohr, P.; Tamm, C. Tetrahedron Lett. 1987, 28, 391.
147
freshly prepared CH
2
N
2
to give a methyl ester in high yield (87% in two steps).
Silylation of newly generated secondary hydroxyl group by TBDPS-Cl and the
subsequent chemoselective desilylation of primary TBS-group by the mild action of
camphorsulfonic acid in MeOH-H
2
O (1:1)
73
at room temperature gave the primary
alcohol in excellent overall yield (84% in two steps). The stereoselective partial
hydrogenation of the triple using Lindlar catalyst in presence of quinoline in EtOAc
afforded the desired Z-alkene (4.15) exclusively in excellent yield (96%). Swern
oxidation
74
of the primary alcohol (4.15) at -78
o
C in CH
2
Cl
2
gave the corresponding
aldehyde (4.16) in excellent yield (95%). Takai olefination
75
of the aldehyde 4.16 by
CrCl
2
and CHI
3
gave the desired 7R-vinyl iodide (4.3) in moderate yield (55%).

4.2.1.4 Synthesis of 7S-vinyl iodide (4.3') for 7S-RvD2
The synthesis of 7S-vinyl iodide (4.3') was accomplished by following the exact same
reaction sequences as described above for 7R-vinly iodide expect that R-glycidol was
used to give the 7S-stereoconfiguration for 7S-vinyl iodide (4.3') for 7S-RvD2. The
reaction sequences and conditions are described in the Scheme 17.

73
Nicolaou, K. C.; Ninkovic, S.; Sarabia, F.; Vourloumis, D.; He, Y.; Vallberg, H.; Finlay, M, R. V.;
Yang, Z. J. Am. Chem. Soc. 1997, 119, 7974.
74
Mancuso, A. J.; Huang, S. L.; Swern, D. J. Org. Chem. 1978, 43, 2480.
75
Takai, K.; Nitta, K.; Utimoto, K. J. Am. Chem. Soc. 1986, 108, 7408
148
O
OTBS
OH
O
O
HO O
O
O
O
O
O
OH
TBSO
O
O
HO
OH
HO
OTBDPS
COOMe
O
OTBDPS
COOMe
OTBDPS
COOMe I
4.11
4.12
4.9 4.10
a
b
Reagents and conditions: (a) DCC, DMAP, CH
2
Cl
2
, rt, overnight, 100%; (b) BF
3
.OEt
2
,
CH
2
Cl
2
,Et
3
N, rt, 1 h, 78%; (c) (i) n-BuLi, BF
3
.OEt
2
,THF,-78
o
C for 15 min, then 5,3 h; (ii) 1N
HCl, THF-H
2
O (1:1), 0
o
C, 1h, 81% in twosteps; (d) (i)1M LiOH, THF-H
2
O (1:1), rt, 3 h; (ii)
CH
2
N
2
, Et
2
O, 1 h, 87% in two steps; (iii) TBDPS-Cl, imidazole, DMAP, CH
2
Cl
2
, overnight, 93%;
(iv) CSA, MeOH:CH
2
Cl
2
(1:1), NEt
3
, rt, 1 h, 90%; (iv) Lindlar catalyst, H
2
gas, quinoline,
EtOAc, rt, 2h, 96%; (e) Swern Oxidation, -78
o
C, 4 h, 95%; (f) CrCl
2
, CHI
3
, THF, 0
o
C, 3 h, 55%.
4.17
4.14' 4.15'
4.16' 4.3'
e
c
d
f
S

Scheme 17. Synthesis of 7S-vinyl iodide (4.3') for 7S-RvD2.

4.2.1.5 Final assembly of 7R-RvD2 and 7S-RvD2 methyl ester
The final assembly of the major building blocks (4.8 and 4.3 & 4.3') and the final
reaction sequences of the synthesis of these two diastereomeric lipid mediators are
presented in Scheme 18. The Pd
0
/Cu
I
mediated cross-coupling reaction of terminal
149
alkyne (4.8) and vinyl iodides (4.3 and 4.3') under standard Sonogashira
76
coupling
conditions using Pd(PPh
3
)
4
and CuI in presence of Et
3
N in benzene at room temperature
afforded stereospecifically the conjugated 8E,12E,14E-trien-10-yn (4.17 and 4.17') in
excellent yield (97%).
OTBDPS
COOMe I
4.3'
4.8
TBSO OTBS
TBSO OTBS
OTBDPS
COOMe
a
4.17'
b
4.18'
c
7S-RvD2 Methyl Ester (4.1')
Reagents and conditions: (a) Pd(Ph
3
)
4
, CuI, C
6
H
6
, Et
3
N, rt, overnight,
97%; (b) TBAF, THF, rt, overnight followed by CH
2
N
2
, ether, rt, 2 h,
88%; (c) Zn (Cu/Ag), H
2
O:MeOH (1:1), 39
o
C, overnight, 87%.
HO OH
OH
COOMe
HO OH
OH
COOMe
OTBDPS
COOMe I
4.3
TBSO OTBS
OTBDPS
COOMe
a
4.17
b
4.18
c
7R-RvD2 Methyl Ester (4.1)
HO OH
OH
COOMe
HO OH
OH
COOMe
R S
R S

Scheme 18. Final assembly of 7R-RvD2 (4.1) and 7S-RvD2 (4.1').

76
(i) Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett. 1975, 16, 4467. (ii) Nicolaou, K. C.;
Ramphal, J. Y.; Petasis, N. A.; Serhan, C. N. Angew. Chem. Int. Ed. 1991, 30, 1100, and references
therein. (iii) Petasis, N. A.; Aritopoulou-Zanze, I.; Fokin, V. V.; Bernasconi, G.; Keledjian, R.; Yang, R.;
Uddin, J.; Nagulapalli, K. C.; Serhan, C. N. Prostaglandins, Leukotrienes and Essential Fatty Acids 2005,
73, 301, and references therein.
150
Fluoride anion mediated desilylation of tris-silyl groups in 4.17 and 4.17' in THF using
TBAF afforded the triols 4.18 and 4.18' along with the formation of corresponding free
carboxylic acids. The free acids were conveniently converted to the corresponding
methyl esters 4.18 and 4.18' by treating them with freshly prepared diazomethane.
Stereospecific reduction of the conjugated 8E,12E,14E-trien-10-yn by activated
Zn(Ag/Cu)-amalgam
77
in MeOH—H
2
O (1:1) at 39
o
C to afford the final products 7R-
RvD2 (4.1) and 7S-RvD2 (4.1') in excellent yields (87% and 85%, respectively), as
shown in Scheme 18. The structure of 7R-RvD2 methyl ester was determined to be
4Z,7R,8E,10Z,12E,14E,16R,17S,19Z-docosahexanoate (4.1) from the analyses of 1 and
2D-NMR spectral data,
1
H-NMR coupling constants, and from the stereochemistry of
the chiral starting materials used. Similarly, the structure of 7S-RvD2 methyl ester was
established to be 4Z,7S,8E,10Z,12E,14E,16R,17S,19Z-docosahexanoate (4.1') from the
similar analyses of 1 and 2D-NMR spectral data,
1
H-NMR coupling constants, and from
the stereochemistry of the chiral starting materials used. The UV,
1
H and
13
C-NMR
spectra of both 7R-RvD2 and 7S-RvD2 are identical, but their HPLC retention time is
different as expected since they are diastereomers.
4.3 Conclusion
In summary, we have accomplished the asymmetric total synthesis of 7R-RvD2 and 7S-
RvD2 in highly convergent fashion using known chiral starting materials. The structure
of 7R-RvD2 methyl ester was determined to be 4Z,7R,8E,10Z,12E,14E, 16R,17S,19Z-
docosahexanoate from the analyses of 1 and 2D-NMR spectral data,
1
H-NMR coupling

77
Boland, W.; Schroer, N.; Sieler, C.; Feigel, M. Helv. Chim. Acta 1987, 70, 1025.
151
constants, and from the stereochemistry of the chiral starting materials used. Similarly,
the structure of 7S-RvD2 methyl ester was established to be
4Z,7S,8E,10Z,12E,14E,16R,17S,19Z-docosahexanoate from the similar analyses. This
highly efficient, flexible route to the final products will allow us to design and synthesis
of number of analogs in appreciable amounts if needed. The stereochemical
assignments, the biological studies, and metabolic inactivation pathways are currently
underway in our collaborator’s lab at Harvard Medical School.

152
4.4 Experimental

O
O
O
4.11

4.4.1 Pent-4-yn-half-ester (4.11): To a solution of DCC (1.0 g, 4.40 mmol) in dry
CH
2
Cl
2
(15 mL) was added the mixture of pent-4-ynoic acid, 4.9 (0.45 g, 4.58 mmol),
3-hydroxymethyl-3-methyloxetane, 4.10 (1.0 g, 10.09 mmol) and DMAP (30 mg, 0.02
mmol) in dry CH
2
Cl
2
(5 mL) at 0
o
C. The DCHU started to precipitate within few
minutes. The solution was then stirred at room temperature under argon for overnight.
The precipitated DCHU was filtered off, and washed with CH
2
Cl
2
. The solvent was
removed using rotary evaporated, and the crude product was purified on a silica column
using 30% EtOAc/hexanes as mobile phase to give pure half-ester 4.11 (0.8 g, 100%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
4.43 (d, J = 6.0 Hz, 2H), 4.30 (d, J = 6.0 Hz, 2H), 4.12
(s, 2H), 2.53 (m, 2H), 2.44 (m, 2H), 1.93 (t, J = 2.8 Hz, 1H), 1.26 (s, 3H);
13
C-NMR
(100 MHz, CDCl
3
) δ
C
171.6, 82.1, 79.3, 79.3, 69.0, 68.7, 38.8, 33.1, 21.0, 14.2.
O
O
O
4.12

4.4.2 Pent-4-yn-OBO-ester (4.12): To a stirred solution of half-ester 4.11 (0.80 g, 4.39
mmol) in dry CH
2
Cl
2
was added BF
3
.OEt
2
(0.14 mL, 1.09 mmol) drop-wise at room
temperature. The reaction mixture was then stirred at room temperature for 1 h. After
an hour Et
3
N (1.0 mL) was added to the reaction mixture to precipitated-out the boron
trifluoride-triethylamine complex. After stirring for 10 min, diethyl ether (20 mL) was
153
added to dilute the solution. The precipitate was filtered off and the solvent was
removed using a rotatory evaporator. The product was purified by column
chromatography on a silica gel pre-treated with 1% Et
3
N in hexanes using 30%
EtOAc/hexanes as the solvent system to give the pure acetylenic OBO-ester 4.12 (0.62
g, 78%).
1
H NMR (400 MHz, CDCl
3
) δ
H
3.84 (s, 6H), 2.28 (m, 2H), 1.88 (m, 2H), 0.76
(s, 3H);
13
C NMR (100 MHz, CDCl
3
) δ
C
108.0, 84.0, 72.0, 72.0, 72.0, 67.8, 35.7, 30.2,
14.4, 12.8.
OH
TBSO
O
O
HO
OH
4.14

4.4.3 7R-8-(t-butyldimethylsilyloxy)-3'-hydroxy-2'-(hydroxymethyl)-2'-
methylpropyl-7-hydroxy-oct-4-ynoate (4.14). To a solution of acetylenic OBO-ester
4.12 (0.45 g, 2.483 mmol) in dry THF (10 mL) at -78
o
C was slowly added n-BuLi
(1.25 mL, 1.6 M solution in hexane, 2.00 mmol). The mixture was stirred for 15 min,
and then added BF
3
.OEt
2
(0.25 mL, 2.00 mmol) solution at -78
o
C and stirred for
additional 15 min. A solution of TBS-protected S-glycidol 4.13 (0.36 g, 1.91 mmol) in
THF (3.0 mL) was slowly added to the reaction mixture at -78
o
C by using a cannula.
The reaction mixture was stirred for 3 h at -78
o
C and then brought to room temperature
and stirred for an additional 40 min. The reaction was quenched with a saturated
solution of NH
4
Cl, extracted with ether (20 mL x 3), washed with brine. The organic
layers were combined, dried over MgSO
4
, and concentrated under reduced pressure.
The crude product was then dissolved in THF-H
2
O (1:1) (3.0 mL), and then was added
1M HCl (0.4 mL, 0.4 mmol) at 0
o
C and stirred for 1 h. The reaction was quenched
154
with saturated aqueous solution of NaHCO
3
, extracted with ether, dried over MgSO
4
,
and then concentrated in vacuo to give the crude triol 4.14. The crude product was
purified on a silica column using 50% EtOAc/hexanes as the solvent system to give the
pure triol 4.14 (0.60 g, 81% in two steps).
1
H –NMR (400 MHz, CDCl
3
) δ
H
4.20 (d, J =
6.4 Hz, 2H), 3.61 (m, 2H), 3.55 (m, 4H), 3.38 (m, 2H), 3.72 (m, 1H), 2.95 (t, J = 4.0 Hz,
1H, -OH), 2.75 (t, J = 4.0 Hz, 1H, -OH), 2.70 (d, J = 4.0 Hz, 1H, -OH), 2.58 – 2.48 (m,
4H), 1.60 (m, 2H), 0.87 (s, 9H), 0.82 (s, 3H), 0.04 (s, 6H).

TBSO
O
OMe
OH
4.20

4.4.4 Methyl (7R)-8-(t-butyldimethylsilyloxy)-7-hydroxyoct-4-ynoate (4.20). To a
solution of ester 4.14 (0.30 g, 0.77 mmol) was in THF-H
2
O (1:1) (4.0 mL) was added
1M solution of LiOH-monohydrate (1.54 mL, 1.54 mmol) at room temperature. The
reaction mixture was stirred for 3 h or until the ester could no longer be detected by
TLC. After the reaction, the pH was of the solution was adjusted to a neutral by very
slowly adding 1N HCl. The product was extracted with ether (15 mL x 3), dried over
MgSO
4
and washed with brine. The crude free acid was then converted to the methyl
ester by using freshly prepared CH
2
N
2
in Et
2
O (10 mL). The esterification was
monitored by a series of TLC, and after completion of the reaction by an hour, the
solution was then bubbled through nitrogen for 10 min to remove excess diazomethane.
The crude product was purified on a silica column using 12% EtOAc/hexanes to give
the pure product 4.20 (0.20 g, 87% in two steps).
1
H-NMR (400 MHz, CDCl
3
) δ
H
3.68
155
(m, 1H), 3.62 (s, 3H), 3.60 (dd, J = 10.0, 3.6 Hz, 1H), 3.53 (dd, J = 10.0, 6.4 Hz, 1H),
2.57 (d, J = 4.8 Hz, 1H), 2.30 (d, J = 4.8 Hz, 1H), 2.43 (m, 4H), 0.82 (s, 9H), 0.01 (s,
6H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
172.7, 104.1, 80.3, 70.3, 65.5, 51.7, 33.5, 25.7,
25.6, 23.3, 14.6, -5.5.
OTBDPS
TBSO
O
OMe
4.21

4.4.5 Methyl (7R)-8-(t-butyldimethylsilyloxy)-7-(t-butyldiphenylsilyloxy)oct-4-
ynoate (4.21). To a solution of TBDPS-Cl (0.13 mL, 0.50 mmol), imidazole (34 mg,
0.50 mmol) and DMAP (2 mg, 0.01 mmol) in dry CH
2
Cl
2
(10 mL) at 0
o
C was added
the alcohol 4.20 (0.1 g, 0.34 mmol) in CH
2
Cl
2
(2.0 mL) through a cannula. The mixture
was warmed to room temperature and stirred for overnight. The reaction was quenched
with a saturated aqueous solution of NH
4
Cl, extracted with ether (20 mL x 3), washed
with brine, dried over MgSO
4
, and concentrated under reduced pressure. The crude was
purified on a silica gel column using 2%EtOAc/hexane as the eluant to give pure 4.21
(0.17 g, 93%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
) δ
H
7.73 (m, 4H), 7.39 (m,
6H), 3.83 (m, 1H), 3.67 (s, 3H), 3.55 (m, 2H), 2.46-2.30 (m, 6H), 1.07 (s, 9H), 0.86 (s,
9H), -0.02 (s, 3H), -0.05 (s, 3H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
172.5, 135.7, 135.8,
135.5, 134.7, 134.1, 134.0, 129.6, 127.6, 127.4, 79.5, 77.8, 72.3, 65.6, 51.7, 33.6, 26.8,
25.8, 23.8, 19.3, 18.2, 14.7, -5.6.

156
OTBDPS
HO
O
OMe
4.22

4.4.6 Methyl (7R)-7-(t-butyldiphenylsilyloxy)-8-hydroxyoct-4-ynoate (4.22). To a
solution of protected diol 4.21 (0.25 g, 0.46 mmol) in a 1:1 mixture of CH
2
Cl
2
:MeOH
(15 mL) was added camphorsulfonic acid (86.3 mg, 0.37 mmol) at room temperature.
The progress of the reaction was monitored by TLC. The reaction was over by an hour,
it was then quenched with Et
3
N (0.30 mL, 2.30 mmol). The solvent was evaporated to
dryness to give a crude mixture, which was then purified on a silica gel column using
15% EtOAc/hexane to afford the title primary alcohol 4.22 (0.18 g, 90%).
1
H-NMR
(400 MHz, CDCl
3
) δ
H
7.67 (m, 4H), 7.39 (m, 6H), 3.87 (m, 1H), 3.64 (s, 3H), 3.61 (m,
2H), 2.41-2.23 (m, 6H), 1.06 (s, 9H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
172.7, 135.8,
135.6, 135.5, 133.4, 129.8, 127.7, 127.6, 80.3, 77.3, 72.3, 65.4, 51.6, 33.5, 26.9, 26.8,
23.7, 19.2, 14.6.

HO
OTBDPS
COOMe
4.15

4.4.7 Methyl (7R, 4Z)-7-(t-butyldiphenylsilyloxy)-8-hydroxyoct-4-enoate (4.15). To
a solution of 4.22 0.24 g, 0.56 mmol) in EtOAc (50 mL) was added 20 mg of Lindlar
catalyst and 2 drops quinoline. The reaction mixture stirred at room temperature under
the static atmosphere of hydrogen gas, and its progress was monitored by TLC. The
reaction was over by 2 h at room temperature. The reaction mixture was then filtered
through celite and solvent was evaporated under reduced pressure. The crude product
157
was purified on a silica column using 22% EtOAc/hexane as the eluant to give the pure
4Z alkene 4.15 (0.235 g, 96%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
) δ
H
7.68
(m, 4H), 7.37 (m, 6H), 5.32 (m, 2H), 3.80 (quintet, J = 4.8 Hz, 1H), 3.63 (s, 3H), 3.49
(m, 2H), 2.34-2.18 (m, 6H), 1.07 (s, 9H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
173.5, 135.8,
135.6, 133.7, 133.6, 129.8, 129.7, 127.6, 127.5, 126.0, 73.3, 65.2, 51.4, 33.6, 31.3, 26.9,
22.5, 19.2.

O
OTBDPS
COOMe
4.16

4.4.8 Methyl (7R, 4Z)-7-(t-butyldiphenylsilyloxy)-8-oxooct-4-enoate (4.16). To a
solution of DMSO (0.13 mL, 1.65 mmol) in dry CH
2
Cl
2
(10 mL) at -78
o
C was slowly
added oxalyl chloride (0.10 mL, 1.10 mmol) and stirred for 15 min, and then added the
alcohol 4.15 (0.23 g, 0.55 mmol) in CH
2
Cl
2
(3 mL) through a cannula, and stirred for 50
min. Et
3
N (0.38 mL, 2.75 mmol) was then added to the reaction mixture, and stirred for
3 h at -78
o
C. The reaction mixture was quenched with a saturated aqueous solution of
NH
4
Cl, extracted with ether (20 mL x 3), washed with brine, dried over anhydrous
MgSO
4
, and concentrated under reduced pressure. The crude product was purified on a
silica column using 20% EtOAc/hexane as the eluant to give the desired aldehyde 4.16
(0.22 g, 95%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
) δ
H
9.53 (d, J = 1.6 Hz,
1H), 7.62 (m, 4H), 7.37 (m, 6H), 5.40 (m, 2H), 4.04 (td, J = 6.4, 1.6 Hz, 1H), 3.62 (s,
3H), 2.46-2.20 (m, 6H), 1.09 (s, 9H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
203.3, 173.3,
135.7, 132.9, 132.8, 130.7, 130.0, 127.8, 124.4, 77.5, 51.5, 33.6, 30.9, 26.9, 22.7, 19.3.
158
OTBDPS
COOMe I
4.3

4.4.9 Methyl (7R, 4Z, 8E)-7-(t-butyldiphenylsilyloxy)-9-iodonona-4, 8-dienoate
(4.3). To a stirring suspension of CrCl
2
(0.37 g, 3.03 mmol) in dry THF (10 mL) at 0
o
C
was added a solution of CHI
3
(0.40 g, 1.01 mmol) and aldehyde 4.16 (0.22 g, 0.51
mmol) in dry THF (10 mL). The reaction mixture was stirred at 0
o
C for 3 h, then
warmed to room temperature and stirred for an additional 1 h. The reaction was
quenched with H
2
O (20 mL), extracted with ether (20 mL x 3), washed with brine, dried
over MgSO
4
, and evaporated to dryness to give a crude product, which was then
purified on a silica column using 3% EtOAc/hexanes as the solvent system to give the
pure vinyl iodide 4.3 (0.15 g, 55%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
) δ
H

7.63 (m, 4H), 7.38 (m, 6H), 6.46 (dd, J = 14.4, 6.8 Hz, 1H), 5.98 (dd, J = 14.4, 1.0 Hz,
1H), 5.34 (m, 2H), 4.09 (q, J = 7.5 Hz, 1H), 3.64 (s, 3H), 2.27-2.16 (m, 6H), 1.06 (s,
9H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
173.4, 147.6, 135.8, 133.6, 133.3, 130.1, 129.7,
127.5, 125.3, 75.5, 51.5, 35.0, 33.8, 26.9, 22.7, 19.2.

HO
OO
4.4

4.4.10 Synthesis of 2R, 3S, 5Z-alkene (4.4). To a stirring suspension of (1-
propyl)triphenylphosphonium bromide, 4.2 (6.63 g, 17.22 mmol) in anhydrous Et
2
O (15
mL) was added drop-wise NaHMDS (15.50 mL of 1M solution, 15.5 mmol) at -78
o
C
over 15 min. The mixture was allowed to warm to 0
o
C over 30 min to give an orange-
159
red solution. The mixture was cooled again at -78
o
C, and a solution of 3.3 (1.50 g, 8.61
mmol) in anhydrous Et
2
O (5.0 mL) was added drop-wise through cannula. The yellow-
colored mixture was stirred at -78
o
C to room temperature over the course of overnight.
The reaction mixture was quenched with saturated aqueous NH
4
Cl solution (30 mL),
extracted with ether (30 mL x 3), washed with brine, dried over MgSO
4
, and
concentrated in vacuo to give a yellow oil. Flash column chromatography on silica gel
using 25% EtOAc/hexanes afforded 4.4 (1.05 g, 61%, 5Z isomer only confirmed by
1
H-
NMR) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
) δ
Η
5.47 (m, 1H), 5.32 (m, 1H),
4.15 (m, 2H), 3.61 (m, 2H), 2.35 (m, 1H), 2.25 (m, 1H), 2.02 (m, 2H), 1.45 (s, 3H), 1.34
(s, 3H), 0.94 (t, J = 7.6 Hz, 3H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
134.3, 123.8, 108.1,
77.8, 76.7, 61.6, 28.1, 27.2, 25.4, 20.7, 14.0.

O
OO
4.5

4.4.11 Synthesis of 2R, 3S, 5Z-aldehyde (4.5). To a suspension of the primary alcohol
4.4 (0.56 g, 2.79 mmol) and NaHCO
3
(5.0 g, 59.52 mmol) in dry CH
2
Cl
2
(15 mL) was
added drop-wise to a solution of Dess-Martin periodinane (2.37 g, 5.59 mmol) in
CH
2
Cl
2
(5 mL) at room temperature. The reaction mixture was stirred for 2 h. The
reaction mixture was then quenched with a 1:1 solution of saturated aqueous Na
2
S
2
O
3

(10 mL). The aqueous phase was extracted with ether (20 mL x 3), washed with brine,
dried over MgSO
4
and concentrated under reduced pressure. A flash column
chromatography on silica gel using 20% EtOAc/Hexane as eluant to yield the aldehyde
160
4.5 (0.40 g, 70%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
) δ
H
9.60 (d, J = 2.8 Hz,
1H), 5.46 (m, 1H), 5.30 (m, 1H), 4.33 (q, J = 6.8 Hz, 1H), 4.26 (dd, J = 6.8, 2.8 Hz, 1H),
2.27 (t, J = 7.6 Hz, 2H), 1.96 (quintet, J = 7.2 Hz, 2H), 1.54 (s, 3H), 1.36 (s, 3H), 0.91
(t, J = 7.2 Hz, 3H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
201.5, 134.9, 122.9, 110.4, 81.8,
78.3, 27.7, 27.3, 25.1, 20.7, 13.8.

OO
4.6
TMS

4.4.12 Synthesis of 4.6. To a suspension of phosphonium salt 3.4 (0.53 g, 1.10 mmol)
in dry THF (5 mL) was added n-BuLi (0.50 mL of 1.6 M solution in hexane, 0.825
mmol) at -78
o
C. The dark red solution was allowed to warm to 0
o
C and stirred for 30
min before re-cooled to -78
o
C. The aldehyde 4.5 (0.11 g, 0.55 mmol) solution in 3 mL
of dry THF was added to the reaction mixture drop wise via a cannula. The dark red
mixture was then brought to room temperature and stirred for 3 h before quenched with
saturated aqueous NH
4
Cl solution, extracted with ether (15 mL x 3) and washed with
brine. The organic layers were combined and dried over anhydrous MgSO
4
, filtered,
and concentrated under reduced pressure. The crude product 4.6 (0.14 g, 80%) was
used for the next reaction without purification.

161
TBSO OTBS
8
TMS
4.23

4.4.13 Synthesis of Z:E mixture of 4.23. The crude product 4.6 (0.14 g, 0.44 mmol)
from the previous reaction was dissolved in 1.75 mL of MeOH and then treated with
1H HCl (1.75 mL, 1.75 mmol) for 40 min at room temperature. The reaction was
quenched with solid NaHCO
3
(one scope), and then the product was extracted with
ether, dried over MgSO
4
, and washed with brine. The crude product was purified on a
silica column using 3% MeOH/CH
2
Cl
2
as the solvent system to give the pure diol (0.11
g, 90%), which was further protected using TBS-OTf (0.44 mL, 1.90 mmol) and 2,6-
lutidine (0.44 mL, 3.8 mmol) in dry CH
2
Cl
2
(10 mL) at room temperature for overnight.
The reaction was quenched with saturated aqueous NH
4
Cl solution, extracted with ether
(20 mL x 3), washed with brine. The organic layers were combined and dried over
MgSO
4
, and concentrated in vacuo. The crude was purified on silica column using 1%
EtOAc/hexane to give the 8Z:8E (3:2) mixture of protected diol 4.23 (0.17 g, 88%).
1
H-
NMR (400 MHz, CDCl
3
) 8Z-isomer: δ
H
6.80 (dd, J = 15.2, 10.8 Hz, 1H), 6.03 (t, J =
10.8 Hz, 1H), 5.59 (d, J = 15.2 Hz, 1H), 5.50 (t, J = 10.8 Hz, 1H), 5.41 (m, 2H), 4.38
(dd, J = 8.8, 4.8 Hz, 1H), 3.63 (m, 1H), 2.19 (m, 2H), 1.99 (m, 2H), 0.94 (t, J = 7.6 Hz,
3H), 0.85 (s, 18H), 0.18 (s, 9H), 0.00 (s 12H); 8E-isomer δ
H
6.62 (dd, J = 14.8, 10.0 Hz,
1H), 6.12 (dd, J = 15.2, 10.8 Hz, 1H), 5.77 (dd, J = 15.2, 7.2 Hz, 1H), 5.55 (d, J = 15.6
Hz, 1H), 5.41 (m, 2H), 3.98 (dd, J = 8.8, 4.8 Hz, 1H), 3.58 (m, 1H), 2.19 (m, 2H), 1.99
(m, 2H), 0.94 (t, J = 7.6 Hz, 3H), 0.85 (s, 18H), 0.18 (s, 9H), 0.00 (s, 12H).
162
TBSO OTBS
TMS
4.24

4.4.14 Isomerization of 4.23. The mixture of 8Z/8E isomers of 4.23 (0.15 g, 0.30
mmol) was dissolved in dry CH
2
Cl
2
(70 mL), then a small crystal of sublimed I
2
(15 mg)
was added to the mixture and stirred at room temperature for overnight (18 h). The
violet color solution was then quenched with a saturated aqueous solution of Na
2
S
2
O
5
,
extracted with ether (25 mL x 3), and washed with brine. The organic layers were
combined, dried over anhydrous MgSO
4
, filtered, and concentrated under reduced
pressure. The residue was purified on a silica gel column using 2% EtOAc/hexane as
the eluant to give the desired 3Z, 8E, 10E isomer 4.24 (0.15 g, 100%).
1
H-NMR (400
MHz, CDCl
3
) δ
H
6.70 (dd, J = 15.6, 10.4 Hz, 1H), 6.19 (dd, J = 15.6, 10.8 Hz, 1H),
5.85 (dd, J = 15.6, 7.2 Hz, 1H), 5.63 (d, J = 15.6 Hz, 1H), 5.44 (m, 2H), 4.05 (dd, J =
6.8, 4.4 Hz, 1H), 3.66 (q, J = 6.8 Hz, 1H), 2.25 (m, 2H), 2.07 (m, 2H), 1.00 (t, J = 7.6
Hz, 3H), 0.92 (s, 18H), 0.25 (s, 6H), 0.07 (s, 9H).

