Synthetic Studies Towards Analogs of Protectin D1

Synthetic Studies Towards Analogs Of Protectin D1

Thesis for the degree Master of Pharmacy

Mai El-khatib

School of Pharmacy Faculty of Mathematics and Natural Sciences

UNIVERSITY OF OSLO 2016

Synthetic Studies Towards Analogs Of Protectin D1

Thesis for the degree Master of Pharmacy

Mai El-khatib

School of Pharmacy Faculty of Mathematics and Natural Sciences

UNIVERSITY OF OSLO 2016

”The secret of life, though, is to fall seven times and to get up eight times.”

- Paulo Coelho

III

IV Acknowledgements

The work presented in this master’s thesis has been undertaken between June 2015 and June 2016 at the school of pharmacy, department of pharmaceutical chemistry, University of Oslo.

First of all, I would like to thank my supervisor Professor Trond Vidar Hansen for his continuous support and motivation throughout the whole year. I am grateful for the inspiring conversations and the suggestions we both exchanged. Your brilliance and love for chemistry has been a great source of motivation.

My sincere gratitude also goes to Associate Professor Anders Vik who has also been motivating me and providing me with insightful comments and encouragement. Without his support I would not be able to conduct this research.

I also wish to acknowledge both Dr. Jørn Tungen and Dr. Marius Aursnes who have managed to make me the lab-nerd I am today and also for sharing their expertise. It was a pleasure working and laughing with you. You always made my day. Thank you.

I would also like to express my dearest gratitude to Mats Wilhelmsen for the continuous support and for the motivation throughout this thesis.

Last but not least, I want to thank my friends and family, especially my parents, Mai and Hisham El-khatib for their love, caring, continuous spiritually support and the encouragement they have given me through my whole life.

This thesis is dedicated to my mom. Our dream is now fulfilled.

Mai El-khatib

Oslo, May 2016

V Abstract

Resolution of the inflammatory process by oxygenated derivatives of eicosapentaenoic acid, docosahexaenoic acid and n-3 docosapentaenoic acid, is an important field of study. It has led to the discovery of certain derivates of the fatty acids, namely specialized pro-resolving mediators. Their ability to induce the resolution of an active inflammatory process and thus prevent several connected diseases is increasingly becoming acknowledged.

Protectin D1 is a potent anti-inflammatory and pro-resolving derivative from docosahexaenoic acid. However, protectin D1 is metabolized rapidly in vivo once biosynthesized. The synthesis of new analogs may result in novel, related compounds. These may in turn prove to be beneficial having structural features, which render them less prone to rapid metabolism, and thus extending the wanted biological activities of the parent, specialized pro-resolving mediator.

The synthesis of two hitherto unknown fatty acid analogs discussed in this thesis is based on the initial synthesis of the three main fragments of each compound. Both analogs are then to be assembled by combining an alpha-, omega- and middle fragment - the two latter building blocks being identical in all cases. In this thesis, these four needed fragments have been successfully synthesized and are ready for further assembly. Future work includes assembly of said fragments into the two wanted protectin D1-analogs and screening for any potential biological effects.

VI Graphical Abstract

The omega-fragment

O OTES OH

The middle fragment

TBSO O S TBSO

Br N S Br O

Alpha fragments

CO2H IPh3P CO2Me

O HO O OH BrPh3P O

O HO O OH IPh3P O

VII Potential assembly of the fragments

TBSO

Br O OTBS + Br CO2Me

IPh3P CO2Me

OTBS OTES

Br CO2Me +

OH OH CO2H

TBSO

Br O OTBS O + O Br O O O IPh3P O

OTBS O OTES O Br O +

OH OH O CO H 2

VIII Abbreviations

AA Arachidonic acid

ALA Alpha-linolenic acid

BLT1 Leukotriene B4 Receptor

ChemR23 Chemokine like receptor 1

COX Cyclooxygenase enzyme

COX-1 Cyclooxygenase-1

COX-2 Cyclooxygenase-2

CSA Camphorsulfonic acid

DDQ 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone

DHA Docosahexaenoic acid

DIBAL-H Diisobutylaluminum hydride

DMAP 4-Dimethylaminopyridine

DMP Dess-Martin periodinane

DMSO Dimethyl sulfoxide

EPA Eicosapentaenenoic acid

GPCR G protein-coupled receptor

HDHA Hydroxydocosahexaenoic acid

HETE Hydroxyeicosatetraenoic acid

HMPA Hexamethylphosphoramide

HpDHA Hydroperoxydocosahexaenoic acid

HpDPA Hydroxyperoxydocosapentaenoic acid

HpETE Hydroperoxyeicosatetraenoic acid

HRMS High-resolution mass spectrometry

LDA Lithium diisopropylamide

LOX Lipooxygenase enzyme

IX LTB4 Leukotriene B4

LXA4 Lipoxin A4

LXB4 Lipoxin B4

MaR Maresin

MRM Multiple reaction monitoring n-3 DPA n-3 docosapentaenoic acid

NaHMDS Sodium bis(trimethylsilyl)amide

NSAID Nonsteroidal anti-inflammatory drug

PCC Pyridinium chlorochromate

PD1 Protectin D1

PGE2 Prostaglandin E2

PMN Polymorph nuclear neutrophil

PUFA Polyunsaturated fatty acid

RedAl® Sodium bis(2-methoxyethoxy)aluminum hydride RvD Resolvin of the D-series

RvE Resolvin of the E-series

Sn(OTf)2 Tin(II) trifluoromethanesulfonate

SPM Specialized pro-resolving lipid mediator

TBAF Tetrabutylammonium fluoride

TBDPSCl tert-Butyl(chloro)diphenylsilane

TBS tert-Butyldimethylsilyl

TBSOTf tert-Butyldimethylsilyl trifluoromethanesulfonate

TES Triethylsilyl

VLDL Very Low Density Lipoprotein

X Table of Contents Acknowledgements………………………………………………………………………..….V Abstract………………………………………………………………………………………VI Graphical abstract…………………………………………………………………………...VII Abbreviations………………………………………………………………………………...IX 1 Introduction ...... 1 1.1 ω-3 Polyunsaturated fatty acids and health benefits ...... 1 1.2 Inflammation ...... 2 1.2.1 Acute inflammation ...... 2 1.2.2 Chronic inflammation ...... 3 1.3 Enzymes involved ...... 4 1.3.1 Lipoxygenases ...... 4 1.3.2 Cyclooxygenases ...... 5 1.4 Specialized pro-resolving mediators ...... 5 1.4.1 Lipoxins ...... 7 1.4.2 E-series Resolvins ...... 8 1.4.3 D-series Resolvins ...... 9 1.4.4 Maresins ...... 11 1.4.5 SPMs derived from n-3 DPA ...... 12 1.5 Protectin D1 ...... 14 1.5.1 Isolation and structure elucidation ...... 15 1.5.2 Further biosynthesis and metabolism ...... 15 1.5.3 Syntheses of Protectin D1 ...... 18 1.6 Synthetic methods ...... 28 1.6.1 Z-selective Wittig reaction ...... 28 1.6.2 Sonogashira coupling reaction ...... 29 1.6.3 Evans-Nagao stereoselective aldol reaction ...... 30 1.7 Aim of study ...... 31 1.7.1 Retrosynthetic analysis of analog 94 ...... 32 1.7.2 Retrosynthetic analysis of analog 95 ...... 33 2 Results and discussion ...... 35 2.1 Synthesis of the ω-fragment 91 ...... 35

2.1.1 Synthesis of 3-methylpent-4-yne-1,3-diol (98) ...... 36 2.1.2 Synthesis of 3,3,9,9-tetraethyl-5-ethynyl-5-methyl-4,8-dioxa-3,9-disilaundecane (99)……...... 37 2.1.3 Synthesis of 3-methyl-3-((triethylsilyl)oxy)pent-4-ynal (100) ...... 38 2.1.4 Synthesis of (Z)-triethyl((3-methylhex-4-en-1-yn-3-yl)oxy)silane (91) ...... 40 2.2 Synthesis of the α –fragment 67 ...... 42 2.2.1 Synthesis of methyl 7-hydroxyhept-4-ynoate (84) ...... 43 2.2.2 Synthesis of methyl (Z)-7-hydroxyhept-4-enoate (85) ...... 44 2.2.3 Synthesis of methyl (Z)-7-(iodotriphenyl-λ5-phosphanyl)hept-4-enoate (67) ..... 46 2.3 Synthesis of the α-fragment 96 ...... 47 2.3.1 Synthesis of tert-butyl 2-(4-hydroxybutoxy)acetate (106) ...... 48 2.3.2 Synthesis of tert-butyl 2-(4-(bromotriphenyl-λ5-phosphanyl)butoxy)acetate (97)……...... 49 2.3.3 Synthesis of tert-butyl 2-(4-(iodotriphenyl-λ5-phosphanyl)butoxy)acetate (96)…… ...... 51 2.4 Synthesis of the middle-fragment 73 and Wittig reaction ...... 53 2.4.1 Synthesis of (R,4E,6E)-7-bromo-3-((tert-butyldimethylsilyl)oxy)-1-((S)-5- isopropyl-2-thioxoimidazolidin-1-yl)hepta-4,6-dien-1-one (73) ...... 54 2.4.2 Attempted Wittig coupling reaction ...... 55 2.4.3 Attempted synthesis of methyl (R,4Z,7Z,11E,13E)-14-bromo-10-((tert- butyldimethylsilyl)oxy)tetradeca-4,7,11,13-tetraenoate (87) ...... 56 3 Summary and future work ...... 59 4 Conclusion ...... 61 5 Experimental chemistry ...... 63 5.1 General ...... 63 5.2 Synthesis of compounds ...... 63 5.2.1 Synthesis of 3-methylpent-4-yne-1,3-diol (98) ...... 63 5.2.2 Synthesis of 3,3,9,9-tetraethyl-5-ethynyl-5-methyl-4,8-dioxa-3,9-disilaundecane (99)…… ...... 64 5.2.3 Synthesis of 3-methyl-3-((triethylsilyl)oxy)pent-4-ynal (100) ...... 65 5.2.4 Synthesis of (Z)-triethyl((3-methylhex-4-en-1-yn-3-yl)oxy)silane (91) ...... 66 5.2.5 Synthesis of methyl 7-hydroxyhept-4-ynoate (84) ...... 67 5.2.6 Synthesis of methyl (Z)-7-hydroxyhept-4-enoate (85) ...... 68 5.2.7 Synthesis of methyl (Z)-7-(iodotriphenyl-λ5-phosphanyl)hept-4-enoate (67) ..... 68

5.2.8 Synthesis of (R,4E,6E)-7-bromo-3-((tert-butyldimethylsilyl)oxy)-1-((S)-5- isopropyl-2-thioxoimidazolidin-1-yl)hepta-4,6-dien-1-one (73) ...... 69 5.2.9 Synthesis of methyl (R,4Z,7Z,11E,13E)-14-bromo-10-((tert- butyldimethylsilyl)oxy)tetradeca-4,7,11,13-tetraenoate (87) ...... 70 5.2.10 Second attempt of the synthesis of methyl (R,4Z,7Z,11E,13E)-14-bromo-10- ((tert-butyldimethylsilyl)oxy)tetradeca-4,7,11,13-tetraenoate (87) ...... 71 5.2.11 Synthesis of tert-butyl 2-(4-hydroxybutoxy)acetate (106) ...... 71 5.2.12 Synthesis of tert-butyl 2-(4-(bromotriphenyl-λ5-phosphanyl)butoxy)acetate (97)……...... 72 5.2.13 Synthesis of tert-butyl 2-(4-(iodotriphenyl-λ5-phosphanyl)butoxy)acetate (96)…… ...... 73 6 References ...... 75 7 Appendix ...... 79 7.1 1H-NMR and 13C-NMR Spectra ...... 79 7.2 MS- and HRMS Characterization ...... 92 7.3 GC Characterization ...... 93

1 Introduction

1.1 ω-3 Polyunsaturated fatty acids and health benefits

The beneficial effects from the intake of essential omega-3 fatty acids has been well established over the past 50 years.1,2 These positive health effects include, but are not limited to, prevention of cardiovascular, neurodegenerative and autoimmune diseases.2,3 The marketed drug Omacor® contains the omega-3 ethyl ester of both eicosapentaenoic acid (1, EPA 20:5) and docosahexaenoic acid (2, DHA 22:6), in 380 mg and 460 mg amount of each fatty acid, respectively.4,5 Since the drug is formulated as ethyl esters, the result is therefore a sustained intestinal absorbance, which is considered beneficial.4 Omacor® reduces the biosynthesis of triglycerides and hence lowers the triglyceride levels in the liver, due to the inhibition of very low-density lipoprotein (VLDL). The risk of developing ischemic heart disease is therefore reduced.5

Although scientists have been aware of the benefits from these fatty acids, the exact cellular and molecular explanation has until recently remained obsolete. Recently, it has been demonstrated that ω-3 polyunsaturated fatty acids (PUFAs) such as EPA (1), DHA (2), arachidonic acid (3, AA 20:4) and n-3 docosapentaenoic acid (4, n-3 DPA 22:5) are precursors for pro-resolving lipid mediators, including resolvins, protectins, maresins and lipoxins.2,6 The pro-resolving lipid mediators are produced in small amounts in the human body to fulfill their requirements, and their precursors need to be obtained in adequate amounts through dietary supplements.7 This matter will be discussed later on.

CO2H CO2H

EPA (1) DHA (2)

CO H CO2H 2

AA (3) n-3 DPA (4)

Figure 1.1 Structures of EPA (1), DHA (2), AA (3) n-3 DPA (4).8

1 1.2 Inflammation

The therapeutic targeting of the inflammatory response is an important field of study.3,9 The inflammation process is part of the human defense system and the immune system’s response against foreign pathogens and tissue damage.3 However, the primary purpose of inflammation is to restore homeostasis and hence constitute a protective biological response to injuries.10 An uncontrolled inflammation process is a unifying component in many chronic diseases including atherosclerosis, asthma, rheumatoid arthritis and type 2 diabetes.11 Inflammation may be divided into acute inflammation and chronic inflammation.

Figure 1.2 An overview illustrating the duration of the inflammatory response and the resolution process. A small selection of the specialized pro-resolving mediators (SPMs) and their functions are also shown. Notice that the maresins and n-3 DPA SPMs are missing. These will be discussed below.3

1.2.1 Acute inflammation

In the early stages of an inflammatory response, the resident cells (tissue macrophage, dendritic cells and epithelial cells) start producing inflammatory mediators, which subsequently increase vessel permeability, vasodilation, blood flow, edema and the recruitment of leukocytes.3,10 Polymorphonuclear leukocytes (PMNs) are the first cells which extravasate into inflamed tissue, and are an important host for the production of the inflammatory mediators, including cytokines, chemokines, lipid mediators and growth

2 factors. An injurious stimulus will trigger the above-mentioned biochemical responses in the body, causing the classic signs of acute inflammation, i.e. swelling, redness, heat and pain.10 If resolution of the underlying initiating factor is not achieved, the acute inflammation may develop into a chronic state of inflammation.6,12

Figure 1.3 Initiation of inflammation stimulates the production of pro-inflammatory lipid mediators, such as

PGE2 (5) and LTB4 (6). When the resolution process starts, lipoxin B4 (7, LXB4), protectin D1 (8, PD1) and resolvin D1 (9, RvD1) among others, are produced.13

1.2.2 Chronic inflammation

Chronic inflammation is defined by the accumulation of lymphocytes, macrophages and plasma cells in tissues, which is collectively called the tissue response.12 The vascular response involves transmigration of lymphocytes and monocytes, and leakage of antibodies into the affected tissue. Monocytes “mature” into macrophages, which produces pro- inflammatory mediators.12 These mediators can regulate the production of collagen in tissues by the activation of fibroblasts. Another interesting effect of these pro-inflammatory mediators is the activation of other macrophages and lymphocytes, which further on perpetuates the inflammatory response.3,12 Chronic inflammatory processes can develop into cardiovascular diseases, rheumatoid arthritis, periodontal diseases, Alzheimer’s disease, asthma and diabetes.3

Figure 1.4 After the initiation of inflammation, there are different pathways such as resolution, abscess formation, wound healing or scarring and chronic inflammation.14

1.3 Enzymes involved

There are several types of enzymes involved in the biosynthesis of novel lipid mediators participating in the resolution of the inflammatory process. The two main groups of enzymes to be discussed further are lipoxygenases (LOX) and cyclooxygenases (COX).

1.3.1 Lipoxygenases

These di-oxygenated groups of enzymes play an essential role in the regulation of inflammation. They catalyze the formation of hydroperoxides from polyunsaturated fatty acids, which further on can be hydrolyzed enzymatically to pro-resolving mediators, such as lipoxins as well as pro-inflammatory compounds, such as leukotrienes.3 LOXs catalyzes four elementary reactions, herein oxygen insertion, peroxy radical reduction, hydrogen abstraction and radical rearrangement. Animal LOX-isoforms are classified based on the position in which they oxidize AA (3), hence giving rise to 5-LOXs, 8-LOXs, 11-LOXs, 12-LOXs and 15-LOXs.15 The major isoform groups found in humans are 5-LOX, 12-LOX and 15-LOX and their phenotypes.16 15-LOX plays an essential role in the promotion of atherosclerosis.16 5-LOX plays an important role in the regulation of asthma since it catalyzes the production of an intermediate, 5-HPETE, which can be converted into pro-inflammatory leukotrienes that

4 have potent bronchoconstrictive effects.16,17 With this in mind, a 5-LOX inhibitor named Zileuton has been made which suppresses the leukotrienes synthesis and causes bronchodilation. Zileuton is therefore used in the treatment of asthma.18 12-LOX can catalyze the biosynthesis of lipoxin A4 (10, LXA4) from AA (3) through the intermediate 15(S)-HETE.

