Novel Chemical Probes for the Cannabinoid System

Research Collection

Doctoral Thesis

Author(s): Westphal, Matthias V.

Publication Date: 2017

Permanent Link: https://doi.org/10.3929/ethz-b-000255663

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ETH Library

DISS. ETH NO. 24543

Novel Chemical Probes for the Cannabinoid System

A dissertation submitted to

ETH ZURICH

for the degree of

DOCTOR OF SCIENCES

presented by

MATTHIAS V. WESTPHAL

M.Sc. LMU Munich

born on 16.07.1987

Citizen of the Federal Republic of Germany

Accepted on the recommendation of

Prof. Dr. Erick M. Carreira, examiner

Prof. Dr. Pablo Rivera-Fuentes, co-examiner

2017

Acknowledgements

Throughout the last three decades I was very lucky to receive support by so many individuals to whom I am greatly indebted.

First and foremost, I wish to thank my family and friends – you are awesome. Many thanks to my mom Regine and dad Rolf, to Günter, Tante Inge, Omama and Opapa for your everlasting support, for believing in me and for the financial safety to focus on my goals. Thank you Veronika and Arabella for being great siblings. And of course thanks to my longtime friends in the Weinheim area. I consider myself very fortunate that we have been keeping in touch throughout the last decade.

During my early education I have had a number of outstanding teachers. I would like to thank Ms. B. Raepple and Ms. R. Pichl for their unique ways of teaching as well as Mr. R. Thieme for dragging me into science.

I wish to express my sincere gratitude to Prof. Erick M. Carreira for giving me the opportunity to pursue PhD studies under his supervision. I very much appreciate his trust in my abilities and the scientific freedom I was allowed to enjoy along with the chance to interact with various external collaborators in the exciting field of cannabinoid research.

I am grateful to Prof. Pablo Rivera-Fuentes for taking over the co-examination of this thesis. Philipp Sondermann, Moritz Hönig, Adrian Bailey, Volker Berg, Dr. James Frank and Dr. David Petrone are most gratefully acknowledged for proof-reading the manuscript.

The work described in this thesis would not have been possible without the help of many individuals outside of ETH. I am deeply indebted to Dr. Uwe Grether for his constant support of the chemical probes project. Elisabeth Zirwes and Dr. Christoph Ullmer are most gratefully acknowledged for biological testing of so many compounds in various assays. Further thanks go to Dr. Wolfgang Guba for homology modelling and to Dr. Mark Rogers-Evans for his support during the early days of the project. Special thanks go to Dr. James Frank, Prof. Dirk Trauner, Prof. Ken Mackie, Prof. Vsevolod Katritch and their coworkers for the exciting collaboration on photoswitchable THC-derivatives. I am grateful to Marjolein Soethoudt and Prof. Mario van der Stelt for evaluating multiple photoactivatable probes. Likewise, Dr. Andrea Martella and Prof. Laura Heitman are gratefully acknowledged for studying receptor binding kinetics. Prof. Dmitry Veprintsev and Tamara Miljus are thanked for pharmacological studies of various tool compounds. A huge thank you goes to Dr. Michael Schafroth and Roman Sarott ii for a great team effort. Roman, I wish you lots of success in further developing and expanding the project. Team cannabinoids!

I want to thank all members of the Carreira lab. You have made this place and the time spent here truly special by any definition. It has been a pleasure and an honor to work with and to learn from you during the past years. In particular, I want to thank my early and late labmates Dr. Stefan Diethelm, Dr. Lorenz Schneider, Emma Robertson, Dr. Patrick Brady (aka Dr. Postdoc), Dr. Mathias fancy-pants Jacobsen, honorary H336-members Nicole Hauser and innovation manager Dr. Hannes Zipfel, Philipp Sondermann, Niels Sievertsen and Moritz Hönig. Thanks for countless coopen-sie sessions, for sharing one musical highlight after the other, and for your support during the ups and downs of our common journey. Further, I want to thank Marco Brandstätter, Dr. Christian Ebner, Dr. Simon Krautwald, Dr. Nikolas Huwyler, Dr. Simon Breitler and Prof. David Sarlah for various semi-scientific activities in- and outside the lab. Thanks to everyone for fun times in general and the very important lunchen-sie sessions in particular – to the G-floor people: anything can happen on Fridays. Thank you Anke Kleint for administrative help and fun chats, and thanks to my students Michael Imhof, Daniel Joss, Sebastian Hecko, Yuki Fuyuki and Franziska Elterlein for your contributions to the project.

ETH Zurich offers a unique infrastructure for research. I would like to thank everyone involved in keeping this place running, in particular the friendly and extremely helpful colleagues of the NMR service, the great teams running the HCI-Shop, waste disposal and glassware cleaning as well as the mass spectrometry service.

I wish all present and future members of the Carreira lab lots of success in your research and the best of luck in your personal activities also known as real life.

iii

Table of Contents Acknowledgements ...... i Abstract ...... v Zusammenfassung ...... vii I. Introduction ...... 1 1 Interrogation of Biological Systems with Chemical Probes ...... 1 1.1 Fluorescent Probes ...... 3

1.2 Covalent Binders ...... 4

2 Photopharmacology ...... 5 3 The Endocannabinoid System ...... 8 4 Aim of this Work ...... 10 II. Novel Chemical Probes for the Cannabinoid Receptor 2 ...... 11 5 Background ...... 13 6 Triazolopyrimidine-Derived Ligands ...... 15 6.1 Electrophilic Probes ...... 20

6.1.1 Synthesis of N-unsubstituted Triazolopyrimidines ...... 21

6.1.2 Synthesis of Fluorosulfonyl Derivatives ...... 22

6.2 Results and Discussion ...... 26

6.2.1 In Vitro Pharmacology ...... 26

6.2.2 Affinity Based Protein Tagging ...... 29

6.2.3 Preliminary Assessment of Receptor Kinetics ...... 31

6.3 Photoactivatable Probes ...... 36

6.3.1 Synthesis of Diazirine Derivatives ...... 37

6.4 Results and Discussion ...... 41

6.4.1 In Vitro Pharmacology ...... 41

6.4.2 Photoaffinity Labeling ...... 44

7 Conclusion and Outlook ...... 47 8 Novel CB2 Selective Cannabinoids ...... 49 8.1 Background ...... 49 iv

8.2 Towards a CB2 Selective Covalent Binder ...... 51

8.2.1 Synthesis and Evaluation of a Hybrid Cannabinoid ...... 53

8.3 Synthesis of Bifunctional Probes ...... 56

8.4 Alternative Access to Amine 128 and Cannabinoids of Increased Polarity ...... 60

8.4.1 Synthesis of Isomerically Pure Resorcinols ...... 61

8.5 Synthesis of AM841 ...... 66

8.6 Photoactivatable, CB2-Selective Cannabinoids ...... 68

8.7 Results and Discussion ...... 69

8.7.1 In Vitro Pharmacology ...... 69

8.7.2 (Photo)-Affinity Labeling ...... 73

8.8 Conclusion and Outlook ...... 78

III. Optical Control of Cannabinoid Receptor 1 ...... 81 9 Background ...... 83 10 Photochromic Tetrahydrocannabinol Derivatives ...... 84 10.1 Synthesis of 3-Br-THC ...... 87

10.2 Synthesis of Photoswitchable 9- Tetrahydrocannabinol Derivatives ...... 89

10.3 Compound Evaluation and Discussion ...... 93

11 Conclusion and Outlook ...... 100 IV. Experimental ...... 102 12 General Methods ...... 103 13 Chemicals ...... 103 14 Analytics ...... 103 15 General Procedures ...... 105 16 Syntheses ...... 105 17 Biological Assays ...... 196 v

Abstract

The identification of 9-tetrahydrocannabinol as the primary psychoactive constituent in preparations of Cannabis sativa resulted in the discovery of the endocannabinoid system (ECS), which is found in all vertebrates. In the current view, the ECS comprises of two G-protein coupled cannabinoid receptors (CB1 and CB2), their endogenous ligands (endocannabinoids) and enzymes related to endocannabinoid metabolism. The ECS has been shown to be involved in many physiological processes of high relevance to human disease. CB1 is mainly expressed in cells of the central nervous system, while CB2 is mainly represented in cells of the immune system. Since their discovery, both receptors have been subject to intense research efforts in order to understand the effects of cannabinoid signaling on a molecular level. Despite impressive progress, not least enabled by specifically developed chemical tools, the full picture of the complex signaling network remains elusive.

Due to the absence of reliable biochemical tools such as specific antibodies, new small molecule derived chemical probes with specifically tailored selectivity and function would be a great addition to the toolbox of practitioners in the field. In a highly collaborative approach of a team composed of pharmacologists, structural biologists and organic chemists, we set out to develop novel tool compounds for the endocannabinoid system.

The first part of this thesis describes the synthesis and pharmacological characterization of highly CB2-selective triazolopyrimidines (I) and non-classical cannabinoids (II) with particular focus on added function in the form of (a) electrophilic functional groups (sulfonyl fluorides, isothiocyanate) and (b) photoactivatable groups (diazirines) for cross-linking, and (c) linker attachment enabling the conjugation of fluorophores and other useful moieties (Figure I).

Figure I. General structures of novel CB2-selective tool compounds.

Our efforts culminated in the development of a photoactivatable affinity probe for CB2 allowing for protein detection in overexpressing cells, in the discovery of a probe with exceptionally long CB2 residence time, and in the evolution of a highly CB2 selective fluoroprobe. vi

In the second part, we describe the dual-catalytic, enantioselective synthesis of a novel 9- tetrahydrocannabinol derivative, namely 3-Br-THC (Figure II).

Figure II. A dual-catalytic, enantioselective strategy for the synthesis of 3-Br-THC.

The versatility of this building block is demonstrated by its elaboration into several photochromic cannabinoids termed azo-THCs (Figure III). Using whole-cell patch clamp electrophysiology, two azo-THCs are identified as photoswitchable ligands for CB1. Furthermore, one photoswitch was evaluated in a cyclic adenosine monophosphate assay and shown to exhibit distinct potencies in the cis and the trans forms.

Figure III. Synthesis of azo-THCs enables optical control of CB1.

vii

Zusammenfassung

Die Identifizierung des 9-Tetrahydrocannabinols als hauptsächliche Ursache der psychoaktiven Wirkung von Cannabis sativa Zubereitungen führte zur Entdeckung des Endocannabinoid Systems (ECS), welches in allen Wirbeltieren vorhanden ist. In der gegenwärtigen Sicht besteht das ECS aus zwei G-Protein gekoppelten Cannabinoid Rezeptoren (CB1 und CB2), deren endogene Liganden (Endocannabinoide) und Enzymen, die im Endocannabinoid Metabolismus eine Rolle spielen. Das ECS ist bei vielen krankheitsrelevanten physiologischen Prozessen involviert. CB1 wird vornehmlich durch Zellen des zentralen Nervensystems exprimiert, während CB2 hauptsächlich in Zellen des Immunsystems zu finden ist. Seit ihrer Entdeckung waren beide Rezeptoren Gegenstand intensiver Forschung mit dem Ziel, die Auswirkungen der durch Cannabinoide aktivierten Signalwege auf molekularer Ebene zu verstehen. Trotz des beeindruckenden Fortschrittts, der nicht zuletzt durch speziell entwickelte chemische Werkzeuge ermöglicht wurde, ist das Gesamtbild dieses komplexen Signalnetzwerks unzureichend verstanden.

Aufgrund der Nichtverfügbarkeit verlässlicher biochemischer Werkzeuge wie beispielsweise spezifische Antikörper, würden neue chemische Sonden mit massgeschneiderter Selektivität und Funktion einen einen wertvollen Beitrag leisten. In einer kollaborativen Herangehensweise und einem Team bestehend aus Pharmakologen, Strukturbiologen und Organischen Chemikern haben wir uns der Entwicklung neuer chemischer Werkzeuge für das Endocannabinoidsystem angenommen.

Der erste Teil der vorliegenden Arbeit beschreibt die Synthese und pharmakologische Charakterisierung von CB2-selektiven Triazolopyrimidinen (I) und nicht-klassischen Cannabinoiden (II) mit besonderem Fokus auf die Implementierung zusätzlicher Funktion in Form von (a) elektrophilen funktionellen Gruppe (Sulfonylfluoride, Thioisocyanate) und (b) photoaktivierbaren Gruppen (Diazirine) zwecks Vernetzung, und (c) einer Linkerbefestigung für die Konjugation von Fluorophoren oder anderen nützlichen Gruppen (Abbildung I).

Figure I. Allgemeine Struktur neuer CB2-selektiver molekularer Werkzeuge.

viii

Unsere Bemühungen führten zur Entwicklung einer photoaktivierbaren Affinitätssonde für CB2 zur Proteindetektion in überexprimierenden Zellen, zur Entdeckung einer Sonde mit ausgesprochen langer CB2 Verweilzeit, und der Evolution einer besonders CB2 selektiven Fluoreszenzsonde.

Im zweiten Teil dieser Arbeit beschreiben wir die dual-katalytische, enantioselektive Synthese des neuen 9-Tetrahydrocannabinol Derivats 3-Br-THC (Abbildung II).

Figure II. Eine dual-katalytische Strategie zur Synthese von 3-Br-THC.

Wir demonstrieren die Vielseitigkeit dieses neuen Bausteins durch dessen Umwandlung in einige photochrome Cannabinoide, genannt azo-THC Derivate (Abbildung III). Mittels «whole-cell patch clamp» Verfahren zeigen wir, dass zwei azo-THC Derivate photoschaltbare CB1 Liganden sind. Des Weiteren wurde ein Ligand im zyklischen Adenosinmonophosphat Assay getestet. Es wurde gezeigt, dass die cis- und trans-Formen unterschiedliche Aktivitäten aufweisen.

Figure III. Die Synthese von azo-THC Derivaten ermöglicht die optische Kontrolle von CB1.

I. Introduction

Interrogation of Biological Systems with Chemical Probes 1

1 Interrogation of Biological Systems with Chemical Probes

The historical border between chemistry and biology as distinct scientific fields has become blurred over the last decades. This process was ignited by researchers of both camps joining forces to follow highly interdisciplinary approaches in order to better understand biological phenomena. In this context, the power of organic synthesis has been of particular importance since it enables the preparation of well-defined molecular entities ranging from rather simple tool compounds to the most complex natural products. Another valuable feature of chemical synthesis is the possibility to introduce almost any functionality into a given target structure, thus enabling manifold follow-up applications (vide infra). The modern approach using bespoke chemical tools to interrogate biological systems has been coined chemical biology.1

The mapping of biological networks at a molecular level is of paramount relevance to the understanding of human disease and drug development programs. The necessity to gain profound knowledge of a drug candidate’s mechanism of action (MoA) brings chemical probes to the scene. A chemical probe has been defined as a «selective small-molecule modulator of a protein’s function that allows the user to ask mechanistic and phenotypic questions about its molecular target in biochemical, cell-based or animal studies.»2 Such probes can greatly facilitate the validation or, equally important, invalidation of a mechanistic hypothesis of drug action. Therefore, early implementation of chemical probes in the drug discovery process is not only crucial for target confidence,3 but can also assist in the making of «go or no-go» decisions in order to attenuate high attrition rates in the clinic.4

To ensure high reliability and significance of biological data collected by use of a tool compound, it should be of the highest quality. Figure 1.1 summarizes some of the general aspects to be considered in the design of chemical probes compared to the requirements of a drug candidate.2 Although certainly valuable, these and other3,5 objective guidelines might not need to be strictly followed in every detail to derive useful molecular probes.6 For example, if there is only limited knowledge of a drug’s mechanism of action, a probe might still be highly

1 A. Miller, J. Tanner, Essentials of Chemical Biology: Structure and Dynamics of Biological Macromolecules, John Wiley & Sons Ltd, Chicester, West Sussex, England, 2008. 2 C. H. Arrowsmith, J. E. Audia, C. Austin, J. Baell, J. Bennett, J. Blagg, C. Bountra, P. E. Brennan, P. J. Brown, M. E. Bunnage, et al., Nat. Chem. Biol. 2015, 11, 536. 3 M. E. Bunnage, E. L. P. Chekler, L. H. Jones, Nat. Chem. Biol. 2013, 9, 195. 4 R. M. Garbaccio, E. R. Parmee, Cell Chem. Biol. 2016, 23, 10. 5 S. V. Frye, Nat. Chem. Biol. 2010, 6, 159. 6 P. Workman, I. Collins, Chem. Biol. 2010, 17, 561.

2 Introduction

Figure 1.1. Different purposes and requirements for chemical probes and drugs. Reprinted by permission from Macmillan Publishers Ltd: Nat. Chem. Biol. 2015, 11, 536, copyright 2015. useful provided it has been shown to not interact with other (known) targets (selectivity criterion). Furthermore, although desirable a probe does not need to be fully optimized with respect to its physico-chemical properties. Of particular interest is the availability of a structurally related but inactive analog, ideally paired with another active compound derived from a different chemotype (i.e. structurally distinct) to reduce the risk of observing an effect due to activation of an unknown target. More specific parameters as defined by researchers at Pfizer Pharmaceuticals are shown in Figure 1.2.3 According to the authors, the selectivity of a probe should be at least 100-fold over target-related proteins and paired with high potency in biochemical activity assays (below 100 nM). The design of the probe may include both covalent (chemical knock-out/knock-in) and non-covalent congeners and should consider parameters such as cell permeability and solubility. Similar to the guidelines above, the use of an inactive control compound is highly recommended (in this example in the form of an inactive enantiomer).

As mentioned, organic synthesis allows researchers to equip molecular tool compounds with additional functionality. This option significantly expands the number of conceivable molecular probes and opens up new avenues for the interrogation of biological systems through the conjugation to fluorescent, radioactive or affinity tags. The important class of (bi-)functional

Interrogation of Biological Systems with Chemical Probes 3 molecular probes has had significant impact on imaging applications, protein detection and target engagement studies.7,8

1.1 Fluorescent Probes

Imaging methods relying on highly selective fluorescent probes offer unique possibilities to map the spatial distribution of target structures within cells and translocalization dynamics with high resolution.9 Likely the most reliable and mature techniques to effect efficient and specific visualization of unmodified proteins are based on antibodies.10 One shortcoming of such immunofluorescence-based strategies is the requirement of generating antibodies against the structure of interest. Especially for rather flexible proteins such as constitutively active G- protein coupled receptors (GPCRs)11 this can be a tedious task with uncertain outcome.

Recently, the field of small-molecule fluorophore conjugates has experienced considerable progress.12 In this approach, small-molecules known to selectively bind the target of interest are conjugated to a fluorophore. In the case of GPCRs the conjugation is usually realized via a linker strategically introduced to a position within the small molecule where it minimally

7 D. Weichert, P. Gmeiner, ACS Chem. Biol. 2015, 10, 1376. 8 G. M. Simon, M. J. Niphakis, B. F. Cravatt, Nat. Chem. Biol. 2013, 9, 200. 9 L. A. Stoddart, L. E. Kilpatrick, S. J. Briddon, S. J. Hill, Neuropharmacology 2015, 98, 48. 10 K. Thorn, Mol. Biol. Cell 2016, 27, 219. 11 A. M. Preininger, J. Meiler, H. E. Hamm, J. Mol. Biol. 2013, 425, 2288. 12 M. Leopoldo, E. Lacivita, F. Berardi, R. Perrone, Drug Discovery Today 2009, 14, 706.

4 Introduction compromises binding affinity and selectivity over other proteins.13 Several reports on such probes for GPCRs have appeared describing efforts towards the fluorescence labeling of 14 15 16 17 adenosine receptor, 2-adrenoceptor, angiotensin receptor, cannabinoid receptor 2, 18 19 13 histamine H3, and muscarinic M3 receptor, and others with varying success. A major hurdle to overcome is the pronounced non-specific binding regularly observed for small-molecule fluorophore conjugates due to their often lipophilic nature.20

1.2 Covalent Binders

Small molecules that covalently bind proteins are highly useful tool compounds for various applications. Selective photoactivatable probes (also called photoaffinity probes) contain structural features required for high affinity and specificity towards a target of interest. An additionally introduced photoreactive group allows for cross-linking via formation of reactive intermediates upon irradiation.21 Three types of photoactivatable groups are commonly employed and include diazirines, aromatic azides and benzophenones.22,23,24

Figure 1.3. Photoactivatable functional groups commonly used in photoaffinity labeling studies.

When irradiated with the corresponding wavelength, these groups are able to form highly reactive carbene, nitrene and diradical intermediates, which can subsequently form a covalent bond to proteins in vicinity.25 In one example of a soluble target protein, diazirine derived

13 A. J. Vernall, S. J. Hill, B. Kellam, Br. J. Pharmacol. 2014, 171, 1073. 14 E. Kozma, P. Suresh Jayasekara, L. Squarcialupi, S. Paoletta, S. Moro, S. Federico, G. Spalluto, K. A. Jacobson, Bioorg. Med. Chem. Lett. 2013, 23, 26. 15 E. Martikkala, M. Lehmusto, M. Lilja, A. Rozwandowicz-Jansen, J. Lunden, T. Tomohiro, P. Hänninen, U. Petäjä-Repo, H. Härmä, Anal. Biochem. 2009, 392, 103. 16 M. A. Giarrusso, M. K. Taylor, J. Ziogas, K. M. Brody, P. E. Macdougall, C. H. Schiesser, Asian J. Org. Chem. 2012, 1, 274. 17 M. Sexton, G. Woodruff, E. A. Horne, Y. H. Lin, G. G. Muccioli, M. Bai, E. Stern, D. J. Bornhop, N. Stella, Chem. Biol. 2011, 18, 563. 18 M. Tomasch, J. S. Schwed, A. Paulke, H. Stark, ACS Med. Chem. Lett. 2013, 4, 269. 19 J. A. Hern, A. H. Baig, G. I. Mashanov, B. Birdsall, J. E. T. Corrie, S. Lazareno, J. E. Molloy, N. J. M. Birdsall, PNAS 2010, 107, 2693. 20 A. Cooper, S. Singh, S. Hook, J. D. A. Tyndall, A. J. Vernall, Pharmacol. Rev. 2017, 69, 316. 21 A. E. Ruoho, H. Kiefer, P. E. Roeder, S. J. Singer, PNAS 1973, 70, 2567. 22 L. Dubinsky, B. P. Krom, M. M. Meijler, Bio. Med. Chem. 2012, 20, 554. 23 G. Dorman, G. D. Prestwich, Biochemistry 1994, 33, 5661. 24 S. A. Fleming, Tetrahedron 1995, 51, 12479. 25 J. Das, Chem. Rev. 2011, 111, 4405.

Photopharmacology 5 probes mimicking general anesthetics have been used to gain insight into the three dimensional structure of a ligand site of adenylate kinase.26

Complementary to photoactivatable probes, the cross-linking between ligand and target can be realized through electrophilic groups present in the ligand, which allow for irreversible or reversible covalent bond formation with nucleophilic amino acids present in the target protein. Such an approach has proven particularly powerful when combined with protein engineering in an approach termed ligand-assisted protein structure (LAPS) to gain structural insight of a target protein.27 The most commonly electrophilic functional groups are α-halo ketone, fluorophosphonate, fluorosulfonyl, enone, isothiocyanate, nitrogen mustards and electrophilic sulfur.7

Figure 1.4. Electrophilic functional groups commonly found in covalently binding ligands.

For example, incubation of the soluble proteome of Staphylococcus aureus with electrophilic -lactones resulted in a selective chemical knock-out of caseinolytic protein protease (ClpP), a serine protease associated with the organism’s virulence and potential target to fight multi- resistant pathogens.28 Covalent ligands have also been used in the field of structural biology as stabilizing agents for GPCRs facilitating crystallogenesis for X-ray crystallographic analysis.29

2 Photopharmacology

The design and realization of functional materials is at the core of synthetic chemistry. Modern drugs, albeit optimized with respect to target binding, selectivity, physico-chemical and pharmacokinetic properties, are often distributed throughout the body following adminstration. To limit a drug’s action to specific regions and thereby attenuate undesired side effects, a compound can be equipped with a molecular switch through the introduction of a photoresponsive element allowing for precise control of activation or inactivation. In the case

26 G. H. Addona, S. S. Husain, T. Stehle, K. W. Miller, J. Biol. Chem. 2002, 277, 25685. 27 N. Zvonok, L. Pandarinathan, J. Williams, M. Johnston, I. Karageorgos, D. R. Janero, S. C. Krishnan, A. Makriyannis, Chem. Biol. 2008, 15, 854. 28 T. Böttcher, S. A. Sieber, J. Am. Chem. Soc. 2008, 130, 14400. 29 D. Weichert, A. C. Kruse, A. Manglik, C. Hiller, C. Zhang, H. Hübner, B. K. Kobilka, P. Gmeiner, PNAS 2014, 111, 10744.

6 Introduction of photocaged ligands, a bioactive pharmacophore is masked with a photolabile protecting group.30 Using light, the active compound can be released with unmet degree of spatial and temporal control. This approach has been particularly useful in the study of neurotransmitter action and dynamics.31,32,33 One example of this approach was realized through the synthesis of caged capsaicin, which was able to activate TRPV1 upon photolysis enabling the time-resolved study of TRPV1 activation (Figure 2.1, b and c).34 Naturally, such an uncaging process is irreversible and once the bioactive compound is set free, the reduction of its action relies mainly on metabolism or cellular transport mechanisms. Therefore, researchers have introduced the concept of photoswitchable ligands. In this approach, the bioactive ligand is modified with a photoresponsive motif. The resulting photochromic ligand (PCL) is able to undergo reversible isomerization between two states (Figure 2.1 d and e). In an ideal setting, the isomerization of a photoswitch is fast, quantitative, and the resulting isomers exhibit distinct pharmacological activities.35,36 One successful example following this approach has been the development of a photoswitchable glutamate derivative enabling optical control of an ionotropic glutamate receptor subtype.37 The strategy has also been utilized to gain optical control over N-methy-D- aspartate receptors using an azobenzene-based photoswitch, which could be cycled between the cis and trans forms of the azobenzene chromophore using light.38 In addition, such ligands might be tethered to the target through bioconjugation39 resulting in a photoswitchable tethered ligand (PTL) (Figure 2.1, f and g) as demonstrated in a work on optical control of metabotropic glutamate receptors.40,41 Furthermore, the concept of photoswitchable tethered ligands has been combined with protein engineering to allow for covalent tethering of a photoswitchable agonist 42 via SNAP tagging to a metabotropic glutamate receptor (mGlu2), which was thus rendered light sensitive.43 Photocontrol of GPCRs is not limited to the use of tools derived from

30 P. Klán, T. Šolomek, C. G. Bochet, A. Blanc, R. Givens, M. Rubina, V. Popik, A. Kostikov, J. Wirz, Chem. Rev. 2013, 113, 119. 31 R. H. Kramer, D. L. Fortin, D. Trauner, Curr. Opin. Neurobiol. 2009, 19, 544. 32 R. Wieboldt, K. R. Gee, L. Niu, D. Ramesh, B. K. Carpenter, G. P. Hess, PNAS 1994, 91, 8752. 33 M. Matsuzaki, G. C. R. Ellis-Davies, T. Nemoto, Y. Miyashita, M. Iino, H. Kasai, Nat. Neurosci. 2001, 4, 1086. 34 B. V. Zemelman, N. Nesnas, G. A. Lee, G. Miesenböck, PNAS 2003, 100, 1352. 35 W. A. Velema, W. Szymanski, B. L. Feringa, J. Am. Chem. Soc. 2014, 136, 2178. 36 J. Broichhagen, J. A. Frank, D. Trauner, Acc. Chem. Res. 2015, 48, 1947. 37 M. Volgraf, P. Gorostiza, S. Szobota, M. R. Helix, E. Y. Isacoff, D. Trauner, J. Am. Chem. Soc. 2007, 129, 260. 38 Laprell, E. Repak, V. Franckevicius, F. Hartrampf, J. Terhag, M. Hollmann, M. Sumser, N. Rebola, D. A. DiGregorio, D. Trauner, Nat. Commun. 2015, 6, 8076. 39 J. Kalia, R. T. Raines, Current Organic Chemistry 2010, 14, 138. 40 J. Levitz, C. Pantoja, B. Gaub, H. Janovjak, A. Reiner, A. Hoagland, D. Schoppik, B. Kane, P. Stawski, A. F. Schier, et al., Nat. Neurosci. 2013, 16, 507. 41 T. Fehrentz, M. Schönberger, D. Trauner, Angew. Chem. Int. Ed. 2011, 50, 12156. 42 A. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel, K. Johnsson, Nat. Biotech. 2003, 21, 86. 43 J. Broichhagen, A. Damijonaitis, J. Levitz, K. R. Sokol, P. Leippe, D. Konrad, E. Y. Isacoff, D. Trauner, ACS Cent. Sci. 2015, 1, 383.

Photopharmacology 7 orthosteric ligands, but has also been showcased for a photoswitchable allosteric modulator of 44 the metabotropic glutamate receptor mGlu5. Recently, a photoswitchable antibiotic was reported that exhibited significantly enhanced antibacterial activity in the cis-form.45 Emerging molecular targets in the promising field of photopharmacology have been reviewed recently.46

Figure 2.1. Three strategies to gain optical control over protein function. a) A ligand binds to a generic receptor and triggers a biological response. b) A caged ligand is broken apart with light, thus releasing its active form. c) Caged capsaicin used to stimulate TRPV1 channels. d) A photoswitchable ligand acts on a receptor. e) 4-GluAzo, a photochromic ligand (PCL) subtype selective for GluR5 kainate receptor. f) A covalently attached photoswitchable tethered ligand (PTL) enabling optical control of a receptor.

44 S. Pittolo, X. Gómez-Santacana, K. Eckelt, X. Rovira, J. Dalton, C. Goudet, J.-P. Pin, A. Llobet, J. Giraldo, A. Llebaria, et al., Nat. Chem. Biol. 2014, 10, 813. 45 W. A. Velema, J. P. van der Berg, M. J. Hansen, W. Szymanski, A. J. M. Driessen, B. L. Feringa, Nat. Chem. 2013, 5, 924. 46 M. M. Lerch, M. J. Hansen, G. M. van Dam, W. Szymanski, B. L. Feringa, Angew. Chem. Int. Ed. 2016, 55, 10978.

Figure 2.2. A photoswitchable antibiotic with increased acitivity in the cis form.

3 The Endocannabinoid System

The consumption of Cannabis sativa preparations either for medical or recreational purposes has been common practice for millennia across many cultures around the world.47 In the 1960’s,

MECHOULAM and GAONI identified 9-tetrahydrocannabinol (9-THC) as the major psychoactive ingredient of such preparations.48 Their work laid the cornerstone for the discovery of a complex signaling system found in all vertebrates,49 which is nowadays known as the endocannabinoid system (ECS). In 1990, MATSUDA and colleagues were the first to clone cannabinoid receptor 1 and to identify it as a G-protein coupled receptor that leads to adenylyl cyclase inhibition upon activation by cannabinoids.50 In search of endogenous ligands for the

CB1 receptor, DEVANE et al. identified an amide derivative of arachidonic acid now termed anandamide.51 This report was followed by the cloning of a peripheral cannabinoid receptor in 1993 (CB2)52 and identification of a second endogenous ligand (2-AG, 1995).53 In 1996,

CRAVATT and colleagues identified a serine hydrolase (fatty acid amide hydrolase, FAAH)54 capable of rapidly degrade anandamide and other fatty acid amides. In todays view, the ECS comprises of the two G-protein coupled cannabinoid receptors (CB1 and CB2), their endogenous ligands (endocannabinoids) and various enzymes related to endocannabinoid metabolism (FAAH, monoacylglycerol lipase, MAGL; diacylglycerol lipase, DAGL). The ECS has been shown to be involved in many physiological processes of high relevance to human

47 E. B. Russo, H.-E. Jiang, X. Li, A. Sutton, A. Carboni, F. del Bianco, G. Mandolino, D. J. Potter, Y.-X. Zhao, S. Bera, et al., J. Exp. Bot. 2008, 59, 4171. 48 Y. Gaoni, R. Mechoulam, J. Am. Chem. Soc. 1964, 86, 1646. 49 M. R. Elphick, M. Egertová, in The Cannabinoid Receptors (Ed.: P.H. Reggio), Humana Press, 2009, pp. 123. 50 L. A. Matsuda, S. J. Lolait, M. J. Brownstein, A. C. Young, T. I. Bonner, Nature 1990, 346, 561. 51 W. A. Devane, L. Hanuš, A. Breuer, R. G. Pertwee, L. A. Stevenson, G. Griffin, D. Gibson, A. Mandelbaum, A. Etinger, R. Mechoulam, Science 1992, 258, 1946. 52 S. Munro, K. L. Thomas, M. Abu-Shaar, Nature 1993, 365, 61. 53 R. Mechoulam, S. Ben-Shabat, L. Hanus, M. Ligumsky, N. E. Kaminski, A. R. Schatz, A. Gopher, S. Almog, B. R. Martin, D. R. Compton, et al., Biochem. Pharmacol. 1995, 50, 83. 54 B. F. Cravatt, D. K. Giang, S. P. Mayfield, D. L. Boger, R. A. Lerner, N. B. Gilula, Nature 1996, 384, 83.

The Endocannabinoid System 9 disease.55 Some examples being pain, inflammation, addiction, stroke, neurodegenerative and cardiovascular disorders.56 CB1 is the most abundant GPCR in cells of the central nervous system and primarily signals via Gi/o proteins upon activation, resulting in reduced activity of adenylyl cyclase and stimulation of mitogen-activated protein kinase. Further downstream effects include closure of calcium channels as well as activation of GIRK channels via GThe latter results in hyperpolarization of the cell (due to potassium efflux) leading to activation of 57 kinases. CB2 also signals mainly via Gi/o but is mainly found in cells of the immune system. CB2 is highly upregulated in inflamed tissue, also in the brain, suggesting a protective role of the receptor for the organism.58 The main signaling pathways of the endocannabinoid system are summarized in Figure 3.1.59 The endocannabinoids anandamide (AEA) and 2- arachidonoylglycerol (2-AG) are each synthesized by two enzymes. In the case of AEA, N-acyl transferase transforms membrane phospholipids (PL) into N-arachidonoyl- phosphatidylethanolamie (NAPE), which then gets hydrolyzed (the phosphate ester) by phospholipase D to release anandamide. 2-AG on the other hand is produced from phosphoinositides (PIPX) by phospholipase C-mediated hydrolysis to 1,2-diacylgycerole, and further through the action of diacylglycerole lipase (DGL or DAGL). After release into the extracellular space, the endocannabinoids can be either degraded by fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), or alternatively be taken up into surrounding cells. Both endocannabinoids can activate the cannabinoid receptors, resulting in adenylyl cyclase inhibition and stimulation of mitogen-activated protein kinase.

Most components of the endocannabinoid system have been considered potential drug targets. One approach under investigation has been the inhibition of endocannabinoid catabolism to increase cannabinoid receptor activation level without the need for exogenous agonists.60 Along these lines, an irreversible FAAH inhibitor was successfully showcased in a mouse model of 61 inflammatory and non-inflammatory pain.

55 P. Pacher, G. Kunos, FEBS J 2013, 280, 1918. 56 A. Cooper, S. Singh, S. Hook, J. D. A. Tyndall, A. J. Vernall, Pharmacol. Rev. 2017, 69, 316. 57 D. Piomelli, Nat. Rev. Neurosci. 2003, 4, 873. 58 C. Benito, R. M. Tolón, M. R. Pazos, E. Núñez, A. I. Castillo, J. Romero, Br. J. Pharmacol. 2008, 153, 277. 59 M. Beltramo, in An Introduction to Pain and Its Relation to Nervous System Disorders (Ed.: A.A. Battaglia), John Wiley & Sons, Ltd, 2016, pp. 169. 60 M. M. Mulvihill, D. K. Nomura, Life Sciences 2013, 92, 492. 61 K. Ahn, S. E. Smith, M. B. Liimatta, D. Beidler, N. Sadagopan, D. T. Dudley, T. Young, P. Wren, Y. Zhang, S. Swaney, et al., J. Pharmacol. Exp. Ther. 2011, 338, 114.

10 Introduction

4 Aim of this Work

The field of cannabinoid receptor research would greatly benefit from specific chemical tools, which allow researchers to further investigate the mechanism of action of CB1 and CB2, to detect receptor expression in distinct cells/tissues, to elucidate their three dimensional structures and their links to function, and to understand on a molecular level the various downstream effects of receptor modulation in a tissue dependent context.

The main part of the present thesis describes a highly interdisciplinary approach towards CB2 selective chemical probes. The novel probes include (putative) covalent binders, photoaffinity probes and fluorescent probes synthesized and biologically tested in collaborating laboratories. The testing included radioligand binding assays (binding affinity CB1/CB2), the study of receptor kinetics (CB2 target residence time), photo-crosslinking experiments (photoaffinity probes) and fluorescence-enabled applications.

Another goal of the project was to develop photoswitchable tetrahydrocannabinol derivatives as CB1 ligands to be cycled between two distinct isomeric forms. To this end, a synthesis of 3- Br-THC was developed. This novel building block proved ideally set up for subsequent cross couplings with a number of azobenzenes. Thus derived azo-THC derivatives were then evaluated in vitro by our collaborators for their light-dependent biological effects using various read outs (cAMP assay, electrophysiology).

II. Novel Chemical Probes for the Cannabinoid Receptor 2

Background 13

5 Background

Despite growing evidence that CB2 plays an important role in a vast number of physiological processes and its involvement in various disease states, the mechanism of action of this GPCR is still poorly understood. This is in part due to the lack of appropriate (bio)-chemical tools such as specific antibodies, selective fluorescent probes and selective irreversible ligands. This chapter describes our efforts towards highly selective chemical probes for use in CB2 research.

Over the last two decades, a large number of tool compounds for the cannabinoid system have been developed.62 Many of these compounds are non-selective modulators of both receptors CB1 and CB2. A collection of such ligands along with naturally occurring cannabinoid receptor ligands is shown in Figure 5.1. Recently, a subset of these ligands (CP55940, WIN55212-2, JWH-015, JWH-133, AM-1241, HU-308 and HU-910) has been pharmacologically characterized in a head-to-head comparative study as “the most widely used CB2R ligands”.63

Among the naturally occurring cannabinoid receptor ligands, the phytocannabinoid 9-THC is probably known best for its psychotropic activity upon consumption of Cannabis sativa preparations. Pharmacologically, 9-THC (Figure 5.1, A) is a partial agonist of both receptors with slightly higher efficacy at CB1. In contrast, the phytocannabinoid -caryophyllene has recently emerged as a rare example of a naturally occurring CB2 selective full agonist with 64,65 relatively high affinity (hCB2 Ki ≈ 150 nM). The two most important endocannabinoids (i.e. compounds synthesized by the organisms itselves) are N-arachidonoylethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG). AEA is a partial agonist of both CB1 and CB2, while 2-AG is a full agonist of both receptors and is much more abundant (Figure 5.1, B).66

The non-classical cannabinoid CP55940 is one of the most widely used compounds in CBR pharmacology. In its tritiated form it is a standard ligand used in radioligand binding assays. The compound exhibits very high affinity for both CB1 and CB2 and fully agonizes both

62 For a very recent review of chemical tools for lipid-binding class A GPCRs including the cannabinoid receptors, see: A. Cooper, S. Singh, S. Hook, J. D. A. Tyndall, A. J. Vernall, Pharmacol. Rev. 2017, 69, 316. 63 M. Soethoudt, U. Grether, J. Fingerle, T. W. Grim, F. Fezza, L. de Petrocellis, C. Ullmer, B. Rothenhäusler, C. Perret, N. van Gils, et al., Nat. Commun. 2017, 8, 13958. 64 J. Gertsch, M. Leonti, S. Raduner, I. Racz, J.-Z. Chen, X.-Q. Xie, K.-H. Altmann, M. Karsak, A. Zimmer, PNAS 2008, 105, 9099. 65 Two more examples being N-alkylamide partial agonists from Echinacea, see a) J. Gertsch, R. G. Pertwee, V. Di Marzo, Br J Pharmacol 2010, 160, 523; b) S. Raduner, A. Majewska, J.-Z. Chen, X.-Q. Xie, J. Hamon, B. Faller, K.-H. Altmann, J. Gertsch, J. Biol. Chem. 2006, 281, 14192. 66 F. Fezza, M. Maccarrone, in Cannabinoids, John Wiley & Sons, Ltd, 2014, pp. 53.

14 Novel Chemical Probes for the Cannabinoid Receptor 2 receptors. WIN55212-2 is a full agonist of both receptors with slight preference for CB2 (ca. 19-fold). SR144528 is a high affinity CB2 selective inverse agonist (hCB2 Ki < 1 nM). JWH- 015, JWH-133, AM-1241, HU-308 and HU-910 on the other hand are CB2 selective agonists, with the cannabinoids exhibiting higher affinity and selectivity for CB2 than the two N-alkyl- 3-aroylindoles (Figure 5.1, C).

Figure 5.1. Bioactive compounds engaging with cannabinoid receptors. Figures in parentheses describe the first occurrence in the literature of synthetic compounds or the year of discovery/linkage to the endocannabinoid system. A: Phytocannabinoids 9-THC and -caryophyllene. B: Endocannabinoids anandamide and 2-AG. C: Synthetic cannabinoid receptor 2 modulators most commonly used in pharmacology studies.

Triazolopyrimidine-Derived Ligands 15

6 Triazolopyrimidine-Derived Ligands

The chemical starting point of our efforts towards CB2 selective probes was a class of compounds sharing a triazolopyrimidine core. Derivatives of this chemotype had emerged as highly CB2 selective agonists from a high-throughput screen performed at F. Hoffmann–La Roche.67 Thanks to the presence of three independent exit vectors, derivatives based on this modular scaffold could be optimized with respect to physico-chemical parameters (for example solubility, metabolic stability), CB2 binding affinity and selectivity over CB1 (Figure 6.1, right). The optimization process was supported by molecular modelling using a homology model of activated bovine rhodopsin.68 The lead optimization resulted in the development of 1

(Figure 6.1, left), a full agonist of CB2 in cAMP assay with subnanomolar EC50 and complete selectivity over CB1 along with well-balanced physico-chemical properties. The early safety profile of this compound allowed its further evaluation in mice for its effect in an ischemia- reperfusion model of kidney damage as well as a model for renal fibrosis (unilateral ureter obstruction). In both experiments, administration of 1 led to significant attenuation of kidney damage as assessed by various kidney markers and histochemical determination of collagen-I deposition.

Figure 6.1. Prototypical CB2 selective agonist based on the triazolopyrimidine core,67 which is shown on the right together with available exit vectors for diversification.

Functional Activity Switch and General Synthesis As outlined in Figure 6.1, the substituent at C-5 is critical in determining the functional activity of triazolopyrimidine derivatives. During the exploration of structure activity relationships in the context of our collaborator’s drug discovery program, it became apparent that larger substituents lead to a switch of functional activity from agonism to inverse agonism. An example of such a pair of compounds from an early stage of our own efforts towards CB2 selective photoaffinity probes is shown in Figure 6.2.

67 M. Nettekoven, J.-M. Adam, S. Bendels, C. Bissantz, J. Fingerle, U. Grether, S. Grüner, W. Guba, A. Kimbara, G. Ottaviani, et al., ChemMedChem 2016, 11, 179. 68 The model has recently been refined after two independent reports of CB1 crystal structures.

16 Novel Chemical Probes for the Cannabinoid Receptor 2

Figure 6.2. Structures and in vitro pharmacology data of tert-butyl and difluorobenzyl substituted analogs.

Both compounds 2 and 3 are potent CB2 binders with Ki values below 50 nM. The cAMP assay reveals a fundamental difference of their ability to modulate the CB2 receptor. While 2 is a very potent full agonist (CP55940 used as reference), its difluorobenzyl counterpart 3 is an inverse agonist with slightly decreased potency. The distinct functional activity of tert-butyl (agonist) and difluorobenzyl (inverse agonist) substituted compounds proved very reliable across a large number of compounds.69 Similarly consistent, 3,3-difluoropyrrolidine derivatives exhibited higher affinity towards CB2, while the respective (S)-3-hydroxypyrrolidine counterparts were slightly more selective over CB1 with only little loss in affinity. The possibility to switch a compound’s functional activity rendered this chemotype highly interesting since both functional versions of any novel tool compound would be accessible.

The synthesis of triazolopyrimidine derived CB2 ligands was adapted from a patent procedure as outlined in Scheme 6.1.70 The strategy relied on construction of the heterocyclic core by [3+2]-cycloaddtion of a benzyl azide with 2-cyanoacetamide followed by condensation with a suitable acid chloride or nitrile to give compounds of type 8. Following dehydration/chlorination, the pyrrolidine unit was introduced via SNAr reaction to afford the final products (10). Alternatively, a dummy benzyl group was removed and the resulting N- unsubstituted triazolopyrimidine 11 was alkylated with suitably functionalized benzyl (pseudo) halides to give two regioisomeric products (12, 13). The isomers could be readily separated by

69 The determination of functional activity in cAMP assay is much more labor-intensive than radioligand binding assays to test for binding affinity. Therefore, the latter was used as primary criterion in the assessment of newly synthesized compounds. In addition, binding affinity was considered the most important parameter with respect to possible applications of the new tools. Accordingly, functional activity was not determined for every single compound. 70 J.-M. Adam, C. Bissantz, U. Grether, A. Kimbara, M. Nettekoven, S. Roever, M. Rogers-Evans (F. Hoffmann- La Roche), [1,2,3]Triazolo[4,5-d]pyrimidine derivatives as agonists of the cannabinoid receptor 2, WO2013068306A1, 2013.

Triazolopyrimidine-Derived Ligands 17 column chromatography to afford two compounds for biological testing. In the vast majority of cases, the isomer with the newly introduced benzyl substituent attached to N-3 was more potent than its N-2 counterpart.

Scheme 6.1. General synthesis of triazolopyrimidines. Final products are framed.

NMR Spectroscopy of Triazolopyrimidines

The targeted triazolopyrimidines exhibited unexpected behavior with respect to their nuclear magnetic resonance (NMR) spectra. It became apparent that the nitrogen atom of the pyrrolidine moiety is planar (sp2) as expected so that the lone pair is in conjugation with the heteroaromatic ring. Due to the unsymmetrically substituted five-membered ring, this situation gives rise to two diastereomeric (rotameric) products, in which the substituent of the pyrrolidine ring is either closer to or further away from the triazole ring. For 3-substituted pyrrolidines, the ratio of the two rotamers was found to be ca. 1.3:1 at room temperature, which corresponds to a free enthalpy difference of ca. 0.65 kJ·mol-1 or 0.16 kcal·mol-1. The presence of two distinct rotamers leads to a distance dependent doubling of resonances in the spectra, with no or undetectable influence on nuclei remote from the pyrrolidine ring. Figure 6.3 shows 1H and 13C spectra (close-up of pyrrolidine signals) of 14 as a representative example in comparison with 15 bearing an unsubstituted pyrrolidine. As can be seen, the latter compound exhibits distinct resonance signals for all four non-equivalent methylene units of the pyrrolidine moiety, while the 3,3-difluoropyrrolidine substituent shows six signals that partly overlap due to the presence of two distinct rotamers around the Cheteroaryl-Npyrrolidine bond.

18 Novel Chemical Probes for the Cannabinoid Receptor 2

Figure 6.3. 1H resonances of 3,3-difluoropyrrolidine (14, top) and pyrrolidine (15, bottom) substituted triazolopyrimidines.

This characteristic behaviour, together with inherently long relaxation times of the quaternary carbon atoms in the triazolopyrimidine core, complicates spectra interpretation. The recording of 13C NMR spectra demands very long acquisition times especially in the case of fluorinated analogs that exhibit further splitting of 13C resonances due to spin-spin coupling with 19F nuclei (Figure 6.4).

Triazolopyrimidine-Derived Ligands 19

Figure 6.4. 13C NMR resonances of the 3,3-difluoropyrrolidine unit of 14. Methylene units adjacent to 2 13 1 CF2 show JC-F ≈ 33 Hz. Inlet shows C signals of the CF2 group (both rotamers) with JC-F ≈ 248 Hz.

Lastly, in the case of difluorobenzyl substituted 3- hydroxypyrrolidine derivatives (see on the right) of which both rotamers exhibit C1 symmetry, we observed additional splitting of the CF2 resonances. We attribute this to the diastereotopic nature of the fluorine atoms resulting in an AB-spin system. The similar chemical shifts of the diastereotopic 19F nuclei lead to a strong roof effect that could be observed in a number of spectra.

In the case of derivatives prepared via alkylation of triazolopyrimidines (Scheme 6.1), the connectivity of both regioisomeric products was determined by heteronuclear multiple bond correlation (HMBC) spectroscopy. This two-dimensional NMR method allowed the differentiation of N-3 substituted derivatives from N-2 substituted ones, since the former are 3 expected to show a cross peak of the benzylic protons with the ring carbon ( JC-H coupling) as indicated on the right. It was found that N-3 substituted compounds generally eluted first during chromatography on silica gel and their benzylic protons consistently resonated slightly upfield (i.e. exhibited a lower chemical shift ) compared to the N-2 counterparts.

20 Novel Chemical Probes for the Cannabinoid Receptor 2

In conclusion, the recorded NMR spectra of this compound class exhibit a surprisingly high level of complexity. The presence of a nearly 1:1 mixture precluded the routine assignment of individual peaks to a distinct isomer by relative intensities. This was not problematic, since an unequivocal assignment of all resonances to the respective rotamer was not considered a huge knowledge profit. The integrals (1H NMR) were consistent when the benzylic methylene group usually appearing as a single peak was used as internal reference (2 H). Therefore, two- dimensional HMBC spectra were recorded for select compounds to clarify the connectivity of regioisomers isolated from alkyation reactions, while one-dimensional NMR spectra served more the purpose of impurity detection and as fingerprints of the compounds.

6.1 Electrophilic Probes

In search of electrophilic probes, we chose to functionalize the benzyl substituent attached to N-3 of the triazolopyrimidine core with a fluorosulfonyl group in ortho position.71 The choice of the ortho position was based on SAR knowledge of our collaborators clearly indicating a wide variety of groups to be tolerated at this position. Furthermore, molecular modelling using a homology model of CB2 suggested this part of triazolopyrimidine-derived ligands to be close to Cys(40) as potential reacting partner (Figure 6.5). Additionally, in accordance to the model it seemed reasonable that linker elongation towards the extracellular space from the benzyl moiety would be possible in order to attach dyes, affinity tags or other functional moieties.

71 For a recent review, see A. Narayanan, L. H. Jones, Chem. Sci. 2015, 6, 2650.

Triazolopyrimidine-Derived Ligands 21

Figure 6.5. ortho-Fluorosulfonyl substituted triazolopyrimidine 14 docked into a homology model of cannabinoid receptor 2. Top points to extracellular space. Cysteines present in wild-type protein are labeled.72

6.1.1 Synthesis of N-unsubstituted Triazolopyrimidines tert-Butyl-substituted triazolopyrimidines 15 and 16 are known compounds.70,73 The corresponding difluorobenzyl derivatives were synthesized according to Scheme 6.2.

Scheme 6.2. Synthesis of N-unsubstituted difluorobenzyl triazolopyrimidines. Reagents and conditions: a) NaN3, MeCN, reflux, workup; then 2-cyanoacetamide, NaOEt, EtOH, reflux, 80%; b) PhCF2CN, K2CO3, DMF, 80 °C, 65%; c) POCl3, DMF (cat.), 80 °C, 93%; d) 3,3-difluoropyrrolidine·HCl, NEt3, CH2Cl2, rt. 82%; e) (S)-3-hydroxypyrrolidine, NEt3, CH2Cl2, rt. 95%; f) anisole–TFA, 65 °C, 50%; g) TFA, 70 °C; then K2CO3, MeOH–THF, rt, 67%.

72 This model was kindly provided by Dr. W. Guba, F. Hoffmann–La Roche, Basel. 73 The material was provided by our collaborators at F. Hoffmann–La Roche, Basel.

22 Novel Chemical Probes for the Cannabinoid Receptor 2

6.1.2 Synthesis of Fluorosulfonyl Derivatives

The synthesis of fluorosulfonyl derivatives was accomplished by either constructing the triazolopyrimidine core from a benzyl (pseudo) halide following a sequence involving nucleophilic displacement with sodium azide, subsequent [3+2]-cycloaddition and condensation or alternatively by alkylation of N-unsubstituted triazolopyrimidines to give two regioisomeric products, which could be readily separated by chromatography.

The latter strategy was applied to the preparation of 3,3-difluoropyrrolidine and (S)-3- hydroxypyrrolidine substituted sulfonyl fluorides 25-28. Radical bromination of o-methyl benzenesulfonyl fluoride under WOHL-ZIEGLER conditions delivered the corresponding benzyl bromide in 80% yield. This building block was subsequently employed in an alkylation reaction of unsubstituted triazolopyrimidines 15 and 16 to yield two pairs of separable regioisomeric products. The third possible regioisomer was never observed in this and related alkylation reactions of N-unsubstituted triazolopyrimidines, likely due to severe steric interaction with the pyrrolidine substituent.

Scheme 6.3. Synthesis of fluorosulfonyl derivatives. Reagents and conditions: a) NBS, AIBN (10 mol%), MeCN, 80 °C, 80%; b) 15, NEt3, DMF, rt, 41% 25, 50% 26; c) 16, NEt3, DMF, rt, 28% 27, 25% 28.

The corresponding difluorobenzyl substituted derivatives (inverse agonists series) were prepared by stepwise construction of the triazolopyrimidine ring (Scheme 6.4). Known o- benzylmercaptobenzyl chloride74 (29) was reacted with sodium azide in DMSO. Without isolation, the resulting benzylic azide underwent a cycloaddition reaction with cyanoacetamide (6) in presence of sodium hydroxide. The product could be conveniently precipitated by the

74 G. W. Stacy, F. W. Villaescusa, T. E. Wollner, J. Org. Chem. 1965, 30, 4074.

Triazolopyrimidine-Derived Ligands 23 addition of water to deliver 31 after filtration in 87% yield. Subsequent condensation with 2,2- difluoro-2-phenylacetonitrile (30) in DMF (90 °C) afforded 32 (43% yield). The thiobenzyl ether was then oxidized to the corresponding sulfonyl chloride by 1,3-dichloro-5,5- dimethylhydantoin in a mixture of MeCN, water and acetic acid,75 directly converted into the respective sulfonyl fluoride76 (KF, wet acetone), and elaborated into heteroaryl chloride 33 using in situ generated VILSMEIER reagent (33% over 3 steps). 33 readily reacted with pyrrolidines 35 and 36 in the presence of NEt3 to yield difluorobenzyl substituted sulfonyl fluorides 34 (87% yield) and 37 (72% yield).

Scheme 6.4. Synthesis of fluorosulfonyl substituted difluorobenzyl derivatives. Reagents and conditions: a) NaN3, 2-cyanoacetamide, NaOH, H2O–DMSO, rt, 87%; b) PhCF2CN, K2CO3, DMF, 90 °C, 43%; c) i. 1,3-dichloro-5,5-dimethylhydantoin, MeCN–H2O–AcOH, –10 °C to 0 °C; ii. KF, acetone–H2O, rt; iii. (COCl)2, DMF, CH2Cl2, reflux, 33%; e) pyrrolidine, NEt3, CH2Cl2, rt, 72% for 3- hydroxypyrrolidine, 87% for 3,3-difluoropyyrolidine.

In order to probe for a potential dependence of putatively irreversible binding on the distance between the electrophilic warhead and nucleophilic amino acids within the receptor, elongated analogs bearing an additional methylene spacer between the aromatic ring and the fluorosulfonyl group were prepared (Scheme 6.5). To this end, 2-methylbenzyl chloride (38) was heated with thiourea in EtOH. Following removal of the solvent, the resulting thiouronium salt was converted to the corresponding sulfonyl chloride in a mixture of aq. hydrochloric acid and MeCN using N-chlorosuccinimide as oxidant. The crude sulfonyl chloride was converted into sulfonyl fluoride 39 by KF in wet acetone. Benzylic radical bromination under WOHL-

75 Y.-M. Pu, A. Christesen, Y.-Y. Ku, Tetrahedron Lett. 2010, 51, 418. 76 J. W. Clader, W. Billard, H. Binch, L.-Y. Chen, G. Crosby, R. A. Duffy, J. Ford, J. A. Kozlowski, J. E. Lachowicz, S. Li, et al., Bioorg. Med. Chem. 2004, 12, 319.

24 Novel Chemical Probes for the Cannabinoid Receptor 2

ZIEGLER conditions afforded alkylating agent 40 (75% yield), which was reacted with triazolopyrimidines 15 and 16 to afford two pairs of regioisomeric products (41-44) ready for biological testing.

Scheme 6.5. Synthesis of elongated sulfonyl fluoride derivatives. Reagents and conditions: a) i. thiourea, EtOH, reflux; then NCS, aq. HCl–MeCN; ii. KF, acetone–H2O, 46%; b) NBS, benzoyl peroxide (10 mol%), CCl4, reflux, 75%; c) 15, NEt3, DMF, 18% for 41, 15% for 42; 16, DIPEA, DMF, 33% for 43, 19% for 44.

Methylsulfone Control Compounds

Since any experiment relying on the potentially covalent bond between ligand and receptor would ideally include negative control compounds lacking any cross-linking functionality, close analogs of the described fluorosulfonyl derivatives were prepared. In order to stay structurally close to the parent probes, it was decided to swap the fluorine atom for a methyl group (sulfonyl fluoride to methyl sulfone). The synthesis of these control compounds is outlined in Scheme 6.6. Known benzyl alcohol 45 was synthesized in three steps and subsequently mesylated to afford 46 (86% yield). Benzyl pseudohalide 46 underwent nucleophilic displacement with sodium azide and was further elaborated into triazole 47 by cycloaddition with 2-cyanoacetamide (6) (64% yield). 47 was condensed with either pivaloyl chloride or 2,2-difluoro-2-phenylacetonitrile to yield the respective amides (48, 49), which were further transformed into the corresponding heteroaryl chlorides (50, 51). Both building blocks were then reacted with 3,3-difluoropyrrolidine and (S)-3-hydroxypyrrolidine to afford compounds 52-55.

Triazolopyrimidine-Derived Ligands 25

Scheme 6.6. Synthesis of methylsulfone control compounds. Reagents and conditions: a) MsCl, NEt3, CH2Cl2, 0 °C, 86%; b) NaN3, 2-cyanoacetamide, NaOH, H2O–DMSO, rt, 64%; c) PhCF2CN, K2CO3, DMF, 80 °C, 73%; d) PivCl, pyridine, DMAA, 90 °C; then KHCO3, 155 °C, 71%; e) (COCl)2, PhMe– DMF, 91% for 51, 91% for 50; f) 3,3-difluoropyrrolidine hydrochloride, NEt3, CH2Cl2, rt, 86% (54); (S)-3-hydroxypyrrolidine, NEt3, CH2Cl2, 84% (55); g) 3,3-difluoropyrrolidine hydrochloride, NEt3, CH2Cl2, rt, 87% (52); (S)-3-hydroxypyrrolidine, NEt3, CH2Cl2, 97% (53).

Bifunctional Fluorosulfonyl Derivatives

The electrophilic triazolopyrimidines described up to now collectively shared a sulfonyl fluoride warhead. In order to expand the applicability of these tool compounds to more elaborate experiments like affinity-based protein tagging, an additional handle for derivatization was introduced in the form of a terminal alkyne. Structure-activity relationship data generated in the course of our collaborator’s drug discovery project suggested that o,o- disubstituted benzyl groups are well tolerated by the receptor. Therefore, we set out to prepare alkylating reagent 59-Br from 2,5-dibromotoluene (56) as shown in Scheme 6.7. Known

26 Novel Chemical Probes for the Cannabinoid Receptor 2 benzylthioether 5777 was oxidized and further converted into sulfonyl fluoride 58 (50% over both steps). SONOGASHIRA coupling with trimethylsilyl acetylene and subsequent radical bromination then afforded 59-Br.

Scheme 6.7. Synthesis of trifunctional building block 59-Br. Reagents and conditions: a) DIPEA, Pd2(dba)3 (2.4 mol%), Xantphos (4.8 mol%), 1,4-dioxane, reflux, 72%; b) SO2Cl2, H2O, AcOH, CH2Cl2, 0 °C to rt, 54%; c) KF, acetone–H2O, 92%; d) trimethylsilyl acetylene, CuI (15 mol%), PdCl2(PPh3)2 (10 mol%), DIPEA, MeCN, 50 °C, 85%; e) NBS, AIBN (13 mol%), MeCN, 80 °C, 48%.

The primary regioisomeric products after alkylation of triazolopyrimidine 15 with 59-Br (Scheme 6.8) were readily separated after TMS-deprotection of the acetylene moiety using

NEt3·3HF to afford alkyne-functionalized sulfonylfluorides 60 and 61.

Scheme 6.8. Synthesis of alkyne-functionalized fluorosulfonyl derivatives. Reagents and conditions: a) NEt3, DMF, rt; b) NEt3·3HF, THF, 0 °C, 12% (60), 15% (61) over 2 steps.

6.2 Results and Discussion

6.2.1 In Vitro Pharmacology

The newly synthesized electrophilic probes were evaluated in radioligand binding assays for their affinity towards both hCB1 and hCB2 using membrane preparations of CHO cells overexpressing the corresponding receptor and tritiated CP55940 as the radioligand. Selected compounds were further investigated in cAMP assays using CHO cells overexpressing CB1 or CB2, respectively. The data obtained is summarized in Table 6.1.

77 M. Follmann, V. Wehner, J. Meneyrol, J.-M. Altenburger, F. Petit, G. Lassalle, J.-P. Herault (Sanofi-Aventis), Chlorothiophene-amides as inhibitors of coagulation factors xa and thrombin, WO2009103440A1, 2009.

Triazolopyrimidine-Derived Ligands 27

In the experiment, the observed Ki values of the positive controls Rimonabant (CB1 antagonist) and JWH-133 (CB2 agonist) often differed from the published values of 5.6 nM and 3.4 nM, respectively. Therefore, as an estimate for a possible over- or underestimation of experimental

Ki values of novel compounds, the figures in parentheses denote experimental Ki multiplied by the ratio of the published Ki of the control compound to the measured value obtained in the respective experiment (normalization to published Ki values of control compounds). This would allow for a ranking of affinity values determined in separate radioligand binding assays. The basis of discussion here is the actual value obtained in the respective assay, keeping a possible deviation in mind with occasional reference to the control compounds.