TBSO OTBS
4.8

4.4.15 6S, 7R, 3Z, 8E, 10E-6,7-bis(t-butyldimethylsilyloxy)-dedeca-3,8,10-trien-12-
yn (4.8). To a solution of TMS protected alkyne 4.24 (0.15 g, 0.30 mmol) in MeOH
(with few drops of CH
2
Cl
2
to dissolve the alkyne) was added a scoop of Na
2
CO
3
, and
stirred for overnight. The cloudy mixture was filtered through cotton to remove solid
163
Na
2
CO
3
. The filtrate was concentrated in vacuo, and then added H
2
O (10 mL),
extracted with ether (15 mL x 3), washed with brine, dried over MgSO
4
. The crude was
purified over silica column using 1% EtOAc/hexane as the eluant to afford the alkyne
4.8 (0.13 g, 100%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.71 (dd, J = 15.6, 10.8 Hz, 1H),
6.20 (dd, J = 15.6, 10.8 Hz, 1H), 5.86 (dd, J = 15.6, 7.2 Hz, 1H), 5.57 (dd, J = 15.6, 2.2
Hz, 1H), 5.43 (m, 2H), 4.06 (dd, J = 7.2, 4.4 Hz, 1H), 3.66 (q, J = 6.5 Hz, 1H), 3.05 (d,
J = 2.2 Hz, 1H), 2.25 (m, 2H), 2.06 (m, 2H), 0.99 (t, J = 7.6 Hz, 3H), 0.93 (s, 9H), 0.90
(s, 9H), 0.08 (s, 3H), 0.07 (s, 3H), 0.06 (s, 3H), 0.04 (s, 3H);
13
C-NMR (100 MHz,
CDCl
3
) δ
C
143.1, 137.9, 133.3, 130.1, 125.0, 109.4, 82.9, 79.4, 76.5, 76.3, 31.6, 25.9,
20.8, 18.1, 18.0, 14.2, -4.2, -4.2, -4.4, -4.7.

TBSO OTBS
OTBDPS
COOMe
4.17

4.4.16 Methyl (7R, 16R, 17S, 4Z, 8E, 12E, 14E, 19Z)-16, 17-bis(t-
butyldimethylsilyloxy)-7-(t-butyldiphenylsilyloxy)-docosa-4, 8, 12, 19-pentaen-10-
ynoate (4.17). To a solution of vinyl iodide 4.3 (80 mg, 0.145 mmol) in benzene (2 mL)
was added Et
3
N (0.1 mL, 0.725 mmol) and then the alkyne 4.8 (76.09 mg, 0.175 mmol)
in benzene (2.0 mL) by a cannula. The mixture was freeze-thaw at -78
o
C to remove
oxygen. The reaction mixture was warmed to room temperature followed by the
addition of Pd(Ph
3
)
4
(16.75 mg, 0.0145 mmol) and CuI (5.52 mg, 0.029 mmol). The
reaction mixture was protected from the light by warping the flask with the aluminum
164
foil. The reaction mixture was then stirred at room temperature for overnight. The
reaction was quenched with saturated aqueous solution of NH
4
Cl, extracted with ether
(15 mL x 3), washed with brine. The organic layers were combined, dried over MgSO
4
,
and concentrated in vacuo to give a crude product. The crude was purified on a silica
column using 3% EtOAc/hexane to give the tris-protected alkyne precursor of 7S-RvD2,
4.17 (120 mg, 97%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
7.63 (m, 4H), 7.35 (m, 6H), 6.56
(dd, J = 15.2, 10.8 Hz, 1H), 6.15 (dd, J = 15.2, 10.8 Hz, 1H), 6.07 (dd, J = 15.2, 5.6 Hz,
1H), 5.76 (dd, J = 15.2, 6.4 Hz, 1H), 5.68 (d, J = 15.2 Hz, 1H), 5.73 (d, J = 15.2 Hz,
1H), 5.42-5.27 (m, 4H), 4.22 (m, 1H), 3.99 (dd, J = 7.6, 4.8 Hz, 1H), 3.62 (s, 3H), 3.60
(m, 1H), 2.24-2.08 (m, 8H), 2.00 (q, J = 7.6 Hz, 2H), 1.05 (s, 9H), 0.94 (t, J = 7.6 Hz,
3H), 0.86 (s, 9H), 0.84 (s, 9H), 0.02 (s, 3H), 0.01 (s, 3H), 0.00 (s, 3H), -0.01 (s, 3H), -
0.01 (s, 3H), -0.02 (s, 3H).

HO OH
OH
COOMe
4.18

4.4.17 Methyl (7R, 16R, 17S, 4Z, 8E, 12E, 14E, 19Z)-7, 16, 17-trihydroxy-docosa-4,
8, 12, 19-pentaen-10-ynoate (4.18). To a solution of tris-protected triol 4.17 (120 mg,
0.14 mmol) in THF (6 mL) at 0
o
C was added TBAF (0.84 mL of 1M solution in THF,
0.84 mmol). The reaction mixture was stirred for overnight at room temperature, and
then quenched with saturated aqueous solution of NH
4
Cl, extracted with ether (15 mL x
3), washed with brine, and dried over MgSO
4
. The combined ether extract was then
165
treated with freshly prepared diazomethane to convert the free acid to the methyl ester.
The solution was then bubbled with nitrogen to remove excess diazomethane. The
crude product was purified over a silica column using 3% MeOH/CH
2
Cl
2
to afford 4.18
(48.08 mg measured by UV, 88%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.56 (dd, J = 15.2,
10.8 Hz, 1H), 6.31 (dd, J = 15.2, 10.8 Hz, 1H), 6.12 (dd, J = 15.6, 5.6 Hz, 1H), 5.85 (d,
J = 15.6 Hz, 1H), 5.82 (dd, J = 15.6, 6.4 Hz, 1H), 5.71 (d, J = 15.6 Hz, 1H), 5.54-5.28
(m, 4H), 4.19 (m, 2H), 3.68 (m, 1H), 3.63 (s, 3H), 2.70 (brs, 1H, -OH), 2.57 (brs, 1H, -
OH), 2.42 (brs, 1H, -OH), 2.36-2.08 (m, 6H), 2.00 (m, 2H), 0.92 (t, J = 7.6 Hz, 3H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
173.8, 144.8, 140.5, 135.1, 133.2, 131.9, 131.2, 125.7,
123.9, 112.0, 109.9, 90.6, 89.4, 74.5, 73.9, 71.3, 51.7, 34.9, 33.5, 29.9, 22.6, 20.7, 14.1.

HO OH
OH
COOMe
7R-RvD2 Methyl Ester (4.1)

4.4.18 Methyl 7R, 16R, 17S-trihydroxy-docosa-4Z, 8E, 10Z, 12E, 19Z-pentaenoate
(4.1). The stereoselective hydrogenation of triple bond (10 mg scale) was done by using
activated Zinc as Zn(Cu/Ag) amalgam in MeOH—H
2
O (1:1) at 39
o
C to give the final
7R-RvD2 (4.1) in excellent yield (87%). The procedure for this stereoselective partial
hydrogenation was described in Chapter 3.
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.71 (dd, J
= 15.2, 9.2 Hz, 1H), 6.69 (dd, J = 15.2, 10.0 Hz, 1H), 6.37 (dd, J = 15.2, 10.0 Hz, 1H),
6.24 (dd, J = 15.2, 10.8 Hz, 1H), 6.00 (q, J = 10.0 Hz, 2H), 5.78 (dd, J = 15.2, 8.8 Hz,
166
1H), 5.76 (dd, J = 14.8, 6.0 Hz, 1H), 5.58 (m, 3H), 5.35 (m, 1H), 4.26 (m, 2H), 4.20 (m,
1H), 3.70 (m, 1H), 3.65 (s, 3H), 2.37-2.02 (m, 8H), 0.93 (t, J = 7.6 Hz, 3H);
13
C-NMR
(100 MHz, CDCl
3
) δ
C
173.8, 136.9, 135.3, 133.1, 132.7, 131.3, 131.1, 129.4, 129.4,
128.7, 126.1, 125.4, 124.0, 74.9, 73.9, 71.7, 51.7, 35.3, 33.6, 29.9, 22.7, 20.7, 14.2.

167
1.99 2.00 1.99
1.90
1.94
0.73
3.08
4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 4.11.

13
C-NMR (100 MHz, CDCl
3
) of 4.11.

O
O
O
4.11
160 140 120 100 80 60 40 20 PPM
168
6.00
1.88
2.73
3.07
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of 4.12.

13
C-NMR (100 MHz, CDCl
3
) of 4.12.

O
O
O
4.12
100 80 60 40 20 PPM
169
4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 4.14.

O
OH
TBSO
O
OH
HO
4.14
170
0.83
3.00
0.95
1.27
0.65
4.09
1.74
10.04
5.46
3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 4.19.

13
C-NMR (100 MHz, CDCl
3
) of 4.19.

OMe
OH
TBSO
O
4.19
150 100 50 0PPM
171
4.00
6.04
0.88
2.62
1.38
9.05
8.20
4.89
3.46
0.48
0.81
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 4.20.

OMe
OTBDPS
TBSO
O
4.20
172
4.00
5.86
1.03
4.95
5.40
9.61
7 6 5 4 3 2 1 PPM
1
H-NMR (400 MHz, CDCl
3
) of 4.21.

13
C-NMR (100 MHz, CDCl
3
) of 4.21.

OMe
OTBDPS
HO
O
4.21
160 140 120 100 80 60 40 20 PPM
173

4.00
5.92
1.91
0.95
3.01
1.98
6.87
9.08
8 7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of 4.15.

13
C-NMR (100 MHz, CDCl
3
) of 4.15.

OTBDPS
HO
COOMe
4.15
160 140 120 100 80 60 40 20 PPM
174
0.63
4.00
5.99
1.84
0.83
3.02
6.27
9.56
9 8 7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of 4.16.

13
C-NMR (100 MHz, CDCl
3
) of 4.16.

OTBDPS
O
COOMe
4.16
200 180 160 140 120 100 80 60 40 20 PPM
175
0.00
4.00
5.84
0.86 0.83
1.94
0.96
2.86
6.05
9.32
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of 4.3.

13
C-NMR (100 MHz, CDCl
3
) of 4.3.

OTBDPS
COOMe I
4.3
160 140 120 100 80 60 40 20 PPM
176
1.02 0.95
1.86
2.05
0.97 1.03
3.02 2.98
3.43
3.00
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of 4.4.

13
C-NMR (100 MHz, CDCl
3
) of 4.4.

OO
HO
4.4
13 4. 3 2 5
12 3. 7 6 8
10 8. 1 2 6
77 .78 4
77 .28 8
77 .00 0
76 .71 0
61 .62 7
28 .06 5
27 .23 8
25 .38 1
20 .72 5
13 .97 2
140 120 100 80 60 40 20 PPM
177

0.53
0.96
0.87
1.59
1.76
1.96
2.64
3.27
3.00
8 6 4 2 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 4.5.

200 150 100 50 PPM

13
C-NMR (100 MHz, CDCl
3
) of 4.5.

OO
O
4.5
178
1.00
0.67
1.67
0.74
5.12
0.85
0.49
1.25
2.59 2.52
4.73
24.82
10.01
14.22
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 4.22.
TBSO OTBS
TMS
8E and 8Z mixture
8
4.22
179
1.00 1.03 1.02 0.99
2.07
0.87 0.91
1.80 1.76
2.94
17.90
6.86
10.36
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 4.23.

TBSO OTBS
TMS
4.23
180
1.00 1.04 1.03 1.05
2.01
0.87 0.91
0.60
1.95 1.87
3.10
18.15
10.72
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 4.8.

140 120 100 80 60 40 20 0 PPM

13
C-NMR (100 MHz, CDCl
3
) of 4.8.

TBSO OTBS
4.8
181
4.00
5.91
1.09
2.20
3.03
4.27
0.99 0.92
3.76
9.64
9.17
2.99
18.62
10.93
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 4.17.

CO
2
Me
OTBDPS
TBSO OTBS
4.17
182
1.05 1.11 1.10
2.13
1.08
4.44
2.15
4.14
0.84 0.99 1.00
6.65
2.37 2.41
3.00
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of 4.18.

160 140 120 100 80 60 40 20 PPM

13
C-NMR (100 MHz, CDCl
3
) of 4.18.

CO
2
Me
OH
HO OH
4.18
183

2.16
1.08
1.21
2.20 2.25
3.43
1.39
2.18
1.08
3.24
6.86
6.79
3.00
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of 4.1.

160 140 120 100 80 60 40 20 PPM

13
C-NMR (100 MHz, CDCl
3
) of 4.1.

CO
2
Me
OH
HO OH
4.1
140 135 130 125 120 PPM
2.16
1.08
1.21
2.20 2.25
3.43
1.39
6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 PPM
184
Chapter 5. Synthetic Efforts towards the First Total Synthesis
of Resolvin D3 (RvD3) and Aspirin-Triggered Resolvin D3
(AT-RvD3)

5.1 Introduction
Epidemiological and dietary studies strongly suggested that omega-3 fatty acids have
tremendous health benefits for number of diseases,
78
including inflammation, and
inflammation associated diseases, cancer, heart disease, Alzheimer’s disease,
pathological retinal angiogenesis
79
and neuroprotective activity in brain damage.
80
The
biochemical mechanism of the beneficial actions of omega-3 remains to be established,
until recently Serhan and colleagues discovered that endogenous anti-inflammatory
lipid mediators are generated during the resolution phase of inflammation, which are
short-lived autacoids derived from omega-3 fatty acids, such as DHA and EPA.
81

Production of these mediators can be enhanced by aspirin to give the corresponding
epimers.
4
Their basic structures were determined from the LC-UV-MS/MS-based
lipidomic analysis. These novel families of lipid mediators were termed resolvin (Rv)
and protectins, because they are generated during the resolution phase of inflammation

78
for a recent review, please see Calder, P. C. Prostaglandins Leukot. Essent. Fatty Acids 2006, 75, 197,
and references therein.
79
Connor, K. M.; SanGiovanni, J. P.; Lofqvist, C.; Aderman, C. M.; Chen, J.; Higuchi, A.; Hong, S.;
Pravada, E. A.; Majchrzak, S.; Carper, D.; Hellstorm, A.; Kang, J. X.; Chew, E. Y.; Salem, N. Jr.; Serhan,
C. N.; Simith, L. E. Nature Medicine 2007, 13, 868, and references therein.
80
for a review, please see Bazan, N. G. Curr. Opin. Clin. Nutr. Metab. Care 2007, 10, 136, and
references therein.
81
Serhan, C. N.; Clish, C. B.; Brannon, J.; Colgan, S. P.; Chiang, N.; Gronert, K. J. Exp. Med. 2000, 192,
1197. (ii) Serhan, C. N.; Hong, S.; Gronert, K.; Colgan, S. P.; Devchand, P. R.; Mirick, G.; Moussignac,
R. L. J. Exp. Med. 2002, 196, 1025. (iii) Hong, S.; Gronert, K.; Devchand, P.; Moussignac, R. L.; Serhan,
C. N. J. Biol. Chem. 2003, 278, 14677.
185
in stereochemically pure forms and act to resolve inflammation.
4
Aspirin acetylates
COX-2, but the enzyme is still active on DHA and EPA to give the aspirin-triggered
resolvins (AT-Rv). A detailed description of their biosynthesis and biological activities
are described in Chapter 1 of this dissertation. Resolvin D3 (RvD3) and aspirin-
triggered resolvin D3 (AT-RvD3) were biosynthesized in mouse exudates via cell-cell
interactions during the resolution phase of inflammation (see Chapter 1). Although the
basic structure of RvD3 and AT-RvD3 were determined by lipidomic analyses and
biosynthetic considerations, however, the absolute stereochemical assignments such as
R- and S-configurations at hydroxyl group bearing carbon centers, and precise Z/E-
configuration of the double bonds remained to be established. A substantial amount of
RvD3 and AT-RvD3 in their pure forms are needed for further chemical and biological
studies. Since they produced in subnanogram quantities in vivo, therefore, it was
necessary to accomplish their asymmetric total synthesis to assign their absolute
stereochemistry by matching their physical and most importantly biological properties
with the biogenic RvD3 and AT-RvD3, and to study their further biological activities,
to establish a structure-activity relationship (SAR), and finally to design and synthesize
metabolically stable & biologically superior analogs. In this chapter of this dissertation,
we described the synthetic efforts towards the first asymmetric total synthesis of RvD3
and AT-RvD3 herein below.

186
5.2 Results and Discussion
5.2.1 First Total Synthesis of RvD3 and AT-RvD3
Resolvin D3 (RvD3) and AT-RvD3 are epimers derived from DHA without or with
actions of aspirin, differ the stereochemistry at C-17 as shown in Figure 18. These two
lipid mediators (RvD3 and AT-RvD3 are structurally very similar to resolvin E1 (RvE1)
derived from EPA as shown in Figure 18. We have already accomplished the total
synthesis of RvE1, and our strategy was heavily relied on the Pd
0
/Cu
I
medicated cross-
coupling reaction, and Wittig reaction. When we embarked on the first total synthesis of
RvD3 and AT-RvD3, we thought we could exploit the strategy of RvE1 to synthesize
these two novel lipid mediators.

OH
COOH
OH
OH
OH
COOH
OH
OH
RvD3 AT-RvD3
OH OH
OH
COOH
RvE1
S R
R

Figure 18. Structures of RvE1, RvD3 and AT-RvD3.
187

As shown in the Scheme 19, we have employed the similar disassembled strategy as we
did for RvE1 to construct the entire carbon frame-work for RvD3 and AT-RvD3. In our
carefully constructed synthetic plan, we decided to employ 5Z and 13Z-double bonds
from the bis-acetylenic precursor of RvD3 and AT-RvD3 in the last step by selective
hydrogenation using Zn (Cu/Ag) amalgam to avoid Z/E-isomerization and losses from
over-hydrogenation. The key retrosynthetic disconnection of our approach to RvD3 and
AT-RvD3 involved the formation of two carbon—carbon bonds using Pd
0
/Cu
I
mediated
cross-coupling of terminal alkynes and vinyl halides (iodide and bromide), called
Sonogashira coupling as depicted in Figure 19. The 4S-stereochemistry of the hydroxyl
group at C-4 was planned to introduce from the S-α-butyrolactone-α-carboxylic acid
(5.3). The stereochemistries of the rest of the chiral centers at C-11 and C-17 were
introduced from chiral starting material such commercially available glycidols with
known chirality, as shown in Figure 19. The major advantages of our

188
OH
COOMe
OH
OH
Chiral Pool Chiral Glycidol
Chiral Glycidol
Selective Reduction
Selective Reduction Lindlar Hydrogenation
HWE
Pd
0
/Cu
I
Coupling
Pd
0
/Cu
I
Coupling
COOMe
OTBS
O
OH
O
O
TMS OTBS
Br OTBS
I
RvD3 (5.1)
5.2 2.3
3.5 (for RvD3)
5.3
OTBS
I
3.5' (for AT-RvD3)
or

Figure 19. Retrosynthetic analysis of RvD3 and AT-RvD3.

approach are; it is highly convergent with less number of linear steps, involved only the
connections of three major building blocks (5.2, 2.3 and 3.5 or 3.5'), two of them (2.3
and 3.5 or 3.5') are already in hands synthesized for RvE1 (see Chapter 2 for 2.3), and
RvD1 and AT-RvD1 (see Chapter 3 for 3.5 or 3.5'); like RvE1, this strategy allowed us
to create the two Z-double bonds simultaneously by a very mild selective reduction of
triple bonds, and this retrosynthetic plan has a high degree of control over the geometry
of three Z- and three E-double bonds. The first asymmetric total synthesis of RvD3 and
AT-RvD3 by employing the above-mentioned convergent strategy is described below.

189
Having two key intermediates; the bottom vinyl iodide (3.5 for RvD3 and 3.5' for AT-
RvD3) and the middle vinyl bromide (2.3) in hands, the synthesis of RvD3 and AT-
RvD3 were began with the synthesis of top terminal alkyne (5.2) from an optically pure
commercially available S-α-butyrolactone-α-carboxylic acid (5.3), as depicted in
Scheme 19. The acid catalyzed methanolsis of 5.3 under reflux condition in dry MeOH
yielded a mixture of diester (5.4)
82
and the corresponding five membered lactone (7:3
ratio detected by
1
H-NMR). The lactone was then converted to the hydroxydiester 5.4
by NaOMe catalyzed hydrolysis of the lactone in excess MeOH for overnight. The
hydroxydiester 5.4 was obtained in excellent yield (91% overall yield two steps). The
ester group adjacent to the hydroxyl group was chemoselectively reduced with borane-
dimethyl sulfide complex in dry THF at 10-15
o
C to give the corresponding diol 5.5 in
excellent yield (95%).
83
The hydroxyl groups in the dihydroxyester 5.5 were silylated
with TBS-groups by TBS-Cl in presence of imidazole and DMAP in dry CH
2
Cl
2
to give
the bis-TBS-protected methyl ester 5.6 in excellent yield (86%). Chemoselective
desilylation of primary TBS-group by the mild action camphorsulfonic acid in MeOH-
CH
2
Cl
2
(1:1) at 0
o
C to afford the primary alcohol 5.7 in high yield (78%)
84
as shown in
Scheme 19.

82
(i) Keck, G. E.; Andrus, M. B.; Romer, D. R. J. Org. Chem. 1991, 56, 417. (ii) Ashoorzadeh, A.;
Caprio, V. Synlett 2005, 2, 346.
83
(i) Saito, S.; Hasegawa, T.; Inaba, M.; Nishida, R.; Fuji, T.; Nomizu, S.; Moriwake, T. Chem. Lett.
1984, 1389. (ii) Fox, M. E.; Jackson, M.; Lennon, I. C.; McCague, R. J. Org. Chem. 2005, 70, 1227.
84
Nicolaou, K. C.; Ninkovic, S.; Sarabia, F.; Vourloumis, D.; He, Y.; Vallberg, H.; Finlay, M. R. V.;
Yang, Z. J. Am. Chem. Soc. 1997, 119, 7974
190
O
O OH
O
MeOOC COOMe
OH
COOMe
OH
HO
COOMe
OTBS
TBSO
COOMe
OTBS
HO
COOMe
OTBS
O
COOMe
OTBS
5.3 5.4
5.5
5.2
5.6
5.7 5.8
Reagents and conditions: (a) (i) HCl (conc.), dry MeOH, reflux, overnight;
(ii) MeONa (cat.), MeOH, rt, overnight, 91%; (b) BH
3
.DMS, NaBH
4
(cat.),
THF, 10-15
o
C, 2h,95%; (c) TBS-Cl,imidazole,DMAP, DMF, rt, overnight,
86%; (d) CSA, NEt
3
, MeOH-CH
2
Cl
2
(1:1), 0
o
C, 1h, 78%; (e) Swern oxidation,
CH
2
Cl
2
,-78
o
C, 4h, 99%; (f) Bestmann-Ohira reagent, anhydrous K
2
CO
3
,
MeOH, 0
o
C, 5h, 45%.
a
b
c
d
e
f

Scheme 19. Synthesis of top terminal alkyne (5.2).

Swern oxidation
85
on the resulting primary alcohol 5.7 afforded the corresponding
aldehyde (5.8) in excellent yield (99%) as shown in Scheme 19. The resulting aldehyde
5.8 was converted to the corresponding alkyne 5.2 by the treatment of 5.8 with

85
Mancuso, A. J.; Huang, S. L.; Swern, D. J. Org. Chem. 1978, 43, 2480.
191
Bestman—Ohira reagent,
86
which was prepared by following the literature
procedure.
9,87
The alkynation gave very low yield (45%), which was because wet
MeOH was used for the reaction. This reaction gives very good yield in anhydrous
conditions.

COOMe
OTBS
TMS OTBS
COOMe
5.2
5.9
5.10
5.11
OTBS
Reagents and conditions: (a) Pd(Ph
3
)
4
,CuI,C
6
H
6
,Et
3
N, rt,
overnight, 91%; (b) I
2
(catalytic), CH
2
Cl
2
, rt, 3 days, 75%; (c)
K
2
CO
3
, MeOH, rt, overnight, 98%.
a
b
c
2.3
TMS OTBS
Br
TMS OTBS
COOMe
OTBS
OTBS
COOMe
OTBS

Scheme 20. Synthesis of bis-alkyne intermediate (5.11) for RvD3 and AT-RvD3.

Having achieved the synthesis of all three key intermediates (5.2, 2.3 and 3.5 & 3.5'),
we now planned to carry-out their connections by carbon—carbon bond-forming

86
(i) Ohira, S. Synth. Commun. 1989, 19, 561. (ii) Mueller, S.; Liepold, B.; Roth, G. J.; Bestmann, H. J.
Synlett 1996, 521.
87
(i) Ghosh, A. K.; Bischoff, A.; Cappiello, J. Eur. J. Org. Chem. 2003, 821. (ii) Goundry, W. R. F.
Tetrahedron 2003, 59, 1719.
192
reactions to build the entire carbon frame-work of RvD3 and AT-RvD3. As depicted in
Scheme 20, the Pd
0
/Cu
I
mediated cross-coupling reaction of terminal alkyne (5.2) and
vinyl-bromide (2.3) (for the synthesis 2.3 please see Chapter 2, Section 2.2.3) using
standard Sonogashira coupling
88
conditions afforded the 7E/7Z-diasteromers of bis-
alkyne 5.9 with excellent yield (91%) as shown in Scheme 20. The 7Z-isomer was,
however, easily isomerized to the corresponding 7E-isomer with catalytic amount of
sublimed iodine in dry CH
2
Cl
2
in very good yield (75%). The Na
2
CO
3
mediated
desilylation of TMS-group furnished the terminal 7E, 9E-bis-alkyne 5.11 with excellent
yield (98%).

The final assembly of the bis-alkyne (5.11) and the vinyl iodide 3.5 for RvD3 and 3.5'
for AT-RvD3 using Sonogashira coupling, and subsequent desilylation and
hydrogenation are yet to be done.

88
Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett. 1975, 16, 4467.
193
5.11
OTBS
COOMe
OTBS
OTBS
I
3.5
OTBS
I
3.5'
OTBS
COOMe
OTBS
OTBS
OTBS
COOMe
OTBS
OTBS
OH
COOMe
OH
OH
OH
COOMe
OH
OH
OH
COOMe
OH
OH
OH
COOMe
OH
OH
RvD3 Methyl Ester (5.1) AT-RvD3 Methyl Ester (5.1')
S
R
Yet to be done Yet to be done

Scheme 21. Synthetic steps for RvD3 and AT-RvD3 are yet to be done.

