Total Synthesis and SAR Studies

Research Collection

Doctoral Thesis

Resorcylic lactone L-783277 as a new lead structure for kinase inhibition total synthesis and SAR studies

Author(s): Hofmann, Tatjana

Publication Date: 2009

Permanent Link: https://doi.org/10.3929/ethz-a-005826434

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ETH Library Diss. ETH No. 18336

Resorcylic Lactone L-783277 as a New Lead Structure for Kinase Inhibition – Total Synthesis and SAR Studies

A dissertation submitted to the Swiss Federal Institute of Technology Zurich

For the degree of Doctor of Sciences ETH Zurich

Presented by Tatjana Hofmann

Dipl. Chem. Johann Wolfgang Goethe-Universität Frankfurt am Main Born March 29, 1978 Citizen of the Federal Republic of Germany

Accepted on the recommendation of

Prof. Dr. Karl-Heinz Altmann, examiner Prof. Dr. P. August Schubiger, co-examiner

Zurich, 2009

Dedicated to my parents

I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale. Marie Curie (1867 - 1934)

The great tragedy of Science – the slaying of a beautiful hypothesis by an ugly fact. Thomas H. Huxley (1825 - 1895)

Acknowledgments First of all I would like to thank Prof. Karl-Heinz Altmann for giving me the opportunity to accomplish my PhD thesis in his research group. I had always the freedom to come up with my own ideas and suggestions and I did really appreciate his guidance, support and interest in my project. I am grateful to Prof. Pius August Schubiger for being the co-examiner of my thesis. Furthermore, he did not hesitate to support me when I kindly asked him to act as a referee for funding bodies. In addition I would like to thank Prof. Michael Detmar and Benjamin Vigl for our collaboration. My personal thanks go Silvia Anthoine Dietrich, Dr. Cotinica Hamel, Fabienne Zdenka Gaugaz, Didier Zurwerra and Dr. Evgeny Prusov. I was fortunate to work together with these highly skilled chemists and understanding as well as supporting characters. Thanks to all of them I enjoyed my time at the ETH and in Switzerland. Kurt Hauenstein is a patient, very helpful, hard-working and highly skilled technician. Thanks to him, I learned a lot of practical skills and I really appreciate that. During my PhD thesis I was responsible for the supervision of three undergraduate students during their “Semesterarbeit” as well as their diploma and master theses. Fabienne Zdenka Gaugaz is a highly talented and motivated student, who owns an indefatigable positive mind and charisma. I am really proud of her return to the group and that she started her own PhD thesis in 2008. Heike Kirchner stayed with me for her external diploma thesis and I appreciated her being a passionate and hardworking student, even if she had to overcome several synthesis problems. Luca Fransioli was my last student and he did a fantastic job. He almost finished two synthesis projects with his straightforward and painstaking working attitude. I wish him all the best for his future and I envy his new colleagues. I would also like to thank Dr. Pascal Furet (Novartis Institute for Biomedical Research, Basel) for modeling studies and Dr. Doriano Fabbro (Novartis Institute for Biomedical Research, Basel) for the biological evaluation of my compounds. I am grateful to Novartis for a one year Doctoral Fellowship in 2006. Special thanks go to the Altmann research group. We have always had an enjoyable and fruitful working atmosphere. In conclusion, I would like to thank Nicola Feyen for her patience while teaching me English and Tobias Ross, Simone Jeger, Martina Adams, Annette Rincker, Nadine Neumann, my sister Stephanie, my dad and Karin for their emotional support.

Curriculum Vitae

Personal Details Name Tatjana Hofmann Date of birth 29th March 1978 Place of birth Fulda, Germany Nationality German

Employment 2005-present PhD student in the group of Prof. Dr. Karl-Heinz Altmann, Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences (D-CHAB), ETH Zürich, Switzerland  “Resorcylic Lactone L-783277 as a New Lead Structure for Kinase Inhibition - Total Synthesis and SAR Studies“  Supervision of Masters students and leading practical courses for undergraduate students  Responsible for NMR service and maintenance 2004-2004 Diploma student in the group of Prof. Dr. Engels, Department of Organic Chemistry and Chemical Biology (OCCB), Johann Wolfgang Goethe University, Frankfurt am Main, Germany  “Synthesis of Minor Groove Labelled 2’-Deoxyguanosines”

Education 2000-2004 Johann Wolfgang Goethe University, Frankfurt am Main, Germany  Graduated with diploma in chemistry (equivalent to Masters Degree) - Grade awarded equivalent to first class honors  1st state examination in botany 1997-2000 Provadis GmbH, Industrie Park Höchst, Frankfurt am Main, Germany  Vocational training as laboratory assistant (qualification awarded by German Chamber of Commerce and Industry) 1988-1997 Albert - Einstein Schule, Gymnasium in Schwalbach am Taunus, Germany  “Abitur“ (equivalent to A levels)

Awards 2006-2007 Novartis Doctoral Fellowship for 2006

Work Experience 11-12/2004 Novartis Pharma AG, Basel, Switzerland  Synthesis of low-molecular weight kinase inhibitors 02-03/2000 Aventis Pharma Deutschland AG, Frankfurt am Main, Germany  Metabolism and Arthritis: lead optimisation of relevant candidates

Publications Tatjana Hofmann, Karl-Heinz Altmann Resorcylic Acid Lactones as New Lead Structures for Kinase Inhibition Comptes Rendus Chimie 2008, 11, 1318-1335

Tatjana Hofmann, Karl-Heinz Altmann Total Synthesis of the Resorcylic Lactone-Based Kinase Inhibitor L-783277 Synlett 2008, 10, 1500-1504

Tatjana Hofmann, Karin Zweig; Joachim W. Engels A New Synthetic Approach for the Synthesis of N2-Modified Guanosines Synthesis 2005, 11, 1797-1800

Oral Presentations Tatjana Hofmann, Karl-Heinz Altmann Resorcylic Lactone L-783277 as a New Lead Structure for Kinase Inhibition - Total Synthesis and SAR Studies Frontiers in Medicinal Chemistry 2009, Heidelberg, Germany 2009

Tatjana Hofmann, Karl-Heinz Altmann Total Synthesis of the Resorcylic Lactone-based Kinase Inhibitor L-783277 Fall Session 2008, Swiss Chemical Society Meeting 2008; Zurich, Switzerland 2008

Tatjana Hofmann, Karl-Heinz Altmann Studies on the Total Synthesis of Resorcylic Lactone L-783277 - A New Lead Structure for Kinase Inhibition Doktorandentag - Fall Session 2007, ETH Zürich, Switzerland 2007

Poster Presentations Tatjana Hofmann, Karl-Heinz Altmann Studies on the Total Synthesis of Resorcylic Lactone L-783277 - A New Lead Structure for Kinase Inhibition Swiss Chemical Society Meeting 2007; Lausanne, Switzerland 2007

Hofmann, Tatjana; Karl-Heinz Altmann Resorcylic Lactone L-783277 as a New Lead Structure for Kinase Inhibition - Total Synthesis and SAR Studies Frontiers in Medicinal Chemistry 2007, Berlin, Germany 2007

Table of Contents Abstract...... I Zusammenfassung...... IV List of Abbreviations, Acronyms and Symbols ...... VII 1. Introduction ...... 1 1.1. Resorcylic Acid Lactones (RALs) ...... 1 1.1.1. Biosynthesis of RALs...... 2 1.1.2. Biological Activity of RALs ...... 2 1.2. MAP Kinase Pathways ...... 5 1.3. Clinically Approved Kinase Inhibitors ...... 7 1.4. Total Syntheses of Cis-Enone Containing RALs ...... 11 1.4.1. LL-Z1640-2 and Hypothemycin ...... 11 1.4.2. Radicicol A...... 20 1.4.3. Aigialomycin D ...... 23 1.5. Conclusions...... 34 2. Aims and Scope ...... 35 2.1. Retrosynthetic Analysis of L-783277 ...... 35 2.1.1. First Generation Approach...... 35 2.1.2. Second Generation Approach ...... 37 2.1.3. Still-Gennari Approach...... 38 2.2. Retrosynthetic Analysis of the Dideoxy Analog D6...... 40 2.3. Retrosynthetic Analysis of the Phenyl Analog P6...... 40 2.3.1. Alkyne Metathesis Approach ...... 41 2.3.2. Macrolactonization Approach ...... 42 2.4. Conclusions...... 43 3. Results and Discussions...... 44 3.1. Total Synthesis of L-783277...... 44 3.1.1. First Generation Approach...... 44 3.1.1.1. Synthesis of the Resorcylic Acid Moiety ...... 44 3.1.1.2. Synthesis of the C7’-C11’ Fragment ...... 45 3.1.1.3. Synthesis of the C1’-C6’ Fragment...... 45 3.1.1.4. Assembly of Fragments C1’-C6’ and C7’-C11’ ...... 48 3.1.1.5. Methyl Ester Cleavage...... 51 3.1.1.6. Suzuki Couplings with Free Carboxylic Acids...... 53 3.1.1.7. Strategy Variation: Change of the Ester Group ...... 54 3.1.2. Second Generation Approach ...... 55 3.1.2.1. Synthesis of the C1’-C11’ Fragment ...... 55 3.1.2.2. Variation of the Synthesis of the C1’-C6’ Fragment...... 56 3.1.2.3. Assembly of Building Blocks ...... 57 3.1.3. Improved Enantioselective Approach ...... 59 3.1.3.1. Enantioselective Addition of the C1’-C6’ Acetylene to the C7’-C11’ Aldehyde...... 60

3.1.3.2. Determination of the Absolute Configuration ...... 61 3.1.3.3. Completion of the Total Synthesis ...... 62 3.1.4. Still-Gennari Approach...... 63 3.1.4.1. Still-Gennari Olefination ...... 63 3.1.4.2. Synthesis of the C1’-C6’ Fragment...... 65 3.1.4.3. Coupling of the Phosphonate to the Resorcylic Moiety ...... 66 3.1.4.4. Coupling of the C1’-C6’ Fragment to the Resorcylic Moiety...... 67 3.2. Synthesis of Dideoxy Analog D6 ...... 68 3.3. Synthesis of Phenyl Analog P6 ...... 70 3.3.1. Alkyne Metathesis Approach ...... 70 3.3.1.1. Synthesis of Individual Fragments ...... 73 3.3.1.2. Coupling of Fragments...... 76 3.3.1.3. Variation of Strategy...... 77 3.3.2. Macrolactonization Approach ...... 80 3.3.2.1. Synthesis of the C1’-C11’ Fragment ...... 80 3.3.2.2. Assembly of Fragments ...... 80 3.3.2.3. Change of Protecting Groups ...... 82 3.4. Stability Measurements ...... 84 3.4.1. Chemical Stability ...... 84 3.4.2. Blood Plasma Stability ...... 85 3.5. Biological evaluation...... 86 3.5.1. In Vitro Kinase Inhibition ...... 86 3.5.2. Cellular Activity ...... 87 3.5.2.1. Inhibition of Cellular Proliferation ...... 87 3.5.2.2. Tube Formation Studies...... 88 3.5.3. In Vivo Evaluation ...... 90 4. Conclusions...... 91 4.1. Total Synthesis of the Natural Product L-783277...... 91 4.1.1. Macrolactonization-Based Approaches ...... 91 4.1.2. Still-Gennari Approach...... 92 4.2. Synthesis of Analogs...... 93 4.2.1. Synthesis of the Dideoxy Analog D6 ...... 93 4.2.2. Synthesis of the Phenyl Analog P6...... 93 4.3. Biological Evaluation ...... 94 5. Outlook...... 95 6. Experimental Section...... 96 6.1. General Methods...... 96 6.1.1. Melting Points ...... 96 6.1.2. Optical Rotations ...... 96 6.1.3. NMR Measurements...... 96 6.1.4. IR Measurements ...... 97 6.1.5. Mass Measurements ...... 97 6.1.6. HPLC Measurements ...... 97 6.2. Experimental Procedures and Analytical Data ...... 97 6.2.1. Synthesis of the Natural Product L-783277 ...... 97 6.2.1.1. Preparation of Compounds described in Chapter 3.1.1.1...... 97 6.2.1.2. Preparation of Compounds described in Chapter 3.1.1.2...... 102 6.2.1.3. Preparation of Compounds described in Chapter 3.1.1.3...... 105 6.2.1.4. Preparation of Compounds described in Chapter 3.1.1.4...... 111 6.2.1.5. Preparation of Compounds described in Chapter 3.1.1.6...... 116 6.2.1.6. Preparation of Compounds described in Chapter 3.1.1.7...... 116 6.2.1.7. Preparation of Compounds described in Chapter 3.1.2.1...... 119 6.2.1.8. Preparation of Compounds described in Chapter 3.1.2.2...... 125 6.2.1.9. Preparation of Compounds described in Chapter 3.1.2.3...... 129 6.2.1.10. Preparation of Compounds described in Chapter 3.1.3.1...... 139 6.2.1.11. Preparation of Compounds described in Chapter 3.1.3.2...... 141 6.2.1.12. Preparation of Compounds described in Chapter 3.1.3.3...... 143 6.2.1.13. Preparation of Compounds described in Chapter 3.1.4.2...... 146 6.2.1.14. Preparation of Compounds described in Chapter 3.1.4.4...... 147 6.2.2. Synthesis of the Dideoxy Analog D6 ...... 153 6.2.3. Synthesis of the Phenyl Analog P6...... 160 6.2.3.1. Preparation of Compounds described in Chapter 3.3.1.1...... 160 6.2.3.2. Preparation of Compounds described in Chapter 3.3.1.2...... 169 6.2.3.3. Preparation of Compounds described in Chapter 3.3.1.3...... 170 6.2.3.4. Preparation of Compounds described in Chapter 3.3.2.1...... 176 6.2.3.5. Preparation of Compounds described in Chapter 3.3.2.2...... 179 6.2.3.6. Preparation of Compounds described in Chapter 3.3.2.3...... 183 6.2.3.7. pH Stability ...... 190 6.2.3.8. Blood Plasma Stability ...... 190 6.2.3.9. Inhibition of Cellular Proliferation ...... 191 7. Bibliography ...... 193

Abstract

The inhibition of disease-relevant kinases leads to interference with cellular signaling pathways or cell cycle progression and represents a new paradigm in modern drug discovery. In particular, several kinase inhibitors have been successfully developed in recent years for the clinical treatment of different types of cancers. While most of these agents are low-molecular-weight synthetic molecules based on different types of heteroaromatic or urea scaffolds, a number of naturally occurring resorcylic acid lactones (RALs) have recently emerged as alternative new lead structures for kinase inhibition. Most of the members of this family of natural products exhibit a cis-enone moiety as part of their macrolactone ring. A 1,4-addition of an active site cysteine residue to the -carbon of the , -unsaturated carbonyl system is responsible for the high potency kinase inhibition of these compounds. One of the most potent representatives is L-783277 (6) which is depicted in Fig. 1.

OH O

O O HO OH L-783277 (6) Figure 1. Resorcylic lactone L-783277 (6).

Total syntheses have been successfully achieved for several RALs. However, no efforts on the total synthesis of 6 had been reported before our own work, although 6 is a highly potent inhibitor of the Ser/Thr kinase Mek1 (IC50 = 4 nM).

The goal of this research project was the development of an efficient enantioselective synthesis of 6 and the characterization of its biological activity with respect to the selectivity of kinase inhibition and its effects on human cancer cells. In a second step, the chemistry developed for the preparation of 6 was planned to be used for the synthesis of a limited number of analogs for SAR and biophysical studies. The initial approach to 6 had to be abandoned due to instability of advanced intermediates. Thus, it was impossible to cleave the ester group in 173 or 177 (Fig. 2) under conditions that did not lead to the destruction of the molecule. The investigation of different ester groups or different protecting groups for the C4’/C5’ hydroxyl groups did not allow to overcome the lack of chemical stability of these advanced intermediates.

OTES

OH O 11' 8' 1 3 OR 5' O O 51'TBSO OTBS 173 :R=Me 177 :R =CH2CH2Si(CH 3)3 Figure 2. Advanced intermediates 173 and 177.

As illustrated in Fig. 3, a new strategy was developed, which successfully led to the natural product L-783277 (6). The design of the second generation approach was based on the convergent assembly of three key intermediates 129R, 191 and 176, which were assembled through addition of the lithiated alkyne 129R to aldehyde 191, followed by Suzuki coupling of 176 with the MOM-protected addition product 192. The resulting protected linear precursor for the macrolactonization, 194, was partially hydrogenated, transformed into the respective seco-acid and cyclized under Mitsunobu conditions. One of the key features of this strategy towards 6 was the late introduction of the ketone moiety at C6’ through selective allylic oxidation of the deprotected intermediate 198.

OTBS

O OH O OTBS Si + + O O O O Br OMOM O O 129R 191 192 176

Si OTBS

OH O OH O OH O

O O O OH OH O OMOM O OH O O O O OH OH 194 198 L-783277 (6)

Figure 3. Successful synthetic approach to L-783277 (6).

Only one of the diastereomers 198 (which arise as a consequence of the non- selective addition of the lithiated alkyne 129R to aldehyde 191) was cleanly converted into the final product by selective allylic oxidation. As a consequence the enantioselective coupling of key intermediates 129R and 191 was developed, leading to an enantioselective and highly efficient total synthesis of the natural product 6. In addition, a third approach that aims at ring closure based on the Still-Gennari reaction has been investigated. Based on the synthetic approach developed for 6 the total syntheses of two selected analogs of 6 have been successfully accomplished (Fig. 4). The dideoxy analog D6

II lacks the C4’ and C5’ hydroxyl groups, while the phenyl analog P6 exhibits a phenylene moiety instead of a Z-configured double bond between C7’ and C8’.

OH O OH O

O O

O O O O HO D6 P6 OH Figure 4. Selected analogs of L-783277 (6).

6 and its dideoxy analog D6 were tested against a panel of 34 kinases (collaboration with Dr. Doriano Fabbro, Novartis Institute for Biomedical Research, Basel). L-

783277 (6) effectively inhibits VEGFR-2, Mek2, PDGFR and MK5, with IC50 values of 8 nM, 15 nM, 87 nM and 640 nM, respectively. These kinases are known to be involved in cancer relevant and inflammatory signaling pathways of cells. Moreover,

6 inhibited Erk2, Tyk2 and cKit with M IC50 values, while IC50 values for all other kinases tested were above 10 M. Interestingly, D6 also inhibited VEGFR-2, but with approx. 60-fold lower potency than 6. Additionally, 6 was tested in proliferation experiments using primary lymphatic endothelial cells, (collaboration with Prof. Michael Detmar and Benjamin Vigl, ETH Zurich), whose proliferation was induced by VEGF-A. In these experiments the proliferation of cells could be effectively inhibited by 6. In in vitro capillary-tube formation studies (collaboration with Prof. James Lorens and Dr. Lasse Evensen, University of Bergen) 6 showed an inhibitory effect on tube formation of human umbilical vein endothelial cells.

In conclusion, we have accomplished the first total synthesis of the resorcylic lactone kinase inhibitor L-783277 (6) in an enantioselective and highly efficient manner. Based on the chemistry developed for the preparation of 6, we have completed the total syntheses of two selected analogs. First results of the biological activity of 6 and analog D6 show selective inhibition of a subset of kinases, which are involved in inflammation and cancer-relevant signaling pathways. The applicability of our approach to analog synthesis has been proven. Our strategy thus grants access to novel analog structures for SAR and biophysical studies around this potent lead structure for anticancer and anti-inflammatory drug discovery.

III

Zusammenfassung

Die Hemmung krankheitsrelevanter Kinasen beeinflusst zelluläre Signalwege oder den Ablauf des Zellzyklus und bildet einen neuen Ansatz in der modernen Arzneimittelforschung. Inbesondere im Bereich der Onkologie sind in den letzten Jahren einige Kinasehemmer erfolgreich für die klinische Anwendung entwickelt worden. Die meisten dieser Verbindungen sind niedermolekulare synthetische Moleküle, bei denen es sich in der Regel um Heteroaromaten oder Harnstoffderivate handelt. Daneben sind einige natürlich vorkommende resorzyklische Laktone (RALs) als neue und alternative Strukturen für die Hemmung von Kinasen in Erscheinung getreten. Die meisten Mitglieder dieser Naturstofffamilie besitzen ein Z-Enon als Teil ihres Laktonringes. Für die hohe Wirksamkeit der Kinasehemmung dieser Verbindungen ist eine 1,4-Addition eines Cysteins im aktiven Zentrum des Proteins an den -Kohlenstoff des ,-ungesättigten Carbonylsystems verantwortlich. Ein hochpotenter Vertreter diese Gruppe ist L-783277 (6), dessen Struktur in Abbildung 1 gezeigt ist.

OH O

O O HO OH L-783277 (6) Abbildung 1: Struktur von L-783277 (6), ein Vertreter der resorzyklischen Laktone.

Für einige Vertreter der resorzyklischen Laktone wurden erfolgreich Totalsynthesen entwickelt. Obwohl 6 ein sehr potenter Kinasehemmer der Ser/Thr-Kinase Mek1 (IC50 = 4 nM) ist, wurden zu diesem Vertreter der RALs noch keine synthetischen Arbeiten veröffentlicht.

Das Ziel dieses Forschungsprojektes war es, eine effiziente und enantioselektive Totalsynthese von 6 zu entwickeln. Desweiteren sollte die biologische Aktivität von 6 im Hinblick auf seine Kinasehemmung und seine Wirkung auf Krebszellen näher charakterisiert werden. Basierend auf der entwickelten Synthesestrategie für 6, sollte anschließend eine gewisse Anzahl von Analog- strukturen für SAR und biophysikalische Studien synthetisiert werden. Die zunächst verfolgte Synthesestrategie für die Totalsynthese von 6 musste aufgrund unzureichender Stabilität fortgeschrittener Zwischenprodukte aufgegeben werden. So war die Spaltung der Estergruppe in den Verbindung 173 und 177 (Abbildung 2) nicht möglich und entsprechende Versuche führten unter verschiedenen Reaktionsbedingungen immer wieder zur Zersetzung der Moleküle. Trotz der Verwendung anderer Estergruppen und unterschiedlicher Schutzgruppen- strategien für die C4’- und C5’-Hydroxylgruppen, konnte die chemische Labilität dieser fortgeschrittenen Zwischenprodukte nicht überwunden werden.

IV OTES

OH O 11' 8' 1 3 OR 5' O O 51'TBSO OTBS 173 :R=Me 177 :R =CH2CH2Si(CH 3)3 Abbildung 2: Intermediate 173 und 177.

Abbildung 3 zeigt die Synthesestrategie, die schließlich zur erfolgreichen Total- synthese des Naturstoffs L-783277 führte. Das Synthesedesign der zweiten Generation basierte auf der konvergenten Verknüpfung der drei Bausteine 129R, 191 und 176. Die Addition des lithiierten Alkins 129R an den Aldehyd 191 lieferte das Schlüsselintermediat 192. In der anschliessenden Suzuki Kupplung mit 176 wird das Intermediat 194 gebildet. Dieser vollständig geschütze lineare Vorläufer der Makro- laktonisierung wurde zunächst teilweise hydriert, in die entsprechende Hydroxysäure überführt und dann mittels einer Mitsunobu Reaktion zyklisiert. Einer der Schlüssel- schritte dieser Synthesestrategie ist das späte Einführen des Ketons in die C6’- Position durch selektive allylische Oxidation des entschützen Zwischenprodukts 198.

OTBS

O OH O OTBS Si + + O O O O Br OMOM O O 129R 191 192 176

Si OTBS

OH O OH O OH O

O O O OH OH O OMOM O OH O O O O OH OH 194 198 L-783277 (6)

Abbildung 3: Erfolgreiche Synthese des Naturstoffs L-783277 (6).

Nur eines der beiden möglichen Diastereomere von 198 (welche aufgrund der unselektiven Addition des lithiierten Alkins 129R an den Aldehyd 191 entstehen), konnte ohne erheblichen Anteil an Nebenprodukten durch allylische Oxidation zur Endverbindung umgesetzt werden. Aus diesem Grund wurde die enantioselektive Kupplung der Bausteine 129R und 191 entwickelt. Dies ermöglichte die enantioselektive und sehr effiziente Totalsynthese des Naturstoffs 6. Zusätzlich wurde eine weitere Ringschlussmethode basierend auf der Still-Gennari Reaktion in Hinblick auf ihre Anwendbarkeit für die Synthese von 6 untersucht, wobei dieses Projekt noch nicht abgeschlossen wurde.

Basierend auf der entwickelten Totalsynthese von 6 wurden zwei seiner Analoga erfolgreich synthetisiert (Abbildung 4). Das Dideoxyanalogon D6 besitzt keine Hydroxylgruppen in der C4’ und C5’ Position, während das Phenylanalogon P6 anstatt der Z-konfigurierten Doppelbindung einen mit der C7’-C8’ Bindung annelierten Phenylrest aufweist.

OH O OH O

O O

O O O O HO D6 P6 OH Abbildung 4: Ausgewählte Analoga von L-783277 (6).

6 und sein Dideoxyanalogon D6 wurden gegen eine Reihe von 34 Kinasen getestet, (in Zusammenarbeit mit Dr. Doriano Fabbro, Novartis Institut für Biomedizinische

Forschung, Basel). 6 hemmt VEGFR-2, Mek2, PDGFR and MK5 mit IC50 Werten von 8 nM, 15 nM, 87 nM bzw. 640 nM. Es ist bekannt, dass diese Kinasen in bestimmten Signalwegen von Zellen bei Krebs und auch bei entzündlichen Erkrankungen eine wichtige Rolle spielen. Darüber hinaus weist 6 gegen Erk2, Tyk2 und cKit IC50 Werte im unteren mikromolaren Bereich auf. Für alle anderen getesteten Kinasen wurden IC50 Werte von über 10 M ermittelt. Interessanterweise inhibiert ebenfalls D6 VEGFR-2, wenn auch mit einer 60-fach geringeren Aktivität als 6. Zusätzlich wurde 6 in Zellproliferationsexperimenten mit primären lymphatischen Endothelzellen untersucht (in Zusammenarbeit mit Prof. Michael Detmar und Benjamin Vigl, ETH Zürich), wobei das Wachstum der Zellen durch VEGF-A induziert wurde. In diesen Experimenten konnte gezeigt werden, dass das Zellwachstum durch 6 wirksam reduziert werden konnte. In sogenannen in vitro “capillary tube formation“ Studien (in Zusammenarbeit mit Prof. James Lorens und Dr. Lasse Evensen, Universität Bergen) zeigte 6 eine inhibierende Wirkung auf die Gefässbildung von menschlichen Nabelschnur- endothelzellen.

Abschließend kann festgestellt werden, dass die erste Totalsynthese des resorzyklischen Kinasehemmers L-783277 (6) erfolgreich realisiert werden konnte und dies in einer enantioselektiven und sehr effizienten Weise. Basierend auf der entwickelten Synthesestrategie, wurden zwei Analoga erfolgreich synthetisiert. Erste Studien zur biologischen Aktivität von 6 und dem Analogon D6 zeigen eine selektive Inhibierung gewisser Kinasen, die in entzündungs- und krebsrelevanten Zellsignal- wegen eine Rolle spielen. Die Anwendbarkeit unserer Synthesestrategie für die Herstellung von Analogstrukturen konnte gezeigt werden. Somit ermöglicht die von uns entwickelte Strategie den Zugang zu Analogstrukturen für SAR und biophysikalische Studien als eine mögliche Grundlage für die Entwicklung neuer Wirkstoffe in der Onkologie und gegen Entzündungskrankheiten.

VI List of Abbreviations, Acronyms and Symbols A abs absolute Ac acetyl aq aqueous aromatic ar ATP adenosine triphosphate

B 9-BBN 9-borabicyclo[3.3.1]nonane bp boiling point br broad (NMR) Bu butyl C CAN ceric ammonium nitrate cat catalytic CD3 CD3 antigen (cluster of differentiation) CDK1/5 cyclin-dependent kinases 1 and 5 CML chronic myeloid leukaemia CNS central nervous system CoA coenzyme A conc concentrated COSY correlation spectroscopy CSA camphor sulfonic acid Cys cysteine

D  delta; chemical shift in ppm (NMR) d doublet (NMR) Da Dalton DCC N,N'-dicyclohexylcarbodiimide DDQ 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone de diastereomeric excess DEAD diethyl azodicarboxylate DET diethyl tartrate DHP 3,4-dihydro-2H-pyran DIAD diisopropyl azodicarboxylate DIEA N,N-diisopropylethylamine DDQ 2,3-dichloro-5,6-dicyano-1,4-benzoquinone DIBAL-H diisobutylaluminium hydride DMAP 4-dimethylaminopyridine

VII

DME dimethoxyethane DMF N,N-dimethylformamide DMP Dess-Martin periodinane DMSO dimethylsulfoxide dr diastereomeric ratio

E E trans ee enantiomeric excess EDB ethylene dibromide EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride EGFR epidermal growth factor receptor EI electron impact Enz enzyme equiv equivalent Erk extracellular signal-regulated kinases ESI electrospray ionization Et ethyl EU European Union

F FC flash chromatography FDA Food and Drug Administration (USA) Fig. figure

G g gram(s) GISTs gastrointestinal stromal tumors

H h hour(s) HIF-1 hypoxia-inducible factor-1, alpha subunit HMDS hexamethyldisilazane HMPA hexamethylphosphoric acid triamide HPLC high performance liquid chromatography HRMS high resolution mass spectrometry Hsp90 heat shock protein 90 HUVEC human umbilical vein endothelial cells HWE Horner-Wadsworth-Emmons reaction Hz Hertz

VIII I IBX 2-iodoxybenzoic acid

IC50 half maximal inhibitory concentration IR infrared iPr isopropyl

J J coupling constant (NMR) JNK c-Jun amino-terminal kinases

K k kilo

L L liter(s) LAH lithium aluminium hydride Lck leukocyte-specific protein tyrosine kinase LDA lithium diisopropylamide

M m multiplet (NMR) m meta M molar MAA Marketing Authorization Application mAb monoclonal antibodies MAPK mitogen activated protein kinase MAPKKK mitogen activated protein kinase kinase kinase MKK equals: Mek, MAP2K mCPBA meta-perbenzoic acid Me methyl Mek1 equals: mitogen activated protein kinase kinase 1 (MAPKK1) MEM β-methoxyethoxymethyl ether MeOH methanol Mes mesyl min minutes

MnO2 manganese dioxide mg milligram μg microgram MOM methoxymethyl MS multiple sclerosis mTOR mammalian target of rapamycin

mw microwave m/z mass to charge ratio (MS)

N N normal NBS N-bromosuccinimide nM nanomolar NME N-methylephedrine NMO N-methylmorpholine-N-Oxide NMR nuclear magnetic resonance NOESY nuclear Overhauser effect spectroscopy NSCLC non-small cell lung cancer

O o ortho

P p para p38 a class of mitogen-activated protein kinases PCC pyridinium chlorochromate PDGFR platelet-derived growth factor Ph phenyl Piv pivaloyl PKA protein kinase A PKC protein kinase C PKS polyketide synthase PMA p-methoxyamphetamine PMB p-methoxybenzyl PPTS pyridinium para-toluene sulfonate pTSA para-toluene sulfonic acid py pyridine

Q q quartet (NMR) quant quantitative R Raf rapidly growing fibrosarcoma or rat fibrosarcoma RCAM ring-closing alkyne metathesis RCC renal cell carcinoma RCM ring-closing metathesis

X R retention factor (TLC) f rt room temperature (ca. 23°C)

S s singulet (NMR) SAR structure-activity relationship sat. saturated Ser serine SOS son of sevenless Src (gene) family of proto-oncogenic tyrosine kinases

T t triplet (NMR) TAS-F tris(dimethylamino)-sulfur(trimethylsilyl) difluoride TBAF tetrabutylammonium fluoride TBAI tetrabutylammonium iodide TBDMS tert-butyldimethylsilyl TBDPS tert-butyldiphenylsilyl TBS tert-butyldimethylsilyl T-cells thymus cells TES triethylsilyl TfO trifluoromethane sulfonate TFP tri-2-furylphosphine THF tetrahydrofuran Thr threonine TIPS triisopropylsilyl TLC thin layer chromatography TMS trimethylsilyl TMSE -trimethylsilyl ethyl TMSOK potassium trimethylsilanolate Tos (Ts) p-toluenesulfonyl

U UV ultraviolet

V VEGFR vascular endothelial growth factor receptor

Z Z cis

XII Introduction

1. Introduction Natural products are chemical compounds or substances produced by living organisms found in nature. They may be extracted from tissues of terrestrial plants, marine organisms or microorganism fermentation broths. Most of the natural products are secondary metabolites featuring complex structures and they usually possess a pharmacological or biological activity, which can be used for pharmaceutical drug discovery and drug design. In the period of from 1970 to 2006 a total number of 24 unique natural products were discovered that led to an approved drug1 and almost half of the drugs approved since 1994 are based on natural products.2 As natural products are able to interact with many specific targets within the cell,3 they are finding increasing use as probes to interrogate biological systems as part of chemical genomics and related research.

1.1. Resorcylic Acid Lactones (RALs) Resorcylic acid lactones (RALs) are polyketide natural products with a 14-membered macrocyclic ring containing a resorcylic acid moiety and they have been known for decades. In 1953, radicicol (1, also called monorden, Fig. 1) was isolated as the first member of this family of natural products. It was obtained from a mould which belongs to the species Monosporium bonorden and had been reported to have antifungal properties at that time.4 Zearalenone (2), an anabolic and marked uterotrophic active metabolite, was isolated from extracts of the fungus Gibberella zeae in 1962,5 followed by isolation of LL-Z1640-2 (3) (isolated from an unidentified fungus in 19786), and hypothemycin (5), an antibiotic metabolite isolated from Hypomyces trichothecoides in 1980 (Fig.1).7 Apart from zearalenone (2), which was used as a bovine growth stimulant and today is known and employed as an estrogenic mycotoxin,8 this group of natural products did not arouse much interest within the organic chemistry community for many years.

OH O OH O OH O O O O O HO O O Cl HO O HO O R OH

radicicol (1) zearalenone (2) LL-Z1640-2 (3); R = H radicicol A (4); R = OMe

OH O OH O OH O O O O HO O O O O O HO HO OH OH OH OH hypothemycin (5) L-783277 (6) aigialomycin D (7) Figure 1. Selected members of the family of resorcylic acid lactones (RALs).

1 Introduction

1.1.1. Biosynthesis of RALs RALs are mycotoxins produced by a variety of different fungal strains via polyketide biosynthesis. The type 1 fungal polyketide synthases (PKSs) are large multidomain enzymes that catalyze the condensation of units of thioacetates or -malonates. Different modules of the multidomain enzyme further process the product of each condensation step. In that way the initially formed -keto esters can be reduced and dehydrated (Fig. 2).9

PKS O S OH O

O O O O HO S CoA HO O

R R Figure 2. Biosynthesis of the resorcylic acid lactones.

The proposed biosynthesis of zearalenone (2) includes two PKSs. One of them is responsible for the assembly of five acetate units and further processing to the adequate oxidation state of each carbon. The second PKS is responsible for the remaining three rounds of condensation without carbonyl reduction. The poly--keto ester part cyclizes to an aromatic resorcylic acid ester. The lactone ring is formed via a cyclization module on the second PKS with concomitant release from the enzyme.10 The diversity of functionality present around the members of the RAL- family is due to the different levels of processsing during the first five condensation steps.11 Since the fungal polyketide synthases are widespread in fungi, RAL-type metabolites have been isolated from different fungal strains. This has aided the revision of the initially proposed erroneous structures of radicicol (1) and hypothemycin (5). Thus, the correct structure of 5 was established after re-isolation from Coriolus versicolor by Agatsuma et al. in 1993.12 This was the time when the RALs produced new interest by discoveries concerning their broad range of biological activity.

1.1.2. Biological Activity of RALs As mentioned above, radicicol (1) was first reported to have mild sedative and a moderate antibiotic activity.13 In 1992 Kwon et al. reported that 1 reversed the Src- transformed morphology of fibroblasts and they ascribed this effect to the inhibition of the oncogenic kinase Src.14,15 In 1998 it was revealed that 1 is a potent and selective inhibitor of Hsp90.16,17 As many protein kinases depend on functional Hsp90 for proper folding, 1 indirectly affects cellular protein kinase activity and, thus, it had initially been thought to be a kinase inhibitor. The heat shock protein 90 (Hsp90) is a molecular chaperone responsible for the folding and maturation of proteins. It is upregulated in response to stress, e. g. elevated temperatures. It is one of the most abundant proteins in cells as it accounts for 1-2 % of their protein mass.18 The ubiquitous functions of Hsp90 include assisting in protein folding, cell signaling and

2 Introduction tumor repression. Therefore, it plays an important role in pathologies ranging from cancer to neurodegenerative diseases and it has recently emerged as one of the most exciting therapeutic targets.19,20 Although 1 does not resemble ATP from a structural point of view, it was found to be a competitive and non-covalent ligand for the ATP binding site of Hsp90.21 Even though 1 is one of the highest affinity ligands for Hsp90 (19 nM), it possesses two pharmacological limitations. The conjugated dienone and the strained allylic epoxide of 1 are highly chemically reactive moieties. These two different reaction sites of 1 seem to be the reason for its effectiveness at the cellular level, but also its failure in animal models due to metabolic instability. These limitations were partly overcome by converting the ketone to an oxime or the corresponding methylated oxime, which prevents the enone to function as a Michael acceptor. The oxime-derivative was shown to be active in mouse xenografts models.22,23 However, the clinical development of these compounds was abandoned due to toxicity, which may be related to the alkylating properties of the epoxide moiety.24 While radicicol (1) does not have any noticeable kinase inhibitory activity, the cis- enone containing RALs hypothemycin (5), LL-Z1640-2 (3) and L-783277 (6) have been reported to irreversibly inhibit mitogen activated protein kinases (MAP kinases) in an ATP-competitive manner.25,26 Inhibitors of MAP kinases are of particular interest, as the MAP kinases relay, amplify and integrate signals from a variety of extracellular stimuli. The importance and the role of the MAP kinase pathway will be further discussed in chapter 1.2. of this thesis. Direct kinase inhibition by cis-enone containing RALs was first reported by Zhao et al. in 1999 for hypothemycin (5) and L-783277 (6).25 Both RALs were found to inhibit

Mek1 with IC50 values of 15 nM (5) and 4 nM (6), respectively. Moreover, it was revealed that L-783277 (6) is a time-dependant, ATP-competitive irreversible inhibitor of Mek, a reversible and moderate inhibitor of Lck (IC50 value of 750 nM), but it does not inhibit Raf, PKC and PKA activities. Interestingly, the 7’,8’-trans analog of 6 was found to be significantly less active against Mek1 (IC50 value of 300 nM). The authors suggested that the inhibition of Mek was most likely due to a covalent interaction between the , unsaturated ketone of L-783277 (6) and the active site of Mek. This hypothesis has recently been corroborated by a study by Schirmer et al.27 By a structural-bioinformatics analysis a conserved cysteine residue in the ATP binding site of kinases inhibited by hypothemycin could be identified, which is only present in a subset of 46 kinases out of the kinome database.27 In a screening panel of 124 kinases hypothemycin inhibited 18 out of 19 enzymes that contained this Cys residue (Cys166 in human Erk2). The formation of a covalent Erk2-hypothemycin adduct (Fig. 3) could be proven by mass spectrometry of a tryptic protein digest after incubation with [3H]-hypothemycin. Recently, the 2.5 Å crystal structure of Erk2 with covalently bound hypothemycin (5) was determined.28

3 Introduction

OH O OH O S-Enz 1 9' 3 O 7' Enz-SH O 1' 5' O O O O 5 O HO O HO OH OH hypothemycin (5) Figure 3. Structure of the covalent Michael adduct between hypothemycin (5) and a Cys-thiol of a protein kinase.27

Prior to this work, Ohori et al. had already published crystallographic data of the Erk2-LL-Z1640-2 (3) adduct,29 by which it was demonstrated that a covalent bond was formed between Cys166 of Erk2 and the C8’ position of the inhibitor LL-Z1640-2 (3). When LL-Z1640-2 (3) was first reported in 1978, it was found not to be associated with any interesting biological activity to it.6 In 2003, it was shown however, that LL- Z1640-2 (3) is a potent and irreversible ATP-competetive inhibitor of TAK1 with an

IC50 value of 8 nM. In contrast, radicicol (1) and zearalenone (2) showed no 26 noteworthy inhibitory effects with IC50’s greater than 10 M. TAK1 is a mitogen activated protein kinase (MAPKKK) which is involved in the p38 MAP kinase signaling cascade of proinflammmatory signals. Furthermore, it could be shown that 3 was 50-fold less active against Mek1 (411 nM) and had no inhibitory effect on MEKK1 or ASK1MAPKKKs. Importantly, the authors were also able to show that 3 effectively suppresses inflammation in vivo. It should be noted here, that the animal model which was used by Ninomiya-Tsuji et al.26 resembles our in vivo evaluation of L-783277 (6) (see chapter 3.5.3.) and was based on topical application of the compound on swollen mouse ears suffering from chemically induced inflammation. Hypothemycin (5) exhibits antiproliferative activity against a number of human cancer cell lines in vitro12 and inhibits tumor growth in vivo.30 Additionally, 5 was found to inhibit T-cell proliferation after stimulation with anti-CD3 mAb/PMA and to modulate the production of cytokines during T-cell activation.31 Hypothemycin (5) was also able to reduce levels of phosphorylated Erk1/2 in PMA-treated T-cells. But it was not determined whether this effect was due to direct kinase inhibition or else was secondary to the compound’s interaction with other none-kinase targets. The latest data on kinase inhibition of the RAL family were published by Winssinger et al., who showed that radicicol A (4) inhibits VEGFR2, VEGFR3, Flt3 and also PDGFR in the low nanomolar range.32 It should be noted, however, that 4 (Fig. 1) had been previously reported to be a general phosphorylation inhibitor of cellular proteins and was a likely kinase inhibitor.33,34 Aigialomycin D (7) was isolated together with hypothemycin (5) from the mangrove fungus Aigialus parvus in Thailand.35 Both 5 and 7 were found to have moderate antimalarial activity in the low micromolar range and to be cytotoxic at similar concentrations. The biological activity of 7 has also been investigated by Winssinger et al., who synthesized selected analogs.36 7 was found to inhibit CDK1 and CDK5 with IC50’s of 5.7 M and 5.8 M, respectively. GSK-3 was inhibited with an IC50

4 Introduction value at 14 M, but surprisingly 7 was not an inhibitor of the Plasmodium homologue of GSK-3. Over the last two decades the inhibition of kinase function has developed into a major paradigm for the discovery of new drugs, in particular in oncology,37,38,39 but also against inflammatory diseases,40,41 and for the treatment of cardiovascular42,43 and CNS-based disorders44,45. All low-molecular-weight protein kinase inhibitors, which have been approved for clinical use so far, are synthetic molecules containing aromatic heterocycles and a variety of hydrogen bond donor and acceptor moieties.46 These compounds will be further discussed in chapter 1.3. It should already be noted, that there is no structural homology between these low-molecular-weight protein kinase inhibitors and the RALs. Moreover, most of these clinically approved synthetic compounds suffer from insufficient solubility and oral bioavailability.47 In conclusion, the diverse biological activity of natural and synthetic resorcylic lactones makes these compounds into representatives of a new attractive scaffold class and a privileged starting point for the discovery of new kinase inhibitors.

1.2. MAP Kinase Pathways The MAP kinases were discovered 20 years ago and together with their upstream regulators they belong to the most extensively studied signal transduction molecules.48 Sturgill and Ray detected an insulin-activated protein (Ser/Thr) kinase activity in extracts of 3T3-L1 adipocytes, capable of phosphorylating a microtubule- associated protein-2 (MAP-2).49 When it was found that tyrosine phosphorylation of the MAP-2 kinase polypeptide was also stimulated by many growth factors and by active phorbol esters, the acronym “MAP” was re-assigned from the term “microtubule-associated protein” to “mitogen-activated protein”. Its activation by insulin and growth factors through a tyrosine-specific phosphorylation suggested that “MAP” might be an important downstream effector of cell signaling and this idea revived interest in this kinase.48 Today, the MAP kinase pathways are known to be ubiquitous signaling modules by which cells transduce extracellular stimuli into intracellular responses. MAP kinases are major components of pathways controlling embryogenesis, cell differentiation, and cell death.50 Multicellular organisms have three well-characterized subfamilies of MAPKs that control a variety of physiological processes. These enzymes are regulated by a three-step-serial phosphorylation system in which one activated kinase phoshorylates and thereby activates the next kinase within the cascade. The extracellular-regulated kinases (Erks) function in the control of cell division. For this reason inhibitors of these enzymes are being sought for cancer treatment.37,38,39 The c-Jun amino-terminal kinases (JNKs) act as regulators of transcription and their inhibitors are presumed to be effective in rheumatoid arthritis.51,52,40 Furthermore, the p38 MAPKs have been shown to be activated by inflammatory cytokines and environmental stresses, thus contributing to diseases like asthma and autoimmunity.41,53

5 Introduction

Fig. 4 gives an overview of the MAP phosphorelay systems. The modules shown are single representatives of pathway connections for the respective MAPK phosphorylation system. For example, there are three Raf proteins (c-Raf1, B-Raf, A- Raf), two MKKs (MKK1 and MKK2), and two Erks (Erk1 and Erk2) that can constitute the respective MAP phosphorylation cascade depending on the growth factor or stimulus. The Erk, JNK and p38 pathways are the best understood pathways of the MAP phosphorelay systems. This schematic overview demonstrates the complexity of the MAPK phosphorylation system which is not understood in every detail so far.53

Stimulus Growth Integrin Oxidative IL-1 factor stress

Activator RasGTP Rac1 Src TRAF6- TAB1/2

MKKK C-Raf1 MEKK1 MEKK2 TAK1

MKK MKK1 MKK4 MKK5 MKK6

MAPK ERK1 JNK1 ERK5 p38

Substrate p90RSK c-Jun MEF2 MNK1

Figure 4. Schematic overview of the MAPK phosphorylation systems.53

Altogether fourteen MAP kinase genes have been identified in the human genome, which define seven MAP kinase signaling pathways. MAP kinases can be classified into conventional or atypical enzymes, depending on their ability to get phosphorylated and thus activated by members of the MAP kinase kinase/Mek family. The conventional MAP kinases (Erk1/Erk2, p38s, JNKs and Erk5) are all substrates of the MAPKKs and are much better understood than the atypical MAP kinases which include Erk3/Erk4, NLK and Erk7.54 The Ras-Raf-Mek-Erk pathway is schematically shown in the second column of Fig. 4 and as an example is outlined in more detail in Fig. 5. The deregulation of this pathway is implicated in the development of numerous types of cancers.55 The binding of epidermal growth factor (EGF) induces receptor dimerisation and autophosphorylation on tyrosine residues. The EGF dimer serves as a docking

6 Introduction partner for the Grb2-SOS complex, which in turn activates the small G-protein Ras by stimulating the exchange of guanosine diphosphate (GDP) to guanosine triphosphate (GTP). This exchange induces a conformational change in Ras, which enables its binding to Raf. Activated Raf activates Mek (also known as MAPK/MKK1) by phosphorylation, which in turn phosphorylates and activates extracellular-signal- regulated kinase (Erk1). Activated Erk has many substrates in the cytosol and is able to induce cell proliferation, differentiation and apoptosis. For example, Erk can control gene expression by entering the nucleus and phosphorylating transcription factors such as Elk1.56

Figure 5. Ras-Raf-Mek-Erk signaling pathway.56

As the Erk pathway is often up-regulated in human tumors it represents an attractive target for anticancer drug development.57 Overall, extensive research on the inhibition of dysregulated cellular signaling pathways so far has led to nine different low-molecular-weight protein kinase inhibitors to be approved for the clinical treatment of different types of cancers. These compounds will be discussed in more detail in chapter 1.3.

1.3. Clinically Approved Kinase Inhibitors Chronic myeloid leukemia (CML) is a form of leukemia characterized by increased proliferation of predominantly myeloid cells in the bone marrow and the accumulation

7 Introduction of these cells in the blood. CML is a clonal bone marrow stem cell disorder. This dysfunction occurs most commonly in the middle-aged and elderly and accounts for 15-20 % of all cases of adult leukemias in Western populations.58 The underlying cause of CML is a characteristic reciprocal translocation between chromosomes 9 and 22, which cytogenetically results in the Philadelphia chromosome (Ph) and molecularly gives rise to the chimeric BCR-ABL1 gene.59 The protein product of this hybrid gene is either a 210 kDa (in 33 % of patients) or a 190 kDa (in 66 % of patients) tyrosine kinase, which is constitutively activated. The RNA-transcript of BCR-ABL does not require activation by other cellular signaling proteins. The BCR- ABL kinase drives the pathogenesis of BCR-ABL1-positive leukemias by activating a cascade of proteins which play critical roles in cellular signal transduction and transformation.60 As a consequence, the BCR-ABL tyrosine kinase represents a therapeutic target for the treatment of CML. Imatinib (8) was the first ABL tyrosine kinase inhibitor approved for clinical use (Fig. 6). The compound is marketed by Novartis as Gleevec® (USA) or Glivec® (Europe/Australia). Furthermore, 8 is the first member of a new class of agents that act by inhibiting particular tyrosine kinase enzymes, instead of non-specifically inhibiting rapidly dividing cells. 8 is also used in the treatment of gastrointestinal stromal tumors (GISTs) and a number of other malignancies.61

N N N OH HN N N N N N N N H N NH S HN F3C NH O Cl dasatinib (10) O N O N N

imatinib (8) nilotinib (9) HN N N Figure 6. Structures of the kinase inhibitors imatinib (8), nilotinib (9) and dasatinib (10).

To date, imatinib (8) is still the first-choice treatment for patients with chronic myeloid leukemia in chronic phase, because of its high response rate. However, there are CML cases which show primary resistance to 8 or where relapse occurs after an initial response.62 Nilotinib (9, Tasigna®) and dasatinib (10, Sprycel®) are second- generation BCR-ABL inhibitors, which have shown significant activity in imatinib- resistant CML cases.60 The epidermal growth factor receptor (EGFR) is a member of the erbB family of receptor tyrosine kinases, which also includes HER2 (erbB2), HER3 (erbB3) and HER4 (erbB4). Activation of these cell surface receptors promotes signaling cascades, which lead to uncontrolled cell growth, differentiation, cell survival, cell cycle progression and angiogenesis.63 Gefitinib (11), also known under its trade name Iressa®, was the first selective inhibitor of the EGFR’s tyrosine kinase domain 8 Introduction

(Fig. 7). 11 was launched in 2002 as non-small cell lung cancer (NSCLC) treatment, but in 2005 it failed to reach statistical significance in a phase III trial. However, in 2008, AstraZeneca filed an MAA in the EU seeking approval for the oral treatment of locally advanced or metastatic NSCLC in patients who have been pretreated with platinum-containing chemotherapy. AstraZeneca continues the clinical development of 11, including phase II trials as monotherapy for the treatment of adrenocortical carcinoma and phase II trials for the treatment of salivary glands cancer. Acting in a similar manner to erlotinib (12), gefitinib (11) selectively targets the mutant proteins in malignant cells.63 Erlotinib (12) (Tarceva®) is used to treat NSCLC and pancreatic cancer. 12 binds to the adenosine triphosphate (ATP) binding site of the receptor in a reversible fashion and has shown a survival benefit in the treatment of lung cancer in phase III trials.64 It should be noted that the mode of action described for 12 resembles all approved kinase inhibitor drugs, except for temsirolimus, which will be discussed below.

F O N O O HN Cl O N N O O N HN O N erlotinib (12)

gefitinib (11)

O N N H

N sunitinib (13) F H O N H N

N Cl NH O HN O F3C NH O HN N O O N H O Cl S O lapatinib (14) F sorafenib (15) Figure 7. Structures of the kinase inhibitors gefitinib (11), erlotinib (12), sunitinib (13), lapatinib (14) and sorafenib (15).

Sunitinib (13), also known under its trade name Sutent®, is an oral, small-molecule, multi-targeted tyrosine kinase inhibitor that has potent anti-angiogenic and antitumor activities. 13 was approved by the FDA for the treatment of renal cell carcinoma (RCC) and imatinib-resistant gastrointestinal stromal tumors (GISTs) in 2006. 13 inhibits cellular signaling by targeting multiple receptor tyrosine kinases, which include all platelet-derived growth factor receptors (PDGF-R) and vascular

9 Introduction endothelial growth factor receptors (VEGF-R), which play important roles in both tumor angiogenesis and tumor cell proliferation.65 Lapatinib (14) is an orally active chemotherapeutic drug, which is used for the treatment of breast cancer. 14 inhibits the tyrosine kinase activity associated with two oncogenes, EGFR (epidermal growth factor receptor) and HER2/neu (Human EGFR type 2). HER2/neu-positive tumors account for approximately 20 % of all breast cancers and are generally associated with poor prognosis.66 14 inhibits receptor signaling by binding to the ATP-binding pocket of the EGFR/HER2 protein kinase domains, preventing auto-phosphorylation and subsequent activation of the signaling cascade.67 To conclude the discussion on clinically relevant low-molecular-weight inhibitors, sorafenib (15, Nexavar®) is an inhibitor of several protein kinases including Raf kinase, PDGF, VEGF receptor 2 and 3 kinases and cKit, the receptor for stem cell factor.68 In contrast to the drugs discussed above, which are all purely synthetic in origin, temsirolimus (16) (Torisel®) is a natural product-derived inhibitor of the mammalian target of rapamycin (mTOR), which is implicated in multiple tumor-promoting intracellular signaling pathways.69 Torisel® is an intravenous drug for the treatment of renal cell carcinoma (RCC) that was approved by the FDA and the European Medicines Agency (EMEA) in 2007. Temsirolimus (16) is an ester of the natural product rapamycin (sirolimus, Rapamune®), which itself is used as an immunosuppressive drug.69 In terms of mechanism of action, as an anticancer agent, temsirolimus works by inhibiting mTOR-driven cell proliferation69 and directed against MS,70 temsirolimus has the potential to block the inflammatory responses related to autoimmune diseases by blocking T-cell proliferation. Sirolimus and temsirolimus (16) form a complex with the intracellular protein FKBP12 that blocks the activation of a kinase cascade that is crucial for cell-cycle progression. These agents thus prevent cell division in new cells, such as in proliferating T and B lymphocytes.70

O OH

O N O O O O O HO O OH

O O O

temsirolimus (16) Figure 8. Structure of temsirolimus (16).

10 Introduction

The serine/threonine specific kinase mTOR exhibits a highly conserved structure. Activation of the mTOR pathway is mediated through a series of complex signaling interactions, which leads to enhanced translation of ribosomal proteins and elongation factors. Furthermore, the activation of mTOR results in the downstream production of hypoxia-inducible factor-1 (HIF-1), which regulates the transcription of genes that stimulate cell growth and angiogenesis.71 In addition to low-molecular-weight kinase inhibitors, antibodies against cell surface receptors containing an intracellular tyrosine kinase domain or against the corresponding ligands have become an important part of cancer treatment. For example, cetuximab, which is marketed under the trade name Erbitux®, is a chimeric monoclonal antibody that blocks the epidermal growth factor receptor (EGFR) and is used for the treatment of metastatic colorectal cancer.72

1.4. Total Syntheses of Cis-Enone Containing RALs As the cis-enone-containing RALs exhibit interesting biological activity without showing structural similarities to traditional kinase inhibitor scaffolds, they represent a promising and attractive new class of lead structures for kinase inhibition. As indicated above, these compounds offer a “built-in” specificity, as their inhibitory potential appears to be related to the formation of a covalent bond with an active site Cys residue of the respective kinases. While this feature may also be regarded as a limitation, as it restricts the number of kinases that could be susceptible to inhibition to ca. 10 % of the human kinome,27 many of the kinases from this group are highly relevant targets for drug discovery. Covalent enzyme inhibitors are often viewed with skepticism as potential drug candidates; however, as has been pointed out recently, covalent enzyme inhibition is not an infrequent mode of action even among marketed drugs.27 The discovery of the interesting biological activities of this natural product family has led to drastically increased interest in the chemistry of RALs in the recent past. In this chapter the existing total syntheses of cis-enone-containing RALs will be briefly discussed, except for the total synthesis of L-783277,73 which was developed within this thesis and will be discussed in detail in subsequent chapters. Furthermore, our group has recently published a review which deals with the biology and chemistry of cis-enone-containing RALs.74

1.4.1. LL-Z1640-2 and Hypothemycin The first total synthesis of a cis-enone containing RAL was reported by Tatsuta and co-workers in 2001 for LL-1640-2 (3).75 As depicted in Scheme 1, the key elements of the retrosynthesis for LL-1640-2 (3) included the Mukaiyama macrolactonization reaction of a protected seco acid LL-I, introduction of the E double bond between C1’ and C2’ through reductive double bond isomerization of the Z-configured allylic carbonate LL-II, and the formation of the C6/C1’ bond through Sonogashira coupling

11 Introduction between the aromatic part LL-III and the alkyne LL-IV. The latter was envisioned to be accessible from LL-V (D-ribose).

OH O OPG O OH

O OH OPG OPG OH (S) O O O 3 OH LL-I OPG

OPG O O OPG O O OH OPG OOH O OPG O OH OPG' O OPG' O I OPG OPG HO OH OPG OPG OH LL-II LL-III LL-IV LL-V (= D-ribose) Scheme 1. Retrosynthesis of LL-Z1640-2 (3) according to Tatsuta et al.75 The synthesis of 3 is summarized in Scheme 2 and started with the addition of lithiated TMS-acetylene to tris-MOM-protected D-ribose 17. This was followed by pivaloylation of the primary hydroxyl group and removal of the TMS group with TBAF to provide the propargylic alcohol 18 in 48 % overall yield (from 17). Although the stereochemical outcome of the acetylide addition remains insignificant, due to the reductive removal of the propargylic hydroxyl group later in the synthesis, it should be noted that the reaction proceeded with high selectivity. Alkyne 18 underwent smooth

Sonogashira coupling with iodide 19 in the presence of Pd(OAc)2, CuI, and Ph3P to furnish, after ethoxycarbonylation of the free hydroxyl group, compound 20 in excellent overall yield (83 %). The aromatic iodide 19 was prepared according to a procedure previously developed by Hegedus,76 presumably from 2-hydroxy-4- methoxy-benzoic acid methyl ester, but the preparation of 19 is not described in detail.75 After partial reduction of the C1’/C2’-triple bond in 20 using a 77 Pd/BaCO3/quinoline-system, subsequent hydrogenolysis under Tsuji conditions gave the desired E olefin in almost quantitative yield. Removal of the pivaloyl protecting group followed by Swern oxidation of the resulting free alcohol and dibromo-olefination of the corresponding aldehyde gave dibromo- olefin 22, which was further elaborated into propargylic alcohol 23 through treatment with n-BuLi and reaction of the lithiated acetylene formed in situ with (S)-propylene oxide. Seco ester 23 already incorporates the entire carbon framework of LL-Z1640-2 (3) and was further converted into the immediate macrocyclization precursor 24 via partial hydrogenation of the triple bond under Lindlar conditions and subsequent cleavage of the methyl ester with NaOH. Macrolactonization of 24 according to the method of Mukaiyama78 followed by global deprotection with 5 % HCl/MeOH gave the macrolactone 26, which was obtained in 36 % overall yield for the four-step sequence from 23. The transformation of this intermediate into LL-Z1640-2 (3) required selective oxidation of the allylic hydroxyl group at C6’, which could be accomplished with Dess-Martin periodinane (62 % yield) or DDQ, although the latter gave 1 in only 20 % yield. Although this is not explicitly discussed by Tatsuta, it

12 Introduction appears that several other oxidation methods failed to deliver the desired product. The synthesis depicted in Scheme 2 provided the target compound LL-Z1640-2 (3) in 3 % overall yield for a longest linear sequence of 19 steps (from D-ribose).

MOMO O

O MOMO O OOH 19 O PivO HO O I i-iii iv,v MOMO OMOM MOMO OMOM O MOMO OMOM OPiv OMOM 20 OMOM O O OMOM 17 O 18

MOMO O O MOMO O Br O vi vii - x OMOM Br O O OMOM O xi O OPiv O OMOM MOMO OMOM 21 22 OMOM

HO + - MOMO O HO MOMO O NClI 25 O OMOM xii, xiiiOH OMOM xiv, xv

O O MOMO OMOM MOMO OMOM 23 24

OH O OH O O xvi O OH OH O OH O O OH OH 26 3

Scheme 2. Reagents and conditions: i. TMS-acetylene, n-BuLi, BF3·Et2O, THF, -78°C → rt. ii. PivCl, pyridine, 0°C, 1h, 48 % (2 steps). iii. TBAF, AcOH/THF, 2h, quant. iv. Pd(OAc)2, CuI, Ph3P, Et3N, 2h, 85 %. v. ClC(O)OEt, pyridine, 0°C, 1h, 98 %. vi. H2, Pd/BaCO3, quinoline, EtOH, 30min. vii. Pd(dba)2CHCl3, nBu3P, HCOONH4, dioxane, 95°C, 1h, 96 % (2 steps). viii. MeONa, MeOH, 50°C, 3h, 95 %. ix. (COCl)2, DMSO, Et3N, CH2Cl2, -78°C → rt. x. CBr4, Ph3P, CH2Cl2, 0°C, 15min, 85 % (2 steps). xi. (S)-propylene oxide, n-BuLi, BF3·Et2O, THF, -78°C → rt, 45 %. xii. H2, Pd/BaCO3, quinoline, AcOEt, 30min. xiii. 2M NaOH/MeOH-dioxane, 90°C. xiv. Et3N, MeCN, 50°C, 1h, 47 % (3 steps). xv. 75 5 % HCl/MeOH, 50°C, 2h, 76 %. xvi. Dess-Martin periodinane, CH2Cl2, 15min, 62 %.

An alternative approach to the synthesis of LL-Z1640-2 (3) has been reported by Sellès & Lett, who investigated the synthesis of 3 and its conversion into hypothemycin (5). According to Sellès & Lett’s retrosynthetic analysis79 (Scheme 3), the macrocyclic framework of hypothemycin (5) and LL-Z1640-2 (3) could be constructed either through Mitsunobu-based macrolactonization from seco acid H-I or through a Suzuki-type macrocyclization of ester H-I’. Both H-I and H-I’ would be derived from o-bromo benzoic acid H-II and either the 10’R or 10’S acetylene H-III through Mitsunobu-based esterification or Suzuki coupling, respectively. As the Mitsunobu-based macrolactonizations proceed with inversion of configuration, the precursor H-I needed to have the opposite configuration (10’-R) as the natural

13 Introduction product. However, the synthesis of the two diastereoisomeric versions of H-III would be achieved in a convergent manner from two fragments comprising C1’-C7’ and C8’-C10’, respectively.

OH O OH OH O OH O OPG (R) OH O Br OH O O O H-I H-II O OH or O O O OH O O(H, PG) OH (S) 5 O O O OPG O Br OPG O O H-I' H-III (10' R or S) Scheme 3: Retrosynthesis of hypothemycin (5) according to Sellès and Lett.79

The synthesis of the aromatic precursor H-II, as in Tatsuta’s case, was based on previous work by Hegedus and co-workers76 and involved O-TBS protection of 4- methoxy salicylic acid 27 followed by conversion to the diethyl amide and ortho bromination to provide the o-bromo diethylamide 28 in 74 % overall yield (Scheme 4).79 The latter was transformed to the corresponding methyl ester 29 by treatment with Meerwein salt and subsequent hydrolysis of the resulting (O-deprotected) imino ester. Saponification of 29 with NaOH in DME at reflux gave carboxylic acid 30 in quantitative yield. Interestingly, the saponification of 29 worked without any detectable decarboxylation which may occur in salicylic acid derivatives with a free phenolic hydroxyl group under more forcing conditions.76

OH OH TBSO N OH OR

O i- iii O iv O

O O O 29 :R=Me 27 28 v 30 :R=H

Scheme 4:. Reagents and conditions: i. TBSCl, iPr2NEt, DMF, 2h. ii. Et2NAlMe2, reflux, overnight, 98 % (2 steps). iii. t-BuLi/pentane (1.08 equiv), Et2O, -78°C, 10min, then Br2 (1.06 equiv.), 75 %. iv. Me3OBF4 (1.3 equiv), CH2Cl2, overnight; v. aq sat. Na2CO3/MeOH 1/1, 6h, 76 %. v. Conc. NaOH/DME 1/1, reflux, overnight; or TMSOK (2 equiv), DME, reflux, overnight, quant.79

The synthesis of precursors H-III is summarized in Scheme 5 starting with mono-TBS protected 2-butene-1,4-diol 31, which was obtained from 2-butyne-1,4-diol via Red-Al reduction and TBS-protection in 60 % overall yield.79 Conversion of alcohol 31 to the corresponding iodide followed by alkylation of TMS-acetylene and selective cleavage of the TBS-ether with DDQ gave allylic alcohol 32, which underwent Sharpless asymmetric epoxidation in 85 % yield and with >90 % ee.

14 Introduction

i-iv v,vi OTBS OH O NHPh HO O Me Si Me Si O 313 323 33

O O O viiO viii - x xi - xiii O OTBS O

O Me Si O Me3Si OH 3 34 35 36

TBSO RO

xiv xv, xvi O O

Me3Si O OH O OPMB

37 38 :R=TBS xvii 39 :R=H

Scheme 5. Reagents and conditions: i. MesCl, Et3N, CH2Cl2, -10°C → rt, 30min. ii. NaI, acetone, 1h, 86 % (2 steps). iii. TMS-acetylene, n-BuLi, THF/HMPA 10/1, -78°C → rt, 4h, 90 %. iv. DDQ (5 mol %), MeCN/H2O 9/1, 2h, 74 %. v. Ti(OiPr)4, (+)-DET, CH2Cl2, tBuOOH, -25°C, overnight, 85 %, > 90 % ee. vi. PhNCO, CH2Cl2/pyridine, 1h, quant. vii. BF3·Et2O, Et2O, -20°C, 2h, then 1N H2SO4, overnight, 91 %. viii. MeONa, MeOH, 8h, then Dowex 50 WX8 column eluted with MeOH, 93 %. ix. TBSCl, imidazole, DMF, 1h. x. 2-methoxypropene, TsOH (cat), CH2Cl2, 1h, 73 % (2 steps). xi. n-BuLi/hexane, Et2O, TMSCl (1.1 equiv), -30°C → -10°C. xii. DDQ (8 mol %), MeCN/H2O 9/1, 2h, 71 % (2 steps). xiii. (COCl)2, DMSO, Et3N, CH2Cl2, -78°C → 0°C, 30min, quant. xiv. 34, t-BuLi/pentane, Et2O, -78°C → 0°C, 56 % (10’ S); 77 % (10’ R). xv. K2CO3 (1.4 equiv), MeOH, 5h. xvi. PMBO(NH=)CCCl3, CF3SO3H 79 (0.004 equiv), Et2O, 4h, 72 % (10’ S); 74 % (10’ R) (2 steps). xvii. TBAF, THF, 10h, 97 %.

The epoxy alcohol was transformed to the phenyl carbamate 33, which upon treatment with BF3·Et2O gave the cyclic carbonate 34, thus establishing the chiral centers at C4’ and C5’ (hypothemycin/LL-Z1640-2 numbering) in a highly efficient manner (78 % overall yield for the 3-step sequence from achiral allylic alcohol 32). Base-catalyzed carbonate methanolysis followed by appropriate protecting group manipulations led to intermediate 35, which was further elaborated into aldehyde 36 via reprotection of the terminal acetylene, TBS removal and Swern oxidation. In order to avoid side reactions in the addition of the lithiated vinyl iodide 43 to aldehyde 36, protection of the alkyne moiety was required. The addition proceeded in 55 % and 77 % yield for the S- and R-enantiomers of 43, respectively. Vinyl iodide 43 was obtained from TMS-acetylene and (R)- or (S)-propylene oxide in 5 steps and excellent overall yield (59 %) through Lewis acid-catalyzed epoxide opening followed by TBS-protection, removal of the TMS group from the terminal alkyne moiety, conversion to the iodo alkyne and finally hydroboration with Sia2BH and protonolysis (Scheme 6).

15 Introduction

O i, iiSiMe3 iii, iv I v TBSO TBSO TBSO I 40 41 42 43

Scheme 6. Reagents and conditions: i. TMS-acetylene, n-BuLi, Et2O, -78°C, then (R)- or (S)- propylene oxide (40R or 40S) and further addition of BF3·Et2O (1.0 equiv) in 50min, -78°C, 89 %. ii. TBS-Cl, imidazole, DMF. iii. K2CO3, MeOH, 5h, 86 % (2 steps). iv. n-BuLi, THF/hexane, -78°C, 15min, 79 then I2, THF, 86 %. v. a) Sia2BH, THF, -20°C → 0°C, 3h; b) AcOH, 65°C, 3h, 89 %.

The two products 37 obtained from the addition of the lithiated vinyl iodide 43 to aldehyde 36 (Scheme 5) were converted to the free terminal acetylenes 38 and 39. The latter corresponds to the (C10’) hydroxyl-unprotected version of precursor H-III in Scheme 3. Steglich esterification of alcohol 39 with carboxylic acid 30 proceeded smoothly and furnished the desired macrocyclization substrate 44 in 76 % yield (Scheme 7).80

OH O OH O OH O iiiO O OH O O O Br OPMB O OPMB O Br O O 30 44 45

Scheme 7. Reagents and conditions: i. 30 (1 equiv), DCC (2.2 equiv), DMAP (1.2 equiv), CH2Cl2, 5h, 76 %. ii. a) Sia2BH (3.9 equiv), THF, -20°C → rt, 2h; b) addition of acetone (3 equiv), 2M aq K3PO4 (2 eq), −10°C → rt; c) addition of the mixture to a solution of 4 mol % [Pd(OAc)2+4TFP] in DME (i.e. 79,80 substrate concentration = 0.034 M in DME/H2O ~ 30/1), refl, 6h, 15 %.

Unfortunately, the intramolecular Suzuki-reaction with the vinyl-borane derived from 44 gave the desired macrolactone 45 only in low yield as part of complex reaction mixtures (15 % under the optimized conditions shown for Scheme 7). Notably, model substrate 46 (Fig. 9) gave the corresponding macrolactone in only 9 % yield, while none of the desired cyclization product was observed with the least constrained substrate 47 (Fig. 9). For this reason Sellès & Lett abandoned the Suzuki-based intramolecular macrocyclization approach.