Sulfonyl fluorine 25 exhibited high affinity for CB2 with a Ki of 16 nM and circa 60-fold selectivity over CB1. Surprisingly, its difluorobenzyl counterpart 34 (supposedly the inverse agonist version) showed only moderate affinity (630 nM) to CB2 and none to CB1. In the respective cAMP assay, tert-butyl analog 25 emerged as full agonist with an EC50 of 1 nM and almost 600-fold selectivity over CB1. In line with its low affinity, 34 was inactive at both receptors (possibly a low affinity neutral antagonist of CB2). tert-Butyl derivative 27 also showed a hCB2 Ki of 16 nM but with complete selectivity over CB1 (hCB1 Ki > 10 M). The respective difluorobenzyl ligand 37 exhibited low affinity to both receptors. N-2 substituted 28 and 36 (both with the sulfonyl fluoride group directly attached to the benzene ring), 44 and 42 (with an additional methylene linker between aromatic ring and sulfonyl moiety) were only moderate binders (no interaction of 44) but with no affinity towards CB1. Elongated 3- hydroxypyrrolidine derivative 43 was highly selective over CB1 with hCB2 Ki of ca. 280 nM, while the difluoropyrrolidine substituted ligand 41 exhibited higher binding affinity (hCB2 Ki = 11 nM) with good selectivity over CB1 (ca. 180-fold). Methylsulfone control compounds 52- 55 all shared high affinity for CB2 with medium to high selectivity, while 55 was only a moderate CB2 binder (hCB2 Ki ca. 350 nM). Lastly, bifunctional o,o-disubstituted sulfonyl fluorides 60 and 61 emerged as promising affinity probes with hCB2 Ki values of < 1 nM and 42 nM (normalized to positive control).

28 Novel Chemical Probes for the Cannabinoid Receptor 2

Table 6.1 hCB1 and hCB2 affinities and select cAMP data of novel mono- and bifunctional fluorosulfonyl derivatized triazolopyrimidines and methylsulfonyl control compounds.a

hCB1 Ki = 931 nM (2605 nM) hCB1 Ki > 10000 nM hCB2 Ki = 16 nM (1 nM) hCB2 Ki = 630 nM (27 nM)

hCB1 cAMP EC50 = 591 nM (83%) hCB1 cAMP EC50 > 10000 nM (inactive) hCB2 cAMP EC50 = 1.1 nM (99%) hCB2 cAMP EC50 > 10000 nM (inactive)

hCB1 Ki > 10000 nM hCB1 Ki = 3289 nM (692 nM) hCB2 Ki = 16 nM (2 nM) hCB2 Ki = 1127 nM (76 nM)

hCB1 Ki > 10000 nM hCB1 Ki > 10000 nM hCB2 Ki = 1165 nM (139 nM) hCB2 Ki = 283 nM (32 nM)

hCB1 Ki > 10000 nM hCB1 Ki > 10000 nM hCB2 Ki > 10000 nM hCB2 Ki = 637 nM (72 nM)

hCB1 Ki > 10000 nM hCB1 Ki = 1956 nM (3911 nM) hCB2 Ki = 361 nM hCB2 Ki = 11 nM (1 nM)

Triazolopyrimidine-Derived Ligands 29

hCB1 Ki = 37 nM (72 nM) hCB1 Ki = 2000 nM (3862 nM) hCB2 Ki = 2 nM (< 1 nM) hCB2 Ki = 25 nM (1 nM)

hCB2 cAMP EC50 = 11 nM (105%) hCB2 cAMP EC50 = 1213 nM (61%) hCB2 cAMP EC50 = 0.090 nM (101%) hCB2 cAMP EC50 = 0.13 nM (103%)

hCB1 Ki = 436 nM (841 nM) hCB1 Ki > 10000 nM hCB2 Ki = 16 nM (1 nM) hCB2 Ki = 347 nM (15 nM)

hCB1 Ki = 311 nM (3163 nM) hCB1 Ki > 10000 nM hCB2 Ki = 1 nM (<1 nM) hCB2 Ki = 421 nM (42 nM) a For binding affinities, figures in parentheses are Ki values normalized to control compounds Rimonabant (Ki hCB1 = 5.6 nM)78 and JWH-133 (Ki hCB2 = 3.4 nM)79 co-determined in the respective experiment and set to published value. For cAMP data, figures in parentheses denote the compound’s efficacy relative to CP55940.

6.2.2 Affinity Based Protein Tagging80

Binfunctional sulfonyl fluorides 60 and 61 were evaluated for their ability to bind CB2 in a selective and irreversible manner. In theory, the compounds should undergo reaction with a cysteine of CB2 located in proximity to the binding pocket to form a stable thiosulfonate linkage.

78 M. Rinaldi-Carmona, F. Barth, M. Héaulme, D. Shire, B. Calandra, C. Congy, S. Martinez, J. Maruani, G. Néliat, D. Caput, et al., FEBS Letters 1994, 350, 240. 79 J. W. Huffman, J. Liddle, S. Yu, M. M. Aung, M. E. Abood, J. L. Wiley, B. R. Martin, Bioorg. Med. Chem. 1999, 7, 2905. 80 The labeling experiments were perfomed by M. Soethoudt, Leiden University.

30 Novel Chemical Probes for the Cannabinoid Receptor 2

In the experiment, CB2 overexpressing membrane preparations of CHO cells were incubated with sulfonyl fluorides 60 and 61 (2 M) in the presence or absence of excess competitor CP55940 (20 M). An azide functionalized dye (Cy5-azide) was then added along with a copper catalyst preparation (“click mix”) to effect the desired cycloaddition. Unlike in other experiments (see later chapters on photoaffinity probes), the addition of reducing agents was omitted to minimize the risk of rupturing the desired thiosulfonate linkage. The proteins were then resolved by electrophoresis on polyacrylamide gel under denaturing conditions (SDS- PAGE) and labeled proteins were visualized by fluorescence imaging. The results are shown in Figure 6.6.

Probe - LEI121 60 61 (2 µM) CP55940 - - + - + - + (20 µM) lane 1 2 3 4 5 6 7

55 kDa

35 kDa

Figure 6.6. Cy5-conjugated proteins from CB2 overexpressing CHO membranes after affinity labeling using LEI121 (a photoaffinity probe, irradiated at 350 nm) and bifunctional sulfonylfluorides 60 (lanes 4 and 5) and 61 (lanes 6 and 7) at 2 M in the presence (+) or absence (-) of CP55940 as competitor (20 M). The proteins were resolved by electrophoresis on polyacrylamide gel (SDS-PAGE) and visualized by fluorescence imaging.

Lanes 2 and 3 in the gel show the positive control from a photoaffinityl labeling experiment (photoaffinity probe LEI121 was developed and will be disclosed by M. Soethoudt and Prof. M. van der Stelt at Leiden University). Lane 2 indicates clean labeling of a protein between the markers for 35 kDa and 55 kDa, which is in good agreement with the expected mass of ca. 40 kDa for the 360 amino acid CB2 protein (without considering glycosylations and other

Triazolopyrimidine-Derived Ligands 31 posttranslational modifications). As evident from lane 3, labeling of CB2 with LEI121 is sensitive to the presence of high affinity competitor CP55940, which allows the conclusion that both compounds either bind to the same site in the receptor, or that CP55940 leads to a structural change of CB2 which either does not bind LEI121, or alternatively leads to inefficient covalent bond formation of the reactive species generated upon irradiation. In contrast, sulfonyl fluorides 60 and 61 were designed to undergo a covalent bond forming reaction with CB2 without the need for photoactivation. Despite the very high affinity of 60 as assessed by radioligand binding assay, no fluorescent band in the expected region could be detected (lanes 4 and 5). Interestingly, 60 selectively labeled a protein whose identity is unknown at this time. This off- target labeling was not dependent on the presence of CP55940. 61 only exhibited very weak, non-selective labeling of unidentified proteins (lanes 6 and 7). Collectively, the findings allow speculation on the nature of the interaction between CB2 and compounds 60 and 61. Although no labeling event could be detected, the absence of the expected band does not rule out a covalent binding event. Firstly, the putative thiosulfonate could undergo hydrolysis during the manipulations following membrane incubation. Secondly, the order of events could have a significant impact on the outcome of the labeling experiments, in which the dye was attached via [3+2] cycloaddition on the intact proteins (before denaturation). It is therefore possible that the alkyne might have been buried in the binding pocket of the receptor, which may have prevented it from participating in the crucial cycloaddition. As follow-up experiment, the order of events should be changed to the order of incubation, washing, denaturation, click-chemistry and SDS-PAGE.

6.2.3 Preliminary Assessment of Receptor Kinetics81

Current lead-optimization of a bioactive compound acting at a known target often focuses on increasing binding affinity measured as Ki, which describes the ratio of free and target-bound ligand at equilibrium (i.e. dissociation constant of the ligand-target complex). Recently, the importance of receptor-ligand binding kinetics has become increasingly important. Recently, the assessment of kinetic parameters has been implemented more frequently in the early drug discovery process.82 Quantification of the dynamic interaction between drug candidates in terms of kon (the rate constant for complex association), koff (the rate constant of dissociation) and target residence time (RT), defined as 1/koff can give insight into structural determinants of binding kinetics (structure-kinetics relationships), complementing the important and more

81 The experiments described in this section were performed by Dr. Andrea Martella, Leiden University. 82 D. Guo, L. H. Heitman, A. P. IJzerman, ACS Med. Chem. Lett. 2016, 7, 819.

32 Novel Chemical Probes for the Cannabinoid Receptor 2 classical approach of determining parameters of drug-target interaction under equilibrium conditions such as binding affinity (Ki) or efficacy (EC50, IC50), commonly known as structure- activity relationships.83

Figure 6.7. Top: dual point competition association assay, in which an unlabeled competitor is co- incubated with a radioligand in a kinetic association experiment, and followed for two or more time points. a: radioligand without competitor; b: unlabeled competitor dissociates slower than the radioligand; c: unlabeled competitor dissociates faster than the radioligand. Bottom: dual point Ki shift, in which an unlabeled competitor is co-incubated with a radioligand for either a short (here 30 min) and a longer (here 10 h) time. A slowly dissociating compound will show a more pronounced Ki shift (squares) compared to a fast dissociating compound (triangles). Reprinted by permission from D. Guo, J. M. Hillger, A. P. IJzerman, L. H. Heitman, Med. Res. Rev. 2014, 34, 856. Copyright © 2014 by John Wiley & Sons, Inc.

In search of a covalent binder (or a compound with long target residence time), we set out to determine kinetic parameters for selected ligands. Compounds 25, 27, 43 and 41 were chosen due to their promising binding affinity and tested for incubation time dependent Ki shifts (12 min/4 h). First, a qualitative dual point experiment (Figure 6.7) was conducted in order to identify encouraging candidates for further investigation.84 The results presented in Figure 6.8 allow for a qualitative assessment of koff relative to the radiolabel: a larger Ki hints at a more slowly dissociating ligand compared to the radioligand.85,86

83 R. A. Copeland, Nat. Rev. Drug Discov. 2016, 15, 87. 84 This initial pre-selection experiment was executed along with a number of other ligands under investigation in the laboratories of our collaborators. 85 D. Guo, J. M. Hillger, A. P. IJzerman, L. H. Heitman, Med. Res. Rev. 2014, 34, 856. 86 The radioligand [3H]-RO6957022 itself was under active development at Leiden University in collaboration with Fa. Hoffmann–La Roche. Its kinetic characterization and validation as a CB2 specific radiolabel with low non-specific binding has been published: A. Martella, H. Sijben, A. C. Rufer, U. Grether, J. Fingerle, C. Ullmer, T. Hartung, A. P. IJzerman, M. van der Stelt, L. H. Heitman, Mol. Pharmacol. 2017, 92, 389.

Triazolopyrimidine-Derived Ligands 33

R O257 1 9 27R O 949

1 2 0 1 2 0 g

g 1 2 m in

d d

1 0 0 1 2 m i n n 1 0 0

n i

i 4 h r s

2 4 h r s

2 0

0 8 0 8 0

6 O

O 6 0 6 0

]

[

4 0

c 4 0

e p

p 2 0 2 0

% % 0 0 T B -9 -8 -7 -6 N S B T B -9 -8 -7 -6 -5 N S B

[C m p d ] ( lo g M ) [C m p d ] (lo g M )

R41O 95 1 43R O 953 1 2 0 1 2 0

g 1 2 m in g 1 2 m in

n n

i i

d d

n 1 0 0 n 1 0 0

i 4 h r s i 4 h r s

b b

2 2

0 8 0 0 8 0

7 7

5 5

9 9

6 6

O 6 0 O 6 0

R R

] ]

H H

3 3

[ [

c 4 0 c 4 0

i i

f f

i i

c c

e e

p 2 0 p 2 0

s s

% % 0 0 T B -9 -8 -7 -6 -5 N S B T B -7 -6 -5 -4 N S B

[C m p d ] (lo g M ) [C m p d ] (lo g M )

Figure 6.8. Dual point Ki shift of selected compounds. Shown is specific radioligand binding measured at two concentrations (pKi ± 1) after 12 min (blue) and 4 h (green) incubation. A leftwards shift indicates a compound dissociates more slowly than the radiolabel.

Qualitatively, dual point Ki shift measurements indicated 25 to be the slowest dissociating ligand in the tested series, followed by 41 and 27. 43 did not exhibit a significant Ki. To confirm the pre-selection experiment, full curve Ki shifts were recorded for compounds 25, 41 and 27 (Figure 6.9). In the experiment, the relative trend seen in the dual point measurement was confirmed. 25 exhibited the most pronouncedi shift (Ki = 0.41), followed by 41

(Ki = 0.29) and 27 (Ki = 0.21).

34 Novel Chemical Probes for the Cannabinoid Receptor 2

R O 7 1 9 R O 9 4 9

1 2 0 25 1 2 0 27

i d

d 1 0 0 1 2 m i n 1 0 0 1 2 m i n

2 4 h rs 4 h rs 2 2 8 0

8 0 0

9 6 6 3 6 0 3

6 0 O

O [ H ]R O 6 9 5 7 0 2 2 = 5 9 7 5 3 d p m [ H ]R O 6 9 5 7 0 2 2 = 5 9 7 5 3 d p m

R ]

] E n d . C o n c .= 3 .3 4 n M E n d . C o n c .= 3 .3 4 n M

[

4 0 4 0

c e

e 2 0 2 0

% % 0 0 T B -1 0 -9 -8 -7 -6 T B -9 -8 -7 -6 -5 lo g [R O 7 1 9 ] (M ) lo g [R O 9 4 9 ] (M )

R O 9 5 1

1 2 0 41

n i

d 1 0 0 1 2 m i n

i b Ligand Ki 2 4 h rs

2 8 0

5 9 6 6 0 3

O [ H ]R O 6 9 5 7 0 2 2 = 5 9 7 5 3 d p m 25 0.41 R

] E n d . C o n c .= 3 .3 4 n M

3 [

4 0

i f

i 27 0.21 c

e 2 0

% 0 41 0.29 T B -9 -8 -7 -6 -5 lo g [R O 9 5 1 ] (M )

Figure 6.9. Full curve Ki shift of selected compounds. Shown is specific radioligand binding measured at multiple concentrations after 12 min (blue) and 4 h (green) incubation. A leftward shift indicates a compound dissociates more slowly than the radiolabel. Experimentally derived Ki shifts are shown in the table.

Due to its marked Ki shift, the target residence time of sulfonyl fluoride 25 was determined in a multipoint competition association assay. In the experiment, membrane preparations of CB2- overexpressing CHO cells were coincubated with both 25 and radiolabel. Specific binding was then determined after defined time intervals. The data presented in Figure 6.10 allows the 87 calculation of kon, koff, RT and the kinetic Kd = koff / kon as follows:

-1 -1 -1 compound kon (M min ) koff (min ) RT (min) 25 1.56 ± 0.63 · 105 0.0125 ± 0.005 ~ 80

87 Calculations performed by A. Martella as described in A. Martella, H. Sijben, A. C. Rufer, U. Grether, J. Fingerle, C. Ullmer, T. Hartung, A. P. IJzerman, M. van der Stelt, L. H. Heitman, Mol. Pharmacol. 2017, 92, 389.

Triazolopyrimidine-Derived Ligands 35

Radioligand only 70 nM 25 140 nM 25 210140 nM 25 140 nM

Figure 6.10. Competition association assay of 25 at various concentrations using radioligand [3H]- RO6957022. DPM (disintegrations of radioisotope per minute) is correlated to CPM (counts per minute detected by the scintillation counter) via a hardware-specific factor termed efficiency describing the ratio of CPM to DPM.

25 exhibited a target residence time of ~80 min. To put this figure into perspective, the following finding needs to be considered. A comprehensive study of residence times in the Leiden laboratories covering numerous CB2 ligands of structurally distinct classes revealed that CB2 residence times are rather short across various chemotypes with maximum RT values of ~10 minutes.88 In this light, the result for sulfonyl fluoride 25 stands out as exceptionally high.

Theoretically, a covalent binder should exhibit an infinite residence time, since koff approaches (equals) zero. If the sulfonyl fluoride group of 25 reacts with a suitable nucleophilic amino acid of the receptor, the resulting covalent bond (thiosulfonate, sulfonate, sulfonamide) might hydrolyze over time, explaining the finite residence time.

Lastly, the preliminary kinetic assessment of electrophilic triazolopyrimidines concluded with wash-out experiments using compounds 54 and 34. Both triazolopyrimidines feature the difluorobenzyl group which was shown to trigger inverse agonism, but differ in their N-3 substitution. While 34 is decorated with a sulfonyl fluoride group, a methyl sulfone is present in 54. Surprisingly, this rather small structural change led to significantly altered binding affinities towards both cannabinoid receptors (hCB2 Ki = 630 nM and 16 nM for 34 and 54, respectively). In the wash-out experiment, membrane preparations of CB2-overexpressing

CHO cells were first incubated with the two ligands at 10-fold Ki and subsequently washed extensively to remove any unbound ligand. Determination of the receptor density (Bmax) and correction for non-specific binding then allows for the estimation of accessible binding sites. In

88 A. Martella et al. - personal communication of unpublished results.

36 Novel Chemical Probes for the Cannabinoid Receptor 2 theory, the specific binding should approach zero if no binding site was available as would be expected upon incubation with a covalent ligand. The results for the pair of ligands are shown in Figure 6.11. Total specific binding of membranes incubated with methyl sulfone 54 and subsequent washout corresponded to those treated with vehicle only. Interestingly, membranes incubated with sulfonyl fluoride 34 exhibited markedly reduced total specific binding of about 30% compared to control. This result is no proof for the formation of a covalent ligand-receptor interaction but attests the compound at least a high wash-resistance (corresponding to a long target residence time). Although in this specific case it cannot be excluded that the obtained result originates from the high concentration of 34 (6.3 M), which could lead to substantial ligand sequestration in the membranes that might not be overcompensated by extensive washing.

Figure 6.11. Wash-out experiments with difluorobenzyl derivatives of the inverse agonist series. Determination of Bmax after incubation with methyl sulfone 54 and sulfonyl fluoride 34 (both compounds from the inverse agonist series) at 10x Ki and subsequent extensive washing. A decrease in specific binding indicates fewer binding sites available for the radioligand. TBU: total binding (unspecific) of radioligand. TB: total specific binding of radioligand (TBU corrected for non-specific binding (NSB) determined in presence of excess non-tritiated ligand).

6.3 Photoactivatable Probes

The previous chapter described the synthesis and in vitro evaluation of electrophilic probes with the potential for covalent binding of CB2 via nucleophilic amino acids. Another objective of the project was to develop CB2 selective photoaffinity probes as a specific detection tool for the receptor. To realize this goal, a number of bifunctional ligands exhibiting both a photoactivatable diazirine and an alkyne as functional handle were synthesized. Both aliphatic as well as trifluoromethylaryl-substituted diazirine derivatives were prepared and evaluated in radioligand binding assays as well as photoaffinity studies.

Triazolopyrimidine-Derived Ligands 37

6.3.1 Synthesis of Diazirine Derivatives

Ortho-ortho disubstituted derivatives

As shown by the data presented in the previous chapter, o,o-disubstituted triazolopyrimidines are well tolerated by CB2. Therefore, it was decided to keep this substitution pattern within the benzyl substituent and to prepare o-alkynyl-o-diazirinyl derivatives for use in photoaffinity labeling studies. To this end, an alkylating reagent incorporating all needed functional groups (62) was devised and prepared as outlined in Scheme 6.9. Palladium catalyzed carboxylate- directed C–H functionalization of benzoic acid as developed by YU89 delivered 2,6- diiodobenzoic acid. Since subsequent reduction with BH3·DMS proceeded sluggishly, it was decided to follow a two-step sequence. Conversion to the acid chloride using oxalyl chloride and catalytic DMF, extractive workup and NaBH4 reduction in MeCN afforded benzyl alcohl 62-a in 80% yield from the acid. After PMB-protection,90 iodine-magnesium exchange using the KNOCHEL method (iPrMgCl·LiCl)91 yielded the corresponding aryl GRIGNARD reagent, which was reacted with N-methoxy-N-methyl-2,2,2-trifluoroacetamide to generate trifluoromethyl aryl ketone 62-c (58% yield). Oxime formation and subsequent tosylation set the stage for the introduction of the diazirine group. Refluxing tosylated oxime

Scheme 6.9. Synthesis of o,o-disubstituted bifunctional diazirine building block 62. Reagents and conditions: a) Pd(OAc)2 (5 mol%), PhI(OAc)2, I2, DMF, 100 °C, 66%; b) i. (COCl)2, CH2Cl2, reflux; ii. NaBH4, MeCN, rt, 80%; c) NaH, PMBCl, DMF, 0 °C, 88%; d) iPrMgCl·LiCl, THF, –20 °C, then 67, 58%; e) HONH2·HCl, pyridine, EtOH, 80 °C, 90%; f) TsCl, NEt3, DMAP (10 mol%), CH2Cl2, rt, 58%; g) NH3, Et2O; then I2, NEt3, MeOH, 40%; h) CuI (20 mol%), PdCl2(PPh3)2 (10 mol%), trimethylsilyl acetylene, DMF, rt, 85%; i) K2CO3, MeOH, rt, 93%; j) i. DDQ, CH2Cl2–H2O, 0 °C; ii. NEt3, MsCl, CH2Cl2, 0 °C, 87%.

89 T.-S. Mei, R. Giri, N. Maugel, J.-Q. Yu, Angew. Chem. Int. Ed. 2008, 47, 5215. 90 THP and TBS protecting groups were not tolerated in the subsequent metalation/acylation step. 91 For a comprehensive review of halogen-metal exchanges, see D. Tilly, F. Chevallier, F. Mongin, P. C. Gros, Chem. Rev. 2014, 114, 1207.

38 Novel Chemical Probes for the Cannabinoid Receptor 2

62-d in a mixture of liquid NH3 and Et2O afforded an intermediate diaziridine, which was directly oxidized with iodine in NEt3 buffered methanol to give 62-e in 40% yield.

SONOGASHIRA coupling with trimethylsilyl acetylene and subsequent potassium carbonate mediated silyl removal delivered 62-f (79% over both steps). Reaction of the asymmetric dibenzyl ether with DDQ selectively cleaved off the more electron-rich p-methoxyarene and yielded the corresponding benzyl alcohol along with 4-methoxybenzaldehyde (the product of oxidative ether cleavage), which coeluted during purification on silica gel. The aldehyde side product could be readily separated after mesylation of the benzylic alcohol to yield 62 in 87% yield over two steps.

Benzyl mesylate 62 was then used for alkylation of two pairs of triazolopyrimidines (agonist and inverse agonist series with both 3,3-difluoro- and 3-hydroxypyrrolidine substituents) to prepare the eight compounds (63-70) shown in Scheme 6.10.

Scheme 6.10. Synthesis of bifunctional trifluoromethylaryl diazirine derivatives. Reagents and conditions: triazolopyrimidine (15, 16, 20, 22), DIPEA or NEt3, CH2Cl2, 38% (63), 36% (64), 23% (65), 29% (66), 29% (67), 51% (68), 34% (69), 45% (70).

Aliphatic Diazirines In addition to the trifluoromethyl substituted diazirines described in the previous chapter, two bifunctional photoaffinity probes bearing an aliphatic diazirine as part of the pyrrolidine substituent were synthesized. The spirocyclic motif was precedented and shown to successfully

Triazolopyrimidine-Derived Ligands 39 undergo photo cross linking in the context of a peptide based photoaffinity probe prepared by the ROBINSON group.92 In their work, the synthesis of photoproline 71 (both enantiomers accessible) enabled its incorporation into a class of peptide antibiotics by solid phase synthesis.93 The tool compounds thus derived led to the identification of the primary target of the parent antibiotic. We applied the synthetic strategy to N-Boc-3-oxo-pyrrolidine (72) to prepare building block 74. Treatment of 72 with ammonia and hydroxylamine O-sulfonic acid as nitrene equivalent and subsequent oxidation of the intermediate diaziridine by iodine in methanol (Scheme 6.11) afforded 73. Boc deprotection with HCl in dioxane afforded pyrrolidinium hydrochloride 74 for use in SNAr reactions.

Scheme 6.11. Synthesis of spirocyclic, diazirine functionalized pyrrolidine hydrochloride. Reagents and conditions: a) NH3, Et2O, reflux (–30 °C); then hydroxylamine O-sulfonic acid, MeOH–Et2O, –78 °C to rt; then I2, NEt3, MeOH, rt, 15%; b) HCl, dioxane, 92%.

Two photoaffinity probes with a tert-butyl (agonist) and a difluorobenzyl substituent (inverse agonist) were synthesized in order to probe for a potential dependence of labeling efficiency (if successful) on functional activity. To this end, known triazole 75 was condensed with pivaloyl chloride. Following SONOGASHIRA coupling with trimethylsilyl acetylene and desilylation, a two-step sequence involving chlorination and SNAr with 74 afforded the agonist version of the aliphatic diazirine derivative 78. It was later found that the sequence could be carried out conveniently starting from 2-ethynylbenzyl methanesulfonate 79. Nucleophilic displacement and subsequent cycloaddtion, condensation with PhCF2CN, chlorination and SNAr afforded the respective inverse agonist 82 (Scheme 6.12).

92 N. Srinivas, P. Jetter, B. J. Ueberbacher, M. Werneburg, K. Zerbe, J. Steinmann, B. V. der Meijden, F. Bernardini, A. Lederer, R. L. A. Dias, et al., Science 2010, 327, 1010. 93 B. van der Meijden, J. A. Robinson, Arkivoc 2011, 2011, 130.

40 Novel Chemical Probes for the Cannabinoid Receptor 2

Scheme 6.12. Synthesis of bifunctional aliphatic diazirine derivatives. Reagents and conditions: a) PivCl, pyridine, DMAA, 80 °C; then KHCO3, 155 °C, 76%; b) Pd(PhCN)2Cl2 (5 mol%), CuI (5 mol%), P(tBu)3·HBF4 (10 mol%), diisopropylamine, PhMe, 50 °C, 65%; c) K2CO3, MeOH, rt, 97%; d) (COCl)2, DMF (cat.), CH2Cl2, rt, 90%; e) 74, NEt3, CH2Cl2, rt, 99%; f) 2-cyanoacetamide, NaN3, NaOH, DMSO–H2O, 72%; g) i. PhCF2CN, K2CO3, DMF, 90 °C; ii. (COCl)2, DMF (cat.), PhMe, 65 °C, 57%; h) 74, NEt3, CH2Cl2, rt, 78%.

In 2013, YAO and coworkers described minimalist linker constructs exhibiting functionalities for both photo cross-linking (diazirine) and subsequent coupling to azide functionalized reporter groups via copper catalyzed cycloaddition of an alkyne (Scheme 6.13, A).94 These constructs might be used to turn any biologically active compound into a potential photoaffinity probe. Since its appearance, the minimalist linker concept has been validated and successfully applied to affinity based protein profiling by various groups.95,96,97 In search of a suitable exit vector for attachment of the minimalist linker construct, screening the SAR data of our collaborators suggested several possibilities, one of which being to derivatize a class of compounds that exhibited an oxadiazole rather than a benzene ring attached to N-3. These compounds stood out due to their very high CB2 affinity. Known lactone 83 is readily available from tetronic acid via dehydration of the corresponding dioxime.98 84 was prepared according to YAO’s protocol. Trimethylaluminum mediated amide formation was followed by mesylation

94 Z. Li, P. Hao, L. Li, C. Y. J. Tan, X. Cheng, G. Y. J. Chen, S. K. Sze, H.-M. Shen, S. Q. Yao, Angew. Chem. Int. Ed. 2013, 52, 8551. The same group published a similar photoactivatable construct featuring a cyclopropene motif for inverse electron demand DIELS-ALDER reactions: Z. Li, D. Wang, L. Li, S. Pan, Z. Na, C. Y. J. Tan, S. Q. Yao, J. Am. Chem. Soc. 2014, 136, 9990. 95 P. Kleiner, W. Heydenreuter, M. Stahl, V. S. Korotkov, S. A. Sieber, Angew. Chem. Int. Ed. 2017, 56, 1396. 96 B. D. Horning, R. M. Suciu, D. A. Ghadiri, O. A. Ulanovskaya, M. L. Matthews, K. M. Lum, K. M. Backus, S. J. Brown, H. Rosen, B. F. Cravatt, J. Am. Chem. Soc. 2016, 138, 13335. 97 S. Zhuang, Q. Li, L. Cai, C. Wang, X. Lei, ACS Cent. Sci. 2017, 3, 501. 98 A. Combs, A. Takvorian, W. Zhu, R. Sparks, N-hydroxyamidinoheterocycles as modulators of indoleamine 2,3- dioxygenase, US20070185165 A1, 2007.

Triazolopyrimidine-Derived Ligands 41 and afforded minimalist linker substituted oxadiazole 85 in 74% yield over two steps. As before, alkylation of triazolopyrimidines 15 and 16 afforded two pairs of regioisomeric photoaffinity probes of the agonist series.

Scheme 6.13. A: Minimalist linker building blocks introduced by YAO.94 B: Synthesis of minimalist linker derived triazolopyrimidines. Reagents and conditions: a) AlMe3, PhMe, 0 °C to rt, 93%; b) MsCl, NEt3, CH2Cl2, 0 °C, 80%; c) 15, NEt3, DMF, rt, 19% (88), 17% (89); d) 16, NEt3, DMF, 13% (90), 27% (91).

6.4 Results and Discussion

6.4.1 In Vitro Pharmacology

Gratifyingly, the o,o-disubstituted triazolopyrimidine derivatives collectively exhibited very promising in vitro profiles as assessed by radioligand binding assay on CB2 overexpressing CHO membranes. The binding affinity of ligands linked to the benzyl motif via N-3 was determined to be below 12 nM in the agonist series (63, 65) and below 58 nM (67, 69) in the inverse agonist series. Moreover, 3-hydroxypyrrolidines (65, 69) exhibited complete selectivity over CB1 (hCB1 Ki > 10 µM). The N-2 isomers were generally less potent binders.

Photoaffinity probes bearing aliphatic diazirines were very potent and reasonably selective with hCB2 Ki values of 10 nM (78, agonist series) and 19 nM (82, inverse agonist series). The minimalist linker derived triazolopyrimidines 88, 89, 90 and 91 collectively did not bind to CB1

42 Novel Chemical Probes for the Cannabinoid Receptor 2 whilst exhibiting moderate binding affinity for CB2 in the N-3 substitutend series. In a functional cAMP assay, 78 emerged as a very potent full agonist of CB2 with an EC50 in the picomolar range, and a full agonist of CB1 (EC50 = 17 nM). 88 on the other hand exhibited CB2 agonism only (hCB2 EC50 = 3.4 nM) with no CB1 activity (hCB1 EC50 > 10 M).

Table 6.2. hCB1 and hCB2 affinities and selected cAMP data of bifunctional photoaffinity probes based on the triazolopyrimidine scaffold.a

hCB1 Ki = 147 nM (1494 nM) hCB1 Ki = 583 nM (5935 nM) hCB2 Ki = 9 nM (1 nM) hCB2 Ki = 52 nM (5 nM)

hCB1 Ki > 10000 nM hCB1 Ki > 10000 nM hCB2 Ki = 12 nM (1 nM) hCB2 Ki = 99 nM (10 nM)

hCB1 Ki = 3393 nM (> 10000 nM) hCB1 Ki > 10000 nM hCB2 Ki = 31 nM (3 nM) hCB2 Ki = 1074 nM (107 nM)

hCB1 Ki > 10000 nM hCB1 Ki = 5459 nM (> 10000 nM) hCB2 Ki = 58 nM (51 nM) hCB2 Ki = 358 nM (312 nM)

Triazolopyrimidine-Derived Ligands 43

hCB1 Ki = 132 nM (352 nM) hCB1 Ki > 10000 nM hCB2 Ki = 10 nM (1 nM) hCB2 Ki = 200 nM (23 nM)

hCB1 cAMP EC50 = 17 nM (95%) hCB1 cAMP EC50 > 10000 nM (inactive) hCB2 cAMP EC50 = 0.03 nM (98%) hCB2 cAMP EC50 = 3.4 nM (95%)

hCB1 Ki = 465 nM (98 nM) hCB1 Ki > 10000 nM hCB2 Ki = 19 nM (1 nM) hCB2 Ki = 933 nM (105 nM)

hCB1 Ki > 10000 nM hCB1 Ki > 10000 nM hCB2 Ki = 559 nM (63 nM) hCB2 Ki = 2861 nM (323 nM) a For binding affinities, the figures in parentheses are Ki values normalized to control compounds 78 79 Rimonabant (Ki hCB1 = 5.6 nM) and JWH-133 (Ki hCB2 = 3.4 nM) codetermined in the respective experiment and set to published value. For cAMP data, figures in parentheses denote the compound’s efficacy relative to CP55940.

44 Novel Chemical Probes for the Cannabinoid Receptor 2

6.4.2 Photoaffinity Labeling99

The newly synthesized photoaffinity probes were evaluated for their ability to selectively label CB2 protein upon irradiation with 350 nm light. Similarly to the sulfonyl fluoride based affinity labels discussed in Chapter 6.1.2, membrane preparations of CB2 overexpressing CHO cells were incubated with photoaffinity probes at varying concentrations and then irradiated with 350 nm light. Subsequently, a “click mix” containing a copper(II) source, sodium ascorbate (reductant), THPTA (tris(3-hydroxypropyltriazolylmethyl)amine, a ligand for copper) and a cyanine dye (Cy5-azide) was added and the mixture was aged for 1 h at rt. After addition of solubilization buffer, aliquots of the various samples were subjected to electrophoresis on polyacrylamide gel under denaturing conditions (SDS-PAGE). Proteins conjugated to Cy5 were visualized by fluorescence imaging.

Figure 6.12 shows the results for compounds 78, 88, 82 (lanes 4 - 13) along with positive control LEI121 (lanes 2 and 3). Albeit with low efficiency, the photoprobes indeed exhibited weak labeling of the target protein which was diminished in the presence of CP55940 as orthosteric competitor. However, the low labeling efficiency along with off-target activity render these compounds less than optimal for applications in more complex settings using for example endogenously expressing cells, in which much less CB2 protein would be present.

LEI121 78 78 88 88 82 Probe - (2 µM) (5 µM) (10 µM) (5 µM) (10 µM (5 µM) CP55940 - + - + - + - + - + - + - (20 µM) lane 1 2 3 4 5 6 7 8 9 10 11 12 13

55 kDa

35 kDa

Figure 6.12. Cy5-conjugated proteins from CB2 overexpressing CHO membranes after photoaffinity labeling using LEI121 and diazirines 78, 88 and 82 (2 and 5 M) in the presence (+) or absence (-) of

99 These experiments were performed by Marjolein Soethoudt at Leiden University.

Triazolopyrimidine-Derived Ligands 45

CP55940 as competitor. The proteins were resolved by electrophoresis on polyacrylamide gel (SDS- PAGE) and visualized by fluorescence imaging.

The evaluation of compounds 70 and 69 is presented in Figure 6.13 (lanes 6 – 13) together with a high concentration run using 82 (lanes 4 and 5) and positive control LEI121 (lanes 2 and 3). Somewhat surprising with respect to their encouraging binding affinities, none of the compounds showed the expected band for a protein of ca. 40 kDa mass. Possible explanations for this are discussed in Chapter 7.

LEI121 82 70 70 69 69 Probe - (2 µM) (10 µM) (5 µM) (10 µM) (5 µM (10 µM) CP55940 - - + - + - + - + - + - + (20 µM) lane 1 2 3 4 5 6 7 8 9 10 11 12 13

55 kDa

35 kDa

Figure 6.13. Cy5-conjugated proteins from CB2 overexpressing CHO membranes after photoaffinity labeling using LEI121 and diazirines 82 (10 M), 70 and 69 (2 and 5 M) in the presence (+) or absence (-) of CP55940 as competitor. The proteins were resolved by electrophoresis on polyacrylamide gel (SDS-PAGE) and visualized by fluorescence imaging.

The same observation was made for compounds 64, 63, 67 (Figure 6.14), 65 and 66 (Figure 6.15). At all tested concentrations, the compounds showed rather weak off-target labeling at best.

46 Novel Chemical Probes for the Cannabinoid Receptor 2

LEI121 64 64 63 63 67 Probe - (2 µM) (5 µM) (10 µM) (5 µM) (10 µM) (5 µM) CP55940 - - + - + - + - + - + - + (20 µM) lane 1 2 3 4 5 6 7 8 9 10 11 12 13

55 kDa

35 kDa

Figure 6.14. Cy5-conjugated proteins from CB2 overexpressing CHO membranes after photoaffinity labeling using LEI121 and diazirines 64, 63 (5 and 10 M) and 67 (5 M) in the presence (+) or absence (-) of CP55940 as competitor. The proteins were resolved by electrophoresis on polyacrylamide gel (SDS-PAGE) and visualized by fluorescence imaging.

LEI121 67 65 65 66 66 Probe - (2 µM) (10 µM) (5 µM) (10 µM) (5 µM (10 µM) CP55940 - - + - + - + - + - + - + (20 µM) lane 1 2 3 4 5 6 7 8 9 10 11 12 13

55 kDa

35 kDa

Figure 6.15. Cy5-conjugated proteins from CB2 overexpressing CHO membranes after photoaffinity labeling using LEI121 and diazirines 67 (10 M), 65 and 66 (5 and 10 M) in the presence (+) or absence (-) of CP55940 as competitor. The proteins were resolved by electrophoresis on polyacrylamide gel (SDS-PAGE) and visualized by fluorescence imaging.

Conclusion and Outlook 47

7 Conclusion and Outlook

In Chapter 6, the synthesis and in vitro pharmacological characterization of various triazolopyrimidine derivatives was described. More specifically, we focused on the preparation of functionally distinct CB2 ligands by choice of the respective C-5 substituent of the heterocyclic core structure. Furthermore, a variety of functional groups commonly used in (photo)-affinity tagging were introduced and combined with an additional handle in the form of an alkyne to allow for conjugation to a reporter tag such as a fluorophore. The prepared ligands can be classified into the following categories: i) monofunctional sulfonyl fluorides (and methylsulfone control compounds); ii) bifunctional alkyne-substituted sulfonyl fluorides; and iii) bifunctional alkyne-substituted diazirines.

In a collaborative effort, the novel tool compounds were evaluated in a radioligand binding assay using tritiated CP55940 for their binding affinities towards hCB1 and hCB2, respectively. In the sulfonyl fluoride series, seven out of the twelve compounds emerged as very potent CB2 binders with hCB2 Ki < 50 nM, five of which showed at least 27-fold selectivity over CB1 (up to no measurable interaction with CB1). In the diazirine series, six out of the fourteen compounds prepared exhibited hCB2 Ki values below 58 nm along with good to excellent selectivity over CB1.

The new probes were then subjected to (photo)-affinity labeling experiments using membrane preparations of CB2 overexpressing CHO cells. Three triazolopyrimidine-derived diazirines (78, 88, 82) emerged as photoactivatable probes that undergo covalent cross linking upon irradiation with light (350 nm), albeit with low efficiency. Interestingly, sulfonyl fluoride 60 selectively labeled an unknown protein (Figure 6.6). o,o-Difunctionalized photoaffinity probes 63-70 emerged as ineffective labeling agents under the experimental conditions despite their high affinity (and selectivity) towards CB2 throughout the series. We see three possible explanations for this outcome. First, the carbene formed upon irradiation is not in proximity to an amino acid to undergo the required insertion reaction and is quenched by solvent instead. Second, the carbene might get trapped internally by N-2 to form an ylide, whose reactivity is largely attenuated with respect to insertion reactions and should eventually get quenched by surrounding water. Third, the order of events could be detrimental to the detection of successful labeling events. The alkyne might not be accessible to undergo copper-catalyzed cycloaddition with the azide-functionalized dye due to its positioning within the binding pocket. Reversing the order of events to follow the steps i) irradiation/cross-linking, ii) denaturation of proteins to set free the alkyne followed by iii) click reaction with the dye might solve this problem.

48 Novel Chemical Probes for the Cannabinoid Receptor 2

Preliminary data on binding kinetics of sulfonyl fluoride derived triazolopyrimidines led to the identification of putative covalent binders in both the agonist and inverse agonist series. 25 exhibited an exceptionally long (but still finite) residence time of about 80 minutes. 34 showed significant wash-resistance in wash-out experiments. Further experiments (kinetics, mass spectrometric analysis of whole protein or peptides upon digestion) are needed to corroborate the assumption of a covalent interaction between the ligands and CB2.

Novel CB2 Selective Cannabinoids 49

8 Novel CB2 Selective Cannabinoids

Chapter 6 describes efforts directed towards the development of CB2 selective, covalent probes based on the triazolopyrimidine scaffold, a compound class chosen for its drug-like properties. In order to maximize the chance to identify a potent covalent binder, we investigated an additional approach relying on a structurally distinct scaffold of the non-classical cannabinoid type.

8.1 Background

This chapter introduces select (bifunctional) tool compounds that have been used in CB2 research. Probes that have significantly contributed to the understanding of cannabinoid receptor biology over the years as well as more recent advancements are covered. Figure 8.1 shows a collection of selective and non-selective chemical probes100 acting on CB2. Introduced in 2005 by the MAKRIYANNIS group, AM841 features an electrophilic isothiocyanate group at the end of its 1,1-dimethylheptyl sidechain. The compound was described to covalently bind to both CB1101 and CB2102 via Cys6.47, an amino acid present in both receptors and part of the CWxP motif that is highly conserved among class A GPCRs.103 Pharmacologically, AM841 is an extremely potent, non-selective full agonist of both receptors. Remarkably, the introduction of the isothiocyanate group increases the potency by a factor of ~40 compared to the parent compound AM4056.104 HU-308 and its enantiomer HU-433 do not feature a functional group for covalent binding but are very potent CB2 agonists with complete selectivity over CB1 105 (hCB1 Ki > 10 M). Interestingly, while HU-308 exhibited significantly higher affinity for CB2 than its enantiomer HU-433 in radioligand binding assays, the latter was much more active in vivo. This counterintuitive finding was suggested to result from slightly different binding poses of the enantiomers leading to the distinct ability to compete with the tritiated radioligand [3H]CP55940. Both HU-308 and HU-210, which is a very potent 8-THC derivative that agonizes both receptors, have been elongated via the allylic alcohol to furnish biotinylated

100 In this context the term chemical probe is used for a tool compound allowing for interrogation of the biological system at hand. This includes selective modulators (agonists, inverse agonists, antagonists) as well as (bi)- functional probes such as covalent binders, fluorescently labeled ligands and photoactivatable crosslinkers. 101 R. P. Picone, A. D. Khanolkar, W. Xu, L. A. Ayotte, G. A. Thakur, D. P. Hurst, M. E. Abood, P. H. Reggio, D. J. Fournier, A. Makriyannis, Mol. Pharmacol. 2005, 68, 1623. 102 D. W. Szymanski, M. Papanastasiou, K. Melchior, N. Zvonok, R. W. Mercier, D. R. Janero, G. A. Thakur, S. Cha, B. Wu, B. Karger, et al., J. Proteome Res. 2011, 10, 4789. 103 M. Olivella, G. Caltabiano, A. Cordomí, BMC Structural Biology 2013, 13, 3. 104 Due to its unprecedented potency, AM841 is also termed a megagonist. 105 R. Smoum, S. Baraghithy, M. Chourasia, A. Breuer, N. Mussai, M. Attar-Namdar, N. M. Kogan, B. Raphael, D. Bolognini, M. G. Cascio, et al., PNAS 2015, 112, 8774.

50 Novel Chemical Probes for the Cannabinoid Receptor 2 compounds 92 (CB2 selective) and 93 (not selective) for subsequent attachment of streptavidin conjugated fluorescent dyes.106 Recently, BAI described near-infrared fluorescent probe NIR760-XLP, which was derived from a series of CB2 selective inverse agonists based on the 7-oxopyrazolopyrimidine scaffold.107 Unfortunately, the fluorescent conjugate suffered from significant non-specific binding but was shown to preferentially bind CB2 over CB1.108 A similar strategy was pursued by STELLA and BORNHOP, who derivatized the CB2 selective antagonist SR144528109 to produce readily conjugatable MBC94.110 The near-infrared fluorescent conjugate NIR-mbc94 was shown to bind to primary microglia (endogenously expressing CB2) derived from wildtype mice, while no specific binding was observed to microglia cells from CB2 knockout mice.111

Lastly, recent developments towards CB2 selective probes for use in positron emission tomography (PET) should be mentioned. MOLDOVAN and HORTI prepared 18F- 94 and applied it in a mouse model of neuroinflammation.112 Due to low metabolic stability of 94 the researchers later switched to a different chemotype.113 GRETHER and AMETAMEY developed pyridine derivative 95 as 11C-PET probe. The compound exhibits high selectivity for CB2 combined with favorable physico-chemical properties and was successfully applied in a rodent model of neuroinflammation.114

Despite these recent advancements in the development of CB2 selective chemical probes, the tools available to the scientific community can only be classified as less than optimal for more complex applications both in vitro and in vivo.

106 L. Martín-Couce, M. Martín-Fontecha, Ó. Palomares, L. Mestre, A. Cordomí, M. Hernangomez, S. Palma, L. Pardo, C. Guaza, M. L. López-Rodríguez, et al., Angew. Chem. Int. Ed. 2012, 51, 6896. 107 M. Aghazadeh Tabrizi, P. G. Baraldi, G. Saponaro, A. R. Moorman, R. Romagnoli, D. Preti, S. Baraldi, E. Ruggiero, C. Tintori, T. Tuccinardi, et al., J. Med. Chem. 2013, 56, 4482. 108 X. Ling, S. Zhang, P. Shao, W. Li, L. Yang, Y. Ding, C. Xu, N. Stella, M. Bai, Biomaterials 2015, 57, 169. 109 M. Rinaldi-Carmona, F. Barth, J. Millan, J.-M. Derocq, P. Casellas, C. Congy, D. Oustric, M. Sarran, M. Bouaboula, B. Calandra, et al., J. Pharmacol. Exp. Ther. 1998, 284, 644. 110 M. Bai, M. Sexton, N. Stella, D. J. Bornhop, Bioconjugate Chem. 2008, 19, 988. 111 M. Sexton, G. Woodruff, E. Horne, Y. Lin, G. Muccioli, M. Bai, E. Stern, D. Bornhop, N. Stella, Chemistry & Biology 2011, 18, 563. 112 R.-P. Moldovan, R. Teodoro, Y. Gao, W. Deuther-Conrad, M. Kranz, Y. Wang, H. Kuwabara, M. Nakano, H. Valentine, S. Fischer, et al., J. Med. Chem. 2016, 59, 7840. 113 R.-P. Moldovan, W. Deuther-Conrad, A. G. Horti, P. Brust, Molecules 2017, 22, 77. 114 R. Slavik, U. Grether, A. Müller Herde, L. Gobbi, J. Fingerle, C. Ullmer, S. D. Krämer, R. Schibli, L. Mu, S. M. Ametamey, J. Med. Chem. 2015, 58, 4266.

Novel CB2 Selective Cannabinoids 51

Figure 8.1. Chemical structures, names (where applicable) and brief description of select probes in CB2 research. Selectivity with respect to CB1/CB2 binding.

8.2 Towards a CB2 Selective Covalent Binder

A chemical probe that irreversibly binds CB2 in a highly selective fashion and exhibits further functionality for conjugation would be a highly desirable addition to the arsenal of tools available to researchers in the field. In search of such a probe, the question arose whether or not straightforward combination of select structural features of both compounds AM841 and HU- 308 would lead to a useful platform for further development into multifunctional or fluorescent

52 Novel Chemical Probes for the Cannabinoid Receptor 2 probes (Figure 8.2). AM841 features all important pharmacophores typical for classical cannabinoids resulting in highly efficient binding to the CBRs. These pharmacophores include the northern aliphatic hydroxyl group (NAH), the phenolic hydroxyl group (PH) and an aliphatic sidechain (SC). Importantly, AM841 exhibits additional functionality in the form of an isothiocyanate group at the end of the aliphatic side chain. This electrophilic group has been claimed to irreversibly bind to both receptors CB1 and CB2 via formation of a covalent bond to a Cys6.47. In contrast, HU-308 is a member of the class of non-classical cannabinoids characterized by the lack of a tricylic benzopyran ring system. The alkylation of the phenolic hydroxyl group has been shown to dramatically decrease CB1 but not CB2 binding.115 Interestingly, assuming a fixed orientation of the NAH within the binding pocket, the absolute configuration of the benzylic stereocenter is opposite in both compounds.

Based on this analysis, a hybrid probe was conceived that would feature the pinene derived, dimethylated core structure of HU-308 for CB2 selectivity, and the isothiocyanate terminated 1,1-dimethylheptyl sidechain for covalent binding. The ideal outcome of a labeling experiment with hybrid cannabinoid 96 (and its derivatives) in the presence of both receptors would be a selective covalent bond formation with only CB2, thus potentially enabling attractive applications in pharmacology, structural biology, and imaging.

Figure 8.2. Design of a putatively covalent, CB2 selective hybrid of AM841 and HU-308. Key structural elements of AM841 and HU-308 combined in 96 are highlighted.

115 A. Poso, J. W. Huffman, Br. J. Pharmacol. 2008, 153, 335.

Novel CB2 Selective Cannabinoids 53

8.2.1 Synthesis and Evaluation of a Hybrid Cannabinoid

Naturally, the initial route to hybrid cannabinoid 96 resembled parts of the syntheses of

AM4056 (the parent compound of AM841), and HU-308, which both rely on a FRIEDEL-

CRAFTS alkylation of a resorcinol derivative (98) with an allylic alcohol (97) derived from either enantiomer of pinene or myrtenol (Scheme 8.1).116

Scheme 8.1. FRIEDEL-CRAFTS alkylation of a resorcinol derivative en route to cannabinoids.

Conveniently, the reaction outcome was shown to be a function of the acidic promoter in use 8 and thus can be directed to yield either the initial product (TsOH·H2O) or the respective  - tetrahydrocannabinol derivative (BF3·Et2O). The formation of the latter can be rationalized by acid catalyzed rearrangement of the pinene portion to an intermediate cannabidiol derivative and subsequent ring closure. 117

TIUS’ synthesis of resorcinol 101 involved formation of ketone 102 via the corresponding

WEINREB amide, TiMe2Cl2 mediated introduction of the geminal dimethyl group as described 118 119 by REETZ and subsequent triple ether cleavage with BBr3 (Scheme 8.2, A).

Recently, the FÜRSTNER group described an efficient iron catalyzed acylation of GRIGNARD reagents to afford ketones in high yields.120 In order to test this method, known acid chloride

103 was prepared from the corresponding carboxylic acid. GRIGNARD reagent 104 was prepared by lithium chloride promoted insertion of magnesium into the respective alkyl bromide as originally described for aryl halides.121 Gratifyingly, when the organometallic was added to a

THF solution of Fe(acac)3 (3 mol%) and 103 at –78 °C, rapid and clean ketone

116 To the best of the author´s knowledge, the synthesis of AM841 has not been published in detail. For a route developed in the context of this thesis, see Chapter 8.5. 117 R. Mechoulam, N. Lander, A. University, J. Zahalka, Tetrahedron: Asymmetry 1990, 1, 315. 118 M. T. Reetz, J. Westermann, S.-H. Kyung, Chem. Ber. 1985, 118, 1050. 119 a) M. A. Tius, Chem. Commun. 1997, 1867. b) P. E. Harrington, I. A. Stergiades, J. Erickson, A. Makriyannis, M. A. Tius, J. Org. Chem. 2000, 65, 6576. c) C. Chu, A. Ramamurthy, A. Makriyannis, M. A. Tius, J. Org. Chem. 2003, 68, 55. 120 B. Scheiper, M. Bonnekessel, H. Krause, A. Fürstner, J. Org. Chem. 2004, 69, 3943. 121 F. M. Piller, P. Appukkuttan, A. Gavryushin, M. Helm, P. Knochel, Angew. Chem. Int. Ed. 2008, 47, 6802.

54 Novel Chemical Probes for the Cannabinoid Receptor 2

Scheme 8.2. A: Synthesis of resorcinol derivative 101 as reported by TIUS. Reagents and conditions: a) (COCl)2, DMF (cat.), CH2Cl2; then N,O-dimethylhydroxylamine·HCl, pyridine, 96%; b) 6- phenoxyhexylmagnesium bromide, THF, 0 °C, 75%; c) TiCl4, ZnMe2, CH2Cl2, –30 °C to rt; d) BBr3, CH2Cl2, –78 °C to rt, 84% over 2 steps. B: Alternative Fe(acac)3 catalyzed cross coupling for the synthesis of 102. Reagents and conditions: a) 6-phenoxyhexylmagnesium chloride·LiCl, Fe(acac)3 (3 mol%), THF, –78 °C, 90%. formation occurred. The transformation proved very robust as made evident by a 40 mmol scale reaction, which delivered 102 in 90% isolated yield (~12.3 g). Although published with 84% yield, the subsequent conversion of 102 into its geminal dimethyl derivative 105 using in situ 122 generated TiMe2Cl2 proceeded sluggishly in our hands (34% yield). Nevertheless, enough material could be isolated to continue the synthetic sequence. According to TLC, the following

BBr3 mediated triple ether cleavage proceeded very cleanly and afforded material with almost quantitative mass recovery after chromatography on silica. However, inspection of 1H and 13C NMR spectra revealed the presence of a second, minor species. Based on two-dimensional NMR spectroscopy (COSY, HSQC) this impurity was identified as isomeric secondary alkyl bromide 108. This finding has not been reported for the substrate at hand but could be substantiated by comparison of the spectroscopic data with known 2-bromooctane (109)123 (Figure 8.3). Interestingly, the reprint of a 13C NMR spectrum of closely related compound 110 synthesized by TIUS using the same BBr3 procedure also suggests the presence of the respective isomeric secondary bromide.124

122 The supporting information to the following reference also describes problems during an attempted REETZ reaction on similar substrates: C. Zanato, A. Pelagalli, K. F. M. Marwick, M. Piras, S. Dall’Angelo, A. Spinaci, R. G. Pertwee, D. J. A. Wyllie, G. E. Hardingham, M. Zanda, Org. Biomol. Chem. 2017, 15, 2086. 123 a) E. C. Ashby, C. O. Welder, J. Org. Chem. 1998, 63, 7707. b) Y. Liu, Y. Xu, S. H. Jung, J. Chae, Synlett 2012, 23, 2692. 124 C. Chu, A. Ramamurthy, A. Makriyannis, M. A. Tius, J. Org. Chem. 2003, 68, 55.

Novel CB2 Selective Cannabinoids 55

Figure 8.3. A: Selected NMR signals of 2-bromooctane and side product 108. B: TIUS’ close analog of 101 showing similar resonances in 13C NMR spectrum indicative of secondary bromide formation.

As the ratio of constitutional isomers was ~9:1 in favor of desired primary alkyl bromide 101 (and this ratio was expected to increase over the following steps) and in order to stay close to the reported synthesis of the parent probes, it was decided to continue with this material to quickly access hybrid 96 for a first biological assessment. Later efforts directed at the synthesis of less lipophilic cannabinoids led to a modification of the described synthetic sequence and delivered isomerically pure resorcinol 101 and various derivatives (Chapter 8.4). With resorcinol 101 and known allylic alcohol 97 in hand, the synthesis of hybrid 96 could be completed in five steps as summarized in Scheme 8.3. Coupling of the two fragments by p- toluenesulfonic acid catalyzed FRIEDEL-CRAFTS alkylation proceeded cleanly to afford 111 in 77-86% yield. In order to avoid potential halide scrambling, dimethyl sulfate was preferred over methyl iodide in the subsequent double alkylation of the two phenolic hydroxyl groups. The transformation was readily effected in presence of K2CO3 to afford the desired product (112)

Scheme 8.3. Synthesis of hybrid 96. Reagents and conditions: a) TsOH·H2O (0.28 equiv), CH2Cl2, rt, 86%; b) Me2SO4, K2CO3, acetone, rt, 73%; c) DIBAL-H, CH2Cl2, 0 °C, 94%; d) NaN3, DMF, rt, 99%; e) PPh3, CS2, THF, rt, 84%.

56 Novel Chemical Probes for the Cannabinoid Receptor 2 in 71-74% yield. Reductive pivalate ester cleavage using DIBAL-H, and subsequent nucleophilic bromide displacement with sodium azide gave rise to 114, which could be elaborated into hybrid 96 by PPh3 and carbon disulfide (73% from 112).

8.3 Synthesis of Bifunctional Probes

The development of affinity labels for any given target can help in various stages of the drug discovery process. Such probes can be used in experiments aiming at target confidence (by direct evidence of target engangement), structural information (crystallogenesis, mutation studies), or off-target detection, and therefore are highly desirable in the context of drug development. Generally, in addition to the key pharmacophores found in the parent structures that provide high affinity and selectivity for a target protein, affinity probes usually exhibit at least two additional motifs. One functional group allows for (irreversible) binding of the probe to its target protein while the second one enables follow-up manipulation or detection (reporter tag such as a fluorophore) of the formed probe-target complex. As described in Chapter 1.2, the irreversible binding might be realized through a reactive, usually electrophilic warhead or alternatively through the use of photoactivatable groups forming reactive species upon irradiation. Many combinations of functional groups have been explored in the context of activity-based protein profiling.125,126

The alkyne group is presumably the most commonly used functionality in this context. It is stable under physiological conditions, but still allows for rapid and robust functionalization in aqueous solution through copper catalyzed cycloaddition reactions with organic azides.127 The latter can then be used to introduce an affinity tag (such as biotin) for isolation and purification of the target protein or alternatively for visualization of the complex by fluorescence microscopy (fluorescent tags).

To further explore the biological activity of this construct as a function of increasingly large substituents attached to the core structure, we targeted an array of putatively CB2 selective affinity probes (both electrophilic and photoactivatable) based on the novel hybrid cannabinoid 96. Along these lines, modification of 96 via the allylic alcohol appeared to be the most attractive route to derive bifunctional probes. Alkylation of 96 under phase transfer

125 D. Greenbaum, A. Baruch, L. Hayrapetian, Z. Darula, A. Burlingame, K. F. Medzihradszky, M. Bogyo, Mol Cell Proteomics 2002, 1, 60. 126 G. C. Adam, E. J. Sorensen, B. F. Cravatt, Mol. Cell Proteomics 2002, 1, 781. 127 A. E. Speers, G. C. Adam, B. F. Cravatt, J. Am. Chem. Soc. 2003, 125, 4686.

Novel CB2 Selective Cannabinoids 57 conditions128 afforded the putatively photoactivatable129 bifunctional probe 115 in 36% yield along with 31% reisolated starting material. Subsequent elaboration into the corresponding isothiocyanate cleanly yielded 116 (81%).

Scheme 8.4. Synthesis of two bifunctional cannabinoids. Reagent and conditions: a) propargyl bromide, Bu4NHSO4 (0.2 equiv), 50% aq. NaOH–PhMe, rt, 36%; b) PPh3, CS2, THF, rt, 81%.

A radioligand binding assay performed in the laboratories of our collaborators revealed very high CB2 affinity of our newly synthesized analogs along with increased selectivity over CB1. This promising result led us to the question whether extension along the allylic alcohol with a longer chain would be tolerated by the receptor. A linker of sufficient length to reach the intercellular space would open up new avenues for the development of putatively covalent, fluorescent probes. To this end, and with results of MARTIN-COUCE130 in mind, building block 117 was targeted, as it would allow for convenient functionalization of 96 via ester formation. The fluorophore nitrobenzoxadiazole was chosen due to its ready availability at low cost and straightforward introduction via SNAr reaction of 4-chloro-7-nitrobenzofurazan with amine nucleophiles. The piperazine unit was envisioned to enhance solubility of the fluorescent probes while the rest of the linker should guarantee sufficient length to reach out of the binding pocket of the receptor.

The synthesis of 117 commenced with amide formation between 4-bromobutanoic acid (118) and methyl 6-aminohexanoate using ethyl chloroformate as activating agent in presence of N- methyl morpholine (Scheme 8.5). Subsequent alkylation of 119 required the use of catalytic sodium iodide and elevated temperatures to yield 120 in 38% yield. Interestingly, when the corresponding alkyl iodide 124 (derived via FINKELSTEIN reaction) was combined with

128 M. Barbazanges, C. Meyer, J. Cossy, Org. Lett. 2007, 9, 3245. 129 Although aliphatic azides are less widely used as photoactivatable cross linkers, a tetrahydrocannabinol derived probe was used successfully for labeling of CB1: R. P. Picone, D. J. Fournier, A. Makriyannis, J. Pept. Res. 2002, 60, 348. 130 L. Martín-Couce, M. Martín-Fontecha, Ó. Palomares, L. Mestre, A. Cordomí, M. Hernangomez, S. Palma, L. Pardo, C. Guaza, M. L. López-Rodríguez, et al., Angew. Chem. Int. Ed. 2012, 51, 6896.

58 Novel Chemical Probes for the Cannabinoid Receptor 2 secondary amine 121, clean formation of 122 was observed as judged by LCMS analysis (for a mechanistic rational, see Scheme 8.6).

Scheme 8.5. Synthesis of fluorescent building block 117. Inlet shows sideproducts observed during execution of the sequence. Reagents and conditions: a) N-methyl morpholine, ethyl chloroformate, methyl 6-aminohexanoate hydrochloride, CHCl3, rt, 24%; b) 121, KI (10 mol%), DMF, 90 °C, 38%; c) Novozyme 435, THF–H2O (pH 7), rt, 75%.

The methyl ester hydrolysis of 120 was first attempted using LiOH·H2O (5 equiv). Surprisingly, clean formation of 123 resulting from hydrolysis the aryl-piperazine bond was observed.131 A convenient solution to this problem was found in the use of an immobilized hydrolytic enzyme (Candida antarctica lipase on acrylic resin), which had previously been applied to ester cleavages of base sensitive substrates by a former group member.132 Treatment of methyl ester 120 with the commercial biocatalyst in a mixture of THF and aqueous phosphate buffer (pH 7) at room temperature resulted in clean formation of the desired product isolated in 75% yield.