5.3 Conclusion
The synthetic efforts towards the first total synthesis of RvD3 and AT-RvD3 were
described here in this chapter. We have successfully synthesized the all key
intermediates with precise control of stereochemistry. The final Sonogashira coupling
for both RvD3 and AT-RvD3 are yet to be done.
194
5.4 Experimental

MeOOC COOMe
OH
5.4

5.4.1 2S-Dimethyl 2-hydroxypentanedioate (5.4). A solution of (S)-(+)-5-oxo-2-
tetrahydrofurancarboxylic acid, 5.3 (5.0 g, 38.43 mmol) and 4 drops of concentrated
HCl in dry MeOH (40 mL) was heated to reflux for overnight. The mixture was cooled
to 0
o
C, and then a solid NaHCO
3
was slowly added to neutralize the solution checked
by a pH paper. The solution was filtered, and the filtrate was concentrated in vacuo,
and then added CH
2
Cl
2
(40 mL) and aqueous solution of NH
4
Cl (40 mL). The aqueous
phase was extracted with CH
2
Cl
2
(40 mL x 3), washed with brine, dried over MgSO
4
,
and concentrated in vacuo. The crude was then purified on silica column using 50%
EtOAc/hexanes as the solvent system to give two products: the desired diester 5.4 (70%)
and a five-membered lactone (30%), which was then converted to the diester 5.4 by
treating it with a catalytic amount of MeONa in MeOH for overnight. A total of 6.1 g of
desired dimethyl ester 5.4 (91%) was obtained.
1
H-NMR (400 MHz, CDCl
3
) δ
H
4.15
(dd, J = 8.0, 4.4 Hz, 1H), 3.69 (s, 3H), 3.58 (s, 3H), 2.46-2.31 (m, 2H), 2.07 (m, 1H),
1.85 (m, 1H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
174.9, 173.5, 69.3, 52.4, 51.5, 29.2, 29.1.

COOMe
OH
HO
5.5

195
5.4.2 4S-Methyl-4,5-dihydroxypentanoate (5.5). To a solution of dimethyl diester 5.4
(2.0 g, 11.35 mmol) in dry THF (15 mL) at 10-15
o
C was added BH
3
.DMS complex
(1.18 mL, 12.48 mmol) over 20 min maintaining the temperature at 10-15
o
C using a
cold water bath. The solution was stirred at the same temperature for an hour. A
catalytic amount of NaBH
4
(23.6 mg, 0.62 mmol) was slowly added to the reaction
mixture and kept the temperature below 20
o
C, and stirred for 1 h. The reaction was
quenched by slowly adding dry MeOH (10 mL), and stirred for an additional 30 min.
The solvent was evaporated to give the crude diol, which was then directly purified on a
silica column using neat EtOAc as the solvent system to give the pure diol 5.5 (1.61 g,
95%) as a colorless oil.
1
H-NMR (400 MHz, acetone-d
6
) δ
H
3.67 (m, 1H), 3.61 (m, 3H),
3.45 (m, 2H), 2.51-2.35 (m, 2H), 1.84-1.76 (m, 1H), 1.65-1.56 (m, 1H);
13
C-NMR (100
MHz, CDCl
3
) δ
C
174.6, 71.8, 67.2, 51.6, 30.9, 29.2.

COOMe
OTBS
TBSO
5.6

5.4.3 4S-Methyl-4,5-bis(t-butyldimethylsilyloxy)pentanoate (5.6). To a mixture of
imidazole (2.21 g, 32.60 mmol), TBS-Cl (4.10 g, 32.60 mmol), and DMAP (66 mg,
0.54 mmol) in anhydrous DMF (13 mL) at 0
o
C was added the diol 5.5 (1.61 g, 10.86
mmol) in anhydrous DMF (2 mL) by a cannula. The reaction mixture was warmed to
room temperature, and stirred for overnight. The reaction mixture was quenched with a
saturated aqueous solution of NH
4
Cl, extracted with ether (20 mL x 3), washed with
brine, dried over MgSO
4
, and concentrated under reduced pressure to give a crude
196
product, which was then purified on silica column using 4% EtOAc/hexanes as the
eluant to give the title compound 5.6 (3.50 g, 86%) as a colorless oil.
1
H-NMR (400
MHz, CDCl
3
) δ
H
3.68 (m, 1H), 3.65 (s, 3H), 3.51 (dd, J = 10.0, 5.2 Hz, 1H), 3.37 (dd, J
= 10.0, 6.8 Hz, 1H), 2.40-2.30 (m, 2H), 1.95-1.87 (m, 1H), 1.72-1.63 (m, 1H), 0.87 (s,
9H), 0.86 (s, 9H), 0.03 (s, 6H), 0.02 (s, 3H), 0.01 (s, 3H).

COOMe
OTBS
HO
5.7

5.4.4 4S-Methyl-4-(t-butyldimethylsilyloxy)-5-hydroxypentanoate (5.7). To a
solution of bis-TBS protected diol 5.6 (2.50 g, 6.63 mmol) in a 1:1 mixture of
CH
2
Cl
2
:MeOH (30 mL) was added camphorsulfonic acid (1.23 g, 5.3 mmol) at 0
o
C.
The reaction progress was monitored by TLC. The reaction was over by 20 min, it was
then quenched with Et
3
N (0.92 mL, 6.63 mmol). The solvent was evaporated to dryness,
and he the crude product was directly purified on a silica gel column using 30%
EtOAc/hexanes as the solvent system to afford the primary alcohol 5.7 (1.35 g, 78%) as
a colorless oil.
1
H-NMR (250 MHz, CDCl
3
) δ
H
3.77 (m, 1H), 3.47 (dd, J = 9.0, 4.5 Hz,
2H), 2.35 (td, J = 7.8, 3.0 Hz, 2H), 1.86-1.78 (m, 2H), 0.87 (s, 9H), 0.06 (s, 3H).

COOMe
OTBS
O
5.8

5.4.5 4S-Methyl-4-(t-butyldimethylsilyloxy)-5-oxopentanoate (5.8). To a solution of
DMSO (0.88 mL, 11.43 mmol) in dry CH
2
Cl
2
(25 mL) at -78
o
C was slowly added
197
oxalyl chloride (0.66 mL, 7.62 mmol) and stirred for 15 min, and then added the
primary alcohol 5.7 (1.0 g, 3.81 mmol) in CH
2
Cl
2
(5 mL) through a cannula, and stirred
for 50 min. Et
3
N (2.65 mL, 19.05 mmol) was then added to the reaction mixture, and
stirred for 3 h at -78
o
C. The reaction mixture was then allowed to come to room
temperature and stirred for an additional 30 min. The reaction was quenched with a
saturated aqueous solution of NH
4
Cl, extracted with ether (30 mL x 3), washed with
brine, dried over anhydrous MgSO
4
, and concentrated under reduced pressure. The
crude product was purified on a silica column using 20%EtOAc/hexane as the eluant to
give the aldehyde 5.8 (0.98 g, 95%) as a colorless oil.
1
H-NMR (250 MHz, CDCl
3
) δ
H

9.57 (d, J = 1.5 Hz, 1H), 4.05 (m, 1H), 3.65 (s, 3H), 2.40 (m, 2H), 1.95 (m, 2H), 0.89 (s,
9H), 0.06 (s, 3H), 0.04 (s, 3H).

COOMe
OTBS
5.2

5.4.6 4S-Methyl-4-(t-butyldimethylsilyloxy)-hex-5-ynoate (5.2). To a stirred
solution of the aldehyde 5.8 (0.60 g, 2.30 mmol) and Bestmann-Ohira reagent
(dimethyl-1-diazo-2-oxopropylphosphonate) 5.12 (1.10 g, 5.76 mmol) in MeOH (20
mL) at 0
o
C was added anhydrous K
2
CO
3
(0.95 g, 6.9 mmol). The cooling was
removed upon the end of the addition. The reaction was monitored by TLC, which was
completed by 5 h at room temperature. The mixture was diluted with ether (10 mL),
and quenched with H
2
O (10 mL), and extracted with ether (20 mL x 3). The combined
ether extracts were washed with a saturated aqueous solution of NaHCO
3
and then with
198
brine, and then dried over MgSO
4
. The solvent was concentrated in vacuo to give the
crude alkyne, which was then purified on a silica column using 15% EtOAc/hexanes as
the solvent system to furnish the pure terminal alkyne 5.2 (0.26 g, 45%).
1
H-NMR (400
MHz, CDCl
3
) δ
H
4.42 (td, J = 6.5, 2.0 Hz, 1H), 3.64 (s, 3H), 2.47 (t, J = 7.5 Hz, 2H),
2.35 (d, J = 2.0 Hz, 1H), 1.97 (m, 2H), 0.86 (s, 9H), 0.01 (s, 3H), 0.06 (s, 3H).

P
O
N
2
O
OMe
OMe
5.12

5.4.7 Preparation of Bestmann-Ohira reagent (5.12). Sodium hydride (0.53 g of
60% in oil, 13.24 mmol) was washed with dry THF (5 mL x 2). To a cold suspension
(0-5
o
C) of NaH in dry THF (30 mL) was added dimethylacetylmethylphosphonate (2.0
g, 12.04 mmol) in dry THF (10 mL) via cannula. After stirring for an hour at 0
o
C,
TsN
3
(2.61 g, 13.24 mmol) in dry THF (5 mL) was added via a syringe. The reaction
mixture was stirred for an hour at 1 h, checked TLC, and found that the reaction was
over by then. The mixture was diluted with EtOAc (15 mL), and filtered through celite.
The filtrate was collected, concentrated under vacuo to give a crude product, which was
then purified on silica column using neat EtOAc as the mobile phase to give the pure
Bestmann-Ohira reagent 5.12 (1.41 g, 71%).
1
Η−NMR (250 ΜΗz, CDCl
3
) δ
H
3.84 (s,
3H), 3.79 (s, 3H), 2.33 (s, 3H).

199
TMS OTBS
COOMe
5.9
OTBS

5.4.8 Methyl 4S, 11R, 7(E/Z), 9E, 4,11-bis(t-butyldimethylsilyloxy)-14-
(trimethylsilyl)- tetradeca-7,9-dien-5,13-diynoate (5.9). To a solution of vinyl
bromide 2.3 (324 mg, 0.84 mmol) in benzene (4 mL) and Et
3
N (0.89 mL, 6.4 mmol)
was added the alkyne 5.2 (165 mg, 0.64 mmol) in benzene (1.0 mL) via a cannula. The
mixture was freeze-thaw several times at -78
o
C to remove oxygen. The reaction
mixture was then warmed to room temperature followed by the addition of Pd(Ph
3
)
4
(74
mg, 0.064 mmol) and CuI (24 mg, 0.128 mmol). The reaction mixture was protected
from the light by warping the flask with the aluminum foil. The reaction mixture was
then stirred at room temperature for overnight. The reaction was quenched with
saturated aqueous solution of NH
4
Cl, extracted with ether (20 mL x 3), washed with
brine. The organic layers were combined, dried over MgSO
4
, and concentrated in
vacuo to give the crude product, which was then purified on a silica column using 1%
EtOAc/hexanes to give the protected alkyne 5.9 (327 mg, 91%).
1
H-NMR (250 MHz,
CDCl
3
) δ
H
6.67 (dd, J = 14.8, 10.5 Hz, 1H), 6.33 (t, J = 10.5 Hz, 1H), 5.86 (dd, J = 14.8,
6.0 Hz, 1H), 5.43 (d, J = 10.5 Hz, 1H), 4.60 (m, 1H), 4.33 (q, J = 7.0 Hz, 1H), 3.66 (s,
3H), 2.51-2.37 (m, 4H), 2.01 (m, 2H), 0.89 (s, 9H), 0.88 (s, 9H), 0.12 (s, 9H), 0.10 (s,
3H), 0.09 (s, 3H), 0.08 (s, 3H), 0.05 (s, 3H).

200
TMS OTBS
COOMe
5.10
OTBS

5.4.9 Methyl 4S, 11R, 7E, 9E, 4, 11-bis(t-butyldimethylsilyloxy)-14-(trimethylsilyl)-
tetradeca-7,9-dien-5,13-diynoate (5.10). The mixture of 7Z/7E isomers of 5.9 (0.22 g,
0.39 mmol) was dissolved in dry CH
2
Cl
2
(80 mL), then a small crystal of sublimed I
2

(20 mg) was added to the mixture and stirred at room temperature for three days under
the sunlight. The violet color solution was then quenched with a saturated aqueous
solution of Na
2
S
2
O
5
, extracted with ether (40 mL x 3), and washed with brine. The
organic layers were combined, dried over anhydrous MgSO
4
, filtered, and concentrated
under reduced pressure. The residue was purified on a silica gel column using 2%
EtOAc/hexane as the eluant to give the desired 7E, 9E isomer, 5.10 (0.165 mg, 75%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.80 (dd, J = 14.8, 10.5 Hz, 1H), 6.42 (d, J = 14.0 Hz,
1H), 6.36 (dd, J = 14.8, 6.0 Hz, 1H), 5.91 (dd, J = 14.8, 10.5 Hz, 1H), 4.61 (m, 1H),
4.28 (q, J = 7.0 Hz, 1H), 3.61 (s, 3H), 2.41-2.37 (m, 4H), 1.98 (m, 2H), 0.84 (s, 18H),
0.11 (s, 9H), 0.10 (s, 3H), 0.05 (s, 9H), 0.03 (s, 3H), 0.02 (s, 3H).

201
0.92
3.01 3.00
2.11
1.05 1.05
5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 PPM

1
H-NMR (400 MHz, CDCl
3
) of 5.4.
180 160 140 120 100 80 60 40 PPM

13
C-NMR (100 MHz, CDCl
3
) of 5.4.

MeOOC COOMe
OH
5.4
202

2.00
2.42
0.98 1.01
1.48
3.28
3.5 3.0 2.5 2.0 1.5 1.0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 5.5.

COOMe
OH
HO
5.5
203
3.62
1.00 0.97
1.88
0.94 0.96
17.41
9.96
4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 5.6.

COOMe
OTBS
TBSO
5.6
204
0.94
3.00
2.15
3.75
10.79
5.93
2.25
4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 5.7.

COOMe
OTBS
HO
5.7
205
0.69
1.23
3.00
2.21
2.59
11.28
6.26
8 6 4 2 0PPM

1
H-NMR (400 MHz, CDCl
3
) of 5.8.

COOMe
OTBS
O
5.8
206
0.91
3.00
2.29
0.68
2.37
10.16
5.60
4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 5.2.

OTBS
COOMe
5.2
207
0.86 0.97
0.72 0.73
0.97 0.98
3.00
5.02
2.15
22.11
21.22
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 5.11.

OTBS
COOMe
OTBS TMS
5.11
208
Chapter 6. First Total Synthesis of Resolvin D4 (RvD4) and
Aspirin-Triggered Resolvin D4 (AT-RvD4)

6.1 Introduction
The D-series of resolvins are formed from docosahexaenoic acid (DHA) during the
resolution phase of inflammation with or without aspirin. The proposed biosynthetic
pathways are described in the Chapter 1. The endogenous DHA converted in vitro
involve the lipoxygenase (LOX) product 17S-H(p)DHA, which is rapidly transformed
by the action of LOX in human PMN into two epoxides 7S, 8-epoxy-17S-HDHA, and
4-epoxy-17S-HDHA. The enzymatic ring opening of these two novel epoxides
produced bioactive lipid mediators denoted as 17S-resolvin D series (RvD1—RvD4).
When treated with aspirin, it also impacts the formation of resolvin D series by
catalytically switching COX-2 activity to a LOX like mechanism that generates 17R-
H(p)-DHA. The subsequent epoxidation followed by enzymatic ring of the epoxides
produced another novel class of lipid mediators named aspirin-triggered resolvin D-
series (AT-RvD1—AT-RvD4).

209
OH
COOH
OH
OH
OH
COOH
OH
OH
SR
RvD4 (6.1) AT-RvD4 (6.1')

Figure 20. Structures of RvD4 (6.1) and AT-RvD4 (6.1').

RvD4 and AT-RvD4 are C-17 epimers generated in stereochemically pure form in very
minute amount, and possess interesting and distinct structures consisting of a
conjugated triene and a conjugated diene chromosphores. The basic structures of RvD4
and AT-RvD4 were elucidated from the LC-UV-MS/MS-based lipidomic analysis, and
a part of absolute stereochemistry was predicted from the consideration of biosynthetic
pathways. To finish the complete stereochemical assignments, and for their further
structural and biological studies, we have under taken a project of the total synthesis of
these lipid mediators. In this chapter, we describe the first total synthesis of RvD4 and
AT-RvD4.
6.2 Results and Discussion
6.2.1 Retrosynthetic Analysis of RvD4 and AT-RvD4
The initial retrosynthetic analysis of RvD4 and AT-RvD4 is depicted in Figure 21. The
carbon frame-work of RvD4 (6.1) and AT-RvD4 (6.1') are little-bit different than other
lipid mediators of D-series. They have a very interesting and distinct structures
consisting of a conjugated triene and a conjugated diene chromosphores, which
210
demanded a careful, and somewhat different strategy. Like other resolvins, there are
several challenges for the synthesis of either RvD4 (6.1) or AT-RvD4 (6.1'). First—the
construction of conjugated polyene carbon skeleton with correct geometry. In our
carefully constructed synthetic plan, we envisaged to generate 10Z and 13Z-double
bonds from a stereoselective mild reduction of triple bonds in the last step to avoid Z/E-
isomerization, and to avoid further complications in handling. The isolated double bond
19Z-double bond was planned to introduce from the stereospecific partial hydrogenation
of a triple bond using Lindlar catalyst. Second—the introduction of correct
stereochemistry at the hydroxyl group bearing chiral centers. In our plan, like other
resolvins, we envisioned to incorporate the 4S- and 5R-configurations from a chiral pool
such as D-(-)-erythrose (6.8) and 17S-stereoconfiguration in RvD4 (6.1) from R-
glycidol, and 17R-configuration in AT-RvD4 (6.1') from S-glycidol as shown in Figure
21. Third—a convergent strategy is required not only for the efficient total synthesis,
but also for the creation of series of biologically superior and enzymatically stable
analogs or mimetics for potential drug-discovery. Based-on above-mentioned logics,
the retrosynthetic disassembly is depicted in Figure 21.

211
OH
COOMe
OH
OH
Natural Chiral Pool Wittig
Pd
0
/Cu
I
Coupling
Chiral Glycidol
Selective Reduction
Lindlar Hydrogenation
Cu
I
Mediated Coupling
Selective Reduction
PPh
3
Br
TMS
O
OH
HO OH
Ph
3
P COOMe
D-(-)-Erythrose (6.7)
RvD4 (6.1)
OTBS
I
Br
OTBS TBSO
COOMe
O
OTBS TBSO
COOMe
OTBS TBSO
COOMe
3.5 (for RvD3)
6.2
6.4 6.3
3.4 6.5
6.6
OTBS
I
3.5' (for AT-RvD3)
O
HO
O
OH
R-glycidol for RvD4
S-glycidol for AT-RvD4
R
S
or

Figure 21. First retrosynthetic analysis of RvD4 and AT-RvD4.

6.2.2 Synthesis of aldehyde 6.5
The synthesis of aldehyde 6.5 with 4S and 5R-stereoconfigurations was started with a
Wittig reaction between a pseudo-aldehyde in D-(-) erythrose (6.7) and Wittig reagent,
methyl (triphenylphosphoranilidene)-acetate (6.6) in THF at 65
o
C to give a trihydroxy
212
2E-alkene as shown in Scheme 22. The absolute stereochemistry at the secondary
hydroxyl groups was derived from the commercially available sugar D-(-)-erythrose
(6.7).

O
OH
HO OH
OTBS TBSO
COOMe
O
Ph
3
P
OMe
O
OTBS TBSO
TBSO
6.6
6.7
OH HO
COOMe
HO
OTBS TBSO
COOMe
TBSO
OTBS TBSO
COOMe
HO
OH HO
HO
O
HO
O
OH
6.8
6.10
6.9 6.11
6.12
6.14
6.5
a
bd
ce
f
g
Reagents and conditions: (a) THF, 65
o
C, overnight, 97%; (b) Pd/C (5%), MeOH,
H
2
gas, rt, overnight, 100% (6.9 : 6.10 = 4:1); (c) TBS-Cl, imidazole, DMAP, DMF,
rt, overnight, (13:14 = 1:4); (d) TBS-Cl, imidazole, DMAP, DMF, rt, overnight, 99%;
(e) Pd/C (5%), EtOAc, H
2
gas, rt,overnight,100%; (f) CSA,MeOH:CH
2
Cl
2
(1:1), 0
o
C, 1h, 65%; (g) Swern Oxidation, CH
2
Cl
2
, -78
o
C, 4h, 97%.
OMe
O
OMe
O
O
TBSO
O
OTBS
6.13 (80%)

Scheme 22. Synthesis of aldehyde 6.5 for RvD4 and AT-RvD4.

The trihydroxy alkene (6.8) obtained from the Wittig reaction was subjected to catalytic
hydrogenation with Pd/C (5% Pd on carbon) in MeOH. Unfortunately, the catalytic
hydrogenation afforded a mixture of chromatographically separable triol (6.9) and a six-
213
membered lactone (6.10) in 4:1 ratio. The triol was then subjected to tris-silylation by
TBS-Cl, imidazole, and DMAP to give the TBS-protected triol 6.12, but again
unfortunately yielded the protected lactone 6.13 as major product (80%) via a base-
induced lactonization to form a thermodynamically stable six-membered lactone.
Having difficulties in getting the uncyclized triol 6.12, we now planned to protect the
hydroxyl groups before hydrogenation to give protected triol with a 2E-double bond in
the molecule. The silylation reaction proceeded smoothly to give the product 6.11 in
excellent yield (99%). The 2E-alkene was subjected to hydrogenation to give the
desired tris-TBS-protected triol 6.12, however, this hydrogenation step was very solvent
sensitive. In our experimental conditions with catalytic Pd/C (5% on carbon) at room
temperature for overnight, if the reaction carried-out in MeOH, all three TBS-groups
were totally desilylated by the cleavage of silyl ethers. In search for a suitable condition
for the hydrogenation of double-bonds in presence of silyl ethers, we have found a
literature procedure where authors described a remarkable solvent effect toward the
Pd/C-catalyzed of silyl ethers.
89
We have found that EtOAc is a suitable solvent for this
substrate for hydrogenation, and the reaction proceeded smoothly in quantitative yield.
Chemoselective desilylation of the primary TBS-group in 6.12 by the mild action of
camphorsulfonic acid at 0
o
C in MeOH—CH
2
Cl
2
(1:1) afforded the primary unprotected
alcohol (6.14) in good yield (65%).
90
Swern-oxidation
91
of the primary alcohol in 6.14

89
Sajiki, H.; Ikawa, T.; Hattori, K; Hirota, K. Chem. Commun. 2003, 654.
90
Nicolaou, K. C.; Ninkovic, S.; Sarabia, F.; Vourloumis, D.; He, Y.; Vallberg, H.; Finlay, M. R. V.;
Yang, Z. J. Am. Chem. Soc. 1997, 119, 7974.
91
Mancuso, A. J.; Huang, S. L.; Swern, D. J. Org. Chem. 1978, 43, 2480.
214
yielded the desired aldehyde 6.5 in excellent yield (97%) with 4S and 5R-
stereoconfigurations at the chiral centers.
6.2.3 Wittig reaction between the aldehyde and the Wittig reagent
The Wittig reagent 3.4 was prepared from the commercially available 2-pentene-4-yn-
1-ol by the following a set of standard transformations described in Chapter 3 (Section
3.2.1.4).

PPh
3
Br
OTBS TBSO
COOMe
O
TMS
COOMe
TBSO OTBS
COOMe
TBSO OTBS
TMS
COOMe
TBSO OTBS
3.4
6.5
TMS
6.15
6.16
6.3
Reagents and conditions: (a) n-BuLi, -78
o
C, THF, 3 h, 90%; (b) I
2
,
CH
2
Cl
2
, rt, 2 days, 95%; (c) Na
2
CO
3
, MeOH, rt, overnight, 96%.
a
b
c

Scheme 23. Synthesis of terminal alkyne 6.3 for RvD4 and AT-RvD4.

215
Having both the aldehyde (6.5) and the Wittig reagent (3.4) in hands, we now focused
on their coupling using n-BuLi in dry THF at -78
o
C. The reaction afforded the single
6Z,8E-isomer (6.15) in excellent yield (90%). The 6Z,8E-isomer (6.15) was easily
converted to the desired 6E,8E-isomer (6.16) with the catalytic amount of sublimed I
2
in
dry CH
2
Cl
2
. The Na
2
CO
3
mediated desilylation of TMS-group from 6.16 afforded the
terminal alkyne 6.3 in excellent yield (96%).
6.2.4 Coupling of propargyl bromide and terminal alkyne
The synthesis of terminal bis-alkyne intermediate 6.2 was accomplished by K
2
CO
3

mediated CuI-catalyzed cross-coupling of propargyl bromide 6.4 and terminal alkyne
6.3 in presence of NaI in DMF
92
as shown in Scheme 24.

COOMe
TBSO OTBS
Br
TMS
COOMe
TBSO OTBS
TMS
6.3
6.4
CuI, NaI, K
2
CO
3
DMF, rt, 89%
6.17
COOMe
TBSO OTBS
6.2
Na
2
CO
3
, MeOH
90%

Scheme 24. Synthesis of bis-alkyne intermediate 6.2.

92
Ivanov, I. V.; Groza, N. V.; Malchenko, G. M.; Myagkova, G. I.; Schewe, T. Bioorg. Khim. 1997, 23,
519, Russ. J. Bioorg. Chem. 1997, 23, 841.
216

The reaction proceeded smoothly, and afforded the TMS-protected bis-alkyne 6.17 in
excellent yield (89%). The Na
2
CO
3
mediated desilylation of TMS-group in MeOH
from 6.17 afforded the terminal alkyne 6.2 in excellent yield (90%).
6.2.5 Final assembly of the RvD4 and AT-RvD4 by Sonogashira coupling
The synthesis of vinyl iodides 3.5 and 3.5' were accomplished in very high yields from
R-glycidol and S-glycidol, respectively. Their in detailed synthetic steps were described
in Chapter 3 for the total synthesis of RvD1 and AT-RvD1 (please see Section 3.2.1.5
for 3.5 and Section 3.2.2 for 3.5').

217
COOMe
TBSO OTBS
6.2
OTBS
I
3.5
OTBS
I
3.5'
OTBS
COOMe
OTBS
OTBS
OTBS
COOMe
OTBS
OTBS
OH
COOMe
OH
OH
OH
COOMe
OH
OH
OH
COOMe
OH
OH
OH
COOMe
OH
OH
RvD4 Methyl Ester (6.1) AT-RvD4 Methyl Ester (6.1')
Reagents and conditions: (a) variations of Sonogashira conditions.
S R
Did not work Did not work a a

Scheme 25. Final Sonogashira coupling of vinyl iodides and terminal alkyne.

Our plan was to use Pd
0
/Cu
I
mediated coupling reaction as the last carbon—carbon
bond-forming reaction to have complete carbon frame-work of RvD4 and AT-RvD4 as
we did for other resolvins described in this dissertation. Unfortunately, Sonogashira
coupling reaction between the terminal alkyne 6.2 and the vinyl iodide 3.5 or 3.5' did
not work-out at all under variety of different conditions such as stoichiometry, solvent,
base and ligands. The reason for failing in Sonogashira coupling might be the terminal
alkyne substrate, where two alkyne groups are separated by a methylene group, which
218
could be very acidic causing the problem, or CuI might be interacting with both the
alkyne bonds preventing it participating in the coupling reaction.
6.2.6 Revised synthetic strategy for RvD4 and AT-RvD4
Since the Sonogashira coupling in the final carbon—carbon bond-forming reaction
didn’t work-out, we have revised our strategy to accomplish the total synthesis of RvD4
and AT-RvD4. We planned to run the CuI mediated coupling reaction of propargyl
bromide and terminal alkyne as the last carbon—carbon bond-forming reaction. The
retrosynthetic plan is depicted in the Figure 22.

OH
COOMe
OH
OH
Pd
0
/Cu
I
Coupling
Selective Reduction
Cu
I
Mediated Coupling
Selective Reduction
OTBS
Br
6.19 3.5
RvD3 (6.1)
OTBS
I HO
OTBS TBSO
COOMe
6.18 6.3

Figure 22. Revised retrosynthetic strategy for RvD4 and AT-RvD4.

219
Sonogashira coupling reaction between the vinyl iodide 3.5 and propargyl alcohol 6.19
under standard conditions afforded the propargyl alcohol, which was then converted to
the corresponding propargyl bromide 6.18 as shown in Scheme 26.