OH O OH O O O

O Br OPMB O Br OPMB 46 47 Figure 9. Model substrates 46 and 47for the intramolecular Suzuki-reaction.80

Fortunately, they obtained more satisfactory results for a macrolactonization-based approach to the hypothemycin/LL-Z1640-2 macrocycle (Scheme 8).80 In contrast to the intramolecular setting, the intermolecular Suzuki reaction between the aryl bromide 29 and the vinyl-borane derived in situ from acetylene 38 provided the coupling product in 76 % yield. Subsequent TBS-removal and ester saponification gave partially protected seco acid 48 which was cyclized to 49 under Mitsunobu conditions in 67 % yield. Removal of the PMB protecting group from 49 gave an inseparable 60/40 mixture of diastereoisomeric alcohols 50, whose oxidation to the

16 Introduction required ketone proved to be a significant challenge. Oxidation could not be achieved with either activated MnO2 or DDQ, but was finally accomplished with PCC in the presence of 2,5-dimethyl-pyrazole81,82 in 62 % yield for the mixture of diastereomers 50.

OH O OH O OH OH O O O i - iiiOH O OPMB iv O O Br O O OPMB O O 29 48 49

OH O OH O OH O v O vi, viiO viii O O OH OH O OH O O O O O OH O OH 50 3 5

Scheme 8. Reagents and conditions: i. a) 38, Sia2BH (2 equiv), THF, -25°C → rt, 2h; b) addition of 2M aq K3PO4 (2 equiv); c) addition of the mixture to a solution of 29 (1.2 equiv) and 15 mol % [Pd(OAc)2+4TFP] in DME (DME/H2O ~ 7/1), reflux, 8h, 71 %. ii. TBAF, THF, 6h, 93 %. iii. 2N aq NaOH (13 equiv)/MeOH 1/3, reflux, overnight, 71 %. iv. 39 (0.007 M), toluene, Ph3P (2 equiv.), DEAD (2 equiv), rt, 15min, 67 %. v. DDQ, CH2Cl2/pH 7 buffer 9/1, 30min, 94 % (3/2 mixture of isomers at C6’). vi. PCC (3 equiv), 2,5-dimethylpyrazole (10 equiv), CH2Cl2, 0°C, 6h, 62 %. vii. pTsOH (0.5 equiv), CH2Cl2/MeOH 1/1, 3.5h, 76 %. viii. mCPBA/NaHCO3 (3 equiv), -20°C → 0°C, 4h, then pH 7 work-up, 17 %.79,80

Interestingly, the major isomer was quantitatively converted to the enone, whereas a large part of the minor isomer was recovered unchanged (23 % yield based on the starting mixture of diastereoisomers). In subsequent experiments the minor isomer was shown to react only slowly under the above Parish conditions81,82 and after 24 h it gave only the 7’,8’-E-isomer 51 in 50 % yield (Scheme 9).

OH O

OH O O

O i O O O O OH O O O 50 51 (minor isomer)

Scheme 9. Reagents and conditions: i. PCC (3 equiv), 2,5-dimethylpyrazole (10 equiv), CH2Cl2, 0°C, 24h, 50 %.80

On the other hand, Jones oxidation of the 60/40 mixture of 50 gave the desired enone in 74 % yield (on a millimolar scale). It is also noteworthy that Sellès & Lett were unable to achieve selective allylic oxidation of the free triol 26 (derived from 50 by treatment with TosOH/MeOH; see also Scheme 2) either under Parish conditions or with MnO2. The latter finding is in contradiction with the work of Tatsuta (vide supra). Conversion of 50 into LL-Z1640-2 (3) was finally accomplished in 76 % yield by deprotection of the acetonide with TosOH/CH2Cl2/MeOH at room temperature. Virtually no isomerization of the C7’/C8’ double bond was observed under these

17 Introduction conditions and the remainder of the material was recovered unchanged (23 % yield of recovered starting material). The final transformation of LL-Z1640-2 (3) into hypothemycin (5) was severely hampered by the low reactivity of the C1’/C2’-double bond and could only be achieved in 17 % yield with mCPBA/NaHCO3 (30 % of starting material recovered).80 However, the epoxidation reaction was highly stereoselective (and also regioselective) and provided hypothemycin (5) as the only isolable epoxide isomer as had been predicted by Sellès & Lett at the outset of ther studies.79 In summary, the synthesis developed by Sellès & Lett provided LL-Z1640-2 (3) and hypothemycin (5) in overall yields of 1 % and 0.17 %, respectively, in 26 (27) steps for the longest linear sequence (from 2-butyne-1,4-diol). Although Sellès & Lett’s approach to LL-Z1640-2 (3) seems to be more convergent than the strategy of Tatsuta, the former includes a significantly higher number of steps for the longest linear sequence and is characterized by a lower overall yield. Thus, Tatsuta’s synthesis of LL-Z1640-2 (3) is more efficient, which is due to the use of D-ribose as a chiral starting material, which more than compensates for the less convergent character of the overall strategy. Only one total synthesis of hypothemycin (5) has been reported in the literature so far, but a number of semisynthetic derivatives of this natural product and their biological assessment have been recently reported by a group at Kosan.83 Methylation of 5 with TMS-diazomethane produced the methyl ethers 52a, 52b, and 52c in a 1:2:3 ratio (Scheme 10). Unfortunately, no yields for these transformations are reported by Hearn et al.83 The in vitro evaluation of compounds 52a-c in cancer cell proliferation assays revealed that the methylation of the hydroxyl group on C5’ does not significantly affect antiproliferative activity in comparison to hypothemycin (5), while monomethylation of the C4’ hydroxyl group or dimethylation lead to a profound loss in potency.83

OH O OH O

O i O

O O O O O HO O R2O OH OR1 5 52a :R1=H, R2=Me

52b :R1=Me, R2=H

52c :R1=R2=Me 83 Scheme 10. Reagents and conditions: i. TMSCHN2, HBF4, CH2Cl2, 52a:52b:52c = 3:1:2.

The Kosan group has also isolated a naturally occurring derivative of hypothemycin (from Hypomyces subiculosus), which lacks the methyl substituent on the C4 phenol group (Scheme 11; 4-O-demethylhypothemycin (53)).84 A series of aryl-alkyl ether derivatives were obtained from 53 employing Mitsunobu chemistry. The products 54a-d were designed to have enhanced polarity and improved aqueous solubility in comparison to hypothemycin (5) (Scheme 11).83 More importantly, most of the derivatives 54a-d, like 4-O-demethylhypothemycin (53), showed similar antiproliferative activity as hypothemycin (5), which indicates that substituents on C4-O

18 Introduction do not interfere with target protein binding. Therefore, it can be assumed that this position could be used to modulate the physicochemical properties of 5 without impairing the desired biological activity. However, it should be noted that no kinase inhibition data have been reported for 53 or any of the semisynthetic derivatives shown in Schemes 10 and 11.

O 54a :R = N

O H N OH O OH O N 54b :R= O i O O O OH OH HO O O O O O N OH R OH 54c :R = O 53

N 54d :R= N 83 Scheme 11. Reagents and conditions: i. ROH, Ph3P, DEAD, THF.

Besides the syntheses discussed above, a third macrolactonization-based approach to LL-Z1640-2 (3) has been reported by Winssinger and co-workers in the context of their work on radicicol A (4), which will be discussed in chapter 1.4.2. In contrast, a conceptually different strategy for the synthesis of 3 has been proposed by Henry et al. that would rely on ring-closure between C1’ and C2’ through ring- closing olefin metathesis (RCM).85 While this strategy has not been fully implemented so far, Henry et al. have reported the synthesis of the requisite triene precursor for the RCM-based ring-closure (61), using tartaric acid as the source of chirality at C4’ and C5’ of the aliphatic part of (3) (Scheme 12).85

O O OTBS TBSO O O O TBSO i - iii iv v, vi O O TBSO O O O O O O 55 56 57 58

OH O

OH 64 O OH O vii - x TBSO xi, xii HO xiii O O O OHO PM BO O O O PM BO O 59 60 61

Scheme 12. Reagents and conditions: i. LAH, THF. ii. NaH, TBSCl, THF, 89 % (2 steps). iii. Swern oxidation, 100 %. iv. a) CBr4, Ph3P, CH2Cl2; b) n-BuLi, Et2O, 79 %. v. (S)-propylene oxide, n-BuLi, BF3•Et2O, THF, 76 %. vi. TBSCl, Et3N, CH2Cl2, 97 %. vii. H2, Pd/BaSO4, quinoline, 100 %. viii. HF•py/pyridine, 65 %. ix. Swern oxidation. x. CH2=CHCH2MgBr, THF, 96 % (2 steps). xi. NaH, PMBCl, TBAI, THF, 100 %. xii. HF•py/pyridine, 97 %. xiii. 64, DCC, 67 %.85

19 Introduction

Thus, protected tartaric acid derivative 55 was first transformed into the known aldehyde 5686 through LAH reduction, mono-TBS protection of the resulting diol and oxidation of the free hydroxyl group under Swern conditions in 76 % overall yield. 56 was converted to acetylene 57, which, after lithiation with n-BuLi, was reacted with (S)-propylene oxide; subsequent TBS-protection of the resulting secondary alcohol gave bis-TBS-protected diol 58. The latter was elaborated into diene 59 via partial hydrogenation of the triple bond followed by selective cleavage of the primary TBS- ether with HF•pyridine, oxidation of the alcohol to the aldehyde and allylation with

CH2=CHCH2MgBr in 62 % overall yield. The hydroxyl group formed in the allylation step was protected as a PMB ether and subsequent removal of the TBS-group furnished the free secondary alcohol 60, whose DCC-mediated esterification with carboxylic acid 64 provided the desired triene 61 in 67 % yield.

OH O TBSO O OH O i-iii iv,v OH O OH

O OH O OTf O 62 63 64

Scheme 13. Reagents and conditions: i. TMS-CH2N2, Et2O, 90 %. ii. TBSCl, Et3N, CH2Cl2, 80 %. iii. 85 Tf2O, pyridine, 100 %. iv. nBu3SnCH=CH2, Pd(Ph3P)4, CH2Cl2, 100 %. v. NaOH, dioxane, 96 %.

Acid 64 was obtained in 5 steps from commercially available methyl 2,4,6- trihydroxybenzoate (62), which was initially converted to triflate 63 through selective methylation at O-4 with TMS-diazomethane, mono-TBS protection, and reaction of the unprotected hydroxyl group with triflic anhydride. Subsequent Stille coupling with

Bu3SnCH=CH2 followed by ester saponification with NaOH gave 64 in 69 % overall yield (based on 62). No attempts on RCM with triene 61 have been reported so far, but it remains to be seen how efficient this reaction can be in light of potential competition from six- membered ring formation through RCM between the terminal double bond in the aliphatic portion of 61 and the C7’ and C8’ double bond (hypothemycin/LL-Z1640-2 numbering). Bajwa & Jennings encountered similar difficulties while synthesizing aigialomycin D (7) based on a RCM-approach, which is discussed in detail in chapter 1.4.3.

1.4.2. Radicicol A The first total synthesis of radicicol A (4) was reported only recently by Winssinger and co-workers partly based on methodology that they had previously developed in the course of their work on aigialomycin (7) (see below).33 As illustrated by the retrosynthesis depicted in Scheme 14, Winssinger’s approach to radicicol A (4), as for the various other syntheses discussed above for LL-Z1640-2 (3), was to rely on macrocyclization through ester bond formation under Mitsunobu conditions. However, in contrast to all previous syntheses of cis-enone-containing resorcylic acid lactones, the construction of the seco acid RA-I (Scheme 14) does not involve formation of a C6-C1’ aryl-alkyl bond. Instead, C1’ is part of the aromatic building

20 Introduction block RA-II and the C1’-C2’ E double bond is formed through alkylation of this precursor with an alkyl iodide intermediate RA-III and subsequent oxidation/elimination. The C2’-C10’ fragment RA-III was envisioned to be assembled from appropriately protected building blocks RA-IV and RA-V, thus leading to a highly convergent synthesis of radicicol A (4). An additional important feature of Winssinger’s strategy was the projected use of a fluorinated protecting group for fragment RA-V, which enables the application of fluorous isolation technologies87 up to the penultimate step before macrolactonization.

OH O O O OH

O OH OPG OH O (R) O O O O OH O O 4 RA-I

O O O OPG' F PGO O PG''O O FPGO O + I OPG O + I O SePh O RA-II RA-III RA-IV RA-V

Scheme 14. Retrosynthetic analysis for radicicol A (4) according to Winssinger.33

As shown in Scheme 15, the aromatic building block 66 was obtained from benzoic acid 65 via esterification with 2-TMS-ethanol followed by deprotonation at the benzylic position (LDA) and reaction with (PhSe)2 in 88 % overall yield. It should be noted that the 2-methoxy substituent in 66 functions as a reversibly protected OH group that needed to be liberated at a later stage in the synthesis.

O O O O

SiMe3 OH i, ii O

O O O O SePh 65 66

Scheme 15. Reagents and conditions: i. a) (COCl)2, DMF (cat), CH2Cl2, 0°C, 1h; b) 2-(trimethylsilyl) ethanol, Et3N, DMAP (cat), 23°C, 1h, 98 %. ii. LDA (2.0 equiv), (PhSe)2 (1.0 equiv), THF, -78°C, 1h, 90 %.33

The protection of the 2-hydroxyl group as a methyl ether was dictated by the enhanced acid sensitivity of commonly used acid-labile protecting groups (such as MEM, EOM, PMB) in the presence of a 5-methoxy substituent, which also makes the aryl moiety highly prone to oxidation.33 This problem does not exist in resorcylic lactones that are unsubstituted at the 5-position. The synthesis of an appropriately protected C7’-C10’ fragment RA-V started with the protection of homoallylic alcohol 67 with a fluorous-tagged version of the

21 Introduction conventional PMB group88 followed by cross metathesis with a 2-fold excess of vinyl- borolane 68 (Scheme 16). Subsequently, the obtained E-vinyl-borolane 69 was converted into the desired Z-vinyl-bromide 70 in 72 % overall yield from 67.89

O B O 68 O i, ii iii B HO O O O Br 67C6F13C3H6O 69C6F13C3H6O 70

OC3H6C6F13 SiMe3 O O O

iv - vii viii, ix O O x, xi O O O C6F13C3H6O I OEOM O O OOEOM 71 72

HO OH O OH O O O O O OH O xii, xiiiOH xiv OH

O O OH O O OH OH O OOEOM O O 73 74 4

Scheme 16. Reagents and conditions: i. p-(C6F13C3H6O)BnO(NH=)CCl3, CSA (cat), CH2Cl2, 12h, 92 %. ii. 68 (2.0 equiv), Grubbs II (2.5 mol %), toluene, 80°C, 12h, 92 %. iii. Br2 (1.0 equiv, 1M in CH2Cl2), Et2O, -20°C, 10min; then NaOMe (2.2 equiv, 1M in MeOH), -20°C, 30min, 89 %. iv. t-BuLi, THF/Et2O, -100°C, 15min, then 76 (1.0 equiv), -100°C, 15min, 88 %. v. EOMCl (8.0 equiv), iPr2NEt (8.0 equiv), TBAI (cat), DMF, 12h, 96 %. vi. TBAF, THF, 12h, 92 %. vii. Ph3P, I2, imidazole, THF, 0°C, 1h, 91 %. viii. 66 (1.0 equiv), LDA (2.0 equiv), THF/HMPA 10/1, -78°C, 20min, 89 %. ix. H2O2 (2.0 F equiv), THF, 2h, 80 %. x. DDQ, CH2Cl2/H2O 2/1, 2h, 77 %. xi. TBAF, THF, 2h, 87 %. xii. R -Ph3P (2.0 F equiv), R -DEAD (2.0 equiv), toluene (10 mM), 2h, 81 % (> 80 % purity). xiii. BCl3, CH2Cl2, 0°C, F 15min, 88 % (purified by PTLC). xiv. PS-IBX (3.0 equiv), CH2Cl2, 1h, 90 %. R = C6F13C3H6; PS = polystyrene.33

Addition of lithiated 70 to aldehyde 76 (obtained in 3 steps from acetal-protected 2- deoxy-D-ribose 75, Scheme 17) gave 88 % yield of a 3/1 mixture of allylic alcohols that was further elaborated into the corresponding mixture of alkyl iodides 71 (Scheme 16) through EOM-protection of the newly formed hydroxyl group, cleavage of the primary TBS-ether, and treatment of the free alcohol with I2/Ph3P/imidazole. Alkyl iodide 71 was reacted with the lithiated selenide 66; subsequent oxidation of the phenylselenyl ether and elimination led to the desired E olefin 72 in high yield (64 % for the 3-step sequence from 71).

22 Introduction

HO O O i-iii TBDPSO O O O 75 O 76

Scheme 17. Reagents and conditions: i. LAH, THF, 0°C → rt, 2h, 95 %. ii. TBDPSCl, imidazole, DMF, 2h, 66 %. iii. PS-IBX (3.0 equiv), CH2Cl2, 2h, 100 %. PS-IBX = Polymer-supported 2-iodoxybenzoic acid.33

Consecutive removal of the fluorous-tagged PMB and the TMS-ethyl protecting groups led to seco acid 73, which could be cyclized under Mitsunobu conditions in excellent yield (81 %). Quite remarkably, no conventional work-up was required in any of the steps leading from 67 to 72 and all products could be recovered by a simple washing/elution sequence on fluorous silica gel. Likewise, the use of fluorous- tagged versions of Ph3P as well as DEAD as Mitsunobu reagents enabled easy separation of by-products formed in the cyclization step. Treatment of the cyclization product with BBr3 in CH2Cl2 for 15 min at 0°C allowed the removal of all remaining protecting groups in a single step, including the selective cleavage of the methyl ether in the 2-position of the aromatic ring. Completion of the synthesis required the selective oxidation of the allylic hydroxyl group in triol 74, which was accomplished with polymer-bound IBX90 to provide the target compound radicicol A (4) in excellent yield (90 %). Overall, radicicol A (4) was obtained from (R)-4-hydroxy-1-pentene in 14 steps with a total yield of 15 %.

O O O O SiMe SiMe O 3 O 3

O O SePh Cl SePh 77 78 Figure 10. Aromatic building blocks 77 and 78, which substituted 66.33

Employing building blocks 77 and 78 (Fig. 10) as substitutes for 66, the chemistry developed for the synthesis of radicicol A (4, Scheme 16) has also been used by Winssinger and co-workers to prepare LL-Z1640-2 (3) and a 5-chloro analog of 4 with similar efficiency.33

1.4.3. Aigialomycin D As indicated above, part of the methodology employed by Winssinger and co- workers in their synthesis of radicicol A (4) had already been developed in the course of their work on aigialomycin D (7), which has led to the identification of this natural product as a protein kinase inhibitor.36 However, the first synthesis of aigialomycin D (7) had been reported by Danishefsky and co-workers two years earlier, when the kinase-inhibitory properties of the compound had not yet been revealed.91,92 Danishefsky’s synthesis of aigialomycin D (7) is summarized in Scheme 18 and features an “ynolide” Diels-Alder reaction between alkyne 84 and diene 85 for the build-up of the aromatic ring as a key defining element. In contrast to all other

23 Introduction syntheses described above, the aromatic moiety was not introduced as a discrete separate building block, but was only generated after macrocycle formation.

HO O OPiv OPiv v-viii i, ii iii, iv O O O O TBSO O 75 O 79 O 80 O 81 O

O OH O O HO O O 67 (O C)3Co ix x, xi (OC)3Co xii, xiii

TBSO O TBSO O O 82 O 83 O TBSO 84 O

TMSO

OH O MOMO O 85 OTMS O O xiv xv - xvii xviii HO MOMO 7 2' TBSO O O 86O 87 O

+ - Scheme 18. Reagents and conditions: i. KHMDS, Ph3P CH3I , THF, -78°C → rt, 68 %. ii. PivCl, Et3N, DMAP, CH2Cl2, 90 %. iii. a) 9-BBN, 0°C → rt; b) NaOH, H2O2, 88 %. iv. SO3·Py, DMSO, CH2Cl2, Et3N. v. a) Propargyl-bromide, Zn, THF; b) TBSOTf, 89 % (3 steps). vi. MeONa, 88 %. vii. SO3·Py, DMSO, + - CH2Cl2, Et3N. viii. KHMDS, Ph3P CH3I , THF, -78°C → rt, 86 % (2 steps). ix. n-BuLi, dry ice, -78°C → rt. x. 67, DIAD, Ph3P, toluene, 85 % (2 steps). xi. (Co)2(CO)8, toluene, 94 %. xii. Grubbs II, CH2Cl2, 2’- (R)-isomer, 38 %; 2’-(S)-isomer, 42 %. xiii. CAN, -10°C, 2’-(R)-83, 94 %; 2’-(S)-83, 95 %. xiv. 140°C, neat 85, 2d, 2’-(R)-84, 74 %; 2’-(S)-84, 84 %. xv. MOMCl, iPr2NEt, 2’-(R)-isomer, 78 %; 2’-(S)-isomer, 83 %. xvi. HF•py, 2’-(R)-isomer, 78 %; 2’-(S)-isomer, 87 %. xvii. [PhC(CF3)2O]2SPh2, 90 % from 2’-(R)- isomer; 87 % from 2’-(S)-isomer. xviii. 0.5 N HCl, 69 %.91,92

The synthesis of macrolactone 84 starts with acetonide-protected 2-deoxy-D-ribose 75 which was further elaborated into aldehyde 80 through Wittig olefination and pivaloylation to give terminal olefin 79 followed by hydroboration and oxidation. Subsequent Reformatsky-type nucleophilic propargylation with propargyl bromide and Zn gave a mixture of secondary alcohols that were protected as TBS-ethers. Subsequent removal of the pivaloyl protecting group, oxidation, and Wittig olefination furnished acetylene 81. The latter was deprotonated and reacted with dry ice to give the crucial carboxylic acid 82. Mitsunobu esterification of 82 with (R)-4-hydroxy-1- pentene 67 followed by reaction of the ester with Co2(CO)8 provided the protected diene 83, which underwent smooth RCM with 2nd generation Grubbs catalyst to provide the desired E-olefin in 70 % total yield, which is the combined yield of the individual diastereoisomeric alcohols that were separated at this stage. It should be noted that no product formation was observed with the uncomplexed (“free”) triple bond; thus, Co-complexation of the alkyne moiety proved to be absolutely crucial for the success of the RCM. Decomplexation of the RCM products with CAN proved to be straightforward and furnished the desired ynolide(s) 84 in excellent yield(s).

24 Introduction

Acetylene(s) 84 underwent highly efficient Diels-Alder reaction with diene 85, resulting in the formation of resorcylic acid lactone(s) 86 in high yield(s). Protection of the phenolic hydroxyl groups as MOM-ethers followed by TBS-removal and dehydration with Martin sulfurane93 gave protected diene 87, which was converted to aigialomycin D (7) by treatment with 0.5 N HCl/MeOH in 69 % yield. Overall, the total yield of aigialomycin D (7) for the 19-step sequence from 2-deoxy-D-ribose was 5 %. As illustrated in Scheme 19, Winssinger’s synthesis of aigialomycin D (7) follows a more conventional strategy for the introduction of the resorcylic acid moiety than Danishefsky’s approach, but it also relies on RCM for macrocycle formation.36 As indicated above, the build-up of the requisite diene precursor is based on similar principles as have already been discussed for the synthesis of radicicol A (4). Specifically, this involves the alkylation of benzyl selenide 89 with alkyl bromide 96 to furnish the phenylselenide 90. Benzyl selenide 89 was obtained from benzoic acid 88 through Mitsunobu-based esterification with (R)-2-hydroxy-1-pentene 67 followed by EOM-protection of the phenolic hydroxyl groups and phenylselenylation at the benzylic position.

HO OH O 67 EOMO O EO MO O

OH i - iiiO iv O

HO EOMO EOMO O 88 89 SePh 90 SePh O

EO MO O OH O O vvi,viiO SePh EOMO HO O OH 91O 7 OH

Scheme 19. Reagents and conditions: i. PS-DEAD, (R)-(+)-4-hydroxy-1-pentene (67), m-ClPh3P, CH2Cl2, 0.5h, 83 %. ii. EOMCl, iPr2EtN, TBAI (cat), DMF, 80°C, 5h, 95 %. iii. a) LDA, THF, -78°C; b) (PhSe)2, 2h, 75 %. iv. LDA (2.0 equiv), 96 (1.0 equiv), THF/HMPA 10/1, -78°C, 20min, 75 %. v. Grubbs II (0.05 equiv), toluene, 80°C, 12h, 92 %. vi. H2O2 (2.0 equiv), THF, 23°C, 3h, 82 %. vii. PS- 36 SO3H, MeOH, 50°C, 2h, quant. PS = polymer-supported.

The synthesis of compound 96 is summarized in Scheme 20 and the initial steps involve cross-metathesis of 5-bromo-1-pentene with 2-butene-1,4-diol 93 to produce allylic alcohol 94 with an E/Z ratio of > 25:1. The latter was submitted to Sharpless asymmetric epoxidation, which proceeded in excellent yield (85 %), and was followed by oxidation and Wittig olefination to give intermediate 95. Lewis-acid-catalyzed

(Sc(OTf)3) epoxide opening furnished the desired anti-diol, which was converted to acetonide 96, the desired electrophile for the alkylation of selenide 89. The alkylation product 90 (Scheme 19) was cyclized with 2nd generation Grubbs catalyst under equilibrating conditions, which gave the desired C7’-C8’ E-alkene with > 10/1 selectivity in 92 % yield. Oxidation of the selenide followed by base-induced elimination of phenylselenic acid gave fully protected aigialomycin, which was

25 Introduction converted to aigialomycin D (7) by treatment with polymer-sulfonic acid in quantitative yield (23 % total yield for the longest linear sequence (13 steps from 88)). Importantly, the order of the RCM and oxidation/elimination steps is crucial for the good yields of the last steps of Winssinger’s synthesis of aigialomycin D (7). When the RCM was attempted on a substrate that already incorporated a double bond between C1’ and C2’, significant amounts of the undesired 6-membered cyclization product were observed arising from RCM between the C1’-C2’ double bond and the terminal double bond adjacent to the dioxolane ring. This again indicates the difficulty of a side reaction, namely formation of a six-membered ring, occurring in RCM-based approaches.

HO93 OH i ii - iv v, vi OH Br Br Br Br O O 92 94 95 96 O

Scheme 20. Reagents and conditions: i. 92 (1.0 equiv), 2-buten-1,4-diol (93) (2.0 equiv), Hoveyda- Grubbs catalyst II (0.01 equiv), CH2Cl2, 4h, 97 %. ii. a) L-(+) diethyl tartrate (0.12 equiv), Ti(OiPr)4 (0.1 equiv), tBuOOH (1.52 equiv), CH2Cl2, -40°C, 30 min; b) + 94 (1.0 equiv), -2°C, 12h, 85 %. iii. SO3·py, CH2Cl2/DMSO, 0°C, 30min. iv. Ph3P=CH2, THF, -10°C, 10min, 70 % (2 steps). v. Sc(OTf)3 (0.2 equiv), THF/H2O 10/1, 2.5h, 100 %. vi. Dimethoxypropane, TsOH·H2O (cat), CH2Cl2, 12h, 70 %. Grubbs– Hoveyda catalyst II=1,3-(Bis-(mesityl)-2-imidazolidinylidene)dichloro-(o-isopropoxyphenyl-methylene) ruthenium.36

Having successfully completed the total synthesis of aigialomycin D (7) by solution methods, Winssinger and co-workers have also extended the chemistry depicted in Scheme 19 to the solid-phase synthesis of a small library of aigialomycin analogs.36 99 (which corresponds to intermediate 89 in Scheme 19) could be elaborated into polymer-bound macrocycles in a way completely analogous to the synthesis depicted in Scheme 19 and employing a series of different alkyl bromides 100 (Scheme 21). Cleavage of the cyclization products from the solid-phase could be achieved either through oxidation/elimination or through radical reduction, thus leading to two different product series, 102 and 103, with or without a double bond between C1’ and C2’, respectively. Unfortunately, and in contrast to aigialomycin D (7), none of the analogs investigated showed any meaningful inhibition of CDK1, CDK5, or GSK3.36

26 Introduction

OH O OH O EOMO O

O i O ii O

HO 97HO 98 EOMO 99 Cl S S

EOMO O OH O Br R iii, iv 100 O v, vi O

EOMO HO S 102 RR 101

vii, vi S OH O S = O PS/DVB HO 103 R'

Scheme 21. Reagents and conditions: i. PS-SH (1.0 equiv SH), 97 (1.1 equiv), iPr2NEt (1.0 equiv), DMF, 60°C, 12h, 82 % (mass gain). ii. EOMCl, DBU, TBAI (cat), DMF, 12h, ~96 % (mass gain; 80 % over two steps based on radical cleavage: AIBN (cat), nBu3SnH (5.0 equiv), toluene, 150°C, microwave, 10min). iii. LDA (6.0 equiv), 100 (2.0 equiv), THF/HMPA 10:1, -78°C, 20min, quant. (based on radical cleavage or oxidation/elimination (H2O2 (4.0 equiv), CH2Cl2/HFIP 1/1, 12h; then toluene, 80°C, 3 h). iv. Grubbs II, CH2Cl2, 120°C, microwave, 25min, 100 % (based on radical cleavage). v. H2O2 (4.0 equiv), CH2Cl2/HFIP 1/1, 12h; then toluene, 80°C, 3h, > 90 %. vi. PS-SO3H, MeOH, 45°C, 3h, > 90 %. vii. AIBN (cat), Bu3SnH (5.0 equiv), toluene, 150°C, microwave, 10min, > 98 %. For substituents R that constitute or contain protected functional groups R’ represents the deprotected substituent, otherwise R = R’.36

More recently, total syntheses of aigialomycin D (7) have also been reported by Lu et al.94 and Vu et al.95 and a total synthesis of epi-aigialomycin D has been published by Bajwa & Jennings.96 The synthesis of Vu et al. is conceptually similar to Winssinger’s approach, as the ring closure was achieved through RCM of a suitable diene 111 (Scheme 24).95 The synthesis of the aromatic moiety starts with methyl 2,4- dihydroxy-6-methylbenzoate 104, which was MOM-protected and saponified to give acid 105 (Scheme 22). Via Mitsunobu-based esterification the aromatic part 106 was completed over 3 steps and 85 % yield.

OH MOMO MOMO O CO Me CO H 2 i, ii 2 iii O

HO MOMO MOMO 104 105 106

Scheme 22. Reagents and conditions: i. MOMCl, NaH, THF, quant. ii. KOH, MeOH/H2O (1:1), reflux; 95 then HOAc, pH 6, 99 %. iii. DIAD, Ph3P, 67, THF, 86 %.

The aliphatic moiety 110 was synthesized starting from commercially available D-(-)- erythronolactone acetonide 107 as a source of chirality (Scheme 23). After reduction

27 Introduction with DIBAL-H and subsequent chain elongation using a commercial Wittig-reagent, both double bond isomers of 108 were obtained. Hydrogenation of the double bond, oxidation of the terminal hydroxyl group and Wittig reaction furnished alkene 109, which was transformed into the required Weinreb amide 110 (36 % yield over 6 steps).

O O O O O O O O i, ii iii-v vi HO O CO Et CO Et N O 2 2 O O 107 108 109 110

Scheme 23. Reagents and conditions: i. DIBAL-H, CH2Cl2, -78°C; 92 %. ii. Ph3P=CHCO2Et, PhCO2H (0.2 mol %), CH2Cl2, reflux, E/Z=1:2, 73 %. iii. H2 (1 atm), 10 % Pd/C (cat.), EtOH, 97 %. iv. PCC, + - . NaOAc, 4 A° MS, CH2Cl2, rt, 80 %. v. Ph3P CH3Br , n-BuLi, THF, -30°C to rt, 71 %. vi. MeONHMe HCl, i-PrMgCl, THF, -20°C to rt, 96 %.95

The precursor for the RCM, 111, was obtained by benzylic acylation of ester 106 with Weinreb-amide 110. Remarkably, diene 111 could be cyclized to 112 under microwave conditions in 98 % yield and with complete E selectivity, while conventional RCM conditions led to a 5.7/1 mixture of E/Z-isomers (Scheme 24).95 Reduction of the keto group followed by dehydration with Martin sulfurane93 and global deprotection under acidic conditions completed the synthesis of aigialomycin D (7) in a highly efficient and stereospecific manner.

O MOMO O 110 MOMO O N O O O i O O ii

MOMO MOMO 106 O O 111 O

MOMO O OH O

O iii-vi O MOMO HO

O O OH O OH 112 7

Scheme 24. Reagents and conditions: i. LDA, -78°C, THF; then 110, 82 %. ii. CH2Cl2 (0.005 M), Grubbs II catalyst (10 mol %), MW irradiation, 100°C, 30min, 98 %, E only. iii. NaBH4, MeOH/H2O (4:1), rt, quant. iv. MsCl, Et3N, DMAP (10 mol %), CH2Cl2. v. DBU, toluene, reflux, 74 % over two steps. vi. 1 N HCl/MeOH (1:1), rt, 48h, 91 %.95

In contrast to the RCM-based approaches of Winssinger and Vu, Lu et al. have prepared aigialomycin D (7) via macrolactonization under Yamaguchi conditions and the two E-double bonds were introduced using Kocienski-modified Julia coupling conditions.97,98 The aromatic moiety was prepared starting from MOM-protected

28 Introduction benzylic alcohol 113, which was brominated using NBS and oxidized to give benzaldehyde 115 (Scheme 25).

OMOM OMOM OMOM Br Br i ii OH OH O MOMO MOMO MOMO 113 114 115

Scheme 25. Reagents and conditions: i. NBS, CHCl3, rt, 30min, 98 %. ii. PCC, NaOAc, CH2Cl2, rt, 4h, 85 %.94

In contrast to Vu’s synthesis, Lu et al. did not use a chiral pool compound for preparing the aliphatic part of aigialomycin D (7). The commercially available prop-2- yn-1-ol 116 was elongated and subsequently reduced to the E-alkene 117 (Scheme 26). The required chirality was introduced via a Sharpless asymmetric epoxidation reaction99 in 89 % yield and with excellent selectivity (91 % ee). The epoxy alcohol obtained from 117 was regioselectively opened with benzoic acid in a titanium- assisted reaction,100 which was followed by ester cleavage with EtMgBr.101 Protection of the primary hydroxyl group gave intermediate 118. After acetonide protection of the remaining free hydroxyl groups and removal of the benzyl group, 1-phenyl-1H- tetrazole-5-thiol could be reacted with the alcohol under Mitsunobu conditions. Finally, oxidation of the resultant sulfide furnished sulfone 119 in 27 % yield over 10 steps.

OPiv N OPiv N i, ii iii-vi vii-x N OH BnO BnO N S OH OH O OH Ph O O O 116 117 118 119

Scheme 26. Reagents and conditions: i. BnOCH2CH2CH2I, BuLi, HMPA, -78°C, 8h, 70 %. ii. LiAlH4, THF, reflux, 1h, 96 %. iii. Ti(O-iPr)4, TBHP,(-)-DIPT, cat. CaH2, CH2Cl2, -25°C, 12h, 89 % and 91 % ee. iv. Ti(O-iPr)4, PhCO2H, CH2Cl2, rt, 15min, 75 %. v. EtMgBr, ether, rt, 1h, 95 %. vi. PivCl, Et3N, DMAP, CH2Cl2, rt, 10h, 88 %. vii. 2-methoxypropene, PPTS, CH2Cl2, rt, 5h, 95 %. viii. 10 % Pd/C, H2, EtOH, rt, 4h, 95 %. ix. 1-phenyl-1H-tetrazole-5-thiol, DIAD, PPh3, THF, 0°C to rt, 1h. x. m-CPBA, 94 NaHCO3, rt, CH2Cl2, 12h, 81 % for two steps.

The C1’-C2’ E-double bond was introduced by coupling of aldehyde 115 to sulfone 119 (Scheme 27). After cleavage of the primary pivaloyl ester and oxidation the C7’- C8’ E-double bond was introduced under Kocienski-modified Julia coupling conditions (Scheme 27).97,98 Interestingly, and in contrast to all other macrolactonization-based approaches discussed before, the carboxyl group of the resorcylic acid portion of 123 was introduced only late in the synthesis through metalation of bromine 122 and subsequent carboxylation with CO2. The macrocycle was closed under Yamaguchi conditions and subsequent global deprotection gave aigialomycin D (7).

29 Introduction

N N OTBS N N OMOM S Ph O O Br OPiv 121 i ii-iv 115 119 MOMO O 120 O

MOMO TBSO MOMO O OH O Br v-vii O viii O

MOMO O MOMO HO O O OH 122 123 O 7 OH

Scheme 27. Reagents and conditions: i. KHMDS, DME, -60°C to rt, 3h, 68 %. ii. DIBAL-H, CH2Cl2, -78°C, 1h, 96 %. iii. Dess-Martin periodinane, CH2Cl2, rt, 2h, 85 %. iv. 121, KHDMS, DME, -60°C to rt, 3h, 58 %. v. TBAF, THF, rt, 8h, 95 %. vi. n-BuLi, CO2, THF, -78°C to rt, 2h, 83 %. vii. 2,4,6- trichlorobenzoylchloride, Et3N, THF, rt, 2h, then DMAP, toluene, reflux, 36h, 51 %. viii. 0.5M HCl, 94 H2O/MeOH, rt, 2d, 70 %.

Bajwa & Jennings were not able to synthesize aigialomycin D (7), but it is worth noting the stereochemical outcome of their RCM-approach, which provided the C6’- epi-analog of 7. First of all they synthesized the aromatic part starting from triflate 124, which had been published by Danishefsky et al. (Scheme 28).102 Styrene 125 was synthesized via a Suzuki-Miyaura coupling using Molander’s conditions.103

O O O O

O i O

BnO OTf BnO 124 125

Scheme 28. Reagents and conditions: i. potassium vinyl trifluoroborate (1.1 equiv), Et3N (1.3 equiv), 96 Pd(dppf)Cl2 (10 mol %), EtOH, 80°C, 16h, 77 %.

The aliphatic framework of aigialomycin D (7) was synthesized from the TBDPS- protected glycidol derivative 126, which was elongated and protected as its MOM- ether (Scheme 29). Deprotection of the primary hydroxyl group and oxidation to aldehyde 128 furnished the C1’-C6’ part of 7.

O i, ii iii, iv OTBDPS OTBDPS O OMOM OMOM 126 127 128

Scheme 29. Reagents and conditions: i. Li2CuCl4 (2 mol %), allylMgBr (1.2 equiv), THF, -30°C, 0.25h, 87 %.ii. MOM-Cl (2.0 equiv), DIPEA (1.5 equiv), CH2Cl2, rt, 8h, 93 %. iii. TBAF (1.5 equiv), THF, rt, 96 16h, 96 %. iv. TPAP (10 mol %), NMO (3.0 equiv), CH2Cl2, 0°C, 1.5h, 74 %.

30 Introduction

The C7’-C11’ part of 7 was synthesized starting from the known TBS-protected propargylic alcohol 129104, which was efficiently coupled to aldehyde 128. The alkyne 130 was selectively reduced (E:Z; 15:1) using Red-Al and the resulting allylic alcohol was oxidized to enone 131. Subsequent enantioselective reduction of the ketone (dr 6:1), followed by simultaneous cleavage of both protecting groups under acidic conditions and final acetonide protection of the resultant mixture of diols gave the protected aliphatic fragment 132 (Scheme 30).

TBSO TBSO HO OTBS i ii, iii iv- vi

OH O O OMOM OMOM O 129 130 131 132 Scheme 30. Reagents and conditions: i. n-BuLi (1.1 equiv), 129 (1.0 equiv), THF, -78°C, 1.5h, then 128 (0.7 equiv), THF, -78°C, 1.5h, 71 %. ii. Red-Al (3.2 equiv), THF, 0°C, 48h, 72 %. iii. TPAP (10 mol %), NMO (3.0 equiv), CH2Cl2, 0°C, 1.5h, 92 %. iv. Red-Al (1.2 equiv), toluene, 0°C, 0.5h, 84 %. v. HCl, MeOH, 50°C, 0.5h, 100 %. vi. DMP (25 equiv), PPTS (2 mol %), CH2Cl2, rt, 5.0h, 62 %.96

Alcohol 132 (6:1 mixture of diastereomers) was directly reacted with the protected aromatic acid 125 to provide a mixture of esters 133 and 134. Surprisingly, the RCM with this 6/1 mixture of trienes 133/134 produced none of the 14-membered macrocycle derived from 133, while 134 was almost completely converted to macrolide 136 (Scheme 31).96 The major product isolated from the reaction mixture was diene 135, which was obtained in 84 % yield and which is the result of 6- membered (rather than 14-membered) ring formation in the RCM.

OH O OH O O O i O O O BnO BnO BnO O O O O 125 133 134

OH O OH O OH O iii O O O

BnO BnO HO

O OH 135 136 epi-7 O OH

Scheme 31. Reagents and conditions: i. NaH (1.2 equiv), 132 (1.3 equiv), THF/DMF 1:1, rt, 5h, 78 % nd of 133:134 in a ratio of 6:1. ii. Grubb’s 2 generation catalyst (5 mol %), CH2Cl2, 50°C, 16h, 84 % of 96 135 and 13 % of 136. (iii.) BBr3 (4.0 equiv), CH2Cl2, -78°C, 1.0h, 74 %.

31 Introduction

For triene 133 this is the sole reaction path, whereas 14-membered ring formation is clearly preferred for 134, thus highlighting the importance of conformational effects in RCM-based cyclization reactions. The latest published total synthesis of aigialomycin D (7) is based on a completely new macrocyclization approach, namely a Ni-catalyzed cyclization as the key transformation.105 As depicted in Scheme 32 aigialomycin D (7) would be derived after deprotection of A-I, which in turn would be accessible via an intramolecular diastereoselective coupling of an alkynyl silane to an appropriate -silyloxy aldehyde.

OH O MOMO O MOMO O TMS

O O O TMS HO MOMO MOMO

OH OPG' O OH OPG OPG 7A-I A-II Scheme 32. Retrosynthesis of aigialomycin (7) according to Montgomery et al.105 The aromatic part of aigialomycin D (7) was prepared from the known resorcylic acid 13776 (Scheme 33). Protection of 137 as its bis-methoxymethyl ether, followed by ester hydrolysis and Mitsunobu esterification with alcohol 138 furnished compound 139 in 52 % over 3 steps.

OH O i-iii MOMO O O O HO HO I MOMO I 138 137 139

Scheme 33. Reagents and conditions: i. MOM-Cl, i-PrNEt2, 87 %. ii. NaOTMS, 86 %. iii. 138, DEAD, 105 PPh3, 70 %.

To complete the aliphatic framework of (7) the known diol 140106 was TBS-protected and further converted to the alkenyl boronic acid 141. Pd-catalyzed cross-coupling of boronic acid 141 with aryl iodide 139 afforded 142 in 90 % yield (Scheme 34). To prepare the substrate for the Ni-catalyzed cyclization the terminal alkynyl moiety of 142 was TMS-protected followed by selective cleavage of the primary TBS ether and subsequent Dess-Martin oxidation. Surprisingly, when the alkynyl silane cyclization precursor 143 was subjected to the reaction with Et3SiH as reducing agent, . Ni(COD)2, IMes HCl 144 and tBuOK in a mixture of THF and water, the main product obtained was 145 in 61 % yield. None of the desired macrocycle was formed under these reaction conditions. It should be noted that the reaction leading to product 145 is analogous to known Nil-catalyzed silyl triflate-promoted couplings of aldehydes and alkenes that involve loss of HOTf.105

32 Introduction

MOMO O

i,ii iii O OH (HO) B 2 OTBS OH MOMO OTBS 140 141 142 OTBS OTBS

MOMO O TMS MOMO O TMS

iv-vi O vii O

MOMO MOMO - Cl OTES 143 O NN+ 145 OTBS OTBS 144

Scheme 34. Reagents and conditions: i. TBS-Cl, imidazole, 97 %. ii. catecholborane, 9-BBN (8mol %); then NaOH, 65 %. iii. 139, Pd(PPh3)4 (35mol %), 90 %. iv. LDA, TMS-Cl, 49 %. v. HF•py. vi. . DMP, 56 % over 2 steps. vii. Et3SiH (5eq), Ni(COD)2 (25mol %), IMes HCl (25mol %), t-BuOK 105 (25mol %), THF:H2O (99:1), 61 %.

Although the authors knew from their prior studies that free terminal alkynes undergo intermolecular couplings with -silyloxy aldehydes with considerably lower diastereoselection than alkynyl silanes, they nonetheless transformed intermediate 142 into the aldehyde 146 (Scheme 35).

MOMO O MOMO O

O i,ii O iii

MOMO MOMO

142 OTBS 146 O OTBS OTBS

MOMO O OH O iv O O

MOMO HO OTES Y 147 X OTBS OH 7 :(X=OH,Y=H) epi-7 :(X=H,Y=OH)

Scheme 35. Reagents and conditions: i. HF•py, 66 %. ii. DMP, 81 %. iii. Et3SiH (5eq), Ni(COD)2, IMes.HCl, t-BuOK (25 mol % each), 61 %. iv. aq HCl, MeOH; then HPLC separation: 7, 46 %; epi-7, 44 %).105

In constrast to 143, substrate 146 underwent Ni-catalyzed macrocyclization in 61 % yield, providing the 14-membered macrocycle 147 as a mixture of diastereomers in a ratio close to 1:1. After global deprotection aigialomycin D (7) and its epimer (epi-7) could be separated via HPLC to give 46 % of 7 and 44 % of its epimer epi-7.

33 Introduction

1.5. Conclusions The discovery of the kinase-inhibitory activity of a number of natural resorcylic acid lactones has led to a broadened interest in the chemistry of these systems, as they represent an entirely new scaffold for kinase inhibition without structural similarities to conventional kinase inhibitors. Especially the cis-enone-containing compounds 3-6 are of particular interest in this context, as they all have been shown to be highly potent protein kinase inhibitors. As of today, total syntheses for all of these natural products have been developed; of which the first total synthesis of L-783277 (6) was developed within this thesis. Based on these strategies, improved analogs and more extensive structure-activity studies should be feasible. Interestingly, all successful approaches to the synthesis of cis-enone-containing resorcylic acid lactones to date are macrolactonization-based and alternative modes of ring closure have rarely been investigated. In contrast, attempts on ring-closure through RCM or a Ni-catalyzed cyclization reaction have been described for aigialomycin D (7). However, it remains to be seen, if efficient non-macrolactonization-based strategies can be developed for cis-enone-containing resorcylic acid lactones. Furthermore, all theses investigations will hopefully lead to the development of a variety of analog structures which will eventually lead to resorcylic-acid-lactone-based drug candidates.

34 Aims and Scope

2. Aims and Scope In light of their interesting biological properties, cis-enone containing RALs have also become important targets for total synthesis. By the time when this research project started, total syntheses had been published for all of the RAL members shown in Fig. 1 except for radicicol A33 and L-783277 (6). Especially for the RAL members most closely related structurally to L-783277 (6), i. e. hypothemycin,79,80 and LL-Z1640- 2,75,77 total syntheses had been successfully achieved. By contrast, no efforts on the total synthesis of L-783277 (6) had been reported, although 6 is a highly potent inhibitor of the Ser/Thr kinase Mek1 (IC50 = 4 nM) and (although to a lesser extent) also of Lck.25 However, this finding has not been specifically followed up in the literature.

OH O

O O HO 6 OH

The goal of this research project is to develop an efficient enantioselective synthesis of 6. The approach should be convergent, in order to limit the number of steps in the longest linear sequence and to achieve a minimum level of complexity. Besides the development of a synthetic approach to 6 it is planned to characterize its biological activity in more detail with respect to the selectivity of kinase inhibition and effects on human cancer cells. In a second step the chemistry developed for the preparation of 6 should be used for the synthesis of a limited number of analogs for structure-activity relationship (SAR) and also biophysical studies, in order to assess the usefulness of 6 as a potential lead structure for drug discovery. The dideoxy analog (D6) and the phenyl analog (P6) of L-783277 were chosen as first target structures for analog synthesis, in order to test the potential of the developed strategy to 6 for the synthesis of analogs. Moreover, the biological evaluation of theses two compounds would provide first valuable information about the importance of individual structural features for biological activity.

OH O OH O

O O

O O O O HO D6 P6 OH

2.1. Retrosynthetic Analysis of L-783277

2.1.1. First Generation Approach The initial strategy for the synthesis of macrolactone 6 is summarized in the retrosynthetic analysis depicted in Scheme 36 and is based on the consecutive

35 Aims and Scope assembly of three key fragments, intermediates L-II, L-IV and L-V. This approach should also allow the rapid synthesis of analogs by the simple replacement of individual building blocks. According to this strategy, the final ring closure will be achieved through a Yamaguchi-type macrolactonization of an appropriately protected seco acid L-I, which may either contain a double or a triple bond between C7’ and C8’. The cis- enone moiety will intentionally be introduced at a very late stage of the synthesis, because it is known that the Z-double bond easily isomerizes to an E-configured double bond under acidic as well as basic conditions.79,80 This will be followed by simple deprotection of the carboxylate and the C10’-OH group or by partial hydrogenation of the triple bond followed by protecting group removal, respectively.

Yamaguchi macrolactonization PGO OH O OH O 10' 8' X -X-Y- = 3 1 7' O S OH OPG OPG Y -X-Y- = O O O O 5 1' 3' 5' 6' OH OPG 6 L-I Suzuki coupling

PGO OH O PGO O OPG OPG + PGO + R O Hal O OPG OPG alkyne acylation L-II L-III L-IV L-V

asymm etric epoxide dihydroxylation opening

HO 4-pentyn-1-ol (S)-propylene oxide 148 40S Scheme 36. Initial retrosynthetic approach to 6. (PG = protecting groups or H). Protecting groups may vary independently.

The linear precursor L-I for the macrolactoniszation will be obtained through Pd- catalyzed Suzuki coupling between an aryl halide L-II and olefin L-III. The latter should in turn be available from an appropriately functionalized version of L-IV and alkyne L-V via addition of a Li-acetylide or Sonogashira coupling. The aromatic building block L-II will be obtained following literature procedures79,80, while intermediate L-IV will be accessed in a stereoselective fashion through asymmetric Sharpless dihydroxylation107,108 of the corresponding olefin. The latter will be derived from 4-pentyn-1-ol (148). Intermediate L-V will be synthesized by treatment of commercially available (S)-propylene oxide (40S) with the ethylenediamine complex of lithium acetylide.109

36 Aims and Scope

2.1.2. Second Generation Approach The first generation approach outlined above had to be abandoned for two reasons. Intermediate L-I (Scheme 36) was planned to feature a methyl ester moiety (173, see below), which turned out not to be cleavable in the presence of the other protecting groups employed. Finally, neither the change of the hydroxyl-protecting groups for 4’- OH, 5’-OH and 10’-OH, nor the change to a TMS ethyl ester group (177, see below) led to a successful synthesis of 6.

OTES

OH O 11' 8' 1 3 OR 5' O O 51'TBSO OTBS 173 :R=Me 177 :R =CH2CH2Si(CH 3)3

The other weak point of the first generation approach proved to be the asymmetric dihydroxylation of the cis-olefin derived from 148 (i. e. 159, Scheme 44). Cis-olefins are known to give generally lower ee’s in AD-mix-mediated dihydroxylations than the corresponding trans-isomers.110

O THPO O 159

However, the synthesis was initially continued in spite of the unsatisfactory stereochemical outcome of only 44 % ee, but had to be abandoned due to the problems with the ester cleavage in 173 and 177. Our second generation retrosynthesis was based on the experiences and knowledge that were gained in our original approach. In particular, the chemistry developed for the synthesis of the resorcylic acid moiety LL-III and the propylene oxide-derived intermediate LL-VI could be retained (Scheme 37). Compared to our first generation approach an alternative route to intermediate LL-V was pursued that relied on commercially available isopropylidene-D-erythrono-1,4- lactone (107), as the source for chirality at C4’ and C5’. It should be noted that this approach was designed before Vu et al.95 had published their total synthesis of aigialomycin D (7), in which they built up the aliphatic framework of 7 starting from the same chiral starting material 107. As illustrated by the retrosynthesis shown in Scheme 37, one of the key features of this new strategy towards 6 would be the late introduction of the ketone moiety at C6’ through selective allylic oxidation of intermediate LL-I, which had been already realized by Tatsuta et al. and Winssinger et al. in the course of their work on LL-Z1640-1 (3)75 and radicicol A (4)33.

37 Aims and Scope

Mitsunobu macrolactonization SiMe3 OH O OH O OH O OPG S 8' 3 O OH 7' O OH O OPG OPG R O O O OH O 5 1' 3' OH OH OPG 6LL-I LL-II oxidation Suzuki hydrogenation coupling

PGO OH O nucleophilic addition SiMe3 OPGO OPG O OPG + O Hal + OPG OPG OPG LL-III LL-IV LL-V LL-VI epoxide opening O O O

O O (R)-propylene oxide 40R isopropylidene-D- erythrono-1,4-lactone 107

Scheme 37. Second generation retrosynthesis approach to 6.73 (PG = protecting groups or H). Protecting groups may vary independently.

The macrocycle would be obtained from intermediate LL-II via partial hydrogenation of the triple bond, simultaneous removal of the C10’-OH and the carboxylic acid protecting group, and Mitsunobu-based macrolactonization. This ring closure method has been intensively investigated by Sellès & Lett in the course of their work on LL- Z1640-2 (3) and hypothemycin (5).79,80 LL-II was envisaged to be the result of a Suzuki coupling between an ortho-halo ester LL-III and the protected C1’-C11’ fragment LL-IV, which would in turn be accessible through addition of the acetylide anion derived from LL-VI to aldehyde LL-V. Thus, the disconnections from the first retrosynthetic approach were retained as well as the successfully established reactions for the assembly of the key intermediates.

2.1.3. Still-Gennari Approach As all successful approaches to the synthesis of cis-enone-containing resorcylic acid lactones to date are macrolactonization-based and alternative modes of ring closure have rarely been investigated, we decided to explore a novel synthetic approach to 6. Although the Still-Gennari olefination is not a typical macrocyclization method,111 it could give access to the cis-enone configuration of 6. In 1983 Still and Gennari developed conditions that provide access to Z-alkenes with excellent stereoselectivity.112 The reaction of -keto phosphonates featuring electron- withdrawing groups (e. g. trifluoroethyl) and aldehydes under strongly dissociating

38 Aims and Scope conditions (KHMDS and 18-crown-6 in THF) affords Z-alkenes almost exclusively. Furthermore, the use of the Still-Gennari olefination as a cyclization method would allow introducing the cis-enone moiety at a very late stage of the synthesis. Sellès and Lett reported a distinct lability of the cis-enone under acidic as well as basic condition.79,80 Thus, this approach would also reduce the risk of isomerization to the corresponding E-double bond, if it was possible to cleave the corresponding protecting groups for the C-4’ and C-5’ hydroxyl groups after the ring closure. For this reason, the stability of 6 was tested by incubation with a 0.02 M solution of pTsOH in THF, similar to conditions which would be required for the acetonide cleavage. There was no degradation obtained within 6 h incubation time. The retrosynthetic analysis depicted in Scheme 38 is again based on the consecutive assembly of three key fragments, intermediates LLL-III, LLL-IV and LLL-V. The linear precursor of the macrocycle, LLL-I, is envisioned to be closed via an intramolecular Still-Gennari olefination. Subsequent global deprotection would furnish the natural product L-783277 (6).

Mitsunobu reaction OH O OH O O 8' Still-Gennari S olefination 3 O 7' O O O OH O CF P 3 O O O 5 1' 3' PGO O OH OPG CF 3 6 LLL-I

SiMe OH O 3 OH O SiMe3 O OPG O O O O O O CF OPG O CF P 3 + P 3 O O O Hal O OPG CF 3 OPG CF3 Suzuki LLL-II LLL-IV LLL-V coupling +

O O HO OPG HO OH LLL-III (R)-butane-1,3-diol O O 149 isopropylidene-D- erythrono-1,4-lactone 107 Scheme 38. Retrosynthetic approach to 6 featuring the Still-Gennari variant of the Wittig reaction as ring-closing method. (PG = protecting groups or H). Protecting groups may vary independently.

Intermediate LLL-I would be derived from LLL-II through cleavage of the fluoride TMS ethyl ester and subsequent Mitsunobu reaction with a suitably protected diol LLL-III, which would in turn be accessible from commercially available (R)-butane- 1,3-diol 149 by mono-protection. LLL-II could be synthesized via Suzuki coupling of the aromatic building block LLL-IV and the trifluoroethyl phosphonate LLL-V. The latter could be synthesized starting from the commercially available isopropylidene- D-erythrono-1,4-lactone 107.

39 Aims and Scope

2.2. Retrosynthetic Analysis of the Dideoxy Analog D6 The dideoxy analog of L-783277 (D6) was chosen to be synthesized for two reasons. On one hand this would allow to use the synthetic strategy developed for the natural product 6 and thus to test its potential for the synthesis of analogs. On the other hand this analog would provide valuable information about the importance of the vicinal diol moiety for the biological activity of 6. The retrosynthetic analysis of dideoxy analog D6 as depicted in Scheme 39 is based on the previously developed route to the natural product 6: The macrocycle was planned to be closed by a Mitsunobu-type macrolactonization and the ketone moiety was planned to be introduced at the very end of the synthesis.

Mitsunobu macrolactonization SiMe3 OH O OH O OPG S O O OPG R O O O D6 D6-I oxidation Suzuki hydrogenation coupling

OH O PGO nucleophilic addition SiMe O 3 O OPG + + O Hal OPG D6-II D6-III D6-IV D6-V epoxide opening

O OH

5-hexen-1-ol (R)-propylene oxide 150 40R

Scheme 39. Retrosynthetic approach to D6. (PG = protecting groups or H). Protecting groups may vary independently.

The linear macrolactonisation precursor D6-I was envisioned to be assembled via Suzuki coupling of the aromatic fragment D6-II and key intermediate D6-III, followed by Z-specific hydrogenation of the triple bond. D6-III would in turn be accessible through addition of the acetylide anion derived from D6-V to aldehyde D6-IV. The C1’-C6’ fragment of D6 would be synthesized from 5-hexen-1-ol 150.

2.3. Retrosynthetic Analysis of the Phenyl Analog P6 In light of the observation by Sellès and Lett79,80 that the Z-configured double bond in 7’,8’-cis-enone containing RAL’s isomerizes easily, together with the fact that the 7’,8’-trans analog of L-783277 (6) proved to be significantly less active against Mek1 25 than 6 (IC50 value of 300 nM vs. 4 nM for 6), the 7’,8’-phenyl analog of L-783277

40 Aims and Scope

(P6) was chosen as a target for synthesis. Modeling studies at the Novartis Institute for Biomedical Research in Basel (collaboration with Dr. Pascal Furet) have shown that Mek has a hydrophobic domain in the area of the 7’-8’ double bond. Replacing the cis-configured double bond of 6 with a phenylene moiety could give rise to a higher binding affinity in comparison to the natural product. It is conceivable that the binding affinity gained by interactions of the phenylene moiety with the hydrophobic domain in the binding pocket of a kinase might lead to measurable enzyme inhibition, even in the absence of covalent bond formation and thus in the absence of a Cys residue from the active site. Thus, analog D6 could be a new lead structure for non- covalent kinase inhibition.

2.3.1. Alkyne Metathesis Approach As alternative modes of ring closure have rarely been investigated for the cis-enone containing RAL members, we decided to investigate the ring-closing alkyne metathesis (RCAM) as a possible cyclization method for phenyl analog P6, leading to the retrosynthetic analysis of P6 as shown in Scheme 40.

Steglich esterification

OH O cis-selective OH O OH O 8' hydrogenation 3 O O O OH 7' O O O OPG O OPG 5 1' asymmetric OH dihydroxylation P6 P6-AI RCM P6-AII

nucleophilic opening O H O O Br O PGO O + + O OPG 2-bromo- (S)-propylene benzaldehyde oxide Grignard P6-AIII P6-AIV reaction 152 40S iron-mediated coupling

O O X O +

O OTf X = Cl, Br 151 P6-AV Scheme 40. Retrosynthetic analysis of the phenyl analog P6: Alkyne metathesis approach. (PG = protecting groups or H). Protecting groups may vary independently.

The final target structure would be obtained through cis-selective hydrogenation of the triple bond and subsequent asymmetric dihydroxylation to obtain the anti-diol. It has to be noted that this approach was investigated at a time when the unsatisfactory stereochemical outcome of the asymmetric dihydroxylation of the cis-olefin 159 (first

41 Aims and Scope generation approach, Scheme 36) had not been recognized. P6-AI would be obtained via RCAM from the linear precursor P6-AII, which would be obtained by Steglich esterification of the carboxylic acid derived from P6-AIII and the C10’- deprotected alcohol derived from P6-AIV. P6-AIII was envisioned to be synthesized through an iron-mediated coupling113 of the known aryl triflate 151114 and the Grignard reagent derived from halo-alkyne P6-AV. The phenylene moiety P6-AIV could be synthesized starting from commercially available 2-bromobenzaldehyde 152, which would be reacted with 1- propinylmagnesium bromide. The expected mixture of diastereomers will be protected to enable lithiation of the o-position and subsequent opening of (S)- propylene oxide (40S).

2.3.2. Macrolactonization Approach Following the RCAM-based strategy outlined above, intermediate P6-AIII could be synthesized, but only in small amounts. In general, the synthesis of several intermediates turned out to be low yielding and inefficient, which led to the revision of the retrosynthesis of P6, after the chemistry for the synthesis of the natural product L- 783277 (6) had been developed.73 As illustrated in Scheme 41 the adapted retrosynthesis of P6 is based on the consecutive assembly of three building blocks, namely P6-MII, P6-MIV and P6-MV. The macrocycle P6 is envisioned to be closed via Mitsunobu macrolactonization. In contrast to the retrosynthesis shown in Scheme 37 the ketone moiety is planned to be already in place before ring closure. The linear precursor P6-MI is accessible by a Suzuki coupling of the aromatic building block P6-MII to key intermediate P6-MIII. The latter would be obtained by the addition of the lithiated aromatic iodide P6-MV to a suitably protected aldehyde P6-MIV, which should again be accessible from the commercially available isopropylidene-D-erythrono-1,4-lactone 107. It was planned to oxidize the C6’-isomers already at this stage of the synthesis, which would e. g. facilitate the interpretation of the NMR spectra. The aromatic iodine P6-MV would be accessible via opening of (R)-propylene oxide (40R) with mono- lithiated 1,2-diiodobenzene 153.

42 Aims and Scope

Mitsunobu SiMe3 macrolactonization OH O OH O OPG O O

O O O O HO PGO OH OPG P6 P6-MI Suzuki coupling

OH O O SiMe OPG O 3 + + O PGO OPG PGO OPG R O Hal OPG nucleophilic I addition and P6-MII P6-MIIIoxidation P6-MIV P6-MV nucleophilic opening

O I O O + O I O 1,2-diiodo- (R)-propylene isopropylidene-D- benzene oxide erythrono-1,4-lactone 153 40R 107 Scheme 41. Retrosynthetic approach to the phenyl analog P6: Macrolactonization approach. (PG = protecting groups or H). Protecting groups may vary independently.

2.4. Conclusions One of the most important objectives when generating these retrosynthetic analyses has been the design of convergent approaches, in order to limit the number of steps for the longest linear sequence. This usually provides better overall yields and reduces complexity. Convergent approaches also allow the exchange of individual building blocks, such that new analogs of a natural product can be generated without having to develop new chemistry for each individual new structure. A second objective was the development of new chemistry, in order to expand the scope of the methodology that is available for the synthesis of cis-enone containing RALs. All the approaches outlined above satisfy the convergency criteria. Key reactions, like the Mitsunobu-based macrolactonization, Suzuki coupling, as well as the use of different esters of the same resorcylic acid building block are common to all approaches.

43 Results and Discussions

3. Results and Discussions

3.1. Total Synthesis of L-783277 At the time when this research project was started Sellès & Lett79,80 had already synthesized LL-Z1640-2 (3) and hypothemycin (5) and Tatsuta and co-workers75 had accomplished the synthesis of LL-Z1640-2 (3). All of this work is contained in three short communications and no experimental details have been published for any of these syntheses.

3.1.1. First Generation Approach

3.1.1.1. Synthesis of the Resorcylic Acid Moiety As our retrosynthetic analysis led to three building blocks, of which the aromatic part had already been previously reported by Sellès and Lett, this approach started with the synthesis of the resorcylic moiety according to the published work.79 As indicated above, this building block (i. e. 29a/b) has been used in almost all our approaches, except for the alkyne metathesis approach to the phenyl analog P6. The synthesis of the aromatic building block is summarized in Scheme 42 and started with TBS-protection of commercially available 2-hydroxy-4-methoxybenzoic acid 27. The doubly TBS-protected intermediate 154 allowed selective and quantitative conversion to the corresponding amide 155. Otherwise, this transformation could only be achieved in a lower yielding (around 45 %) two step- sequence in which the carboxylic acid is converted to its acyl chloride (without previous TBS-protection of the phenolic group) and further reacted with diethyl amine and catalytic amounts of DMAP.115 As the phenolic OH-group has to be protected for the o-lithiation, the approach depicted in Scheme 42 was favored.

Et N O O COOH O OTBS Et2N O 2 O TBSO X HO i TBSO ii TBSO HO X iii iv

O O O O O X=Br: 28a X=Br: 29a 27 154 155 X=I: 28b X=I: 29b

Scheme 42. Reagents and conditions: i. TBS-Cl, DIEA, DMF, rt, overnight; quant. ii. Et2NAlMe2 (from Me3Al and Et2NH), toluene, -10°C → rt, 45min; then 154, reflux, 20h; quant. iii. 28a: t-BuLi in pentane, -78°C → -40°C, 30min; then Br2, Et2O, -78°C → rt; 68 %. 28b: t-BuLi in pentane, -78°C → -40°C, + - 30min; then ICl, Et2O, -78°C → rt; 40 %. iv. 29a: Me3O BF4 , CH2Cl2, rt, 4h; then evaporation, sat. + - NaHCO3/MeOH (1:1), rt, overnight; 66 %. 29b: Me3O BF4 , CH2Cl2, rt, 4h; then evaporation, sat. NaHCO3 / MeOH (1:1), rt, overnight; 65 %.

According to Sellès and Lett the TBS protection of the phenolic OH-group is necessary for o-selective lithiation, as it effectively suppresses the formation of the m-substituted halide. Thus, o-lithiation of 154, followed by quenching with bromine or iodine provided 28a and 28b with good selectivity. The m-substituted product was

44 Results and Discussions only observed in 7-10 % in both cases, as determined by NMR-spectroscopy. The 2- OTBS group also allows conversion of amides 28 into methyl esters 29 in good yields. Reacting 28a with Meerwein Salt and subsequent hydrolysis of the resulting imidate salt gave the desired methyl ester 29a in 66 % yield over 4 steps. The iodinated aromatic building block 29b was also synthesised, in order to investigate its usability for the Suzuki coupling with intermediate L-III (Scheme 36), which is one of the key reactions of the projected total synthesis.

3.1.1.2. Synthesis of the C7’-C11’ Fragment Initially it was planned to carry out the macrolactonization of the linear precursor L-I (Scheme 36) under Yamaguchi conditions,116 which requires the S-configuration of the C7’-C11’ fragment. The synthesis of the TES-protected or the TBS-protected (S)- 4-pentyn-2-ol (156S and 129S, respectively) was accomplished according to literature procedures.109 The first step involved opening of the commercially available (S)-(-)-propylene oxide 40S with the Li-acetylene diamine complex (Scheme 43).

OH O i ii OR

R=TBS: 129S 40S 138S R=TES: 156S

Scheme 43. Reagents and conditions: i. Li-≡-H*H2N-CH2-CH2-NH2, DMSO, rt, overnight; 64 %. ii. 129S: TBS-Cl, imidazole, CH2Cl2, 0°C → rt overnight; 71 %. 156S: TES-OTf, 2,6-lutidine, CH2Cl2, -78°C → rt 3.5h; quant.

Silyl-protection of the free alcohol 138S provided the required C7’-C11’ fragments 129S and 156S in 52 % and 64 % yield, respectively over 2 steps. It has to be pointed out that the intermediate as well as the silyl-protected products are volatile; therefore, distillation or FC using low-boiling solvents (e. g., pentane or Et2O) are required for the purification of these compounds.

3.1.1.3. Synthesis of the C1’-C6’ Fragment As illustrated in Scheme 44, our initial strategy aimed at the synthesis of acid chloride 165 as a suitably activated version of key intermediate L-IV (Scheme 36). Acid chloride 165 was prepared starting from commercially available 4-pentyn-1-ol 148, which was protected as its THP ether followed by chain extension with methyl chloroformate.117 Z-specific hydrogenation of the resulting alkyne 158 using Lindlar catalyst provided 159 as the substrate for the enantioselective hydroxylation reaction with AD-mix  according to Sharpless which proceeded in quantitative yield to give 160.

45 Results and Discussions

O i O ii HO iii O O O THPO O THPO 148 157 158 159

HO O HO O O iv v vi O THPO HO HO O O O OH OH O 160 161 162

O O O O O O vii viii ix O OH Cl O O O

163 164 165

Scheme 44. Reagents and conditions: i. CSA, dihydropyran, CH2Cl2, 0°C → rt, 3h; quant. ii. 16, n- BuLi, -78°C, 3.5h; then methyl chloroformate, THF, -36°C, 1h; quant. iii. Lindlar catalyst, H2, EtOAc, rt, 1h; 92 %. iv. AD-mix , methanesulfonamide, tBuOH/H2O (1:1), 0°C, overnight; quant; 44 % ee. v. p- TsOH (cat) MeOH, rt, overnight; 93 %. vi. 2,2-dimethoxypropane, CSA, rt, 1.5h; 81 %. vii. 2-NO2- . PhSeCN, n-Bu3P, rt, 1h; then NaHCO3, 30 % H2O2, rt, 19h; 81 %. viii. LiOH H2O, MeOH, rt, overnight; 85 %. ix. oxalyl chloride, DMF (cat), reflux, 1h. As the anti-diol was required, the substrate for the asymmetric dihydroxylation reaction had to be cis-configured.107,108 Fig. 11 shows an empirical model for the Sharpless asymmetric dihydroxylation of cis-disubstituted olefins, which allows the appropriate choice of the required reagent (AD-mix  or ).107 This empirical model shows an aliphatic group as one substituent and a cis-oriented phenyl ring as a neighbouring substituent. In addition, Sharpless & Wang also investigated substrates featuring ester groups instead of an aliphatic chain or a cyclohexane moiety instead of a phenyl ring, of which the combination of an ester group and a phenyl ring as substituents gave the highest ee’s of 78-80 %. By comparison, a substrate featuring a methyl group and a cyclohexane ring as the 1,2-substituents only gave 56 % ee. As the interaction/complexation of the olefin-substituents with the chiral ligands

(DHQ)2PHAL/(DHQD)2PHAL or DHQ-IND/DHQD-IND are responsible for the stereochemical outcome of the reaction, the unsatisfactory stereochemical outcome of systems lacking a phenyl substituent may be explained by the insufficient interaction between the chiral ligands and the substrate. According to the empirical model shown in Fig. 11, the attack of the hydroxylating reagent has to come from the -face, in order to obtain the (S,S)-anti-diol 160 and therefore AD-mix  was predicted to be the appropriate reagent for our case.