LXA4 (10) enhances the resolution process and prevents the development of a prolonged state of inflammation.3

1.3.2 Cyclooxygenases

The COX pathway can produce both pro- and anti-inflammatory mediators from AA (3), including prostaglandins, prostacyclins and thromboxanes.6,19 COX exists in two isomers, namely COX-1 and COX-2.19 COX-1 produces anti-inflammatory mediators, which maintains vascular homeostasis, renal and gastrointestinal blood flow. Nevertheless, it can also produce mediators that play a severe role in the maintenance of platelet function and antithrombogenesis.6,19 On the other hand, COX-2 produces pro-inflammatory mediators which are involved in fever, pain, uterine contractions and ovulation, to mention some.19 Inhibition of COX-2 by the usage of NSAIDs or glucocorticoids, has been an interesting field of study for the treatment of inflammation.19 SPMs and related analogs thereof have shown beneficial anti-inflammatory effects and may develop into a new potential group of drug candidates to treat inflammation.13

1.4 Specialized pro-resolving mediators

The highly coordinated biochemical and metabolic processes which are collectively referred to as the resolution stage, starts after a few hours of the initiation of the inflammatory response, as shown in Figure 1.2. During the resolution stage, the production of the anti- inflammatory lipid mediators derived from AA (3) and ω-3 polyunsaturated fatty acids, are switched on. However, the production of the pro-inflammatory mediators, such as prostaglandins and leukotrienes, are switched of, as briefly illustrated in Figure 1.3.12,20

SPMs are appreciated for their potent role in inducing resolution of inflammation.11,12 Their ability to act as agonists on G protein-coupled receptors (GPCR), inhibit leukocyte trafficking, promote cessation of neutrophil influx and regulate the release of pro- inflammatory mediators, is vital to the resolution of an active inflammatory process.6,11 SPMs

5 regulate the release of pro-inflammatory mediators such as eicosanoids, chemokines, cytokines and adipokines.6 They result in early cessation of inflammatory diseases such as Alzheimer’s disease, Parkinson’s disease, rheumatoid arthritis, asthma, cystic fibrosis and inflammatory bowel diseases.3,9,21

Figure 1.5 An overview showing the precursors of SPMs and their biological activity. The stereochemistry is determined by direct comparison with synthetic material and is of paramount importance for the biological activity. Note that the maresins and the SPMs derived from n-3 DPA (4) are missing, and will be discussed later on.14

The SPMs are derived from EPA (1), DHA (2) and n-3 DPA (4).3,11 AA (3) gives rise to the classic eicosanoids, herein both the pro-resolution mediators LXA4 (10) and LXB4 (7), and the pro-inflammatory mediators prostaglandins, leukotrienes and thromboxanes.11 EPA (1) is the precursor for the production of E-series resolvins. DHA (2) on the other hand, is the precursor for the D-series resolvins, maresins and protectins.11,14 Recent research has shown that the alpha-linolenic acid (ALA) derivate n-3 DPA (4) is also a precursor for several potent anti- inflammatory mediators, as shown in Scheme 1.1.22

6 Arachidonic acid (AA) Eicosapentaenoic acid (EPA) Docosahexaenoic acid (DHA) n-3 Docosapentaenoic acid (DPA)

Prostaglandines Leukotrienes Thromoxanes Lipoxins E-series Protectins D-series Maresins n-3 DPA n-3 DPA n-3DPA resolvins resolvins protectins resolvins maresins

PD1 MaR1 PD1 MaR1n-3 DPA LXA4 LXB4 RvE1 RvE2 RvE3 n-3 DPA MaR2n-3 DPA RvD1 n-3 DPA RvD1, RvD2, RvD3, RvD4, RvD2 n-3 DPA RvD5, RvD6 RvD5 n-3 DPA

Classic eicosanoids Specialized Pro-resolving Mediators

Scheme 1.1 Overview of the production of SPMs and classic eicosanoids.23

1.4.1 Lipoxins

24 LXA4 (10) and LXB4 (7) contain a trihydroxytetraene moiety and are bioactive eicosanoids. As the first pro-resolution lipid mediators discovered by Samuelsson and his group in 1984,25,26 their role in the resolution of inflammation is well established.24 They are primarily derived from AA (3), but further research has shown that some lipoxins may also be derived from EPA (1).23 They are classified as classic eicosanoids and not SPMS, as shown in Scheme 1.1.

CO H OH OH 2

CO2H OH OH

OH OH

LXA4 (10) LXB4 (7)

27 Figure 1.6 Structures of LXA4 (10) and LXB4 (7).

Their biosynthesis starts during the progression of inflammation. Prostaglandins induce enzymes that are capable of stimulating the switch from leukotriene production to lipoxin production.23 At nanomolar concentrations, lipoxins play a dominant role in the resolution of inflammation due to their biological effects.24 Some of their effects include limiting PMNs

7 infiltration, adhesion of endothelial cells and tissue damage, reducing pain signals, inhibiting the production of cytokines and stimulating phagocytosis. This facilitates the resolution of inflammation. Discovering a method for stimulating the production of lipoxins is therefore quite attractive as a therapeutic target.3

1.4.2 E-series Resolvins

The trihydroxypentaene E-series resolvins, as shown in Figure 1.2, were the first SPMs to be reported.28 Their source of origin is EPA (1), hence the name E-series resolvins. Their effects appear to arise through the action on G protein-coupled receptors (GPCRs), mainly chemR23 and BLT1.3 Consequently, by binding to chemR23 as agonists, they can inhibit leukocyte migration and increase phagocytic activity of macrophages, which are crucial biochemical actions that are well appreciated in the resolution of the inflammation process.3,28-30 RvE1 (11) antagonize the BLT1 receptor, which in turn suppress the leukocyte trafficking by 29 inhibiting the production of LTB4 (6). Serhan and co-workers examined the potency of RvE1 (11) by giving indomethacin (100ng/mouse) and RvE1 (11, 100 ng/mouse), to mice with Zymosan-induced peritonitis, which resulted in 25% and 50∼60% inhibition of leukocyte recruitment, respectively.31 By comparing synthetic RvE1 (11) with locally administrated dexamethasone and aspirin, they also found out that RvE1 (11) is a potent inhibitor with regards to inhibiting leukocyte infiltration at nanomolar level.3

The biosynthesis of RvE1 (11), RvE2 (12) and RvE3 (13) commences with the formation of the intermediate 18R-HpETE (14) from EPA (1), either by acetylated COX-2 or cytochrome p450. Furthermore, this intermediate is transformed by 5-LOX and either hydrolyzed enzymatically or reduced to give RvE1 (11) and RvE2 (12), respectively. On the other hand, epoxide formation and non-enzymatic hydrolysis of 18R-HpETE (14) gives RvE3 (13), as illustrated in Scheme 1.2.28,29,32

8 COX-2 or EPA (1) Microbial P450 HOO

CO2H 1. 5-LOX 2. Enzymatic hydrolysis 18R-HpETE (14) 1. Epoxide formation OH 2. Non-enzymatic hydrolysis

1. 5-LOX HO 2. Reduction OH

CO2H CO2H RvE1 (11) OH HO OH

OH RvE3 (13)

CO2H

RvE2 (12) Scheme 1.2 Biosynthesis of RvE1 (11), RvE2 (12) and RvE3 (13) through enzyme hydrolysis, reduction and 23 non-enzymatic hydrolysis, respectively.

Resolvins of the E-series have shown beneficial health effects, especially in patients with dry eye syndrome and a drug candidate based on this class of SPMs displays promising results in the treatment of said condition.11

1.4.3 D-series Resolvins

Resolvins of the D-series are formed from DHA (2) by the oxygenation of the 17th-position of DHA (2) by 15-LOX. This leads to the intermediates 17S-HpDHA (15), 7S,8S-epoxide (16) and 4S,5S-epoxide (17) which further on can be transformed into six types of D-series Resolvins, as shown in Scheme 1.3.28

9 CO2H 15-LOX Peroxidase DHA (2) 17S-HDHA O2

HOO 5-LOX 5-LOX O2 O2 17S-HpDHA (15)

HO HOO HOO CO H CO2H 2 Peroxidase CO2H

HO Peroxidase HOO HO RvD5 (22) -H2O

-H2O

HO CO2H O CO2H O CO2H

HO HO HO

7S,8S-Epoxide (16) 4S,5S-Epoxide (17) RvD6 (23) Hydrolase +H2O HO HO Hydrolase +H2O CO2H CO2H OH OH

HO HO RvD1 (18) RvD2 (19) OH HO CO2H HO CO2H OH

HO HO

RvD3 (20) RvD4 (21)

Scheme 1.3 Biosynthesis of D-series resolvins, including enzymes involved and the structures of RvD1 - RvD6 (18-23).3,23

Their ability to inhibit PMN transendothelial migration, which is one of the first responses to inflammation, is quite important, especially since it is achieved at 10 nM concentration.3 In addition, resolvins of the D-series can also limit leukocyte infiltration and induce phagocytosis.3 As these effects are of importance to control the progress of inflammation, D- series resolvins, mostly RvD1 (18) and RvD2 (19), may be able to prevent several diseases such as ocular peritonitis, sepsis, kidney ischemia-reperfusion and peritonitis.14

10 1.4.4 Maresins

While EPA (2) constitutes the origin of resolvins of the E-series, DHA (1) generates resolvins of the D-series, protectins and maresins.28 The maresins are SPMs derived from DHA (2) and was reported in 2009.23 However, MaR2 (24) was discovered five years after MaR1 (25), but they share the similar pathway in the biosynthesis.23 Macrophages biosynthesize MaR1 (25), hence the name, and can stimulate macrophage M2. The M2 phenotype is associated with anti-inflammatory functions and thus MaR1 (25) play an essential role in the resolution process.14 M2 macrophages produce more of the SPMs and less of the pro-inflammatory 14 mediators such as LTB4 (6) and prostacyclins. Similar to resolvins and protectins, maresins also block PMN infiltration and induce macrophage apoptosis.24 Maresins, however, are more potent than D-series resolvins in stimulating human macrophage efferocytosis at 1 nM concentrations.33

The biosynthesis of MaR1 (25) and MaR2 (24) follows a similar pathway, firstly through the intermediate 14S-HpDHA (26), which is converted to a 13S,14S-epoxide (27). Subsequently, the epoxide can either undergo enzymatic hydrolysis and give dihydroxylation at C-7 and C- 14 and yield MaR1 (25) after enzymatic hydrolysis, or alternatively open the epoxide and give the diol at C-13 and C-14, as shown in Scheme 1.4.6,23,24

11 12-LOX DHA (2)

OOH Macrophage CO2H

14S-HpDHA (26)

13 14 O

CO2H 13S,14S-epoxy intermediate (27) Enzymatic Enzymatic hydrolysis hydrolysis

OH OH HO

CO2H CO2H

MaR1 (25) MaR2 (24)

Scheme 1.4 Biosynthesis of MaR1 (25) and MaR2 (24).23

Recently, Serhan and co-workers discovered a new family of maresins, namely maresin conjugates, which promote tissue regeneration and resolution of inflammation.34,35 They also reported a potent group of molecules containing DHA (2) and sulfido-conjugates (14-series sulfido-conjugated mediators), which stimulate resolution of infection, bacterial phagocytosis and promote tissue regeneration.36

1.4.5 SPMs derived from n-3 DPA

Recently, a group of novel SPMs derived from n-3 DPA (4) has been isolated from human macrophages, a discovery made in 2013 by Serhan and co-workers.22,23

12 n-3 DPA (4) differs from the DHA (2) by the lack of the C-4 double bond, and is an intermediate in the transformation of ALA to EPA (1) and DHA (2). SPMs derived from n-3 DPA (4) are most likely produced when the level of n-3 DPA (4) is elevated. They resemble same pro-resolving actions as their sister analogs derived from DHA (2), thus indicating that the C-4 – C-5 cis-olefin is not of significant importance for the pro-inflammatory effects.23 They are referred to as n-3 DPA-maresins, -resolvins and -protectins.23

The biosynthesis of MaR1n-3 DPA (28) and PD1n-3 DPA (29) commences with the conversion of n-3 DPA (4) to 14S-HpDPA (30) and 17S-HpDPA (31) through 12-LOX and 17-LOX mediated oxidation, respectively. The resulting hydroperoxides are converted to the 13S,14S- epoxide (32) (MaR1n-3 DPA, 28) and most likely 16S,17S-epoxide (33) (PD1n-3 DPA, 29).

Through a lipoxygenation step, the epoxides are further hydrolyzed enzymatically to MaR1n-3 37 DPA (28) and PD1n-3 DPA (29), as shown in Scheme 1.5. Although several structures of the oxygenated n-3 DPAs have been identified, the stereochemical configurations present in these compounds are still to be determined.23

13 O

OOH OH CO2H CO H 2 O H 14S-HpDPA (30) 13S,14S-epoxy n-3 DPA (32)

12-LOX

CO2H CO2H

MaR1n-3 DPA (28) n-3 DPA (4)

17-LOX

CO2H

O OH

CO H O 2 H HOO

17S-HpDPA (31) 16S,17S-epoxy DPA (33)

HO2C

PD1n-3 DPA (29)

23 Scheme 1.5 The biosynthesis of MaR1n-3 DPA (28) and PD1n-3 DPA (29) from n-3 DPA (4).

1.5 Protectin D1

Protectin D1 (8, PD1) is a potent anti-inflammatory and pro-resolving lipid mediator. The potency was determined by Serhan and co-workers by radiolabeling PD1 (8) in order to 3 observe specific binding to human neutrophils, which resulted in a Kd of ∼ 25 nM. The DHA derivate PD1 (8) was first discovered and isolated by Serhan and his group in 2002.23 Interestingly, they also confirmed that PD1 (8) is more potent than indomethacin in reducing PMN infiltration in murine peritonitis, as they observed ∼40% more inhibition with a dose as small as 100 ng/mouse. In vitro studies proved that human PMN transmigration was reduced by 35-45% with a concentration of 10-100 nM PD1.38

14 Occasionally, PD1 (8) is referred to as neuroprotectin D1 (Figure 1.5) since it has been found in neural tissue. However, considering the fact that PD1 (8) has been detected in several other tissues as well, the prefix neuro is rarely used now.3,23

1.5.1 Isolation and structure elucidation

In the isolation and structure elucidation of PD1 (8) it was important to establish the correct stereochemistry. The methods used to establish the absolute configurations of the stereogenic centers as well as the olefin geometry was biosynthesis studies, matching synthetized material with defined stereochemistry compound, and lastly by observing PD1s (8) actions in several biological systems.39 Multiple reaction monitoring (MRM) and NMR were used for further structural elucidation and to match authentic material from inflammatory exudates with compounds made using synthetic organic chemistry.39

1.5.2 Further biosynthesis and metabolism

The biosynthesis of PD1 (8) starts with the formation of 17S-HpDHA (15), a result of either an oxidation or 15-LOX actions on DHA (2).3 Furthermore, this unstable intermediate forms the epoxide quickly, which requires enzymatic transformations in order to obtain the correct double bond geometry in PD1 (8).3 The determination of the double bond geometry and the chirality of the C-10, showed that the most active compound has an E,E,Z-triene and the configuration R at the C-10.3 The intermediate formed after the enzymatic hydrolysis is of a carbocation-type intermediate, since it gives the formation of the E,E,Z-triene and not the E,E,Z-triene. This proves that the former is governed by enzymatic actions and the latter by non-enzymatic actions.23 Recently, Professor Hansen and his group were able to confirm that the 16S,17S-epoxide (34) intermediate is indeed an intermediate in the biosynthesis of PD1 (8). This was proved by synthetizing the epoxide intermediate, followed by incubation with human macrophages, which successfully converted the intermediate into PD1 (8) by 15- LOX.40

15 CO2H

15-LO DHA (2) O2 - H2O O HOO CO2H

17S-HpDHA 16S,17S-epoxy DHA derivate (15) (34)

Enzyme hydrolysis

OH CO2H OH CO2H

PD1 (8)

Scheme 1.6 Outline of the biosynthesis of PD1 (8) showing the carbocation intermediate, which results in the given double bound geometry.3,23

Although the biosynthesis has been well established, there is still a lack of research on the PD1 metabolism.41 Recently, the LIPCHEM group were able to identify and synthetize a ω-22 monohydroxylated metabolite of PD1 (8), named 22-OH-PD1 (35), which was the first metabolite of PD1 (8) to be isolated and synthetized.23 The synthesis was achieved in nine steps, and the compound exhibited potent anti-inflammatory actions resembling those of PD1 (8).41 The enzymes involved in the ω-oxidation are CYP1 monooxygenases, as with other SPMs.41 Usually ω-oxidations in other mediators reduce the biological activity by the conversion to less active metabolites.42-44 In PD1 (8), on the other hand, the biological activity of the metabolites seem to have similar biological activity, which is advantageous for possible new drug candidates.41

16 HO

OH CO2H OH CO2H

OH OH

PD1 (8) 22-OH-PD1 (35)

Scheme 1.7 The ω-oxidation of PD1 (8), resulting in the metabolite 22-OH-PD1 (35), through oxidation with CYP1 monooxygenases.41

PUFAs can usually be metabolized by mainly two types of enzymes, LOX and P450 monooxygenases.45 The former yields hydroperoxy-intermediates, while the latter can yield four different oxidation products, depending on where the oxidation occurs.45 Mainly, P450 monooxygenases can either give an internal hydroxy metabolite, a terminal hydroxyl metabolite or epoxy metabolites which are converted to the dihydroxy metabolites, as briefly shown in Scheme 1.8.45

LOX OOH

R R1 R R1

P450 P450 P450 monooxygenase monooxygenase monooxygenase

O HO OH H2O OH R 1 R1 R R 1 R R OH Internal hydroxy metabolite Terminal hydroxy metabolite Epoxy metabolite Dihydroxy metabolite

Scheme 1.8 Possible metabolism reactions of lipoxygenases and P450 monooxygenases on PUFAs.45

Next, the secondary alcohol in C-17 may be metabolized by oxidation, which is mediated by eicosaoxidoreductase enzymes to the corresponding ketone, like in RvE1 (11).3,43 By introducing an additional methyl group in the ω-6 position, a tertiary alcohol is obtained and metabolism by oxidation of the alcohol becomes unachievable. Alternatively, since β- oxidation at C-3 may occur, such as in 3-oxa-lipoxins and 3-oxa-leukotrienes, introducing oxygen in the C-3 position may help avoid the previously mentioned oxidation.46-48

17 Consequently, the aim of this study was to synthetize such analogs, which will be discussed further in section 1.7.

1.5.3 Syntheses of Protectin D1

Kobayashi and Ogawa published the first total synthesis of PD1 (8) in 2011 based on a Suzuki-Miyaura cross coupling reaction.49 Petasis and co-workers introduced a new synthesis in 2012, which mainly included Sonogashira cross coupling reaction.39 Alternatively, Professor Hansen and his group published a stereoselective synthesis of PD1 (8) in 2014, mainly by using Evans-Nagao aldol-, Z-selective Wittig- and Sonogashira reactions.50 Later in 2014, Spur and Rodrigues also assembled their key fragments using Sonogashira reaction, and achieved a new synthesis of PD1 (8),51 as explained in more detail below.