Scheme 8.6. Mechanistic rational for the formation of 122 during the synthesis of 120 from alkyl iodide 124.

With fluorescently labeled carboxylic acid 117 in hand, attention was turned to the synthesis of CB2 selective, putatively covalent fluoroprobes via ester formation with 96. The reaction could

131 The rate of basic hydrolysis of NBD-proline derivatives has been documented, see L. Johnson, S. Lagerkvist, P. Lindroth, M. Ahnoff, K. Martinsson, Anal. Chem. 1982, 54, 939. 132 Julian Egger, ETH Dissertation No. 21363, Eidgenössische Technische Hochschule Zürich, Switzerland, 2013.

Novel CB2 Selective Cannabinoids 59 be effected by use of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as coupling reagent and delivered ester 127 in 90% yield (Scheme 8.7).

Scheme 8.7. Ester formation leading to bifunctional fluoroprobe 127.

In order to reduce the risk of rapid enzymatic hydrolysis of the ester linkage in a biological environment, two additional analogs were targeted, in which this vulnerable group was exchanged for an amide. In addition to the presumably higher stability against hydrolysis, this functional group exchange was exciting for a surprising reason: despite decades of research, examples of derivatives bearing an amide portion in this region of classical and non-classical cannabinoids are virtually unknown,133 as opposed to a small number of amine analogs that had been evaluated solely in vivo in mice134 and baboons.135 Interestingly, the amine derived cannabinoids evaluated in these studies exhibited only a fraction of the effects observed after administration of THC, attesting low cannabimimetic activity to these compounds. Since these studies were carried out before the discovery and cloning of cannabinoid receptors, no data on binding affinity to either receptor was reported.

Amine 128 was considered to be a straightforward intermediate for the synthesis of amide- derived analogs that could be prepared from allylic alcohol 113 in three steps (Scheme 8.8).

First, 113 was subjected to MITSUNOBU conditions in the presence of phthalimide to afford the

133 An acetamide derivative of 8-THC was reported to be analgesically inactive in mice as assessed by the hot- plate test: R. S. Wilson, B. R. Martin, W. L. Dewey, J. Med. Chem. 1979, 22, 879. 134 D. R. Compton, P. J. Little, B. R. Martin, J. W. Gilman, J. K. Saha, V. S. Jorapur, H. P. Sard, R. K. Razdan, J. Med. Chem. 1990, 33, 1437. 135 H. Edery, G. Porath, R. Mechoulam, N. Lander, M. Srebnik, N. Lewis, J. Med. Chem. 1984, 27, 1370.

60 Novel Chemical Probes for the Cannabinoid Receptor 2 protected amine. Bromide displacement using sodium azide and subsequent hydrazinolysis in the presence of excess crotyl alcohol as diimide scavenger136 afforded the desired amine 128.

Scheme 8.8. Synthesis of allylic amine 128. Reagents and conditions: a) DIAD, PPh3, phthalimide, THF, rt, 62%; b) NaN3, DMF, rt, 88%; c) N2H4·H2O, crotyl alcohol, EtOH, 75 °C, 73%.

Having secured a synthetic route to amine 128, attention was turned to the synthesis of the amide analogs discussed above (Scheme 8.9). EDC·HCl successfully mediated the coupling of amine 128 and fluorophore labeled acid 117 to yield amide analog 129. Subsequent conversion of the azide into the corresponding isothiocyanate was effected by PPh3 and CS2 and afforded putatively covalent fluoroprobe 130.

Scheme 8.9. Synthesis of fluorescent cannabinoids. Reagents and conditions: a) 117, EDC·HCl, NEt3, THF, rt, 56%; b) PPh3, CS2, THF, rt, 74%.

8.4 Alternative Access to Amine 128 and Cannabinoids of Increased Polarity

Promising biological data of our novel amide derivatives (Chapter 8.7) demanded the development of a more convergent approach to secure convenient access to amine 128. To this end, N-protected allylic alcohol was synthesized in four steps from (R)--pinene (Scheme

8.10). CrO3 mediated oxidation in presence of N-hydroxyphthalimide afforded verbenone in 44% yield (4.9 g).137 Subsequent radical bromination using N-bromosuccinimide proceeded

136 B. E. Maryanoff, M. N. Greco, H.-C. Zhang, P. Andrade-Gordon, J. A. Kauffman, K. C. Nicolaou, A. Liu, P. H. Brungs, J. Am. Chem. Soc. 1995, 117, 1225. 137 P. Marwah, H. A. Lardy, Process for Effecting Allylic Oxidation Using Dicarboxylic Acid Imides and Chromium Reagents, US Patent US6384251 B1, 2002.

Novel CB2 Selective Cannabinoids 61 sluggishly in acetonitrile138 with concomitant formation of multiple side products, but could be 139 successfully carried out in CCl4 as published. Nucleophilic displacement by potassium phthalimide yielded enone 131 in 85% yield. The bromination–displacement sequence could also be carried out without intermediate purification of bromo-verbenone to deliver enone 131 in essentially the same yield over two steps (56%). LUCHE reduction gave rise to allylic alcohol

132 and set the stage for subsequent FRIEDEL-CRAFTS alkylation in analogy to the synthetic sequence described before (vide supra).

Scheme 8.10. Synthesis of N-phthalimide protected allylic alcohol 132. Reagents and conditions: a) CrO3, N-hydroxyphthalimide, acetone, rt, 44%; b) NBS, dibenzoyl peroxide, CCl4, reflux, 62%; c) potassium phthalimide, DMF, rt, 85%; d) CeCl3·7H2O, NaBH4, MeOH, –78 °C, 93%.

8.4.1 Synthesis of Isomerically Pure Resorcinols

As described in Chapter 8.2.1, triple ether cleavage of 105 afforded the desired linear alkyl bromide along with acceptable amounts (~10%) of the respective constitutively isomeric secondary alkyl bromide (108). Although the ratio of desired to undesired isomer increased during the following steps, this finding clearly warranted an improved strategy towards the resorcinol building blocks. Mechanistically, the formation of said side product might arise from HBr elimination and subsequent Markovnikov addition to the resulting terminal olefin, or from a (concerted) 1,2-hydride shift followed by bromide trapping of the resulting secondary carbocation. Unfortunately, performing the transformation in the presence of homogenous

(DIPEA) or heterogenous (K2CO3) base (CH2Cl2, –78 °C to 0 °C) was not effective in suppressing the undesired reaction pathway.

Another concern at this stage of the project was the rather high lipophilicity of cannabinoids, as this parameter correlates with non-specific binding to cellular membranes which in turn might result in decreased signal to noise ratio in fluorescence-based applications. Among the vast amount of structure-activity data available for THC derivatives, one particular patent on “water-soluble cannabinoids” caught our attention.140 In this work, various THC analogs

138 In the author’s experience, CCl4 can often be substituted by MeCN in radical reactions. 139 O. V. Ardashov, E. V. Khaid, O. S. Mikhal’chenko, D. V. Korchagina, K. P. Volcho, N. F. Salakhutdinov, Russ Chem Bull 2013, 62, 171. 140 B. R. Martin, R. K. Razdan, A. Mahadevan, Water Soluble Cannabinoids, WO2006012176A1, 2008.

62 Novel Chemical Probes for the Cannabinoid Receptor 2 bearing very polar functional groups are reported along with binding data for both CB1 and CB2. The results show that amongst other groups, tertiary, secondary and also primary amides are well tolerated. Accordingly, we sought for a more flexible route to resorcinol derivatives that would both circumvent contamination with isomeric side product and allow for introduction of polar groups into the aliphatic sidechain.

Scalability and forging the quaternary center in dimethylheptyl substituted resorcinols were considered the two main concerns of the synthesis. A relatively recent method for sp2-sp3 cross coupling was developed by AGGARWAL and coworkers.141,142 In this approach, an electron rich aryl lithium species 133 is reacted with a (chiral) secondary or tertiary boronic ester 134 in THF. The resulting ate-species 135 is then treated with a solution of NBS in MeOH, resulting in a stereospecific migration of the (chiral) alkyl moiety with concomitant deborylation and bromide elimination to yield the cross-coupled product 137 (Figure 8.4, A).

Two boronic esters 138 and 139 were prepared from known 140, which is accessible from commercial 141 by reaction with isopropyl magnesium chloride143 and α-bromination.144

MATTESON rearrangement employing the corresponding GRIGNARD reagents delivered chloro- and OTBS-substituted tertiary boronic esters 138 and 139. (Figure 8.4, B). Chloride 138 and TBS protected 139 were then subjected to oxidative cross-coupling with the aryl lithium species derived from 143 under slightly modified conditions.145 Gratifyingly, both boronic esters successfully underwent the coupling reaction to afford the desired material. Unfortunately, both products were contaminated with inseparable debrominated 143 resulting from hydrolysis of the intermediate aryl lithium species. In the case of TBS protected 139, this undesired side product could be easily removed after deprotection (AcCl in MeOH) to afford pure alcohol 144 in 57% (160 mg) from 143. The coupling and desilylation could also be carried out in a one- pot fashion without intermediate purification to give the product in 46% yield (0.5 g). Attempts to directly convert the silylated intermediate into 101 using BBr3 again resulted in formation of the secondary bromide.146 Therefore, we followed a two-step procedure consisting of the described desilylation and double demethylation using neat MeMgI at 170 °C to furnish the

141 A. Bonet, M. Odachowski, D. Leonori, S. Essafi, V. K. Aggarwal, Nat Chem 2014, 6, 584. 142 M. Odachowski, A. Bonet, S. Essafi, P. Conti-Ramsden, J. N. Harvey, D. Leonori, V. K. Aggarwal, J. Am. Chem. Soc. 2016, 138, 9521. 143 M. Myslinska, G. L. Heise, D. J. Walsh, Tetrahedron Letters 2012, 53, 2937. 144 B. Potter, E. K. Edelstein, J. P. Morken, Org. Lett. 2016, 18, 3286. 145 The original report prescribes the addition of a freshly prepared solution of NBS in MeOH. Due to the low solubility of NBS in MeOH, we found it more convenient to first add MeOH to the boronate followed by portionwise addition of the oxidant. This ensures a constantly high concentration of MeOH facilitating the deboronation. 146 S. Kim, J. H. Park, J. Org. Chem. 1988, 53, 3111.

Novel CB2 Selective Cannabinoids 63 corresponding triol 144. APPEL reaction then afforded isomerically pure 101 without any detectable secondary bromide.

Figure 8.4. A: AGGARWAL’s oxidative sp2-sp3 coupling of electron rich aryl lithiums with alkylboronates.141 B: Synthesis of isomerically pure bromoresorcinol 101. Reagents and conditions: a) isopropylmagnesium chloride, THF, –78 °C to rt, 65%; b) Br2, CCl4, rt, 91%; c) RMgBr·LiCl, THF, –78 °C to rt, 65% for R=OTBS, 93% for R=Cl; d) n-BuLi, 143, THF, –78 °C; then 139, –78 °C to rt; then MeOH, NBS, –78 °C to rt; e) AcCl, MeOH, rt, 57% from 143; f) MeMgI, neat, 170 °C, 83%; g) PPh3, CBr4, THF, 0 °C, to rt, 83%.

Although this route nicely demonstrated the practicability of the AGGARWAL coupling, overall step count, scalability of the oxidative coupling and the cumbersome double demethylation rendered this approach less attractive. This would be even more pronounced with respect to the conceived cannabinoids of increased polarity, as introduction of an amide in the side chain would require additional oxidation steps. Therefore, another strategy was devised that would deliver resorcinol derivatives with the terminal carbon of the sidechain in the desired oxidation state. The new synthesis commenced with chemoselective GRIGNARD addition of 145 to ketoester 146, a known compound available in one step from 2-acetylcyclohexanone by Fe(III) catalyzed retro-CLAISEN condensation, in 92% yield.147 The geminal dimethyl group was then introduced by reaction of tertiary alcohol 147 with SOCl2 and treatment of the resulting tertiary 148 alkyl chloride with AlMe3 in CH2Cl2 according to a literature procedure. In line with the original report of this method, the crude product contained ca. 10% of inseparable olefin 148, which could be easily removed by selective osmium(VIII) catalyzed double bond cleavage149

147 S. Biswas, S. Maiti, U. Jana, Eur. J. Org. Chem. 2010, 2010, 2861. 148 J. A. Hartsel, D. T. Craft, Q.-H. Chen, M. Ma, P. R. Carlier, J. Org. Chem. 2012, 77, 3127. 149 B. R. Travis, R. S. Narayan, B. Borhan, J. Am. Chem. Soc. 2002, 124, 3824.

64 Novel Chemical Probes for the Cannabinoid Receptor 2 to afford 149 in 42% yield from tertiary alcohol 147. BBr3 mediated cleavage of the two methyl ethers completed the synthesis of 150 in almost quantitative yield (Figure 8.5, A).

The same strategy was applied to the synthesis of chlorinated resorcinol 151. GRIGNARD addition of 153 to ketone 152 furnished tertiary alcohol 154 in 60% yield. Treatment with concentrated hydrochloric acid cleanly furnished the corresponding tertiary chloride, which after simple workup was reacted with AlMe3 to forge the geminal dimethyl group. Again, the olefinic side product 156 (ca. 10%) could be removed after oxidative double bond cleavage and

155 was isolated in 67% yield (3.61 g) from 154. BBr3 effected double ether cleavage to yield resorcinol 151 in 93% yield. This revised strategy greatly facilitated the synthesis of larger quantities of isomerically pure resorcinols 150 and 151.

Figure 8.5. A: Synthesis of ester functionalized resorcinol 150. Reagents and conditions: a) 145, THF, –78 °C to rt, 92%; b) SOCl2, neat, 0 °C; then AlMe3, CH2Cl2–hexane, –78 °C to rt; then OsO4 (cat.), Oxone, DMF, rt, 42% from 154; c) BBr3, CH2Cl2, –78 °C to rt, 97%. B: Synthesis of chlorinated resorcinol 151. Reagents and conditions: a) 153, THC, –78 °C to rt, 60%; b) HCl conc., rt; then AlMe3, CH2Cl2, –78 °C to rt; then OsO4 (cat.), Oxone, DMF, rt, 67% from 154; c) BBr3, CH2Cl2, –78 °C to rt, 93%.

With N-phthalimide protected allyl alcohol 132 and resorcinols in hand, the synthesis of allylic amines 128 and 157 could be achieved in four and five steps, respectively (Scheme 8.6). The

Novel CB2 Selective Cannabinoids 65 conversion of ester 158 into the primary amide required a two-step sequence consisting of selective enzymatic hydrolysis and amide formation.

Figure 8.6. A: Synthesis of 128 from N-phthalimide allylic alcohol. Reagents and conditions: a) TsOH·H2O, CH2Cl2, 65%; b) NaN3, DMF, rt; c) dimethyl sulfate, K2CO3, acetone, rt, 64% over two steps; d) N2H4·H2O, crotyl alcohol, EtOH, reflux, 73%. B: Synthesis of 157. Reagents and conditions: a) TsOH·H2O, CH2Cl2, 67%; b) dimethyl sulfate, K2CO3, acetone, rt, 91%; c) Novozyme 435, THF– H2O (pH 7), rt, 98%; d) HATU, DIPEA, NH4Cl, DMF, rt, 87%; e) N2H4·H2O, crotyl alcohol, EtOH, reflux, 92%.

In the light of the unexpected biological behavior of HU-308 and HU-433 reported by

MECHOULAM (Chapter 8.1), 151 was applied to the synthesis of ent-96 (i.e. isothiocyanate substituted HU-433) essentially following the synthetic strategy presented in Scheme 8.3.

66 Novel Chemical Probes for the Cannabinoid Receptor 2

8.5 Synthesis of AM841

To test for a ligand’s ability to bind the receptor in a covalent fashion, MAKRIYANNIS and coworkers incubated membrane preparations of cells expressing the respective receptor (CB1 or CB2) with putatively covalent probes. After several washing steps to remove unbound ligand, the so-treated preparations were used for determination of specific binding of tritiated

CP55940 (Bmax). Covalent binders would lead to significantly reduced Bmax due to the decreased number of unoccupied binding sites compared to vehicle control.101

We planned to evaluate the novel putatively covalent probes described above side by side with AM841 as positive control. Surprisingly, the synthesis of AM841 had not been disclosed150 but likely mirrors the strategy used for the preparation of closely related HU-243,151 which differs from our target only by the isothiocyanate group. Accordingly, our synthesis commenced with

FRIEDEL-CRAFTS reaction of ester functionalized resorcinol 150, which was readily available from an earlier campaign towards less hydrophobic cannabinoids (Chapter 8.4), with known pinene derivative 165 in presence of excess BF3·Et2O. Reduction using LAH then furnished triol 167 (-H: HU-210). In the originally reported synthesis of HU-243, the allylic alcohol

(HU-210) was diastereoselectively hydrogenated using WILKINSON’s catalyst in ethyl acetate. The procedure yielded the product in a diastereomeric ratio of 19:1 in favor of the desired diastereoisomer, in which the hydroxymethyl substituent is in the equatorial position of the cyclohexane ring.152 Attempts to effect stereoselective reduction of 167 in ethyl butyrate (1 atm

H2, 120 °C) or ethyl acetate (autoclave, 7 bar H2, 100 °C) resulted in the formation of the desired product in only 14% yield and as a 2:1 mixture of diastereomers. Other identified products were the corresponding aldehyde and its decarbonylated congener. To overcome the low stereoselectivity of the rhodium catalyzed hydrogenation, the strategy was adapted to first cleanly generate aldehyde 168 by iridium catalyzed redox isomerization as developed by

MAZET.153 The initial 3:1 mixture of diastereomeric aldehydes could be epimerized to the thermodynamically preferred isomer, in which all substituents are in equatorial positions, as

150 Shortly before finalization of this thesis, two crystal structures of activated CB1 in complex with agonists were published along with the synthesis of AM841, which is shown at the end of this chapter. For the original paper, see T. Hua, K. Vemuri, S. P. Nikas, R. B. Laprairie, Y. Wu, L. Qu, M. Pu, A. Korde, S. Jiang, J.-H. Ho, et al., Nature 2017, 547, 468. 151 W. A. Devane, A. Breuer, T. Sheskin, T. U. C. Jaerbe, M. S. Eisen, R. Mechoulam, J. Med. Chem. 1992, 35, 2065. 152 The exact setup was not described, but it seems likely that the authors used an autoclave (“[…] under hydrogen introduced at atmospheric pressure”). 153 H. Li, C. Mazet, Acc. Chem. Res. 2016, 49, 1232.

Novel CB2 Selective Cannabinoids 67 previously shown on similar systems.154 The primary alcohol in 168 was converted into the corresponding iodide 169 via APPEL reaction. Reduction of the aldehyde with NaBH4 in methanol yielded intermediate 170, which could be elaborated into AM841 by nucleophilic displacement and conversion of the resulting azide into an isothiocyanate group (PPh3, CS2).

Scheme 8.11. Synthesis of AM841. Reagents and conditions: a) BF3·Et2O, CH2Cl2, rt; b) LAH, THF, 0 °C, 40% over two steps; c) [Ir(COD)(Cy3P)(Py)]BArF (5 mol%), H2 for 2 min, then degas; then allyl alcohol, THF, 40 °C; then K2CO3, MeOH–THF, rt, 86-93%; d) PPh3, I2, imidazole, CH2Cl2, rt, 79%; e) NaBH4, MeOH, 0 °C, 75%; f) NaN3, DMSO, rt, 96%; g) PPh3, CS2, THF, rt, 84%.

Addendum

Shortly before finalization of this thesis, a joint work by MAKRIYANNIS, ZHAO, BOHN and LIU was published describing two crystal structures of activated human CB1 in complex with agonists AM11542 and AM841, respectively.150 Although published as covalent ligand for CB1 already in 2005,101 this recent paper for the first time describes the synthesis of AM841 as shown in Scheme 8.12.

Strikingly, the authors do not comment on a covalent bond between cysteine 6.47 and the electrophilic isothiocyanate of AM841. Structural features are described focusing on the crystal structure of CB1 in complex with AM11542 (no isothiocyanate). AM841, formerly described as a covalent binder, is now termed a washing-resistant ligand. Since the crystallographic data

154 For a similar epimerization, see C. Chu, A. Ramamurthy, A. Makriyannis, M. A. Tius, J. Org. Chem. 2003, 68, 55.

68 Novel Chemical Probes for the Cannabinoid Receptor 2 is not yet publicly available, we can only speculate at this time about the nature of AM841’s interaction with CB1. It is important to note, that if AM841 should not interact covalently with CB1, the same does not necessarily hold true for CB2, for which a covalent interaction was shown by mass spectrometric analysis of the peptide fragments following protein digestion.155

For the synthesis itself, the reported 1H NMR shifts are in very good agreement with the data obtained for our material. With respect to 13C NMR, although resolved in our spectrum, the researchers do not report a signal for C-6 (dibenzopyran numbering) likely due to overlap with solvent resonances (CDCl3). Interestingly, the researchers report the shift of the isothiocyanate carbon (130.1 ppm), which is not present in the reprint of the spectrum and which we could only detect by HMBC spectroscopy (129.5 ppm).156 All other chemical shifts match our data considering a systematic shift of 0.2 ppm due to different referencing.

Scheme 8.12. MAKRIYANNIS’ synthesis of AM841. Reagents and conditions: a) TMSOTf, CH2Cl2– MeNO2, 0 °C to rt, 71%; b) TBSCl, imidazole, DMAP, DMF, rt, 85%; c) ClPPh3CH2OMe, KMHDS, THF, 0 °C to rt, 73%; d) Cl3CCOOH, CH2Cl2, rt, 95%; e) K2CO3, EtOH, rt, 84%; f) NaBH4, EtOH, 0 °C, 98%; g) TBAF, THF, –40 °C, 96%; h) tetramethylguanidinium azide, CHCl3–MeNO2, rt, 84%; i) PPh3, CS2, THF, 76%.

8.6 Photoactivatable, CB2-Selective Cannabinoids

In order to expand the arsenal of photoaffinity probes synthesized in the context of this thesis, amines 128 and 157 were coupled to YAO’s acid 86 (Scheme 8.13, B). The EDC mediated amide formation with either amine delivered the product in 75% yield.

155 D. W. Szymanski, M. Papanastasiou, K. Melchior, N. Zvonok, R. W. Mercier, D. R. Janero, G. A. Thakur, S. Cha, B. Wu, B. Karger, et al., J. Proteome Res. 2011, 10, 4789. 156 The near-silence of isothiocyanate carbon in 13C NMR spectroscopy is a common phenomenon, see R. Glaser, R. Hillebrand, W. Wycoff, C. Camasta, K. S. Gates, J. Org. Chem. 2015, 80, 4360.

Novel CB2 Selective Cannabinoids 69

Scheme 8.13. Application of the minimalist linker concept. Synthesis of photoprobes 176 and 177. Reagent and conditions: a) 86, EDC·HCl, HOBt, DIPEA, DMF, rt, 75% for 176, 75% for 177.

8.7 Results and Discussion

The chemical probes described in the previous chapters were biologically evaluated in the laboratories of our collaborators. The primary assessment included determination of binding affinity (Ki) for both CB1 and CB2 by competitive radioligand binding assay against tritiated CP55940. Promising compounds were further evaluated for their physico-chemical properties and in cAMP assay. Bifunctional photoactivatable probes were tested for their ability to covalently label the target in CB2 overexpressing cells upon irradiation with UV light and subsequent conjugation to a fluorescent dye (Cy5-azide) via copper catalyzed alkyne-azide cycloaddition.

8.7.1 In Vitro Pharmacology

Table 8.1 lists the binding data obtained for the compounds described in this part of the present work. In addition to the primary data and to allow for better comparability of compounds tested in independent experiments, the figures in parentheses are normalized to the respective positive control set to match their published Ki values.

70 Novel Chemical Probes for the Cannabinoid Receptor 2

Table 8.1. hCB1 and hCB2 affinities and select cAMP data of novel mono- and bifunctional cannabinoids.a

hCB1 Ki = 642 nM (135 nM) hCB1 Ki = 763 nM (160 nM) hCB2 Ki = 6 nM (< 1 nM) hCB2 Ki = 5 nM (<1 nM)

hCB1 Ki > 10000 nM hCB1 Ki > 10000 nM hCB2 Ki = 85 nM (6 nM) hCB2 Ki = 38 nM (3 nM)

hCB1 Ki = 2240 nM (471 nM) hCB1 Ki > 10000 nM hCB2 Ki = 14 nM (< 1 nM) hCB2 Ki = 4 nM (< 1 nM)

hCB1 cAMP EC50 > 10000 nM (inactive) hCB1 cAMP EC50 > 10000 nM (inactive) hCB2 cAMP EC50 = 1.9 nM (96%) hCB2 cAMP EC50 = 0.7 nM (98%)

hCB1 Ki > 10000 nM hCB1 Ki > 10000 nM hCB2 Ki = 4 nM (< 1 nM) hCB2 Ki = 1470 nM (64 nM)

hCB1 Ki < 1 nM hCB1 Ki <1 nM hCB2 Ki < 1 nM hCB2 Ki < 1 nM

Novel CB2 Selective Cannabinoids 71

hCB1 Ki <1 nM hCB1 Ki = 2528 nM (5698 nM) hCB2 Ki < 1 nM hCB2 Ki = 60 nM (3 nM)

hCB1 Ki = 400 nM (902 nM) hCB1 Ki = 5022 nM (>10000 nM) hCB2 Ki = 107 nM (5 nM) hCB2 Ki = 11 nM (< 1 nM)

hCB1 cAMP EC50 = 109 nM (105%) hCB1 cAMP EC50 > 10000 nM (inactive) hCB2 cAMP EC50 = 24 nM (95%) hCB2 cAMP EC50 = 1.9 nM (96%)

hCB1 = 3875 nM (8732 nM) hCB1 > 10000 nM hCB2 = 33 nM (2 nM) hCB2 = 132 nM (7 nM) a For binding affinities, figures in parentheses are Ki values normalized to control compounds 78 79 Rimonabant (Ki hCB1 = 5.6 nM) and JWH-133 (Ki hCB2 = 3.4 nM) codetermined in the respective experiment and set to published value. For cAMP data, figures in parentheses denote the compound’s efficacy relative to CP55940.

Discussion

Although exhibiting high selectivity for CB2 with affinities in the one-digit nanomolar range, the analogs 113, 114 and 96 were found to also interact with CB1. This is somewhat surprising since the compounds differ from HU-308 (reported with complete selectivity) only in one additional substituent at the end of the aliphatic side chain, a position that was shown to tolerate a wide range of substituents. Nevertheless, this data supports the concept of a CB2 selective, putatively covalent hybrid borrowing features from both HU-308 and AM841. When the allylic alcohol was propargylated to furnish bifunctional probes 115 (a potential photoprobe) and 116 (a putative covalent binder), high affinity for CB2 was retained along with complete selectivity over CB1 in agreement with data of MARTIN-COUCE and colleagues,106 who described a CB2 selective analog elongated via the same exit vector, but without the functionality in the aliphatic side chain. Gratifyingly, the longer linker used to furnish fluorescent probes 127, 176 and 130

72 Novel Chemical Probes for the Cannabinoid Receptor 2 was also well tolerated as the compounds exhibited high affinity towards CB2 along with no detectable interaction with CB1. A very interesting finding was the large increase in binding affinity by a factor of ~300 (> 60 corrected for control compounds) when the ester linkage was exchanged for a secondary amide in 176 and 130. As mentioned in Chapter 8.3, amide substituents at this position have been neglected in almost all SAR studies to date in the fields of classical and non-classical cannabinoids. To the best of our knowledge, the combination of extremely high CB2 affinity of fluorescent probes 176 and 130 (both 4 nM, 0.2 nM normalized to control) along with their complete selectivity over CB1 (> 10 M) is unmet in the field, rendering this chemotype the most promising basis to date for the development of CB2 specific probes that require linkage to relatively bulky functionalities such as fluorophores. It should be emphasized that identification of a suitable exit vector for further functionalization of any given bioactive compound without losing substantial binding affinity is not a trivial task.157

As expected, AM841 behaved as non-selective binder of both CB1 and CB2 with very high affinity (< 1 nM). This characteristic is shared by its close azide analog 171 and interestingly also by the respective primary alcohol (168-red). A database search for cannabinoids with a hydroxyl terminated 1,1-dimethylheptyl sidechain resulted in zero hits. Therefore, this novel sidechain opens up new possibilities for the synthesis of potent ligands, especially with respect to the development of less lipophilic cannabinoids.

Photoprobe 176 (terminal azide) exhibited a significantly increased (ca. 10-fold, >5-fold corrected for controls) binding preference for CB2 compared to 177 (terminal primary amide), suggesting that introduction of polar residues at the terminus of the aliphatic sidechain leads to decreased CB2 affinity along with reduced selectivity of HU-308 derivatives over CB1, although more analogs are needed to corroborate this hypothesis.

Lastly, three compounds of the enantiomeric series of HU-308 (i.e. HU-433 derivatives) were evaluated. In agreement with MECHOULAM’s report,105 the binding binding affinities of ent- 113-Cl (60 nM), azide ent-114 (33 nM) and isothiocyanate ent-96 (132 nM) derivatives were lower compared to the bromide 113 (6 nM), azide 114 (5 nM) and isothiocyanate 96 (14 nM) derived from HU-308. A preliminary washout/radioligand saturation assay, that should indicate a diminished Bmax after incubation with a covalent binder and extensive washing, revealed no

157 For an unsuccessful attempt starting from a high-affinity compound, see A. S. Yates, S. W. Doughty, D. A. Kendall, B. Kellam, Bioorg. Med. Chem. Lett. 2005, 15, 3758.

Novel CB2 Selective Cannabinoids 73 covalent interaction of 96 (which was the motivation to synthesize its enantiomer, experiments pending).

8.7.2 (Photo)-Affinity Labeling158

As described for triazolopyrimidine based photoactivatable and electrophilic probes, the cannabinoid derived tool compounds described above were evaluated for their ability to label CB2 either upon irradiation or due to electrophilic isothiocyanate group. Figure 8.7 shows the affinity labeling experiment with 60 (a repetition at 10 M) (lanes 4 and 5) and electrophilic fluoroprobe 130 (lanes 6 to 9). Despite its exceptional affinity for CB2 in the radioligand binding assay, the latter did not exhibit the expected band for a protein of ca. 40 kDa. The repetition experiment with 60 again revealed clean off-target labeling of an unknown protein of much lower mass.

The results for positive control LEI121, 115 (alkyne, azide) and 116 (alkyne, isothiocyanate) in presence of various competitors (Figure 8.8). The azide was irradiated with 300 nm light, instead of the otherwise applied 350 nm, but did not lead to any selective binding of CB2. Isothiocyanate analog 116 reacted unselectively with a large number of proteins (possibly also CB2) but not in a useful manner. This finding is somewhat unexpected in light of the results of compound 130, which shares the same isothiocyanate in the dimethylheptyl sidechain (and actually a higher binding affinity) but could not be used to visualize any covalently modified protein.

Next, minimalist linker derived non-classical cannabinoids 176 (Figure 8.9) and 177 (Figure 8.10) were evaluated. Gratifyingly, in these experiments 176 emerged as selective photoaffinity label of CB2. The labeling efficiency was concentration dependent. When applied at 100 nM concentration, only little cross linking could be registered, while treatment of the membranes at 2 M and 10 M concentration led to a significantly increased fluorescence intensity of the corresponding CB2 band, which could be outcompeted by the presence of excess CP55940. Primary amide analog 177 did not show any CB2 labeling.

158 Experimental data and figures in this chapter were provided by M. Soethoudt, Leiden University.

74 Novel Chemical Probes for the Cannabinoid Receptor 2

LEI121 60 130 130 Probe - (2 µM) (10 µM) (0.04 µM) (10 µM) CP55940 - - + - + - + - + (20 µM) lane 1 2 3 4 5 6 7 8 9

55 kDa

35 kDa

Figure 8.7. Fluorophore-conjugated proteins from CB2 overexpressing CHO membranes after photoaffinity labeling using LEI121 (lanes 2 and 3), bifunctional fluorosulfonyl 60 (10 M) (lanes 4 and 5) and electrophilic fluoroprobe 130 (0.04 and 10 M) (lanes 6-9) in the presence or absence of CP55940 as competitor. The proteins were resolved by electrophoresis on polyacrylamide gel (SDS- PAGE) and visualized by fluorescence imaging.

LEI121 115 116 Probe (2 µM) - + + + + + + + + + + Competitor - - 1 2 3 - 1 2 - 1 2 (20 µM) UV 350 nm 300 nm No UV lane 1 2 3 4 5 6 7 8 9 10 11

55 kDa

35 kDa

Figure 8.8. Fluorophore-conjugated proteins from CB2 overexpressing CHO membranes after photoaffinity labeling at 300 nm (azide activation) or affinity labeling (isothiocyanate) using LEI121 (2 M, lanes 2-5, activation at 350 nm), bifunctional alkylazide 115 (2 M, lanes 6-8) and bifunctional isothiocyanate 116 (2 M, lanes 9-11) in the presence of competitors 1 (CP55940), 2 (HU-308) or 3 (a triazolopyrimidine). The proteins were resolved by electrophoresis on polyacrylamide gel (SDS-PAGE) and visualized by fluorescence imaging.

Novel CB2 Selective Cannabinoids 75

LEI121 176 176 176 Probe - (2 µM) (0.1 µM) (2 µM) (10 µM) CP55940 - - + - + - + - + (20 µM) lane 1 2 3 4 5 6 7 8 9 10 11 12 13

55 kDa

35 kDa

Figure 8.9. Cy5-conjugated proteins from CB2 overexpressing CHO membranes after photoaffinity labeling using LEI121 (lanes 2 and 3) and diazirine 176 at various concentrations in the presence (+) or absence (-) of CP55940 as competitor (lanes 8-13). Lanes 4-7 show results of other compounds. The proteins were resolved by electrophoresis on polyacrylamide gel (SDS-PAGE) and visualized by fluorescence imaging. LEI121 177 177 177 Probe - (2 µM) (0.1 µM) (2 µM) (10 µM) CP55940 - - + - + - + - + (20 µM) 1 2 3 4 5 6 7 8 9

55 kDa

35 kDa

Figure 8.10. Cy5-conjugated proteins from CB2 overexpressing CHO membranes after photoaffinity labeling using LEI121 (lanes 2 and 3) and diazirine 177 at various concentrations in the presence (+) or absence (-) of CP55940 as competitor (lanes 4-9). The proteins were resolved by electrophoresis on polyacrylamide gel (SDS-PAGE) and visualized by fluorescence imaging.

76 Novel Chemical Probes for the Cannabinoid Receptor 2

The specificity of 176 was further probed in a number of control experiments (Figure 8.11) including: no CHO membrane (lane 1), heat denatured proteins (lane 2), probe omitted (lane 3), no UV irradiation (lane 4), no click mix (lane 5), no competitor (lane 6), various competitors (20 M) in the order of CP55940, SR144538, AM630, HU-308, HU-910 and JWH-133 (lanes 7-12). Interestingly, the photo probe could be outcompeted only by CP55940 and to some extent by SR144538 under the chosen conditions, which possibly reflects a relative ranking of binding affinities. The presence of CB2 antagonist AM630 led to a more pronounced off-target labeling of an unknown protein.

Quantification of fluorescence intensity of the control experiments gave the results summarized in Figure 8.12. Treatment of wild-type CHO membranes (not expressing CB2) with the photo probe did not result in any specific binding. The same was observed when proteins were heat denatured before use in the procedure, as well as when one crucial component of the experimental procedure (addition of photo probe, irradiation, and addition of click-mix) was omitted. The addition of 4-fold excess CP55940 led to almost quantitative depletion of CB2 labeling, while a decrease of roughly 50% was observed in the presence of SR144538. The other CB2 ligands (AM630, HU-308, HU-910 and JWH-133) when added in 4-fold excess did not lead to a significant change in labeling efficiency, suggesting them so be weaker CB2 binders than the photo probe itself (11 nM, <1 nM corrected for control JWH-133 in the same binding assay).

Novel CB2 Selective Cannabinoids 77

CB2 - + membranes Active + - + protein

176 (5 µM) + - +

UV + - + Click mix + - + Competitor - 4 1 2 6 5 7 (20 µM) lane 1 2 3 4 5 6 7 8 9 10 11 12

55 kDa

35 kDa

Figure 8.11. Cy5-conjugated proteins from CB2 overexpressing CHO membranes after photoaffinity labeling using diazirine 176 (5 M). Control experiments from left to right: wild-type CHO membranes (lane 1), heat denatured proteins (lane 2), no photoaffinity probe (lane 3), no UV irradiation (lane 4), no click mix (lane 5), no competitor (lane 6), various competitors (20 M) (lanes 7-12). The proteins were resolved by electrophoresis on polyacrylamide gel (SDS-PAGE) and visualized by fluorescence imaging.

Figure 8.12. Relative fluorescence of CB2 bands in lanes 1–12 (Figure 8.11). MW5-319 is 176.

78 Novel Chemical Probes for the Cannabinoid Receptor 2

8.8 Conclusion and Outlook

In this chapter, we described the synthesis and in vitro pharmacological characterization of functionalized cannabinoids based on MECHOULAM’s compound HU-308 and its enantiomer HU-433 combined with an electrophilic isothiocyanate as found in AM841. The novel compounds are highly CB2 selective with dissociation constants (hCB2 Ki) down to the single- digit nanomolar range as assessed by radioligand binding assay. Interestingly, we found that elongation via the northern aliphatic hydroxyl group to yield ether or ester derivatives reinforced the selectivity over CB1 as compared to compounds with a free primary alcohol. An additional important finding was that exchange of the ester linkage in 127 for the corresponding amide resulted in significantly increased CB2 binding affinity, while complete selectivity over CB1 was retained. Borrowing from literature precedence, this exit vector was used for the attachment of a fluorophore functionalized linker via amide bond formation, resulting in the discovery of 130, the most selective and highest affinity CB2 fluorescent probe to date. We believe that the identification of a linker long enough to reach out of the binding pocket into the extracellular space together with the high binding affinity of the amide linked core will facilitate the development of highly useful tool compounds for CB2 research.

Since the high lipophilicity of cannabinoid derived probes can hamper their usefulness in cellular applications due to non-specific binding, a route to analogs of increased polarity has been developed. The new strategy not only allows for scalable and modular synthetic access to distinctly substituted resorcinols, but also circumvents a problematic step of an earlier strategy that yielded a mixture of constitutionally isomeric alkyl bromides.

In addition, we present an efficient and flexible strategy towards AM841, a prominent tool compound in CBR research of which the synthesis had not been disclosed until recently. Key step of the present route is an iridium catalyzed redox isomerization of an allylic alcohol to yield the corresponding aldehyde, which is reduced after base mediated epimerization to afford the equatorial hydroxymethyl substituent.

Coupling of amine 128 to a minimalist linker featuring both an alkyne and a photoactivatable diazirine afforded photoaffinity label 176. This compound was shown to effectively label the target in CB2 overexpressing cells upon irradiation with light (350 nm) as shown by subsequent attachment of an azide functionalized fluorophore via copper catalyzed cycloaddition. Importantly, the specificity of the labeling event was showcased by control experiments conducted in the presence of potent CB2 modulators, showing decreased labeling in the presence of CP55940 and SR144538 as quantified by fluorescence intensity.

Novel CB2 Selective Cannabinoids 79

In search of less lipophilic cannabinoids, amide analog 177 was prepared by the coupling of amine 157 to the minimalist linker construct. Interestingly, although the compound exhibited a binding affinity towards hCB2 of 100 nM (likely better considering the positive control JWH- 133 in the same radioligand binding assay) it failed to label the receptor in the respective photoaffinity labeling experiment. This finding might result from the structural change (azide to amide) possibly leading to a different binding pose within the receptor being detrimental for cross linking. A rather unlikely explanation of the counterintuitive result relies on the azide as reactive group. Although more common for aryl azides, a THC derivative bearing a terminal azide in the pentyl sidechain was claimed to undergo cross-linking to CB1 upon irradiation with UV light without further discussion of the resulting ligand-receptor interaction, which is described as wash-resistant.159 The authors of a contribution to a monography on organic azides state that «[,,,] light and heat induced decomposition of alkyl azide does not produce alkyl nitrenes, which can be intercepted in respectable yields with a bimolecular trap.»160 Therefore, and as alternative to an unlikely nitrene insertion, an imine as initial product of photolysis (or the respective aldehyde) might form a reasonably stable product with amino acids of the receptor. With cross-linking to Cy5 and subsequent gel electrophoresis, a covalent nature of the interaction of ligand 176 with CB2 upon photoactivation could be unequivocally demonstrated. The failure of the very close analog 177 to undergo photoaffinity labeling might result from a different binding pose (compared to 176) exhibiting a deleterious effect on cross linking efficiency.

159 S. H. Burstein, C. A. Audette, A. Charalambous, S. A. Doyle, Y. Guo, S. A. Hunter, A. Makriyannis, Biochem. Biophys. Res. Com. 1991, 176, 492. 160 N. Gritsan, M. Platz, in Organic Azides (Eds.: S. Bräse, K. Banert), John Wiley & Sons, Ltd, 2009, pp. 311 ff.

III. Optical Control of Cannabinoid Receptor 1

Background 83

9 Background

This part of the present thesis describes the enantioselective synthesis of a novel building block 3-bromo tetrahydrocannabinol (3-Br-THC) and its elaboration into seven derivatives featuring a photoswitchable diazobenzene moiety.161 Key step in the synthetic sequence is a dual catalytic α-allylation of an aldehyde using a chiral iridium complex in combination with a chiral amine. The azo-tetrahydrocannabinol derivatives (azo-THC’s) were evaluated for their responsiveness to light (E/Z isomerization of the diazene) and further for their ability to modulate CB1 in a light-dependent fashion as assessed by electrophysiology and luminescence based cyclic adenosine monophosphate (cAMP) assay.

As detailed in the introductory part of the present work, the cannabinoid receptors are involved in a vast number of biological processes. CB1 is known to engage with the naturally occurring phytocannabinoids such as 9-THC (a phytocannabinoid produced by the plant Cannabis sativa), the arachidonic acid derivatives anandamide and 2-AG (lipids known as endocannabinoids), and a large number of synthetic ligands originating from drug discovery efforts. This testifies the importance and promise of CB1 as target for the treatment of disease. Despite its fundamental significance for many mammalian body functions, the exact role of CB1 in specific tissues is poorly understood. Therefore, tools allowing for precise spatiotemporal control of CB1 activation and inactivation are highly desirable and might help to successfully decipher the role of CB1 in physiological processes, especially in the context of human disease.

The use of photoresponsive ligands to study cannabinoid receptors has been largely neglected. This is surprising, since such an approach would offer the opportunity to gain insight into receptor signaling upon precisely controlled ligand release. In this context, one report on a photopharmacological approach to control CB1 function stands out. HEINBOCKEL et al. have prepared a derivative of anandamide (Figure 9.1), in which the amide nitrogen is linked to a photolabile protecting group that is further conjugated to a polyethylene glycol unit to enhance water solubility.162 The caged endocannabinoid 178 neither interacts with CB1, nor is it processed by the enzymatic machinery of the endocannabinoid system. Removal of the unnatural N-subsituent by photolysis cleanly released anandamide. The rapid uncaging process was estimated to reach quantitative conversion after less than 100 s irradiation time (half-life

161 The work described in this chapter was conducted in collaboration with Michael A. Schafroth, a fellow PhD student in the Carreira group, and Dr. James A. Frank (LMU Munich). 162 T. Heinbockel, D. H. Brager, C. G. Reich, J. Zhao, S. Muralidharan, B. E. Alger, J. P. Y. Kao, J. Neurosci. 2005, 25, 9449.

84 t1/2 ≈ 19 s). This valuable tool was then successfully applied in a study of endocannabinoid signaling dynamics.

Figure 9.1. Release of anandamide by photolysis of the caged derivative 178.

Since its discovery, a number of racemic, diastereoselective and enantioselective syntheses of

9-THC have appeared in the literature.163 In 2014, SCHAFROTH and CARREIRA published an efficient approach to all possible stereoisomers of 9-tetrahydrocannabinol.164 In this work, the strategic use of a dual-catalytic, diastereodivergent α-allylation of a linear aldehyde enabled the synthesis of any desired stereoisomer by choice of the respective chiral catalysts (Scheme 9.1).

Shortly after publication of this manuscript, the TRAUNER and CARREIRA groups joined forces to develop photochromic ligands enabling optical control of CB1.

Scheme 9.1. Iridium catalyzed -allylation of an aldehyde as key step of a stereodivergent approach to all possible isomers of THC as published by SCHAFROTH et al.

10 Photochromic Tetrahydrocannabinol Derivatives

Over the last decades, a large number of tetrahydrocannabinol derivatives were prepared and tested for cannabinoid activity (in vivo and later in vitro). The structure activity relationship of

163 For a recent review, see M. A. Schafroth, E. M. Carreira, in Phytocannabinoids: Unraveling the Complex Chemistry and Pharmacology of Cannabis Sativa (Eds.: A.D. Kinghorn, H. Falk, S. Gibbons, J. Kobayashi), Springer International Publishing, Cham, 2017, pp. 37. 164 M. A. Schafroth, G. Zuccarello, S. Krautwald, D. Sarlah, E. M. Carreira, Angew. Chem. Int. Ed. 2014, 53, 13898.

Photochromic Tetrahydrocannabinol Derivatives 85

CB1 agonists today is quite well established.165,166 The substitution of the C-3 pentyl chain by longer or bulkier alkyl groups was shown to be tolerated in terms of their binding affinity (Scheme 10.1). For example, substitution at C-3 with a 1,1-dimethylheptyl sidechain led to a 9-THC analog 183 with pronounced potency in cAMP assay.167 The incorporation of adamantyl or naphtyl groups were also well-tolerated (184, 185), leading to analogs of slightly

9 168,169 higher affinity compared to  -THC.

Scheme 10.1. Representative THC-derived cannabinoids with differing C-3 residues.

The precedence of these cannabinoids with bulky C-3-substituents strongly suggested that incorporation of a sterically demanding photoswitch may be tolerated by the receptor. We chose azobenzene as the photochromic component, both for its ease of synthesis and its optical properties, which can be tuned by variation of the substitution pattern to effect red-shifting of the absorption wavelength.170,171,172

Seven final compounds incorporating an azobenzene motif were contemplated. In azo-THC-1, the diazo-group is directly attached to the aromatic THC core. azo-THC-2–7 possess a phenyl spacer with diazo-groups in the ortho-, meta- and para-positions, respectively, and were prepared both as unsubstituted (azo-THC-2–4) and p-diethylamino (azo-THC-5–7) analogs. The latter three compounds were synthesized to tune the probes´ optical properties towards

165 D. R. Compton, K. C. Rice, B. R. De Costa, R. K. Razdan, L. S. Melvin, M. R. Johnson, B. R. Martin, J. Pharmacol. Exp. Ther. 1993, 265, 218. 166 E. W. Bow, J. M. Rimoldi, Perspect. Medicin. Chem. 2016, 2016, 17. 167 M.-H. Rhee, Z. Vogel, J. Barg, M. Bayewitch, R. Levy, L. Hanuš, A. Breuer, R. Mechoulam, J. Med. Chem. 1997, 40, 3228. 168 D. P. Papahatjis, V. R. Nahmias, T. Andreou, P. Fan, A. Makriyannis, Bioorg. Med. Chem. Lett. 2006, 16, 1616. 169 M. Rinaldi-Carmona, F. Barth, M. Héaulme, D. Shire, B. Calandra, C. Congy, S. Martinez, J. Maruani, G. Néliat, D. Caput, et al., FEBS Letters 1994, 350, 240. 170 W. A. Velema, W. Szymanski, B. L. Feringa, J. Am. Chem. Soc. 2014, 136, 2178. 171 A. A. Beharry, G. A. Woolley, Chem. Soc. Rev. 2011, 40, 4422. 172 S. Samanta, A. A. Beharry, O. Sadovski, T. M. McCormick, A. Babalhavaeji, V. Tropepe, G. A. Woolley, J. Am. Chem. Soc. 2013, 135, 9777.

86 longer irradiation wavelengths, since substitution with electron donating groups on the azobenzene leads to a red shift of the absorption maximum.

The synthetic strategy towards the contemplated azo-THCs was based on one central building block, namely 3-Br-THC, which would be transformed into the target structures by cross coupling chemistry (Scheme 10.2).

Scheme 10.2. 3-Br-THC as central building block in the synthesis of azo-THCs.

In addition, 3-Br-THC was considered a versatile intermediate for the synthesis of THC-derived libraries, various natural products of the machaeriol/machaeridiol class as well as perrottetinene (Figure 10.1). The latter stands out for its cis configuration of the two adjacent stereocenters, which should be accessible using the described dual catalytic approach as was shown in the case of THC.

Figure 10.1. Natural products potentially accessible from 3-Br-THC.

Photochromic Tetrahydrocannabinol Derivatives 87

10.1 Synthesis of 3-Br-THC

The synthesis of 3-Br-THC (Scheme 10.3) commenced with Ir-catalyzed allylation of aldehyde 187 using (S)-PN-olefin ligand 181 and (S)-Jørgensen-Hayashi catalyst 180 to give 188 in 52% yield (dr > 20:1, ee > 99%). Ring closing metathesis (Grubbs II catalyst) and subsequent oxidative esterification (iodine, KOH, MeOH) followed by treatment with excess methyl magnesium iodide furnished tertiary alcohol 191 in 76% yield from 188. The last ring was closed via intramolecular SNAr with KHMDS and afforded chromane 192 in 90% yield. Final removal of the methyl group with excess sodium thioethanolate in DMF yielded 3-Br-THC.

Scheme 10.3. Synthesis of 3-Br-THC. Reagent and conditions. a) [Ir(COD)Cl]2 (3 mol%), (S)-PN- olefin 181 (12 mol%), (S)-Jørgensen–Hayashi catalyst 180 (15 mol%), Zn(OTf)2 (5 mol%), 5- methylhex-5-enal (187), DCE, rt, 52%; b) HG-II (3 mol%), CH2Cl2, rt, 91%. (c) I2, KOH, MeOH, 0 °C, 88%; d) MeMgI, Et2O, 0 °C, 95%; e) KHMDS, THF–PhMe, 65 °C, 90%; f) NaSEt, DMF, 140 °C, 86%.

The 1H NMR spectra of the deprotected material suggested the presence of a minor co-product, which exhibited a characteristic resonance signal (=3.51 ppm) shifted downfield relative to the benzylic proton of the desired product (=3.15 ppm). The comparison of these signals with those of the benzylic protons of cis- and trans-THC,164 which resonate at =3.56 ppm and=3.20 ppm, respectively, suggested that epimerization occurred to a small extent during the demethylation reaction (dr > 10:1).

During the early stages of this project, another synthesis of nominal 3-Br-THC (Scheme 10.4) was reported by DETHE et al. that relied on a BF3·Et2O-mediated FRIEDEL-CRAFTS reaction of 1-bromo-3,5-dihydroxybenzene with monoterpenoid derivative 193.173 However, we noted that NMR spectroscopic data of our material prepared via the dual catalysis approach differed

173 D. H. Dethe, R. D. Erande, S. Mahapatra, S. Das, V. K. B, Chem. Commun. 2015, 51, 2871.

88 significantly from DETHE´s published data. Importantly, we were able to confirm our structure by converting 3-Br-THC into known 9-tetrahydrocannabinol methyl ether (196).174 The NMR data of our material perfectly matched KOBAYASHI’s reported values, confirming the structural identity of 3-Br-THC prepared by the dual catalysis approach. FRIEDEL-CRAFTS reactions of resorcinol derivatives en route to THC analogs are known to yield regioisomeric products with respect to aromatic ring substitution.175 Therefore, we speculate that DETHE and colleagues isolated 1-Br-THC.

Scheme 10.4. A: DETHE´s reported synthesis of 9-3-Br-THC and 8-THC. B: Conversion of 3-Br-THC into known intermediate 196.

Table 10.1. NMR data of 196 compared with KOBAYASHI’s report.a

1H NMR 13C NMR Literature (300 MHz) Control 196 (400 MHz) Literature Control 196 (75 MHz) (101 MHz) 0.89 (t, J = 7 Hz, 3 H) 0.90 (s, J = 7 Hz, 3H) 14.2 14.2 1.08 (s, 3 H) 1.08 (s, 3H) 19.3 19.3 1.41 (s, 3 H) 1.41 (s, 3H) 22.7 22.7 1.67 (s, 3H) 1.67 (s, 3H) 23.6 23.6 1.2–1.8 (m, 7 H) 1.27 – 1.37 (m, 4H) 25.3 25.3 1.37 – 1.40 (m, 1H) 27.7 27.7 1.56 – 1.64 (m, 2H) 31.0 31.0 1.84–1.98 (m, 2 H) 1.63 – 1.72 (m, 1H) 31.4 31.4

174 A. D. William, Y. Kobayashi, J. Org. Chem. 2002, 67, 8771. 175 R. K. Razdan, H. C. Dalzell, G. R. Handrick, J. Am. Chem. Soc. 1974, 96, 5860.

Photochromic Tetrahydrocannabinol Derivatives 89

1.86 – 1.95 (m, 1H) 31.8 31.8 2.09–2.20 (m, 2 H) 2.10 – 2.19 (m, 2H) 34.1 34.1 2.50 (t, J = 8 Hz, 2 H) 2.50 (dd, J = 8.8, 6.8 Hz, 36.2 36.2 2H) 3.17 (d, J = 11 Hz, 1 3.17 (d, J = 11.0 Hz, 1H) 46.0 46.1 H), 3.84 (s, 3 H) 3.84 (br s, 3H) 55.3 55.3 6.23 (br s, 1 H) 6.23 (br s, 1H) 77.3 77.1a 6.27 (br s, 1 H) 6.26 (br s, 1H) 103.0 103.1 6.31 (br s, 1 H) 6.31 (br s, 1H) 110.2 110.4 110.4 110.5 124.8 125.0 133.4 133.6 142.5 142.7 154.3 154.5 158.3 158.5 a) Signal overlaps in 13C spectrum with residual solvent peak and was therefore assigned by 2D spectroscopy (HMBC).

10.2 Synthesis of Photoswitchable 9- Tetrahydrocannabinol Derivatives

Having secured synthetic access to 3-Br-THC, we then focused on the synthesis of azo-THCs as outlined in Scheme 10.5. To this end, the phenol was first TBS protected. The resulting intermediate was subjected to BUCHWALD-HARTWIG coupling with N-Boc-hydrazine to afford 197 in 66% over both steps. After Boc-removal, the resulting hydrazine was oxidized by ambient air in bicarbonate buffered methanol. Desilylation then afforded azo-THC-1 in 48% yield.

Scheme 10.5. Synthesis of azo-THC-1. Reagents and conditions: a) TBSOTf, 2,6-lutidine, rt, 74%; b) Pd(OAc)2 (10 mol%), P(tBu)3·HBF4 (10 mol%), Cs2CO3, PhMe, 110 °C, 89%; c) 2,6-lutidine, TMSOTf, CH2Cl2, 0 °C; NaHCO3, air, MeOH, rt, 52%; d) TBAF, THF, 0 °C, 93%.

Next, we focused our attention on the preparation of the remaining azo-THCs by direct cross- coupling of the unprotected 3-Br-THC with boronic acid derivatives. The synthesis of meta- and para-substituted trifluoroborates was accomplished following a literature procedure via the

90 corresponding boronic acid pinacol esters (Scheme 10.6).176 201 and 207 were prepared via

MILLS condensation of commercially available 199 and 205 with nitrosobenzene and subsequent conversion into the potassium trifluoroborate salts. The respective para- diethylamino substituted building blocks were prepared by diazocoupling of 4-iodoaniline 202 and 3-bromoaniline 208 with diethylaniline using a protocol developed by WANNER.177 In the case of 204, the intermediate diazobenzene was metalated with n-BuLi under cryogenic conditions, trapped with trimethyl borate and directly converted into the trifluoroborate. For 178 211, MIYAURA borylation of aryl bromide 209 followed by treatment with KHF2 afforded the desired building block. Strikingly, an attempt to generate the ortho-substituted building blocks by the same sequence failed. First, the formation of the ortho-boronic ester proceeded very sluggishly. And second, subsequent treatment of the boronic acid with KHF2 did not result in any conversion of the starting material, even at elevated temperatures (70 °C). Since the lithiation of ortho-iodo substituted azobenzenes was precendented,179 we focused our attention on the respective triolborates (213, 216). The compounds were prepared by halogen metal exchange, trapping of the resulting organolithium with trimethyl borate and subsequent treatment with the respective triol.180 After isolation by filtration, the triolborates could be used without further purification. It was later recognized that the ability of triolborates to undergo cross coupling reactions without added base was crucial for the synthesis of azo-THC-4 and azo-THC-7.

176 J. H. Harvey, B. K. Butler, D. Trauner, Tetrahedron Lett. 2007, 48, 1661. 177 T. A. Lutz, P. Spanner, K. T. Wanner, Tetrahedron 2016, 72, 1579. 178 T. Ishiyama, M. Murata, N. Miyaura, J. Org. Chem. 1995, 60, 7508. 179 N. Kano, J. Yoshino, T. Kawashima, Org. Lett. 2005, 7, 3909. 180 Y. Yamamoto, M. Takizawa, X.-Q. Yu, N. Miyaura, Angew. Chem. Int. Ed. 2008, 47, 928.

Photochromic Tetrahydrocannabinol Derivatives 91

Scheme 10.6. Synthesis of boronic acid building blocks for SUZUKI cross coupling en route to azo- THCs. Reagents and conditions: a) nitrosobenzene, AcOH, 90 °C, 43%; b) KHF2, MeCN–H2O, rt, 86%; c) HCl, NaNO2; then sulfamic acid; then NaOAc, diethylaniline, EtOH–H2O, 0 °C, 88%; d) n-BuLi, Et2O, –110 °C; then B(OMe)3; then KHF2, 74%; e) nitrosobenzene, AcOH, 90 °C, 33%; f) KHF2, MeCN–H2O, rt, 97%; g) HCl, NaNO2; then sulfamic acid; then NaOAc, diethylaniline, EtOH–H2O, 0 °C, 79%; h) PdCl2(dppf) (5 mol%), KOAc, B2pin2, DMSO, 80 °C, 85%; i) KHF2, MeCN–H2O, rt, 92%; j) n-BuLi, Et2O, –100 °C; then B(OMe)3 ,–100 °C to –20 °C; then 1,1,1-tris(hydroxymethyl)ethane;.k) HCl, NaNO2; then sulfamic acid; then NaOAc, diethylaniline, EtOH–H2O, 0 °C, 87%; l) n-BuLi, Et2O, –100 °C; then B(OMe)3 ,–100 °C to –20 °C; then 1,1,1-tris(hydroxymethyl)ethane. azo-THC-2–7 were synthesized as shown in Scheme 10.7. In each case, 3-Br-THC was subjected to palladium-catalyzed SUZUKI cross coupling with the respective boronic acid derivative. Conveniently, application of TRAUNER’s protocol for the reaction with potassium trifluoroborates (PdCl2(dppf), Cs2CO3, MeOH, 65 °C) furnished the corresponding targets in 53–85% yield. Interestingly, the reaction of ortho-substituted triolborates using the same reaction conditions led to substantial decomposition, likely due to base-mediated decomposition via an electrocyclization pathway. A database search revealed that the desired structural motif (diaryldiazene with a free 3-hydroxyphenyl substituent in ortho-position) had

92 virtually no precedence, as opposed to the respective ether derivatives. Gratifyingly, when the cross coupling was carried out without added base in a mixture of DMF and water (65 °C), both products azo-THC-4 and azo-THC-7 could be isolated in acceptable yield (38% and 30%, respectively). This showcases the usefulness of triolborates for the synthesis of sensitive compounds.

Scheme 10.7. Synthesis of azo-THC-2–7 by SUZUKI cross coupling of 3-Br-THC with various boronic acid derivatives. Reagents and conditions: a) 213/216, PdCl2(dppf) (5 mol%), DMF–H2O, 65 °C, 38% for azo-THC-4, 30% for azo-THC-7; b) 207/211, PdCl2(dppf), Cs2CO3, MeOH, 65 °C, 70% for azo- THC-3, 85% for azo-THC-6; c) 201/204, PdCl2(dppf), Cs2CO3, MeOH, 65 °C, 53% for azo-THC-2, 74% for azo-THC-5.

Photochromic Tetrahydrocannabinol Derivatives 93

10.3 Compound Evaluation and Discussion181

The novel azo-THCs were evaluated for their ability to modulate CB1 in a light-dependent fashion by two distinct readouts. First, the use of whole-cell electrophysiology on CB1- expressing AtT-20 cells transfected with G-protein coupled inwardly rectifying potassium

+ 182 channels allowed the recording of K currents upon CB1 activation coupled via G subunits. Second, by performing a bioluminescence-based cAMP assay of the individual E and (enriched) Z isomers.

Whole-cell Electrophysiology

AtT-20 cells stably expressing CB1 were transiently transfected with genes encoding for G- protein coupled inwardly rectifying potassium channels (GIRK1/2).183,184 GIRK channels can

185 couple to CB1 via G subunits of heterotrimeric G proteins. Upon activation, GIRK channels become permeable for potassium ions, which then diffuse along their concentration gradient from the intracellular space to the outside, resulting in hyperpolarization of the cell. This process can be followed by whole-cell patch clamp electrophysiology, which measures currents crossing the plasma membrane.186 To test the viability of the assay, the effect of CP55940 (a well-known full agonist of CB1) was first evaluated as positive control. Indeed, when CP55940 was applied at 100 nM concentration, an inward current was registered while the membrane potential was kept at –60 mV (Figure 10.2).

181 The biological evaluation (whole-cell patch clamp electrophysiology and cAMP assays) of azo-THCs described in this section was carried out by Dr. James Frank, LMU Munich. 182 Q. Lei, M. B. Jones, E. M. Talley, A. D. Schrier, W. E. McIntire, J. C. Garrison, D. A. Bayliss, PNAS 2000, 97, 9771. 183 The cells were kindly provided by Prof. Ken Mackie, Indiana University. 184 K. Mackie, Y. Lai, R. Westenbroek, R. Mitchell, J. Neurosci. 1995, 15, 6552. 185 S. D. McAllister, G. Griffin, L. S. Satin, M. E. Abood, J. Pharmacol. Exp. Ther. 1999, 291, 618. 186 E. A. Johnson, S. Oldfield, E. Braksator, A. Gonzalez-Cuello, D. Couch, K. J. Hall, S. J. Mundell, C. P. Bailey, E. Kelly, G. Henderson, Mol. Pharmacol. 2006, 70, 676.