-face "HOOH" DHQD-IND-()-attack

OH O THPO O AD-mix  CH3 Ph THPO O O H H OH cis-olefin anti-diol 160

AD-mix   (DHQ)2PHAL + K2OsO2(OH)4 +K3Fe(CN)6   -face "HOOH" DHQ-IND-( )-attack AD-mix   (DHQD)2PHAL + K2OsO2(OH)4 +K3Fe(CN)6

Figure 11. Empirical model of the Sharpless asymmetric dihydroxylation of cis-disubstituted olefins.107

46 Results and Discussions

Unfortunately, compound 160 was obtained with an ee of only 44 % as determined by chiral HPLC using a Chiralpak AD-H column after conversion of 161 into the acetonide 162 (no clean separation of enantiomers could be accomplished with 160 or 161). This outcome may not be surprising, as cis-olefins are known to give generally lower ee’s in AD-mix-mediated dihydroxylations than the corresponding trans-isomers.110 It should also be noted that the absolute configuration of 160 was not rigorously established, but is believed to be as shown on the basis of the empirical model discussed above. Nevertheless, at this point the synthesis was continued with the mixture of isomers, hoping that the separation of diastereomers would be possible at a later stage of the synthesis. After deprotection of 160 the 1,2-diol moiety was selectively protected as an acetonide, which allowed introduction of the terminal double bond via Grieco- Sharpless olefination118,119 which is an elimination reaction of an aliphatic primary alcohol through a selenide to a terminal alkene (Fig. 12). The tributylphosphine first reacts with o-nitrophenylselenocyanate to form a selenophosphonium salt which reacts with the alcohol providing an oxaphosphonium salt.118 Reaction of the aryl selenium anion with the oxaphosphonium species provides the corresponding alkyl aryl selenide plus tributylphosphine oxide. In the second step the selenide is oxidized with hydrogen peroxide to a selenoxide and spontaneous elimination takes place with expulsion of a selenol in a fashion similar to that of the Cope elimination.120

R Bu3P OH NO NO 2 NO 2 2 - Se SN2 Se Se CN CN + + R PBu PBu 3 O 3

NO 2 [O] syn-elimination + Se R + O PBu3 R OH Se Se R - NO 2 H O

Figure 12. Mechanism of the Grieco-Sharpless olefination.118

Saponification of 163 using standard conditions gave the free acid 164 which was converted to the acyl chloride 165 by refluxing in oxalyl chloride (Scheme 44). This material was used without purification and characterization to investigate the feasibility of a Sonogashira coupling with acyl chloride 165.121 Unfortunately, the Sonogashira coupling of 165 with 129S gave very low yields (s. below, “Assembly of Fragments C1’-C6’ and C7’-C11’”). This was assumed to be caused by the instability of the acetonide moiety under the conditions of acid chloride formation. For this reason an alternative approach for the activation of acid 164 was pursued, which involved conversion of ester 163 into the Weinreb amide 166 (Scheme 45).

47 Results and Discussions

O O O O i O O N O O 163 166 Scheme 45. Reagents and conditions: i. HCl•HN(OMe)Me, n-BuLi, -78°C → rt, 20min; then at -78°C addition of 163, 15min; 84 %. The elaboration of 148 into 166 was performed on a multi-g scale in 47 % overall yield for 8 steps.

3.1.1.4. Assembly of Fragments C1’-C6’ and C7’-C11’ As indicated above, initial attempts on the Sonogashira coupling between the presumed acid chloride 165 and alkyne 129S gave low yields, which we surmised to be related to the instability of the acetonide protecting group under the condtions of acid chloride formation. As the purported 165 was not purified and characterized, it is not clear wether the bad yield obtained in the coupling step is due to the coupling itself or due to a low conversion of acid 164 into the desired chloride 165. At the same time, it was also found that the product 167 that was obtained in low yield from the Sonogashira coupling had suffered from epimerization at the centre  to the keto group (Scheme 46). While the use of Weinreb amide 166 as coupling substrate gave a significant improvement in yield, unfortunately, it did not alleviate the epimerization problem: Work-up of the reaction initially provided a mixture of both epimers 168 and 167 which was completely converted to 167 during purification by FC. This conclusion is based on the comparison of the 1H-NMR spectra of the material before and after FC. A significant shift in signals was observed only for the 4’/5’ region of the molecule. Based on interpretion of the NMR-spectra it was presumed that the 5’-position of 168 had suffered from epimerization, which may be due to the (enforced) unfavorable syn-arrangement of the bulky substituents on the 5-membered acetonide ring. For this reason we adopted an alternative protecting group strategy.

OTBS

O i TBSO + Cl O O O O 129S 165 167 O

OTBS OTBS

O ii O TBSO + N + 129S 166 O O O O O O O O

168 167

Scheme 46: Reagents and conditions: i. Pd(PPh3)2Cl2, CuI, Et3N, rt, 15h; traces. ii. 129S, n-BuLi, -10°C, 20min; then 166, -78°C, overnight; 40 %. 48 Results and Discussions

We decided that the hydroxyl groups of intermediate L-IV (Scheme 36) should not be linked in a cyclic structure. Thus, ester 163 was deprotected under standard conditions and the obtained diol 169 was converted to its bis-TBS derivative 170, followed by transformation to the Weinreb amide 171 (Scheme 47) It is worth noting that the direct deprotection/protection of Weinreb amide 166 proved not to be feasible, due to its sensitivity to the required acidic conditions for acetonide cleavage.

O O O O i ii iii O O O O N O O HO OH TBSO OTBS TBSO OTBS

163 169 170 171

Scheme 47: Reagents and conditions: i. p-TsOH, MeOH, rt, 3h; 93 %. ii. TBS-OTf, lutidine; CH2Cl2, -78°C → rt, 3h; 97 %. iii. LiN(OMe)Me, -78°C, 15min; 97 %.

As the C’-10 protecting group would have to be cleaved selectively next to the C4’/C5’ TBS groups, the following coupling attempts were carried out with the TES- protected alkyne 156S. Coupling of 156S and 171 under optimized conditions did indeed provide the desired ketone 172 without epimerization in 89 % yield (Scheme 48).

OTES O O TBSO O OTES i HO Br ii + O + N TBSO OTBS O O OTBS 156S 171 172 29a

OTES OH OH

OH O OH O OH O iii O O OH

O O O O O O TBSO TBSO TBSO OTBS OTBS OTBS 173 174 175 Scheme 48: Reagents and conditions: i. 156S, n-BuLi, -10°C, 20min; then 171, -78°C → -18°C, overnight; 89 %. ii. 172, 9-BBN, THF, rt, 2h; then 2 M K3PO4; then added to a solution of 29a, [Pd(OAc)2 + 4 TFP], DME, 65°C, 3h , then rt overnight; 69 %. iii. THF/HOAc/H2O (2:2:1), rt, 3h; 88 %.

The Suzuki-Miyaura coupling of alkene 172 and aromatic halides 29a and 29b, respectively, was first attempted with more conventional catalysts such as 1,1'-bis-

[(diphenylphosphino)ferrocene]dichloro-palladium (II) [PdCl2(dppf)] or tetrakis (triphenylphosphine)palladium (0) [Pd(PPh3)4], but the desired product 173 (Scheme 48) was only obtained in yields between 15 % to 41 % (Table 1). In most cases dehalogenated products or unreacted aromatic starting material was recovered.

However, it was found that the use of [Pd(OAc)2 + 4TFP] (generated in situ from Pd(II)(OAc)2 and tri-2-furylphosphine) could strongly benefit the coupling yield.

49 Results and Discussions

Farina and Krishnan have investigated the effects of tri-2-furylphosphine and triphenylarsine ligands on Pd in the Stille reaction.122 These two catalyst/ligand systems are known to exhibit improved air-stability in comparison to [Pd(PPh3)4] and were found to lead to large rate acceleration. The authors assumed that the catalyst/ligand system decisively influences the transmetalation step of the cross coupling reaction which is the rate-determining step in the catalytic-cycle.

Table 1: Optimisation of the Suzuki coupling of the aromatic part 29 and alkene 172.

Aromatic Part Catalyst/Ligand Base Yield

29b [PdCl2(dppf)2], Ph3As Cs2CO3 33 %

29b [Pd(PPh3)4] Cs2CO3 10 %

29a [PdCl2(dppf)2, Ph3As Cs2CO3 41 %

29a [Pd(OAc)2 + 4 TFP] K3PO4 69 %

In 1979, Suzuki and Miyaura reported the stereoselective synthesis of arylated E- alkenes by the reaction of 1-alkenylboranes with arylhalides in the presence of a Pd catalyst.123 The Pd-catalyzed cross-coupling reaction between organoboron compounds and organic halides or triflates provides a powerful and generally applicable method for the formation of carbon-carbon bonds and is known as the Suzuki cross-coupling (Fig. 13).124

Pd(0) cat R1-B(R)2 R2-X R1-R2 X-B(R)2 base,ligand product

R = alkyl, allyl, alkenyl, alkynyl, aryl R = alkenyl, aryl, alkyl 1 2 base =Na2CO3,Ba(OH)2,K3PO4, R =alkyl,OH,O-alkyl X =Cl,Br,I,OTf,OPO(OR)2 Cs2CO3,K2CO3,TlOH,KF, + - CsF, Bu4F, NaOH, M ( O-alkyl) Figure 13. Scope of the Suzuki cross-coupling.124

The mechanism of the Suzuki cross-coupling is analogous to the catalytic cycle for various other cross-coupling reactions.125 It consists of four distinct steps (Fig. 14). Firstly, the Pd(0) species undergoes an oxidative addition to an organic halide. Subsequently, the anion attached to the Pd is exchanged for the anion of the base, a step that is called metathesis. This is followed by the transmetalation between Pd(II) and the alkylboron-ate complex, and reductive elimination to form the C-C single bond and simultaneous regeneration of Pd(0). Although organoboronic acids do not transmetalate to Pd(II)-complexes, the corresponding boron-ate complexes readily undergo transmetalation. The quaternization of the boron atom with an anion increases the nucleophilicity of the alkyl group which accelerates its transfer to the Pd in the transmetalation step. In addition, very bulky and electron-rich ligands (e. g.

P(tBu)3 or TFP) increase the reactivity of otherwise unreactive substrates (e. g. aryl chlorides) by accelerating the rate of the oxidative addition step.126

50 Results and Discussions

R1-R2 R2-X LnPd(0)

reductive oxidative elimination addition

L + - R1-B(R)2 M ( OR) organoborane base

R1 X Ln-1Pd(II) LnPd(II) R2 R2 OR R B(R) transmetalation 1 2 boron-ate complex

L+ RO B(R)2 M+(-OR) OR metathesis OR L Pd(II) n M+(-X) R2 Figure 14. Mechanism of the Suzuki cross-coupling.124

The Suzuki-Miyaura coupling of 172 and 29a with [Pd(OAc)2 + 4TFP] as the catalyst, after extensive optimization, gave the desired coupling product 173 in 69 % yield. It may be speculated that this catalyst/ligand system has a suitable electron-density for the o-halogented salicylic acid derivates, thereby accelerating the oxidative as well as the transmetalation step. The [Pd(OAc)2 + 4TFP] system exhibits improved air- stability, but it should be noted that the solvent (DME) was always degassed carefully, which proved to be a crucial for good reproducibility. The coupling product 173 was selectively deprotected and afforded 174 in excellent yield (88 %).

3.1.1.5. Methyl Ester Cleavage

The methyl ester cleavage turned out to be problematic which we felt could have been caused by steric crowding around the aromatic methyl ester group and/or the sensitivity of the enyne moiety towards strong bases and acids. Table 2 gives an overview of different attempts to cleave the aromatic methyl ester moiety of intermediates 173 and 174. The substrates 173 and 174 appeared to be too sensitive for the saponification conditions that are required for the aromatic methyl ester to be cleaved, as we observed either no cleavage of the ester or decomposition.

To overcome the stability problem, we investigated the reaction of 173/174 with bis(tri-n-butyltin)oxide (BBTO) as a mild, neutral, and selective reagent for cleavage of esters.127 In a control experiment BBTO led to quantitative deprotection of benzoic acid methyl ester, whereas the corresponding deprotection of the isolated building block 29a led only to 10 % of product formation; none of the desired product was obtained in the attempted deprotection of 173. Interestingly, this deprotection method is known to have two major limitations, which are steric hindrance around the carbonyl carbon and the presence of fluoroalkyl groups.127 This suggests that the 51 Results and Discussions carbonyl carbon of the intermediates 173/174 is not accessible for sterically demanding nucleophiles such as BBTO.

Table 2. Screening of reaction conditions for methyl ester cleavage in 173 and 174. Substrate 173 Substrate 174 LiOH, THF/water, rt → TMSOK, DME, 110°C, 5-120min, mw → no reaction, loss of TES decomposition TMSOK, toluene, 60°C → 2N NaOH/MeOH reflux overnight → decomposition no conversion; migration of TBS-groups bis(tri-n-butyltin)oxide (BBTO) toluene, LiI, pyridine, reflux overnight → 80°C or 110°C → decomposition no conversion pig liver esterase (PLE), 5 % acetone in PLE, 5 % acetone in phosphate buffer, phosphate buffer, pH = 7.2, 38°C, 4d → pH = 7.2, 38°C, 4d → 15 % impure product; not reproducible oiled out; no conversion

Base-catalyzed hydrolysis using alkali metal hydroxides or carbonates in aqueous methanol remains the most common method for cleaving simple esters, with the stability of the substrate to basic conditions as the main limitations. In more complex substrates lithium iodide can be used, in order to selectively cleave methyl esters.128 By performing the reaction in an aprotic solvent it is possible to make carbonyl attack an unfavorable pathway. Instead, the attack on the -carbon atom of the alcohol component of the ester predominates. As the ester is more accessible at this position in comparison to the more sterically demanding carbonyl atom, this nucleophilic version of ester cleavage might have been the method of choice.129 Unfortunately, only decomposition was observed when 173/174 were treated with LiI in pyridine was used. Enzymes often show a reduced acceptance for aromatic substrates, with pig liver esterase (PLE) showing the broadest substrate-specificity.130 In test reactions PLE did indeed accept the aromatic ester 29a as a substrate; in contrast the lipase from Candida culindracea (CCL) did not cleave the methyl ester at all. Unfortunately, all enzyme-based attempts to cleave the methyl esters of 173 or 174 remained unsuccessful with both lipases (PLE and CCL) under a variety of conditions. One problem seemed to be substrate acceptance in general, but an additional contributing factor may have been the very poor solubility of the substrates in phosphate buffer. Even the addition of acetone as a co-solvent did not improve the situation. As only traces of acid 175 were obtained in a non-reproducible fashion, this approach was abandoned.

52 Results and Discussions

3.1.1.6. Suzuki Couplings with Free Carboxylic Acids In order to avoid the methyl ester cleavage step, Suzuki couplings of unprotected carboxylic acids were investigated (Scheme 49). Bumagin & Bykov reported such 131 Pd-catalyzed couplings of halobenzoic acids with arylboronic acids. Pd salts PdX2 (X = Cl or OAc) were found to be the most effective catalysts for the cross coupling with phenyl boronic acids in coupling reactions with aryl bromides and iodides. Several test reactions with free benzoic acids were carried out under the conditions reported by Bumagin & Bykov. The first entry in Scheme 49 shows the conversion of ester 29b to acid 30b. It was found that the resorcylic acid methyl ester moiety of the aromatic part was easily cleaved with potassium trimethylsilanolate (TMSOK) in 1,2-dimethoxyethane at 110°C in a microwave reactor.

O O O OH

HO I TMSOK, DME, HO I 110 °C, mw, 2h quant O O 29b 30b

O OH 3 mol% Pd(OAc)2 O 3equivNa2CO3 HO Br H O, rt OH OH 2 B + O OH 0% O 30a

O OH 3 mol% Pd(OAc)2 O Br 5equivNaOH OH H 2O, rt OH B + OH 0% O

O OH 3 mol% Pd(OAc)2 O 2.5 equiv Na2CO3 I H O, rt OH + 2 OH B OH 58 %

O OH O OH 3mol%Pd(OAc)2 2.5 equiv Na CO HO I 2 3 HO H2O, rt OH B + OH 35 % O O 30b Scheme 49. Conversion of 29a to 30b and Suzuki couplings with free carboxylic acids.

The results of the Suzuki trials show that the coupling is generally possible with an aryl iodide, although the yields are low. Unfortunately, the Suzuki coupling of the 53 Results and Discussions alkene 172 with free 2-iodo-benzoic acid 30b did not afford the desired product and therefore this approach was also abandoned.

3.1.1.7. Strategy Variation: Change of the Ester Group In the next step we investigated variations of the carboxylic acid protecting group. To this end the acid labile tert-butyl ester and the base labile 2-(p-toluene-sulfonyl)ethyl (TSE) esters of acid 30a (Scheme 50) were synthesized. These esters were coupled to the alkene 172, but the basic labile TSE ester was already cleaved under the Suzuki conditions and no product was obtained. The Suzuki coupling of the alkene 172 and the tert-butyl ester of 29a worked in low yield (10 %), but the cleavage of the tert-butyl ester turned out to be impossible under mild conditions.132 Cleavage attempts were carried out at 0°C, at rt and at last under reflux conditions for two h with trimethylsilyl trifluoromethanesulfonate (TMS-OTf). No cleavage of the ester moiety occurred under these conditions, only loss of the TES protecting group was observed. After a careful analysis of the remaining options it was felt that the -trimethylsilyl ethyl (TMSE) ester would be the ester of choice. TMS ethyl esters undergo a fragmentation reaction upon treatment with TBAF to give ethylene fluorotrimethyl- silane and the tetrabutylammonium salt of the corresponding acid. In addition, the attack of the reagent occurs at a position remote from the crowded carbonyl carbon, which is an important characteristic for our sterical demanding substrate. Thus, methyl ester 29a was converted to the free acid under the optimized conditions developed for the cleavage of 29b (Scheme 49). The resulting acid 30a was easily esterified using Steglich conditions to afford the TMS ethyl ester 176. Subsequent Suzuki coupling to alkene 172 gave the fully protected linear precursor 177 in excellent yield (Scheme 50).

OTES O O O OH O O Si HO Br HO Br HO Br i ii iii OTBS

O O O O OTBS 29a 30a 176 172

Si Si OTES OH OH

OH O OH O OH O

O OTBS iv O OH v OH OH

O O O O O O OTBS OH OH 177 178 179

Scheme 50. Reagents and conditions: i. TMSOK, DME, mw 110°C, 2h; then acidic work-up, 95 %. ii. HOCH2CH2Si(CH3)3, DCC, DMAP, CH2Cl2, 48h, 88 %. iii. 172, 9-BBN, THF, rt, 2h; then 2 M K3PO4; added to a solution of 176, [Pd(OAc)2 + 4 TFP], DME, 6h, reflux; 70 %. iv. HF•py, THF, rt, 2h; 88 %. v. TBAF, p-TSOH, rt, 24h, not defined.

54 Results and Discussions

Initially it was planned to remove the TES group and the TMS ethyl ester without cleavage of the two TBS protecting groups, but this approach was found not to be feasible. Although the deprotection of a -trimethylsilyl ethyl ester in the presence of secondary TBS protecting groups has been employed by Nicolaou et al. in the synthesis of epothilone B analogues133, this was not possible in our case. However, there is also an example reported in literature in which an aromatic TBDPS group was found to be less stable than the TMS ethyl ester.134 The reactivity of the TBS groups is strongly dependent on the substrate and in our case the TBS groups were at least as labile as the TMS ethyl ester. Various deprotection conditions for TMS ethyl esters were investigated, which included acidic conditions as well as fluoride-containing reagents. Ester cleavage was carried out at low temperature and with different numbers of equivalents of fluoride containing reagents, but the conversion of the TMS ethyl ester was very slow and not selective. Finally it was found that the ester cleavage was only possible when TBAF was used. When buffered TBAF (addition of pTsOH) was used, hardly any TMS ethyl ester cleavage was observed. Even tris(dimethylamino)sulfur(trimethylsilyl) difluoride (TAS-F) did not improve the selectivity, although it is a mild and neutral fluoride containing reagent.135 If global deprotection could have been achieved in a clean reaction, it would have been an option to carry out a 1,2-selective acetonide protection and to investigate the macrolactonization of this substrate, although there would have been the risk of epimerization at the C5’. Finally, HF•pyridine was found to cleave all hydroxyl protecting groups to afford 178, but the final TMS ethyl ester cleavage to 179 was not reproducible. In the end we decided to abandon this approach. Based on the experience we had gained in the two years working on the first approach a new strategy was developed that finally led to the natural product 6.73

3.1.2. Second Generation Approach As mentioned above the second generation retrosynthetic analysis took into account experiences and knowledge gained in the first generation approach. Thus, the disconnection of the target structure into three building blocks and the use of previously established key reactions for their assembly were retained in our second generation approach (Scheme 37). By contrast, an alternative route to the intermediate C1’-C11’ fragment was pursued that relied on isopropylidene-D- erythrono-1,4-lactone (107) as a chiral and commercially available starting material. Another important new feature of the second approach is the late introduction of the ketone moiety at the C6’ position. We hoped that this would provide intermediates exhibiting a different chemical behavior in comparison to the ynone structure of the first generation approach.

3.1.2.1. Synthesis of the C1’-C11’ Fragment The synthesis of the aliphatic framework of L-783277 (6) started with the reduction of the lactone 107 to the corresponding lactol 180 followed by chain elongation using

55 Results and Discussions the commercially available Wittig reagent carboethoxy-methylene triphenylphosphorane (Scheme 51). The synthesis of the isopropylidene-D-erythrono-1,4- lactone (107) derived fragment is similar to a reaction sequence published by Toba et al.136 The double bond of both isomers of 181 was reduced and the resulting alcohol 182 was oxidized to the aldehyde 183 under Swern conditions. Thus, the synthesis of intermediate 183 could be achieved in 68 % yield over 4 steps.

O O O O O OH OEt OH OEt O OEt O i OH ii iii iv O O O O O O O O O O

107 180 181 182 183

Scheme 51. Reagents and conditions: i. DIBAL-H, Et2O, -78°C, 2h, 95 %. ii. Ph3PCHCOOEt, dioxane, reflux, 7h, 91 %; 1.1:1 E/Z-isomer. iii. H2, Pd/C, EtOH, 4h, 94 %. iv. (COCl)2, DMSO, Et3N, CH2Cl2, -78°C, 1h, 79 %.

The other half of the aliphatic framework of 6 derives from 129R which was synthesized according to Scheme 43, but starting from R-propylene oxide 40R. The C7’-C11’ fragment was coupled to the C1’-6’ fragment by lithiation of the alkyne 129R followed by addition to the aldehyde 183. Unfortunately, the yield of the coupling of the intermediates 129R and 183 did not exceed 57 % (Scheme 52). This was due to side reactions of the lithiated species on account of the ester function of 183. Although MOM-protection of 184 and the subsequent reduction of the ethyl ester 185 worked in acceptable yields, this route was not further pursued due to the side reactions associated with the presence of an ester function in 183.

OTBS OTBS OTBS

i ii iii TBSO O O EtO OH EtO OMOM HO OMOM 129R OO OO OO

184 185 186 Scheme 52. Reagents and conditions: i. 129R, 2 eq n-BuLi, -78°C, 45min; then addition of 183, -78°C, overnight, 57 %. ii. MOM-Cl, (i-Pr)2NEt, Bu4NI, DMF, 20h, 80 %. iii. DIBAL-H, CH2Cl2, -78°C, 2h, 80 %; 4:1 mixrure of isomers, separable at this point.

3.1.2.2. Variation of the Synthesis of the C1’-C6’ Fragment To overcome the side reaction associated with the presence of an ester function in 183 the C1’-C6’ fragment was synthesized by introducing the required terminal double bond before coupling with intermediate 129R. The reaction sequence in Scheme 51 was followed up to the stage of the saturated alcohol 182, which was protected as TBS ether to afford ethyl ester 187. The latter could be reduced to alcohol 188 in satisfactory yields (Scheme 53).

56 Results and Discussions

O O OH OEt OTBS OEt OTBS OH OTBS OH O i ii iii iv v

OO OO OO OO OO OO

182 187 188 189190 191

Scheme 53. Reagents and conditions: i. TBS-OTf, CH2Cl2, 1h, 93 %. ii. DIBAL-H, toluene, 2h, 85 %. iii. 2-NO2-PhSeCN, Bu3P; then NaHCO3, 30 % H2O2, 19h, 81 %. iv. TBAF, THF, 0°C → rt, 0.5h, 90 %. v. (COCl)2, DMSO, Et3N, CH2Cl2, -78°C, 1h, 74 %.

The terminal hydroxyl group was converted to a terminal double bond under Grieco- Sharpless conditions. The TBS group of the resulting alkene 189 was removed and alcohol 190 was subsequently oxidized to aldehyde 191, which was ready to be coupled to building block 129R.

3.1.2.3. Assembly of Building Blocks The addition of intermediates 129R to 191 was successfully carried out in 87 % yield (Scheme 54). The mixture of diastereomers turned out not to be separable by FC. Therefore, the mixture of alcohols 192 was protected as MOM-ether using MOM-Cl and DIPEA in the presence of TBAI as a catalyst. The Suzuki coupling of alkene 193 and the aromatic building block 176 (see Scheme 50) was accomplished in excellent yield and led to the fully protected linear macrolactonization precursor 194. The triple bond of 194 could be hydrogenated to the desired Z-double bond using Lindlar catalyst. Fortunately, at this stage the C6’-isomers of 195 could be separated by FC, when fine silica gel (15-40 m) was used. The seperated C6’-isomers were taken through the remainder of the synthesis individually. Thus, simultaneous cleavage of the TMS-ethyl ester and the TBS-ether moieties with TBAF gave the seco acids 196 (in quantitative yield for both isomers), which were cyclized under Mitsunobu conditions (with concomitant inversion of configuration at C10’). Subsequent deprotection with sulfonic acid resin gave the fully deprotected macrolactones 197, which were both submitted to oxidation with polymer-bound IBX. The latter was purchased from Novabiochem, but it can also be prepared according to Sorg et al.137 Under the oxidation conditions the major C6’-isomer of 198 led to a 1:4 mixture of L- 783277 (6) and a second mono-oxidized product, which could be assigned as the C5’ oxidized analogue 6a (82 % total yield after FC).

OH O

O OH HO O 6a

In contrast, the minor C6’-isomer of 198 gave L-783277 (6) in 93 % yield and 91 % HPLC purity, together with 8 % of a second mono-oxidized product that could not be separated by TLC or FC. Purification of a sample by HPLC gave L-783277 (6) with > 95 % final purity. The NMR-spectra of this material were indistinguishable from those

57 Results and Discussions of L-783277 (6) from natural sources, which was kindly provided by Frank Petersen (Novartis, Basel).

OTBS OTBS OTBS

Si i ii iii OH O TBSO O 129R OH O O O O O O O O O O O 192 193 194

Si OH O OTBS OH O OH iv v vi O OH

O O O O O O O O O O 195 196

OH O OH O OH O vii viii O O O

O O O O OH O O O HO HO O OH OH 197 198 6 Scheme 54. Reagents and conditions: i. n-BuLi, -78°C, 1.5h; then addition of 191, -78°C, 3h, 87 % (2/1 mixture of isomers). ii. MOM-Cl, (i-Pr)2NEt, Bu4NI, DMF, 19h, 87 %. iii. 193, 9-BBN, THF, rt, 2h; then. 176, 2 M K3PO4, [Pd(OAc)2 + 4 TFP], DME, reflux, 5.5h, 81 %. iv. H2, Lindlar catalyst, AcOEt, 3h, 90 %; separation of isomers (1.9/1). v. TBAF, THF, rt, 15h, quant (major isomer); quant (minor isomer). vi. DIAD, Ph3P, toluene, 25min, 59 % (major isomer); 74 % (minor isomer). vii. Sulfonic acid resin, MeOH, reflux, 2.5h, 78 % (major isomer); 46 % (minor isomer). viii. IBX (polymer supported), CH2Cl2, rt, 6h; from major isomer of 198: 1:4 product mixture of L-783277 (6) and a second mono- oxidized species (major product), 82 % (total yield); from minor isomer of 198: 93 % of 6 (91 % purity).73

Interestingly, no differences in oxidation selectivity between C6’-isomers appear to be operative in the case of radicicol A (4), for which Winssinger and co-workers have reported a > 90 % yield for the polymer-bound IBX-mediated oxidation of the C6’- isomeric mixture of triol precursors corresponding to 197 (see Scheme 16).33 Different oxidizing agents had been investigated for the allylic oxidation of 197 to 6, before the conditions shown in Scheme 54 could be established. When DMP, IBX, or

MnO2 were used as oxidizing agents, overoxidation took place immediately. This finding is in contrast to the results reported by Tatsuta et al. for their work on LL- Z1640-2 (3), where the allylic hydroxyl group of the 4’/5’-unprotected substrate was oxidized using DMP.75 For this reason we started to investigate the allylic oxidation with polymer-bound IBX according to Winssinger et al.33 In summary, we have accomplished the first total synthesis of the resorcylic lactone kinase inhibitor L-783277 (6), which represents an alternative lead structure for anticancer or anti-inflammatory drug discovery.73 At the same time, the efficiency of the critical steps of the synthesis still remained to be improved. The use of an

58 Results and Discussions enantioselective method for the synthesis of 192 could give rise to the isomer, which we had shown to be easily convertible to the natural product 6.

3.1.3. Improved Enantioselective Approach The most critical step in our total synthesis of L-783277 (6) was the addition of the acetylene 129R to the aldehyde 191, because the two diastereomers formed during this reaction could not be elaborated into the final product with equal efficiency. An enantioselective version of this addition would be a suitable approach to improve the overall yield and practicability of the synthesis of 6. Chiral secondary propargylic alcohols in general are versatile and highly valuable building blocks for asymmetric synthesis and they can be obtained by the direct asymmetric addition of metal alkynylides to aldehydes.138 The need for a practical and general synthetic access to chiral propargylic alcohols formed under mild reaction conditions led to the development of a method using Zn(OTf)2 and N-methyl ephedrine (NME) as a chiral reagent by Carreira in 2000.139 This reaction encompasses a wide scope for both aldehydes 199 and alkynes 200,140 providing the products 201 in useful yields and with excellent enantioselectivities (Scheme 55).

Zn(OTf)2, Et3N OH O (+)-NME + 2 1 R1 H R toluene, rt R 1-20 h R2 199 200 201

R1 = alkyl, aryl, alkenyl 52-99% yield R2 = alkyl, aryl, SiR3 90-99% ee Scheme 55. Direct asymmetric addition of zinc alkynylides to aldehydes developed by Carreira et al.139

The nucleophilic Zn-alkynylides are generated in situ at room temperature from terminal alkynes without the need for a separate preparative activation step. From a mechanistic point of view the Lewis acid Zn(OTf)2 is suggested to coordinate to the triple bond, thereby forming a -complex. As a consequence, the terminal C-H bond is labilized to such an extent that weak amine bases effect deprotonation and concomitant formation of the corresponding Zn-acetylide (Fig. 15). This mechanistic model is supported by experimental data which were obtained in extensive React-IR studies.141

R 1 Zn(OTf) Zn(OTf)2 Et3N R 1 H R 1 H +

Zn(OTf)2 Et3NH(OTf)

Figure 15. Formation of Zn-acetylide from acetylene in the presence of Et3N and Zn(OTf)2.

In 2001 Carreira also developed a catalytic version of the addition of Zn-alkynylides to aldehydes.142 For the successful implementation of the catalytic process the reaction temperature had to be increased up to 60°C, so that the kinetic barrier for protonation of the initially formed Zn-alkynylide complex could be overcome. Thus,

59 Results and Discussions the turnover of the reaction became reasonable and the products were also obtained with good enantioselectivities (> 90 %). In light of its versatility and mildness, it is not surprising that the above method for the enantioselective preparation of secondary propargylic alcohols has been used for the synthesis of a number of natural products; e. g., the Zn(OTf)2 mediated addition of alkynes to aldehydes features prominently in Carreira’s syntheses of epothilone A and B,143 leucascandrolide A,144 as well as 35-deoxy amphotericin B methyl ester145.

In addition, the enantioselective Zn(OTf)2-mediated addition of alkynes to aldehydes has been employed by other research groups, e. g. in the total syntheses of (+)- gigantecin,146 epoxomicin,147 (-)-panacene148, longimicin D149, FR901464,150 and (+)- desoxygaliellalactone151.

3.1.3.1. Enantioselective Addition of the C1’-C6’ Acetylene to the C7’-C11’ Aldehyde

The Zn(OTf)2-mediated coupling of the C7’-C11’ fragment 129R (also see Scheme 43) with the C1’-C6’ fragment 191 (see Scheme 53) constituted the key step in our second generation approach to 6, as the branched aldehyde seemed to be a suitable substrate for this reaction. The enantioselective addition of alkyne 129R to aldehyde 191 was carried out according to literature procedures in the presence of the chiral additives (-)-NME or (+)-NME to afford the minor as well as the major isomer of 192 (Scheme 56).145 As the stoichiometric approach is more widely used in total synthesis than the catalytic procedure, a ca. two fold excess of Zn(OTf)2, NME and base was used for the reaction (Scheme 56).

OTBS

Zn(OTf)2 9' 11' O Et3N (-)-NME TBSO to lue ne 3' O O 6' 129R 46 % 1' OH O O 191

192a

OTBS 9' Zn(OTf)2 11' O Et3N (+)-NME TBSO to lue ne 3' 6' O O 129R 26 % 1' OH 24:1 O O 191 major:minor isomer 192b Scheme 56. Enantioselective zinc triflate mediated additions of alkyne 129R to aldehyde 191.145

60 Results and Discussions

Before the Zn(OTf)2 was used in the reaction it was dried under high vacuum (0.03 mbar) at 144°C for 2 h, vigorously stirring the solid with an appropriate magnetic stirrer in order to obtain a fine powder. After cooling to room temperature the chiral additive, (-)-NME or (+)-NME, was added. The solids were suspended in toluene at rt and Et3N was added. The resulting milky mixture was stirred at rt for two h and then treated with alkyne 129R in toluene. After 45 min at rt a solution of aldehyde 191 in toluene was added. The reaction mixture changed its color from pale yellow to yellow and was stirred overnight, while the colour of the solution changed to a final red-brown. Work-up and purification by FC furnished either diastereomer in moderate (192a, 46 %) or low (192b, 26 %) yield, but with excellent diastereoselectivity Based on comparison of their NMR spectra with those of the product in the non- selective approach to 192 (Scheme 54), the identity of the minor and major isomer in the non-selective addition reaction could be assigned. Thus, the minor isomer obtained in the non-selective reaction corresponds to 192a and the major isomer to 192b (Scheme 56). Interestingly, there seems to be a match/match situation in the case of 192a, because the yield is moderate and the other diastereomer could not be detected by H-NMR. In contrast, the 192b could be synthesized only in 26 % yield and the rate of 192a was detectable by H-NMR.

3.1.3.2. Determination of the Absolute Configuration As 192b could not be used for the synthesis of 6 (and therefore was expandable), it was employed to assign the absolute configuration of the newly formed stereocenter at C6’ by Mosher ester analysis.152 Following literature procedures 192b was converted into its R- and S-Mosher ester (Fig. 16).153

OTBS

-0.06 9' 11' -0.05

R2 MeO Ph R 2 Ph OMe R O R O 0.07 5' 6' 0.00 1 1 0.02 1' CF3 CF 3 OH H O H O O O (R)-MT PA (S)-MT PA 192b Figure 16. Mosher ester analysis of 192b.153

1 With  defined as S - R, the analysis of the H-NMR spectra indicated a positive shift for all the protons of the C1’-C5’ part, which means that they are situated in front of the paper plane in Fig. 16 and thus correspond to R1. In contrast, negative values were observed for the propargylic C7’-C11’ part, which indicates that these carbons are situated behind the paper plane and correspond to R2 in Fig. 16. Thus, 192b could be unambiguously assigned as the (6’R)-isomer by applying the Mosher ester analysis.

61 Results and Discussions

3.1.3.3. Completion of the Total Synthesis The 6’S-isomer 192a was carried through the remainder of the synthesis (Scheme

57). As discussed before, the enantioselective Zn(OTf)2-mediated addition of alkyne 129R to aldehyde 191 furnished the minor isomer 192a with acceptable yield (46 %) and excellent diastereoselectivity, as the other diastereomer could not be detected by 1H-NMR. The single isomer could be cleanly protected as its MOM-ether, which was coupled to the resorcylic moiety 176 under optimized Suzuki coupling conditions to obtain 194a in 81 % yield (Scheme 57). The triple bond of the coupling product 194a was cis-selectively reduced using Lindlar catalyst. Simultaneous cleavage of the TMS ethyl ester and the TBS-group led to the linear macrolactonization precursor 196a, which was cyclized under Mitsunobu conditions. Global deprotection under standard conditions finally led to triol 198a which was submitted to oxidation with polymer-bound IBX.

OTBS OTBS OTBS

Si i ii iii OH O TBSO O 129R OH O O O O O O O O O O O 192a 193a 194a

Si OH O OTBS OH O OH iv v vi O OH

O O O O O O O O O O 195a 196a

OH O OH O OH O vii viii O O O

O O O O OH O O O HO HO O OH OH 197a 198a 6

Scheme 57. Reagents and conditions: i. 129R, Zn(OTf)2, (-)-NME, Et3N, toluene, rt, 45min; then 191, rt, 16h, 46 %. ii. MOM-Cl, (i-Pr)2NEt, Bu4NI, DMF, 19h, 80 %. iii. 193a, 9-BBN, THF, rt, 2h; then. 176, 2 M K3PO4, [Pd(OAc)2 + 4 TFP], DME, reflux, 5.5h, 68 %. iv. H2, Lindlar catalyst, AcOEt, 3h, 86 %. v. TBAF, THF, rt, 15h, 99 %. vi. DIAD, Ph3P, toluene, 25min, 64 %. vi. Sulfonic acid resin, MeOH, reflux, 2.5h, 71 %. vii. IBX (polymer supported), CH2Cl2, rt, 1.5h; 91 % of 6 (88 % purity); after HPLC purification 52 % of 6 (> 95 % purity).

The allylic alcohol was smoothly converted to the desired enone, thus completing the total synthesis of L-783277 (6) in an enantioselective and efficient manner (in 27 % yield over 7 linear steps from the acetylide addition product 192a). The HPLC purification of the final product turned out to be difficult, as the natural product is only sparingly soluble even in DMSO. For analytical purposes the solubility is sufficient, but for preparative purification by RP-HPLC the poor solubility hampers the separation. We assume that the macrolactone partly crystallizes out when it is 62 Results and Discussions injected, so it smears over the column, which corrupts the separation. For this reason, attempts were made to improve the purification of the final compound by FC by using more and a finer grade silica gel (15-40 m) than was usually employed in FC-based separations. In this way, the purity of the crude oxidation product could be increased up to 96 %. As the natural product seems to be stable on silica gel, the purification by FC is preferred to purification by preparative HPLC.

3.1.4. Still-Gennari Approach As all successful approaches to the synthesis of cis-enone-containing resorcylic acid lactones to date are macrolactonization-based and alternative modes of ring closure have rarely been investigated, we decided to explore the Still-Gennari olefination as a possible and novel ring-closing method for the synthesis of L-783277 (6). This project was performed by Luca Fransioli as the second part of his master thesis after he had successfully synthesized the dideoxy analog D6.154

3.1.4.1. Still-Gennari Olefination Macrolides are among the most important classes of natural products; due to their diverse biological activities they aroused substantial interest in their chemistry which grants access to these challenging structures.155 Macrocycles are generally synthesized from smaller molecules and the resulting linear precusor is then cyclized in an intramolecular reaction. This can be realized by high dilution chemistry using low concentrations of the linear precursors, in order to avoid polymerization, and the activation of one of the reacting groups of the linear precursor.111 Most of the macrolactonization-based cyclization methods are characterized by an activation of the acid group, e. g. by converting it into an activated ester according to Corey- Nicolaou, Mukaiyama or Keck; into a mixed anhydride, e. g. according to Yamaguchi, or into the corresponding acyl halide.111 The left part of Fig. 17 shows the general pathway of an acid activation-based macrolactonization method. In this particular case, the linear seco acid is reacted with with 2,4,6-trichlorobenzoyl chloride in the presence of Et3N to form a mixed anhydride, which is diluted and added to a refluxing solution of DMAP. The latter is a known as a catalyst for acyl transfer reactions. DMAP regeoselectively attacks the mixed anhydride at the less hindered carbonyl group, producing an acyl-substituted DMAP. The deprotonated alcohol reacts with the carbonyl group of the highly activated intermediate and the macrolactone is formed.111 In contrast, the Mitsunobu-based macrolactonization is among the alcohol activation- based macrolactonization methods and proceeds under inversion of configuration.156

In the first step PPh3 reacts with DEAD or DIAD in an irreversible addition and forms a zwitterionic adduct which deprotonates the acid. The alcohol reacts with the protonated DEAD/PPh3 adduct and forms the key oxyphosphonium ion in situ which is attacked by the carboxylate anion to give the respective macrolactone.

63 Results and Discussions

HO OH O O Cl

O- Cl Cl N

NMe2 -O O

O O P(Ph) N 3

- O NM e2

O O Figure 17. General pathways of acid activation-based (left pathway: Yamaguchi esterification) and alcohol activation-based (right pathway: Mitsunobu-based reaction) macrolactonization methods.

Other important cyclization methods are based on the intramolecular formation of C- C, C=C or even C≡C bonds. The formation of C-C bond includes C-nucleophilic additions to carbonyl groups, alkene-acetal couplings, alkylations of carbanions or enols and many others.157 Among the methods for the intramolecular formation of C=C bonds are the Wittig reaction, which is based on the reaction of an aldehyde with a phosphonium ylide. In contrast, the trans-selective Horner-Wadsworth- Emmons (HWE) reaction and the cis-selective Still-Gennari reaction are based on the reaction of an aldehyde with a phosphonate stabilized carbanion. In recent years ring closing alkene and alkyne metathesis have become powerful methods for the intramolecular formation of C=C or C≡C bonds and these methods have been applied in many cases in the total synthesis of natural products.158 As our alternative approach to 6 envisioned macrocyclization through the Still- Gennari variant of the HWE reaction, the mechanism of this reaction shall be briefly discussed here. The reaction of phosphonates featuring electron-withdrawing groups (e. g. trifluoroethyl) and aldehydes under strongly dissociating conditions (KHMDS and 18-crown-6 in THF) affords Z-alkenes almost exclusively. In contrast, the classical HWE-olefination is a reaction of stabilized phosphonate carbanions with aldehydes (or ketones) which predominantly produces E-alkenes. As indicated above, the Still-Gennari variant of the HWE reaction grants access to Z-alkenes with excellent stereoselectivity by modifying the phosphonate reagent.112 The reaction is highly selective as Z/E ratios are generally obtained in the range from 100:0 to 4:1.159 The assumed mechanism of the Still-Gennari reaction is depicted in Fig. 18, but it has to be noted that it is not fully understood so far.124 In a first step phosphonate I is deprotonated by a base which leads to formation of the phosphonate carbanion II, which in a second step attacks the carbonyl group of the aldehyde III. The rate determining step (RDS) is the formation of intermediate Vanti, which is faster than the

64 Results and Discussions

formation of Vsyn, because the energy of the transition state for IVanti is lower than of the transition state leading to IVsyn, due to sterical reasons (indicated in red). The chelated adduct Vanti then rearranges to oxaphosphetane VIanti. The following elimination step is faster than the initial addition, which essentially becomes irreversible unlike in the case of the conventional HWE olefination.160 As a consequence, the formation of the (Z)-stereoisomer is predominant. In addition, the use of a metal-complexing agent (usually 18-crown-6) is essential in order to minimize coordination of metal cation to the carbanion.124,161,162

M O O -HBase O O O O O O M 1 P 1 P 1 P 1 P R O R2 R O R2 R O R2 R O R2 R1O R1O R1O R1O H Base M M R1 = trifluoroalkyl I II R2 = alkyl

M k M k M O O anti addition O O O syn addition O O (slow) + (slower) 1 P 1 P 1 P R O OR2 R O OR2 3 R O OR2 OH R H 1 OH R1O R1O R O H R3 R3 H IVanti II III IVsyn phosphonate carbanion aldehyde kanti k RDS syn (fast) (fast)

R1O P O R1O P O R1O R1O COOR2 COOR2 H H O- O- 3 O O A R kcis k R3 H R2O P R2O P trans (fast) 2 O O (fast) R O R2O V V anti M O syn 3 3 M O 1 OR1 R R OR OP OP OR1 OR1 2 2 2 COOR COOR R3 COOR R3 COOR2

VIcis (Z)-Alken (E)-Alken VItrans cis-oxaphosphetane major minor trans-oxaphosphetane Figure 18. Mechanism of the Still-Gennari reaction.124

The Still-Gennari reaction can also be used for intramolecular cyclizations. Exemplarily this has been demonstrated in the total syntheses of phorboxazol A163 with a selectivity of Z:E = 4:1 and dictyostatin164 with even a better selectivity of Z:E = 6.5:1.

3.1.4.2. Synthesis of the C1’-C6’ Fragment The synthesis of the C1’-C6’ fragment started again from isopropyliden-D- erythronolactone 107 which was converted to aldehyde 191 according to the route outlined in Scheme 51 and 53. To obtain the desired trifluoroethylphosphonate 204 aldehyde 191 was oxidized to acid 202 using Pinnick-based oxidation conditions. The

65 Results and Discussions

Pinnick oxidation is a very mild method to oxidize aliphatic, aromatic, saturated or 165 unsaturated aldehydes. The oxidizing species is sodium chlorite (NaClO2); as hypochlorous acid (HClO) is formed as a by-product of the oxidation process, a scavenger (most often isobutene or 2-methyl-2-butene) is used in order to avoid side reactions. The acid was smoothly transformed to its acid chloride 203 using the Ghosez reagent:166 203 was directly converted to the phosphonate 204 according to the procedure of Paterson et al.167

CF3 O O O O O O P O CF3 H i OH ii Cl iii

OO OO OO OO

191 202 203 204 Scheme 58. Reagents and conditions: i. 2-methyl-2-butene, t-BuOH, NaClO2, NaH2PO4, H2O, rt, 1h, quant. ii. Me2C=C(NMe2)Cl, CH2Cl2, rt, 1h, quant. iii. MeP(O)(OCH2CF3)2, LiHMDS, -98°C, 1h, 23 % over 3 steps.167 The most challenging issue in this sequence was the volatility of aldehyde 191 as well as acid chloride 203, which precluded the drying of these intermediates in high vacuum. As a result, the overall yield of the reaction sequence shown in Scheme 58 was only moderate. However, no attempts were made at optimizing the yield for 204, as the primary objective of this project was the evaluation of the applicability of the Still-Gennari approach to the synthesis of 6.

3.1.4.3. Coupling of the Phosphonate to the Resorcylic Moiety Suzuki coupling of the phosphonate 204 with the aromatic bromide 176 (Scheme 50) was carried out under the previously developed optimized conditions (Scheme 59).

Si O OH O CF3 O P O O CF3 O O O P O i O CF3 O O OO O CF3

204 205

Scheme 59. Reagents and conditions: i. 204, 9-BBN, THF, rt, 2h; then 176, 2 M K3PO4, [Pd(OAc)2 + 4 TFP], DME, reflux, 5.5h, 0 %. Unfortunately, no product was obtained which was probably due to the basic conditions required for the second part of the Suzuki coupling, as the hydroboration step had worked perfectly. Regarding the results we obtained for the coupling attempt, we assume that the phosphonate may have been saponified under the basic conditions. To overcome this problem, we decided to change the order in which the building blocks were to be assembled.

66 Results and Discussions

3.1.4.4. Coupling of the C1’-C6’ Fragment to the Resorcylic Moiety As the phosphonate turned out be too labile for the basic conditions required for the Suzuki coupling, we decided to couple fragment C1’-C6’ 190 to the aromatic bromide 176 (Scheme 60). The intermediate 206 was then to be further transformed into the phosphonate 205. However, the Suzuki coupling only gave low yields (33 %).

Si OH O OH i O OO O OH O O 190 206

Scheme 60. Reagents and conditions: i. 190, 9-BBN, THF, rt, 2h; then 176, 2 M K3PO4, [Pd(OAc)2 + 4 TFP], DME, reflux, 6h, 33 %. Again, the volatility of the C1’-C6’ fragment prohibited its drying under high vacuum. For this reason, only low conversion in the hydroboration step was observed, which is responsible for the low yield of the overall coupling reaction. Furthermore, the trifurylphosphine could hardly be separated from the product 206. As we had always observed low yields for all Suzuki coupling attempts with substrates exhibiting an unprotected hydroxyl group, we decided to protect the hydroxyl group of intermediate. After a careful analysis of all possible protecting groups for the free alcohol function that would be orthogonal with the TMS-ethyl ester, intermediate 190 was protected as PMB ether. As this protecting group is stable under basic conditions and is cleavable without the use of a fluoride source, it should be stable under Suzuki coupling conditions and be cleavable after the coupling without affecting the TMS- ethyl ester group.

Si OH O

OH i OPMB ii O iii

OO OO O OPMB O O

190 207 208

Si Si 210: R=H OH O OH O vi iv O v O O 211: R=OH vii O OH O R 212: R=Cl O O viii O O 213: R=CH2PO(OCH2CF3)2 209

Scheme 61. Reagents and conditions: i. pCH3OC6H4CH2OC(CCl3)=NH, PPTS , cyclohexane, 0°C → rt, 18h, 53 %; ii. 207, 9-BBN, THF, rt, 2h; then 176, 2 M K3PO4, [Pd(OAc)2 + 4 TFP], DME, reflux, 4h, 89 %. iii: DDQ, CH2Cl2/H2O (19:1), 6.5h, -5°C → 0°C, 69 %. iv. (COCl)2, DMSO, CH2Cl2, -78°C, 1h; then TEA, -78°C → 0°C, 67 %. v. 2-methyl-2-butene, t-BuOH, NaClO2, NaH2PO4, H2O, rt, 1h, quant. vi. Me2C=C(NMe2)Cl, CH2Cl2, rt, 1h, quant. vii. MeP(O)(OCH2CF3)2, LiHMDS, THF, -98°C, 1h, 34 % over 3 steps. 67 Results and Discussions

Alcohol 190 was finally PMB protected with freshly synthesized p-methoxybenzyl trichloroacetimidate, after several attempts using p-methoxybenzyl bromide and sodium hydride had failed (Scheme 61). The Suzuki coupling of intermediate 207 with bromide 176 gave 208 in excellent yield (89 %).

The subsequent removal of the PMB group with DDQ in a mixture of CH2Cl2 and water at room temperature168, but ca. 10 % of C5’-racemized product was observed under these conditions. The racemization could be efficiently suppressed at lower reaction temperature which is shown in Table 3. When DDQ in a CH2Cl2/buffer mixture (pH 7.5) was used, racemization was completely prevented.169 The oxidation of PMB ether 208 with CAN170 did not lead to any of the desired alcohol; rather, the starting material decomposed within several minutes.

Table 3. Reaction conditions for the PMB-deprotection of 208. temperature / reagent solvent yield racemization reaction time

DDQ CH2Cl2 / H2O 19:1 rt / 2h 79 % 10 %

DDQ CH2Cl2 / H2O 19:1 -5°C → 0°C / 6.5h 69 % only traces CH Cl / Na HPO DDQ 2 2 2 3 0°C → rt / 2h 58 % - buffer 19:1 (pH 7.5)

CAN CH3CN/H2O 4:1 0°C / 10min 0 % -

The deprotected intermediate 209 was oxidized to aldehyde 210. Interestingly, the oxidation with Dess-Martin periodinane (DMP) only led only to a 23 % yield. Using polymer-bound IBX the yield of the oxidation could be improved to 46 %, but the reaction time was exceedingly too long (72 h). Ultimately, the oxidation was carried out under Swern conditions and the aldehyde 210 could be obtained in 67 % yield (Scheme 61). 210 was oxidized to the corresponding acid 211 via Pinnick oxidation, followed by conversion to the acid chloride 212 and finally reaction of 212 with bis- (2,2,2-trifluoroethyl) methylphosphonate to give 213 in 34 % yield for the three step sequence from aldehyde 210. The final three-step conversion into the phosphonate still has to be improved, but in principle the route is feasible. For reasons of time this sub-project could not be finished within this thesis.

3.2. Synthesis of Dideoxy Analog D6 The dideoxy analogue of L-783277 (6) was chosen to be synthesized for two reasons. On one hand this would allow to use the synthetic strategy developed for the natural product 6 and thus to test its potential for the synthesis of analogues. On the other hand this analogue would provide valuable information about the importance of the vicinal diol moiety for biological activity. This project was also part of Luca Fransioli’s master thesis.

68 Results and Discussions

As the retrosynthetic analysis of the dideoxy analogue D6 (Scheme 39) is based on the previously developed route to the natural product 6, the aromatic building block 176 (see Scheme 42 and 50) and the C7’-C11’ 156R (see Scheme 43) were synthesized as discussed above. In contrast, the synthesis for the C1’-C6’ fragment had to be newly developed as the C4’ and C5’ hydroxyl groups were now omitted. The synthesis of D6 started with commercially available 5-hexen-1-ol 150 which was oxidized to its aldehyde 214 using Swern conditions (Scheme 62). Although the Swern reaction worked in quantitative yield, 214 had to be handled carefully as it decomposes slowly at room temperature and is highly volatile.

OTES OTES

i ii iii OH O OTES 150 214 156R OH O O 215 216

Scheme 62. Reagents and conditions: i. (COCl)2, DMSO, Et3N, CH2Cl2, -78°C, 1h, quant. ii. 156R, n- BuLi, THF, -10°C, 20min; then 214, -78°C → -18°C, 1.5h, 51 %; 1:1.1 mixture of diastereomers. iii. MOM-Cl, (i-Pr)2NEt, Bu4NI, DMF, 19h, 87 %.

The addition of lithiated alkyne 156R to aldehyde 214 still gave at least moderate yields. As the C6’-isomers 215 were not separable by FC, the ca. 1:1 mixture was carried on through the synthesis. MOM protection furnished the C1’-C6’ fragment 216 in 44 % yield over three steps from alcohol 150.

OTES OTES

Si Si OH O OH O OTES i ii O O

O O O O O O O O 216 217 218

OH O OH OH O iii iv v OH O

O O O O O O 219 220

OH O OH O vi O O

O OH O O 221 D6

Scheme 63. Reagents and conditions: i. 216, 9-BBN, THF, rt, 2h; then. 176, 2 M K3PO4, [Pd(OAc)2 + 4 TFP], DME, reflux, 1.5h, 83 %. ii. H2, Lindlar catalyst, AcOEt, 3h, 92 %. iii. TBAF, THF, rt, 2h, 90 %. iv. DEAD, Ph3P, toluene/THF, 1h, 64 %. v. Sulfonic acid resin, MeOH, reflux, 3h, 73 %. vi. DMP, CH2Cl2, 4.5h, 72 %. The assembly of building blocks was carried out in the same order and using the same key transformations that had been developed for the natural product 6 69 Results and Discussions

(Scheme 63). Key intermediate 216 was attached to bromide 176 via Suzuki coupling to yield intermediate 217 in excellent yield (83 %), which was followed by Z-specific hydrogenation using the Lindlar catalyst. The seco acid 219 was obtained from alkene 218 through simultaneous cleavage of the fluoride labile TMS ethyl ester and the TBS ether using TBAF as a fluoride source. The macrocycle 220 was obtained via Mitsunobu macrolactonization of the linear precursor 219. It has to be noted that the cyclization was slower than in the case of the natural product, possibly due to a lack of pre-orientation of 219 that might be provided by the presence of the acetonide ring in seco acid 196 (Scheme 57) in the case of the natural product. Cleavage of the MOM ether and subsequent oxidation of the hydroxyl group completed the synthesis of the dideoxy analogue D6, which was obtained in 23 % total yield over 6 linear steps from olefin 216.

3.3. Synthesis of Phenyl Analog P6 The phenyl analog P6 was chosen as a target for synthesis, because it retains the Z geometry of the natural product for the C7’/C8’ bond, but cannot serve as an acceptor for a 1,4-addition of a protein nucleophile.

OH O

O O HO OH P6

Modeling studies at the Novartis Institute for Biomedical Research in Basel (Dr. Pascal Furet, unpublished results) indicate that a hydrophobic pocket exists in the ATP binding site of Mek that could accommodate the bulky phenyl moiety. Thus, this analogue could help to understand, whether the intrinsic affinity of L-783277 (6) for certain kinases could be enhanced to an extent that would allow inhibition without a covalent interaction. In this context it should be remembered that L-783277 (6) has been reported to inhibit LCK with sub-M activity without forming a covalent adduct with the kinase. The synthesis of phenyl analogue P6 was carried out according to two independent approaches, of which the alkyne metathesis-based approach was the subject of Fabienne Zdenka Gaugaz’s and Heike Kirchner’s diploma theses.171,172

3.3.1. Alkyne Metathesis Approach The metal-catalyzed rearrangement of carbon-carbon double bonds is called alkene (olefin) metathesis. In 1955 Anderson & Merckling173 reported a double-bond rearrangement, but olefin metathesis as we know it today was only introduced in 1967 by Calderon and co-workers.174 There are different variants of olefin metathesis reactions, such as ring-opening metathesis polymerization (ROMP), ring-closing metathesis (RCM), acyclic diene metathesis polymerization (ADMET), ring-opening metathesis (ROM), and cross-metathesis (CM or XMET).124 These olefin metathesis reactions grant access to molecules and polymers that would be difficult to obtain by other means. Due to intolerance of the available catalysts to functional groups, the 70 Results and Discussions olefin metathesis could not be used for the synthesis of complex organic molecules at the time of its discovery and for many years to come. In the more recent past, however, alkene metathesis has become a reliable and widely used synthetic method, especially in the context of total synthesis. This development can be ascribed to new catalyst systems exhibiting high activity and excellent functional- group tolerance.158 The three commercially available catalysts which are most routinely used by organic chemists are shown in Fig. 19.

iPr iPr PCy3 N N Mes Mes F 3C N Me Cl Me Ph Ru Cl O Mo Cl Ph Ru F 3C Me Cl Ph O PCy3 PCy3 CF F3C 3 Me 222 223 224 Figure 19. Commonly used alkene metathesis catalysts.158

The Mo-based catalyst 222 was introduced in 1990 by Schrock,175 and represented a groundbreaking advance in catalyst design from the time when the first Wo-carbenes were developed by Katz and co-workers.176,177 Catalyst 222 can be used for a wide variety of alkene substrates and is especially suited for the use with sterically crowded systems.178,179 Unfortunately, this catalyst also has a number of drawbacks, such as a pronounced sensitivity to oxygen, moisture, and also to certain polar or protic functional groups. A more general and practical group metathesis catalysts are Ru-based carbene complexes that were first introduced by Grubbs and co-workers and initially optimized to 223.180 This catalyst is less active than the Schrock Mo- based systems, but it exhibits much greater functional-group tolerance. For this reason, 223 has been applied in the total synthesis of natural and designed products by far more often than 222. The replacement of one of the phosphine ligands with a N-heterocyclic carbene ligand increases the catalytic activity, thermal stability and functional-group tolerance of the complex.181 Thus, 224 is the “second-generation” catalyst which was designed based on its predecessor 223. Although these catalysts are well-defined and afford good yields in a wide range of reactions, the geometrical outcome of the reaction is not predictable or controllable. Often mixtures of Z and E isomers are obtained and the separation of these is usually difficult. For example, in the synthesis of epothilone A the 16-membered ring was formed via RCM and the geometrical outcome became a key issue.182 The metal-catalyzed rearrangement of carbon-carbon triple bonds is called alkyne metathesis. Despite the mechanistic similarities between alkyne and alkene metathesis, the carbene-type catalysts 222, 223 and 224 do not catalyze the corresponding alkyne-metathesis reactions. For the alkyne metathesis other transition-metal-based catalyst systems have been developed. The most commonly used alkyne metathesis catalysts are shown in Fig. 20.

71 Results and Discussions

Me [Mo(CO)6] / ArOH (tBuO)3W Me Me 225 226

Me Me Me Me

tBu tBu Cl Me N N CH2Cl2 Me N N Mo tBu Mo tBu

But N Me But N Me Me Me

Me Me 227 228 Figure 20. Commonly used alkyne metathesis catalysts.158

Mortreux and co-workers achieved the first homogenously catalyzed metathesis reaction of a C-C triple bond in 1974. The initially used catalyst system was formed in 183 situ from [Mo(CO)6]/resorcinol at 100°C. Moreover, mixtures of Mo(CO)6 and a number of phenolic additives (e.g. 4-chlorophenol) have been investigated. These systems generate one or more not catalytically active species in situ that are not well defined. On one hand this catalyst system has a user-friendly nature, but unfortunately it exhibits a limited tolerance of polar functional groups and elevated temperatures (ca. 140-150°C) are required for its formation and catalytic activity.158 In the 1980s the well-defined Schrock Wo carbyne complex 226 was shown to catalyze the metathesis of terminal alkynes accompanied by the formation of gaseous acetylene.184 At the same time, Mori and co-workers were able to achieve cross-metathesis of internal alkynes in the presence of a Mortreux-type catalyst,185 while the conditions for ring-closing alkyne metathesis were developed in Fürstner’s group.186 In 1999 the monochloro-Mo-complex 228 was developed which is formed in situ by the activation of the corresponding trisamido complex 227 with CH2Cl2 as a chlorine source.187 The Wo- and Mo-complexes 226 and 228 complement each in terms of scope, activity, and functional group tolerance. In comparison with the Mortreux- based catalysts 225 they have been proven to be more reliable and robust.158 The mechanism of the alkyne cross metathesis (Fig. 21) was first proposed by Katz and co-workers in 1975.176 In the proposed alkylidyne mechanism metalla- cyclobutadienes are formed from acetylenes and alkylidyne complexes, analogous to metallacyclobutanes that are formed in alkene metathesis from olefins and metal alkylidene complexes. By cycloreversion along the alternative metallocycle bonds the metathesis product is obtained. This mechanism was later experimentally established by Schrock.188

72 Results and Discussions

MR R 2 R 2 R2 2 M M M + +

R1 R1 R1 R 1 R 1 R1 R1 R1

M=Mo(OR)3,W(OR)3 Figure 21. Alkylidyne mechanism of alkyne metathesis.176,182

As one drawback of the RCM is the lack of control of double bond geometry, Fürstner investigated the combination of RCAM followed by Lindlar reduction of the resulting cycloalkynes as a stereoselective route to macrocyclic Z-alkenes.189 This two-step reaction sequence is a tool for the design of synthetic approaches to complex structures featuring a Z-configured double bond as part of a larger ring. According to our initial retrosynthetic approach to phenyl analog P6 (Scheme 40), the required anti-diol was to be obtained through asymmetric dihydroxylation of a suitable cyclic Z-alkene precursor. The stereoselective incorporation of the required Z double bond was envisaged to be achieved via RCAM followed by Lindlar reduction; thus, the suitability of RCAM as a cyclization method in the synthesis of P6 was investigated.

3.3.1.1. Synthesis of Individual Fragments The C1-C6 aromatic building block 151 was synthesized according to Kamisuki et al. who had developed a 3 step synthesis with 47 % overall yield of this derivative as part of their work on dehydroaltenusin.114 Synthesis of the aryltriflate 151 started from commercially available 2,4,6-trihydroxybenzoic acid 229 which was reacted with thionyl chloride (SOCl2) in the presence of N,N-dimethylaminopyridine (DMAP) in acetone to give acetonide 230 in 48 % yield, which was in the same range as described by Kamisuki (56 %). Danishefsky published an alternative (but not a more efficient) route to 230 using trifluoroacetic acid (TFA) and trifluoroacetic anhydride (TFAA) in acetone.102 Regioselective protection of the 4-hydroxy group of 230 was accomplished using Mitsunobu conditions with diisopropyl azodicarboxylate (DIAD) and triphenyl-phosphine (PPh3) in the presence of methanol. The monomethyl ether 231 was obtained in only 48 % yield in contrast to Kamisuki (89 %). After treatment of

231 with triflic anhydride (Tf2O) in pyridine the corresponding triflate 151 was obtained in 99 % yield (27 % overall yield from 229).

OH O O O O O O O

OH i O ii O iii O

HO OH HO OH O OH O OTf 229 230 231 151

Scheme 64.: Reactions and conditions: i. DMAP, acetone, SOCl2, DME, 0°C → rt, 1.5h, 48 %. ii. MeOH, Ph3P, DIAD, THF, 0°C → rt, 5h, 56 %. iii. Tf2O, pyridine, -10°C → 0°C, 3.5h, 99 %.

The synthesis of C1’-C6’ fragment started from commercially available 5-chloropent- 1-yne 148 by protection as its THP-ether under standard conditions (Scheme 65). Alkyne 232 was methylated by lithiation and quenching with methyl iodide. 73 Results and Discussions

Intermediate 233 was deprotected using p-TsOH in MeOH. Problems were encountered in the subsequent conversion of alcohol 234 into bromide 235 by a modified Appel reaction.190 The side-product of this reaction is bromoform which could be removed neither by extraction nor by distillation. It could only be separated by FC, whereby the fractions were checked by 1H-NMR. In spite of these difficulties, bromide 235 was obtained in 63 % yieldover 4 steps from 148.

i ii HO THPO THPO 148 232 233

iii iv HO Br 234 235

Scheme 65. Reactions and conditions: i. DHP, CSA, CH2Cl2, 0°C → rt, 2.5h, 95 %. ii. n-BuLi 1.6 M in hexane, THF, 0°C, 15min; then CH3I, HMPA, -78°C → rt, 2h, 96 %. iii. p-TsOH (cat), MeOH, rt, overnight, 89 %. iv. CBr4, Ph3P, CH2Cl2, 0°C → rt, overnight, 78 %.

The synthesis of the C7’-C11’ fragment is shown in Scheme 66. The addition of commercially available 1-propynyl magnesiumbromide to 2-bromo-benzaldehyde 152 gave a mixture of diastereomers in a ratio of 1:1. Intermediate 236 was TBS- protected following standard procedures.

O H HO TBSO TBSO

Br Br Br OH i ii iii

152 236 237a 238a

Scheme 66. Reactions and conditions: i. H3C-≡-MgBr (0.5 M in THF), THF, 0°C → rt, 2h, quant. ii. TBS-Cl, imidazole, CH2Cl2, rt, 18h, quant. iii. Mg, BrCH2CH2Br, THF, 70°C < 1min, rt, 2h; then CuI, 40S, -30°C → rt, overnight, 13 %.

Surprisingly, problems were encountered again for the conversion of arylbromide 237a into alcohol 238a, although a similar transformation had been published in 1990 by Kaliakoudas et al.191 They reported the coupling of a Normant-cuprate (which was formed from 2,6-dimethoxyphenyl magnesiumbromide and a catalytic amount of CuI) to propylene oxide in 63 % yield. However, in our case the Grignard reagent derived from 237a in situ turned out to be so reactive that 80 % of the product formed was a homodimer of 237a.

TBSO

homodimer of 237a

The reaction was investigated with CuI in catalytic and stoichiometric amounts, as well as with an excess of 237a with regard to (S)-propylene oxide 40S. Results previously obtained in our group on other substates had shown that the formation of 74 Results and Discussions homodimers of the Grignard reagent could be efficiently suppressed by the slow addition of a mixture of EDB and halide to Mg. However, when a mixture of arylbromide 237a and EDB was added very slowly (within 4 h) to Mg, followed by addition of CuI and (S)-propylene oxide (40S), a quantitative amount of homodimer was formed, presumably as a result of an Ullmann reaction.192 In the presence of copper aryl halides (I > Br >> Cl) can be easily coupled to each other. Theses difficulties prompted us to change our original strategy and attempt a direct lithiation approach. It was planned to lithiate the aryl bromide which would open the epoxide in a regioselective manner.193

HO RO RO

Br Br OH i ii

236 237a R = TBS 238a R = TBS 237b R = TIPS 238b R = TIPS 237c R = PMB 238c R = PMB

Scheme 67. Reactions and conditions: i. a) TBS-Cl, imidazole, CH2Cl2, 0°C → rt, 19h, 94 %. b) TIPS- OTf, 2,6-lutidine, CH2Cl2, -78°C → rt, 6h, 98 %. c) C6H5CH2OC(CCl3)=NH, PPTS, cyclohexane, 0°C → rt, 19h, 61 %; ii: a) t-BuLi, Et2O, 15 min; then BF3•OEt2, 40S, -78°C → rt, 4h, 238a 0 %, 238b 15 %; 238c 0 %.

As the stability of the hydroxyl protecting group turned out to be crucial, intermediate 237 was synthesized bearing three different protecting groups (Scheme 67). Unfortunately, all lithiation approaches failed (237a, 237c) or proved to deliver the desired product only in low yields (in the case of 237b, from which 238b was obtained in 15 % yield). As the lithiation approach and the Normant-cuprate approach both gave unsatisfactory results, we also investigated the possible introduction of the C9’-C11’ fragment via Pd-catalyzed cross-coupling.194 The coupling worked only in moderate yields (see Scheme 68), but no attempts have been carried out to optimize the reaction.