OTBS
HO
6.19 3.5 OTBS
I HO
6.20
OTBS
Br
6.18
Pd(Ph
3
)
4
, CuI, NEt
3
, C
6
H
6
,
rt, overnight, 90%
NBS, PPh
3
, CH
2
Cl
2
, 0
o
C, 40 min, 55%

Scheme 26. Synthesis of propargyl bromide for RvD4.

220
COOMe
TBSO OTBS
6.3 OTBS
Br
6.18 OTBS
Br
6.18'
OTBS
COOMe
OTBS
OTBS
OTBS
COOMe
OTBS
OTBS
OH
COOMe
OH
OH
OH
COOMe
OH
OH
OH
COOMe
OH
OH
OH
COOMe
OH
OH
RvD4 Methyl Ester (6.1) AT-RvD4 Methyl Ester (6.1')
a a
Reagents and conditions: (a) NaI, CuI, K
2
CO
3
, DMF,rt (b)
TBAF, THF, rt; (c) Zn (Cu/Ag), H
2
O:MeOH (1:1), 40
o
C.
yet to be done yet to be done
S R

Scheme 27. Final assembly of RvD4 and AT-RvD4 which are yet to be done.

The CuI mediated final carbon—carbon bond forming reaction of terminal alkyne 6.3
and propargyl bromide 6.18 to build the entire carbon frame-work of RvD4 (and AT-
RvD4) was planned as shown in Scheme 27, which are yet to be done.
6.3 Conclusion
We have described the progress towards the total synthesis of RvD4 and AT-RvD4.
Since our initial plan didn’t work out, we have revised our plan, and made a significant
progress towards completing their total synthesis. Only carbon—carbon bond forming
reaction remains to be done.
221
6.4 Experimental

OH HO
HO
6.8
OMe
O
6.4.1 Methyl (4S, 5R, 2E)-4,5,6-trihydroxyhex-2-enoate (6.8). To a suspension of D-
(-)-erythrose, 6.7 (2.0 g, 16.65 mmol) in dry THF (20 mL) under argon was added
methyl (triphenylphosphoranylidene) acetate 6.6 (5.67 g, 16.65 mmol). The flask was
sealed and heated at 65
o
C for overnight. The resulting yellow solution was
concentrated in vacuo to give a crude mixture of the desired triol and
triphenylphosphine oxide. The crude was then purified on a silica column using 12%
MeOH/CH
2
Cl
2
to yield the pure product 6.8 (2.8 g, 97%) with exclusively 2E double-
bond geometry.
1
H-NMR (400 MHz, CD
3
OD) δ
H
7.13 (dd, J = 16.0, 4.8 Hz, 1H), 6.09
(dd, J = 16.0, 2.0 Hz, 1H), 4.22 (td, J = 8.0, 1.6 Hz, 1H), 3.72 (s, 3H), 3.66 (dd, J = 11.0,
4.4 Hz, 1H), 3.59 (dd, J = 11.0, 6.0 Hz, 1H), 3.54 (m, 1H).
OH HO
HO
O
HO
O
OH
6.10 6.9
OMe
O

6.4.2 Methyl (4S, 5R, 2E)-4,5,6-trihydroxyhexanoate (6.9). To a solution of 6.8 (2.8
g, 15.90 mmol) in MeOH (35 mL) was added one scope of 5% Pd(0)/C. The reaction
mixture was stirred under the static atmosphere of H
2
gas at room temperature for
overnight. The Pd/C was filtered off, and the filtrate was evaporated under reduced
pressure to give a viscous liquid, which was a mixture 4:1 mixture of 6.9 and 6.10.

222

OTBS TBSO
TBSO
6.11
OMe
O
6.4.3 Methyl (4S, 5R, 2E)-4,5,6-tris(t-butyldimethylsilyloxy)hex-2-enoate (6.11). To
a mixture of imidazole (3.08 g, 45.41 mmol), TBS-Cl (6.80 g, 45.41 mmol), and DMAP
(69 mg, 0.6 mmol) in anhydrous DMF (12 mL) at 0
o
C was added a solution of
trihydroxy ester 6.8 (2.0 g, 11.35 mmol) in anhydrous DMF (3.0 mL). The reaction
mixture was warmed to room temperature, and stirred for overnight. The reaction
mixture was quenched with a saturated aqueous solution of NH
4
Cl, extracted with ether
(30 mL x 3), washed with brine, dried over MgSO
4
, and concentrated under reduced
pressure to give a crude product, which was then purified on silica column using 3%
EtOAc/hexanes as the eluant to afford the pure 6.11 (5.80 g, 99%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.98 (dd, J = 15.2, 5.2 Hz, 1H), 5.95 (dd, J = 15.2, 1.5
Hz, 1H), 4.38 (td, J = 5.6, 1.6 Hz, 1H), 3.70 (s, 3H), 3.66 (m, 1H), 3.54 (dd, J = 10.4,
6.0 Hz, 1H), 3.49 (dd, J = 10.4, 5.6 Hz, 1H), 0.88 (s, 9H), 0.87 (s, 9H), 0.83 (s, 9H),
0.03 (s, 3H), 0.02 (s, 6H), 0.01 (s, 3H), 0.00 (s, 3H), -0.02 (s, 3H);
13
C-NMR (100 MHz,
CDCl
3
) δ
C
168.8, 148.7, 120.9, 77.3, 73.2, 64.3, 51.4, 25.9, 25.8, 25.6, 18.2, 18.0, -2.9, -
4.5, -4.6, -4.7, -4.8, -5.4.

OTBS TBSO
TBSO
6.12
OMe
O
6.4.4 Methyl (4S, 5R)-4,5,6-tris(t-butyldimethylsilyloxy)hexanoate (6.12). To a
solution of 6.11 (3.75 g, 7.22 mmol) in EtOAc (50 mL) was added one scope of 5%
Pd(0)/C. The reaction mixture was stirred under the static atmosphere of H
2
gas at
223
room temperature for overnight. The reaction mixture was filtered through celite and
Pd/C was filtered off. The filtrate was evaporated under reduced pressure to afford pure
6.12 (3.76, 100%) as a viscous liquid.
1
H-NMR (400 MHz, CDCl
3
) δ
H
3.73 (q, J = 4.0
Hz, 1H), 3.62 (m, 3H), 3.60 (m, 1H), 3.54 (dd, J = 10.4, 6.0 Hz, 1H), 3.41 (dd, J = 10.4,
5.6 Hz, 1H), 2.45-2.27 (m, 2H), 1.88-1.70 (m, 2H), 0.85 (s, 18H), 0.84 (s, 9H), 0.04 (s,
3H), 0.03 (s, 6H), 0.01 (s, 3H), 0.00 (s, 6H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
174.4,
77.1, 72.7, 64.9, 30.1, 27.2, 25.9, 25.8, 25.7, 18.3, 18.2, 18.1, -3.0, -4.1, -4.4, -4.7, -4.9,
-5.5.

OTBS TBSO
HO
6.14
OMe
O
6.4.5 Methyl (4S, 5R)-4,5-bis(t-butyldimethylsilyloxy)-6-hydroxyhexanoate (6.14).
To a solution of protected triol 6.12 (2.0 g, 3.83 mmol) in a 1:1 mixture of
CH
2
Cl
2
:MeOH (30 mL) was added camphorsulfonic acid (0.71 g, 3.07 mmol) at 0
o
C.
The progress of the reaction was monitored by TLC. The reaction was over by an hour,
it was then quenched with Et
3
N (0.53 mL, 3.83 mmol). The solvent was evaporated
under reduced pressure to give a crude mixture, which was then purified on a silica gel
column using 8% EtOAc/hexanes to afford the primary alcohol 6.14 (1.0 g, 65%).
1
H-
NMR (400 MHz, CDCl
3
) δ
H
3.77 (q, J = 4.8 Hz, 1H), 3.65 (s, 3H), 3.57 (m, 2H), 3.63
(m, 1H), 2.43-2.30 (m, 2H), 1.97 (dd, J = 6.8, 4.0 Hz, 1H), 1.86 (dd, J = 6.8, 5.2 Hz,
1H), 1.83 (t, J = 5.0 Hz, -OH), 0.88 (s, 9H), 0.87 (s, 9H), 0.08 (s, 6H), 0.07 (s, 3H), 0.04
224
(s, 3H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
174.1, 75.0, 72.6, 63.6, 51.5, 29.1, 28.2, 25.8,
18.0, -4.5, -4.6, -4.7, -4.8.

OTBS TBSO
O
6.5
OMe
O
6.4.6 Methyl (4S, 5R)-4,5-bis(t-butyldimethylsilyloxy)-6-oxohexanoate (6.5). To a
solution of DMSO (0.46 mL, 5.90 mmol) in dry CH
2
Cl
2
(25 mL) at -78
o
C was slowly
added oxalyl chloride (0.35 mL, 3.92 mmol) and stirred for 15 min, and then added the
alcohol 6.14 (0.80 g, 1.96 mmol) in CH
2
Cl
2
(5 mL) through a cannula, and stirred for 50
min. Et
3
N (1.36 mL, 9.8 mmol) was then added to the reaction mixture by a syringe,
and stirred for 3 h at -78
o
C. The reaction mixture was then brought to room
temperature, and stirred for an additional 25 min. The reaction was quenched with a
saturated aqueous solution of NH
4
Cl, extracted with ether (30 mL x 3), washed with
brine, dried over anhydrous MgSO
4
, and concentrated under reduced pressure. The
crude product was purified on a silica column using 9% EtOAc/hexanes as the eluant to
give the pure aldehyde 6.5 (0.77 g, 97%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
)
δ
H
9.58 (d, J = 2.4 Hz, 1H), 3.94 (m, 1H), 3.86 (dd, J = 4.0, 2.4 Hz, 1H), 3.65 (s, 3H),
2.35 (t, J = 7.6 Hz, 2H), 1.97-1.78 (m, 2H), 0.89 (s, 9H), 0.85 (s, 9H), 0.06 (s, 3H), 0.05
(s, 6H), 0.04 (s, 3H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
203.3, 173.6, 80.8, 74.0, 51.6,
29.5, 28.3, 25.8, 18.3, 18.2, -4.4, -4.7, -4.9, -4.9.

225
COOMe
TBSO OTBS TMS
6.15

6.4.7 Methyl (4S, 5R, 6Z, 8E)-4,5-bis(t-butyldimethylsilyloxy)-11-(trimethylsilyl)-
undeca-6,8-dien-10-ynoate (6.15). To a suspension of phosphonium salt 3.4 (1.75 g,
3.65 mmol) in dry THF (15 mL) was added n-BuLi (1.6 mL of 1.6 M solution in hexane,
2.56 mmol) at -78
o
C. The dark red solution was allowed to warm to 0
o
C and stirred
for 30 min before re-cooled at -78
o
C. The aldehyde 6.5 (0.74 g, 1.83 mmol) solution in
5 mL THF was added to the reaction mixture dropwise via a cannula. The dark red
mixture was then brought to room temperature and stirred for 3 h before quenched with
saturated aqueous NH
4
Cl solution, extracted with ether (25 mL x 3) and washed with
brine. The organic layers were combined and dried over anhydrous MgSO
4
, filtered,
and concentrated under reduced pressure. The residue was purified over a silica column
using 2% EtOAc/hexanes as the eluant to afford 6.15 (0.86 g, 90%) with exclusively 6Z,
8E double bonds geometry confirmed by NMR analysis.
1
H-NMR (400 MHz, CDCl
3
)
δ
H
6.80 (dd, J = 15.6, 11.2 Hz, 1H), 6.03 (t, J = 11.2 Hz, 1H), 5.60 (d, J = 15.6 Hz, 1H),
5.42 (t, J = 9.2 Hz, 1H), 4.35 (ddd, J = 8.0, 4.0, 1.5 Hz, 1H), 3.65 (s, 3H), 3.62 (m, 1H),
2.40 (t, J = 7.6 Hz, 2H), 1.82 (m, 2H), 0.85 (s, 9H), 0.84 (s, 9H), 0.18 (s, 9H), 0.03 (s,
3H), 0.02 (s, 3H), 0.01 (s, 3H), -0.02 (s, 3H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
174.3,
137.8, 135.5, 129.0, 112.7, 104.3, 97.8, 75.1, 72.0, 51.5, 29.3, 28.2, 26.0, 25.9, 18.1, -
0.08, -3.9, -4.0, -4.8.

226
COOMe
TBSO OTBS
TMS
6.16

6.4.8 Methyl (4S, 5R, 6E, 8E)-4,5-bis(t-butyldimethylsilyloxy)-11-(trimethylsilyl)-
undeca-6,8-dien-10-ynoate (6.16). To a solution of 6Z, 8E isomer, 6.15 (0.75 g, 1.43
mmol) in dry CH
2
Cl
2
(135 mL) was added a small crystal of sublimed I
2
(15 mg), and
stirred under the light at room temperature for 2 days. The violet color solution was
then quenched with a saturated aqueous solution of Na
2
S
2
O
5
, extracted with ether (40
mL x 3), and washed with brine. The organic layers were combined, dried over
anhydrous MgSO
4
, filtered, and concentrated under reduced pressure. The residue was
purified on a silica gel column using 2% EtOAc/hexanes as the eluant to give the pure
6E, 8E isomer 6.16 (0.72 g, 96%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
) δ
H

6.60 (dd, J = 15.6, 10.8 Hz, 1H), 6.14 (dd, J = 15.6, 10.8 Hz, 1H), 5.73 (dd, J = 15.6,
6.8 Hz, 1H), 5.57 (d, J = 15.6 Hz, 1H), 3.95 (q, J = 4.4 Hz, 1H), 3.64 (s, 3H), 3.60 (q, J
= 4.8 Hz, 1H), 2.35 (t, J = 7.6 Hz, 2H), 1.79 (m, 2H), 0.85 (s, 9H), 0.84 (s, 9H), 0.17 (s,
9H), 0.02 (s, 3H), 0.00 (s, 3H), -0.01 (s, 3H), -0.04 (s, 3H);
13
C-NMR (100 MHz, CDCl
3
)
δ
C
174.3, 142.1, 137.5, 130.7, 110.9, 104.3, 97.1, 76.5, 75.0, 51.5, 34.1, 29.4, 28.2, 25.9,
22.3, 18.2, 18.1, 14.1, -0.1, -4.0, -4.1, -4.7, -4.8.

COOMe
TBSO OTBS
6.3

6.4.9 Methyl (4S, 5R, 6E, 8E)-4,5-bis(t-butyldimethylsilyloxy)undeca-6,8-dien-10-
ynoate (6.3). To a solution of TMS-protected alkyne 6.16 (0.35 g, 0.66 mmol) in
227
MeOH (15 mL) was added a scoop of Na
2
CO
3
, and stirred for overnight. The cloudy
mixture was concentrated in vacuo to remove MeOH, and then added H
2
O (10 mL) to
quench the reaction, extracted with ether (15 mL x 3), washed with brine, dried over
MgSO
4
. The crude was purified over silica column using 5% EtOAc/hexanes as the
eluant to afford the alkyne 6.3 (0.29 g, 96%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.61 (dd,
J = 15.6, 10.8 Hz, 1H), 6.14 (dd, J = 15.6, 10.8 Hz, 1H), 5.73 (dd, J = 14.8, 7.2 Hz, 1H),
5.51 (dd, J = 15.2, 2.0 Hz, 1H), 3.95 (t, J = 5.0 Hz, 1H), 3.62 (s, 3H), 3.60 (m, 1H), 2.99
(d, J = 2.0 Hz, 1H), 2.34 (t, J = 7.6 Hz, 2H), 1.78 (m, 2H), 0.85 (s, 9H), 0.83 (s, 9H),
0.02 (s, 3H), 0.01 (s, 3H), -0.01 (s, 3H), -0.03 (s, 3H);
13
C-NMR (100 MHz, CDCl
3
) δ
C

174.1, 142.8, 137.8, 130.3, 109.8, 82.8, 79.6, 76.4, 74.9, 51.5, 29.4, 28.2, 25.9, 18.2,
18.1, -4.1, -4.2, -4.7, -4.8.

COOMe
TBSO OTBS
TMS
6.17

6.4.10 Methyl (4S, 5R, 6E, 8E)-4,5-bis(t-butyldimethylsilyloxy)-14-(trimethylsilyl)-
tetradeca-6,8-dien-10,13-diynoate (6.17). To a suspension of anhydrous K
2
CO
3

(171.2 mg, 1.24 mmol), CuI (236 mg, 1.24 mmol) and NaI (186 mg, 1.24 mmol) in
anhydrous DMF (5 mL) under argon atmosphere were added the propargyl bromide 6.4
(177 mg, 0.92 mmol) and the terminal alkyne 6.3 (280 mg, 0.62 mmol) in anhydrous
DMF (2 mL) by a cannula. The resulting reaction mixture was stirred at room
temperature for overnight (~8 h). The reaction was quenched with a saturated aqueous
solution of NH
4
Cl, extracted with ether (20 mL x 3), dried over MgSO
4
, and
228
concentrated in vacuo to give the crude product, which was then purified on a silica
column using 3% EtOAc/hexanes as the solvent system to afford the pure TMS
protected bis-alkyne 6.17 (309 mg, 89%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
)
δ
H
6.52 (dd, J = 15.6, 10.4 Hz, 1H), 6.12 (dd, J = 15.2, 11.2 Hz, 1H), 5.68 (dd, J = 14.8,
6.8 Hz, 1H), 5.52 (d, J = 15.2 Hz, 1H), 3.94 (t, J = 5.6 Hz, 1H), 3.62 (s, 3H), 3.59 (q, J
= 5.2 Hz, 1H), 3.33 (d, J = 2.0 Hz, 2H), 2.35 (t, J = 7.6 Hz, 2H), 1.78 (m, 2H), 0.85 (s,
9H), 0.83 (s, 9H), 0.13 (s, 9H), 0.01 (s, 3H), 0.00 (s, 3H), -0.01 (s, 3H), -0.04 (s, 3H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
174.3, 141.0, 136.8, 130.7, 110.9, 99.5, 85.6, 85.2,
79.9, 76.5, 75.0, 51.5, 29.4, 28.2, 25.9, 18.2, 18.1, 11.7, -0.13, -4.1, -4.2, -4.7, -4.8.

COOMe
TBSO OTBS
6.2

6.4.11 Methyl (4S, 5R, 6E, 8E)-4,5-bis(t-butyldimethylsilyloxy)tetradeca-6,8-dien-
10,13-diynoate (6.2). To a solution of TMS-protected bis-alkyne 6.17 (0.30 g, 0.53
mmol) in MeOH (15 mL) was added a scoop of Na
2
CO
3
, and stirred for overnight. The
cloudy mixture was concentrated in vacuo to remove MeOH, and then added H
2
O (10
mL) to quench the reaction, extracted with ether (20 mL x 3), washed with brine, dried
over MgSO
4
. The crude was purified over silica column using 2% EtOAc/hexanes as
the eluant to afford the terminal alkyne 6.2 (0.23 g, 90%).
1
H-NMR (400 MHz, CDCl
3
)
δ
H
6.64 (dd, J = 16.0, 10.8 Hz, 1H), 6.16 (dd, J = 14.8, 10.8 Hz, 1H), 5.75 (dd, J = 15.2,
6.8 Hz, 1H), 5.54 (d, J = 15.2 Hz, 1H), 3.96 (t, J = 5.6 Hz, 1H), 3.64 (s, 3H), 3.59 (q, J
= 5.2 Hz, 1H), 2.36 (t, J = 7.6 Hz, 2H), 1.97 (d, J = 2.0 Hz, 2H), 1.79 (m, 2H), 0.86 (s,
229
9H), 0.84 (s, 9H), 0.02 (s, 3H), 0.00 (s, 3H), -0.01 (s, 3H), -0.03 (s, 3H);
13
C-NMR (100
MHz, CDCl
3
) δ
C
174.2, 143.8, 138.1, 130.4, 109.9, 109.8, 81.1, 79.5, 76.4, 75.0, 64.5,
51.5, 29.4, 28.2, 25.9, 18.1, 18.0, 4.7, -4.1, -4.2, -4.7, -4.8.

230
0.89 0.86
0.97
3.00
1.37
1.97
7 6 5 4 3 2 PPM

1
H-NMR (400 MHz, CDCl
3
) of 6.8.

OH HO
COOMe
HO
6.8
231
0.91 0.84 0.83
4.00
1.59
31.65
18.91
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 6.11.

160 140 120 100 80 60 40 20 0 PPM

13
C-NMR (100 MHz, CDCl
3
) of 6.11.

OTBS TBSO
TBSO
COOMe
6.11
232
1.05
4.00
0.98 0.95
1.97 1.94
28.56
16.77
4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 6.12.

160 140 120 100 80 60 40 20 0 PPM

13
C-NMR (100 MHz, CDCl
3
) of 6.12.

OTBS TBSO
COOMe
TBSO
6.12
233
1.08
4.00
2.00 1.99
0.88
1.97
19.04
11.42
4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 6.14.

150 100 50 0 PPM

13
C-NMR (100 MHz, CDCl
3
) of 6.14.
OTBS TBSO
COOMe
HO
6.14
234

0.894
0.849
0.060
0.056
0.041
0.75
0.96 0.99
3.00
1.99 1.98
19.17
11.30
8 6 4 2 0PPM

1
H-NMR (400 MHz, CDCl
3
) of 6.5.

OTBS TBSO
COOMe
O
6.5
235
200 150 100 50 0PPM

13
C-NMR (100 MHz, CDCl
3
) of 6.5.

1.00 1.01 0.97 0.96 0.93
3.84
1.93 1.97
17.91
7.59
10.70
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 6.15.
COOMe
TBSO OTBS TMS
6.15
236

160 140 120 100 80 60 40 20 0 PPM

13
C-NMR (100 MHz, CDCl
3
) of 6.15.

237
1.00 0.96 0.95 0.96 0.96
4.40
2.27 2.24
1.16
19.29
8.98
12.14
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 6.16.

150 100 50 0PPM

13
C-NMR (100 MHz, CDCl
3
) of 6.16.

COOMe
TBSO OTBS
TMS 6.16
238
1.00 1.00 0.98 0.97 0.89
4.01
0.70
2.04 2.06
18.99
11.47
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 6.3.

160 140 120 100 80 60 40 20 0 PPM

13
C-NMR (100 MHz, CDCl
3
) of 6.3.
COOMe
TBSO OTBS
6.3
239
1.00 1.22 1.18 1.07 1.06
4.67
1.67
2.44 2.44
23.21
9.18
13.86
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 6.17.

160 140 120 100 80 60 40 20 0 PPM

13
C-NMR (400 MHz, CDCl
3
) of 6.17.
COOMe
TBSO OTBS
TMS
6.17
240

7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 6.2.

COOMe
TBSO OTBS
6.2
241
Chapter 7. Metabolic Inactivation of Lipoxins and Resolvins
and Design & Synthesis of their Biostable Analogs

7.1 Introduction
The E- and D-series of resolvins are lipid mediators derived from EPA and DHA,
respectively. These lipid mediators are generated, and act locally at the sites of
inflammation,
93
where they down-regulate polymorphonuclear leukocyte (PMN)
infiltration and promote resolution much like their arachidonic acid-derived cousins
lipoxins (LXs).
94
The major PUFA derived lipid mediators including LXs are generated
in very minute amounts in a stereochemically pure form in specific cell-types via well
regulated enzymatic processes, and undergo enzyme-mediated conversion to inactive
metabolites, thereby modulating their local concentration and hence their bioactions.
95

The metabolic inactivation of these lipid mediators is an important component of their
mechanism of actions. Here below is a description of their metabolic inactivation
pathways. Based-on those pathways, we have designed and synthesized a number of
metabolically stable analogs. The synthesis and bioactions of these enzymatically
stable analogs are also described herein.

93
(i) Hong, S.; Gronert, K.; Devchand, P.; Moussignac, R. L.; Serhan, C. N. J. Biol. Chem. 2003, 278,
14677. (ii) Marcheselli, V. L.; Hong, S.; Lukiw, W. J.; Hua-Tian, X.; Gronert, K.; Musto, A.; Hardy, M.;
Gimenez, J. M.; Chiang, N.; Serhan, C. N.; Bazan, N. G. J. Biol. Chem. 2003, 278, 43807.
94
For a recent review, please see Bannenberg, G.; Arita, M.; Serhan, C. N. The Scientific World Journal
2007, 7, 1440, and references therein.
95
(i) Funk, C. D. Science 1987, 237, 1171. (ii) Serhan, C. N. Prostaglandins Other Lipid Mediat. 2002,
68, 433. (iii) Serhan, C. N.; Fiore, S.; Brezinski, D. A.; Lynch, S. Biochemistry 1993, 32, 6313. (iv) Clish,
C. B.; Levy, B. D.; Chiang, N.; Tai, H. H.; Serhan, C. N. J. Biol. Chem. 2000, 275, 25372. (v) Maddox, J.;
Serhan, C. N. J. Exp. Med. 1996, 183, 137.
242
7.2 Metabolic inactivation lipoxins and design and synthesis of biostable
lipoxin analogs

7.2.1 Metabolic inactivation pathways of lipoxins
Characteristic of autacoids, lipoxins (LXs), leukotrienes (LTs) and PGs are rapidly
metabolized following biosynthesis and bioaction as shown in the Figure 23.
96

Figure 23. Enzymatic oxido/reductase mediated inactivation of lipoxins and related
lipid mediators. Petasis et. al. Prostaglandins, Leukotrienes and Essential Fatty Acids
2005, 73, 301.
LXs are converted by specific leukocytes of the monocyte/macrophage lineage to 15-
oxo-lipoxins by 15-PGDH, which catalyzes the dehydrogenation of the 15-OH group as

96
For a recent review, please see Petasis, N. A.; Akritopoulou-Zane, I.; Fokin, V. V.; Bernasconi, G.;
Keledjian, R.; Yang, R.; Uddin, J.; Nagulapalli, K. C.; Serhan, C. N. Prostaglandins, Leukotrienes and
Essential Fatty Acids 2005, 73, 301, and references therein.
243
shown in the Figure 23.
97
The 15-oxo-lipoxin is then irreversibly reduced to 13,14-
dehydro-15-oxo-lipoxin by LTB
4
12-hydroxy dehydrogenase/prostaglandin reductase
(LTB
4
12-HD/PGR).
98
Both 15-oxo-lipoxin and 13,14-dehydro-15-oxo-lopoxin can
undergo further reduction to the corresponding achiral secondary alcohols, mediated by
15-PGDH.
5
All of these metabolites are biologically either inactive or less potent. LXs
and LTs can also undergo cytochrome P-450 mediated omega-oxidation at C-20 to
inactive metabolites as shown in the Figure 24.
4

Figure 24. Metabolic inactivation of LXA
4
and LTB
4
. Petasis et. al. Prostaglandins,
Leukotrienes and Essential Fatty Acids 2005, 73, 301.

97
Serhan, C. N.; Fiore, S.; Brezinski, D. A.; Lynch, S. Biochemistry 1993, 32, 6313. (ii) Maddox, J.;
Serhan, C. N. J. Exp. Med. 1996, 183, 137. (iii) Serhan, C. N.; Maddox, J. F.; Petasis, N. A.;
Akritopoulou-Zane, I.; Papayianni, A.; Brady, H. R.; Colgan, S. P.; Madara, J. L. Biochemistry 1995, 34,
14609. (iv) Yokomizo, T.; Izumi, T.; Takahashi, T.; Kasama, T.; Kobayashi, Y.; Sato, F.; Taketani, Y.;
Shimuzu, T. J. Biol. Chem. 1993, 268, 18128.s
98
Clish, C. B.; Levy, B. D.; Chiang, N.; Tai, H. H.; Serhan, C. N. J. Biol. Chem. 2000, 275, 25372.
244
The metabolic inactivation pathways of LXs mentioned-above suggesting that these
anti-inflammatory, pro-resolving lipid mediators are highly susceptible to enzyme
mediated conversion to less potent or inactive metabolites that do not effectively
compete at the recombinant LXA
4
receptor and hence lost their functional ability to
inhibit PMN responses at the site of inflammation. The most vulnerable parts of their
structures are the 15-hydroxyl group, and the omega-carbon. Considering these
pathways, it is possible for us, the synthetic group, to design and synthesize stable
lipoxins analogs with increased half-lives and potent bioactions. Such biostable analogs
would facilitate the therapeutic potential of these pro-resolving and anti-inflammatory
eicosanoids.
7.2.2 Design of Lipoxin analogs
Soon after the discovery of lipoxins, Nicolaou group was at the forefront for their total
synthesis.
99
Despite the total synthesis of natural lipoxins and their stereoisomers, there
was no systematic study on the synthesis of their biostable analogs prior to the initiative
taken from our group. Over the years, our group has systematically designed and
accomplished the synthesis of a number of LXs analogs and their mimetics with
increased biostability and enhanced bioactivity as shown in Figure 25.