1.5.3.1 Kobayashi and Ogawa’s strategy

The iodide-fragment 36 in Kobayashi’s synthesis was prepared several steps. The synthesis commenced with the propargyl alcohol 37. The iodo-functional group was installed to 37 and yielded compound 38. Further deprotection of 38 yielded an aldehyde 39, which was further treated with the Wittig-salt 40 in a Z-selective Wittig reaction. With esterification of the alcohol 41 and deprotection of 42, the fragment 36 was obtained, as illustrated in Scheme 1.9.23,49

18 OPMB 1. Pd cat. Bu3SnH 1. DDQ I OPMB I O OTBDPS 2. I 2. PCC 2 OTBDPS OTBDPS

37 38 39

1. NaHMDS,

IPh3P

TBSO 1. SO3 Py DMSO 40 2. NaClO2, 2. PPTS tert-BuOH, NaH2PO4 I CO2Me I OH OTBDPS 3. CH2N2 OTBDPS 42 41

TBAF THF

I CO2Me OH 36

Scheme 1.9 Outline of the construction of the iodide fragment 36.49

To make the next intermediate, propargyl alcohol 43 was used and first reduced and oxidized yielding an aldehyde intermediate. The aldehyde was propargylated by zinc and TiCl4 in catalytic amounts, affording alcohol 44. Later, a Sharpless asymmetric epoxidation was used in a kinetic resolution step, followed by bromine and TBAF to obtain to install the brominated compound 45. The protected alcohol 46 was further cross-coupled in a Sonogashira coupling reaction and hydroborated the terminal acetylene 47 to give the borane 48, as outlined in Scheme 1.10. The borane 48 was achieved in 11 steps and 23% overall yield. 23,49

19 1. tert-BuOOH, 1. RedAl R D-(-)-DIPT, Ti(O-i-Pr) TMS 4 Br HO 2. PCC 2. Br2, -78 ° °C TMS OH 3. Propargyl bromide, 3. TBAF, THF, -78 °C Zn, TiCl4 OH 43 44 45

TBSCl, Imidazole

1. TMS-acetylene, Pd(PPh3)4, CuI BSia Br 2 Sia2BH PrNH2, benzene

THF, 0 °C 2. H2, Pd/BaSO4 3. K CO , MeOH OTBS OTBS OTBS 2 3

48 47 46

Scheme 1.10 Outline of the syntheses of the intermediate 48.49

The synthesis was based on a Suzuki-Miyaura cross-coupling reaction between compound 42 and 48 and afforded the whole carbon skeleton (Scheme 1.11). Furthermore, deprotection and basic hydrolysis yielded PD1 (8), in 20 steps totally.23,49

BSia2

OTBS 1. TBAF, THF, 48 Pd(PPh3)4, OTBS 0 °C 2 N NaOH CO2Me PD1 + (8) THF 2. 2 N LiOH, MeOH, H2O, rt. I CO2Me OH OTBS 49 42

Scheme 1.11 Outline of the final construction of the carbon skeleton 49 in Kobayashi’s synthesis of PD1 (8).23,49

1.5.3.2 Petasis’ strategy

As previously mentioned, Petasis’ synthesis was based on a Sonogashira cross coupling reaction.39 In brief, they established the precursor for the E,E,Z-triene, with a Sonogashira cross-coupling reaction. The essential ω-fragment used in the above-mentioned cross coupling reaction was prepared by the addition of the anion of 1-butyne 50 to TBS-protected (S)- glycidol 51, affording the alkyne 52. Next, silylation, deprotection and reduction afforded the diol 53, which was oxidized to the corresponding aldehyde.39 Lastly, the conversion of the

20 aldehyde to the acetylene, employing the Corey-Fuchs homologation, afforded the ω-end fragment 54 (Scheme 1.12).

50 1. TBDPSCl, n-BuLi OTBDPS OH imidazole, DMAP + OH OTBS BF3 OEt2 2. CSA O 52 3. H2, Lindlar's cat. 53 OTBS

1. (COCl)2 51 DMSO, Et3N 2. Ph3P, CBr4 3. n-BuLi, Et2O

OTBDPS

Scheme 1.12 Outline of the synthesis of the ω-fragment 54, which is further used in the Sonogashira cross- coupling reaction.23,39

The iodide 55 was prepared briefly using the same reactions as in the construction of the above-mentioned acetylene 54. First the TBS-protected (R)-glycidol 56 was reacted with alkyne 57 and yielded the diol 58. Compound 59 was further transformed by applying the Appel reaction and Lindlar’s reduction, generating the Z-olefin 60. After Swern oxidation, the Takai reaction and the Wittig reaction were applied, in order to yield the targeted intermediate 55, as shown in Scheme 1.13.13,23

21 TBSO O 1. NBS, PPh , TBSO 56 1. n-BuLi, BF OEt 3 3 2 0 °C, CH Cl -78 °C, THF 2 2 + HO TBDPSO OH 2. TBSOTF, lutidine, 2. TBDPSCl, imidazole, TBDPSO 0 °C, CH2Cl2 OTBS DMAP, CH2Cl2 3. CuI, NaI, K2CO3, 3. CSA, CH2Cl2/MeOH 57 58 methyl 4-pentynoate

59 CO2Me

1. CSA, CH2Cl2/MeOH 2. H2, Lindlar cat. quinoline I 1. (COCl)2, DMSO, Et N, -78 C CO Me 3 ° CO Me 2 HO 2 OTBDPS 2. Ph3P=CHCHO, TBDPSO 55 toluene, Δ 60 3. CHI3, CrCl2, THF

Scheme 1.13 Outline of the synthesis of the second key intermediate used in the Sonogashira cross-coupling reaction.23,39

Subsequently, the Boland reagent, Zn(Cu/Ag) was applied in order to obtain reduction of the conjugated alkyne 61 and to establish the correct absolute stereochemistry in a cis-selective fashion.23,39 A basic hydrolysis of compound 61 was applied, simply using aqueous sodium hydroxide, which finally furnished PD1 (8), as illustrated in Scheme 1.14.13,23

OTBDPS

Pd(PPh3)4, 54 CuI CO2Me Benzene I +

OTBDPS CO2Me TBDPSO TBDPSO 61

55 1. TBAF, THF, rt. 2. Zn(Cu/Ag), MeOH/H2O 3. NaOH, H2O

PD1 (8) Scheme 1.14 Outline of the final steps in Petasis’ synthesis of PD1 (8).23,39

22 1.5.3.3 Spur and Rodriguez’ strategy

Spur and Rodriguez reported a total synthesis of PD1 (8) in 2014, which also relied on the Sonogashira cross-coupling reaction between two main fragments.51 The first fragment, which was made in three steps, involving the opening of acetal-protected D-ribose 62 and Z- selective Wittig reaction yielding the alkene 63. Subsequently, the Appel reaction was applied in order to furnish the corresponding iodide, which later on was deprotonated and eliminated using LDA to obtain the desired intermediate 64 as illustrated in Scheme 15.23,51

OH 1. NaHMDS, 1. I , PPh , O 2 3 THF, -78 °C imidazole, toluene HO O O 2. CH3(CH2)2PPh3I, 2. LDA, THF, O O -78 °C to 0 °C -78 °C OH

62 63 64

Scheme 1.15 Outline of the synthesis of the C15-C22 intermediate 64 in Spur and Rodriguez’ synthesis of PD1 (8).23

The α-fragment was constructed using the alcohol 65, which is an enantiopure compound making it easier to obtain the stereogenic center in C-10 position. From 65, the aldehyde 66 was obtained using the Swern reaction, and subsequently reacted further with the Wittig salt

67 to yield the diene 68. The acetal-protecting group was then cleaved using CuCl2⋅2H2O and a protecting group were introduced, to give the corresponding alcohol 69. Further on, Dess- Martin oxidation, Wittig homologation of the aldehyde and Takai reaction furnished the vinyl iodide 70, as shown in Scheme 1.16.23,51

23 OH O CO2M O 67 O e (COCl)2, DMSO O O O O Et3N, -78 °C NaHMDS, THF, -78 °C 68 65 66 1. CuCl2 2H2O, MeCN, 0 °C 2.TBSCl, imidazole, DMAP 3. HF py, THF, 0 oC

I 1.DMP, CH Cl CO Me 2 2 CO Me 2 HO 2 2. Ph P=CHCHO, toluene OTBS 70 3 OTBS 69 3. CrCl2, CHI3, THF, 0 °C

67: IPh3P CO2Me

Scheme 1.16 Outline of the synthesis of the key fragment 70 in Spur and Rodriguez’ synthesis.51

Finally, fragment 64 and 70 were cross-coupled using the Sonogashira reaction, to complete the carbon-chain of PD1 (8). The last step included desilyation of 71, cis-reduction and ester hydrolysis giving PD1 (8), as shown in Scheme 1.17. The synthesis was completed in 12 steps and resulted in a 0,02% overall yield.51

CO2Me CO Me OTBS 2 70 Pd(PPh3)4 CuI + HO OTBS Piperidine 71 Benzene 1. TBAF, THF, 0 °C OH 2. Zn(Cu/Ag), MeOH/H2O 3. 1 N LiOH, H2O 64

PD1 (8)

Scheme 1.17 Outline of the final steps of Spur and Rodriguez’ synthesis.23

1.5.3.4 Hansen’s strategy

Professor Hansen and co-workers developed a stereoselective total synthesis of PD1 (8) in 2014. The main fragments were the terminal alkyne 72, the aldehyde 73 and the Wittig-salt

24 67,50 as shown in the retrosynthetic analysis in Scheme 1.18. The total synthesis of PD1 (8) was done in eight steps from the aldehyde 78 and obtained an overall yield of 15%.50

Sonogashira and Z-selective reduction Z-selective OH OH Wittig

CO H (S) (R) 2 8

OTBS OTBS + + Br O IPh3P CO2Me Evans-Nagao 72 73 aldol 67

O S O CO2H Br O + N S +

78 79a 83 82

Scheme 1.18 Hansen’s retrosynthetic analysis of PD1 (8) reported in 2014.50

The synthesis commenced with the preparation of alkyne 72 from 1-butyne 74 and THP- protected (S)-glycidol 75,50 as shown in Scheme 1.19.

O OTBS + OTHP

1-butyne (74) (S)-THP glycidol (75) 72

Scheme 1.19 A brief outline of the synthesis of the terminal alkyne 72, a key fragment in the synthesis of PD1 (8) reported by Hansen and his co-workers.50

The aldehyde 73 was obtained by treating pyridinum-1-sulfonate 76 with aqueous potassium hydroxide, yielding the potassium salt of the aldehyde 77, which was transformed further into the aldehyde 78 using bromine and triphenylphosphine. Further on, the aldehyde 78 was

25 reacted with thiazolidinone 79a in an Evans-Nagao aldol reaction, to obtain the intermediate 80a in a 15.3:1 diastereomeric ratio, as determined by HPLC and 1H-NMR analysis. The TBSO-protected alcohol 81 was then reduced with DIBAL-H in order to afford the aldehyde 73,50 as illustrated in Scheme 1.20.

1. KOH (aq.) Br2, PPh3, N KO O Br O -20 °C to rt. CH2Cl2, 0 °C SO3

76 77 78

O S OH O S 1. (i-Pr)2NEt, TiCl4 N S Br N S CH2Cl2, -78 °C 2. aldehyde 78 R R 54-86% 79a: R = i-Pr 80a: R = i-Pr 79b: R = Ph 80b: R = Ph 79c: R = Bn 80c: R = Bn TBSOTf, 2,6-lutidine CH2Cl2, -78 °C 97%

OTBS TBSO O S DIBAL-H,

Br O Br N S CH2Cl2, -78 °C 73 81

Scheme 1.20 Outline of the synthesis of the main fragment 73 in Hansen’s strategy.50

The next step included the synthesis of the Wittig-salt 67. First, the dianion of 4-pentynoic acid 82 was reacted with ethylene oxide 83 to obtain the 7-hydroxy-hept-4-ynoic acid, which was esterified immediately using MeOH and catalytic amounts of H2SO4 yielding 84. Subsequently, the alkyne in 84 was reduced using Lindlar’s catalyst, which afforded the Z- alkene 85. The iodide 86 was prepared using the Appel reaction. The iodide 87 was in turn treated with triphenylphosphine in acetonitrile, affording the Wittig-salt 67. The corresponding ylide of the Wittig-salt was obtained by treatment with NaHMDS, and it was then reacted with aldehyde 73 in THF at -78 °C, affording the tetraene ester 87 (Scheme 1.21).50

26 CO2H 1. n-BuLi, HMPA, THF, 0 °C CO2Me 2. ethylene oxide (83) HO 82 84 3. MeOH, cat. H2SO4

H2, Lindlar´s catalyst, hexanes/EtOAc

I2, PPh3, I CO2Me HO CO2Me CH Cl , imidazole 86 2 2 85

PPh3, MeCN, Δ

1. NaHMDS, IPh3P CO2Me 67 HMPA, THF, -78 °C 2. aldehyde 73

OTBS

Br CO2Me 87

Scheme 1.21 Outline of the synthesis of the Wittig-salt 67 and the tetraene ester 87.50

The alkyne 72 was reacted with 87 in a Sonogashira cross-coupling reaction to afford the whole carbon skeleton of PD1 (8).50 This afforded the bis-hydroxyl-protected methyl ester 88, which was deprotected using five equivalents TBAF in THF at 0 °C, yielding the diol 89. The diol 89 was then reduced using the hydrogenation reaction, providing the hexaene 90. A simple esterification and acidic work-up furnished PD1 (8) in 78% yield (Scheme 1.22).50

27 OTBS

Br CO2Me 87

Pd(PPh3)4, CuI, Et2NH, alkyne 91

OTBS CO Me TBSO 2 88

TBAF, THF,0 °C

OH CO Me HO 2 89

1. H2, Lindlar´s catalyst EtOAc/pyridine/1-octene

OH OH

CO2R R = Me: 90 LiOH (aq.), MeOH, 0 °C R = H: protectin D1 (8)

Scheme 1.22 Outline of the final steps of Hansen’s synthesis of PD1 (8).50

1.6 Synthetic methods

The approaches intended to be deployed in the final synthetic steps of the two wanted analogs of PD1 (8), was the Z-selective Wittig reaction, Sonogashira reaction and Evans-Nagao aldol reaction.

1.6.1 Z-selective Wittig reaction

The Wittig reaction is one of the most used methods in organic chemistry to create carbon- carbon double bonds with defined geometry.52 Wittig and Geissler discovered the reaction in 1953, when they were able to react benzophenone with metyhlentripenylphosporane, resulting in 1,1-diphenylethylen. Hence, a new method to synthesize olefins was discovered. This method made it possible to create pure isomeric compounds, a great discovery since the

28 earlier used methods generally gave a mixture of the isomeric compounds. Another advantage of the Wittig reaction is that the reaction can be carried out under comparatively mild conditions, constituting a major advance in synthetic methodology in light of the of harsh 53 conditions which were previously often used. The mechanism for the Wittig reaction is based on the nucleophilic addition of the phosphonium ylide to the electrophilic carbonyl compound, aldehydes and ketones in general, which forms a four-membered ring.53,54 Ylides can be classified as stabile ylides, semi-stabile ylides and non-stabile ylides, which is to a large extent determined by the R-group directly connected to the phosphonium.54 The stability of the ylide dictates the stereochemistry of the alkene. The E-isomer is not favored, and is formed fast and reversibly. The Z-isomer on the other hand, is favored and is formed slow and irreversibly.54 Several factors increase the yield of the Z-isomer, such as carrying out the reaction at low temperatures, avoiding lithium as cation and using a highly diluted reaction mixture.23,55

H H H 2 H Ph3P R 1 Ph P R1 + R2 R R2 3 O O Ph P O R1 H 3

H H H H 1 + R 2 Ph3P O R R1 R2 Ph3P O

Scheme 1.23 The reaction mechanism of the Wittig reaction.56

The Z-selective Wittig reaction was applied in order to forge the double bond between the Wittig-salt 67 and the aldehyde 73, as shown in Scheme 2.14,50 and in the last synthetic step in the preparation of the ω-fragment.

1.6.2 Sonogashira coupling reaction

The Sonogashira coupling reaction is one of the most used methods for the formation of Csp2 – 57,58 Csp bonds, and has been used widely in different areas, including medicinal chemistry. In order to make this coupling possible, one must use a metal catalyst, often Palladium (Pd) and Copper (Cu) as a co-catalyst.59

29 The Sonogashira coupling reaction was discovered in 1975 and before its discovery, it was nearly impossible to obtain alkylation from halides and alkynes in one step.60

R1-X 0 Pd L2

R1 R2

R1 L L Pd R2 R1 Pd X L L L 2 R1 Pd R L

Cu+X- Cu R2

+ - R3N HX

H R2 H R2 Cu+X- R N 3

Scheme 1.24 The reaction mechanism of the Sonogashira coupling reaction.58,61

The reaction mechanism is quite complex, and starts with the oxidative addition of the catalyst to the aryl halide. Subsequently, this complex gets connected with the deprotonated terminal alkyne, in a step called transmetallation. Later, reductive elimination occurs and results in the wanted product (Scheme 1.24). 57 In this thesis, the Sonogashira coupling reaction was supposed to be applied for the coupling between alkyne 91 and bromide tetraene ester 87, as illustrated in Scheme 1.22.

1.6.3 Evans-Nagao stereoselective aldol reaction

The aldol reaction was discovered by Wurtz and Borodin in the 1870s,23 and is one of the most widely used reactions and approaches for the formation of carbon-carbon bounds.62 In brief, this can be obtained by the addition of an enolate donor, obtained by either lithium-, boron-, titanium- or silyl-based Lewis acids, to a carbonyl acceptor.62 Using chiral starting

30 materials or auxiliaries, one may often control the desired configuration of the stereogenic centre.62 The formation of Z-enolates favor the formation of syn-diastereomers, while E-enolate favour the formation of anti-diastereomers,23 thus explaining the stereochemistry of the alcohol in 80a. Evans used acyl oxazolidinone auxiliaries in order to obtain enantiopure alcohols,63 such as the one afforded in 80a. The stereogenic orientation of the alcohol minimize steric interactions.23 Nagao and co-workers elaborated this strategy and discovered in 1986 that using 1,2 equivalents of Sn(OTf)2 and 1-N-ethylpiperidine at -50 °C can generate products of high stereoselectivity, as shown in Scheme 1.25.64,65 Subsequently, the thiazolidinethione auxiliaries can be removed using DIBAL-H and afford aldehydes,23 as shown in Scheme 1.20.

S O S O OH 1. Sn(OTf)2, N-ethylpiperidine S N S N Ph 2. Cinnamaldehyde CH2Cl2 -50 °C

92 93

Scheme 1.25 Outline of the Nagao acetate aldol reaction from compound 92 to 93.13

1.7 Aim of study

The treatment of inflammation as well as enhancing the resolution of the inflammatory process, is highly appreciated in order to avoid the development of many chronic diseases. The SPMs play an essential role for this purpose, but are unfortunately biosynthesized in small amounts and rapidly metabolized. Therefore, the synthesis of analogs with the emphasis of extending the biological activity, is of high interest. By making an analog with a methyl group (Scheme 1.26) at C-17 position, the secondary alcohol will become a tertiary alcohol, which inhibits metabolism by oxidation. Moreover, altering the alpha-fragment in PD1 (8), will result in an analog with an oxygen atom in the C-3 position, thus avoiding β-oxidation.