A B

Figure 10.2. Validation of the electrophysiological setup. Application of CP55940 (100 nM) led to activation of GIRK channels. The cellular membrane potential was held at –60 mV. A: Addition of CP55940 leads to hyperpolarization. B: Inward-current is increased in the presence of CP55940 in voltage ramps and C: IV steps (500 ms) between –140 and –20 mV (20 mV increments) showed increased currents after CP55940 application.

When applied at a concentration of 5 M, azo-THC-1 led to a slowly increasing inward current (Figure 10.3, A), which could not be modulated by cycling between the cis and trans isomers by irradiation with 360 nm and 450 nm light, respectively. In a voltage-ramp experiment (–140 to –20 mV over 2 s), the presence of azo-THC-1 resulted in increased currents, indicating activation of CB1 and coupling to GIRK channels (Figure 10.3, B). As already expected from Figure 10.3 A, IV-steps between –140 mV and –20 mV (20 mV increments) looked very similarly for both the cis and the trans isomer of azo-THC-1. In conclusion, azo-THC-1 did not affect CB1 activity in a light-dependent manner.

Photochromic Tetrahydrocannabinol Derivatives 95

A B

Figure 10.3. A: Application of azo-THC-1 (5 M) resulted in hyperpolarization without appreciable changes upon irradiation with 360 nm/450 nm. The membrane potential was held at –60 mV. B: azo- THC-1 (trans-isomer) leads to increased inward current in a voltage ramp (–140 mV to –20 mV). C: IV-steps (500 ms) between –140 and –20 mV (20 mV increments) of azo-THC-1 indicates no significant change in efficacy between the trans and cis forms.

We then tested azo-THC-2 using the same setup (Figure 10.4). Upon addition of the ligand, a small increase of an inward current was recorded, which could not be modulated by light- induced isomerization from cis to trans over multiple cycles. As can be deduced from voltage ramp experiments (Figure 10.4 right), the addition of azo-THC-2 led to slightly increased currents compared to vehicle, which were very similar for both isomers (cis and trans) and much weaker than the currents recorded in presence of CP55940.

The situation was similar for diethylamino substituted compounds azo-THC-5–7, which collectively did not induce an appreciable current at concentrations up to 20 M (data not shown). This indicates that further substitution in the para-position of the terminal aromatic is detrimental to binding affinity and results in non-active ligands.

before 5 µM azo-THC-2 (trans) 2 µM azo-THC-2 5 µM azo-THC-2 (cis) 100 nM CP55940

Figure 10.4. Left: Recorded currents upon addition azo-THC-2 to an AtT-20 cell over time. Membrane potential was held at –60 mV. Right: Voltage ramps (I-V diagram) between –140 mV to –20 mV in the absence of ligand, in the presence of azo-THC-2 (5 M, trans and cis, respectively) and CP55940 (100 nM).

We then subjected azo-THC-3 to the electrophysiology experiment, in which addition of the ligand (black bar) led to a slowly increasing inward current (Figure 10.5). Upon irradiation with 360 nm light (trans to cis isomerization) we were pleased to observe a robust inward current, which decreased immediately upon a second isomerization step (cis to trans) using 450 nm light. The cycling process could be repeated multiple times over more than 10 minutes accompanied by a decreasing current amplitude likely due to desensitization (Figure 10.6).187

Figure 10.5. I-t diagram using azo-THC-3 (2 M) shows light-dependent inward currents. Membrane potential held at –60 mV.

187 W. Jin, S. Brown, J. P. Roche, C. Hsieh, J. P. Celver, A. Kovoor, C. Chavkin, K. Mackie, J. Neurosci. 1999, 19, 3773.

Photochromic Tetrahydrocannabinol Derivatives 97

Figure 10.6. Recorded currents upon repeated photoisomerization of azo-THC-3 (2 M) over prolonged time and many photoswitching cycles. Membrane potential held at –60 mV.

The increased current evoked by the cis isomer of azo-THC-3 was further confirmed in IV steps between –120 mV and –20 mV (20 mV increments) as shown in Figure 10.7. The currents resulting from cis-azo-THC-3 were in the same range as when CP55940 was applied, indicating full activation of CB1 by the photoswitch.

Figure 10.7. IV-steps using azo-THC-3 (2 M) between –120 mV and –20 mV (20 mV increments).

Importantly, addition of the CB1-selective antagonist Rimonabant (2 M) led to complete depletion of any photosensitive inward current after an induction period of circa two minutes. This finding strongly supports that the observed effects are indeed mediated by CB1 and not due to a direct interaction between the photoswitchable ligand and GIRK (Figure 10.8).

Figure 10.8. Light-dependent KIR current induced by azo-THC-3 before and after treatment with CB1 antagonist rimonabant (RIM, 2 M). The cellular membrane potential was held at –60 mV.

The last compound of the series, azo-THC-4, also emerged as CB1 agonist that activated the receptor in a light-dependent manner (Figure 10.9). Interestingly, this photoswitchable ligand turned out to activate CB1 more strongly in the trans-form, rather than the cis-isomer in the case of azo-THC-3.

Figure 10.9. Light-dependent KIR current induced by azo-THC-4 (2 M). The cellular membrane potential was held at –60 mV.

The robust inward current evoked by trans-azo-THC-4 was readily reduced upon photoisomerization to the cis-isomer (360 nm), but could be restored by another isomerization back to the trans-form (450 nm). Measuring current-voltage steps between –120 to 20 mV confirmed this result (Figure 10.10).

Photochromic Tetrahydrocannabinol Derivatives 99

Figure 10.10. IV-relationship in presence trans- or cis-azo-THC-4 (2 nM). Currents are normalized to the maximum effect evoked by CP55940 (100 nM).

Cyclic Adenosine Monophosphate Assay

CB1 signals via Gαi upon activation and therefore is negatively coupled to adenylyl 188,189 cyclase. Consequently, receptor activation results in decreased cellular cyclic adenosine monophosphate (cAMP) levels when compared to controls (untreated cell or co-incubated in the presence of antagonists). We investigated the effect of both cis- and trans-azo-THC-3 on forskolin-stimulated (10 M) cAMP levels in AtT-20(CB1) cells in a proprietary bioluminescence-based assay (Promega) (Figure 10.11).190 The assay was carried out with compounds kept either in the dark or pretreated with 365 nm irradiation (trans to cis isomerization). As expected from the electrophysiology experiments, cis-azo-THC-3 was more potent than its trans isomer with EC50 values of 71 ± 15 nM (cis) and 200 ± 11 nM (trans), translating into a potency change by a factor of 3 upon isomerization. Interestingly, both isomers of azo-THC-3 resulted in cAMP levels at least as low as with CP55940 (both at 100 nM) suggesting both isomers could be considered full agonists. As expected, the CB1 antagonist Rimonabant (2 M) decreased the effect of all compounds. In conclusion, these data suggest that azo-THC-3 light-dependently reduces forskolin-stimulated cAMP synthesis in

AtT-20(CB1) cells via Gαi mediated inhibition of adenylyl cylase.

188 A. C. Howlett, R. M. Fleming, Mol Pharmacol 1984, 26, 532. 189 L. A. Matsuda, S. J. Lolait, M. J. Brownstein, A. C. Young, T. I. Bonner, Nature 1990, 346, 561. 190 cAMP-GloTM (Promega).

100

Figure 10.11. A: Normalized luminescence evoked by azo-THC-3 in forskolin-stimulated cAMP assay in AtT-20(CB1) cells. Compounds were either kept in the dark (trans) or pre-irradiated with 365 nm light (cis). Higher luminescence indicates decreased cAMP concentration. B: CB1 antagonist Rimonabant (2 M) reduced the effects of agonists CP55940 (100 nM) and both isomers of azo-THC- 3 (100 nM). Experiments were performed in triplicates. Values are normalized to luminescence evoked by cells treated with forskolin only (10 M).

11 Conclusion and Outlook

An efficient enantioselective synthesis of 3-Br-THC was accomplished relying on dual- catalytic α-allylation of an aldehyde. The developed strategy can likely be applied to the diastereodivergent synthesis of all stereoisomers of this valuable building block. Therefore, 3- Br-THCs exhibiting all stereochemical permutations should prove as useful linchpins for the synthesis of various related natural products and the preparation of THC-derived compound libraries.

The versatility of this novel building block was showcased by its elaboration into various THC- derivatives incorporating a diazobenzene motif allowing for reversible photoisomerization between two distinct states (cis and trans). In combination with whole-cell electrophysiology, our synthetic approach enabled the identification of two photoswitchable CB1 agonists, azo- THC-3 and azo-THC-4, which were both shown to activate GIRK channels coupled to CB1

(via Gβ in a light-dependent fashion. The finding that azo-THC-3 is more active in the cis form, while azo-THC-4 is more active in the trans form provides flexibility for the planning of future applications of the two compounds. Furthermore, azo-THC-3 was shown to effect CB1 signaling via Gαi, resulting in adenylyl cyclase inhibition as shown in a bioluminescence based cAMP assay. It is important to note that the EC50 values determined for the individual cis and trans isomers differed by a factor of three. Future studies may involve development of analogs with even more pronounced bioactivity differences between cis and trans isomers. The precise

Conclusion and Outlook 101 spatiotemporal control of receptor activation offered by azo-THC-3 and azo-THC-4 is likely to enrich the understanding of CB1 pharmacology in response to cannabinoid ligands.

The extension of the principles derived from this work to the development of receptor-selective photoswitchable ligands for CB1 and CB2 seems straightforward, and further synthetic efforts towards this goal have been initiated.

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IV. Experimental

103

12 General Methods

Non-aqueous reactions were performed under an inert atmosphere of dry nitrogen in flame dried glassware sealed with a rubber septum unless otherwise noted. The protecting gas was passed over a column of CaCl2 and supplied through a glass manifold. Reactions were stirred magnetically and monitored by thin layer chromatography (TLC). Analytical thin layer chromatography was performed using MERCK SILICA GEL F254 TLC glass plates and visualized by ultraviolet light (UV). Additionally, TLC plates were stained with aqueous potassium permanganate (KMnO4) [1.5 g KMnO4, 200 mL H2O, 10 g K2CO3, 2.5 mL 1 M

NaOH aq.] or “Seebach’s magic stain” (Seebach) [2.5 g phosphomolybdic acid, 1.0 g Ce(SO4)2,

94 mL H2O, 6 mL conc. H2SO4]. Concentration under reduced pressure (= in vacuo) was performed by rotator evaporation at 40-45 °C at the appropriate pressure. Chromatographic purification was performed as flash chromatography on FLUKA silica gel 60 Å (230-400 mesh) at 0.3 – 0.5 bar over-pressure. Yields refer to the purified compound.

13 Chemicals

All chemicals and solvents were purchased from ABCR, ACROS, ALDRICH, COMBI-BLOCKS,

FLUOROCHEM, FLUKA, MERCK, FISHER-SCIENTIFIC, TCI, STREM or LANCASTER and were used as received from the commercial supplier without further purification unless mentioned otherwise. THF, Et2O, CH2Cl2, MeCN and toluene were dried on a LC TECHNOLOGY

SOLUTIONS SP-1 solvent purification system under N2. Deuterated solvents were obtained from

ARMAR CHEMICALS, Döttingen, Switzerland. Diisoproylamine and pyridine were distilled from

KOH and NEt3 was distilled from calcium hydride under an atmosphere of dry nitrogen.

14 Analytics

Nuclear Magnetic Resonance (NMR) spectra were recorded on VARIAN MERCURY (300 MHz),

BRUKER AV and DRX (400 MHz), BRUKER DRX and DRXII (500 MHz) or BRUKER AVIII (600 MHz with cryoprobe) spectrometers. Measurements were carried out at ambient temperature (ca. 22 °C). Chemical shifts (δ) are reported in ppm with the residual solvent signal as internal standard (chloroform at 7.26 and 77.16 ppm for 1H and 13C NMR spectroscopy, respectively), unless otherwise noted. The data is reported as (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet or unresolved, b = broad signal, app = apparent, coupling constant(s) in Hz, integration). 13C NMR spectra were recorded with broadband 1H decoupling. Service measurements were performed by the NMR service team of the Laboratorium für Organische

104

Chemie at ETH Zürich by Mr. René Arnold, Mr. Rainer Frankenstein and Mr. Philipp Zumbrunnen under direction of Dr. Marc-Olivier Ebert.

Infrared (IR) spectra were recorded on a PERKIN ELMER TWO-FT-IR (UATR) as thin films. Absorptions are given in wavenumbers (cm–1).

Mass spectrometry (MS) analyses were performed as high resolution EI measurements on a

WATERS MICROMASS AUTOSPEC ULTIMA at 70 eV, as high resolution ESI measurements on a

BRUKER DALTONICS MAXIS (UHR-TOF) instrument or as MALDI on a BRUKER SOLARIX– MALDI-FTICR-MS instrument by the mass spectrometry service of the Laboratorium für Organische Chemie at ETH Zürich by Mr. Louis Bertschi, Mr. Oswald Greter, Mr. Rolf Häfliger and Dr. Xiangyang Zhang.

105

15 General Procedures

GP1: Cycloaddition of benzyl azide with 2-cyanoacetamide

The transformation is handled open to air.

The benzyl (pseudo)halide (1 equiv) was dissolved in DMSO (1 volume) to give a ca. 1 M solution. DIPEA (0.1 equiv) was added to scavenge any potentially formed hydrazoic acid. Sodium azide (1.05 equiv) was added (note: depending on the leaving group, the nucleophilic displacement reaction can be quite exothermic, add in portions). Upon consumption of the starting material, the mixture (often a suspension due to salt precipitate) was transferred to a solution of 2-cyanoacetamide (1.5 equiv), DMSO (1 volume) and 15% aq. NaOH (1.5 equiv). Note: the cycloaddition is usually quite exotherm, adjust the addition accordingly. The reaction is usually very fast (less than 1 h), but can be left overnight without problems. Upon full consumption of intermediate benzyl azide, water (3 volumes) is added slowly (exotherm) leading to precipitation of the product. After filtration, the filtercake is washed with water,

EtOH and Et2O to yield the product after drying.

GP2: Grignard formation

LiCl (1.25 equiv) is added to a SCHLENK flask and flame-dried under high vacuum (evacuation, flame-drying under dynamic vacuum, N2 backfill, repeat 2x). Mg (2.50 equiv) is added and the flask is evacuated and backfilled with N2 again. After cooling, a fifth of total amount THF (1 volume to give a theoretical 1 M solution) is added, followed by 1,2-dibromoethane (50 L). When the exothermic reaction is over, the residual THF is added along with alkyl or aryl bromide (1.00 equiv). The concentration of active GRIGNARD reagent is determined by titration against iodine.191

16 Syntheses Synthesis of 18

The title compound was prepared according to GP1 from 4-methoxybenzyl chloride (6.0 g, 38.3 mmol, 1.0 equiv) and isolated as beige solid (7.6 g, 30.8 mmol, 80%).

191 A. Krasovskiy, P. Knochel, Synthesis 2006, 2006, 890.

106

1H NMR (400 MHz, DMSO) δ 7.43 (s, 1H), 7.24 – 7.16 (m, 2H), 7.07 (s, 1H), 6.95 – 6.88 (m, 2H), 6.36 (s, 2H), 5.33 (s, 2H), 3.72 (s, 3H). 13C NMR (101 MHz, DMSO) δ 164.7, 159.3, 145.0, 129.5, 128.3, 122.2, 114.4, 55.6, 48.3. IR (neat) 3393, 3304, 3161, 1669, 1632, 1615, -1 1569, 1513, 1298, 1245, 1179, 1027, 782 cm . HRMS (ESI+): m/z calcd for C11H13N5NaO2 [M+Na]+ 270.0961, found 270.0961.

Synthesis of 5-(difluoro(phenyl)methyl)-3-(4-methoxybenzyl)-3,4-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one

18 (2.0 g, 8.1 mmol, 1.0 equiv) was dissolved in DMF (27 mL), combined with K2CO3 (5.6 g,

40.4 mmol, 5.0 equiv) and PhCF2CN (1.86 g, 12.1 mmol, 1.5 equiv). The flask was sealed and heated at 80 °C overnight. LCMS indicated residual starting material. PhCF2CN (0.9 g, 5.9 mmol, 0.7 equiv) was added and stirring continued at 80 °C for 3 h. The reaction mixture was cooled to rt and neutralized by the addition of aq. HCl (2 M HCl) until pH = 7. The precipitate was collected by filtration, washed with water, EtOH and ether to give the title compound as colorless solid (1.1 g). The washings were diluted with EtOAc, washed with 5% aq. LiCl and brine, dried over MgSO4, filtered and concentrated. Flash chromatography on silica (20% acetone in PhMe gradient to 50%, 10% increments) afforded another crop (0.9 g) of the title compound (2.0 g combined, 5.2 mmol, 65%).

1H NMR (400 MHz, DMSO) δ 13.57 (s, 1H), 7.77 – 7.66 (m, 2H), 7.57 (td, J = 10.0, 9.0, 4.4 Hz, 3H), 7.27 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.1 Hz, 2H), 5.64 (s, 2H), 3.72 (s, 3H). 13C NMR (101 MHz, DMSO) δ 159.2, 155.5, 153.7 (t, J = 33.6 Hz), 147.4, 132.9 (d, J = 26.4 Hz), 131.3, 129.9, 129.4, 129.0, 128.8, 126.8, 125.7 (t, J = 5.6 Hz), 114.1, 55.1, 49.8. 19F NMR (377 MHz, DMSO) δ -95.8. IR (neat) 3076, 3017, 2977, 2916, 2844, 1722, 1613, 1593, 1564, 1533, 1515, 1454, 1386, 1295, 1255, 1180, 1166, 1117, 1029, 1011, 905, 822, 798, 759, 692, 551 cm-1. + HRMS (ESI+): m/z calcd for C19H15F2N5NaO2 [M+Na] 406.1086, found 406.1088.

107

Synthesis of 7-chloro-5-(difluoro(phenyl)methyl)-3-(4-methoxybenzyl)-3H- [1,2,3]triazolo[4,5-d]pyrimidine

5-(Difluoro(phenyl)methyl)-3-(4-methoxybenzyl)-3,4-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (1.24 g, 3.23 mmol, 1.00 equiv) was combined with POCl3 (9.3 mL) and a drop of DMF. The mixture was stirred at 80 °C overnight. The volatiles were removed and the residue was purified by flash chromatography on silica (10% EtOAc in hexanes) to afford the title compound as colorless solid (1.21 g, 3.01 mmol, 93%).

1 Rf = 0.37 (20% EtOAc in hexanes; KMnO4, UV). H NMR (400 MHz, CDCl3) δ 7.76 – 7.69 (m, 2H), 7.50 – 7.41 (m, 5H), 6.89 – 6.80 (m, 2H), 5.83 (s, 2H), 3.78 (s, 3H). 13C NMR (101

MHz, CDCl3) δ 160.9 (t, J = 32.5 Hz), 160.2, 155.1, 149.9, 135.2 (t, J = 27.0 Hz), 133.9, 130.7 (t, J = 1.7 Hz), 130.6, 128.6, 126.0 (t, J = 6.0 Hz), 125.7, 116.6 (t, J = 247.8 Hz), 114.5, 55.5, 19 51.5. F NMR (377 MHz, CDCl3) δ -98.1. IR (neat) 1612, 1586, 1568, 1514, 1453, 1339, 1251, 1179, 1160, 1105, 1065, 1005, 971, 1871, 806, 771, 698 cm-1. HRMS (ESI+): m/z calcd + for C19H15ClF2N5O [M+H] 402.0928, found 402.0932.

Synthesis of 19

7-Chloro-5-(difluoro(phenyl)methyl)-3-(4-methoxybenzyl)-3H-[1,2,3]triazolo[4,5- d]pyrimidine (622 mg, 1.55 mmol, 1.00 equiv) and 3,3-difluoropyrrolidine hydrochloride

(244 mg, 1.70 mmol, 1.10 equiv) were dissolved in CH2Cl2 (3.1 mL). NEt3 (0.32 mL, 2.3 mmol, 1.5 equiv) was added dropwise and the mixture was stirred for 30 min. The reaction mixture was diluted with CH2Cl2 (100 mL), washed with water and brine (20 mL each), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (5% EtOAc in hexanes to 10%) afforded the title compound as colorless solid (600 mg, 1.27 mmol, 82%).

A mixture of rotamers (ca. 1:1.2), all signals reported. 1 Rf = 0.29 (20% EtOAc in hexanes; Seebach, UV). H NMR (400 MHz, CDCl3) δ 7.74 – 7.67 (m, 2H), 7.41 (d, J = 2.0 Hz, 4H), 7.40 (d, J = 2.2 Hz, 1H), 6.85 – 6.79 (m, 2H), 5.70 (s, 2H),

108

4.56 (t, J = 12.6 Hz, 1H), 4.50 (t, J = 7.5 Hz, 1H), 4.11 (t, J = 12.8 Hz, 1H), 4.04 (t, J = 7.5 Hz, 1H), 3.77 (s, 3H), 2.59 (tt, J = 13.1, 7.1 Hz, 1H), 2.48 (app dq, J = 13.8, 7.5, 6.8 Hz, 1H). 13C

NMR (101 MHz, CDCl3) δ 161.1 (t, J = 30.6 Hz), 159.8, 153.0, 150.1, 150.0, 136.5 (t, J = 27.4 Hz), 130.4, 130.1 (d, J = 2.0 Hz), 128.2, 128.2, 127.4 (t, J = 248.3 Hz), 127.0, 126.7 (t, J = 247.6 Hz), 126.0 (td, J = 6.0, 2.6 Hz), 125.1, 117.1 (t, J = 246.1 Hz), 117.1 (t, J = 246.1 Hz), 114.3, 55.6 (t, J = 33.1 Hz), 55.4, 53.9 (t, J = 33.1 Hz), 50.4, 47.1, 45.0, 34.6 (t, J = 24.3 Hz), 19 32.9 (t, J = 23.8 Hz). F NMR (377 MHz, CDCl3) δ -98.8, -98.9, -101.3, -102.0. IR (neat) 2959, 2898, 1605, 1576, 1514, 1349, 1250, 1178, 1123, 1088, 1034, 1009, 912, 699 cm-1. + HRMS (ESI+): m/z calcd for C23H21F4N6O [M+H] 473.1707, found 473.1710.

Synthesis of 20

19 (200 mg, 0.423 mmol, 1.00 equiv) was combined with anisole (1.4 mL, 12.8 mmol, 30 equiv) and TFA (6.5 mL, 85 mmol, 200 equiv) and stirred at 65 °C for 5 h. The reaction mixture was concentrated, diluted with EtOAc (200 mL) and washed with aq. NaOH (1 Mm 50 mL). The aq. washing was backextracted with EtOAc (2x 20 mL). The combined organic fractions were washed with brine, dried over MgSO4, filtered and concentrated. Flash chromatography on silica (50% EtOAc in hexanes to 100%) afforded product contaminated with unidentified byproduct. The solid was triturated with EtOH, filtered and the filtercake was washed with EtOH to yield the title compound as tan solid (23 mg). The washings were concentrated and the residue triturated with 20% EtOAc in hexanes with sonication. Filtration and washing with 20% EtOAc in hexanes afforded a second crop of the title compound (74 mg combined, 0.210 mmol, 50%).

1H NMR (400 MHz, DMSO) δ 7.71 – 7.60 (m, 2H), 7.54 – 7.43 (m, 3H), 4.57 (t, J = 12.7 Hz, 1H), 4.41 (t, J = 7.7 Hz, 1H), 4.09 (t, J = 12.8 Hz, 1H), 3.93 (t, J = 7.3 Hz, 1H), 2.75 – 2.54 (m, 2H), 2.50 (d, J = 2.9 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 159.9 (t, J = 29.8 Hz), 152.9, 152.3, 136.3 (t, J = 27.0 Hz), 130.7, 128.9, 128.6, 127.9, 126.0 (t, J = 5.9 Hz), 124.0, 117.6 (t, J = 245.1 Hz), 55.4 (t, J = 32.8 Hz), 53.7 (d, J = 33.1 Hz), 47.5, 45.4, 33.9 (t, J = 23.8 Hz), 32.3 (t, J = 22.9 Hz). 19F NMR (376 MHz, DMSO) δ -96.2, -96.3, -100.4, -100.9. IR (neat) 2961 (br), 1606, 1524, 1381, 1249, 1137, 1119, 1014, 922, 805, 773, 699 cm-1. HRMS (ESI+): m/z + calcd for C15H13F4N6 [M+H] 353.1132, found 353.1132.

109

Synthesis of 21

18 (272 mg, 0.677 mmol, 1.00 equiv) was dissolved in CH2Cl2 (1.3 mL). NEt3 (0.14 mL, 1.0 equiv, 1.5 equiv) and (S)-3-hydroxypyrrolidine (65 mg, 0.745 mmol, 1.10 equiv) were added and it was stirred at rt for 45 min. The reaction mixture was diluted with CH2Cl2

(100 mL), washed with water and brine (20 mL each), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (50% EtOAc in hexanes) afforded the title compound as colorless foam (291 mg, 0.643 mmol, 95%).

A mixture of rotamers (ca. 1:1), all signals reported. 1 Rf = 0.22 (50% EtOAc in hexanes; KMnO4, UV). H NMR (600 MHz, CDCl3) δ 7.76 – 7.69 (m, 2H), 7.43 – 7.37 (m, 5H), 6.83 – 6.79 (m, 2H), 5.67 (app d, J = 2.6 Hz, 2H), 4.74 – 4.62 (m, 1H), 4.49 – 4.36 (m, 1H), 4.29 – 4.15 (m, 1H), 4.03 – 3.79 (m, 2H), 3.76 (d, J = 0.7 Hz, 13 3H), 2.24 – 2.07 (m, 2H), 1.77 (br s, 1H). C NMR (151 MHz, CDCl3) δ 161.0 (app td, J = 30.1, 12.1 Hz), 159.7, 153.1 (app d, J = 3.9 Hz), 150.0 (app d, J = 4.8 Hz), 136.8 (t, J = 27.2 Hz), 130.3, 129.9, 128.1, 127.3 (app d, J = 2.4 Hz), 126.1 (app td, J = 6.0, 3.2 Hz), 125.2 (app d, J = 17.3 Hz), 117.3 (t, J = 246.2 Hz), 114.2, 71.3, 69.7, 57.7, 56.0, 55.4, 50.2, 47.3, 45.7, 19 34.7, 32.8. F NMR (470 MHz, CDCl3) δ -98.7, -98.7, -98.8. IR (neat) 3411 (br), 2946, 1609, -1 + 1514, 1250, 1091, 699 cm . HRMS (ESI+): m/z calcd for C23H23F2N6O2 [M+H] 453.1845, found 453.1850.

Synthesis of 22

21 (141 mg, 0.312 mmol, 1.00 euqiv) was dissolved in TFA (4.8 mL) and heated to 70 °C for 3 h. LCMS indicated full consumption of starting material, the formation of product and of the ester. The mixture was lyophilized. The residue was dissolved in MeOH (3 mL) and THF

(3 mL), treated with K2CO3 (215 mg, 1.56 mmol, 5.0 equiv) and stirred for 30 min. The mix was neutralized with HCl and concentrated (coevaporation with EtOH). The residual solid was dry loaded on a silica column. Elution with 5% MeOH in EtOAc (to 10%, to 30%) afforded a

110 colorless solid (220 mg). This was partitioned between EtOAc and sat. NaHCO3. The aq. phase was removed, the organic phase washed with brine, dried over MgSO4, filtered and concentrated to give the title compound as yellowish solid (69 mg, 0.208 mmol, 67%).

A mixture of rotamers (ca. 1:1), all signals reported. 1H NMR (500 MHz, MeOD) δ 7.70 – 7.62 (m, 2H), 7.41 – 7.36 (m, 3H), 4.58 (ddt, J = 46.7, 4.3, 2.2 Hz, 1H), 4.40 – 4.28 (m, 1H), 4.23 – 4.11 (m, 1H), 3.93 – 3.71 (m, 2H), 2.28 – 2.02 (m, 2H). 13C NMR (151 MHz, MeOD) δ 162.1 (app td, J = 30.1, 6.1 Hz), 154.3 (app d, J = 7.5 Hz), 153.1 (app d, J = 11.1 Hz), 139.0 – 137.5 (m), 131.0 (t, J = 1.6 Hz), 129.2, 126.8 (app td, J = 6.0, 2.0 Hz), 125.2 (app d, J = 12.2 Hz), 118.4 (t, J = 245.4 Hz), 71.7, 70.1, 58.5, 56.7, 46.7, 35.1, 33.3. 19F NMR (470 MHz, MeOD) δ -99.75, -99.80, -99.83, -99.86. IR (neat) 3395 (br), 2948, 1606, 1525, 1452, 1385, 1247, 1102, 1013, 942, 916, 750, 967 cm-1. HRMS (ESI+): m/z + calcd for C15H15F2N6O [M+H] 333.1270, found 333.1272.

Synthesis of 24

The sulfonyl fluoride (174 mg, 1.00 mmol, 1.0 equiv) was combined with NBS (214 mg,

1.20 equiv, 1.2 equiv), benzoyl peroxide (32 mg, 0.10 mmol, 0.1 equiv) and CCl4 (5 mL). The flask was capped and stirred at 80 °C overnight. The volatiles were removed in vacuo. Flash chromatography on silica (1.5% EtOAc in hexanes) afforded the title compound as colorless solid (115 mg, 0.454 mmol, 45%).

1 Rf = 0.32 (10% EtOAc in hexanes; KMnO4, UV). H NMR (400 MHz, CDCl3) δ 8.09 (app dd, J = 8.0, 1.2 Hz, 1H), 7.80 – 7.71 (m, 2H), 7.56 (app ddt, J = 8.2, 6.8, 1.7 Hz, 1H), 4.87 (s, 2H). 13 C NMR (101 MHz, CDCl3) δ 138.3, 136.1, 133.7, 131.6 (d, J = 24.0 Hz), 130.9 (d, J = 1.5 19 -1 Hz), 129.4, 27.7. F NMR (376 MHz, CDCl3) δ 66.1. IR (neat) 1401, 1210, 761, 597 cm . + HRMS (EI+): m/z calcd for C7H6BrFO2S [M] 251.9256, found 251.9251.

Synthesis of 25 and 26

111

The triazolopyrimidine (50 mg, 0.177 mmol, 1.0 equiv) was combined with the benzyl bromide

(49 mg, 0.195 mmol, 1.1 mmol) and DMF (0.4 mL). NEt3 (49 L, 0.354 mmol, 2.0 equiv) was added and it was stirred for 45 min at rt. The mixture was diluted with EtOAc (100 mL) and washed with 5% aq. LiCl (2x 20 mL) and brine (20 mL). It was dried over MgSO4, filtered and concentrated. Flash chromatography on silica (15% EtOAc in hexanes) afforded 25 (33 mg, 0.073 mmol, 41%) as colorless oil and 26 (40 mg, 0.088 mmol, 50%) as colorless oil that solidified upon standing.

25: a mixture of rotamers (1:1.3), all signals reported. 1 Rf = 0.33 (20% EtOAc in hexanes; KMnO4, UV). H NMR (400 MHz, CDCl3) δ 8.16 (dd, J = 7.9, 1.4 Hz, 1H), 7.62 (td, J = 7.7, 1.5 Hz, 1H), 7.54 (tt, J = 7.8, 1.4 Hz, 1H), 7.08 (d, J = 7.7 Hz, 1H), 6.20 (s, 2H), 4.66 – 4.51 (m, 2H), 4.22 – 4.06 (m, 2H), 2.58 (dtt, J = 42.7, 13.9, 7.5 13 Hz, 2H), 1.33 (s, 9H). C NMR (101 MHz, CDCl3) δ 176.7, 152.5, 151.4, 131.5, 131.2, 130.8 (d, J = 1.5 Hz), 130.1, 128.9, 127.7 (d, J = 249.0 Hz), 127.1 (d, J = 279.0 Hz), 123.9, 55.5 (t, J = 32.8 Hz), 53.8 (t, J = 32.7 Hz), 46.9, 46.4 (d, J = 1.3 Hz), 44.6, 39.8, 34.7 (t, J = 24.4 Hz), 19 33.0 (t, J = 23.9 Hz), 29.7. F NMR (282 MHz, CDCl3) δ 64.9, 64.9, -101.3, -101.9. IR (neat) 2961, 1601, 1579, 1481, 1405, 1318, 1210, 1126, 927, 781, 757, 590 cm-1. HRMS (ESI+): m/z + calcd for C19H22F3N6O2S [M+H] 455.1472, found 455.1470.

26: a mixture of rotamers (1:1.3), all signals reported. 1 Rf = 0.20 (20% EtOAc in hexanes; KMnO4, UV). H NMR (400 MHz, CDCl3) δ 8.15 (app dd, J = 8.0, 1.3 Hz, 1H), 7.67 (app td, J = 7.7, 1.4 Hz, 1H), 7.58 (app tt, J = 7.7, 1.4 Hz, 1H), 7.18 (app dd, J = 15.3, 7.8 Hz, 1H), 6.26 (s, 2H), 4.46 – 4.30 (m, 2H), 4.20 – 4.04 (m, 2H), 2.65 – 13 2.45 (m, 2H), 1.40 (s, 9H). C NMR (101 MHz, CDCl3) δ 176.2, 160.3, 152.9, 136.1, 134.9, 131.5, 131.2, 131.0, 130.9, 130.8, 129.5, 127.6 (t, J = 248.0 Hz), 127.0 (t, J = 247.0 Hz), 126.0, 56.8, 55.3 (t, J = 32.8 Hz), 53.8 (t, J = 33.0 Hz), 46.6, 44.7, 39.9, 34.7 (t, J = 24.1 Hz), 33.0 (t, 19 J = 23.7 Hz), 29.7. F NMR (282 MHz, CDCl3) δ 65.7, 65.6, -101.1, -101.8. IR (neat) 2960, 1591, 1565, 1483, 1449, 1406, 1341, 1211, 1164, 1128, 782, 753, 732, 600, 583 cm-1. HRMS + (ESI+): m/z calcd for C19H22F3N6O2S [M+H] 455.1472, found 455.1472.

Synthesis of 27 and 28

112

The triazolopyrimidine (50 mg, 0.191 mmol, 1.0 equiv) was combined with the benzyl bromide

(53 mg, 0.210 mmol, 1.1 mmol) and DMF (0.4 mL). NEt3 (40 L, 0.286 mmol, 1.5 equiv) was added and it was stirred for 1 h at rt. The mixture was diluted with EtOAc (100 mL) and washed with 5% aq. LiCl (2x 20 mL) and brine (20 mL). It was dried over MgSO4, filtered and concentrated. Flash chromatography on silica (50% EtOAc in hexanes + 10% AcOH) afforded 27 (23 mg, 0.053 mmol, 28%) and 28 (21 mg, 0.048 mmol, 25%) as colorless foams.

27: a mixture of rotamers (1:1), all signals reported. 1 Rf = 0.29 (50% EtOAc in hexanes; KMnO4, UV). H NMR (400 MHz, CDCl3) δ 8.15 (app dd, J = 7.9, 1.4 Hz, 1H), 7.60 (app td, J = 7.7, 1.5 Hz, 1H), 7.55 – 7.50 (m, 1H), 7.04 (app d, J = 7.8 Hz, 1H), 6.18 (s, 2H), 4.80 – 4.66 (m, 1H), 4.54 – 4.39 (m, 1H), 4.35 – 4.21 (m, 1H), 4.09 13 – 3.83 (m, 2H), 2.28 – 2.10 (m, 2H), 1.33 (app d, J = 3.2 Hz, 9H). C NMR (101 MHz, CDCl3) δ 176.5, 176.5, 152.6, 151.3, 136.6, 135.9, 131.3, 131.1, 130.7 (d, J = 1.5 Hz), 130.0, 128.7, 124.1, 124.0, 71.5, 69.8, 57.5, 55.7, 47.0, 46.3, 45.3, 39.8, 34.9, 32.9, 29.7, 25.0. 19F NMR (377

MHz, CDCl3) δ 64.8 (app d, J = 7.0 Hz). IR (neat) 3399, 2958, 1603, 1578, 1482, 1406, 1317, -1 + 1211, 780, 760, 591 cm . HRMS (ESI+): m/z calcd for C19H24FN6O3S [M+H] 435.1609, found 435.1607.

28: a mixture of rotamers (1:1.3), all signals reported. 1 Rf = 0.29 (50% EtOAc in hexanes; KMnO4, UV). H NMR (400 MHz, CDCl3) δ 8.1 (app d, J = 7.9 Hz, 1H), 7.6 (app t, J = 7.7 Hz, 1H), 7.6 – 7.5 (m, 1H), 7.1 (app d, J = 7.8 Hz, 1H), 6.2 (app d, J = 6.9 Hz, 2H), 4.8 – 4.7 (m, 1H), 4.3 – 3.8 (m, 4H), 2.2 – 2.1 (m, 2H), 1.4 (app d, J = 13 4.7 Hz, 9H). C NMR (101 MHz, CDCl3) δ 176.2, 176.2, 160.2, 160.1, 153.1, 153.0, 136.1, 135.3, 135.3, 131.3, 131.0, 130.7, 130.7, 130.7, 129.3, 126.5, 126.4, 71.4, 69.7, 57.1, 56.6, 55.8, 19 46.8, 45.3, 39.8, 34.8, 33.0, 29.8, 29.7. F NMR (377 MHz, CDCl3) δ 65.6 (app d, J = 7.0 Hz). IR (neat) 3324, 2958, 1594, 1563, 1483, 1449, 1407, 1397, 1327, 1212, 793, 760, 733, 601, -1 + 584 cm . HRMS (ESI+): m/z calcd for C19H24FN6O3S [M+H] 435.1609, found 435.1608.

Synthesis of 31

According to GP1 from benzyl(2-(chloromethyl)phenyl)sulfane (3.39 g, 13.6 mmol) to give 31 (4.03 g, 11.9 mmol, 87%) as colorless solid.

113

1H NMR (400 MHz, DMSO) δ 7.52 – 7.42 (m, 2H), 7.40 – 7.22 (m, 6H), 7.19 (app t, J = 7.5 Hz, 1H), 7.12 (s, 1H), 6.64 (app d, J = 7.6 Hz, 1H), 6.39 (s, 2H), 5.41 (s, 2H), 4.23 (s, 2H). 13C NMR (101 MHz, DMSO) δ 164.3, 145.3, 137.2, 135.4, 134.3, 130.3, 129.0, 128.4, 128.2, 127.2, 127.0, 126.7, 121.6, 46.8, 37.5. IR (neat) 3434, 3317, 3250 (br), 2098, 1654, 1629, 1556, -1 + 1514, 1240, 1019, 751, 705 cm . HRMS (ESI+): m/z calcd for C17H17N5NaOS [M+Na] 362.1046, found 362.1051.

Synthesis of 32

31 (679 mg, 2.00 mmol, 1.00 equiv) was dissolved in DMF (4.00 mL). 2,2-difluoro-2- phenylacetonitrile (459 mg, 3.00 mmol, 1.50 equiv) and K2CO3 (1.38 g, 10.0 mmol, 5.00 equiv) were added. The flask was capped and then heated to 90 °C overnight. The reaction mix was cooled to rt and water (20 mL) was added. The precipitate was collected by filtration (washing with water). The material was futher purified by flash chromatography on silica (20% acetone in toluene) to give the title compound as yellow solid (406 mg, 0.845 mmol, 43%).

1 Rf = 0.38 (30% acetone in toluene; UV, KMnO4). H NMR (600 MHz, Acetone) δ 7.73 – 7.71 (m, 2H), 7.67 – 7.64 (m, 2H), 7.59 – 7.49 (m, 5H), 7.33 – 7.29 (m, 1H), 7.26 – 7.19 (m, 5H), 5.80 (s, 2H), 4.14 (s, 2H). 13C NMR (151 MHz, Acetone) δ 166.4 (t, J = 31.3 Hz), 155.9, 155.0 (t, J = 33.7 Hz), 149.2, 138.4, 136.8, 136.0, 134.8 (t, J = 25.6 Hz), 134.3 (t, J = 26.2 Hz), 133.5, 132.1, 131.6, 130.6, 130.4, 129.9, 129.8, 129.6, 129.4, 129.2, 128.4, 128.0, 126.7 (t, J = 5.9 Hz), 126.2 (t, J = 6.2 Hz), 116.8 (t, J = 247.2 Hz), 115.8 (t, J = 251.7 Hz), 49.4, 40.2. 19F NMR (377 MHz, Acetone) δ -98.0. IR 3188 (br), 3065, 1714, 1453, 1264, 1116, 1066, 697 cm-1. + HRMS (ESI+): m/z calcd for C25H20 F2N5OS [M+H] 476.1351, found 476.1347.

Synthesis of 33

32 (150 mg, 0.315 mmol, 1.00 equiv) was dissolved in a mixture of MeCN (6.4 mL), AcOH (0.080 mL) and water (0.16 mL) and cooled to –10 °C. 1,3-Dichloro-5,5-dimethylhydantoin (124 mg, 0.631 mmol, 2.00 equiv) was added. The reaction mix was stirred for 1.5 h at that temperature, then diluted with CH2Cl2 (100 mL) and washed with brine, dried over MgSO4,

114 filtered and concentrated. The crude sulfonyl chloride was dissolved in acetone (1.0 mL). KF (92 mg, 1.58 mmol) and water (53 µL) were added and it was stirred overnight at rt. The reaction mix was diluted with CH2Cl2, filtered, dried over MgSO4, filtered again and concentrated. The crude sulfonyl fluoride was dissolved in CH2Cl2 (1.6 mL). DMF (25 µL) was added, followed by (COCl)2 (56 µL, 0.64 mmol, 2.0 equiv) and the reaction mixture was heated to reflux overnight. TLC indicated incomplete conversion. Another portion (COCl)2 (56 µL, 0.64 mmol, 2.0 equiv) was added to drive the reaction to completion. After 2 h, the mix was cooled to rt, diluted with EtOAc (100 mL), washed with halfsat. aq. sodium bicarbonate, 5% aq. LiCl, and brine, dried over MgSO4, filtered and concentrated. Flash chromatography on silica (10% EtOAc in hexanes to 15% to 20%) afforded the title compound (48 mg, 0.11 mmol, 33% over 3 steps) as yellow oil.

1 Rf = 0.23 (20% EtOAc in hexanes; UV, weak KMnO4). H NMR (400 MHz, CDCl3) δ 8.21 (dd, J = 7.9, 1.5 Hz, 1H), 7.69 (ddt, J = 7.7, 3.5, 2.1 Hz, 4H), 7.68 – 7.59 (m, 2H), 7.48 – 7.37 13 (m, 3H), 7.21 (dd, J = 7.7, 1.3 Hz, 1H), 6.36 (s, 2H). C NMR (101 MHz, CDCl3) δ 161.5 (t, J = 32.8 Hz), 155.5, 150.4, 136.1, 134.8 (t, J = 26.9 Hz), 133.7, 133.6, 131.7 (d, J = 24.4 Hz), 131.1 (d, J = 1.4 Hz), 130.8, 130.6, 129.8, 128.6, 125.9 (t, J = 6.0 Hz), 116.3 (t, J = 248.3 Hz), 19 47.8. F NMR (377 MHz, CDCl3) δ 66.0, -98.3. IR 2922, 2852, 1730, 1588, 1569, 1407, 1262, -1 1212, 1074, 1005, 923, 789, 768, 697 cm . HRMS (ESI+): m/z calcd for C18H12ClF3N5O2S [M+H]+ 454.0347, found 454.0352.

Synthesis of 34

A solution of NEt3 (4.7 mg, 0.046 mmol, 2.1 equiv) in CH2Cl2 (0.1 mL) was added to a suspension of 33 (10 mg, 0.022 mmol, 1.0 equiv) and 3,3-difluoropyrrolidine·HCl (3.3 mg,

0.023 mmol, 1.1 equiv) in CH2Cl2 (0.1 mL). The reaction mixture was stirred until TLC indicated full consumption of starting material, then directly subjected to flash chromatography on silica to afforded the title compound (10 mg, 0.019 mmol, 87%) as colorless foam. ca. 1.15:1 mixture of rotamers. All signals reported. 1 Rf = 0.26 (hexanes:CH2Cl2:EtOAc 6:3:1; UV, KMnO4). H NMR (500 MHz, CDCl3) δ 8.18 (app dd, J = 7.8, 1.6 Hz, 1H), 7.70 – 7.65 (m, 2H), 7.64 – 7.54 (m, 2H), 7.43 – 7.36 (m, 3H),

115

7.04 – 7.00 (m, 1H), 6.25 (d, J = 1.3 Hz, 2H), 4.63 – 4.52 (m, 2H), 4.20 – 4.06 (m, 2H), 2.68 – 13 2.47 (m, 2H). C NMR (151 MHz, CDCl3) δ 161.8 (d, J = 30.8 Hz), 161.8 (d, J = 30.8 Hz), 153.1, 153.0, 136.3 (t, J = 27.2 Hz), 136.3 (t, J = 27.2 Hz), 136.0, 135.4, 131.5 (d, J = 24.1 Hz), 131.5 (d, J = 24.1 Hz), 131.0, 130.2, 130.1, 129.2, 128.3, 128.3, 129.1 – 125.6 (m), 126.6 (t, J = 247.8 Hz), 126.0, 125.9, 125.9, 124.8, 124.8, 55.7 (t, J = 33.2 Hz), 54.0 (t, J = 33.1 Hz), 47.3 (t, J = 3.1 Hz), 46.9, 45.2 (t, J = 2.9 Hz), 34.6 (t, J = 24.3 Hz), 32.9 (t, J = 23.9 Hz). 19F NMR

(470 MHz, CDCl3) δ 65.5, 65.5, -99.0, -99.2, -101.3, -102.0. IR 2925, 1609, 1578, 1406, 1212, -1 + 1124, 1088, 759 cm . HRMS (ESI+): m/z calcd for C22H18 F5N6O2S [M+H] 525.1127, found 525.1129.

Synthesis of 37

A solution of NEt3 (6.7 mg, 0.066 mmol, 1.5 equiv) in CH2Cl2 (0.1 mL) was added to 33 (20 mg, 0.044 mmol, 1.0 equiv). Then, a solution of (S)-pyrrolidin-3-ol (4.2 mg, 0.048 mmol,

1.1 equiv) in CH2Cl2 (0.1 mL) was added. The reaction mixture was stirred for 30 min at rt, then directly subjected to flash chromatography on silica (50% EtOAc in hexanes) to afforded the title compound (16 mg, 0.032 mmol, 72%) as colorless oil.

Ca. 1:1 mixture of rotamers. All signals reported. 1 Rf = 0.40 (10% acetone in CH2Cl2; UV, KMnO4). H NMR (400 MHz, CDCl3) δ 8.16 (app dd, J = 7.7, 1.6 Hz, 1H), 7.69 (app td, J = 6.9, 2.2 Hz, 2H), 7.62 – 7.51 (m, 2H), 7.43 – 7.34 (m, 3H), 7.00 – 6.94 (m, 1H), 6.23 (s, 2H), 4.80 – 4.66 (m, 1H), 4.56 – 4.18 (m, 2H), 4.09 – 3.81 13 (m, 2H), 2.28 – 2.10 (m, 2H), 1.96 – 1.76 (2x br s, 1H). C NMR (101 MHz, CDCl3) δ 161.7 (t, J = 30.4 Hz), 161.7 (t, J = 30.5 Hz), 153.1, 153.1, 150.9, 150.9, 136.5 (t, J = 26.8 Hz), 136.0, 135.8, 135.8, 131.3 (d, J = 24.1 Hz), 130.9, 130.0, 130.0, 129.0, 128.2, 126.0 (t, J = 6.1 Hz), 126.0 (t, J = 6.0 Hz), 125.1, 124.9, 117.1 (t, J = 246.5 Hz), 71.3, 69.6, 57.8, 56.2, 47.5, 46.8, 45.8, 34.8, 32.9. 19F NMR (377 MHz, CDCl3) δ 65.4, 65.3, -98.9, -98.9, -99.0 (2 rotamers, benzylic F atoms diastereotopic). IR 3413 (br), 2928, 1611, 1586, 1405, 1212, 761, 299, 592 -1 + cm . HRMS (ESI+): m/z calcd for C22H20 F3N6O3S [M+H] 505.1264, found 505.1261.

Synthesis of 39

116

1-(Chloromethyl)-2-methylbenzene (3.35 g, 23.8 mmol, 1.00 equiv) and thiourea (1.81 g, 23.8 mmol, 1.00 equiv) were combined with EtOH (24 mL) and heated to reflux (oilbath 90 °C) for 1 h. The solvent was removed under reduced pressure to leave a colorless solid. MeCN (34 mL) and aq. HCl (2 M, 6.9 mL) were added and it was stirred until most of the solid was dissolved. NCS (12.7 g, 95.0 mmol, 4.00 equiv) was then added in portions at such a rate that the temperature did not rise above 23 °C (internal temperature ranged between 12 °C and 23 °C, help of an icebath if needed). After completion of the addition the neon-yellow mix was stirred for another 30 min (no cooling, internal temperature 12-16 °C). It was then poured into an addition funnel containing water (100 mL). The transfer was quantitated with the help of ether.

The aq. phase was then extracted with Et2O (3x 50 mL). The combined organics were washed with sat. aq. sodium bicarbonate (30 mL) and brine (30 mL), dried over MgSO4, filtered and concentrated to give of crude material (5.84 g, 79% pure by NMR, 95%), which was taken on as such for the formation of the sulfonyl fluoride.

The crude was dissolved in a mix of acetone (77 mL) and water (23 mL). Potassium fluoride (2.77 g, 47.6 mmol, 2.00 equiv) was added and it was stirred overnight at rt. The mix was poured into a separation funnel filled with water (300 mL), extracted with Et2O (3x 100 mL), washed with sat. aq. sodium bicarbonate, water and brine (30 mL each), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (5% EtOAc in hexanes) afforded the sulfonyl fluoride (2.04 g, 10.4 mmol, 46%) as colorless oil that solidified overnight.

1 Rf = 0.47 (10% EtOAc in hexanes; UV, KMnO4). H NMR (400 MHz, CDCl3) δ 7.41 – 7.32 (m, 2H), 7.30 – 7.25 (m, 2H), 4.66 (d, J = 2.9 Hz, 2H), 2.46 (s, 3H). 13C NMR (101 MHz,

CDCl3) δ 138.4, 131.9 (d, J = 1.3 Hz), 131.4, 130.3, 127.0, 124.1, 54.5 (d, J = 17.3 Hz), 19.6 19 (d, J = 1.3 Hz). F NMR (282 MHz, CDCl3) δ = 52.5. IR (neat) 2951, 1498, 1466, 1398, 1202, -1 + 1179, 792, 783, 725, 616, 567 cm . HRMS (EI+): m/z calcd for C8H9FO2S [M] 188.0307, found 188.0302.

Synthesis of 40

39 (188 mg, 1.00 mmol, 1.00 equiv), NBS (196 mg, 1.10 mmol, 1.10 equiv) and benzoyl peroxide (32 mg, 0.10 mmol, 0.10 equiv) were combined with CCl4 (5.0 mL) and heated to

117 reflux for 3 h. The reaction mix was cooled to rt. Celite was added and the volatiles were removed. Flash chromatography on silica (15% CH2Cl2 in hexanes) afforded the title compound as colorless solid (94% pure by NMR, 213 mg, 0.750 mmol, 75%). NMR indicates a small amount of residual starting material (ca. 6%).

1 Rf = 0.16 (20% CH2Cl2 in hexanes; UV, KMnO4). H NMR (400 MHz, CDCl3) δ 7.48 – 7.39 13 (m, 4H), 4.85 (d, J = 2.2 Hz, 2H), 4.63 (s, 2H). C NMR (101 MHz, CDCl3) δ 138.0, 133.1 (d, J = 1.1 Hz), 131.5, 130.9, 130.0, 124.9, 53.7 (d, J = 17.8 Hz), 30.7 (d, J = 1.2 Hz). IR (neat) 3032, 2988, 2939, 2855, 1496, 1455, 1394, 1201, 802, 776 cm-1. HRMS (EI+): m/z calcd for + C8H8BrFO2S [M] 265.9412, found 265.9407.

Synthesis of 41 and 42

15 (50.0 mg, 0.177 mmol, 1.00 equiv) and 40 (52.0 mg, 0.183 mmol, 1.03 equiv) were dissolved in DMF (0.6 mL). NEt3 (30 µl, 0.22 mmol, 1.2 equiv) was added at rt and stirred for 1 h, when TLC (20% EtOAc in hexanes) of a miniworkup (5% aq. LiCl / EtOAc) indicated almost complete consumption of the benzyl bromide. The reaction mix was diluted with EtOAc

(15 mL), washed with 5% aq. LiCl (3x 1 mL) and brine (2 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (10% EtOAc in hexanes to 15% to 20%) afforded 41 (15 mg, 0.032 mmol, 18%) as colorless oil and 42 (12 mg, 0.026 mmol, 14%) as colorless solid.

Data for 41, 1.27:1 mixture of rotamers. All signals reported. 1 Rf = 0.35 (20% EtOAc in hexanes; KMnO4, UV). H NMR (600 MHz, CDCl3) δ 7.82 (dd, J = 7.6, 1.2 Hz, 1H), 7.45 (ddd, J = 7.7, 6.6, 2.2 Hz, 1H), 7.42 – 7.37 (m, 2H), 5.86 (s, 2H), 5.43 (d, J = 1.8 Hz, 2H), 4.57 – 4.44 (m, 2H), 4.17 – 4.02 (m, 2H), 2.64 – 2.44 (m, 2H), 1.40 (d, J = 13 3.1 Hz, 9H). C NMR (151 MHz, CDCl3) δ 176.3, 152.4, 152.4, 150.3, 150.2, 135.6, 132.9, 132.7, 130.8, 129.8, 127.6 (t, J = 248.2 Hz), 127.0 (t, J = 247.2 Hz), 125.1, 123.9, 123.9, 55.5 (t, J = 32.8 Hz), 54.4, 54.3, 53.7 (t, J = 32.8 Hz), 47.1, 46.8, 44.6, 39.9, 39.9, 34.6 (t, J = 24.2 19 Hz), 33.0 (t, J = 23.8 Hz), 29.8. F NMR (376 MHz, CDCl3) δ = 52.4, -101.3, -101.9. IR (neat) 2961, 2927, 1601, 1579, 1483, 1405, 1319, 1207, 1126, 807, 732, 540 cm-1. HRMS (ESI+): + m/z calcd for C20H24F3N6O2S [M+H] 469.1628, found 469.1624.

118

Data for 42, 1.25:1 mixture of rotamers. All signals reported. 1 Rf = 0.22 (20% EtOAc in hexanes; KMnO4, UV). H NMR (600 MHz, CDCl3) δ 7.60 (br, 1H), 7.48 – 7.40 (m, 3H), 5.93 (s, 2H), 5.21 (br, 2H), 4.47 – 4.33 (m, 2H), 4.17 – 4.02 (m, 2H), 2.67 13 – 2.45 (m, 2H), 1.38 (s, 9H). C NMR (151 MHz, CDCl3) δ 176.0, 160.1, 152.8, 134.5, 133.2, 132.3, 130.9, 130.3, 127.7 (t, J = 248.3 Hz), 126.9 (t, J = 247.3 Hz), 125.5, 125.4, 57.8, 55.4 (t, J = 32.8 Hz), 54.3, 54.2, 53.8 (t, J = 32.7 Hz), 46.6, 44.7, 39.8, 34.7 (t, J = 24.1 Hz), 33.0 (t, J 19 = 23.5 Hz), 29.7. F NMR (376 MHz, CDCl3) δ = 52.6, -101.0, -101.8. IR (neat) 2959, 2928, 1593, 1566, 1484, 1405, 1340, 1206, 1165, 1129, 810, 731 cm-1. HRMS (ESI+): m/z calcd for + C20H24F3N6O2S [M+H] 469.1628, found 469.1623.

Synthesis of 43 and 44

16 (55 mg, 0.21 mmol, 1.0 equiv) and 40 (60 mg, 0.21 mmol, 1.0 equiv) were dissolved in DMF (0.7 mL). DIPEA (44 L, 0.25 mmol, 1.2 equiv) was added and it was stirred overnight at rt. The reaction mix was diluted with EtOAc (60 mL), washed with 5% aq. LiCl (2x 10 mL) and brine (10 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (40% EtOAc in hexanes + 10% AcOH) followed by washing of the respective combined fractions with sat. aq. sodium bicarbonate, drying over MgSO4, filtration and concentration afforded 43 (31 mg, 0.069 mmol, 33%) and 44 (18 mg, 0.040 mmol, 19%) as colorless oils.

Data for 43, 1:1 mixture of rotamers. All signals reported. 1 Rf = 0.36 (40% EtOAc in hexanes + 10% AcOH; UV, KMnO4). H NMR (400 MHz, CDCl3) δ 7.84 – 7.78 (m, 1H), 7.47 – 7.33 (m, 3H), 5.83 (s, 2H), 5.52 – 5.41 (m, 2H), 4.74 – 4.61 (m, 1H), 4.46 – 4.31 (m, 1H), 4.29 – 4.13 (m, 1H), 4.04 – 3.79 (m, 2H), 2.24 – 2.06 (m, 2H), 1.40 13 (d, J = 3.2 Hz, 9H). C NMR (101 MHz, CDCl3) δ 176.1, 152.5, 150.1, 135.8, 132.8, 132.7, 130.7, 129.7, 125.1, 124.1, 124.0, 71.4, 69.8, 57.4, 55.6, 54.5, 54.3, 47.0, 46.9, 45.2, 39.8, 39.8, 19 34.8, 32.8, 29.9. F NMR (282 MHz, CDCl3) δ = 52.3. IR (neat) 3403 (br), 2959, 2928, 1602, 1576, 1483, 1403, 1317, 1205, 910, 506, 730, 541 cm-1. HRMS (ESI+): m/z calcd for + C20H26FN6O3S [M+H] 449.1766, found 449.1767.

Data for 44, 1:1 mixture of rotamers. All signals reported.

119

1 Rf = 0.20 (40% EtOAc in hexanes + 10% AcOH; UV, KMnO4). H NMR (400 MHz, CDCl3) δ 7.63 – 7.55 (m, 1H), 7.46 – 7.37 (m, 3H), 5.90 (s, 0.5H), 5.88 (s, 0.5H), 5.27 – 5.15 (m, 2H), 4.78 – 4.65 (m, 1H), 4.34 – 4.07 (m, 2H), 4.05 – 3.80 (m, 2H), 2.26 – 2.09 (m, 2H), 1.37 (d, J 13 = 3.6 Hz, 9H). C NMR (101 MHz, CDCl3) δ 176.1, 160.1, 153.0, 134.7, 133.1, 132.2, 130.8, 130.2, 125.8, 125.8, 125.5, 71.5, 69.7, 57.6, 57.0, 55.8, 54.2 (d, J = 17.2 Hz), 46.8, 45.3, 39.8, 19 34.8, 33.0, 29.7. F NMR (282 MHz, CDCl3) δ = 52.5. IR (neat) 3343, 2958, 1596, 1563, -1 + 1405, 1327, 1205, 910, 809, 731 cm . HRMS (ESI+): m/z calcd for C20H26FN6O3S [M+H] 449.1766, found 449.1764.

Synthesis of 46

45 (3.20 g, 17.2 mmol, 1.00 equiv) and NEt3 (3.6 mL, 26 mmol, 1.5 equiv) were dissolved in

CH2Cl2 (60 mL) and cooled to 0 °C. MsCl (1.47 mL, 18.9 mL, 1.10 equiv) was added dropwise and it was stirred for 15 min when TLC indicated full consumption of starting material. The reaction was quenched by the addition of water, transferred to a separation funnel (rinsing with

CH2Cl2). The organics were washed with water (50 mL) and brine (50 mL), dried over MgSO4, filtered and concentrated to give crude material. Flash chromatography on silica (40% EtOAc in hexanes) afforded the title compound (3.91 g, 14.8 mmol, 86%) as colorless oil.

1 Rf = 0.43 (50% EtOAc in hexanes; UV, KMnO4). H NMR (400 MHz, CDCl3) δ 8.07 (app d, J = 7.8, 0.9 Hz, 1H), 7.72 – 7.64 (m, 2H), 7.63 – 7.57 (m, 1H), 5.68 (s, 2H), 3.14 (s, 3H), 3.11 13 (s, 3H). C NMR (101 MHz, CDCl3) δ 138.8, 134.4, 132.9, 131.7, 130.3, 130.3, 68.5, 45.3, 37.8. IR 3029, 2938, 1353, 1306, 1174, 1153, 1129, 957, 939, 820, 751, 548, 528, 515 cm-1. + HRMS (MALDI): m/z calcd for C9H12NaO5S2 [M] 287.0018, found 287.0018.

Synthesis of 47

According to GP1 from 46 (3.86 g, 14.6 mmol) to give the title compound (2.76 g, 9.35 mmol, 64%) as a colorless solid.

1 H NMR (400 MHz, CDCl3) δ 8.03 (app d, J = 7.9 Hz, 1H), 7.65 (app dt, J = 33.4, 7.6 Hz, 2H), 7.54 (s, 1H), 7.17 (s, 1H), 6.70 (app d, J = 7.8 Hz, 1H), 6.50 (s, 2H), 5.88 (s, 2H), 3.37 (s, 3H).

120

13 C NMR (101 MHz, CDCl3) δ 164.2, 145.6, 137.8, 135.0, 134.3, 129.7, 128.7, 127.9, 121.8, 45.7, 44.1. IR 3453, 3408, 3320, 3162, 1654, 1629, 1568, 1516, 1301, 1291, 1251, 1149, 1128, -1 + 961, 783, 751 cm . HRMS (ESI+): m/z calcd for C11H14N5O3S [M+H] 296.0812, found 296.0820.

Synthesis of 48

According to GP1 from 47 (1.00 g, 3.39 mmol) to give 48 (870 mg, 2.41 mmol, 71%) as beige solid.

1H NMR (400 MHz, DMSO) δ 12.23 (s, 1H), 8.04 (dd, J = 7.8, 1.6 Hz, 1H), 7.73 – 7.60 (m, 2H), 7.23 (dd, J = 7.6, 1.5 Hz, 1H), 6.21 (s, 2H), 3.41 (s, 3H), 1.31 (s, 9H). 13C NMR (101 MHz, DMSO) δ 168.8, 156.4, 148.8, 138.4, 134.3, 134.1, 130.1, 129.4, 129.1, 127.9, 46.0, 44.3, 37.7, 27.8. IR 3191, 3123, 2965, 1694, 1573, 1373, 1308, 1150, 1083, 956, 777, 743, 538, 531 -1 + cm . HRMS (MALDI): m/z calcd for C16H20N5O3S [M+H] 362.1281, found 362.1280.