99
For a full synthetic review on LXs, please see Nicolaou, K. C.; Ramphal, Y. P.; Petasis, N. A.; Serhan,
C. N. Angew. Chem. Int. Ed. Engl. 1991, 30, 1119, and references therein.
245
OH HO
COOMe
OH HO
COOMe
Me OH
OH HO
OH
COOMe
OH HO
OH
O
COOMe
OH HO
OH
O
CONMe
2
OH HO
OH
O
COOMe
F
OH HO
OH
COOMe
OH HO
OH
O
COOMe
OH HO
OH
O
COOMe
CF
3

Figure 25. Structures of lipoxins analogs with the modifications in the omega-end.
The modifications on lipoxins structures were made based-on the metabolic inactivation
pathways, and structure-activity relationship as shown in Figure 26.

Figure 26. Design of Lipoxin analogs. Petasis et. al. Prostaglandins, Leukotrienes and
Essential Fatty Acids 2005, 73, 301
246
7.2.3 Synthesis of p-F-phenoxy-15-epi-Lipoxin (ATLa)
The retrosynthetic analysis of p-F-phenoxy-15-epi-lipoxin (ATLa) shown in Figure 27.
Like the synthesis of other lipid mediators, we have planned to construct the last
carbon—carbon bond by the Pd
0
/Cu
I
mediated cross-coupling reaction of terminal
alkyne (7.2) and p-F-phenoxy-vinyl iodide (7.3).
OH HO
OH
O
Chiral pool
Wittig
Pd
0
/Cu
I
coupling
Selective
hydrogenation
7.1
COOMe
F
OTBS TBSO
I
OTBS
O
COOMe
F
7.2 7.3

Figure 27. Retrosynthetic analysis of p-F-Phenoxy-epi-LXA
4
.

The synthesis of terminal alkyne was described in this dissertation. The synthesis of p-
F-phenoxy-vinyl iodide (7.3) is described below.

247
The synthesis of vinyl iodide (7.3) was started with the CsF catalyzed ring opening of S-
glycidol (7.5) by p-fluorophenol (7.4) as shown in Scheme 28. This nucleophilic
reaction of p-F-phenol (7.4) with optically active S-glycidol (7.5) was first carried out in
EtOH with catalytic amount of CsF by following a literature procedure,
100
but the
reaction yielded a mixture of chromatographically separable region-isomers.
OH F
O
OH
OH
HO O F
OH
TBSO O F
OTBDPS
HO O F
OTBDPS
O
O F
OTBS
O F
Reagents and Conditions: (a) CsF (catalytic), DMF, 90
o
C, 2 days, 90% (97% ee);
(b) TBS-Cl, imidazole, DMAP, CH
2
Cl
2
, rt, overnight, 90%; (c) (i) TBDPS-Cl,
imidazole, DMAP, CH
2
Cl
2
, rt, overnight, 98%; (ii) CSA, Et
3
N, MeOH-CH
2
Cl
2
(1:1),
rt, 30 min, 99%; (d) Trichloroisocyanuric acid, TEMPO, CH
2
Cl
2
, 0
o
C --> rt, 20 min,
83%; (e)(i) CrCl
2
,CHI
3
,THF,0
o
C, 3h; (ii) TBAF, THF, rt, overnight, 55% in two
steps; (iii) TBS-OTf, lutidine, CH
2
Cl
2
, rt, overnight, 88%.
a
b
c
d
e
S
I
S
7.3
7.4 7.5 7.6
7.7 7.8
7.9

Scheme 28. Synthesis of p-F-phenoxy-vinyl iodide (7.3).
When the same nucleophilic ring opening of optically active S-glycidol (7.5) by p-F-
phenol (7.4) was tried in DMF with catalytic amount of CsF, the attack occurred only at

100
Kitaori, K.; Furukawa, Y.; Yoshimoto, H.; Otera, J. Tetrahedron 1999, 55, 14381.
248
the least-substituted site of the glycidol afforded the desired diol in excellent yield
(90%). The enantiomeric excess (ee) of the diol was determined to 97% by using a
chiral column following a literature procedure.
101
Selective silylation of primary
alcohol with TBS-group surprisingly afforded only the primary alcohol protected
product (7.7) in excellent yield (90%). The silylation of the secondary alcohol group by
TBDPS-group yielded the bis-silylated product in excellent yield (98%). The
chemoselective desilylation of the primary TBS-group by the mild action of
camphorsulfonic acid furnished the desired primary alcohol (7.8) in excellent yield
(99%). The oxidation of the primary alcohol group to the corresponding aldehyde (7.9)
was done by trichloroisocyanuric acid and catalytic amount of TEMPO in CH
2
Cl
2
.
102

This oxidation method has several advantages over the traditional Swern oxidation
103

and Dess-Martin periodinane.
104
Swern oxidation, even though a favorable and reliable
method for oxidation, however, suffers serious drawbacks from the use of activated
dimethyl sulfoxide as a reagent and very low temperature (-78
o
C), and mainly the
generation of very stinky dimethyl sulfide as a by-product. Moreover, the Swern
oxidation is not chemoselective. The Dess-Martin periodinane reagent is very
expensive, and the reagent can get bad very easily. The trichloroisocyanuric acid is a
cheap reagent, and the reaction can carried-out at room temperature, and it is a very

101
Kuwabe, S-I.; Torraca, K. E.; Buchwald, S. L. J. Am. Chem. Soc. 2001, 123, 12202.
102
Luca, L. D.; Giacomelli, G.; Porcheddu, A. Org. Lett. 2001, 3, 3041.
103
Mancuso, A. J.; Huang, S. L.; Swern, D. J. Org. Chem. 1978, 43, 2480.
104
(i) Dess, D. B.; Marin, J. C. J. Org. Chem. 1983, 48, 4155. (ii) Dess, D. B.; Martin, J. C. J. Am. Chem.
Soc. 1991, 113, 7277.
249
quick reaction. The Takai olefination on the aldehyde (7.9) afforded the desired p-F-
phenoxy-vinyl iodide (7.3) in moderate yield.
105

Having terminal alkyne (7.2) and p-F-phenoxy-vinyl iodide (7.3) in hands, we then
focused on their Pd
0
/Cu
I
mediated coupling using standard Sonogashira condition with
Pd(PPh
3
)
4
and CuI in presence of Et
3
N in benzene at room temperature. The coupling
reaction proceeded smoothly and afforded the desired product in excellent yield (98%)
as shown in Scheme 29.

105
Takai, K.; Nitta, K.; Utimoto, K. J. Am. Chem. Soc. 1986, 108, 7408.
250
OTBS TBSO
7.2 7.3
7.10
a
7.11
b
c
p-F-Phenoxy-15-epi- LXA
4
Methyl ester (7.1)
Reagents and conditions: (a) Pd(Ph
3
)
4
, CuI, C
6
H
6
, Et
3
N, rt, overnight, 98%;
(b) TBAF, THF, rt, overnight followed by CH
2
N
2
, ether, rt, 2 h, 97%;
(c) Zn (Cu/Ag), H
2
O:MeOH (1:1), 35
o
C, overnight, 89%.
OTBS TBSO
OTBS
O F
I
O
OTBS
F
OH HO
O
OH
F
OH HO
O
OH
F
COOMe
COOMe
COOMe
COOMe

Scheme 29. Final assembly of p-F-phenoxy-LXA
4
methyl ester (7.1).
The fluoride anion induced desilylation of TBS-groups followed by the stereoselective
reduction of the triple bond using activated zinc, Zn(Cu/Ag) in MeOH-H
2
O (1:1) at 35
o
C afforded the final product of p-F-phenoxy-15-epi-LXA
4
methyl ester (ATLa, 7.1) in
excellent yield (89%). The structure of 7.1 was fully characterized from the analysis of
UV, mass, and 1 and 2D-NMR spectral data.
251
7.3 Metabolic Inactivation of Resolvin E1 (RvE1) and Synthesis of
Biostable p-F-phenoxy-RvE1 Analog
7.3.1 Metabolic Inactivation Pathways of RvE1
The resolvin E1 (RvE1) is a lipid mediator derived from EPA during the resolution
phase of inflammation and act locally to stop leukocyte recruitment and promote
resolution to tissue homeostasis. The complete absolute stereochemistry of resolvin E1
was determined to be 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic
acid.
106
RvE1 is very potent in both in vitro and in vivo systems, where it showed
potent counterregulatory and tissue-protective roles. The metabolic inactivation
pathways of RvE1 were unknown until recently we and our collaborator in Harvard
Medical School discovered its enzymatic inactivation pathways,
107
which is described
below.
Enzymatic conversion of RvE1—The arachidonic acid derived lipid mediators,
lipoxins were the substrate of the enzyme 15-PGDH.
5
To determine the enzymatic
conversion of resolvin E1, it was incubated with recombinant human 15-PGDH and
NAD
+
by thinking that RvE1 might be the substrate for this enzyme as it was for
lipoxins. The enzymatic reaction was monitored by UV at 340 nm as shown in the
Figure 28. The increase of absorbance at 340 nm indicates the formation of NADH,
means RvE1 is also a substrate of 15-PGDH.

106
Arita, M.; Bianchini, F.; Aliberti, F.; Sher, A.; Chiang, N.; Hong, S.; Yang, R.; Petasis, N. A.; Serhan,
C. N. J. Exp. Med. 2005, 201, 713.
107
Arita, M.; Oh, S. F.; Chonan, T.; Hong, S.; Elangovan, S.; Sun, Y-P.; Uddin, J.; Petasis, N. A.; Serhan,
C. N. J. Biol. Chem. 2006, 281, 22847.
252

Figure 28. Reaction progress monitored by UV at 340 nm.
J. Biol. Chem. 2006, 281, 22847.

Identification of the enzymatic conversion products of RvE1—The 15-PGDH and
RvE1 reaction products was purified by solid extraction (Sep-Pak C18), and then
further purified by reversed-phase HPLC. A LC-UV-MS/MS based analysis was
employed to determine whether 15-PGDH could catalyze the conversion of RvE1 to
oxo-metabolites as it did for lipoxins. The results are shown in Figure 29. The results
presented in the Figure 29 clearly demonstrated that RvE1 is enzymatically further
metabolized via NAD
+
-dependent oxidation of C-18 hydroxyl to generate 18-oxo-RvE1.
253

Figure 29. LC-ESI-MS/MS chromatograms and spectra of RvE1
and its metabolites. J. Biol. Chem. 2006, 281, 22847.

The conversion of 18-OH-RvE1 to 18-oxo-RvE1 also switched the chromophore from
diene to dienone. This extended conjugation lowered the HOMO—LUMO energy gap,
and consequently the absorption maximum was red-shifted from 234 nm to 270 nm as
shown in Figure 29. The structure of 18-oxo-RvE1 was elucidated by the MS/MS
fragmentation analysis, which was further confirmed by isolating the 18-oxo-RvE1 and
254
then treated with diazomethane followed by methoxyamine to from the methoxime and
analyzed by GC-MS. The results obtained from the GC-MS analysis, as shown in
Figure 30, confirmed the structure of 18-oxo-RvE1 as the metabolite obtained from
RvE1.

Figure 30. GC-MS spectrum of the derivatized methoximation product of 18-oxo-RvE1.
J. Biol. Chem. 2006, 281, 22847.

RvE1 Metabolism in Cells and Tissues—To address whether oxidation of the 18-OH
group is also the major pathway of RvE1 metabolism in cells and tissues, our
collaborator performed RvE1 incubations with isolated mouse lung and isolated human
PMN. The results obtained from HPLC-UV-MS/MS analysis are shown in Figure 31.
The results clearly demonstrated that RvE1 undergoes initial metabolic pathways that
255
are tissue specific. The 18-OH oxidation was the major route in the lung (Panel A) and
the omega-oxidation to form 20-hydroxy-RvE1 was a major route in human PMN
(Panel B).

Figure 31. RvE1 metabolism in cells and tissues.
J. Biol. Chem. 2006, 281, 22847.
Biological Activities of the RvE1 Metabolites—To determine the bioactions of RvE1
metabolites, the isolated 18-oxo-RvE1 was assessed for its ability to regulate leukocyte
infiltration in vivo using the zymosan-induced peritonitis. The results are presented in
the Figure 32.

256

Figure 32. Biological activities of major RvE1 metabolite, 18-oxo-RvE1.
J. Biol. Chem. 2006, 281, 22847.

The results shown in the Figure 32 demonstrated that 18-oxo-RvE1 is essentially devoid
of anti-inflammatory activity, thus confirming that conversion of RvE1 to 18-oxo-RvE1
is a mode of metabolic inactivation of RvE1.
7.3.2 Synthesis of p-F-phenoxy-RvE1 analog to prevent metabolic
inactivation
Because RvE1 conversion to the inactive 18-oxo-RvE1 metabolite, we have planned to
synthesize p-F-phenoxy-RvE1 analog to prevent omega-oxidation as well as to slow
down the 18-OH oxidation. This design approach and rationale is similar to the one
used for ATLa.
108
The synthesis of p-F-phenoxy-RvE1 methyl ester is described below.
The synthesis of terminal alkyne (2.21) and the p-F-phenoxy vinyl iodide (7.3) were
described previously in this dissertation. For terminal alkyne (2.21) please see Chapter

108
Takano, T.; Clish, C. B.; Gronert, K.; Petasis, N. A.; Serhan, C. N. J. Clin. Investig. 1998, 101, 819.
257
2 (Section 2.2.5), and p-F-phenoxy vinyl iodide (7.3) please see Section 7.2.3 of this
Chapter. Having both intermediates in hands, the final Sonogashira coupling was done
using the standard conditions as described in Scheme 30.
OTBS O
OMe
2.21
OTBS
7.3
OTBS O
OMe
OTBS
7.12
7.13
p-F-Phenoxy-RvE1 Methyl Ester (7.14)
a
b
c
Reagenst and conditions: (a) Pd(Ph
3
)
4
, CuI, NEt
3
, C
6
H
6
, rt,
overnight, 75%; (b) TBAF, THF, 0
o
C to rt, overnight followed by
CH
2
N
2
, ether, 2h, 96%; (c) Zn (Cu/Ag), H
2
O-MeOH (1:1), 40
o
C,
overnight, 75%.
OTBS
O F
I
O
OTBS
F
OH O
OMe
OH
O
OH
F
OH O
OMe
OH
O
OH
F

Scheme 30. Final assembly of p-F-Phenoxy-RvE1 (7.14) analog.

258
The desilylation of TBS-groups by fluoride anion followed by stereoselective partial
reduction by activated Zinc, Zn(Cu/Ag) in MeOH-H
2
O (1:1) at 40
o
C yielded the final
product (7.14) in excellent yield as shown in Scheme 30. The final compound was
purified by a reversed-phase HPLC on ODS column using H
2
O-MeOH as the eluent.
The structure of p-F-phenoxy-RvE1 methyl ester (7.14) was elucidated by an extensive
analysis of 1 and 2D-NMR data.
7.3.3 Biological Activities of p-F-phenoxy-RvE1 Analog
The novel p-F-pheoxy-RvE1 analog was then tested for its anti-inflammatory actions
and compared with the parent RvE1 molecule. This analog is designed to protect from
both omega-oxidation and rapid dehydrogenation at C-18. This novel analog is found
to be resistant to rapid oxidation by recombinant dehydrogenase as shown in the Figure
33 (Panel B and C).
259

Figure 33.
1
H-NMR spectrum (panel A), and bioconversion rate of RvE1 analog.
J. Biol. Chem. 2006, 281, 22847.

To determine whether the designed RvE1 analog retained the anti-inflammatory activity
of native RvE1, we directly compared their bioactions, and found that RvE1 analog is as
potent as native RvE1 in stopping total leukocyte infiltration and specifically PMN
infiltration into murine peritonium.
15
The designed RvE1 analog which prevent rapid
degradation to inactive metabolites can be useful to further expand the roles of resolvins
in inflammation and their therapeutic potential for the treatment of inflammation and
inflammation associated diseases.

260
7.4 Metabolic Inactivation of Resolvin D1 (RvD1) and AT-RvD1 and
Synthesis of their Biostable Analogs

7.4.1 Metabolic inactivation pathways of RvD1 and AT-RvD1
109

Resolvin D1 (RvD1) and Aspirin-Triggered Resolvin D1 (AT-RvD1) are a novel family
of lipid mediators derived from DHA.
110
Both RvD1 and AT-RvD1 are generated via a
series of well-regulated enzymatic pathways, and act locally at the sites of inflammation
much like their arachidonic acid-derived cousins, lipoxins (LXs). As discussed above,
LXs and Resolvin E1 are rapidly inactivated by 15-prostagalndin
dehydrogenase/eicosanoid oxidoreductase (15-PGDH).
5,111
Since their generation, and
bioactions are similar, therefore, we were interested to see whether RvD1 and AT-RvD1
are also the substrates for the same enzymatic conversion to inactive metabolites.

Conversion rate of RvD1 and AT-RvD1, and their comparison with LXA
4
—Resolvin
D1 (RvD1), AT-RvD1 and LXA
4
were incubated with 15-PGDH and NAD
+
. The
enzymatic reactions were monitored spectrophotometrically by the formation of NADH
from NAD
+
at 340 nm. The results are shown in the Figure 34.

109
Sun, Y-P.; Oh, S. F.; Uddin, J.; Yang, R.; Gotlinger, K.; Campbell, E.; Colgan, S. P.; Petasis, N. A.;
Serhan, C. N. J. Biol. Chem. 2007, 282, 9323.
110
Serhan, C. N.; Hong, S.; Gronert, K.; Colgan, S. P.; Devchand, P. R.; Mirick, G.; Moussignac, R.-L. J.
Exp. Med. 2002, 196, 1025.
111
Arita, M.; Oh, S. F.; Chonan, T.; Hong, S.; Elangovan, S.; Sun, Y-P.; Uddin, J.; Petasis, N. A.; Serhan,
C. N. J. Biol. Chem. 2006, 281, 22847.

261

Figure 34. Conversion of RvD1, AT-RvD1 and LXA4 by 15-PGDH.
J. Biol. Chem. 2007, 282, 9323.

It is evident from the results (Figure 34) that LXA
4
was converted most rapidly and
RvD1 was converted to a similar extent within 25 min. However, AT-RvD1 was
interestingly resistant to rapid conversion in these incubations. RvD1 and AT-RvD1 are
epimers, differ only the absolute stereoconfiguration at C-17, 17S for RvD1 and 17R for
AT-RvD1. These conversion results of RvD1 and AT-RvD1 follow the trend found
with LXA
4
and aspirin-triggered LXA
4
(15-epi-LXA4)
112
suggesting that the 15-PGDH
enzyme may preferentially act on S-configured omega-proximal hydroxyl groups rather
than their R-configured counterparts.

112
Serhan, C. N.; Maddox, J. F.; Petasis, N. A.; Akritopoulou-Zane, I.; Papayianni, A.; Brady, H. R.;
Colgan, S. P.; Madara, J. L. Biochemistry 1995, 34, 14609.
262
Determination of the structures of RvD1 and AT-RvD1 metabolites—After the
enzymatic conversion of RvD1 and AT-RvD1, the reactions were stopped with cold
MeOH, and the samples were extracted with C18 solid phase extraction. The extracted
metabolites were analyzed by RP-HPLC, UV and MS/MS spectra. The results are
presented in Figure 35.

Figure 35. Analysis of RvD1 metabolites. J. Biol. Chem. 2007, 282, 9323.

HPLC-chromatogram of the metabolites showed (Figure 35) two distinct products. One
was eluted at 15.9 min and another one at 17.6 min. UV absorption spectra of the
metabolites were very interesting. The native RvD1 has a conjugated tetraene moiety,
which has a characteristic triplet chromophore with λ
max
of 301 nm (Figure 35). But
263
both metabolites have the red-shifted single broad UV absorbance at λ
max
351 nm. This
significant red-shift in UV indicates that each RvD1 metabolite has a potential single
ketone in conjugation with tetraene moiety-containing product. Three potential
metabolites are possible from the enzymatic conversion of RvD1—7-oxo-RvD1, 8-oxo-
RvD1 and 17-oxo-RvD1. Formation of 7-oxo product would retain the conjugated
tetraene moiety, would have given the characteristic triplet in UV with λ
max
301 nm.
Absence of the conjugated tetraene characteristic UV absorbance at 301 nm in
metabolites indicated that 7-oxo-RvD1 is not a metabolic product of RvD1. Both 8-
oxo-RvD1 and 17-oxo-RvD1 would extend the conjugation with the newly generated
ketone group, lowering HOMO—LUMO gap, thereby red-shifting the absorption
maxima at λ
max
351 nm. The red-shifted UV absorbance of metabolites clearly suggests
that two metabolites identified in HPLC were 8-oxo-RvD1 and 17-oxo-RvD1 (Figure
35). To further confirm their structures, both metabolites were purified by RP-HPLC,
and subject to MS/MS analysis. The results are presented in Figure 35. The MS/MS
fragmentation patterns confirmed the structures of metabolites as 8-oxo-RvD1 and 17-
oxo-RvD1 (Figure 7.12). 17-oxo-RvD1 was also produced from RvD1 in murine lung
and the structure was characterized using the same criteria and MS/MS analysis as
shown in Figure 36.
264

Figure 36. RvD1 metabolism in murine lung.
J. Biol. Chem. 2007, 282, 9323.

7.4.2 Bioactions of RvD1 metabolites
Having identified the structures of metabolic products of RvD1 as 8-oxo-RvD1 and 17-
oxo-RvD1, we next sought their in vivo anti-inflammatory actions. The results obtained
from acute inflammation are presented in Figure 37. The data clearly showed that 8-
oxo-RvD1 limited PMN infiltration in murine peritonitis by 41%, which was
comparable to native RvD1 (44%). But the 17-oxo-RvD1 didn’t reduce the PMN
265
infiltration in vivo. When compared with the actions of RvD1, 8-oxo-RvD1 was
effective, whereas 17-oxo-RvD1 was significantly less bioactive (Figure 37).

Figure 37. Bioactions of RvD1 and its metabolites 17-oxo-RvD1
and 8-oxo-RvD1. J. Biol. Chem. 2007, 282, 9323.

The inactivity of 17-oxo-RvD1 suggests that 15-PGDH is an inactivating enzyme for
RvD1 and AT-RvD1, which is consistent with the data found for other lipid mediators
such as lipoxins and RvE1.

266
7.4.3 Design and synthesis of biostable AT-RvD1 analogs
AT-RvD1 analog design—Resolvin D1 metabolism pathways are similar to lipoxins
and resolvin E1 (RvE1). These lipid mediators are autocoids that are generated in a
well-regulated manner, act locally at the sites of inflammation, and are inactivated
quickly. We have found that conversion of RvD1 to 17-oxo-RvD1 is the potential
inactivation pathway in vivo, and the conversion rate for AT-RvD1 is much slower than
RvD1, therefore we have decided to design AT-RvD1 analogs such as p-F-phenoxy-
AT-RvD1 and 17R/S-RvD1. These analogs might give longer half-lives in vivo by
preventing the 17-oxidation to inactive metabolites.

Retrosynthetic analysis of AT-RvD1 analogs—The retrosynthetic disassembly of the p-
F-phenoxy-AT-RvD1 (7.16) and 17R/S-RvD1 (7.15) are depicted in Figure 38. Like
the synthesis of AT-RvD1, we have envisaged to introduce the conjugated 13Z-double
bond in the last step by a stereoselective partial reduction of a triple bond to avoid
isomerization. We have planned to perform the Pd
0
/Cu
I
mediated cross-coupling
reaction of terminal alkyne and vinyl iodide as the last carbon—carbon bond-forming
reaction as shown in Figure 38. The rest is same as the synthesis of native AT-RvD1.

267
OH HO
COOMe
O
OH
F
OH HO
COOMe
Me OH
Pd
0
/Cu
I
cross coupling
OTBS TBSO
COOMe
3.20
Me OTBS
Br
O F
I
7.17 7.3
7.15
7.16
OTBS

Figure 38. Retrosynthetic analysis of AT-RvD1 analogs.

The terminal alkyne 3.20 is the common intermediate synthesized for RvD1 and AT-
RvD1. A detailed description of its synthesis was described in Chapter 3 (Section
3.2.1.6) of this dissertation. The synthesis of p-F-phenoxy-vinyl iodide from (7.3) from
S-glycidol was described in this Chapter (Scheme 7.2). And now we have to synthesize
only the racemic-vinyl bromide (7.17), which is described below.

Synthesis of racemic vinyl bromide (7.17)—The synthesis of racemic vinyl bromide
(7.17) is shown in Scheme 31. The synthesis was started with the stereoselective partial
hydrogenation of hept-4-yn-2-ol (10) by using Lindlar catalyst. The reaction yielded
only 4Z-proudct in EtOAc, however, when MeOH was used as solvent, a mixture of
4Z/4E-isomers was obtained. The 2-hydroxyl group was then oxidized to the
corresponding keto group. At first we employed the reliable Swern oxidation, the
reaction worked fine, but the
1
H-NMR data showed a mixture of compound. One
268
compound was the 4Z, 2-keto compound, and other one was an α,β-unsaturated keto
compound, which might formed during the reaction via the acid induced migration of
the double bond to form a more stable α,β-unsaturated compound.

OH OH
O
TMS
HO Me
Me OTBS
Me OTBS
a
b
c
d
e
Reagents and conditions: (a) Lindlar catalyst, quinoline, H
2
gas, EtOAc
rt, 3h, 100%; (b) Dess-Martin periodinane, NaHCO
3
, DCM, rt, 2h, 90%;
(c) TMS-acetylene, n-BuLi, THF, -78
o
C, 2h; (d) (i) TBAF, THF, rt,
overnight, 75% in two steps; (ii) TBS-OTf, lutidine, DCM, rt, overnight,
100%; (e) Cp
2
Zr(H)Cl, NBS, THF, rt, 2h, 85%.
Br
7.17
7.18 7.19
7.20 7.21
7.22

Scheme 31. Synthesis of racemic vinyl bromide for 17R/S-RvD1.

After having difficulties in Swern oxidation, we then tried Dess-Martin periodinane
using NaHCO
3
in the reaction mixture as shown in Scheme 31. The reaction worked
fine with excellent yield (90%) and no side product was obtained. The base induced
nucleophilic addition of TMS-acetylene to the keto group yielded the racemic
secondary alcohol 7.21. The silylation of the newly generated secondary hydroxyl
269
group by TBS-group followed by desilylation of TMS-group by Na
2
CO
3
gave the
terminal alkyne 7.22. The terminal alkyne 7.22 was then employed a hydrozirconation
using Schwartz’s reagent
113
bis(cyclopentadienyl)zirconium chloride hydride followed
by a bromolysis with NBS to give the vinyl bromide (7.17) in excellent yield (85%)
with exclusively 1E-geomtry.

Final assembly of AT-RvD1 analogs—Having achieved the synthesis of terminal
alkyne (3.20), p-F-phenoxy-vinyl iodide (7.3) and racemic-vinyl bromide (7.17), we
now move on to their carbon—carbon bond-forming reactions to construct the entire
molecules. The cross-coupling reaction of terminal alkyne (3.20) and vinyl halides (7.3
and 7.17) for both p-F-phenoxy-AT-RvD1 and 17R/S-RvD1 were done using standard
Sonogashira condition as described in Scheme 32. The desilytion of TBS-groups by
TBAF gave the corresponding trihydroxy alkyne precursors. The stereoselective partial
reduction using activated zinc gave the final products in excellent yields. The final
products, p-F-phenoxy-AT-RvD1 (7.16) and 17R/S-RvD1 (7.15) methyl esters were
purified by reversed-phase HPLC on ODS using MeOH-H
2
O as the eluent. Their
structures were characterized by the extensive analysis of
1
H and
13
C-NMR data. Both
of the AT-RvD1 analogs were sent to our collaborator’s lab at Harvard Medical School,

113
(i) Buchwald, S. L.; LaMaire, S. J.; Nielsen, R. B.; Watson, B. T.; King, S. M. Org. Synth. 1993, 71,
77. (ii) Buchwald, S. L.; LaMaire, S. J.; Nielsend, R.; Watson, B. T.; King, S. M. Tetrahedron Lett. 1987,
28, 3895.