31 1.7.1 Retrosynthetic analysis of analog 94

Sonogashira and Z-selective reduction Z-selective Wittig OH OH CO H (R) 2 94

OTBS OTES + Br O + IPh3P CO2Me Z-selective wittig Evans-Nagao Alkylation 91 aldol 73 67

O S OTES + O + CO2H PPh Br + Br O N S 3 O Grignard

103 100 78 79a 83 82

O MgBr + OH

101 102 Scheme 1.26 Retrosynthetic analysis of PD1-analog 94.

32 1.7.2 Retrosynthetic analysis of analog 95

Sonogashira and Z-selective reduction Z-selective Wittig

OH OH O CO2H

OTBS OTES + + O CO2H Br O IPh3P Z-selective wittig Evans-Nagao aldol 91 73 96a

O O IPh3P O 96

S OTES O O HO + + Br PPh3Br O Br O N S OH O Grignard

103 100 78 79a 104 105

O MgBr + OH 101 102

Scheme 1.27 Retrosynthetic analysis of PD1-analog 95.

The justification for using this synthetic strategy, based on this retrosynthetic analysis was to maintain several structural features present in the analogs 94 and 95. Firstly, there is one stereogenic center in the C17 position in the form of a secondary alcohol, which might undergo dehydration to yield the corresponding conjugated alkene. Secondly, the E,E,Z-triene between C-11 and C-15, must remain in the same preferred configuration and hence it is of high importance to avoid unwanted transformation of this moiety. Lastly, the tertiary alcohol and the methyl group must be introduced at the ω-6 position. With these considerations in

33 mind, both the analogs were divided into three key fragments, namely the ω-fragment 91, the middle fragment 73 and last but not least the α-fragment 67 and the α-fragment 96. In analog 94, a methyl group is introduced in C-17 position to give a tertiary alcohol, which will prevent this carbinol atom from being metabolized by oxidation. Furthermore, in analog 95, an oxygen atom is introduced into the C-3 position to avoid β-oxidation.

Hence, the aim of this thesis was: Ø To synthesize PD1-analouges, introducing a methyl-group at ω-6 position and oxygen in the β-position to the acid. Ø To investigate the biological effects and potencies of the desired analogs.

34 2 Results and discussion

The five fragments 67, 73, 91, 96 and 97 were prepared as outlined in section 5.2. The final assembly of the two fragments 67 and 73 was unfortunately unsuccessful, as further described in section 2.4.2 and 2.4.3. Assigned spectra for each of the compounds, which were successfully synthesized, are found in the appendix.

TBSO OTES IPh P CO Me 3 2 Br O 91 67 73 O O O O IPh3P O BrPh3P O

96 97 Figure 2.1 Overview of the successfully synthetized fragments needed for the total synthesis of analogs 94 and 95.

2.1 Synthesis of the ω-fragment 91

The fragment 91 was prepared in four steps as outlined in Scheme 2.1, using a procedure developed by Dr. J.M. Nolsøe in the LIPCHEM group at the University of Oslo. This produced 91 in 8% overall yield. NMR-spectra of every synthetic step leading towards the ω- fragment are explained thoroughly and were compared with the unpublished spectra recorded by the LIPCHEM group.66

Ethynylmagnesium O TESOTf, 2,6-Lutidine bromide 101 OH OTES OH HO THF CH2Cl2 TESO 0 °C to rt. -78 °C to rt. 102 98 99 51% 59%

DMSO NaHMDS Oxalyl chloride OTES Wittig-salt 103 OTES

CH2Cl2 O THF/HMPA Et3N -78 °C to 20 °C -78 °C to rt. 100 91 38% 71% Scheme 2.1 Outline of the synthesis of the ω-fragment 91.

35 In the preparation of fragment 91 destined for the ω-end, a Grignard reaction was conducted to introduce the tertiary alcohol and the alkyne moiety in 98. After TES-protection of the alcohols, the primary silyl ether in compound 99 was oxidized to the corresponding aldehyde 100 using oxalyl chloride and DMSO in a Swern oxidation. Subsequently, the unstable aldehyde 100 was reacted with the non-stabilized ylide obtained from the addition of the base NaHMDS, in a Wittig reaction. Each step is described in more detail in section 5.2.1 – 5.2.4.

2.1.1 Synthesis of 3-methylpent-4-yne-1,3-diol (98)

Ethynylmagnesium bromide 101 was used as a nucleophilic component to react with the electrophilic carbonyl in 102, in order to produce compound 98.

O Ethynylmagnesium bromide 101 OH OH THF HO 0 °C to rt.

102 59% 98 Scheme 2.2 Outline of the synthesis of the tertiary alcohol 98.

A large excess of commercial ethynylmagnesium bromide (101) was added dropwise to the ketone 102 in THF at 0 °C. After aqueous work up, the crude product was purified by flash chromatography on silica gel, resulting in the desired compound 98 in a 51% yield as a clear yellow oil. This reaction was performed twice in order to obtain sufficient amounts of the compound. The highest yield attained using this procedure was 51%. The product was analyzed by 1H- and 13C-NMR, and the results are found in the appendix (Figure 7.1 and 7.2).

2.1.1.1 NMR interpretation

In the 1H-NMR spectrum of 98 (Figure 7.1), the singlet at 1.54 ppm (3H) arises from the methyl group adjacent to the tertiary alcohol. The multiplets that occur at 1.83 ppm (1H) and 2.00 ppm (1H), should correspond to the two diastereotopic protons in the methylene group next to the tertiary alcohol. The singlet at 2.50 ppm (1H) is in the area where one would expect to observe the proton on the terminal alkyne. The broad singlet at 2.88 ppm (≈2H) arises from the two alcohol groups. The methylene group shifted downfield and directly bound to the primary alcohol gives two multiplets at 3.92 (1H) and 4.17 (1H). The two

36 protons in this methylene group are diastereotopic, and hence resonate at different frequencies.

1.54 4.17 2.88 31.1 3.92 61.1 OH HO 69.1 1.83, 87.5 2.50 2.88 2.00 72.4 43.8

Figure 2.2 The structure shows proposed chemical shifts for 1H-NMR (blue) and 13C-NMR (black) for compound 98.

The 13C-NMR spectrum (Figure 7.2) shows six signals, which is in agreement with the number of carbon atoms present in the molecule. The signal at 31.1 ppm is most likely caused by the methyl group adjacent to the tertiary alcohol. The signals at 43.8 ppm and 61.1 ppm stem from the two methylene groups, where the latter appears more downfield due to the deshielding effects caused by the primary alcohol present in the molecule. The signal at 69.1 ppm most likely corresponds to the tetrasubstituted carbon atom. The signals at 72.4 ppm and 87.5 ppm are present in the area where we would expect to observe carbon atoms in alkynes.

2.1.2 Synthesis of 3,3,9,9-tetraethyl-5-ethynyl-5-methyl-4,8-dioxa-3,9- disilaundecane (99)

In the second step of the synthesis of the ω-fragment 91, both alcohols were protected as TES- ethers in order to avoid unwanted, subsequent reactions.

TESOTf, 2,6-Lutidine OH OTES HO CH2Cl2 TESO -78 °C to rt.

98 59% 99

Scheme 2.3 Outline of the synthesis of the diprotected alcohol 99.

67 The sterically hindered base 2,6-lutidine (pKaH 6,6) was added to the diol 98, which was dissolved in CH2Cl2 at -78 °C and added TESOTf. After aqueous work-up the crude product was purified twice using flash chromatography, providing compound 99 in a 59% yield as a

37 clear oil. The product was analyzed by 1H- and 13C-NMR, as shown in the appendix (Figure 7.3 and 7.4)

2.1.2.1 NMR interpretation

In the 1H-NMR spectrum of 99 (Figure 7.3), the signal at 0.79-0.51 ppm (m, 12H) and the signal 0.96 ppm (td, J = 7.9, 3.1 Hz, 18H) stem from the six methylene groups and the six methyl groups in the two TES-groups, respectively. The singlet at 1.48 ppm (3H) accounts for the protons in the methyl group bound to the tertiary carbon atom. The signal at 1.94 ppm (dt, J = 8.1, 6.4 Hz, 2H) and at 3.84 ppm (t, 2H) arise from the two methylene groups. The latter appears downfield due to the deshielding effect caused by the directly adjacent silyl ether. The singlet at 2.41 ppm (1H) is in the area expected for the proton on the terminal alkyne.

CH2 in TES: 0.79-0.51 and 4.6 1.48 CH3 in TES: 0.96 and 7.1 3.84 31.6 59.7 OTES 88.0 TESO 67.6 1.94 2.41 47.8 72.1

Figure 2.3 The structure shows proposed chemical shifts for 1H-NMR (blue) and 13C-NMR (black) for compound 99.

The 13C-NMR spectrum (Figure 7.4) shows eight signals, which is in agreement with the number of carbon atoms present in the molecule. The signals at 4.6 ppm and 7.1 ppm are most likely caused by the methylene and methyl groups in the two TES-groups. The signal at 31.6 ppm arises from the remaining methyl group. The signals at 47.8 ppm and 59.7 ppm show the two methylene groups present in the molecule, in which the latter signal appears downfield due to the deshielding effects from the silyl ether. Next, the signal appearing at 67.6 ppm resonates for the tetrasubstituted carbon atom present in the compound 99. Lastly, the signals at 72.1 ppm and 88.0 ppm are from the alkyne. Both NMR-spectra show traces of heptane.68

2.1.3 Synthesis of 3-methyl-3-((triethylsilyl)oxy)pent-4-ynal (100)

The primary TES-protected alcohol 99 was deprotected in situ and subsequently oxidized using the Swern oxidation conditions.

38 DMSO OTES Oxalyl chloride OTES TESO O CH2Cl2 Et3N -78 °C to 20 °C

99 38% 100 Scheme 2.4 Outline of the synthesis of the aldehyde 100.

DMSO, dissolved in dry CH2Cl2 at -78 °C, was added oxalyl chloride slowly. Next, the silyl ether 99 was added. The reaction mixture was stirred for one hour before the temperature was regulated to -20 °C. After 45 minutes the reaction mixture was re-cooled to -78 °C and Et3N was added. After aqueous work-up, the crude product was purified by flash chromatography, affording the desired compound 100 in 38% yield as a clear yellow oil. The product was analyzed by 1H- and 13C-NMR, and the results are presented in the appendix (Figure 7.5 and 7.6).

There are many explanations for the resulting low yield. Firstly, the reactant may not have been converted completely to the final product. However, the TLC-analysis conducted at the end of the process indicated that no starting material was present, hence the above-mentioned explanation is most likely not applicable. Secondly, other unwanted reactions may occur, and unwanted byproducts would consequently lower the yield. Nevertheless, loss of product may also occur during the purification, extraction and separation procedures. Lastly, there may be impurities present in the reactants, which will not participate in the reaction.

2.1.3.1 NMR interpretation

In the 1H-NMR spectrum of compound 100 (Figure 7.5), the multiplet at 0.69 ppm (6H) and the triplet at 0.96 ppm (9H), accrue from the methylene groups and methyl groups, respectively, present in the TES-group. However, the singlet at 1.58 ppm (3H) shows the methyl group. Furthermore, the singlet at 2.57 ppm (1H) shows the proton on the terminal alkyne. The deshielded doublet that appears downfield at 2.62 ppm (J = 2.9 Hz, 2H) arises from the methylene group adjacent to the aldehyde. Finally, the triplet at 9.89 ppm (J = 2.9 Hz, 1H) is in the area expected for the hydrogen part of the aldehyde functional group.

39 CH2 in TES: 0.69 and 7.0 1.58 CH3 in TES: 0.96 and 6.1 31.6 9.89 201.7 OTES 86.6 O 66.4 2.62 2.57 57.3 73.9 100

Figure 2.4 The structure shows proposed chemical shifts for 1H-NMR (blue) and 13C-NMR (black) for compound 100.

The 13C-NMR spectrum (Figure 7.6) shows eight signals, which is in agreement with the number of carbon atoms present in the molecule. The signals at 6.1 ppm and 7.0 ppm are most likely from the methylene groups and methyl groups in the TES-group. The signal at 31.6 ppm shows the methyl group present in the molecule. Next, the signal shifted downfield at 57.3 ppm most likely corresponds to the methylene group adjacent to the aldehyde functionality. The signal at 66.4 ppm is probably caused by the tetrasubstituted carbon atom. The signals at 73.9 ppm and 86.6 ppm are in the area expected for carbons in the alkyne group. The remaining signal at 201.7 ppm shows the aldehyde. However, both NMR-spectra show minor impurities such as heptane and acetone. Traces of water was also observed in the 1H-NMR spectrum (Figure 7.5).68

2.1.4 Synthesis of (Z)-triethyl((3-methylhex-4-en-1-yn-3-yl)oxy)silane (91)

The last step in the synthesis of the ω-fragment was based on a Z-selective Wittig reaction between the commercially available Wittig-salt 103 and aldehyde 100.

NaHMDS OTES Wittig-salt 103 OTES

O THF/HMPA -78 °C to rt.

100 71% 91 Scheme 2.5 Outline of the Wittig reaction in the final step of the preparation of the ω-fragment 91.

The Wittig-salt 103 was suspended in dry THF and added HMPA. After evacuating and flushing with argon three times, the strong base NaHMDS was added at -78 °C to obtain the non-stabilized ylide, and brought to ambient temperature for one hour. It was important to avoid any access to oxygen in the reaction mixture, in order to avoid the formation of the potential byproducts. Lastly, the aldehyde 100 (dissolved in dry THF) was added dropwise at -78 °C. The reaction mixture was slowly stirred for 18 hours, allowing it to obtain room

40 temperature. After aqueous work-up, the crude product was purified by flash chromatography, and the compound 91 was obtained in 71% yield as a clear oil. The identity of the product was confirmed by 1H-NMR and 13C-NMR analysis, as shown in the appendix (Figure 7.7 and 7.8).

2.1.4.1 NMR interpretation

In the 1H-NMR spectrum (Figure 7.7), the multiplets at 0.76-0.61 ppm (6H) and 0.97 ppm (9H) stem from the methylene groups and methyl groups in the TES-group, respectively. However, the latter signal overlaps with the signal from the methyl group bound to the tertiary carbon, which explains the integration for 12 protons for this signal. Furthermore, the doublet at 1.43 ppm (J = 1.2 Hz, 3H) shows the other methyl group present in the molecule. The multiplets at 2.06 ppm (2H) and 2.51 ppm – 2.32 ppm (3H) shows the two allylic methylene groups. The signal for the proton in the terminal alkyne also occurs in the multiplet signal at 2.51 ppm – 2.32 ppm, which explains the integration for three protons. The multiplet at 5.65 ppm – 5.36 ppm (2H) corresponds to the two protons in the Z-alkene. No signal from the E- isomer was observed in the 1H-NMR spectrum.

CH2 in TES: 0.76-0.61 and 7.1 CH3 in TES: 0.97 and 6.3 5.56- 5.56- 0.97 5.36 5.36 30.6 1.43 134.3 124.1 OTES 14.3 88.5 69.0 2.06 2.51-2.32 2.51-2.32 21.0 42.9 72.0 91

Figure 2.5 The structure shows proposed chemical shifts for 1H-NMR (blue) and 13C-NMR (black) for compound 91.

The 13C-NMR spectrum (Figure 7.8) shows 11 signals, which is in full agreement with the number of carbon atoms present in the molecule. The signals at 6.3 ppm and 7.1 ppm are most likely, as previously mentioned, from the methylene groups and methyl groups in the TES- group. The signals at 14.3 ppm and 30.6 ppm arise from the two methyl groups, and the signals at 21.0 ppm and 42.9 ppm show the two methylene groups present in the molecule. Next, the signal at 69.0 ppm is most likely from tetrasubstituted carbon atom and the signals at 72.0 ppm and 88.5 ppm resonates for the alkyne group. Lastly, the signals at 124.1 ppm and 134.3 ppm are in the expected area for carbons in the alkene group.

41 2.1.4.2 Analytical experiments

GC-analysis was conducted in order to determine the purity of the synthetized compound. The compound 91 proved to be of >98% purity (chromatogram 7.1), which is considered to be pure enough for the intended subsequent coupling reactions. MS characterization shows a base peak of 275.2 mass/charge (Figure 7.26), corresponding with the molecular mass of the sodium adduct of the ω-fragment 91. The measured HRMS (HRESTOFMS) spectra (Figure 7.27) shows a peak at 275.1801 m/z, which is close to the calculated mass of the sodium adduct of the compound 91. The error is 0.3 ppm, and is clearly within the normal accepted 10.0 ppm error criteria. The reported MS-spectrum is in accordance with the previous unpublished MS-spectrum of compound 91.66

2.2 Synthesis of the α –fragment 67

The second fragment derived from the retrosynthetic analysis (Scheme 1.26), namely the α- fragment 67, was achieved through a four step process starting from 4-pentynoic acid (82) as previously reported by Hansen et. al,23 see Scheme 1.21. This afforded 67 in a 24% overall yield. Each step is described in more detail in section 5.2.5 - 5.2.7.

1. n-BuLi, 0 °C 2. ethylene oxide (83) H2 HMPA/Heptane/THF CO2Me Lindlar's catalyst CO2H HO 3. cat. H2SO4, MeOH Heptane/EtOAc 25% 98% 82 84

1. I2, PPh3 Imidazole, CH2Cl2 IPh P CO Me HO CO2Me 3 2 2. PPh3, MeCN, Δ 85 97% 67 Scheme 2.6 Outline of the synthesis of the first α-fragment 67.

The preparation of the first α-fragment 67 started with the alkylation of the dianion of 82 with ethylene oxide 83, which was subjected to further esterification in methanol in the presence of sulphuric acid. Subsequently, the alkyne 84 was reduced using Lindlar’s catalyst and lastly converted from the primary alcohol in the alkene 85 to the corresponding Wittig-salt 67.

42 2.2.1 Synthesis of methyl 7-hydroxyhept-4-ynoate (84)

The first step in the synthesis of the α-fragment started with an alkylation reaction followed by an esterification to obtain compound 84.

1. n-BuLi, 0 °C 2. ethylene oxide (83) HMPA/Heptane/THF CO2Me CO2H HO 3. cat. H2SO4, MeOH

82 25%25 25% 84

Scheme 2.7 Outline of the synthesis of the methyl ester 84.