Synthesis of 49

47 (500 mg, 1.7 mmol, 1.00 equiv) was added to a solution of PhCF2CN (389 mg, 2.54 mmol,

1.5 equiv)) in DMF (5.6 mL). K2CO3 (1.2 g, 8.5 mmol, 5 equiv) was added, the flask was sealed and heated at 80 °C for 4 h. The reaction mixture was cooled to rt and poured on icewater (100 mL). It was neutralized with aq. HCl (2 M). The resulting precipitate was filtered, washed with water, EtOH, and ether to afford the title compound as colorless solid (530 mg, 1.23 mmol, 73%).

IR 3076, 3014, 2976, 2920, 1728, 1589, 1567, 1531, 1308, 1262, 1153, 1099, 1011, 906, 749, -1 + 690, 535 cm . HRMS (MALDI): m/z calcd for C16H20N5O3S [M+H] 432.0964, found 432.0960.

Synthesis of 50

121

48 (250 mg, 0.692 mmol, 1.00 equiv) was dissolved in a mixture of CH2Cl2 (1 mL), toluene

(1 mL) and DMF (2 mL). (COCl)2 (121 L, 1.38 mmol, 2.00 equiv) was added and it was stirred overnight at rt. The reaction mixture was diluted with EtOAc (80 mL), washed with 5% aq. LiCl (2x 20 mL) and brine (20 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (25% EtOAc in hexanes) afforded the title compound (240 mg, 0.632 mmol, 91%) as colorless foam.

1 Rf = 0.32 (25% EtOAc in hexanes; UV, weak KMnO4). H NMR (400 MHz, CDCl3) δ 8.16 – 8.10 (m, 1H), 7.59 – 7.50 (m, 2H), 7.40 – 7.34 (m, 1H), 6.42 (s, 2H), 3.39 (s, 3H), 1.43 (s, 10H). 13 C NMR (101 MHz, CDCl3) δ 177.3, 153.6, 150.5, 138.8, 134.5, 134.1, 132.3, 130.9, 130.3, 129.6, 46.8, 45.3, 40.4, 29.7. IR 2967, 2927, 1592, 1561, 1402, 1310, 1149, 1129, 1075, 991, -1 + 885, 736, 531 cm . HRMS (ESI+): m/z calcd for C16H19ClN5O2S [M+H] 380.0942, found 380.0941.

Synthesis of 51

49 (200 mg, 0.464 mmol, 1.00 equiv) was dissolved in a mixture of DMF (2 mL) and toluene

(2 mL). (COCl)2 (81 L, 0.93 mmol, 2.0 equiv) was added slowly. After the reaction mixture was stirred for 2 h at rt, it was diluted with EtOAc (100 mL), washed with 5% aq. LiCl (3x

20 mL) and brine (20 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (20% EtOAc in hexanes to 50%) afforded the title compound (190 mg, 0.422 mmol, 91%) as colorless foam.

1 Rf = 0.13 (20% EtOAc in hexanes; UV). H NMR (400 MHz, CDCl3) δ 8.15 – 8.10 (m, 1H), 7.63 (app dd, J = 7.7, 1.9 Hz, 2H), 7.57 – 7.52 (m, 2H), 7.47 – 7.33 (m, 4H), 6.49 (s, 2H), 3.40 13 (s, 3H). C NMR (101 MHz, CDCl3) δ 161.2 (t, J = 32.7 Hz), 155.6, 150.1, 139.0, 134.8 (d, J = 27.0 Hz), 134.5, 133.8, 133.3, 130.9, 130.8, 130.4, 129.8, 128.7, 125.8 (t, J = 6.0 Hz), 116.5 19 (t, J = 247.9 Hz), 47.2, 45.3. F NMR (376 MHz, CDCl3) δ -97.9. IR 3018, 2928, 1733, 1587,

122

1570, 1311, 1262, 1153, 1131, 1076, 1005, 973, 783, 771, 748, 599, 540, 533 cm-1. HRMS + (ESI+): m/z calcd for C19H15 ClF2N5O2S [M+H] 450.0598, found 450.0603.

Synthesis of 52

50 (30 mg, 0.079 mmol, 1.00 equiv) and 3,3-difluoropyrrolidine·HCl (13 mg, 0.12 mmol,

1.5 equiv) were suspended in CH2Cl2 (0.5 mL). NEt3 (28 L, 0.20 mmol, 2.5 equiv) was added. After 1 h, the reaction mix was diluted with EtOAc (20 mL), washed with aq. HCl (1 M, 2x

5 mL) and brine (5 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (20% EtOAc in hexanes) afforded the title compound (31 mg, 0.069 mmol, 87%) as colorless oil.

Mixture of rotamers (ca. 1.2:1). All signals reported. 1 Rf = 0.34 (25% EtOAc in hexanes; UV, KMnO4). H NMR (400 MHz, CDCl3) δ 8.11 (dd, J = 7.6, 1.7 Hz, 1H), 7.55 – 7.46 (m, 2H), 7.44 (d, J = 7.5 Hz, 1H), 6.29 (s, 2H), 4.62 – 4.49 (m, 2H), 4.20 – 4.05 (m, 2H), 3.48 (s, 3H), 2.69 – 2.45 (m, 2H), 1.34 (s, 9H). 13C NMR (101 MHz,

CDCl3) δ 176.5, 152.4, 151.0, 138.6, 135.5, 134.3, 130.9, 129.9, 129.0, 127.7 (d, J = 248.3 Hz), 127.0 (t, J = 247.1 Hz), 124.0, 55.5 (t, J = 33.2 Hz), 53.8 (t, J = 32.7 Hz), 46.8, 45.9, 45.3, 44.6, 19 39.9, 34.6 (t, J = 24.3 Hz), 33.0 (t, J = 23.8 Hz), 29.8. F NMR (376 MHz, CDCl3) δ -101.3, - 101.8. IR 2960, 2926, 1599, 1578, 1481, 1391, 1153, 1126, 920, 750, 732, 540, 532 cm-1. + HRMS (ESI+): m/z calcd for C20H25F2N6O2S [M+H] 451.1722, found 451.1724.

Synthesis of 53

50 (30 mg, 0.079 mmol, 1.00 equiv) and (S)-pyrrolidin-3-ol (10 mg, 0.12 mmol, 1.5 equiv) were dissolved in CH2Cl2 (0.5 mL). NEt3 (17 L, 0.12 mmol, 1.5 equiv) was added. After 1 h,

123 the reaction mix was concentrated. Flash chromatography on silica (70% EtOAc in hexanes) afforded the title compound (33 mg, 0.077 mmol, 97%) as colorless oil.

Mixture of rotamers (ca. 1.1:1). All signals reported. 1 Rf = 0.46 (70% EtOAc in hexanes; UV, KMnO4). H NMR (400 MHz, CDCl3) δ 8.11 (dd, J = 7.6, 1.7 Hz, 1H), 7.55 – 7.46 (m, 2H), 7.44 (d, J = 7.5 Hz, 1H), 6.29 (s, 2H), 4.62 – 4.49 (m, 2H), 4.20 – 4.05 (m, 2H), 3.48 (s, 3H), 2.69 – 2.45 (m, 2H), 1.34 (s, 9H). 13C NMR (101 MHz,

CDCl3) δ 176.4, 176.3, 152.6, 152.6, 150.9, 138.5, 135.7, 135.7, 134.2, 131.0, 130.9, 129.8, 128.9, 124.2, 124.1, 71.4, 69.7, 57.4, 55.6, 47.0, 45.8, 45.3, 39.8, 39.8, 34.8, 32.8, 29.8. IR 3429, 2958, 2925, 1602, 1575, 1482, 1310, 1152, 913, 732, 540, 532 cm-1. HRMS (ESI+): m/z + calcd for C20H27N6O3S [M+H] 431.1860, found 431.1858.

Synthesis of 54

51 (30 mg, 0.067 mmol, 1.0 equiv) and 3,3-difluoropyrrolidine·HCl (8.6 mg, 0.080 mmol,

1.2 equiv) were combined with CH2Cl2 (0.5 mL) and NEt3 (17 L, 0.17 mmol, 2.5 equiv) was added. After 30 min the reaction mixture was concentrated. Flash chromatography on silica (30% EtOAc in hexanes) afforded the title compound (30 mg, 0.058 mmol, 86%) as colorless oil.

Mixture of rotamers (ca. 1.2:1). All signals reported. 1 Rf = 0.30 (30% EtOAc in hexanes; UV, KMnO4). H NMR (400 MHz, Acetone) δ 8.13 – 8.08 (m, 1H), 7.69 – 7.58 (m, 4H), 7.45 (dd, J = 5.0, 2.2 Hz, 3H), 7.32 – 7.25 (m, 1H), 6.38 (s, 2H), 4.69 – 4.53 (m, 2H), 4.20 – 4.03 (m, 2H), 3.50 (s, 3H), 2.81 – 2.59 (m, 2H). 13C NMR (101 MHz, Acetone) δ 161.7 (t, J = 30.3 Hz), 161.7 (t, J = 30.6 Hz), 153.9, 153.9, 151.1, 151.1, 140.1, 137.2 (d, J = 27.1 Hz), 137.2 (t, J = 27.2 Hz), 135.8, 135.7, 135.0, 131.1, 131.0, 130.7, 129.9, 129.2, 128.9 (d, J = 247.4 Hz), 128.1 (t, J = 246.0 Hz), 126.6 (t, J = 6.0 Hz), 117.9 (d, J = 245.7 Hz), 117.9 (t, J = 245.5 Hz), 56.1 (t, J = 33.2 Hz), 54.4 (t, J = 33.2 Hz), 48.1 (t, J = 3.4 Hz), 47.0, 46.0 (t, J = 3.3 Hz), 45.2, 34.7 (t, J = 24.0 Hz), 33.0 (t, J = 23.6 Hz). 19F NMR (377 MHz, Acetone) δ -98.6, -98.8, -102.2, -102.9. IR 3008, 2924, 1610, 1578, 1350, 1312, 1153,

124

-1 + 1127, 752 cm . HRMS (ESI+): m/z calcd for C23H21F4N6O2S [M+H] 521.1377, found 521.1380.

Synthesis of 55

51 (29 mg, 0.064 mmol, 1.0 equiv) and (S)-pyrrolidin-3-ol (6.7 mg, 0.077 mmol, 1.2 equiv) were combined with CH2Cl2 (0.3 mL) and NEt3 (13 L, 0.097 mmol, 1.5 equiv) was added. After 30 min the reaction mixture was concentrated. Flash chromatography on silica (70% EtOAc in hexanes) afforded the title compound (27 mg, 0.054 mmol, 84%) as colorless solid.

A rotamer mixture of ca. 1.2:1. All signals reported. 1 Rf = 0.34 (70% EtOAc in hexanes; UV, KMnO4). H NMR (400 MHz, Acetone) δ 8.12 – 8.08 (m, 1H), 7.68 – 7.63 (m, 2H), 7.63 – 7.59 (m, 2H), 7.46 – 7.40 (m, 3H), 7.31 – 7.27 (m, 1H), 6.35 (s, 2H), 4.75 – 4.60 (m, 1H), 4.48 – 4.16 (m, 3H), 3.98 – 3.74 (m, 2H), 3.51 (d, J = 0.7 Hz, 3H), 2.34 – 2.07 (m, 2H). 13C NMR (101 MHz, Acetone) δ 161.7 (t, J = 30.1 Hz), 161.7 (t, J = 30.1 Hz), 153.9, 153.8, 151.0, 151.0, 140.0, 137.4 (t, J = 27.2 Hz), 136.0 (d, J = 1.4 Hz), 134.9, 131.0, 130.9, 130.6, 129.8, 129.1, 126.6 (t, J = 6.1 Hz), 126.5 (t, J = 6.0 Hz), 125.8, 125.6, 118.0 (t, J = 245.4 Hz), 71.2, 69.5, 58.5, 56.8, 48.4, 46.8, 46.6, 45.2, 35.0, 33.2. 19F NMR (377 MHz, Acetone) δ -98.5, -98.6, -98.6. IR 3436 (br), 2925, 1698, 1611, 1576, 1525, 1310, 1153 cm-1. + HRMS (ESI+): m/z calcd for C23H23F2N6O3S [M+H] 501.1515, found 501.1519.

Synthesis of 57

The title compound was prepared according to a patent procedure (WO2009/103440 A1, 2009) from 1,3-dibromo-2-methylbenzene (4.41 g, 17.6 mmol, 1.00 equiv), DIPEA (4.9 mL,

28.2 mmol, 1.6 equiv), Xantphos (490 mg, 0.85 mmol, 4.8 mol%) and Pd2(dba)3 (388 mg, 0.423 mmol, 2.4 mol%) and isolated as colorless solid.

1 Rf = 0.46 (hexanes; weak UV, KMnO4). H NMR (400 MHz, CDCl3) δ 7.37 (dd, J = 8.0, 1.1 Hz, 1H), 7.30 – 7.21 (m, 5H), 7.19 (dd, J = 7.8, 1.2 Hz, 1H), 6.93 (td, J = 7.9, 0.7 Hz, 1H), 4.04 13 (s, 2H), 2.44 (s, 3H). C NMR (101 MHz, CDCl3) δ 138.0, 137.4, 136.8, 130.6, 129.0, 128.7,

125

128.4, 127.5, 127.3, 125.9, 39.1, 20.7. IR (neat) 3056, 3029, 2919, 1554, 1492, 1452, 1427, -1 + 1070, 762, 734, 714, 694, 489 cm . HRMS (ESI+): m/z calcd for C14H13BrS [M+H] 291.9916, found 267.8955.

Synthesis of 3-bromo-2-methylbenzenesulfonyl chloride

According to Patent WO2009/103440 A1, 2009, Page/Page column 85 from thioether (1.46 g, 5.0 mmol) to give the product as colorless solid (727 mg, 2.7 mmol, 54%) after flash chromatography on silica (0.6% EtOAc in hexanes).

1 H NMR (400 MHz, CDCl3) δ 8.07 (dd, J = 8.1, 1.3 Hz, 1H), 7.92 (dd, J = 8.0, 1.2 Hz, 1H), 13 7.28 (td, J = 8.0, 0.8 Hz, 1H), 2.87 (s, 3H). C NMR (101 MHz, CDCl3) δ 144.6, 139.5, 137.8, 128.8, 128.2, 127.5, 20.4. IR (neat) 2930, 2862, 2102, 1732, 1605, 1572, 1453, 1411, 1380, -1 + 1239, 1123 cm . HRMS (MALDI): m/z calcd for C7H6BrClO2S [M] 267.8955, found 267.8955.

Synthesis of 58

3-Bromo-2-methylbenzenesulfonyl chloride (727 mg, 2.70 mmol, 1.00 equiv) was dissolved in acetone (8.5 mL). KF (783 mg, 13.5 mmol, 5.00 equiv) was added followed by water (0.45 mL) at rt. After stirring overnight, the reaction mixture was concentrated in a stream of nitrogen, then partitioned between EtOAc (100 mL) and water (20 mL). The aq. phase was discarded.

The organics were washed with brine (20 mL), dried over MgSO4, filtered and concentrated to leave the product (628 mg, 2.48 mmol, 92%) as colorless solid.

1 Rf = 0.16 (1% EtOAc in hexanes; weak UV, KMnO4). H NMR (400 MHz, CDCl3) δ 8.03 (dd, J = 8.1, 1.2 Hz, 1H), 7.93 (dd, J = 8.0, 1.2 Hz, 1H), 7.28 (dd, J = 8.1 Hz, 1H), 2.76 (s, 3H). 13C

NMR (101 MHz, CDCl3) δ 139.7, 138.7 (d, J = 1.0 Hz), 134.3 (d, J = 22.9 Hz), 129.5 (d, J = 1.8 Hz), 128.3 (d, J = 1.2 Hz), 127.6 (d, J = 1.1 Hz), 20.6 (d, J = 1.8 Hz). 19F NMR (376 MHz, - CDCl3) δ 60.3. IR 3103, 1436, 1400, 1387, 1217, 1200, 806, 773, 734, 694, 607, 558, 471 cm 1 + . HRMS (MALDI): m/z calcd for C7H6 BrFO2S [M] 251.9250, found 251.9250.

Synthesis of 59

126

58 (628 mg, 2.48 mmol, 1.00 equiv), (PPh3)2PdCl2 (174 mg, 248 mmmol, 0.10 equiv) and CuI (372 mg, 0.372 mmol, 0.15 equiv) were placed in a 25 mL pear-shaped flask. After evacuation and backfilling with nitrogen (2x), MeCN (12.4 mL) was added, followed by DIPEA (0.87 mL, 5.0 mmol, 2.0 equiv). Nitrogen was bubbled through the black solution for 5 min, before TMS- acetylene (0.70 mL, 5.0 mmol, 2.0 equiv) was added. The flask was capped and placed in a preheated oilbath (50 °C). After 3h, an aliquot (0.1 mL, filtered over celite and concentrated) was analyzed by NMR and indicated residual starting material. Additional TMS-acetylene (0.70 mL, 5.0 mmol, 2.0 equiv) was added and stirring was continued at 50 °C overnight. After 24 h, the reaction mixture was cooled to rt, filtered over celite, and extracted (water/EtOAc).

The organics were washed with water and brine, dried over MgSO4, filtered and concentrated. Flash chromatography on silica (0.7% EtOAc in hexanes) afforded the target compound (610 mg, 2.12 mmol, 85% corrected) containing starting material (6% by NMR) as brown oil that solidified upon standing.

1 Rf = 0.18 (1% EtOAc in hexanes; UV, KMnO4). H NMR (400 MHz, CDCl3) δ 7.99 (dd, J = 8.1, 1.6 Hz, 1H), 7.78 (dd, J = 7.8, 1.3 Hz, 1H), 7.35 (dd, J = 8.0 Hz, 1H), 2.80 (s, 3H), 0.28 (s, 13 9H). C NMR (101 MHz, CDCl3) δ 141.4, 138.9, 133.4 (d, J = 22.6 Hz), 129.9, 127.1, 126.2, 19 102.5, 101.3, 18.4, -0.1. F NMR (376 MHz, CDCl3) δ 60.0. IR 2962, 2164, 1456, 1438, 1405, -1 1251, 1215, 883, 843, 807, 762, 680, 590, 580 cm . HRMS (MALDI): m/z calcd for C12H15 + FO2SSi [M] 270.0541, found 270.0541.

Synthesis of 60 and 61

59-Br (30 mg, 0.086 mmol, 1.00 equiv) and NEt3 (18 L, 0.13 mmol, 1.5 equiv) were dissolved in DMF (0.43 mL) and 15 (27 mg, 0.094 mmol, 1.1 equiv) was added at rt. After 30 min, the reaction mixture was diluted with EtOAc (100 mL), washed with 5% aq. LiCl (2x 20 mL) and brine (20 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (5% EtOAc in hexanes) afforded a mix of inseparable regioisomers (14 mg, 0.025 mmol, 30%). The mixture of regioisomers (14 mg, 0.025 mmol, 1.0 equiv) was dissolved in THF (0.4 mL) and cooled to 0 °C. A solution of NEt3·3HF (8.2 mg, 0.051 mmol, 2.0 equiv) in THF (0.1 mL)

127 was added. TLC indicated full conversion after 4 h. The reaction mix was diluted with EtOAc

(15 mL), washed with sat. sodium bicarbonate and brine (3 mL each), dried over MgSO4, filtered and concentrated. Preparative TLC (25% EtOAc in hexanes) afforded 60 (5 mg, 0.01 mmol, 41%) and 61 (6 mg, 0.013 mmol, 49%) as colorless oils.

Data for 60. A mixture of rotamers (ca. 1.3:1). All signals reported. 1 Rf = 0.33 (20% EtOAc in hexanes; UV, KMnO4). H NMR (600 MHz, CDCl3) δ 8.20 (dd, J = 8.1, 1.3 Hz, 1H), 7.89 (dd, J = 7.8, 1.3 Hz, 1H), 7.62 (app td, J = 8.0, 1.0 Hz, 1H), 6.24 (s, 2H), 4.58 – 4.45 (m, 2H), 4.16 – 4.02 (m, 2H), 3.21 (s, 1H), 2.53 (dtt, J = 58.9, 13.8, 7.4 Hz, 2H), 13 1.29 (s, 9H). C NMR (151 MHz, CDCl3) δ 175.8, 152.4, 151.2, 139.8, 136.5, 134.8 (d, J = 24.3 Hz), 130.9, 129.4, 127.8, 127.1 (t, J = 248.0 Hz), 123.5, 85.7, 78.9, 55.4 (t, J = 32.6 Hz), 53.7 (t, J = 32.7 Hz), 46.7, 45.9, 44.5, 39.7, 34.7 (t, J = 24.1 Hz), 33.0 (t, J = 23.6 Hz), 29.7. 19F

NMR (565 MHz, CDCl3) δ 66.2, -101.2, -101.7. IR 3302, 2961, 2927, 2899, 1770, 1759, 1601, -1 + 1580, 1410, 1323, 1216, 1127, 774 cm . HRMS (ESI+): m/z calcd for C21H22F3N6O2S [M+H] 479.1472, found 479.1474.

Data for 61. A mixture of rotamers (ca. 1.3:1). All signals reported. 1 Rf = 0.21 (20% EtOAc in hexanes; UV, KMnO4). H NMR (600 MHz, CDCl3) δ 8.19 (d, J = 8.6 Hz, 1H), 7.96 (dd, J = 7.8, 1.3 Hz, 1H), 7.66 (app t, J = 8.1 Hz, 1H), 6.40 (s, 2H), 4.37 – 4.22 (m, 2H), 4.17 – 4.01 (m, 2H), 3.36 (s, 1H), 2.61 – 2.45 (m, 2H), 1.37 (s, 9H). 13C NMR

(151 MHz, CDCl3) δ 175.6, 159.8, 152.8, 140.0, 134.7, 134.5 (d, J = 24.4 Hz), 131.0, 130.0, 128.5, 127.8 (t, J = 250.0 Hz), 127.1 (t, J = 247.5 Hz), 125.3, 85.9, 78.9, 55.2 (t, J = 32.6 Hz), 54.7, 53.7 (t, J = 32.6 Hz), 46.4, 44.6, 39.8, 34.7 (t, J = 24.3 Hz), 33.0 (t, J = 23.9 Hz), 29.7. 19F

NMR (565 MHz, CDCl3) δ 66.6, -101.0, -101.7. IR 3299, 2980, 2961, 1770, 1759, 1596, 1567, -1 + 1410, 1241, 1219, 1130, 776, 590 cm . HRMS (ESI+): m/z calcd for C21H22F3N6O2S [M+H] 479.1472, found 479.1467.

Synthesis of 62-a

2,6-Diiodobenzoic acid (11.29 g, 30.20 mmol, 1.00 equiv) was suspended in CH2Cl2 (60 mL) and DMF (117 L, 1.51 mmol, 0.05 equiv) was added. (COCl)2 (2.70 mL, 30.8 mmol, 1.02 equiv) was added at rt. After bubbling ceased, the mix was heated to reflux for 30 min

(TLC indicated clean conversion to acid chloride). The mix was further diluted with CH2Cl2

(100 mL), washed with sat. aq. sodium bicarbonate and brine (50 mL each), dried over MgSO4, filtered and concentrated. The crude acid chloride was dissolved in a mixture of acetonitrile (15

128 mL) and THF (15 mL) and NaBH4 (2.29 g, 60.4 mmol, 2.00 equiv) was added in portions at rt (exotherm, temp rises to ca. 45 °C). When TLC indicated full conversion of intermediate acid chloride (ca. 30 min), MeOH (1.22 mL, 30.2 mmol, 1.00 equiv) was added slowly, followed by sat. NH4Cl (50 mL) and water (50 mL). Extration with EtOAc (3x100 mL), washing with brine

(1x 100 mL), drying over MgSO4, filtration and concentration followed by flash chromatography on silica (15% EtOAc in hexanes to 20% to 50%) afforded the title compound (8.60 g, 23.9 mmol, 79%) as colorless solid.

1 Rf = 0.43 (20% EtOAc in hexanes; UV, KMnO4). H NMR (400 MHz, CDCl3) δ 7.85 (d, J = 7.9 Hz, 2H), 6.62 (t, J = 7.9 Hz, 1H), 5.03 (d, J = 6.9 Hz, 2H), 2.16 (t, J = 6.9 Hz, 1H). 13C

NMR (101 MHz, CDCl3) δ 143.6, 140.4, 131.5, 99.8, 75.0. IR 3349, 2959, 1541, 1419, 1187, -1 + 1034, 1006, 775, 682 cm . HRMS (MALDI): m/z calcd for C7H6I2NaO [M+Na] 382.8399, found 382.8400.

Synthesis of 62-b

(2,6-diiodophenyl)methanol (2.72 g, 7.56 mmol, 1.00 equiv) was dissolved in DMF (12.6 mL) and cooled to 0 °C. NaH (60% suspension in mineral oil, 332 mg, 8.31 mmol, 1.10 equiv) was added in portions and stirred for 20 min. PMB-Cl (0.670 mL, 4.92 mmol, 1.10 equiv) was added and the cooling bath was removed. After 25 min, TLC of a miniworkup (5% aq. LiCl/EtOAc) indicated full consumption of starting material. The reaction was quenched by the addition of sat. NH4Cl (5 mL). Extraction with EtOAc (3x 50 mL), washing with 5% aq. LiCl

(50 mL) and brine (50 mL), drying over MgSO4, filtration and concentration. Flash chromatography on silica (5% EtOAc in hexanes) afforded the title compound (3.18 g, 6.62 mmol, 88%) as colorless solid.

1 Rf = 0.22 (5% EtOAc in hexanes; UV, KMnO4). H NMR (400 MHz, CDCl3) δ 7.85 (d, J = 7.9 Hz, 2H), 6.62 (t, J = 7.9 Hz, 1H), 5.03 (app d, J = 6.9 Hz, 2H), 2.16 (app t, J = 6.9 Hz, 1H). 13C

NMR (101 MHz, CDCl3) δ 159.5, 141.6, 140.3, 131.4, 130.2, 129.9, 113.9, 100.8, 81.0, 72.9, 55.4. IR 2998, 2952, 2906, 2853, 2834, 1612, 1544, 1513, 1423, 1248, 1079, 1035, 818, 771, 683 cm-1.

Synthesis of 62-c

129

62-b (1.90 g, 3.96 mmol, 1.00 equiv) was dissolved in THF (8 mL) and cooled to –30 °C. A THF solution of iPrMgCl·LiCl (0.87 M, 5.00 mL, 4.35 mmol, 1.10 equiv) was added and the resulting mixture was stirred for 2.5 h (temperature was allowed to rise to –20 °C). 2,2,2- Trifluoro-N-methoxy-N-methylacetamide (684 mg, 4.35 mmol, 1.10 equiv) was added and it was stirred overnight (allowed to reach room temperature). The reaction was quenched by the addition of sat. NH4Cl. Extraction with EtOAc, washing with brine, drying over MgSO4, filtration and concentration followed by flash chromatography on silica afforded the title compound (1.04 g, 2.31 mmol, 58%) as colorless oil.

1 Rf = 0.38 (50% CH2Cl2 in hexanes; UV, KMnO4). H NMR (400 MHz, CDCl3) δ 7.95 (dd, J = 7.9, 1.2 Hz, 1H), 7.35 (dd, J = 7.6, 1.0 Hz, 1H), 7.25 (d, J = 8.1 Hz, 2H), 7.09 (t, J = 7.8 Hz, 1H), 6.90 (d, J = 8.7 Hz, 2H), 4.57 (s, 2H), 4.48 (s, 2H), 3.82 (s, 3H). 13C NMR (101 MHz,

CDCl3) δ 185.9 (q, J = 36.4 Hz), 159.7, 142.1, 140.4, 134.0, 130.1, 129.0, 128.4, 127.5, 116.3 19 (q, J = 291.1 Hz), 114.0, 96.7, 74.5, 73.0, 55.4. F NMR (376 MHz, CDCl3) δ -76.16. IR 2936, 2866, 2838, 1737, 1710, 1312, 1513, 1248, 1207, 1174, 1141, 1113, 1034, 947, 818, 762, 749, 715, 688, 571, 521 cm-1.

Synthesis of 2,2,2-trifluoro-1-(3-iodo-2-(((4-methoxybenzyl)oxy)methyl)phenyl)ethan-1- one oxime

62-c (2.98 g, 6.62 mmol, 1.00 equiv) was dissolved in EtOH (20 mL). Hydroxylamine hydrochloride (0.55 g, 7.9 mmol, 1.2 equiv) and pyridine (0.8 mL, 9.9 mmol, 1.5 equiv) were added and it was stirred at 80 °C for 3 h. The volatiles were removed. The residue was taken up in EtOAc (150 mL), washed with water and brine (30 mL) each, dried over MgSO4, filtered and concentrated. Flash chromatography on silica afforded the title compound as colorless oil (2.78 g, 5.98 mmol, 90%).

Synthesis of 62-d

2,2,2-trifluoro-1-(3-iodo-2-(((4-methoxybenzyl)oxy)methyl)phenyl)ethan-1-one oxime

(2.78 g, 5.98 mmol, 1.0 equiv) was dissolved in CH2Cl2 (12 mL) at rt. DMAP (73 mg,

0.60 mmol, 0.1 equiv), NEt3 (1.25 mL, 9.0 mmol, 1.5 equiv) and TsCl (1.25 g, 6.6 mmol, 1.1 equiv) were added successively and the reaction mixture was stirred for 30 min. It was

130 diluted with CH2Cl2 (100 mL), washed with water and brine (20 mL each), dried over MgSO4, filtered and concentrated. Flash chromatography on silica afforded the title compound as colorless oil (1.8 g, 2.91 mmol, 49%) as a 2:1 mixture of diastereomers.

All signals reported. 1 H NMR (400 MHz, CDCl3) δ 7.95 (ddd, J = 15.0, 7.8, 1.4 Hz, 2H), 7.90 – 7.86 (m, 3H), 7.40 – 7.31 (m, 3H), 7.18 – 7.13 (m, 3H), 7.13 – 7.07 (m, 2H), 7.02 (dt, J = 15.5, 7.7 Hz, 2H), 6.89 – 6.83 (m, 3H), 4.42 (s, 3H), 4.19 (s, 4H), 3.82 (s, 3H), 3.81 (s, 2H), 2.43 (d, J = 17.3 Hz, 5H). 13 C NMR (101 MHz, CDCl3) δ 159.5, 159.4, 156.4 (q, J = 33.7 Hz), 146.2, 145.9, 142.3, 142.1, 140.8, 139.4, 131.9, 131.5, 130.7, 130.0, 129.9, 129.9, 129.8, 129.4, 129.3, 129.2, 129.2, 129.0, 128.9, 128.0, 128.0, 117.2 (q, J = 282.6 Hz), 113.9, 113.8, 99.5, 98.3, 74.8, 74.2, 72.9, 72.4, 19 55.4 (d, J = 1.8 Hz), 21.9, 21.8. F NMR (376 MHz, CDCl3) δ -63.9, -66.6. HRMS (ESI+): + m/z calcd for C24H25F3IN2O5S [M+NH4] 637.0476, found 637.0468.

Synthesis of 62-e

62-d (1.8 g, 2.91 mmol, 1.00 equiv) was dissolved in Et2O in a glass pressure vessel and cooled below -30 °C using a dry ice/acetone bath under a constant flow of nitrogen (open vessel). Then ammonia (10 mL) was condensed into the vessel (dry ice condenser). The vessel was sealed and allowed to warm to room temperature. After 4 h, it was cooled to –78 °C, opened and allowed to reach rt under a stream of nitrogen. The residue was partitioned between water and ether. The layers were separated and the aqueous phase was further extracted with Et2O (3x 20 mL). The combined organic fractions were washed with water and brine (50 mL each), dried over MgSO4, filtered and concentrated. The residue was taken up in MeOH (10 mL) and NEt3 (2.0 mL, 14.5 mmol, 5.0 equiv) was added. While stirring, the mixture was titrated with a saturated solution of I2 in MeOH, until a dark brown color persisted. The mixture was diluted with water (200 mL) and extracted with EtOAc (3x 40 mL). The combined organic fractions were washed with sat. Na2S2O3 (10 mL), water and brine (20 mL each), dried over MgSO4, filtered and concentrated. The residue was chromatographed on silica (5% EtOAc in hexanes) to afford the title compound as light-yellow oil (540 mg, 1.17 mmol, 40%).

1 Rf = 0.57 (20% EtOAc in hexanes; UV, KMnO4. H NMR (300 MHz, CDCl3) δ 7.96 (dt, J = 8.0, 1.0 Hz, 1H), 7.65 (d, J = 7.7 Hz, 1H), 7.45 – 7.36 (m, 2H), 7.05 (td, J = 7.8, 0.8 Hz, 1H),

131

6.93 – 6.85 (m, 2H), 4.88 (s, 2H), 4.67 (s, 2H), 3.81 (d, J = 0.8 Hz, 3H). IR 2935, 2838, 1615, 1515, 1328, 1247, 1198, 1180, 1156, 1083, 949, 832, 741, 674 cm-1.

Synthesis of 3-(2-(((4-methoxybenzyl)oxy)methyl)-3-((trimethylsilyl)ethynyl)phenyl)-3- (trifluoromethyl)-3H-diazirine

62-e (196 mg, 0.424 mmol, 1.00 equiv), Pd(PPh3)2Cl2 (30 mg, 0.042 mmol, 0.10 equiv), CuI (16 mg, 0.085 mmol, 0.20 equiv) were combined in a round-bottom-flask. After evacuation and backfilling with N2 (3x), degassed DMF (1.4 mL) and NEt3 (118 µL, 0.848 mmol, 2.00 equiv) were added. TMS-acetylene (89 µL, 0.64 mmol, 1.5 equiv) was added and the mixture was stirred at rt for 20 min (TLC of a miniworkup 5% aq. LiCl/EtOAc indicates full conversion of starting material). The reaction mixture was diluted with 5% aq. LiCl and extracted with Et2O.

The combined organics were washed with 5% aq. LiCl and brine, dried over MgSO4, filtered and concentrated. Flash chromatography on silica (2% EtOAc in hexanes) afforded the title compound (146 mg, 0.338 mmol, 80%) as yellow oil.

The same reaction starting with 667 mg ArI afforded 432 mg product (85%).

1 Rf = 0.15 (2% EtOAc in hexanes; UV, KMnO4). H NMR (600 MHz, CDCl3) δ 7.65 (d, J = 7.8 Hz, 1H), 7.57 (dd, J = 7.8, 1.3 Hz, 1H), 7.39 – 7.33 (m, 2H), 7.33 (app t, J = 7.8 Hz, 1H), 6.92 – 6.86 (m, 2H), 4.94 (s, 2H), 4.65 (s, 2H), 3.81 (s, 3H), 0.25 (s, 9H). 13C NMR (151 MHz,

CDCl3) δ 159.4, 141.4, 135.2, 131.5, 130.3, 129.8, 129.0, 128.4, 126.0, 122.2 (q, J = 275.0 Hz), 19 113.9, 102.2, 99.9, 73.5, 67.6, 55.4, 28.0 (q, J = 41.8 Hz), 0.0. F NMR (565 MHz, CDCl3) δ -68.2. IR 2959, 2904, 2857, 2838, 2158, 1614, 1514, 1332, 1248, 1191, 1152, 1071, 1037, 868, -1 + 841, 759, 726 cm . HRMS (MALDI): m/z calcd for C22H27F3N3O2Si [M+NH4] 450.1819, found 450.1815.

Synthesis of 62-f

3-(2-(((4-methoxybenzyl)oxy)methyl)-3-((trimethylsilyl)ethynyl)phenyl)-3-(trifluoromethyl)-

3H-diazirine (408 mg, 0.943 mmol, 1.00 equiv) was dissolved in MeOH (2.7 mL) and K2CO3 (143 mg, 1.04 mmol, 1.10 equiv) was added. After 1 h 45 min, the reaction mixture was diluted with sat. NH4Cl (50 mL). Extraction with Et2O (3x 30 mL), washing with brine, drying over

132

MgSO4, filtration and concentration followed by flash chromatography on silica (3% EtOAc in hexanes) afforded the title compound (113 mg, 0.314 mmol, 93%) as light yellow oil.

1 Rf = 0.25 (5% EtOAc in hexanes; UV, KMnO4). H NMR (300 MHz, CDCl3) δ 7.70 (dd, J = 7.8, 1.3 Hz, 1H), 7.62 (dd, J = 7.8, 1.4 Hz, 1H), 7.43 – 7.37 (m, 2H), 7.36 (t, J = 7.7 Hz, 1H), 6.97 – 6.86 (m, 2H), 4.97 (s, 2H), 4.68 (s, 2H), 3.82 (s, 3H), 3.29 (s, 1H). 13C NMR (75 MHz, CDCl3) δ 159.4, 141.7, 135.5, 131.8, 130.2, 129.9, 129.1, 128.5, 125.1, 122.1 (q, J = 275.0 Hz), 113.8, 82.4, 80.9, 73.3, 67.3, 55.3, 27.9 (q, J = 41.8 Hz). 19F NMR (282 MHz, CDCl3) δ -68.2. IR 3298, 2934, 2856, 1614, 1514, 1330, 1246, 1190, 1150, 1070, 1035, 854, 820, 806, 796, -1 + 748, 716, 644 cm . HRMS (MALDI): m/z calcd for C19H15F3N2NaO2 [M+Na] 383.0978, found 383.0975. Synthesis of 62

62-e (318 mg, 0.883 mmol, 1.00 equiv) was dissolved in CH2Cl2 (8 mL) and water (0.8 mL) at 0 °C. DDQ (301 mg, 1.324 mmol, 1.5 equiv) was added and it was stirred 2 h at 0 °C, and further 1.5 h at rt. Sat. aq. sodium bicarbonate was added and the mixture was extraction with

CH2Cl2 (3x 20 mL). The combined organic fractions were dried over MgSO4, filtered and concentrated. Filtration over silica (20% EtOAc in hexanes) gave a mixture of intermediate benzyl alcohol and p-anisaldehyde. This mixture was dissolved in CH2Cl2 (8 mL) and cooled to 0 °C. NEt3 (0.19 mL, 1.32 mmol, 1.5 equiv) was added followed by MsCl (76 L, 0.97 mmol, 1.1 equiv). After 20 min, the reaction was quenched by the addition of sat. aq. sodium bicarbonate, extracted with CH2Cl2, dried over MgSO4, filtered and concentrated. Flash chromatography on silica (15% EtOAc in hexanes) afforded the title compound as colorless oil (244 mg, 0.767 mmol, 87% over both steps), that turned red upon standing.

1 Rf = 0.24 (20% EtOAc in hexanes; UV, KMnO4). H NMR (300 MHz, CDCl3) δ 7.77 – 7.71 (m, 1H), 7.67 (dd, J = 7.8, 1.4 Hz, 1H), 7.48 (t, J = 7.8 Hz, 1H), 5.69 (s, 2H), 3.47 (s, 1H), 3.13 13 (s, 3H). C NMR (75 MHz, CDCl3) δ 136.3, 135.9, 132.2, 131.0, 128.9, 125.9, 121.8 (q, J = 19 275.0 Hz), 84.4, 79.6, 65.8, 37.6, 27.4 (q, J = 42.4 Hz). F NMR (282 MHz, CDCl3) δ -67.9. IR 3279, 1622, 1468, 1360, 1332, 1249, 1194, 1177, 1158, 944, 826, 712, 527 cm-1. HRMS + (MALDI): m/z calcd for C12H9F3N2NaO3S [M+Na] 341.0178, found 341.0180.

Synthesis of 63 and 64

133

62 (23 mg, 0.072 mmol, 1.0 equiv) and 15 (25 mg, 0.087 mmol, 1.20 equiv) were dissolved in

DMF (0.5 mL) and NEt3 (20 L, 0.015 mmol, 2.0 equiv) was added. The reaction mixture was stirred overnight at rt, diluted with EtOAc (60 mL), washed with 5% aq. LiCl (2x 20 mL) and brine (20 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (5% EtOAc in hexanes) afforded 63 (14 mg, 0.028 mmol, 38%) as colorless oil and 64 (13 mg, 0.026 mmol, 36%) as light-purple solid.

Data for 63. Mixture of rotamers 1.3:1. All signals reported. 1 Rf = 0.30 (10% EtOAc in hexanes; UV, KMnO4). H NMR (600 MHz, CDCl3) δ 7.75 (dd, J = 7.9, 1.3 Hz, 1H), 7.62 (dd, J = 7.8, 1.3 Hz, 1H), 7.44 (app t, J = 7.8 Hz, 1H), 6.20 (s, 2H), 4.59 – 4.46 (m, 2H), 4.18 – 4.04 (m, 2H), 3.10 (s, 1H), 2.63 – 2.44 (m, 2H), 1.29 (s, 9H). 13C NMR

(151 MHz, CDCl3) δ 175.8, 152.4, 151.3, 138.5, 135.8, 132.0, 129.8, 128.7, 127.8 (t, J = 247.9 Hz), 127.2 (t, J = 247.9 Hz), 125.6, 123.5, 122.1 (q, J = 274.9 Hz), 83.9, 80.0, 55.5 (t, J = 32.7 Hz), 53.7 (t, J = 32.8 Hz), 46.7, 46.0, 44.5, 39.7, 34.7 (t, J = 24.2 Hz), 33.0 (t, J = 23.6 Hz), 19 29.7, 27.4 (q, J = 41.9 Hz). F NMR (471 MHz, CDCl3) δ -68.0, -101.2, -101.7. IR 3306, 2960, 2928, 1600, 1579, 1483, 1324, 1248, 1199, 1191, 1157, 1127, 928, 807, 716 cm-1. HRMS + (ESI+): m/z calcd for C23H22F5N8 [M+H] 505.1882, found 505.1881.

Data for 64. Mixture of rotamers 1.2:1. All signals reported. 1 Rf = 0.20 (10% EtOAc in hexanes; UV, KMnO4). H NMR (600 MHz, CDCl3) δ δ 7.76 (dd, J = 7.8, 1.4 Hz, 1H), 7.67 (dd, J = 7.8, 1.4 Hz, 1H), 7.48 (app t, J = 7.8 Hz, 1H), 6.31 (s, 2H), 4.32 – 4.17 (m, 2H), 4.16 – 4.01 (m, 2H), 3.24 (s, 1H), 2.59 – 2.44 (m, 2H), 1.38 (s, 9H). 13C

NMR (151 MHz, CDCl3) δ 175.5, 159.9, 152.8, 136.9, 135.9, 132.2, 130.4, 128.8, 127.8 (t, J = 248.4 Hz), 127.1 (t, J = 248.0 Hz), 126.3, 125.2, 122.0 (q, J = 275.0 Hz), 84.2, 79.9, 55.4, 55.2 (t, J = 32.9 Hz), 53.7 (t, J = 32.6 Hz), 46.3, 44.5, 39.8, 34.7 (t, J = 24.2 Hz), 33.0 (t, J = 23.6 19 Hz), 29.7, 27.3 (q, J = 42.0 Hz). F NMR (471 MHz, CDCl3) δ -68.0, -101.0, -101.7. IR 3304, 2961, 2929, 1595, 1567, 1486, 1387, 1336, 1248, 1191, 1159, 1130, 932, 718 cm-1. HRMS + (ESI+): m/z calcd for C23H22F5N8 [M+H] 505.1882, found 505.1882.

Synthesis of 65 and 66

134

62 (23 mg, 0.072 mmol, 1.0 equiv) and 16 (23 mg, 0.087 mmol, 1.20 equiv) were dissolved in

DMF (0.5 mL) and NEt3 (15 L, 0.011 mmol, 1.5 equiv) was added. The reaction mixture was stirred overnight at rt, diluted with EtOAc (60 mL), washed with 5% aq. LiCl (2x 20 mL) and brine (20 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (30% EtOAc in hexanes + 5% AcOH; fractions containing product were pooled, washed with sat. sodium bicarbonate and brine, then dried over MgSO4, filtered and concentrated) afforded 65 (8 mg, 0.017 mmol, 23%) as colorless oil and 66 (10 mg, 0.021 mmol, 29%) as tan wax.

Data for 65. Mixture of rotamers 1.3:1. All signals reported. 1 Rf = 0.38 (30% EtOAc in hexanes + 5% AcOH; UV, KMnO4). H NMR (600 MHz, CDCl3) δ 7.74 (dd, J = 7.9, 1.3 Hz, 1H), 7.61 (dd, J = 7.8, 1.3 Hz, 1H), 7.42 (app t, J = 7.8 Hz, 1H), 6.18 (s, 2H), 4.68 (app d, J = 41.8 Hz, 1H), 4.49 – 4.33 (m, 1H), 4.29 – 4.18 (m, 1H), 4.06 – 3.83 (m, 2H), 3.09 (s, 1H), 2.26 – 2.07 (m, 2H), 1.29 (app d, J = 4.6 Hz, 9H). 13C NMR (151 MHz,

CDCl3) δ 175.6, 175.5, 152.6, 151.2, 138.8, 135.8, 132.0, 129.7, 128.7, 125.6, 123.8, 123.7, 122.1 (q, J = 274.9 Hz), 83.9, 80.0, 71.6, 70.0, 57.4, 55.6, 46.8, 45.9, 45.1, 39.7, 34.9, 32.9, 19 29.8, 27.4 (q, J = 41.9 Hz). F NMR (565 MHz, CDCl3) δ -68.0. IR 3409 (br), 3306, 2958, -1 2927, 2869, 1603, 1577, 1326, 1191, 1156 cm . HRMS (ESI+): m/z calcd for C23H24F3N8O [M+H]+ 485.2020, found 485.2018.

Data for 66. Mixture of rotamers 1.2:1. All signals reported. 1 Rf = 0.17 (30% EtOAc in hexanes + 5% AcOH; UV, KMnO4). H NMR (600 MHz, CDCl3) δ 7.75 (dd, J = 7.9, 1.3 Hz, 1H), 7.65 (dd, J = 7.8, 1.4 Hz, 1H), 7.46 (app t, J = 7.8 Hz, 1H), 6.29 (s, 2H), 4.67 (app d, J = 22.2 Hz, 1H), 4.16 – 3.82 (m, 4H), 3.23 (s, 1H), 2.21 – 2.05 (m, 2H), 13 1.38 (app d, J = 4.7 Hz, 9H). C NMR (151 MHz, CDCl3) δ 175.5, 159.8, 153.1, 137.1, 135.8, 132.1, 130.2, 128.8, 126.3, 125.6, 125.5, 122.0 (q, J = 275.0 Hz), 84.2, 79.9, 71.5, 69.8, 56.9, 55.7, 55.3, 46.5, 45.1, 39.7, 34.8, 33.1, 32.1, 29.8, 27.3 (q, J = 42.0 Hz). 19F NMR (565 MHz,

CDCl3) δ -68.0. IR 3304, 2961, 2929, 1595, 1567, 1486, 1387, 1336, 1248, 1191, 1159, 1130, -1 + 932, 718 cm . HRMS (ESI+): m/z calcd for C23H24F3N8O [M+H] 485.2020, found 485.2015.

Synthesis of 67 and 68

135

62 (27 mg, 0.085 mmol, 1.3 equiv) and 20 (23 mg, 0.065 mmol, 1.00 equiv) were dissolved in

DMF (0.3 mL) and NEt3 (18 L, 0.13 mmol, 2.0 equiv) was added. The reaction mixture was stirred overnight at rt, diluted with EtOAc (60 mL), washed with 5% aq. LiCl (2x 20 mL) and brine (20 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (10% EtOAc in hexanes) afforded 67 (11 mg, 0.019 mmol, 29%) as light-yellow oil and 68 (19 mg, 0.033 mmol, 51%) as light-red oil.

Data for 67. Mixture of rotamers 1.2:1. All signals reported. 1 Rf = 0.51 (20% EtOAc in hexanes; UV, KMnO4). H NMR (600 MHz, CDCl3) δ 7.77 (dd, J = 7.8, 1.3 Hz, 1H), 7.69 – 7.65 (m, 2H), 7.59 (dd, J = 7.8, 1.3 Hz, 1H), 7.46 (app t, J = 7.8 Hz, 1H), 7.43 – 7.38 (m, 3H), 6.21 (app d, J = 1.8 Hz, 2H), 4.59 – 4.48 (m, 2H), 4.17 – 4.05 (m, 13 2H), 2.86 (s, 1H), 2.63 – 2.45 (m, 2H). C NMR (151 MHz, CDCl3) δ 161.1 (t, J = 30.4 Hz), 161.1 (t, J = 30.6 Hz), 153.0, 152.9, 150.8, 150.8, 138.0, 136.6 (t, J = 27.3 Hz), 136.5 (t, J = 27.3 Hz), 135.9, 132.1, 130.1, 130.1, 128.8, 128.3, 128.3, 127.4 (t, J = 248.3 Hz), 126.7 (t, J = 247.4 Hz), 125.9 (t, J = 6.0 Hz), 125.9 (t, J = 6.0 Hz), 125.4, 125.4, 124.4, 124.3, 122.0 (q, J = 274.7 Hz), 117.0 (t, J = 246.6 Hz), 117.0 (t, J = 246.8 Hz), 84.4, 79.6, 55.6 (t, J = 33.1 Hz), 54.0 (t, J = 33.0 Hz), 47.1, 46.3, 45.1, 34.6 (t, J = 24.3 Hz), 32.9 (t, J = 23.9 Hz), 27.3 (q, J = 42.0 19 Hz). F NMR (565 MHz, CDCl3) δ -68.0, -99.3, -99.4, -101.3, -101.9. IR 3299, 2931, 1607, -1 + 1577, 1248, 1200, 1156, 699 cm . HRMS (ESI+): m/z calcd for C26H18F7N8 [M+H] 575.1537, found 575.1537.

Data for 68. Mixture of rotamers 1.2:1. All signals reported. 1 Rf = 0.39 (20% EtOAc in hexanes; UV, KMnO4). H NMR (600 MHz, CDCl3) δ 7.77 (dd, J = 7.8, 3.1 Hz, 1H), 7.76 – 7.72 (m, 2H), 7.67 (d, J = 7.8 Hz, 1H), 7.50 (app td, J = 7.8, 2.7 Hz, 1H), 7.40 – 7.37 (m, 3H), 6.34 (s, 2H), 4.29 – 4.17 (m, 2H), 4.13 – 4.01 (m, 2H), 3.23 (s, 1H), 13 2.59 – 2.44 (m, 2H). C NMR (151 MHz, CDCl3) δ 161.0 (t, J = 30.2 Hz), 161.0 (t, J = 30.3 Hz), 158.8, 158.7, 153.7, 153.7, 136.7 (t, J = 27.3 Hz), 136.6 (t, J = 27.3 Hz), 136.4, 136.4, 136.0, 135.9, 132.3, 132.2, 130.7, 130.6, 129.9, 127.4 (t, J = 248.7 Hz), 126.6 (t, J = 248.7 Hz), 126.3, 126.3, 126.1, 126.1 (t, J = 6.0 Hz), 126.1 (t, J = 6.0 Hz), 121.9 (q, J = 275.0 Hz), 117.1 (t, J = 246.4 Hz), 117.0 (t, J = 246.5 Hz), 84.4, 84.4, 79.7, 79.7, 55.8 (d, J = 3.3 Hz), 55.3 (t, J

136

= 33.1 Hz), 53.9 (t, J = 32.9 Hz), 46.7, 45.1, 34.6 (t, J = 24.3 Hz), 32.8 (d, J = 23.8 Hz), 27.3 19 (q, J = 42.0 Hz). F NMR (565 MHz, CDCl3) δ -67.9, -99.5, -99.6, -101.1, -101.9. IR 3301, 1600, 1562, 1395, 1248, 1201, 1192, 1156, 1119, 1002, 918, 807, 719, 699 cm-1. HRMS + (ESI+): m/z calcd for C26H18F7N8 [M+H] 575.1537, found 575.1538.

Synthesis of 69 and 70

22 (32 mg, 0.096 mmol, 1.0 equiv) and 62 (31 mg, 0.098 mmol, 1.01 equiv) were dissolved in DMF (0.5 mL) and DIPEA (25 L, 0.14 mmol, 1.5 equiv) was added. The reaction mixture was stirred overnight at rt, diluted with EtOAc (50 mL), washed with 5% aq. LiCl (2x 10 mL) and brine (10 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (10% EtOAc in hexanes + 20% AcOH) afforded 69 (18 mg, 0.032 mmol, 34%) as colorless and 70 (24 mg, 0.043 mmol, 45%) as pink wax.

Data for 69. Mixture of rotamers 1.2:1. All signals reported. 1 Rf = 0.23 (10% EtOAc in hexanes + 20% AcOH; UV, KMnO4). H NMR (600 MHz, CDCl3) δ 7.76 (dd, J = 7.8, 1.4 Hz, 1H), 7.71 – 7.67 (m, 2H), 7.57 (dd, J = 7.7, 1.4 Hz, 1H), 7.44 (app t, J = 7.8 Hz, 1H), 7.41 – 7.36 (m, 3H), 6.19 (s, 2H), 4.69 (app dd, J = 38.1, 3.1 Hz, 1H), 4.49 – 4.35 (m, 1H), 4.28 – 4.16 (m, 1H), 4.07 – 3.83 (m, 2H), 2.84 (s, 1H), 2.23 – 2.19 (m, 1H), 13 2.14 – 2.10 (m, 1H). C NMR (151 MHz, CDCl3) δ 161.1 (t, J = 30.1 Hz), 161.0 (t, J = 30.2 Hz), 153.1, 150.7, 150.7, 138.3, 138.2, 136.8 (t, J = 27.4 Hz), 135.8, 132.1, 130.0, 130.0, 128.8, 128.2, 126.0 (t, J = 6.0 Hz), 126.0 (t, J = 6.0 Hz), 125.4, 124.6, 124.5, 122.1 (q, J = 274.9 Hz), 117.1 (t, J = 246.4 Hz), 84.4, 79.7, 71.4, 69.7, 57.7, 56.0, 47.3, 46.1, 45.7, 34.8, 32.9, 27.3 (q, 19 J = 41.9 Hz). F NMR (565 MHz, CDCl3) δ -68.0, -98.7, -99.2, -99.3, -99.7. IR 3416 (br), -1 + 3297, 2949, 1610, 1574, 1248, 1156 cm . HRMS (ESI+): m/z calcd for C26H20F5N8O [M+H] 555.1675, found 555.1676.

Data for 70. Mixture of rotamers 1.1:1. All signals reported. 1 Rf = 0.13 (10% EtOAc in hexanes + 20% AcOH; UV, KMnO4). H NMR (600 MHz, CDCl3) δ 7.77 – 7.71 (m, 3H), 7.66 – 7.62 (m, 1H), 7.49 – 7.44 (m, 1H), 7.35 (app ddd, J = 9.1, 4.5, 2.1 Hz, 3H), 6.32 – 6.29 (m, 2H), 4.69 – 4.62 (m, 1H), 4.13 – 3.75 (m, 5H), 3.23 (d, J = 1.6 Hz, 13 1H), 2.16 – 2.04 (m, 2H). C NMR (151 MHz, CDCl3) δ 161.1 (t, J = 29.8 Hz), 161.0 (t, J = 29.9 Hz), 158.6, 158.5, 153.7, 153.7, 136.8 (d, J = 27.3 Hz), 136.8 (d, J = 27.3 Hz), 136.7,

137

136.6, 135.9, 135.9, 132.1, 130.5, 129.8, 128.8, 128.8, 128.1, 126.6, 126.5, 126.2, 126.2, 126.1 (t, J = 6.0 Hz), 126.1 (t, J = 6.2 Hz), 121.9 (q, J = 275.0 Hz), 117.2 (t, J = 246.1 Hz), 114.2, 84.5, 84.4, 79.7, 71.2, 69.5, 57.1, 56.1, 55.6, 47.1, 45.7, 34.6, 32.9, 27.3 (q, J = 41.8 Hz). 19F NMR (470 MHz, CDCl3) δ -67.9, -99.2, -99.3, -99.4. IR 3364 (br), 3299, 2926, 2860, 1604, 1559, 1395, 1334, 1248, 1191, 1155, 1107, 726, 699 cm-1. HRMS (ESI+): m/z calcd for + C26H20F5N8O [M+H] 555.1675, found 555.1677.

Synthesis of 73

N-Boc-3-oxopyrrolidine (5.55 g, 30.0 mmol, 1.00 equiv) was dissolved in Et2O (5 mL) and cooled to –78 °C. Ammonia (ca. 100 mL) was condensed into the flask. The solution was refluxed (dry ice condenser) for 6 h, then cooled to –78 °C. A solution of hydroxylamine-O- sulfonic acid (3.73 g, 33.0 mmol, 1.1 equiv) in MeOH (20 mL) was added via addition funnel. Upon completion of the addition, the reaction mixture was refluxed for another 1.5 h and then allowed to warm to rt overnight. The resulting suspension was filtered. The filter cake was washed with MeOH (2x 30 mL). The filtrate turned cloudy during the washing and was filtered again (same frit), washing with MeOH (50 mL). Triethylamine (4 mL) was added to the filtrate and it was concentrated. Another portion of MeOH (50 mL) was added, and it was concentrated again (to remove any residual ammonia). MeOH was added (100 mL) and it was cooled to 0 °C. The yellow solution was titrated with a solution of iodine in MeOH (ca. 7 g/100 mL) until an orange color remained. Celite was added and the mix was concentrated. Flash column chromatography on silica (10% EtOAc in hexanes) afforded the title compound as yellow oil (876 mg, 4.44 mmol, 15%).

1 Rf = 0.49 (20% EtOAc in hexanes; UV, KMnO4). H NMR (300 MHz, CDCl3) δ 3.63 (t, J = 13 7.4 Hz, 2H), 2.99 (s, 2H), 1.67 (t, J = 7.4 Hz, 2H), 1.45 (s, 9H). C NMR (75 MHz, CDCl3) δ 154.0, 80.1, 48.2, 44.4, 29.3, 28.7, 28.5. IR 2977, 2873, 1697, 1478, 1398, 1366, 1352, 1220, -1 + 1166, 1103, 984, 885, 772 cm . HRMS (ESI+): m/z calcd for C26H20F5N8O [M+H] 555.1675, found 555.1677.

Synthesis of 74

73 (272 mg, 1.38 mmol, 1.00 equiv) was combined with a solution of HCl in dioxane (4 M, 3.45 mL, 13.8 mmol, 10.0 equiv). After 30 min, the resulting solid was collected by filtration,

138 washed with Et2O (2x 2 mL) and dried to give the title compound as colorless solid (170 mg, 1.27 mmol, 92%).

1 13 H NMR (400 MHz, H2O) δ 3.69 (t, J = 7.6 Hz, 2H), 3.10 (s, 2H), 1.90 (t, J = 7.6 Hz, 2H). C

NMR (101 MHz, H2O) δ 48.0, 47.7, 33.4, 29.0. IR 2883, 2724, 2602, 2504, 2447, 2211, 1615, 1575, 1443, 1424, 1272, 1211, 1018, 888, 615, 518 cm-1.

Synthesis of 75

The title compound was prepared according to GP1 from 2-bromobenzyl bromide (5.00 g, 20.0 mmol, 1.00 equiv), sodium azide (1.37 g, 21.0 mmol, 1.05 equiv) and 2-cyanoacetamide (2.52 g, 30.0 mmol, 1.50 equiv) and isolated as colorless solid (5.50 g, 18.6 mmol, 93%).

1H NMR (400 MHz, DMSO) δ 7.69 (dd, J = 7.9, 1.3 Hz, 1H), 7.50 (s, 1H), 7.35 (td, J = 7.5, 1.4 Hz, 1H), 7.27 (td, J = 7.7, 1.8 Hz, 1H), 7.14 (s, 1H), 6.65 (dd, J = 7.7, 1.7 Hz, 1H), 6.48 (s, 2H), 5.44 (s, 2H). 13C NMR (101 MHz, DMSO) δ 164.3, 145.4, 134.9, 132.7, 129.6, 128.1, + 128.0, 121.9, 121.6, 48.8. HRMS (ESI+): m/z calcd for C10H11BrN5O [M+H] 296.0141, found 296.0144.

Synthesis of 76

The title compound was prepared according to GP2 from 75 (2.00 g, 6.75 mmol, 1.00 equiv) and PivCl (1.25 mL, 10.1 mmol, 1.5 equiv) and isolated as colorless solid (1.85 g, 5.11 mmol, 76%).

1 H NMR (400 MHz, CDCl3) δ 11.38 (s, 1H), 7.61 (dd, J = 7.9, 1.3 Hz, 1H), 7.27 (ddd, J = 8.2, 13 7.0, 1.3 Hz, 1H), 7.24 – 7.17 (m, 2H), 5.86 (s, 2H), 1.47 (s, 9H). C NMR (101 MHz, CDCl3) δ 168.7, 157.5, 149.7, 134.0, 133.2, 130.5, 130.2, 128.0, 127.9, 123.6, 50.5, 38.4, 28.6.

Synthesis of 77

139

76 (307 mg, 0.848 mmol, 1.00 equiv) was combined with Pd(PhCN)2Cl2 (16 mg, 0.042 mmol,

5 mol%), CuI (8 mg, 0.042 mmol, 5 mol%) and P(tBu)3·HBF4 (25 mg, 0.085 mmol, 10 mol%).

The flask was evacuated and backfilled with nitrogen (3x), before PhMe (4.7 mL) and HNiPr2 (0.19 mL, 1.4 mmol, 1.6 equiv) were added. Nitrogen was bubbled through the solution for 5 min. Trimethylsilyl acetylene (0.24 mL, 1.7 mmol, 2.0 equiv) was added, the flask was sealed and heated to 50 °C for 24 h. The solution was filtered over celite and concentrated. Flash chromatography on silica (20% EtOAc in hexanes to 50%) afforded the silylated intermediate along with unidentified contaminants (210 mg total mass). The intermediate was dissolved in

MeOH (2.8 mL) and K2CO3 (382 mg, 2.77 mmol, ca. 5 equiv) was added. The reaction mixture was stirred for 5 h before the volatiles were removed under reduced pressure. The residue was partitioned between CH2Cl2 (20 mL) and water (20 mL). The layers were separated and the aq. phase was extracted with CH2Cl2 (2x 20 mL). The combined organic fractions were washed with brine, dried over MgSO4, filtered and concentrated. Flash chromatography on silica

(CH2Cl2:hexanes:EtOAc 2:2:1 to EtOAc:CH2Cl2 3:7 afforded the title compound as colorless solid (135 mg, 0.439 mmol, 52% over both steps).

1 Rf = 0.50 (50% EtOAc in hexanes; UV, KMnO4). H NMR (400 MHz, CDCl3) δ 11.35 (s, 1H), 7.60 – 7.54 (m, 1H), 7.36 – 7.30 (m, 2H), 7.30 – 7.26 (m, 1H), 5.96 (s, 2H), 3.41 (s, 1H), 1.48 13 (s, 9H). C NMR (101 MHz, CDCl3) δ 168.5, 157.6, 149.6, 136.7, 133.2, 129.3, 128.7, 128.4, 128.0, 121.7, 83.0, 80.9, 48.8, 38.3, 28.6. IR 3196, 3117, 2974, 1698, 1571, 1176, 760 cm-1. + HRMS (ESI+): m/z calcd for C17H18 N5O [M+H] 479.1515, found 479.1513.

Synthesis of 5-(tert-butyl)-7-chloro-3-(2-ethynylbenzyl)-3H-[1,2,3]triazolo[4,5- d]pyrimidine

77 (100 mg, 0.33 mmol, 1.00 equiv) was dissolved in CH2Cl2 (1 mL) at 0 °C. DMF (25 L, 0.33 mmol, 1.00 equiv) was added followed by oxalyl chloride (43L, 0.49 mmol, 1.5 equiv).

The mixture was allowed to stir at rt for 3 h before it was diluted with CH2Cl2 (50 mL). The mixture was washed with sat. sodium bicarbonate and brine (20 mL each), dried over MgSO4, filtered and concentrated to yield the product as colorless solid (95 mg, 0.29 mmol, 90%).

140

1 H NMR (400 MHz, CDCl3) δ 7.57 – 7.52 (m, 1H), 7.37 – 7.27 (m, 3H), 6.06 (s, 2H), 3.38 (s, 13 1H), 1.44 (s, 9H). C NMR (101 MHz, CDCl3) δ 176.6, 153.2, 150.6, 136.3, 133.3, 132.2, 129.4, 129.2, 128.7, 122.0, 83.2, 80.8, 49.4, 40.4, 29.7.

Synthesis of 78

5-(tert-Butyl)-7-chloro-3-(2-ethynylbenzyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine (40 mg,

0.123 mmol, 1.00 equiv) was combined with 74 (20 mg, 0.147 mmol, 1.2 equiv) and CH2Cl2

(1.2 mL). NEt3 (34 mL, 0.246 mmol, 2.0 equiv) was added and it was stirred at rt for 30 min before CH2Cl2 (50 mL) was added. The solution was washed with water and brine (20 mL each), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (5% EtOAc in hexanes) afforded the title compound as colorless solid (47 mg, 0.122 mmol, 99%).

Mixture of rotamers (ca. 1.2:1). All signals reported. 1 H NMR (500 MHz, CDCl3) δ 7.53 (dd, J = 6.1, 2.5 Hz, 1H), 7.29 – 7.21 (m, 3H), 5.95 (d, J = 4.9 Hz, 2H), 4.62 (t, J = 7.4 Hz, 1H), 4.22 – 4.12 (m, 1H), 3.94 (s, 1H), 3.49 (s, 1H), 3.39 (s, 13 1H), 1.92 (dt, J = 53.1, 7.5 Hz, 2H), 1.40 – 1.31 (m, 9H). C NMR (126 MHz, CDCl3) δ 175.9, 152.3, 151.2, 137.7, 133.0, 129.2, 128.8, 128.1, 121.7, 82.9, 81.1, 50.9, 49.0, 48.3, 47.6, 45.4, 39.8, 32.4, 31.4, 29.8, 29.8, 29.4, 27.8. IR (neat) 3299, 2959, 2925, 2865, 1599, 1579, 1482, -1 + 1326, 1219, 759 cm . HRMS (ESI+): m/z calcd for C21H23N8 [M+H] 387.2040, found 387.2036.

Synthesis of 80

According to GP1 to give the title compound (1.8 g, 7.5 mmol, 72%) as beige solid.

1H NMR (400 MHz, DMSO) δ 7.55 (d, J = 7.3, 1.7 Hz, 1H), 7.48 (s, 1H), 7.42 – 7.29 (m, 2H), 7.12 (s, 1H), 6.72 – 6.66 (m, 1H), 6.42 (s, 2H), 5.52 (s, 2H), 4.56 (s, 1H). 13C NMR (101 MHz, DMSO) δ 164.3, 145.4, 137.9, 132.5, 129.3, 127.7, 126.3, 121.6, 120.2, 86.4, 80.6, 47.1. IR

141

(neat) 3412, 3286, 3153, 1634, 1570, 1515, 1430, 1251, 796, 754 cm-1. HRMS (ESI+): m/z + calcd for C12H11N5NaO [M+Na] 264.0856, found 264.0855.

Synthesis of 81

80 (482 mg, 2.00 equiv, 1.00 equiv) was combined with PhCF2CN (487 mg, 3.18 mmol, 1.59 equiv), K2CO3 (1.38 g, 10.0 mmol, 5.00 equiv) and DMF (6.7 mL). The flask was sealed and heated at 90 °C for 3 h. After cooling to rt, aq. HCl (1 M, 25 mL) was added and the resulting precipitate was isolated by filtration. The filtercake was transferred to another round-bottom- flask and dried by azeotropic removal of water (3x 10 mL PhMe). The residue was dissolved in PhMe (6.7 mL). Three drops of DMF were added, followed by oxalyl chloride (0.35 mL, 4.00 mmol, 2.00 equiv). The reaction mixture was heated to 65 °C for 3 h. After cooling to rt, EtOAc was added (100 mL). The resulting solution was washed with sat. aq. sodium bicarbonate and brine (30 mL each), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (5% EtOAc in hexanes to 10%) afforded the title compound as colorless solid (450 mg, 1.14 mmol, 57% over both steps).

1 Rf = 0.26 (10% EtOAc in hexanes; KMnO4, UV). H NMR (400 MHz, CDCl3) δ 7.75 – 7.68 (m, 2H), 7.56 – 7.48 (m, 1H), 7.45 – 7.36 (m, 4H), 7.35 – 7.28 (m, 2H), 6.08 (s, 2H), 3.27 (s, 13 1H). C NMR (101 MHz, CDCl3) δ 160.8 (t, J = 32.5 Hz), 155.0, 150.3 (d, J = 1.3 Hz), 135.6, 135.1 (t, J = 27.0 Hz), 133.5, 133.3, 130.6 (t, J = 1.7 Hz), 129.6, 129.4, 129.0, 128.6, 125.9 (t, 19 J = 6.0 Hz), 122.1, 116.5 (t, J = 247.9 Hz), 83.7, 80.4, 49.9. F NMR (376 MHz, CDCl3) δ - 97.96. IR (neat) 3291, 1586, 1567, 1488, 1452, 1402, 1338, 1261, 1216, 1161, 1101, 1063, 1004, 970, 910, 870, 806, 759, 732, 721, 696, 668, 628, 578 cm-1. HRMS (ESI+): m/z calcd for + C20H13ClF2N5 [M+H] 396.0822, found 396.0821.

Synthesis of 82

142

A solution of NEt3 (19 mg, 0.19 mmol, 1.5 equiv) in CH2Cl2 (0.1 mL) was added to a suspension of 81 (50 mg, 0.13 mmol, 1.0 equiv) and 3,3-difluoropyrrolidine·HCl (25 mg,

0.19 mmol, 1.5 equiv) in CH2Cl2 (0.5 mL). The reaction mixture was stirred until TLC indicated full consumption of starting material, then directly subjected to flash chromatography on silica to afforded the title compound (45 mg, 0.10 mmol, 78%) as colorless oil.

1.1:1 mixture of rotamers. All signals reported. 1 Rf = 0.33 (20% EtOAc in hexanes; UV, KMnO4). H NMR (600 MHz, CDCl3) δ 7.74 – 7.69 (m, 1H), 7.68 – 7.65 (m, 1H), 7.54 – 7.49 (m, 1H), 7.43 – 7.35 (m, 3H), 7.29 – 7.24 (m, 2H), 7.22 – 7.16 (m, 1H), 5.96 (s, 2H), 4.62 (t, J = 7.5 Hz, 1H), 4.18 – 4.14 (m, 1H), 3.93 (s, 1H), 3.48 (s, 1H), 3.29 (s, 1H), 2.00 – 1.95 (m, 1H), 1.89 – 1.85 (m, 1H). 13C NMR (151 MHz,

CDCl3) δ 161.2 (t, J = 30.4 Hz), 161.1 (t, J = 30.5 Hz), 152.8, 152.8, 150.7, 150.6, 137.1, 137.0, 136.6 (t, J = 27.2 Hz), 136.5 (t, J = 27.2 Hz), 133.1, 130.0, 129.3, 128.9, 128.9, 128.4, 128.2, 126.0 (t, J = 6.0 Hz), 125.9 (t, J = 6.0 Hz), 124.9, 124.6, 121.8, 121.8, 117.1 (t, J = 246.5 Hz), 117.0 (t, J = 246.6 Hz), 83.3, 80.8 (d, J = 2.6 Hz), 51.0, 49.2, 48.8, 48.0, 45.9, 32.2, 31.0, 29.3, 27.6. 19F NMR (282 MHz, CDCl3) δ -98.9, -99.1. IR 3301, 1602, 1574, 1518, 1471, 1451, 1353, 1261, 1087, 1065, 1008, 947, 911, 760, 725, 698, 639 cm-1. HRMS (ESI+): m/z calcd for + C24H18F2N8Na [M+Na] 479.1515, found 479.1513.

Synthesis of N-(2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethyl)-4-(hydroxymethyl)-1,2,5- oxadiazole-3-carboxamide

2-(3-(But-3-yn-1-yl)-3H-diazirin-3-yl)ethan-1-amine94 (96 mg, 0.700 mmol, 1.00 equiv) was dissolved in PhMe (2.3 mL) and cooled to 0 °C. AlMe3 in PhMe (2 M, 0.370 mL, 0.740 mmol, 1.06 equiv) was added dropwise. Upon completion of the addition, the mixture was stirred for 30 min, before a solution of furo[3,4-c][1,2,5]oxadiazol-4(6H)-one (106 mg, 0.840 mmol, 1.2 equiv) in PhMe (1.0 mL) was added. The cooling bath was removed and the mixture was allowed to warm to rt. A precipitate formed. THF (1.0 mL) was added and the resulting solution was stirred at rt for 1 h. The reaction was quenched by the addition of sat. aq. Rochelle's salt (2 mL) and water (2 mL). EtOAc (5 mL) was added and it was stirred vigorously for for 1h. The mixture was extracted with EtOAc (3x 5 mL EtOAc). The combined organic extracts were washed with brine (15 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (25% EtOAc in hexanes) afforded the title compound as slightly yellow oil (171 mg, 0.650 mmol, 93%).

143

1 Rf = 0.23 (25% EtOAc in hexanes; UV, KMnO4). H NMR (600 MHz, CDCl3) δ 7.19 (s, 1H), 4.94 (d, J = 7.1 Hz, 2H), 4.55 (t, J = 7.4 Hz, 1H), 3.36 (q, J = 6.6 Hz, 2H), 2.08 – 2.02 (m, 3H), 13 1.86 (t, J = 6.8 Hz, 2H), 1.68 (t, J = 7.1 Hz, 2H). C NMR (151 MHz, CDCl3) δ 158.1, 154.5, 148.8, 82.5, 69.7, 54.2, 34.9, 32.3, 32.0, 26.5, 13.2. IR 3300 (br) 3294, 2929, 1667, 1564, 1548, -1 + 1436, 1173, 1048, 1020, 968, 907, 642 cm . HRMS (ESI+): m/z calcd for C11H14N5O3 [M+H] 264.1091, found 264.1096.

Synthesis of 85

N-(2-(3-(But-3-yn-1-yl)-3H-diazirin-3-yl)ethyl)-4-(hydroxymethyl)-1,2,5-oxadiazole-3- carboxamide (87 mg, 0.330 mmol, 1.00 equiv) and NEt3 (69 L, 0.500 mmol, 1.50 equiv) were dissolved in CH2Cl2 (1.1 mL) and cooled to 0 °C. MsCl (31 L, 0.400 mmol, 1.20 equiv) was added and the reaction was stirred for 30 min. The reaction was quenched by the addition of MeOH (a few drops). The volatiles were removed in vacuo. Flash chromatography on silica (30% EtOAc in hexanes to 50%) afforded the title compound as colorless oil (90 mg, 0.264 mmol, 80%).

1 Rf = 0.31 (40% EtOAc in hexanes; UV, KMnO4). H NMR (600 MHz, CDCl3) δ 6.94 (s, 1H), 5.65 (s, 2H), 3.36 – 3.31 (m, 2H), 3.18 (s, 3H), 2.06 – 2.02 (m, 3H), 1.85 (t, J = 6.8 Hz, 2H), 13 1.71 – 1.66 (m, 2H). C NMR (151 MHz, CDCl3) δ 156.6, 150.2, 147.7, 82.7, 69.8, 59.4, 38.3, 34.8, 32.5, 32.2, 26.7, 13.3. IR 3374, 3293, 2938, 1680, 1547, 1440, 1355, 1256, 1173, 923, -1 + 905, 809, 644, 526 cm . HRMS (ESI+): m/z calcd for C12H15N5NaO5S [M+Na] 364.0686, found 364.0692.

Synthesis of 88 and 89

15 (37 mg, 0.132 mmol, 1.00 equiv) was combined with 85 (45 mg, 0.132 mmol, 1.00 equiv) and DMF (0.4 mL). NEt3 (37 L, 0.264 mmol, 2.00 equiv) was added and it was stirred for 5 h.

144

The reaction mixture was diluted with EtOAc (100 mL) and the resulting solution was washed with 5% aq. LiCl and brine (20 mL each). It was dried over MgSO4, filtered and concentrated. Flash chromatography on silica (10% EtOAc in hexanes to 25%) afforded 88 (13 mg, 0.025 mmol, 19%) as colorless oil and 89 (12 mg, 0.023 mmol, 17%) as yellow oil.

Data for 88. A mixture of rotamers (1.3:1). All signals reported. 1 Rf = 0.16 (20% EtOAc in hexanes; UV, KMnO4). H NMR (600 MHz, CDCl3) δ 6.98 (t, J = 6.1 Hz, 1H), 6.19 (s, 2H), 4.62 – 4.49 (m, 2H), 4.19 – 4.04 (m, 2H), 3.34 (td, J = 6.9, 6.1 Hz, 2H), 2.56 (dtt, J = 63.1, 13.8, 7.4 Hz, 2H), 2.07 – 2.03 (m, 3H), 1.85 (t, J = 6.8 Hz, 2H), 1.71 – 13 1.67 (m, 2H), 1.32 (d, J = 2.7 Hz, 9H). C NMR (151 MHz, CDCl3) δ 176.5, 157.0, 152.4, 151.1, 151.1, 151.0, 147.8, 127.7 (t, J = 248.2 Hz), 127.1 (t, J = 247.2 Hz), 123.7 (d, J = 3.8 Hz), 82.7, 69.8, 55.5 (t, J = 32.8 Hz), 53.8 (t, J = 32.8 Hz), 46.8, 44.6, 40.0, 39.8 (d, J = 7.0 Hz), 34.8, 34.7 (t, J = 24.5 Hz), 33.0 (t, J = 23.7 Hz), 32.6, 32.2, 29.7, 26.7, 13.3. 19F NMR

(470 MHz, CDCl3) δ -101.21, -101.80. IR 3306, 2960, 1684, 1602, 1581, 1546, 1482, 1322, -1 1219, 1166, 1127, 986, 927, 738, 640 cm . HRMS (ESI+): m/z calcd for C23H28F2N11O2 [M+H]+ 528.2390, found 528.2387.

Data for 89. A mixture of rotamers (1.3:1). All signals reported. 1 H NMR (600 MHz, CDCl3) δ 7.01 (t, J = 6.2 Hz, 1H), 6.26 (s, 2H), 4.47 – 4.31 (m, 2H), 4.17 – 4.04 (m, 2H), 3.30 (q, J = 6.6 Hz, 2H), 2.55 (ddt, J = 49.2, 13.5, 6.4 Hz, 2H), 2.04 – 2.00 (m, 13 3H), 1.80 (t, J = 6.9 Hz, 2H), 1.66 (t, J = 7.1 Hz, 2H), 1.38 (s, 9H). C NMR (151 MHz, CDCl3) δ 176.1, 159.9 (d, J = 15.3 Hz), 156.7, 152.9, 149.7, 148.1, 127.7 (t, J = 248.3 Hz), 127.0 (t, J = 247.2 Hz), 126.1 – 125.7 (m), 123.7, 123.5, 82.7, 69.8, 55.3 (t, J = 32.7 Hz), 53.8 (t, J = 32.7 Hz), 49.3, 46.6, 44.7, 39.9, 34.8, 34.7 (t, J = 26.3 Hz), 33.0 (t, J = 23.5 Hz), 32.5, 32.1, 29.6, 19 26.6, 13.3. F NMR (470 MHz, CDCl3) δ -100.99, -101.73. IR 3306, 2960, 1682, 1593, 1567, 1485, 1345, 1271, 1165, 1128, 927, 810, 731, 650 cm-1. HRMS (ESI+): m/z calcd for + C23H28F2N11O2 [M+H] 528.2390, found 528.2387.

Synthesis of 90 and 91

145

16 (31 mg, 0.117 mmol, 1.00 equiv) was combined with 85 (40 mg, 0.117 mmol, 1.00 equiv) and DMF (0.4 mL). NEt3 (33 L, 0.234 mmol, 2.00 equiv) was added and it was stirred for 5 h. The reaction mixture was diluted with EtOAc (100 mL) and the resulting solution was washed with 5% aq. LiCl and brine (20 mL each). It was dried over MgSO4, filtered and concentrated. Flash chromatography on silica (40% EtOAc in cyclohexane, 2x) afforded 90 (8 mg, 0.016 mmol, 13%) and 89 (16 mg, 0.032 mmol, 27%) as colorless oils.

Data for 90. A mixture of rotamers (1:1). All signals reported. 1 Rf = 0.28 (50% EtOAc in hexanes; UV, KMnO4). H NMR (600 MHz, CDCl3) δ 6.99 (t, J = 6.1 Hz, 1H), 6.17 (s, 2H), 4.71 (d, J = 49.1 Hz, 1H), 4.50 – 4.36 (m, 1H), 4.31 – 4.20 (m, 1H), 4.07 – 3.84 (m, 2H), 3.34 (td, J = 6.8, 6.0 Hz, 2H), 2.26 – 2.09 (m, 2H), 2.06 – 2.03 (m, 3H), 1.84 (t, J = 6.9 Hz, 2H), 1.71 – 1.67 (m, 2H), 1.32 (d, J = 4.6 Hz, 9H). 13C NMR (151 MHz,

CDCl3) δ 157.04, 152.60 (app d, J = 3.4 Hz), 151.05, 147.87, 124.01, 123.91, 82.69, 71.54, 69.88, 69.81, 57.48, 55.66, 46.97, 45.21, 39.94, 39.73 (app d, J = 3.7 Hz), 34.87, 34.81, 32.86, 32.58, 32.19, 29.77, 26.68, 13.35. IR 3320 (br), 3302, 2957, 2927, 1684, 1605, 1579, 1483, -1 + 1326, 1221, 1100, 733 cm . HRMS (ESI+): m/z calcd for C23H30N11O3 [M+H] 508.2528, found 508.2525.

Data for 91. A mixture of rotamers (1:1). All signals reported. 1 Rf = 0.21 (50% EtOAc in hexanes; UV, KMnO4). H NMR (600 MHz, CDCl3) δ 7.08 (app dt, J = 13.8, 5.9 Hz, 1H), 6.23 (app d, J = 7.6 Hz, 2H), 4.75 – 4.64 (m, 1H), 4.30 – 4.06 (m, 2H), 4.04 – 3.83 (m, 2H), 3.28 (app td, J = 6.9, 6.0 Hz, 2H), 2.22 – 2.09 (m, 2H), 2.04 – 1.99 (m, 3H), 1.78 (t, J = 6.9 Hz, 2H), 1.65 (t, J = 7.2 Hz, 2H), 1.37 (app d, J = 5.8 Hz, 9H). 13C NMR

(151 MHz, CDCl3) δ 176.1 (app d, J = 6.9 Hz), 159.9 (app d, J = 9.5 Hz), 156.8, 153.0 (app d, J = 6.7 Hz), 149.8 (app d, J = 5.4 Hz), 148.1 (app d, J = 2.6 Hz), 126.3 (app d, J = 13.8 Hz), 82.7, 71.4, 69.8 (app d, J = 3.6 Hz), 57.1, 55.8, 49.1 (app d, J = 6.9 Hz), 46.8, 45.3, 39.8 (app d, J = 3.7 Hz), 34.8, 33.0, 32.5, 32.1, 29.7, 26.7, 13.3. IR 3302 (br) 2958, 2928, 1681, 1597, -1 + 1565, 1439, 1387, 1328, 1173, 732 cm . HRMS (ESI+): m/z calcd for C23H30N11O3 [M+H] 508.2528, found 508.2524.

Synthesis of 102

Grignard formation:

146

LiCl (4.12 g, 97.0 mmol, 1.53 equiv) was flame dried under high-vacuum (backfilling with nitrogen, 3 cycles). Magnesium (2.84 g, 117 mmol, 1.84 equiv) was added to the flask, which was again evacuated and refilled with nitrogen. After cooling to ambient temperature, THF (80 mL) was added. The resulting suspension was stirred until most LiCl was dissolved. 6- Bromohexyloxybenzene (16.3 g, 63.5 mmol, 1.00 equiv) was added slowly (exothermic) via syringe and the resulting reaction mixture was stirred for 3 h. Titration against iodine192 indicated a concentration of 0.55 M.

Cross coupling: 3,5-Dimethoxybenzoyl chloride (8.02 g, 40.0 mmol, 1.00 equiv) was dissolved in THF

(130 mL). Fe(acac)3 (0.42 g, 1.2 mmol, 0.030 equiv) was added and the red solution was cooled to –78 °C. A cooled solution of the GRIGNARD reagent (0 °C, lower temperature leads to precipitation) in THF (0.55 M, 80 mL, 44 mmol, 1.1 equiv) was added slowly via cannula over 30 min. After completion of the addition, the dark brown reaction mixture was stirred for another 30 min, before the cooling bath was removed. After additional 15 min, the brown turbid mixture had turned into a homogenous solution. The reaction was quenched by the addition of sat. aq. NH4Cl and further diluted with water. The organics were extracted with Et2O (3x), combined, and washed with brine. Drying over MgSO4, filtration and concentration followed by flash chromatography on silica (7.5% EtOAc in hexanes) afforded the title compound as colorless solid (12.3 g, 35.8 mmol, 90%).

The NMR spectroscopic data were full in agreement with literature values.193 1 H NMR (400 MHz, CDCl3) δ = 7.31 – 7.25 (m, 2H), 7.10 (d, J = 2.3 Hz, 2H), 6.95 – 6.88 (m, 3H), 6.65 (t, J = 2.3 Hz, 1H), 3.96 (t, J = 6.5 Hz, 2H), 3.83 (s, 6H), 2.94 (t, J = 7.3 Hz, 2H), 13 1.86 – 1.73 (m, 4H), 1.58 – 1.40 (m, 4H). C NMR (101 MHz, CDCl3) δ = 200.0, 160.9, 159.1, 139.1, 129.5, 120.5, 114.5, 106.0, 105.1, 67.7, 55.6, 38.6, 29.2, 29.1, 26.0, 24.4.

Synthesis of 111

TsOH·H2O (66 mg, 0.35 mmol, 0.28 eq.) was coevaporated with toluene (2x 5 mL). Then, a solution of 101 (389 mg, 1.23 mmol, 1.00 eq.) in CH2Cl2 (40 mL) was added. After flushing

192 A. Krasovskiy, P. Knochel, Synthesis 2006, 2006, 890. 193 C. Chu, A. Ramamurthy, A. Makriyannis, M. A. Tius, J. Org. Chem. 2003, 68, 55.

147 the flask with N2, a solution of 97 (318 mg, 1.26 mmol, 1.02 eq.) in CH2Cl2 (10 mL) was added at rt. TLC analysis after 1 h indicated full conversion of the starting material. Half saturated aq.

NaHCO3 was added (40 mL) and the organic phase was separated. The aqueous phase was extracted with CH2Cl2 (1x 15 mL). The combined organic fractions were washed with brine

(20 mL), dried with MgSO4, filtered and concentrated. Flash chromatography on silica (5% EtOAc in hexanes) afforded the title compound as colorless oil (567 mg, 1.03 mmol, 84%).

1 Rf = 0.26 (10% EtOAc in hexanes; KMnO4, UV). H NMR (400 MHz, CDCl3) δ 6.33 (s, 2H), 6.01 (s, 1H), 5.80 (s, 2H), 4.68 – 4.47 (m, 2H), 4.02 (s, 1H), 3.37 (t, J = 6.8 Hz, 2H), 2.37 (ddd, J = 9.6, 6.2, 5.0 Hz, 1H), 2.33 – 2.28 (m, 2H), 1.83 – 1.75 (m, 2H), 1.54 – 1.46 (m, 3H), 1.42 – 1.30 (m, 2H), 1.34 (s, 3H), 1.23 (s, 9H), 1.22 (s, 2H), 1.20 (s, 6H), 1.14 – 1.04 (m, 2H), 0.98 (s, 13 3H). C NMR (101 MHz, CDCl3) δ = 178.7, 155.1, 150.4, 149.5, 120.4, 111.7, 106.5, 66.6, 47.4, 44.3, 44.3, 44.2, 41.0, 39.1, 37.9, 37.5, 34.2, 32.9, 29.5, 28.8, 28.2, 28.2, 27.4, 26.0, 24.6, 20.9. IR (neat) 3450 (br), 2933, 2868, 1706, 1625, 1574, 1429, 1282, 1160, 1024, 909, 731 cm-

1 + ퟐퟒ . HRMS (MALDI): m/z calcd for C30H46BrO4 [M+H] 549.2574, found 549.2574. [] 퐃 =

+54.2 (c = 1.27, CHCl3).

Synthesis of 112

111 (1.16 g, 2.11 mmol, 1.00 equiv) was dissolved in acetone (4.2 mL) and K2CO3 (758 mg, 5.49 mmol, 2.60 equiv) was added. Dimethyl sulfate (504 µL, 5.28 mmol, 2.50 equiv) was added and it was stirred for 20 h. The reaction mix was diluted with Et2O (100 mL), washed with water (20 mL) and brine (20 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (3% EtOAc in hexanes) afforded the title compound as colorless oil (895 mg, 1.55 mmol, 73%).

1 Rf = 0.55 (10% EtOAc in hexanes; KMnO4, UV). H NMR (400 MHz, CDCl3) δ 6.47 (s, 2H), 5.77 (s, 1H), 4.61 – 4.47 (m, 2H), 3.99 (s, 1H), 3.74 (s, 6H), 3.37 (t, J = 6.8 Hz, 2H), 2.20 – 2.13 (m, 2H), 2.08 – 2.03 (m, 1H), 1.84 – 1.76 (m, 2H), 1.74 – 1.69 (m, 1H), 1.60 – 1.53 (m, 2H), 1.44 – 1.33 (m, 2H), 1.29 (s, 3H), 1.27 (s, 6H), 1.29 – 1.18 (m, 2H), 1.21 (s, 9H), 1.17 – 13 1.08 (m, 2H), 0.97 (s, 3H). C NMR (101 MHz, CDCl3) δ = 178.6, 158.5, 149.4, 137.3, 126.4, 117.7, 102.8, 67.6, 55.9, 47.5, 44.6, 43.9, 41.0, 39.0, 38.1, 37.7, 34.1, 32.9, 29.6, 29.0, 28.2, 27.7, 27.5, 26.5, 24.7, 21.2. IR (neat) 2932, 2866, 1726, 1606, 1573, 1462, 1411, 1281, 1239,

148

-1 + 1123, 1151 cm . HRMS (MALDI): m/z calcd for C32H49BrO4 [M] 576.2809, found 576.2808.

ퟐퟓ [] 퐃 = +71.8 (c = 1.27, CHCl3).

Synthesis of 113

112 (895 mg, 1.55 mmol) was dissolved in CH2Cl2 (7.7 mL) at 0 °C. A solution of DIBAL in hexanes (1 M, 3.87 mL, 3.87 mmol) was added slowly via syringe. TLC after 10 minutes indicated full consumption of starting material. The reaction was quenched by the addition of EtOAc (10 mL) and a saturated aq. solution of Rochelle's salt (5 mL). The mixture was vigorously stirred for 20 min and then extracted with EtOAc (3x 30 mL). The combined organics were washed with brine (20 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (13% EtOAc in hexanes) afforded the title compound as colorless oil (660 mg, 1.34 mmol, 86%).

1 Rf = 0.28 (20% EtOAc in hexanes; KMnO4, weakly UV). H NMR (400 MHz, CDCl3) δ 6.48 (s, 2H), 5.71 (s, 1H), 4.07 (s, 2H), 4.01 (app p, J = 2.0 Hz, 1H), 3.75 (s, 6H), 3.37 (t, J = 6.8 Hz, 2H), 2.25 – 2.18 (m, 2H), 2.10 – 2.04 (m, 1H), 1.85 – 1.75 (m, 2H), 1.73 – 1.70 (m, 1H), 1.60 – 1.54 (m, 2H), 1.43 – 1.35 (m, 2H), 1.31 (s, 3H), 1.32 – 1.20 (m, 2H), 1.27 (s, 6H), 1.17 – 1.07 13 (m, 2H), 0.97 (s, 3H). C NMR (101 MHz, CDCl3) δ = 158.5, 149.3, 142.0, 123.8, 117.8, 102.9, 66.8, 55.9, 47.5, 44.5, 43.9, 41.0, 38.1, 37.6, 34.2, 32.9, 29.6, 29.0, 29.0, 28.2, 28.1, 26.4, 24.7, 21.2. IR (neat) 3402 (br), 2931, 2861, 1605, 1572, 1462, 1410, 1238, 1120, 833 cm-1.

+ ퟐퟑ 25 HRMS (MALDI): m/z calcd for C27H41BrO3 [M] 492.2234, found 492.2235. [] 퐃 D = +86.8

(c = 1.96, CHCl3).

Synthesis of 114

113 (134 mg, 0.272 mmol, 1.00 equiv) was dissolved in DMF (1.00 mL) and sodium azide (88 mg, 1.36 mmol, 5.00 equiv) was added. After stirring overnight at rt, the reaction mix was diluted with Et2O (50 mL), washed with water (10 mL) and brine (2x 10 mL), dried over

149

MgSO4, filtered and concentrated. Flash chromatography on silica (15% EtOAc in hexanes) afforded the title compound as colorless oil (111 mg, 0.244 mmol, 90%).

1 Rf = 0.45 (25% EtOAc in hexanes; KMnO4, weak UV). H NMR (400 MHz, CDCl3) δ 6.49 (s, 2H), 5.74 – 5.68 (m, 1H), 4.11 – 4.06 (m, 2H), 4.03 – 4.00 (m, 1H), 3.75 (s, 6H), 3.22 (t, J = 6.9 Hz, 2H), 2.26 – 2.18 (m, 2H), 2.11 – 2.05 (m, 1H), 1.76 – 1.70 (m, 1H), 1.63 – 1.50 (m, 4H), 1.49 – 1.37 (m, 2H), 1.32 (s, 3H), 1.28 (s, 6H), 1.35 – 1.24 (m, 2H), 1.18 – 1.08 (m, 2H), 13 0.98 (s, 3H). C NMR (101 MHz, CDCl3) δ = 158.5, 149.3, 142.0, 123.7, 117.8, 102.8, 66.7, 55.9, 51.5, 47.5, 44.5, 43.9, 40.9, 38.0, 37.6, 29.9, 29.0, 28.9, 28.0, 26.7, 26.4, 24.6, 21.1. IR (neat) 3413 (br), 2933, 2863, 2096, 1606, 1572, 1463, 1411, 1239, 1122 cm-1. HRMS

+ ퟐퟓ (MALDI): m/z calcd for C27H41N3O3 [M] 455.3142, found 455.3141. [] 퐃 = +101.5 (c = 0.84,

CHCl3).

Synthesis of 96

114 (150 mg, 0.329 mmol, 1.00 equiv) was dissolved in THF (1.00 mL). After the flask was flushed with N2, CS2 (200 L, 3.32 mmol, 10.1 equiv) was added and it was stirred for 2 min, before PPh3 (90.0 mg, 0.343 mmol, 1.04 equiv) was added at once. The reaction was stirred for 8 h at rt, then 4 h at 45 °C. After cooling to rt, isolute was added and the volatiles were removed. Flash chromatography on silica (15% EtOAc in hexanes) yielded product contaminated with triphenylphosphine oxide. A second flash chromatography on silica (CH2Cl2 to 10% EtOAc in

CH2Cl2) afforded the title compound as colorless oil (130 mg, 0.276 mmol, 84%).

1 Rf = 0.21 (20% EtOAc in hexanes; KMnO4, UV). H NMR (400 MHz, CDCl3) δ 6.48 (s, 2H), 5.70 (s, 1H), 4.07 (s, 2H), 4.02 – 3.99 (m, 1H), 3.75 (s, 6H), 3.47 (t, J = 6.6 Hz, 2H), 2.25 – 2.18 (m, 2H), 2.10 – 2.04 (m, 1H), 1.71 (d, J = 7.5 Hz, 1H), 1.66 – 1.53 (m, 4H), 1.41 – 1.32 (m, 2H), 1.31 (s, 3H), 1.28 (s, 6H), 1.26 – 1.20 (m, 2H), 1.18 (s, 1H), 1.16 – 1.07 (m, 2H), 0.97 13 (s, 3H). C NMR (101 MHz, CDCl3) δ = 158.6, 149.3, 142.1, 123.8, 117.8, 102.9, 66.8, 56.0, 47.5, 45.2, 44.6, 43.9, 41.0, 38.1, 37.6, 30.1, 29.6, 29.0, 28.1, 26.6, 26.4, 24.6, 21.2. IR (neat) 3423 (br), 2933, 2862, 2180, 2099, 1605, 1572, 1452, 1410, 1238, 1122 cm-1. HRMS

+ ퟐퟓ (MALDI): m/z calcd for C28H41NO3S [M] 471.2802, found 471.2803. [] 퐃 = +97.6 (c = 0.80,

CHCl3).

150

Synthesis of 115

114 (26 mg, 0.057 mmol, 1.0 equiv) was dissolved in toluene (0.45 mL) and propargyl bromide (80% in toluene, 17 mg, 0.114 mmol, 2.0 equiv) was added at rt. Then 35% aq. NaOH (0.45 mL) and Bu4NHSO4 (4.0 mg, 0.012 mmol) were added and it was stirred vigorously for 3 h.

The reaction mix was diluted with hexanes, washed with water and brine, dried over MgSO4, filtered and concentrated. Flash chromatography on silica (5% EtOAc in hexanes) afforded the title compound as colorless oil (10 mg, 0.020 mmol, 36%) and recovered starting material (8 mg, 0.02 mmol, 31%).

1 Rf = 0.27 (5% EtOAc in hexanes; KMnO4, UV). H NMR (400 MHz, CDCl3) δ 6.48 (s, 2H), 5.80 (s, 1H), 4.23 – 4.12 (m, 2H), 4.11 – 3.98 (m, 3H), 3.75 (s, 6H), 3.22 (t, J = 6.9 Hz, 2H), 2.41 (t, J = 2.4 Hz, 1H), 2.29 – 2.24 (m, 1H), 2.23 – 2.16 (m, 1H), 2.08 – 2.03 (m, 1H), 1.69 (d, J = 8.4 Hz, 1H), 1.61 – 1.50 (m, 4H), 1.30 (s, 3H), 1.27 (s, 6H), 1.36 – 1.19 (m, 4H), 1.17 – 13 1.07 (m, 2H), 0.95 (s, 3H). C NMR (101 MHz, CDCl3) δ = 158.5, 149.3, 138.7, 127.5, 117.6, 102.7, 80.6, 74.1, 73.0, 55.9, 55.9, 51.6, 47.7, 44.6, 43.5, 41.0, 38.1, 37.8, 30.0, 29.0, 29.0, 27.8, 26.8, 26.4, 24.7, 21.1. IR (neat) 3305, 2933, 2862, 2096, 1606, 1572, 1464, 1411, 1239, 1121, -1 + 1067 cm . HRMS (MALDI): m/z calcd for C30H43N3O3Na [M+Na] 516.3197, found

ퟐퟓ 516.3201. [] 퐃 = +68.3 (c = 0.50, CHCl3).

Synthesis of 116

CS2 (0.25 mL of a solution of 50 L neat CS2 in 0.80 mL THF, ca. 0.27 mmol, 22 equiv) was added to the alkyl azide (6.0 mg, 0.012 mmol, 1.0 equiv). PPh3 (4.0 mg, 0.015 mmol, 1.26 equiv) was added and the reaction mix heated to 40 °C for 8 h. The volatiles were removed and the crude was purified by flash chromatography on silica (40% CH2Cl2 in hexanes to 50%) to give material contaminated with triphenylphosphine sulfide. A second flash chromatography

151 on silica (5% EtOAc in hexanes) afforded the title compound as colorless solid (5.0 mg, 0.0098 mmol, 81%).

1 Rf = 0.20 (40% CH2Cl2 in hexanes; KMnO4, UV). H NMR (400 MHz, CDCl3) δ 6.48 (s, 2H), 5.80 (s, 1H), 4.23 – 4.12 (m, 2H), 4.11 – 3.99 (m, 3H), 3.75 (s, 6H), 3.47 (t, J = 6.6 Hz, 2H), 2.41 (t, J = 2.4 Hz, 1H), 2.29 – 2.24 (m, 1H), 2.23 – 2.16 (m, 1H), 2.06 (ddd, J = 7.8, 3.9, 2.0 Hz, 1H), 1.69 (d, J = 8.5 Hz, 1H), 1.66 – 1.61 (m, 2H), 1.61 – 1.53 (m, 2H), 1.36 (tt, J = 8.8, 1.6 Hz, 2H), 1.29 (s, 3H), 1.28 (s, 6H), 1.27 – 1.20 (m, 2H), 1.16 – 1.07 (m, 2H), 0.95 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ = 158.5, 149.3, 138.8, 127.5, 117.6, 102.7, 80.6, 74.1, 73.0, 56.0, 55.9, 47.7, 45.2, 44.6, 43.5, 41.0, 38.1, 37.8, 30.1, 29.6, 29.0, 27.8, 26.6, 26.4, 24.6, 21.1. IR (neat) 3293, 2933, 2860, 2185, 2100, 1605, 1572, 1452, 1411, 1239, 1121 cm-1. HRMS

+ ퟐퟒ (MALDI): m/z calcd for C31H43NO3S [M] 509.2958, found 509.2957. [] 퐃 = +49.3 (c = 0.467,

CHCl3).

Synthesis of 119

4-Bromobutanoic acid (2.96 g, 17.7 mmol) was dissolved in CHCl3 (90 mL) and cooled to 0 °C. Then N-Me-morpholine (2.0 mL, 18 mmol, 1.2 equiv) was added and it was stirred for 10 minutes, before ethyl chloroformate (1.80 ml, 18.7 mmol, 1.1 equiv) was added via syringe. It was stirred for 45 minutes at 0 °C. Then solid methyl 6-aminohexanoate hydrochloride (3.54 g, 19.5 mmol, 1.10 equiv) was added at once. It was stirred for 5 min before N-methyl-morpholine (4.0 mL, 36 mmol, 2.0 equiv) was added dropwisely. It was stirred overnight (0 °C to rt). The mix was diluted with CH2Cl2 (200 mL), washed with 1 M aq. HCl, water and brine (50 mL each), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (50% EtOAc in hexanes) afforded 119 (1.26 g, 4.28 mmol, 24%) as colorless oil that solidified overnight.

1 Rf = 0.19 (50% EtOAc in hexanes; KMnO4). H NMR (400 MHz, CDCl3) δ 5.69 (br s, 1H), 3.65 (s, 3H), 3.58 (t, J = 6.2 Hz, 2H), 3.28 – 3.20 (m, 2H), 2.33 (t, J = 7.1 Hz, 2H), 2.30 (t, J = 7.4 Hz, 2H), 2.13 – 2.05 (m, 2H), 1.67 – 1.58 (m, 2H), 1.50 (tt, J = 7.7, 6.6 Hz, 2H), 1.38 – 1.29 (m, 2H).

13 C NMR (101 MHz, CDCl3) 174.2, 171.7, 51.6, 44.7, 39.4, 33.9, 33.4, 29.3, 28.3, 26.4, 24.5. IR (neat) 3319, 2940, 2867, 1731, 1638, 1535, 1735, 1193, 1166, 980 cm-1. HRMS (MALDI): + m/z calcd for C11H21BrNO3 [M] 294.0699, found 294.0699.

Synthesis of 120

152

119 (324 mg, 1.10 mmol, 1.10 equiv) was dissolved in THF (2.00 mL) and combined with 121 (249 mg, 1.00 mmol, 1.00 equiv). The suspension was stirred at rt for 10 min, then diluted successively with DMF (ca. 2 mL) to dissolve the material. After 30 min at rt, TLC indicated no conversion. KI (17 mg, 0.10 mmol, 0.1 equiv) was added and stirring was continued at 90 °C for 5 h. The reaction mixture was cooled to rt and diluted with EtOAc (100 mL). The organics were washed with 5% aq. LiCl (2x 20 mL) and brine (1x 20 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (5% MeOH in CH2Cl2) afforded 120 (175 mg, 0.378 mmol, 38%) as orange solid.

1 Rf = 0.19 (5% MeOH in CH2Cl2; orange spot, UV, KMnO4). H NMR (400 MHz, CDCl3) δ 8.41 (d, J = 8.9 Hz, 1H), 6.30 (d, J = 9.0 Hz, 1H), 5.71 (s, 1H), 4.12 (t, J = 5.1 Hz, 4H), 3.66 (s, 3H), 3.26 (td, J = 7.1, 5.8 Hz, 2H), 2.80 – 2.68 (m, 4H), 2.51 (t, J = 7.1 Hz, 2H), 2.31 (t, J = 7.4 Hz, 2H), 2.27 (t, J = 7.2 Hz, 2H), 1.90 (app p, J = 7.2 Hz, 2H), 1.64 (app p, J = 7.4 Hz, 2H), 13 1.52 (tt, J = 7.9, 6.5 Hz, 2H), 1.40 – 1.30 (m, 2H). C NMR (101 MHz, CDCl3) δ = 174.2, 172.4, 145.2, 145.0, 144.9, 135.2, 123.8, 102.8, 57.4, 52.7, 51.7, 49.4, 39.4, 34.2, 34.0, 29.5, 26.5, 24.6, 22.4. IR 3290, 3096, 2931, 2860, 1733, 1639, 1610, 1548, 1487, 1438, 1296, 1250, -1 + 1166, 997 cm . HRMS (ESI+): m/z calcd for C21H31N6O6Na [M+H] 463.2300, found 463.2303.

Synthesis of 117

120 (29 mg, 0.063 mmol) was dissolved in a mixture of aqueous phosphate buffer pH7 (2.7 mL) and THF (0.6 mL). Novozyme (26 mg, 0.063 mmol) (lipase on acrylic resin from Candida Antarctica) was added and the reaction mix was stirred at rt. After 3.5 h, it was filtered over celite and concentrated. The residue was purified by preparative HPLC to give the title compound (32 mg, 0.047 mmol, 75%) as orange oil.

153

1 H NMR (400 MHz, CD3CN) δ 8.52 (d, J = 8.8 Hz, 1H), 7.50 (br, 4H), 7.10 (s, 1H), 6.62 (d, J = 8.9 Hz, 1H), 4.88 (br, 1H), 3.74 (br, 4H), 3.37 (br, 1H), 3.21 (td, J = 6.4, 2.3 Hz, 4H), 2.57 – 2.44 (m, 2H), 2.30 (t, J = 7.3 Hz, 2H), 2.07 – 1.99 (m, 2H), 1.97 (app p, J = 2.5 Hz, 2H), 1.59 13 (m, J = 7.5 Hz, 2H), 1.56 – 1.47 (m, 2H), 1.40 – 1.31 (m, 2H). C NMR (101 MHz, CD3CN) δ = 175.4, 174.9, 146.2, 145.8, 145.6, 136.5, 126.1, 106.0, 58.2, 51.9, 47.3, 40.1, 34.7, 34.2, 29.4, 26.8, 25.1, 20.4. IR 3301 (br), 3095, 2935, 2868, 2614 (br), 1667, 1616, 1544, 1441, 1301, -1 + 1253, 1195, 1132 cm . HRMS (MALDI): m/z calcd for C20H29N6O6 [M+H] 449.2143, found 449.2138.

Synthesis of 127

96 (7.0 mg, 0.015 mmol, 1.0 equiv) was dissolved in dry THF (0.1 mL) and cooled to 0 °C.

NEt3 (20 L, 0.14 mmol, 9.7 equiv) and DMAP (4.0 mg, 0.033 mmol, 2.2 equiv) were added followed by a solution of 117 (16 mg, 0.024 mmol) in dry THF (0.15 mL). After the addition of EDC (12 mg, 0.063 mmol, 0.063 mmol, 4.2 equiv), the cooling bath was removed and the mixture was stirred at rt for 3 h. The reaction mixture was diluted with EtOAc (100 mL), washed with water (10 mL) and brine (2x 10 mL), dried over MgSO4, filtered and concentrated.

Flash chromatography on silica (50% EtOAc in CH2Cl2 + 7% MeOH) afforded material slightly contaminated with a fluorescent impurity according to TLC. Preparative TLC (50% EtOAc in

CH2Cl2 + 10% MeOH) yielded the product as orange oil (12 mg, 0.013 mmol, 90%).

1 Rf = 0.18 (50% EtOAc in CH2Cl2 + 5% MeOH; KMnO4, UV). H NMR (400 MHz, CDCl3) δ 8.41 (d, J = 8.9 Hz, 1H), 6.47 (s, 2H), 6.29 (d, J = 9.0 Hz, 1H), 5.78 – 5.75 (m, 1H), 5.68 (s, 1H), 4.59 – 4.50 (m, 2H), 4.17 – 4.06 (m, 4H), 3.98 (s, 1H), 3.73 (s, 6H), 3.46 (t, J = 6.6 Hz, 2H), 3.31 – 3.21 (m, 2H), 2.71 (s, 4H), 2.48 (s, 2H), 2.33 (t, J = 7.3 Hz, 2H), 2.25 (t, J = 7.2 Hz, 2H), 2.20 – 2.12 (m, 2H), 2.08 – 2.02 (m, 1H), 1.94 – 1.84 (m, 2H), 1.70 (d, J = 7.6 Hz, 1H), 1.64 (m, J = 14.9, 7.2, 2.1 Hz, 4H), 1.60 – 1.48 (m, 4H), 1.41 – 1.32 (m, 4H), 1.28 (s, 3H), 1.27 13 (s, 6H), 1.32 – 1.19 (m, 2H), 1.17 – 1.06 (m, 2H), 0.94 (s, 3H). C NMR (101 MHz, CDCl3) δ = 173.7, 172.4, 158.5, 149.4, 145.2, 145.0, 144.9, 137.1, 135.2, 127.0, 117.5, 102.8, 102.8, 102.7, 67.7, 57.4, 55.9, 52.7, 49.5, 47.4, 45.2, 44.5, 44.0, 41.0, 39.4, 38.1, 37.7, 34.4, 34.3, 30.1, 29.6, 29.5, 29.0, 27.8, 26.6, 26.5, 26.4, 24.7, 24.6, 22.5, 21.1. IR (neat) 3313, 2932, 2862, 2181,

154

2105, 1729, 1649, 1609, 1545, 1296, 1248, 1122, 997 cm-1. HRMS (ESI+): m/z calcd for

+ ퟐퟑ C48H68N7O8S [M+H] 902.4845, found 902.4844. [] 퐃 = +24.4 (c = 0.475, CHCl3).

Synthesis of 2-(((1S,4S,5S)-4-(4-(8-bromo-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6- dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)isoindoline-1,3-dione

113 (110 mg, 0.223 mmol, 1.00 equiv) was dissolved in in THF (1.70 mL) and cooled to 0 °C.

PPh3 (76.0 mg, 0.290 mmol, 1.30 equiv), phthalimide (42.0 mg, 0.285 mmol, 1.28 equiv) and a solution of DIAD (59 L, 0.30 mmol, 1.3 equiv) in THF (0.5 mL) were added sequentially. The cooling bath was removed and it was stirred at rt. TLC after 1.5 h (20% EtOAc in hexanes) indicated full consumption of starting material. The mixture was diluted with diethylether

(50 mL), washed with sat. sodium bicarbonate and brine (10 mL each), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (5% EtOAc in hexanes) afforded the title compound as colorless oil (86 mg, 0.14 mmol, 62%).

1 Rf = 0.28 (10% EtOAc in hexanes; KMnO4, UV). H NMR (400 MHz, CDCl3) δ 7.84 (dd, J = 5.5, 3.0 Hz, 2H), 7.70 (dd, J = 5.5, 3.1 Hz, 2H), 6.43 (s, 2H), 5.57 – 5.53 (m, 1H), 4.34 – 4.19 (m, 2H), 3.94 (t, J = 2.5 Hz, 1H), 3.71 (s, 6H), 3.36 (t, J = 6.8 Hz, 2H), 2.19 – 2.11 (m, 2H), 2.04 – 1.98 (m, 1H), 1.83 – 1.74 (m, 2H), 1.72 (d, J = 7.9 Hz, 1H), 1.59 – 1.50 (m, 2H), 1.36 (ddt, J = 9.0, 7.3, 6.0 Hz, 2H), 1.26 (s, 3H), 1.24 (s, 6H), 1.22 – 1.17 (m, 2H), 1.14 – 1.04 (m, 13 2H), 0.93 (s, 3H). C NMR (101 MHz, CDCl3) δ 168.3, 158.6, 149.3, 136.1, 133.9, 132.4, 123.7, 123.3, 117.6, 102.6, 55.8, 47.6, 44.5, 44.3, 42.6, 41.2, 38.1, 37.5, 34.2, 32.9, 29.6, 29.0, 28.2, 27.7, 26.4, 24.6, 20.9. IR (neat) 2931, 2861, 1773, 1716, 1605, 1572, 1390, 1120 cm-1.

+ ퟐퟓ HRMS (MALDI): m/z calcd for C35H45BrNO4 [M+H] 622.2526, found 622.2541. [] 퐃 =

+58.3 (c = 1.0, CHCl3).

Synthesis of 2-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6- dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)isoindoline-1,3-dione

155

113 (29 mg, 0.047 mmol, 1.0 equiv) was dissolved in DMF (0.31 mL), combined with NaN3 (5 equiv) and stirred for 1.5 h. The reaction mix was diluted with Et2O (50 mL), washed with 5% aq. LiCl (3x 3 mL) and brine (1x 5 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (40% CH2Cl2 in hexanes to 100%) afforded the title compound as colorless oil (24 mg, 0.041 mmol, 88%).

1 Rf = 0.48 (20% EtOAc in hexanes; KMnO4, UV). H NMR (400 MHz, CDCl3) δ 7.84 (dd, J = 5.4, 3.0 Hz, 2H), 7.70 (dd, J = 5.5, 3.0 Hz, 2H), 6.43 (s, 2H), 5.55 (dd, J = 2.9, 1.5 Hz, 1H), 4.34 – 4.18 (m, 2H), 3.96 – 3.93 (m, 1H), 3.71 (s, 6H), 3.21 (t, J = 6.9 Hz, 2H), 2.20 – 2.11 (m, 2H), 2.01 (ddt, J = 5.9, 4.2, 1.9 Hz, 1H), 1.73 (d, J = 8.0 Hz, 1H), 1.57 – 1.50 (m, 4H), 1.26 (s, 3H), 1.24 (s, 6H), 1.35 – 1.16 (m, 4H), 1.14 – 1.05 (m, 2H), 0.94 (s, 3H). 13C NMR (101 MHz,

CDCl3) δ 168.3, 158.5, 149.3, 136.0, 133.9, 132.4, 123.7, 123.2, 117.6, 102.6, 55.8, 51.6, 47.6, 44.6, 44.3, 42.6, 41.2, 38.1, 37.5, 30.0, 29.0, 29.0, 29.0, 27.7, 26.7, 26.4, 24.7, 20.9. IR (neat) 2932, 2862, 2095, 1772, 1716, 1605, 1572, 1390, 1239, 1121 cm-1. HRMS (MALDI): m/z calcd

+ ퟐퟒ for C35H44N4O4 [M] 584.3357, found 584.3357. [] 퐃 = +72.3 (c = 0.733, CHCl3).

Synthesis of 128

The alkyl azide (24 mg, 0.041 mmol, 1.0 equiv) was dissolved in EtOH (0.4 mL) and combined with solutions of E/Z-crotyl-alcohol (44 mg, 0.62 mmol, 15 equiv) and hydrazine·H2O (21 mg, 0.41 mmol, 10 equiv) in EtOH (0.2 mL each). The flask was flushed with nitrogen, capped, and then heated to 75 °C. After 1.5 h the mixture was cooled to rt, filtered (rinsing with EtOH) and concentrated. Flash chromatography on silica (10% MeOH in CH2Cl2 + 1% NEt3) afforded contaminated material. Preparative TLC (same eluent) yielded the title compound as colorless oil (12 mg, 0.026 mmol, 64%).

1 Rf = 0.29 (10% MeOH in CH2Cl2 + 1% NEt3; KMnO4, UV). H NMR (400 MHz, CDCl3) δ 6.47 (s, 2H), 5.56 (dd, J = 2.8, 1.5 Hz, 1H), 3.98 (t, J = 2.3 Hz, 1H), 3.74 (s, 6H), 3.27 – 3.19 (m, 4H), 2.22 – 2.16 (m, 1H), 2.13 (td, J = 5.7, 1.4 Hz, 1H), 2.06 (tt, J = 6.0, 1.6 Hz, 1H), 1.98 (br, 2H), 1.71 (d, J = 8.3 Hz, 1H), 1.56 (ddd, J = 14.7, 6.2, 4.1 Hz, 4H), 1.37 – 1.31 (m, 2H), 1.30 (s, 3H), 1.27 (s, 6H), 1.25 – 1.18 (m, 2H), 1.16 – 1.07 (m, 2H), 0.96 (s, 3H). 13C NMR

(101 MHz, CDCl3) δ 158.6, 149.2, 143.1, 121.4, 118.1, 102.9, 56.0, 51.6, 47.6, 47.5, 44.6, 44.6,

156

41.0, 38.1, 37.6, 30.0, 29.0, 29.0, 29.0, 28.1, 26.8, 26.5, 24.7, 21.2. IR (neat) 2932, 2095, 1605, -1 1572, 1463, 1411, 1239, 1122, 833 cm . HRMS (MALDI): m/z calcd for C27H40N3O2 [M-

+ ퟐퟓ NH2] 438.3115, found 438.3115. [] 퐃 = +104.5 (c = 0.6, CHCl3).

Synthesis of 129

117 (19 mg, 0.028 mmol, 1.1 equiv) was dissolved in THF (0.3 mL) combined with NEt3 (20 L, 0.14 mmol, 5.4 equiv). EDC·HCl (6.2 mg, 0.032 mmol, 1.2 equiv) was added and it was stirred for 5 minutes at rt, before a solution of 128 (12 mg, 0.026 mmol, 1.0 equiv) in THF (0.3 mL) was added. The mixture was concentrated under a stream of nitrogen to a volume of about 0.3 mL and stirred at rt. After 3 h, the mixture was diluted with EtOAc (100 mL), washed with water (10 mL) and brine (2x 10 mL), dried over MgSO4, filtered and concentrated. The residue was purified by preparative TLC (CH2Cl2:hexanes:MeOH 10:3:1) to afford the title compound (13 mg, 0.015 mmol, 56%) as orange oil.

1 Rf = 0.37 (CH2Cl2:hexanes:MeOH 10:3:1; orange spot, KMnO4, UV). H NMR (400 MHz,

CDCl3) δ 8.41 (d, J = 8.9 Hz, 1H), 6.47 (s, 2H), 6.29 (d, J = 9.0 Hz, 1H), 5.86 (s, 1H), 5.60 (dt, J = 2.9, 1.5 Hz, 1H), 5.37 (t, J = 5.7 Hz, 1H), 4.11 (t, J = 4.9 Hz, 4H), 3.99 – 3.93 (m, 1H), 3.93 – 3.77 (m, 2H), 3.73 (s, 6H), 3.26 (app q, J = 6.7 Hz, 2H), 3.21 (t, J = 6.9 Hz, 2H), 2.70 (t, J = 4.9 Hz, 4H), 2.47 (t, J = 7.1 Hz, 2H), 2.25 (t, J = 7.3 Hz, 2H), 2.22 – 2.13 (m, 3H), 2.10 – 2.03 (m, 2H), 1.89 (q, J = 7.1 Hz, 2H), 1.67 (t, J = 8.4 Hz, 3H), 1.59 – 1.49 (m, 4H), 1.43 – 1.28 (m, 6H), 1.27 (s, 3H), 1.26 (d, J = 4.9 Hz, 6H), 1.21 (dd, J = 9.2, 7.3 Hz, 2H), 1.15 – 1.06 (m, 2H), 13 0.94 (s, 3H). C NMR (101 MHz, CDCl3) δ 172.7, 172.6, 158.5, 149.5, 145.3, 145.0, 144.9, 138.5, 135.3, 124.1, 123.7, 117.6, 102.8, 102.7, 57.4, 55.9, 52.7, 51.6, 49.5, 47.4, 44.6, 44.5, 44.5, 40.9, 39.3, 38.1, 37.6, 36.6, 34.3, 30.0, 29.3, 29.0, 29.0, 28.0, 26.7, 26.5, 26.4, 25.2, 24.7, 22.6, 21.2. IR (neat) 3301, 3094, 2931, 2861, 2095, 1644, 1609, 1543, 1294, 1247, 1121, 997, -1 + 912, 733 cm . HRMS (ESI+): m/z calcd for C47H69N10O7 [M+H] 885.5345, found 885.5345.

Synthesis of 130

157

129 (12 mg, 0.014 mmol, 1.0 equiv) was dissolved in THF (0.3 mL) and combined with CS2

(16 L, 0.27 mmol, 20 equiv). PPh3 (4.3 mg, 0.016 mmol, 1.2 equiv) was added and the reaction mix was heated to 40 °C for 5 h. After cooling to rt, the residue was directly purified by preparative TLC (10:3:1 CH2Cl2:hexanes:MeOH) to afford the title compound as orange oil (9 mg, 0.014 mmol, 74%).

1 Rf = 0.37 (CH2Cl2:hexanes:MeOH 10:3:1; orange spot, KMnO4, UV). H NMR (400 MHz,

CDCl3) δ 8.42 (d, J = 8.9 Hz, 1H), 6.47 (s, 2H), 6.30 (d, J = 9.0 Hz, 1H), 5.89 (s, 1H), 5.63 – 5.57 (m, 1H), 5.38 (t, J = 5.6 Hz, 1H), 4.13 (t, J = 5.0 Hz, 4H), 3.96 (t, J = 2.2 Hz, 1H), 3.92 – 3.78 (m, 2H), 3.73 (s, 6H), 3.46 (t, J = 6.6 Hz, 2H), 3.26 (q, J = 6.7 Hz, 2H), 2.73 (s, 4H), 2.50 (s, 2H), 2.26 (t, J = 7.2 Hz, 2H), 2.23 – 2.13 (m, 3H), 2.09 – 2.03 (m, 2H), 1.90 (app p, J = 7.1 Hz, 2H), 1.71 – 1.60 (m, 5H), 1.55 (ddd, J = 14.6, 8.7, 6.0 Hz, 4H), 1.44 – 1.31 (m, 4H), 1.27 (s, 3H), 1.27 (s, 6H), 1.32 – 1.17 (m, 6H), 1.17 – 1.05 (m, 2H), 0.94 (s, 3H). 13C NMR (101

MHz, CDCl3) δ 172.7, 172.5, 158.5, 149.4, 145.2, 145.0, 144.9, 138.5, 135.2, 124.1, 123.9, 117.6, 102.8, 102.8, 57.4, 56.0, 52.7, 49.4, 47.4, 45.2, 44.6, 44.5, 40.9, 39.3, 38.1, 37.6, 36.6, 34.2, 30.0, 29.6, 29.3, 29.0, 28.0, 26.6, 26.5, 26.4, 25.2, 24.6, 22.5, 21.2. IR (neat) 3301, 3094, 2932, 2861, 2181, 2098, 1645, 1610, 1545, 1296, 1249, 1122, 997 cm-1. HRMS (ESI+): m/z + calcd for C48H69N8O7S [M+H] 901.5004, found 901.5008.

Synthesis of 131

130 (2.00 g, 8.73 mmol, 1.00 equiv) was dissolved in DMF (17 mL) at rt. Potassium phthalimide (1.70 g, 9.17 mmol, 1.05 equiv) was added and it was stirred for 3 h. Water was added and the mixture was extracted with EtOAc (3x 20 mL). The combined organic fractions were washed with 5% aq. LiCl (2x 20 mL) and brine (20 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica afforded the title compound as colorless oil (2.2 g, 7.5 mmol, 85%).

158

1 H NMR (400 MHz, CDCl3) δ 7.92 – 7.86 (m, 2H), 7.80 – 7.74 (m, 2H), 5.66 – 5.63 (m, 1H), 4.53 (dd, J = 17.6, 1.8 Hz, 1H), 4.38 (dd, J = 17.6, 1.8 Hz, 1H), 2.86 (dt, J = 9.3, 5.5 Hz, 1H), 2.67 (td, J = 5.8, 1.7 Hz, 1H), 2.54 (td, J = 5.9, 1.6 Hz, 1H), 2.16 (d, J = 9.3 Hz, 1H), 1.51 (s, 13 3H), 1.03 (s, 3H). C NMR (101 MHz, CDCl3) δ 202.8, 167.7, 165.6, 134.5, 131.9, 123.8, 120.2, 58.2, 54.7, 46.5, 41.4, 40.9, 26.7, 22.2.

Synthesis of 132

CeCl3 (1.82 g, 4.89 mmol, 1.00 equiv) was dissolved in THF–MeOH (1:1, 50 mL) and NaBH4 (0.191 g, 5.04 mmol, 1.03 equiv) was added at rt. After 15 minutes, the cloudy mix was cooled to –78 °C and a precooled (–78 °C) solution of the enone (1.45 g, 4.89 mmol, 1.00 equiv) in

THF–MeOH (1:1, 10 mL) was added via cannula. After 1h, another portion NaBH4 (0.191 g, 5.04 mmol, 1.03 equiv) was added and stirring was continued at –78 °C for 4h. Acetone was added to quench the reaction. Extraction (water/EtOAc 3x 10 mL), washing with brine, drying over MgSO4, filtration and concentration. Flash chromatography on silica (20% EtOAc in hexanes to 25%) afforded the title compound as light yellow solid (1.35 g, 4.54 mmol, 93%).