270
and the biological studies are currently underway.
OTBS TBSO
COOMe
3.20
Me OTBS
Br
O
OTBS
F
I
7.17 7.3
OTBS TBSO
COOMe
O
OTBS
F
OTBS TBSO
COOMe
Me OTBS
OH HO
COOMe
O
OH
F
OH HO
COOMe
Me OH
OH HO
COOMe
O
OH
F
OH HO
COOMe
Me OH
7.23 7.25
7.24 7.26
17R/S-RvD1 Methyl Ester (7.15) p-F-Phenoxy-AT-RvD1 Methyl Ester (7.16)
Reagents and conditions: (a) Pd(Ph
3
)
4
, CuI, C
6
H
6
,
Et
3
N, rt, overnight, 85%; (b) TBAF, THF, rt, overnight
followed by CH
2
N
2
, ether, rt, 2 h, 99%; (c) Zn
(Cu/Ag), H
2
O:MeOH (1:1), 40
o
C, overnight, 82%.
a
b
c
Reagents and conditions: (a) Pd(Ph
3
)
4
, CuI, C
6
H
6
,
Et
3
N, rt, overnight, 93%; (b) TBAF, THF, rt, overnight
followed by CH
2
N
2
, ether, rt, 2 h, 95%; (c) Zn
(Cu/Ag), H
2
O:MeOH (1:1), 38
o
C, overnight, 83%.
a
b
c

Scheme 32. Final assembly of AT-RvD1 analogs.

7.5 Conclusion
The lipoxins and resolvins are potent lipid mediators derived from AA, DHA and EPA.
These mediators are not only anti-inflammatory, but also promote resolution back to the
non-inflammed state. The identification of the resolvins, protectins as well as their
271
arachidonic acid derived cousins, lipoxins as endogenous pro-resolving, anti-
inflammatory lipid mediators provide the evidence that resolution of tissues to
homeostasis is a biochemically active process
114
and not a passive one as was once
believed. The identification of their metabolic inactivation pathways provided the basis
for the design of their biostable analogs. We have synthesized a number of analogs, and
biological studies on these analogs gave promising results, which might illuminate the
therapeutic potential of these lipid mediators for the treatment of inflammation and
inflammation associated diseases.

114
(i) Bannenberg, G. L.; Chiang, N.; Ariel, A.; Arita, M.; Tjonahen, E.; Gotlinger, K. H.; Hong, S.;
Serhan, C. N. J. Immunol. 2005, 174, 4345. (ii) Serhan, C.N.; Savill, J. Nat. Immunol. 2005, 6, 1191.
272
7.6 Experimental

OH
HO O F
7.6

7.6.1 2S, 3-(4-fluorophenoxy)propane-1,2-diol (7.6). To a solution of fluorophenol
(7.4, 1.36 g, 12.16 mmol) in anhydrous DMF (5.0 mL) was added CsF (12, 0.103 g,
0.68 mmol). The reaction mixture was stirred for 1h at room temperature and S-
glycidol (7.5, 1.0 g, 13.51 mmol, 97% ee) was added. The reaction mixture was stirred
at 90
o
C for 2 days under argon atmosphere. After cooling to room temperature,
distilled water (10 mL) was added. The product was extracted with EtOAc (30 mL x 3),
and washed with brine. The combined organic layers were dried over anhydrous
MgSO
4
, filtered, and concentrated in vacuo to give the crude product as white crystals.
The crude product was purified by a column chromatography on silica gel eluting with
hexane-EtOAc (1:1) to afford 2.03 g (90%) of title compound 7.6. The enantiomeric
excess (ee) of the product 7.6 was found to be 97% (same as the starting glycidol 7.5),
which was determined by chiral HPLC analysis using Chiracel OD, 15%
isopropanol/hexanes, 1.0 mL/min, 210 nm, t
R
(minor) = 9.008 min and t
R
(major) =
9.942 min.
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.91 (dd, J = 8.4, 7.6 Hz, 2H), 6.78 (dd, J =
9.2, 4.4 Hz, 2H), 4.05 (quintet, J = 6.0 Hz. 1H), 3.90 (d, J = 5.6 Hz, 2H), 3.77 (dd, J =
11.2, 4.0 Hz, 1H), 3.67 (dd, J = 11.2, 6.4 Hz, 1H);
13
C-NMR (100 MHz, CDCl
3
) δ
C

157.4 (d, J
C-F
= 237.5 Hz), 154.4, 116.0, 115.8, 115.5, 115.4, 70.5, 69.6, 63.5.

273
OH
TBSO O F
7.7

7.6.2 2R-1-(t-butyldimethylsilyloxy)-3-(4-fluorophenoxy)propan-2-ol (7.7). To a
mixture of imidazole (0.74 g, 10.90 mmol), TBS-Cl (1.64 g, 10.90 mmol), and DMAP
(67 mg, 0.55 mmol) in dry CH
2
Cl
2
(30 mL) at 0
o
C was added the diol 7.6 (2.03 g,
10.90 mmol). The reaction mixture was warmed to room temperature, and stirred for
overnight. The reaction mixture was quenched with a saturated aqueous solution of
NH
4
Cl, extracted with ether (30 mL x 3), washed with brine, dried over MgSO
4
, and
concentrated under reduced pressure to give a crude product. The crude product was
purified on silica column using 10% EtOAc/hexane as the eluant to give the title
monoprotected alcohol 7.7 (3.0 g, 90%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
)
δ
H
6.98 (dd, J = 9.2, 8.4 Hz, 2H), 6.86 (dd, J = 9.2, 4.4 Hz, 2H), 4.02 (quintet, J = 5.2
Hz, 1H), 3.97 (d, J = 5.6 Hz, 2H), 3.77 (m, 2H), 0.91 (s, 9H), 0.09 (s, 3H), 0.08 (s, 3H).

OTBDPS
TBSO O F
7.27

7.6.3 2R-1-(t-butyldimethylsilyloxy)-2-(t-butyldiphenylsilyloxy)-3-(4-fluorophenoxy)
propane (7.27). To a solution of TBDPS-Cl (3.10 mL, 11.98 mmol), imidazole (0.81 g,
11.98 mmol) and DMAP (60 mg, 0.50 mmol) in dry CH
2
Cl
2
(50 mL) at 0
o
C was added
the alcohol 7.7 (3.0 g, 9.98 mmol) in CH
2
Cl
2
(5.0 mL) through a cannula. The mixture
was warmed to room temperature and stirred for overnight. The reaction was quenched
with a saturated aqueous solution of NH
4
Cl, extracted with ether (50 mL x 3), washed
274
with brine, dried over MgSO
4
, and concentrated under reduced pressure. The crude was
purified on a silica gel column using 2% EtOAc/hexane as the eluant to afford the
protected diol 7.27 (5.3 g, 98%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
7.67 (m, 4H), 7.43-
7.28 (m, 6H), 6.88 (t, J = 8.8 Hz, 2H), 6.64 (dd, J = 8.8, 4.4 Hz, 2H), 4.02 (quintet, J =
5.6 Hz, 1H), 3.97 ( dd, J = 9.6, 4.4 Hz, 1H), 3.85 (dd, J = 9.6, 6.0 Hz, 1H), 3.59 (m, 2H),
1.04 (s, 9H), 0.00 (s, 9H), -0.08 (s, 3H), -0.11 (s, 3H).

OTBDPS
HO O F
7.8

7.6.4 2S-2-(t-butyldiphenylsilyloxy)-3-(4-fluorophenoxy)propan-1-ol (7.8). To a
solution of bis-silylated protected diol 7.27 (6.0 g, 11.13 mmol) in a 1:1 mixture of
CH
2
Cl
2
:MeOH (40 mL) was added camphorsulfonic acid (2.50 g, 11.13 mmol) at room
temperature. The reaction progress was monitored by TLC. The reaction was over by
30 min, it was then quenched with Et
3
N (1.55 mL, 11.13 mmol). The solvent was
evaporated to dryness, and then added a saturated aqueous solution of NH
4
Cl, extracted
with ether (50 mL x 3), washed with brine, and dried over MgSO
4
. The crude product
was purified on a silica gel column using 10% EtOAc/hexane to afford the primary
alcohol 7.8 (4.6 g, 99%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
) δ
H
7.68 (m,
4H), 7.39 (m, 6H), 6.85 (dd, J = 9.2, 8.8 Hz, 2H), 6.56 (dd, J = 8.8, 4.4 Hz, 2H), 4.08
(m, 1H), 3.92 (dd, J = 9.6, 6.4 Hz, 1H), 3.83 (dd, J = 9.6, 5.6 Hz, 1H), 3.68 (d, J = 3.6
Hz, 2H), 1.07 (s, 9H).

275
OTBDPS
O O F
7.9

7.6.5 2R-2-(t-butyldiphenylsilyloxy)-3-(4-fluorophenoxy)propanal (7.9). To a
solution of primary alcohol 7.8 (2.58 g, 6.07 mmol) in dry CH
2
Cl
2
(25 mL) was added
the trichloroisocyanuric acid (1.48 g, 6.38 mmol) at 0
o
C. The reaction mixture was
stirred at 0
o
C, followed by the careful addition of TEMPO (9.5 mg, 0.061 mmol)
[Caution! very exothermic reaction, if not careful the reaction mixture might overflow].
After the addition, the reaction mixture was warmed to room temperature, and
monitored the reaction progress by TLC. The reaction was over by 20 min. The
reaction mixture was then filtered on celite, added a saturated solution of NaHCO
3
to
the filtrate, extracted with ether (35 mL x 3), washed with brine, dried over MgSO
4
, and
concentrated in vacuo. The crude product was purified on a silica column using 10%
EtOAc/hexanes as eluant to give the pure aldehyde 7.9 (1.72 g, 83%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
) δ
H
9.72 (s, 1H), 7.65 (m, 4H), 7.37 (m, 6H), 6.90 (dd, J =
9.6, 8.4 Hz, 2H), 6.66 (dd, J = 8.8, 4.4 Hz, 2H), 4.30 (m, 1H), 4.04 (m, 2H), 1.11 (s, 9H).

OH
O F
7.28
I

7.6.6 2S, 3E, 1-(4-fluorophenoxy)-4-iodo-but-3-en-2-ol (7.28). To a stirring
suspension of CrCl
2
(4.6 g, 37.84 mmol) in dry THF (25 mL) at 0
o
C was added a
solution of CHI
3
(3.7 g, 9.46 mmol) and aldehyde 7.9 (2.0 g, 4.73 mmol) in dry THF (5
mL). The reaction mixture was stirred at 0
o
C for 3 h, then warmed to room
276
temperature and stirred for an additional 1 h. The reaction was quenched with H
2
O (20
mL), extracted with pentane (30 mL x 3), washed with brine, dried over MgSO
4
, and
evaporated to dryness to give a crude product. The crude was then dissolved in THF (5
mL), was added 1.0 M solution of TBAF (7.09 mL, 7.09 mmol) to the flask at 0
o
C and
stirred for 2 h. The reaction was quenched with a saturated aqueous solution of NH
4
Cl
(15 mL), extracted with ether (15 mL x 3), washed with brine, dried over MgSO
4
, and
concentrated under reduced pressure. The crude product was purified on a silica
column using 10% EtOAc/hexane to give a pure vinyl iodide 7.28 (0.88 g, 55% in 2
steps).
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.98 (dd, J = 9.6, 8.4 Hz, 2H), 6.84 (dd, J = 8.8,
4.4 Hz, 2H), 6.63 (d, J = 13.6 Hz, 1H), 6.65 (m, 1H), 3.97 (dd, J = 9.6, 3.6 Hz, 1H),
3.84 (dd, J = 9.6, 7.6 Hz, 1H).

OTBS
O F
7.3
I

7.6.7 2S, 3E, 2-(t-butyldimethylsilyloxy)-1-(4-fluorophenoxy)-4-iodobut-3-ene (7.3).
To a solution of alcohol 7.28 (0.88 g, 2.85 mmol) in dry CH
2
Cl
2
(15 mL) was added
2,6-lutidine (0.86 mL, 7.42 mmol) at 0
o
C followed by the addition of TBS-OTf (0.85
mL, 3.71 mmol). The reaction mixture was stirred at room temperature for overnight.
The reaction was quenched with a saturated aqueous solution of NH
4
Cl, extracted with
pentane (20 mL x 3), washed with brine, and dried over MgSO
4
. The crude product
was purified on a silica column using 1% EtOAc/hexanes-pentane (1:1) as the eluant to
give pure vinyl iodide 7.3 (0.93 g, 88%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
)
277
δ
H
6.94 (dd, J = 9.6, 8.4 Hz, 2H), 6.78 (dd, J = 8.8, 4.4 Hz, 2H), 6.63 (dd, J = 14.8, 5.2
Hz, 1H), 6.43 (dd, J = 14.8, 1.6 Hz, 1H), 4.42 (m, 1H), 3.78 (m, 2H), 0.88 (s, 9H), 0.07
(s, 3H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
163.4, 160.9, 159.4, 149.9, 120.7, 120.5, 120.2,
120.1, 82.9, 78.4, 76.7, 30.6, 23.1, 0.00.

OTBS O
OMe
OTBS
7.12
O
OTBS
F

7.6.8 p-F-phenoxy tris-TBS protected alkyne precursor of RvE1 (7.12). To a
solution of p-fluorophenoxy vinyl iodide 7.3 (195 mg, 0.47 mmol) in benzene (4 mL)
was added Et
3
N (0.3 mL, 1.8 mmol) and then the terminal alkyne 2.21 (180 mg, 0.36
mmol) in benzene (1.0 mL) by a cannula. The mixture was freeze-thaw several times at
-78
o
C to remove oxygen. The reaction mixture was warmed to room temperature
followed by the addition of Pd(Ph
3
)
4
(35 mg, 0.03 mmol) and CuI (11 mg, 0.06 mmol).
The reaction mixture was protected from the light by warping the flask with the
aluminum foil. The reaction mixture was then stirred at room temperature for overnight.
The reaction was quenched with saturated aqueous solution of NH
4
Cl, extracted with
ether (15 mL x 3), washed with brine. The organic layers were combined, dried over
MgSO
4
, and concentrated in vacuo to give a crude product. The crude was purified on a
silica column using 2% EtOAc/hexane to give the protected alkyne precursor of p-
fluorophenoxy RvE1, 7.12 (215 mg, 75%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.93 (t, J =
8.0 Hz, 2H), 6.77 (dd, J = 9.2, 4.4 Hz, 2H), 6.42 (dd, J = 14.4, 10.4 Hz, 1H), 6.23 (dd, J
= 14.8, 10.8 Hz, 1H), 6.11 (dd, J = 16.0, 6.0 Hz, 1H), 5.82 (d, J = 15.6 Hz, 1H), 6.00
278
(dd, J = 15.6, 6.0 Hz, 1H), 5.57 (d, J = 16.0 Hz, 1H), 4.52 (m, 1H), 4.43 (m, 1H), 4.32
(m, 1H), 3.78 (m, 2H), 3.64 (s, 3H), 2.51 (m, 2H), 2.32 (m, 2H), 1.74 (m, 2H), 1.58 (m,
2H), 0.88 (s, 9H), 0.87 (s, 9H), 0.86 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H), 0.06 (s, 3H),
0.05 (s, 3H), 0.04 (s, 3H), 0.03 (s, 3H).

7.13
OH O
OMe
OH
O
OH
F

7.6.9 p-F-phenoxy bis-alkyne precursor of RvE1 (7.13). To a solution of tris-TBS
protected p-fluorophenoxy RvE1 7.12 (100 mg, 0.125 mmol) in THF (10 mL) at 0
o
C
was added TBAF (0.75 mL of 1M solution in THF, 0.75 mmol). The reaction mixture
was stirred for overnight at room temperature, and then quenched with saturated
aqueous solution of NH
4
Cl, extracted with ether (20 mL x 3), washed with brine, and
dried over MgSO
4
. The combined ether extract was then treated with freshly prepared
diazomethane to convert the free acid to the methyl ester. The solution was then
bubbled with nitrogen to remove excess diazomethane. The crude product was purified
over a silica column using 2% MeOH/CH
2
Cl
2
to afford 7.13 (55 mg measured by UV,
96%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.95 (t, J = 9.6 Hz, 2H), 6.82 (dd, J = 9.6, 4.4
Hz, 2H), 6.55 (dd, J = 15.6, 10.4 Hz, 1H), 6.31 (dd, J = 15.2, 10.8 Hz, 1H), 6.12 (dd, J
= 15.2, 5.2 Hz, 1H), 5.86 (d, J = 15.6 Hz, 1H), 5.84 (dd, J = 15.2, 6.0 Hz, 1H), 5.62 (d,
J = 14.4 Hz, 1H), 4.54 (m, 1H), 4.50 (m, 1H), 4.34 (q, J = 4.8 Hz, 1H), 3.94 (dd, J =
10.0, 4.0 Hz, 1H), 3.80 (dd, J = 10.4, 7.2 Hz, 1H), 3.65 (s, 3H), 2.58 (td, J = 6.0, 1.6 Hz,
279
2H), 2.36 (t, J = 6.4 Hz, 2H), 1.75 (m, 4H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
174.0,
157.5 (d, J
C-F
= 247.5 Hz), 154.3, 141.1, 139.9, 136.5, 130.1, 116.0, 115.8, 115.6, 112.1,
111.4, 92.6, 87.0, 84.0, 80.8, 71.9, 70.3, 70.1, 62.4, 51.6, 36.9, 33.5, 28.5, 20.5.

p-F-Phenoxy-RvE1 Methyl Ester (7.14)
OH O
OMe
OH
O
OH
F

7.6.10 Hydrogenation of p-F-phenoxy bis-alkyne precursor of RvE1 methyl ester.
The hydrogenation of p-F-phenoxy bis alkyne precursor of RvE1 (7.13) was done by
using activated Zinc as Zn(Cu/Ag) in MeOH—H
2
O (1:1) at 40
o
C using the same
procedure as described previously in this dissertation. The hydrogenated product was
purified by a reversed-phase HPLC on ODS using MeOH—H
2
O as the mobile phase to
give ultra pure p-F-phenoxy-RvE1 analog (7.14).
1
H-NMR (400 MHz, acetone-d
6
) δ
H

7.04 (t, J = 8.8 Hz, 2H), 6.96 (dd, J = 9.2, 4.8 Hz, 2H), 6.72 (dd, J = 15.6, 10.8 Hz, 1H),
6.59 (dd, J = 14.8, 11.0 Hz, 1H), 6.34 (dd, J = 14.8, 10.8 Hz, 1H), 6.25 (dd, J = 14.8,
10.8 Hz, 1H), 6.10 (t, J = 10.4 Hz, 1H), 6.02 (t, J = 10.8 Hz, 1H), 5.82 (dd, J = 14.8, 5.6
Hz, 1H), 5.81 (dd, J = 14.8, 6.0 Hz, 1H), 5.53 (dt, J = 10.8, 7.6 Hz, 1H), 5.39 (t, J = 8.8
Hz, 1H), 4.57 (m, 2H), 4.37 (m, 1H, -OH), 4.21 (m, 1H), 3.97 (dd, J = 9.6, 4.4 Hz, 1H),
3.89 (dd, J = 9.2, 7.2 Hz, 1H), 3.81 (m, 1H, -OH), 3.60 (s, 3H), 3.15 (m, 1H, -OH), 2.45
(t, J = 6.0 Hz, 2H), 2.33 (t, J = 7.2 Hz, 2H), 1.73-1.53 (m, 3H), 1.39 (m, 1H).

280
7.25
OTBS TBSO
COOMe
O
OTBS
F

7.6.11 Methyl (7S, 8R, 17S, 4Z, 9E, 11E, 15E)-tris(t-butyldimethylsilyloxy)-18-(4-
fluorophenoxy)octadeca-4, 9, 11, 15-tetraen-13-ynoate (7.25). To a solution of 4-
fluorophenoxy vinyl iodide 7.3 (111.4 mg, 0.26 mmol) in benzene (4 mL) was added
Et
3
N (0.14 mL, 1.01 mmol) and the alkyne 3.20 (100 mg, 0.20 mmol). The mixture
was freeze-thaw at -78
o
C to remove oxygen. The reaction mixture was then warmed to
room temperature, followed by the addition of Pd(Ph
3
)
4
and CuI. The reaction vessel
was protected from light by wrapping the flask with aluminum foil. The reaction
mixture was then stirred at room temperature for overnight. The reaction was quenched
with saturated aqueous solution of NH
4
Cl, extracted with ether (15 mL x 3), washed
with brine. The combined organic extract was dried over MgSO
4
, concentrated under
reduced pressure to give a crude product. The crude oil was purified on a silica column
using 2% EtOAc/hexane to the tri-TBS protected p-fluorophenoxy AT-RvD1, 7.25 (148
mg, 93%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.94 (t, J = 8.8 Hz, 2H), 6.78 (dd, J = 8.8,
4.4 Hz, 2H), 6.57 (dd, J = 15.6, 11.2 Hz, 1H), 6.20 (dd, J = 15.6, 4.8 Hz, 1H), 6.16 (dd,
J = 15.6, 10. 8 Hz, 1H), 5.98 (d, J = 15.6 Hz, 1H), 5.75 (dd, J = 15.6, 7.2 Hz, 1H), 5.67
(dd, J = 15.6, 2.4 Hz, 1H), 5.44 (m, 1H), 5.39 (m, 1H), 4.56 (m, 1H), 3.98 (m, 1H), 3.79
(m, 1H), 3.59 (m, 1H), 3.65 (s, 3H), 2.33 (m, 4H), 2.22 (m, 2H), 0.89 (s, 9H), 0.86 (s,
9H), 0.84 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H), 0.01 (s, 3H), 0.00 (s, 3H), -0.01 (s, 3H), -
0.03 (s, 3H).
281
7.26
OH HO
COOMe
O
OH
F

7.6.12 Methyl (7S, 8R, 17S, 4Z, 9E, 11E, 15E)-7, 8, 17-trihydroxy-18-(4-
fluorophenoxy)-octadeca-4, 9, 11, 15-tetraen-13-ynoate (7.26). To a solution of
protected triol 7.25 (148 mg, 0.19 mmol) in THF (6 mL) at 0
o
C was added TBAF (1.13
mL of 1M solution in THF, 1.13 mmol). The reaction mixture was stirred for overnight
at room temperature, and then quenched with saturated aqueous solution of NH
4
Cl,
extracted with ether (15 mL x 3), washed with brine, and dried over MgSO
4
. The
combined ether extract was then treated with freshly prepared diazomethane to convert
the free acid to the corresponding methyl ester. The solution was then bubbled with
nitrogen to remove the excess amount of diazomethane. The crude product was purified
over a silica column using 3% MeOH/CH
2
Cl
2
to afford 7.26 (80.2 mg, 95%).
1
H-NMR
(400 MHz, CDCl
3
) δ
H
6.94 (dd, J = 9.2, 8.0 Hz, 2H), 6.80 (dd, J = 8.8, 4.4 Hz, 2H),
6.57 (dd, J = 15.6, 10.4 Hz, 1H), 6.33 (dd, J = 15.6, 10.8 Hz, 1H), 6.15 (dd, J = 15.6,
5.6 Hz, 1H), 6.01 (dt, J = 15.6, 2.0 Hz, 1H), 5.84 (dd, J = 15.6, 5.6 Hz, 1H), 5.72 (dd, J
= 15.6, 2.0 Hz,1H), 5.44 (m, 2H), 4.56 (m, 1H), 4.19 (m, 1H), 3.93 (dd, J = 10.0, 3.6 Hz,
1H), 3.81 (dd, J = 9.6, 7.6 Hz, 1H), 3.68 (m, 1H), 3.62 (s, 3H), 2.90 (brs, 1H, -OH),
2.82 (brs, 2H, -OH), 2.37 (m, 5H), 2.15 (m, 1H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
174.0,
157.5 (d, J
C-F
= 238.5 Hz), 154.3, 141.0, 140.0, 133.9, 131.5, 130.8, 126.7, 116.0, 115.8,
115.7, 115.6, 112.2, 111.7, 90.3, 90.1, 74.6, 73.8, 71.9, 70.3, 51.7, 33.4, 29.9, 22.5.

282
p-F-Phenoxy-AT-RvD1 Methyl ester (7.16)
17
OH HO
COOMe
O
OH
F

7.6.13 Methyl (7S, 8R, 17S, 4Z, 9E, 11E, 13Z, 15E)-7, 8, 17-trihydroxy-18-(4-
fluorophenoxy)-octadeca-4, 9, 11, 13, 15-pentaenoate (7.16). Stereoselective partial
hydrogenation of the triple bond was done using activated Zinc as Zn(Cu/Ag) at 38 oC
in MeOH—H
2
O (1:1) by following the procedure as described previously in this
dissertation. The amount of 7.26 was started with 4.0 mg, and got 3.32 mg ultra pure
hydrogenated product 7.16 (yield 83%). The final product was purified by HPLC using
Phenomenex C18 ODS column, solvent system, 33% H2O/MeOH.
1
H-NMR (400 MHz,
CDCl
3
) δ
H
6.96 (t, J = 8.8 Hz, 2H), 6.83 (dd, J = 8.8, 4.4 Hz, 2H), 6.70 (dd, J = 14.8,
10.4 Hz, 1H), 6.39 (dd, J = 14.8, 10.8 Hz, 1H), 6.27 (dd, J = 14.8, 10.4 Hz, 1H), 6.04 (q,
J = 10.8 Hz, 1H), 5.80 (dd, J = 15.6, 7.2 Hz, 1H), 5.78 (dd, J = 15.6, 5.6 Hz, 1H), 5.46
(m, 2H), 4.62 (m, 1H), 4.21 (m, 1H), 4.70 (m, 1H), 3.97 (dd, J = 10.0, 4.0 Hz, 1H), 3.84
(dd, J = 9.2, 7.6 Hz, 1H), 3.64 (s, 3H), 2.41-2.15 (m, 6H);
13
C-NMR (100 MHz, CDCl
3
)
174.0, 157.5 (d, J
C-F
= 238.5 Hz), 154.5, 133.3, 132.7, 132.0, 131.6, 130.9, 130.3, 128.8,
128.4, 127.6, 126.7, 116.0, 115.8, 115.7, 115.6, 74.9, 73.8, 72.3, 70.8, 51.7, 33.4, 30.0,
22.6.
OH
7.19

7.6.14 4Z-Hept-4-en-2-ol (7.19). To a solution of 7.18 (2.0 g, 17.84 mmol) in EtOAc
(150 mL) was added 200 mg of Lindlar catalyst and 3 drops of quinoline. The mixture
283
was stirred at room temperature under the static atmosphere of hydrogen. The reaction
progress was monitored by TLC, and it was over by 3 h. The mixture was then filtered
through celite and the solvent was evaporated under reduced pressure. The crude
product was purified on a silica column using 10% EtOAc/hexane as the eluant to give
the product 7.19 as colorless oil with the Z-double bond.
1
H-NMR (400 MHz, CDCl
3
)
δ
H
5.51 (m, 1H), 5.32 (m, 1H), 3.76 (sextet, J = 6.4 Hz, 1H), 2.24-2.12 (m, 2H), 2.06-
2.00 (m, 2H), 1.14 (d, J = 8.0 Hz, 3H), 0.92 (t, J = 7.6 Hz, 3H);
13
C-NMR (100 MHz,
CDCl
3
) δ
C
134.8, 124.4, 67.5, 36.9, 22.6, 20.6, 14.1.