RO RO

Br i

237a R = TBS 239a R = TBS 237b R = TIPS 239b R = TIPS 237c R = PMB 239c R =PMB

Scheme 68. Reactions and conditions: i. Bu3SnOMe, isopropenyl acetate, 2'-(diphenylphosphino)- N,N-dimethylbiphenyl-2-amine, Pd2(dba)3, toluene, 100°C; 239a 22h, 21 %; 239b 3.5h, 28 %; 239c 22h, 21 %.

The next step would have been the enantioselective reduction of the ketone using a Corey-Bakshi-Shibata (CBS) protocol. Corey and co-workers could show that catalytic oxazaborolidines (formed from borane (BH3) and chiral amino alcohols) lead to a rapid and enantioselective reduction of ketones.195

75 Results and Discussions

3.3.1.2. Coupling of Fragments The coupling of triflate 151 and aliphatic halide 235 was envisioned to be carried out as an iron-catalyzed cross-coupling according to Fürstner et al.113 This method seemed to perfectly suit for our needs, as it allows the coupling of triflates and aliphatic Grignard reagents under mild conditions. The two step-sequence of the iron- mediated cross-coupling to produce intermediate 240, followed by deprotection to the carboxylic acid 241 is depicted in Scheme 69. Unfortunately, the coupling of triflate 151 and aliphatic halide 235 was not as straightdorward as we had hoped for. Several experiments were carried out, in order to identify, if the problem was associated with the triflate or with the aliphatic halide (see also Table 4).

O O O O OH OH iii O O O

O OTf O O 151 240 241

Scheme 69. Reactions and conditions: i. 235, Mg, EDB, THF, rt, 3h; then 151, Fe(acac)3, THF, NMP, 0°C, 1h, 54 %; ii: 2 M KOH/EtOH 1:1, 65°C, overnight, 85 %.

As shown in Table 4 the coupling of triflate 151 to commercially available hexylmagnesium bromide 242 gave the coupling product only in moderate yield (37 %). Furthermore, the coupling of 151 and Grignard reagents derived from the aliphatic bromide 235 and chloride 235a were investigated. The aliphatic chloride 235a was synthesized by methylation of lithiated 5-chloropent-1-yne in 82 % yield. In the case of 235a the formation of the Grignard species was not successful and therefore, no coupling product was obtained. In general, coupling of 151 with Grignard reagents derived from bromide 235 worked, but the in situ formed Grignard reagent was so active that mostly homo-coupling was the main reaction path. Slow addition (4 h) of a dilute mixture of 235 and EDB in THF to Mg powder in THF improved the yield of the coupling from 28 % to 36 %.At the same time the slow addition also caused other problems. Thus, after 3 hours, the Grignard reagent derived from 235 precipitated. It was found that this precipitate could be re-dissolved by further addition of THF. This modification led to a 54 % yield for the coupling reaction. Unfortunately, this reaction was not always reproducible. In addition, the iron-mediated coupling was carried out with 2,6-dichloropyridine 243 and hexylmagnesium bromide 242, as this coupling has been described by Leitner in his PhD thesis.196 Interestingly, the test coupling of these commercially available substrates worked in 77 % yield, thus confirming the results reported by Leitner. For this reason, we speculated that the reactivity of triflate 151 and the Grignard reagent derived from 235 were unbalanced, because the triflate 151 turned out to be not reactive enough and the in situ formed Grignard reagent was prone to homo- coupling, due to its high reactivity.

76 Results and Discussions

Table 4: Iron-catalyzed coupling attempts. starting material C1’-C6’ fragment yield

O O C6H13MgBr 37 % O 242 O OTf 151

151 Cl 0 % 235a

151 Br 54 % 235

C6H13MgBr 77 % Cl N Cl 242 243

The reproducibility problems with the iron-mediated coupling led us to abandon this approach and to investigate an alternative strategy for the synthesis of 241 that would involve the Pd-mediated coupling of triflate 151 with a suitable C1’-C6’ fragment.

3.3.1.3. Variation of Strategy In the alternative approach the C1-C6’ fragment was envisioned to be synthesized as shown in Scheme 70. To enhance the yield for the assembly of fragments, we decided to use a high yielding Sonogashira coupling197 for the attachment of the C1’- C6’ segment to the resorcylic acid moiety. In a second step the methylated alkyne moiety, that was required for the planned construction of the macrocycle via alkyne metathesis, would then be introduced in a subsequent step after the Sonogashira coupling. Commercially available but-3-yn-1-ol was coupled to triflate 151 using a Sonogashira cross-coupling protocol. The coupling product 244 was obtained in excellent yield (80 %) and was completely hydrogenated over Pd on charcoal (Pd/C) in MeOH. Primary alcohol 245 was oxidized to aldehyde 246 under Swern conditions.198 It was planned to perform a one-carbon homologation of aldehyde 246 to its corresponding terminal alkyne using carbon tetrabromide (Corey-Fuchs alkyne synthesis).199 The first step of the homologation gave dibromo olefin 247 in 76 % yield. Thus, 247 could be efficiently synthesized from triflate 151 in 48 % yield over four steps. The second step of the Corey-Fuchs homologation normally involves treatment of a bromoolefin with two equivalents of n-BuLi which leads to Li-halogen exchange and elimination, followed by simple hydrolysis.

77 Results and Discussions

O O O O O O

O i O iiO iii

MeO OTf MeO MeO OH 151 244OH 245

O O O O O O

O iv O v O Br MeO O MeO MeO 246 247Br 240

Scheme 70. Reactions and conditions: i. PdCl2(PPh3)2, CuI, Et3N, but-3-yn-1-ol, DMF, 70°C, 5h, 80 %. ii. Pd/C, MeOH, rt, 19h, 85 %. iii. (CO)2Cl2, DMSO, CH2Cl2, -75°C, 1h; then Et3N, -10°C, 93 %. iv. CBr4, PPh3, CH2Cl2, 0°C, 1h, 76 %. v: lithium-halogen exchange, then quenching with MeI.

As indicated by the dotted arrow in Scheme 70 the second step of the Corey-Fuchs homologation turned out to be problematic. Table 5 gives an overview on the different conditions that were investigated. Although the Li-halogen exchange could be tracked by TLC, no product was obtained after direct quenching of the Li-acetylide with methyl iodide. Inspection of the NMR spectra of the main side product indicated that the acetonide protecting group had been attacked. When the more sterically hindered base LDA was used in the conversion of 247 to the corresponding alkyne, the latter could be obtained in 76 % yield (entry 4, Table 5).

Table 5. Reaction conditions for the second step of the Corey-Fuchs homologation.

entry conditions yield

1 n-BuLi (2 eq), MeI (1.5 eq), THF, -78°C → rt, 18h 0 % 240

2 n-BuLi (2 eq), MeI (1.5 eq), THF, -78°C → 0 C, 1.5h 0 % 240

3 n-BuLi (2 eq), MeI (1.5 eq), THF, -78°C, 1h 0 % 240

4 LDA (3 eq), THF, -78°C, 2.5h 76 % 249

However, even though LDA was always freshly prepared from n-BuLi and diisopropylamine according to a literature procedure,200 this approach seemed not to be reproducible. At the same time we tested the Ohira-Bestmann modification of the Seyferth-Gilbert homologation, which entails a one-pot conversion of aldehydes to the corresponding terminal alkynes using -diazaphosphonates under basic conditions.201 As shown in Scheme 71, aldehyde 246 could be reacted with dimethyl

1-diazo-2-oxopropyl-phosphonate 248 and K2CO3 to give terminal alkyne 249 in 57 % yield. Methylation of the latter was achieved by deprotonation with LDA and subsequent quenching with methyl iodide. It has to be noted that the temperature has to be controlled carefully in this reaction, in order to prevent opening of the acetonide. Finally, the C1-C6’ fragment 241 was obtained in an efficient and reproducible manner by cleavage of the acetonide under standard conditions.

78 Results and Discussions

OO O O O O P O i O + O O N+ O O N- O 246 248 249

O O OH OH ii iii O O

O O 240 241

Scheme 71. Reactions and conditions: i. 248, K2CO3, MeOH, 0°C (1.5h) → rt (30min), 57 %; ii: LDA, MeI in HMPA, THF, -78°C → rt, 19h, 86 %. iii. 2 M KOH/EtOH (1:1), reflux, 14h, 85 %

Coupling of the C1-C6’ fragment 241 and the C7’-C11’ fragment 237a was carried out under Steglich esterification conditions. DCC or EDC (a water-soluble DCC analog202) in the presence of DMAP both gave ca. 30 % yield of the desired ester 250 (Scheme 72). Esterifications in the syntheses of RAL’s are mostly carried out under Mitsunobu conditions and it may be speculated that the free phenolic group of the aromatic C1-C6 fragment hampers the esterification of the adjacent carboxylate. It is conceivable that the benzoic acid is not easily converted to the appropriate actived ester, due to the presence of a resonance-stabilized hydrogen bond between the phenolic hydroxyl group and the carboxylic acid moiety. However, usage of a Mitsunobu-based esterification would have required the opposite configuration at C- 10’.

OH O OH O HO i O OH O OTBS O OTBS

241 237a 250

Scheme 72. Reactions and conditions: i. DCC, DMAP, CH2Cl2, 0°C → rt, overnight, 32 %.

After the protection of the phenolic OH-group of intermediate 250 the ring-closing alkyne metathesis could have been investigated. The following steps such as cis- selective reduction of the triple bond, asymmetric dihydroxylation, global deprotection and subsequent benzylic oxidation the C6’-hydroxyl group should have led to the target molecule P6. However, due to a lack of time these last steps could not be accomplished within the diploma theses of Fabienne Zdenka Gaugaz and Heike Kirchner. In addition, the unsatisfactory stereochemical outcome of the Sharpless-dihydroxylation in the first generation approach to 6 (Scheme 44), which became known only when work on P6 was already in progress, raised serious questions about the feasibility of this

79 Results and Discussions transformation in the context of a macrocyclic precursor. We therefore turned our attention to the macrolactonization-based approach described below.

3.3.2. Macrolactonization Approach As depicted in the retrosynthetic analysis shown in Scheme 41, the aromatic fragment 176 (Scheme 50) and the C1’-C6’ derived fragment 190 (Scheme 53) had already been synthesized as part of the total synthesis of L-783277 (6). What remained to be accomplished was the development of a suitable C7’-C11’ fragment (in analogy to P6-MV, Scheme 41) and the assembly of fragments to produce P6.

3.3.2.1. Synthesis of the C1’-C11’ Fragment The synthesis of the C7’-C11’ fragment P6-MV started with lithiation of 1,2- diiodobenzene (153) and subsequent opening of (R)-propylene oxide 40R (Scheme 73).203 The lithiation of 153 turned out to be a critical step that was prone to side reactions. Lowering the temperature from -78°C to -90°C, however, and the use of toluene as the solvent (instead of THF) improved the yield from 38 % to 79 %. Protection of the alcohol 251 with TBS-Cl provided intermediate 252 in excellent yield (Scheme 73). It should be noted that 251 was successfully protected with TBS-OTf on small scale, but that protection on larger scale led to epimerization.

TBSO TBSO

i ii iii iv I OH OTBS OH O I I I O O O O 153 251 252 253 254

Scheme 73. Reactions and conditions: i. n-BuLi, toluene, -90°C, 1h; then 40R, BF3•OEt2, -90°C, 90min, 79 %. ii. TBS-Cl, DIEA, DMF, 14h, 92 %. iii. n-BuLi, THF, -78°C, 2h; then 190, THF, -78°C, 2h, 58 %. iv. DMP, CH2Cl2, rt, 5 h, 77 %.

Iodide 252 was lithiated and aldehyde 190 (Scheme 53) was added dropwise to the reaction mixture; producing a mixture of diastereomers 253 in 58 % yield (Scheme 73). Initially it was planned to oxidize the C-6’ hydroxyl function at this stage of the synthesis. Interestingly, the two isomers were not converted to the desired ketone 254 with equal efficiency, which reflects the observations we had made in the course of the synthesis of L-783277 (6) for the allylic oxidation of intermediate 196 (Scheme 54). It may be speculated that the difference in reactivity is due to steric hindrance, with the acetonide shielding one side of the molecule more than the other side. After several attempts at the oxidation of 253, using MnO2, TPAP, IBX, or DMP-solution

(ca. 15 % in CH2Cl2), it was found that the direct use of the 15 % DMP solution as the solvent (without further dilution) gave rise to ketone 254 in satisfactory yields.

3.3.2.2. Assembly of Fragments With the required fragments in hand, the resorcylic fragment 176 was coupled to the C1’-C11’ fragment 254 in high yield using our proven Suzuki coupling conditions (Scheme 74). After the simultaneous cleavage of the TMS ethyl ester and the TBS 80 Results and Discussions ether an undesired hemiketal formation occurred. Unfortunately, the hemiketal state appears to be strongly favored for 256, which effectively prevented the macrolactonization of this intermediate.

OTBS OH O OTBS

i OTMSE ii

O O O O O O O

254 255

OH O OH OH O OH OH

O O O O O O OH O O

256

Scheme 74. Reagents and conditions: i. 9-BBN, THF, rt, 2h; then 176, 2 M K3PO4, [Pd(OAc)2 + 4 TFP], DME, reflux, 2.5h, 75 %. ii. TBAF, THF, 14h, 81 %.

To avoid hemiketal formation, we first focused on protecting the keto group of intermediate 255. However, the ketone was not reactive enough; at the same time an elimination of the TBS group occurred. For this reason, we went two steps back in the sequence and decided to assemble the C1’-C11’ fragment with an alcohol oxidation state at the C6’ position. As depicted in Scheme 75 the C6’-hydroxy group of 253 was protected as triisopropyl (TIPS) ether, because there was hope that the TBS ether may be cleavable in the presence of the TIPS ether, which should have a slightly higher stability.204 The protection of 253 was slow, but the product could be obtained in high yield (81 %). The Suzuki coupling of 257 with 176 afforded the protected linear precursor 258 in 70 % yield. Unfortunately, it was not possible to cleave the TBS ether without simultaneous cleavage of the TIPS group. TBAF was again needed as the fluoride source for the cleavage of the TMS ethyl ester and under these conditions the two silyl protecting groups could not be differentiated. We did, however, investigate the macrolactonization of diol 259 and the main product could indeed be assigned as the desired 14-membered macrocycle, although some 10-membered ring formation was also observed. Unfortunately, the macrolactonization was not reproducible. For this reason this strategy was not further pursued.

81 Results and Discussions

TBSO TBSO

iii

OH OTIPS O O O O 253 257

OH O OTBS OH O OH

OTMSE iii OH

O OTIPS O OH O O O O 258 259

Scheme 75. Reagents and conditions: i. TIPS-OTf, 2,6-lutidine, CH2Cl2, -78°C., 2h; then rt, 10h, 81 %. ii. 9-BBN, THF, rt, 2h; then. 176, 2 M K3PO4, [Pd(OAc)2 + 4 TFP], DME, reflux, 7h, 70 %. iii. TBAF, THF, 4h, 88 %.

Only a limited choice of suitable protecting groups was available for the C-6’ position, as the protecting group had to be stable under the basic conditions of the Pd- mediated Suzuki coupling. The PMB group seemed to be an alternative, but it could not be introduced in 253. Using p-methoxybenzyl trichloroacetimidate and PPTS only’ fragments of 253 were observed. In contrast, when PMB-Cl and NaH were used in order to protect the C6’-hydroxyl group, no conversion to the desired PMB-ether was observed. In conclusion, the easiest modification seemed to be the change of the protecting group of the carboxyl group. Although methyl ester protection had caused extensive problems in the first generation approach of the total synthesis of L-783277 (6), we decided to change back from the fluoride labile TMS ethyl ester to the corresponding methyl ester. This decision was based on the assumption that the problems we had encountered in the synthesis of 6 had been primarily related to the lability of the ynone part of intermediate 174 (Scheme 48). Thus, it was interesting to see if the methyl ester would be cleavable in the absence of this structural element. This would provide information about the impact of the ynone moiety on chemical stability of 173 and 174.

3.3.2.3. Change of Protecting Groups As illustrated in Scheme 76 in addition to the (re)incoporation of the methyl ester group, the other protecting groups also had to be changed in order to maintain orthogonality. Accordingly, the protected linear macrolactonization precursor 263 features a TES protecting group in the C10’-position and a TBS group in the C6’- position. The use of, e. g., HF•pyridine is a non-basic fluoride source or slightly acidic conditions were expected to allow the selective cleavage of the TES group and provide the desired seco acid for the macrolactonization. The synthesis started again from commercially available 1,2-diiodobenzene 153, which was carefully lithiated at low temperature and reacted with (R)-propylene oxide 40R. Alcohol 251 was TES-protected under carefully optimized conditions. It has to 82 Results and Discussions be noted that at slightly higher reaction temperature than -78°C epimerization at the C5’ position was observed. The use of TES-OTf instead of TES-Cl led to decomposition in the protection step. In the next step 260 was again carefully lithiated and aldehyde 191 (Scheme 53) was added slowly to the reaction mixture. As expected, two isomers were obtained in a ratio of 2:1. Subsequently, the alcohol was protected as its TBS ether. In the following, only those fractions obtained in the FC purification of 262 that contained mostly the major isomer were carried through the remainder of the synthesis (which simplified the interpretation of NMR spectra).

TESO

i ii iii iv I OH OTES OH O I I I O 153 251 260 261

TESO OH O OTES OH O OH

v O vi O

OTBS O OTBSO OTBS O O O O O O 262 263 264

OH O OH OH O

vii OH viii O ix

O OTBS O OTBS O O O O 265 266

OH O OH O OH O

O x O xi O

O OH O OH O O HO HO O OH OH 267O 268 P6

Scheme 76. Reagents and conditions: i. n-BuLi, toluene, -85°C, 1h; then 40R, -85°C, 2h, 75 %. ii. TES-Cl, DIPEA, DMF, 15h, 98 %. iii. n-BuLi, THF, -85°C, 2h; then 191, THF, -80°C, 12h, 72 % in a 2:1 ratio of isomers. iv. TBS-OTf, DIPEA, CH2Cl2, -78°C, 12h, 94 %. v. 9-BBN, THF, rt, 1.5h; then 29a, 2 M K3PO4, [Pd(OAc)2 + 4 TFP], DME, reflux, 6h, 76 %. vi. THF/AcOH/H2O 2:2:1, 1.5h, rt, 81 %. vii. 2 N NaOH (13eq)/MeOH (1:3), reflux, 8h, 53 %. viii. DIAD, Ph3P, toluene, rt, 25min, 35 %. ix. TBAF, THF, rt, 14h, 97 %. x. Sulfonic acid resin, MeOH, reflux, 1h, 82 %. xi. DMP, CH2Cl2, rt, 20min, 62 % crude.

Unfortunately, intermediate 262 showed a distinct tendency to epimerize when the reaction temperature exceeded -78°C. For this reason, only a small excess of TBS- triflate could be used. Interestingly, TBS-Cl was not reactive enough to protect the C6’-alcohol.

83 Results and Discussions

Alkene 262 was coupled to the resorcylic acid moiety 29a (see Scheme 42) under optimized Suzuki coupling conditions, which furnished the fully-protected linear precursor 263 in excellent yield. The TES ether could be easily cleaved under acidic conditions using aqueous acetic acid in THF. Unfortunately, almost 30 % of a byproduct was formed, of which the TBS-group had migrated to the OH-group at position C10’. Methyl ester cleavage in 264 was carried out under various conditions. LiOH in THF gave no cleavage at all, while no selectivity between ester cleavage and TBS- deprotection was obtained with TMSOK in a microwave reactor. Finally, it was found that ester cleavage is favored with NaOH in MeOH under reflux conditions. The yield of the Mitsunobu macrolactonization was lower than expected, although the same conditions and reagents were used as for the synthesis of the natural product L-783277 (6). The synthesis was continued with the cleavage of the TBS ether of intermediate 266 using TBAF in THF. Part of 267 was submitted to a DMP oxidation. After five minutes the transformation still appeared feasible, but after one hour TLC showed complete decomposition. In addition, the reaction mixture had changed its color from colorless to a cherry-like red, which might point to the formation of a chinone-like structure. It seems that the steric demand of the acetonide prevents the oxidation at position C6’ and instead side reactions occur which lead to decomposition of the molecule. To decrease steric crowding the acetonide in 267 was cleaved using the optimized conditions that had been developed in the synthesis of L-783277 (6). The crude intermediate 268 was submitted to oxidation with IBX (polymer supported), but after one hour no reaction had occurred. As the polymer-bound IBX seemed to be not reactive enough, it was removed by filtration. The starting material 268 was resubmitted to a DMP (non-polymer bound) oxidation. Benzylic alcohol 268 was converted in a spot-to-spot manner to a single product, which showed the expected mass for P6. The crude material was purified by FC and stored in the freezer. Unfortunately, the material decomposed upon storage.

3.4. Stability Measurements

3.4.1. Chemical Stability As the cis-configured double bond of L-783277 has been reported to isomerize under basic as well as acidic conditions,79,80 its stability was tested at different pH-values (1, 3 and 5) (Fig. 22). L-783277 (6) was dissolved in DMSO and samples were incubated with different hydrochloric acid (HCl) solutions. After distinct time points (0.25 h, 1 h, 3.5 h, 6 h, 24 h) the mixtures were analyzed by HPLC to follow the isomerization process (and possibly other degradative changes).

84 Results and Discussions

100.00

90.00

pH 1 80.00 pH 3 pH 5

70.00

% of L-783277 remaining 60.00

50.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 t [h]

Figure 22. pH-stability of L-783277 (6).

As depicted in Fig. 22 L-783277 (6) shows a moderate rate of conversion to other products at pH 3 and 5, while the pH-stability of 6 drops rapidly at pH 1. At pH 3 and pH 5 the sole product formed from 6 is the corresponding E-isomer, as determined by mass spectrometry and HPLC-coinjection with an authentic sample of E-6. In contrast, additional unidentified minor degradation products were observed at pH 1, apart from the isomerization of the 7’/8’ double bond.

3.4.2. Blood Plasma Stability From a structural and a biochemical point of view the natural product L-783277 (6) represents a potent new lead structure for kinase inhibition. However, for the development of a drug the ADMET (administration, distribution, metabolism, excretion and toxicity) profile of a compound is equally important. The metabolic stability of L-783277 (6) was tested in human plasma. 6 was incubated with human serum at 37°C and the metabolic stability was monitored by HPLC over a period of 6.5 h. Fig. 23 shows the time-dependant changes in the relative concentration of 6. After 6.5 h incubation time only 50 % of the parent compound was detected. In analogy to the chemical stability studies, only one major metabolite was found, which could be identified as the E-isomer of the natural product 6 by mass spectrometry and HPLC- coinjection. In a control sample, the recovery rate of L-783277 (6) at time point 0 was found to be ≥ 99 %.

85 Results and Discussions

100.00

90.00

80.00 L-783277 70.00 E-isomer -6)

E 60.00

50.00

40.00

% of total (6 + 30.00

20.00

10.00

0.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 t [h]

Figure 23. Blood plasma stability of L-783277 (6).

L-783277 (6) shows moderate stability in human plasma. However, the only metabolite that could be identified was the E-isomer of 6. Quite notably, the ester moiety of 6 is not susceptible to hydrolysis to any significant extent. While E-6 is a significantly less potent kinase inhibitor than 6, it should be noted that it is not 25 inactive, e. g., E-6 has been reported to inhibit Mek1 with an IC50 of 300 nM.

3.5. Biological evaluation

3.5.1. In Vitro Kinase Inhibition The natural product L-783277 (6), the dideoxy analogue D6 and the two precursors for the selective allylic oxidation 198a and 198b were tested against a panel of 34 kinases (collaboration with Dr. Doriano Fabbro at the Novartis Institute for Biomedical Research, in Basel). With the exception of Erk2, MK5 and Mek2 the in vitro kinase inhibition assays were performed with recombinant glutathione S-transferase-fused kinase domains, employing [-33P]ATP as a phosphate donor and polyGluTyr-(4:1) peptide as acceptor.205 For Erk2 (purchased from Proquinase) the Erktide KRELVEPLTPSGEAPNQALLR was used as the peptide substrate. For Mek2 (1-393 with N-terminal GST; produced at Novartis) Erk kinase dead from Proquinase was the substrate (Dr. Doriano Fabbro, personal communication). Within the panel of 34 kinases investigated, only 6 were inhibited by L-783277 (6) at concentrations < 10 M; inhibition of 1 kinase was observed for the dideoxy analog

D6, while 198a and 198b showed no measurable inhibitory activity. The relevant IC50 values are summarized in Table 6.

86 Results and Discussions

Table 6. Biological activity of L-783277 (6), D6, 198a, 198b against various kinases. In vitro kinase inhibition assays were performed with the recombinant glutathione S-transferase-fused kinase domains. The

relevant inhibition values are listed as IC50 values in M. Kinases L-783277 (6) D6 198a 198b Erk2 1.8 > 10 > 10 > 10 KDR (VEGFR-2) 0.008 0.5 > 10 > 10 MK5 0.64 > 10 > 10 > 10 PDGFR 0.087 > 10 > 10 > 10 Tyk2 6.8 > 10 > 10 > 10 c-Kit 6.0 > 10 > 10 > 10

L-783277 (6) effectively inhibits VEGFR-2, PDGFR and MK5, with IC50 values of 8 nM, 87 nM and 640 nM, respectively. These kinases are known to be involved in cancer-relevant and inflammatory signaling pathways of cells and their ATP binding sites all contain a Cys residue corresponding to Cys166 in Erk2. Moreover, 6 inhibited Erk2, Tyk2 and c-Kit with low M IC50 values. L-783277 (6) was also tested against Mek2 at Upstate (compound submitted by Dr. Doriano Fabbro) and found to exhibit an IC50 value of 15 nM, thus confirming the original observation by Zhao et al.25 Lastly, the compound was also tested against Flt-3 (ITD) (collaboration with Dr.

Jens Pohlmann at Basilea Pharmaceuica, Basel), which was inhibited with an IC50 of 23 nM. Again, Flt-3 is one of those kinases whose ATP binding site contains a Cys residue corresponding to Erk Cys166.

While a direct comparison of IC50 values generated in different laboratories is difficult, especially in the case of a covalent inhibitor such as L-783277 (6), it is clear that 6 shows a highly selective kinase inhibition profile, with inhibition of a number of disease-relevant kinases. These data clearly warrant the further investigation of this lead compound in future SAR studies Interestingly, D6 was also found to inhibit KDR/VEGFR-2, but with ca. 60-fold lower potency than 6. A similar decrease in potency was observed against Mek2 (IC50 = 6.8 M, Upstate).These observations are in line with recent findings reported for hypothemycin (5), where methylation of the hydroxyl group on C5’ does not significantly affect antiproliferative activity (compared to hypothemycin), while monomethylation of the C4’ hydroxyl group or dimethylation lead to a profound loss in potency.83 This suggests that perhaps only one OH group is required for full kinase inhibitory activity and this question should be investigated in future studies.

3.5.2. Cellular Activity

3.5.2.1. Inhibition of Cellular Proliferation L-783277 (6) was tested in proliferation experiments, which were carried out by

Benjamin Vigl (group of Prof. Detmar, ETH Zürich). IC50 values were determined for the inhibition of proliferation of primary lymphatic endothelial cells, either in the presence or absence of vascular endothelial growth factor (VEGF-A). In these experiments it could be shown that the proliferation of cells could be effectively

87 Results and Discussions inhibited by L-783277 (6). However, cell proliferation of cells was inhibited in the presence as well as the absence of VEGF-A; thus, the origin of the antiproliferative activity cannot solely be related to the inhibition of the kinase activity of VEGF-R, although this may be an important component of the overall cellular effect. The graph shown in Fig. 24 is a representative example of various experiments that were carried out. The cell number is shown on the y-axis and the inhibitor concentration on the x-axis.

IC50 on LECs 070507s mean  SD of triplicates

18000

16000 ICIC5050 = =9,023 9.02 nMnM RR2=0,932 = 0.93 14000

prolif. counts 12000

10000

0 1 2 3 4 -2 -1 log10 nM PFK277-397 pos ctl pos neg ctl log 10 nM L-783277

Figure 24: Exemplary graph for proliferation assays.

Table 7 gives an overview of cellular data we have obtained in cell proliferation experiments on HL-60 and PHLEC cells (in collaboration with Benjamin Vigl and Dr. Jürg Gertsch). The proliferation of cells could be efficiently suppressed by 6. The dideoxy analog D6 also inhibited the proliferation of HL-60 cells, but only to a moderate extent. Table 7. Inhibition of cell proliferation by L-783277 (6) and D6 on HL-60 and PHLEC cells. The inhibition values are listed as IC50 values in M.

L-783277 (6) D6

HL-60 (72h) 0.745 8.9

PHLEC (-VEGF) (72h)* 0.088 N. D.

PHLEC (+VEGF) (72h) * 0.035 N. D.

3.5.2.2. Tube Formation Studies The capillary-like tube formation assay of cells is a commonly used method for the assessment of angiogenesis in vitro.206,207 The inhibition of tube formation by L- 783277 (6) (i. e. its anti-angiogenic activity) was investigated in human umbilical vein endothelial cells (HUVEC) in the laboratory of Prof. James Lorens at the University of Bergen, Norway (experiments performed by Dr. Lasse Evensen).

88 Results and Discussions

L-783277 (6) was added at time point of seeding (day 0) or day 5 with a concentration of 100 nM. The bars in the charts are averages of the three replicates. Every plate has their DMSO control and was compared to that. L-783277 (6) inhibited the formation of tubes, when the cells were incubated with the compound on day 0 (Fig. 25). In contrast, 6 had no noteworthy effect on established capillary-like networks, which was tested by incubating the cells with the compound on day 5.

L-783277 (6) 100 nM

day 0 addition

day 5 addition

Figure 25: Pictures of the capillary tube forming assay using HUVECs. Cells are stimulated with VEGF on day 0 and were incubated with 6 (100 nM) on day 0 and day 5. The pictures were taken 72 h after incubation of cells with 6.

The quantitative analysis of the data depicted in Fig. 25 involves the determination of the total tube length in a predefined section and the results of this analysis are shown in Fig. 26. When incubated with the cells on day 0, L-783277 (6) inhibits VEGF- induced tube formation.

DMSO 25 % L-783277 100 nM Figure 26: Computed quantification of the capillary tube formation assay.

In contrast, the effects observed after incubation on day 5 (when a cellular network has already been established and further network growth is not anymore dependent on VEGF) the effects are clearly less pronounced. Thus, in agreement with its 89 Results and Discussions

VEGFR-inihbitory properties 6 is able to inihibit VEGF-dependent processes with better efficiency than VEGF-independent events.

3.5.3. In Vivo Evaluation L-783277 (6) was also investigated in a mouse model of chronic inflammation with a psoriasis-like phenotype.208,209 The corresponding mouse strain K14-VEGF-A expresses VEGF-A under the control of the keratinocyte-specific promoter K14. Once these mice develop an inflammatory response, they are unable to down-regulate it and, therefore, develop a chronic inflammatory condition which is manifest as ear swelling and can be assessed by measuring ear thickness. Treatment with 6 was topical and involved the application of a 1 mg/ml solution of 6 in a 1:1 mixture of EtOH and acetone to the swollen ear twice daily for 16 days. No difference in ear swelling could be detected on day 16 between animals treated with 6 and those of an untreated control group. Since the chronic inflammation driven by the above transgene might be stronger than would be the case in a chronic or acute setting, 6 was also tested in a model of oxazolone-induced acute inflammation in wild type FVB mice. Again, no difference was observed between the mice treated with 6 and the vehicle control group. It still needs to be clarified, whether compound 6 exhibits sufficient skin penetration for a therapeutic effect to occur. For this reason, no firm conclusions can be drawn from these first in vivo experiments.

90 Conclusions

4. Conclusions

4.1. Total Synthesis of the Natural Product L-783277 We have accomplished the first total synthesis of the resorcylic lactone kinase inhibitor L-783277 (6), which represents an interesting lead structure for anticancer and anti-inflammatory drug discovery. Two different routes have been investigated, of which the macrolactonization-based approach has already granted efficient access to L-783277 in a highly enantioselective manner. The Still-Gennari approach pursues a novel general route to cis-enone containing resorcylic lactones and could be completed in six remaining steps based on the advanced intermediate prepared in this thesis.

OH O

O O HO 6 OH

4.1.1. Macrolactonization-Based Approaches The first generation approach was based on the consecutive assembly of three fragments via alkyne acylation, Suzuki coupling and Yamaguchi macrolactonization. This approach was successfully implemented up to a fully protected linear macrolactonization precursor. The anti-diol moiety was planned to be introduced via asymmetric Sharpless-dihydroxylation of a cis-configured double bond. Although this transformation could be optimized in terms of yield, the enantiomeric excess was unsatisfactory, as the two enantiomers were only obtained in a ratio of 2:1. Due to the instability of the advanced intermediate 173 under the reaction conditions that were required for methyl ester cleavage of the resorcylic moiety, the overall protecting group strategy was modified, including the change of the ester moiety to a fluoride sensitive TMS ethyl ester group. However, the latter could not be selectively cleaved in the presence of the 4’/5’-OTBS groups. While this first generation approach ultimately did not lead to L-783277, it has to be highlighted that within this part of the work syntheses of the resorcylic fragment and the C1’-C7’ fragment were developed that could also be used in all following approaches.

OTES

OH O

O O TBSO OTBS 173 : R = Me 177 : R = CH2CH2Si(CH3)3

The second generation approach was designed based on the experience and knowledge we had gained upon implementation of our first generation strategy. One

91 Conclusions of the key features of this new approach was the late introduction of the C6’-ketone through selective allylic oxidation. At the same time, the second generation approach was again characterized by the consecutive assembly of three fragments, including the addition of a lithiated alkyne to an aldehyde, Suzuki coupling and Mitsunobu- based macrolactonization. As mentioned above, the resorcylic fragment and the C1’- C7’ fragment could be synthesized according to the methods developed in the first generation approach. As the asymmetric dihydroxylation had not been as selective as expected, an alternative route to the C1’-C6’ fragment was pursued that relied on a chiral pool precursor (isopropylene-D-erythrono-lactone 107). The total synthesis of 6 was successfully accomplished up to the key step of the selective allylic oxidation. Due to the non-selective nature of the acetylide addition to the C1’-C6’ aldehyde two isomeric triols had to be processed in the last step of the synthesis. Unfortunately, only the minor C6’-isomer 198a (Scheme 54) was cleanly converted to the natural product (6). Thus, the first total synthesis of 6 was successfully accomplished, albeit in only moderate overall yield for the elaboration of the mixture of acetylide addition products 192 (Scheme 54) into the final product. In retrospect, the critical step of the total synthesis of L-783277 (6) had been the addition of the acetylene to the aldehyde, because the two diastereomers formed during this reaction would not be converted to the final product with equal efficiency. For this reason, an enantioselective version of this addition was asked for, in order to improve the overall yield and practicality of our approach to 6. By the use of a Zn- mediated asymmetric addition of the acetylene to the aldehyde we gained access to the desired isomer, which was easily and cleanly converted to the natural product 6 through selective allylic oxidation in the last step of the synthesis. Following this enantioselective approach the absolute configuration of the crucial C6’ stereocenter could be assigned to be (S) for the more reactive isomer in the oxidation step. Based on the stereoselctive acetylide addition, L-783277 (6) could be synthesized in a highly enantioselective and efficient manner.

4.1.2. Still-Gennari Approach As all successful approaches to the synthesis of cis-enone containing resorcylic acid lactones to date are macrolactonization-based and alternative modes of ring-closure have rarely been investigated, we decided to investigate the Still-Gennari olefination as a suitable and novel cyclization method for L-783277 (6). Besides the Still-Gennari cyclization, this approach was based on the consecutive assembly of three fragments via Suzuki coupling and Mitsunobu reaction. This approach was successfully accomplished up to intermediate 213 (Scheme 61). The latter already features the required phosphonate-ester for the Still-Gennari olefination. For completion of the synthesis of 6 the fluoride-labile TMS ethyl ester would have to be cleaved, the C8’- C11’ fragment would have to be introduced, via Mitsunobu reaction, followed by deprotection and oxidation to the aldehyde. Thereafter, the next step would be the cyclization via Still-Gennari conditions. Deprotection of the acetonide would furnish L- 783277 (6) in six steps from previously prepared intermediates.

92 Conclusions

4.2. Synthesis of Analogs Based on the chemistry developed for the preparation of L-783277 (6) we have completed the syntheses of two selected analogs, D6 and P6, which confirms the applicability of our approach for structure-activity relationship (SAR) and also biophysical studies, in order to assess the usefulness of L-783277 as a potential lead structure for drug discovery.

OH O OH O

O O

O O O O HO D6 P6 OH

4.2.1. Synthesis of the Dideoxy Analog D6 The retrosynthetic approach to the dideoxy analog D6 was based on the consecutive assembly of three key intermediates via addition of a lithiathed alkyne to an aldehyde, Suzuki cross-coupling and Mitsunobu-based macrolactonization. The synthesis was carried out in analogy to the total synthesis of 6 except for the C1’-C6’ fragment which lacks the two C4’ and C5’ hydroxyl groups. The required modified C1’-C6’ fragment could be prepared in one step, although the synthesis was slightly hampered by the volatility and instability of the aldehyde which was added to the lithiated alkyne. The mixture of diastereomers obtained in the acetylide addition step was carried through the remainder of the synthesis to provide the desired D6 in excellent yield. The total synthesis of a first selected analog, following the chemistry developed for the synthesis of 6, which differs from 6 only in one key fragment, proves its general applicability.

4.2.2. Synthesis of the Phenyl Analog P6 The synthesis of a second analog, P6, was pursued following two independent routes, one of which envisaged ring-closure through alkyne metathesis. In addition, this approach to P6 was based on the consecutive assembly of three key intermediates via iron-mediated cross-coupling and Steglich esterification. Firstly, the synthesis of the C6’-C11’ moiety turned out to be difficult. Secondly, the iron- mediated cross-coupling proved to be not reproducible. Therefore, a Sonogashira cross-coupling was investigated for the coupling of the resorcylic moiety to the C1’- C6’ fragment. The required protected alkyne could be introduced using Bestmann’s reagent. Finally, the linear precursor for the alkyne metathesis could be synthesized using Steglich esterification conditions. The remaining five steps of the synthesis of P6 would have envolved the protection of the phenolic OH-group, alkyne metathesis, cis-selective reduction of the triple bond, followed by asymmetric dihydroxylation and global deprotection. The unsatisfactory stereochemical outcome of the Sharpless- dihydroxylation in the first generation approach to 6 (Scheme 44) prompted us to revise the synthetic strategy to P6.

93 Conclusions

The revised retrosynthetic approach for the synthesis of P6 was again based on the consecutive assembly of three key intermediates via addition of a lithiathed aryl iodide to an aldehyde, Suzuki coupling and Mitsunobu-based macrolactonization. It was initially planned to introduce the C6’ ketone moiety at an early stage of the synthesis. In comparison to the previous alkyne metathesis-based approach, a highly efficient synthetic route to the C7’-C11’ fragment was developed starting from 1,2- diiodobenzene. In general, the envisioned synthetic route could be followed through to the deprotected linear precursor for the macrolactonization. However, the spontaneous and non-reversible formation of a six-membered hemiketal inhibited the macrocyclization. Accordingly, the initial strategy had to be changed and the C6’ hydroxyl group was carried through the synthesis up to the very last step. In the end, an overall change of hydroxyl protecting groups and the resorcylic ester led to the C6’ deprotected macrocycle, with an OH-group in the C6’ position. The desired benzylic oxidation of the C4’/C5’ acetonide-protected macrocycle turned out not to be feasible, due to complete decomposition. As a consequence the benzylic oxidation of the fully deprotected intermediate had to be investigated. Finally, the use of DMP as oxidizing agent afforded the desired phenyl analog P6.

4.3. Biological Evaluation First results of the biological activity of L-783277 (6) and D6 are available. 6 and D6 show selective inhibition of a subset of kinases, which are involved in inflammatory and cancer-relevant signaling pathways. In cell proliferation experiments 6 effectively inhibited the VEGF-induced proliferation of lymphatic endothelial cells and the proliferation of HL-60 cells. Moreover, in tube formation studies on HUVECs it could be shown that 6 is able to inhibit VEGF-dependent processes, but 6 has no notable effect on network formation which is a VEGF-independent event. First in vivo experiments with 6 in inflammation models did not show any beneficial effect of the compound. However, it remains to be determined, if 6 shows a sufficient level of skin penetration for a therapeutic effect to be even possible under the experimental conditions used.

94 Outlook

5. Outlook We have developed the first enantioselective and high-yielding total synthesis of L- 783277 (6) by which we also gained access to two selected analogs. There are three different motivations, of which one is the investigation of new cyclization methods for the total synthesis of RAL members. This interest will be addressed by accomplishment of the Still-Gennari approach to L-783277 which has been almost completed. Secondly, the analogous lactam of the natural product L-783277 (6) would be an interesting target structure, as esters are often hydrolysed by esterases and thereby decrease the in vivo half-life of the respective drug. Closing, there are various structural features of L-783277 which can be exchanged or modified in order to investigate their impact on kinase inhibition. By modifying the resorcylic moiety, the C4’/C5’ hydroxyl groups or the cis-configured double bond we will establish the basis for extensive structure-activity relationship (SAR) and biophysical studies. This work established a base for exploring the structural diversity around this new lead structure for kinase inhibition.

95 Experimental Section

6. Experimental Section

6.1. General Methods All non-aqueous reactions were carried out using heatgun-dried glassware under a gas flow of dry argon unless otherwise noted. All absolute solvents had HPLC quality and were obtained over molecular sieves. If not indicated otherwise, reactions were magnetically stirred and monitored by thin layer chromatography using Merck Silica Gel 60 F254 plates and visualized by fluorescence quenching under UV light. In addition, TLC plates were stained using ceric ammonium molybdate or potassium permanganate stain. Chromatographic purification of products (flash chromatography) was performed on Merck Silica Gel 60 (230-400 mesh) using a forced flow of eluant at 0.3-0.5 bar. Concentration under reduced pressure was performed by rotary evaporation at 40°C at the appropriate pressure, unless otherwise stated. Purified compounds were further dried for 3-12 h under high vacuum (0.01-0.02 Torr). Yields refer to chromatographically purified and spectroscopically pure compounds, unless otherwise stated. Chemical names were generated with AutoNom 2.02 (Beilstein Informationssysteme GmbH) or ACD Name 8.0 (Advanced Chemistry Development, Inc., (ACD/Labs). In contrast, in most cases the assignment of NMR signals is based on RAL numbering which is indicated in the corresponding picture.

6.1.1. Melting Points Melting points were measured on a Büchi B-540 apparatus. All melting points were measured in open capillaries and are uncorrected.

6.1.2. Optical Rotations Optical rotations were measured on a Jasco P-1020 polarimeter operating at the T sodium D line with a suitable cell, and are reported as follows: []D , concentration (g/100 ml), and solvent.

6.1.3. NMR Measurements NMR spectra were recorded either on a Bruker AV spectrometer operating at 400 MHz and 100 MHz for 1H and 13C acquisitions, respectively, or on a Bruker DRX 500 spectrometer operating at 500 MHz and 125 MHz for 1H and 13C acquisitions, respectively. Chemical shifts () are reported in ppm relative to TMS with the solvent resonance as the internal standard relative to chloroform ( 7.26), methanol ( 3.31), DMSO ( 2.50), dichloromethane ( 5.32) and acetone ( 2.05) for 1H, and chloroform ( 77.2) methanol ( 49.0), DMSO ( 39.5), dichloromethane ( 53.8) and acetone ( 29.8) for 13C. All 13C spectra were measured with complete proton decoupling. Data are reported as follows: s = singlet, bs = broad singlet, d = doublet, bd = broad duplet; t = triplet, q = quartet, m = multiplet; coupling constants in Hz. NMR data of diastereomeric mixtures are generally described for the major isomer, otherwise it is indicated.

96 Experimental Section

6.1.4. IR Measurements IR spectra were recorded on a Jasco FT/IR-6200 spectrometer and all absorptions are given in wavenumbers (cm-1).

6.1.5. Mass Measurements Mass measurements were recorded by the MS service at ETH Zürich. Mass spectral data were collected using a Ultima Micromass AutoSpec spectrometer (EI), a Bruker ReflexII spectrometer (ESI) or a Ultima IonSpec pectrometer (MALDI) as indicated.

6.1.6. HPLC Measurements Analytical RP-HPLC was performed on a Waters Symmetry column (C18, 3.5 m, 4.6 x 100 mm) at a detection wavelength of 254 nm, with a solvent system and gradient as follows: eluent A was acetonitrile and eluent B was water. The gradient was from 5 % eluent A to 20 % eluent A in 0-12 min. Preparative RP-HPLC was performed on a Waters Symmetry column (C18, 5.0 m, 19 x 100 mm) at a detection wavelength of 254 nm,, with a solvent system and gradient as follows: eluent A was acetonitrile and eluent B was water. The gradient was from 5 % eluent A to 20 % eluent A in 0-20 min.

6.2. Experimental Procedures and Analytical Data

6.2.1. Synthesis of the Natural Product L-783277

6.2.1.1. Preparation of Compounds described in Chapter 3.1.1.1.

O OTBS

TBSO 1

3 5

154 tert-Butyl(dimethyl)silyl 2-{[tert-butyl(dimethyl)silyl]oxy}-4-methoxybenzoate 2-Hydroxy-4-methoxybenzoic acid 27 (3 g, 17.85 mmol) was dissolved in 20 ml abs DMF. DIPEA (8.33 g, 11.22 ml, 64.26 mmol, 3.6 equiv) and TBS-Cl (6.57 g, 44.78 mmol, 2.5 equiv) were added to the reaction mixture, which was stirred overnight. The pink solution was diluted with sat. NaHCO3 solution and extracted with

EtOAc (3 x 150 ml). The combined organic layers were washed with diluted NaHCO3- solution (2 x 100 ml), water and brine, dried over anhydrous Na2SO4, filtered and evaporated in vacuo. 154 (7.07 g, quant) was obtained as rose oil.

Rf = 0.88 (hexane/EtOAc 1:1).

97 Experimental Section

1 H-NMR (400 MHz, MeOD-d4):  = 7.76 (d, J = 9.2 Hz, 1H, H-7) ; 6.60 (dd, J = 2.4 Hz and 9.2 Hz, 1H, H-6); 6.42 (d, J = 2.4 Hz, 1H, H-4); 3.81 (s, 3H, 4-OCH3); 1.02 (s,

18H, Si-C(CH3)3); 0.34 (s, 6H, Si-(CH3)2); 0.21 (s, 6H, Si-(CH3)2). 13 C-NMR (100 MHz, MeOD-d4):  = 165.10 (C-1); 163.16 (C-5); 130.70 (C-7); 106.61 (C-3); 106.11 (C-6); 105.67 (C-2); 99.38 (C-4); 53.76 (O-CH3); 24.16 (Si-C(CH3)3);

16.99 (Si-C(CH3)3); -7.92 (Si-(CH3)2).

IR (film): max = 2946, 2844, 2682, 2552, 2497, 1619, 1438, 1355, 1244, 1202, 1148, 1024, 959, 840, 777 cm-1. + HRMS (MALDI): calcd for C20H37O4Si2 [M-H] : 397.2152, found: 397.2152.

Et2N O

TBSO 1

3 5 O

155 2-(tert-Butyldimethylsilyloxy)-N,N-diethyl-4-methoxybenzamide Trimethylaluminium (11.6 ml, 22.60 mmol, 4 equiv) was slowly added at -10°C to a solution of diethylamine (2.7 ml, 25.43 mmol, 4.5 equiv) in toluene (8 ml), which was allowed to warm to rt and stirred for additional 45 min. 154 (2.24 g, 5.65 mmol) was dissolved in toluene (6 ml) and was slowly added at rt to the solution of Et2NAlMe2. After refluxing the mixture overnight, it was quenched at 0°C by adding brine. The organic layer was washed with water (2 x 100 ml) and brine (1 x 100 ml), dried over

Na2SO4, filtered and evaporated in vacuo. 155 (1.91 g, quant) was obtained as colorless oil.

Rf = 0.27 (hexane/EtOAc 2:1). 1 H-NMR (400 MHz, CDCl3):  = 6.92 (d, J = 2.4 Hz, 1H, H-6); 6.31 (d, J = 2.4 Hz, 1H,

H-4); 3.72 (s, 3H, 4-OCH3); 3.05-3.38 (m, 4H, N-CH2CH3); 1.21 (t, J = 7.6 Hz, 3H, N-

CH2CH3); 1.07 (t, J = 7.8 Hz, 3H, N-CH2CH3); 0.91 (s, 9H, Si-C(CH3)3); 0.21 (s, 3H,

Si-(CH3)2); 0.17 (s, 3H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 167.99 (C-1); 160.29 (C-5); 152.62 (C-2); 116.60 (C-

3); 106.07 (C-6); 100.87 (C-4); 94.00 (C-7); 55.54 (4-OCH3); 43.12 (N-CH2CH3);

39.29 (N-CH2CH3); 25.51 (Si-C(CH3)3); 18.07 (Si-C(CH3)3); 13.95 (N-CH2CH3); 12.68

(N-CH2CH3); -4.03 (Si-(CH3)2); -4.70 (Si-(CH3)2).

IR (film): max = 2930, 2857, 1632, 1606, 1470, 1428, 1286, 1254, 1164, 980, 837, 782 cm-1. + HRMS (EI): calcd for C14H22NO3Si [M-C4H9] : 280.1364, found: 280.1363.

98 Experimental Section

Et2N O

TBSO 1 Br

3 5 O 28a 2-Bromo-6-(tert-butyldimethylsilyloxy)-N,N-diethyl-4-methoxybenzamide

A solution of 155 (7.20 g, 21.35 mmol) in Et2O (140 ml) was collod to -78°C and t- BuLi (14.5 ml, 24.55 mmol) was added dropwise. The reaction mixture was allowed to warm to -40°C and was further stirred for 30 min at this temperature. After cooling down to -78°C bromine was added dropwise (1.26 ml, 24.55 mmol). The reaction mixture was stirred for 15 min at -78°C and subsequently it was slowly warmed to rt.

The organic layer was washed with diluted NaHSO3 solution (2 x 100 ml) and with sat. NaHCO3 solution (2 x 100 ml). The organic layer was dried over MgSO4, filtered and evaporated in vacuo to give an orange oil. The crude was purified by FC

(hexane/EtOAc 10:1 + Et3N → 8:1→ 5:1→ 2:1) and 6.04 g (14.55 mmol, 68 %) of 28a as a yellow oil were obtained.

Rf = 0.52 (hexane/EtOAc 2:1) 1 H-NMR (400 MHz, MeOD-d4):  = 6.83-6.84 (d, 1H, H-5); 6.41-6.42 (d, 1H, H-3); 3.79 (s, 3H, 4-OCH3); 3.30-3.35 (m, 2H, N-CH2CH3); 3.19-3.23 (m, 2H, N-CH2CH3);

1.24-1.28 (t, J = 7.2 Hz, 3H, N-CH2CH3); 1.10-1.14 (t, J = 7.2 Hz, 3H, N-CH2CH3);

0.97 (s, 9H, Si-C(CH3)3); 0.30 (s, 3H, Si-(CH3)2); 0.24 (s, 3H, Si-(CH3)2). 13 C-NMR (100 MHz, MeOD-d4):  = 168.9 (C=O); 162.5 (C-4); 154.8 (C-2); 124.4 (C-

1); 121.5 (C-6); 111.7 (C-5); 105.9 (C-3); 56.2 (4-OCH3); 44.8 (N-CH2CH3) 40.9 (N-

CH2CH3); 26.0 (Si-C(CH3)3); 19.0 (Si-C(CH3)3); 14.2 (N-CH2CH3); 13.1 (N-CH2CH3); -4.0 (Si(CH3)2); -4.6 (Si(CH3)2). -1 IR (film): max = 2930, 2857, 1638, 1597, 1557, 1420, 1282, 1154, 994, 840, 782 cm + HRMS (EI): calcd for C14H21BrNO3Si [M-C4H9] : 358.0469; found: 358.0469.

Et2N O

TBSO 1 I

O 28b 2-(tert-Butyldimethylsilyloxy)-N,N-diethyl-6-iodo-4-methoxybenzamide 155 (370 mg, 1.09 mmol, 1 equiv) was dissolved in 3 ml abs ether and cooled to - 78°C, followed by slow addition of t-BuLi solution (0.7 ml, 1.20 mmol, 1.09 equiv). The mixture was allowed to warm up to -40°C over a period of one hour and was stirred at this temperature for two more h. The solution was cooled to -78°C again

99 Experimental Section and a solution of ICl (211 mg, 1.30 mmol, 1.15 equiv) in 3 ml abs ether was added dropwise. After 30 min stirring at -78°C the solution was allowed to warm up to rt overnight. The mixture was diluted with 7 ml ether and quenched with 50 ml of brine, which was extracted with ether (3 x 50 ml). The organic phase was washed with

NaHCO3 solution (2 x 10 ml) and the combined organic layers were dried over anhydrous Na2SO4, filtered and evaporated in vacuo. A brown oil was obtained which was purified by FC (hexane/EtOAc 4:1, + 0.2 % Et3N) to give 28b (174 mg, 34 %).

Rf = 0.44 (hexane/EtOAc 4:1). 1 H-NMR (400 MHz, CDCl3):  = 6.92 (d, J = 2.4 Hz, 1H, H-6). 6.31 (d, J = 2.4 Hz, 1H,

H-4); 3.72 (s, 3H, 4-OCH3); 3.05-3.38 (m, 4H, N-CH2CH3); 1.21 (t, J = 7.6 Hz, 3H, N- CH2CH3); 1.07 (t, J = 7.8 Hz, 3H, N-CH2CH3); 0.91 (s, 9H, Si-C(CH3)3); 0.21 (s, 3H,

Si-(CH3)2); 0.17 (s, 3H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 168.0 (C-1); 160.3 (C-5); 152.6 (C-2); 116.6 (C-3); 106.1 (C-6); 100.9 (C-4); 94.0 (C-7); 55.5 (4-OCH3); 43.1 (N-CH2CH3); 39.3 (N-

CH2CH3); 25.5 (Si-C(CH3)3); 18.1 (Si-C(CH3)3); 14.0 (N-CH2CH3); 12.7 (N-CH2CH3);

-4.0 (Si-(CH3)2); -4.7 (Si-(CH3)2). + HRMS (EI): calcd for C17H27INO3Si [M-CH3] : 448.0800, found: 448.0798.

O O

HO 1 Br

3 5

O 29a 2-Bromo-6-hydroxy-4-methoxy-benzoic acid methyl ester To a solution of trimethyloxonium tetrafluoroborate (269 mg; 1.82 mmol, 1.05 equiv) in 5 ml CH2Cl2 at rt and under argon was added 28a (720 mg, 1.73 mmol, 1 equiv) in

2 ml CH2Cl2. The mixture was stirred for 12 h and it was concentrated in vacuo to yield a light brown oil. The oil was dissolved in 10 ml of a 1:1 solution of MeOH and a sat. Na2CO3 solution which gave a suspension. The reaction mixture was further stirred over the week-end at rt. The mixture was extracted with 50 ml of ether and the layers were separated. The aqueous layer was acidified to pH 2 with a 1N HCl solution and it was extracted with two 50 ml portions of ether. The combined organic layers were dried over MgSO4, filtered and evaporated in vacuo. The crude was purified by FC (hexane/EtOAc 5:1) to give pale yellow crystals of 29a (297 mg, 66 %).

Rf = 0.63 (hexane/EtOAc 2:1). 1 H-NMR (400 MHz, MeOD-d4):  = 6.73 (d, J = 2.3 Hz, 1H, H-5); 6.43 (d, J = 2.2 Hz, 1H, H-3); 3.89 (s, 3H, 1-OCH3); 3.78 (s, 3H, 4-OCH3).

100 Experimental Section

13 C-NMR (100 MHz, MeOD-d4):  = 170.1 (C=O); 165.4 (C-2); 164.0 (C-4); 123.6 (C- 6); 115.2 (C-5); 106.6 (C-1); 100.8 (C-3); 55.8 (1-OCH3); 52.3 (4-OCH3).

IR (film): max = 3101, 2953, 2847, 1644, 1613, 1561, 1434, 1330, 1252, 1205, 1153, 981, 795 cm-1. + HRMS (EI): calcd for C9H9BrO4 [M-H] : 259.9679; found: 259.9678. Melting point: 106°C.

O O

HO 1 I

3 5

O 29b Methyl 2-iodo-6-hydroxy-4-methoxybenzoate To a solution of trimethyloxonium tetrafluoroborate (49 mg, 0.34 mmol, 1.03 equiv) in

2 ml CH2Cl2 at rt and under argon was added 28b (153 mg, 1.33 mmol, 1 equiv) in

2 ml CH2Cl2 and the mixture was stirred overnight. The mixture was concentrated in vacuo to yield an orange oil which was dissolved in 2 ml of a 1:1 solution of MeOH and a sat. Na2CO3 solution. The mixture was stirred overnight at rt. The aqueous layer was acidified to pH 2 with a 10 % HCl solution and was extracted with two 100 ml portions of ether. The combined organic layers were dried over ahydrous

Na2SO4, filtered and evaporated in vacuo. The crude was purified by FC (hexane/EtOAc 4:1) to give 29b (66 mg, 65 %).

Rf = 0.52 (hexane/EtOAc 3:1). 1 H-NMR (400 MHz, MeOD-d4):  = 7.01 (d, J = 2.4 Hz, 1H, H-6); 6.44 (d, J = 2.4 Hz,

1H, H-4); 3.88 (s, 3H, 1-OCH3); 3.77 (s, 3H, 4-OCH3). 13 C-NMR (100 MHz, MeOD-d4):  = 170.33 (C-5); 164.06 (C-1); 160.55 (C-3); 119.59

(C-6); 118.01 (C-2); 102.31 (C-4); 94.06 (C-7); 56.10 (1-OCH3); 52.53 (4-OCH3). + HRMS (EI): calcd for C9H9IO4 [M-H] : 307.9541; found: 307.9540.

101 Experimental Section

6.2.1.2. Preparation of Compounds described in Chapter 3.1.1.2. The C7’-C11’ Fragment was synthesized according to literature procedures which also contain analytical data for all compounds.109

138S (S)-Pent-4-yn-2-ol210 To a suspension of solid lithium acetylide ethylenediamine complex (3.17 g, 34.46 mmol, 2 equiv) in 15 ml abs DMSO was added (S)-propylene oxide 40S (1,22 ml, 17.23 mmol, 1 equiv) dropwise at 5°C. After stirring at rt for 30 h the brown solution was diluted with saturated NH4Cl solution (30 ml). The quenched reaction mixture was extracted with Et2O (3 x 30 ml) and the combined organic layers were dried over MgSO4, filtered and evaporated in vacuo (up to 100 mbar; 40°C water bath). An orange oil was obtained which was further purified by a distillation at atmospheric pressure (bp: 126°C) to give 138S (0.93 g, 64 %).

Rf = 0.5 (hexane/EtOAc 1:1). 1 H-NMR (400 MHz, CDCl3):  = 4.01-3.94 (m, 1H, H-2); 2.44-2.29 (m, 2H, H-3); 2.06 (t, J = 4 Hz, 1H, H-5); 1.85 (brs, 1H, -OH); 1.27 (d, J = 8 Hz, 3H, H-1). 13 C-NMR (100 MHz, CDCl3):  = 81.0 (C-4); 71.0 (C-5); 66.4 (C-2); 29.1 (C-3); 22.4 (C-1).

138R (R)-Pent-4-yn-2-ol210 To a suspension of solid lithium acetylide ethylenediamine complex (3.00 g, 32.61 mmol, 2 equiv) in 15 ml abs DMSO was added (S)-propylene oxide 40R (1,15 ml, 16.20 mmol, 1 equiv) dropwise at 5°C. After stirring at rt for 30 h the brown solution was diluted with sat. NH4Cl solution (30 ml). The quenched reaction mixture was extracted with Et2O (3 x 30 mL) and the combined organic layers were dried over MgSO4, filtered and evaporated in vacuo (up to 100 mbar; 40°C water bath). An orange oil was obtained which was further purified by a distillation at atmospheric pressure (bp: 126°C) to give 138R (0.87 g, 64 %).

Rf = 0.50 (hexane/EtOAc 1:1). 1 H-NMR (400 MHz, CDCl3):  = 4.01-3.94 (m, 1H, H-2); 2.44-2.29 (m, 2H, H-3); 2.06- 2.07 (t, J = 3.4 Hz, 1H, H-5); 1.92 (s, 1H, -OH); 1.26-1.28 (d, J = 6.5 Hz, 3H, H-1).

102 Experimental Section

13 C-NMR (100 MHz, CDCl3):  = 81.0 (C-4); 71.0 (C-5); 66.4 (C-2); 29.1 (C-3); 22.4 (C-1).

OTES

156S (S)-Triethyl(pent-4-yn-2-yloxy)silane

138S (880 mg, 10.47 mmol, 1 equiv) was dissolved in 15 ml of abs CH2Cl2 and the 2,6-lutidine (2.44 ml, 20.94 mmol, 2 equiv) was added. The reaction mixture was cooled to -78°C and a solution of triethylsilyl fluoromethanesulfonate (3.6 ml,

15.70 mmol, 1.5 equiv) in 7 ml of abs CH2Cl2 was added and the reaction was further stirred for 15 min at -78°C. The reaction mixture was allowed to warm up to rt. After 3 h stirring at rt 20 ml of brine were added and the reaction mixture was extracted with CH2Cl2 (2 x 15 ml). The organic phase was washed with sat. NH4Cl solution and sat. NaHCO3 solution, dried over MgSO4 and evaporated in vacuo (up to 500 mbar).

The crude product was purified by column chromatography with neat CH2Cl2 or pentane to give 156S (1.54 g, 75 %).

Rf = 0.79 (CH2Cl2). 1 H-NMR (400 MHz, CDCl3):  = 4.00-3.93 (m, 1 H, H-2) ; 2.40-2.22 (m, 2H, H-3); 1.98 (t, J = 2.6 Hz, 1H, H-5); 1.26 (d, J = 6.0 Hz, 3H, H-1); 0.96 (t, J = 8.0 Hz, 9H, Si-

CH2CH3); 0.61 (q, J = 7.6 Hz, 6H, Si-CH2). 13 C-NMR (100 MHz, CDCl3):  = 82.0 (C-4); 70.0 (C-5); 67.4 (C-2); 29.6 (C-3); 23.4

(C-1); 7.0 (Si-CH2CH3); 4.98 (Si-CH2CH3).

OTES

156R (R)-Triethyl(pent-4-yn-2-yloxy)silane

138R (1.76 g, 2094 mmol, 1 equiv) was dissolved in 30 ml of abs CH2Cl2 and 2,6- lutidine (4.88 ml, 41.88 mmol, 2 equiv) was added. The reaction mixture was cooled to -78°C and a solution of triethylsilyl fluoromethanesulfonate (7.2 ml, 31.40 mmol,

1.5 equiv) in 14 ml of abs CH2Cl2 was added and the reaction was further stirred for 15 min at -78°C. The reaction mixture was allowed to warm up to rt. After 3 h stirring at rt 20 ml of brine were added and the reaction mixture was extracted with CH2Cl2

(2 x 30 ml). The organic phase was washed with sat. NH4Cl solution and sat.

NaHCO3 solution, dried over MgSO4 and evaporated in vacuo (up to 500 mbar). The crude product was purified by FC with neat CH2Cl2 or pentane to give 156R (3.00 g, 73 %).

Rf = 0.79 (CH2Cl2).

103 Experimental Section

20 []D = +4.95° (c = 0.99, CHCl3). 1 H-NMR (400 MHz, CDCl3):  = 3.97-3.92 (m, 1H, H-2) ; 2.40-2.22 (m, 2H, H-3); 1.98 (t, J = 2.8 Hz, 1H, H-5) ; 1.28-1.22 (d, J = 6.2 Hz, 3H, H-1); 0.64-0.58 (q, J = 7.8 Hz,

6H, SiCH3CH2); 0.99-0.93 (t, J = 7.8 Hz, 9H, SiCH3CH2). 13 C-NMR (100 MHz, CDCl3):  = 81.9 (C-4); 69.9 (C-5); 67.4 (C-2); 29.6 (C-3); 23.4

(C-1); 6.9 (Si-CH2CH3); 5.0 (Si-CH3CH2).

IR (film): max = 3315, 2954, 2941, 2914, 2877, 1240, 1127, 1099, 1083, 1002, 741, -1 735, 725 cm . HRMS (EI): no signal found.

OTBS

129S (S)-tert-Butyldimethyl(pent-4-yn-2-yloxy)silane109

138S (1.44 g, 17.16 mmol, 1 equiv) was dissolved in 10 ml of abs CH2Cl2, imidazole (4.69 g, 68.90 mmol, 4 equiv) and TBS-Cl (5.19 g, 34.46 mmol, 2 equiv) were added to the solution at 0°C, which was stirred at rt overnight. 200 ml of water were added to the reaction mixture which was extracted with CH2Cl2 (3 x 100 ml).The combined organic layers were washed with water (3 x 100 ml) and once with brine (1 x 100 ml), dried over MgSO4, filtered and evaporated in vacuo (up to 500 mbar) to give an orange oil. The crude oil was purified by FC (pentane/ Et2O 100:0 → 33:1 → 2:1) to give a colorless oil (2.42 g, 71 %).

Rf = 0.22 (pentane). 1 H-NMR (400 MHz, CDCl3):  = 4.00-3.92 (m, 1H, H-2) ; 2.38-2.218 (m, 2H, H-3); 1.97 (t, J = 2.8 Hz, 1H, H-5); 1.23 (d, J = 6.4 Hz, 3H, H-1); 0.89 (s, 9H, Si-tert-butyl);

0.08 (s, 3H, Si-CH3); 0.07 (s, 3H, Si-CH3). 13 C-NMR (100 MHz, CDCl3):  = 82.1 (C-4); 69.9 (C-5); 67.7 (C-2); 29.5 (C-3); 26.0 (Si-tert-butyl); 23.4 (C-1); 18.3 (Si-C quaternary); -4.5 (Si-CH3); -4.6 (Si-CH3).

IR (film): max = 3315, 2957, 2930, 2858, 2361, 1472, 1378, 1255, 1128, 1100, 1083, 1002, 835, 774, 634 cm-1.

OTBS

129R (R)-tert-Butyldimethyl(pent-4-yn-2-yloxy)silane109

138S (1.00 g, 11.90 mmol, 1 equiv) was dissolved in 10 ml of abs CH2Cl2, imidazole (3.24 g, 47.60 mmol, 4 equiv) and TBS-Cl (3.59 g, 23.79 mmol, 2 equiv) were added to the solution at 0°C, which was stirred at rt overnight. 200 ml of water were added 104 Experimental Section

to the reaction mixture which was extracted with CH2Cl2 (3 x 100 ml).The combined organic layers were washed with water (3 x 100 ml) and once with brine (1 x 100 ml), dried over MgSO4, filtered and evaporated in vacuo (up to 500 mbar) to give an orange oil. The crude oil was purified by FC (pentane/ Et2O 100:0 → 33:1 → 2:1) to give a colorless oil (1.65 g, 70 %).

Rf = 0.22 (pentane). 20 []D = +0.83° ± 0.02° (c = 1.045, CHCl3) 1 H-NMR (400 MHz, CDCl3):  = 3.99-3.94 (m, 1H, H-2); 2.38-2.21 (m, 2H, H-3); 1.97 (t, J = 2.7 Hz, 1H, H-5); 1.24 (d, J = 5.7 Hz, 3H, H-1); 0.89 (s, 9H, Si-tert-butyl); 0.08

(s, 3H, Si-CH3); 0.07 (s, 3H, Si-CH3). 13 C-NMR (100 MHz, CDCl3):  = 82.1 (C-4); 69.8 (C-5); 67.7 (C-2); 29.5 (C-3); 26.0

(Si-tert-butyl); 23.4 (C-1); 18.3 (Si-C quaternary); -4.5 (Si-CH3); -4.6 (Si-CH3).

IR (film): max = 3315, 2957, 2930, 2858, 2361, 1472, 1378, 1255, 1128, 1100, 1083, 1002, 835, 774, 634 cm-1.

6.2.1.3. Preparation of Compounds described in Chapter 3.1.1.3.

9 O 7 5 3 1 11 O 157 2-(Pent-4-ynyloxy)tetrahydro-2H-pyran117 To a solution of 4-pentyn-1-ol 148 (8.65 g, 97.64 mmol, 1 equiv) and 3,4-dihydro-2H- pyran (9.51 g, 107.41, 1.1 equiv) in 20 ml abs CH2Cl2 was added (+)-CSA (227 mg, 0.98 mmol, 0.01 equiv). The reaction mixture was warmed up to rt and stirred for 3 h.

The reaction was quenched with sat. NaHCO3 solution and extracted with CH2Cl2

(2 x 50 ml). The combined organic layers were dried over MgSO4, filtered and evaporated in vacuo. The crude product was purified by FC (hexane/EtOAc 30:1 → 10:1) to give 157 (16.4 g, quantitative) as acolorless liquid.

Rf = 0.35 (hexane/EtOAc 10:1). 1 H-NMR (400 MHz, CDCl3):  = 4.60 (t, 1H, J = 2.8 Hz, H-8); 3.89-3.80 (m, 2H, O-

CH2); 3.54-3.53 (m, 2H, O-CH2)); 2.33-2.29 (m, 2H, H-3); 1.94 (t, J = 2.8 Hz, 1H, H- 1); 1.84-1.49 (m, 8H, H-4, H-10, H-11, H-12). 13 C-NMR (100 MHz, CDCl3):  = 99.0 (C-7); 84.2 (C-2); 68.6 (C-1); 66.0 (C-5); 62.4 (C-9); 31.8 (C-12); 28.9 (C-4); 25.7 (C-10); 19.7 (C-11); 15.5 (C-3). - HRMS (EI): calcd for C10H15O2 [M-H] : 167.1067, found: 167.1071.