1 H NMR (400 MHz, CDCl3) δ 7.85 (dd, J = 5.4, 3.1 Hz, 2H), 7.73 (dd, J = 5.5, 3.1 Hz, 2H), 5.49 (dt, J = 3.1, 1.6 Hz, 1H), 4.50 – 4.45 (m, 1H), 4.24 (app qt, J = 15.9, 1.7 Hz, 2H), 2.47 (ddd, J = 9.2, 6.3, 5.3 Hz, 1H), 2.29 (tdd, J = 5.8, 3.3, 1.9 Hz, 1H), 2.14 (td, J = 5.4, 1.5 Hz, 1H), 1.68 (s, 1H), 1.35 (d, J = 9.3 Hz, 1H), 1.33 (s, 3H), 1.03 (s, 3H). 13C NMR (101 MHz,

CDCl3) δ 168.1, 144.8, 134.2, 132.1, 123.5, 121.4, 73.1, 48.4, 44.8, 41.8, 39.2, 35.8, 26.8, 22.8. IR 3472 (br), 2920, 1771, 1707, 1422, 1389, 727, 713 cm-1. HRMS (ESI+): m/z calcd for + C18H19NNaO3 [M+Na] 320.1257, found 320.1257.

Synthesis of 138

140 (7.47 g, 30.0 mmol, 1.00 equiv) was dissolved in THF (30 mL) and cooled to –78 °C. A solution of 6-chlorohexylmagnesium bromide·LiCl (1.0 M, 30.0 mL, 30.0 mmol, 1.00 equiv) was added slowly via cannula. The resulting mixture was stirred overnight (thereby allowed to reach rt). The reaction mixture was diluted with ether (200 mL) and water (400 mL) was added. The aqueous phase was extracted with ether (3x 100 mL). The combined organic fractions were

159 washed with brine (100 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (2% Et2O in pentane) afforded the title compound as colorless oil (6.48 g, 22.5 mmol, 93%).

1 Rf = 0.35 (5% Et2O in pentane; Seebach). H NMR (400 MHz, CDCl3) δ 3.52 (t, J = 6.8 Hz, 2H), 1.81 – 1.70 (m, 2H), 1.47 – 1.36 (m, 2H), 1.33 – 1.23 (m, 6H), 1.22 (s, 12H), 0.90 (s, 6H). 13 13 C NMR C NMR (101 MHz, CDCl3) δ 83.0, 45.3, 41.2, 32.8, 29.9, 27.0, 26.4, 25.0, 24.8. IR 2978, 2932, 2859, 1475, 1370, 1389, 1306, 1142, 852, 693 cm-1. HRMS (EI+): m/z calcd + for C14H27BClO2 [M-CH3] 271.1793, found 273.1773.

Synthesis of 139

140 (1.75 g, 7.03 mmol, 1.00 equiv) was dissolved in THF (13 mL) and cooled to –78 °C. A solution of (6-((tert-butyldimethylsilyl)oxy)hexyl)magnesium bromide·LiCl (0.56 M, 11.3 mL, 6.33 mmol, 0.90 equiv) was added slowly via syringe. The resulting reaction mixture was stirred overnight (thereby allowed to reach rt). The reaction was quenched by the addition of sat. aq. NH4Cl (15 mL). The organic material was extracted with EtOAc (3x 20 mL). The combined organic fractions were washed with brine, dried over MgSO4, filtered and concentrated in vacuo. Flash chromatography on silica (1% Et2O in hexanes to 2%) afforded the title compound as colorless oil (1.58 g, 4.11 mmol, 65%).

1 Rf = 0.15 (3% Et2O in hexanes; KMnO4). H NMR (400 MHz, CDCl3) δ 3.59 (t, J = 6.7 Hz, 2H), 1.57 – 1.46 (m, 2H), 1.35 – 1.23 (m, 8H), 1.22 (s, 12H), 0.90 (s, 6H), 0.89 (s, 9H), 0.04 (s, 13 6H). C NMR (101 MHz, CDCl3) δ 83.0, 63.5, 41.4, 33.0, 30.5, 26.6, 26.1, 25.9, 25.1, 24.8, 18.5, -5.1. IR 2930, 2858, 1474, 1370, 1306, 1255, 1140, 1102, 835, 775 cm-1. HRMS (ESI+): + m/z calcd for C21H46BO3Si [M+H] 385.3308, found 385.3308.

Synthesis of 144

BuLi (1.6 M in hexanes, 2.8 mL, 4.5 mmol, 1.13 equiv) was added to a –78 °C solution of 1- Br-3,5-dimethoxybenzene (1.03 g, 4.74 mmol, 1.2 equiv) in THF (15 mL). After 1h, a solution of the boronic ester (1.52 g, 3.95 mmol, 1.00 equiv) in THF (7.5 mL) was added and it was stirred for 50 min at –78 °C, then another 30 min without cooling bath. The mixture was cooled

160 back to –78 °C and MeOH (15 mL) was added. After 10 min, NBS (774 mg, 4.35 mmol,

1.10 equiv) was added as a solid and the reaction mix was stirred for 75 min. Aq. sat. Na2S2O3 (0.5 mL) was added and the mixture was allowed to reach rt, before conc. HCl (0.5 mL) was added. After 1 h at rt, water (400 mL) was added the the mixture was extracted with EtOAc (3x 100 mL). The combined organic fractions were washed with sat. aq. sodium bicarbonate

(50 mL) and brine (50 mL), dried over MgSO4, filtered and concentrated in vacuo. Flash chromatography on silica (10% EtOAc in hexanes to 15%) afforded the title compound as colorless oil (505 mg, 1.80 mmol, 46%).

1 Rf = 0.48 (50% EtOAc in hexanes; Uv, KMnO4). H NMR (400 MHz, CDCl3) δ 6.48 (d, J = 2.3 Hz, 2H), 6.30 (t, J = 2.2 Hz, 1H), 3.79 (s, 6H), 3.59 (t, J = 6.6 Hz, 2H), 1.59 – 1.53 (m, 2H), 1.52 – 1.45 (m, 2H), 1.29 (tdd, J = 4.2, 3.4, 1.2 Hz, 2H), 1.25 (s, 6H), 1.24 – 1.19 (m, 2H), 1.12 13 – 1.03 (m, 2H). C NMR (101 MHz, CDCl3) δ 160.6, 152.6, 104.8, 96.7, 77.2, 63.1, 55.3, 44.5, 38.1, 32.9, 30.2, 29.1, 25.7, 24.7. IR 3350 (br), 2931, 2858, 1595, 1456, 1422, 1204, 1154, -1 + 1054, 699 cm . HRMS (ESI+): m/z calcd for C17H29O3 [M+H] 281.2111, found 281.2113.

Synthesis of 5-(8-hydroxy-2-methyloctan-2-yl)benzene-1,3-diol

A freshly prepared solution of MeMgI (2.5 M in Et2O, 0.69 mL, 1.7 mmol, 3.3 equiv) was added to a solution of 144 (147 mg, 0.524 mmol, 1.00 equiv) in Et2O at rt. The mixture was heated to 170 °C under a stream of nitrogen. When dry, vacuum was applied (100 mbar) and the temperature was kept for 2 h. After cooling to rt, the residue was diluted with dry Et2O, then quenched by the addition of sat. aq. NH4Cl. After dilution with water, the resulting mixture was extracted with EtOAc (3x 10 mL). The combined organic fraction were washed with brine

(10 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (50% EtOAc in hexanes) afforded the title compound as light-brown solid (110 mg, 0.436 mmol, 83%).

1 Rf = 0.18 (50% EtOAc in hexanes; weak UV, KMnO4). H NMR (400 MHz, MeOD) δ 6.29 (d, J = 2.2 Hz, 2H), 6.08 (t, J = 2.1 Hz, 1H), 3.50 (t, J = 6.6 Hz, 2H), 1.58 – 1.52 (m, 2H), 1.46 (tt, J = 7.9, 6.1 Hz, 2H), 1.33 – 1.22 (m, 4H), 1.22 (s, 6H), 1.09 (dtt, J = 13.9, 7.7, 2.6 Hz, 2H). 13C NMR (101 MHz, MeOD) δ 159.0, 153.3, 105.7, 100.6, 63.0, 45.5, 38.5, 33.6, 31.2, 29.5, 26.8, 25.8. IR 3302 (br), 2932, 2859, 1600, 1465, 1332, 1152, 995, 843, 699 cm-1. HRMS (ESI+): + m/z calcd for C15H24O3 [M+H] 253.1798, found 253.1799.

161

Synthesis of 101

PPh3 (257 mg, 0.981 mmol, 1.1 equiv) was added to a solution of the triol (225 mg,

0.892 mmol, 1.0 equiv) and CBr4 (355 mg, 1.07 mmol, 1.2 equiv) in THF (9 mL) at 0 °C. After 10 minutes, the cooling bath was removed and it was stirred at rt for 1 h. Water (50 mL) was added the the organic material was extracted with EtOAc (3x 15 mL). The combined organic fractions were washed with brine (10 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (20% EtOAc in hexanes) afforded the title compound as colorless oil.

The spectra did not indicate any contamination with the respective secondary alkyl bromide and matched the literature data.

1 H NMR (300 MHz, CDCl3) δ 6.38 (d, J = 2.2 Hz, 2H), 6.18 (t, J = 2.2 Hz, 1H), 3.37 (t, J = 6.8 Hz, 2H), 1.86 – 1.73 (m, 2H), 1.58 – 1.48 (m, 2H), 1.44 – 1.30 (m, 2H), 1.23 (s, 2H), 1.22 (s, 6H), 1.13 – 1.01 (m, 2H).

Synthesis of 147

1-Bromo-3,5-dimethoxybenzene was converted to the corresponding GRIGNARD reagent according to GP2. The GRIGNARD reagent (0.7 M in THF, 10.2 mL, 8.4 mmol, 1.05 equiv) was added slowly to a solution of the ketoester in THF at –78 °C and stirred at that temperature for 30 min before the cooling bath was removed and stirring continued at for 1 h. The reaction was quenched by the addition of sat. aq. NH4Cl. Extraction with Et2O (3x 30 mL), washing with brine (30 mL), drying over MgSO4, filtration and concentration followed by flash chromatography on silica (10% EtOAc in hexanes) afforded the title compound as colorless oil (2.38 g, 7.34 mmol, 92%).

1 Rf = 0.38 (35% EtOAc in hexanes; UV, Seebach, KMnO4). H NMR (400 MHz, CDCl3) δ 6.57 (d, J = 2.3 Hz, 2H), 6.34 (t, J = 2.3 Hz, 1H), 4.09 (q, J = 7.2 Hz, 2H), 3.79 (s, 6H), 2.23 (t, J = 7.5 Hz, 2H), 1.83 – 1.69 (m, 2H), 1.61 – 1.53 (m, 2H), 1.51 (s, 3H), 1.33 – 1.25 (m, 4H), 1.23 13 (t, J = 7.1 Hz, 3H). C NMR (101 MHz, CDCl3) δ 173.9, 160.8, 150.9, 103.4, 98.2, 74.9, 60.3, 55.4, 43.9, 34.4, 30.3, 29.5, 24.9, 23.7, 14.4. IR 3500 (br), 2938, 1731, 1595, 1457, 1424, 1204,

162

-1 + 1152, 1046, 844, 702 cm . HRMS (ESI+): m/z calcd for C18H28NaO5 [M+Na] 347.1829, found 347.1831.

Synthesis of 149

The tertiary alcohol (2.36 g, 7.27 mmol, 1.00 equiv) was cooled to 0 °C and SOCl2 (1.33 mL, 18.2 mmol, 2.5 equiv) was added. After 1.5 h, the volatiles were removed in vacuo and the residue was dissolved in CH2Cl2 (12 mL). After cooling to –78 °C, a solution of AlMe3 (1.40 mL, 14.6 mmol, 2.0 equiv) in hexanes (5 mL) was added. The reaction mixture was stirred overnight (thereby allowed to reach rt). The solution was cooled with the help of an icebath, while dilute aq. HCl (1 M, 60 mL) was added. The organic material was extracted with

CH2Cl2 (3x 20 mL). The combined organic fractions were washed with brine (20 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (5% EtOAc in hexanes) afforded a mixture of the desired product along with inseparable olefinic side product (1.74 g).

The material was dissolved in DMF (23 mL) and OsO4 (2.5% in tBuOH, 3.05 mL, ca. 0.2 mmol) was added. After stirring for 5 min, oxone (1.15 g) was added. After 3 h, sodium sulfite (127 mg, 1.0 mmol) was added and it was stirred for 1 h. Water (400 mL) was added the the organic material was extracted with EtOAc (3x 100 mL). The combined organic fractions were washed witn aq. HCl (1 M, 2x 50 mL), 5% aq. LiCl (50 mL) and brine (50 mL), dried over MgSO4, filtered and concentrated in vacuo. Flash chromatography on silica (6% EtOAc in hexanes) afforded the title compound as colorless oil (980 mg, 3.03 mmol, 42%).

1 Rf = 0.50 (20% EtOAc in hexanes; very weak UV, Seebach). H NMR (400 MHz, CDCl3) δ 6.47 (d, J = 2.2 Hz, 2H), 6.30 (t, J = 2.2 Hz, 1H), 4.10 (q, J = 7.1 Hz, 2H), 3.79 (s, 6H), 2.22 (t, J = 7.6 Hz, 2H), 1.59 – 1.52 (m, 4H), 1.25 (s, 6H), 1.23 (d, J = 7.1 Hz, 3H), 1.31 – 1.16 (m, 13 2H), 1.12 – 1.03 (m, 2H). C NMR (101 MHz, CDCl3) δ 174.0, 160.6, 152.5, 104.8, 96.8, 60.3, 55.4, 44.4, 38.1, 34.5, 29.9, 29.1, 25.0, 24.5, 14.4. IR 2934, 1733, 1595, 1457, 1422, 1204, -1 + 1154, 1053, 700 cm . HRMS (ESI+): m/z calcd for C19H31O4 [M+H] 323.2217, found 323.2221.

Synthesis of 150

163

BBr3 (0.58 mL, 6.1 mmol, 2.2 equiv) was added to a solution of the substrate (0.90 g, 2.8 mmol,

1.0 equiv) in CH2Cl2 at –78 °C. 15 min after the addition, the cooling bath was removed and it was stirred at rt for 1.5 h. The reaction was quenched by the addition of ethanol (3.3 mL, 56 mmol, 20 equiv) while cooling with the dry ice–acetone bath. The resulting mixture was poured into a separation funnel filled with ice water. The organics were extracted with CH2Cl2

(3x 30 mL). The combined organics were washed with brine (30 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (25% EtOAc in hexanes) afforded the title compound as colorless oil (0.80 mg, 2.7 mmol, 97%), which turned yellow-brown upon standing.

1 H NMR (400 MHz, CDCl3) δ = 6.37 (d, J = 2.2 Hz, 2H), 6.20 (t, J = 2.2 Hz, 1H), 5.73 (br s, 2H), 4.12 (q, J = 7.1 Hz, 2H), 2.23 (t, J = 7.6 Hz, 2H), 1.60 – 1.52 (m, 2H), 1.52 – 1.47 (m, 2H), 1.24 (t, J = 7.1 Hz, 3H), 1.24 – 1.20 (m, 2H), 1.19 (s, 6H), 1.10 – 1.01 (m, 2H). 13C NMR (101

MHz, CDCl3) δ = 175.0, 156.7, 153.1, 105.9, 100.2, 60.7, 43.9, 37.8, 34.5, 29.6, 29.1, 24.8, 24.1, 14.3. IR 3376 (br), 2934, 2862, 1702, 1599, 1508, 1464, 1437, 1372, 1332, 1150, 994, -1 + 842, 700 cm . HRMS (ESI+): m/z calcd for C17H26NaO4 [M+Na] 317.1723, found 317.1731.

Synthesis of 155

154 (5.39 g, 17.9 mmol, 1.00 equiv) was combined with conc. HCl (100 mL) and stirred overnight at rt. The reaction mixture was diluted with water and the organic material was extracted with CH2Cl2 (3x 50 mL). The combined organic extracts were washed with brine

(50 mL), dried over MgSO4, filtered and concentrated to leave the crude tertiary alkyl chloride

(5.4 g). The residue was dissolved in CH2Cl2 (110 mL) and cooled to –78 °C. AlMe3 (3.34 mL, 33.8 mmol, ca. 2 equiv) was added and the resulting yellow solution was stirred for 15 min, before it was warmed 0 °C. After stirring at 0 °C for 2 h, the mixture was allowed to reach rt. After 4 h, the reaction mixture was cooled to 0 °C and dilute aq. HCl (1 M, 100 mL) was added dropwise via addition funnel. The organic layer was separated and the aqueous phase extracted with CH2Cl2 (50 mL). The combined organic fractions were washed with brine, dried over 1 MgSO4, filtered and concentrated in vacuo to yield crude product (5.13 g). H NMR indicated ca. 10% elimination product. The residue was dissolved in DMF (34 mL) and OsO4 (2.5% in tBuOH, 1.5 mL) was added. After stirring at rt for 5 min, oxone (4.22 g) was added and it was stirred until LCMS indicated full conversion of the olefinic side product. Sodium sulfite

164

(0.756 g, 6.0 mmol) was added and the mixture was stirred for 1 h. The mixture was diluted with water (300 mL) and extracted with Et2O (3x 50 mL). The combined organic fractions were washed with sat. aq. LiCl (2x 50 mL) and brine (2x 50 mL), dried over MgSO4, filtered and concentrated under reduced pressure. Flash chromatography on silica (5% EtOAc in hexanes) afforded the title compound as colorless oil (3.61 g, 12.1 mmol, 67%).

1 H NMR (400 MHz, CDCl3) δ 6.48 (d, J = 2.3 Hz, 2H), 6.30 (t, J = 2.2 Hz, 1H), 3.80 (s, 6H), 3.49 (t, J = 6.8 Hz, 2H), 1.75 – 1.66 (m, 2H), 1.59 – 1.53 (m, 2H), 1.36 (ddt, J = 9.2, 7.2, 6.2 13 Hz, 2H), 1.26 (s, 6H), 1.25 – 1.18 (m, 2H), 1.13 – 1.03 (m, 2H). C NMR (101 MHz, CDCl3) δ 160.6, 152.5, 104.8, 96.7, 55.4, 45.3, 44.5, 38.1, 32.8, 29.7, 29.1, 26.9, 24.7. IR 2934, 3859, -1 1595, 1456, 1422, 1205, 1155, 1054, 833, 700 cm . HRMS (EI+): m/z calcd for C17H27ClO2 [M]+ 298.1700, found 298.1694.

Synthesis of 151

155 (2.37 g, 7.93 mmol, 1.00 equiv) was dissolved in CH2Cl2 (80 mL) and the mixture was cooled to –78 °C. BBr3 (1.69 mL, 17.8 mmol, 2.3 equiv) was added slowly. The yellow solution was stirred at –78 °C for 1 h and was then allowed to warm to rt. After stirring at rt for 2.5 h, the reaction was quenched by addition of aq. sat. NaHCO3. After separation of the organic layer, the aq. phase was extracted with EtOAc (3x 50 mL). The combined organic extracts were washed with brine, dried over MgSO4, filtered and concentrated in vacuo. Flash chromatography on silica (10% EtOAc in hexanes to 20%) afforded the title compound (2.01 g, 7.39 mmol, 93%) as colorless oil.

1 H NMR (400 MHz, CDCl3) δ 6.37 (d, J = 2.2 Hz, 2H), 6.17 (t, J = 2.2 Hz, 1H), 4.67 (s, 2H), 3.49 (t, J = 6.7 Hz, 2H), 1.70 (dt, J = 14.7, 6.8 Hz, 2H), 1.57 – 1.50 (m, 2H), 1.36 (dtd, J = 9.3, 7.2, 5.6 Hz, 2H), 1.28 – 1.16 (m, 2H), 1.23 (s, 6H), 1.11 – 1.02 (m, 2H). 13C NMR (101 MHz,

CDCl3) δ 156.5, 153.4, 106.0, 100.1, 45.4, 44.4, 37.9, 32.7, 29.6, 29.0, 26.9, 24.6. IR 3335 (br), 2933, 2859, 1598, 1465, 1331, 1151, 993, 843, 699 cm-1. HRMS (EI+): m/z calcd for + C15H23ClO2 [M] 270.1387, found 270.1381.

Synthesis of ((1R,4R,5R)-4-(4-(8-chloro-2-methyloctan-2-yl)-2,6-dihydroxyphenyl)-6,6- dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl pivalate

165

97 (1.8 g, 7.1 mmol, 1.1 equiv) and 151 (1.8 g, 6.7 mmol, 1.0 equiv) were dissolved in CH2Cl2

(270 mL). TsOH·H2O (354 mg, 1.86 mmol, 0.28 equiv) was added and the mixture was stirred at rt for 40 min. Sat. aq. sodium bicarbonate (100 mL) was added and the layers were separated.

The aq. phase was extracted with CH2Cl2 (100 mL). The combined organic layers were washed with brine, dried over MgSO4, filtered and concentrated in vacuo. Flash chromatography on silica (5% EtOAc in hexanes to 10%) afforded the title compound as colorless oil (2.25 g, 4.45 mmol, 67%).

1 H NMR (400 MHz, CDCl3) δ 6.33 (s, 2H), 6.01 (dt, J = 3.1, 1.6 Hz, 1H), 5.75 (s, 2H), 4.67 – 4.47 (m, 2H), 4.04 – 3.99 (m, 1H), 3.49 (t, J = 6.8 Hz, 2H), 2.37 (ddd, J = 9.6, 6.2, 5.0 Hz, 1H), 2.30 (dt, J = 5.8, 1.3 Hz, 2H), 1.70 (dt, J = 14.6, 6.8 Hz, 2H), 1.54 – 1.47 (m, 3H), 1.41 – 1.32 (m, 5H), 1.21 (d, J = 12.8 Hz, 17H), 1.14 – 1.06 (m, 2H), 0.98 (s, 3H). 13C NMR (101 MHz,

CDCl3) δ 178.7, 155.1, 150.4, 149.5, 120.4, 111.7, 106.5, 66.6, 47.4, 45.3, 44.3, 44.2, 41.0, 39.1, 37.9, 37.5, 32.8, 29.7, 28.9, 28.2, 27.4, 26.9, 26.0, 24.6, 20.9. IR 3452 (br), 2933, 2869, 1728, 1706, 1626, 1575, 1429, 1283, 1162, 1026, 757 cm-1. HRMS (ESI+): m/z calcd for

+ ퟐퟒ C30H46ClO4 [M+H] 505.3079, found 505.3075. [] 퐃 = –47 (c = 2.0, CHCl3).

Synthesis of ((1R,4R,5R)-4-(4-(8-chloro-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6- dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl pivalate

((1R,4R,5R)-4-(4-(8-chloro-2-methyloctan-2-yl)-2,6-dihydroxyphenyl)-6,6- dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl pivalate (2.23 g, 4.41 mmol, 1.0 equiv) was combined with acetone (9 mL), K2CO3 (1.83 g, 13.2 mmol, 3.0 equiv) and dimethyl sulfate (1.23 mL, 13.2 mmol, 3.0 equiv) and stirred at rt overnight. The reaction mixture was diluted with Et2O (200 mL), washed with water (50 mL) and brine (50 mL), dried over MgSO4, filtered and concentrated (rotavap in a fumehood). Flash chromatography on silica afforded the title compound as colorless oil (1.65 g, 3.09 mmol, 70%).

166

1 H NMR (400 MHz, CDCl3) δ 6.47 (s, 2H), 5.78 (dt, J = 2.8, 1.4 Hz, 1H), 4.61 – 4.48 (m, 2H), 4.00 (s, 1H), 3.74 (s, 6H), 3.50 (t, J = 6.7 Hz, 2H), 2.17 (dd, J = 7.3, 5.6 Hz, 2H), 2.06 (tt, J = 5.8, 1.9 Hz, 1H), 1.77 – 1.67 (m, 3H), 1.61 – 1.54 (m, 2H), 1.43 – 1.34 (m, 2H), 1.30 (s, 3H), 1.27 (s, 6H), 1.26 – 1.23 (m, 2H), 1.23 (s, 9H), 1.18 – 1.09 (m, 2H), 0.98 (s, 3H). 13C NMR

(101 MHz, CDCl3) δ 178.6, 158.5, 149.4, 137.3, 126.4, 117.7, 102.8, 67.6, 55.9, 47.5, 45.3, 44.6, 43.9, 41.0, 39.0, 38.1, 37.7, 32.8, 29.7, 29.0, 29.0, 27.7, 27.5, 26.9, 26.5, 24.7, 21.2. IR 2933, 2865, 1726, 1606, 1573, 1463, 1411, 1281, 1239, 1152, 1122, 834 cm-1. HRMS (ESI+):

+ ퟐퟑ m/z calcd for C32H50ClO4 [M+H] 533.3392, found 533.3396. [] 퐃 = –79.0 (c = 1.0, CHCl3).

Synthesis of ent-113-Cl

((1R,4R,5R)-4-(4-(8-Chloro-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6- dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl pivalate (1.64 g, 3.08 mmol, 1.00 equiv) was dissolved in CH2Cl2 (31 mL) and cooled to 0 °C. DIBAL (1 M in hexanes, 6.5 mL, 6.5 mmol, 2.1 equiv) was added via syringe and the solution was stirred at 0 °C for 15 min. Sat. aq. Rochelle’s salt (10 mL) was added and the mixture was vigorously stirred at rt until the phases separated cleanly. The organic phase was separated and the aq. layer extracted with ether. The combined organic layers were washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure. Flash chromatography on silica (10 % EtOAc in hexanes) afforded the title compound as colorless oil (1.20 g, 2.67 mmol, 87%).

1 H NMR (400 MHz, CDCl3) δ 6.48 (s, 2H), 5.73 – 5.69 (m, 1H), 4.11 – 4.05 (m, 2H), 4.03 – 3.99 (m, 1H), 3.74 (s, 6H), 3.50 (t, J = 6.7 Hz, 2H), 2.26 – 2.18 (m, 2H), 2.07 (tq, J = 6.4, 2.3 Hz, 1H), 1.76 – 1.67 (m, 3H), 1.60 – 1.54 (m, 2H), 1.38 (ddt, J = 9.3, 7.3, 6.3 Hz, 2H), 1.31 (s, 3H), 1.27 (s, 6H), 1.27 – 1.20 (m, 2H), 1.17 – 1.08 (m, 2H), 0.97 (s, 3H). 13C NMR (101 MHz,

CDCl3) δ 158.6, 149.4, 142.1, 123.9, 117.8, 102.9, 66.8, 55.9, 47.5, 45.3, 44.6, 43.9, 41.0, 38.1, 37.6, 32.8, 29.7, 29.1, 29.0, 28.1, 26.9, 26.4, 24.7, 21.2. IR 3391 (br), 2933, 2862, 1606, 1572, -1 + 1463, 1411, 1238, 1121, 833 cm . HRMS (ESI+): m/z calcd for C27H42ClO3 [M+H] 449.2817,

ퟐퟒ found 449.2820. [] 퐃 = –93.3 (c = 0.75, CHCl3).

Synthesis of ent-114

167

The alkyl chloride (1.25 g, 2.78 mmol, 1.00 equiv) was combined with DMSO (5 mL) and

NaN3 (0.905 g, 13.9 mmol, 5.00 equiv) and the resulting reaction mixture was stirred at 50 °C overnight. Water (200 mL) was added and the organic material was extracted with Et2O (3x

50 mL). The combined extracts were washed with brine (50 mL), dried over MgSO4, filtered and concentrated to afford the pure product (1.22 g, 2.68 mmol, 96%).

1 H NMR (400 MHz, CDCl3) δ 6.48 (s, 2H), 5.70 (s, 1H), 4.07 (s, 2H), 4.00 (t, J = 2.3 Hz, 1H), 3.74 (s, 6H), 3.22 (t, J = 6.9 Hz, 2H), 2.21 (dd, J = 7.3, 5.8 Hz, 2H), 2.07 (tt, J = 5.9, 1.9 Hz, 1H), 1.71 (d, J = 7.5 Hz, 1H), 1.60 – 1.50 (m, 4H), 1.31 (s, 3H), 1.27 (s, 6H), 1.26 (s, 4H), 1.16 13 – 1.07 (m, 2H), 0.97 (s, 3H). C NMR (101 MHz, CDCl3) δ 158.6, 149.4, 142.1, 123.9, 117.8, 102.9, 66.8, 56.0, 51.6, 47.5, 44.6, 44.0, 41.0, 38.1, 37.6, 30.0, 29.0, 28.1, 26.8, 26.4, 24.7, 21.2. IR 3402 (br), 2933, 2863, 2095, 1606, 1572, 1463, 1411, 1239, 1122, 833, 670 cm-1. HRMS

+ ퟐퟑ (ESI+): m/z calcd for C27H41N3NaO3 [M+Na] 478.3040, found 478.3039. [] 퐃 = –88.8 (c =

5.0, CHCl3).

Synthesis of ent-96

ent-114 (120 mg, 0.263 mmol, 1.00 equiv) was dissolved in THF (0.8 mL) and CS2 (0.32 mL,

5.3 mmol, 20 equiv) was added. PPh3 (76 mg, 0.29 mmol, 1.1 equiv) was added and the resulting mixture was stirred at 40 °C overnight. The volatiles were removed in vacuo (rotavap in fumehood). Flash chromatography on silica (10% EtOAc in hexane) afforded the title compound as yellow oil (120 mg, 0.254 mmol, 97%).

1 H NMR (400 MHz, CDCl3) δ 6.48 (s, 2H), 5.70 (s, 1H), 4.10 – 4.05 (m, 2H), 4.03 – 3.99 (m, 1H), 3.75 (s, 6H), 3.47 (t, J = 6.6 Hz, 2H), 2.21 (dd, J = 7.3, 5.8 Hz, 2H), 2.07 (td, J = 5.7, 4.8, 2.9 Hz, 1H), 1.71 (d, J = 7.5 Hz, 1H), 1.63 (dt, J = 14.6, 6.8 Hz, 2H), 1.59 – 1.50 (m, 2H), 1.36 (dq, J = 9.4, 6.8 Hz, 2H), 1.31 (s, 3H), 1.28 (s, 6H), 1.27 – 1.20 (m, 2H), 1.17 – 1.08 (m, 2H), 13 0.97 (s, 3H). C NMR (101 MHz, CDCl3) δ 158.6, 149.3, 142.1, 123.9, 117.8, 102.9, 66.8,

168

56.0, 47.5, 45.2, 44.6, 44.0, 41.0, 38.1, 37.6, 30.1, 29.6, 29.0, 28.1, 26.6, 26.4, 24.6, 21.2. IR 3391 (br), 2932, 2861, 2181, 2101, 1605, 1572, 1452, 1410, 1239, 1122, 986, 834 cm-1. HRMS

+ ퟐퟓ (ESI+): m/z calcd for C28H41NNaO3S [M+Na] 494.2699, found 494.2695. [] 퐃 = –78.5 (c =

2.0, CHCl3).

Synthesis of 162

The allylic alcohol (231 mg, 0.776 mmol, 1.05 equiv) and resorcinol derivative (233 mg,

0.739 mmol, 1.00 equiv) were dissolved in CH2Cl2 (30 mL). TsOH·H2O (39 mg, 0.21 mmol,

0.28 equiv) was added. The reaction was stirred for 2 h, then diluted with addition CH2Cl2

(100 mL), washed with sat. aq. NaHCO3 and brine (each 30 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (10% EtOAc in hexanes) afforded the title compound as colorless foam (287 mg, 0.483 mmol, 65%).

1 Rf = 0.32 (25% EtOAc in hexanes; UV, KMnO4, Seebach). H NMR (400 MHz, CDCl3) δ 7.88 (dd, J = 5.5, 3.0 Hz, 2H), 7.73 (dd, J = 5.5, 3.0 Hz, 2H), 6.36 (s, 2H), 5.97 (s, 1H), 4.45 (ddd, J = 15.5, 3.1, 1.9 Hz, 1H), 4.22 (dt, J = 15.5, 1.4 Hz, 1H), 3.99 (s, 1H), 3.36 (t, J = 6.9 Hz, 2H), 2.34 – 2.21 (m, 3H), 1.82 – 1.73 (m, 2H), 1.48 (td, J = 8.5, 5.3 Hz, 3H), 1.39 – 1.32 (m, 2H), 1.31 (s, 3H), 1.28 – 1.20 (m, 2H), 1.18 (s, 6H), 1.12 – 1.02 (m, 2H), 0.99 (s, 3H). 13C NMR

(101 MHz, CDCl3) δ 168.5, 150.3, 148.1, 134.3, 132.1, 123.7, 120.6, 111.6, 106.6, 47.3, 44.3, 44.3, 43.3, 41.3, 37.8, 37.5, 34.2, 33.0, 29.6, 28.9, 28.9, 28.2, 28.2, 26.0, 24.6, 20.7. IR 3462 (br), 2929, 1771, 1707, 1627, 1574, 1426, 1393, 1341, 729 cm-1.

Synthesis of 164

162 (119 mg, 0.200 mmol, 1.00 equiv) was combined with NaN3 (65 mg, 1.00 equiv, 5.00 equiv) and DMF (1 mL) at rt and stirred overnight. Water (50 mL) was added and the mixture was extracted with Et2O (3x 50 mL). The combined organic extracts were washed with brine (20 mL), dried over MgSO4, filtered and concentrated.The crude material was dissolved in acetone (1 mL) and combined with dimethyl sulfate (57 L, 0.60 mmol, 3.0 equiv) and

K2CO3 (97 mg, 0.70 mmol, 3.5 equiv). The reaction mixture was stirred for 12 h at rt. Solvent

169 was removed under a stream of nitrogen. Water was added and the mixture was extracted with

Et2O (3x 50 mL). The combined organic extracts were washed with brine (20 mL), dried over

MgSO4, filtered and concentrated. Flash chromatography on silica afforded the title compound as colorless oil (75 mg, 0.128 mmol, 64%). Spectral data matched with material prepared en route to 128.

Synthesis of 157

160 (53 mg, 79% purity, 0.055 mmol, 1.0 equiv) was combined with EtOH (1.4 mL), E/Z- crotyl alcohol (89 µL, 1.04 mmol, ca. 15 equiv), and N2H4·H2O (34 µL, 0.70 mmol, ca. 10 equiv). The flask was capped and placed in an oilbath preheated to 75 °C. After 1.5 h, the cloudy mixture was allowed to cool to rt. The precipitate was removed by filtration (rinsing with EtOH). The filtrate was concentrated. Flash chromatography on silica (5% MeOH in

DCM, 2% NEt3) afforded 157 (20 mg, 0.045 mmol, 82%) as colorless oil.

1 H NMR (400 MHz, MeOH-d4) δ 6.54 (s, 2H), 5.86 – 5.78 (m, 1H), 4.08 – 4.02 (m, 1H), 3.74 (s, 6H), 3.50 – 3.38 (m, 2H), 2.29 – 2.22 (m, 1H), 2.22 – 2.18 (m, 1H), 2.13 (t, J = 7.5 Hz, 2H), 2.06 – 2.00 (m, 1H), 1.78 (d, J = 8.5 Hz, 1H), 1.66 – 1.59 (m, 2H), 1.59 – 1.49 (m, 2H), 1.33 (s, 3H), 1.28 (s, 6H), 1.27 – 1.22 (m, 2H), 1.17 – 1.06 (m, 2H), 0.99 (s, 3H). 13C NMR (101

MHz, MeOH-d4) δ 179.3, 159.7, 150.7, 137.4, 128.3, 117.8, 103.7, 56.1, 48.5, 45.8, 45.6, 45.3, 41.9, 39.0, 38.8, 36.5, 31.0, 29.5, 28.5, 26.8, 26.6, 25.7, 21.3. HRMS (ESI+): m/z calcd for + C27H43N2O3 [M+H] 443.3268, found 443.3270.

Synthesis of 158

161 (395 mg, 0.688 mmol, 1.00 equiv) was combined with acetone (4 mL) and K2CO3 (476 mg, 3.44 mmol, 5.0 equiv). Dimethyl sulfate (0.197 mL, 2.07 mmol, 3.0 equiv) was added and the suspension was vigorously stirred at rt overnight. The mixture was diluted with diethylether (10 mL) and filtered over celite. The filtrate was concentrated (rotavap in a fumehood). Flash

170 chromatography on silica (10% EtOAc in hexanes) afforded the title compound (376 mg, 0.625 mmol, 91%) as colorless oil.

1 H NMR (400 MHz, CDCl3) δ 7.84 (dd, J = 5.4, 3.0 Hz, 2H), 7.70 (dd, J = 5.4, 3.0 Hz, 2H), 6.42 (s, 2H), 5.55 (s, 1H), 4.34 – 4.18 (m, 2H), 4.09 (q, J = 7.1 Hz, 2H), 3.94 (t, J = 2.5 Hz, 1H), 3.71 (s, 6H), 2.22 (t, J = 7.5 Hz, 2H), 2.18 – 2.11 (m, 2H), 2.04 – 1.98 (m, 1H), 1.72 (d, J = 7.8 Hz, 1H), 1.63 – 1.50 (m, 4H), 1.28 – 1.20 (m, 14H), 1.15 – 1.04 (m, 2H), 0.93 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 173.9, 168.3, 158.5, 149.2, 136.0, 133.9, 132.4, 123.7, 123.2, 117.5, 102.5, 60.3, 55.8, 47.6, 44.5, 44.3, 42.6, 41.2, 38.1, 37.5, 34.5, 29.9, 29.0, 29.0, 27.7, + 26.4, 25.0, 24.5, 20.9, 14.4. HRMS (ESI+): m/z calcd for C37H48NO6 [M+H] 602.3476, found 602.3476.

Synthesis of 159

158 (290 mg, 0.482 mmol, 1.00 equiv) was combined with THF (2.1 mL) and aq. phosphate buffer pH = 7.0 (7.5 mL). Novozyme 435 (on acrylic resin) was added and the mixture was stirred for 3 h. The reaction mixture was filtered (quantitated with THF) and the filtrate diluted with water (200 mL). Extraction with EtOAc (3x 30 mL) and washing of the combined organic fractions with brine (20 mL), followed by drying over MgSO4, filtration and concentration afforded the title compound as slightly yellow oil (270 mg, 0.471 mmol, 98%).

1 H NMR (400 MHz, CDCl3) δ 7.87 – 7.82 (m, 2H), 7.72 – 7.67 (m, 2H), 6.43 (s, 2H), 5.57 – 5.54 (m, 1H), 4.35 – 4.19 (m, 2H), 3.98 – 3.92 (m, 1H), 3.72 (s, 6H), 2.28 (t, J = 7.5 Hz, 2H), 2.19 – 2.11 (m, 2H), 2.01 (tt, J = 5.8, 1.8 Hz, 1H), 1.73 (d, J = 7.9 Hz, 1H), 1.62 – 1.51 (m, 13 4H), 1.31 – 1.20 (m, 11H), 1.16 – 1.06 (m, 2H), 0.94 (s, 3H). C NMR (101 MHz, CDCl3) δ 179.9, 168.3, 158.5, 149.2, 136.0, 133.9, 132.4, 123.7, 123.2, 117.6, 102.5, 55.8, 47.5, 44.4, 44.3, 42.6, 41.2, 38.0, 37.5, 34.1, 29.8, 29.0, 27.7, 26.4, 24.6, 24.4, 20.9. IR 2933, 2864, 1772, -1 1713, 1605, 1572, 1391, 1239, 1119, 731 cm . HRMS (ESI+): m/z calcd for C35H43NNaO6 [M+Na]+ 596.2983, found 596.2984.

Synthesis of 160

171

159 (53 mg, 0.092 mmol, 1.0 equiv) was dissolved in DMF (0.9 mL). DIPEA (81 µL, 0.46 mmol, 5.0 equiv) and HATU (53 mg, 0.14 mmol, 1.5 equiv) were added and the mixture was stirred for 15 min. NH4Cl (12 mg, 0.23 mmol, 2.5 equiv) was added and the mixture was stirred for 2 h. Water (10 mL) was added and the organic material was extracted with EtOAc (3x 10 mL). The combined organic fractions were washed with 5% aq. LiCl and brine, dried with Na2SO4, filtered and concentrated. Flash chromatography on silica (75% EtOAc in hexanes) afforded the title compound (contaminants tetramethylurea, DMF; 60 mg, 79% purity, 0.083 mmol, 89%) as colorless oil.

1 H NMR (400 MHz, CD3CN) δ 7.86 – 7.75 (m, 4H), 6.51 (s, 2H), 5.98 (s, 1H), 5.51 (dt, J = 2.9, 1.4 Hz, 1H), 5.47 (s, 1H), 4.18 (dd, J = 2.3, 1.6 Hz, 2H), 3.89 (t, J = 2.5 Hz, 1H), 3.68 (s, 6H), 2.19 – 2.12 (m, 2H), 2.04 (t, J = 7.5 Hz, 2H), 1.94 (p, J = 2.5 Hz, 1H), 1.66 – 1.61 (m, 1H), 1.61 – 1.55 (m, 2H), 1.51 – 1.41 (m, 2H), 1.24 (d, J = 2.0 Hz, 9H), 1.19 (dt, J = 8.8, 7.3 13 Hz, 2H), 1.06 (ddd, J = 12.8, 6.5, 3.3 Hz, 2H), 0.88 (s, 3H). C NMR (101 MHz, CD3CN) δ 175.9, 169.1, 159.4, 150.5, 137.4, 135.1, 133.1, 124.2, 123.8, 117.9, 103.8, 56.3, 48.4, 45.0, 44.6, 43.0, 41.5, 38.3, 36.1, 30.6, 29.3, 29.2, 28.0, 26.5, 26.2, 25.3, 21.0 cm-1. HRMS (ESI+): + m/z calcd for C35H45N2O5 [M+H] 573.3323, found 573.3318.

Synthesis of 161

150 (330 mg, 1.03 mmol, 1.00 equiv) and 132 (337 mg, 1.13 mmol, 1.10 equiv) were dissolved in CH2Cl2 (20 mL). TsOH·H2O (49 mg, 0.26 mmol, 0.25 equiv) was added and the mixture was stirred at rt for 1.5 h. Solid NaHCO3 (ca. 0.5 g) was added and it was stirred for 5 min, before

MgSO4 (ca. 0.5 g) was added. After a few minutes, the mixture was filtered and concentrated. Flash chromatography on silica (15% EtOAc in hexanes) afforded the title compound (431 mg, 0.75 mmol, 67%) as colorless foam.

172

1 Rf = 0.66 (50% EtOAc in hexanes; UV, KMnO4, Seebach). H NMR (400 MHz, CDCl3) δ 7.88 (dd, J = 5.5, 3.0 Hz, 2H), 7.72 (dd, J = 5.5, 3.1 Hz, 2H), 6.35 (s, 2H), 5.97 (s, 1H), 4.44 (ddd, J = 15.5, 3.0, 1.9 Hz, 1H), 4.21 (dt, J = 15.5, 1.4 Hz, 1H), 4.11 (q, J = 7.1 Hz, 2H), 4.00 (s, 1H), 2.33 – 2.18 (m, 5H), 1.61 – 1.52 (m, 2H), 1.52 – 1.42 (m, 3H), 1.30 (s, 3H), 1.24 (t, J = 7.1 Hz, 3H), 1.20 (d, J = 2.2 Hz, 2H), 1.17 (s, 6H), 1.12 – 1.04 (m, 2H), 0.98 (s, 3H). 13C NMR (101

MHz, CDCl3) δ 174.4, 168.5, 150.1, 148.0, 134.3, 132.1, 123.7, 120.7, 111.7, 106.6, 60.4, 47.3, 44.3, 43.9, 43.3, 41.3, 37.8, 37.5, 34.5, 29.7, 29.1, 28.9, 28.2, 26.0, 24.9, 24.2, 20.7, 14.4. IR -1 + 3459 (br), 2933, 1715, 1427, 1323 cm . HRMS (ESI+): m/z calcd for C35H44NO6 [M+H] 574.3163, found 574.3169.

Synthesis of 167

165 (410 mg, 1.63 mmol, 1.05 equiv) and 150 (455 mg, 1.55 mmol, 1.00 equiv) were combined with CH2Cl2 (77 mL) and cooled to –25 °C (external temperature control). BF3·Et2O (1.0 mL, 8.2 mmol, 5.3 equiv) was added dropwisely, turning the colorless solution yellow. TLC after 10 min indicated almost full conversion. The reaction mixture was stirred for a total of 35 min before it was poured into a separation funnel filled with icewater. The phases were separated and the aqueous phase extracted once with CH2Cl2. The combined organics were washed with brine, dried over MgSO4, filtered and concentrated. Flash chromatography on silica (15% Et2O in hexanes) afforded the title compound (0.49 g, 0.93 mmol, 60% uncorrected) along with an inseparable and unidentified byproduct. The material was dissolved in THF (5 mL) and cooled to 0 °C. LAH (4 M in Et2O, 0.7 mL, 2.8 mmol, 3.0 equiv) was added dropwise. After 30 min, the reaction was quenched by the addition of EtOAc (3 mL). A saturated aqueous solution of Rochelle’s salt was added and it was rigorously stirred until a clear phase separation occurred. The organics were extracted with EtOAc (3x 20 mL). The combined organics were washed with brine, dried over MgSO4, filtered and concentrated. Flash chromatography on silica (50% EtOAc in hexanes) afforded the title compound as colorless foam (246 mg, 0.611 mmol, 66% over two steps).

1 H NMR (400 MHz, CDCl3) δ = 6.34 (d, J = 1.7 Hz, 1H), 6.27 (d, J = 1.8 Hz, 1H), 5.71 (d, J = 4.7 Hz, 1H), 4.10 – 3.99 (m, 2H), 3.60 (t, J = 6.5 Hz, 2H), 3.47 (dd, J = 16.2, 4.7 Hz, 1H), 2.68 (td, J = 11.1, 4.8 Hz, 1H), 2.24 – 2.16 (m, 1H), 1.89 – 1.76 (m, 3H), 1.52 – 1.42 (m, 4H), 1.37 (s, 3H), 1.34 – 1.25 (m, 2H), 1.25 – 1.18 (m, 2H), 1.17 (s, 3H), 1.14 (s, 3H), 1.07 (s, 2H), 1.06

173

13 (s, 3H). C NMR (101 MHz, CDCl3) δ = 155.1, 154.4, 149.9, 138.4, 121.7, 110.1, 107.7, 105.8, 76.6, 67.1, 63.2, 45.2, 43.9, 37.4, 32.5, 31.6, 31.5, 29.8, 29.1, 29.0, 27.8, 27.7, 25.4, 24.3, 18.6. IR 3327 (br), 2932, 2859, 1622, 1575, 1415, 1331, 1267, 1187, 1084, 1047, 1009, 910,

-1 + ퟐퟑ 732 cm . HRMS (ESI+): m/z calcd for C25H39O4 [M+H] 403.2843, found 403.2849. [] 퐃 = –

157 (c = 2.0, CHCl3).

Synthesis of 168

Crabtree–Pfaltz catalyst [Ir(COD)(Cy3P)(Py)]BArF (9 mg, 0.006 mmol, 6 mol%) was dissolved in THF (1.5 mL). Hydrogen was bubbled through the solution for 3 min, turning the orange solution colorless. Excess hydrogen was removed by degassing using the freeze-pump- thaw technique (3 cycles, N2). 167 (43 mg, 0.11 mmol, 1.0 equiv) was added under nitrogen. The flask was sealed and placed in a waterbath preheated to 40 °C. After 2.5 h, the reaction was allowed to cool to rt, and two drops of acetic acid were added. After 5 min, the solution was transferred to a round-bottom-flask (transfer quantitated with MeOH (9 mL). K2CO3 (295 mg, 2.14 mmol, 20.0 equiv) was added and it was stirred at rt for 4 h. Water (200 mL) was added and the organics were extracted with EtOAc (3x 30 mL). The combined organics were washed with brine (slow phase separation). The combined aqueous washings were extracted once with

CH2Cl2 (30 mL). All organics were combined, dried over MgSO4, filtered and concentrated. Flash chromatography on silica (40% EtOAc in hexanes) afforded the title compound as colorless oil (40 mg, 0.099 mmol, 93%) in a diastereomeric ratio > 15:1 (before isomerization ca. 1:3 as determined in separate experiment without the isomerization step).

Note: an attempt to avoid the tedious extraction by dilution with Et2O (excess) and filtration over celite in order to remove the precipitated salts led to almost complete decomposition of the product upon concentration.

1 H NMR (400 MHz, CDCl3) δ = 9.65 (d, J = 1.4 Hz, 1H), 6.33 (d, J = 1.8 Hz, 1H), 6.23 (d, J = 1.9 Hz, 1H), 6.15 (br s, 1H), 3.63 (t, J = 6.4 Hz, 2H), 3.57 – 3.49 (m, 1H), 2.58 – 2.45 (m, 2H), 2.16 – 2.08 (m, 1H), 2.03 – 1.95 (m, 1H), 1.86 (br s, 1H), 1.54 – 1.44 (m, 6H), 1.39 (s, 3H), 1.37 – 1.20 (m, 6H), 1.18 (s, 3H), 1.17 (s, 3H), 1.08 (s, 3H), 1.15 – 0.96 (m, 4H). 13C NMR

(101 MHz, CDCl3) δ = 204.8, 155.0, 154.6, 150.1, 108.9, 107.7, 105.6, 76.9, 63.2, 50.5, 48.9, 43.8, 37.4, 35.0, 32.4, 29.9, 29.8, 29.1, 28.9, 27.8, 27.0, 26.2, 25.3, 24.2, 19.2. IR 3355 (br),

174

2932, 2860, 1718, 1622, 1575, 1415, 1334, 1141, 1040, 756 cm-1. HRMS (MALDI+): m/z

+ ퟐퟑ calcd for C25H38O4 [M] 402.2765, found 402.2766. [] 퐃 = –85 (c = 1.0, CHCl3).

Synthesis of 168-red

168 was prepared as described but isolated without performing the isomerization step (dr 1:3).

This material (40 mg, 0.099 mmol, 1.00 equiv) was dissolved in MeOH (10 mL) and K2CO3 (275 mg, 1.99 mmol, 20 equiv) was added. The reaction was stirred at rt overnight. AcOH

(0.28 mL, 5.0 mmol, 50 equiv) was added at 0 °C and it was stirred for 15 min. NaBH4 (36 mg, 0.959 mmol, ca. 10 equiv) was added and the reaction mixture was stirred for 1 h. Water (100 mL) was added and the mix was extracted with EtOAc (3x 20 mL). The combined organic fractions were washed with brine (20 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (50% EtOAc in hexanes) afforded the title compound as colorless foam (34 mg, 0.084 mmol, 85%).

1 H NMR (400 MHz, CDCl3) δ 6.32 (d, J = 1.8 Hz, 1H), 6.25 (d, J = 1.8 Hz, 1H), 3.60 (t, J = 6.5 Hz, 2H), 3.55 – 3.45 (m, 2H), 3.29 (d, J = 12.8 Hz, 1H), 2.45 (td, J = 11.1, 2.7 Hz, 1H), 1.95 – 1.86 (m, 2H), 1.83 – 1.73 (m, 1H), 1.52 – 1.42 (m, 5H), 1.36 (s, 3H), 1.32 – 1.24 (m, 2H), 1.24 – 1.13 (m, 8H), 1.13 – 1.05 (m, 4H), 1.02 (s, 3H), 0.77 (q, J = 11.9 Hz, 1H). 13C

NMR (101 MHz, CDCl3) δ 155.0, 154.6, 149.7, 109.9, 107.6, 105.8, 77.0, 68.6, 63.2, 49.5, 43.9, 40.6, 37.4, 35.1, 33.4, 32.5, 29.9, 29.8, 29.1, 29.0, 27.9, 27.6, 25.4, 24.3, 19.2. IR 3337 (br), 2930, 1623, 1573, 1414, 1332, 1141, 1038, 845, 754, 666 cm-1. HRMS (ESI+): m/z calcd

+ ퟐퟑ for C25H41O4 [M+H] 405.2999, found 405.3000. [] 퐃 = –65 (c = 1.0, CHCl3).

Synthesis of 169

168 (9.0 mg, 0.022 mmol, 1.0 equiv) and PPh3 (7.0 mg, 0.027 mmol, 1.2 equiv) were combined with CH2Cl2 (0.4 mL). Iodine (6.2 mg, 0.025 mmol, 1.1 equiv) was added and the resulting mixture was stirred until TLC analysis indicated full consumption of the starting material (ca.

30 min). The solution was diluted with Et2O (30 mL), washed with a 5:1 mixture of water and

175 saturated aqueous Na2S2O3 solution (10 mL), and brine (10 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica afforded the title compound as colorless oil (9.0 mg, 0.018 mmol, 79%).

1 H NMR (400 MHz, CDCl3) δ = 9.66 (d, J = 1.4 Hz, 1H), 6.36 (d, J = 1.7 Hz, 1H), 6.19 (d, J = 1.9 Hz, 1H), 4.73 (br s, 1H), 3.53 – 3.46 (m, 1H), 3.15 (t, J = 7.0 Hz, 2H), 2.58 – 2.45 (m, 2H), 2.17 – 2.08 (m, 1H), 2.04 – 1.97 (m, 1H), 1.80 – 1.71 (m, 2H), 1.54 – 1.43 (m, 4H), 1.40 (s, 3H), 1.38 – 1.27 (m, 3H), 1.27 – 1.22 (m, 2H), 1.21 (s, 6H), 1.19 – 1.15 (m, 1H), 1.10 (s, 3H), 13 1.08 – 1.04 (m, 2H). C NMR (101 MHz, CDCl3) δ = 204.3, 154.8, 154.5, 150.3, 109.0, 108.2, 105.5, 76.9, 50.5, 48.9, 44.4, 37.5, 35.0, 33.7, 30.5, 30.0, 29.3, 28.9, 28.8, 27.9, 27.0, 26.2, 24.6, 19.2, 7.5. IR 3409 (br), 2931, 2860, 1718, 1621, 1573, 1459, 1414, 1333, 1270, 1243, 1189, -1 + 1141, 1038 cm . HRMS (MALDI+): m/z calcd for C25H37IO3 [M] 512.1782, found 512.1782.

Synthesis of 170

169 (8.0 mg, 0.016 mmol, 1.0 equiv) was dissolved in MeOH (1.0 mL) and cooled to 0 °C.

NaBH4 (0.9 mg, 0.023 mmol, 1.5 equiv) was added and the resulting mixture was stirred until TLC analysis indicated full consumption of the starting material. The solution was diluted with

Et2O (30 mL), washed with aqueous HCl (1 M, 10 mL), and brine (10 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica afforded the title compound as light- yellow oil (6.0 mg, 0.018 mmol, 75%).

1 H NMR (500 MHz, CDCl3) δ = 6.35 (d, J = 1.9 Hz, 1H), 6.19 (d, J = 1.9 Hz, 1H), 4.77 (br s, 1H), 3.56 – 3.49 (m, 2H), 3.22 – 3.18 (m, 1H), 3.14 (t, J = 7.1 Hz, 2H), 2.48 (td, J = 11.1, 2.8 Hz, 1H), 2.00 – 1.95 (m, 1H), 1.95 – 1.90 (m, 1H), 1.75 (dt, J = 14.6, 7.1 Hz, 3H), 1.51 – 1.47 (m, 3H), 1.39 (s, 3H), 1.36 – 1.25 (m, 3H), 1.21 (s, 2H), 1.20 (s, 6H), 1.17 – 1.11 (m, 2H), 1.09 13 (s, 3H), 1.08 – 1.03 (m, 2H), 0.87 – 0.79 (m, 1H). C NMR (126 MHz, CDCl3) δ = 154.7, 154.4, 149.7, 109.7, 107.9, 105.4, 76.9, 68.6, 49.4, 44.3, 40.5, 37.3, 35.0, 33.5, 33.2, 30.4, 29.7, 29.2, 28.7, 28.7, 27.8, 27.5, 24.4, 19.1, 7.4. IR 3345 (br), 2931, 2862, 1619, 1573, 1464, 1414, -1 + 1332, 1271, 1138, 1038, 974, 838 cm . HRMS (ESI+): m/z calcd for C25H40IO3 [M+H] 515.2017, found 515.2015.

Synthesis of 171

176

170 (5.0 mg, 0.0097 mmol, 1.0 equiv) was dissolved in DMSO (0.3 mL). NaN3 (14 mg,

0.22 mmol, 22 equiv) was added. The flask was evacuated and backfilld with N2 (3x). After

1 h, the mixture was diluted with Et2O (30 mL) and washed with water (10 mL) and brine

(10 mL). The organics were dried over MgSO4, filtered and concentrated. Flash chromatography on silica (30% EtOAc in hexanes) afforded the title compound (4.0 mg, 0.0093 mmol, 95%).

1 Rf = 0.41 (50% EtOAc in hexanes; weak UV, Seebach). H NMR (400 MHz, CDCl3) δ = 6.35 (d, J = 1.7 Hz, 1H), 6.19 (d, J = 1.7 Hz, 1H), 4.87 (br s, 1H), 3.57 – 3.49 (m, 2H), 3.22 (t, J = 6.9 Hz, 2H), 3.21 (d, J = 14.6 Hz, 1H), 2.48 (td, J = 11.1, 2.8 Hz, 1H), 2.00 – 1.90 (m, 2H), 1.83 – 1.72 (m, 1H), 1.58 – 1.44 (m, 5H), 1.39 (s, 3H), 1.34 – 1.22 (m, 4H), 1.20 (s, 6H), 1.18 – 1.10 13 (m, 2H), 1.09 (s, 3H), 1.08 (s, 2H), 0.83 (dd, J = 11.9 Hz, 1H). C NMR (101 MHz, CDCl3) δ = 154.8, 154.6, 149.9, 109.8, 108.1, 105.5, 77.1, 68.7, 51.7, 49.5, 44.4, 40.7, 37.4, 35.1, 33.3, 29.9, 28.9, 28.9, 28.9, 27.9, 27.6, 26.7, 24.6, 19.2. IR 3352 (br), 2932, 2861, 2095, 1621, 1573, 1462, 1414, 1384, 1332, 1270, 1138, 1039, 975, 842, 730 cm-1. HRMS (ESI+): m/z calcd for + C25H40N3O3 [M+H] 430.3064, found 430.3065.

Synthesis of AM841

171 (4.0 mg, 0.0093 mmol, 1.0 equiv) was dissolved in THF (0.4 mL). PPh3 (3.7 mg,

0.014 mmol, 1.5 equiv) was added, followed by CS2 (13 L, 0.21 mmol, 23 equiv). The flask was sealed and the reaction mixture was stirred overnight at rt. The solvent was evaporated in a stream of nitrogen and the residue purified by flash chromatography on silica (20% EtOAc in hexanes) to afford the title compound (3.5 mg, 0.0079 mmol, 85%).

1 H NMR (500 MHz, CDCl3) δ = 6.35 (d, J = 1.8 Hz, 1H), 6.20 (d, J = 1.8 Hz, 1H), 4.77 (br s, 1H), 3.57 – 3.49 (m, 2H), 3.46 (t, J = 6.6 Hz, 2H), 3.22 – 3.17 (m, 1H), 2.48 (td, J = 11.1, 2.8 Hz, 1H), 2.00 – 1.95 (m, 1H), 1.94 – 1.90 (m, 1H), 1.77 (td, J = 8.2, 7.1, 4.0 Hz, 1H), 1.65 – 1.58 (m, 2H), 1.52 – 1.47 (m, 3H), 1.39 (s, 3H), 1.36 – 1.30 (m, 2H), 1.27 – 1.17 (m, 2H), 1.20

177

(s, 6H), 1.17 – 1.11 (m, 2H), 1.09 (s, 3H), 1.12 – 1.02 (m, 2H), 0.87 – 0.79 (m, 1H). 13C NMR

(126 MHz, CDCl3) δ 154.8, 154.6, 149.8, 109.8, 108.1, 105.5, 77.1, 68.7, 49.5, 45.2, 44.3, 40.7, 37.4, 35.1, 33.3, 30.0, 29.9, 29.5, 28.9, 28.9, 27.9, 27.6, 26.5, 24.5, 19.2. IR 3326 (br), 2931, 2860, 2184, 2098, 1621, 1573, 1451, 1414, 1332, 1271, 1138, 1039, 975, 731 cm-1. HRMS

+ ퟐퟑ (ESI+): m/z calcd for C26H40NO3S [M+H] 446.2723, found 446.2727. [] 퐃 = –82 (c = 0.12,

CHCl3).

Synthesis of 176

86 (6.0 mg, 0.036 mmol, 1.5 equiv), 128 (11 mg, 0.024 mmol, 1.0 equiv) and HOBt (7.0 mg, 0.036 mmol, 1.5 equiv) were combined with DMF (0.12 mL). The flask was evacuated and backfilled with nitrogen three times, before DIPEA (9 L, 0.05 mmol, 2 equiv) and EDC·HCl (7.0 mg, 0.036 mmol, 1.5 equiv) were added. The reaction mixture was stirred in the dark overnight. The mix was diluted with EtOAc (100 mL), washed with 5% aq. LiCl (2x 30 mL) and brine (30 mL), dried over MgSO4, filtered and concentrated in vacuo. Flash chromatography on silica (20% EtOAc in hexanes) afforded the title compound (11 mg, 0.018 mmol, 75%) as colorless oil.

1 Rf = 0.14 (20% EtOAc in hexanes; UV, KMnO4). H NMR (300 MHz, CDCl3) δ 6.47 (s, 2H), 5.61 (s, 1H), 5.36 – 5.26 (m, 1H), 4.00 – 3.94 (m, 1H), 3.88 – 3.82 (m, 2H), 3.74 (s, 6H), 3.22 (t, J = 6.9 Hz, 2H), 2.22 – 2.13 (m, 1H), 2.11 – 2.01 (m, 2H), 2.01 – 1.95 (m, 2H), 1.95 – 1.90 (m, 2H), 1.90 – 1.82 (m, 2H), 1.71 – 1.61 (m, 3H), 1.61 – 1.48 (m, 4H), 1.27 (d, J = 3.8 Hz, 13 13H), 1.18 – 1.05 (m, 2H), 0.95 (s, 3H). C NMR (75 MHz, CDCl3) δ 170.9, 158.5, 149.4, 138.3, 124.2, 117.6, 102.8, 82.9, 69.3, 55.9, 51.6, 47.4, 44.8, 44.6, 44.5, 40.9, 38.1, 37.6, 32.6, 30.6, 30.0, 29.0, 29.0, 28.7, 28.0, 28.0, 26.8, 26.4, 24.7, 21.2, 13.5. IR 3306, 2931, 2861, 2095, 1645, 1605, 1571, 1451, 1411, 1238, 1121, 669, 640 cm-1. HRMS (EI+): m/z calcd for + C35H51N6O3 [M+H] 603.4017, found 603.4018.

Synthesis of 177

178

86 (6.7 mg, 0.041 mmol, 1.5 equiv), 157 (12 mg, 0.027 mmol, 1.0 equiv), EDC·HCl (7.8 mg, 0.041 mmol, 1.5 equiv) and HOBt (7.8 mg, 0.041 mmol, 1.5equiv) were combined with DMF (0.27 mL) and stirred for 5 min, before DIPEA (9 L, 0.05 mmol, 2 equiv) was added. It was stirred for 1.5 h, then diluted with EtOAc (60 mL), washed with 5% aq. LiCl (2x 20 mL) and brine (20 mL). It was dried over MgSO4, filtered and concentrated in vacuo. Flash chromatography on silica (50% EtOAc in hexanes to 100%) afforded the title compound (12 mg, 0.020 mmol, 75%) as colorless oil.

1 Rf = 0.29 (EtOAc; UV, Seebach). H NMR (400 MHz, CDCl3) δ 6.46 (s, 2H), 5.60 (s, 1H), 5.42 – 5.31 (m, 2H), 3.96 (t, J = 2.3 Hz, 1H), 3.87 – 3.82 (m, 2H), 3.73 (s, 6H), 2.21 – 2.12 (m, 3H), 2.11 – 1.99 (m, 4H), 1.99 – 1.90 (m, 3H), 1.89 – 1.82 (m, 2H), 1.70 – 1.63 (m, 3H), 1.63 – 1.53 (m, 4H), 1.27 (d, J = 6.4 Hz, 11H), 1.16 – 1.07 (m, 2H), 0.94 (s, 3H). 13C NMR (101

MHz, CDCl3) δ 175.5, 171.0, 158.5, 149.4, 138.4, 124.1, 117.6, 102.8, 82.9, 69.3, 55.9, 47.4, 44.8, 44.5, 44.4, 41.0, 38.1, 37.6, 35.9, 32.6, 30.6, 29.9, 29.0, 29.0, 28.7, 28.0, 28.0, 26.4, 25.4, 24.5, 21.2, 13.5. IR 3463, 3302, 2932, 1651, 1606, 1571, 1451, 1410, 1238, 1121 cm-1. HRMS + (EI+): m/z calcd for C35H51N4O4 [M+H] 591.3905, found 591.3904.

Synthesis of 186

To a solution of 4-bromo-2-fluoro-6-methoxybenzaldehyde (7.00 g, 30.0 mmol, 1.00 equiv) in

THF (30 mL) at 0 °C was added a solution of vinylmagnesium chloride in THF (1.6 M, 21.0 mL, 33.6 mmol, 1.10 equiv). After 30 min the reaction was quenched by addition of saturated, aqueous NH4Cl and the mixture was extracted with EtOAc. The combined organic extracts were dried over Na2SO4, filtered and concentrated. Purification by flash chromatography (hexanes/EtOAc 9:1, then 6:1) yielded the product as colorless oil (7.20 g, 27.6 mmol, 92% yield).

1 H NMR (400 MHz, CDCl3)  = 6.91 (dd, J = 9.2, 1.8 Hz, 1H), 6.85 (t, J = 1.6 Hz, 1H), 6.14 (dddd, J = 17.0, 10.3, 5.6, 1.0 Hz, 1H), 5.54 – 5.44 (m, 1H), 5.24 – 5.17 (m, 1H), 5.13 (dt, J = 10.4, 1.4 Hz, 1H),

13 3.87 (s, 3H), 3.34 – 3.04 (m, 1H). C NMR (101 MHz, CDCl3)  = 160.4 (d, J = 248.6 Hz), 158.8 (d,

179

J = 8.7 Hz), 138.8, 121.7 (d, J = 13.3 Hz), 117.5 (d, J = 16.6 Hz), 115.0, 112.6 (d, J = 26.9 Hz), 111.1 (d, J = 3.2 Hz), 67.8 (d, J = 5.1 Hz), 56.5. IR 3431, 3087, 2942, 1600, 1578, 1463, 1447, 1409, 1207,

+ 1080, 925, 851, 824. EI-MS calcd for C10H10BrFO2 [M] 259.9843; found 259.9850.

Synthesis of 188

[{Ir(cod)Cl}2] (154 mg, 0.230 mmol, 0.03 equiv) and ligand (S)-L (482 mg, 0.950 mmol,

0.124 equiv) were dissolved in 1,2-dichloroethane (15 mL) under an atmosphere of N2. The mixture was vigorously stirred for 15 min. The resulting dark red solution was added to a mixture of allylic alcohol 186 (2.00 g, 7.66 mmol, 1.0 equiv) and 5-methylhex-5-enal (187) (2.58 g, 23.0 mmol, 3.0 equiv), affording a yellow solution. To this solution were added

Jørgensen-Hayashi catalyst (S)-A (687 mg, 1.15 mmol, 0.15 equiv) and Zn(OTf)2 (139 mg, 0.83 mmol, 0.05 equiv). The vessel was purged with nitrogen and the resulting solution was stirred at rt for 24 h. After removal of the volatiles, a 1H NMR spectrum of the crude mixture was recorded to determine the diastereomeric ratio of the product. The mixture was then directly loaded on silica and purified by flash column chromatography (5:2 hexanes/CH2Cl2) to give the product as colorless oil (1.41 g, 3.97 mmol, 52% yield, dr > 20:1). A small sample of the aldehyde was reduced using sodium borohydride and the resulting primary alcohol was then used to determine the enantiomeric excess by SFC analysis (>99 %ee).

1 H NMR (400 MHz, CDCl3)  = 9.41 (dd, J = 4.0, 1.1 Hz, 1H), 6.88 (dd, J = 9.6, 1.8 Hz, 1H), 6.80 (t, J = 1.6 Hz, 1H), 6.13 (dddd, J = 17.0, 10.0, 9.0, 2.4 Hz, 1H), 5.23 – 5.15 (m, 1H), 5.15 – 5.09 (m, 1H), 4.81 – 4.67 (m, 2H), 4.03 (t, J = 9.7 Hz, 1H), 3.84 (s, 3H), 2.97 – 2.86 (m, 1H),

13 2.13 – 1.92 (m, 2H), 1.87 – 1.74 (m, 2H), 1.72 (s, 3H). C NMR (101 MHz, CDCl3)  = 203.6 , 160.9 (d, J = 247.6 Hz), 158.3 (d, J = 9.7 Hz), 144.9 , 136.2 (d, J = 2.6 Hz), 120.9 (d, J = 13.6 Hz), 117.7 , 116.2 (d, J = 16.8 Hz), 112.4 (d, J = 27.7 Hz), 110.9 (d, J = 3.0 Hz), 110.6 , 56.2 ,

19 53.1 (d, J = 2.3 Hz), 40.4 , 34.9 , 26.2 , 22.3. F NMR (376 MHz, CDCl3)  = –111.85. IR

180

3079, 2940, 1725, 1599, 1577, 1463, 1446, 1410, 1096, 921, 890, 856. ESI-MS calcd for

+ ퟐퟓ C17H20BrFNaO2 [M+Na] 377.0523; found 377.0518. [] 퐃 = +58.6 (c = 2.0, CHCl3). SFC > 99% enantiomeric excess (Chiralpak OJ-H; flow: 2.00 mL/min; 7.52 min (minor), 8.75 min

(major); 99.5% CO2, 0.5% MeOH at 120 bar, 25 °C). SFC of re-isolated starting material > 99% enantiomeric excess (Waters Trefoil; flow: 2.00 min/min; 1.75 min (major), 1.90

(minor); 98.0% CO2, 2.0% MeOH at 220 bar, 40 °C)

Synthesis of 189

188 (1.40 g, 3.94 mmol, 1.00 equiv) was dissolved in CH2Cl2 (40 mL) and the solution was degassed by bubbling nitrogen through it for 15 min. Grubbs 2nd gen. catalyst (100 mg, 0.12 mmol, 0.03 equiv) was added to the solution and the reaction mixture was stirred at rt for 16 h. After complete consumption of the starting material the reaction was directly purified by flash column chromatography

(hexanes/CH2Cl2 7:3) to yield the product as colorless oil (1.17 g, 3.58 mmol, 91% yield).

1 H NMR (400 MHz, CDCl3)  = 9.53 (dd, J = 2.5, 0.7 Hz, 1H), 6.84 (dd, J = 9.8, 1.9 Hz, 1H), 6.78 (t, J = 1.6 Hz, 1H), 5.27 – 5.13 (m, 1H), 4.13 – 4.06 (m, 1H), 3.79 (s, 3H), 2.85 – 2.75 (m, 1H), 2.22 – 2.11 (m, 1H), 2.10 – 1.99 (m, 2H), 1.75 – 1.64 (m, 4H). 13C NMR (101 MHz,

CDCl3)  = 204.4, 162.01 (d, J = 249.8 Hz), 159.0 (d, J = 9.8 Hz), 133.6 (d, J = 1.7 Hz), 122.1 (d, J = 1.2 Hz), 120.6 (d, J = 13.5 Hz), 118.4 (d, J = 14.7 Hz), 112.6 (d, J = 27.0 Hz), 110.7 (d, J = 3.1 Hz), 56.4 , 50.7 (d, J = 2.4 Hz), 32.1 (d, J = 1.0 Hz), 28.9 , 23.5 (d, J = 4.9 Hz). 19F NMR

(376 MHz, CDCl3)  = –111.34. IR 2926, 2836, 2721, 1723, 1598, 1575, 1463, 1445, 1409,

+ ퟐퟓ 1084, 851, 824. ESI-MS calcd for C15H16BrFNaO2 [M+Na] 349.0210; found 349.0207. [] 퐃 =

–103.4 (c = 2.0, CHCl3).

Synthesis of 190

To a solution of 189 (1.00 g, 3.06 mmol, 1.0 equiv) in MeOH (30 mL) was simultaneously added a solution of iodine in MeOH (0.78 M, 15.3 mL, 11.9 mmol, 3.9 equiv) and a solution of

KOH in MeOH (0.78 M, 31.3 mL, 24.5 mmol, 8 equiv) at 0 °C. After 30 min, the reaction was

181 quenched by addition of saturated, aqueous sodium thiosulfate solution and the mixture was extracted with CH2Cl2. The combined organic extracts were washed with brine, dried over

MgSO4 and concentrated. The product was purified by flash chromatography (hexanes/CH2Cl2 1:1) to give the product as slightly yellow oil (0.96 g, 2.69 mmol, 88% yield).

1 H NMR (400 MHz, CDCl3)  = 6.82 (dd, J = 9.7, 1.8 Hz, 1H), 6.77 (t, J = 1.6 Hz, 1H), 5.13 (dq, J = 2.4, 1.3 Hz, 1H), 4.13 (ddp, J = 10.5, 4.3, 2.2 Hz, 1H), 3.78 (s, 3H), 3.51 (s, 3H), 2.97 (ddd, J = 12.9, 10.6, 2.9 Hz, 1H), 2.24 – 2.11 (m, 1H), 2.11 – 1.97 (m, 2H), 1.92 – 1.79 (m,

13 1H), 1.68 (s, 3H). C NMR (101 MHz, CDCl3)  = 175.93 , 161.95 (d, J = 249.3 Hz), 159.44 (d, J = 9.8 Hz), 133.03 (d, J = 1.4 Hz), 122.50 (d, J = 0.9 Hz), 120.25 (d, J = 13.6 Hz), 119.03 (d, J = 15.1 Hz), 112.35 (d, J = 27.3 Hz), 110.77 (d, J = 3.1 Hz), 56.49 , 51.57 , 43.78 (d, J =

19 2.9 Hz), 34.69 (d, J = 1.4 Hz), 29.46 , 26.98 , 23.43. F NMR (377 MHz, CDCl3)  = –112.10. IR 2948, 2838, 1733, 1599, 1576, 1435, 1410, 1260, 1163, 1093, 1085, 852, 824. ESI-MS + 25 calcd for C16H18BrFNaO3 [M+Na] 379.0316; found 379.0316. [α] D = –137.8 (c = 2.0,

CHCl3).

Synthesis of 191

To a solution of the ester 190 (925 mg, 2.59 mmol, 1.00 equiv) in Et2O (13 mL) at 0 °C was added freshly prepared MeMgI in Et2O (2.5 M, 6.5 mL, 13 mmol, 5.0 equiv). The cooling bath was removed. Upon consumption of the starting material the reaction was quenched by addition of saturated aqueous NH4Cl solution. The aqueous layer was extracted with EtOAc. The combined organic extracts were washed with brine, dried over MgSO4, filtered and concentrated. Flash chromatography on silica (hexanes/EtOAc 8:1) afforded the title compound as slightly yellow oil (880 mg, 2.46 mmol, 95%).

1 H NMR (500 MHz, CDCl3)  = 6.82 (dd, J = 9.9, 1.9 Hz, 1H), 6.77 (t, J = 1.6 Hz, 1H), 5.00 – 4.95 (m, 1H), 3.89 – 3.83 (m, 1H), 3.82 (s, 3H), 2.22 – 2.09 (m, 2H), 2.00 – 1.90 (m, 2H), 1.65 (dt, J = 2.5, 1.2 Hz, 3H), 1.45 – 1.36 (m, 1H), 1.14 (s, 3H), 1.06 (s, 3H). 13C NMR (126

MHz, CDCl3)  = 161.7 (d, J = 249.1 Hz), 158.3 (d, J = 6.7 Hz), 134.0 (d, J = 1.6 Hz), 123.4, 122.8 (d, J = 14.5 Hz), 119.6 (d, J = 13.5 Hz), 112.8 (d, J = 27.2 Hz), 110.8, 73.9, 56.5, 47.7,

19 33.5, 30.4, 28.1, 26.7, 25.9, 23.3. F NMR (377 MHz, CDCl3)  = –109.60. IR 3593, 3441,

182

2964, 2927, 1597, 1574, 1409, 1207, 1090, 854, 824. ESI-MS calcd for C17H22BrFNaO2 + 25 [M+Na] 379.0679; found 379.0677. [α] D = –61.0 (c = 2.0, CHCl3).

Synthesis of 192

To a solution of the tertiary alcohol 191 (650 mg, 1.82 mmol, 1.00 equiv) in THF (18 mL) was added a solution of KHMDS in PhMe (5.5 mL, 2.73 mmol, 1.5 equiv) and the mixture was stirred at 65 °C for 2 h. After complete consumption of starting material remaining KHMDS was quenched by addition of sat. aq. NH4Cl. The aqueous layer was extracted with EtOAc, dried over Na2SO4 and concentrated under reduced pressure. The product was purified by flash chromatography (hexanes/CH2Cl2 9:1) to give the product (550 mg, 1.63 mmol, 90% yield).

1 H NMR (400 MHz, CDCl3)  = 6.63 (d, J = 1.9 Hz, 1H), 6.54 (d, J = 1.9 Hz, 1H), 6.15 – 6.12 (m, 1H), 3.83 (s, 3H), 3.17 – 3.06 (m, 1H), 2.19 – 2.10 (m, 2H), 1.94 – 1.86 (m, 1H), 1.70 –

13 1.61 (m, 4H), 1.40 (s, 3H), 1.38 – 1.32 (m, 1H), 1.05 (s, 3H). C NMR (101 MHz, CDCl3)  = 159.2, 155.5, 134.0, 124.2, 120.3, 114.0, 112.5, 106.3, 77.9, 55.7, 45.8, 34.0, 31.3, 27.6, 25.2, 23.5, 19.3. IR 3407, 2975, 2939, 1583, 1567, 1409, 1218, 1177, 1117, 901, 833, 759. ESI-MS + 25 calcd for C17H22BrNaO2 [M+H] 337.0798; found 337.0796. [α] D = –62.3 (c = 2.0, CHCl3).

Structural Confirmation of 192: Synthesis of known 196

Pentylmagnesium chloride (2 M in THF, 50 L, 0.10 mmol, 2.2 equiv) was added to a solution of 192 (15 mg, 0.044 mmol, 1.0 equiv) and Pd(PPh3)4 (5.1 mg, 0.0045 mmol, 0.10 equiv) in THF (0.9 mL) at rt. The reaction mixture was stirred overnight. Excess Grignard reagent was quenched by the addition of sat. aq. NH4Cl. The organic extracts were extracted with Et2O. The combined organic extracts were washed with brine, dried over MgSO4, filtered and concentrated. Flash chromatography on silica afforded the title compound as colorless oil (12 mg, 0.037 mmol, 82%). The NMR spectroscopic data was in perfect agreement with

KOBAYASHI’s report (see main body of the thesis).

Synthesis of 3-Br-THC

183

To a solution of 192 (275 mg, 0.815 mmol, 1.00 equiv) in N,N-dimethylformamide (16 mL) was added technical grade (~90%) sodium ethanethiolate (762 mg, 8.15 mmol. 10 equiv) and the mixture was heated at 140 °C for 3 h. After complete consumption of starting material, remaining NaSEt was quenched by addition of sat. aq. NH4Cl (1 mL). The aqueous layer was extracted with EtOAc, washed with sat. aq. LiCl, dried over Na2SO4 and concentrated under reduced pressure. The product was purified by flash chromatography (hexanes/CH2Cl2 3:2) to give Br-THC (227 mg, 0.702 mmol, 86% yield).

1 H NMR (400 MHz, CDCl3)  = 6.60 (d, J = 2.0 Hz, 1H), 6.47 (d, J = 1.9 Hz, 1H), 6.22 – 6.19 (m, 1H), 4.91 (s, 1H), 3.19 – 3.11 (m, 1H), 2.20 – 2.13 (m, 2H), 1.95 – 1.87 (m, 1H), 1.70 –

13 1.66 (m, 4H), 1.45 – 1.35 (m, 4H), 1.07 (s, 3H). C NMR (101 MHz, CDCl3)  = 156.0, 155.1, 135.1, 123.0, 119.9, 113.9, 111.2, 110.7, 78.1, 45.7, 33.7, 31.2, 27.6, 25.1, 23.5, 19.4. IR 3401, + 2976, 2929, 1599, 1577, 1411, 1181, 1113, 1041, 831, 761. ESI-MS calcd for C16H19BrO2 [M] 25 322.0563; found 322.0563. [α] D = –111.1 (c = 1.0, CHCl3).

Synthesis of TBS-3-Br-THC

3-Br-THC (25 mg, 0.077 mmol, 1.0 equiv) and 2,6-lutidine (14 L, 0.12 mmol, 1.5 equiv) were dissolved in CH2Cl2 (0.4 mL) and cooled to –78 °C. TBSOTf (20 L, 0.085 mmol, 1.1 equiv) was added. After 30 min, no conversion was observed on TLC. The reaction mixture was warmed to 0 °C. 2,6-Lutidine (37 L, 0.12 mmol, 4.0 equiv) and TBSOTf (55 L, 0.23 mmol, 3.0 equiv) were added and the reaction was stirred overnight at rt. After quenching with sat. aq. sodium bicarbonate, the organic extracts were extracted with Et2O. The combined organic extracts were washed with brine, dried over Na2SO4, filtered and concentrated. Flash chromatography on silica (hexanes to 2.5% EtOAc in hexanes) afforded the title compound as yellow oil (25 mg, 0.057 mmol, 74% yield).

1 H NMR (400 MHz, CDCl3)  = 6.62 (d, J = 2.0 Hz, 1H), 6.49 (d, J = 2.0 Hz, 1H), 6.27 – 6.22 (m, 1H), 3.10 – 3.02 (m, 1H), 2.18 – 2.11 (m, 2H), 1.95 – 1.86 (m, 1H), 1.65 – 1.63 (m, 3H), 1.70 – 1.52 (m, 1H), 1.39 (s, 3H), 1.44 – 1.30 (m, 1H), 1.05 (s, 3H), 1.01 (s, 9H), 0.28 (s, 3H),

184

13 0.17 (s, 3H). C NMR (101 MHz, CDCl3)  = 155.8, 155.4, 133.3, 124.6, 119.6, 115.3, 114.5, 114.2, 77.8, 45.7, 34.1, 31.2, 27.5, 25.9, 25.2, 23.2, 19.3, 18.4, -3.6, -4.2. IR 2957, 2929, 2859, 1582, 1571, 1464, 1409, 1253, 1180, 1116, 1064, 934, 924, 834, 780. HRMS (ESI+): m/z calcd

+ ퟐퟐ for C22H34BrO2Si [M+H] 437.1506, found 437.1509. [] 퐃 = –119 (c = 1.0, CHCl3).

Synthesis of 197

TBS-3-Br-THC (35 mg, 0.080 mmol, 1.0 equiv), tris(tert-butyl)phosphonium tetrafluoroborate (2.3 mg. 0.0080 mmol, 0.10 mmol), Pd(OAc)2 (1.8 mg, 0.0080 mmol, 0.10 equiv), tert-butyl 1-phenylhydrazinecarboxylate (22 mg, 0.10 mmol, 1.3 equiv) and

Cs2CO3 (37 mg, 0.11 mmol, 1.4 equiv) were combined in a 10 mL flask, which was evacuated and backfilled with N2 three times. Degassed PhMe (0.5 mL) was added. The flask was sealed and heated at 110 °C overnight. After cooling to rt, EtOAc (20 mL) was added. The organic extracts were washed with water and brine (2 mL each), dried over Na2SO4, filtered and concentrated. Flash chromatography on silica (15:1 hexanes/EtOAc) afforded the title compound as brown oil (40 mg, 0.071 mmol, 89% yield).

1 H NMR (400 MHz, CDCl3)  = 7.62 – 7.54 (m, 2H), 7.29 (dd, J = 8.7, 7.3 Hz, 2H), 7.13 – 7.05 (m, 1H), 6.29 – 6.26 (m, 1H), 6.20 (s, 1H), 5.94 (d, J = 2.3 Hz, 1H), 5.87 (d, J = 2.3 Hz, 1H), 3.04 (dt, J = 10.2, 2.4 Hz, 1H), 2.17 – 2.08 (m, 2H), 1.94 – 1.83 (m, 1H), 1.65 – 1.61 (m, 1H), 1.63 – 1.61 (m, 3H), 1.42 (s, 9H), 1.36 (s, 3H), 1.35 – 1.31 (m, 1H), 1.04 (s, 3H), 0.96 (s,

13 9H), 0.17 (s, 3H), 0.09 (s, 3H). C NMR (101 MHz, CDCl3) 155.7, 155.5, 154.1, 147.8, 143.0, 132.4, 128.5, 125.7, 124.5, 121.9, 108.9, 97.2, 95.8, 82.3, 77.2, 46.0, 34.0, 31.3, 28.3, 27.6, 26.0, 25.3, 23.2, 19.3, 18.4, -3.6, -4.1. IR 3344, 2958, 2929, 2859, 1717, 1616, 1598, 1482, 1368, 1304, 1252, 1182, 1155, 1066, 839, 779, 753, 691 HRMS (ESI+): m/z calcd for

+ ퟐퟑ C33H49N2O4Si [M+H] 565.3456, found 565.3463. [] 퐃 = –72 (c = 1.0, CHCl3).

Synthesis of 198

185

197 (33 mg, 0.058 mmol, 1.0 equiv) and 2,6-lutidine (34 L, 0.29 mmol, 5.0 equiv) were combined with dry CH2Cl2 (0.6 mL) and cooled to 0 °C. TMSOTf (42 L, 0.23 mmol,

4.0 equiv) was added and it was stirred at 0 °C for 1 h. NaHCO3 (49 mg, 0.58 mmol, 10 equiv) was added, followed by MeOH (3 mL). The mixture was filtered over celite and concentrated.

The residue was dissolved in MeOH (3 mL) and NaHCO3 (49 mg, 0.58 mmol, 10 equiv) was added. The yellow mixture was stirred open to air overnight, before it was diluted with EtOAc.

The organic extracts were washed with water and brine, dried over Na2SO4, filtered and concentrated. Flash chromatography on silica (gradient hexanes to hexanes/EtOAc 5:1) afforded the title compound as orange oil (14 mg, 0.030 mmol, 52% yield).

1 H NMR (400 MHz, CDCl3)  7.89 – 7.84 (m, 2H), 7.53 – 7.43 (m, 3H), 7.06 (d, J = 1.9 Hz, 1H), 6.99 (d, J = 1.9 Hz, 1H), 6.35 – 6.32 (m, 1H), 3.27 – 3.16 (m, 1H), 2.21 – 2.13 (m, 2H), 1.94 (dtd, J = 12.5, 4.8, 4.3, 2.4 Hz, 1H), 1.73 (ddd, J = 13.0, 11.1, 2.2 Hz, 1H), 1.68 – 1.66 (m, 3H), 1.45 (s, 3H), 1.43 – 1.39 (m, 1H), 1.11 (s, 3H), 1.04 (s, 9H), 0.35 (s, 3H), 0.22 (s, 3H). 13C

NMR (101 MHz, CDCl3)  155.4, 155.3, 152.8, 152.3, 133.3, 130.8, 129.1, 124.7, 123.0, 119.4, 106.1, 105.3, 77.5, 45.8, 34.7, 31.2, 27.6, 26.0, 25.3, 23.2, 19.3, 18.5, -3.5, -4.1. IR 2930, 2859, 1576, 1419, 1326, 1180, 1117, 1096, 1066, 838, 780, 690 HRMS (ESI+): m/z calcd for

+ ퟐퟐ C28H39N2O2Si [M+H] 463.2775, found 463.2778. [] 퐃 = –284 (c = 0.5, CHCl3).

Synthesis of azo-THC-1

198 (0.010 g, 0.022 mmol, 1.0 equiv) was dissolved in THF (0.2 mL) and cooled to 0 °C. A solution of TBAF in THF (1 M, 0.060 mL, 0.060 mmol, 2.7 equiv) was added. After 15 min, sat. aq. NaHCO3 and water (1.5 mL each) was added. The organic extracts were extracted with

CH2Cl2 (3x 20 mL), dried over Na2SO4, filtered and concentrated. Flash chromatography on silica (hexanes/EtOAc 10:1 to EtOAc) afforded the title compound as orange oil (7 mg, 0.02 mmol, 93% yield).

1 H NMR (400 MHz, CDCl3) 7.89 – 7.84 (m, 2H), 7.53 – 7.44 (m, 3H), 7.08 (d, J = 1.9 Hz, 1H), 6.90 (d, J = 1.9 Hz, 1H), 6.33 – 6.30 (m, 1H), 5.00 (s, 1H), 3.34 – 3.27 (m, 1H), 2.23 – 2.17 (m, 2H), 1.99 – 1.92 (m, 1H), 1.81 – 1.73 (m, 1H), 1.72 – 1.70 (m, 3H), 1.46 (s, 3H), 1.43

186

13 (d, J = 9.5 Hz, 1H), 1.13 (s, 3H). C NMR (101 MHz, CDCl3) 155.7, 155.0, 152.7, 152.4, 135.0, 131.0, 129.2, 123.2, 123.0, 107.1, 100.4, 77.9, 45.7, 34.3, 31.3, 29.9, 27.7, 25.2, 23.6, 19.5. IR 3377, 2926, 1580, 1446, 1421, 1326, 1181, 1133, 1114, 1054, 854, 767, 690 HRMS

+ ퟐퟐ (ESI+): m/z calcd for C22H25N2O2 [M+H] 349.1911, found 349.1911. [] 퐃 = –281 (c = 0.35,

CHCl3).

Synthesis of azo-THC-2

3-Br-THC (0.010 g, 0.031 mmmol, 1.0 equiv), 201 (14 mg, 0.049 mmol, 1.6 equiv),

PdCl2(dppf) (1.1 mg, 0.0015 mmol, 0.049 equiv) and Cs2CO3 (0.030 g, 0.093 mmol, 3.0 equiv) were combined with MeOH (0.5 mL). The flask was capped with a septum. After the atmosphere was replaced with N2 by evacuation/backfilling through a needle, the reaction mixture was placed in an oilbath preheated to 65 °C. After 1 h, TLC analysis indicated full consumption of starting material. The mixture was cooled to rt, diluted with Et2O (5 mL) and filtered over celite. Concentration followed by flash chromatography on silica (10% Et2O in hexanes) afforded the title compound as orange oil (7.0 mg, 0.016 mmol, 53% yield, ca. 10:1 dr).

1 Rf = 0.22 (25% Et2O in hexanes; UV, Seebach’s magic). H NMR (300 MHz, CDCl3)  7.99 – 7.89 (m, 4H), 7.74 – 7.63 (m, 2H), 7.57 – 7.46 (m, 3H), 6.77 (d, J = 1.7 Hz, 1H), 6.63 (d, J = 1.8 Hz, 1H), 6.36 – 6.30 (m, 1H), 4.97 (br s, 1H), 3.34 – 3.24 (m, 1H), 2.25 – 2.15 (m, 2H), 2.02 – 1.90 (m, 1H), 1.81 – 1.72 (m, 1H), 1.71 (dt, J = 2.4, 1.3 Hz, 3H), 1.46 (s, 3H), 1.50 –

13 1.40 (m, 1H), 1.15 (s, 3H). C NMR (75 MHz, CDCl3)  155.6, 155.0, 152.9, 151.9, 143.2, 139.8, 135.0, 131.1, 129.2, 127.5, 123.4, 123.3, 123.0, 111.9, 109.3, 106.2, 77.8, 45.9, 33.9, 31.3, 27.7, 25.2, 23.6, 19.5. IR 3374 (br), 3065, 2974, 2928, 2870, 1617, 1600, 1582, 1562, 1575, 1425, 1347, 1268, 1235, 1184, 1133, 1114, 1043, 918, 830, 768, 734, 638 cm-1. HRMS

+ ퟐퟓ (ESI+): m/z calcd for C28H29N2O2 [M+H] 425.2224, found 425.2227. [] 퐃 = –83.6 (c = 0.25,

CHCl3).

Synthesis of 206

187

3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (1.10 g, 5.02 mmol, 1.0 equiv) and nitrosobenzene (0.80 g, 7.5 mmol, 1.5 equiv) were dissolved in AcOH (10 mL) and heated to 90 °C for 3 h. After cooling to rt, the reaction mix was diluted with water (200 mL) and extracted with CH2Cl2 (3x 50 mL). The combined organic extracts were washed with sat. aq.

NaHCO3, water and brine (20 mL each), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (40% CH2Cl2 in hexanes) afforded two fractions. Fraction one (690 mg) contained product together with minor impurities, fraction two contained clean product as orange oil, that solidified upon standing (513 mg, 1.67 mmol, 33% yield).

1 Rf = 0.36 (50% CH2Cl2 in hexanes; yellow spot, UV, KMnO4). H NMR (400 MHz, CDCl3) 8.38 – 8.36 (m, 1H), 8.02 (ddd, J = 7.9, 2.1, 1.3 Hz, 1H), 7.97 – 7.91 (m, 3H), 7.57 – 7.45

13 (m, 4H), 1.39 (s, 12H). C NMR (101 MHz, CDCl3)  = 152.8, 152.2, 137.4, 131.0, 129.4, 129.2, 128.7, 125.5, 123.0, 84.2, 25.1. IR (neat) 3060, 2978, 2830, 1603, 1421, 1354, 1324, -1 + 1139, 699, 691 cm . HRMS (MALDI): m/z calcd for C18H21BN2O2 [M] 308.1694, found 308.1691.

Synthesis of 207

In a polyethylene flask, 206 (0.310 g, 1.00 mmol, 1.00 equiv) was dissolved in a 6:1 mixture of MeCN and water (8 mL) and potassium bifluoride (236 mg, 3.02 mmol, 3.02 equiv) was added.

After 3 h, NaHCO3 (127 mg, 1.51 mmol, 1.51 equiv) was added and it was stirred for 5 min. The mixture was transferred to a round-bottom-flask and the volatiles were removed. The resulting solid was extracted with acetone (4x 3 mL) and the extract was filtered over celite. Concentration afforded an orange oil. Addition of acetone followed by diethyl ether did not lead to precipitation. After some experimentation, the product could be precipitated from diethyl ether by the addition of MeCN to give 11 as orange solid (0.280 g, 0.972 mmol, 97% yield).

188

1H NMR (400 MHz, d6-acetone)  = 8.12 (s, 1H), 7.90 (d, J = 7.3 Hz, 2H), 7.66 (t, J = 7.6 Hz, 2H), 7.60 – 7.51 (m, 2H), 7.52 – 7.47 (m, 1H), 7.31 (t, J = 7.2 Hz, 1H). 13C NMR (101 MHz, d6-acetone)  = 153.9, 152.5, 136.2, 131.2, 130.0, 127.9, 127.7, 123.2, 120.1. 19F NMR (377 MHz, d6-acetone)  = –137.7. IR 1410, 1244, 1198, 1142, 936, 839, 799, 763, 698, 686, 606, -1 - 531 cm . HRMS (ESI-): m/z calcd for C12H9BF3N2 [M] 249.0819, found 249.0819.

Synthesis of azo-THC-3

Cs2CO3 (0.12 g, 0.37 mmol, 3.0 equiv), 207 (54 mg, 0.19 mmol, 1.5 equiv) and PdCl2(dppf) (5 mg, 0.007 mmol, 0.06 equiv) were placed in a round-bottom flask, which was evacuated and backfilled with N2 (3x). A solution of 3-Br-THC (40 mg, 0.12 mmol, 1.0 equiv) in degassed MeOH (1.2 mL) was added. The flask was sealed and placed in an oilbath preheated to 65 °C.

After 2 h, the reaction mixture was cooled to room temperature, diluted with Et2O, filtered over celite and concentrated. Flash chromatography on silica (10% Et2O in hexanes) afforded the E- isomer of the desired product. The column was eluted with EtOAc. Fractions containing cis- isomer were pooled with trans-isomer and concentrated. Further purification by preparative

TLC (30% Et2O in hexanes) afforded the title compound as orange wax (37 mg, 0.087 mmol, 70% yield, ca. 15:1 dr).

1 H NMR (400 MHz, CDCl3)  = 8.11 (t, J = 1.9 Hz, 1H), 7.96 – 7.93 (m, 2H), 7.87 (ddd, J = 7.9, 2.0, 1.2 Hz, 1H), 7.63 (dt, J = 7.8, 1.4 Hz, 1H), 7.52 (dddd, J = 13.4, 8.5, 4.5, 2.2 Hz, 4H), 6.80 (d, J = 1.8 Hz, 1H), 6.63 (d, J = 1.8 Hz, 1H), 6.36 (t, J = 1.7 Hz, 1H), 5.20 (s, 1H), 3.33 – 3.27 (m, 1H), 2.20 (dt, J = 8.6, 3.1 Hz, 2H), 1.95 (dtt, J = 12.5, 4.3, 2.3 Hz, 1H), 1.77 (ddd, J = 13.0, 11.0, 2.2 Hz, 1H), 1.71 (d, J = 1.0 Hz, 3H), 1.47 (s, 3H), 1.45 – 1.42 (m, 1H), 1.15 (s, 3H).

13 C NMR (101 MHz, CDCl3)  = 155.6, 155.0, 153.1, 152.8, 141.6, 140.0, 134.9, 131.2, 129.5, 129.4, 129.2, 123.5, 123.0, 122.3, 120.9, 111.6, 109.2, 106.3, 77.8, 45.9, 33.9, 31.3, 27.7, 25.2, 23.5, 19.5. IR 3382 (br), 3062, 2974, 2928, 2870, 1619, 1566, 1400, 1346, 1185, 1131, 1044, -1 + 846, 797, 761, 692 cm . HRMS (ESI+): m/z calcd for C28H29N2O2 [M+H] 425.2224, found ퟐퟑ 425.2223. [] 퐃 = –150 (c = 1.00, CHCl3).

Synthesis of azo-THC-4

189

2-Iodoazobenzene (0.31 g, 1.0 mmol, 1.0 equiv) was dissolved in dry Et2O (8 mL) and cooled to –100 °C (internal temperature control). n-BuLi (1.6 M in hexanes, 0.69 mL, 1.1 mmol, 1.1 equiv) was added dropwise. Upon completion of the addition, the reaction mixture was stirred for additional 20 min before a solution of trimethyl borate (125 mg, 1.20 mmol,

1.2 equiv) in Et2O (1 mL) was added. The cooling bath was removed. After 2 h, 2- (hydroxymethyl)-2-methylpropane-1,3-diol (0.12 g, 1.0 mmol, 1.0 equiv) was added and the resulting mixture was stirred overnight at rt. The volatiles were removed and Et2O (5 mL) was added. The suspension was sonicated for 2 min. Filtration and washing with additional Et2O afforded 213 as orange solid (0.16 g, 0.51 mmol, 50%). The material was used without further purification.

3-Br-THC (0.020 g, 0.062 mmol, 1.0 equiv) and lithium triolborate 213 (22 mg, 0.068 mmol,

1.1 equiv) were combined with DMF (0.5 mL) and water (0.1 mL). PdCl2(dppf) (2 mg, 0.002 mmol, 5 mol%) was added, the flask was capped and placed in an oilbath preheated to 65 °C. After 1 h, an additional portion of triolborate (22 mg, 0.068 mmol, 1.1 equiv) was added and it was stirred for further 3h, before a final portion of triolborate (22 mg, 0.068 mmol, 1.1 equiv) was added. When TLC analysis indicated full consumption of starting material, the reaction mixture was cooled to rt, diluted with Et2O (5 mL) and filtered over celite. It was further diluted with Et2O (30 mL) and washed with 5% aq. LiCl (3x 15 mL) and brine (15 mL).

The organic phase was dried over MgSO4, filtered and concentrated. Flash chromatography on silica (5% Et2O in pentane) afforded the title compound as orange oil (0.010 g, 0.024 mmol, 38%).

1 H NMR (600 MHz, C6D6)  = 7.99 – 7.94 (m, 2H), 7.91 – 7.87 (m, 1H), 7.49 – 7.46 (m, 1H), 7.14 (dd, J = 7.4, 1.7 Hz, 1H), 7.12 (dd, J = 7.7, 1.7 Hz, 1H), 7.11 – 7.06 (m, 2H), 7.04 – 7.02 (m, 1H), 7.02 – 7.01 (m, 1H), 6.74 – 6.72 (m, 1H), 6.03 (d, J = 1.8 Hz, 1H), 4.19 – 4.16 (m, 1H), 3.39 – 3.34 (m, 1H), 1.92 – 1.86 (m, 2H), 1.74 (ddd, J = 13.0, 11.2, 2.2 Hz, 1H), 1.68 (dq, J = 2.3, 1.0 Hz, 3H), 1.56 – 1.51 (m, 1H), 1.27 (s, 3H), 1.09 – 1.04 (m, 1H), 0.97 (s, 3H). 13C

NMR (151 MHz, C6D6)  = 155.4, 154.5, 153.6, 150.4, 141.7, 139.0, 133.1, 131.1, 131.0, 130.9, 129.3, 128.0, 125.0, 123.8, 116.5, 113.3, 111.4, 111.0, 77.2, 45.9, 34.5, 31.4, 27.7, 25.2, 23.6, 19.5. IR 3370 (br), 3061, 2974, 2928, 2870, 2831, 1618, 1579, 1561, 1406, 1184, 1042, 919,

190

+ 910, 774, 733, 688. HRMS (ESI+): m/z calcd for C28H29N2O2 [M+H] 425.2224, found 425.2225.

Synthesis of 203

4-Iodoaniline (1.47 g, 6.71 mmol, 1.00 equiv) was dissolved in EtOH and aq. HCl (2 M, 10 mL, 20 mmol, 3 equiv) and cooled to 0 °C. A solution of sodium nitrite (0.51 g, 7.4 mmol, 1.1 equiv) in water (8 mL) was added dropwise over 10 min. 5 min after completion of the addition, sulfamic acid (130 mg, 1.34 mmol, 0.2 equiv) was added to quench excess nitrite. In a second flask, diethylaniline (1.00 g, 6.71 mmol, 1.00 equiv) was combined with EtOH (110 mmol), water (55 mL) and sodium acetate (1.83 g, 13.4 mmol, 2.00 equiv) and cooled to 0 °C. To this was added the diazonium salt suspension at once and stirring continued for 1.5 h at 0 °C. The precipitated was collected by filtration, washed with icecold water and dried to yield the the title compound as orange solid (2.23 g, 5.88 mmol, 88%).

1 H NMR (400 MHz, CDCl3) δ 7.88 – 7.82 (m, 2H), 7.82 – 7.77 (m, 2H), 7.61 – 7.55 (m, 2H), 6.75 – 6.69 (m, 2H), 3.46 (q, J = 7.1 Hz, 4H), 1.23 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, -1 CDCl3) δ 152.8, 150.5, 143.1, 138.2, 125.7, 124.0, 111.1, 95.0, 44.9, 12.8 cm . IR 2970, 1596,

1513, 1402, 1351, 1270, 1134, 1077, 1002, 829. HRMS (ESI+): m/z calcd for C16H19IN3 [M+H]+ 380.0618, found 380.0619.

Synthesis of 204

203 (758 mg, 2.00 mmol, 1.00 equiv) was dissolved in Et2O (20 mL) and cooled to -100 °C (internal temp). As material crashed out during cooling, THF (15 mL) was added. When the temp was stable at -100 °C, BuLi (1.6 M, 1.38 mL, 2.20 mmol, 1.10 equiv) was added dropwise (each drop leading to a black precipiate). The dark suspension was stirred at that temperature for another 10 minutes, before a solution of B(OMe)3 in THF (2 mL) was added. The cooling bath was removed and the mixture allowed to warm to 0 °C, whereby it turned into a dark red solution (-40 °C), and then into a suspension again (-20 °C). A solution of KHF2 in water (5 mL) was added (resulting in a slight exotherm, temperature increased to 10 °C) turning the

191 suspension into a dark-red/black solution). The solvent was removed under reduced pressure. The residue was dissolved in a minimum amount of acetone and precipitated by the addition of

Et2O to yield the title compound as brown solid (532 mg, 1.48 mmol, 74%).

1H NMR (400 MHz, Acetone) δ 7.86 – 7.72 (m, 2H), 7.62 (s, 4H), 6.87 – 6.74 (m, 2H), 3.50 (q, J = 7.0 Hz, 4H), 1.21 (t, J = 7.0 Hz, 6H). 13C NMR (101 MHz, Acetone) δ 133.0, 125.4, 121.1, 111.9, 45.1, 12.9 (not all carbon atoms detected). 19F NMR (376 MHz, Acetone) δ 34.68. IR 2979, 1601, 1515, 1403, 1356, 1215, 1194, 1141, 956, 837 cm-1. HRMS (ESI-): m/z calcd - for C16H18BF3N3 [M] 320.1554, found 320.1551.

Synthesis of azo-THC-5

The compound was prepared in analogy to the procedure described for the preparation of azo- THC-2 from 3-Br-THC (16 mg, 0.050 mmol, 1.00 equiv) and 204 (27 mg, 0.75 mmol,

1.5 equiv) and isolated by preparative thin layer chromatography (hexanes:CH2Cl2:acetone 7:2:1) as dark red oil (13 mg, 0.026 mmol, 64%).

1 H NMR (400 MHz, CDCl3) δ 7.86 (dd, J = 9.9, 7.8 Hz, 4H), 7.67 – 7.58 (m, 2H), 6.75 (d, J = 1.8 Hz, 1H), 6.73 (d, J = 8.9 Hz, 2H), 6.59 (d, J = 1.8 Hz, 1H), 6.35 (t, J = 1.7 Hz, 1H), 5.23 (s, 1H), 3.46 (q, J = 7.1 Hz, 4H), 3.29 (dt, J = 10.9, 2.3 Hz, 1H), 2.24 – 2.15 (m, 2H), 1.95 (dtt, J = 12.4, 4.3, 2.3 Hz, 1H), 1.80 – 1.73 (m, 1H), 1.70 (dd, J = 2.3, 1.3 Hz, 3H), 1.46 (s, 3H), 1.51 – 1.38 (m, 1H), 1.23 (t, J = 7.1 Hz, 6H), 1.14 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 155.5, 155.0, 152.6, 150.3, 143.4, 141.3, 140.1, 134.7, 127.4, 125.5, 123.5, 122.6, 111.4, 111.2, 109.1, 106.2, 77.7, 45.9, 44.9, 33.9, 31.3, 27.7, 25.2, 23.5, 19.5, 12.8. IR 3370, 2972, 2927, 1597, 1559, 1515, 1401, 1390, 1351, 1270, 1137, 1077, 1044, 919, 826, 732. HRMS (ESI+): m/z + calcd for C32H38N3O2 [M+H] 496.2959, found 496.2956.

Synthesis of 209

192

The title compound was prepared in analogy to the synthesis of 203 from 3-Br-aniline (876 mg, 5.1 mmol, 1.0 equiv) and diethylaniline (600 mg, 4.0 mmol, 0.8 equiv) and isolated as red oil after flash chromatography on silica (20% CH2Cl2 in hexanes) in 79% yield.

1 H NMR (400 MHz, CDCl3) δ 7.98 (t, J = 1.9 Hz, 1H), 7.89 – 7.84 (m, 2H), 7.78 (ddd, J = 7.9, 1.8, 1.0 Hz, 1H), 7.48 (ddd, J = 7.9, 2.0, 1.0 Hz, 1H), 7.34 (t, J = 7.9 Hz, 1H), 6.76 – 6.70 (m, 13 2H), 3.46 (q, J = 7.1 Hz, 4H), 1.24 (t, J = 7.1 Hz, 6H). C NMR (101 MHz, CDCl3) δ 154.4, 150.5, 142.9, 131.6, 130.2, 125.7, 123.9, 123.0, 122.2, 111.0, 44.8, 12.7. IR 3001, 1599, 1515, -1 + 1393, 1354, 1270, 1137, 821 cm . HRMS (ESI+): m/z calcd for C16H19BrN3 [M+H] 332.0757, found 332.0763.

Synthesis of 210

209 (847 mg, 2.55 mmol, 1.00 equiv) was combined with PdCl2(dppf) (56 mg, 0.076 mmol,

3 mol%), KOAc (751 mg, 7.65 mmol, 3.00 equiv), B2pin2 (712 mg, 2.80 mmol, 1.10 equiv) and DMSO (8.5 mL) and heated at 80 °C overnight. After cooling, the mixture was diluted with water (200 mL) and extracted with EtOAc (3x 50 mL). The combined organic extracts were washed with brine (50 mL), dried over MgSO4, filtered and concentrated. Flash chromatography on silica (50% CH2Cl2 in hexanes to pure CH2Cl2) afforded the title compound as organge solid (820 mg, 2.16 mmol, 85%).

1 H NMR (400 MHz, CDCl3) δ 8.26 (s, 1H), 7.91 (ddd, J = 8.0, 2.2, 1.3 Hz, 1H), 7.89 – 7.84 (m, 2H), 7.81 (dt, J = 7.2, 1.2 Hz, 1H), 7.47 (ddd, J = 7.9, 7.3, 0.5 Hz, 1H), 3.45 (q, J = 7.1 Hz, 13 4H), 1.37 (s, 12H), 1.23 (t, J = 7.1 Hz, 6H). C NMR (101 MHz, CDCl3) δ 152.7, 150.1, 143.2, 135.5, 128.6, 128.4, 125.3, 124.7, 110.9, 83.9, 44.7, 25.0, 24.9, 12.7. IR 2976, 1599, 1515, -1 + 1397, 1353, 1142, 822, 703 cm . HRMS (ESI+): m/z calcd for C22H31BN3O2 [M+H] 380.2508, found 380.2508.

Synthesis of 211

193

210 (387 mg, 1.02 mmol, 1.00 equiv) was combined with MeCN (7 mL), water (1.2 mL) and

KHF2 (239 mg, 3.06 mmol, 3.00 equiv) and stirred for 1 h. NaHCO3 (129 mg, 1.53 mmol, 1.5 equiv) was added and the solvent was removed under reduced pressure. The resulting solid was extracted with acetone (4x 3 mL), and the extract was filtered over celite. Concentration afforded an orange oil. The oil was dissolved in acetone and the product precipitated by the addition of Et2O. The precipitate was collected by filtration to give the product as an orange solid (337 mg, 0.94 mmol, 92%).

1H NMR (400 MHz, Acetone) δ 8.05 – 8.01 (m, 1H), 7.83 – 7.78 (m, 2H), 7.55 (dd, J = 7.9, 2.1 Hz, 2H), 7.24 (t, J = 7.5 Hz, 1H), 6.85 – 6.78 (m, 2H), 3.50 (q, J = 7.0 Hz, 4H), 1.21 (t, J = 7.0 Hz, 6H). 13C NMR (101 MHz, Acetone) δ 152.9, 150.6, 144.1, 134.3, 134.3, 127.6, 127.2, 127.2, 125.4, 119.4, 111.9, 45.1, 12.9. 19F NMR (376 MHz, Acetone) δ 34.7. IR 3623, 2973, 1693, 1600, 1515, 1397, 1355, 1271, 1222, 1152, 1008, 821 cm-1. HRMS (ESI-): m/z calcd for - C16H18BF3N3 [M] 320.1554, found 320.1558.

194

Synthesis of azo-THC-6

The compound was prepared in analogy to the procedure described for the preparation of azo- THC-2 from 3-Br-THC (30 mg, 0.093 mmol, 1.00 equiv) and 211 (50 mg, 0.139 mmol,

1.5 equiv) and isolated by flash chromatography on silica (10% Et2O in hexanes) as orange oil (22 mg, 0.044 mmol, 48%).

1 Rf = 0.37 (20% EtOAc in hexanes; UV, red spot, CAN). H NMR (400 MHz, CDCl3) δ 8.02 (t, J = 1.8 Hz, 1H), 7.88 (d, J = 9.2 Hz, 2H), 7.77 (ddd, J = 7.7, 2.0, 1.3 Hz, 1H), 7.52 (dt, J = 7.7, 1.5 Hz, 1H), 7.46 (t, J = 7.7 Hz, 1H), 6.79 (d, J = 1.8 Hz, 1H), 6.76 – 6.71 (m, 2H), 6.62 (d, J = 1.8 Hz, 1H), 6.38 – 6.35 (m, 1H), 5.30 – 5.25 (m, 1H), 3.45 (q, J = 7.1 Hz, 4H), 3.29 (dt, J = 10.9, 2.3 Hz, 1H), 2.19 (dd, J = 6.6, 3.2 Hz, 2H), 1.98 – 1.91 (m, 1H), 1.76 (ddd, J = 12.9, 10.9, 2.2 Hz, 1H), 1.70 (dd, J = 2.4, 1.3 Hz, 3H), 1.46 (s, 3H), 1.48 – 1.42 (m, 1H), 1.23 (t, J = 13 7.1 Hz, 6H), 1.14 (s, 3H). C NMR (101 MHz, CDCl3) δ 155.3, 154.8, 153.6, 150.2, 143.1, 141.2, 140.3, 134.5, 129.1, 127.5, 125.4, 123.5, 121.4, 120.3, 111.2, 111.0, 109.0, 106.2, 77.5, 45.8, 44.7, 33.8, 31.2, 27.6, 25.0, 23.4, 19.4, 12.7. IR 3380 (br), 2973, 2929, 1598, 1565, 1515, -1 + 1397, 1353, 1271, 1137, 821 cm . HRMS (ESI+): m/z calcd for C32H38N3O2 [M+H] 496.2959, ퟐퟒ found 496.2959. [] 퐃 = –184 (c = 0.58, CHCl3).

Synthesis of 215

The title compound was prepared in analogy to the synthesis of 203 from 2-iodoaniline (1.28 g, 5.84 mmol, 1.00 equiv) and diethylaniline (872 mg, 5.84 mmol, 1.00 equiv) and isolated as dark red oil after flash chromatography on silica (20% CH2Cl2 in hexanes) in 79% yield.

1 H NMR (400 MHz, CDCl3) δ 7.97 (dd, J = 7.9, 1.3 Hz, 1H), 7.95 – 7.91 (m, 2H), 7.60 (dd, J = 8.0, 1.6 Hz, 1H), 7.41 – 7.35 (m, 1H), 7.05 (ddd, J = 7.9, 7.2, 1.6 Hz, 1H), 6.77 – 6.71 (m, 13 2H), 3.46 (q, J = 7.1 Hz, 4H), 1.24 (t, J = 7.1 Hz, 6H). C NMR (101 MHz, CDCl3) δ 152.0, 150.7, 143.3, 139.6, 130.5, 128.9, 126.3, 117.4, 111.1, 101.4, 44.9, 12.8. IR 2971, 1594, 1513, 1451, 1391, 1351, 1270, 1249, 1138, 1105, 1077, 1014, 821, 759 cm-1. HRMS (ESI+): m/z + calcd for C16H19IN3 [M+H] 380.0618, found 380.0617.

195

Synthesis of azo-THC-7

215 (0.37 g, 1.0 mmol, 1.0 equiv) was dissolved in dry Et2O (8 mL) and cooled to –100 °C

(internal temperature control). n-BuLi (1.6 M in hexanes, 0.69 mL, 1.1 mmol, 1.1 equiv) was added dropwise. Upon completion of the addition, the reaction mixture was stirred for additional 20 min before a solution of trimethyl borate (125 mg, 1.20 mmol, 1.2 equiv) in Et2O (1 mL) was added. The cooling bath was removed. After 2 h, 2-(hydroxymethyl)-2- methylpropane-1,3-diol (0.12 g, 1.0 mmol, 1.0 equiv) was added and the resulting mixture was stirred overnight at rt. The volatiles were removed and Et2O (5 mL) was added. The suspension was sonicated for 2 min. Filtration and washing with additional Et2O afforded 216 as orange solid (0.176 g, 0.46 mmol, 46%). The material was used without further purification.

3-Br-THC (0.024 g, 0.074 mmol, 1.0 equiv) and lithium triolborate 216 (32 mg, 0.082 mmol,

1.1 equiv) were combined with DMF (0.6 mL) and water (0.1 mL). PdCl2(dppf) (2 mg, 0.002 mmol, 5 mol%) was added, the flask was capped and placed in an oilbath preheated to 65 °C. After 1 h, an additional portion of triolborate (32 mg, 0.088 mmol, 1.1 equiv) was added and it was stirred for further 3 h. The reaction mixture was cooled to rt, diluted with Et2O (5 mL) and filtered over celite. It was further diluted with Et2O (30 mL) and washed with 5% aq. LiCl

(3x 15 mL) and brine (15 mL). The organic phase was dried over MgSO4, filtered and concentrated. Preparative thin layer chromatography (7% acetone in hexanes, developed three times) and flash chromatography on silica (pipette column, 10% acetone in hexanes) afforded the title compound as orange oil (0.011 g, 0.022 mmol, 30%).

1 H NMR (300 MHz, CDCl3) δ 7.78 (d, J = 9.2 Hz, 2H), 7.68 – 7.59 (m, 1H), 7.53 – 7.46 (m, 1H), 7.41 – 7.33 (m, 2H), 6.69 (d, J = 9.2 Hz, 2H), 6.63 (d, J = 1.7 Hz, 1H), 6.42 – 6.38 (m, 1H), 6.37 (d, J = 1.7 Hz, 1H), 4.92 (s, 1H), 3.44 (q, J = 7.1 Hz, 4H), 3.35 – 3.26 (m, 1H), 2.25 – 2.15 (m, 2H), 2.02 – 1.90 (m, 1H), 1.78 (ddd, J = 13.0, 11.0, 2.2 Hz, 1H), 1.71 (dd, J = 2.3, 1.3 Hz, 3H), 1.54 – 1.36 (m, 1H), 1.44 (s, 3H), 1.22 (t, J = 7.1 Hz, 6H), 1.15 (s, 3H). 13C NMR

(75 MHz, CDCl3) δ 154.6, 153.6, 150.6, 150.0, 143.8, 138.9, 138.8, 134.4, 130.5, 129.1, 128.0, 125.8, 123.9, 116.3, 112.7, 111.1, 111.0, 110.7, 77.4, 45.9, 44.8, 34.0, 31.4, 27.7, 25.3, 23.6,

196

19.6, 12.8. IR 3370, 2979, 2926, 1598, 1565, 1515, 1397, 1354, 1270, 1246, 1144, 1045, 922, -1 + 821, 831 cm . HRMS (ESI+): m/z calcd for C32H38N3O2 [M+H] 496.2959, found 496.2954.

17 Biological Assays Two-step covalent SDS-PAGE visualization of LEI121 labeling of CB2R194 From ref. 194: WT CHO and CB2R membrane aliquots were prepared as described previously.195 Protein concentrations of both membrane aliquots were diluted to 2.8 µg/µL or to an otherwise stated concentration. Membranes were homogenized for 20 seconds with a Heidolph Silent crusher at 25.000 rpm, and benzonase was added (1:10.000 dilution from working stock of 2500000 U/ mL). 18 µL of protein was added per well of a 96-well flatbottom plate and 1 µL 20x concentrated competitor or MilliQ water with the same % of DMSO was added, but the samples without UV were kept in Eppendorf tubes protected with alumina foil. Samples with inactive protein were denatured with 2 µL 10% SDS before addition of competitor. After incubation of 30 min at rt, 1 µL 40 µM LEI121 or MilliQ water with the same % of DMSO was added, and the protein was again incubated for 30 min at rt. The samples were then diluted with 30 µL 50 mM Hepes buffer and irradiated for 5 min with CaproboxTM, preset at 350 nm. The ligation reaction was then performed with 5 µL click master mix per sample, which is prepared as follows: 2.5 µL 10 mM CuSO4 and 1.5 µL 100 mM NaAsc were mixed together until the the copper is fully reduced (visible by the change from the rusty brown color to bright yellow), then 0.5 µL 10 mM THPTA and 0.5 µL 0.4 mM CY5-N3 (14) was added. Samples without click mix received MilliQ with the same % of DMSO. After incubation in the dark for 1 hr, the protein was denatured with 18 µL (or 16 µL in case of the already denatured sample) 4x Laemmli sample buffer, and the samples were resolved on a 12.5% acrylamide gel (12 µL per sample per well). When PNGase F was used, 1 µL of 500000 U/mL or 1 µL MilliQ was added after the ligation step, followed by incubation at 37°C for 1 hr, before denaturation and gel electrophoresis.

Radioligand binding assays196

194 These experiments were performed by Marjolein Soethoudt at Leiden University. More details can be found in a joint publication: M. Soethoudt, S. C. Stolze, M. V. Westphal, L. van Stralen, A. Martella, E. J. van Rooden, W. Guba, Z. V. Varga, H. Deng, S. I. van Kasteren, U. Grether, A. P. IJzerman, P. Pacher, E. M. Carreira, H. S. Overkleeft, A. Ioan-Facsinay, L. H. Heitman, M. van der Stelt, J. Am. Chem. Soc. 2018, Article ASAP, doi: 10.1021/jacs.7b11281. 195 M. Soethoudt, U. Grether, J. Fingerle, T. W. Grim, F. Fezza, L. de Petrocellis, C. Ullmer, B. Rothenhäusler, C. Perret, N. van Gils, D. Finlay, C. MacDonald, A. Chicca, M. D. Gens, J. Stuart, H. de Vries, N. Mastrangelo, L. Xia, G. Alachouzos, M. P. Baggelaar, A. Martella, E. D. Mock, H. Deng, L. H. Heitman, M. Connor, V. Di Marzo, J. Gertsch, A. H. Lichtman, M. Maccarrone, P. Pacher, M. Glass, M. van der Stelt, Nat. Commun. 2017, 8, 13958. 196 These experiments were performed in the laboratory of Dr. Christoph Ullmer, F. Hoffmann-La Roche.

197

Binding assays were performed by displacement with [3H]-CP55940 using membrane fractions of CHO cells expressing recombinant human CB2R or CB1R (hCB2R and hCB1R respectively).

Mouse spleen was used as a source of CB2R (Perkin Elmer). Membrane aliquots containing 5 mg (CHOK1hCB1_bgal) or 1 mg (CHOK1hCB2_bgal) of membrane protein in 100 µL assay buffer (50 mM Tris-HCl, 5 mM MgCl2, 0.1% bovine serum albumin (BSA), pH 7.4) were 3 incubated at 30 °C for 1 h, in presence of 3.5 nM [ H]CP55940 (CHOK1hCB1_bgal) or 1.5 nM 3 [ H]CP55940 (CHOK1hCB2_bgal). Incubation was terminated by rapid filtration performed on GF/C filters (Whatman International, Maidstone, UK), presoaked for 30 min with 0.25% polyethylene imine (PEI), using a Brandel harvester (Brandel, Gaithersburg, MD, USA). Filter- bound radioactivity was determined by scintillation spectrometry using a Tri-Carb 2900 TR liquid scintillation counter (Perkin Elmer, Boston, MA, USA). Nonspecific binding was determined in the presence of 1 mM ‘cold’ agonist. Ligand Ki values were calculated from a single experiment using triplicates of 10 different concentrations of the compound.

CB2 binding kinetics197 Adapted from ref. 197: Competition association experiments with [3H]RO6957022. The kinetic parameters of unlabeled competitor ligands were determined using the competition association assay as described by Motulsky and Mahan.198 CHO-K1_hCB2 membranes (1.5 µg per well) were incubated in assay buffer at 25 °C with a fixed amount of [3H]RO6957022 (3 nM) at different time points between 0 and 90 min in either absence (control) or presence of an unlabeled competing ligand. Assay validation was performed by homologous competition association (see ref. 197, results section, Fig. 3). IC50 concentrations of unlabeled competitor ligands were used to obtain approximately 50% displacement of the radioligand after 90 min incubation with [3H]RO6957022. Appropriate vehicle controls (i.e. DMSO, ethanol and Tocrisolve™) were used according to the solvent used for each ligand. To prevent degradation of the endocannabinoids during the assay, 1 µM of phenylmethylsulfonyl fluoride (PMSF) was added to membrane preparations 30 min in advance of the assay. Harvesting and counting procedures were performed as described in ref. 197, section “Saturation binding experiments with [3H]RO6957022”.

CB2 washout with subsequent determination of total specific binding (Bmax) of radiolabel197

197 These experiments were performed by. Dr. Andrea Martella at Leiden University. For a validation of the respective radioligand, see A. Martella, H. Sijben, A. C. Rufer, U. Grether, J. Fingerle, C. Ullmer, T. Hartung, A. P. IJzerman, M. van der Stelt, L. H. Heitman, Mol. Pharmacol. 2017, 92, 389. 198 H. J. Motulsky, L. C. Mahan, Mol. Pharmacol. 1984, 25, 1.

198

Membrane preparations of CB2 overexpressing CHO cells are incubated with excess CB2 binders followed by extensive washing steps to remove any unbound ligand. Subsequent determination of total specific binding of a radiolabeled compound and comparison with data from the same experiment without preincubation allows for an estimation of reversible or irreversible binding of CB2 ligands. Total specific binding of radiolabel should approach zero if preincubation is performed with an irreversible binder.