O
7.20

7.6.15 4Z-Hept-4-en-2-one (7.20). To a solution of Dess-Martin periodinane (4.46 g,
10.50 mmol) in CH
2
Cl
2
(10 mL) was added NaHCO
3
(4.0 g) at room temperature. A
solution of primary alcohol 7.19 (1.0 g, 8.76 mmol) in dry CH
2
Cl
2
(5 mL) was
canulated to the suspension of Dess-Martin periodinane and NaHCO
3
at room
temperature. The reaction mixture was stirred at room temperature for 2 h. The
reaction mixture was then quenched with a saturated aqueous solution of NH
4
Cl. The
aqueous phase was extracted with ether (20 mL x 3), washed with brine, dried over
MgSO
4
. The ether extract was concentrated under reduced pressure. A flash column
chromatography on silica gel using 10% ether/pentane as the solvent system to give the
ketone 7.20 (0.88 g, 90%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
) δ
H
5.53 –
5.47 (m, 1H), 5.44 – 5.39 (m, 1H), 3.07 (d, J = 7.2 Hz, 2H), 2.05 (s, 3H), 0.88 (t, J = 7.6
Hz, 3H).
284
TMS
HO Me
7.21

7.6.16 5Z, 3-Methyl-1-(trimethylsilyl)oct-5-en-1-yn-3-ol (7.21). To a solution of
TMS-acetylene (0.98 g, 10.03 mmol) in dry THF (20 mL) at -78
o
C was slowly added
n-BuLi (5.0 mL of 1.6 M solution in hexane, 8.02 mmol). The mixture was stirred for
40 min at -78
o
C. After then the ketone 7.20 (0.75 g, 6.69 mmol) in dry THF (5.0 mL)
was added to the mixture at -78
o
C by a canula. The reaction mixture was stirred at -78
o
C for 1 h, and then removed the dry-ice bath to warm to the room temperature, and
stirred for an additional hour. The reaction was quenched with a saturated aqueous
solution of NH
4
Cl, extracted with ether (40 mL x 3), washed with brine. The organic
layers were combined, dried over MgSO
4
, and concentrated under vacuum to give the
crude product 7.21 (1.3 g), which was used for the next reaction without purification.
Crude
1
H-NMR (400 MHz, CDCl
3
) δ
H
5.70-5.63 (m, 1H), 5.57 – 5.50 (m, 1H), 2.47-
2.41 (m, 2H), 2.13-2.09 (m, 2H), 1.50 (s, 3H), 1.00 (t, J = 7.6 Hz, 3H), 0.18 (s, 9H).

Me OH
7.29

7.6.17 5Z, 3-Methyl-oct-5-en-1-yn-3-ol (7.29). To a solution of 7.21 (1.3 g, 6.18 mmol)
in dry THF (15 mL) at 0
o
C was added TBAF (10.03 mL of 1M solution in THF, 10.03
mmol). The reaction mixture was stirred for overnight at room temperature. The
reaction was quenched with saturated aqueous solution of NH
4
Cl, extracted with ether
(20 mL x 3), washed with brine, and dried over MgSO
4
. The combined ether extract
285
was concentrated under reduced pressure to give a crude oil, which was then purified by
a flash column on silica gel using 11% ether/pentane-hexane (1:1) as the solvent system
to give the pure 7.29 (0.69 g, 75% in two steps) as a colorless oil.
1
H-NMR (400 MHz,
CDCl
3
) δ
H
5.64 - 5.58 (m, 1H), 5.52 - 5.46 (m, 1H), 2.47 - 2.34 (m, 2H), 2.41 (s, 1H),
2.08 - 2.00 (m, 2H), 1.46 (s, 3H), 0.39 (t, J = 7.6 Hz, 3H);
13
C-NMR (100 MHz, CDCl
3
)
δ
C
136.2, 122.7, 87.5, 71.2, 67.3, 50.0, 29.0, 20.7, 14.1.

Me OTBS
7.22

7.6.18 5Z, 3-Methyl, 3-(t-butyldimethylsilyloxy)oct-5-en-1-yn (7.22). To a solution
of tertiary alcohol 7.29 (0.55 g, 3.98 mmol) in dry CH
2
Cl
2
(10 mL) was added 2,6-
lutidine (2.3 mL, 19.9 mmol) at room temperature. The mixture was then cooled at 0
o
C,
and then was added TBS-OTf (1.82 mL, 7.96 mmol). The reaction mixture was stirred
at room temperature for overnight. The reaction was quenched with a saturated aqueous
solution of NH
4
Cl, extracted with ether, washed with brine, and dried over MgSO
4
. The
crude product was purified on a silica column using only pentane as the eluant to give
20 (1.0 g, 100%) as a colorless.
1
H-NMR (400 MHz, CDCl
3
) δ
H
5.59 (m, 2H), 2.51 (s,
1H), 2.49 (m, 2H), 2.16 (m, 2H), 1.52 (s, 3H), 1.06 (t, J = 7.6 Hz, 3H), 0.96 (s, 9H),
0.27 (s, 6H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
134.0, 124.0, 88.2, 71.9, 68.8, 42.6, 30.4,
25.7, 20.8, 18.0, 14.2, -2.9, -3.09.

286
Me OTBS
Br
7.17

7.6.19 1E, 5Z, 1-bromo-3-Methyl, 3-(t-butyldimethylsilyloxy)oct-1, 5-diene (7.17).
To a suspension of Cp
2
Zr(H)Cl (515 mg, 2.0 mmol) in dry THF (8.0 mL) at room
temperature was added a solution of terminal alkyne 7.22 (0.50 g, 1.98 mmol) in THF
(2.0 mL) by a cannula. The reaction was protected from light and stirred for an hour at
which time it turned to a clear orange solution. At this point, NBS (356 mg, 2.0 mmol)
was slowly added and a yellow solution was produced indicated that the reaction was
over. The reaction mixture was then poured into aqueous solution of NaHCO
3
and
extracted with ether. The combined ether extracts were washed with brine, dried over
MgSO
4
and concentrated in vacuo. Flash column chromatography on silica column
using only pentane as the solvent system gave the vinyl bromide 7.17 (0.56 g).
1
H-
NMR (400 MHz, CDCl
3
) δ
H
6.20 (s, 2H), 5.45 (m, 1H), 5.35 (m, 1H), 2.23 (m, 2H),
2.00 (m, 2H), 1.28 (s, 3H), 0.94 (t, J = 7.6 Hz, 3H), 0.86 (s, 9H), 0.07 (s, 3H), 0.07 (s,
3H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
144.6, 134.0, 123.7, 104.8, 76.6, 46.9, 41.2, 27.0,
25.8, 20.8, 18.2, 14.1, -2.1.

7.23
OTBS TBSO
COOMe
Me OTBS

7.6.20 Methyl (7S, 8R, 17R/S, 4Z, 9E, 11E, 15E, 19Z)-17-methyl-tris(t-
butyldimethylsilyloxy)docosa-4, 9, 11, 15, 19-pentaen-13-ynoate (7.23). To a
287
solution of vinyl bromide 7.17 (97 mg, 0.92 mmol) in benzene (4 mL) was added Et
3
N
(0.11 mL, 0.81 mmol) and the alkyne 3.20 (80 mg, 0.16 mmol). The mixture was
freeze-thaw at -78
o
C to remove oxygen. The reaction mixture was warmed to room
temperature followed by the addition of Pd(Ph
3
)
4
and CuI, and protected from light.
The reaction mixture was then stirred at room temperature for overnight. The reaction
was quenched with saturated aqueous solution of NH
4
Cl, extracted with ether (15 mL x
3), washed with brine. The combined organic extract was dried over MgSO
4
,
concentrated under reduced pressure to give a crude product. The crude oil was
purified on a silica column using 2% EtOAc/hexane to the tris-TBS protected alkyne
precursor of 17R/S-RvD1, 7.23 (99 mg, 85%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.55 (dd,
J = 15.6, 10.8 Hz, 1H), 6.15 (dd, J = 16.0, 10.8 Hz, 1H), 6.12 (d, J = 16.0 Hz, 1H), 5.73
(dd, J = 15.6, 7.2 Hz, 1H), 5.80 (dd, J = 16.0, 2.4 Hz, 1H), 5.67 (dd, J = 15.6, 2.0 Hz,
1H), 5.49 – 5.30 (m, 4H), 3.98 (dd, J = 7.2, 6.0 Hz, 1H), 3.65 (s, 3H), 3.59 (q, J = 6.0
Hz, 1H), 2.32 (m, 4H), 2.54 – 2.11 (m, 4H), 2.01 – 1.97 (m, 2H), 1.28 (s, 3H), 0.94 (t, J
= 7.6 Hz, 3H), 0.87 (s, 9H), 0.86 (s, 9H), 0.84 (s, 9H), 0.07 (s, 3H), 0.06 (s, 3H), 0.02 (s,
3H), 0.00 (s, 6H), -0.02 (s, 3H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
150.2, 140.8, 136.8,
133.7, 130.8, 128.9, 127.5, 124.0, 111.0, 107.4, 90.8, 89.1, 76.6, 76.2, 75.5, 51.5, 47.0,
41.3, 33.9, 31.5, 27.3, 25.9, 23.0, 20.8, 18.3, 18.2, 18.1, 14.1, -2.1, -4.1, -4.4, -4.7.

288
7.24
OH HO
COOMe
Me OH

7.6.21 Methyl (7S, 8R, 17R/S, 4Z, 9E, 11E, 15E, 19Z)-17-methyl-7, 8, 17-
trihydroxy-docosa-4, 9, 11, 15, 19-pentaen-13-ynoate (7.24). To a solution of TBS-
protected triol 7.23 (90 mg, 0.12 mmol) in THF (5 mL) at 0
o
C was added TBAF (0.72
mL of 1M solution in THF, 0.72 mmol). The reaction mixture was stirred for overnight
at room temperature, and then quenched with saturated aqueous solution of NH
4
Cl,
extracted with ether (15 mL x 3), washed with brine, and dried over MgSO
4
. The
combined ether extract was then treated with freshly prepared diazomethane to convert
the free acid to the methyl ester. The solution was then bubbled with nitrogen to
remove excess diazomethane. The crude product was purified over a silica column
using 3% MeOH/CH
2
Cl
2
to afford 7.24 (48 mg, 100%).
1
H-NMR (400 MHz, CDCl
3
)
δ
H
6.55 (dd, J = 15.6, 10.8 Hz, 1H), 6.32 (dd, J = 15.6, 10.8 Hz, 1H), 6.17 (d, J = 16.0
Hz, 1H), 5.83 (d, J = 15.6 Hz, 1H), 5.82 (dd, J = 16.0, 8.0 Hz, 1H), 5.71 (dd, J = 15.6,
2.0 Hz, 1H), 5.60 – 5.54 (m, 1H), 5.44 (m, 2H), 5.32 – 5.26 (m, 1H), 4.18 (m, 1H), 3.67
(m, 1H), 3.63 (s, 3H), 2.38 – 2.10 (m, 8H), 2.04 – 1.98 (m, 2H), 1.26 (s, 3H), 0.92 (t, J
= 7.6 Hz, 3H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
174.0, 149.4, 140.4, 136.2, 133.4, 131.6,
130.8, 126.7, 122.5, 112.0, 107.8, 90.8, 89.1, 74.6, 73.8, 72.9, 51.7, 39.8, 33.4, 29.9,
27.4, 22.5, 20.6, 14.1.
289
OH HO
COOMe
17R/S-RvD1 Methyl ester (7.15)
Me OH
17

7.6.22 Methyl (7S, 8R, 17R/S, 4Z, 9E, 11E, 15E, 19Z)-17-methyl-7, 8, 17-
trihydroxy-docosa-4, 9, 11, 15, 19-21-hexaenoate (7.15). The stereoselective
hydrogenation was done using activated Zinc as previously described in this dissertation.
The final product (7.15) was purified by a prep-HPLC on ODS using MeOH—H
2
O as
the mobile phase. The structure of the 17R/S-RvD1 methyl ester was characterized
from the analysis of NMR and UV data.
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.72 (dd, J =
14.8, 10.0 Hz, 1H), 6.70 (dd, J = 15.6, 10.4 Hz, 1H, 6.38 (dd, J = 14.8, 10.4 Hz, 1H),
6.24 (dd, J = 14.8, 10.4 Hz, 1H), 5.99 (m, 2H), 5.82 (d, J = 15.6 Hz, 1H), 5.79 (dd, J =
14.8, 7.2 Hz, 1H), 5.58 (m, 1H), 5.47 (m, 2H), 5.33 (m, 2H), 4.21 (m, 1H), 3.71 (m, 1H),
3.65 (s, 3H), 2.45 – 2.15 (m, 8H), 2.05 (m, 2H), 1.31 (s, 3H), 0.95 (t, J = 7.6 Hz, 3H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
173.9, 141.7, 136.0, 133.0, 132.5, 131.3, 130.9, 129.6,
128.9, 128.8, 126.8, 123.0, 122.8, 75.0, 73.8, 72.9, 51.7, 40.2, 33.5, 30.0, 27.8, 22.6,
20.7, 14.2

290
2.02 2.00
1.17
2.10
1.05 1.07
7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 PPM

1
H-NMR (400 MHz, CDCl
3
) of 7.6.

160 140 120 100 80 60 PPM

13
C-NMR (100 MHz, CDCl
3
) of 7.6.

OH
HO O F
7.6
291
2.00
2.11
3.12
2.20
9.57
5.79
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of 7.7.

OH
TBSO O F
7.7
292

4.20
6.35
2.00 2.04 1.97
1.02
1.94
9.40 9.48
5.41
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 7.28.

OTBDPS
TBSO O F
7.28
293
4.08
6.13
1.95 2.00
1.16
1.08 1.09
2.08
9.38
7 6 5 4 3 2 1 PPM

OTBDPS
HO O F
7.8
294

0.95
4.21
6.31
2.01 2.00
0.98
2.11
9.60
0.00
9 8 7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of 7.9.

OTBDPS
O
O F
7.9
295

1.87
1.82
1.56
0.83
0.95
1.00
7 6 5 4 3 PPM

1
H-NMR (400 MHz, CDCl
3
) of 7.27.

OH
O F
I
7.27
296
2.00 2.01
0.92 0.89
0.97
2.20
10.28
6.04
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 7.3.

160 140 120 100 80 60 40 20 0 PPM

13
C-NMR (100 MHz, CDCl
3
) of 7.3.
OTBS
O F
I
7.3
297

7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 7.25.

7.0 6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 PPM
OTBS TBSO
COOMe
O
OTBS
F
7.25
298
2.00
2.16
1.06
1.11
1.13
1.03
1.09
1.04
2.28
1.14
1.07 1.07
1.14
1.10
3.33
5.96
1.32
3.03
7 6 5 4 3 2 PPM

1
H-NMR (400 MHz, CDCl
3
) of 7.26.

160 140 120 100 80 60 40 20 PPM

13
C-NMR (100 MHz, CDCl
3
) of 7.26.
7.0 6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 PPM
OH HO
COOMe
O
OH
F
7.26
299

2.00
3.21
1.14 1.15
1.21
2.18 2.19
2.28
1.04 1.06
1.18 1.16
1.13
3.13
2.08
6.86
1.19
7 6 5 4 3 2 PPM

1
H-NMR (400 MHz, CDCl
3
) of 7.16.

160 140 120 100 80 60 40 20 PPM

13
C-NMR (100 MHz, CDCl
3
) of 7.16.
7.0 6.8 6.6 6.4 6.2 6.0 5.8 5.6 PPM
136 134 132 130 128 126 124 PPM
OH HO
COOMe
O
OH
F
7.16
300

1.00 1.00 1.02
1.95
2.35
3.02
3.44
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of 7.19.

140 120 100 80 60 40 20 PPM

13
C-NMR (100 MHz, CDCl
3
) of 7.19.

OH
7.19
301

1.00 0.98
1.59
3.10 3.15
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of 7.20.

O
7.20
302
1.00
1.61
2.15
3.29
2.73
8.83
1.05
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 7.21.

140 120 100 80 60 40 20 0 PPM

13
C-NMR (100 MHz, CDCl
3
) of 7.21.

TMS
HO Me
7.21
303

1.00 1.00
2.04
2.98
3.26 3.27
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of 7.29.

120 100 80 60 40 20 PPM

13
C-NMR (100 MHz, CDCl
3
) of 7.22.
Me OH
7.29
304

2.88
1.92
3.33
3.02
10.67
6.12
2.00
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of 7.22.

120 100 80 60 40 20 0 PPM

13
C-NMR (100 MHz, CDCl
3
) of 7.22.

Me OTBS
7.22
305
1.63
1.00
0.94
1.64
2.12
2.94
9.71
5.50
3.24
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 7.17.

140 120 100 80 60 40 20 0 PPM

13
C-NMR (100 MHz, CDCl
3
) of 7.17.

Me OTBS
Br
7.17
306
0.90
2.00
2.98
4.26
1.00
3.23
1.07
4.30 4.43
2.07
3.13
2.82
29.62
17.07
7 6 5 4 3 2 1 0 PPM

1
H-NMR (400 MHz, CDCl
3
) of 7.23.

160 140 120 100 80 60 40 20 0 PPM

13
C-NMR (100 MHz, CDCl
3
) of 7.23.

2.00
4.26
0.90
2.94
6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 PPM
OTBS TBSO
COOMe
Me OTBS
7.23
307
0.96
1.06 1.05
1.97
1.01
1.13
2.18
1.06
1.01
1.16
3.00
7.39
1.44
2.56
2.65
2.78
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of 7.24.

160 140 120 100 80 60 40 20 PPM

13
C-NMR (100 MHz, CDCl
3
) of 7.24.

OH HO
COOMe
Me OH
7.24
0.96
1.06 1.05
1.97
1.01
1.13
2.18
1.06
6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 PPM
308
2.05
1.06
1.14
2.04 2.06
1.05
2.08
1.04
0.97 0.94
3.00
1.42
2.07
2.91
3.11
4.11
3.60
7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of 7.15.

160 140 120 100 80 60 40 20 PPM

13
C-NMR (100 MHz, CDCl
3
) of 7.15.

2.05
1.06
1.14
2.04 2.06
1.05
2.08
1.04
6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 PPM
OH HO
COOMe
Me OH
7.15
309
Chapter 8. Design and Synthesis of Anticancer Small
Molecules

8 Introduction
Cancer is a group of many related diseases of cells. Cancer develops when cells in a
part of the body is begin to grow abnormally in out of control fashion. Normal cells
grow, divide and die in an orderly fashion, however, cancer cells are aggressive,
continue to grow, divide and never die, invasive and sometimes metastatic. Although
cancers are diverse and heterogeneous in nature, but most cancers have at least two
things in common, suppressed apoptosis, the programmed cell death, and deregulated
cell proliferation.
8.1 Apoptosis
Apoptosis or programmed cell death is a normal component of the development of
multicellular organisms to destroy cells that represent a threat to the integrity of the
organism.
115
Cells die in response to a variety of stimuli and during apoptosis they do so
in a controlled, regulated fashion, which makes apoptosis distinct from another form of
cell death called necrosis. Inducing cancerous cells to undergo apoptosis is a very
important and attractive strategy to manage cancers.
116
Apoptosis can be triggered by
either intrinsic (mitochondria) or extrinsic (death receptor). In both pathways, upon

115
(i) Strasser, A.; O’Connor, L.; Dixit, V. M. Annu. Rev. Biochem. 2000, 69, 217. (ii) Jiang, X.; Wang,
X. Annu. Rev. Biochem. 2004, 73, 87. (iii) Earnshaw, W. C.; Martins, L. M.; Kaufmann, S. H. Annu. Rev.
Biochem. 1999, 68, 383.
116
(i) Evan, G. I.; Vousden, K. H. Nature 2001, 411, 342. (ii) Nicholas, D. W. Nature 2000, 407, 810.
310
receiving specific signals instructing the cells to undergo apoptosis a number of
distinctive changes occur in the cell. Both pathways share the same central effectors,
caspases, which carryout the breakdown or cleavage of both structural and functional
elements of the cell that required for normal cellular function. Caspases are generally
inactive, but typically activated in the early stages of apoptosis through the cleavage of
either caspase 9 (intrinsic) or caspase 8 (extrinsic) upon formation of apoptosome or
activation of death receptors.
117
There are number of ways through which apoptosis can
be induced in cells to mange cancers. One of the ways is to down-regulate the anti-
apoptotic proteins such as survivin, Bcl-2 or over-expression of pro-apoptotic proteins
such as BAD.

Survivin—Survivin is a structurally unique member of the inhibitors of apoptosis (IAP)
family of proteins.
118
Certain IAP proteins including survivin have been shown to
target a downstream step in apoptosis by associating with initiator and effector caspases
and preventing their proteolytic processing and catalytic activity.
4
Survivin was found
abundantly expressed in fetal skin,
119
but undetectable in most normal skin.
120

Interference of survivin expression/function using antisense or a dormant negative
mutant caused spontaneous apoptosis in keratinocytes
6
implicates its role as anti-
apoptotic protein. Although the precise mechanism of survivin in apoptosis is not

117
Hengartner, M. O. Nature 2000, 407, 770.
118
(i) Ambrosini, G.; Adida, C.; Altieri, D. C. Nat. Med. 1997, 3, 917. (ii) Deveraux, Q. L.; Redd, J. C.
Genes Dev. 1999, 13, 239.
119
Adida, C.; Crotty, P. L.; McGrath, J.; Berrebi, D.; Diebold, J.; Altieri, D. C. Am. J. Pathol 1998, 152,
43.
120
Grossman, D.; McNiff, J. M.; Li, F.; Altieri, D. C. Lab. Invest. 1999, 79, 1121.
311
completely elucidated, however, it is very conclusive for its role in inhibiting apoptosis.
In vitro over-expression of survivin has shown to inhibit cell death
121
and in vivo over-
expressing of survivin in transgenic mice resulted the inhibition of apoptosis.
122

Because of survivin’s role in apoptosis, and down-regulation of its expression could be
an interesting of way of combating cancer.
8.2 Regulations of Cell Cycle
The 2001 Nobel Prize in Physiology or Medicine was awarded to Lee Hartwell, Paul
Nurse, and Tim Hunt for their ground-breaking work on cell cycle regulation. Cell
cycle, which is essential for cell proliferation, is a series of well-regulated events that
take place leading to its replication. It has been well know that loss of control of cell
cycle regulation can lead to undesired cell divisions and eventually tumorgenesis.
2,123

Cell cycle consists of four distinct phases—G1 phase, S phase, G2 phase and M phase.
Each phase of cell cycle is briefly described below.
124

G1 phase — This the first phase of within interphase, from the end of the previous M
phase till the beginning of DNA synthesis (S phase). At this stage, a cell increases in
size, synthesizes RNA and proteins to ensure enough material for entering S phase. In
G1 phase, cyclin D is unregulated, which complexes with CDK4 or CDK6, and induces

121
Hoffman, W. H.; Biade, S.; Zilfou, J. T.; Chen, J.; Murphy, M. J. Biol. Chem. 2002, 277, 3247.
122
Grossman, D.; Kim, P. J. Schechner, J. S.; Altieri, D. C. Prod. Natl. Acad. Sci. USA 2001, 98, 635.
123
(i) Kastan, M. B.; Bartek, J. Nature 2004, 432, 316. (ii) Massague, J. Nature 2004, 432, 298. (iii)
Hartwell, L. H.; Kastan, M. B. Science 1994, 266, 1821.
124
(i) Stillman, B. Science 1996, 274, 1659. (ii) Elledge, S. J. Science 1996, 274, 1664. (iii) Murray, A. W.
Nature 1992, 359, 599. (iv) Nasmyth, K. Science 1996, 274, 1643.
312
cyclin E/CDK2 complex in late G1. There is a so called G1 check-point at this stage to
ensure everything is ready for DNA synthesis in S phase.

S phase — This is called the synthetic phase, where the entire genomic DNA of a cell
duplicates, and all the chromosomes are replicated at this phase.
G2 phase — After DNA synthesis is done, a cell enters G2 phase which lasts until the
cell enters M phase. At this stage, cell continues to grow and significant protein
synthesis occurs in preparation for cell division, mainly involving the synthesis of
microtubules, which requires during the M phase. Inhibition of protein synthesis during
G2 phase prevents the cell from undergoing mitosis, hence cell proliferation. A check-
point, G2 check-point, is there to determine the cell is ready to enter M phase.

M phase — Cell enters M phase with all necessary ingredients to grow, and divide.
Once the nuclear division is completed, two identical daughter cells are formed. A
check-point called metaphase check-point is there to ensure the cell is ready for cell
division.

G0 phase — In this phase a cell leaves the cell cycle and quit dividing. This could be a
permanent or a temporary period.
For the regulation of cell cycle, the activities of cyclin dependent kinases (CDKs)
complexes with their regulatory cyclins have to be precisely controlled as shown in
Figure 39. CDKs are key regulators of cell-division cycle. It became clear that cyclins-
313
CDK complexes and other cell cycle regulators are mechanistically involved in the
development of cancer.
125
Potent bioactive and selective small molecule mediated
inhibition of the cell cycle progression remains a principle aim for the development of
novel therapeutics for the treatment of cancer.

Figure 39. Regulation of cell cycle (copied from internet).

125
Jacks, T.; Weinberg, R. A. Science 1998, 280, 1035.
314
8.3 Cycloxygenase 2 (COX-2) and Cancer
Cycloxygenases (COX-1 and COX-2) are the enzymes that catalyze the conversion of
arachidonic acid to pro-inflammatory lipid mediators such as prostaglandins.
126
Two
isoforms, COX-1 and COX-2 are well known. COX-1 believed to be constitutively
express in most tissues, and is important for balancing many physiological processes
such gastric and renal protection, and platelet function. COX-2 is inducible during
inflammation, angiogenesis and neoplasia.
12
For over a period of time, a considerable
research has been done to find its role in cancer development, cancer cell growth and
cell survival. In a seminal paper in the journal Cell, authors showed that the expression
of COX-2 in early mouse intestinal polyps, and knocking out of COX-2 gene
dramatically reduced the number of polyps. These data clearly showed the significant
role of COX-2 in carcinogenesis.
127
A significant amount of research has also been
demonstrated that prostaglandins and other COX-2 generated downstream mediators
which promote cancer cell proliferation and survival in an autocrine and paracrine
manner.
128
Furthermore, different types of human tumors, including colon, pancreatic,
prostate and head and neck cancer, have been shown to express elevated level of COX-
2. Considering the above-mentioned research enhancing COX-2 roles in carcinogenesis.

126
Smith, W. L.; DeWitt, D. L.; Garavito, R. M. Annu. Rev. Biochem. 2000, 69, 145.
127
Oshima, M.; Dinchuk, J. E.; Kargman, S. L.; Oshima, H. et. al. Cell 1996, 87, 803.
128
(i) Hla, T.; Ristimaki, A.; Appelby, S.; Barriocanal, J. G. Ann. N Y Acad. Sci. 1993, 696, 197. (ii)
Dubois, R. N.; Abramson, S. B.; Crofford, L.; Gupta, R. A. Faseb J. 1998, 12, 1063. (iii) Taketo, M. M. J.
Natl. Cancer Inst. 1998, 90, 1529.
315
8.4 COX-2 Inhibitors as Anticancer Agents
A number of COX-2 inhibitors are examined by the National Cancer Institute (NCI) and
others for their potential to prevent to treat cancer. Epidemiological and clinical studies
have been shown that people who regularly take non-steroidal anti-inflammatory drugs
(NSAIDs), such as aspirin and ibuprofen to treat arthritis, have lower rates of
colorectoral cancer and death due to colorectal cancer.
129
Non-selective NSAIDs block
both COX-1 and COX-2, but inhibition of COX-1 induces certain medical problems,
like gastrointestinal effect (GI effect) that occur when NSAIDs are taken regularly for
long periods of time. Therefore, scientists have discovered NSAIDs that block only
COX-2, such as celecoxib (Celebrex®) rofecoxib (Vioxx®) or valdecoxib (Bextra®).
These drugs are second generation NSAIDs with less GI effects, since they are more
selective; do not block COX-1.

Celebrex as an anticancer agent — Celebrex was developed as a selective COX-2
inhibitor by Pfizer, Inc. for the treatment of arthritis. In addition to that it has been most
successful in treating cancer, and is approved by FDA for the treatment of patients with
familial adenomatous polyposis (FAP). Early clinical trials and epidemiologic studies
suggest that celecoxib has both chemotherapeutic and chemopreventative potential.
130

129
Marx, J. Science 2001, 291, 581.
130
(i) Akhmedkhanov, A.; Toniolo, P.; Zeleniuch-Jacquotte, A.; Koenig, K. L.; Shore, R. E. Br. J. Cancer
2002, 87, 49. (ii) Harris, R. E.; Beebe-Donk, J.; Schuller, H. M. Oncol. Rep. 2002, 9, 693. (iii) Moysich,
K. B.; Menezes, A.; Ronsani, A.; Swede, H., Reid, M. E.; Cummings, K. M. BMC cancer 2002, 2, 31. (iv)
Muscat, J. E.; Chen, S. Q.; Richie Jr., J. P.; Altorki, N. K.; Citron, M.; Olson, S. Cancer 2003, 97, 1732.
(v) Phillips, R. K. S.; Wallace, M. H.; Lynch, P. M.; Hawk, E.; Gordon, G. B.; Sunders, B. P. Gut 2002,
50, 857.
316
Although many of the anticancer effects of celecoxib may be related to the inhibition of
COX-2, however, the underlying molecular mechanism of its antineoplastic properties
is poorly understood.
131
There are a number of reports describing the effect of
celecoxib as an anticancer and pro-apoptotic agent without any apparent involvement of
COX-2.
132

The objectives of my research project were to design and synthesize small molecules to
further investigate the COX-2 independent anticancer activities of celecoxib, and to
facilitate the development of novel anticancer agents based on the findings that are
optimized to inhibit the newly identified targets.