Commercially available 4-pentynoic acid (82) was dissolved in dry HMPA and n-BuLi was added dropwise at 0 °C. Next, ethylene oxide (83) was added to the dianion obtained from the previous step. The reaction mixture was allowed to reach ambient temperature overnight, before the esterification with catalytic amounts of MeOH and H2SO4 was performed. After refluxing overnight, the crude product was purified using flash chromatography, which afforded the desired methyl ester 84 in 25% yield over two steps as an yellow oil. The identity of the product was confirmed by 1H-NMR and 13C-NMR analysis, as shown in the appendix (Figure 7.9 and 7.10).

2.2.1.1 NMR interpretation

In the 1H-NMR spectrum (Figure 7.9), the multiplet at 2.42-0.23 ppm (2H) and the overlapping multiplet at 2.54-2.43 ppm (4H) arise from three methylene groups present in the molecule. However, the last methylene group appears downfield as a triplet at 3.65 ppm (J = 6.2 Hz, 2H), due to deshielding effects by the directly attached primary alcohol. Lastly, the singlet at 3.69 ppm (3H) corresponds to the deshielded methyl group in the ester.

2.54-2.43 172.7 3.65 14.9 61.3 CO2Me 3.69 80.5 33.8 51.9 HO 77.9 2.42-2.35 2.54-2.43 23.2 84

Figure 2.6 The structure shows proposed chemical shifts for 1H-NMR (blue) and 13C-NMR (black) for compound 84.

43 The 13C-NMR spectrum (Figure 7.10) shows eight signals, which is in agreement with the number of carbon atoms present in the molecule. The signals at 14.9 ppm and 23.2 ppm are most likely from the two methylene groups attached to the alkyne. The signals at 33.8 ppm may arise from the methylene group next to the carbonyl. Next, the signal at 51.9 ppm most likely arises from the methyl group directly attached to oxygen in the ester. The signal at 61.3 ppm corresponds to the deshielded methylene group attached to the alcohol. Furthermore, the signals at 77.9 ppm and 80.5 ppm are most likely from the two carbons in the alkyne. Lastly, the signal at 172.7 ppm is in the expected area for the carbon in the carbonyl group.

Apart from small amounts of impurities from acetone, the obtained NMR-spectra were in accordance with previously reported data.69

2.2.2 Synthesis of methyl (Z)-7-hydroxyhept-4-enoate (85)

The stereoselective reduction of 84 using Lindlar’s catalyst proved troublesome. Several attempts were performed before a successful reduction protocol was achieved.

H2 CO2Me Lindlar's catalyst HO HO CO2Me Heptane/EtOAc 84 98% 85

Scheme 2.8 Outline of the Lindlar’s reduction of the alkyne 84.

Methyl 7-hydroxyhept-4-ynoate (84), dissolved in 30% EtOAc in heptane was evacuated and filled with hydrogen gas to ensure an oxygen free environment. Lindlar’s catalyst was added and reaction mixture was stirred for 20 hours. The alkene 85 was obtained in 98% yield as pale yellow oil. The identity of the product was confirmed by 1H-NMR and 13C-NMR analysis, as shown in the appendix (Figure 7.11 and 7.12).

The Lindlar reaction was performed five times, of which the last two were successful. In the first three attempts, no reduction was observed (NMR-analysis). In the two final and successful attempts, a brand new batch of Lindlar’s catalyst was used, opposed to an old batch used in the initial three. Hence, the quality of the catalyst can most likely explain the failure

44 observed in the first three attempts. All attempts were analyzed continually, using AgNO3- 1 impregnated TLC plates and H-NMR. AgNO3-dipped TLC plates were used because the silver ions form complexes with π-bonds in a varied matter, which will reveal and separate the alkyne from the alkene. However, 1H-NMR will reveal whether an alkene was formed or not, by potentially providing signals between 5 ppm and 7 ppm. These signals were observed in the last two attempts, as seen in the appendix (Figure 7.11).

2.2.2.1 NMR interpretation

In the 1H-NMR spectrum of 85 (Figure 7.11), the signals at 1.82-1.49 ppm (m, 2H), 2.37-2.33 ppm (m, 2H) and 2.40 ppm (d, J = 3.2 Hz, 2H), most likely arise from three methylene groups present in the molecule. The doublet at 3.65 ppm (J = 6.3 Hz, 2H) should correspond to the deshielded methylene group adjacent to the primary alcohol. Next, the singlet at 3.67 ppm (3H) shows the deshielded methyl group in the ester. Lastly, the multiplet at 5.56-5.39 ppm (2H) is in the area where one would expect to observe protons in alkenes.

5.56-5.39 3.65 130.9 5.56-5.39 3.67 62.3 127.3 173.8 51.8 HO CO2Me

3 x CH2: 1.82-1.49, 2.37-2.33, 2.40 22.9, 31.0, 34.0 85 Figure 2.7 The structure shows proposed chemical shifts 1H-NMR (blue) and 13C-NMR (black) for compound 85.

The 13C-NMR spectrum (Figure 7.12) shows eight signals, which is in agreement with the number of carbons present in the molecule. The signals at 22.9 ppm, 31.0 ppm and 34.0 ppm are most likely from three methylene groups present in the molecule. The signal at 51.8 ppm most likely stem from the methyl group in the ester. Next, the signal at 62.3 ppm shows the methylene group adjacent to the alcohol, which appears downfield due to deshielding effects of the primary alcohol. Furthermore, the signals at 127.3 ppm and 130.9 ppm are in the area where one would expect to observe alkenes. Lastly, the signal arising at 173.8 ppm corresponds to the carbonyl carbon in the ester moiety.

The obtained 1H-NMR spectrum should have reported a coupling constant for the alkene signal in order to determine the configuration of the double bound. However, Lindlar catalyst

45 reduces alkynes stereoselectively to cis-alkenes via syn addition.70 Hence the obtained NMR- spectra are in accordance with the previously reported data in the literature.13

2.2.3 Synthesis of methyl (Z)-7-(iodotriphenyl-λ5-phosphanyl)hept-4-enoate (67)

Compound 67 was prepared from the alcohol 85 over two steps. First an Appel reaction was performed in order to obtain the corresponding iodide, which was subsequently converted to the desired Wittig-salt 67.

1. I2, PPh3 Imidazole, CH2Cl2 IPh P CO Me HO CO2Me 3 2 2. PPh3, MeCN, Δ 85 97% 67 Scheme 2.9 Outline of the final step in the synthesis of the first α-fragment 67.

The Appel reaction (iodine, triphenylphosphine and imidazole in CH2Cl2) provided the corresponding alkyl iodide, which was immediately reacted with triphenylphosphine in refluxing acetonitrile. This afforded the Wittig-salt 67 after purification by flash chromatography in 97% yield over two steps as a pale yellow oil. The identity of the product was confirmed by 1H-NMR and 13C-NMR analysis, as shown in the appendix (Figure 7.13 and 7.14).

2.2.3.1 NMR interpretation

In the 1H-NMR spectrum of 67 (Figure 7.13), the multiplet at 7.69-7.60 ppm (15H) arises from the protons in the three aromatic rings in the triphenylphosphine group. The multiplets at 5.80-5.54 ppm (1H) and 5.40-5.32 ppm (1H) correspond to the protons on the alkene. Next, the signals at 3.87-3.72 ppm (m, 2H), 2.55-2.40 ppm (m, 2H), 2.31 ppm (t, J = 7,0 Hz, 2H) and 2.16 ppm (t, J = 6,9 Hz, 2H) show the four methylene groups present in the molecule. Furthermore, the singlet appearing at 3.62 ppm (3H) is most likely caused by the deshielded methyl group in the ester.

46 5.40- 5.80- 3.87- 2.16 5.32 5.52 3.72 23.3 127.7 130.4 33.5 3.62 53.6 IPh3P 2.31 2.55- CO2Me 7.96-7.90 20.6 2.40 173.6 118.2, 130.7, 22.8 134.0, 135.3 67 Figure 2.8 The structure shows proposed chemical shifts for 1H-NMR (blue) and 13C-NMR (black) for compound 67.

The 13C-NMR spectrum (Figure 7.14) shows 12 signals, which is in agreement with the expected number of signals. As coupling is a through-bond effect, the largest coupling constants are seen for the carbon atoms nearest the phosphorus. Hence, the signals arising at 1’ 1 118.2 ppm (d, JCP = 85.8 Hz) and 23.3 ppm (d, JCP = 48.3 Hz) account for the directly attached carbon atoms in the aromatic rings and in the aliphatic region, respectively. The 2’ 3’ signals arising at 130.7 ppm (d, JCP = 12.5 Hz), 134.0 ppm (d, JCP = 10.0 Hz) and 135.3 4’ ppm (d, JCP = 3.0 Hz) are in the area where we would expect aromatic ring carbon atoms to 3 resonate. The signals appearing at 127.7 ppm (d, JCP = 15.7 Hz) and 130.4 ppm are characteristic for the olefinic carbons. Next, the signal shifted downfield at 53.6 ppm 2 corresponds to the methyl group in the ester. However, the three signals at 20.6 ppm (d, JCP = 3.7 Hz), 22.8 ppm and 33.5 ppm, correspond to the three remaining methylene groups. Lastly, the remaining signal at 173.6 shows the carbon in the carbonyl group.

The obtained NMR-spectra are in agreement with those previously reported in the literature.69

2.3 Synthesis of the α-fragment 96

Next, the other α-fragment 96 from the retrosynthetic analysis (Scheme 1.27), was achieved in three steps from 104 in a 44% overall yield. Each step of the synthesis is described in more detail in section 5.2.11 – 5.2.13.

47 NaH O tert-butyl 2-bromoacetate (105) HO O OH HO O THF 0 °C 104 106 52% 1. PBr3, Et2O 1. I2, PPh3 2. PPh3, MeCN, Δ Imidazole, CH2Cl2 2% 2. PPh3, MeCN, Δ 85%

O O O O IPh P O BrPh3P O 3 97 96 Scheme 2.10 Outline of the synthesis of the α-fragment 96.

The synthesis commenced with the reaction between the anion of 104 and the alpha-halo ester moiety in the tert-butyl acetate 105 group, which successfully resulted in compound 106. Lastly, the primary alcohol was converted to the corresponding bromide 97 and iodide Wittig- salt 96. The bromide-salt 97 was attained in low yields and the iodide-salt 96 was therefore more preferable to synthetize, as the yield was significantly higher.

2.3.1 Synthesis of tert-butyl 2-(4-hydroxybutoxy)acetate (106)

The desired compound 106 desired was obtained by deprotonating one hydroxyl group in the diol 104 and alkylating it with tert-butyl 2-bromoacetate (105).

NaH O tert-butyl 2-bromoacetate (105) HO O OH HO O THF 0 C 104 ° 106 52%

Scheme 2.11 Outline of the synthesis of the desired alcohol 106.

The commercially available diol 104 was dissolved in dry THF and NaH was added at 0 °C. After one hour, tert-butyl 2-bromoacetae (105) was added dropwise and the reaction was allowed to reach ambient temperature overnight. After aqueous work-up, the crude product was purified by flash chromatography, affording the desired compound 106 in 52% yield, as a viscous, yellow oil. The product was analyzed by 1H- and 13C-NMR, as shown in the appendix (Figure 7.18 and 7.19).

48 2.3.1.1 NMR interpretation

In the 1H-NMR spectrum of 106 (Figure 7.18), the singlet at 1.48 ppm (9H) arises from the three methyl groups in the tert-butyl group. Next, the multiplet at 1.77-1.64 ppm (4H) most likely arises from two methylene groups in the molecule. The triplet at 3.56 ppm (J = 5,5 Hz, 2H) should correspond to the deshielded methylene group adjacent to the oxygen in the ether moiety. Furthermore, the triplet at 3.68 ppm (J = 5,7 Hz, 2H) is most likely consistent with the methylene group adjacent to the primary alcohol. Lastly, the singlet at 3.96 ppm (2H) may correlate with the methylene group between the ether group and the carbonyl.

1.48 30.0 1.48 3.68 1.77-1.64 O 30.0 62.8 26.7 O 169.7 HO O 81.8 1.48 1.77-1.64 3.56 3.96 30.0 28.2 68.9 71.7

106

Figure 2.9 The structure shows proposed chemical shifts for 1H-NMR (blue) and 13C-NMR (black) for compound 106.

The 13C-NMR spectrum (Figure 7.19) shows eight signals, which is in agreement with the expected number of signals. The signals at 26.7 ppm and 28.2 ppm may occur due to two of the methylene groups present in the molecule. Next, the signal appearing at 30.0 ppm is most likely arising from the methyl groups in the tert-butyl group. Furthermore, the signals at 62.8 ppm, 68.9 ppm and 71.7 ppm stem from the deshielded carbons in the three methylene groups adjacent to the oxygen. The signal at 81.8 ppm is most likely caused by the deshielded carbon atom in the tert-butyl group. Finally, the signal appearing downfield at 169.7 ppm is in the area where one would expect to observe the carbon in the carbonyl.

2.3.2 Synthesis of tert-butyl 2-(4-(bromotriphenyl-λ5- phosphanyl)butoxy)acetate (97)

The syntheses of the corresponding alkyl bromide and the related Wittig-salt 97 were performed in two steps.

O 1. PBr3, Et2O O O O HO O BrPh P O 2. PPh3, MeCN, Δ 3 106 2% 97 Scheme 2.12 Outline of the two-step synthesis towards the Wittig-salt 97.

49 Firstly, the primary alcohol 106 was dissolved in dry Et2O and PBr3 was added at 0 °C. After aqueous work-up, the crude product (53 mg) was dissolved in MeCN and added triphenylphosphine. The reaction mixture was refluxed overnight, and the crude product was purified by flash chromatography to give the desired product 97 in 2% yield, over two steps as a pale, viscous and yellow oil. The product was analyzed by 1H- and 13C-NMR, as presented in the appendix (Figure 7.20 and 7.21).

2.3.2.1 NMR interpretation

In the 1H-NMR spectrum of 97 (Figure 7.20), the singlet arising at 1.46 ppm (9H) corresponds to the three methyl groups in the tert-butyl group. The overlapping multiplet at 1.85 ppm (4H) most likely occur due to two of the methylene groups present in the molecule. On the other hand, the overlapping multiplet at 3.58 ppm (4H) most likely corresponds to two of the methylene groups present in the compound 97, as shown in Figure 2.9. The singlet at 3.98 ppm (2H), should be congruous with the deshielded methylene groups. Lastly, the multiplet arising at 8.15-7.68 ppm (15H) is in the area where one would expect to observe aromatic protons from the triphenyl moiety.

1.46 28.3 3.58 1.85 O 1.46 28.3 22.1 20.7 O BrPh P 171.9 O 82.9 1.46 3 1,85 3,58 3.98 28.3 8.15-7.68 30.6 69.3 70.8 120.0, 131.5, 134.9, 136.2

Figure 2.10 The structure shows proposed chemical shifts for 1H-NMR (blue) and 13C-NMR (black) for compound 97.

The 13C-NMR spectrum (Figure 7.21) shows 12 signals, which is in agreement with the 1’ expected number of signals. The signals arising at 120.0 ppm (d, JCP = 86.4 Hz), and 22.1 1 ppm (d, JCP = 51.6 Hz) account for the directly attached carbon atoms in the aromatic rings and in the aliphatic region, respectively. As with compound 67, the closer the carbon atom is to the phosphorus nucleus, the larger the coupling constant will appear. Hence, the signals 2’ 3’ 4’ arising at 131.5 ppm (d, JCP = 12.6 Hz), 134.9 ppm (d, JCP = 9.9 ) and 136.2 ppm (d, JCP = 3.1 Hz), Hz), are in the area where one would expect aromatic rings to resonate. The signals

50 2 3 appearing at 30.6 ppm (d, JCP = 17.1 Hz) and 20.7 ppm (d, JCP = 4.1 Hz) arise from two of the methylene groups nearest the phosphorus nucleus. Next, the signals shifted downfield at 69.3 ppm and 70.8 correspond to the deshielded methylene groups. The signals at 28.3 ppm and 82.9 ppm account for the methyl groups and the deshielded carbon atom in the tert-butyl group, respectively. Finally, the signal appearing at 171.9 ppm is in the area where we would expect to observe the carbon in the carbonyl to resonate. Both NMR-spectra show solvent 71 impurities caused by CH2Cl2 and acetone.

However, the main reason for the poor yield obtained in this synthesis could possibly be due to the extraction step. During the extraction of the bromide, formation of an emulsion was observed, which may have resulted in a substantial loss of the desired compound 97. The iodide and the corresponding Wittig-salt 96 was therefore synthetized and obtained in higher yields, as described in the next section.

2.3.3 Synthesis of tert-butyl 2-(4-(iodotriphenyl-λ5- phosphanyl)butoxy)acetate (96)

The Appel reaction was utilized to make the iodide 107, which was quickly converted to the corresponding Wittig-salt 96, due to potential stability issues.

O 1. I2, PPh3 O Imidazole, CH Cl O 2 2 O HO O IPh3P O 2. PPh3, MeCN, Δ

106 85% 96

Scheme 2.13 Outline of the synthesis of the desired Wittig-salt 96.

The primary alcohol 106 was dissolved in dry CH2Cl2, followed by the addition of triphenylphosphine and imidazole. After placing the reaction mixture in a cooling bath, iodine was added and the mixture stirred for 45 minutes. After aqueous work-up, the crude product was purified by flash chromatography and the obtained product was a viscous clear oil, which was directly converted to the Wittig-salt 96 by dissolving the obtained iodide in MeCN and adding triphenylphosphine. After refluxing the reaction mixture for 12 hours, the crude product was purified by flash chromatography to give the desired compound in 85% yield

51 over two steps, as a viscous, yellow oil. Both the iodide 107 and the Wittig-salt 96 were analyzed by 1H- and 13C-NMR, as presented in the appendix (Figure 7.22 - 7.25).

2.3.3.1 NMR interpretation of the iodide 107

In the 1H-NMR spectrum of the iodide 107 (Figure 7.22), the singlet at 1.48 ppm (s, 9H) arises from the three methyl groups in the tert-butyl group. Next, the multiplet at 1.73 ppm (m, 2H) and the singlet at 1.94 ppm (m, 2H) most likely arise from two of the methylene groups in the molecule. The signal at 3.24 ppm (t, J = 6,9 Hz, 2H) should correspond to the methylene group adjacent to the iodide. Furthermore, the triplet at 3.53 ppm (t, J = 6,1 Hz, 2H) is most likely consonant with the methylene group adjacent to the oxygen. Lastly, the singlet at 3.94 ppm (s, 2H) may correspond to the methylene group between the ether group and the carbonyl.

1.48 30.0 3.24 1.73 O 62.7 26.7 1.48 O 30.0 I 169.7 O 81.8 1.48 1.94 3.53 3.94 30.0 28.2 68.9 71.7

107

Figure 2.11 The structure shows proposed chemical shifts for 1H-NMR (blue) and 13C-NMR (black) of the iodide 107.

The 13C-NMR spectrum (Figure 7.23) shows eight signals, which is in agreement with the expected number of signals. The signals at 26.7 ppm and 28.2 ppm may occur due to two of the methylene groups present in the molecule. Next, the signal appearing at 30.0 ppm is most likely arising from the methyl groups in the tert-butyl group. Furthermore, the signals at 62.7 ppm, 68.9 ppm and 71.7 ppm arise from the deshielded carbons in the three methylene groups adjacent to the oxygen and the iodide. The signal at 81.8 ppm is most likely caused by the deshielded carbon atom in the tert-butyl group. Finally, the signal appearing downfield at 169.7 ppm is in the area where one would expect to observe the carbon in the carbonyl.

2.3.3.2 NMR interpretation for the Wittig-salt 96

In the 1H-NMR spectra of the corresponding Wittig-salt 96 (Figure 7.24), the multiplet arising at 1.60 – 1.42 ppm (9H) corresponds to the three methyl groups in the tert-butyl group. The overlapping multiplet at 2.16 – 1.72 ppm (4H) most likely occur due to two of the methylene

52 groups present in the molecule. However, the multiplet at 3.75 – 3.53 ppm (4H) should correspond to two deshielded methylene groups. The multiplet at 4.05 ppm (2H) is most likely from the last deshielded methylene group. Lastly, the multiplet at 8.08 – 7.72 ppm (15H) corresponds to the protons present in the triphenylphosphine group.

1.60-1.42 28.4 2.16-1.72 O 1.60-1.42 22.3 20.7 28.4 O 1.60-1.42 IPh3P 171.9 O 82.9 2.16-1.72 69.3 70.9 28.4 8.08-7.72 30.6 120.0, 131.5, 134.9, 136.2 3 x Deshielded methylene groups: 3.75-3.53, 4.05 96

Figure 2.12 The structure shows proposed chemical shifts for 1H-NMR (blue) and 13C-NMR (black) for compound 96.

The 13C-NMR spectrum (Figure 7.25) shows 12 signals, which is in agreement with the 1’ expected number of signals. The signals at 120.0 ppm (d, JCP = 86.4 Hz), 131.5 ppm (d, 2’ 3’ 4’ JCP = 12.9 Hz), 134.9 ppm (d, JCP = 10.4 Hz) and 136.2 ppm (d, JCP = 3.0 Hz) resonate for the carbon atoms in the aromatic rings in triphenylphosphine, in the order closest to the 1 2 phosphorus atom. However, the signals at 22.3 ppm (d, JCP = 51.4 Hz), 30.6 ppm (d, JCP = 3 16.9 Hz) and 20.7 ppm (d, JCP = 4.1 Hz) in the aliphatic region corresponds to the methylene groups, in the order closest to phosphorus. Next, the signals shifted downfield at 69.3 ppm and 70.9 ppm corresponds to the deshielded methylene groups. The signals appearing at 28.4 ppm and 82.9 ppm stem from the methyl groups and the tetrasubstituted carbon atom in the tert-butyl group, respectively. Lastly, the signal appearing at 171.9 ppm resonates for the carbon in the ester group.

Characterization and analytical techniques could unfortunately not be performed due to the limited timeframe available.

2.4 Synthesis of the middle-fragment 73 and Wittig reaction

The middle-fragment 73 from both retrosynthetic analyses (Scheme 1.26 and 1.27), was achieved by a DIBAL-H reduction in one step from 81. The synthesis is described more detailed in section 5.2.8.

TBSO O S DIBAL-H TBSO -78 °C Br N S Br O CH2Cl2 81 73 52% 1. NaHMDS, HMPA, THF, -78 °C 2. Wittig-salt 67

OTBS

Br CO2Me 87 Scheme 2.14 Outline of the synthesis of the middle-fragment 73 and the Wittig coupling reaction.

2.4.1 Synthesis of (R,4E,6E)-7-bromo-3-((tert-butyldimethylsilyl)oxy)-1- ((S)-5-isopropyl-2-thioxoimidazolidin-1-yl)hepta-4,6-dien-1-one (73)

Compound 81 had already been prepared in our research group as shown in Scheme 1.20. The vinyl bromide 81 was dissolved in dry CH2Cl2 and added DIBAL-H at -78 °C until the removal of the entire auxiliary was achieved by reduction to the corresponding aldehyde 73. After flash chromatography, the desired compound was obtained in 52% yield as a yellow oil. The DIBAL-H reduction was performed three times in total. Note that only the highest yield obtained is reported in this thesis. The product was analyzed by 1H- and 13C-NMR as shown in the appendix (Figure 7.15 and 7.16).

TBSO O S DIBAL-H TBSO -78 °C Br N S Br O CH2Cl2 81 73 52%

Scheme 2.15 Outline of the synthesis of the middle-fragment 73.

2.4.1.1 NMR interpretation

In the 1H-NMR spectrum of compound 73 (Figure 7.15), the multiplet that occurs at 0.15 to - 0.14 ppm (6H) and the singlet at 0.88 ppm (9H) arise from the five methyl groups in the TBS-

54 group. The multiplet at 2.73-2.40 ppm (2H) corresponds to the methylene group adjacent to the aldehyde. Next, the singlet at 5.30 ppm (1H) shows the proton in the carbinol atom directly attached to the TBS-group, which explains the downfield appearance due to deshielding. The signals arising at 5.76 ppm (dd, J = 15.2, 6.7 Hz, 1H), 6.13 ppm (dd, J = 14.0, 10.6 Hz, 1H), 6.34 ppm (dd, J = 13.5, 10.8 Hz, 1H) and 6.69 ppm (dd, J = 13.5, 10.9 Hz, 1H) arises from the four protons in the trans alkenes. This can be confirmed by the coupling constants, which are in the expected area for trans alkenes having two vicinal protons. Aldehyde proton typically resonates in the 9.5-10.5 ppm region, which may explain the last triplet at 9.76 ppm (J = 2.3 Hz, 1H).

TBSO 9.76 5.30 68.2 200.7

Br 2.73-2.40 O 51.2 dimethyl in TBS: 0.15- -0.14 and -4.6 tert-butyl in TBS: 0.88 and 17.9, 25.6 Alkene groups: 5.76, 6.13, 6.34, 6.69 and 109.3, 127.3, 135.9, 136.3

73 Figure 2.13 The structure shows proposed chemical shifts for 1H-NMR (blue) and 13C-NMR (black) for compound 73.

The 13C-NMR spectrum (Figure 7.16) shows ten signals, which is in agreement with the number of carbon atoms expected. The signals at -4.6 ppm, 17.9 ppm and 25.6 ppm are most likely from the five methyl groups and the tetrasubstituted carbon in the TBS-group. Furthermore, the signal at 51.2 is most likely from the methylene group adjacent to the aldehyde. The signal at 68.2 ppm probably arises from the carbon directly attached to the TBS-group. Next, the signals at 109.3 ppm, 127.3 ppm, 135.9 ppm and 136.3 ppm shows the two alkenes present in the molecule. Lastly, the remaining signal at 200.7 ppm shows the aldehyde. The obtained NMR-spectra are in agreement with those reported in the literature.13

2.4.2 Attempted Wittig coupling reaction

The first attempt of the assembly of compound 67 and compound 73 was performed, as previously reported, by Hansen and co-workers.23 The attempted synthesis is described in more detail in section 5.2.9 and 5.2.10.

55 1. NaHMDS, HMPA, THF, OTBS -78 °C IPh3P CO2Me Br CO2Me 67 87 2. Aldehyde 73 Scheme 2.16 Outline of the attempted assembly of fragment 67 and 73.

2.4.3 Attempted synthesis of methyl (R,4Z,7Z,11E,13E)-14-bromo-10-((tert- butyldimethylsilyl)oxy)tetradeca-4,7,11,13-tetraenoate (87)

The Wittig-salt 67 was dissolved in HMPA and dry THF and cooled to -78 °C before the base NaHMDS was added. Subsequently, after evacuation of the reaction flask and flushing with argon, the freshly prepared 73 was added and the reaction mixture was stirred for 24 h. After preparation of the sample, TLC-analysis was performed to determine if the olefination reaction had been successful. This could unfortunately not be observed. The obtained product was analyzed by 1H-NMR (Figure 7.17) and the product identity was not confirmed.

1. NaHMDS, HMPA, THF, -78 °C OTBS

IPh3P CO2Me Br CO2Me 67 2. Aldehyde 73 87

1. NaHMDS, HMPA, THF, -78 °C 2. aldehyde 73 Br O 108 Scheme 2.17 Outline of the attempted synthesis of compound 87.

The compound that was obtained during this process is probably 108. This might be explained by the fact that the Wittig-salt was not soluble in the reaction medium, leading to excess NaHMDS base present at the time when the aldehyde was added. This may in turn lead to elimination of the OTBS moiety via an E1cB mechanism, giving aldehyde 108 in substantial amounts. This leads us most likely to the formation of the conjugated compound 108.

Alternatively, after the deprotonation of one of the α-hydrogens, a Claisen condensation of the ester moiety present in the Wittig-salt 67 may have occurred, affording β-ketoesters. Dimerization of the Wittig-fragment may also occur in the presence of oxygen and might be a contributing factor to the observed outcome72. However, these two later suggestions are less

56 probable since the 1H-NMR nearly confirmed the identity of compound 108 and which was formed in high yield.

2.4.3.1 NMR interpretation of compound 108 In the 1H-NMR (Figure 7.17), the signals at 6.23 ppm (dd, J = 15.3, 7.9 Hz, 1H), 6.70 – 6.40 ppm (m, 1H), 6.84 ppm (d, J = 10.6 Hz, 1H), 7.20 – 6.99 ppm (m, 1H) and 7.85 – 7.63 ppm (m, 2H) are most likely arising from the trans triene. Next, the signal at 9.59 ppm (d, J = 7.9 Hz, 1H) is in the area where we would expect to observe the proton in an aldehyde group.

9.59 Br O H in alkenes: 6.23, 6.70-6.40, 6.48, 7.20-6.99, 7.85-7.63

108

Figure 2.14 The structure shows proposed chemical shifts for 1H-NMR (blue) and 13C-NMR (black) for compound 108.

58 3 Summary and future work

The decisive factor when it comes to not finalizing this project, was the limited time available to complete all the synthetic work. The overall aim of this project was to perform a total synthesis of the defined PD1-analogs 94 and 95, which possibly would show to enhance and sustain the biological effects observed by PD1 (8), by avoiding early and rapid metabolism in vivo. No biological testing could be performed, as the intended total synthesis could not be completed within the allocated timeframe. However, the main fragments necessary for the final coupling reactions, which would yield the end products, are all synthetized. These fragments include the ω-fragment 91, the middle fragment 73 and the two α-fragments 67 and 96.

In the synthetic pathway to the desired PD1-analog 94, the three necessary intermediary fragments were successfully synthetized and their identity have been confirmed. The remaining reaction steps to complete the synthesis are the Wittig olefination, Sonogashira coupling reaction to afford compound 109, deprotection of the alcohols, Lindlar’s reduction of the alkyne 110 and finally hydrolysis of the ester 111 (Scheme 3.1).

1. NaHMDS, OTBS HMPA, THF, -78 °C IPh3P CO2Me Br CO2Me 67 2. aldehyde 73 87

Pd(PPh3)4, CuI, Et2NH, alkyne 91

OH OTBS HO CO Me TBSO CO M 2 e 2 110 TBAF, THF, 0 °C 109

1. H2, Lindlar´s catalyst EtOAc/pyridine/1-octene

LiOH (aq.), OH OH MeOH, 0 °C 94 CO2R

111

Scheme 3.1 Outline of the final steps of the synthesis of analog 94.

59 The remaining reaction steps for the completion of the synthesis of analog 95, are nearly identical to the previous analog 94 (Scheme 3.2).

O 1. NaHMDS, OTBS O O HMPA, THF, -78 °C O IPh3P O Br 112 O 2. aldehyde 73 96

Pd(PPh3)4, CuI, Et2NH, alkyne 91

OH O OTBS O O O HO O TBSO O 114 113 TBAF, THF, 0 °C

1. H2, Lindlar´s catalyst EtOAc/pyridine/1-octene

LiOH (aq.), OH OH O MeOH, 0 °C O 95 O 115

Scheme 3.2 Outline of the final steps in the synthesis of analog 95.

60 4 Conclusion

Analogs of novel SPMs will be useful for the development of a potential new class of anti- inflammatory drugs in the future. Their well-studied and essential biological effects are thought to be important in the treatment of several inflammatory diseases. The SPMs have shown to be highly potent and this further adds to the huge potential of new drugs in this therapeutic area.

Reported herein are the synthesis of the ω-fragment 91, two α-fragments 67 and 96 and a middle fragment 73, as final intermediates for a potential new drug lead. Depending on the various results after the assembly of the different fragments, potential future work on the development of new analogs, and hopefully new drug candidates, will need to be evaluated.

62 5 Experimental chemistry

5.1 General

Unless noted otherwise, all commercially available solvents and reagents were used as purchased without any further purification. Thin layer chromatography (TLC) was performed on silica gel 60 F254 aluminum-backed plates fabricated by Merck. Flash column chromatography was performed on silica gel 60 (40-63 µm, Fluka) produced by Merck. NMR spectra were recorded using Bruker AVII400 or Bruker AVIII400 (400 MHz 1H, 100 MHz 13C and 300 MHz).

Coupling constants (J) are reported in Hertz and chemical shifts are reported in parts per 1 13 million (δ), relative to CDCl3 (7,27 ppm for H-NMR and 77,00 ppm for C-NMR) and 1 13 MeOD-d4 (3,31 ppm and 4,78 ppm for H-NMR J = 1,7, and 49,15 ppm for C-NMR).

Mass spectra were recorded at 70 eV on a Waters Prospec Q, with EI, ES or CI as methods of ionization. High-resolution-mass-spectra were recorded at the department of chemistry, University of Oslo, on Waters Prospec Q, using ES or EI as methods of ionization,

GC-analyses were performed on an Agilent 7820A with a FID-detector and HP-5 capillary column, with helium as the carrier gas and by applying the conditions stated.

5.2 Synthesis of compounds

5.2.1 Synthesis of 3-methylpent-4-yne-1,3-diol (98)

O Ethynylmagnesium bromide 101 OH OH THF HO 0 °C to rt.

102 98

63 The ketone 102 (2,00 g, 22,7 mmol) was placed in a 250 mL round bottomed flask and dissolved in dry THF (100 mL) and cooled to 0 °C. Ethynylmagnesium bromide 101 (91 mL, 0,75 mol) was added dropwise. When the deprotonation of the alcohol were complete, the mixture became a milky suspension. As more Grignard reagent was added, the mixture became transparent. When the addition was completed, the mixture turned to a yellow solution, which was allowed to attain ambient temperature over 48 hours. The reaction mixture was cooled to 0 °C and quenched by addition of a saturated aqueous solution of

NaH2PO4 (10 mL). The aqueous phase was extracted with Et2O (2 x 15 mL), the combined organic extracts were dried (NaSO4), and the solvent was evaporated in vacuo. The residue was purified by flash chromatography (silica gel, EtOAc/Hept 1:1) and the product was obtained as a clear yellow oil. Yield: 1,01 g (51%). TLC (heptane/EtOAc, 1:1, visualized with

KMnO4-stain): Rf = 0,19. The reported spectroscopic data are in full agreement with those reported in the unpublished protocol of our group.66 1H-NMR (400 MHz, Chloroform-d) δ 4.17 (m, 1H), 3.92 (m, 1H), 2.88 (s, 2H), 2.50 (s, 1H), 2.00 (m, 1H), 1.83 (m, 1H), 1.54 (s, 3H). 13C-NMR (101 MHz, Chloroform-d) δ 87.5, 72.4, 69.1, 61.1, 43.8, 31.1.

5.2.2 Synthesis of 3,3,9,9-tetraethyl-5-ethynyl-5-methyl-4,8-dioxa-3,9- disilaundecane (99)

OH TESOTf, 2,6-Lutidine OTES HO CH2Cl2 TESO -78 °C to rt.

98 99

3-Methylpent-4-yne-1,3-diol (98) (0,94 g, 8,21 mmol) was placed in a 250 mL round bottomed flask, and dissolved in CH2Cl2 (93,7 mL) and cooled to -78 °C. After 10 minutes, 2,6-lutidine (5,54 g, 51,7 mmol) was added and then TESOTf (6,51 g, 24,6 mmol) was added slowly. The mixture was allowed to attain ambient temperature for 48 hours. The mixture was quenched by addition of saturated aqueous solution NH4Cl (25 mL) and diluted with CH2Cl2

(25 mL). The phases were separated and the aqueous phase was extracted with CH2Cl2 (3 x

20 mL). The combined organic phase was dried over Na2SO4, and the solvent evaporated in vacuo. The residue was purified with flash chromatography (silica gel, Hept/EtOAc 95:5 to Hept/EtOAc 95,5:0,5) and the product was obtained as a clear oil. Yield: 0,56 g (59%). The

64 reported spectroscopic data are in full agreement with those reported in the unpublished protocol of our group.66 1H-NMR (400 MHz, Chloroform-d) δ 3.84 (t, 2H), 2.41 (s, 1H), 1.94 (d J = 8.1, 6.4 Hz, 2H), 1.48 (s, 3H), 0.96 (td, J = 7.9, 3.1 Hz, 18H), 0.79 – 0.51 (m, 12H). 13 C-NMR (101 MHz, CDCl3) δ 88.0, 72.1, 67.6, 59.7, 47.8, 31.6, 7.1, 4.6.

5.2.3 Synthesis of 3-methyl-3-((triethylsilyl)oxy)pent-4-ynal (100)

DMSO OTES Oxalyl chloride OTES TESO O CH2Cl2 Et3N -78 °C to 20 °C

99 100

DMSO (1,2 mL) was placed in a 100 mL round bottomed flask, and dissolved in dry CH2Cl2 (4 mL) and cooled to -78 °C. Oxalyl chloride (0,72 mL, 8,4 mmol) was added carefully dropwise. After 15 minutes, a solution of silyl ether 99 (0,66 g, 1,93 mmol) in CH2Cl2 (3,0 mL) was added to the flask. The mixture was allowed to stir for one hour. After one hour the flask was taken to -20 °C and the reaction mixture was stirred at that temperature for 45 minutes. Later, the reaction was cooled to -78 °C and Et3N (28,9 mmol, 4 mL) was added in one go. The mixture became a milky suspension. The mixture was heated to ambient temperature, treated with water and diluted with CH2Cl2 (20 mL). The phases were separated and the aqueous phase was extracted with CH2Cl2 (4 x 15 mL). The organic phases were combined and dried over Na2SO4 for 20 minutes. The solvent was evaporated in in vacuo to give the crude product, as a clear yellow liquid. The residue was purified by column flash chromatography on silica (silica gel, Hept/Et2O 95:5), which afforded a yellow oil. Yield:

0,25 g (38%). TLC (Hept/EtOAc 1:1, visualized with KMnO4-stain): Rf = 0,78. The reported spectroscopic data are in full agreement with those reported in the unpublished protocol of our group.66 1H-NMR (400 MHz, Chloroform-d) δ 9.89 (t, J = 2.9 Hz, 1H), 2.62 (d, J = 2.9 Hz, 13 2H), 2.57 (s, 1H), 1.58 (s, 3H), 0.96 (t, 9H), 0.69 (m, 6H). C-NMR (101 MHz, CDCl3) δ 201.7, 86.6, 73.9, 66.4, 57.3, 31.6, 7.0, 6.1.

65 5.2.4 Synthesis of (Z)-triethyl((3-methylhex-4-en-1-yn-3-yl)oxy)silane (91)

NaHMDS OTES Wittig-salt 103 OTES

O THF/HMPA -78 °C to rt. 100 91

The Wittig-salt 103 (0,304 g, 0,79 mmol) was suspended in dry THF (7,5 mL). HMPA (1,24 mL, 7,13 mmol) was added and the suspension was degassed and purged three times with argon. Subsequently, the suspension was cooled to -78 °C and then NaHMDS (1,31 mL, 6,43 mmol) was added in a dropwise manner. The reaction mixture changed color during the addition from yellow to green. To ensure homogeneity, the reaction mixture was brought to ambient temperature for one hour. The reaction mixture was recooled down to -78 °C and the aldehyde 100 (0,170 g, 0,75 mmol), dissolved in dry THF (1,9 mL), were added dropwise. The reaction mixture was allowed to attain ambient temperature over 18 hours. Later, the mixture was quenched with phosphate buffer (pH = 7,2, 4 mL, 0,955 g/mL, saturated). The mixture was diluted with water and extracted with Et2O (4 x 20 mL). The organic phases were combined and dried over Na2SO4, filtered and evaporated in vacuo. The residue was purified with flash chromatography on silica (Hept/Et2O 95:5). The purified product was obtained as a clear oil. Yield: 121 mg (71%). TLC (Hept/Et2O, 95:5, visualized with KMnO4-stain): Rf=0,82. The reported spectroscopic data are in full agreement with those reported in the unpublished protocol of our group.66 1H-NMR (400 MHz, Chloroform-d) δ 5.65 – 5.36 (m, 2H), 2.51 – 2.32 (m, 3H), 2.06 (m, 2H), 1.43 (d, J = 1.2 Hz, 3H), 0.97 (m, 13 12H), 0.76 – 0.61 (m, 6H). C-NMR (101 MHz, CDCl3) δ 134.3, 124.1, 88.5, 72.0, 69.0, 42.9, 30.6, 21.0, 14.3, 7.1, 6.3. HRESTOFMS m/z 275.1801 [M + Na]+ (calculated for

C15H28NaOSi, 275.1802). The chemical purity (98,2%) was determined by GC analysis.

Initial temperature 100 °C, rate 2 °C/min to 150 °C, then 6 °C/min to 300 °C, tr(major) = 8.08 min and tr(minor) = 7.58 min.

66 5.2.5 Synthesis of methyl 7-hydroxyhept-4-ynoate (84)

1. n-BuLi, 0 °C 2. ethylene oxide (83) HMPA/Heptane/THF CO2Me CO2H HO 3. cat. H2SO4, MeOH

82 25%25 84

4-Pentynoic acid (82) (5,00 g, 51,0 mmol, 1 equiv.) was placed in a 500 mL round bottomed flask and dissolved in dry HMPA (100 mL). At 0 °C, n-BuLi (44,9 mL, 2,5 M, 2,2 equiv.) was added in a dropwise manner and the reaction mixture attained a black color. The reaction mixture was then stirred for one hour before ethylene oxide (83) in THF (25 mL, 2,5-3,3 M) was added slowly at 0 °C. The reaction mixture was allowed to attain ambient temperature and was then stirred for 24 hours. The yellow reaction mixture was cooled to 0 °C and water (150 mL) was added. Then, concentrated HCl was added dropwise until the mixture was acidic to litmus. Later, the reaction mixture was extracted with Et2O (3 x 100 mL) and EtOAc

(2 x 100 mL). The organic phases were combined and dried over MgSO4, filtered and concentrated in vacuo to give the acid contaminated with HMPA.

The mixture was dissolved in dry MeOH (250 mL) and a few drops of H2SO4. The reaction mixture was refluxed for 12 hours and allowed to attain ambient temperature, before solid

NaHCO3 (5,00 g) and saturated aqueous NaHCO3 (50 mL) were added. The aqueous phase was extracted with Et2O (4 x 100 mL), and the organic phases were combined dried over

Na2SO4, and concentrated in vacuo. The crude product was purified by column chromatography on silica (Hept/EtOAc 7:3) to give the product as a yellow oil. Yield: 1,82 g

(25% for two steps starting from 77). TLC (Hept/EtOAc 5:5, visualized with KMnO4-dip): Rf = 0,27. The reported spectroscopic data are in full agreement with those reported in the literature.50,69 1H-NMR (400 MHz, Chloroform-d) δ 3.69 (s, 3H), 3.65 (t, J = 6.2 Hz, 2H), 2.54 – 2.43 (m, 4H), 2.42 – 2.35 (m, 2H). 13C-NMR (101 MHz, Chloroform-d) δ 172.7, 80.5, 77.9, 61.3, 51.9, 33.8, 23.2, 14.9.

67 5.2.6 Synthesis of methyl (Z)-7-hydroxyhept-4-enoate (85)

H2 CO2Me Lindlar's catalyst HO HO CO2Me Heptane/EtOAc 84 85

Methyl 7-hydroxyhept-4-ynoate (84) (200 mg, 1,28 mmol) was dissolved in 30% EtOAc in heptane (10 mL) in a 50 mL round bottomed flask. The flask was evacuated and filled with argon gas three times. Lindlar’s catalyst (50 mg) was added and the reaction flask was evacuated and filled with hydrogen gas. The reaction mixture was stirred for 20 h. The alkene was filtered through a layer of celite and then concentrated in vacuo to give the desired product as a pale yellow oil. Yield: 195 mg (98%). TLC (heptane/EtOAc 5:5, KMnO4 stain):

Rf = 0,25. The reported spectroscopic data are in full agreement with those reported in the literature.13,50 1H-NMR (400 MHz, Chloroform-d) δ 5.56 – 5.39 (m, 2H), 3.67 (s, 3H), 3.65 (d, J = 6.3 Hz, 2H), 2.40 (d, J = 3.2 Hz, 2H), 2.37 – 2.33 (m, 2H), 1.82 – 1.49 (m, 2H). 13C-NMR (101 MHz, Chloroform-d) δ 173.8, 130.9, 127.3, 62.3, 51.8, 34.0, 31.0, 22.9.

5.2.7 Synthesis of methyl (Z)-7-(iodotriphenyl-λ5-phosphanyl)hept-4-enoate (67)

1. I2, PPh3 Imidazole, CH2Cl2 IPh P CO Me HO CO2Me 3 2 2. PPh3, MeCN, Δ 85 67

The alkene 85 (700 mg, 4,43 mmol) was dissolved in dry CH2Cl2 (47 mL) in a round- bottomed flask, followed by addition of triphenylphosphine (1,78 g, 6,81 mmol) and imidazole (467 mg, 6,86 mmol). The reaction flask was placed in a cooling bath (ice, water and salt) for 15 minutes before iodine (1,75 g, 6,88 mmol) was added in one portion with rapid stirring. The reaction mixture attained a yellow color. The reaction mixture was allowed to stir for 15 minutes before the cooling bath was removed and the reaction mixture stirred at ambient temperature for another 2 h. A saturated solution of aqueous Na2SO3 (4,7 mL) was

68 added and the aqueous phase was extracted with CH2Cl2 (2 x 5 mL), dried over Na2SO4, filtered and concentrated in vacuo, which resulted in a viscous yellow oil. The oil was run through a short silica column (92:8 Hept/EtOAc) to yield a pale yellow oil. The resulting yellow oil was dissolved in dry MeCN (47 mL), and triphenylphosphine (1,98 g, 7,57 mmol) was added and the reaction mixture was refluxed for 12 hours under argon atmosphere. The reaction mixture was cooled, concentrated in vacuo and purified by flash chromatography

(CH2Cl2 until all triphenylphosphine was out of the column followed by 5% MeOH in

CH2Cl2) to yield the desired product as a viscous clear pale yellow oil. Yield: 2,27 g (97%).

TLC (CH2Cl2/MeOH 95:5, KMnO4 stain): Rf = 0,23. The reported spectroscopic data are in full agreement with those reported in the literature.50,69 1H-NMR (400 MHz, Chloroform-d) δ 7.96 – 7.60 (m, 15H), 5.80 – 5.54 (m, 1H), 5.40 – 5.32 (m, 1H), 3.87 – 3.72 (m, 2H), 3.62 (s, 3H), 2.55 – 2.40 (m, 2H), 2.31 (t, J = 7,0 Hz, 2H), 2.16 (t, J = 6,9 Hz, 2H). 13C-NMR (101 4’ 3’ MHz, Chloroform-d) δ 173.6, 135.3 (d, JCP = 3.0 Hz), 134.0 (d, JCP = 10.0 Hz), 130.7 (d, 2’ 3 1’ JCP = 12.5 Hz), 130.4, 127.7 (d, JCP = 15.7 Hz), 118.2 (d, JCP = 85.8 Hz), 53.6, 33.5, 23.3 1 2 (d, JCP = 48.3 Hz), 22.8, 20.6 (d, JCP = 3.7 Hz).

5.2.8 Synthesis of (R,4E,6E)-7-bromo-3-((tert-butyldimethylsilyl)oxy)-1- ((S)-5-isopropyl-2-thioxoimidazolidin-1-yl)hepta-4,6-dien-1-one (73)

TBSO O S DIBAL-H TBSO -78 °C Br N S Br O CH2Cl2 81 73

Vinyl bromide 81 (246 mg, 0,513 mmol) was dissolved in dry CH2Cl2 (10 mL). DIBAL-H (0,62 mL, 1M in hexane) was added in a dropwise manner at -78 °C. The reaction mixture was stirred for one hour. The mixture was tested with TLC (Hept/EtOAc 9:1) and some starting material was observed. More DIBAL-H (0,12 mL) was added and the reaction mixture was stirred for one more hour. TLC-analysis indicated that there still was starting material present. Therefore more DIBAL-H (0,06 mL) was added and the reaction mixture was again stirred for 30 minutes. Once again, the TLC-analysis indicated the presence of starting material, and more DIBAL-H (0,06 mL) was added. Finally, the reaction mixture was quenched with saturated aqueous NaHCO3 (5 mL). The mixture was stirred for 30 minutes

69 after the cooling bath was removed. Solid K-Na tartrat tetrahydrate (0,5 g) was added and stirring was continued for 30 minutes. Et2O (30 mL) was added, the phases were separated, and the aqueous layer was extracted with diethyl ether (3 x 20 mL). The combined organic phase was dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by flash chromatography (silica gel, heptane/EtOAc 95:5), and resulted in the desired product as a yellow oil. Yield: 128 mg (52%). TLC (Hept/EtOAc 9:1, KMnO4-stain):

Rf=0,25. The reported spectroscopic data are in full agreement with those reported in the literature.73 1H-NMR (400 MHz, Chloroform-d) δ 9.76 (t, J = 2.3 Hz, 1H), 6.69 (dd, J = 13.5, 10.9 Hz, 1H), 6.34 (dd, J = 13.5, 10.8 Hz, 1H), 6.13 (dd, J = 14.0, 10.6 Hz, 1H), 5.76 (dd, J = 15.2, 6.7 Hz, 1H), 5.30 (s, 1H), 2.73 – 2.40 (m, 2H), 0.88 (s, 9H), 0.15 – -0.14 (m, 6H). 13C-

NMR (101 MHz, CDCl3) δ 200.7, 136.3, 135.9, 127.3, 109.3, 68.2, 51.2, 25.6, 17.9, -4.6.

5.2.9 Synthesis of methyl (R,4Z,7Z,11E,13E)-14-bromo-10-((tert- butyldimethylsilyl)oxy)tetradeca-4,7,11,13-tetraenoate (87)

1. NaHMDS, HMPA, THF, OTBS -78 °C IPh3P CO2Me Br CO2Me 67 87 2. Aldehyde 73

Wittig salt 67 (247 mg, 0,46 mmol) was dissolved in THF (4,1 mL) and added molecular sieves and HMPA (0,7 mL). The reaction mixture was cooled to -78 °C, and NaHMDS (0,7 mL, 0,6 M in toluene, 1.0 equiv.) was added slowly and the reaction mixture attained a yellow color. The reaction flask was evacuated three times. The reaction mixture was stirred for one hour at 0 °C. Aldehyde 73 (120 mg, 0,47 mmol) was added at -78 °C, and the reaction mixture was allowed to attain room temperature while stirring for 24 hours in ice/acetone bath. The reaction mixture was quenched with phosphate buffer (4,3 mL, pH = 7,2, 0,955 g/mL, saturated), added Et2O (6,5 mL) and the phases were separated. The aqueous phase was extracted with Et2O (2 x 6,5 mL) and concentrated in vacuo. TLC (Hept/EtOAc 95:5, CAM- stain): Rf=0,12. The crude product was analyzed with NMR and the reported spectrum was deviant from those reported in the literature.50 1H-NMR (400 MHz, Chloroform-d) δ 9.59 (d, J = 7.9 Hz, 1H), 7.85 – 7.63 (m, 2H), 7.20 – 6.99 (m, 1H), 6.84 (d, J = 10.6 Hz, 1H), 6.70 – 6.40 (m, 1H), 6.23 (dd, J = 15.3, 7.9 Hz, 1H).

70 5.2.10 Second attempt of the synthesis of methyl (R,4Z,7Z,11E,13E)-14- bromo-10-((tert-butyldimethylsilyl)oxy)tetradeca-4,7,11,13-tetraenoate (87)

1. NaHMDS, HMPA, THF, OTBS -78 °C IPh3P CO2Me Br CO2Me 67 87 2. Aldehyde 73

Wittig salt 67 (149 mg, 0,28 mmol) was dissolved in THF (2,5 mL) and added molecular sieves and HMPA (0,4 mL). The reaction mixture was cooled to -78 °C, and NaHMDS (0,4 mL, 0,6 M in toluene, 1.0 equiv.) was added slowly and the reaction mixture attained a yellow color. The reaction flask was evacuated three times. The reaction mixture was stirred for three hours at 0 °C as more THF (3x50 mL) was added to solve the Wittig salt 67. The Wittig-salt was still not resolved, and more HMPA (1,5 mL) was added. Aldehyde 73 (72 mg, 0,28 mmol) was added at -78 °C, and the reaction mixture was allowed to attain room temperature while stirring for 22 hours in ice/acetone bath. The reaction mixture was quenched with phosphate buffer (2,6 mL, pH = 7,2, 0,955 g/mL, saturated), added Et2O (3,9 mL) and the phases were separated. The aqueous phase was extracted with Et2O (2 x 3,9 mL) and concentrated in vacuo. TLC (Hept/EtOAc 95:5, CAM-stain): Rf=0,13. The crude product was analyzed with NMR and identity of the product was not confirmed, as the NMR-spectrum was identical to the one from the first attempt.

5.2.11 Synthesis of tert-butyl 2-(4-hydroxybutoxy)acetate (106)

NaH O tert-butyl 2-bromoacetate (105) HO O OH HO O THF 0 °C 104 106

Butan-1,4-diol (104) (1,50 g, 16,7 mmol) was dissolved in dry THF (30 mL) at 0 °C under argon atmosphere. NaH (0,69 g, 28,8 mmol, 60% dispersion in oil) was added and the reaction mixture was allowed to obtain room temperature for one hour. The reaction mixture was recooled to 0 °C, and tert-butyl 2-bromoacetate (105) (4,33 g, 22,1 mmol) was added

71 dropwise. After 18 hours the reaction was quenched with NH4Cl. The aqueous phase was extracted with Et2O (3 x 20 mL), and the combined organic phases was washed with brine and dried over Na2SO4. The solvent was evaporated in vacuo. The residue was purified with flash chromatography (silica gel, Hept/EtOAc 6:4) and the product obtained was a viscous 1 yellow oil. Yield: 782 mg (52%). TLC (Hept/EtOAc 6:4, KMnO4-stain): Rf = 0,24. H-NMR (400 MHz, Chloroform-d) δ 3.96 (s, 2H), 3.68 (t, J = 5,7 Hz, 2H), 3.56 (t, J = 5,5 Hz, 2H), 1.77 – 1.64 (m, 4H), 1.48 (s, 9H). 13C-NMR (101 MHz, Chloroform-d) δ 169.7, 81.8, 71.7, 68.9, 62.8, 30.0, 28.2, 26.7.

5.2.12 Synthesis of tert-butyl 2-(4-(bromotriphenyl-λ5- phosphanyl)butoxy)acetate (97)

O 1. PBr3, Et2O O O O HO O BrPh P O 2. PPh3, MeCN, Δ 3 106 97

The alcohol 106 (210 mg, 1,03 mmol) was placed in a 25 ml round bottomed flask and dissolved in dry Et2O (6,2 mL) and cooled to 0 °C. PBr3 (0,1 mL, 1,05 mmol) was added dropwise at 0 °C, and the reaction mixture was stirred for three hours. When the bromination of the alcohol was complete, the reaction mixture attained an orange color. The reaction mixture was first diluted with Et2O (30 mL) before it was quenched with saturated NaHCO3

(2 x 10 mL). The aqueous phase was extracted with Et2O (1 x 10 mL) and the combined organic phase was washed with saturated NaCl (1 x 10 mL), dried (MgSO4), and the solvent was evaporated in vacuo. Yield: 53 mg (25%). TLC (Hept/EtOAc 6:4, visualized by KMnO4):

Rf = 0,69. The resulting yellow oil was dissolved in MeCN (1,6 mL) and triphenylphosphine (69 mg, 0,3 mmol) was added. The reaction mixture was refluxed overnight, cooled, and concentrated in vacuo. The crude product was purified by flash chromatography, first with

CH2Cl2 until all the triphenylphosphine was out of the column, followed by 20% MeOH in

CH2Cl2. The obtained product was a viscous pale oil. Yield: 12 mg (2% over two steps from 1 107). TLC (CH2Cl2/MeOH 8:2, visualized by KMnO4): Rf = 0,33. H-NMR (400 MHz,

Methanol-d4) δ 8.15 – 7.68 (m, 15H), 3.98 (s, 2H), 3.58 (m, 4H), 1.85 (td, J = 6.5, 3.5 Hz, 13 4’ 4H), 1.46 (s, 9H). C-NMR (101 MHz, Methanol-d4) δ 171.9, 136.2 (d, JCP = 3.1 Hz), 134.9

72 3’ 2’ 1’ (d, JCP = 9.9 Hz), 131.5 (d, JCP = 12.6 Hz), 120.0 (d, JCP = 86.4 Hz), 82.9, 70.8, 69.3, 30.6 2 1 3 (d, JCP = 17.1 Hz), 28.3, 22.1 (d, JCP = 51.6 Hz), 20.7 (d, JCP = 4.1 Hz).

5.2.13 Synthesis of tert-butyl 2-(4-(iodotriphenyl-λ5- phosphanyl)butoxy)acetate (96)

1. I , PPh O 2 3 O Imidazole, CH2Cl2 O O HO O IPh3P O 2. PPh3, MeCN, Δ 106 96

The primary alcohol 106 (408 mg, 2,0 mmol) was dissolved in dry CH2Cl2 (20 mL), and triphenylphosphine (786 mg, 3,0 mmol) and imidazole (211 mg, 3,1 mmol) was added. The reaction flask was evacuated three times, before being placed in a cooling bath (ice, water, salt). 15 minutes later, iodine (787 mg, 3,10 mmol) was added in one portion and with rapid stirring for 15 minutes. The cooling bath was then removed and the reaction mixture was allowed to attain ambient temperature for 40 minutes. The reaction was then quenched with saturated solution of Na2SO3 (20 mL), and the aqueous phase was extracted with CH2Cl2 (2 mL x 20). The combined organic phase was dried over MgSO4 and concentrated in vacuo to yield a white solid compound. The crude product was run through a short silica column (silica gel, Hept/EtOAc 92:8) and resulted in a viscous clear oil. Yield: 495 mg (64%). TLC 1 (Hept/EtOAc 92:8, visualized by KMnO4): Rf = 0,26. H-NMR (300 MHz, Chloroform-d) δ 3.94 (s, 2H), 3.53 (t, J = 6,1 Hz, 2H), 3.24 (t, J = 6,9 Hz, 2H), 1.94 (m, 2H), 1.73 (m, 2H), 1.48 (s, 9H). 13C-NMR (101 MHz, Chloroform-d) δ 169.7, 81.8, 71.7, 68.9, 62.7, 30.0, 28.2, 26.7. Next the iodide 107 was dissolved in dry MeCN (17 mL) and added triphenylphosphine (704 mg, 2,7 mmol). The reaction flask was evacuated four times, and the reaction mixture was refluxed for 12 hours under argon atmosphere. The reaction mixture was then cooled, concentrated in vacuo and purified by flash chromatography (CH2Cl2 until all triphenylphosphine was out of the column followed by 10% MeOH in CH2Cl2) to yield the desired product as a viscous yellow oil. Yield: 975 mg (85% over two steps from 106). TLC

(CH2Cl2/MeOH 9:1, visualized by KMnO4): Rf = 0,26. NMR confirmed the identity of 96. 1 H-NMR (400 MHz, Methanol-d4) δ 8.08 – 7.72 (m, 15H), 4.05 (m, 2H), 3.75 – 3.53 (m, 4H),

73 13 2.16 – 1.72 (m, 4H), 1.60 – 1.42 (m, 9H). C-NMR (101 MHz, Methanol-d4) δ 171.9, 136.2 4’ 3’ 2’ 1’ (d, JCP = 3.0 Hz), 134.9 (d, JCP = 10.4 Hz), 131.5 (d, JCP = 12.9 Hz), 120.0 (d, JCP = 86.4 2 1 3 Hz), 82.9, 70.9, 69.3, 30.6 (d, JCP = 16.9 Hz), 28.4, 22.3 (d, JCP = 51.4 Hz), 20.7 (d, JCP = 4.1 Hz).

74 6 References

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78 7 Appendix

7.1 1H-NMR and 13C-NMR Spectra

ME-001.10.ﬁd PROTON CDCl3 {D:\uio\AVII400-05} mmelkhat 14 26000

H3C OH 24000 8 7 2 4 HO 3 5 1 CH 22000 6

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79 ME-001.11.ﬁd 3 3 3 l l l

C C C 75000 C13CPD CDCl3 {D:\uio\AVII400-05} mmelkhat 14 D D D C C C

8 0 8 6 9 6 7 5 7 4 8 4 1 3 0 0 8 0 ...... 7 7 7 7 2 9 1 3 1 8 7 7 7 7 6 6 4 3 70000

H3C OH 8 7 65000 2 4 HO 3 5 1 CH 6 60000

55000

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90 85 80 75 70 65 60 55 50 45 40 35 30 25 f1 (ppm) Figure 7.2 13C-NMR spectrum of compound 98.

ME-002.10.ﬁd PROTON CDCl3 {D:\uio\AVII400-05} mmelkhat 104

11000

H3C OTES 8 2 4 10000 OSET 3 5 CH 6

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0 1 9 0 4 2 0 2 2 . . 0 9 0 0 . . . . 8 2

2 0 2 3 1 1 -1000

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 f1 (ppm) Figure 7.3 1H-NMR spectrum of compound 99.

80 ME-002.11.ﬁd 3 3 3 l l l C13CPD CDCl3 {D:\uio\AVII400-05} mmelkhaC t C 10C 4 2000 D D D C C C

6 8 6 4 5 0 1 6 6 9 4 1 8 0 6 7 7 5 1 2 ...... 1 6 7 7 7 6 2 7 9 7 1 . .

8 7 7 7 7 6 5 4 3 7 4 1900

1800 H3C OTES 8 2 4 1700 OSET 3 5 CH 6 1600

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100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 -10 f1 (ppm) Figure 7.4 13C-NMR spectrum of compound 99.

ME-003 (2).10.ﬁd PROTON CDCl3 {D:\uio\AVII400-05} mmelkhat 29 15000

14000

H3C OTES 8 4 1 13000 O 3 2 5 CH 6 12000

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10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 f1 (ppm) Figure 7.5 1H-NMR spectrum of compound 100.

81 ME-003 (2).10.ﬁd PROTON CDCl3 {D:\uio\AVII400-05} mmelkhat 29 15000

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-1000 1 1 4 0 1 4 9 0 9 1 0 7 ...... 0 2 0 3 9 5 -2000

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 f1 (ppm) Figure 7.6 13C-NMR spectrum of compound 100.

ME 4 (H).10.ﬁd PROTON CDCl3 {D:\uio\AVII400-05} mmelkhat 90 7500

H3C OTES 7000 9

H3C 5 4 2 7 6 3 1 6500 CH 8

6000

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4 -500 0 0 2 8 8 7 . 0 8 0 5 9 . . . . 1 . 2 2 2 3 1 5

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 f1 (ppm) Figure 7.7 1H-NMR spectrum of compound 91.

82 23000

ME 4 (H).11.ﬁd 3 3 3 l l l C13CPD CDCl3 {D:\uio\AVII400-05} mmelkhat 90 C C C D D D C C C 4 8 22000 3 0 9 8 6 4 5 7 6 6 8 4 . . 4 4 1 8 9 9 8 5 9 3 4 8 4 4 ...... 1 2 3 2 8 7 7 6 1 8 2 0 0 4 . . 1 1 8 7 7 7 7 6 4 3 2 1 7 6 21000

20000

19000 H3C OTES 9

H3C 5 4 2 18000 7 6 3 1 CH 17000 8

16000

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140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 f1 (ppm) Figure 7.8 13C-NMR spectrum of compound 91.

ME-005 (H1) 65000 PROTON CDCl3 {D:\uio\AVII400-05} mmelkhat 20

O 60000 9

2 8 CH3 11 3 1 O 55000 10 6 4

HO 5 7

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45000

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0 0 3 7 3 0 1 2 1 . . . .

3 2 4 2 -5000

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 f1 (ppm) Figure 7.9 1H-NMR spectrum of compound 84.

83 ME-005 (C1) 3 3 3 l l l

C13CPD CDCl3 {D:\uio\AVII400-05} mmelkhat 19 C C C 70000 D D D C C C 5 0 3 7 0 8 6 4 0 3 9 4 2 0 0 . 8 1 8 4 1 6 2 1 2 5 2 6 5 3 ...... 7 0 8 7 7 7 1 2 4 2 3 5 4 4 1 8 7 7 7 7 6 5 3 3 2 1 1 1 65000 O 9

2 8 60000 CH3 11 3 1 O 10 6 4 HO 5 55000 7

50000

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180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 f1 (ppm) Figure 7.10 13C-NMR spectrum of compound 84.

ME10NY.10.ﬁd 28000 O 2

1 4 7 OH 26000 9 O 3 5 6 8 10

CH3 24000 11

22000

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0 0 8 7 6 7 2 0 0 5 9 1 7

...... -2000 2 3 1 1 2 1

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 f1 (ppm) Figure 7.11 1H-NMR spectrum of compound 85.

84 ME-010.11.ﬁd 3 3 3 l l l C13CPD CDCl3 {D:\uio\AVII400-05} mmelkhat 3 C C C D D D C C C 0 0 1 8 9 3 8 6 4 9 5 7 5 3 . . . 4 1 8 2 7 9 9 9 3 0 7 ......

7 3 2 7 7 6 2 1 3 0 2 45000 1 1 1 7 7 7 6 5 3 3 2

O 2 40000 1 4 7 OH 9 O 3 5 6 8 10

CH3 11 35000

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210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm) Figure 7.12 13C-NMR spectrum of compound 85.

8500 ME-011(CogH).10.ﬁd PROTON CDCl3 {D:\uio\AVII400-05} mmelkhat 5

8000 26 27 25 7500 28 O 24 I 3 29 30 17 16

2 5 8 7000 H3C P 12 10 11 15 O 4 6 7 9 1 13 14 18 6500 23 19

22 6000 20 21

5500

5000

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6 -500 3 0 2 4 2 5 8 5 . 0 0 0 0 0 0 2 5 ...... 1 1 1 2 3 2 2 2

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 f1 (ppm) Figure 7.13 1H-NMR spectrum of compound 67.

85 80000

ME-011(CogH).11.ﬁd 3 3 3 l l l C13CPD CDCl3 {D:\uio\AVII400-05} mmelkhat 5 C C C D D D C C C 9 8 5 0 0 6 3 6 1 6 7 2 5 2 2 0 9 7 6 3 8 6 6 8 8 6 4 5 8 8 0 3 1 7 ...... 4 1 8 5 4 5 1 8 6 5 3 5 5 4 3 0 0 0 7 7 8 7 ......

7 3 3 3 3 3 3 3 2 2 1 1 7 7 6 3 3 3 3 2 0 0 75000 1 1 1 1 1 1 1 1 1 1 1 1 7 7 7 5 3 2 2 2 2 2

70000 26 27 25 65000 28 O 24 I 2 29 30 17 16

1 4 7 60000 P 12 11 15 O 3 5 6 8 9 13 14 18 55000 CH3 23 10 19

22 50000 20 21

45000

40000

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170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 f1 (ppm) Figure 7.14 13C-NMR spectrum of compound 67.

ME-012 (H).10.ﬁd PROTON CDCl3 {D:\uio\AVII400-05} mmelkhat 52 16000

TBSO 15000

2 4 6 8 Br 3 5 7 O 1 9 14000

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11000

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1 2 5 0 1 7 1 0 0 -1000 9 0 9 0 0 1 9 0 4 ...... 0 1 0 1 1 1 1 9 6

10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 f1 (ppm) Figure 7.15 1H-NMR spectrum of compound 73.

86 50000

ME-012 (H).11.ﬁd 3 3 3 l l l C13CPD CDCl3 {D:\uio\AVII400-05} mmelkhat 52 C C C D D D C C C 2 5 6 8 7 7 2 8 2 2 6 4 2 4 5 6 3 5 . . . . . 1 8 5 2 1 5 9 5 0 6 5 7 9 ...... 0 3 3 2 0 7 6 6 8 1 5 7 4 2 1 1 1 1 7 7 7 6 5 2 1 -

45000 TBSO

2 4 6 8 Br 3 5 7 O 1 9 40000

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210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm) Figure 7.16 13C-NMR spectrum of compound 73.

ME-013 (H u ﬂash).10.ﬁd 28000

26000 2 4 6 8 Br 3 5 7 O 1 9 24000

22000

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0 1 0 5 6 6 4 5 0 7 3 4 6

...... -2000 0 2 0 0 1 0

16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 -3 f1 (ppm) Figure 7.17 1H-NMR spectrum of the resulting compound 108 in the attempted Wittig reaction.

87 ME-016/10

O CH3 70000 3 12 CH3 13 9 7 O 1 11 5 HO 8 6 2 O CH3 65000 10 4 14

60000

55000

50000

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0 4 0 5 4 5 9 0 9 0 7

. . . . . -5000 1 2 1 4 8

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 f1 (ppm) Figure 7.18 1H-NMR spectrum of compound 106.

ME-016.11.ﬁd 3 3 3 l l l C13CPD CDCl3 {D:\uio\AVII400-05} mmelkhat 49 C C C D D D C C C 4 7 1 8 6 4 3 3 7 4 4 1 . 8 4 1 8 7 9 7 0 2 7 9 ...... 6 1 7 7 6 1 8 2 0 8 6

1 8 7 7 7 7 6 6 3 2 2 6.0E+09

O CH3 5.5E+09 3 12 CH3 13 9 7 O 1 11 5 HO 8 6 2 O CH3 10 4 14 5.0E+09

4.5E+09

4.0E+09

3.5E+09

3.0E+09

2.5E+09

2.0E+09

1.5E+09

1.0E+09

5.0E+08

0.0E+00

-5.0E+08

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 f1 (ppm) Figure 7.19 13C-NMR spectrum of compound 106.

88 ME-019/10

340000

24 320000 23 25

22 300000 26 O CH3 21 3 11 17 16 CH3 12 280000 9 7 1 10 18 O 15 5 P 8 6 2 O CH3 14 4 13 19 20 32 260000 Br 27 33 31

28 240000 30 29 220000

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-20000 5 6 3 6 8 6 . 8 1 0 6 5 . . . . 1 1 4 4 8

16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 -3 f1 (ppm) Figure 7.20 1H-NMR spectrum of compound 97.

ME-019/11 D D D D D D D 70000 O O O O O O O e e e e e e e M M M M M M M 0 6 3 1 1 5 2 4 8 9 2 2 9 8 5 4 4 8 9 6 4 3 1 0 9 7 6 8 1 3 5 4 7 3 5 ...... 8 7 2 6 4 2 0 7 5 3 6 5 3 3 8 6 6 1 6 6 4 4 1 1 0 ...... 9 7 3 3 3 3 3 3 2 2 0 9 9 9 9 9 8 8 8 0 0 8 2 1 0 0 1 1 1 1 1 1 1 1 1 1 8 7 6 4 4 4 4 4 4 4 3 3 2 2 2 2 2 65000

24 23 60000 25

22 26 O CH3 21 3 11 17 16 55000 CH3 12 9 7 1 10 18 O 15 5 P 8 6 2 O CH3 14 4 13 50000 19 20 32 Br 27 33 31

28 45000 30 29

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180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 f1 (ppm) Figure 7.21 13C-NMR spectrum of compound 97.

89 Mai iodid.1.ﬁd 9000 O CH3 3 12 CH3 13 9 7 O 1 11 8500 5 I 8 6 2 O CH3 10 4 14 8000

7500

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6500

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7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 f1 (ppm) Figure 7.22 1H-NMR spectrum of compound 107.

ME-21.11.ﬁd 3 3 3 l l l C13CPD CDCl3 {D:\uio\AVII400-05} mmelkhat 47 C C C D D D 85000 C C C 4 7 0 8 6 4 2 2 4 1 3 9 . 8 4 1 8 7 9 7 0 2 6 9 ...... 6 1 7 7 6 1 8 2 0 8 6 1 8 7 7 7 7 6 6 3 2 2 80000

O CH3 3 12 75000 CH3 13 9 7 O 1 11 5 8 6 2 70000 I O CH3 10 4 14

65000

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170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 f1 (ppm)

Figure 7.23 13C-NMR spectrum of compound 107.

90 Me-023wittig.10.ﬁd PROTON128 MeOD {D:\uio\AVII400-05} mmelkhat 109 50000 24 23 25

22 26 O CH3 45000 21 3 11 17 16 CH3 12 9 7 1 10 18 O 15 5 P 8 6 2 O CH3 14 4 13 19 20 32 40000 I 27 33 31

28 30 29 35000

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0 9 1 6 2 9 0 . 9 3 9 0 5 . . . . 1 1 4 3 9

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 f1 (ppm) Figure 7.24 1H-NMR spectrum of compound 96.

Me-023wittig.11.ﬁd D D D D D D D D D D D D D D D D

O O O O O O O O O O O O O O O O 170000

C13CPD MeOD {D:\uio\AVII400-05} mmelkhat 109 e e e e e e e e e e e e e e e e M M M M M M M M M M M M M M M M 8 6 3 6 5 9 6 5 9 8 2 2 9 8 5 4 4 5 8 8 4 8 4 6 3 6 3 1 4 2 0 3 1 9 1 7 6 2 5 0 2 1 0 6 ...... 8 8 3 6 6 4 4 2 2 2 0 0 0 8 8 7 6 5 3 7 5 4 5 0 7 6 1 6 6 4 4 1 1 0 9 ...... 7 3 3 3 3 3 3 2 1 2 0 9 9 9 9 9 9 9 9 9 9 9 8 8 8 8 8 8 0 0 8 2 2 0 0

1 1 1 1 1 1 1 1 1 8 7 6 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 2 2 2 2 2 160000

24 23 150000 25

22 26 140000 O CH3 21 3 11 17 16 CH3 12 9 7 1 10 18 O 15 5 130000 P 8 6 2 O CH3 14 4 13 19 20 32 I 27 120000 33 31

28 30 110000 29

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220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm) Figure 7.25 13C-NMR spectrum of compound 96.

91 7.2 MS- and HRMS Characterization

Figure 7.26 MS-spectrum of compound 91.

Figure 7.27 HRMS-spectrum of compound 91.

92 7.3 GC Characterization

Chromatogram 7.1 G-chromatogram of compound 91.