105 Experimental Section

O 5 1 O THPO 158 Methyl 6-(tetrahydro-2H-pyran-2-yloxy)hex-2-ynoate117 To a cold (-78°C) solution of 157 (16.62 g, 98.90 mmol, 1 equiv) in 130 ml abs THF was added n-BuLi (65 ml, 103.80 mmol, 1.05 equiv) dropwise over a period of 1.5 h. After stirring for 2 h at -78°C the solution was transferred dropwise to a cold (-35°C) solution of methyl chloroformate (23 ml, 297 mmol, 3 equiv) in 25 ml abs THF over a period of 1.25 h. After stirring for 1 hour at -30°C the cooling bath was removed and the reaction mixture was allowed to warm up to rt overnight. The reaction mixture was poured into 300 ml of a sat. NH4Cl solution and extracted with ether (2 x 300 ml), washed with brine, dried over anhydrous Na2SO4, filtered and evaporated in vacuo. 158 (22.36 g, quantitative) was obtained as a pale yellow liquid.

Rf = 0.17 (hexane/EtOAc 10:1). 1 H-NMR (400 MHz, CDCl3):  = 4.57 (t, J = 4.0 Hz, 1H, H-8); 3.86-3.76 (m, 2H, O-

CH2); 3.74 (O-CH3); 3.52-3.42 (m, 2H, O-CH2); 1.94 (dt, J = 4.0 Hz, 2H, H-4); 1.88- 1.46 (m, 8H, H-5,11,12,13). 13 C-NMR (100 MHz, CDCl3):  = 154.3 (C-1); 99.0 (C-8); 89.3 (C-3); 73.1 (C-2); 65.6

(C-6); 62.4 (C-10); 52.7 (O-CH3); 30.7 (C-13); 28.0 (C-5); 25.5 (C-11); 19.6 (C-12); 15.8 (C-4). - HRMS (EI): calcd for C12H17O4 [M-H] : 225.1122, found: 225.1118.

3 5 O THPO O 1 159 (Z)-Methyl 6-(tetrahydro-2H-pyran-2-yloxy)hex-2-enoate To a solution of 158 (2.74 g, 12.1 mmol, 1 equiv) in 80 ml EtOAc was added Lindlar’s catalyst (258 mg, 0.12 mmol, 0.01 equiv). The suspension was vigorously stirred under a hydrogen atmosphere. Every 15 min the flask was flushed with argon and the reaction was controlled by MS and TLC. After 45 min the reaction was complete and it was filtered through a pad of Celite and washed with EtOAc. The evaporation of the filtrate gave a colorless oil which was was purified by FC (hexane/EtOAc 8:1) to give 159 (2.54 g, 92 %).

Rf = 0.26 (hexane/EtOAc 7:1). 1 H-NMR (400 MHz, CDCl3):  = 6.28-6.21 (m, 1 H, H-3); 5.79-5.75 (dt, J = 11.6 Hz, 1H, H-2); 4.57 (t, 1H, J = 4.4 Hz, H-8); 3.78-3.72 (m, 2H, O-CH2); 3.68 (s, 3H, O-

CH3); 3.50-3.36 (m, 2H, O-CH2); 2.71-2.69 (tq, J = 7.2 Hz, 2H, H-4); 1.83-1.47 (m, 8H, H-5, H-11, H-12, H-13).

106 Experimental Section

13 C-NMR (100 MHz, CDCl3):  = 167.00 (C-1); 150.4 (C-3); 119.7 (C-2); 99.0 (C-8); 67.1 (C-6); 62.4 (C-10); 51.18 (O-CH3); 30.8 (C-13); 29.3 (C-5); 26.1 (C-4); 25.6 (C- 11); 19.7 (C-12). + HRMS (EI): calcd for C12H20NaO4 [M-Na] : 251.1259, found: 251.2012.

HO O 5 3 1 THPO O OH 160 (2S,3S)-2,3-Dihydroxy-6-(tetrahydro-pyran-2-yloxy)-hexanoic acid methyl ester To a mechanically stirred solution of AD-Mix-beta (49.61 g) in 348 ml of a mixture of tert-butyl alcohol/water (1:1) at room temperature was added methanesulfonamide (3.34 g, 35.07 mmol, 1 equiv). The yellow mixture (2 phases) was cooled to 0°C, at which point some salt precipitated. Olefin 159 (8.04 g, 35.07 mmol, 1 equiv) was added at once and it was stirred at 0°C overnight. Na2SO3 was added to the reaction mixture at 0°C. After warming up to rt the mixture was stirred for additional 4 h. The two phases were separated and the aqueous phase was extracted with CH2Cl2 (150 ml) and EtOAc (150 ml). The combined organic phases were dried over MgSO4 and filtered and loaded on Celite. The crude product was purified by FC (hexane/tOAc 1:5 → 1:10) to give 160 (9.24 g, quant).

Rf = 0.34 (hexane/EtOAc 1:5). 1 H-NMR (400 MHz, MeOD-d4):  = 4.59 (t, J = 4.0 Hz, 1H, H-8); 3.81-3.71 (m, 1 H, H-

3); 4.08 (d, J = 4.0 Hz, 1H, H-2); 3.89-3.72 (m, 2H, O-CH2); 3.71 (s, 3H, O-CH3); 3.53-3.39 (m, 2H, O-CH2); 1.86-1.47 (m, 10H, H-4, H-5, H-11, H-12, H-13). 13 C-NMR (100 MHz, MeOD-d4):  = 167.0 (C-1); 150.4 (C-3); 119.7 (C-2); 74.1 (C-8);

68.7 (C-6); 63.6 (C-10); 52.5 (O-CH3); 32.0 (C-13); 30.3 (C-11); 27.2 (C-5); 26.8 (C- 4); 20.8 (C-12). + HRMS (ESI): calcd for C12H22NaO6 [M-Na] : 285.1309, found: 285.1315.

HO O 5 3 HO 1 O OH 161 (2S,3S)-2,3,6-Trihydroxy-hexanoic acid methyl ester 160 (10.19 g, 38.88 mmol, 1 equiv) was dissolved in 300 ml of MeOH and p-TsOH (296 mg, 1.56 mmol, 0.04 equiv) was added. The solution was stirred overnight at rt. The reaction mixture was evaporated in vacuo and the crude product was purified by

FC (CH2Cl2/MeOH 10:1 → 10:2) to give 161 (6.92 g, 93 %).

Rf = 0.14 (CH2Cl2/MeOH 10:1).

107 Experimental Section

1 H-NMR (400 MHz, MeOD-d4):  = 1.49-1.78 (m, 2 H, H-4,5); 3.56-3.59 (t, 2 H, J = 4.0 Hz, H-6); 3.75 (s, 3 H, O-CH3); 3.75-3.78 (m, 2 H, H-3); 4.07 (d, 1 H, J = 8.0 Hz, H-2). 13 C-NMR (100 MHz, MeOD-d4):  = 20.8 (C-12); 26.8 (C-4); 27.2 (C-5); 30.3 (C-11); 32.0 (C-13); 52.5 (O-CH3); 63.6 (C-10); 68.7 (C-6); 74.1 (C-8); 119.7 (C-2); 150.4 (C- 3); 167.0 (C-1). + HRMS (ESI): calcd for C7H14NaO5 [M-Na] : 201.07334, found: 201.0734.

O O 5 3 1 HO O O 162 (4S,5S)-5-(3-Hydroxy-propyl)-2,2-dimethyl-[1,3]dioxolane-4-carboxylic acid methyl ester Triol 161 (143 mg, 0.80 mmol, 1 equiv) was dissolved in 0.6 ml 2,2- dimethoxypropane and the p-TsOH (8 mg, 0.04 mmol,0.05 equiv) was added. The homogenous mixture was stirred for 4.5 h. The solution was diluted with CH2Cl2 and washed with sat. NaHCO3 solution (2 x 20 ml) and brine (2 x 20 ml). The combined organic layers were dried over anhydrous Na2SO4, filtered and evaporated in vacuo.

The crude product was purified by FC (CH2Cl2/MeOH 50:1 + 0.1 % Et3N) to give 162 (141 mg, 81 %).

Rf = 0.18 (CH2Cl2/MeOH 20:1). 1 H-NMR (400 MHz, CDCl3):  = 4.60 (d, J = 8.0 Hz, 1H, H-2); 4.38-4.33 (m, 2H, H-3); 1.38 (s, 3H, CH3); 3.76 (s, 3H, O-CH3); 3.69-3.66 (t, J = 8.0 Hz, 2H, 6-H); 1.78-1.67

(m, 1H, H-4, H-5);1.61 (s, 3H, CH3); 1.53-1.44 (m, 1H, H-4, H-5). 13 C-NMR (100 MHz, CDCl3):  = 170.9 (C-1); 110.8 (C(CH3)2); 77.9 (C-3); 77.6 (C-2); 62.5 (C-6); 52.1 (O-CH3); 35.8 (CH3); 29.8 (C-5); 27.1 (C-4); 27.0 (CH3). + HRMS (ESI): calcd for C10H18NaO5 [M-Na] : 241.1046, found: 241.1042.

O O

5 3 1 O O 163 (4S,5S)-5-Allyl-2,2-dimethyl-[1,3]dioxolane-4-carboxylic acid methyl ester The reaction was carried out under dry conditions and anargon atmosphere. To a solution of 162 (3.2 g, 14.67 mmol, 1 equiv) and 2-nitrophenyl selenocyanate (10.66 g, 46.95 mmol, 3.2 equiv) in 60 ml abs THF was added tri-n-butylphosphine (11.7 ml, 46.95 mmol, 3.2 equiv) dropwise, so that the temperature did not exceed

35°C. The mixture was stirred at rt for 2 h. NaHCO3 (37.9 g) was added to the

108 Experimental Section

mixture and subsequently H2O2 solution (52 ml, 30 %) was added dropwise, again such that the temperature did not exceed 35°C. After 19 h at rt 250 ml of brine was added to the reaction mixture, which was extracted with Et2O (3 x 300 ml). The combined organic phases were dried over MgSO4, filtered and evaporated in vacuo. The crude product was directly loaded on Celite and purified by FC (hexane/EtOAc 25:1) to give 163 (2.38 g, 81 %).

Rf = 0.25 (hexane/EtOAc 10:1). 1 H-NMR (400 MHz, CDCl3):  = 5.89-5.79 (m, 1H, H-5); 5.17-5.09 (m, 2H, H-6); 4.61

(d, J = 6.7 Hz, 1H, H-2); 4.41-4.36 (m, 1H, H-3); 3.75 (s, 3H, O-CH3); 2.37-2.04 (m,

2H, H-4); 1.61 (s, 3H, CH3); 1.38 (s, 3H, CH3). 13 C-NMR (100 MHz, CDCl3):  = 170.7 (C-1); 133.8 (C-5); 117.8 (C-6); 110.8

(C(CH3)2); 77.3 (C-2 and C-3); 75.6 (N-O-CH3); 52.0 (O-CH3); 34.9 (C-4); 25.7 (CH3);

27.1 (CH3). + HRMS (EI): calcd for C9H13O4 [M-CH3] : 185.0809, found: 185.0811.

O O

5 3 1 OH O

164 (4S,5S)-5-Allyl-2,2-dimethyl-[1,3]dioxolane-4-carboxylic acid 163 (122 mg, 0.61 mmol, 1 equiv) was dissolved in 1.2 ml methanol. Lithium hydroxide monohydrate (32 mg, 0.76 mmol, 1.25 equiv) was added and the mixture was stirred overnight at rt. The solution was evaporated in vacuo. The yellow oil was dissolved in EtOAc and water. The aqueous phase was acidified with 2 % KHSO4 solution to pH = 5 and saturated with solid NaCl. The aqueous phase was extracted with EtOAc (20 x 20 ml) and Et2O (7 x 20 ml). The combined organic phases were dried over MgSO4, filtered and evaporated in vacuo to give 164 (96 mg, 85 %). 1 H-NMR (400 MHz, MeOD-d4):  = 5.85-5.82 (m, 1H, =CH); 5.14-5.04 (m, 2H, =CH2);

4.59 (d, J = 7.2 Hz, 1H, -CH); 4.37-4.42 (m, 1H, -CH); 2.18-2.43 (m, 2H, CH2);

1.53 (s, 3H, CH3); 1.35 (s, 3H, CH3). 13 C-NMR (100 MHz, MeOD-d4):  = 25.7 (CH3); 27.4 (CH3); 36.2 (CH2); 66.9 (-CH);

78.3 (-CH); 108.5 (C(CH3)2); 111.4 (=CH2); 117.6 (=CH); 135.5 (C=O).

109 Experimental Section

O O

5 3 1 Cl O 165 (4S,5S)-5-Allyl-2,2-dimethyl-[1,3]dioxolane-4-carbonyl chloride 164 (81 mg, 0.44 mmol, 1 equiv; coevaporated with toluene) was dissolved in 1.3 ml of abs toluene and cooled to 0°C. Oxalyl chloride (95 l, 1.09 mmol, 2.5 equiv) was added and one drop of DMF. After 1 hour at rt the color had changed from colorless to red. The solvent was evaporated in vacuo. Under an argon atmosphere three 2 ml portions of toluene were added to the red residue, which were successively evaporated in vacuo. the crude material was used as it was for the next step.

O O O 5 3 1 N O 166 (4S,5S)-5-Allyl-2,2-dimethyl-[1,3]dioxolane-4-carboxylic acid methoxy-methyl- amide Methyl ester 163 (100 mg, 0.50 mmol, 1 equiv) was dissolved in 3 ml of abs THF and the solution was cooled to -78°C. In a second three-necked flask was placed N,O- dimethylhydroxylamine hydrochloride (244 mg, 2.50 mmol, 5 equiv) in 7 ml abs THF. The solution was also cooled to -78°C and n-BuLi (3.1 ml, 5.00 mmol, 5 equiv) was slowly added via syringe. The reaction mixture was warmed to rt, stirred for 20 min and again cooled to -78°C. The cold solution of 163 was slowly added via a flexible Teflon tube to the mixture. The reaction mixture was stirred and monitored by TLC for completion. After 15 min it was quenched at -78°C by addition of 3 ml sat. NH4Cl solution. The mixture was warmed to rt and the aqueous phase was separated and extracted with EtOAc (3 x 20 ml). The combined organic phases were washed with brine (50 ml), dried over MgSO4, filtered and concentrated in vacuo. The crude was purified by FC (hexane/EtOAc 10:1 → 8:1) to give 166 (95 mg, 84 %).

Rf = 0.29 (hexane/EtOAc 1:1). 1 H-NMR (400 MHz, CDCl3):  = 5.87-5.77 (m, 1H, H-5); 5.13-5.06 (m, 2H, H-6); 4.94

(d, J = 4 Hz, 1H, H-2) ; 4.47-4.42 (q, J = 4.0 Hz1H, H-3); 3.71 (s, 3H, N-O-CH3);

3.18 (s, 3H, N-CH3); 2.37-2.04 (t, J = 8.0 Hz, 2H, H-4); 1.62 (s, 3H, CH3); 1.39 (s, 3H,

CH3). 13 C-NMR (100 MHz, CDCl3):  = 170.1 (C-1); 134.4 (C-5); 117.4 (C-6); 110.2 (C(CH3)2); 77.4 (C-3); 77.2 (C-2); 75.6 (N-O-CH3); 61.4 (C-4); 35.5 (N-CH3); 27.5

(CH3); 25.9 (CH3). + HRMS (EI): calcd for C11H19NO4 [M-H] : 229.1309, found: 229.1308.

110 Experimental Section

6.2.1.4. Preparation of Compounds described in Chapter 3.1.1.4.

O 3 1 O 5 HO OH 169 (2S,3S)-2,3-Dihydroxy-hex-5-enoic acid methyl ester 163 (100 mg, 0.50 mmol, 1 equiv) was dissolved in 5 ml MeOH and p- toluenesulphonic acid monohydrate (48 mg, 0.25 mmol, 0.5 equiv) was added. The solution was stirred at rt. After 3 h the starting material was completely converted into the product. The crude product was directly put on Celite and purified by FC (hexane/EtOAc 1:1) to give 169 (74 mg, 93 %).

Rf = 0.22 (hexane/EtOAc 1:1). 1 H-NMR (400 MHz, CDCl3):  = 5.89-5.79 (m, 1H, H-5); 5.19-5.12 (m, 2H, H-6); 4.27-

4.25 (dd, J = 4 Hz, 1H, H-2); 3.96-3.91 (m, 1H, H-3); 3.83 (s, 3H, O-CH3); 2.35-2.30 (m, 2H, H-4). 13 C-NMR (100 MHz, CDCl3):  = 173.2 (C-1); 134.0 (C-5); 118.6 (C-6); 73.5 (C-2);

72.5 (C-3); 52.9 (O-CH3); 36.7 (C-4).

O 3 1 O 5 TBSO OTBS 170 (2S,3S)-2,3-Bis-(tert-butyl-dimethyl-silanyloxy)-hex-5-enoic acid methyl ester

169 (1.92 g, 12.0 mmol, 1 equiv) was dissolved in 50 ml of CH2Cl2 and 2,6-lutidine (7.7 ml, 66.0 mmol, 5.5 equiv) was added. The mixture was cooled to -78°C. To the cold mixture was added a solution of TBS-OTf (8.3 ml, 36.0 mmol, 3 equiv) in 30 ml of CH2Cl2 and the reaction was stirred for 10 min at -78°C, bevor it was slowly warmed up to rt. After 3 h stirring at rt the reaction was complete. 150 ml of sat.

NaHCO3 were added to the reaction mixture and it was extracted with CH2Cl2 (3x

200 ml). The organic phase was washed with water (100 ml), sat. NH4Cl solution

(100 ml), brine (100 ml), dried over MgSO4 and concentrated in vacuo. The crude product was purified by FC (hexane/EtOAc 100:1 → 50:1 → 20:1) to give 170 (4.52 g, 97 %).

Rf = 0.68 (hexane/EtOAc 10:1). 1 H-NMR (400 MHz, CDCl3):  = 5.88-5.78 (m, 1H, H-5); 5.11-5.06 (m, 2H, H-6); 4.09

(d, J = 4 Hz, 1H, H-2); 3.96-3.91 (q, J = 4 Hz, 1H, H-3); 3.69 (s, 3H, OCH3); 2.39-2.36

111 Experimental Section

(m, 2H, H-4); 0.90 (s, 9H, Si-C(CH3)3); 0.86 (s, 9H, Si-C(CH3)3); 0.06 (s, 3H, Si- (CH3)2); 0.05 (s, 3H, Si-(CH3)2); 0.04 (s, 3H, Si-(CH3)2); 0.03 (s, 3H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 172.9 (C-1); 134.2 (C-5); 117.9 (C-6); 75.4 (C-2);

74.4 (C-3); 51.7 (OCH3); 37.6 (C-4); 25.9 (Si-C(CH3)3); 25.9 (Si-C(CH3)3); 18.3 (Si- C(CH3)3); 18.2 (Si-C(CH3)3); -4.3 (Si-(CH3)2); -4.7 (Si-(CH3)2); -5.0 (Si-(CH3)2); -5.1

(Si-(CH3)2). + HRMS (EI): calcd for C15H31O4Si2 [M-C4H9] : 331.1756, found: 331.1756.

O 5 3 O 1 N TBSO OTBS 171 (2S,3S)-2,3-Bis-(tert-butyl-dimethyl-silanyloxy)-hex-5-enoic acid methoxymethyl-amide Methyl ester 170 (140 mg, 0.36 mmol, 1 equiv) was dissolved in 7 ml of abs THF and the solution was cooled to -78°C. In a second three-necked flask was placed N,O- dimethylhydroxylamine hydrochloride (180 mg, 1.84 mmol, 5.1 equiv) in 15 ml abs THF. The solution was cooled to -78°C and n-BuLi (2.3 ml, 3.68 mmol, 10.2 equiv) was slowly added via syringe. The reaction mixture was warmed to rt, stirred at this temperature for 30 min and it was again cooled to -78°C. The solution of 170 was slowly added to the mixture via a flexible Teflon tube. The reaction mixture was monitored for completion by TLC. After 1.25 h the reaction mixture was quenched at -

78°C by addition of 10 ml sat. NH4Cl solution. The solution was warmed to rt and the aqueous phase was separated and extracted with EtOAc (3 x 10 ml). The combined organic phases were washed with brine (20 ml), dried over MgSO4, filtered and concentrated in vacuo. The crude product was purified by FC (hexane /EtOAc 50:1 → 20:1) to give 171 (147mg, 97 %).

Rf = 0.32 (hexane/EtOAc 10:1). 1 H-NMR (400 MHz, CDCl3):  = 5.98-5.87 (m, 1H, H-5); 5.12-5.09 (m, 2H, H-6); 4.50

(bd, J = 8 Hz, 1H, H-2); 4.06-4.02 (m, 2H, H-3); 3.72 (s, 3H, N-O-CH3); 3.16 (s, 3H,

N-CH3); 2.49-2.36 (m, 2H, H-4); 0.88 (s, 9H, Si-C(CH3)3); 0.85 (s, 9H, Si-C(CH3)3); 0.07 (s, 3H, Si-(CH3)2); 0.05 (s, 3H, Si-(CH3)2); 0.02 (s, 3H, Si-(CH3)2); 0.00 (s, 3H,

Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 173.38 (C-1); 134.11 (C-5); 117.76 (C-6); 77.39 (C- 2); 73.17 (C-3); 61.72 (N-OCH3); 37.22 (C-4); 34.13 (N-CH3); 25.98 (Si-C(CH3)3);

25.93 (Si-C(CH3)3); 18.27 (Si-C(CH3)3); -4.59 (Si-(CH3)2); -4.64 (Si-(CH3)2); -4.71 (Si-

(CH3)2); -4.92 (Si-(CH3)2). + HRMS (MALDI): calcd for C20H44O4Si2 [M-H] : 418.2731, found: 418.2731.

112 Experimental Section

OTES

11 9

TBSO 7 135 O OTBS 172 (4S,5S,10S)-4,5-Bis-(tert-butyl-dimethyl-silanyloxy)-10-triethylsilanyloxy-undec- 1-en-7-yn-6-one To a cold (-10°C) solution of 156S (641 mg, 3.24 mmol, 1.5 equiv) in abs THF (40 ml) was added n-BuLi (2.0 ml, 3.24 ml, 1.5 equiv) dropwise over a period of 20 min and it was further stirred for 20 min at 0°C. The solution of lithiated alkyne 156S was cooled to -78°C and it was slowly added to a precooled solution (-78°C) of 171 (0.90 g, 2.16 mmol, 1 equiv) in 30 ml abs THF over a period of 1 hour via Teflon tube. The solution was allowed to warm up to -18°C and stored overnight in a freezer at that temperature. Then the reaction mixture was quenched at that temperature by adding sat. aqueous NH4Cl solution. The mixture was diluted with 10 ml of Et2O. The layers were separated and the aqueous phase was extracted once with Et2O. The combined organic phases were washed with brine (50 ml) and further dried over

MgSO4, filtered and evaporated in vacuo. The crude product was purified by FC (hexane/EtOAc 60:1).

Rf = 0.44 (hexane/EtOAc 10:1). 1 H-NMR (400 MHz, CDCl3):  = 5.86-5.75 (m, 1H, H-2); 5.11-5.05 (m, 2H, H-1); 4.07- 3.99 (m, 3H, H-10, H-4, H-5); 2.60-2.41 (m, 2H, H-9); 2.37-2.34 (m, 2H, H-3); 1.28 (d,

J = 8.0 Hz, 3H, H-11); 0.96 (t, J = 8.0 Hz, 9H, Si-CH2CH3); 0.93 (s, 9H, Si-C(CH3)3);

0.87 (s, 9H, Si-C(CH3)3); 0.60 (q, J = 8.0 Hz, 6H, Si-CH2CH3); 0.07 (2s, 6H, Si- (CH3)2); 0.06 (2s, 6H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 189.1 (C-6); 134.3 (C-2); 118.0 (C-1); 94.3 (C-8); 81.9 (C-7); 81.8 (C-5); 74.6 (C-4); 66.8 (C-10); 37.6 (C-3) ; 30.3 (C-9); 26.0 (Si-

C(CH3)3); 25.9 (Si-C(CH3)3); 23.6 (C-11); 18.3 (Si-C(CH3)3); 18.2 (Si-C(CH3)3); -4.7

(Si-CH2CH3); 6.9 (Si-CH2CH3); 4.9 (Si-(CH3)2); -4.2 (Si-(CH3)2); -4.4 (Si-(CH3)2); -4.6

(Si-(CH3)2). + HRMS (MALDI): calcd for C20H44NO4Si2 [M-H] : 555.3643, found: 555.3705.

113 Experimental Section

OTES

11' 9' OH O 1 3 O 7' 1' 3' 5' O O 5 TBSO OTBS 173 2-[(4S,5S,10S)-4,5-Bis-(tert-butyl-dimethyl-silanyloxy)-6-oxo-10- triethylsilanyloxy-undec-7-ynyl]-6-hydroxy-4-methoxy-benzoic acid methyl ester To a solution of 172 (81 mg, 0.15 mmol, 1 equiv; dried twice with abs toluene) in 2 ml abs THF was added a 0.5 M solution of 9-BBN (0.41 ml, 0.20 mmol, 1.4 equiv) and the mixture was stirred for 2 h at rt. Then a 2 M solution of K3PO4 (146 l, 0.29 mmol, 2 equiv) was added to the mixture of 172 (solution A). In a separate flask, 29a (41 mg, 0.16 mmol, 1.1 equiv) was added to a mixture of trifurylphosphine (25 mg,

0.11 mmol, 0.74 equiv) and [Pd(OAc)2] (7 mg, 0.03 mmol, 0.22 equiv) in 1ml of DME (solution B) and stirred for 5 min (the colour changed from red to yellow). Then solution A was added dropwise to solution B at rt. The reaction mixture was stirred 2 h at rt and refluxed for another 2.5 h. Overnight the mixture was allowed to cool to rt and its color changed from yellow to orange. 5 ml of water were added, followed by 5 ml of EtOAc. The aqueous solution was extracted with EtOAc (2 x 5 ml) and the combined organic extracts were washed with brine (20 ml), dried over MgSO4 and directly put on Celite. The crude product was purified by FC (hexane/EtOAc 100:0 → 80:1 → 50:1) to give 173 (74 mg, 69 %).

Rf = 0.34 (hexane/EtOAc 10:1). 1 H-NMR (400 MHz, CDCl3):  = 11.76 (s, 1H, 2-OH); 6.33 (d, J = 2.4 Hz, 1H, H-3); 6.25 (d, J = 2.8 Hz, 1H, H-5); 4.02-3.95 (m, 3H, H-10’, H-4’, H-5’); 3.91 (s, 3H, 1-

OCH3); 3.79 (s, 3H, 4-OCH3); 2.97-2.78 (m, 2H, H-1’); 2.59-2.40 (m, 2H, H-9’); 1.62- 1.552 (m, 4H, H-2’ and H-3’); 1.26 (m, 3H, H-11’); 0.95 (t, J = 8.0 Hz, 9H, Si-

CH2CH3); 0.90 (s, 9H, Si-C(CH3)3); 0.85 (s, 9H, Si-C(CH3)3); 0.60 (q, J = 7.6 Hz, 6H,

Si-CH2CH3); 0.06 (2s, 6H, Si-(CH3)2); 0.04 (s, 3H, Si-(CH3)2); 0.02 (s, 3H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 189.2 (C6’); 172.1 (C-4); 165.8 (1-(C=O)OCH3); 164.1 (C-2); 147.4 (C-6); 110.9 (C-5); 104.8 (C-1); 101.0 (C-3); 99.2 (C-7’); 94.4 (C-

8’); 82.1 (C-5’); 74.7 (C-4’); 66.8 (C-10’); 55.4 (4-OCH3); 52.1 (1-OCH3); 37.1 (C-1’); 32.9 (C-3’); 30.3 (C-9’); 26.9 (C-2’); 26.0 (Si-C(CH3)3); 25.9 (Si-C(CH3)3); 23.7 (C-11);

18.4 (Si-C(CH3)3); 18.2 (Si-C(CH3)3); 6.9 (Si-CH2CH3); 4.9 (Si-CH2CH3); -4.3 (Si-

(CH3)2); -4.4 (Si-(CH3)2); -4.6 (Si-(CH3)2); -4.8 (Si-(CH3)2). + HRMS (MALDI): calcd for C38H68NaO8Si3 [M-Na] : 759.4114, found: 759.4127.

114 Experimental Section

11' 9' OH O 1 3 O 7' 1' 3' 5' O O 5 TBSO OTBS 174 2-[(4S,5S,10S)-4,5-Bis-(tert-butyl-dimethyl-silanyloxy)-10-hydroxy-6-oxo-undec- 7-ynyl]-6-hydroxy-4-methoxy-benzoic acid methyl ester 173 (17 mg, 0.023 mmol) was dissolved in 0.5 ml of a 2:2:1 mixture of

THF/HOAc/H2O. After 2 h stirring at rt the reaction was complete. To the solution was added sat. NaHCO3 solution until pH = 7 was obtained. EtOAc was added to the mixture and the phases were separated. The aqueous phase was further extracted with EtOAc (3 x 5 ml). The combined organic phases were washed with brine, dried over MgSO4, filtered and concentrated in vacuo. The crude product was purified by FC (hexane/EtOAc 10:1 → 8:1 → 5:1) to give 174 (12.3 mg, 88 %).

Rf = 0.21 (hexane/EtOAc 3:1). 1 H-NMR (400 MHz, CDCl3):  = 11.75 (s, 1H, 2-OH); 6.33 (d, J = 2.8 Hz, 1H, H-3); 6.26 (d, J = 2.8 Hz, 1H, H-5); 4.07-3.97 (m, 3H, H-10’, H-4’, H-5’); 3.92 (s, 3H, 1-

OCH3); 3.80 (s, 3H, 4-OCH3); 2.92-2.78 (m, 2H, H-1’); 2.54 (d, J = 4 Hz, 2H, H-9’); 1.65-1.55 (m, 4H, H-2’ and H-3’); 1.30 (d, J = 4 Hz, 3H, H-11’); 0.90 (s, 9H, Si-

C(CH3)3); 0.85 (s, 9H, Si-C(CH3)3); 0.06 (s, 6H, Si-(CH3)2); 0.04 (s, 3H, Si-(CH3)2); 0.02 (s, 3H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 189.2 (C6’); 172.0 (C-4); 165.8 (1-(C=O)OCH3); 164.1 (C-2); 147.3 (C-6); 111.1 (C-5); 104.7 (C-1) ; 99.3 (C-3); 93.6 (C-7’); 82.2 (C-

8’); 82.0 (C-5’); 74.8 (C-4’); 66.1 (C-10’); 55.4 (4-OCH3); 52.1 (1-OCH3); 37.0 (C-1’);

33.0 (C-3’); 29.8 (C-9’); 26.8 (C-2’); 26.0 (Si-C(CH3)3); 25.9 (Si-C(CH3)3); 22.8 (C-

11’); 18.4 (Si-C(CH3)3); 18.2 (Si-C(CH3)3); -4.3 (Si-(CH3)2); -4.4 (Si-(CH3)2); -4.6 (Si-

(CH3)2); -4.9 (Si-(CH3)2). + HRMS (MALDI): calcd for C32H58NO8Si2 [M-NH4] : 640.3695, found: 640.3691.

115 Experimental Section

6.2.1.5. Preparation of Compounds described in Chapter 3.1.1.6.

O OH

HO 1 I

O 30b 2-Bromo-6-hydroxy-4-methoxybenzoic acid To a solution of 29b (34 mg, 0.11 mmol, 1 equiv) in 3 ml abs DME was added potassium trimethylsilanolat (28 mg, 0.22 mmol, 2.00 equiv) and 400 μl of abs DMF. The reaction mixture was heated for 120 min at 110°C. The solution was acidified (pH 3) with 1N HCl solution and extracted with ethyl acteate. The organic phase was dried over MgSO4, filtered and evaporated in vacuo. The crude product 30b (26 mg, 81 %) was obtained as white crystals.

Rf = 0.16 (CH2Cl2/MeOH 5:1). 1 H-NMR (400 MHz, CDCl3):  = 11.48 (s, 1H, COOH); 11.40 (brs, 1H, 2-OH); 7.27 (d, J = 4 Hz, 1H, H-6); 6.48 (d, J = 4 Hz, 1H, H-4); 3.82 (s, 3H, 4-OCH3). 13 C-NMR (100 MHz, CDCl3):  = 172.1 (C-1); 166.0 (C-3); 165.1 (C-5); 123.9 (C-6);

101.8 (C-2); 101.8 (C-4); 95.6 (C-7) 56.0 (4-OCH3). + MS (ESI): calcd for C8H7IO4 [M-H] : 293.94, found: 293.91.

6.2.1.6. Preparation of Compounds described in Chapter 3.1.1.7.

O OH

HO 1 Br

O 30a 2-Bromo-6-hydroxy-4-methoxybenzoic acid To a solution of 29a (2.53 g, 9.74 mmol, 1 equiv) in 20 ml abs DME was added potassium trimethylsilanolate (3.57 g, 27.82 mmol, 2.86 equiv) and 400 μl of abs DMF. The reaction mixture was heated for 120 min at 110°C. The solution was acidified (pH 3) with 1N HCl solution and extracted with EtOAc. The organic phase was dried over MgSO4, filtered and evaporated in vacuo. The crude product 30a (2.34 g, 95 %) was obtained as pale rose crystals.

Rf = 0.33 (CH2Cl2/MeOH 5:1).

116 Experimental Section

1 H-NMR (400 MHz, CDCl3):  = 6.84 (d, J = 4.0 Hz, 1H, H-5); 6.45 (d, J = 4.0 Hz, 1H, H-3); 3.82 (s, 3H, 4-OCH3). 13 C-NMR (100 MHz, CDCl3):  = 170.1 (C=O); 165.4 (C-2); 164.0 (C-4); 123.6 (C-6);

115.2 (C-5); 106.6 (C-1); 100.8 (C-3); 55.8 (4-OCH3).

IR (film): max = 3385, 3069, 2832, 2536, 1599, 1443, 1241, 1200, 1161, 981, 836, 795, 605 cm-1. + HRMS (EI): calcd for C8H7BrO4 [M-H] : 245.9522, found: 245.9523. Melting point: 175°C.

O O   Si HO 1 Br

O 176 2-(Trimethylsilyl)ethyl 2-bromo-6-hydroxy-4-methoxybenzoate A solution of -trimethylsilylethanol (51 mg, 0.43 mmol, 1.5 equiv), DCC (71 mg, 0.34 mmol, 1.2 equiv), DMAP (18 mg, 0.14 mmol, 0.5 equiv) and 30a (70 mg,

0.29 mmol, 1 equiv) in 3 ml abs CH2Cl2 was stirred under argon at rt for two days. The reaction mixture was put on Celite and concentrated in vacuo. Purification of the residue by FC (hexane/EtOAc 100:1 → 80:1 → 30:1) provided 176 (84 mg, 88 %) as colorless oil.

Rf = 0.33 (hexane/EtOAc 20:1). 1 H-NMR (400 MHz, CDCl3):  = 11.69 (s, 1H, 2-OH); 6.80 (d, J = 4 Hz, 1H, H-5); 6.42 (d, J = 4 Hz, 1H, H-3); 4.49-4.44 (m, 2H, H-); 3.80 (s, 3H, 4-OCH3); 1.25-1.19 (m,

2H, H-); 0.07 (s, 9H, Si-(CH3)3). 13 C-NMR (100 MHz, CDCl3):  = 169.8 (C-4); 165.3 (C=O); 163.8 (C-2); 123.8 (C-6); 115.1 (C-5); 106.9 (C-1); 100.8 (C-3); 64.6 (C-); 55.8 (4-OCH3); 17.6 (C-β), -1.4 (Si-

(CH3)3).

IR (film): max = 2955, 2898, 2843, 1651, 1604, 1567, 1319, 1244, 1150, 1038, 983, 833, 692 cm-1. + HRMS (EI): calcd for C8H7BrO4 [M-H] : 346.0231, found: 346.0230.

117 Experimental Section

OTES

Si 9' 11' OH O   1 3 O OTBS 7' 1' 3' 5' O O 5 177 OTBS 2-[(4S,5S,10S)-4,5-Bis-(tert-butyl-dimethyl-silanyloxy)-6-oxo-10- triethylsilanyloxy-undec-7-ynyl]-6-hydroxy-4-methoxy-benzoic acid 2- trimethylsilanyl-ethyl ester To a solution of 172 (1.1 g, 1.99 mmol, 1.1 equiv; dried twice with abs toluene) in 17 ml abs THF was added a 0.5 M solution of 9-BBN (6.0 ml, 2.98 mmol, 1.5 equiv) and the mixture was stirred for 2 h at rt. Then a 2 M solution of K3PO4 (1.8 ml, 3.61 mmol, 2 equiv) was added to the mixture of 172 (solution A). In a separate flask, 176 (625 mg, 1.81 mmol, 1.0 equiv) was added to a mixture of trifurylphosphine

(252 mg, 1.08 mmol, 0.6 equiv) and [Pd(OAc)2] (61 mg, 0.27 mmol, 0.15 equiv) in 13 ml of degassed DME (solution B) and stirred for 5 min (the colour changed from red to yellow). Solution A was added dropwise to solution B at rt. The reaction mixture was refluxed for another 5 h. After cooling the mixture was put on Celite and the crude product was purified by FC (hexane/EtOAc 100:0 → 70:1 → 50:1 → 20:1) to give 177 (1.05 g, 70 %).

Rf = 0.22 (hexane/EtOAc 30:1). 1 H-NMR (400 MHz, CDCl3):  = 11.89 (s, 1H, 2-OH) ; 6.33 (d, J = 2.6 Hz, 1H, H-3); 6.25 (d, J = 2.6 Hz, 1H, H-5); 4.38-4.42 (m, 2H, H-); 3.98-4.01 (m, 3H, H-10’, H-4’,

H-5’); 3.79 (s, 3H, 4-OCH3); 2.87-2.91 (m, 2H, H-1’); 2.40-2.55 (m, 2H, H-9’); 1.55- 1.64 (m, 4H, H-2’ and H-3’); 1.26 (d, J = 5.9 Hz, 3H, H-11’); 0.95 (t, J = 8.0 Hz, 9H,

Si-CH2CH3); 1.13-1.18 (m, 2H, H-); 0.90 (s, 9H, Si-C(CH3)3); 0.85 (s, 9H, Si- C(CH3)3); 0.59 (q, 6H, J = 7.9 Hz, Si-CH2CH3); 0.09 (s, 9H, Si-(CH3)3); 0.05 (s, 3H,

Si-(CH3)2); 0.04 (s, 3H, Si-(CH3)2); 0.03 (s, 3H, Si-(CH3)2); 0.01 (s, 3H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 189.1 (C6’); 171.9 (1-(C=O)); 165.8 (C-4); 163.9 (C- 2); 147.4 (C-6); 110.9 (C-5); 105.0 (C-1); 101.0 (C-3); 99.2 (C-8’); 82.1 (C-5’); 81.8

(C-7’); 74.8 (C-4’); 66.8 (C-10’); 64.0 (-C); 55.4 (4-OCH3); 36.9 (C-1’); 32.9 (C-3’);

30.3 (C-9’); 26.9 (C-2’); 26.0 (Si-C(CH3)3); 25.9 (Si-C(CH3)3); 23.7 (C-11’); 18.4 (Si- C(CH3)3); 18.2 (Si-C(CH3)3); 17.7 (-C); 6.9 (Si-CH2CH3); 4.9 (Si-CH2CH3); -1.4 (Si-

(CH3)3); -4.2 (Si-(CH3)2) ; -4.3 (Si-(CH3)2); -4.6 (Si-(CH3)2) ; -4.8 (Si-(CH3)2). + HRMS (MALDI): calcd for C42H78NaO8Si4 [M-Na] : 845.4672, found: 845.4681.

118 Experimental Section

Si 9' 11' OH O   1 3 O OH 7' 1' 3' 5' O O 5 178 OH 2-Hydroxy-4-methoxy-6-((4S,5S,10S)-4,5,10-trihydroxy-6-oxo-undec-7-ynyl)- benzoic acid 2-trimethylsilanyl-ethyl ester 177 (37 mg, 0.05 mmol, 1 equiv) was dissolved in 288 l of abs THF in a polyethylene vessel. Hydrogen fluoride-pyridine (123 ml, 1.37 mmol, 26 equiv) was added to the solution. The reaction was monitored by TLC. After 1 day stirring at rt the TBS groups had been completely removed and the reaction mixture was quenched by adding it dropwise to a sat. NaHCO3 solution (pH = 7), EtOAc was added and the phases were separated. The aqueous phase was extracted with EtOAc (2 x 5 ml). The combined organic phases were washed twice with saturated

CuSO4 solution to remove the pyridine. The organic phase was dried over MgSO4, filtered and evaporated in vacuo. The crude product was purified by FC

(CH2Cl2/MeOH 20:1) to give 178 (21.9 mg, 88 %).

Rf = 0.28 (CH2Cl2/MeOH 10:1). 1 H-NMR (400 MHz, CDCl3):  = 11.76 (s, 1H, 2-OH); 6.33 (d, J = 2.6 Hz, 1H, H-5); 6.27 (d, J = 2.6 Hz, 1H, H-3); 4.39-4.44 (m, 2H, H-); 4.33 (t, J = 4.5 Hz, 1H, H-5’);

4.0-4.09 (m, 2H, H-10’, H-4’); 3.79 (s, 3H, 4-OCH3); 2.89-2.93 (m, 2H, H-1’); 2.52- 2.66 (m, 2H, H-9’); 1.59-1.84 (m, 2H, H-2’); 1.48-1.66 (m, 2H, H-3’) 1.29 (d, J = 6.3

Hz, 3H, H-11’); 1.12-1.17 (m, 2H, H-); 0.08 (s, 9H, Si-(CH3)3). 13 C-NMR (100 MHz, CDCl3):  = 187.1 (C6’); 171.6 (1-(C=O)); 165.7 (C-4); 163.9 (C- 2); 147.3 (C-6); 110.9 (C-3); 105.1 (C-1); 99.2 (C-5); 98.3 (C-8’); 81.7 (C-5’); 80.5 (C-

7’); 73.1 (C-4’); 65.9 (C-10’); 64.0 (-C); 55.4 (4-OCH3); 36.5 (C-1’); 32.8 (C-3’); 29.8

(C-9’); 28.0 (C-2’); 23.0 (C-11’); 17.7 (-C); -1.4 (Si-(CH3)3). + HRMS (ESI): calcd for C24H36O8Si [M-H] : 480.2179, found: 479.2175.

6.2.1.7. Preparation of Compounds described in Chapter 3.1.2.1.

O 1 3 OH O O

180 (3aR,6aR)-2,2-Dimethyl-tetrahydro-furo[3,4-d][1,3]dioxol-4-ol To a stirred ethereal solution (70 ml) of (-)-2,3-O-isopropylidene-D-erythronolactone (500 mg, 3.16 mmol, 1 equiv) was added dropwise a 1.7 M solution of DIBAL-H in

119 Experimental Section toluene (2.8 ml, 4.75 mmol, 1.5 equiv) at -78°C. After 2 h, the reaction mixture was quenched by the addition of saturated Rochelle’s salt solution (45 ml) and stirred overnight while warming to rt. The separated aqueous phase was extracted with Et2O (3 x 5 ml), and the combined organic layers were dried and evaporated to provide the crude product, which was further purified by FC (hexane/EtOAc 5:1 → 2:1 → 1:1 →1:2) to give 180 (350 mg, 95 %) as a colorless oil.

Rf = 0.36 (hexane/EtOAc 1:1). 1 H-NMR (400 MHz, CDCl3):  = 5.43 (bs, 1H, H-1); 4.83-4.86 (m, 1H, H-3); 4.58 (d, J

= 5.6 Hz, 1H, H-2); 4.10-3.97 (m, 2H, H-4); 1.48 (s, 3H, C(CH3)2); 1.33 (s, 3H,

C(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 112.5 (C(CH3)2); 102.0 (C-1); 85.3 (C-2); 80.1 (C-3);

72.2 (C-4); 26.4 (C(CH3)2); 24.9 (C(CH3)2). -1 IR (film): max = 3419, 2985, 2942, 2882, 1645, 1375, 1208, 1063, 985, 854 cm . + HRMS (EI): calcd for C6H9O4 [M-CH3] : 145.0486, found: 145.0486.

O OH O 1 5 3 O O

181 (E)-3-((4S,5R)-5-Hydroxymethyl-2,2-dimethyl-[1,3]dioxolan-4-yl)-acrylic acid ethyl ester 180 (0.67 g, 4.29 mmol, 1 equiv) was dissolved in 4 ml abs toluene. Carbethoxy- methylene triphenylphosphorane (1.55 g, 4.45 mmol, 1.06 equiv) was added to the reaction mixture, which was refluxed for 7 h. The crude product was directly put on Celite and purified by FC (hexane/EtOAc 2:1 → 1:1 → 1:2) to give the E and Z- isomer of 181 (0.87 g, 91 %) in a ratio of 1.1/1. E-isomer:

Rf = 0.20 (hexane/Et2O 1:2). 20 []D = +2.32° ± 0.198° (c = 0.660, CHCl3) 1 H-NMR (400 MHz, CDCl3):  = 6.88 (dd, 1H, J = 15.6 Hz, H-3); 6.12 (dd, 1H, J = 15.4 Hz, H-2); 4.80 (m, 1H, H-4); 4.36 (q, J = 7.2 Hz, 2H, H-5); 4.19 (q, J = 7.2 Hz,

2H, CH2CH3); 3.56 (d, 2H, J = 6.1 Hz, 6-H); 1.52 (s, 3H, C(CH3)2); 1.29 (t, J = 7.1 Hz,

3H, CH2CH3); 1.40 (s, 3H, C(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 166.0 (C-1); 142.2 (C-3); 123.3 (C-2); 109.7

(C(CH3)2); 78.4 (C-5); 76.1 (C-4); 62.0 (C-6); 60.8 (CH2CH3); 27.8 (C(CH3)2); 25.4

(C(CH3)2); 14.3 (CH2CH3).

120 Experimental Section

IR (film): max = 3466, 2986, 2937, 1715, 1659, 1457, 1372, 1256, 1162, 1036, 858, 517 cm-1. + HRMS (EI): calcd for C10H15O5 [M-CH3] : 215.0914, found: 215.0803.

Z-isomer:

Rf = 0.28 (hexane/Et2O 1:2). 20 []D = +146.31° ± 0.06° (c = 0.886, CHCl3). 1 H-NMR (400 MHz, CDCl3):  = 6.38 (dd, J = 11.5 Hz, 1H, H-3); 5.92 (dd, J = 11.7 Hz, 1H, H-2); 5.59 (dt, J = 7.0 Hz, 1H, H-4); 4.55-4.59 (m, 2H, H-5); 4.17 (q, J = 7.3

Hz, 2H, CH2CH3); 3.62-3.44 (m, 2H, 6-H); 1.52 (s, 3H, C(CH3)2); 1.29 (t, J = 7.1 Hz, 3H, CH2CH3); 1.40 (s, 3H, C(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 14.3 (CH2CH3); 24.8 (C(CH3)2); 27.5 (C(CH3)2); 60.8

(CH2CH3); 61.7 (C-6); 75.0 (C-4); 79.0 (C-5); 109.0 (C(CH3)2); 121.2 (C-2); 147.4 (C- 3); 166.2 (C-1).

IR (film): max = 3466, 2986, 2937, 1715, 1659, 1457, 1372, 1256, 1162, 1036, 858, 517 cm-1. + HRMS (EI): calcd for C10H15O5 [M-CH3] : 215.0914, found: 215.0877.

O OH O 1 5 3 O O

182 3-((4S,5R)-5-Hydroxymethyl-2,2-dimethyl-[1,3]dioxolan-4-yl)-propionic acid ethyl ester 181 (2.62 g, 11.39 mmol, 1 equiv) was dissolved in 250 ml ethanol and Pd/C (240 mg, 0.23 mmol, 0.02 equiv) was added. The heterogenous mixture was evacuated and vented three times with argon, afterwards the mixture was evacuated and vented three times with hydrogen. The mixture was stirred 2 h at rt. Then it was filtered over cotton and Celite and evaporated in vacuo. The crude product was purified by FC (hexane/Et2O 1:1 → 1:2 → neat Et2O) to give 182 (2.49 g, 94 %).

Rf = 0.17 (hexane/Et2O 1:2). 20 []D = +146.31° ± 0.06° (c = 0.886, CHCl3). 1 H-NMR (400 MHz, CDCl3):  = 4.20-4.16 (m, 2H, H-4 and H-5); 4.13 (q, J = 7.1 Hz,

2H, CH2CH3); 3.65 (d, J = 5.3 Hz, 2H, H-6); 2.57-2.36 (m, 2H, H-3); 1.86-1.81 (m, 2H, H-2); 1.45 (s, 3H, C(CH3)2); 1.35 (s, 3H, C(CH3)2); 1.25 (t, J = 7.3 Hz, 3H, CH2CH3).

121 Experimental Section

13 C-NMR (100 MHz, CDCl3):  = 173.3 (C-1); 108.4 (C(CH3)2); 77.9 (C-5); 76.1 (C-4); 61.8 (C-6); 60.6 (CH2CH3); 31.3 (C-3); 28.2 (C(CH3)2); 25.6 (C(CH3)2); 24.8 (C-2);

14.3 (CH2CH3). -1 IR (film): max = 2985, 2937, 1731, 1372, 1216, 1161, 1037, 849, 515 cm . + HRMS (EI): calcd for C10H17O5 [M-CH3] : 217.1071, found: 217.1068.

O O O 1 5 3 O O

183 3-((4S,5S)-5-Formyl-2,2-dimethyl-[1,3]dioxolan-4-yl)-propionic acid ethyl ester

To a solution of oxalyl chloride (253 l, 2.90 mmol, 1.5 equiv) in 9 ml of CH2Cl2 was added DMSO (411 ml, 5.80 mmol, 3 equiv) at -78°C within 5 min. The solution was stirred at -78°C for 10 min. A solution of 182 (449 mg, 1.93 mmol, 1 equiv) in 5 ml of

CH2Cl2 was added dropwise within 20 min. The mixture was stirred at -78°C for 1 hour, and 0.85 ml of Et3N was added dropwise at -78°C within 10 min. The mixture was allowed to warm to -10°C, followed by addition of 10 ml of water and 10 ml of

CH2Cl2. The phases were separated and the organic phase was washed with CH2Cl2 (2 x 50 ml), dried over MgSO4, filtered and evaporated in vacuo. The residue was purified by FC (hexane/Et2O 1:1.5 plus 0.15 % Et3N → 1:2) to give 183 (352 mg, 79 %).

Rf = 0.17 (hexane/Et2O 1:2). 1 H-NMR (400 MHz, CDCl3):  = 9.63 (d, J = 2.4 Hz, 1H, H-1); 4.50 (dd, J = 7.2 Hz,

2H, H-2); 4.40-4.35 (m, 1H, H-3); 4.05 (q, J = 7.2 Hz, 2H, CH2CH3); 2.41-2.37 (m, 2H, H-5); 1.85-1.58 (m, 2H, H-4); 1.32 (s, 3H, C(CH3)2); 1.47 (s, 3H, C(CH3)2); 1.18 (t, J =

7.8 Hz, 3H, CH2CH3).

122 Experimental Section

OTBS

9 11

O 7 3 5 O 1 OH OO

184 3-{(4S,5R)-5-[(R)-5-(tert-Butyl-dimethyl-silanyloxy)-1-hydroxy-hex-2-ynyl]-2,2- dimethyl-[1,3]dioxolan-4-yl}-propionic acid ethyl ester To a cold (-78°C) solution of 129R (275 mg, 1.39 mmol, 2.2 equiv) in 1.6 ml abs THF was added n-BuLi (0.79 ml, 1.26 mmol, 2. equiv) over a period of 5 min and it was stirred for 45 min longer at -78°C. To the prepared solution of lithiated alkyne was added a solution of 183 (145 mg, 0.63 mmol, 1 equiv) in 0.6 ml abs THF via syringe. The reaction was further stirred for 2.5 h at that temperature and it was quenched at that temperature by adding saturated aqueous NH4Cl solution. The mixture was diluted with 5 ml Et2O. The layers were separated and the aqueous phase was extracted twice with Et2O (2 x 10 ml). The combined organic phases were dried over

MgSO4, filtered and evaporated in vacuo. The crude was purified by FC (hexane/EtOAc 10:1 → 5:1) to give 184 (52 mg, 57 %) in a ratio of 4:1.

Rf = 0.46 (hexane/EtOAc 2:1). 1 H-NMR (400 MHz, CDCl3):  = 4.35-4.32 (m, 1H, H-6); 4.22-4.17 (m, 1H, H-4); 4.11

(q, J = 7.3 Hz, 2H, CH2CH3); 4.11-4.07 (m, 1H, H-5); 3.97-3.92 (m, 1H, H-10); 2.58- 2.24 (m, 2H, H-2); 2.24-2.23 (m, 2H, H-9); 2.08-1.83 (m, 2H, H-3); 1.46 (s, 3H,

C(CH3)2); 1.36 (s, 3H, C(CH3)2); 1.26 (t, J = 7.2 Hz, 3H, CH2CH3); 1.21 (d, J = 6.1 Hz;

3H, H-11); 0.88 (2s, 9H, Si-C(CH3)3); 0.07 (s, 3H, Si-(CH3)2); 0.06 (s, 3H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 173.2 (C-1); 108.9 (C(CH3)2); 85.1 (C-7); 80.6 (C-5);

79.2 (C-8); 76.3 (C-4); 67.6 (C-10); 61.5 (C-6); 60.5 (CH2CH3); 31.35 (C-2); 29.8 (C-

9); 28.2 (C(CH3)2); 26.0 (Si-C(CH3)3); 25.7 (C-3); 25.1 (C(CH3)2); 23.5 (C-11); 18.2 (Si-C(CH3)3); 14.4 (CH2CH3); -4.5 (Si-(CH3)2); -4.6 (Si-(CH3)2). + HRMS (ESI): calcd for C22H40NaO6Si [M-Na] : 451.2492, found: 451.2494.

123 Experimental Section

OTBS

9 11

O 7 3 5 O 1 OMOM OO

185 3-{(4S,5S)-5-[(R)-5-(tert-Butyl-dimethyl-silanyloxy)-1-methoxymethoxy-hex-2- ynyl]-2,2-dimethyl-[1,3]dioxolan-4-yl}-propionic acid ethyl ester

184 (27 mg, 0.06 mmol, 1 equiv) was dissolved in anhydrous CH2Cl2 (8.3 ml) and DIPEA (868 l, 4.98 mmol, 79 equiv) and molecular sieves (278 mg) were added. The mixture was stirred for 30 min under argon at room temperature. MOM-Cl (378 l, 4.98 mmol, 79 equiv) was added and the reaction mixture was further stirred for 19 h. The crude product was directly put on Celite and purified by FC (hexane/EtOAc 10:1 → 5:1 → 3:1) to give 185 (24 mg, 80 %).

Rf = 0.35 (hexane/EtOAc 5:1). 1 H-NMR (400 MHz, CDCl3):  = 4.98 (d, J = 6.7 Hz, 1H, CH2OCH3); 4.68 (d, J =

6.7 Hz, 1H, CH2OCH3); 4.26 (m, 1H, H-6); 4.18-4.11 (m, 4H, H-4, H-5, CH2CH3);

3.75-3.88 (m, 1H, H-10); 2.58-2.24 (m, 2H, H-2); 3.41 (s, 1H, CH2OCH3); 2.62-2.50 (m, 1H, H-2); 2.41-2.25 (m, 3H, H-2, H-9); 2.10-1.98 (m, 1H, H-3); 1.91-1.78 (m, 1H,

H-3); 1.46 (s, 3H, C(CH3)2); 1.36 (s, 3H, C(CH3)2); 1.25 (t, J = 7.2 Hz, 3H, CH2CH3);

1.22 (d, J = 6.1 Hz; 3H, H-11); 0.88 (2s, 9H, Si-C(CH3)3); 0.06 (s, 6H, Si-(CH3)2). + HRMS (ESI): calcd for C24H44NaO7Si [M-Na] : 495.2754, found: 495.2750.

OTBS

9 11

7 3 5 HO 1 OMOM OO

186 3-{(4S,5S)-5-[(R)-5-(tert-Butyl-dimethyl-silanyloxy)-1-methoxymethoxy-hex-2- ynyl]-2,2-dimethyl-[1,3]dioxolan-4-yl}-propan-1-ol

To a solution of 185 (23 mg, 0.05 mmol) in abs CH2Cl2 (1 ml) was added a 1.7 molar solution of DIBAL-H in toluene dropwise at -78°C and the mixture was stirred at this temperature for 2 h under argon. The reaction mixture was quenched with MeOH, poured into saturated Rochelle’s salt solution and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4, filtered and concentrated in vacuo.

124 Experimental Section

The crude product was purified by FC (hexane/EtOAc 5:1 → 2:1) to give 186 (17 mg, 80 %).

Rf = 0.21 (hexane/EtOAc 2:1). 1 H-NMR (400 MHz, CDCl3):  = 4.99 (d, J = 6.7 Hz, 1H, CH2OCH3); 4.61 (d, J = 6.7 Hz, 1H, CH2OCH3); 4.39-4.44 (m, 1H, H-6); 4.28-4.20 (m, 1H, H-5); 4.18-4.15 (m,

1H, H-4); 3.98-3.89 (m, 1H, H-10); 3.73-3.64 (m, 2H, H-1); 3.39 (s, 1H, CH2OCH3);

2.46-2.2.25 (m, 2H, H-9); 1.98-1.70 (m, 4H, H-2, H-3); 1.52 (s, 3H, C(CH3)2); 1.46 (s, 3H, C(CH3)2); 1.25 (t, J = 7.2 Hz, 3H, CH2CH3); 1.23 (d, J = 6.0 Hz; 3H, H-11); 0.86

(2s, 9H, Si-C(CH3)3); 0.06 (s, 6H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 109.1 (C(CH3)2); 94.0 (CH2OCH3); 85.9 (C-7); 79.6 (C-5); 77.8 (C-4); 77.2 (C-8); 67.5 (C-10); 65.2 (C-6); 62.9 (C-1); 55.9 (CH2OCH3);

30.1 (C-2); 29.8 (C-9); 28.3 (C(CH3)2); 26.8 (C-3); 25.9 (C(CH3)2 and Si-C(CH3)3);

23.4 (C-11); 18.2 (Si-C(CH3)3); -4.5 (Si-(CH3)2); -4.7 (Si-(CH3)2). + HRMS (ESI): calcd for C22H42NaO6Si [M-Na] : 453.2648, found: 495.2646.

6.2.1.8. Preparation of Compounds described in Chapter 3.1.2.2.

O OTBS O 1 3 5 OO

187 3-[(4S,5R)-5-(tert-Butyl-dimethyl-silanyloxymethyl)-2,2-dimethyl-[1,3]dioxolan-4- yl]-propionic acid ethyl ester

182 (109 mg, 0.47 mmol, 1 equiv) was dissolved in 2 ml of CH2Cl2 and 2,6-lutidine (100 l, 0.86 mmol, 1.4 equiv) was added and the solution was cooled to -78°C. To the cold mixture was added dropwise TBS-OTf (148 l, 0.65 mmol, 1.8 equiv) and the reaction was further stirred for 1 hour at -78°C. The reaction was quenched by adding of 7 ml of sat. NaHCO3 solution and it was extracted with CH2Cl2 (3 x 15 ml).

The organic phase was dried over MgSO4, filtered and concentrated in vacuo. The crude product was purified by FC (hexane/EtOAc) to give 187 (150 mg, 93 %).

Rf = 0.27 (hexane/EtOAc 10:1). 20 []D = -16.81° ± 0.07° (c = 0.958, CHCl3). 1 H-NMR (400 MHz, CDCl3):  = 4.17-4.08 (m, 3H, CH2CH3 plus H-4 and H-5); 3.70- 3.60 (m, 2H, H-6); 2.56-2.38 (m, 2H, H-2); 2.00-1.80 (m, 2H, H-3); 1.40 (s, 3H,

C(CH3)2); 1.32 (s, 3H, C(CH3)2); 1.25 (t, J = 7.1 Hz, 3H, CH2CH3); 0.89 (s, 9H, Si-

C(CH3)3); 0.06 (s, 6H, Si-(CH3)2).

125 Experimental Section

13 C-NMR (100 MHz, CDCl3):  = 173.5 (C-1); 108.1 (C(CH3)2); 77.9 (C-5); 76.8 C-4); 61.9 (C-6); 60.5 (CH2CH3); 31.5 (C-2); 28.3 (C(CH3)2); 26.0 (Si-C(CH3)3); 25.7

(C(CH3)2); 24.9 (C-3); 18.4 (Si-C(CH3)3); 14.4 (CH2CH3); -5.3 (Si-(CH3)2); -5.3 (Si-

(CH3)2).

IR (film): max = 2985, 2931, 2858, 1737, 1471, 1370, 1252, 1080, 836, 776, 513 cm-1. + HRMS (EI): calcd for C10H17O5 [M-C4H9] : 289.1471, found: 289.1465.

OTBS OH 1 5 3 OO

188 3-[(4S,5R)-5-(tert-Butyl-dimethyl-silanyloxymethyl)-2,2-dimethyl-[1,3]dioxolan-4- yl]-propan-1-ol

To a solution of 187 (150 mg, 0.43 mmol, 1equiv) in 6 ml abs CH2Cl2 was added a 1.7 M solution of DIBAL in toluene (560 l, 0.95 mmol, 2.2 equiv) at -78°C and the mixture was stirred at this temperature for 2 h. The reaction mixture was quenched with MeOH and was subsequently poured into saturated Rochelle’s salt solution. After one hour stirring at rt the mixture was extracted with EtOAc (2 x 10 ml). The organic layer was washed with brine, dried over MgSO4, filtered and concentrated in vacuo. The crude product was purified by FC (hexane/EtOAc 10:1 → 5:1 → 2:1) to give 188 (112 mg, 85 %).

Rf = 0.09 (hexane/EtOAc 5:1). 20 []D = -8.61° ± 0.05° (c = 0.784, CHCl3). 1 H-NMR (400 MHz, CDCl3):  = 4.19-4.07 (m, 2H, H-2 and H-3); 3.70-3.58 (m, 4H, H- 1 and H-6); 2.56-2.38 (m, 2H, H-2); 1.82-1.58 (m, 4H, H-4 and H-5); 1.42 (s, 3H,

C(CH3)2); 1.34 (s, 3H, C(CH3)2); 0.89 (s, 9H, Si-C(CH3)3); 0.06 (s, 6H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 108.0 (C(CH3)2); 78.2 (C-2); 77.8 (C-3); 62.9 (C-6);

62.2 (C-1); 28.3 (C-4 and C-5); 26.1 (C(CH3)2); 26.0 (Si-C(CH3)3); 25.7 (C(CH3)2);

18.4 (Si-C(CH3)3); -5.2 (Si-(CH3)2); -5.3 (Si-(CH3)2).

IR (film): max = 3417, 2985, 2930, 2858, 1472, 1370, 1251, 1217, 1093, 1054, 836, 775, 514 cm-1. + HRMS (EI): calcd for C14H29O4Si [M-CH3] : 289.1835, found: 289.1826.

126 Experimental Section

OTBS 1 5 3 OO

189 ((4R,5S)-5-Allyl-2,2-dimethyl-[1,3]dioxolan-4-ylmethoxy)-tert-butyl-dimethyl- silane To a solution of 188 (80 mg, 0.27 mmol, 1 equiv) and 2-nitrophenyl selenocyanate (309 mg, 1.36 mmol, 5 equiv) in 2.5 ml abs THF tri-n-butylphosphine (336 l, 1.36 mmol, 5 equiv) was added dropwise, so that the temperature did not exceed

35°C. The mixture was stirred for 2 h at rt. NaHCO3 (0.76 g) was added to the mixture and 1 ml of a 30 % H2O2 solution was added dropwise, again such that the temperature did not exceed 35°C. After 19 h at rt 5 ml of 5 % KHSO4 solution was added to the mixture, which was extracted with Et2O (3 x 5 ml). The combined organic phases were dried over MgSO4, filtered and evaporated in vacuo. The crude product was directly loaded on Celite and purified by FC (hexane/EtOAc 100:1 → 70:1 → 50:1) to give 189 (61 mg, 81 %).

Rf = 0.70 (hexane/EtOAc 10:1). 20 []D = -21.28° ± 0.08° (c = 0.868, CHCl3). 1 H-NMR (400 MHz, CDCl3):  = 5.92-5.84 (m, 1H, H-5); 5.15-5.06 (m, 2H, H-6); 4.23- 4.18 (m, 1H, H-3); 4.13-4.08 (m, 1H, H-2); 3.71-3.61 (m, 2H, H-1); 2.45-2.28 (m, 2H,

H-4); 1.43 (s, 3H, C(CH3)2); 1.34 (s, 3H, C(CH3)2); 0.89 (s, 9H, Si-C(CH3)3); 0.06 (s,

6H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 135.38 (C-5) ; 116.91 (C-6); 108.07 (C(CH3)2); 77.94

(C-2); 77.20 (C-3); 62.04 (C-1); 33.99 (C-4); 28.29 (C(CH3)2); 26.03 (Si-C(CH3)3);

25.66 (C(CH3)2); 18.41 (Si-C(CH3)3); -5.25 (Si-(CH3)2); -5.31 (Si-(CH3)2).

IR (film): max = 3078, 2985, 2931, 2858, 1472, 1368, 1253, 1216, 1096, 833, 775, 514 cm-1. + HRMS (EI): calcd for C15H30NaO3Si [M-Na] : 309.1862, found: 309.1856.

OH 1 5 3 OO

190 ((4R,5S)-5-Allyl-2,2-dimethyl-[1,3]dioxolan-4-yl)-methanol The reaction was carried out under an argon atmosphere. To a stirred solution of 189 (30 mg, 0.11 mmol, 1 equiv) in 2 ml abs THF at 0°C was added a 1 M solution of tetra-n-butylammonium fluoride in THF (210 l, 0.21 mmol, 2 equiv). After 0.5 h

127 Experimental Section stirring at 0°C, the reaction mixture was allowed to come to rt and it was further stirred for 0.5 h. Saturated NH4Cl solution was added and the mixture was extracted with Et2O (3 x 10 ml). The combined organic extracts were dried over MgSO4, filtered and evaporated in vacuo. The crude product was purified by FC (hexane/EtOAc 5:1) to give 190 (16 mg, 90 %).

Rf = 0.09 (hexane/EtOAc 5:1). 20 []D = +10.29° ± 0.11° (c = 1.105, CHCl3). 1 H-NMR (400 MHz, CDCl3):  = 5.89-5.78 (m, 1H, H-5); 5.17-5.09 (m, 2H, H-6); 4.27- 4.23 (m, 1H, H-3); 4.18 (q, J = 5.9 Hz, 1H, H-2); 3.64 (d, J = 5.8 Hz, 2H, H-1); 2.24-

2.44 (m, 2H, H-4); 1.48 (s, 3H, C(CH3)2) 1.37 (s, 3H, C(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 134.4 (C-5); 117.5 (C-6); 108.4 (C(CH3)2); 77.9 (C-

2); 76.4 (C-3); 61.8 (C-1); 33.8 (C-4); 25.6 (C(CH3)2); 24.8 (C(CH3)2).

IR (film): max = 3443, 3078, 2985, 2937, 2878, 1643, 1370, 1216, 1166, 1038, 915, 836, 518 cm-1. + HRMS (EI): calcd for C8H13O2 [M-O(CH3)2] : 141.0910, found: 141.0905.

O 1 5 3 OO

191 (4S,5S)-5-Allyl-2,2-dimethyl-[1,3]dioxolane-4-carbaldehyde

To a solution of oxalyl chloride (250 ml, 2.66 mmol, 1.6 equiv) in 8 ml of CH2Cl2 was added DMSO (402 ml, 5.32 mmol, 3.2 equiv) at -78°C within 5 min. The solution was stirred at -78°C for 10 min. Then a solution of 190 (305 mg, 1.77 mmol, 1 equiv) in

4.5 ml CH2Cl2 was added dropwise within 20 min. The mixture was stirred at -78°C for 1 hour, and 0.78 ml of Et3N were added dropwise at -78°C within 10 min. The mixture was allowed to warm to -10°C, followed by addition of 10 ml of water and 10 ml of CH2Cl2. The phases were separated and the organic phase was washed with brine (2 x 15 ml), dried over MgSO4, filtered and evaporated in vacuo. The residue was purified by FC (hexane/EtOAc 5:1 + 0.15 % Et3N) to give 191 (222 mg, 74 %).

Rf = 0.21 (hexane/EtOAc 5:1). 1 H-NMR (400 MHz, CD2Cl2):  = 9.64 (d, J = 3.1 Hz, 1H, H-1); 5.88-5.78 (m, 1H, H- 5); 5.16-5.09 (m, 2H, H-6); 4.44-4.39 (m, 1H, H-3); 4.30 (dd, J = 7.1 Hz and 3.0 Hz,

2H, H-2); 2.33-2.20 (m, 2H, H-4); 1.56 (s, 3H, CH(CH3)2); 1.40 (s, 3H, C(CH3)2). 13 C-NMR (100 MHz, CD2Cl2):  = 201.8 (C-1); 133.8 (C-5); 117.7 (C-6); 110.6

(C(CH3)2); 82.0 (C-2); 78.0 (C-3); 34.2 (C-4); 27.3 (C(CH3)2); 25.1 (C(CH3)2).

IR (film): max = 3080, 2987, 2938, 1733, 1643, 1381, 1217, 1065, 918, 835, 510 cm-1.

128 Experimental Section

+ HRMS (ESI): calcd for C9H14NaO3 [M-Na] : 193.0835, found: 193.0835.

6.2.1.9. Preparation of Compounds described in Chapter 3.1.2.3.

OTBS

9 11

7 135 OH O O 192 (R)-1-((4R,5S)-5-Allyl-2,2-dimethyl-[1,3]dioxolan-4-yl)-5-(tert-butyl-dimethyl- silanyloxy)-hex-2-yn-1-ol To a cold (-78°C) solution of 129R (1.23 g, 6.22 mmol, 1.2 equiv) in 40 ml abs THF was added n-BuLi (3.9 ml, 6.22 mmol, 1.2 equiv) dropwise over a period of 10 min and the mixture was stirred for 30 min at -10°C. The solution was again cooled to -78°C. To the solution of the lithiated alkyne was added a pre-cooled solution (-78°C) of 191 (882 mg, 5.19 mmol, 1 equiv) in 25 ml abs THF over a period of 10 min via syringe. After 1.5 h stirring at -78°C the solution was allowed to warm to -10°C and it was stirred for 0.5 h at that temperature. The reaction mixture was allowed to warm to rt and the reaction was quenched by adding saturated aqueous

NH4Cl solution. The layers were separated and the aqueous phase was extracted with Et2O (2 x 50 ml). The combined organic phases were dried over MgSO4, filtered and evaporated in vacuo. The crude product was purified by FC (hexane/Et2O 6:1 → 4:1) to give a diastereomeric mixture of 192 (1.66 g, 87 %) in a ratio of 2:1.

Rf = 0.08 (hexane/Et2O 4:1). 1 H-NMR (400 MHz, CDCl3):  = 5.92-5.82 (m, 1H, H-2); 5.17-5.09 (m, 2H, H-1); 4.36- 4.33 (m, 1H, H-6); 4.27-4.22 (m, 1H, H-4); 4.10 (dd, J = 6.0 Hz,1H, H-5); 3.96-3.92 (m, 1H, H-10); 2.54-2.49 (m, 1H, H-3); 2.47 (d, J = 4.15 Hz, 1H, 6-OH); 2.42-2.36 (m,

2H, H-3 and H-9); 2.32-2.25 (m, 1H, H-9); 1.49 (s, 3H, C(CH3)2); 1.38 (s, 3H,

C(CH3)2); 1.21 (d, J = 6.0 Hz, 3H, H-11); 0.88 (s, 3H, Si-C(CH3)3); 0.06 (s, 3H, Si-

(CH3)2); 0.05 (s, 3H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 134.8 (C-2); 117.3 (C-1); 108.9 (C(CH3)2); 84.9 (C- 8); 80.5 (C-5); 79.4 (C-7); 76.8 (C-4); 67.5 (C-10); 61.4 (C-6); 34.1 (C-3); 29.8 (C-9);

28.1 (C(CH3)2); 26.0 (Si-C(CH3)3); 25.5 (C(CH3)2); 23.5 (C-11); 18.2 (Si-C(CH3)3);

-4.5 (Si-(CH3)2); -4.6 (Si-(CH3)2).

IR (film): max = 2930, 2858, 2361, 2342, 1473, 1378, 1253, 1126, 1097, 1064, 997, 918, 835, 775 cm-1. + HRMS (ESI): calcd for C20H36NaO4Si [M-Na] : 391.2281, found: 391.2275.

129 Experimental Section

OTBS

9 11

7 135 O O O O 193 [(R)-5-((4S,5S)-5-Allyl-2,2-dimethyl-[1,3]dioxolan-4-yl)-5-methoxymethoxy-1- methyl-pent-3-ynyloxy]-tert-butyl-dimethyl-silane 192 (168 mg, 0.46 mmol, 1 equiv) was dissolved in 7 ml anhydrous DMF, tetrabutylammonium iodide (8 mg, 0.02 mmol, 0.05 equiv), DIPEA (636 l, 3.65 mmol, 5 equiv) and MOM-Cl (277 l, 3.65 mmol, 8 equiv) were added sequentially. The mixture was stirred for 19 h at room temperature. It was quenched by addition of EtOAc and diluted NaHCO3 (pH = 10). The pH was adjusted to pH = 7 using NH4Cl and the aqueous phase was extracted with EtOAc (3 x 15 ml). The organic phase was dried over MgSO4, filtered and evaporated in vacuo. The crude was purified by FC (hexane/EtOAc 50:1 → 20:1) to give 193 (163 mg, 87 %).

Rf = 0.35 (hexane/Et2O 4:1). Analytics for the minor isomer 193a from enriched fractions after FC: 1 H-NMR (400 MHz, CDCl3):  = 5.95-5.84 (m, 1H, H-2); 5.17-5.08 (m, 2H, H-1); 5.00 (d, J = 6.6 Hz, 1H, OCH2OCH3); 4.61 (d, J = 6.7 Hz, 1H, OCH2OCH3); 4.44-4.42 (m, 1H, H-6); 4.30-4.25 (m, 1H, H-4); 4.20-4.17 (m, 1H, H-5); 3.99-3.91 (m, 1H, H-10);

3.39 (s, 3H, OCH2OCH3); 2.54-2.50 (m, 2H, H-3); 2.44 (dd, J = 4.9 Hz, 1H, H-9); 2.30 (dd, J = 7.7 Hz, 1H, H-9); 1.52 (s, 3H, C(CH3)2); 1.37 (s, 3H, C(CH3)2); 1.24 (d, J =

5.8 Hz, 3H, H-11); 0.88 (s, 9H, Si-C(CH3)3); 0.06 (s, 3H, Si-(CH3)2); 0.05 (s, 3H, Si-

(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 135.2 (C-2); 117.1 (C-1); 108.7 (C(CH3)2); 94.1

(OCH2OCH3); 85.8 (C-8); 79.1 (C-5); 77.6 (C-7); 76.9 (C-4); 67.7 (C-10); 65.9 (C-6);

56.3 (OCH2OCH3); 34.0 (C-3); 30.0 (C-9); 27.6 (C(CH3)2); 26.0 (Si-C(CH3)3); 25.5

(C(CH3)2); 23.4 (C-11); 18.2 (Si-C(CH3)3); -4.5 (Si-(CH3)2); -4.6 (Si-(CH3)2).

IR (film): max = 2931, 2858, 2361, 2342, 1473, 1378, 1253, 1217, 1066, 1032, 1098, 998, 917, 835, 775 cm-1. + HRMS (ESI): calcd for C20H36NaO4Si [M-Na] : 435.2537, found: 435.2536.

130 Experimental Section

OTBS

Si 9' 11' OH O   1 3 O 7' 1' 3' 5' O O O 5 O 194 O

2-(3-{(4S,5S)-5-[(R)-5-(tert-Butyl-dimethyl-silanyloxy)-1-methoxymethoxy-hex-2- ynyl]-2,2-dimethyl-[1,3]dioxolan-4-yl}-propyl)-6-hydroxy-4-methoxy-benzoic acid 2-trimethylsilanyl-ethyl ester The reaction was carried out under an argon atmosphere. To a solution of 193 (1.53 g, 3.71 mmol, 1.1 equiv) in 10 ml abs THF was added dropwise a 0.5 M solution of 9-BBN (7.42 ml, 4.97 mmol, 1.4 equiv) in THF, and the mixture was stirred at rt for 1.5 h at rt. Then a 2 M solution of K3PO4 (3.36 ml, 6.75 mmol, 2 equiv) was added to the mixture (solution A). In a separate flask, 176 (1.17 g, 3.1 mmol, 1 equiv) was added to a mixture of trifurylphosphine (470 mg, 2.02 mmol, 0.6 equiv) and

[Pd(OAc)2] (114 mg, 0.51 mmol, 0.15 equiv) in 2 ml of degassed abs DME (solution B) and stirred for 5 min (the colour changed from red to yellow). Solution A was added dropwise to solution B at rt. The reaction mixture was refluxed for 5.5 h and the mixture was directly put on Celite and purified by FC (hexane/EtOAc 100:1 → 50:1 → 20:1 → 10:1) to give 194 (1.86 g, 81 %).

Rf = 0.15 (hexane/EtOAc 10:1). 1 H-NMR (400 MHz, CDCl3):  = 11.85 (s, 1H, 2-OH) ; 6.32 (d, J = 2.6 Hz, 1H, H-3);

6.28 (d, J = 2.7 Hz, 1H, H-5) ; 4.83 (dd, J = 6.6 Hz and 124.4 Hz, 2H, OCH2OCH3); 4.44-4.40 (m, 2H, H-); 4.34-4.31 (m, 1H, H-6’); 4.22-4.18 (m, 1H, H-5’); 4.17-4.10

(m, 1H, H-4’); 3.95-3.88 (m, 1H, H-10’); 3.79 (s, 3H, 4-OCH3); 3.41 (s, 3H,

OCH2OCH3); 3.03-2.83 (m, 2H, H-1’); 2.40-2.22 (m, 2H, H-9’); 1.82-1.90 (m, 2H, H-

2’); 1.79-1.72 (m, 2H, H-3’); 1.44 (s, 3H, C(CH3)2); 1.35 (s, 3H, C(CH3)2); 1.20 (d, J =

6.0 Hz, 3H, H-11’); 1.18-1.15 (m, 2H, H-); 0.87 (s, 9H, Si-C(CH3)3); 0.08 (s, 9H, Si-

(CH3)3); 0.05 (s, 6H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 171.8 (C=O); 165.8 (C-4); 163.9 (C-2); 147.4 (C-6);

110.9 (C-5); 108.8 (C(CH3)2); 105.1 (C-1); 99.0 (C-3); 93.9 (OCH2OCH3); 85.8 (C-8’); 79.6 (C-5’); 77.6 (C-4’); 77.3 (C-7’); 67.5 (C-10’); 65.2 (C-6’); 63.9 (C-); 55.9

(OCH2OCH3); 55.4 (4-OCH3); 36.7 (C-1’); 29.8 (C-9’); 29.8 (C-3’); 28.73 (C-2’); 28.4

(C(CH3)2); 25.9 (Si-C(CH3)3 and C(CH3)2); 23.4 (C-11’); 18.2 (Si-C(CH3)3); 17.8 (C-);

-1.4 (Si-(CH3)3); -4.5(Si-(CH3)2); -4.7 (Si-(CH3)2).

IR (film): max = 2954, 2860, 2342, 1646, 1614, 1250, 1214, 1157, 1099, 1032, 998, 834, 775 cm-1. + HRMS (ESI): calcd for C35H60NaO9Si2 [M-Na] : 703.3668, found: 703.3665.

131 Experimental Section

Si  OH O OTBS  1 9' 3 O 7' 11' 1' 3' 5' O O O 5 O 195 O

2-(3-{(4S,5S)-5-[(Z)-(R)-5-(tert-Butyl-dimethyl-silanyloxy)-1-methoxymethoxy- hex-2-enyl]-2,2-dimethyl-[1,3]dioxolan-4-yl}-propyl)-6-hydroxy-4-methoxy- benzoic acid 2-trimethylsilanyl-ethyl ester To a solution of 194 (192 mg, 0.28 mmol, 1 equiv) in 5 ml EtOAc was added Lindlar’s catalyst (12 mg, 0.006 mmol, 0.02 equiv). The heterogenous suspension was vigorously stirred under a hydrogen balloon, after every 30 min the flask was flushed with argon and the reaction was controlled by MS and TLC. After 2.5 h the reaction was complete. The mixture was directly put on Celite and purified by several flash chromatographies (Fine silica gel: 15-40 m; hexane/Et2O 10:1 → 8:1 → 6:1) to give 113 mg (59 %) of 195b and 45 mg (31 %) of the 195a. Analytics for the major isomer 195b:

Rf = 0.11 (hexane/Et2O 5:1). 20 []D = 30.09° ± 0.05° (c = 0.688, CHCl3). 1 H-NMR (400 MHz, CDCl3):  = 11.84 (s, 1H, 2-OH); 6.32 (d, J = 2.6 Hz, 1H, H-3); 6.25 (d, J = 2.6 Hz, 1H, H-5); 5.81-5.74 (m, 1H, H-8’); 5.35-5.29 (m, 1H, H-7’); 4.63

(dd, J = 6.7 Hz and 59.5 Hz, 2H, OCH2OCH3); 4.45-4.35 (m, 3H, H-6’ and H-); 4.13- 4.09 (m, 1H, H-5’); 4.05-4.00 (m, 1H, H-4’); 3.92-3.87 (m, 1H, H-10’); 3.79 (s, 3H, 4-

OCH3); 3.38 (s, 3H, OCH2OCH3); 2.96-2.83 (m, 2H, H-1’); 2.41-2.08 (m, 2H, H-9’);

1.88-1.67 (m, 2H, H-2’); 1.74-1.53 (m, 2H, H-3’); 1.43 (s, 3H, C(CH3)2); 1.35 (s, 3H,

C(CH3)2); 1.18-1.16 (m, 2H, H-); 1.14 (d, J = 6.1 Hz, 3H, H-11’); 0.88 (s, 9H, Si-

C(CH3)3); 0.08 (s, 9H, Si-(CH3)3); 0.05 (2s, 6H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 171.7 (C=O); 165.8 (C-4); 163.9 (C-2); 147.4 (C-6);

133.5 (C-8’); 126.4 (C-7’); 110.9 (C-5); 108.3 (C(CH3)2); 105.1 (C-1); 99.0 (C-3); 93.0

(OCH2OCH3); 80.2 (C-5’); 77.4 (C-4’); 69.4 (C-6’); 68.1 (C-10’); 63.9 (C-); 55.6

(OCH2OCH3); 55.4 (4-OCH3); 38.1 (C-9’); 36.8 (C-1’); 29.8 (C-3’); 28.7 (C-2’); 28.3

(C(CH3)2); 26.0 (Si-C(CH3)3 and C(CH3)2); 23.8 (C-11’); 18.2 (Si-C(CH3)3); 17.7 (C-);

-1.4 (Si-(CH3)3); -4.4 (Si-(CH3)2); -4.5 (Si-(CH3)2).

IR (film): max = 2954, 2361, 2342, 1647, 1614, 1578, 1457, 1376, 1321, 1251, 1214, 1157, 1030, 834, 774, 759 cm-1. + HRMS (MALDI): calcd for C35H62NaO9Si2 [M-Na] : 705.3825, found: 705.3831.

132 Experimental Section

Analytics for the major isomer 195a:

Rf = 0.13 (hexane/Et2O 5:1). 20 []D = -25.00° ± 0.06° (c = 0.688, CHCl3). 1 H-NMR (400 MHz, CDCl3):  = 11.84 (s, 1 H, 2-OH); 6.31 (d, J = 2.6 Hz, 1H, H-3); 6.29 (d, J = 2.6 Hz, 1H, H-5); 5.87-5.80 (m, 1H, H-8’); 5.41-5.36 (m, 1H, H-7’); 4.66

(d, J = 6.4 Hz, 1H, OCH2OCH3); 4.44-4.39 (m, 4H, H-6’, H- and OCH2OCH3); 4.20- 4.15 (m, 1H, H-5’); 4.05 (t, J = 6.9 Hz, 1H, H-4’); 3.87-3.81 (m, 1H, H-10’); 3.78 (s,

3H, 4-OCH3) ; 3.32 (s, 3H, OCH2OCH3); 3.04-2.85 (m, 2H, H-1’) ; 2.35-2.22 (m, 2H, H-9’); 1.92-1.81 (m, 1H, H-2’); 1.76-1.43 (m, 3H, H-3’ and H-2’); 1.40 (s, 3H,

C(CH3)2); 1.31 (s, 3H, C(CH3)2); 1.21-1.16 ( m, 2H, H-); 1.13 (d, J = 6.1 Hz, 3H, H- 11’); 0.88 (s, 9H, Si-C(CH3)3); 0.08 (s, 9H, Si-(CH3)3); 0.05 (2s, 6H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 172.0 (C=O); 165.8 (C-4); 163.9 (C-2); 147.7 (C-6);

132.9 (C-8’); 126.5 (C-7’); 110.9 (C-5); 107.8 (C(CH3)2); 105.2 (C-1); 99.0 (C-3); 93.4 (OCH2OCH3); 79.1 (C-5’); 77.8 (C-4’); 69.7 (C-6’); 68.6 (C-10’); 63.8 (C-); 56.2

(OCH2OCH3); 55.4 (4-OCH3); 38.1 (C-9’); 36.9 (C-1’); 29.7 (C-3’); 29.0 (C-2’); 28.0

(C(CH3)2); 26.0 (Si-C(CH3)3); 25.5 C(CH3)2); 23.7 (C-11’); 18.3 (Si-C(CH3)3); 17.7 (C- ); -1.4 (Si-(CH3)3); -4.4 (Si-(CH3)2); -4.6 (Si-(CH3)2).

IR (film): max = 2954, 2361, 2343, 1647, 1614, 1578, 1457, 1370, 1320, 1251, 1214, 1157, 1033, 834, 773, 757 cm-1. + HRMS (MALDI): calcd for C35H62NaO9Si2 [M-Na] : 705.3825, found: 705.3831.

OH O OH 1 9' 3 OH 7' 11' 1' 3' 5' O O O 5 O O 196b

2-Hydroxy-6-{3-[(4S,5S)-5-((Z)-(1R,5R)-5-hydroxy-1-methoxymethoxy-hex-2- enyl)-2,2-dimethyl-[1,3]dioxolan-4-yl]-propyl}-4-methoxy-benzoic acid To a stirred solution of 195b (100 mg, 0.15 mmol, 1 equiv) in 2 ml THF at 0°C was added tetra-n-butylammonium fluoride (733 ml, 0.73 mmol, 5 equiv). After 10 min stirring at 0°C, the reaction mixture was allowed to warm to rt and it was further stirred for 15 h. Saturated NH4Cl solution was added and the mixture (pH = 6) was extracted with EtOAc (2 x 10 ml). The combined organic extracts were dried over

MgSO4, filtered and evaporated in vacuo. The crude product was purified by FC (EtOAc /MeOH 20:1) to give 196b (69 mg, quantitative).

Rf = 0.35 (EtOAc/MeOH 10:1). 20 []D = +29.82° ± 0.31° (c = 0.700, CHCl3). 1 H-NMR (400 MHz, CO(CD3)2):  = 6.18-6.16 (m, 2H, H-3 and H-5); 5.81-5.76 (m, 1H, H-8’); 5.35 (t, J = 10.5 Hz, 1H, H-7’); 4.66 (d, J = 6.7 Hz, 1H, OCH2OCH3); 4.49 133 Experimental Section

(d, J = 6.7 Hz, 1H, OCH2OCH3); 4.45-4.41 (m, 1H, H-6’); 4.03-3.98 (m, 2H, H-4’ and H-5’); 1.19 (d, J = 6.1 Hz, 3H, H-11’); 3.96-3.91 (m, 1H, H-10’); 3.75 (s, 3H, 4-OCH3);

3.34 (s, 3H, OCH2OCH3); 3.19-3.03 (m, 2H, H-1’); 2.40-2.17 (m, 2H, H-9’); 1.89-1.55

(m, 4H, H-3’ and H-2’); 1.38 (s, 3H, CH(CH3)2); 1.26 (s, 3H, C(CH3)2). 13 C-NMR (100 MHz, CO(CD3)2):  = 170.9 (C=O); 166.4 (C-2); 162.7 (C-4); 149.0 (C-

6); 23.7 (C-11’); 132.9 (C-8’); 128.2 (C-7’); 110.6 (C-1); 108.9 (C(CH3)2); 108.4 (C-

51); 99.3 (C-3); 93.8 (OCH2OCH3); 81.1 (C-5’); 78.1 (C-4’); 70.3 (C-6’); 67.7 (C-10’); 55.6 (OCH2OCH3); 55.3 (4-OCH3); 38.3 (C-9’); 36.3 (C-1’); covered by acetone-d6

(C-2’ and C-3’); 28.2 (C(CH3)2); 26.3 (C(CH3)2).

IR (film): max = 2971, 2362, 2340, 1583, 1431, 1373, 1282, 1218, 1184, 1046, 880, 843, 670, 651, 599 cm-1. + HRMS (ESI): calcd for C24H36NaO9 [M-Na] : 491.2252, found: 491.2253.

OH O OH 1 9' 3 OH 7' 11' 1' 3' 5' O O O 5 O O 196a 2-Hydroxy-6-{3-[(4S,5S)-5-((Z)-(1S,5R)-5-hydroxy-1-methoxymethoxy-hex-2- enyl)-2,2-dimethyl-[1,3]dioxolan-4-yl]-propyl}-4-methoxy-benzoic acid To a stirred solution of 195a (minor isomer) (55 mg, 0.08 mmol, 1 equiv) in 2 ml THF at 0°C was added tetra-n-butylammonium fluoride (403 l, 0.40 mmol, 5 equiv). After 10 min stirring at 0°C, the reaction mixture was allowed to warm up rt and it was further stirred for 15 h. Saturated NH4Cl solution was added and the mixture (pH = 6) was extracted with EtOAc (2 x 10 ml). The combined organic extracts were dried over MgSO4, filtered and evaporated in vacuo. The crude product was purified by FC (EtOAc /MeOH 20:1) to give 196a (38 mg, quantitative).

Rf = 0.35 (EtOAc/MeOH 10:1). 20 []D = -43.89° ± 0.14° (c = 0.668, CHCl3). 1 H-NMR (400 MHz, CO(CD3)2):  = 6.19-6.17 (m, 2H, H-3 and H-5); 5.82-5.76 (m,

1H, H-8’); 5.37 (t, J = 10.4 Hz, 1H, H-7’); 4.67 (d, J = 6.6 Hz, 1H, OCH2OCH3); 4.49

(t, J = 8.1 Hz, 1H, H-6’); 4.41 (m, J = 6.4 Hz, 1H, OCH2OCH3); 4.17-4.12 (m, 1H, H-

4’); 4.00 (t, J = 6.4 Hz, 1H, H-5’); 3.85-3.80 (m, 1H, H-10’); 3.75 (s, 3H, 4-OCH3);

3.28 (s, 3H, OCH2OCH3); 3.26-3.09 (m, 2H, H-1’); 2.36-2.21 (m, 2H, H-9’); 1.91-1.60

(m, 4H, H-3’ and H-2’); 1.31 (s, 3H, C(CH3)2); 1.23 (s, 3H, C(CH3)2); 1.16 (d, J = 6.1 Hz, 3H, H-11’). 13 C-NMR (100 MHz, CO(CD3)2):  = 172.0 (C=O); 165.8 (C-2); 162.7 (C-4); 147.7 (C-

6); 132.5 (C-8’); 129.8 (C-7’); 108.0 (C-5 and C(CH3)2); 100.0 (C-3); 99.3 (C-1); 93.9

(OCH2OCH3); 79.9 (C-5’); 78.7 (C-4’); 70.2 (C-6’); 67.6 (C-10’); 56.3 (OCH2OCH3);

134 Experimental Section

55.3 (4-OCH3); 38.4 (C-9’); 36.2 (C-1’); 29.0 (C-3’); 28.3 (C(CH3)2); 25.9 (C-2’ and C(CH3)2); 23.4 (C-11’).

IR (film): max = 2961, 2361, 2342, 1582, 1430, 1369, 1271, 1214, 1184, 1058, 1029, 919, 843, 750, 667, 600 cm-1. + HRMS (ESI): calcd for C24H36NaO9 [M-Na] : 491.2252, found: 491.2253.

11' OH O 1 9' 3 O 7' 1' 3' 5' O O O 5 O 197b O

(Z)-(5S,9S,10R,14S)-18-Hydroxy-20-methoxy-10-methoxymethoxy-7,7,14- trimethyl-6,8,15-trioxa-tricyclo[15.4.0.0*5,9*]henicosa-1(17),11,18,20-tetraen-16- one To a solution of 196b (53 mg, 0.12 mmol, 1 equiv) in 17 ml abs toluene was added triphenylphosphine (62 mg, 0.24 mmol, 2 equiv) and DIAD (47 l, 0.24 mmol, 2 equiv). The mixture was stirred at rt for 25 min and directly put on Celite and purified by FC (hexane/EtOAcE 50:1 → 10:1 → 5:1) to give 197b (30 mg, 59 %).

Rf = 0.47 (hexane/EtOAc 1:1). 20 []D = -46.81° ± 0.28° (c = 0.317, CHCl3). 1 H-NMR (400 MHz, CDCl3):  = 12.04 (s, 1H, 2-OH); 6.33 (d, J = 2.6 Hz, 1H, H-3); 6.26 (d, J = 2.6 Hz, 1H, H-5); 5.75-5.69 (m, 1H, H7’); 5.60-5.51 (m, 2H, H-10’); 5.22-

5.17 (m, 1H, H-8’); 4.65 (dd, J = 7.0 Hz, 2H, OCH2OCH3); 4.45 (t, J = 9.2 Hz, 1H, H- 6’); 4.04 (dd, J = 9.2 Hz, 1H, H-5’); 3.90 (q, J = 6.1 Hz, 1H, H-4’); 3.79 (s, 3H, 4-

OCH3); 3.41 (s, 3H, OCH2OCH3); 3.29-3.23 (m, 1H, H-1’); 2.97-2.87 (m, 1H, H-9’); 2.51-2.44 (m, 1H, H-1’); 2.41-2.35 (m, 1H, H-9’); 1.81-1.54 (m, 4H, H-2’ and H-3’);

1.51 (s, 3H, C(CH3)2); 1.44 (d, J = 6.25 Hz, 3H, H-11’); 1.35 (s, 3H, C(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 171.6 (C=O); 166.0 (C-2); 164.2 (C-4); 147.9 (C-6);

133.5 (C-8’); 127.7 (C-7’); 110.8 (C-5); 108.1 (C(CH3)2); 105.3 (C-1); 99.3 (C-3); 93.2

(OCH2OCH3); 80.1 (C-5’); 78.7 (C-4’); 72.1 (C-10’); 69.8 (C-6’); 55.5 (OCH2OCH3);

55.4 (4-OCH3); 36.0 (C-1’); 35.5 (C-9’); 31.6 (C-2’); 31.3 (C-3’); 28.2 (C(CH3)2); 25.7

(C(CH3)2); 21.2 (C-11’).

IR (film): max = 2983, 2361, 2342, 1733, 1645, 1611, 1376, 1252, 1213, 1160, 1144, 1094, 1028, 752, 667 cm-1. + HRMS (ESI): calcd for C24H34NaO8 [M-Na] : 473.21459, found: 473.21441.

135 Experimental Section

11' OH O 1 9' 3 O 7' 1' 3' 5' O O O 5 O 197a O

(Z)-(5S,9S,10S,14S)-18-Hydroxy-20-methoxy-10-methoxymethoxy-7,7,14- trimethyl-6,8,15-trioxa-tricyclo[15.4.0.0*5,9*]henicosa-1(17),11,18,20-tetraen-16- one To a solution of 196a (53 mg, 0.12 mmol, 1 equiv) in 17 ml abs toluene was added triphenylphosphine (62 mg, 0.24 mmol, 2 equiv) and DIAD (47 l, 0.24 mmol, 2 equiv). The mixture was stirred at rt for 25 min and directly put on Celite and purified by FC (hexane/EtOAcE 50:1 → 10:1 → 5:1) to give 197a (30 mg, 59 %).

Rf = 0.47 (hexane/EtOAc 1:1). 1 H-NMR (400 MHz, CDCl3):  = 11.85 (s, 1H, 2-OH); 6.35 (d, J = 2.6 Hz, 1H, H-5); 6.27 (d, J = 2.6 Hz, 1H, H-5); 5.92-6.09 (m, 2H, H-7’ and H-8’); 5.59-5.62 (m, 1H, H-

10’); 4.53-4.65 (dd, J = 7.0 Hz, 2H, OCH2OCH3); 4.43-4.45 (m, 1H, H-6’); 4.39 (d, J = 7.0 Hz, 1H, H-5’); 4.08 (q, J = 7.0 Hz, 1H, H-4’); 3.80 (s, 3H, 4-OCH3); 3.35 (s, 3H,

OCH2OCH3); 3.20-3.27 (m, 1H, H-9’); 2.40-2.47 (m, 1H, H-9’); 2.21-2.36 (m, 2H, H-

1’); 1.53 (s, 3H, C(CH3)2); 1.47 (d, J = 6.5 Hz, 3H, H-11’); 1.36 (s, 3H, C(CH3)2); 1.28- 1.24 (m, 4H, H-2’ and H-3’). 13 C-NMR (100 MHz, CDCl3):  = 171.3 (C=O); 165.6 (C-4); 164.2 (C-2); 147.6 (C-6);

129.7 (C-8’); 127.4 (C-7’); 110.8 (C-5); 107.8 (C(CH3)2); 105.7 (C-1); 99.3 (C-3); 92.5 (OCH2OCH3); 79.1 (C-5’); 76.9 (C-4’); 71.0 (C-10’); 67.3 (C-6’); 55.7 (OCH2OCH3);

55.5 (4-OCH3); 36.3 (C-9’); 30.8 (C-1’); 29.9 (C-3’); 26.9 (C(CH3)2); 24.6 (C(CH3)2); 22.8 (C-2’); 18.0 (C-11’).

IR (film): max = 2934, 2361, 2342, 1732, 1646, 1614, 1457, 1376, 1255, 1210, 1160, 1094, 1033, 752, 670 cm-1. + HRMS (ESI): calcd for C24H34NaO8 [M-Na] : 473.21459, found: 473.21438.

OH O

O OH HO 198b OH (Z)-(7S,11R,12S,13S)-4,11,12,13-Tetrahydroxy-2-methoxy-7-methyl- 7,8,11,12,13,14,15,16-octahydro-6-oxa-benzocyclotetradecen-5-one 197b (13.6 mg, 0.03 mmol, 1 equiv) was dissolved in 1 ml (0.03 M) of abs MeOH and sulfonic acid resin (Novabiochem, 3.1 mmol/g; 33 mg, 0.1 mmol, 3.4 equiv) was added. The mixture was refluxed for 2.5 h. The resin was filtered off and washed with

136 Experimental Section methanol. The filtrate was evaporated and the crude product was purified by FC (EtOAc/MeOH 50:1 → 30:1 → 10:1 to give 198b (8.6 mg, 78 %).

Rf = 0.35 (EtOAc/MeOH 10:1). 20 []D = -81° ± 2° (c = 0.84, CHCl3). 1 H-NMR (400 MHz, CDCl3):  = 12.14 (s, 1H, 2-OH) ; 6.33 (d, J = 2.7 Hz, 1H, H-3); 6.28 (d, J = 2.7 Hz, 1H, H-5); 5.75-5.69 (dt, J = 10.7 Hz, 1H, H8’); 5.52 (dt, J = 11.2 Hz, 1H, H-7’); 5.48-5.40 (m, 2H, H-10’); 4.51 (dd, J = 6.8 Hz, 1H, H-6’); 3.80 (s,

3H, 4-OCH3); 3.66-3.60 (m, 2H, H-4’ and H-5’); 3.19-3.11 (m, 1H, H-1’); 2.97-2.88 (m, 1H, H-9’); 2.59-2.52 (m, 1H, H-1’); 2.41-2.34 (m, 1H, H-9’); 1.81-1.49 (m, 4H, H-2’ and H-3’); 1.42 (d, J = 6.0 Hz, 3H, H-11’). 13 C-NMR (100 MHz, CDCl3):  = 171.7 (C=O); 166.4 (C-2); 164.3 (C-4); 147.5 (C-6); 131.6 (C-8’); 129.9 (C-7’); 109.7 (C-5); 104.8 (C-1); 99.1 (C-3); covered by the solvent peak (C-5’); 73.1 (C-4’); 72.5 (C-10’); 66.8 (C-6’); 55.5 (4-OCH3); 36.4 (C-1’); 35.2 (C-9’); 32.1 (C-3’); 28.4 (C-2’); 21.4 (C-11’).

IR (film): max = 3436, 2940, 2360, 2342, 1637, 1609, 1583, 1460, 1359, 1320, 1263, 1204, 1161, 1119, 1082, 1029, 992, 960, 812 cm-1. + HRMS (EI): calcd for C19H26O7 [M-H] : 366.1673, found: 366.1675.

11' OH O 1 9' 3 O 7' 1' 3' 5' O OH 5 HO OH 198a (Z)-(7S,11S,12S,13S)-4,11,12,13-Tetrahydroxy-2-methoxy-7-methyl- 7,8,11,12,13,14,15,16-octahydro-6-oxa-benzocyclotetradecen-5-one 197a (115 mg, 0.26 mmol, 1 equiv) was dissolved in 1 ml (0.03 M) of abs MeOH and sulfonic acid resin (Novabiochem, 3.1 mmol/g; 247 mg, 0.77 mmol, 3 equiv) was added. The mixture was refluxed for 2.5 h. The resin was filtered off and washed with methanol. The filtrate was evaporated and the crude product was purified by FC (EtOAc/MeOH 50:1 → 30:1 → 10:1 to give 198a (43 mg, 46 %).

Rf = 0.38 (EtOAc/MeOH 10:1). 20 []D = +30.26° ± 0.32° (c = 0.542, MeOH). 1 H-NMR (400 MHz, DMSO-d6):  = 12.14 (s, 1H, 2-OH); 6.30 (d, J = 2.1 Hz, 1H, H-5); 6.23 (d, J = 2.3 Hz, 1H, H-3); 5.55 (dt, J = 11.2 Hz, 1H, H-8’); 5.43 (dt, J = 8.9 Hz, 1H, H-7’); 5.28-5.22 (m, 1H, H-10’): 4.50-4.49 (m, 1H, H-6’); 4.47-4.44 (m, 1H, 6’-

OH); 4.17 (d, J = 7.2 Hz, 1H, 5’-OH); 3.70 (s, 3H, 4-OCH3); 3.32 (bs, 1H, H-5’ covered by water); 3.29 (s, 1H, H-4’); 2.90-2.82 (m, 1H, H-9’); 2.59-2.44 (m, 2H, H- 1’); 2.17-2.10 (m, 1H, H-9’); 1.78-1.72 (m, 2H, H-2’); 1.51-1.43 (m, 1H, H-3’); 4.31 (d, J = 3.9 Hz, 1H, 4’-OH); 1.34 (d, J = 6.4 Hz, 3H, H-11’); 1.17-1.14 (m, 1H, H-3’).

137 Experimental Section

13 C-NMR (100 MHz, DMSO-d6):  = 168.2 (C=O); 160.9 (C-2); 156.8 (C-2); 141.2 (C- 6); 133.2 (C-7’); 122.7 (C-8’); 114.9 (C-1); 104.1 (C-5); 98.6 (C-3); 80.0 (C-4’); 69.4

(C-10’); 68.0 (C-6’); 67.4 (C-5’); 55.0 (4-OCH3); 31.9 (C-9’); 29.9 (C-1’); 29.2 (C-3’); 23.3 (C-2’); 18.0 (C-11’).

IR (film): max = 3436, 2940, 2360, 2342, 1637, 1609, 1583, 1460, 1359, 1320, 1263, 1204, 1161, 1119, 1082, 1029, 992, 960, 812 cm-1. + HRMS (EI): calcd for C19H26NaO7 [M-Na] : 389.15707, found: 389.15719.

11' OH O 1 9' 3 O 7' 1' 3' 5' O O 5 HO 6 OH (Z)-(7S,12S,13S)-4,12,13-Trihydroxy-2-methoxy-7-methyl-7,8,13,14,15,16- hexahydro-12H-6-oxa-benzocyclotetradecene-5,11-dione

A solution of 198a (11.1 mg, 0.03 mmol, 1 equiv) in abs CH2Cl2 (0.91 ml, 7.4 mM) was treated with 73 mg (0.08 mmol, 2.7 equiv) of commercially available IBX resin (Novabiochem, 1.1 mmol/g). The progress of the reaction was monitored by TLC every 15 min and workup was initiated upon complete consumption of starting material (4 h). The resin was removed by filtration, washed several times with

CH2Cl2, and the combined filtrates were evaporated in vacuo. Purification of the residue by FC (EtOAc/MeOH, 20:1) gave 10.3 mg of 6 (93 %, 91 % purity). This material contained 8 % of a second mono-oxidized product (according to MS analysis). Purification by preparative HPLC gave 5.76 mg (52 %) of 6 with > 95 % purity. Preparation from 198b:

A solution of 198b (5 mg, 0.014 mmol, 1 equiv) in abs CH2Cl2 (3.5 ml, 7.4 mM) was treated with 37 mg (0.041 mmol, 3 equiv) of commercially available IBX resin (Novabiochem, 1.1 mmol/g). The progress of the reaction was monitored by TLC every 15 min and workup was initiated upon complete consumption of starting material (75 min). The resin was removed by filtration, washed several times with

CH2Cl2, and the combined filtrates were evaporated in vacuo. Purification of the residue by FC (EtOAc/MeOH, 20:1) gave 4.1 mg (82 %) of 6 and the 5’ oxidized analog in a ratio of 1:4.

Rf = 0.49 (EtOAc/MeOH 20:1). 1 H-NMR (500 MHz, DMSO-d6):  = 6.50 (d, J = 11.8 Hz, 1H, H-7’); 6.30 (d, J = 2.4 Hz, 1H, H-5); 6.28 (d, J = 2.4 Hz, 1H, H-3) , 6.25-6.20 (m, 1H, H-8’), 5.28–5.32 (m, 1H, H-10’), 4.88 (d, J = 4.9 Hz, 1H, 5’-OH); 4.69 (d, J = 6.6 Hz, 1H, 4’-OH); 4.31-

4.29 (m, 1H, H-5’) 3.75-3.72 (m, 1H, H-4’); 3.73 (s, 3H, 4-OCH3); 3.01-3.08 (m, 1H,

138 Experimental Section

H-9’); 2.67-2.61 (m, 2H, H-1’ and H-9’); 2.43-2.37 (m, 1H, H-1’); 1.60-1.56 (m, 1H, H- 2’), 1.40-1.36 (m, 2H, H-3’); 1.32 (d, J = 6.2 Hz, 3H, H-11’); 1.23 (m, 1H, H-2’).

13 C-NMR (125 MHz, DMSO-d6):  = 201.7 (C-6’): 169.7 (1-C=O); 162.6 (C-4); 161.6 (C-2); 145.1 (C-6); 143.0 (C-8’); 127.3 (7’); 108.5 (C-1); 106.9 (C-5); 98.8 (C-3); 81.4

(C-5’); 72.4 (C-10’); 71.19 (C-4’), 55.2 (4-OCH3); 35.7 (C-9’); 34.3 (C-1’); 30.9 (C-3’); 26.9 (C-2’); 19.9 (C-11’). + HRMS (ESI): calcd for C19H25NaO7 [M-Na] : 387.14142, found: 387.14155.

6.2.1.10. Preparation of Compounds described in Chapter 3.1.3.1.

OTBS

911

7 1 35 OH O O 192a

(1S,5R)-1-((4R,5S)-5-Allyl-2,2-dimethyl-[1,3]dioxolan-4-yl)-5-(tert-butyl-dimethyl- silanyloxy)-hex-2-yn-1-ol

Zn(OTf)2 (137 mg, 0.376 mmol, 1.65 equiv) was dried at 144°C under high vacuum (0.06 mbar) for 3 h with vigorous stirring. After cooling to rt, (-)-NME (75 mg, 0.417 mmol, 1.83 equiv) was added and the flask was flushed with argon (3 x). The solids were suspended in toluene (1.7 ml) at rt, and Et3N (63 l, 0.456 mmol, 2.00 equiv) was added. The milky mixture was stirred at rt for 2 h, treated with alkyne 129R (54 mg, 0.373 mmol, 1.20 equiv) in toluene (0.5 ml), followed by rinsing with toluene (2 x 0.3 ml). The reaction was further stirred at rt for 45 min. Then a solution of aldehyde 191 (38.7 mg, 0.228 mmol, 1.00 equiv) in toluene (0.3 ml) was added, followed by rinsing with toluene (2 x 0.3 ml). The reaction mixture changed its colour from pale yellow to yellow. After 17 h at rt, the reaction mixture (red-brown colored) was poured into sat. aqueous NH4Cl (5 ml) and extracted with Et2O (2 x 5 ml). The combined organic phases were dried over MgSO4, filtered and concentrated under reduced pressure. Purification by chromatography on silica gel (hexane/EtOAc 20:1 → 5:1) furnished 192a (39 mg, 46 %) as a clear, colorless oil.

Rf = 0.08 (hexane/Et2O 4:1). 1 H-NMR (400 MHz, CDCl3):  = 5.91-5.84 (m, 1H, H-2); 5.17-5.08 (m, 2H, H-1); 4.43- 4.39 (m, 1H, H-6); 4.30-4.25 (m, 1H, H-4); 4.10 (t, J = 5.7 Hz, 1H, H-5); 3.99-3.91 (m, 1H, H-10); 2.61-2.49 (m, 2H, H-3); 2.44-2.26 (m, 2H, H-9); 2.17 (d, J = 6.5 Hz, 1H, 6-

OH); 1.51 (s, 3H, C(CH3)2); 1.36 (s, 3H, C(CH3)2); 1.23 (d, J = 6.1 Hz, 3H, H-11); 0.88 (s, 3H, Si-C(CH3)3); 0.07 (s, 3H, Si-(CH3)2); 0.06 (s, 3H, Si-(CH3)2).

139 Experimental Section

13 C-NMR (100 MHz, CDCl3):  = 135.1 (C-2); 117.1 (C-1); 108.6 (C(CH3)2); 85.0 (C- 8); 80.3 (C-7); 79.9 (C-5); 76.9 (C-4); 67.7 (C-10); 62.4 (C-6); 34.0 (C-3); 29.9 (C-9);

27.6 (C(CH3)2); 26.0 (Si-C(CH3)3); 25.5 (C(CH3)2); 23.5 (C-11); 18.2 (Si-C(CH3)3);

-4.5 (Si-(CH3)2); -4.6 (Si-(CH3)2).

IR (film): max = 2930, 2858, 2361, 2342, 1473, 1378, 1253, 1126, 1097, 1064, 997, 918, 835, 775 cm-1. + HRMS (ESI): calcd for C20H36NaO4Si [M-Na] : 391.2275, found: 391.2269.

OTBS

911

7 1 35 OH O O 192b

(1R,5R)-1-((4R,5S)-5-Allyl-2,2-dimethyl-[1,3]dioxolan-4-yl)-5-(tert-butyl-dimethyl- silanyloxy)-hex-2-yn-1-ol

Zn(OTf)2 (290 mg, 0.796 mmol, 1.65 equiv.) was dried at 150°C under high vacuum (0.04 mbar) for 3 h with vigorous stirring. After cooling to rt, (+)-NME (158 mg, 0.882 mmol, 1.83 equiv.) was added, and the flask was flushed with argon (3 x). The solids were suspended in toluene (3.6 ml) at rt, and Et3N (134 l, 0.964 mmol, 2.00 equiv.) was added. The milky mixture was stirred at rt for 2 h, treated with alkyne 129R (115 mg, 0.578 mmol, 1.20 equiv.) in toluene (1.0 ml), followed by rinsing with toluene (2 x 0.6 ml). The reaction was further stirred at rt for 45 min. Then a solution of aldehyde 191 (38.7 mg, 0.228 mmol, 1.00 equiv.) in toluene (0.6 ml) was added, followed by rinsing with toluene (2 x 0.6 ml). The reaction mixture changed its colour from pale yellow to yellow. After 17 h at rt, the reaction mixture (red-brown coloured) was poured into saturated aqueous NH4Cl (10 ml) and extracted with Et2O (2 x 10 ml). The combined organic phases were dried over

MgSO4, filtered and concentrated under reduced pressure. Purification by chromatography on silica gel (hexane/EtOAc 20:1 → 5:1) furnished 192b (47 mg, 26 %) as a clear, colorless oil.

2H, H-3 and H-9); 2.32-2.25 (m, 1H, H-9); 1.49 (s, 3H, C(CH3)2); 1.38 (s, 3H,

C(CH3)2); 1.21 (d, J = 6.0 Hz, 3H, H-11); 0.88 (s, 3H, Si-C(CH3)3); 0.06 (s, 3H, Si-

(CH3)2); 0.05 (s, 3H, Si-(CH3)2).

140 Experimental Section

13 C-NMR (100 MHz, CDCl3):  = 134.8 (C-2); 117.3 (C-1); 108.9 (C(CH3)2); 84.9 (C- 8); 80.5 (C-5); 79.4 (C-7); 76.8 (C-4); 67.5 (C-10); 61.4 (C-6); 34.1 (C-3); 29.8 (C-9);

28.1 (C(CH3)2); 26.0 (Si-C(CH3)3); 25.5 (C(CH3)2); 23.5 (C-11); 18.2 (Si-C(CH3)3);

-4.5 (Si-(CH3)2); -4.6 (Si-(CH3)2).

IR (film): max = 2930, 2858, 2361, 2342, 1473, 1378, 1253, 1126, 1097, 1064, 997, 918, 835, 775 cm-1. + HRMS (ESI): calcd for C20H36NaO4Si [M-Na] : 391.2281, found: 391.2275.

6.2.1.11. Preparation of Compounds described in Chapter 3.1.3.2.

OTBS

9 11

7 O (R) 1 3 5 CF O 3 O O Ph O

(R)-MTPA ester (R)-3,3,3-Trifluoro-2-methoxy-2-phenyl-propionic acid (1R,5R)-1-((4S,5S)-5-allyl- 2,2-dimethyl-[1,3]dioxolan-4-yl)-5-(tert-butyl-dimethyl-silanyloxy)-hex-2-ynyl ester

192-6’R (15 mg, 0.04 mmol, 1 equiv) was dissolved in 1 ml abs CH2Cl2. Pyridine (7 l, 0.13 mmol, 3.1 equiv), DMAP (cat) and (S)-MTPA-Cl (13.4 l, 0.08 mmol, 1.9 equiv) were added to the mixture and it was stirred at rt overnight. To the reaction mixture was added water and the layers were separated. The aqueous phase was further extracted with Et2O (2 x 5 ml). The combined organic phases were dried over

MgSO4, filtered and concentrated under reduced pressure. Purification by FC (hexane/EtOAc 20:1) furnished the (R)-MTPA ester (23 mg, 96 %) as a clear colorless oil.

Rf = 0.52 (hexane/EtOAc 5:1). 1 H-NMR (400 MHz, CDCl3):  = 7.60-7.58 (m, 1H, Ar-H); 7.40-7.35 (m, 3H, Ar-H); 5.87-5.79 (m, 1H, H-2); 5.69-5.65 (m, 1H, H-6); 5.14-5.09 (m, 2H, H-1); 4.24-4.20 (m,

2H, H-5 and H-4); 3.96-3.90 (m, 1H, H-10); 3.42 (s, 3H, O-CH3); 2.43-2.27 (m, 4H, H-

3 and H-9); 1.42 (s, 3H, C(CH3)2); 1.27 (s, 3H, C(CH3)2); 1.21 (d, J = 6.0 Hz, 3H, H-

11); 0.88 (s, 3H, Si-C(CH3)3); 0.07 (s, 3H, Si-(CH3)2); 0.06 (s, 3H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 165.5 (C=O); 134.7 (C-2); 129.7 (ar-C); 128.3 (ar- C); 127.8 (ar-C); 124.8 (CF3); 117.4 (C-1); 109.3 (C(CH3)2); 87.0 (C-8); 84.9 (Cq);

84.6 (Cq); 78.1 (C-5); 76.8 (C-4); 75.7 (C-7); 67.3 (C-10); 65.0 (C-6); 55.7 (O-CH3);

34.1 (C-3); 29.8 (C-9); 27.8 (C(CH3)2); 25.9 (Si-C(CH3)3); 25.7 (C(CH3)2); 23.3 (C-11); 18.2 (Si-C(CH3)3); -4.5 (Si-(CH3)2); -4.7 (Si-(CH3)2).

141 Experimental Section

+ HRMS (ESI): calcd for C30H43F3NaO6Si [M-Na] : 607.2673, found: 607.2660.

OTBS

9 11

7 O (S) 3 5 CF 1 O 3 O O O Ph

(S)-MTPA ester (S)-3,3,3-Trifluoro-2-methoxy-2-phenyl-propionic acid (1R,5R)-1-((4S,5S)-5-allyl- 2,2-dimethyl-[1,3]dioxolan-4-yl)-5-(tert-butyl-dimethyl-silanyloxy)-hex-2-ynyl ester

192-6’R (15 mg, 0.04 mmol, 1 equiv) was dissolved in 1 ml abs CH2Cl2. Pyridine (7 l, 0.13 mmol, 3.1 equiv), DMAP (cat.) and (R)-MTPA-Cl (13.4 l, 0.08 mmol, 1.9 equiv) were added to the mixture and it was stirred at rt for 2.5 h. To the reaction mixture was added water and the layers were separated. The aqueous phase was further extracted with Et2O (2 x 5 ml). The combined organic phases were dried over

MgSO4, filtered and concentrated under reduced pressure. Purification by FC (hexane/EtOAc 20:1) furnished the (S)-MTPA ester (19 mg, 79 %) as a clear colorless oil.

Rf = 0.52 (hexane/EtOAc 5:1). 1 H-NMR (400 MHz, CDCl3):  = 7.61-7.60 (m, 1H, Ar-H); 7.40-7.36 (m, 3H, Ar-H); 5.89-5.84 (m, 1H, H-2); 5.69-5.65 (dt, J = 8.6 Hz, 1H, H-6); 5.17-5.11 (m, 2H, H-1);

4.31-4.23 (m, 2H, H-5 and H-4); 3.94-3.86 (m, 1H, H-10); 3.63 (s, 3H, O-CH3); 2.51- 2.49 (m, 1H, H-3); 2.43-2.32 (m, 2H, H-3 and H-9); 2.28-2.21 (m, 1H, H-9); 1.44 (s,

3H, C(CH3)2); 1.33 (s, 3H, C(CH3)2); 1.15 (d, J = 6.1 Hz, 3H, H-11); 0.87 (s, 3H, Si-

C(CH3)3); 0.06 (s, 3H, Si-(CH3)2); 0.05 (s, 3H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 165.6 (C=O); 134.6 (C-2); 129.7 (ar-C); 128.3 (ar-

C); 127.7 (ar-C); 124.8 (CF3); 117.5 (C-1); 109.3 (C(CH3)2); 87.0 (C-8); 85.0 (Cq);

85.2 (Cq); 78.0 (C-5); 76.9 (C-4); 75.2 (C-7); 67.3 (C-10); 65.1 (C-6); 55.9 (O-CH3);

34.3 (C-3); 29.7 (C-9); 28.0 (C(CH3)2); 25.9 (Si-C(CH3)3); 25.9 (C(CH3)2); 23.3 (C-11);

18.2 (Si-C(CH3)3); -4.5 (Si-(CH3)2); -4.7 (Si-(CH3)2). + HRMS (ESI): calcd for C30H43F3NaO6Si [M-Na] : 607.2673, found: 607.2673.

142 Experimental Section

6.2.1.12. Preparation of Compounds described in Chapter 3.1.3.3.

OTBS

9 11

7 35 1 O O O O 193a [(1S,5R)-5-((4S,5S)-5-Allyl-2,2-dimethyl-[1,3]dioxolan-4-yl)-5-methoxymethoxy-1- methyl-pent-3-ynyloxy]-tert-butyl-dimethyl-silane 192a (170 mg, 0.46 mmol, 1 equiv) was dissolved in 7 ml anhydrous DMF, tetrabutylammonium iodide (1.7 mg, 0.005 mmol, 0.01 equiv), and DIPEA (643 l, 3.69 mmol, 5 equiv) and MOM-Cl (281 l, 3.69 mmol, 8 equiv) were added sequentially. The mixture was stirred for 19 h at rt. The reaction was quenched by addition of EtOAc and diluted NaHCO3 (pH = 10). The pH was adjusted to pH = 7 using NH4Cl and the aqueous phase was extracted with EtOAc (3 x 15 ml). The organic phase was dried over MgSO4, filtered and evaporated in vacuo. The crude product was purified by FC (hexane/EtOAc 50:1 → 20:1) to give 193a (151 mg, 80 %).

Rf = 0.35 (hexane/ Et2O 4:1). 1 H-NMR (400 MHz, CDCl3):  = 5.95-5.84 (m, 1H, H-2); 5.17-5.08 (m, 2H, H-1); 5.00

(d, J = 6.6 Hz, 1H, OCH2OCH3); 4.61 (d, J = 6.7 Hz, 1H, OCH2OCH3); 4.44-4.42 (m, 1H, H-6); 4.30-4.25 (m, 1H, H-4); 4.20-4.17 (m, 1H, H-5); 3.99-3.91 (m, 1H, H-10);

3.39 (s, 3H, OCH2OCH3); 2.54-2.50 (m, 2H, H-3); 2.44 (dd, J = 4.9 Hz, 1H, H-9); 2.30

(dd, J = 7.7 Hz, 1H, H-9); 1.52 (s, 3H, C(CH3)2); 1.37 (s, 3H, C(CH3)2); 1.24 (d, J =

5.8 Hz, 3H, H-11); 0.88 (s, 9H, Si-C(CH3)3); 0.06 (s, 3H, Si-(CH3)2); 0.05 (s, 3H, Si-

(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 135.2 (C-2); 117.1 (C-1); 108.7 (C(CH3)2); 94.1

(OCH2OCH3); 85.8 (C-8); 79.1 (C-5); 77.6 (C-7); 76.9 (C-4); 67.7 (C-10); 65.9 (C-6);

56.3 (OCH2OCH3); 34.0 (C-3); 30.0 (C-9); 27.6 (C(CH3)2); 26.0 (Si-C(CH3)3); 25.5

(C(CH3)2); 23.4 (C-11); 18.2 (Si-C(CH3)3); -4.5 (Si-(CH3)2); -4.6 (Si-(CH3)2).

IR (film): max = 2931, 2858, 2361, 2342, 1473, 1378, 1253, 1217, 1066, 1032, 1098, 998, 917, 835, 775 cm-1. + HRMS (ESI): calcd for C20H36NaO4Si [M-Na] : 435.2537, found: 435.2530.

143 Experimental Section

OTBS

Si 11' OH O 9' 1 3 O 7' 1' 3' 5' O O O 5 O O 194a

2-(3-{(4S,5S)-5-[(1S,5R)-5-(tert-Butyl-dimethyl-silanyloxy)-1-methoxymethoxy- hex-2-ynyl]-2,2-dimethyl-[1,3]dioxolan-4-yl}-propyl)-6-hydroxy-4-methoxy- benzoic acid 2-trimethylsilanyl-ethyl ester The reaction was carried out under an argon atmosphere. To a solution of 193a (160 mg, 0.39 mmol, 1.1 equiv) in 1.4 ml abs THF was added dropwise a 0.5 M solution of 9-BBN (1.01 ml, 0.50 mmol, 1.4 equiv) in THF, and the mixture was stirred at rt for 1.5 h at rt. Then a 2 M solution of K3PO4 (358 ml, 0.72 mmol, 2 equiv) was added to the mixture (solution A). In a separate flask 176 (124 mg, 0.36 mmol, 1 equiv) was added to a mixture of trifurylphosphine (50 mg, 0.22 mmol, 0.6 equiv) and [Pd(OAc)2] (12 mg, 0.05 mmol, 0.15 equiv) in 2 ml of degassed abs DME (solution B) and stirred for 5 min (the colour changed from red to yellow). Solution A was added dropwise to solution B at rt. The reaction mixture was refluxed for 5.5 h and the mixture was directly put on Celite and purified by FC (hexane/EtOAcE 100:1 → 50:1 → 20:1 → 10:1) to give 194a (166 mg, 68 %).

Rf = 0.25 (hexane/EtOAc 5:1). 1 H-NMR (400 MHz, CDCl3):  = 11.83 (s, 1H, 2-OH); 6.33 (d, J = 2.5 Hz, 1H, H-5); 6.28 (d, J = 2.6 Hz, 1H, H-3); 4.99 (d, J = 6.5 Hz, 1H, OCH2OCH3); 4.59 (d, J =

6.5 Hz, 1H, OCH2OCH3); 4.45-4.40 (m, 1H, H-); 4.38-4.36 (m, 1H, H-6’); 4.20-4.15 (m, 1H, H-4’); 4.13-4.10 (m, 1H, H-5’); 3.98-3.90 (m, 1H, H-10’); 3.79 (s, 3H, 4-

OCH3); 3.36 (s, 3H, OCH2OCH3); 3.02-2.87 (m, 2H, H-1’); 2.46-2.40 (m, 1H, H-9’); 2.32-2.25 (m, 1H, H-9’), 1.97-1.80 (m, 1H, H-2’); 1.79-1.71 (m, 1H, H-2’); 1.69-1.59

(m, 1H, H-2’); 1.48 (s, 3H, C(CH3)2); 1.34 (s, 3H, C(CH3)2); 1.23 (d, J = 6.1 Hz, 3H, H- 11’); 1.20-1.14 (m, 2H, H-); 0.88 (s, 9H, Si-C(CH3)3); 0.09 (s, 9H, Si-(CH3)3); 0.06 (s,

6H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 171.7 (C=O); 165.8 (C-2); 164.0 (C-4); 147.4 (C-6); 110.9 (C-5); 108.4 (C(CH3)2); 105.1 (C-1); 99.1 (C-3); 94.1 (OCH2OCH3); 85.6 (C-8’); 79.1 (C-5’); 77.7 (C-7’); 77.6 (C-4’); 67.7 (C-10’); 66.1 (C-6’); 63.9 (C-); 56.4

(OCH2OCH3); 55.4 (4-OCH3); 36.8 (C-1’); 32.2 (C-1’); 30.0 (C-3’); 29.2 (C-2’); 27.8

(C(CH3)2); 26.0 (SiC(CH3)3); 25.6 (C(CH3)2); 23.4 (C-11’); 18.2 (Si-C(CH3)3); 17.8 (C-

); -1.4 (Si-(CH3)3); -4.5 (Si-(CH3)2); -4.6 (Si-(CH3)2).

IR (film): max = 2954, 2860, 2342, 1646, 1614, 1250, 1214, 1157, 1099, 1032, 998, 834, 775 cm-1. + HRMS (ESI): calcd for C35H60NaO9Si2 [M-Na] : 703.3668, found: 703.3673.

144 Experimental Section

Si OH O OTBS 1 9' 3 O 7' 11' 1' 3' 5' O O O 5 O O 195a 2-(3-{(4S,5S)-5-[(Z)-(1S,5R)-5-(tert-Butyl-dimethyl-silanyloxy)-1- methoxymethoxy-hex-2-enyl]-2,2-dimethyl-[1,3]dioxolan-4-yl}-propyl)-6- hydroxy-4-methoxy-benzoic acid 2-trimethylsilanyl-ethyl ester To a solution of 194a (156 mg, 0.23 mmol, 1 equiv) in 5 ml EtOAc was added 30 mg of Lindlar’s catalyst (0.014 mmol, 0.06 equiv). The suspension was vigorously stirred under a hydrogen balloon, after every 60 min the flask was flushed with argon and the reaction was monitored by MS and TLC. The mixture was evaporated in vacuo. The crude product was purified by FC (hexane/EtOAc 100:0 → 50:1 → 20:1 → 10:1 → 5:1) to give 195a (134 mg, 86 %).

Rf = 0.11 (hexane/EtOAc 5:1). 20 []D = -25.00° ± 0.06° (c = 0.688, CHCl3). 1 H-NMR (400 MHz, CDCl3):  = 11.84 (s, 1H, 2-OH) ; 6.33 (d, J = 2.5 Hz, 1H, H-3); 6.30 (d, J = 2.7 Hz, 1H, H-5); 5.86-5.81 (m, 1H, H-8’); 5.36-5.41 (m, 1H, H-7’); 4.67

(d, J = 6.6 Hz, 1H, OCH2OCH3); 4.45-4.39 (m, 4H, H-6’, H- and OCH2OCH3); 4.20- 4.16 (m, 1H, H-4’); 4.07-4.04 (m, 1H, H-5’); 3.89-3.82 (m, 1H, H-10’); 3.79 (s, 3H, 4-

OCH3); 3.32 (s, 3H, OCH2OCH3); 3.05-2.98 (m, 1H, H-1’); 2.93-2.86 (m, 1H, H-1’); 2.35-2.20 (m, 2H, H-9’); 1.92-1.83 (m, 1H, H-2’); 1.77-1.58 (m, 3H, H-3’ and H-2’);

1.40 (s, 3H, C(CH3)2); 1.32 (s, 3H, C(CH3)2); 1.20-1.16 (m, 2H, H-); 1.14 (d, J = 6.1 Hz, 3H, H-11’); 0.89 (s, 9H, Si-C(CH3)3); 0.09 (s, 9H, Si-(CH3)3); 0.06 (s, 6 H, Si-

(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 171.8 (C=O); 165.8 (C-2); 163.96 (C-4); 147.5 (C-6); 132.9 (C-8’); 127.5 (C-7’); 110.9 (C-5); 107.9 (C(CH3)2); 105.12(C-1); 99.1 (C-3); 93.5

(OCH2OCH3); 79.2 (C-5’); 77.8 (C-4’); 69.8 (C-6’); 68.6 (C-10’); 63.9 (C-); 56.3

(OCH2OCH3); 55.4 (4-OCH3); 38.1 (C-9’); 36.9 (C-1’); 29.7 (C-3’); 29.0 (C-2’); 28.1

(C(CH3)2); 26.0 (Si-C(CH3)3); 25.5 (C(CH3)2); 23.8 (C-11’); 18.3 (Si-C(CH3)3); 17.7 (C-

); -1.4 (Si-(CH3)3); -4.3 (Si-(CH3)2); -4.6 (Si-(CH3)2).

IR (film): max = 2954, 2361, 2343, 1647, 1614, 1578, 1457, 1370, 1320, 1251, 1214, 1157, 1033, 834, 773, 757 cm-1. + HRMS (MALDI): calcd for C35H62NaO9Si2 [M-Na] : 705.3825, found: 705.3831.

145 Experimental Section

6.2.1.13. Preparation of Compounds described in Chapter 3.1.4.2.

O 1 3 OH 5 OO

202 (4S,5S)-5-Allyl-2,2-dimethyl-1,3-dioxolan-4-carbonic acid To a solution of 191 (119 mg, 0.70 mmol, 1 equiv) in t-BuOH (8 ml) was added 2- methyl-2-butene (743 μl, 7.00 mmol, 10 equiv). A solution of NaClO2 (80 %; 126 mg,

1.40 mmol, 2 equiv) and NaH2PO4 (627 mg, 4.55 mmol, 6.5 equiv) in water (8 ml) was added dropwise to the reaction mixture and it was stirred for 1 hour at rt. Brine

(70 ml) was added to the mixture which was extracted with Et2O (3 x 25 ml). The organic phase was dried over MgSO4, filtered and evaporated in vacuo. The crude product 202 (87 mg, 67 %) was obtained as a yellow oil which was used for the next step without purification.

Rf = 0.20 (EtOAc). 1 H-NMR (400 MHz, CDCl3):  = 5.81-5.91 (m, 1H, H-2); 5.10-5.19 (m, 2H, H-1); 4.57- 4.61 (m, 1H, H-5); 4.40-4.45 (m, 1H, H-4); 2.24-2.49 (m, 2H, H-3) ; 1.61 (s, 3H,

C(CH3)2); 1.40 (s, 3H, C(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 173.4 (C-6); 133.8 (C-2); 118.0 (C-1); 111.0

(C(CH3)2); 77.1 (C-4); 76.6 (C-5); 34.9 (C-3); 27.1 (C(CH3)2); 25.3 (C(CH3)2).

IR (film): max = 3444, 3080, 2985, 2939, 2356, 1728, 1643, 1382, 1215, 1082, 918, 843, 514 cm-1. + HRMS (ESI): calcd for C9H13Na2O4 [M-Na2] : 231.0604, found: 231.0604.

CF3 O O 1 O P 3 O CF3 5 OO

204 Bis(2,2,2-trifluoroethyl) 2-((4S,5S)-5-Allyl-2,2-dimethyl-1,3-dioxolane-4-yl)-2- oxoethylphosphonate

To a solution of 202 (76 mg, 0.41 mmol, 1 equiv) in CH2Cl2 (5 ml) was added 1- chloro-N,N,2-trimethyl-1-propenylamine (108 μl, 0.82 mmol, 2 equiv) and the mixture was stirred for 1 hour at rt. The reaction mixture was evaporated in vacuo and acid

146 Experimental Section chloride 203 was stored under argon. In the meantime a solution of methylphosphonic acid-bis(2,2,2-trifluoroethyl)ester (351 mg, 1.35 mmol, 3.3 equiv) in abs THF (1 ml) was cooled to -98°C and a solution of LiHMDS (1.35 ml, 1.35 mmol, 3.3 equiv) was added. The reaction mixture was stirred for 10 min and subsequently a solution of acid chloride 203 in THF (3 ml) was added to the mixture which was stirred for 1 hour at -98°C. The reaction was quenched by addition of sat.

NH4Cl solution (20 ml) and subsequently extracted with CH2Cl2 (3 x 10 ml). The organic phase was dried over MgSO4, filtered and evaporated in vacuo. The crude product was purified by FC (hexane/EtOAc 50:1 → 25:1 → 10:1 → 5:1 → 1:1) to give 204 (61 mg, 23 % over 3 steps) of a yellow sirup.

Rf = 0.09 (Hexan/EtOAc 5:1), Rf = 0.65 (Hexan/EtOAc 1:1) 1 H-NMR (400 MHz, CDCl3):  = 5.80-5.74 (m, 1H, H-2); 5.14-5.10 (m, 2H, H-1); 4.51-

4.31 (m, 6H, H-4, H-5, P(OCH2CF3)2); 3.76-3.22 (m, 2H, H-7); 2.33-2.07 (m, 2H, H- 3); 1.61 (s, 3H, C(CH3)2); 1.38 (s, 3H, C(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 203.1 (C-6); 133.4 (C-2); 124.0 (CF3); 121.2 (CF3);

118.3 (C-1); 110.7 (C(CH3)2); 82.3 (C-5); 77.8 (C-4); 61.8-63.1 (CH2CF3); 38.4 (C-7); 34.4 (C-3); 26.9 (C(CH3)2); 24.6 (C(CH3)2). + HRMS (ESI): calcd for C14H19NaF6NaO6P [M-Na] : 451.0716, found: 451.0719.

6.2.1.14. Preparation of Compounds described in Chapter 3.1.4.4.

1 3 OPMB 5 OO

207 (4S,5R)-4-Allyl-5-((4-methoxybenzyloxy)methyl)-2,2-dimethyl-1,3-dioxolane To a solution of 190 (779 mg, 4.53 mmol, 1 equiv) and benzyl trichloroacetimidate (2.54 mg, 9.05 mmol, 2 equiv) in cyclohexane (20 ml) was added PPTS (57 mg, 0.23 mmol, 0.05 equiv) at 0°C and the mixture was stirred for 10 min before it was allowed to warmed rt. At this temperature the reaction mixture was stirred for 18 h. The suspension was filtered through Celite. The filtrate was washed with sat.

NaHCO3 solution (2 x 50 ml), dried over MgSO4, filtered and evaporated in vacuo. The crude product was purified by FC (Fine silica gel 0.015-0.040 nm; toluenel/acetone 100:1) to give 207 (706 mg, 53 %) as colorless oil.

Rf = 0.38 (hexane/EtOAc 5:1). 20 []D = -26.4° (c = 0.60, CHCl3). 1 H-NMR (400 MHz, CDCl3):  = 7.27-7.24 (d, J = 8.8 Hz, 2H, ortho-H); 6.89-6.86 (d, J = 8.8 Hz, 2H, meta-H); 5.89-5.89 (m, 1H, H-2); 5.13-5.06 (m, 2H, H-1); 4.54-4.44 (q,

J = 26.7 Hz, 2H, PMB-CH2); 4.29-4.25 (q, J = 12.0 Hz, 1H, H-5); 4.22-4.17 (q, J = 147 Experimental Section

13.4 Hz, 1H, H-4); 3.80 (s, 3H, O-CH3); 3.52-3.44 (m, 2H, H-6); 2.29-2.26 (m, 2H, H- 3); 1.44 (s, 3H, C(CH3)2); 1.34 (s, 3H, C(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 159.4 (p-C); 134.9 (C-2); 130.1 (arom C-1); 129.6

(o-C); 117.1 (C-1); 113.9 (m-C); 108.3 (C(CH3)2); 76.8 (C-4); 76.4 (C-5); 73.3 (PMB- CH2); 68.6 (C-6); 55.4 (O-CH3); 34.2 (C-3); 28.3 (C(CH3)2); 25.7 (C(CH3)2).

IR (film): max = 3075, 2985, 2935, 2909, 2864, 1612, 1512, 1369, 1245, 1172, 1093, 1074, 1035, 820, 580, 513 cm-1. + HRMS (ESI): calcd for C17H24NaO4 [M-Na] : 315.1567, found: 315.1566.

Si OH O  1  3 O 1' 3' 5' O OPMB 5 O O 208 2-(Trimethylsilyl)ethyl 2-hydroxy-4-methoxy-6-(3-((4S,5R)-5-((4- methoxybenzyloxy)methyl)-2,2-dimethyl-1,3-dioxolane-4-yl)propyl)benzoate The reaction was carried out under an argon atmosphere. To a solution of 207 (686 mg, 2.35 mmol, 1.01 equiv) in 13 ml abs THF was added dropwise a 0.5 M solution of 9-BBN (5.98 ml, 2.99 mmol, 1.4 equiv) in THF, and the mixture was stirred at rt for 1 hour. Then a 2 M solution of K3PO4 (2.13 ml, 4.27 mmol, 2 equiv) was added to the mixture (solution A). In a separate flask, 176 (738 mg, 2.13 mmol, 1 equiv) was added to a mixture of trifurylphosphine (297 mg, 1.28 mmol, 0.6 equiv) and [Pd(OAc)2] (72 mg, 0.32 mmol, 0.15 equiv) in 13 ml of degassed abs DME (solution B) and stirred for 5 min (the colour changed from red to yellow). Solution A was added dropwise to solution B at rt. The reaction mixture was refluxed for 4 h and the mixture was directly put on Celite and purified by FC (hexane/EtOAc 100:1 → 50:1 → 20:1 → 10:1) to give 208 (1.07 g (1.91, 89 %).

Rf = 0.25 (hexane/EtOAc 5:1). 1 H-NMR (400 MHz, CDCl3):  = 11.84 (s, 1H, 2-OH); 7.23 (d, J = 9.0 Hz, 2H, o-H); 6.87 (d, J = 9.0 Hz, 2H, m-H); 6.33 (d, J = 2.8 Hz, 1H, H-3); 6.2 (d, J = 2.8 Hz, 1H, H-

5); 4.50-4.38 (m, 4H, PMB-CH2/H-α); 4.24-4.19 (q, J = 6.1 Hz, 1H, H-5’); 4.14-4.08

(m, 1H, H-4’); 3.80 (s, 3H, PMB-OCH3); 3.77 (s, 3H, 4-OCH3); 3.48-3.39 (m, 2H, H-

6’); 3.00-2.81 (m, 2H, H-1’); 1.89-1.41 (m, 4H, H-2’, H-3’); 1.40 (s, 3H, C(CH3)2); 1.32

(s, 3H, C(CH3)2); 1.17-1.12 (m, 2H, H-β); 0.07 (s, 9H, Si(CH3)3). 13 C-NMR (100 MHz, CDCl3):  = 171.7 (C=O); 165.7 (C-2); 163.9 (C-4); 159.4 (p-C); 147.3 (C-6); 130.1 (PMB arom C-1); 129.5 (o-C); 113.9 (m-C); 110.9 (C-5); 108.0

(C(CH3)2); 105.1 (C-1); 99.1 (C-3); 77.4 (C-4’); 76.5 (C-5’); 73.2 (PMB-CH2); 68.8 (C- 6’); 63.8 (C-α); 55.4 (4-OCH3/PMB-CH3); 36.8 (C-1’); 32.2 (C-2’); 26.4 (C-3’); 28.4

(C(CH3)2); 25.7 (C(CH3)2); 17.7 (C-β); -1.4 (Si-(CH3)3).

148 Experimental Section

IR (film): max = 3397, 2925, 2860, 1646, 1612, 1579, 1512, 1414, 1368, 1300, 1429, 1209, 1158, 1038, 836, 516 cm-1. + HRMS (ESI): calcd for C30H44NaO8Si [M-Na] : 583.2698, found: 583.2691.

Si OH O  1  3 O 1' 3' 5' O OH 5 O O 209 2-(Trimethylsilyl)ethyl 2-Hydroxy-6-(3-((4S,5R)-5-(hydroxymethyl)-2,2-dimethyl- 1,3-dioxolane-4-yl)propyl)-4-methoxybenzoate To a solution of 208 (250 mg, 0.45 mmol, 1 equiv) in 10 ml of a mixture of

CH2Cl2/water (19:1) was added DDQ (102 mg, 0.45 mmol, 1 equiv) at -10°C and the mixture was stirred for 2 h, followed by 1.5 h at -5°C and 3 h a 0°C. Sat. NaHCO3 solution (10 ml) was added to the reaction mixture and it was extracted with CH2Cl2 (3 x 10 ml). The organic phase was dried over MgSO4, filtered and evaporated in vacuo. The crude product was purified by FC (hexane/EtOAc 10:1 → 5:1 → 2:1) to give 209 (136 mg, 69 %) as a pale orange sirup.

Rf = 0.32 (hexane/EtOAc 2:1). 20 []D = +17.6° (c = 0.61, CHCl3). 1 H-NMR (400 MHz, CDCl3):  = 11.83 (s, 1H, 2-OH); 6.34 (d, J = 2.7 Hz, 1H, H-3); 6.28 (d, J = 2.7 Hz, 1H, H-5); 4.45-4.40 (m, 2H, H-); 4.16-4.11 (m, 2H, H-4’, H-5’);

3.80 (s, 3H, 4-OCH3); 3.61-3.57 (m, 2H, H-6’); 2.99-2.88 (m, 2H, H-1’); 1.87-1.60 (m,

4H, H-2’, H-3’); 1.46 (s, 3H, C(CH3)2); 1.35 (s, 3H, C(CH3)2); 1.20-1.14 (m, 2H, H-β); 0.09 (s, 9H, Si-(CH3)3). 13 C-NMR (100 MHz, CDCl3):  = 171.7 (C=O); 165.8 (C-2); 164.0 (C-4); 147.2 (C-6);

110.9 (C-5); 108.2 (C(CH3)2); 105.1 (C-1); 99.2 (C-3); 78.0 (C-5’); 77.4 (C-4’); 63.9 (C-); 62.0 (C-6’); 55.4 (4-O-CH3); 36.8 (C-1’); 29.0 (C-2’/C-3’); 25.6 (C(CH3)2); 28.4

(C(CH3)2); 17.8 (C-); -1.4 (Si(CH3)3). -1 IR (film): max = 3480, 2933, 1645, 1613, 1371, 1321, 1250, 1157, 1041, 836 cm . + HRMS (ESI): calcd for C22H36NaO7Si [M-Na] : 463.2123, found: 463.2128.

149 Experimental Section

Si OH O  1  3 O 1' 3' 5' O O 5 O O 210 2-(Trimethylsilyl)ethyl 2-(3-((4S,5S)-5-formyl-2,2-dimethyl-1,3-dioxolane-4- yl)propyl)-6-hydroxy-4-methoxybenzoate

To a solution of oxalyl chloride (43 μl, 0.50 mmol, 1.6 equiv) in abs CH2Cl2 (3.5 ml) was added DMSO (70 μl, 0.99 mmol, 3.2 equiv) at -78°C. The mixture was stirred for 10 min at -78°C and subsequently a solution of 209 (136 mg, 0.31 mmol, 1 equiv) in

CH2Cl2 (3.5 ml) was added. The reaction mixture was stirred for 1 hour at -78°C. TEA (137 μl, 0.99 mmol, 3.2 equiv) was added dropwise and after 10 min the mixture was allowed to warm to rt. Water (15 ml) und CH2Cl2 (15 ml) were added and the aqueous phase was extracted with CH2Cl2 (2 x 10 ml). The organic phase was dried over MgSO4, filtered and evaporated in vacuo. The crude product was purified by FC (hexane/EtOAc 10:1 → 5:1→ 2:1) to give 210 (90 mg, 67 %) as a pale yellow oil.

Rf = 0.64 (hexane/EtOAc 2:1). 20 []D = -1.7° (c = 0.47, CHCl3). 1 H-NMR (400 MHz, CDCl3):  = 11.83 (s, 1H, 2-OH); 9.64 (d, J = 3.2 Hz, 1H, H-6’); 6.34 (d, J = 2.9 Hz, 1H, H-3); 6.27 (d, J = 2.9 Hz, 1H, H-5); 4.43-4.40 (m, 2H, H-);

4.34-4.29 (m, 1H, H-4’); 4.23 (dd, J = 7.1 Hz, 1H, H-5’); 3.79 (s, 3H, 4-OCH3); 2.96- 2.85 (m, 2H, H-1’); 1.84-1.53 (m, 4H, H-2’, H-3’); 1.58 (s, 3H, C(CH3)2); 1.40 (s, 3H,

C(CH3)2); 1.17-1.12 (m, 2H, H-); 0.09 (s, 9H, Si-(CH3)3). 13 C-NMR (100 MHz, CDCl3):  = 202.1 (C-6’); 171.5 (C=O); 165.7 (C-2); 163.8 (C-4); 146.7 (C-6); 110.7 (C-5); 110.5 (C(CH3)2); 104.9 (C-1); 99.1 (C-3); 82.03 (C-5’); 78.6

(C-4’); 63.8 (C-); 55.3 (4-OCH3); 36.4 (C-1’); 29.7 (C-3’); 28.6 (C-2’); 27.7

(C(CH3)2); 25.3 (C(CH3)2); 17.6 (C-β); -1.6 (Si-(CH3)3).

IR (film): max = 2924, 2853, 1734, 1647, 1614, 1579, 1461, 1372, 1252, 1159, 839 cm-1. HRMS (ESI): no signal found.

150 Experimental Section

Si OH O  1  3 O O 1' 3' 5' O OH 5 O O 211 (4S,5S)-5-(3-(3-Hydroxy-5-methoxy-2-((2- (trimethylsilyl)ethoxy)carbonyl)phenyl)propyl)-2,2-dimethyl-1,3-dioxolane-4- carbonic acid To a solution of 210 (82 mg, 0.19 mmol, 1 equiv) in 2.2 ml t-BuOH was added 2- methyl-2-butene (199 μl, 1.87 mmol, 10 equiv). A solution of NaClO2 (80 %; 34 mg, 0.37 mmol, 2.0 equiv) and NaH2PO4·H2O (168 mg, 1.22 mmol, 6.4 equiv) in water (2.2 ml) was added to the mixture which was stirred for 1 hour. Brine (8 ml) was added to the mixture which was extracted with Et2O (3 x 5 ml). The organic phase was dried over MgSO4, filtered and evaporated in vacuo. The crude product 211 (111 mg, quant was used for the next reaction without purification.

Rf = 0.07 (hexane/EtOAc 2:1). 1 H-NMR (400 MHz, CDCl3):  = 11.83 (s, 1H, 2-OH); 6.33 (d, J = 2.6 Hz, 1H, H-3); 6.27 (d, J = 2.6 Hz, 1H, H-5); 4.54 (d, J = 7.2 Hz, 1H, H-5’); 4.45-4.39 (m, 2H, H-);

4.32-4.37 (m, 1H, H-4’); 3.79 (s, 3H, 4-OCH3) ; 2.83-3.00 (m, 2H, H-1’); 1.52-1.86 (m, 4H, H-2’, H-3’) ; 1.58 (s, 6H, C(CH3)2); 1.37 (s, 6H, C(CH3)2); 1.11-1.18 (m, 2H,

H-β); 0.08 (s, 9H, Si(CH3)3). 13 C-NMR (100 MHz, CDCl3):  = 171.7 (C-6’); 171.7 (C=O); 165.7 (C-2); 164.0 (C-4); 147.2 (C-6); 110.8 (C-5); 110.7 (C(CH3)2); 105.1 (C-1); 99.2 (C-3); 77.8 (C-4’); 77.4

(C-5’); 64.0 (C-); 55.4 (4-OCH3); 36.6 (C-1’); 30.5 (C-3’); 28.8 (C-2’); 25.7/27.2

(C(CH3)2); 17.7 (C-β); -1.4 (Si-(CH3)3). -1 IR (film): max = 2839, 1726, 1645, 1612, 1577, 1370, 1251, 1157, 1092, 836 cm . + HRMS (ESI): calcd for C22H34NaO8Si [M-Na] : 477.1915, found: 477.1911.

151 Experimental Section

Si OH O  1  3 O O O 1' 3' 5' P O O CF3 5 O O O CF3 213

2-(Trimethylsilyl)ethyl 2-(3-((4S,5S)-5-(2-(bis(2,2,2- trifluoroethoxy)phosphoryl)acetyl)-2,2-dimethyl-1,3-dioxolane-4-yl)propyl)-6- hydroxy-4-methoxybenzoate

To a solution of 211 (99 mg, 0.17 mmol, 1 equiv) in CH2Cl2 (2 ml) was added 1- chloro-N,N,2-trimethyl-1-propenylamine (44 μl, 0.33 mmol, 2 equiv) and it was stirred for 1 hour at rt gerührt. The mixture was evaporated in vacuo and the residue 212 was dried in high vacuum for 30 min. In the meantime, a solution of methylphosphonic acid bis(2,2,2-trifluoroethyl)ester (143 mg, 0.55 mmol, 3.2 equiv) in THF (0.5 ml) was cooled to -98°C and a 1 M solution of LiHMDS (551 μl, 0.55 mmol, 3.2 equiv) was added dropwise to the mixture which was stirred for 10 min, before a solution of acid chloride 212 in THF (1.2 ml) was added. The mixture was stirred for 1 hour at -98°C. The reaction was quenched by addition of

NH4Cl solution (10 ml) and it was allowed to warm to rt. The mixture was extracted with CH2Cl2 (3 x 4 ml), dried over MgSO4, filtered and evaporated in vacuo. The crude product was purified by FC (hexane/EtOAc 10:1 → 5:1 → 2:1 → 0:1) to give 213 (39 mg, mmol, 34 % over 3 step) as a yellow oil.

Rf = 0.48 (hexane/EtOAc 2:1). 20 []D = -11.7° (c = 0.77, CHCl3). 1 H-NMR (400 MHz, CDCl3):  = 11.80 (s, 1H, 2-OH); 6.33 (d, J = 2.7 Hz, 1H, H-3);

6.26 (d, J = 2.7 Hz, 1H, H-5); 4.51-4.28 (m, 8H, H-4’, H-5’, P(OCH2CF3)2, H-); 3.79

(s, 3H, 4-OCH3); 3.45 (dd, J = 19.4 Hz and 16.9 Hz, 2H, H-7’); 2.95-2.81 (m, 2H, H-

1’); 1.86-1.51 (m, 4H, H-2’, H-3’); 1.58 (s, 2H, C(CH3)2); 1.37 (s, 2H, C(CH3)2); 1.18-

1.11 (m, 2H, H-β); 0.09 (s, 9H, Si-(CH3)3). 13 C-NMR (100 MHz, CDCl3):  = 203.3 (C-6’); 171.6 (C=O); 165.8 (C-2); 164.0 (C-4);

146.9 (C-6); 121.3 (CF3); 124.1 (CF3); 110.9 (C-5); 110.6 (C(CH3)2); 105.0 (C-1); 99.2

(C-3); 82.7 (C-5’); 78.5 (C-4’); 63.9 (C-); 61.9 (CH2CF3); 62.7 (CH2CF3); 55.4 (4- OCH3); 38.3/39.7 (C-7’); 36.4 (C-1’); 30.2 (C-3’); 29.0 (C-2’); 24.7 (C(CH3)2); 27.1

(C(CH3)2); 17.7 (C-β); -1.5 (Si-(CH3)3). -1 IR (film): max = 2954, 1720, 1647, 1613, 1579, 1419, 1252, 1159, 1068, 837 cm . + HRMS (ESI): calcd for C27H39F6NaO10PSi [M-Na] : 719.1846, found: 719.1858.

152 Experimental Section

6.2.2. Synthesis of the Dideoxy Analog D6

1 35 O 214 Hex-5-enal

To a solution of oxalylchloride (2.90 ml, 30.31 mmol, 1.5 equiv) in CH2Cl2 (50 ml) was added DMSO (4.70 ml, 66.61 mmol, 3 equiv) dropwise within 5 min at -78°C. The mixture was stirred for 10 min at -78°C and a solution of 5-hexen-1-ol (150) (2.50 ml,

20.82 mmol, 1 equiv) in CH2Cl2 (7.5 ml) was added dropwise within 10 min. The reaction mixture was stirred for 1 hour at -78°C. TEA (9.20 ml, 66.19 mmol, 3 equiv) was added within 10 min, before it was allowed to warm to 0°C. Water (60 ml) and

CH2Cl2 (60 ml) were added. The aqueous phase was washed with CH2Cl2 (2 x 60 ml) and the combined organic phases were dried over MgSO4, filtered and evaporated in vacuo (up to 700 mbar). The crude product was purified via FC (pentane/Et2O 10:1) to give 214 (2.0 g, quant) as a colorless oil.

Rf = 0.53 (hexane/EtOAc 5:1), Rf = 0.44 (pentane/Et2O 10:1). 1 H-NMR (400 MHz, CDCl3):  = 9.77 (t, J = 1.6 Hz , 1H, H-6); 5.81-5.71 (m, 2H, H-2); 5.05-4.93 (m, 2H, H-1); 2.46-2.42 (m, 2H, H-5); 2.14-2.04 (q, J = 7.4 Hz, 2H, H-3); 1.77-1.63 (m, 2H, H-4). 13 C-NMR (100 MHz, CDCl3):  = 202.5 (C-6); 137.7 (C-2); 115.7 (C-1); 43.3 (C-5); 33.1 (C-3); 21.3 (C-4). -1 IR (film): max = 3075, 2939, 2863, 1727, 1640, 1443, 1356, 1137, 993, 910 cm . + HRMS (EI): calcd for C6H10O [M-H] : 98.0726, found: 98.0724.

OTES

9 11

7 1 35 OH 215 (R)-10-(Triethylsilyloxy)undec-1-en-7-yn-6-ol To a solution of 156R (100 mg, 0.50 mmol, 1 equiv) in abs THF (5 ml) was added n- BuLi (1.6 M in hexane; 380 μl, 0.61 mmol, 1.2 equiv) at -10°C and stirred for 20 min. The mixture was cooled to -78°C and a precooled solution of 214 (60 mg, 0.61 mmol, 1.2 equiv) in abs THF (3.5 ml) was added dropwise. The reaction mixture was stirred for 1.5 h at -78°C, allowed to warm to -18°C. The reaction was quenched at this temperature by addition of sat. NH4Cl solution (10 ml). The mixture was extracted

153 Experimental Section

with Et2O (1 x 10 ml), dried over MgSO4, filtered and evaporated in vacuo. The crude product was purified by FC (hexane/Et2O 4:1 → 2:1) to give 215 (76 mg, 51 %) as a pale yellow oil.

Rf = 0.51 (hexane/Et2O 4:1). 1 H-NMR (400 MHz, CDCl3):  = 5.84-5.77 (m, 1H, H-2); 5.04-4.94 (m, 2H, H-1); 4.36- 4.35 (m, 1H, H-6); 3.95-3.90 (m, 1H, H-10); 2.42-2.24 (m, 2H, H-9); 2.11-2.06 (q, J = 7.6 Hz, 2H, H-3); 1.72-1.65 (m, 2H, H-5); 1.58-1.52 (m, 2H, H-4’); 1.24-1.22 (d, J =

6.0 Hz, 3H, H-11); 0.99-0.94 (t, J = 7.9 Hz, 9H, Si-CH2CH3); 0.58-0.63 (q, J = 7.9 Hz,

6H, Si-CH2CH3). 13 C-NMR (100 MHz, CDCl3):  = 138.6 (C-2); 114.9 (C-1); 82.9 (C-7/C-8); 67.6 (C- 10); 62.7 (C-6); 37.6 (C-5); 33.5 (C-3); 29.8 (C-9); 24.6 (C-4); 23.5 (C-11); 7.0 (Si-

CH2CH3); 5.0 (Si-CH2CH3).

IR (film): max = 3379, 3077, 2955, 2912, 2876, 1813, 1641, 1459, 1377, 1239, 1125, 1096, 1000, 909, 724 cm-1. + HRMS (EI): calcd for C15H27O2Si [M-C2H5] : 267.1775, found: 267.1777.

OTES

9 11

7 1 35 O O 216 (R)-10-(Triethylsilyloxy)undec-1-en-7-yn-6-ol To a solution of 215 (587 mg, 1.98 mmol, 1 equiv) in DMF (60 ml) was added sequentially TBAI (3.7 mg, 0.01 mmol, 0.005 equiv), DIPEA (2.76 ml, 15.85 mmol, 8 equiv) und MOM-Cl (1.20 ml, 15.85 mmol, 8 equiv). The mixture was stirred for

17 h. EtOAc (50 ml) and diluted NaHCO3 solution (50 ml) were added to the mixture.

The pH-value was adjusted to pH 7 by addition of sat. NH4Cl solution. The phases were separated and the aqueous phase was extracted with EtOAc (3 x 50 ml). The organic phase was dried over MgSO4, filtered, and evaporated in vacuo. The crude product was purified by FC (hexane/EtOAc 20:1) to give 216 (614 mg, 91 %) as a colorless oil.

Rf = 0.50 (hexane/EtOAc 10:1). 1 H-NMR (400 MHz, CDCl3):  = 5.84-5.76 (m, 1H, H-2); 5.04-4.97 (m, 2H, H-1); 4.75

(dd, 2H, MOM-CH2); 4.33-4.30 (m, 1H, H-6); 3.95-3.90 (m, 1H, H-10); 3.36 (s, 3H, MOM-CH3); 2.43-2.24 (m, 2H, H-9); 2.12-2.06 (q, J = 7.3 Hz, 2H, H-3); 1.75-1.68 (m, 2H, H-5); 1.61-1.54 (m, 2H, H-4); 1.23 (d, J = 6.3 Hz, 3H, H-11); 0.95 (t, J = 8.2 Hz,

9H, Si-CH2CH3); 0.60 (q, J = 8.2 Hz, 6H, Si-CH2CH3).

154 Experimental Section

13 C-NMR (100 MHz, CDCl3):  = 138.7 (C-2); 114.8 (C-1); 94.0 (MOM-CH2); 83.5 (C- 8); 80.3 (C-7); 67.6 (C-10); 65.8 (C-6’), 55.7 (MOM-CH3); 35.5 (C-5); 33.5 (C-3); 29.9

(C-9); 24.8 (C-4); 23.5 (C-11); 7.0 (Si-CH2CH3); 5.0 (Si-CH2CH3).

IR (film): max = 3077, 2954, 2877, 1641, 1461, 1239, 1097, 1034, 1000,741, 735, 725 cm-1. + HRMS (EI): calcd for C15H27O2Si [M-C2H5] : 311.2050, found: 311.2050.

OTES

 Si 11' OH O 9' 1  3 O 7' 3' 5' O O O 5 1' 217 (R)-2-(Trimethylsilyl)ethyl 2-Hydroxy-4-methoxy-6-(6-(methoxymethoxy)-10- (triethylsilyloxy)undec-7-ynyl)benzoate The reaction was carried out under an argon atmosphere. To a solution of 216 (100 mg, 0.30 mmol, 1.1 equiv) in 0.65 ml abs THF was added dropwise a 0.5 M solution of 9-BBN in THF (0.75 ml, 0.37 mmol, 1.2 equiv), and the mixture was stirred at rt for 1 hour. Then a 2 M solution of K3PO4 (267 l, 0.53 mmol, 2 equiv) was added to the mixture (solution A). In a separate flask 176 (92 mg, 0.26 mmol, 1 equiv) was added to a mixture of trifurylphosphine (37 mg, 0.16 mmol, 0.6 equiv) and [Pd(OAc)2] (9 mg, 0.04 mmol, 0.15 equiv) in 1 ml of degassed abs DME (solution B) and stirred for 5 min (the color changed from red to yellow). Then solution A was added dropwise to solution B at rt. The reaction mixture was refluxed for 1.5 h and directly put on Celite and purified by FC (hexane/Et2O 20:1 → 10:1) to give 217 (133 mg, 83 %) as colorless oil.

Rf = 0.28 (hexane/Et2O 10:1). 1 H-NMR (400 MHz, CDCl3):  = 11.83 (s, 1H, 2-OH); 6.32-6.33 (d, J = 2.9 Hz , 1H, H-

3); 6.27 (d, J = 2.9 Hz, 1H, H-5); 4.71 (dd, 2H, MOM-CH2); 4.39-4.44 (m, 2H, H-);

4.32-4.30 (m, 1H, H-6’); 3.94-3.91 (m, 1H, H-10’); 3.79 (s, 3H, 4-OCH3); 3.36 (s, 3H,

MOM-CH3); 2.88 (t, J = 7.7 Hz, 2H, H-1’); 2.42-2.27 (m, 2H, H-9’); 1.72-1.68 (m, 2H, H-5’); 1.59-1.51 (m, 2H, H-2’); 1.50-1.48 (m, 2H, H-4’); 1.42-1.36 (m, 2H, H-3’); 1.23 (d, J = 7.1 Hz, 3H, H-11’); 1.13-1.17 (m, 2H, H-β); 0.95 (t, J = 8.2 Hz, 9H, Si-

CH2CH3); 0.59 (q, J = 8.2 Hz, 6H, Si-CH2CH3); 0.09 (s, 9H, Si-(CH3)3). 13 C-NMR (100 MHz, CDCl3):  = 171.8 (C=O); 165.7 (C-2); 163.9 (C-4); 147.9 (C-6);

110.8 (C-5); 105.1 (C-1); 99.0 (C-3); 94.0 (MOM-CH2); 83.4 (C-8’); 80.4 (C-7’); 67.6

(C-10’); 65.9 (C-6’); 63.8 (C-); 55.7 (MOM-CH3); 55.4 (4-OCH3); 36.9 (C-1’); 36.1 (C-5’); 32.0 (C-2’); 29.9 (C-9’); 29.6 (C-3’); 25.5 (C-4’); 23.5 (C-11’); 17.7 (C-β); 6.9

(Si-CH2CH3); 5.0 (Si-CH2CH3); -1.4 (Si-(CH3)3).

155 Experimental Section

IR (film): max = 2950, 2875, 1646, 1613, 1576, 1251, 1157, 1038, 837, 743, 732, 726 cm-1. + HRMS (EI): calcd for C32H56NaO7Si2 [M-Na] : 631.3457, found: 631.3457.

 Si OH O OTES 1  9' 11' 3 O 7' 3' 5' O O O 5 1' 218 (R,Z)-2-(Trimethylsilyl)ethyl 2-hydroxy-4-methoxy-6-(6-(methoxymethoxy)-10- (triethylsilyloxy)undec-7-enyl)benzoate To a solution of 217 (924 mg, 1.52 mmol, 1 equiv) in 40 ml EtOAc was added Lindlar’s catalyst (162 mg, 0.08 mmol, 0.05 equiv). The heterogenous suspension was vigorously stirred under a hydrogen balloon, after every 30 min the flask was flushed with argon and the reaction was monitored by MS and TLC. After 3 h the reaction was complete. The mixture was directly put on Celite and purified by FC (hexane/ EtOAc 50:1 → 20:1 → 10:1) to give 218 (853 mg, 92 %).

Rf = 0.35 (hexane/EtOAc 10:1). 1 H-NMR (400 MHz, CDCl3):  = 11.83 (s, 1H, 2-OH); 6.33 (d, 1H, J = 2.6 Hz, H-3); 6.27 (d, 1H, J = 2.6 Hz, H-5); 5.67-5.60 (m, 1H, H-8’); 5.29 (t, 1H, J = 9.8 Hz, H-7’);

4.46-4.67 (m, 2H, MOM-CH2); 4.44-4.39 (m, 2H, H-); 4.36-4.30 (m, 1H, H-6’); 3.88-

3.81 (m, 1H, H-10’); 3.79 (s, 3H, 4-OCH3); 3.34 (s, 3H, MOM-CH3); 2.88 (t, 2H, J = 8.0 Hz, H-1’); 2.31-2.22 (m, 2H, H-9’); 1.65-1.52 (m, 3H, H-2’, H-5’); 1.44-1.26 (m, 5H, H-3’, H-4’, H-5’); 1.26 (d, 3H, J = 2.1 Hz, H-11’); 1.26-1.09 (m, 5H, H-β (2H), 0.95

(t, 9H, J = 8.0 Hz, Si-CH2CH3); 0.59 (q, 6H, J = 8.0 Hz, Si-CH2CH3); 0.09 (s, 9H, Si-

(CH3)3). 13 C-NMR (100 MHz, CDCl3):  = 171.8 (C=O);165.7 (C-2); 163.9 (C-4); 148.0 (C-6);

131.6 (C-7’); 130.1 (C-8’); 110.8 (C-5); 105.1 (C-1); 99.0 (C-3); 93.5 (MOM-CH2);

71.0/71.1 (C-6’); 68.3/68.4 (C-10’); 63.8 (C-α); 55.4 (4-OCH3/MOM-CH3); 37.9/38.0 (C-9’); 37.0 (C-1’); 35.8/35.9 (C-5’); 32.1 (C-2’); 30.0 (C-3’); 25.6/25.8 (C-4’);

23.6/23.7 (C-11’); 17.7 (C-β); 7.0 (Si-CH2CH3); 5.0/5.1 (Si-CH2CH3); -1.4 (Si-(CH3)3).

IR (film): max = 2950, 2875, 1646, 1613, 1577, 1320, 1251, 1204, 1157, 1096, 1037, 837, 743, 725 cm-1. + HRMS (ESI): calcd for C32H58NaO7Si2 [M-Na] : 633.3613, found: 633.3616.

156 Experimental Section

OH O OH 1 9' 11' 3 OH 7' 3' 5' O O O 5 1' 219 (R,Z)-2-Hydroxy-6-(10-hydroxy-6-(methoxymethoxy)undec-7-enyl)-4- methoxybenzoesäure To a solution of 218 (746 mg, 1.22 mmol, 1 equiv) in THF (45 ml) was added TBAF (1 M in THF; 2.4 ml, 2.44 mmol, 2 equiv). The reaction mixture was stirred for 2 h and sat. NH4Cl solution was added. The aqueous phase was extracted with EtOAc (3 x 10 ml). The organic phase was dried over MgSO4, filtered and evaporated in vacuo. The crude product was purified by FC (EtOAc/MeOH 100:0 → 10:1 → 2:1) to give 219 (436 mg, 90 %) as a colorless solid.

Rf = 0.43 (EtOAc/MeOH 10:1). 1 H-NMR (400 MHz, CDCl3):  = 6.25 (s, 1H, H-3/H-5); 5.17 (s, 1H, H-3/H-5); 5.58-

5.45 (m, 1H, H-8’); 5.30-5.22 (m, 1H, H-7’); 4.71-4.43 (m, 2H, MOM-CH2); 4.33-4.28 (m, 1H, H-6’); 3.89-3.81 (m, 1H, H-10’); 3.73 (s, 3H, 4-OCH3); 3.32 (s, 3H, MOM-

CH3); 2.35-2.12 (m, 2H, H-9’); 3.13-2.94 (m, 2H, H-1’); 1.79-1.15 (m, 11H, H-2’, H-3’, H-4’, H-5’, H-11’). 13 C-NMR (100 MHz, CDCl3):  = bad signal/noise ratio. -1 IR (film): max = 3367, 2933, 2877, 1610, 1582, 1430, 1375, 1157, 1034, 843 cm . + HRMS (ESI): calcd for C21H32NaO7 [M-Na] : 419.2040, found: 419.2040.

11' OH O 1 9' 3 O 7' 3' 5' O O O 5 1' 220 (S,Z)-16-Hydroxy-14-methoxy-7-(methoxymethoxy)-3-methyl-3,4,7,8,9,10,11,12- octahydro-1H-benzo[c][1]oxacyclotetradecin-1-on To solution of 219 (427 mg, 1.08 mmol, 1 equiv) in toluene (150 ml, 0.007 M) and

THF (4 ml) as cosolvent were added PPh3 (565 mg, 2.16 mmol, 2 equiv) und DEAD (350 μl, 2.16 mmol, 2 equiv). The reaction mixture was stirred for 1 hour at rt. The crude product was adsorbed on Celite and purified by FC (hexane/EtOAc 10:1 → 5:1 → 3:1). The flask was rinsed with EtOAc and this solution was also applied to the column. 220 (261 mg, 64 %) was obtained as colorless oil.

Rf = 0.80 (hexane/EtOAc 1:1). 1 H-NMR (400 MHz, CDCl3):  = 12.14 (s, 1H, 2-OH); 6.32-6.31 (m, 1H, H-3); 6.29- 6.27 (m, 1H, H-5); 5.60-5.92 (m, 1H, H-8’); 5.51-5.46 (m, 0.4H, H-7’); 5.44-5.37 (m, 1H, H-10’); 5.34-5.28 (m, 0.6H, H-7’); 5.10-5.03 (m, 0.5H, H-10’); 4.53-4.70 (m, 2H,

157 Experimental Section

MOM-CH2); 4.40-4.56 (m, 1H, H-6’); 3.79 (s, 3H, 4-OCH3); 3.37 (s, 3H, MOM-CH3); 3.21-3.07 (m, 1H, H-1’); 2.98-2.88 (m, 1H, H-9’); 2.73-2.51 (m, 1H, H-1’); 2.247-2.27 (m, 1H, H-9’); 1.78-1.72 (m, 1H, H-5’); 1.62-1.22 (m, 10H, H-2’, H-3’, H-4’, H-5’, H- 11’(d, 3H, J = 6.7 Hz)). 13 C-NMR (100 MHz, CDCl3):  = 172.0 (C=O); 166.3 (C-2); 165.4 (C-2); 164.1 (C-4); 164.0 (C-4); 148.2 (C-6); 148.1 (C-6); 133.1 (C-7’); 132.1 (C-7’); 130.7 (C-8’); 127.6 (C-8’); 109.5 (C-5); 109.2 (C-5); 105.8 (C-1); 105.1 (C-1); 98.8 (C-3); 98.7 (C-3); 93.6

(MOM-CH2); 93.5 (MOM-CH2); 72.7 (C-10’); 71.9 (C-10’); 70.6 (C-6’); 69.1 (C-6’);

55.3/55.4 (MOM-CH3/4-OCH3); 36.1 (C-1’); 35.1 (C-9’); 34.4 (C-1’); 33.4/33.5 (C- 5’/C-9’); 30.9 (C-2’); 29.3 (C-2’); 27.4 (C-3’); 27.1 (C-3’); 24.6 (C-4’); 22.8 (C-4’); 21.4 (C-11’); 18.5 (C-11’).

IR (film): max = 2932, 2860, 1736, 1640, 1612, 1576, 1249, 1204, 1158, 1033, 829 cm-1. + HRMS (ESI): calcd for C21H30NaO6 [M-Na] : 401.19346, found: 401.1935.

11' OH O 1 9' 3 O 7' 3' 5' O OH 5 1' 221 (S,Z)-7,16-Dihydroxy-14-methoxy-3-methyl-3,4,7,8,9,10,11,12-octahydro-1H- benzo[c][1]oxacyclotetradecin-1-one To a solution of 220 (235 mg, 0.62 mmol) in MeOH (21 ml) was added sulfonic acid resin (3.1 mmol/g; 220 mg, 0.68 mmol) and the mixture was refluxed for 3 h. The resin was filtered off and washed with MeOH. The filtrate was evaporated in vacuo and the residue was purified by FC (hexane/EtOAc 4:1 → 2:1 → 1:1) to give 221 (152 mg, 73 %) as colorless crystals.

Rf = 0.47 (hexane/EtOAc 1:1). 1 H-NMR (400 MHz, CDCl3):  = 12.09 (s, 0.45H, 2-OH); 11.78 (s, 0.4H, 2-OH); 6.32 (d, J = 2.6 Hz, 1H, H-3); 6.29 (d, J = 2.6 Hz, 1H, H-5); 5.82-5.76 (m, 0.45H, H-8’); 5.65-5.60 (m, 0.5H, H-7’); 5.56-5.50 (m, 0.54H, H-8’); 5.48-5.38 (m, 1.5H, H-10’ (1H), H-7’ (0.5H)); 4.61-4.56 (m, 0.55H, H-6’); 4.48-4.53 (m, 0.45H, H-6’); 3.79 (s, 3H, 4-

OCH3); 3.22-3.06 (m, 1H, H-1’); 2.97-2.84 (m, 1H, H-9’); 2.73-2.53 (m, 1H, H-1’); 2.49-2.42 (m, 0.5H, H-9’); 2.35-2.29 (m, 0.5H, H-9’); 1.79-1.68 (m, 1H, H-5’); 1.67- 1.30 (m, 9.45H, H-2’, H-3’, H-4’, H-5’, H-11 (d, J = 6.6 Hz); 1.22-1.17 (m, 0.55H, H- 4’). 13 C-NMR (100 MHz, CDCl3):  = 171.8 (C=O); 171.3 (C=O); 166.1 (C-2); 165.5 (C-2); 164.1 (C-4); 164.1 (C-4); 148.1 (C-6); 135.7 (C-7’); 134.9 (C-7’); 129.1 (C-8’); 126.0 (C-8’); 109.3 (C-5); 109.3 (C-5); 105.7 (C-1); 105.2 (C-1); 98.8 (C-3); 98.8 (C-3); 72.6

(C-10’); 71.8 (C-10’); 67.8 (C-6’); 66.5 (C-6’); 55.4 (4-OCH3); 36.1 (C-5’); 35.7 (C-5’);

158 Experimental Section

35.4 (C-1’); 35.2 (C-9’); 34.4 (C-1’); 33.3 (C-9’); 30.5 (C-2’); 29.4 (C-2’); 27.4 (C-3’); 27.2 (C-3’); 24.4(C-4’); 22.7 (C-4’); 21.4 (C-11’); 18.4 (C-11’).

IR (film): max = 3440, 2932, 2856, 1739, 1637, 1611, 1575, 1248, 1201, 1158, 1040, 826 cm-1 + HRMS (ESI): calcd for C19H26NaO5 [M-Na] : 357.16725, found: 357.1672. Melting point: 66°C.

11' OH O 1 9' 3 O 7' 3' 5' O O 5 1' D6 (S,Z)-16-Hydroxy-14-methoxy-3-methyl-3,4,9,10,11,12-hexahydro-1H- benzo[c][1]oxacyclotetradecin-1,7(8H)-dione

To a solution of 221 (91 mg, 0.27 mmol, 1 equiv) in CH2Cl2 (5 ml) was added DMP

(15 % in CH2Cl2; 400 μl, 0.19 mmol, 0.7 equiv). The mixture was stirred for 30 min, a second portion of DMP (400 μl, 0.19 mmol, 0.7 equiv) was added, followed by a third portion after 1 hour (330 μl, 0.16 mmol, 0.6 equiv). After 3 h the mixture was directly put on a column and was purified by FC (hexane/EtOAc 5:1) to give D6 (65 mg, 72 %) as colorless crystals.

Rf = 0.18 (hexane/EtOAc 10:1). 20 []D = -24.5° (c = 0.89, CHCl3). 1 H-NMR (400 MHz, CDCl3):  = 6.35-6.32 (m, 1H, H-3); 6.30-6.29 (m, 1H, H-7’); 6.24- 6.23 (m, 1H, H-5); 5.92-5.86 (m, 1H, H-8’); 5.52-5.44 (m, 1H, H-10’); 3.79 (s, 3H, 4-

OCH3); 3.29-3.20 (m, 1H, H-9’); 3.15-3.09 (m, 1H, H-1’); 2.64-2.57 (m, 1H, H-5’); 2.46-2.41 (m, 1H, H-9’); 2.40-2.32 (m, 1H, H-1’); 2.27-2.21 (m, 1H, H-5’); 2.09-2.02 (m, 1H, H-4’); 1.53-1.43 (m, 3H, H-3’, H-2’, H-4’); 1.40 (d, J = 5.7 Hz, 3H, H-11’); 1.36-1.23 (m, 2H, H-3’ und H-2’). 13 C-NMR (100 MHz, CDCl3):  = 204.7 (C=O); 171.6 (C-6’); 166.3 ( C-2); 164.1 (C-4); 147.8 (C-6); 138.9 (C-8’); 132.7 (C-7’); 110.5 (C-5); 104.8 (C-1); 99.1 (C-3); 72.4 (C-

10’); 40.3 (C-5’); 55.4 (4-OCH3); 37.3 (C-1’); 36.3 (C-9’); 31.4 (C-2’); 27.2 (C-3’); 24.0 (C-4’); 21.2 (C-11’).

IR (film): max = 3060, 2928, 2854, 1728, 1685, 1642, 1616, 1575, 1319, 1254, 1205, 1162, 1039, 826 cm-1. + HRMS (ESI): calcd for C19H24NaO5 [M-Na] : 355.15159, found: 355.1516. Melting point: 100°C.

159 Experimental Section

6.2.3. Synthesis of the Phenyl Analog P6

6.2.3.1. Preparation of Compounds described in Chapter 3.3.1.1.

13O O 8a 4 O 4a 7 HO 5 OH 230 5,7-Dihydroxy-2,2-dimethyl-1,3-benzodioxin-4-one

Thionyl chloride of (12.8 ml, 176.36 mmol, 3 equiv) was slowly added to a solution of 2,4,6-trihydroxybenzoic acid 229 (10 g, 58.78 mmol, 1 equiv), acetone (43 ml, 587.81 mmol, 10 equiv) and DMAP (0.7 g, 5.73 mmol, 0.1 equiv) in 60 ml DME at 0°C. The solution was stirred for 1.5 h and slowly warmed to rt. The reaction mixture was poured into sat. NaHCO3 solution, extracted with EtOAc (3 x 50 ml), washed with brine, dried over MgSO4, filtered and concentrated in vacuo. The crude product was purified by FC (hexane/EtOAc 5:1 → 1:1) to give 230 (5.27 g, 48 %) as white crystals.

Rf : 0.53 (hexane/EtOAc 1:1). 1 H-NMR (400 MHz, MeOD-d4):  = 6.01 (d, J = 2.1 Hz, 1H, H-8); 5.92 (d, J = 2.2 Hz,

1H, H-6); 1.70 (s, 6H, C(CH3)2). 13 C-NMR (100 MHz, MeOD-d4):  = 168.4 (C-4); 166.7 (C-7); 164.5 (C-5); 158.8 (C-

8a); 108.1 (C(CH3)2); 98.4 (C-4a); 96.7 (C-6); 93.2 (C-8); 25.8 (C(CH3)2). + HRMS (EI): calcd for C10H10O5 [M-H] : 210.0528, found: 210.0522. Melting point: 195°C; (lit. 200-201°C)114.

13O O 8a 4 O 4a 7 O 5 OH 231 5-Hydroxy-7-methoxy-2,2-dimethyl-1,3-benzodioxine-4-one DIAD (7.6 ml, 38.34 mmol, 1.5 equiv) was added to a solution of 230 (5.36 g, 25.50 mmol, 1.0 equiv) and methanol (1.6 ml, 39.50 mmol, 1.6 equiv) in 80 ml abs THF at 0°C. The solution was allowed to warm to rt and was stirred for 5 h. It was diluted with EtOAc and washed with brine (2 x 50 ml). The aqueous layer was back- extracted with EtOAc (2 x 100 ml). The combined organic layers were dried over

160 Experimental Section

MgSO4, filtered and the solvent was removed in vacuo. Purification by FC (hexane/EtOAc 10:1 → 6:1) yielded 2.74 g (48 %) of 231 as white crystals.

Rf : 0.75 (hexane/EtOAc 1:1). 1 H-NMR (400 MHz, CDCl3):  = 10.45 (1H, s, 5-OH); 6.15 (d, J = 2.3 Hz, 1H, H-8); 6.00 (d, J = 2.3 Hz, 1H, H-6); 3.81 (s, 3H, 7-OCH3); 1.73 (s, 6H, C(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 167.8 (C-4); 165.3 (C-7); 163.3 (C-5); 157.0 (C-8a);

107.1 (C(CH3)2); 95.9 (C-4a); 94.8 (C-6); 93.2 (C-8); 55.9 (7-OCH3); 25.8 (C(CH3)2). + HRMS (EI): calcd for C11H12O5 [M-H] : 224.0685, found: 224.0679. Melting point: 104°C; (lit. 108-109°C)114.

13O O 8a 4 O 4a 7 O 5 OTf 151 7-Methoxy-2,2-dimethyl-5-[(trifluoromethyl)sulfonyl]-1,3-benzodioxin-4-one 5 ml of triflic anhydride (29.72 mmol, 2,7 equiv) were added to a solution of 231 (2.5 g, 11.15 mmole, 1 equiv) in 50 ml pyridine at -10°C. The reaction mixture was stirred for 3.30 h at 0°C. It was poured into ice water, stirred, extracted with Et2O (2 x 50 ml), washed with cold diluted HCl, sat. NaHCO3, diluted HCl, sat. NaHCO3 and brine. The organic layers were dried over MgSO4, filtered and concentrated. 3.87 g (99 %) of 151 as a pure yellow oil were obtained.

Rf: 0.69 (hexane/EtOAc 1:1). 1 H-NMR (400 MHz, CDCl3):  = 6.54 (d, J = 2.4 Hz, 1H, H-8); 6.48 (d, J = 2.4 Hz, 1H,

H-6); 3.88 (s, 3H, 7-OCH3); 1.74 (s, 6H, C(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 165.7 (C-4); 159.0 (C-7); 157.2 (C-8a); 150.1 (C-5);

117.3 (5-Tf); 106.8 (C(CH3)2); 105.5 (C-4a); 101.3 (C-6); 96.8 (C-8); 56.4 (7-OCH3);

25.8 (C(CH3)2). + HRMS (EI): calcd for C12H11F3O7S [M-H] : 356.0178, found: 356.0173. Melting point: 55°C; (lit. 58-59°C)114.

161 Experimental Section

6 O 1 1' 3' 4 2 O 5' 232 2-(Pent-4-ynyloxy)-tetrahydropyran

CSA (80.4 mg, 0.35 mmol) was added to a cold solution of 4-pentyne-1-ol (3.2 ml,

34.62 mmol) and dihydropyran (3.5 ml, 38.37 mmol) in 6 ml CH2Cl2. The solution was allowed to warm to rt and was stirred for 2.5 h.The reaction was quenched with sat.

NaHCO3 and extracted with CH2Cl2 (3 x 30 ml). The combined organic layers were dried over MgSO4, filtered and concentrated. The orange liquid was distilled at 80°C and 0.5 mbar, whereby 5.27 g (91 %) of 7 were obtained as a colorless liquid.

Rf: 0.35 (hexane/EtOAc 9:1). 1 H-NMR (400 MHz, CDCl3):  = 4.57 (t, J = 2.8 Hz, 1H, H-2); 3.87-3.80 (m, 2H, H-1’); 3.52-3.45 (m, 2H, H-6); 2.33-2.29 (m, 2H, H-3’); 1.94 (t, J = 4.2 Hz, 1-H, H-5’); 1.85- 1.50 (m, 8H, H-2’, H-3, H-4, H-5). 13 C-NMR (100 MHz, CDCl3):  = 98.9 (C-2); 84.1 (C-4’); 68.5 (C-5’); 66.0 (C-1’); 62.4 (C-6); 30.8 (C-3); 28.8 (C-2'); 25.6 (C-5); 19.7 (C-4); 15.5 (C-3’). HRMS (EI): no signal found.

6 O 1 1' 3' 4 2 O 5' 233 2-(Hex-4-ynyloxy)tetrahydropyran 21.3 ml of a 1.6 M solution of n-BuLi in hexane (34.00 mmol, 1.1 equiv) was added to a cold solution of 232 (5.2 g, 30.91 mmol, 1 equiv) in 20 ml abs THF. It was stirred for 15 min and cooled to -78°C. A solution of MeI (5.6 ml, 89.96 mmol, 2.9 equiv) in 4 ml HEMPA was added. The reaction mixture was allowed to warm to rt and was stirred for 2 h. It was quenched by addition of water, extracted with pentane (3 x 50 ml) and washed with brine. The combined organic layers were dried over MgSO4, filtered and evaporated in vacuo. Purification by FC (pentane/Et2O 1:0 → 20:1 → 10:1) yielded 5.42 g (96 %) of 233 as a colorless liquid.

Rf: 0.53 (pentane/Et2O 9:1). 1 H-NMR (400 MHz, CDCl3):  = 4.60 (t, J = 2.8 Hz, 1H, H-2); 3.90-3.78 (m, 2H, H-1’); 3.53-3.44 (m, 2H, H-6); 2.27-2.21 (m, 2H, H-3’); 1.86-1.47 (m, 11H, H-2’, H-6’, H-3, H-4, H-5). 13 C-NMR (100 MHz, CDCl3):  = 99.0 (C-2); 78.8 (C-4’); 75.9 (C-5’); 66.3 (C-1’); 62.3 (C-6); 30.9 (C-3); 29.3 (C-2’); 25.7 (C-5); 19.7 (C-4); 15.8 (C-3’); 3.7 (C-6’).

162 Experimental Section

-1 IR (film): max = 2938, 2866, 2355 cm . + HRMS (EI): calcd for C11H18O2 [M-H] : 182.1307, found: 181.1228.

234 4-Hexyn-1-ol p-TsOH (80.4 mg, 0.42 mmol, 0.01 equiv) was added to a solution of 233 (7.7 g, 42.25 mmol, 1 equiv) in 30 ml methanol. The solution was stirred overnight at rt.

Then, the reaction was quenched with sat. NaHCO3 (1 ml) and the pH was adjusted to pH 7 with 1N HCl-solution. The methanol was evaporated in vacuo. The concentrated aqueous solution was diluted with 5 ml of brine and extracted with

CH2Cl2 (3 x 20 ml). The organic layers were dried over MgSO4, filtered and concentrated. The crude residue was purified by FC (hexane/EtOAc 20:1 → 2:1) which gave 234 (3.70 g, 80 %) as a colorless liquid.

Rf: 0.50 (hexane/EtOAc 1:1). 1 H-NMR (400 MHz, CDCl3):  = 3.76-3.75 (q, J = 3.6 Hz, 2H, H-1); 2.28-2.23 (m, 2H, H-3); 1.78-1.76 (m, 3H, H-6); 1.75-1.70 (m, 2H, H-2). 13 C-NMR (100 MHz, CDCl3):  = 78.6 (C-4); 76.4 (C-5); 62.2 (C-1); 31.7 (C-2); 15.5 (C-3); 3.6 (C-6). + HRMS (EI): calcd for C6H10NaO [M-Na] : 121.0629, found: 121.0479.

5 3 Br 1 235 6-Bromohex-2-yne Triphenylphosphine (7.3 g, 27.82 mmol, 1.3 equiv) was added to a cold solution of 234 (2.1 g, 21.40 mmol, 1 equiv) and tetrabromo methane (9.22 g, 27.82 mmol, 1.3 equiv) in 40 ml CH2Cl2. The solution was allowed to warm to rt and was stirred overnight. The solution was concentrated and the residue was purified by FC (neat pentane). The fractions were checked by 1H-NMR. 235 (1.90 g, 78 %) was obtained as a pure light yellow oil.

Rf: 0.41 (pentane). 1 H-NMR (400 MHz, CDCl3):  = 3.54-3.51 (t, J = 6.8 Hz, 2H, H-6); 2.34-2.29 (m, 2H, H-4); 2.03-1.97 (m, 2H, H-5); 1.78 (t, J = 2.4 Hz, 3H, H-1). 13 C-NMR (100 MHz, CDCl3):  = 32.8 (C-5); 32.0 (C-6); 17.6 (C-4); 3.6 (C-1). -1 IR (film): max = 2928, 2859, 2361 cm .

163 Experimental Section

HRMS (EI): no signal found.

3 HO 1

1' Br

5' 3' 236 1-(2-Bromophenyl)-but-2-yn-1-ol 66 ml of a 0.5 M solution of 1-propinyl magnesium bromide in THF (33.00 mmol, equiv) were added to a solution of 2-bromobenzaldehyde (3.2 ml, 27.41 mmol, 1.2 equiv) in 50 ml THF at 0°C. The reaction mixture was stirred for 2 h at rt. It was quenched with water, extracted with Et2O (2 x 50 ml) and washed with brine. The combined organic extracts were dried over MgSO4, filtered and concentrated to yield 6.29 g (quant) of 236 as a yellow oil.

Rf: 0.33 (hexane/EtOAc 5:1). 1 H-NMR (400 MHz, CDCl3):  = 7.78 (dd, J = 7.8 Hz, 1H, H-6’); 7.56 (dd, J = 7.8 Hz, 1H, H-3’); 7.36 (dt, J = 7.5 Hz, 1H, H-5’); 7.19 (dt, J = 7.8 Hz, 1H, H-4’); 5.77-5.75 (m, 1H, H-1); 1.91 (d, J = 2.3 Hz, 3H, H-4). 13 C-NMR (100 MHz, CDCl3):  = 140.2 (C-1’); 133.1 (C-3’); 130.0 (C-4’); 128.7 (C-6’); 128.0 (C-5’); 122.9 (C-2’); 83.6 (C-3); 78.2 (C-2); 64.5 (C-1); 4.0 (C-4). + HRMS (EI): calcd for C10H9BrO [M-H] : 225.9816, found: 225.9808.

3 TBSO 1

1' Br

5' 3' 237a (1-(2-Bromophenyl)but-2-ynyl-1-oxy)(tert-butyl)dimethylsilane 8.2 g of imidazole (120.46 mmol, 3 equiv) and TBS-Cl (18.16 g, 120.49 mmol, 3 equiv) were added to a solution of 236 (9.04 g, 40.16 mmol, 1 equiv) in 50 ml dichloromethane at rt. The solution was stirred for 18 h. It was poured into sat.

NaHCO3, extracted with CH2Cl2 (3 x 30 ml) and washed with brine. The combined organic layers were dried over MgSO4, filtered and concentrated. The liquid was distilled (4 mbar, 100°C). 9.97 g (97 %) of 237a were obtained as an orange liquid.

Rf: 0.76 (hexane/EtOAc 5:1).

164 Experimental Section

1 H-NMR (400 MHz, CDCl3):  = 7.72 (dd, J = 8.4 Hz, 1H, H-6’); 7.50 (dd, J = 8.4 Hz, 1H, H-3’); 7.34 (dt, J = 7.2 Hz, 1H, H-5’); 7.13 (dt, J = 7.6 Hz, 1H, H-4’); 5.68 (q, J = 2.3 Hz, 1H, H-1); 1.83 (d, J = 2.3 Hz, 3H, H-4); 0.92 (t, J = 2.4 Hz, 9H, Si-

C(CH3)3); 0.17 (s, 3H, Si-(CH3)2); 0.12 (s, 3H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 141.9 (C-1’); 132.7 (C-3’); 129.2 (C-4’); 128.3 (C-6’);

127.8 (C-5’); 121.7 (C-2’); 81.7 (C-3); 79.4 (C-2); 64.7 (Si-C(CH3)3); 26.0 (Si-

C(CH3)3); 18.5 (Si-C(CH3)3); 4.0 (C-4); -4.4 (Si-(CH3)2); -4.7 (Si-(CH3)2). -1 IR (film): max = 3065, 2935, 2890, 2857, 2361, 1066, 1030 cm . + HRMS (EI): calcd for C16H23BrOSi [M-C4H9] : 280.9992, found: 280.9994.

3 TIPSO 1

1' Br

5' 3' 237b (1-(2-bromophenyl)but-2-ynyloxy)triisopropylsilane

To a solution 236 (298 mg, 1.33 mmol, 1.0 equiv) in 10 ml CH2Cl2 was added 2.6- lutidine (466 l, 429 mg, 4.00 mmol, 3.0 equiv). The mixture was cooled to -78°C and and TIPS-OTf (538 l, 613 mg, 2.00 mmol, 1.5 equiv) was added dropwise. The mixture was stirred for 20 min at this temperature and further stirred at rt for 6 h. The mixture was poured into sat. NaHCO3 solution, the layers were seperated and the aqueous phase was extracted with CH2Cl2 (3 x 10 ml). The combined organic layers were dried over MgSO4, filtered and concentrated. The crude product was purified by FC (hexane/EtOAc 20:1) to give 237b (499 mg, 98 %) as a pale yellow liquid.

Rf: 0.86 (hexane/EtOAc 5:1). 1 H-NMR (400 MHz, CDCl3):  = 7.76 (dd, J = 7.8 Hz, 1H, H-6’); 7.50 (dd, J = 8.0 Hz, 1H, H-3’); 7.34 (dt, J = 7.2 Hz, 1H, H-5’); 7.13 (dt, J = 7.7 Hz, 1H, H-4’); 5.68 (q,

J = 2.1 Hz, 1H, H-1); 1.80 (d, J = 2.3 Hz, 3H, H-4); 1.11-1.04 (4s, 21H, Si-CH(CH3)2 and Si-CH(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 142.2 (C-1’); 132.3 (C-3’); 128.8 (C-4’); 127.8 (C-6’);

127.7 (C-5’); 121.1 (C-2’); 81.1 (C-3); 79.5 (C-2); 64.4 (C-1); 18.1 (Si-CH(CH3)2); 12.2

(Si-CH(CH3)2); 3.7 (C-4). + HRMS (EI): calcd for C16H23BrOSi [M-C3H6] : 338.0667, found: 339.0597.

165 Experimental Section

O 13' 3 11' O 1 9' 7' 1' Br

5' 3' 237c 1-bromo-2-(1-(4-methoxybenzyloxy)but-2-ynyl)benzene To a solution 236 (204 mg, 0.91 mmol, 1.0 equiv) and benzyl trichloroacetimidate (1.6 g, 6.01 mmol, 6.6 eq) in 10 ml cyclohexane at 0°C was added PPTS (18 mg, 0.07 mmol, 0.08 equiv). The mixture was stirred for 19 h at rt. The mixture was poured into sat. NaHCO3 solution, the layers were seperated. The organic phase was washed with NaHCO3 solution (3 x 10 ml). The combined organic layers were dried over MgSO4, filtered and concentrated. The crude product was purified by FC (hexane/EtOAc 20:1 → 10:1 → 5:1) to give 237c (470 mg, 61 %) as a pale yellow liquid.

Rf: 0.61 (hexane/EtOAc 3:1). 1 H-NMR (400 MHz, CDCl3):  = 7.78 (dd, J = 7.8 Hz, 1H, H-3’); 7.54 (dd, J = 8.0 Hz, 1H, H-6’); 7.29-7.38 (m, 3H, H-5’, H9’, H-13’); 7.16 (dt, J = 7.8 Hz, 1H, H-4’); 6.85- 6.89 (m, 2H, H-10’, H-12’); 5.46 (q, J = 2.1 Hz, 1H, H-1); 4.67 (d, J = 11 Hz, 1H, H-

7’); 4.55 (d, J = 11 Hz, 1H, H-7’); 3.80 (s, 3H, 11’-OCH3); 1.93 (d, J = 2.1 Hz, 3H, H- 4). 13 C-NMR (100 MHz, CDCl3):  = 159.5 (C-11’); 138.7 (C-1’); 132.9 (C-6’); 130.1 (C-9’ and C-13’); 129.9 (C-8’); 129.8 (C-5’); 129.5 (C-3’); 127.8 (C-4’); 123.5 (C-2’); 114.0

(C-10’ and C-12’); 84.1 (C-3); 76.7 (C-2); 70.5 (C-7’); 55.4 (11’-OCH3); 4.0 (C-4). + HRMS (MALDI): calcd for C18H17BrNaO2 [M-Na] : 367.0310, found: 367.0305.

13 11 TBSO 3 9 OH

7 5 1 238a (S)-1-{2-[1-(tert-Butyl-dimethyl-silanyloxy)-but-2-ynyl]-phenyl}-propan-2-ol Mg turnings (0.15 g, 6.17 mmol, 2.1 equiv) were heated under vacuum for 10 min. 237a (1 g, 2.95 mmol, 1 equiv) was dissolved in 1 ml of abs THF and the solution was added to the Mg. EDB (0.25 ml, 2.95 mmol, 1 equiv) was added to the reaction mixture, which was briefly heated with a heat gun. The mixture was stirred at rt for 2 h. Dry CuI (dried at 90°C, 10-3 mbar, 2 h, 12 mg, 0.29 mmol, 0.1 equiv) was added to the Grignard solution at -30°C together with 2 ml of abs THF and the mixture was

166 Experimental Section stirred for 15 min. (S)-propylene oxide 40S (0.23 ml, 3.24 mmol, 1.1 equiv) was added at -30°C. The mixture was allowed to warm to rt and was stirred overnight.

The mixture was diluted with 25 ml NH4Cl solution and extracted with Et2O (3 x 25 ml). The combined organic phases were washed with sat. NH4Cl (100 ml) and brine (100 ml). The extract was dried over MgSO4, filtered and evaporated in vacuo. Purification by FC (hexane/EtOAc 7:1) gave 123.8 mg (13 %) of 238a (1:0.9 mixture of diastereomers) as a yellow liquid.

Rf : 0.39 (hexane/EtOAc 2:1). 1 H-NMR (400 MHz, CDCl3):  = 7.61-7.58 (m, 1H, Ar-H); 7.52-7.50 (m, 1H, Ar-H); 7.25-7.15 (m, 6H, Ar-H); 5.63-5.61 (q, J = 2.2 Hz, 1H, H-10); 5.58-5.56 (q, J = 2.1 Hz, 1H, H-10); 4.00-4.20 (m, 2H, H-2); 3.10-2.80 (m, 4H, H-3); 1.82-1.81 (q, J = 1.3 Hz,

6H, H-13); 1.31 (s, 3H, H-1); 1.30 (s, 3H, H-1); 0.91 (s, 18H, Si-C(CH3)3); 0.12 (s, 3H,

Si-(CH3)2); 0.17 (s, 3H, Si-(CH3)2); 0.17 (s, 3H, Si-(CH3)2); 0.14 (s, 3H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 141.2/140.9 (C-9); 136.3/135.7 (C-4); 130.8/130.7 (C-5); 128.1/127.8 (C-6); 127.6/127.2 (C-8); 126.9 (C-7); 82.5/82.2 (C-12); 80.5/80.4

(C-11); 69.0/68.7 (C-2); 64.0/63.2 (C-10); 41.9 (C-3); 26.1/26.0 (Si-C(CH3)3); 23.8/23.5 (C-1); 18.5 (Si-C(CH3)3); 3.9 (C-13); -4.3/-4.7 (Si-(CH3)2). -1 IR (film): max = 3416, 3066, 3026, 2932, 2890, 2859, 1367 cm . + HRMS (EI): calcd for C15H21O2Si [M- C4H9] : 261.1311, found: 261.1301.

9' TBSO 7' 1 3 1' 3' O 5' 239a 1-{2-[1-(tert-Butyl-dimethyl-silanyloxy)-but-2-inyl]-phenyl}-propan-2-one

To a solution of 237a (51 mg, 0.15 mmol, 1 equiv), Bu3SnOMe (55 mg, 0.17 mmol, 1.2 equiv), isopropenyl acetate (17 mg, 0.17 mmol, 1.2 equiv) and 2'-(diphenylphosphino)-N,N-dimethylbiphenyl-2-amine (2.3 mg, 0.006 mmol, 0.04 equiv) in 1.5 ml toluene was added Pd2(dba)3 (2 mg, 0.002 mmol, 0.01 equiv) at rt. The reaction mixture was stirred for 22 h at 100°C. After evaporation the residue was purified by FC (hexane/EtOAc 50:1 → 30:1) to yield 239a (10 mg, 21 %) of a yellow oil.

Rf: 0.16 (hexane/EtOAc 10:1). 1 H-NMR (400 MHz, CDCl3):  = 7.48-7.45 (m, 1H, Ar-H); 7.28-7.25 (m, 2H, Ar-H); 7.15-7.13 (m, 1H, Ar-H); 5.44 (q, J = 2.2 Hz, 1H, H-7’); 3.97 (dd, J = 16.7 Hz, 2H, H-

1); 2.15 (s, 3H, H-3); 1.79 (d, J = 2.3 Hz, 3H, H-10’); 1.67-1.60 (m, 9H, Si-C(CH3)3);

0.14 (s, 3H, Si-(CH3)2); 0.08 (s, 3H, Si-(CH3)2). + HRMS (EI): calcd for C18H25O2Si [M-CH3] : 301.1619, found: 301.1617. 167 Experimental Section

9' TIPSO 7' 1 3 1' 3' O 5' 239b 1-[2-(1-Triisopropylsilanyloxy-but-2-inyl)-phenyl]-propan-2-one

To a solution of 237b (57 mg, 0.15 mmol, 1 equiv), Bu3SnOMe (52 mg, 0.16 mmol, 1.2 equiv), isopropenyl acetate (16 mg, 0.16 mmol, 1.2 equiv) and 2'-(diphenylphosphino)-N,N-dimethylbiphenyl-2-amine (1.9 mg, 0.005 mmol, 0.04 equiv) in 1.5 ml toluene was added Pd2(dba)3 (1 mg, 0.001 mmol, 0.01 equiv) at rt. The reaction mixture was stirred for 22 h at 100°C. After evaporation the residue was purified by FC (hexane/EtOAc 50:1 → 30:1) to yield 239b (13 mg, 28 %) of a yellow oil.

Rf: 0.35 (hexane/EtOAc 10:1). 1 H-NMR (400 MHz, CDCl3):  = 7.49-7.46 (m, 1H, Ar-H); 7.29-7.24 (m, 2H, Ar-H); 7.16-7.12 (m, 1H, Ar-H); 5.50 (q, J = 2.1 Hz, 1H, H-7’); 4.00 (dd, J = 16.6 Hz, 2H, H- 1); 2.15 (s, 3H, H-3); 1.77 (d, J = 2.2 Hz, 3H, H-10’); 1.10-1.03 (5s, 21H, SiCH

(CH3)2). + HRMS (EI): calcd for C19H27O2Si [M-C3H7] : 315.1775, found: 315.1777.

9' PMBO 7' 1 3 1' 3' O 5' 239c 1-{2-[1-(4-Methoxy-benzyloxy)-but-2-inyl]-phenyl}-propan-2-one

To a solution of 237c (52 mg, 0.15 mmol, 1 equiv), Bu3SnOMe (55 mg, 0.17 mmol, 1.2 equiv), Isopropenylacetat (17 mg, 0.17 mmol, 1.2 equiv) and Buchwald-ligand

(2.3 mg, 0.006 mmol, 0.04 equiv) in 1.5 ml toluene was added Pd2(dba)3 (2 mg, 0.002 mmol, 0.01 equiv) at rt. The reaction mixture was stirred for 22 h at 100°C. After evaporation the obtained residue was purified by flash (hexane/EtOAc 50:1 → 30:1) to yield 239c (10 mg, 21 %) of a yellow oil.

Rf: 0.27 (pentane/Et2O 5:1). 1 H-NMR (400 MHz, CDCl3):  = 7.86-7.84 (m, 1H, Ar-H); 7.31-7.29 (m, 3H, Ar-H); 7.02-7.00 (m, 1H, Ar-H); 6.90-6.88 (m, 3H, Ar-H); 5.04 (s, 3H, H-7’, H-11’); 3.90 (s,

2H, H-1); 3.81 (s, 3H, PMB-OCH3); 2.08 (s, 3H, H-10’); 1.55 (s, 3H, H-3). + HRMS (EI): calcd for C22H25O3 [M-CH3] : 337.1569, found: 337.1594.

168 Experimental Section

6.2.3.2. Preparation of Compounds described in Chapter 3.3.1.2.

1 O O 3 8a 4 4a O O 5' 7 1' 3' 240 5-(Hex-4-ynyl)-7-methoxy-2,2-dimethyl-1,3-benzodioxin-4-one 453 mg (18.63 mmol, 13 equiv) of magnesium powder was added to 5 ml of abs THF. A solution of 235 (1.00 g, 6.21 mmol, 4 equiv) and EDB (0.4 ml, 4.66 mmol, 3 equiv) in 8.9 ml of abs THF was added over 3 h with a syringe pump. The precipitated Grignard reagent was dissolved in 30 ml of abs THF. 151 (506 mg, 1.42 mmol, 1 equiv) was added to a solution of THF (7 ml) and NMP (0.84 ml). Then,

Fe(acac)3 (36 mg, 0.10 mmol, 0.07 equiv) was added and the mixture was cooled to 0°C. The Grignard reagent was added dropwise to this solution which was stirred for 1 more hour at 0°C. The mixture was quenched by addition of 1N HCl (40 ml). The aqueous phase was extracted with Et2O (3 x 50 ml). The combined organic phases were washed with brine, dried over MgSO4, filtered and evaporated in vacuo. Purification by FC (hexane/EtOAc 1:0 → 20:1) gave 223 mg (54 %) of 240 as a light yellow oil.

Rf: 0.61 (hexane/EtOAc 2:1). 1 H-NMR (400 MHz, CDCl3):  = 6.51 (d, J = 2.4 Hz, 1H, H-6); 6.30 (d, J = 2.4 Hz, 1H,

H-8); 3.83 (s, 3H, 7-OCH3); 3.15-3.11 (m, 2H, H-1’), 2.21-2.17 (m, 2H, H-3’), 1.81-

1.77 (m, 5H, H-2’,H-6’), 1.69 (s, 6H, C(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 164.9 (C-4), 160.1 (C-7), 159.3 (C-8a), 149.3 (C-5),

112.6 (C-6), 105.0 (C(CH3)2), 99.6 (C-8), 79.1 (C-4’), 76.1 (C-5’), 55.7 (7-OCH3), 33.8

(C-1’), 30.2 (C-2’), 25.8 (C(CH3)2), 18.7 (C-3’), 3.7 (C-6’); -1 IR (film): max = 2992, 2939, 2853, 1726 cm . + HRMS (EI): calcd for C17H20O4 [M-H] : 288.1362, found: 288.1334.

169 Experimental Section

OH OH 1 3 O

O 5' 5 1' 3' 241 2-(Hex-4-ynyl)-6-hydroxy-4-methoxybenzoic acid A solution of 240 (201.4 mg, 0.79 mmol, 1 equiv) in 30 ml of 2 M KOH:EtOH 1:1 was refluxed overnight. The mixture was diluted with 20 ml water and washed with 50 ml of CH2Cl2. The aqueous phase was acidified to pH 1 with 1N HCl and extracted with EtOAc (2 x 70 ml). The combined organic extracts were washed with brine, dried over MgSO4, filtered and evaporated in vacuo. 128 mg (74 %) of 241 were obtained as light red crystals.

Rf: 0.59 (CH2Cl2/MeOH 5:1). 1 H-NMR (400 MHz, CDCl3):  = 11.53 (s, 1H, 2-OH), 6.38-6.36 (q, J = 2.4 Hz, 2H, H-

3, H-5), 3.82 (s, 3H, 4-OCH3), 3.04 (t, J = 7.2 Hz, 2H, H-1’), 2.16-2.20 (m, 2H, H-3’), 1.83-1.76 (m, 5H, H-2’, H-6’). 13 C-NMR (100 MHz, CDCl3):  = 175.4 (C=O), 166.9 (C-4), 165.1 (C-2), 148.5 (C-6),

111.7 (C-5), 103.4 (C-1), 99.2 (C-3), 79.0 (C-4’), 76.1 (C-5’), 55.6 (4-OCH3), 35.8 (C- 1’), 30.9 (C-2’), 18.8 (C-3’), 3.6 (C-6’). -1 IR (film): max = 2939, 2850, 2362, 1619 cm . + HRMS (EI): calcd for C14H16O4 [M-H] : 248.1049, found: 248.1039.

6.2.3.3. Preparation of Compounds described in Chapter 3.3.1.3.

13O O 8a O 4a 7 O 6 1' OH 244 3' 5-(4-Hydroxy-but-1-inyl)-7-methoxy-2,2-dimethyl-1,3-benzodioxin-4-one Abs DMF (11 ml) was degassed under argon for 10 min. 151 (2 g, 5.61 mmol,

1 equiv), PdCl2(PPh3)2 (392 mg, 0.56 mmol, 0.10 equiv), Et3N (935 l, 6.74 mmol, 1.2 equiv) and but-3-yn-1-ol (1.06 ml, 14.03 mmol, 2.5 equiv) were added sequentially. The mixture was stirred for 5 h at 70°C. After cooling to rt, EtOAc and sat. NH4Cl solution were added. The phases were separated and the organic phase was dried over MgSO4, filtered and evaporated in vacuo. The residue was purified by FC (hexane/EtOAc 10:1 → 5:1 → 2:1 → 1:1) to give 244 (1.24 g, 80 %) as a yellow oil.

Rf: 0.32 (hexane/EtOAc 1:1).

170 Experimental Section

1 H-NMR (400 MHz, CDCl3):  = 6.72 (d, J = 2.5 Hz, 1H, H-6); 6.39 (d, J = 2.5 Hz, 1H, H-8); 3.85 (t, J = 5.4 Hz, 2H, H-4’); 3.84 (s, 3H, 7-OCH3); 2.69 (t, J = 5.4 Hz, 2H, H-

3’); 1.70 (s, 6H, C(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 165.0 (C-7); 159.6 (C-4); 158.5 (C-8a); 127.1 (C-5); 114.7 (C-6); 107.0 (C-4a); 105.7 (C(CH3)2); 101.8 (C-8); 95.3 (C-2’); 81.1 (C-1’); 60.9

(C-4’`); 55.8 (7-OCH3); 25.7 (C(CH3)2); 24.6 (C-3’). + HRMS (MALDI): calcd for C15H16NaO5 [M-Na] : 299.0896, found: 299.0889.

13O O 8a O 4a 7 O OH 6 1' 3' 245 5-(4-Hydroxy-butyl)-7-methoxy-2,2-dimethyl-1,3-benzodioxin-4-one A solution of 244 (1.51 g, 5.47 mmol, 1 equiv) in 25 ml MeOH was three times sequentially evacuated and vented with argon. Pd/C (5 %, 702 mg, 0.33 mmol, 0.06 equiv) was added and the flask was flooded with hydrogen and stirred for 19 h at rt. The mixture was filtered over Celite which was washed with methanol. The filtrate was evaporated in vacuo and the residue was purified by FC (hexane/EtOAc 3:1 → 2:1 → 1:1) to give 245 (1.30 g, 85 %) as a pale yellow oil.

Rf: 0.32 (hexane/EtOAc 1:1). 1 H-NMR (400 MHz, CDCl3):  = 6.47 (d, J = 2.5 Hz, 1H, H-6); 6.30 (d, J = 2.5 Hz, 1H,

H-8); 3.82 (s, 3H, 7-OCH3); 3.72 (t, J = 6.0 Hz, 2H, H-4’); 3.03 (t, J = 7.9 Hz, 2H, H- 1’); 1.68 (s, 6H, C(CH3)2); 1.67-1.65 (m, 4H, H-2’ and H-3’). 13 C-NMR (100 MHz, CDCl3):  = 165.0 (C-7); 160.3 (C-4); 159.3 (C-8a); 149.9 (C-5);

112.3 (C-6); 105.0 (C(CH3)2); 104.6 (C-4a); 99.4 (C-8); 61.9 (C-4’); 55.5 (7-OCH3); 34.0 (C-1’); 32.2 (C-3’); 26.9 (C-2’); 25.6 (C(CH3)2). + HRMS (MALDI): calcd for C15H20NaO5 [M-Na] : 303.1209, found: 303.1202.

171 Experimental Section

13O O 8a O 4a 7 O O 6 1' 3' 246 5-(4-Formyl-butyl)-7-methoxy-2,2-dimethyl-1,3-benzodioxin-4-one

To a solution of oxalylchloride (330 l, 3.85 mmol, 1.5 equiv) in 7 ml abs CH2Cl2 was added dropwise DMSO (546 l, 7.70 mmol, 3 equiv) at -75°C. The mixture was stirred for 10 min at this temperature. Then a solution of 245 (770 mg, 2.75 mmol,

1 equiv) in 7 ml abs CH2Cl2 was added dropwise at -75°C and the mixture was further stirred for 1 h. Et3N (2.14 ml, 15.41 mmol, 6 equiv) was added to the mixture before it was allowed to warm to -10°C. Water (14 ml) and CH2Cl2 (40 ml) were added. The organic phase was washed with brine (2 x 20 ml) and dried over MgSO4, filtered, and evaporated in vacuo. The residue was purified by FC (hexane/EtOAc 10:1 → 5:1 → 3:1 → 2:1) to give 246 (633 mg, 93 %) as a yellow oil.

Rf: 0.58 (hexane/EtOAc 1:1). 1 H-NMR (400 MHz, CDCl3):  = 9.78 (t, J = 1.7 Hz, 1H, H-4’), 6.46 (d, J = 2.5 Hz, 1H, H-6); 6.31 (d, J = 2.5 Hz, 1H, H-8); 3.82 (s, 3H, 7-OCH3); 3.09-3.05 (m, 2H, H-1’); 2.52 (dt, J = 1.7 Hz and 7.3 Hz, 2H, H-3’), 1.98-1.90 (m, 2H, H-2’); 1.68 (s, 6H,

C(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 202.6 (C-4’); 164.9 (C-7); 160.1 (C-4); 159.3 (C-8a);

148.6 (C-5); 112.4 (C-6); 105.0 (C(CH3)2); 104.9 (C-4a); 99.7 (C-8); 55.6 (7-OCH3);

43.5 (C-3’); 33.8 (C-1’); 25.6 (C(CH3)2); 23.4 (C-2’). + HRMS (MALDI): calcd for C15H18NaO5 [M-Na] : 301.1052, found: 301.1044.

13O O 8a O 4a 7 Br O 6 1' 3' Br 247 5-(5,5-Dibromo-pent-4-enyl)-7-methoxy-2,2-dimethyl-1,3-benzodioxin-4-one To a solution of carbon tetrabromide (0.93 ml, 9.53 mmol, 4 equiv) in 7 ml abs

CH2Cl2 was added a solution of Ph3P (4.99 g, 19.06 mmol, 8 equiv) in 7 ml CH2Cl2 at 0°C. Subsequently a solution of 246 (662 mg, 2.38 mmol, 1 equiv) in 7 ml abs CH2Cl2 was added at 0°C and the mixture was stirred for 1 h at this temperature. Water

(7 ml) and CH2Cl2 (10 ml) were added stirring was continued for 10 min at rt. Saturated NaHCO3 solution was added and the phases were separated. The

172 Experimental Section

aqueous phase was extracted with CH2Cl2 (3 x 20 ml). The combined organic phases were washed with sat. NH4Cl solution, dried over MgSO4, filtered, and evaporated in vacuo. The crude product was purified by FC (hexane/EtOAc 10:1 → 5:1 → 3:1 → 2:1) to give 247 (0.78 g, 76 %) as a pale yellow oil.

Rf: 0.43 (hexane/EtOAc 5:1). 1 H-NMR (400 MHz, CDCl3):  = 6.48-6.44 (m, 2H, H-6, H-4’); 6.31 (d, J = 2.5 Hz, 1H,

H-8); 3.83 (s, 3H, 7-OCH3); 3.07-3.03 (m, 2H, H-1’); 2.18 (q, J = 7.3 Hz, 2H, H-3’); 1.77-1.71 (m, 2H, H-2’); 1.69 (s, 6H, C(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 164.9 (C-7); 160.0 (C-4); 159.2 (C-8a); 148.9 (C-5);

138.5 (C-4’); 112.4 (C-6); 104.9 (C(CH3)2); 104.7 (C-4a); 99.5 (C-8); 88.9 (C-5’); 55.6 (7-OCH3); 34.2 (C-1’); 32.9 (C-1’); 28.9 (C-2’); 25.6 (C(CH3)2). + HRMS (MALDI): calcd for C16H18Br2O4 [M-H] : 431.9572, found: 431.9566.

13O O 8a O 4a 7 5' O 6 1' 3' 248 7-Methoxy-2,2-dimethyl-5-pent-4-inyl-1,3-benzodioxin-4-one

To a solution of 247 (52 mg, 0.12 mmol, 1 equiv) in 1.5 ml abs THF was added freshly prepared LDA (0.6 ml, 0.35 mmol, 3 equiv, 0.59 M in THF) at -78°C and the mixture was stirred for 2.5 h at this temperature. The reaction was quenched by addition of sat. NH4Cl solution and extracted with Et2O (2 x 7 ml). The organic phase was dried over MgSO4, filtered and evaporated in vacuo. The crude product was purified by FC (hexane/EtOAc 20:1) to give 248 (25 mg, 76 %) as a pale yellow oil.

Bestmann-approach: To a solution of Bestmann reagent (52 mg, 0.27 mmol, 1.5 equiv) in 2 ml MeOH was added K2CO3 (41 mg, 0.30 mmol, 1.7 equiv) at rt and the mixture was stirred for 15 min. The mixture was cooled to 0°C and it was added dropwise to a solution of 246 (50 mg, 0.18 mmol, 1 equiv) in 1 ml MeOH at 0°C and stirring was continued for

1.5 h at 0°C and 30 min at rt. Et2O (10 ml) and NaHCO3-solution (w = 5 %; 10 ml) were added to the mixture. The organic phase was dried over MgSO4, filtered, and evaporated in vacuo. The crude product was purified by FC (hexane/EtOAc 20:1 → 10:1 → 5:1 → 1:1) to give 248 (24 mg, 57 %) as a pale yellow oil.

Rf: 0.29 (pentane/Et2O 10:1); 0.47 (hexane/EtOAc 3:1). 1 H-NMR (400 MHz, CDCl3):  = 6.51 (d, J = 2.6 Hz, 1H, H-6); 6.31 (d, J = 2.6 Hz, 1H,

H-8); 3.83 (s, 3H, 7-OCH3); 3.19-3.17 (m, 2H, H-1’); 2.25 (dt, J = 2.6 Hz and 7.1 Hz,

173 Experimental Section

2H, H-3’); 1.98 (t, J = 2.6 Hz, 1H, H-5’); 1.80-1.88 (m, 2H, H-2’); 1.68 (s, 6H,

C(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 164.8 (C-7); 160.0 (C-4); 159.2 (C-8a); 148.7 (C-5);

112.5 (C-6); 104.9 (C(CH3)2); 104.8 (C-4a); 99.6 (C-8); 84.3 (C-5’); 68.6 (C-4’); 55.5 (7-OCH3); 33.6 (C-1’); 29.4 (C-2’); 25.6 (C(CH3)2); 18.2 (C-3’). + HRMS (ESI): calcd for C16H18NaO4 [M-Na] : 297.10973, found: 297.10951.

13O O 8a O 4a 7 5' O 6 1' 3' 240 5-(Hex-4-inyl)-7-methoxy-2,2-dimethyl-1,3-benzodioxin-4-one To a solution of 248 (156 mg, 0.57 mmol, 1 equiv) in 3 ml abs THF was added dropwise freshly prepared LDA (1.07 ml, 0.63 mmol, 1.1 equiv, 0.59 M in THF) at -78°C and the mixture was stirred for 15 min at this temperature. A solution of MeI (103 l, 1.66 mmol, 2.9 equiv) in 1.5 ml HMPA was added at -78°C and the mixture was stirred for 1 hour at -78°C and subsequently for 19 h at rt. Water (10 ml) was added to the mixture and it was extracted with pentane (3 x 10 ml). The combined organic phases were washed with brine (10 ml), dried over MgSO4, filtered and evaporated in vacuo. The crude product was purified by FC (pentane/Et2O 10:1) to give 240 (138 mg, 88 %) as a pale yellow oil.

Rf: 0.21 (pentane/Et2O 5:1). 1 H-NMR (400 MHz, CDCl3):  = 6.51 (d, J = 2.5 Hz, 1H, H-6); 6.30 (d, J = 2.6 Hz, 1H,

H-8); 3.83 (s, 3H, 7-OCH3); 3.15-3.11 (m, 2H, H-1’); 2.16-2.25 (m, 2H, H-3’); 1.77-

1.80 (m, 5H, H-2’, H-6’); 1.68 (s, 6H, C(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 164.8 (C-4); 160.0 (C-7); 159.2 (C-8a); 149.1 (C-5);

112.4 (C-6); 104.8 (C(CH3)2); 99.4 (C-8); 98.9 (C-4a); 78.9 (4`-C-4’); 75.9 (C-5’); 55.5

(7-OCH3); 33.7 (C-1’); 30.0 (C-2’); 25.6 (C(CH3)2); 18.5 (C-3’); 3.5 (C-6’). + HRMS (ESI): calcd for C17H20NaO4 [M-Na] : 311.12538, found: 311.12530.

174 Experimental Section

OH O 15' 13' 1 3 O 17' 11'

O 1' 10' OTBS 5 7'

3' 250

(2S)-1-(2-(1-(tert-Butyldimethylsilyloxy)but-2-ynyl)phenyl)propyl-2-(hex-4-ynyl)- 6-hydroxy-4-methoxybenzoate A mixture of acid 6 (25 mg, 0.10 mmol, 1 equiv) and alcohol 13 (32.2 mg, 0.10 mmol,

1 equiv) was co-evaporated with toluene several times. CH2Cl2 (1.2 ml) and DMAP (4.75 mg, 0.04 mmol, 0.25 equiv) were added to the reaction mixture, the solution was cooled down to 0°C, and DCC (27 mg, 0.13 mmol, 1.3 equiv) was added. The solution was stirred overnight at rt. The solution was concentrated and purified by FC (hexane/EtOAc 1:0 → 50:1 → 10:1). 17.9 mg (32 %) of 14 were obtained as white crystals.

Rf: 0.82 (hexane/EtOAc 2:1). 1 H-NMR (400 MHz, CDCl3):  = 11.91 (d, J = 3.6 Hz, 1H, 2-OH); 7.70-7.54 (m, 2H, H- 12’, H-14’); 7.28-7.17 (m, 2H, 13’, 15’); 6.35-6.33 (q, J = 2.6 Hz, 2H, 3, 5); 5.74-5.71 (m, 1H, 10’); 5.59-5.57 (m, 1H, H-18’); 5.51-5.49 (m, 1H, H-18’); 3.81 (s, 3H, 4-

OCH3); 3.41-3.34 (m, 1H, 1’); 3.13-2.90 (m, 3H, H-17’, H-1’); 2.20-2.16 (m, 2H, H-3’);

1.83-1.73 (m, 8H, H-2’, H-6’, H-7’); 1.44-1.39 (m, 3H, 18’-CH3), 0.93 (s, 9H, Si-

C(CH3)3), 0.90 (s, 9H, Si-C(CH3)3), 0.18, 0.16, 0.15, 0.11 (s, 6H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 171.2 (C-20’); 166.0 (C-4); 164.0 (C-2); 146.9 (C-6); 141.3 (C-11’); 133.8 (C-16’); 129.0, 127.8, 127.7, 127.4, 127.2, 126.9 (C12’-C15’); 111.1 (5); 105.2 (1); 99.2 (3); 82.2 (C-8’); 82.0 (C-8’); 80.6 (C-9’); 80.3 (C-9’); 79.0

(C-4’); 76.2 (C-5’); 72.8 (C-18’); 63.0 (C-10’); 62.9 (C-10’); 55.4 (4-OCH3); 38.9 (C-

17’); 38.6 (C-17’); 36.0 (C-1’); 30.9 (C-2’); 27.2 (18’-CH3); 26.0 (Si-C(CH3)3); 19.7 (C-

3’); 19.6 (C-3’); 18.8 (Si-C(CH3)3); 18.5 (Si-C(CH3)3); 3.9 (C-6’); 3.7 (C-7’); -4.4 (Si-

(CH3)2); -4.8 (Si-(CH3)2). -1 IR (film): max = 3061, 2931, 2858, 2352, 1727 cm . + HRMS (EI): calcd for C29H35O5Si [M-C4H9] : 491.2254, found: 491.2255.

175 Experimental Section

6.2.3.4. Preparation of Compounds described in Chapter 3.3.2.1.

5 3

1 OH I 251 (R)-1-(2-Iodo-phenyl)-propan-2-ol A dried three-necked round-bottomed flask was equipped with a digital thermometer and was charged with o-diiodobenzene (4 g, 12.13 mmol, 1.4 equiv) and dry toluene (80 ml). The colorless solution was cooled to -90°C. To the solution was added dropwise n-BuLi (1.6 M in hexane; 7.6 ml, 12.13 mmol, 1.4 equiv) at a rate that the inner temperature was maintained under -90°C. The resulting viscous suspension was stirred for 1 hour, (R)-propylene oxide 40R (610 l, 8.66 mmol, 1 equiv) was added, followed by addition of BF3•OEt2 (1.5 ml, 12.13 mmol, 1.4 equiv) at -90°C. The reaction mixture was stirred for 3 h between -90 and -78°C and quenched with 40 ml NaHCO3 solution at -78°C. The resulting mixture was extracted with EtOAc (3 x 50 ml). The combined organic extracts were washed with brine, dried over MgSO4, filtered and evaporated in vacuo. The residue was purified by FC (hexane/EtOAc 50:1 → 25:1 → 10:1 → 3:1) to give 251 (1.80 g, 79 %) as a colorless oil.

Rf: 0.23 (hexane/EtOAc 3:1). 1 H-NMR (400 MHz, CDCl3):  = 7.84 (d, J = 7.9Hz, 1H, H-6); 7.32-7.24 (m, 2H, H-3 and H-5); 6.92 (dt, J = 1.9 Hz and 7.7 Hz, 1H, H-4); 4.16-4.08 (m, 1H, CH); 2.89 (dq,

J = 4.8 Hz and 13.7 Hz, 2 H, CH2); 1.29 (d, J = 6.3 Hz, 3H, CH3). 13 C-NMR (100 MHz, CDCl3):  = 141.6(C-2); 139.9 (C-6); 131.0 (C-3); 128.5 (C-4);

128.5 (C-5); 101.3 (C-1); 67.8 (CH); 50.1 (CH2); 23.1 (CH3).

IR (film): max = 2967, 2925, 1561, 1465, 1435, 1374, 1114, 1078, , 1040, 1010, 934, 746 cm-1. + HRMS (EI): calcd for C9H11IO [M-H] : 261.9855, found: 261.9850.

5 3

1 OTBS I 252 tert-Butyl-[(R)-2-(2-iodo-phenyl)-1-methyl-ethoxy]-dimethyl-silane 251 (1.75 g, 10.02 mmol, 1 equiv) was dissolved in 13 ml abs DMF. DIEA (3.42 ml, 20.04 mmol, 2 equiv) and TBS-Cl (2.27 g, 15.03 mmol, 1.5 equiv) were added sequentially to the solution which was stirred at rt overnight. The mixture was 176 Experimental Section

quenched by addition of 10 ml of sat. NaHCO3 and it was extracted with EtOAc (3 x 20 ml). The combined organic phase was dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude product was purified by FC (Fine silica gel; hexane/EtOAc 100:0 → 100:1) to give 252 (2.30 g, 92 %).

Rf: 0.80 (hexane/EtOAc 3:1). 1 H-NMR (400 MHz, CDCl3):  = 7.83 (d, J = 7.6 Hz, 1 H, H-6); 7.30-7.24 (m, 2 H, H-3 and H-5); 6.92 (dt, J = 2.2 Hz and 7.7 Hz, 1 H, H-4); 4.19-4.11 (m, 1 H, CH); 2.88-

2.85 (dd, J = 3.3 Hz and 5.2 Hz, 2 H, CH2); 1.26 (d, J = 6.1 Hz, 3H, CH3); 0.86 (s, 9H,

Si-C(CH3)3); -0.07 (s, 3H, Si-CH3); -0.24 (s, 3H, Si-CH3). 13 C-NMR (100 MHz, CDCl3):  = 142.4 (C-2); 139.4 (C-6); 132.1 (C-3); 128.1 (C-4); 128.0 (C-5); 101.1 (C-1); 68.2 (CH); 50.5 (CH2); 26.0 (Si-C(CH3)3); 24.1 (CH3); 18.1

(Si-C(CH3)3); -4.8 (Si-(CH3)2); -5.0 (Si-(CH3)2). HRMS (EI): no signal found.

TBSO 11 9 7 3 5 1 OH O O 253

((4R,5S)-5-Allyl-2,2-dimethyl-[1,3]dioxolan-4-yl)-{2-[(R)-2-(tert-butyl-dimethyl- silanyloxy)-propyl]-phenyl}-methanol To a stirred solution of 252 (133 mg, 0.34 mmol, 1.9 equiv) in abs THF (2 ml) was added n-BuLi (1.6 M in hexane; 210 l, 0.34 mmol, 1.9 equiv) at -78°C. After 2 h 191 (30 mg, 0.18 mmol, 1.0 equiv) in abs THF (1.5 ml) was added dropwise followed by stirring of the mixture for 2 h at -78°C. The mixture was allowed to warm to rt slowly over a period of 10 h. The mixture was treated with NH4Cl solution (2 ml). The layers were separated and the aqueous layer was extracted with Et2O (2 x 1 ml). The combined organic extracts were dried over MgSO4, filtered and evaporated in vacuo. The crude product was purified by FC (hexane/EtOAc 100:1 → 70:1 → 30:1 → 10:1) to give 253 (43 mg, 58 %) as a colorless oil.

Rf: 0.21; 0.28 (hexane/EtOAc 3:1). 1 H-NMR (400 MHz, CDCl3):  = 7.42-7.40 (m, 1H, H-Ar) ; 7.25-7.18 (m, 3H, H-Ar); 5.79-5.72 (m, 1H, H-2); 5.06-5.00 (m, 3H, H-1 and H-6); 4.57 (t, J = 6.0 Hz, 1H, H-5); 4.27-4.22 (m, 1H, H-4); 4.07-4.03 (m, 1H, H-10); 3.31 (d, J = 3.5 Hz, 1H, 6-OH); 2.90 (dd, J = 8.9 Hz and 13.7 Hz, 1H, H-9); 2.69 (dd, J = 4.7 Hz and 10.7 Hz, 1H, H-9);

2.38-2.34 (m, 1H, H-3); 2.07-2.03 (m, 1H, H-3); 1.59 (s, 3H, C(CH3)2); 1.43 (s, 3H,

C(CH3)2); 1.24 (d, J = 6.2 Hz, 3H, H-11’); 0.76 (s, 9H, Si-C(CH3)3); -0.09 (s, 3H, Si-

(CH3)2); -0.31 (s, 3H, Si-(CH3)2).

177 Experimental Section

13 C-NMR (100 MHz, CDCl3):  = 139.5 (C-7’); 137.8 (C-8’); 134.8 (C-2’); 130.7 (ar-C); 128.0 (ar-C); 126.8 (ar-C); 126.5 (ar-C); 117.3 (C-1’); 108.6 (C(CH3)2); 90.0 (C-5’);

77.1 (C-4’); 71.0 (C-10’); 67.9 (C-6’); 42.1 (C-9’); 34.7 (C-3’); 28.0 (CH3); 26.0 (Si-

C(CH3)3); 25.7 (CH3); 24.5 (C-11’); 18.2 (Si-C(CH3)3); -4.9 (Si-(CH3)2); -4.9 (Si- (CH3)2).

IR (film): max = 2955, 2929, 1457, 1375, 1255, 1216, 1127, 1090, 991, 911, 835, 757, 669 cm-1. + HRMS (ESI): calcd for C24H40NaO4Si [M-Na] : 443.2588, found: 443.2587.

TBSO 11 9 7 3 5 1 O O O 254

((4S,5S)-5-Allyl-2,2-dimethyl-[1,3]dioxolan-4-yl)-{2-[(R)-2-(tert-butyl-dimethyl- silanyloxy)-propyl]-phenyl}-methanone 253 (676 mg, 1.61 mmol, 1 equiv) was dissolved in 1 ml Dess-Martin periodinane solution (15 wt % in CH2Cl2; 5 ml, 2.42 mmol, 1.5 equiv) at rt and the reaction mixture was further stirred for 5 h. Afterwards the reaction mixture was directly put on the column and purified by FC (hexane/EtOAc 100:0 → 50:1 → 25:1 → 10:1 → 5:1) to give 254 (520 mg, 77 %).

Rf: 0.45 (hexane/EtOAc 5:1). 1 H-NMR (400 MHz, CDCl3):  = 7.60 (dd, J = 1.1 Hz and 7.7 Hz, 1H, H-Ar); 7.43-7.39 (m, 1H, H-Ar); 7.36-7.28 (m, 2H, H-Ar); 5.85-5.75 (m, 1H, H-2); 5.28 (d, J = 6.8 Hz, 1H, H-5); 5.08-5.03 (m, 2H, H-1); 4.62-4.57 (m, 1H, H-4); 4.15-4.11 (m, 1H, H-10); 3.12 (dd, J = 4.1 Hz and 12.1 Hz, 1H, H-9); 2.71 (dd, J = 8.5 Hz and 13.2 Hz, 1H, H-

9); 2.26-2.16 (m, 2H, H-3) ; 1.55 (s, 3H, C(CH3)2); 1.41 (s, 3H, C(CH3)2); 1.21 (d, J =

6.1 Hz, 3H, H-11’); 0.79 (s, 9H, Si-C(CH3)3); -0.14 (s, 3H, Si-(CH3)2); -0.32 (s, 3H, Si-

(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 199.6 (C-6’); 141.1 (C-7’); 136.0 (C-8’); 134.3 (C-2’); 134.1 (ar-C); 131.5 (ar-C); 128.8 (ar-C); 126.1 (ar-C); 117.8 (C-1’); 110.0 (C(CH3)2);

81.3 (C-5’); 78.1 (C-4’); 69.7 (C-10’); 44.5 (C-9’); 35.8 (C-3’); 27.4 (C(CH3)2); 26.0 (Si-

C(CH3)3); 25.6 (C(CH3)2); 24.4 (C-11’); 18.2 (Si-C(CH3)3); -4.8 (Si-(CH3)2); -5.0 (Si- (CH3)2).

IR (film): max = 2967, 2925, 1772, 1718, 1608, 1458, 1280, 1121, 1030, 956, 744, 693, 629, 604 cm-1. + HRMS (ESI): calcd for C24H38NaO4Si [M-Na] : 441.2432, found: 441.2441.

178 Experimental Section

6.2.3.5. Preparation of Compounds described in Chapter 3.3.2.2.

Si(CH3)3  OH O OTBS  1 3 O 11' 7' 9' 3' 5' O O 51' O O 255 2-[3-((4S,5S)-5-{2-[(R)-2-(tert-Butyl-dimethyl-silanyloxy)-propyl]-benzoyl}-2,2- dimethyl-[1,3]dioxolan-4-yl)-propyl]-6-hydroxy-4-methoxy-benzoic acid 2- trimethylsilanyl-ethyl ester To a solution of 254 (377 mg, 1.08 mmol, 1.1 equiv) in 1.3 ml abs THF was added dropwise a 0.5 M solution of 9-BBN in THF (2.8 ml, 1.37 mmol, 1.4 equiv) and the mixture was stirred for 1.5 h at rt. Then a 2 M solution of K3PO4 (0.87 ml, 1.96 mmol, 2.0 equiv) was added to the mixture (solution A). In a separate flask, 176 (374 mg, 1.08 mmol, 1.1 equiv) was added to a mixture of trifurylphosphine (136 mg,

0.59 mmol, 0.6 equiv) and [Pd(OAc)2] (34 mg, 0.15 mmol, 0.15 equiv) in 3.5 ml degassed abs DME (solution B) and stirred for 5 min (the colour changed from red to yellow). Solution A was added dropwise to solution B at rt. The reaction mixture was refluxed at 73°C for 2.5 h and the mixture was directly put on Celite and it was purified by FC (hexane/EtOAc 100:1 → 50:1 → 20:1 → 10:1 → 5:1) to give 255 (556 mg, 75 %).

Rf: 0.40 (hexane/EtOAc 5:1). 1 H-NMR (400 MHz, CDCl3):  = 11.83 (2-OH); 7.53 (dd, J = 1.2 Hz and 7.8 Hz, 1H, H-Ar); 7.41-7.25 (m, 3H, H-Ar) ; 6.31 (d, J = 2.6 Hz, 1H, H-3); 6.20 (d, J = 2.7 Hz, 1H, H-5); 5.20 (d, J = 7.1 Hz, 1H, H-5’); 4.52-4.48 (m, 1H, H-4’); 4.41-4.37 (m, 2H, H-);

4.15-4.11 (m, 1H, H-10’); 3.78 (s, 3H, 4-OCH3); 3.04 (dd, J = 4.1 Hz and 12.9 Hz, 1H, H-9); 2.93-2.77 (m, 2H, H-1’); 2.72 (dd, J = 8.3 Hz and 12.9 Hz, 1H, H-9); 1.89-1.85

(m, 1H, H-3’); 1.59-1.54 (m, 3H, H-3’ and H-2’); 1.39 (s, 3H, C(CH3)2); 1.51 (s, 3H,

C(CH3)2); 1.19 (d, J = 6.0 Hz, 3H, H-11’); 1.15-1.10 (m, 2H, H-); 0.80 (s, 9H, Si-

C(CH3)3); 0.08 (s, 9H, Si(CH3)3); -0.13 (s, 3H, Si-(CH3)2); -0.31 (s, 3H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 200.8 (C-6’); 171.7 (C-1); 165.8 (C-2); 163.9 (C-4); 147.1 (C-6); 140.6 (C-8’); 136.5 (C-7’); 133.9 (ar-C); 131.3 (ar-C); 128.6 (ar-C); 125.9

(ar-C); 110.8 (C-5); 109.9 (C(CH3)2); 105.1 (C-1); 99.1 (C-3); 81.7 (C-5’); 78.7 (C-4’);

69.7 (C-10’); 55.4 (4-OCH3); 44.4 (C-9’); 36.5 (C-1’); 31.1 (C-2’); 28.8 (C-3’); 27.1

(CH3); 26.0 (Si-C(CH3)3); 25.5 (CH3); 24.3 (C-11’); 18.2 (Si-C(CH3)3); 17.7 (C-); -1.4

(Si-(CH3)3); -4.8 (Si-(CH3)2); -5.0 (Si-(CH3)2).

IR (film): max = 2955, 2928, 1698, 1646, 1615, 1577, 1471, 1373, 1251, 1213, 1158, 834, 758,668 cm-1. + HRMS (ESI): calcd for C37H58NaO8Si2 [M-Na] : 709.3362, found: 709.3588. 179 Experimental Section

OH O 1 9' 3 OH 7' 3' 5' O O 11' 51' O OH O 256 2-Hydroxy-6-(3-{(4S,5S)-5-[2-((R)-2-hydroxy-propyl)-benzoyl]-2,2-dimethyl- [1,3]dioxolan-4-yl}-propyl)-4-methoxy-benzoic acid To a stirred solution of 255 (52 mg, 0.08 mmol, 1 equiv) in THF (2 ml) at rt tetra-n- butylammonium fluoride (1 M solution in THF; 180 l, 0.38 mmol, 2.4 equiv) was added dropwise. The reaction was stirred overnight at rt. Saturated NH4Cl solution was added and the mixture (pH = 6) was extracted with EtOAc (2 x 5 ml). The combined organic extracts were dried over MgSO4, filtered and evaporated in vacuo. The crude was purified by FC (EtOAc/MeOH 100:0 → 100:1) to give 256 (29 mg, 81 %).

Rf: 0.35 (EtOAc/MeOH 10:1). NMR: broad signals, but 13C-NMR does not show the C6’ ketone, in comparison with the 13C-NMR spectra of 255.

IR (film): max = 2931, 1595, 1457, 1370, 1285, 1251, 1157, 1078, 1056, 837, 752, 668 cm-1. + HRMS (ESI): calcd for C26H32NaO8 [M-Na] : 495.1989, found: 495.1977.

TBSO 11 9 7 3 5 1 OTIPS O O 257

(4S,5S)-4-Allyl-5-({2-[(R)-2-(tert-butyl-dimethyl-silanyloxy)-propyl]-phenyl}- triisopropylsilanyloxy-methyl)-2,2-dimethyl-[1,3]dioxolane

253 (150 mg, 0.36 mmol, 1 equiv) was dissolved in 1.5 ml of CH2Cl2 and 2,6-lutidine (104 ml, 0.89 mmol, 2.5 equiv) was added. The mixture was cooled to -78°C. To the cold mixture was added TIPS-OTf and the reaction was stirred for 1 hour at -78°C.

The mixture was allowed to warm to rt and was stirred overnight. Saturated NaHCO3 solution (5 ml) was added to the reaction mixture and it was extracted with CH2Cl2 (3 x 10 ml). The combined organic phases were dried over MgSO4, filtered, and evaporated in vacuo. The crude product was purified by FC (hexane/EtOAc 100:1) to give 257 (166 mg, 81 %).

Rf: 0.35 (EtOAc/MeOH 10:1).

180 Experimental Section

1 H-NMR (400 MHz, CDCl3):  = 7.59-7.14 (m, 4H, Ar-H); 5.80-5.70 (m, 1H, H-2); 5.05-4.97 (m, 3H, H-1 and H-6); 4.54 (bs, 1H, H-5); 4.27-4.12 (m, 1H, H-10); 3.88 (bs, 1H, H-4); 3.07-2.83 (m, 2H, H-9); 2.36-2.28 (m, 1H, H-3); 2.09-2.03 (m, 1H, H-3);

1.52 (s, 3H, C(CH3)2); 1.37 (s, 3H, C(CH3)2); 1.19 (d, J = 6.2 Hz, 3H, H-11); 1.04 (s, 6H, Si-CH(CH3)2); 1.03 (s, 3H, Si-CH(CH3)2); 0.97 (s, 6H, Si-CH(CH3)2); 0.95 (s, 4H,

Si-CH(CH3)2); 0.85 (s, 9H, Si-(CH3)3); 0.01 (s, 3H, Si-(CH3)2); -0.12 (bs, 3H, Si-

(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 135.2 (C-2); 127.6 (ar-C); 127.5 (ar-C); 126.1 (ar-C);

117.1 (C-1); 108.2 (C(CH3)2); 81.9 (C-5); 77.4 (C-4); 69.1 (C-6); 69.0 C-10); 42.2 (C-

9); 34.8 (C-3); 28.2 (C(CH3)2); 26.0 (C(CH3)2 and Si-C(CH3)3); 24.5 (C-11); 18.3 (Si- CH(CH3)2); 18.2 (Si-CH(CH3)2); 18.2 (Si-C(CH3)3); 13.1 (Si-CHCH3); 12.9 (Si-

CHCH3); -4.3 (Si-(CH3)2); -4.5 (Si-(CH3)2).

IR (film): max = 2929, 2865, 1458, 1369, 1254, 1218, 1088, 1054, 988, 911, 883, 834, 774, 678 cm-1. + HRMS (ESI): calcd for C33H60NaO4Si2 [M-Na] : 599.3922, found: 599.3925.

Si(CH3)3  OH O OTBS  1 3 O 11' 7' 9' 3' 5' O OTIPS 51' O O 258 2-{3-[(4S,5S)-5-({2-[(R)-2-(tert-Butyl-dimethyl-silanyloxy)-propyl]-phenyl}- triisopropylsilanyloxy-methyl)-2,2-dimethyl-[1,3]dioxolan-4-yl]-propyl}-6- hydroxy-4-methoxy-benzoic acid 2-trimethylsilanyl-ethyl ester The reaction was carried out under an argon atmosphere. To a solution of 257 (150 mg, 0.26 mmol, 1 equiv) in 0.5 ml abs THF was added dropwise a 0.5 M solution of 9-BBN in THF (0.73 ml, 0.36 mmol, 1.4 equiv) and the mixture was stirred at rt for 1.5 h. Then a 2 M solution of K3PO4 (0.26 ml, 0.52 mmol, 2 equiv) was added to the mixture (solution A). In a separate flask 176 was added to a mixture of trifurylphosphine (36 mg, 0.16 mmol, 0.6 equiv) and [Pd(OAc)2] (9 mg, 0.04 mmol, 0.15 equiv) in 1 ml of degassed abs DME (solution B) and stirred for 5 min (the colour changed from red to yellow). Solution A was added dropwise to solution B at rt. The reaction mixture was refluxed at 73°C for 4.5 h and was directly put on Celite and purified by FC (hexane/EtOAc 100:1 → 80:1 → 60:1 → 40:1→ 20:1) to give 258 (154 mg, 70 %).

Rf: 0.18 (hexane/EtOAc 20:1). 1 H-NMR (400 MHz, CDCl3):  = 11.82 (s, 1H, 2-OH); 7.25-7.07 (m, 4H, Ar-H); 6.32 (d, J = 2.6 Hz, 1H, H-3); 6.20 (d, J = 2.5 Hz, 1H, H-5); 5.00 (bs, 1H, H-6’); 4.47-4.33

(m, 3H, H-10’ and H-); 4.21-4.12 (m, 2H, H-4’ and H-5’); 3.79 (s, 3H, 4-OCH3); 3.13-

181 Experimental Section

3.10 (m, 1H, H-9’); 2.89-2.78 (m, 3H, H-1’ and H-9’); 1.81-1.71 (m, 2H, H-2’); 1.64-

1.55 (m, 1H, H-3’); 1.44 (s, 3H, C(CH3)2); 1.35 (s, 3H, C(CH3)2); 1.29-1.24 (m, 1H, H-

3’); 1.17 (d, J = 5.9 Hz, 3H, H-11’); 1.09 (m, 2H, H-); 1.03 (s, 8H, Si-CH(CH3)2); 1.01

(s, 3H, Si-CH(CH3)2); 0.96 (s, 6H, Si-CH(CH3)2); 0.95 (s, 4H, Si-CH(CH3)2); 0.85 (s, 9H, Si-CH(CH3)2); 0.08 (s, 9H, Si-(CH3)3); 0.01 (s, 3H, Si-(CH3)2); -0.12 (bs, 3H, Si-

(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 171.8 (C=O); 165.7 (C-2); 163.9 (C-4); 147.4 (C-6); 127.5 (?); 127.6 (ar-C); 127.5 (ar-C); 126.1 (ar-C); 110.9 (C-5); 107.8 (C(CH3)2); 105.1 (?);99.0 (C-3); 81.9 (C-5’); 77.4 (C-4’); 69.2 (C-6’); 68.9 C-10’); 63.8 (C-?); 55.4

(4-OCH3); 42.2 (C-9’); 36.5 (C-1’); 29. 8 (C-3’); 27.1 (C(CH3)2) and (C-2’); 26.1 (C(CH3)2); 26.0 (Si-C(CH3)3); 22.8 (C-11’); 18.3 (Si-CH(CH3)2); 18.2 (Si-CH(CH3)2);

18.2 (Si-C(CH3)3); 17.7 (C-); 12.9 (Si-CHCH3); -1.4 (Si-(CH3)3); -4.3 (Si-(CH3)2); -4.5

(Si-(CH3)2).

IR (film): max = 2948, 2865, 1652, 1616, 1508, 1251, 1205, 1158, 1057, 989, 834, 757, 669 cm-1. + HRMS (ESI): calcd for C46H80NaO8Si3 [M-Na] : 867.5053, found: 867.5036.

OH O OH 1 3 OH 11' 7' 9' 3' 5' O OH 51' O O 259 2-Hydroxy-6-[3-((4S,5R)-5-{hydroxy-[2-((R)-2-hydroxy-propyl)-phenyl]-methyl}- 2,2-dimethyl-[1,3]dioxolan-4-yl)-propyl]-4-methoxy-benzoic acid To a stirred solution of 258 (17.5 mg, 0.02 mmol, 1.0 equiv) in THF (0.5 ml) was added tetra-n-butylammonium fluoride (1 M solution in THF; 63 l, 0.04 mmol, 3 equiv) dropwise at rt. The reaction was stirred for 1.5 h. Saturated NH4Cl solution was added and the mixture (pH = 6) was extracted with EtOAc (2 x 5 ml). The combined organic phases were dried over MgSO4, filtered and evaporated in vacuo. The crude product was directly put on the column and purified by FC (EtOAc/MeOH 100:1 → 50:1 → 20:1 → 10:1 → 5:1) to give 259 (8.6 mg, 88 %).

Rf: 0.24 (EtOAc/MeOH 10:1). NMR: broad signals.

IR (film): max = 2962, 2928, 1582, 1457, 1430, 1374, 1271, 1159, 1076, 834, 748, 668 cm-1. + HRMS (ESI): calcd for C46H80NaO8Si3 [M-Na] : 497.2146, found: 497.2148.

182 Experimental Section

6.2.3.6. Preparation of Compounds described in Chapter 3.3.2.3.

5 3

1 OTES I 260 Triethyl-[(R)-2-(2-iodo-phenyl)-1-methyl-ethoxy]-silane 251 (800 mg, 3.05 mmol, 1.0 equiv) was dissolved in 4 ml of DMF. DIEA (1.04 ml, 6.10 mmol, 2.0 equiv) and TES-Cl (775 l, 4.58 mmol, 1.5 equiv) were added to the solution which was stirred at rt overnight. The reaction was quenched by addition of

4 ml of sat. NaHCO3 and it was extracted with EtOAc (3 x 8 ml). The combined organic extracts were dried over MgSO4, filtered and evaporated in vacuo. The crude product was purified by FC (hexane/EtOAc 100:1 → 50:1) to give 260 (1.09 g, 95 %).

Rf: 0.8 (hexane/ EtOAc 10:1). 1 H-NMR (400 MHz, CDCl3):  = 7.80 (dd, J = 8.2 Hz, 1H, H-6); 7.24-7.20 (m, 2H, H-3 and H-5); 6.91-6.86 (m, 1H, H-4); 4.15-4.07 (m, 1H, CH); 2.84 (dd, J = 3.4 Hz and 7.2

Hz, 2H, CH2); 1.20 (d, J = 6.0 Hz, 3H, CH3); 0.86 (t, J = 8.1 Hz, 9H, Si-CH2CH3); 0.53-0.37 (m, 6H, Si-CH2CH3). 13 C-NMR (100 MHz, CDCl3):  = 142.3 (C-2); 139.4 (C-6); 132.0 (C-3); 128.1 (C-4);

128.0 (C-5); 101.1 (C-1); 68.1 (CH); 50.5 (CH2); 24.0 (CH3); 7.0 (Si-CH2CH3); 4.9 (Si- CH2CH3).

IR (film): max = 2953, 2875, 1457, 1375, 1129, 1120, 1059, 1010, 991, 743, 714, 669 cm-1. + HRMS (ESI): calcd for C13H20IOSi [M-C2H5] : 347.0314, found: 347.0323.

TESO 11 9 7 3 5 1 OH O O 261

((4R,5S)-5-Allyl-2,2-dimethyl-[1,3]dioxolan-4-yl)-[2-((R)-2-triethylsilanyloxy- propyl)-phenyl]-methanol To a stirred solution of aryl iodide 260 (885 mg, 2.35 mmol, 2.6 equiv) in THF (9 ml) was added n-BuLi (1.6 M in hexane, 1.38 ml, 2.21 mmol, 2.4 equiv) over one hour at -83°C. After 1 h stirring, a solution of 191 in THF (4 ml) was added dropwise within 30 min. The reaction mixture was placed in a -80°C freezer overnight. The mixture was treated with NH4Cl solution (9 ml). The layers were separated and the aqueous layer was extracted with Et2O (2 x 7 ml). The combined organic extracts were dried

183 Experimental Section

over MgSO4, filtered, and evaporated in vacuo. The crude product was purified by FC (hexane/EtOAc 100:1 → 70:1 → 30:1 → 10:1) to give 261 (265 mg, 72 %, 2:1 mixture of isomers).

Rf: 0.42, 0.48 (hexane/ EtOAc 5:1). 1 H-NMR (400 MHz, CDCl3):  = 7.40-7.38 (m, 1H, Ar-H); 7.25-7.16 (m, 3H, Ar-H); 5.77-5.67 (m, 1H, H-2); 5.04-4.94 (m, 3H, H-1 and H-6); 4.67 (t, J = 6.4 Hz, 1H, H-5); 4.23-4.18 (m, 1H, H-4); 4.06-3.98 (m, 1H, H-10); 3.68 (d, J = 2.7 Hz, 1H, 6-OH); 2.96 (dd, J = 13.5 Hz, 1H, H-9); 2.69 (dd, J = 13.7 Hz, 1H, H-9); 2.34-2.25 (m, 1H, H-3);

1.94-1.87 (m, 1H, H-3); 1.59 (s, 3H, C(CH3)2); 1.45 (s, 3H, C(CH3)2); 1.26 (d, J =

6.1 Hz, 3H, H-11); 0.76 (t, J = 7.8 Hz, 9H, Si-CH2CH3); 0.40 (q, J = 7.8 Hz, 6H, Si- CH2CH3). 13 C-NMR (100 MHz, CDCl3):  = 139.4 (C-7); 138.1 (C-7); 134.9 (C-2); 130.6 (ar-C);

128.2 (ar-C); 126.9 (ar-C); 126.3 (ar-C); 117.2 (C-1); 108.8 (C(CH3)2); 80.0 (C-5); 77.1 (C-4); 70.8 (C-10); 67.7 (C-6); 42.1 C-9); 34.7 (C-3); 28.2 (C(CH3)2); 25.9

(C(CH3)2); 24.5 (C-11); 6.7 (Si-CH2CH3); 4.6 (Si-CH2CH3).

IR (film): max =2955, 2876, 1457, 1375, 1217, 1126, 1057, 1002, 888, 742, 726, 669 cm-1. + HRMS (ESI): calcd for C24H40NaO4Si [M-Na] : 443.2588, found: 443.2583.

TESO 11 9 7 3 5 1 OTBS O O 262

(4S,5S)-4-Allyl-5-{(tert-butyl-dimethyl-silanyloxy)-[2-((R)-2-triethylsilanyloxy- propyl)-phenyl]-methyl}-2,2-dimethyl-[1,3]dioxolane

261 (254 mg, 0.60 mmol, 1.0 equiv) was dissolved in abs CH2Cl2 (9 ml) and 2,6- lutidine (155 ml, 1.33 mmol, 2.2 equiv) was added. The mixture was cooled to -78°C.

To the cold mixture was added a solution of TBS-OTf in 0.8 ml abs CH2Cl2. The reaction mixture was allowed to warm to rt overnight. Sat. NaHCO3 solution was added and the mixture was extracted with CH2Cl2 (3 x 10 ml). The combined organic phases were dried over MgSO4, filtered, and evaporated in vacuo. The crude product was purified by FC (hexane/EtOAc 100:0 → 100:1 → 50:1) to give 262 (302 mg, 94 %, 2:1 mixture of isomers).

Rf: 0.26 (hexane/ EtOAc 20:1). 1 H-NMR (400 MHz, CDCl3):  = 7.51(bs, 1H, Ar-H); 7.20-7.17 (m, 3H, Ar-H); 5.90- 5.79 (m, 1H, H-2); 5.14-5.09 (m, 2H, H-1); 4.99 (d, J = 3.1 Hz, 1H, H-6); 4.23 (bs, 1H, H-5); 4.17-4.04 (m, 2H, H-4 and H-10); 2.91-2.86 (m, 1H, H-9); 2.74-2.69 (m, 1H, H-

9); 2.61-2.53 (m, 1H, H-3); 2.32-2.26 (m, 1H, H-3); 1.61 (s, 3H, C(CH3)2); 1.35 (s, 3H,

C(CH3)2); 1.15 (d, J = 6.0 Hz, 3H, H-11); 0.92 (t, J = 7.9 Hz, 9H, Si-CH2CH3); 0.89 (s, 184 Experimental Section

9H, Si-C(CH3)3); 0.55 (q, J = 7.8 Hz, 6H, Si-CH2CH3); 0.07 (s, 3H, Si(CH3)2); -0.25 (s, 3H, Si-(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 139.66 (C-7); 135.19 (C-8 and C-2); 130.82 (C-Ar);

129.47 (ar-C); 127.36 (ar-C); 126.12 (ar-C); 117.07 (C-1); 108.37 (C(CH3)2);81.07 (C- 5); 77.01 (C-4); 68.83 (C-10 and C-6); 42.57 (C-9); 34.18 (C-3); 27.50 (C(CH3)2);

26.08 (Si-C(CH3)3); 25.85 (C(CH3)2); 24.09 (C-11); 18.35 (Si-C(CH3)3); 6.98 (Si-

CH2CH3); 5.12 (Si-CH2CH3); -3.77 (Si(CH3)2); -4.18 (Si(CH3)2).

IR (film): max = 2955, 2930, 1457, 1376, 1252, 1217, 1072, 1004, 834, 774, 744, 669 cm-1. + HRMS (ESI): calcd for C30H54NaO4Si2 [M-Na] : 557.3453, found: 557.3451.

OH O OTES 1 9' 11' 3 O 7' 3' 5' O OTBS 51' O O 263

2-[3-((4S,5S)-5-{(tert-Butyl-dimethyl-silanyloxy)-[2-((R)-2-triethylsilanyloxy- propyl)-phenyl]-methyl}-2,2-dimethyl-[1,3]dioxolan-4-yl)-propyl]-6-hydroxy-4- methoxy-benzoic acid methyl ester The reaction was carried out under an argon atmosphere. To a solution of 262 (281 mg, 0.53 mmol, 1.0 equiv) in 1 ml abs THF was added dropwise a 0.5 M solution of 9-BBN in THF (1.58 ml, 0,79 mmol, 1.5 equiv) and the mixture was stirred at rt for 1.5 h. Then a 2 M solution of K3PO4 (0.53 ml, 1.05 mmol, 2.0 equiv) was added to the mixture (solution A). In a separate flask 29a (136 mg, 0.53 mmol, 1.0 equiv) was added to a mixture of trifurylphosphine (73 mg, 0.32 mmol, 0.6 equiv) and [Pd(OAc)2] (18 mg, 0.08 mmol, 0.15 equiv) in 3.5 ml of degassed abs DME (solution B) and stirred for 5 min (the colour changed from red to yellow). Solution A was added dropwise to solution B at rt. The reaction mixture was refluxed at 73°C for 6 h and directly put on Celite and purified by FC (hexane/EtOAc 100:1 → 75:1 → 50:1 → 25:1) to give 263 (286 mg, 76 %).

Rf: 0.15 (hexane/ EtOAc 10:1). 1 H-NMR (400 MHz, CDCl3):  = 11.73 (s, 1H, 2-OH); 7.48 (bs, 1H, Ar-H); 7.20-7.17 (m, 3H, Ar-H); 6.33 (d, J = 2.7 Hz, 1H, H-3); 6.27 (d, J = 2.6 Hz, 1H, H-5); 4.96 (bs, 1H, H-6’); 4.24-4.18 (m, 1H, H-5’); 4.15-4.09 (m, 1H, H-10’); 4.04-3.98 (m, 1H, H-4’);

3.88 (s, 3H, 1-OCH3); 3.79 (s, 3H, 4-OCH3); 2.95-2.89 (m, 1H, H-9’); 2.85 (t, J = 7.4 Hz, 2H, H-9’); 2.77-2.67 (m, 1H, H-9’); 1.91-1.78 (m, 2H, H-2’ and H-3’); 1.56 (s, 3H,

C(CH3)2); 1.54-1.47 (m, 2H, H-2’ and H-3’); 1.33 (s, 3H, C(CH3)2); 1.13 (d, J = 6.0 Hz,

3H, H-11’); 0.97-0.90 (m, 9H, Si-CH2CH3); 0.88 (s, 9H, Si-C(CH3)3); 0.57 (q, J =

7.6 Hz, 6H, Si-CH2CH3); 0.05 (s, 3H, Si-(CH3)2); -0.24 (s, 3H, Si-(CH3)2).

185 Experimental Section

13 C-NMR (100 MHz, CDCl3):  = 172.0 (C=O); 165.8 (C-2); 164.2 (C-4); 147.4 (C-6); 139.7 (C-7’); 137.0 (C-8’); 130.7 (ar-C); 129.5 (ar-C); 127.4 (ar-C); 126.1 (ar-C);

110.9 (C-5); 108.1 (C(CH3)2); 104.7 (C-1); 99.0 (C-3); 81.3 (C-5’); 77.4 (C-4’); 69.0

(C-10’); 68.8 (C-6’); 55.4 (4-OCH3); 52.0 (1-OCH3); 42.7 (C-9); 36.9 (C-1’); 29.9 (C- 3’); 29.0 (C-2’), 27.3 (C(CH3)2); 26.1 (Si-C(CH3)3); 25.8 (C(CH3)2); 23.9 (C-11); 18.3

(Si-C(CH3)3); 7.0 (Si-CH2CH3); 5.1 (Si-CH2CH3); -3.8 (Si-(CH3)2); -4.2 (Si-(CH3)2).

IR (film): max = 2953, 2875, 1653, 1616, 1559, 1472, 1377, 1326, 1254, 1214, 1157, 1080, 1003, 835, 712, 669 cm-1. + HRMS (ESI): calcd for C39H64NaO8Si2 [M-Na] : 739.4032, found: 739.4039.

OH O OH 1 9' 11' 3 O 7' 3' 5' O OTBS 51' O O 264

THF/HOAc/H2O. After 1.5 h stirring at rt sat. Na2CO3 solution was added until pH = 6 was obtained. EtOAc was added to the mixture and the phases were separated. The aqueous phase was further extracted with EtOAc (3 x 5 ml). The combined organic extracts were washed with brine, dried over MgSO4, filtered and evaporated in vacuo. The crude product was purified through a short flash column (hexane/EtOAc 50:1 → 20:1 → 10:1 → 5:1 → 2:1) to give 264 (156 mg, 70 %).

Rf: 0.40 (hexane/ EtOAc 2:1). 1 H-NMR (400 MHz, CDCl3):  = 11.73 (s, 1H, 2-OH); 7.48 (bs, 1H, Ar-H); 7.20-7.17 (m, 3H, Ar-H); 6.33 (d, J = 2.7 Hz, 1H, H-3); 6.27 (d, J = 2.6 Hz, 1H, H-5); 4.96 (bs, 1H, H-6’); 4.24-4.18 (m, 1H, H-5’); 4.15-4.09 (m, 1H, H-10’); 4.04-3.98 (m, 1H, H-4’);

3.88 (s, 3H, 1-OCH3); 3.79 (s, 3H, 4-OCH3); 2.95-2.89 (m, 1H, H-9’); 2.85 (t, J = 7.4 Hz, 2H, H-9’); 2.77-2.67 (m, 1H, H-9’); 1.91-1.78 (m, 2H, H-2’ and H-3’); 1.56 (s, 3H,

C(CH3)2); 1.54-1.47 (m, 2H, H-2’ and H-3’); 1.33 (s, 3H, C(CH3)2); 1.13 (d, J = 6.0 Hz,

3H, H-11’); 0.97-0.90 (m, 9H, Si-CH2CH3); 0.88 (s, 9H, Si-C(CH3)3); 0.57 (q, J =

7.6 Hz, 6H, Si-CH2CH3); 0.05 (s, 3H, Si-(CH3)2); -0.24 (s, 3H, Si(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 171.9 (C=O); 165.8 (C-2); 164.1 (C-4); 147.2 (C-6); 140.4 (C-7’); 137.0 (C-8’); 130.5 (C-Ar); 129.5 (ar-C); 127.8 (ar-C); 126.6 (ar-C);

111.0 (C-5); 108.2 (C(CH3)2); 104.7 (C-1); 99.0 (C-3); 81.5 (C-5’); 77.5 (C-4’); 68.6

(C-10’); 60.5 (C-6’); 55.4 (4-OCH3); 52.0 (1-OCH3); 42.3 (C-9’); 36.8 (C-1’); 29.6 (C-

3’); 28.7 (C-2’), 27.6 (C(CH3)2); 26.0 (Si-C(CH3)3); 25.9 (C(CH3)2); 23.6 (C-11’); 18.3 (Si-C(CH3)3); -4.0 (Si-(CH3)2); -4.3 (Si-(CH3)2).

186 Experimental Section

IR (film): max = 2954, 2857, 1614, 1459, 1374, 1303, 1214, 1111, 1052, 953, 905, 834, 775, 673, 516 cm-1. + HRMS (ESI): calcd for C33H50NaO8Si [M-Na] : 625.3167, found: 625.3162.

OH O OH 1 9' 11' 3 OH 7' 3' 5' O OTBS 51' O O 265

2-[3-((4S,5S)-5-{(tert-Butyl-dimethyl-silanyloxy)-[2-((R)-2-hydroxy-propyl)- phenyl]-methyl}-2,2-dimethyl-[1,3]dioxolan-4-yl)-propyl]-6-hydroxy-4-methoxy- benzoic acid 264 (113 mg, 0.19 mmol, 1.0 equiv) was dissolved in 3.8 ml of MeOH and 1.2 ml 2N NaOH was added to the mixture. The reaction mixture was refluxed at 85°C for 8 h. At that point most of the starting material had been consumed and a sideproduct started to appear. The mixture was diluted with EtOAc (20 ml) and the aqueous phase was acidified with 1N HCl to pH 4. The aqueous phase was extracted with

EtOAc (3 x 10 ml). The combined organic extracts were dried over MgSO4, filtered and evaporated in vacuo. The crude product was purified by FC (fine silica gel; neat EtOAc) to give 265 (41 mg, 37 %).

Rf: 0.29 (EtOAc/MeOH 30:1). NMR: broad signals. + HRMS (ESI): calcd for C32H48NaO8Si [M-Na] : 611.3011, found: 611.3009.

11' OH O 1 9' 3 O 7' 1' 3' 5' O OTBS 5 O O 266

(3aS,10R,19aS)-4-{[tert-Butyl(dimethyl)silyl]oxy}-10,13-dihydroxy-15-methoxy- 2,2-dimethyl-3a,4,9,10,17,18,19,19aoctahydro-12H-dibenzo[c,k][1,3]dioxolo[4,5- h]oxacyclotetradecin-12-one To a solution of 265 (40.8 mg, 0.07 mmol, 1.0 equiv) in 9.9 ml abs toluene (0.007 M) was added triphenylphosphine (37 mg, 0.14 mmol, 2.0 equiv) and DIAD (28 l, 0.14 mmol, 2.0 equiv). The mixture was stirred for 2 h at rt. The reaction mixture was directly put on Celite and purified by FC (hexane/EtOAcE 50:1 → 10:1 → 5:1) to give 266 (10.1 mg, 26 %).

Rf: 0.40 (hexane/EtOAc 5:1).

187 Experimental Section

1 H-NMR (400 MHz, CDCl3):  = 11.91 (s, 1H, 2-OH); 7.39 (dd, J = 1.5 Hz and 7.5 Hz, 1H, Ar-H); 7.21-7.12 (m, 3H, Ar-H); 6.26 (d, J = 2.8 Hz, 1H, H-3); 6.12 (d, J = 2.8 Hz, 1H, H-5); 6.11-6.07 (m, 1H, H-10’); 5.09 (d, J = 8.7 Hz, 1H, H-6’); 4.55 (dd, J = 5.2 Hz and 8.5 Hz, 1H, H-5’); 3.73 (s, 3H, 4-OCH3); 3.67-3.63 (m, 1H, H-4’); 3.42 (dd, J = 11.9 Hz and 16.2 Hz, 1H, H-9’); 3.07-3.00 (m, 1H, H-1’); 2.95 (dd, J = 2.9 Hz and 16.2 Hz, 1H, H-9’); 2.98-2.91 (m, 1H, H-1’); 1.59 (d, J = 6.0 Hz, 3H, H-11’); 1.52 (s,

3H, C(CH3)2); 1.50-1.41 (m, 2H, H-3’); 1.39 (s, 3H, C(CH3)2); 1.30-1.24 (m, 1H, H-2’); 0.90-0.84 (m, 1H, H-2’); 0.77 (s, 9H, Si-C(CH3)3); 0.08 (s, 3H, Si(CH3)2); 0.00 (s, 3H,

Si(CH3)2). 13 C-NMR (100 MHz, CDCl3):  = 171.3 (C=O); 165.7 (C-2); 164.1 (C-4); 147.7 (C-6); 139.5 (C-7’); 134.3 (C-8’); 128.4 (ar-C); 127.8 (ar-C); 127.0 (ar-C); 126.7 (ar-C);

110.7 (C-5); 107.9 (C(CH3)2); 105.0 (C-1); 99.2 (C-3); 82.8 (C-5’); 77.5 (C-4’); 69.4

(C-10’); 68.8 (C-6’); 55.4 (4-OCH3); 36.9 (C-9’); 36.7 (C-1’); 33.0 (C-3’); 30.5 (C-2’), 28.6 (C(CH3)2); 26.0 (C(CH3)2); 25.8 (Si-C(CH3)3); 21.5 (C-11’); 18.4 (Si-C(CH3)3);

-3.8 (Si-(CH3)2); -4.4 (Si-(CH3)2).

IR (film): max = 2930, 2856, 2360, 2338, 1646, 1613, 1578, 1460, 1376, 1303, 1253, 1211, 1160, 1108, 1064, 875, 740 cm-1. + HRMS (ESI): calcd for C32H46NaO7Si [M-Na] : 593.2905, found: 593.2908.

11' OH O 1 9' 3 O 7' 1' 3' 5' O OH 5 O O 267

(3aR,10R,19aS)-4,10,13-Trihydroxy-15-methoxy-2,2-dimethyl- 3a,4,9,10,17,18,19,19a-octahydro-12H-dibenzo[c,k][1,3]dioxolo[4,5- h]oxacyclotetradecin-12-one To a stirred solution of 266 (10.1 mg, 0.018 mmol, 1.0 equiv) in abs THF (0.5 ml) at rt tetra-n-butylammonium fluoride (1 M solution in THF; 20 l, 0.020 mmol, 1.1 equiv) was added dropwise. The reaction was stirred overnight. NH4Cl solution (5 ml) was added and the mixture (pH = 6) was extracted with EtOAc (2 x 5 ml). The combined organic phases were dried over MgSO4, filtered, and evaporated in vacuo. The crude product was directly put on the column and purified by FC (hexane/EtOAc 50:1 → 10:1 → 1:1) to give 267 (7.1 mg, 88 %).

Rf: 0.21 (hexane/EtOAc 2:1). 1 H-NMR (400 MHz, CDCl3):  = 11.77 (s, 1H, 2-OH); 7.41-7.34 (m, 2H, Ar-H); 7.24- 7.17 (m, 2H, Ar-H); 6.26 (d, J = 2.7 Hz, 1H, H-3); 6.16 (d, J = 2.7 Hz, 1H, H-5); 6.11- 6.04 (m, 1H, H-10’); 5.06 (dd, J = 1.6 Hz and 8.0 Hz, 1H, H-6’); 4.52 (dd, J = 5.5 Hz and 8.0 Hz, 1H, H-5’); 4.02-3.98 (m, 1H, H-4’); 3.74 (s, 3H, 4-OCH3); 3.50 (dd, J = 11.8 Hz and 16.2 Hz, 1H, H-9’); 3.18-3.11 (m, 1H, H-1’); 3.01 (dd, J = 2.8 Hz and 16.1 Hz, 1H, H-9’); 2.85 (d, J = 2.4 Hz, 1H, 6’-OH); 2.39-2.33 (m, 1H, H-1’); 1.74-1.62 188 Experimental Section

(m, 2H, H-3’); 1.58 (d, J = 6.2 Hz, 3H, H-11’); 1.57 (s, 3H, C(CH3)2); 1.44 (s, 3H, C(CH3)2); 1.43-1.36 (m, 1H, H-2’); 1.12-1.01 (m, 1H, H-2’). 13 C-NMR (100 MHz, CDCl3):  = 171.2 (C=O); 165.5 (C-2); 164.0 (C-4); 147.1 (C-6); 138.4 (C-7’); 136.3 (C-8’); 128.7 (ar-C); 128.1 (ar-C); 127.4 (ar-C); 127.2 (ar-C);

110.6 (C-5); 108.4 (C(CH3)2); 105.3 (C-1); 99.2 (C-3); 80.7 (C-5’); 77.6 (C-4’); 66.7

(C-10’); 66.7 (C-6’); 55.4 (4-OCH3); 36.6 (C-9’); 36.2 (C-1’); 31.0 (C-3’); 29.6 (C-2’);

28.3 (C(CH3)2); 25.9 (C(CH3)2); 21.5 (C-11’).

IR (film): max = 2982, 2360, 1643, 1576, 1424, 1353, 1255, 1159, 1107, 1035, 872, 809, 730, 649 cm-1. + HRMS (ESI): calcd for C26H32NaO7 [M-Na] : 479.2040, found: 479.2028.

11' OH O 1 9' 3 O 7' 1' 3' 5' O OH 5 HO OH 268 (6S,7S,17R)-5,6,7,14,17-Pentahydroxy-12-methoxy-5,6,7,8,9,10,17,18-octahydro- 15H-dibenzo[c,k]oxacyclotetradecin-15-one 267 (7.3 mg, 0.016 mmol, 1.0 equiv) was dissolved in 2 ml of abs MeOH, sulfonic acid resin (3.1 mmol/g; 5.2 mg, 0.016 mmol, 1.0 equiv) was added, and the mixture was refluxed for 1 hour. The resin was filtered off and washed with methanol. The filtrate was evaporated and the crude product was purified by FC (EtOAc/MeOH 100:1 → 10:1) to give 268 (5.5 mg, 82 %). minor isomer:

Rf: 0.28 (EtOAc/MeOH 20:1). 1 H-NMR (400 MHz, CDCl3):  = 11.97 (s, 1H, 2-OH); 7.56 (dd, J = 1.5 Hz and 7.6 Hz, 1H, ar-H); 7.37-7.23 (m, 3H, ar-H); 6.29 (d, J = 2.7 Hz, 1H, H-5); 6.19 (d, J = 2.7 Hz, 1H, H-3); 6.10-5.92 (m, 1H, H-10’); 5.17 (d, J = 5.0 Hz, 1H, H-6’); 4.02 (dd, J = 2.6 Hz and 5.0 Hz, 1H, H-5’); 3.76 (s, 3H, 4-OCH3); 3.51-3.43 (m, 2H, H-4’ and H-9’); 3.07 (dt, J = 4.5 Hz and 12.2 Hz, 1H, H-1’); 2.96 (dd, J = 3.0 Hz and 16.3 Hz, 1H, H-9’); 2.38 (dt, J = 5.0 Hz and 12.4 Hz, 1H, H-1’); 1.86-1.66 (m, 2H, H-3’); 1.57 (d, J = 5.9 Hz, 3H, H-11’); 1.52-1.40 (m, 1H, H-2’); 1.22-1.14 (m, 1H, H-2’). 13 C-NMR (100 MHz, CDCl3):  = 171.5 (C=O); 166.3 (C-2); 164.2 (C-4); 147.3 (C-6); 138.7 (C-7’); 134.9 (C-8’); 128.9 (C-ar); 127.7 (ar-C); 127.5 (ar-C); 127.4 (ar-C); 110.3 (C-5); 104.7 (C-1); 99.3 (C-3); 77.4 (C-5’); 73.5 (C-4’); 71.5 (C-10’); 68.1 (C-6’);

55.4 (4-OCH3); 37.0 (C-1’); 36.7 (C-9’); 33.4 (C-3’); 29.3 (C-2’); 21.9 (C-11’). + HRMS (ESI): calcd for C23H28NaO7 [M-Na] : 439.1727, found: 439.1732.

189 Experimental Section

11' OH O 1 9' 3 O 7' 1' 3' 5' O O 5 HO OH P6 (6S,7S,17S)-6,7,14-Trihydroxy-12-methoxy-17-methyl-7,8,9,10,17,18-hexahydro- 5H-dibenzo[c,k]oxacyclotetradecine-5,15(6H)-dione

268 (4.5 mg, 0.011 mmol, 1.0 equiv) was dissolved in 0.5 ml abs CH2Cl2 and DMP (4.5 mg, 0.011 mmol, 1.0 equiv) was added at once at rt. The conversion was monitored by TLC every 5 min. The reaction mixture was stirred for 20 min. The reaction mixture was directly put on the column and purified by FC (pure EtOAc) to give P6 (2.8 mg, 62 %). The product decomposed upon storage at 7°C.

Rf: 0.48 (EtOAc/MeOH 20:1). MS-ESI: m/z = 413.03 [M-H]-.

6.2.3.7. pH Stability The chemical stability of L-783277 (6) was tested by incubation with hydrochloric acid (HCl) solutions of different pH values (1, 3, 5) at 25°C. 6 was dissoveld in DMSO to give a stock solution with a concentration of 1 mg/ml. 30 l of the stock solution of 6 were incubated with 130 l of each hydrochloric solution. After distinct time points (0.25 h, 1 h, 3.5 h, 6 h, 24 h) the mixtures (4 l) were injected and measured by HPLC to follow the degradation process of 6.

6.2.3.8. Blood Plasma Stability The metabolic stability of L-783277 (6) was tested in human plasma. For this stability study in serum triplicate samples were incubated with a stock solution of 6 (4.05 mg/ml) and denatured by addition of methanol at distinct time points: 0.5 h, 1 h, 3 h, 6.5 h. After centrifugation of the individual samples, the supernatant (3 x 4 l) was injected and the quantity of 6 and its degradation products were measured by HPLC. To determine the recovery rate of 6 a calibration curve was established with 5 different concentrations in a range from 0.1 mg/ml to 1 mg/ml (Fig. 27), which could be used to quantifiy the reextracted 6 after denaturation.

190 Experimental Section

25.000.000 y = 2E+07x + 195893 R2 = 0,9996

20.000.000

15.000.000

10.000.000 % of L-783277 % of

5.000.000

- 0,00 0,20 0,40 0,60 0,80 1,00 1,20 conc [mg/ml]

Figure 27: HPLC calibration curve of L-783277 (6) for the UV absorption in dependence on the compound concentration.

6.2.3.9. Inhibition of Cellular Proliferation The cellular proliferation experiments have been carried out by Benjamin Vigl.

Measurement of cell growth inhibition (EC50 value determination): Human LECs and HUVECs were maintained on fibronectin coated plates in EBM 20 % FCS, 2 mmol/l L-Glutamine, 25 µg/ml cAMP and 10 µg/ml hydrocortisol211 whereas NIH 3T3 mouse fibroblasts, human MCF-7 breast cancer cells and human HaCaT keratinocytes were kept in DMEM 10 % FCS. To quantify proliferation the fluorometric MUH (5- methylumbelliferylheptanoate) assay was used as described in Detmar et al.208 and Kajiya et al.209 1500 to 300 cells/well were seeded to 96 well plates, which in case of endothelial cells were fibronectin coated. After 24 h cells were starved for additional 24 h in medium containing only 2 %FCS. L-783277 (6) was added in the given concentration in half-log dilution steps from a stock concentration of 10 mg/ml in DMSO stored on - 20°C. For endothelial cells VEGF-A was added as a proliferative stimulus at a final concentration of 20 ng/ml. The DMSO concentration was added on all compound containing wells at the concentration found in the highest L-783277 (6) concentration. Cells were incubated for 72 h and washed with PBS twice. 150 l MUH (50 g/ml in PBS) were added for one hour on 37°C. Fluorescence, which is proportional to the number of cells, was recorded after shaking on a SpectraMAX Gemini EM (Buchler Biotech). The background of empty wells was substracted; growth was expressed as percentage of control treatment and plotted as a semi-log dose-response curve in GraphPad Prism 4.01. Sigmoid dose-

191 Experimental Section response curve was calculated. Five technical replicates per condition were made and experiments were repeated twice for each cell type. Values are displayed as average  SD.

Table 8: Inhibition of proliferation of different cell types by L-783277 (6). Cell growth in presence of varying concentrations of L-783277 (6) was determined by MUH assay. Endothelial cells were grown in absence or presence of VEGF (20 ng/ml). EC50 values are provided in nanomolar concentrations. Experiments were repeated twice for each cell type with comparable results. Endothelial cells Non-endothelial cells Cell NIH3 LEC HUVEC HaCaT MCF7 type T3

EC50 no VEGF + VEGF no VEGF + VEGF (nM) 58.2 28.8 33.0 27.0 1712 610 8371

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