8.5 Design and Synthesis of Celebrex Analogs
Celebrex is a COX-2 inhibitor widely used for the treatment of both osteoarthritis and
rheumatoid arthritis, and is a FDA approved drug for the treatment of patients with
familial adenomatous polyposis (FAP). Despite promising results, the underlying
molecular mechanism by which celecoxib exerts its anticancer activity has remained
unclear and somewhat controversial. In order to further investigate the anti-tumor
mechanisms of celecoxib, and to better understand the COX-2’s involvement, we have
design a drug called 2,5-dimethyl celecoxib (DMC), a close structural analog of

131
Gupta, R. A.; Dubois, R. N. Gastroenterology 1998, 114, 1095.
132
(i) Arico, S.; Pattingre, S.; Bauvy, C.; Gane, P.; Barbat, A.; Codogno, P.; Ogier-Denis, E. J. Biol.
Chem. 2002, 277, 27613. (ii) Hanif, R.; Pittas, A.; Feng, Y.; Koutsos, M. I.; Qiao, L.; Staiano-Coico, L.;
Shiff, S. I.; Rigas, B. Biochem. Pharmacol. 1996, 52, 237. (iii) Kardosh, A.; Blumenthal, M.; Wang, W.
J.; Chen, T. C.; Schönthal, A. H. Cancer Biol. Ther. 2004, 3, 9. (iv) Tegeder, I.; Pfeilschifter, J.;
Geisslinger, G. Faseb J. 2001, 15, 2057. (v) Tegeder, I.; Pfeilschifter, J.; Geisslinger, G. Cancer Res.
2000, 60, 6846.
317
celecoxib that lacks the ability to inhibit COX-2. The structure of celecoxib and DMC
is depicted in Figure 40. Celebrex has one methyl group at 4-position of the phenyl ring,
which allows the drug to fit well into the active site of COX-2, and hence block its
catalytic activity, however, when two methyl groups were employed at positions 2 and
5 of the phenyl ring in the analog 2,5-dimethyl celecoxib (DMC) made it too bulky to
fit into the active site of the enzyme, and hence cannot block the COX-2’s catalytic
activity.
133

N
N
H
3
C
S
H
2
N
O
O
CF
3
N
N
S
H
2
N
O
O
CF
3
CH
3
H
3
C
Celecoxib 2,5-Dimehtyl Celecoxib (DMC)
4
2
5

Figure 40. Structure of celecoxib and DMC.

Synthesis of DMC — The biological activity of DMC towards COX-1 and COX-2
enzymes has been reported earlier,
19
and but its synthetic procedure has not been
described in the literature. We have synthesized the drug DMC in our laboratory as
shown in Scheme 33. This is a two steps synthesis started with the enolization of 2,5-

133
Penning, T. D.; Talley, J. J.; Bertenshaw, S. R.; Carter, J. S.; Collins, P. W.; Docter, S.; Graneto, M. J.;
Lee, L. F.; Malecha, J. W.; Miyashiro, J. M.; Rogers, R. S.; Rogier, D. J.; Yu, S. S.; Anderson, G. D.;
Burton, E. G.; Cogbum, J. N.; Gregory, S. A.; Koboldt, C. M.; Perkins, W. E.; Seibert, K.; Veenhuizen, A.
W.; Zhang, Y. Y.; Isakson, P. C. J. Med. Chem. 1997, 40, 1347.
318
dimethyl acetophenone (8.2) with NaOMe in anhydrous MeOH, followed the addition
trifluoroethyl acetate under reflux condition to give the keto-enol (8.3) in excellent yield.
The nucleophilic addition of 4-sulfamoyl phenyl hydrazine.HCl (8.4) to the diketone 8.3
under reflux condition to give the final product 2,5-dimethyl celecoxib (8.1) in excellent
yield (92%). The final product was first purified with a silica column, and then re-
crystallized from minimum amount of EtOAc in hexane. The structure of 2,5-dimethyl
celecoxib was elucidated by extensive NMR (
1
H and
13
C) studies, and its purity was
checked by HPLC. The ultra-pure DMC was then supplied to our collaborator labs at
USC Keck Medical School for its biological studies, which will be discussed later in
this chapter.

319
CH
3
CH
3
CH
3
O CH
3
CH
3
O
CF
3
OH
N N
S
H
2
N O
O
CH
3
H
3
C
CF
3
8.2 8.3
NaOMe, CF
3
CO
2
Et
MeOH, reflux for 24 h
S
O
O
H
2
N
H
N
NH
2
.HCl
EtOH, Reflux
20h
2,5-Dimethyl celecoxib (DMC, 8.1)
8.4

Scheme 33. Synthesis of 2,5-dimethyl celecoxib (DMC).

8.6 Design and Synthesis of Celebrex, DMC and Vioxx Analogs
In order to study the structure-activity relationship of celecoxib, DMC and Vioxx for
their anti-tumor activity, we have designed a number of their analogs as shown in
Figure 41. The structural modifications were done to elucidate the structural basis
underlying the anti-tumor molecular mechanisms of celecoxib, DMC and to optimize
the anticancer activity of DMC. To reach this goal, the sulfonamide (-SO
2
NH
2
)
pharmacophore present in celecoxib and DMC were designed replace by methylsulfonyl
320
(–SO
2
Me) pharmacophore. Vioxx is a COX-2 inhibitor, widely used for the treatment
of arthritis, but doesn’t possess any of celecoxib’s anti-tumor activity.
134
Since Vioxx
has methylsulfonyl (-SO
2
Me) pharmacophore, and has no anticancer activity, we have
decided to introduce methylsulfonyl (-SO
2
Me) to both celecoxib and DMC to see is
there any changes in biological activities. In retrospect, we have designed to
incorporate celecoxib’s methyl sulfonamide (-SO
2
NH
2
) to Vioxx to see similar effect
might exert or not.

N
N
H
3
C
S
H
2
N
O
O
CF
3
N
N
S
H
2
N
O
O
CF
3
CH
3
H
3
C
Celecoxib Dimehtyl Celecoxib (DMC)
O
S
O
O
Me
O
Vioxx
N
N
H
3
C
S
Me
O
O
CF
3
N
N
S
Me
O
O
CF
3
CH
3
H
3
C
Modified Celecoxib Modified DMC
O
S
O
O
H
2
N
O
Modified Vioxx

Figure 41. Structures of Modified Drugs.

134
Kardosh, A.; Wang, W.; Uddin, J.; Petasis, N. A.; Hofman, F. M.; Chen, T. C.; Schönthal, A. H.
Cancer Biol. Therapy 2005, 4, 571.
321
Synthesis of modified celecoxib — The synthesis of sulfonamido functional group
modified celecoxib is similar to the synthesis of celecoxib
19
o DMC.
20
The synthetic
route is depicted in Scheme 34. The synthesis was started with the base-induced
addition of trifluoroethyl acetate to 4-methyl acetophenone (8.5) under reflux condition
for overnight. The addition 4-methylsulfonyl phenyl hydrazine.HCl (8.7) to the
diketone (8.6) under reflux condition yielded the modified celecoxib (8.8) in excellent
yield. The final compound is first purified on a silica column, and the further purified
by re-crystallization from minimum amount of EtOAc in hexane. The structure of
modified celecoxib was elucidated by NMR analysis.

322
CH
3
O O
CF
3
OH
N N
S
Me O
O
CF
3
8.5 8.6
NaOMe, CF
3
CO
2
Et
MeOH, reflux for 24 h
S
Me
H
N
NH
2
.HCl
EtOH, Reflux
20h
Modified celecoxib (8.8)
O
O
H
3
C H
3
C
H
3
C
8.7

Scheme 34. Synthesis of modified celecoxib (8.8).

Synthesis of modified DMC — The modified DMC was synthesized in excellent yield
by following the same procedure as described above for modified celecoxib as shown in
Scheme 35.
323
CH
3
CH
3
CH
3
O CH
3
CH
3
O
CF
3
OH
N N
S
Me O
O
CH
3
H
3
C
CF
3
8.2 8.3
NaOMe, CF
3
CO
2
Et
MeOH, reflux for 24 h
S
Me
H
N
NH
2
.HCl
EtOH, Reflux
20h
Modified DMC (8.10)
O
O
8.9

Scheme 35. Synthesis of modified DMC.

Synthesis of modified Vioxx — The modified Vioxx was synthesized using the reaction
sequence illustrated in Scheme 36.
135
The condensation of phenylacetic acid (8.11)
with phenylacyl bromide (8.12) in presence of triethylamine in MeCN afforded the
phenylacyl phenylacetate 8.13 in excellent yield.
136
The NaH induced intramolecular
cyclization of diketone 8.13 in DMSO yielded the 3,4-diphenyl furanone (8.14) in
excellent yield. The subsequent chlorosulfonation of 8.14 with chlorosulfonic acid in

135
Uddin, J. M.; Rao, P. N.P.; Knaus, E. E. J. Heterocyclic Chem. 2003, 40, 861.
136
Habeeb, A. G.; Rao, P. N. P.; Knaus, E. E. J. Med. Chem. 2001, 44, 3039.
324
chloroform at -5
o
C yielded the single product 8.15. The electrophilic chlorosulfonation
occurs exclusively at the para-position of the C-4 phenyl ring as described in the
literature.
21

OH O O
Br
MeCN, NEt
3
, 1h, rt
O O
O
O
O
NaH, DMSO
1h, rt
O
S
O
O
H
2
N
O
Modified Vioxx (8.16)
ClSO
3
H, -5
o
C
CHCl
3
NH
4
OH, EtOH
O
S
O
O
Cl
O
8.11 8.12 8.13
8.14
8.15

Scheme 36. Synthesis of modified Vioxx (8.16).

The chlorosulfonated product 8.15 was then treated with concentrated ammonium
hydroxide in ethanol to give the sulfonamido Vioxx (8.16) in excellent yield.
325

All three modified drugs were fist purified by column chromatography, and then re-
crystallized from minimum amount of EtOAc in hexane. These drugs were supplied to
our collaborator at USC Keck School of Medicine for their biological studies.

8.7 Anticancer Activities of Celecoxib and DMC
The anti-proliferative effects of celecoxib and DMC were done in our collaborators at
USC Keck School of Medicine. The aim of the project was to study the COX-2’s
involvement in the anti-tumor effects of celecoxib, and to find target(s) other than
COX-2 for its anticancer activities. In order to determine the role of COX-2 in the anti-
tumor effects of celecoxib and other COX-2 inhibitors, our collaborator investigated a
variety of cell lines with varying degree of expression of COX-2. Our collaborators
mainly focused on glioblastoma, lymphoma, drug resistant multiple myeloma. The
results are briefly described below.

8.7.1 COX-2 independent anti-tumor activity of celecoxib on Burkitt’s
lymphoma
137

In order to investigate the involvement COX-2, and to see whether NSAIDs could be
considered for the treatment of Burkitt’s lymphoma, our collaborators determined the
effects of celecoxib, DMC and other NSAIDs on three different Burkitt’s lymphoma
cell lines such as Raji, Ramos and A6876 cells in vitro. The Raji and Ramos lymphoma

137
Kardosh, A.; Wang, W.; Uddin, J.; Petasis, N. A.; Chen, T. C.; Schönthal, A. H. Cancer Biol. &
Therapy 2005, 4, 571.
326
cells were incubated with increasing concentration of celecoxib and DMC. Cell
proliferation were measured using an MTT assay. As shown in Figure 42, DMC,
although lacks COX-2 inhibitory function, but potently mimics the anti-proliferative
properties of celecoxib in vitro in Raji and Ramos cell lines.
23

Figure 42. Cell proliferation in the presence of celecoxib and DMC on Raji and Ramos
cell lines. Copied from Kardosh et. al. Cancer Biol. & Therapy 2005, 4, 571.

The in vivo anti-proliferative effects of celecoxib and DMC were also investigated by
our collaborator. To test the in vivo effects, nude mice were injected subcutaneously
with Raji lymphoma cells, and treated with celecoxib and DMC. As shown in Figure
43, tumor growth was significantly reduced in mice treated with either celecoxib or
DMC with their chow. Both celecoxib and DMC were equally effective in suppressing
tumor growth. These results demonstrate both celecoxib and DMC are able to
327
suppress tumor growth in experimental animals, thus confirming that COX-2 inhibitory
function is not required for their anti-tumor property.
137

Figure 43. Tumor formation in animals treated with celecoxib or DMC.
Copied from Kardosh et. al. Cancer Biol. & Therapy 2005, 4, 571.

Celecoxib and DMC down-regulate cell cycle activity
137
— As described above, DMC
potently mimics the anti-tumor activity of celecoxib both in vitro and in vivo, which
excludes any involvement of COX-2 for their anti-tumor activity. In order to determine
the mechanisms by which these two drugs exert their anti-tumor property, our
collaborator investigated the activity of cyclin dependent kinases (CDKs), which are the
important regulator of cell cycle. As shown in Figure 44, both celecoxib and DMC
effectively down-regulated the expression of cyclin A and cyclin B, important
components of CDKs, which are absolutely required for cell proliferation to occur.
328
Without these cyclins, the enzymatic activity of CDKs was lost, which caused the cells
to stop proliferation.

Figure 44. Cyclin A and cyclin B expression in tumor tissue in vivo.
Copied from Kardosh et. al. Cancer Biol. & Therapy 2005, 4, 571.

By performing a combination of in vitro and vivo studies with Burkitt’s lymphoma cells,
our collaborator demonstrated that inhibition of COX-2 is not necessary at all since
DMC, a non-COX-2 drug, potently mimics all of the anti-proliferative and anti-
tumorigenic properties of the COX-2 inhibitor, celecoxib. Our collaborator showed that
DMC actually effectively inhibits cell proliferation through the down-regulation of
cyclins A and B and ensuing loss of CDKs activity.

329
8.7.2 DMC as a promising drug for multiple myeloma
138

Multiple myeloma (MM) is a cancer of plasma cells, which are immune system cells in
bone marrow to help fight infection and diseases. MM is an incurable cancer, which is
because MM patients frequently develop drug-resistant disease, and ultimately succumb
to death. Our collaborator led by Dr. Schönthal at USC Keck Medical School
hypothesized that celecoxib and DMC could be effective for the treatment of patients
with MM. In fact, it was found that both celecoxib and DMC can inhibit the
proliferation and induce apoptosis even in highly drug-resistant MM cells.
138

Both DMC and celecoxib inhibit cell growth and induced apoptosis via inhibition of
several different components of mitogenic and survival pathways such as STAT3, MAP
kinase kinase (MEK), survivin, NF-kB, and various cyclins, but were independent of
COX-2.
138

10.8 Conclusion
In summary, we have accomplished the synthesis of a number of celecoxib analogs for
testing the COX-2 involvement in anticancer activity of celecoxib, and to find a better
drug for the treatment of different types of cancers. We have found that DMC—despite
lacks COX-2 inhibitory function—is able to mimic essentially all celecoxib’s anticancer
effects, means that COX-2 is not involve for their anticancer activity. Another
important aspect of our study was— as we know prolonged exposure to high dosages of

138
Kardosh, A.; Soriano, N.; Liu, Y-T.; Uddin, J.; Petasis, N. A.; Hofman, F. M.; Chen, T. C.; Schönthal,
A. H. Blood 2005, 106, 4330.
330
COX-2 inhibitors such as Celebrex, Vioxx and Bextra could lead to life-threatening
cardiovascular side-effects. Since DMC potently mimics the anti-tumor activity of
celecoxib, and is not a COX-2 inhibitor, therefore it would be a far better drug for the
treatment of different types of cancers such for the treatment of patients with multiple
myeloma without having any cardiovascular side-effects. The underlying molecular
mechanisms for the anticancer properties of DMC (and celecoxib), and further
biological studies towards identifying the molecular targets for these drugs are currently
underway in our collaborator’s lab at USC Keck Medical School.
331
8.9 Experimental
8.9.1 Synthesis of DMC
Step 1. Synthesis of 1-(2,5-dimethylphenyl)-4,4,4-trifluorobutane-1,3-dione. 2,5-
Dimethylacetophenone (2.00 g, 13.54 mmol) was dissolved in 15.0 mL of dry MeOH,
and then freshly prepared NaOMe (1.54 g, 28.33 mmol) in MeOH was added. The
mixture was stirred for 5 min, and then ethyl trifluoroacetate (2.90 mL, 24.29 mmol)
was added. After refluxing for 24h, the mixture was cooled at room temperature. 10%
HCl (20 mL) was added to the mixture, and then extracted with EtOAc (3 x 20 mL),
washed with brine and dried over MgSO
4
. The organic extract was purified by column
chromatography using 5% EtOAc/hexane to give the tile compound (3.10 g, 91%);
1
H
NMR (CDCl
3
, 400 MHz) δ 7.40 (s, 1H), 7.25 (d, J = 8.0 Hz, 1H), 7.20 (d, J = 8.0 Hz,,
1H), 6.35 (s, 1H), 2.51 (s, 3H), 2.38 (s, 3H);
13
C NMR (CDCl
3
, 100 MHz) δ 191.5,
175.5 (q, J
C-F
= 36.0 Hz), 136.0, 135.4, 133.7, 133.4, 132.2, 129.6, 117.5 (q, J
C-F
=
285.0 Hz), 96.4, 20.9, 20.7.

Step 2. Synthesis of 4-[5-(2,5-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-
benzenesulfonamide (DMC). (4-Sulfamoylphenyl)hydrazine.HCl (2.05 g, 9.17 mmol)
was added to a stirred solution of the compound of step 1 (1.60 g, 6.55 mmol) in 30 mL
of EtOH. The mixture was heated to reflux and stirred for 20 h. After cooling at room
temperature, the mixture was concentrated in vacuo. The residue was dissolved in
EtOAc, washed with H
2
O (20 mL x 2), brine, and dried over MgSO
4
. The EtOAc
extract was concentrated and purified by column chromatography using 30%
332
EtOAc/hexane as the eluent to give DMC (2.10 g, 92%);
1
H NMR (CDCl
3
, 400 MHz)
δ 7.77 (dd, J = 8.8, 2.4 Hz, 2H), 7.38 (dd, J = 8.8, 2.4 Hz, 2H), 7.16 (d, J = 8.0 Hz, 1H),
7.09 (s, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.66 (s, 1H), 5.46 (br.s, 2H), 2.30 (s, 3H), 1.89 (s,
3H);
13
C NMR (CDCl
3
, 100 MHz) δ 144.9, 144.2, 143.8, 142.7, 141.1, 136.3, 133.9,
131.2, 131.0, 130.9, 128.6, 127.6, 124.0, 122.6, 119.9, 107.5, 21.0, 19.4.

8.9.2 Synthesis of Modified DMC
The first step of the synthesis of DMC and modified DMC are same. In second step (4-
Sulfonyl methyl phenyl)hydrazine.HCl used instead of (4-
Sulfamoylphenyl)hydrazine.HCl. The reaction mixture was reflux in EtOH. The
reaction mixture was cooled to room temperature, EtOH was evaporated, and added
H
2
O (20 mL), and then extracted with EtOAc. The crude product was purified on a
silica column using 25% EtOAc/hexane to give final 4-sulfonyl methyl DMC in
excellent yield (63% in two steps). The final product was further purified by re-
crystallized from a minimum quantities of EtOAc in hexane.
1
H-NMR (400 MHz,
CDCl
3
) δ
H
7.83 (d, J = 8.8 Hz, 2H), 7.46 (d, J = 8.8 Hz, 2H), 7.17-7.08 (m, 3H), 6.65 (s,
1H), 3.00 (s, 3H), 2.30 (s, 3H), 1.88 (s, 3H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
144.7,
144.0 (d, J
C-F
= 38.3 Hz), 143.5, 139.2, 136.1, 133.6, 131.0, 130.8, 130.6, 128.4, 123.8,
120.9 (q, J
C-F
= 267.5 Hz), 107.4, 44.3, 20.7, 19.2.

333
8.9.3 Synthesis of Modified Celebrex
The synthesis of modified Celebrex is same as the synthesis of modified DMC. Instead
of using 2,5-dimethyl acetophenone, the 4-methyl acetophenone was used. The final
product was purified on a silica column, and then re-crystallized from minimum amount
of EtOAc in hexane.
1
H-NMR (400 MHz, CDCl
3
) δ
H
7.91 (d, J = 8.8 Hz, 2H), 7.51 (d,
J = 9.2 Hz, 2H), 7.16 (d, J = 8.0 Hz, 2H), 7.10 (d, J = 8.0 Hz, 2H), 6.73 (s, 3H), 3.04 (s,
3H), 2.36 (s, 3H);
13
C-NMR (100 MHz, CDCl
3
) δ
C
145.3, 144.2 (q, J
C-F
= 38.5 Hz)
139.9, 139.7, 129.8, 128.7, 128.5, 125.6, 125.6, 125.0, 120.9 (q, J
C-F
= 267.7 Hz), 106.4,
44.4, 21.3.

8.9.4 Synthesis of Modified Vioxx

O O
O
8.13

Step 1. Synthesis of phenacyl phenylacetate (8.13). Phenylacyl bromide (8.12, 7.3 g,
36.7 mmol) was added slowly to a solution of 4-methylphenylacetic acid (8.11, 5.0 g,
36.7 mmol) in acetonitrile (100 mL) containing Et
3
N (35.8 mL, 256.9 mmol) at room
temperature. The reaction mixture was stirred for an hour at room temperature. After
an hour, the solvent was removed in vacuo, and then 30 mL was added to the flask. The
aqueous layer was extracted with EtOAc (30 mL x 3). The combined ethyl acetate
extract was washed with HCl (dilute), and washed again with brine, and dried over
334
MgSO
4
. The crude product was purified on a silica column using 20% EtOAc/hexane
to give pure product 8.13 as white solid (6.60 g, 70%).
1
H-NMR (400 MHz, CDCl
3
)
was perfect.

O
O
8.14

Step 2. Synthesis of 3,4-diphenyl-2(5H)furanone (8.14).
A solution of phenylacyl phenylacetate (8.13, 6.6 g, 26.0 mmol) in dimethyl sulfoxide
(10 mL) was added drop wise to a stirred suspension of sodium hydride (1.6 g, 69.0
mmol) in dimethyl sulfoxide (10 mL) at 0 oC (Caution! Exothermic). The reaction
mixture was stirred for an hour at room temperature. After stirring an hour, H
2
O (20
mL) was added to the reaction mixture (Caution! Very exothermic!!), and then
extracted the crude product with EtOAc (25 ml x 4). The combined extracts were
washed with water, the dried over MgSO4, and concentrated in vacuo. The crude
product was purified on a silica column using 20% EtOAc/hexane to give the tile
compound 8.14 as white solid (4.0 g, 70%).
1
H-NMR (400 MHz, CDCl
3
) was perfect.

335
O
S
O
O
H
2
N
O
Modified Vioxx (8.16)

Step 3 and 4. Synthesis of modified Vioxx (8.16).
Step 3—Chlorosulfonation: Chlorosulfonic acid (1.5 mL, 22.5 mmol) was added drop-
wise to a solution of 3,4-diphenyl-2(5H)furanone (8.14, 0.59 g, 2.5 mmol) in
chloroform (1.5 mL) at -5 oC with vigorous stirring. After removing the cooling bath,
the reaction mixture was allowed to stir for an hour at room temperature. After an hour,
the reaction mixture was poured into crushed ice (15 g) drop by drop. The mixture was
extracted with EtOAc (25 mL x 3), and then washed with water, and then dried over
MgSO4. The EtOAc extract was concentrated to give the corresponding mono sulfonyl
chloride (8.15) as a brown-syrup. Concentrated ammonium hydroxide (8 mL) was
added to a solution of the sulfonyl chloride (8.15) in 95% EtOH (5 mL). The reaction
mixture was stirred for an hour at room temperature. After an hour, the solvent was
removed in vacuo, added H
2
O (15 mL) to it, and then extracted with EtOAc (15 mL x
3). The combined EtOAc extracts washed with H
2
O, dried over MgSO
4
and then
concentrated to give a crude product. The crude was then purified on silica column
using 50% EtOAc/hexane to give the pure final product (8.16), which was then further
purified by re-crystallization from minimum amount EtOAc-MeOH and excess amount
of hexane to give pure modified Vioxx (8.16).
1
H-NMR (400 MHz, CDCl
3
) δ
H
7.85 (d,
J = 8.0 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 7.45-7.36 (m, 5H), 5.42 (s, 2H), 3.34 (s, 3H).
336
0.97 1.01 1.02
0.95
2.93
3.00
7 6 5 4 3 2 PPM

1
H-NMR (400 MHz, CDCl
3
) of 8.3.

200 150 100 50 0 PPM

13
C-NMR (100 MHz, CDCl
3
) of 8.3.

CH
3
CH
3
O
CF
3
OH
8.3
337
1.99 2.00
0.99
1.96
0.93
1.91
3.04 3.00
8 7 6 5 4 3 2 PPM

1
H-NMR (400 MHz, CDCl
3
) of 8.1.

140 120 100 80 60 40 20 PPM

13
C-NMR (100 MHz, CDCl
3
) of 8.1.
N N
S
H
2
N O
O
CH
3
H
3
C
CF
3
8.1
338
1.86 1.87 1.88 1.92
0.87
3.02 3.03
8 7 6 5 4 3 2 PPM

1
H-NMR (400 MHz, CDCl
3
) of 8.8.

140 120 100 80 60 40 20 PPM

13
C-NMR (100 MHz, CDCl
3
) of 8.8.
N N
S
H
3
C O
O
CF
3
H
3
C
8.8
339

1.96 1.98
0.96
1.96
0.90
3.11
3.04 3.00
8 7 6 5 4 3 2 1 PPM

1
H-NMR (400 MHz, CDCl
3
) of 8.10.

140 120 100 80 60 40 20 PPM

13
C-NMR (100 MHz, CDCl
3
) of 8.10.

N N
S
H
3
C O
O
CH
3
H
3
C
CF
3
8.10
340
2.00 2.06
7.27
2.11
8 7 6 5 4 3 PPM

1
H-NMR (400 MHz, CDCl
3
) of 8.16.

2.00
4.90
2.16
0.86
2.02
8 7 6 5 4 3 2 PPM

1
H-NMR (400 MHz, CDCl
3
) of UMC.

O
S
O
O
H
2
N
O
8.16
N N
S
H
2
N O
O
CF
3
UMC
341
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Abstract (if available)

Abstract This dissertation deals with two distinct projects. First -- Design and synthesis of novel anti-inflammatory lipid mediators -- It is well documented that omega-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) display beneficial actions in many human diseases. The underlying molecular mechanism for these actions remains of tremendous interest, but yet to be established until recently Serhan and colleagues identified a novel class of lipid mediators generated from EPA and DHA during the resolution phase of inflammation via cell-cell interactions that displayed potent anti-inflammatory, pro-resolving activities. The lipid mediators derived from EPA are designated as E-series of resolvins such as RvE1, and the lipid mediators generated from DHA are denoted as D-series of resolvins such as RvD1, RvD2, RvD3 and RvD4. In addition, aspirin triggers the endogenous formation of epimeric series of D-series of resolvins named aspirin-triggered resolvins such as AT-RvD1, AT-RvD2, AT-RvD3 and AT-RvD4. These lipid mediators are generated in very minute quantities in stereochemically pure form and possess potent anti-inflammatory, pro-resolving bioactions.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Uddin, Jasim (author)

Core Title Design and synthesis of novel anti-inflammatory lipid mediators and anticancer small molecules

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 04/29/2008

Defense Date 12/12/2007

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag anticancer,inflammation,lipid mediators,OAI-PMH Harvest,small molecules,total sythesis

Language English

Advisor Petasis, Nicos A. (committee chair), Bau, Robert (committee member), Periana, Roy A. (committee member), Schönthal, Axel H. (committee member)

Creator Email muddin@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m1211

Unique identifier UC159631

Identifier etd-Uddin-20080429 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-78217 (legacy record id),usctheses-m1211 (legacy record id)

Legacy Identifier etd-Uddin-20080429.pdf

Dmrecord 78217

Document Type Dissertation

Rights Uddin, Jasim

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu